Keynote speakers

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Michael Bonkowski*1, Maike Hünninghaus1, Dörte Dibbern2, Robert Koller3, Tim Urich4, ...... Christopher Franco*1, Hoang Xuyen Le1, Ross Ballard2 ...... Lucas Dantas Lopes*, Michele de Cássia Pereira e Silva, Luana Bresciani, Fernando Dini ...... 1APTA -IAC, Brazil, 2Syngenta Company, Brazil, 3Moura Lacerda.
Keynote speakers Sunday 21 June – 15:45-16:15 From the foundations of rhizosphere research in the 19th century to the present high-tech bioanalytical based research approaches Anton Hartmann* Helmholtz Zentrum München, German Research Center for Environmental Health, Department Environmental Sciences, Research Unit Microbe-Plant Interactions, Germany When Lorenz Hiltner coined the term “rhizosphere” in 1904, there was already a profound and in part controversial discussion of concepts and original discoveries about organismic interactions of plants with microbes. In 1866, the botanist Anton de Bary called for the first time pathogenic fungi living inside leaves “endophytes”. In 1878, he suggested the term “symbiosis” for systems, when different types of organisms are living closely together. In 1887, the French botanist M.L.V. Galippe presented evidences for microorganisms living inside healthy plant tissues. One year later, Hellriegel and Wilfarth published their groundbreaking studies on the exceptional mineral N-independence of leguminous plants and the importance of endophytic bacteria within the root nodules. The functional role of symbiotic interactions of Rhizobia with legumes was one major research focus of L. Hiltner in his early research work in the 1890s. Thus, the “rhizosphere” concept about the important roles of rhizosphere microbes for both plant nutrition and health was a synopsis of Hiltner´s own research experience and the current concepts and knowledge. The further development of the rhizosphere concept for plant nutrition and applied soil chemistry was quite retarded, because soil scientists were still thinking on nutrient fluxes to the plant according to equilibrium equations and not according to dynamic gradients of quite different qualities. According to Liebig´s law of limiting nutrient supply, the application of mineral fertilizer brought great improvement of yields, but the involvement of specific rhizosphere processes and microbes were disregarded. The scientific knowledge about specific rhizosphere functions was further developed in the second half of the 20th century. The “green revolution” and the breeding of high yielding crop varieties brought secured crop yields, but specific rhizosphere processes were again neglected and many environmental problems were created. Since about two decades, the application of molecular genetic methods brought even more profound insights into the microbial community structure, localization and interaction in the rhizosphere. A “second green revolution” needs to come to implement the much enhanced (micro)-biological knowledge about root-soil interactions and the prominent role of microberoot interactions to improve plant crop growth and health in an efficient and sustainable way.

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Keynote speakers Sunday 21 June – 16:15-16:45 Back to the Roots: microbiology & chemistry at the root-soil interface Jos Raaijmakers* Netherlands Institute of Ecology, Netherlands Plant roots are colonized by an astounding number of microorganisms that can reach cell densities much greater than the number of plant cells. Various studies have shown that members of the rhizosphere microbiome can have profound effects on seedling vigour, plant growth and development, nutrition and tolerance to pathogens and abiotic stress. For the vast majority of rhizosphere microorganisms, however, there is limited knowledge on the mechanisms involved in modulation of plant growth and plant health. Novel ‘omics technologies have provided more in-depth knowledge of the diversity and functioning of the rhizosphere microbiome and significant advances are being made to uncover mechanisms, genes and metabolites involved in the multitrophic interactions in the rhizosphere. To better understand this intriguing complexity, both reductionists’ and systems ecology approaches are needed to identify the biotic and abiotic factors involved in microbiome assembly and activity. Here, new results are presented on how rhizosphere bacteria impact on root architecture, root growth and tolerance to soil-borne pathogens. For the rhizosphere bacteria, we showed that representatives of the y-Proteobacteria protect plants from pathogen infection by the production of chlorinated peptides and alter root architecture and plant growth via modulation of sulfur assimilation. For the β-Proteobacteria, comparative genomics, genetics, and chemical analyses revealed that specific volatile organic compounds (VOCs) inhibit pathogen growth. An overview will be given on the wealth of yet unknown functions and the metabolic potential of the rhizosphere microbiome.

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Keynote speakers Sunday 21 June – 16:45-17:15 Tracking trade in the rhizosphere Toby Kiers* VU Amsterdam, Netherlands Associating with microbes in the rhizosphere can have both costs and benefits for host plants. Because associations generally involve multiple microbial genotypes varying in mutualistic benefit, a potential tragedy of the commons can arise. How do plants maintain cooperation with the most beneficial rhizosphere microbes over the course of evolution? Specific host mechanisms may be employed that reduce the fitness benefits to microbes from defection. However in some rhizosphere mutualisms, biological markets have evolved in which control is bidirectional. A major aim of our group is to study these markets by quantitatively tracking the trade of carbon and nutrients between the hosts and microbes. We are exploring the use of quantum dot technology to track trading strategies across space and time. Ultimately, we are interested in understanding how cooperation is maintained among plant hosts and their symbiotic microbial communities.

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Keynote speakers Monday 22 June – 08:30-09:00 A microbial green revolution: Breeding microbes to feed the world Ian Sanders* University of Lausanne, Switzerland Today we face an unprecedented challenge to feed the growing global human population that can only be achieved with major changes in how we combine science with agronomy. The green revolution is thought to have saved approximately 1 billion lives and was largely fuelled by effective plant genetic improvement (breeding) programs. All plants live with microbes and it is well known that many rhizosphere microbes can be beneficial for plant growth. However, microbiologists have largely ignored the principals used by plant breeders of taking naturally occurring genetic variation as a base to genetically improve microorganisms to increase crop production. Mycorrhizal fungi form symbioses with all our major crops. They help plants obtain phosphate from the soil; an essential nutrient that limits crop production in the tropics. In recent years, our group, has shown that naturally occurring processes in these fungi can be used to develop genetically novel varieties. These novel fungal genotypes have been shown to induce enormous changes in rice growth (up to five fold) in greenhouse conditions. Cassava is an obvious target for the application of mycorrhizal fungi. Cassava is globally important, annually feeding almost a billion people in 105 countries. It is an important crop for subsistence farming throughout tropical and subtropical regions for smallholder farmers, but especially in sub-Saharan Africa. In this presentation, I will demonstrate that genetic improvement of mycorrhizal fungi in a lab-based in vitro system can be realistically and practically used to achieve large increases in cassava production in real farming situations in tropical regions. Our research demonstrates the power of using microbial genetics to improve food production and that this could potentially lead to more rapid improvements than conventional genetic plant improvement programs. Furthermore, variation in growth response of crop varieties to inoculation with mycorrhizal fungi likely has a genetic basis and responsiveness to these microbes is a trait that could also be incorporated into crop breeding programs leading to further increases.

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Keynote speakers Monday 22 June – 14:00-14:30 Titrating complexity: Root-associated bacterial communities in wild and simplified synthetic ecosystems Jeff Dangl* HHMI and Univ. of North Carolina, United States Plant-roots harbor a rich, non-random bacterial community in their roots, which arguably results when the plant selects a subset of microbes from the surrounding soil, combined with microbe-microbe competition for access to the niche provided by the plant tissue. Evidence from culture independent surveys in wild soils indicates some degree of soil specificity over the bacterial community that assembles on wild-type roots, which suggests fine tuning by the plant in response to different conditions. Fully elucidating the role of the plant in modulating bacterial assembly requires controlling both the bacterial community and the abiotic factors in the system. We have performed microcosm reconstitution experiments by inoculating plants with a complex, but well defined, bacterial communities of dozens of isolates, while systematically varying nutrient composition in a soil-like substrate with standard physical properties. Monitoring of plant phenotypes has established the effect of the bacterial community on plant health, while sequencing allows for unambiguous and accurate quantification of the final communities in the root. We have defined a set of robust bacterial root colonizers, and nutrient perturbation experiments will lead to the identification of bacterial strains that may help the plant to cope with nutrient stress. In parallel, we are performing comparative genomics analysis on root isolates, and matching functional genomic information to bacterial phenotypes (such as colonization) and to plant phenotypes. In summary, our approach will allow us to identify the rules that govern bacterial colonization of the plant in response to a variety of nutrient conditions, and will ultimately lead to a functional characterization of the root microbiome.

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Keynote speakers Monday 22 June – 14:30-15:00 Going underground: the role of roots in ecosystem response to climate change Franciska de Vries* The University of Manchester, United Kingdom Plant-soil interactions are central to determining the impact of climate change on ecosystem functioning. Climate-induced changes in the growth and structure of plant communities will alter the amount and composition of plant-derived carbon entering the soil via litter and root exudates; this in turn can have cascading effects on soil organisms and the processes they perform. Ecologists are increasingly using plant functional traits for predicting the response of plant communities to climate change, and their effect on soil communities and processes. Most research to date has focussed on aboveground traits however, while recent evidence points to belowground traits as important drivers of many ecosystem processes. I will present three lines of evidence that point to the crucial role of plant roots in the response of belowground organisms and processes to drought. First, in a glasshouse experiment, we found that the presence of a plant increased the recovery of several soil microbial and faunal groups after extreme drought. This recovery was linked to the amount of labile C in the soil solution; presumably originating from plant root exudates. In a second glasshouse-based experiment, we found that four species with contrasting root systems differed in their root plasticity under drought, with cascading effects on microbial biomass and soil N and C availability. Finally, I will show results from a field-based mesocosm experiment, in which we imposed a drought on plant communities that varied in abundance of the same four plant species with contrasting root systems. We found that plant communities modified soil communities both directly and indirectly, through roots and their impact on soil abiotic properties, with cascading effects on ecosystem processes. I conclude that plants, through their root traits and belowground C inputs, drive ecosystem response to drought.

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Keynote speakers Tuesday 23 June – 08:30-09:00 Bacterial volatiles and plant health Choong-Min Ryu* KRIBB, South Korea Certain plant growth-promoting rhizobacteria (PGPR) elicit induced systemic resistance (ISR) and plant growth promotion in the absence of physical contact with plants via bacterial olatile compound (BVC) emissions. In this presentation, I review the recent progess made by research into the interactions between PGPR and BVC, focusing on BVC emission by PGPR strains in plants. Particular attention will be given to the mechanisms by which these bacterial species elicit ISR. We provide an overview of recent progress in the elucidation of PGPR BVC interactions from studies utilizing transcriptome, metabolome and proteome analyses. By monitoring defense gene expression patterns, performing 2-dimensional electrophoresis, and studying defense signaling null mutants, salicylic acid and ethylene were found to be key players in plant signaling pathways involved in the ISR response. Bacterial VOCs also confer induced systemic tolerance to abiotic stresses, such as drought and heavy metals. The current analytical approaches for PGPR volatiles profiling is also provided with needed future developments. Furthermore, to assess potential utilization of PGPR VOCs for crop plants, volatile suspensions were applied to crops like pepper and cucumber and were found to be effective at protecting plants against plant pathogens and insect pests in the field. Taken together, these studies provide further insights into the biological and ecological potential of PGPR VOCs for enhancing plant self-immunity and/or adaptation to biotic and abiotic stresses in modern agriculture.

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Keynote speakers Tuesday 23 June 14:00-14:30 Identification and Isolation of Genes that Influence Laccaria Colonization in Populus Gerald Tuskan*1, David Weston1, Mike Robeson1, Dale Pelletier1, Christopher Warren Schadt1, Wellington Muchero1, Rytas Vilgalys2, Jonathan Plett3, Francis Martin3, Anthony Bryan1, Gregory Bonito2, Tae-Hyuk Ahn1, Chongle Pan1, Jessy Labbé1, Timothy Tschaplinski1, Xiaohan Yang1, Hengfu Yin1, Kerrie Barry4, Jeremy Schmutz5 1 Oak Ridge National Laboratory, United States, 2Duke University, United States, 3INRA, Nancy, France, 4Joint Genome Institute, United States, 5HudsonAlpha, United States There is a hidden molecular language that plants and microbes use to communicate. Proteins, metabolites and possibly even RNAs are exchanged between plants, bacteria and fungi. The beneficial, commensal, detrimental or neutral outcome is determined through such signals prior to intimate contact among the communal participants. Small secreted protein discovery, subcellular localization, yeast secretion assays and Laccaria nuclear localization are presented. Results indicate that there are several hundred small proteins produced in Populus that have the ability to move into Laccaria, with a subset that invokes anatomical changes in hyphal growth. In addition, using a GWAS approaches, containing 1084 unrelated Populus genotypes clonally replicated in four contrasting environments, with each genotype resequenced to a minimum 18X depth, for identifying genes involved in Laccaria colonization, we created a candidate gene list, validated this subset in a QTL population and identified a D-mannose lectin kinase that was wholly correlated with successful colonization. Overexpressing the Populus D-mannose kinase in transgenic Arabidopsis resulted in intercellular hyphal growth in Arabidopsis.

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Keynote speakers Tuesday 23 June – 14:30-15:00 Phosphorus acquisition by plants on the world’s most phosphorus-impoverished soils: implications for future crop production Hans Lambers* University of Western Australia, Australia South-western Australian soils are amongst the most heavily leached and phosphorusimpoverished in the world. This region is also a hotspot of higher plant species diversity, and therefore offers unique opportunities to study plant adaptations to nutrient-poor conditions. A large proportion of species from the impoverished environments in Australia cannot produce an association with mycorrhizal fungi, but, instead, produce cluster roots (Proteaceae, Casuarinaceae, Fabaceae) or dauciform roots (Cyperaceae). These specialised roots are a phosphorus-mobilising adaptation in structure and functioning; they release large amounts of exudates (carboxylates) in an exudative burst. Cluster-root-bearing Proteaceae in Australia occur on the most impoverished soils; mycorrhizal species inhabit less impoverished soils in this region. Non-mycorrhizal capillaroid roots (Restionaceae) and sand-binding roots (Anarthriaceae, Haemodoraceae) appear to function in a similar way as cluster roots. Carboxylates not only mobilise phoporus, but also a range of micronutrients, including manganese. In addition to the well-known specialised carboxylate-releasing roots, there are likely others, and a screening of species in plant communities as well as in germplasm collections can be based on an analysis of manganese concentrations in mature leaves. I will explore what traits roots of south-western Australian species have that allow them to function on the world’s most impoverished soils, and compare these with what is known about campos rupestres in Brazil, a region similarly low in available phosphorus and also a hotspot of plant diversity. I will explore which of these traits might be of interest for the development of more phosphorus-efficient crops and for which soils such traits are the most relevant.

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Keynote speakers Wednesday 24 June – 08:30-09:00 Multiscale imaging and modelling of rhizosphere processes Tiina Roose* University of Southampton, United Kingdom In this talk I will describe a state of the art image based model of the soil-root interactions, i.e., a quantitative, model of the rhizosphere based on fundamental scientific laws. This will be realised by a combination of innovative, data rich fusion of structural imaging methods, integration of experimental efforts to both support and challenge modelling capabilities at the scale of underpinning bio-physical processes, and application of mathematically sound homogenisation/scale-up techniques to translate knowledge from rhizosphere to field scale. The specific science question I will address with these techniques is how to translate this knowledge from the single root scale to root system, field and ecosystem scale in order to predict how the climate change, different soil management strategies and plant breeding will influence the soil fertility.

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Keynote speakers Wednesday 24 June – 14:15-14:45 Potato cultivation in the Andes – from plant microbiome characterization to microbial field applications Angela Sessitsch*1, Stefan Pfeiffer2, Jonas Ghyselinck3, Birgit Mitter1, Peter Kromann4, Barbara Doyle Prestwich5, Brigitte Schloter-Hai6, Michael Schloter6, Stéphane Declerck7 1 AIT Austrian Institute of Technology, Austria, 2Wilhelms GmbH, Germany, 3ProDigest, Belgium, 4International Potato Center (CIP), Peru, 5University College Cork, Ireland, 6Helmholtz Zentrum München - German Research Center for Environmental Health, Germany, 7KU Leuven, Belgium The rhizosphere is the biogeochemically most active compartment of the soil, which hosts a rich microflora supporting plant nutrition and health. In frame of a European project we addressed microbiota associated with potato grown in its center of origin, the Central Andes, at high altitudes. We examined rhizosphere microbiomes of potato cultivated in fields at different vegetation stages and at distinct sites at altitudes ranging from 3245 to 4070 m.a.s.l., differing in soil characteristics, climate, and agricultural practices by 454 sequence analysis of 16S rRNA genes. Our analysis revealed a core microbiome, which was consistently found across field sites and followed the same dynamics through plant development. The most dominating bacterial species of the core microbiome were found to be evenly distributed during plant growth, despite significant community changes between the vegetative and the germinative phase of plant development. Furthermore, some taxa were constantly found in rhizosphere communities associated with potatoes cultivated in Europe, indicating a close association with S. tuberosum. Furthermore, a strain collection was established and screened for various plant growthpromoting and disease suppressing activities. Selected strains were tested in greenhouse trials including also aeroponic systems as well as initial field trials. The VALORAM strain collection represents a promising source of future biocontrol applications and will help local industry as well as farmers in the Andes to combat various diseases.

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Monday 22 June – Parallel session Rhizosphere Microbiome 1 Convenors: Philip Poole, University of Oxford, UK and Rodrigo Mendes, Brazilian Agricultural Research Corporation, Embrapa Environment, Brazil

Convenor talk Control of the plant microbiome Philip Poole*, Andrzej Tkacz, Alison East, Vinoy Ramachandran Department of Plant Sciences, University of Oxford, United Kingdom Colonization by bacteria of the rhizosphere is crucial to plant productivity, with plants secreting 10-30% of total photosynthate to the rhizosphere. While rhizosphere colonization is poorly understood, recent advances in genome sequencing and analysis makes it possible to address this in exciting new ways. Microarray and metabolic analysis has been used to dissect the composition of the pea root secretome and map the transcriptional response of bacterial to secreted metabolites. We are now developing a suite of lux biosensors to the presence of specific metabolites that are being used for spatial and temporal mapping of root secretion. In addition they are excellent biosensors to enable identification of plant mutants altered in metabolite secretion. Plants are also excellent models to develop systems of experimental selection to examine how specific microbes are selected. Fingerprinting techniques such as ARISA and high through put sequencing are being used to map community level changes in the rhizosphere microbial community (microbiome). Coming full circle these techniques allow us to examine how natural variation and mutation of key regulatory systems in plants control the rhizosphere microbiome.

Active fungi in the rhizosphere along a chronosequence of abandoned agricultural soils Emilia Hannula*1, Elly Morriën2, Wim H. van der Putten2, Wietse de Boer2, Hans (J.A.) van Veen2 1 Netherlands Institute of Ecology (NIOO-KNAW), Netherlands, 2Netherlands Institute of Ecology NIOO-KNAW, Netherlands Many of the ecosystem functions provided by rhizosphere soils, such as plant growth, organic matter formation, carbon sequestration, nutrient mineralization and pathogen suppression depend on microbial activities. Here, we present results on the size, the structure and diversity of active fungal communities in soils along a chronosequence of ex-arable fields in The Netherlands. These fields are currently undergoing a transition from an arable system into species-diverse grasslands. Earlier research has shown that there is a succession in plant community composition and that soil process rates such as mineralization increase in time, but, the contribution of rhizosphere microbes in this transition is unknown. We hypothesized that the role of fungi would increase with increased time after abandonment and that this would have further consequences to both soil functioning and plant community 12 Rhizosphere Microbiome 1

Monday 22 June – Parallel session development. In order to assess the short-term fate, turnover and retention of recent plantassimilated carbon we studied active rhizosphere communities in intact soil cores from the field that have been exposed to pulse labelling with 13CO2. The fungal contribution to the processes was evaluated using PLFA-SIP and community structure and diversity was analysed using DNA-SIP combined with 454-sequencing. Upon labelling, most of the root-derived 13C was found in fungal biomass. Interestingly, fungi in the cores from long-term abandoned fields received significantly more carbon from the plants than the fungi in the short-term abandoned fields in which bacteria dominated The active 13C-labelled fungi in the rhizosphere were analysed and interaction networks of species were constructed to relate functioning of the rhizosphere community to its network structure. Analysis revealed a shift in fungal communities with time since abandonment resulting in cascading effects on fungal feeders. These results show that fungi and fungal diversity in the rhizosphere of grassland plant communities play an important role in both carbon cycling and ecosystem development.

Effect of nitrogen fertilization on the wheat root microbiome Tim Mauchline*1, Rifat Hayat2, Dariush Rowlands1, Ian Clark1, Penny Hirsch1 1 Rothamsted Research, United Kingdom, 2PMAS Arid Agriculture University, Pakistan The effects of mineral and farmyard manure (FYM) fertilization on bacterial community composition was measured by 16S rRNA gene amplicon sequencing in bulk and wheat rhizosphere soil obtained from the Broadbalk experiment. This experiment at Rothamsted Research was initiated in 1843 and is the oldest continuous arable experiment in the world. It offers a unique possibility to test the interaction between agricultural fertilization practices (including no N, mineral fertilizers and FYM), the wheat rhizosphere microbiome and wheat yields, which are highest in the high mineral N fertilized plots (288 kg N ha) whereas zero N application results in the lowest yields. FYM (35 t ha) and medium N fertilization (144 kg N) result in similar yields. It was found that all treatments could be discriminated from one another in both bulk and rhizosphere soil. Distinctly different rhizosphere soil communities could be detected in seedlings compared to mature plants for all treatments with the exception of FYM. In bulk soil the manure treated plots showed the highest species richness and all other treatments had similar species richness. However, in rhizosphere soil, FYM treatment resulted in the highest species richness and the mineral fertilized treatment revealed lower species richness than treatments with zero N fertilization. Results indicate that species richness in rhizosphere soil is not necessarily correlated with plant productivity as indicated by wheat yields in the field and that that mineral nitrogen fertilizer application results in a simplification of the rhizosphere microbiome.

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Monday 22 June – Parallel session Cross-talk between a diazotrophic bacterium and sugarcane Chanyarat Paungfoo-Lonhienne1, Thierry Lonhienne1, Yun Kit Yeoh1, Bogdan Donose1, Richard Webb1, Jeramy Parsons1, Webber Liao1, Prakash Lakshmanan2, Nicole Robinson1, Philip Hugenholtz1, Susanne Schmidt*1, Mark Ragan1 1 The University of Queensland, Australia, 2Sugar Research Australia, Australia Despite the putative importance of diazotrophic bacteria as contributors of biologically fixed nitrogen (N) to ecosystems and agriculture, their association with non-leguminous plants is not well understood. We have applied functional studies and high-resolution transcriptomic analysis to characterize the association of the native diazotrophic Burkholderia australis (enriched in sugarcane rhizosphere) with sugarcane. B. australis forms a biofilm at the root surface that involves quorum sensing, the formation of extracellular polysaccharides, and a reduced production of immunogenic factors. Through over-production of cytochrome bd ubiquinol oxidase, B. australis creates a microaerobic environment in the rhizosphere favourable for N2 fixation. In response, roots increase the expression of genes involved in adaptation to hypoxia and aerenchyma formation, facilitating the diffusion of oxygen into the root cortex. Aerobic and anaerobic energy pathways of B. australis are highly induced, suggesting high metabolic activity fuelled by photosynthates. There is evidence for a sophisticated interplay between B. australis and sugarcane with the hallmarks of symbiotic biological N2 fixation.

Diverse agricultural soils differentially influence tomato gene-expression and protein profile: A next generation sequencing approach Alessandra Salvioli*1, Matteo Chialva1, Stefania Daghino1, Mara Novero1, Davide Spadaro2, Paolo Bagnaresi3, Silvia Perotto1, Paola Bonfante1 1 University of Torino, Deptartment of Life Sciences and Systems Biology, Italy, 2University of Torino, Deptartment of Agricultural, Forestry and Food Sciences (DISAFA) AGROINNOVA , Italy, 3Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Genomics Research Centre, Italy Root-associated microbiota, in addition to soil characteristics, play a major role in shaping plant physiology and agronomic traits under diverse environmental conditions. We used high throughput RNA and protein sequencing to investigate the global response of Solanum lycopersicum grown on two agricultural soils (coded AL and RO) that were previously characterized as being different for biological and chemo-physical properties. Root colonization by ubiquitous beneficial arbuscular mycorrhizal fungi was significantly different (i.e. much lower in AL) suggesting that the two soils contain two different microbiota. We investigated the root transcriptome and proteome from tomato plants grown in microcosms containing AL or RO soils. RNA-seq experiments were performed; differentially expressed genes and Gene Ontology enriched-categories were computed: 285 differentially expressed genes and 37 enriched GO-terms were found, indicating that the two soils, with their microbes, differentially influence the tomato root transcriptome. Among the most enriched categories were “oxidation-reduction processes” and those related to plant-defense and cellwall. Plotting our dataset in KEGG maps revealed regulation of pathways involved in lignification, brassinosteroid and gibberellin synthesis as well as plant-pathogen interactions. 14 Rhizosphere Microbiome 1

Monday 22 June – Parallel session A global proteome profiling was performed on the same samples used for the transcriptome analysis. A dataset of unique proteins was obtained, 554 proteins being common to both soils. Of those, 208 proteins were up-regulated and 84 down-regulated in AL as compared with RO. 680 proteins were specifically expressed in AL, while 290 were specific to AV. Overall, proteomic data nicely supports the transcriptomic data. According to Gene Ontology enrichment analysis, the most regulated proteins, beside biological processes related to primary metabolism, were involved in response to biotic stimuli, to oxidative stress and to cell wall organization. This combination of -omic approaches provides a first insight on the differential soil impact on plant physiology.

Importance of plant nutrition and health promoting rhizosphere microorganisms in maize John Larsen*1, Miguel Najera Rincon2, Carlos Gonzalez Esquivel1, Alejandro Alarcon3, Juan Jose Peña Cabriales4 1 Universidad Nacional Autonoma de Mexico, Mexico, 2Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Mexico, 3Colegio Postgraduados, Mexico, 4Centro de Investigación y Estudios Avanzados del IPN, Mexico A series of lab, greenhouse and field experiments were performed to examine the importance of natural populations of different functional groups of rhizosphere microorganisms including plant growth promoting bacteria, arbuscular mycorrhizal fungi, yeasts, Trichoderma and entomopathogenic fungi in maize nutrition and root health. All functional groups formed an abundant and diverse part of the rhizosphere microbiome in all nine maize fields included in the present study, in most cases responding to soil physico-chemical characteristics, plant growth stage and maize agroecosystem. A large culture collection of the rhizophere microorganisms examined were obtained and their functional traits evaluated in in vitro assays, revealing common plant nutrition and root health promoting traits. In a greenhouse pot assay maize plants grown in sterile P deficient soil remained stunted throughout the 16week growing period; whereas plants grown in soil re-inoculated with maize rhizosphere soil resulted in fully grown maize plants, which suggest that the rhizosphere microbiome is essential for maize growth in P deficient soils. In a similar pot experiment growing maize in sterile P or N deficient soil revealed that re-inoculation with maize rhizosphere soil in the N deficient soil did not alleviate plants from N stress, whereas again strong plant growth promotion was observed in maize grown in P deficient soil. Most likely the native populations of arbuscular mycorrhizal fungi played a key role in the observed plant growth promotion since their elimination from the inoculum with a filtration method (preventing passage of propagules of arbuscular mycorrhizal fungi) neutralized the effect of the rhizosphere inoculum. In conclusion, natural communities of plant nutrition and root health promoting microorganisms form an abundant and diverse part of the maize rhizosphere biome, which should be conserved and integrated as an important biological resource in maize agroecosystems to minimize the use of agrochemicals.

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Monday 22 June – Parallel session Microbial food web links uncovered - probing carbon fluxes in the maize rhizosphere Michael Bonkowski*1, Maike Hünninghaus1, Dörte Dibbern2, Robert Koller3, Tim Urich4, Tillmann Lueders2 1 University of Cologne, Germany, 2German Research Center for Environmental Health, Germany, 3Forschungszentrum Jülich, Germany, 4University of Vienna, Austria Plants allocate a significant amount of their photosynthetically fixed carbon (C) to promote microbial associations in their rhizosphere. It has been assumed recently that these belowground C-inputs form the major energy source driving the entire food web in soils. However, only few studies to date were able to picture specific microbial food webs depending on fresh root C-inputs, even though great efforts have been made to understand rhizosphere microbiota. It is now well-known that each plant species strongly interacts with a distinct subset of the soil microbial community colonizing its roots, i.e. its rhizosphere “microbiome”, but a detailed understanding of the C flows from the microbiome through the fungal and bacterial energy channels into soil food webs is needed to comprehensively understand terrestrial C cycling. In a plant labeling experiment using combined rRNA-SIP with 13C-labeled rhizodeposits and pyrotag sequencing we traced the C flow from maize (Zea mays L.) roots through communities of rhizosphere fungi, bacteria and heterotrophic protists. We found a highly diverse active microbial community in rhizosphere as well as in bulk soil, but only specific subsets of distinct taxa actively relied on fresh root-C. Mycorrhizal fungi within the Paraglomerales were the determinant factor for the allocation of plant-derived C into soil communities, thereby connecting food webs of rhizosphere and bulk soil. We unravel the organization of the rhizodeposit-associated microbial soil food web, identify specific microbial key players also within rhizosphere bacteria (e.g. Mucilaginibacter, Opitutus, Sphingobium) and protozoa (e.g. Thaumatomonadida, Bicosoecida, Leptomyxida), and detect a highly dynamic succession of plant-derived C through the respective microbes. These insights are crucial to improve our current perspective of the plant-associated microbial functional diversity and of the trophic interactions at the basis of soil food webs.

Increasing interactions among microbial populations underlie successional trajectories of the rhizosphere microbiome of a common annual grass over two seasons Mary Firestone1, Shengjing Shi1, Erin Nuccio*2, Donald Herman1, Ruud Rijkers1, Katerina Estera1, Jiabao Li3, Ulisses da Rocha4, Zhili He3, Jennifer Pett-Ridge2, Eoin Brodie5, Jizhong Zhou3 1 University of California, USA, 2Lawrence Livermore National Laboratory, USA, 3University of Oklahoma, USA, 4Lawrence Berkeley National Lab, USA, 5Lawrence Berkeley National Laboratory, USA It is well known that rhizosphere microbiomes differ from those of surrounding soil; yet we know little about how these root-associated microbial communities change through the plant’s growing season and between seasons. We analyzed the response of soil microorganisms to roots of the common annual grass Avena fatua over two seasons using 16 Rhizosphere Microbiome 1

Monday 22 June – Parallel session high-throughput sequencing of 16S rRNA genes. The rhizosphere microbiomes followed a consistent successional pattern as plants grew, although starting communities were distinct between growing seasons. Succession in the rhizosphere was characterized by a significant decrease in both taxonomic and phylogenetic diversity relative to background soil communities. Taxa that were stimulated in rhizosphere soil (e.g., α-, β-Proteobacteria and Bacteriodetes) displayed phylogenetic clustering, suggesting some evolutionary basis for the response of soil microorganisms to plant roots. To begin disentangling the interactions occurring among rhizosphere inhabitants and to explore environmental controllers of and the topologies of the communities through time, we analyzed co-occurrence of taxa using a Random Matrix Theory Model to construct networks of positive and negative interactions between populations within the community. Interactions between populations became substantially more extensive and complex in rhizosphere soil over time while the interaction patterns of bulk soil were much simpler over time. Analyses of the rhizosphere networks were used to interpret interactions in terms of modular structure, keystone species, Eigen gene network, and interrelatedness of taxa. Documenting and understanding the bases of reproducible patterns of rhizosphere succession and exploring the distribution of rhizosphere competence traits will better enable understanding and prediction of rhizosphere microbiome assembly.

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Monday 22 June – Parallel session Rhizosphere and Climate Change Convenors: Ian Anderson, University of Western Sydney, Australia, and Bruce Hungate, Northern Arizona University, USA

Convenor talk Impacts of climate change factors on ectomycorrhizal and other eucalypt rhizosphere fungi Ian Anderson*1, Jonathan Plett1, Barbara Drigo1, Kerry Keniry1, Francis Martin2, Annegret Kohler2 1 University of Western Sydney, Australia, 2INRA, UMR 1136 INRA-University of Lorraine, France Soil fungi play important roles in forest carbon and nutrient cycles, but relatively little is known about how they will respond to future climate change, especially in the context of southern hemisphere forest ecosystems. Our recent research focus has been to use controlled environment glasshouse and microcosm experiments to investigate the interactive effects of elevated atmospheric CO2, temperature and drought on Australian forest soil fungi, including those that form ectomycorrhizal associations with eucalypts. In a glasshouse experiment, Eucalyptus saligna and E. sideroxylon seedlings were grown in field soil and maintained for 5 months under sub-ambient (280 ppm), ambient (380 ppm) and elevated (640 ppm) atmospheric CO2 conditions at both 26°C and 30°C. Multivariate analyses of molecular data showed a significant (P < 0.035) separation between fungal communities associated with the two different tree species and a clear separation between the communities from the 280, 400 and 640 ppm CO2 treatments at 34oC. This response appeared to be plant-dependent at 280 and 400 ppm CO2, however, all 640 ppm CO2 samples clustered together regardless of tree species. Interestingly, several of the key fungal species identified to respond strongly to the climate change factors were ectomycorrhizal fungi, including Pisolithus sp. so we performed a subsequent microcosm experiment and used transcriptomics to investigate the response of E. grandis (for which a genome sequence is available) to colonization by different Pisolithus isolates under ambient (400 ppm) and elevated (650 ppm) CO2. Our data showed that E. grandis varies in its susceptibility to colonization by different Pisolithus isolates in a manner that is not predictable by geographic origin or the ITS-based phylogeny of the fungal partner. Further, elevated levels of CO2 alter the receptivity of E. grandis to Pisolithus, which is correlated to a dramatic shift in the transcriptomic profile of the root. These data provide a starting point for understanding how future environmental change may alter the signalling between plants and their ectomycorrhizal partners and is a step towards determining the mechanism behind observed shifts in eucalypt-associated fungal communities exposed to elevated levels of atmospheric CO2.

18 Rhizosphere and Climate Change

Monday 22 June – Parallel session Convenor talk Rising CO2, Climate Change, and the Microbial Ecology of Rhizosphere Effects on Soil Carbon Bruce Hungate* Northern Arizona University, United States Much progress has been made bringing physiology and diversity to the understanding of carbon input to ecosystems. Yet the representation of the physiology, diversity, and ecology of carbon losses from soil, controlled by microbial respiration, is rudimentary. Tackling this problem is important to understanding soil carbon persistence, because the sensitivity of soil carbon to external forcings has the potential to mitigate or exacerbate global climate change. The rhizosphere is a critical nexus modulating responses of soil carbon to climate change and rising carbon dioxide, because the breakdown of soil carbon can slow or accelerate in response to inputs of new carbon substrates from root exudation and turnover. Responses to substrate inputs in the rhizosphere are highly idiosyncratic: changes in the rates of soil carbon loss can increase or decline, can be short-lived and quantitatively trivial, or longlasting and dominating decadal-scale patterns of carbon cycling and storage. Mechanisms driving such responses are inscrutable – though many have been invoked, none adequately predict emergent patterns of carbon cycling. Thus, the rhizosphere priming effect is quantitatively important, ubiquitous, and mechanistically inscrutable. One hypothesis accounting for the idiosyncratic nature of rhizosphere priming is that the ecology and biodiversity of soil microorganisms are central modulators, an idea that is particularly challenging to test given current techniques. Here, I discuss two developments in quantitative microbial ecology that have the potential to connect the population biology of rhizosphere microorganisms with the quantitative biogeochemistry of rhizosphere priming. I present examples investigating taxon-specific microbial growth rates and ecosystem-scale biochemistry that provides insight on community-level patterns of carbon anabolism and catabolism during priming and in response to warming, informing our understanding of the microbially-mediated feedbacks between terrestrial ecosystems and the changing atmosphere. This work aims to advance a quantitative understanding of microbial ecosystems, integrating biochemical, population and community ecology with element cycling, and is part of a broader effort in the field of microbial ecology to identify the microbial ecological interactions that influence ecosystem processes.

Can plants self-mitigate rhizosphere nitrous oxide? Liz Baggs*1, Nick Morley1, Eric Paterson2, Antia Villada3, Christos Gougoulias3, Liz Shaw3 1 University of Aberdeen, United Kingdom, 2The James Hutton Institute, United Kingdom, 3University of Reading, United Kingdom Plant-derived carbon is a key driver of rhizosphere processes, which presents management opportunities for mitigation of greenhouse gases. Here we take the example of soil nitrate reducing processes and demonstrate the role of plant-derived carbon in regulating nitrous oxide production and reduction in the rhizosphere. We use stable- and radio-isotopes and a new isotope-fluorescent probe-cell sorting approach to verify (i) the link between plant19 Rhizosphere and Climate Change

Monday 22 June – Parallel session carbon and the activity of the microbial nitrate reducers, (ii) uptake of carbon compounds into pseudomonads, (iii) the role of both composite rhizodeposits and individual compounds in regulating the magnitude of nitrous oxide emission, and (iv) their influence on the gaseous product ratio during denitrification. We examine the implications on net emission of nitrous oxide and consider the likely effect of these relationships on interactions with other greenhouse gas genic processes, the impact on soil carbon stocks and the potential for us to capitalise on plant-driven mitigation.

What will climate change mean for infectious diseases? The rhizosphere perspective Barbara Drigo*, Thomas Jeffries, Brajesh K. Singh, Catriona A. Macdonald, Yui Osanai, Ian C. Anderson Hawkesbury Institute for the Environment, University of Western Sydney, Australia Scientists have long predicted large-scale responses of infectious diseases to climate change, giving rise to a polarizing debate, especially concerning human pathogens for which socioeconomic drivers and control measures can limit the detection of climate-mediated changes. Climate change has already increased the occurrence of diseases in some natural and agricultural ecosystems, but in many cases, outcomes depend on the form of climate change and details of the host-pathogen system. Here, we describe how climate change will affect terrestrial ecosystems and their capacity to reduce infectious diseases. Rhizosphere and bulk soil was collected from grassland, forest and agricultural ecosystems at the Hawkesbury and EUC-FACE climate change field and greenhouse experiments in Western Sydney (Australia). Real-time PCR approaches targeting toxins-encoding genes revealed that elevated CO2 and rainfall patterns intensified the effect of warming by significantly increasing the virulence of soil-borne human pathogens associated with grassland and forest rhizosphere and bulk soils. As opposed to simply increasing the biomass of soil-borne pathogens at ambient CO2 under changes in rainfall patterns and temperature, elevated atmospheric CO2 strongly selected for virulent human pathogens and effected shifts in pathogens composition. 16S rRNA, 18S rRNA and ITS region sequencing with the Illumina Miseq platform revealed the dominance of several opportunistic and true human pathogens in the rhizosphere microbiome, including E.coli 0157:H7, Enterobacteriacae, Chlamydia, Staphylococcus, Salmonella and Clostridium species. The potential mechanisms involved in the interplay between human pathogens in the rhizosphere microbiome are presented in a bioclimatic model of relative microbial abundance that specifically incorporates interactions between biological units.

20 Rhizosphere and Climate Change

Monday 22 June – Parallel session Plants and plant diversity as regulators for nitrous oxide emissions in intensively managed systems Jan Willem Van Groenigen*1, Diego Abalos2, Bandhu Baral3, Thom W. Kuyper1, Gerlinde B. De Deyn1 1 Wageningen University and Research, Netherlands, 2Technical University of Madrid, Spain, 3Nepal Agricultural Research Council, Nepal Intensively managed agricultural soils are the dominant source for emissions of the greenhouse gas nitrous oxide (N2O). The ultimate cause of this is increased nitrogen (N) fertilizer use. Although N fertilizer is applied to nurture plants, a possible role for plants in mitigation strategies for N2O emissions from intensively managed systems has hardly been considered. Here, we show that the concepts of trait-based ecology and nutrient stoichiometry can help to devise novel N2O mitigation strategies. In a first study, we tested whether N2O emissions are dependent on grass species richness and/or species identity. We measured N2O emissions from monocultures and two- and four-species mixtures of common grass species with different root functional traits. We found no relation between plant species richness and N2O emissions. However, emissions were significantly reduced in specific plant species combinations. The species mixture resulting in lowest N2O emissions depended on the soil nutrient status. Reduced emissions up to 44% in multi-species combinations could not be explained by total biomass productivity only; divergence in root traits also played a role. In a second study, we tested whether N/Phosphorus (P) stoichiometry can affect N2O emissions in heavily fertilized silage maize plants and found that N2O emissions decreased with 50% with adequate P fertilization. This could be related to increased plant growth, which reduced soil mineral N concentrations. We conclude that novel, plant-based mitigation strategies can be developed, specifically focusing on (i) selection of grass species combinations with traits that are fine-tuned to local N deposition regimes; and (ii) judicious use of non-N fertilizers in order to satisfy stoichiometric relations. Our results underline the pivotal role that plant ecology plays in the soil biogeochemical cycle.

Root growth dynamics and root exudation patterns in a mixed spruce-beech forest under seasonal water limitation Marie Zwetsloot*, Alex Paya, Taryn Bauerle Cornell University, United States Forests store approximately 50% of all terrestrial carbon with a substantial proportion of assimilated carbon directly allocated to roots. Under current climate change predictions, the degree by which leaf level responses to drought affects root growth dynamics and root exudation patterns may have a significant impact on soil carbon inputs in forests. How different neighboring tree species further influence these root interactions also remains unclear. We hypothesized that 1) tree neighbor identity affects root lifespan and root vertical distribution, 2) hydraulic strategy at the leaf level determines belowground carbon inputs governing root dynamics and root exudation patterns, and 3) fine root age class influences root exudation rates through changes in root membrane permeability. To test this, we utilized a 70 ±4 –year-old forest (Kranzberg experimental forest in Freising, Germany) outfitted with precipitation exclusion infrastructure to investigate the effects of inter- and 21 Rhizosphere and Climate Change

Monday 22 June – Parallel session intra-specific interactions on the growth, placement, survivorship and respiration of Norway spruce (Picea abies, an isohydric species) and European beech (Fagus sylvatica, an anisohydric species) under seasonal water limitation. In addition, we designed a controlled pot experiment to focus on root exudation among root age classes. Results demonstrate vertical root niche partitioning, increased root lifespan and increased root respiration in the intermixing zone of spruce and beech trees in comparison to monotypic plots. Surprisingly, root membrane permeability did not change among fine roots (1-50 days). Instead, nonstructural root carbon reserves and species specific chemical characteristics may be better indicators of the quantity and quality of root exudation, ultimately influencing rhizosphere dynamics. Expected findings under water limitation include reduced root carbon reserves leading to decreases in root production and root exudation. In conclusion, tree neighbor identity and tree hydraulic strategy should be considered as important factors in estimating future climatic effects on root-facilitated carbon cycling in forests.

Role of mycorrhiza in plant N:P stoichiometry under drought in Australian grasslands Pierre Mariotte*, Alberto Canarini, Feike Dijkstra The University of Sydney, Australia Stoichiometric homeostasis represents the ability of an organism to maintain constant chemical elements despite variations in the environment and is a powerful mechanism involved in the maintenance of ecosystem functioning, especially under climate change. For example, drought directly increases soil N:P ratio, and consequently influences plant N:P stoichiometry. However, the degree of plant stoichiometric homeostasis and its regulation under drought is still poorly understood. Here we hypothesize that, due to their benefits to plants for nutrient acquisition, arbuscular mycorrhizal fungi (AMF) have important influences on the ability of plants to maintain N:P stoichiometry. We suggest that stoichiometric homeostasis is dependent on the degree of root colonization by AMF, especially under drought conditions. However, since symbiosis between plant and AMF is dependent of soil P availability, we expect the positive effects of AMF to occur only in low P soils. To test our hypotheses, we carried out a greenhouse pots experiment at the University of Sydney involving multi treatment interactions of low/high soil P, presence/absence of AMF and control/drought on the N:P stoichiometry of two C3 (Austrodanthonia caespitosa, Microlaena stipoides) and two C4 (Bothriochlora macra, Themeda trandra) Australian grassland species growing together in competition. We added 15N in the soil to compare nitrogen acquisition by plants related to the presence of mycorrhiza. At the end of the experiment, we measured plant biomass, 13C and 15N in plant leaves, N:P ratio in soil and plant tissue, together with the percent of root colonization of the four plant species. Undergoing analysis will highlight contrasted stoichiometric homeostasis of plants species, in ambient and drought conditions, and will determine the role of arbuscular fungi in maintaining plant stoichiometry. These findings will improve our knowledge of ecological stoichiometry in grasslands and give new perspectives to understand its regulation processes under climate change in contrasted soil conditions.

22 Rhizosphere and Climate Change

Monday 22 June – Parallel session

Effects of free-air CO2 enrichment on soil nematode assemblage depend on nitrogen fertilization and rice cultivar traits Manqiang Liu*1, Xiaoyun Chen1, Chunwu Zhu2, Zhengkun Hu1, Huixin Li1, Feng Hu1 1 College of Resources and Environmental Sciences, Nanjing Agricultural University, China, 2State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China Exploring the responses of soil biota to interacting global change factors would contribute to understand the mechanisms of belowground ecosystem process. Soil nematodes play important roles in nutrient transformation and plant growth, which could indicate soil food web and functional state. The interactive responses of nematodes to elevated CO2 and agricultural practices such as N fertilization and crop cultivars are, however, not well understood. Herein, using a free-air CO2 enrichment facility, two nitrogen fertilization regimes including low N (0 N kg ha−1) and high (250 kg ha−1) and two rice cultivars with distinct growth enhancement at elevated CO2 (low and high responsive cultivars), were examined in a rice paddy field. Total abundance, trophic group and various ecological indices of nematodes were used to identify the responses of soil food web. We found that the effects of elevated CO2 on nematodes strongly depended on N fertilization and rice cultivars. Elevated CO2 decreased nematode abundance for low responsive cultivar but increased it for high responsive cultivar, particularly in high N plots. Elevated CO2 decreased the proportions of bacterivore and fungivore for both cultivars in low N plots, but increased them for low responsive cultivar in high N plots. The reverse was true for herbivores. Regardless of N fertilization, the proportion of omnivore-predators was increased by elevated CO2 for low responsive cultivar, but the reverse was true for the high responsive cultivar. Elevated CO2 moderately increased nematode channel ratio (NCR), Shannon diversity, maturity index (MI) and structural index (SI) particularly for low responsive cultivar. Our results support there is a substantially interactive response of soil food web to global change and agriculturally managed factors.

23 Rhizosphere and Climate Change

Monday 22 June – Parallel session Root Development Convenors: Philip Benfey, Duke University, USA, and Christa Testerink, University of Amsterdam, the Netherlands

Convenor talk A systems approach to improved root traits Philip Benfey* Duke University, United States Root systems are high value targets for crop improvement due to their potential to boost or stabilize yields, improve disease resistance, and reduce the need for fertilizers. Root system architecture (RSA) describes the spatial organization of the root system, which is critical for its function in challenging environments. We have developed a semi-automated 3D imaging and phenotyping system to identify the genetic basis of root architecture. The integrated system combines hardware, imaging, software and analysis. We automatically reconstructed and phenotyped a well-studied rice mapping population identifying QTLs for RSA traits that control the extent, shape, distribution, and surface size of root networks. Apparent tradeoffs at some QTL clusters were consistent with genetic limitations on ‘ideal’ RSA phenotypes. We recently extended this work to maize where we mapped large effect QTLs for RSA using the NAM mapping population. We are also using X-ray imaging to phenotype roots grown in soil. Thus, our approach can directly aid breeding efforts as well as identify important genes underlying environmentally robust QTLs.

Convenor talk Take it or leave it: How plant roots avoid salt Christa Testerink* University of Amsterdam, Netherlands To deal with drought, high salinity, or nutrient deprivation, plants adjust and optimize their root system architecture. We study the intracellular signalling pathways linking salinity stress to root development and direction of root growth. By quantifying root system architecture (RSA) dynamics of 31 different Arabidopsis accessions in mild salt stress conditions, differential regulation of main and lateral root growth rate and root branching were revealed. Applying ROOT-FIT to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked. RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na+/K+ ratios in shoots of seedlings grown under mild salt stress. In addition to changing their overall RSA, a variety of plant species can also selectively change direction of root growth, to avoid high salt concentrations. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by lipid-dependent clathrin-mediated endocytosis of the PIN-FORMED 2 24 Root Development

Monday 22 June – Parallel session (PIN2) auxin efflux carrier. Our results thus identify a cellular pathway essential for plant responses to salt stress, which may serve to integrate multiple environmental signals to optimize root growth in response to changing conditions.

Natural variation of an exocyst gene switches between distinct root system architectures in Arabidopsis Wolfgang Busch*, Takehiko Ogura, Christian Goeschl, Daniele Filiault, Radka Slovak, Santosh B. Satbhai Gregor Mendel Institute, Austria Despite their inability to move, vascular plants have colonized the vast majority of the earth’s land surface. A key to this success is that plants are modular organisms that can tune their organ number as well as the growth of these organs. This capacity allows for a remarkable plasticity of plant architecture. Under the ground, Root System Architecture (RSA) determines crucial parameters for plant survival such as the ability to take up water, to forage the soil for nutrients and to anchor the plant. RSA is mainly shaped by the processes of root branching, root elongation and root growth direction, all of which are regulated by the plant hormone auxin. However, the same hormone is an ubiquitous regulator of almost every aspect of plant growth and development at the molecular, cellular, tissue and organ levels. It is not clear which genetic and molecular mechanisms allow RSA to be shaped without affecting a plethora of other processes including shoot architecture formation. Using natural variation and genome wide association studies in A. thaliana, we identify a novel auxin signaling component, a component of the exocyst system, which exclusively regulates RSA by modulating lateral root density and root growth direction. We show that this exocyst gene regulates the dynamic localization of a specific PIN auxin efflux carrier in columella cells. We further show that allelic variation of the exocyst gene switches between two RSA types and can increase resistance to drought conditions demonstrating an adaptive value of RSA.

Discovery of new bacterial traits involved in plant growth promotion and induced systemic resistance by the rhizobacterium Pseudomonas fluorescens Xu Cheng*1, Judith van de Mortel2, Desalegn Etalo3, Ester Dekkers2, Mieke WoltersArts4, Dragana Kocevski2, Jos Raaijmakers3 1 Wageningen University, Netherlands, 2Laboratory of Phytopathology, Wageningen University, Netherlands, 3Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO), Netherlands, 4Molecular Plant Physiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Netherlands Rhizobacterial strain Pseudomonas fluorescens SS101 (Pf.SS101) induces multiple metabolic, transcriptional and phenotypic changes in Arabidopsis thaliana (Arabidopsis), including alteration of root architecture, enhancement of shoot and root biomass, enhanced greening and induced systemic resistance (ISR) against bacterial leaf pathogens and insect pests. To identify the underlying bacterial traits involved in plant growth promotion (PGP), induction of lateral root formation and ISR, targeted and untargeted approaches were adopted. None of the well-known PGP mechanisms, including the production of indole-acetic acid and volatile 25 Root Development

Monday 22 June – Parallel session organic compounds, appeared to play a major role in the plant phenotypic changes induced by Pf.SS101. Untargeted analyses by individual screening of a library of 7488 random transposon mutants of Pf.SS101 led to the selection of 21 mutants defective in PGP and ISR. Cloning of the disrupted genes, genetic complementation, site-directed mutagenesis and transcriptional analyses suggested that several of the genes involved in PGP and ISR are associated with amino acid biosynthesis, glucose utilization, transcription or sulfur assimilation. Subsequent whole-genome transcriptome analyses of Arabidopsis seedlings indicated that modulation of the plants’ sulfur metabolism is one of the key mechanisms involved in Pf.SS101-induced plant growth promotion and induced systemic resistance.

Revealing a role for plant hormones in root growth responses to excess metals Stefanie De Smet*, Ann Cuypers, Jaco Vangronsveld, Tony Remans Hasselt University, Belgium Plants possess a high level of plasticity, especially in the root system. This enables the plants to adjust their growth and development according to a changing environment in order to survive. The positioning of the roots is the first factor that determines metal uptake and subsequent plant growth. Plants are subject to the toxic effects of metals and the physiology and molecular mechanisms of metal uptake, sequestration and detoxification are under intense investigation. However, root system development under these circumstances is poorly understood. We found that roots of Arabidopsis thaliana exposed to cadmium (Cd), copper (Cu) or zinc (Zn) showed different morphological responses. Exposure to Cd or excess Cu both stimulated the outgrowth of lateral roots, but Cd inhibited the elongation of these lateral roots more severely than Cu did. Zn inhibited both lateral root outgrowth and elongation. Microscopic analysis of lateral root formation, using a cyclinB-GUS reporter line, showed that Zn exposure affects both emergence of lateral root primordia and activation of the meristems. This indicates the existence of metal-specific interference with the intrinsic pathways of lateral root development, outgrowth and elongation. As lateral root development is strongly connected with the action of phytohormones, and given reports of altered phytohormone concentrations or gradients during stress (including metal stress), we are investigating the involvement of phytohormones in the metal-specific root responses to Cd, Cu and Zn. Analysis of root architecture after Cd, Cu or Zn exposure of the abscisic acid insensitive mutant abi4-1 indicated that ABI4 may be involved in the inhibition of lateral root emergence after Zn exposure, and also in root developmental responses to low Cd concentrations. Analysis of further phytohormone insensitive or signaling mutants may bring more insight in the mechanisms of metal -specific alterations in root architecture.

26 Root Development

Monday 22 June – Parallel session Root endodermal differentiation requires the transcription factor MYB36 Louisa Liberman*1, Erin E. Sparks1, Miguel Moreno-Risueño2, Jalean Petricka3, Philip N. Benfey1 1 Duke University, United States, 2Universidad Politecnica de Madrid, Spain, 3Carleton College, United States The plant root endodermis regulates water and ion flow into and out of the central vasculature. Studies investigating endodermal function note that microbes rarely colonize this cell type, suggesting that the endodermis may also play a regulatory role in plantmicrobe interactions. Given the importance of this cell-type, endodermal specification has been extensively studied in Arabidopsis thaliana. The transcription factors SCARECROW and SHORTROOT regulate specification of the bi-potent stem cell that gives rise to the cortical and endodermal progenitors. Subsequent progenitor proliferation and differentiation generate mature endodermis, marked by a cell wall modification called the Casparian Strip. We identified a transcription factor, MYB36 that regulates the transition from proliferation to differentiation in the endodermis. myb36 mutants display delayed and abnormal Casparian Strip deposition. We show that SCARECROW directly activates MYB36 expression, which in turn directly regulates essential Casparian Strip formation genes. In addition, MYB36 represses extra divisions within the endodermis. Our results demonstrate that MYB36 is a critical positive regulator of differentiation and negative regulator of cell proliferation. Additionally myb36 mutant roots appear to be hypersensitive to growth-promoting bacteria, supporting the hypothesis that the Casparian strip functions to regulate plant-bacterial interactions.

Stimulation of tobacco growth by Bacillus vallismortis EXTN-1 via volatile organic compounds Kyungseok Park*, Yong-Soon Park, Swarnalee Dutta National Academy of Agricultural Science, South Korea It has been known that Bacillus vallismortis strain EXTN-1 (EXTN-1), one of the plant growthpromoting rhizobacteria (PGPR), is implicated in promoting plant growth and eliciting induced systemic resistance against diverse pathogens in important agricultural crop species. However, effect of certain volatile organic compounds (VOCs) released by EXTN-1 on the plant growth promotion is still unclear. To confirm whether VOCs from EXTN-1 are key component in the regulation of plant growth, we assessed plant growth using I-plate system, which can inhibit physical contacts between tobacco plants and EXTN-1. The growth of tobacco plants was significantly stimulated by VOCs from EXTN-1. Specifically, the levels of fresh and dry weight of plants were 10 and 7 times higher in King’s B (KB) media, respectively, when compared to control, suggesting that plant growth promotion is regulated by VOCs released from EXTN-1. For more evidence, we performed solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) analysis to identify the specific VOCs that affected on plant growth, resulting that at least 10 individual volatiles were characterized and 3-hydroxy-2-butanone was relatively highly indentified. The bioassay revealed that plant growth promotion was enhanced by 3-hydroxy-2-butanone in a broad range of concentrations. Collectively, our study strongly suggests that tobacco growth promotion is facilitated by EXTN1 via VOCs, especially 3-hydroxy-2-butanone. 27 Root Development

Monday 22 June – Parallel session Penicillium bilaii increases maize root growth and P uptake from sewage sludge Beatriz Gómez-Muñoz*, Lars S. Jensen, Andreas de Neergaard, Jakob Magid University of Copenhagen, Denmark Penillium bilaii may act as an enhancer of phosphorus availability to plants, as it has been shown to increase plant root length and the number of roots hairs. Therefore, the combined application of P. bilaii with organic wastes (e.g. sewage sludge rich in phosphorus) could increase phosphorus use efficiency and uptake in plants. In this work we tested the effect of P. bilaii as P-solubilizer from sewage sludge on maize using a Rhizobox design, allowing monitoring of the root development over time. Maize was sown in 1) soil (control) and in 2) soil with a sewage sludge ‘hot spot’ (40 mg total P kg-1 soil), 3) soil inoculated with P. bilaii, and soil with the combination of sludge and P. bilaii with inoculation either 4) coated on the seed or 5) mixed into the sewage sludge ‘hot spot’. Finally, triple superphosphate (same P rate and placement as 2) was added as positive control (6). The experiment lasted 27 days, in a growth chamber. After 9 days of growth, the shoot and root length of the plants treated with P. bilaii and triple superphosphate were significantly higher than the control, whereas at the end of experiment, the shoots length and biomass were higher in treatments with phosphorus applied (2, 4, 5, 6), and P. bilaii inoculation alone did not increase the shoot biomass. In contrast, several root parameters (weight, length, surface area, forks) were significantly increased by P. bilaii application alone, and in combination with sewage sludge. Inoculation of the hot spot was significantly less effective, than inoculation of the seed. Our data show that P. bilaii can significantly increase root growth and development, and that the effect is larger in combination with a placed phosphorous substrate. Further data on plant nutrient concentrations will be presented at the conference.

28 Root Development

Monday 22 June – Parallel session Nutrient Acquisition 1 Convenors: Hans Lambers, the University of Western Australia, Australia, and Jianbo Shen, China Agricultural University, China

Rhizosphere microbial activities and mycorrhizal status of maize genotypes as affected by phosphorus fertilization under field conditions Ran Erel1, Annette Bérard2, Lidia Campos-Soriano3, Line Capowiez2, Claude Doussan2, Ghislain Sévenier2, Didier Arnal1, Marcel Bach3, Gérard Souche1, Blanca San Segundo3, Philippe Hinsinger*1 1 INRA, UMR Eco&Sols, France, 2INRA, UMR1114 EMMAH, France, 3Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB. Edifici CRAG, Campus UAB,Bellaterra, Spain Because they are primarily carbon-limited, it is generally assumed that soil microbial communities can be stimulated in the rhizosphere. It is also generally accepted that arbuscular mycorrhizal (AM) colonization of roots is depressed by P fertilization. We tested these hypotheses in a long term field trial that was designed to evaluate the potential interaction of 23 genotypes of maize with P levels. This experiment was carried out at INRAAuzeville (France), comparing two extreme treatments: P0, which had not been fertilized for 45 years and P4, which had received 3- to 4-fold the annual P offtake by crops. Plants were harvested at the 6-8-leaf stage. The level of root colonization by AM fungi was examined by microscopy observations (trypan-blue) and quantified by qPCR targeting three AM species. Rhizosphere and bulk soil were assayed for various microbial activities. Substrate-induced respiration was measured with the MicroRespTM technique. Microbial biomass and respiration significantly increased in the rhizosphere, the greatest microbial activity being found in P4. In the bulk soil however, microbial activity was not modified by phosphorus level. In contrast, alkaline phosphatase activity was similar in rhizosphere and bulk soil, being slightly smaller in P0. In accordance, acid phosphatase activity was also lowest for P0. Although the total length of fine roots was increased under phosphorus-deficient conditions, these roots remained susceptible to colonization by AM fungi, whatever the method used. Interestingly, for some of the maize genotypes, the intensity of AM colonization was even higher in P4 soil compared to P0 soil. The consistent higher microbial activity in rhizosphere of high P soil suggests that co-limitation of soil microbial communities by phosphorus and carbon may occur in the unfertilized soil. These results obtained under field conditions also challenge the hypothesis that the AM colonization of roots is depressed by P fertilization.

29 Nutrient Acquisition 1

Monday 22 June – Parallel session Growth condition and P mobilizing properties of cluster roots of Helicia cochinchinensis (Proteaceae) at Miyajima Island, Japan Jun Wasaki*, Taiki Yamauchi, Jin Takahashi, Hayato Maruyama, Shinji Uchida, Seiji Mukai, Hiromi Tsubota Hiroshima University, Japan Some specific plants form bottle brush-like root structures, so-called cluster roots, during P deficiency. The family Proteaceae are well known group for cluster root-forming plants. Helicia cochinchinensis Lour. is only one species present in Japan among the Proteaceae, and is distributed in the range from southwest Japan to the Indochina Peninsula. It is still unknown what properties of soils distributing H. cochinchinensis and whether this species forms cluster roots. The aim of this study is to investigate the growth conditions, morphological properties of roots, and phosphate mobilizing abilities of H. cochinchinensis growing in Miyajima Island, Hiroshima, Japan. Available P in soils growing H. cochinchinensis grew was very poor; ranged 0.46 – 3.7 mg-P/kg-soil (Olsen-P). We found cluster roots from P deficit H. cochinchinensis in natural and hydroponic conditions. It was shown that acid phosphatase activity was increased in the rhizosphere soil. Activity staining revealed a higher activity of acid phosphatase in half-part from root tip of each rootlet. Strong decrease of pH in the rhizosphere of matured cluster roots was also shown, suggesting that the cluster root enhanced organic acid exudation like other Proteaceae plants. P concentration of leaves of H. cochinchinensis was low level; those of mature and senesced leaves ranged 0.34 - 0.69 mgP/gDW and 0.15 - 0.29 mg-P/gDW, respectively. This range was some extent high than that of other Proteaceae plants growing under very low P soils in Western Australia, but it was very low level among general plant species. It was concluded that the P mobilizing capacity of cluster roots and the tolerance to low P content supported the growth of H. cochinchinensis under low P soil at Miyajima Island.

Morphological variations are main adaptive responses in maize roots to low phosphorus availability Chunjian Li, Haitao Liu* China Agricultural University, China Plants show different adaptive responses in roots to phosphorus (P) deficiency, including morphological and physiological traits. To clarify the adaptive mechanisms of maize roots to low P availability, hydroponic experiments under P-sufficient (HP, 250 µM) and P-deficient (LP, 1 µM) conditions were conducted, with two legumes (Vicia faba L. and Lupinus albus L.) as references. The results showed that the LP treatment increased the root biomass, root to shoot dry weight ratio (R/S), and total root length (TRL) of maize and faba bean with more dramatic changes in maize than in faba bean in the R/S and TRL. However, the same treatment for 12 d did not cause any variation in shoot and root growth in white lupin. Root exudation of carboxylates including malate and citrate and acid phosphatase (APase) activity on maize root surface showed no significant difference, while dramatically increased in both legumes when they grew under LP, compared with that under HP. Rhizospheric alkalization (agar technique) or net H+ influx (ion-selective electrode technique) on maize root surface was observed regardless of P supply levels. By contrast, increased rhizospheric acidification or net H+ efflux on root surface of both legumes was observed under LP compared with that 30 Nutrient Acquisition 1

Monday 22 June – Parallel session under HP. The P inflow rate (mg P m-1 root length d-1) and P uptake efficiency (mg P m-1 root length) in legumes were remarkable high than those in maize under LP. However, P use efficiency (mg dry weight mg-1 P) of maize was much higher compared with both legumes. The results suggest that differing from the coupled morphological and physiological responses in roots of white lupin and faba bean, morphological variations in maize roots are the main adaptive responses to low phosphorus availability.

Impact of bio-effectors to promote maize growth and nutrient uptake from alternative phosphorous fertilizers in organically managed soils Cécile Thonar1, Sarah Symanczik1, Florian Walder1, Alessandro Piccolo2, Vincenza Cozzolino2, Martin Kulhánek3, Andreas de Neergaard4, Jonas Duus Stevens Lekfeldt5, Martin Rex6, Markus Weinmann7, Günter Neumann7, Paul Mäder*1 1 FiBL (Research Institute of Organic Agriculture), Switzerland, 2CERMANU-University of Naples Federico II, Italy, 3Department of Agroenvironmental Chemistry and Plant Nutrition, Czech University of Life Sciences, Czech Republic, 4Department of Plant and Environmental Sciences, University of Copenhagen, Denmark, 5Plant and Environmental Sciences, University of Copenhagen, Denmark, 6Arbeitsgemeinschaft Hüttenkalk e.V., Germany, 7Institute of Crop Science 340h, University of Hohenheim, Germany Agricultural production is challenged by the limitation of non-renewable resources. The growing interest in low-input systems supports the endeavor to reduce the input of mineral fertilizers. Instead, alternative fertilizers are used which often have a lower availability of key macronutrients, especially phosphorus (P). Biological inoculants, so-called bio-effectors (BEs), can be combined to these fertilizers to improve the nutrient use efficiency. Their use in lowinput systems aims at improving the plant availability of mineral nutrients, for instance via an extended rooting system and the stimulation of mycorrhizal associations. In addition, BEs have the potential to solubilize sparingly available sources of mineral nutrients and mineralize organically bound forms of P and N. The goal of this study was to assess the potential efficacy of BEs in combination with alternative fertilizers (e.g. composted manure, digestate, green compost) to promote plant growth and nutrient uptake in soils typical for various European regions. Pot experiments were conducted in Czech Republic, Denmark, Germany, Italy and Switzerland where the same variety of maize was grown in local soils deficient in P in combination with alternative fertilizers and the same set of BE (Trichoderma, Pseudomonas and Bacillus strains). Common guidelines for pot experiment implementation and performance were developed to allow data comparison and soils and fertilizers were analysed by the same laboratories. Efficiency of BEs to improve maize growth and nutrient uptake differed mainly according to the soil origin. Promising results were mostly obtained with BEs in combination with organic fertilizers such as composted animal manures and fresh digestate of organic wastes. In any of the experiments, the nutrient use efficiency of mineral recycling fertilizers was improved by BE inoculation. At the conference, plant performances data will be presented together with soil and fertilizer parameters associated with successful BE inoculation.

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Monday 22 June – Parallel session

Cluster roots of Embothrium coccineum (Proteaceae) affect enzyme activities and phosphorus lability in rhizosphere soil Mabel Delgado*1, Alejandra Zúñiga-Feest2, Leonardo Almonacid3, Hans Lambers4, Fernando Borie5 1 Universidad Austral de Chile, Chile, 2Universidad Austral de Chile, Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias., Chile, 3Universidad de la Frontera, Chile, 4University of Western Australia, School of Plant Biology, Australia, 5Universidad de la Frontera, Ciencias Químicas, Chile Cluster roots release organic compounds into the rhizospere, enhancing a plant’s capacity to acquire scarcely-available nutrients. Microorganisms may assimilate these organic compound and nutrients that are released or mobilised by plants, generating competition for nutrient uptake. Mechanisms limiting microbial degradation of citrate exuded from cluster roots have been reported for Lupinus albus. However, it is not known if similar mechanisms operate in other cluster-root producing species. Our aim was to determine the effect of cluster roots of Embothrium coccineum, growing under natural conditions, on soil enzyme activities and phosphorus lability in its rhizosphere. We determined enzyme activities: acid phosphatase, dehydrogenase and β-glucosidase, and the rate of hydrolysis of fluorescein diacetate, as well as phosphorus fractions in the cluster root rhizosphere at different cluster-root developmental stages (juvenile, mature, semisenescent, senescent), in the non-cluster root rhizosphere, and in bulk soil. In addition, we measured the concentrations of total phosphorus and manganese in roots. Our results showed that rhizosphere of senescing cluster roots presented the highest phosphatase, β-glucosidase and dehydrogenase activities, and fastest rate of hydrolysis of fluorescein diacetate, being 2.6, 4.6-, 3.3- and 25.8-fold greater, respectively, than those in the rhizosphere of mature cluster roots. The phosphorus fractionation showed that the inorganic phosphorus fraction was 15% greater in the rhizosphere of mature cluster roots than in that of other stages. Mature cluster roots showed the highest total phosphorus concentration, suggesting the fastest phosphorus uptake. We concluded that cluster roots of E. coccineum modified their rhizosphere depending on their development stage, decreasing soil enzymatic activities at their mature stage. In addition, mature cluster roots increased the inorganic phosphorus fraction in their rhizosphere, allowing the highest total root phosphorus concentration at this developmental stage.

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Monday 22 June – Parallel session

Does wheat regulate adaptations of soil phosphorus acquisition synchronously with increasing shoot P concentration? Qi Shen*, Zhihui Wen, Haigang Li, Jianbo Shen China Agricultural University, China The adaptations of root growth and rhizosphere processes for soil phosphorus (P) acquisition have been investigated intensively in wheat (Triticum aestivum L.). However, the variations of regulation for these adaptations with increasing shoot P concentration have been neglected. A pot experiment was carried out to focus on this question in the greenhouse. A critical level of shoot P concentration for shoot biomass was observed at 4.63 mg g-1, at which wheat attained the maximum biomass and below which shoot biomass decreased because of P limitation. In contrast, the critical level of shoot P concentration for total root length (TRL), specific root length (SRL) and the proportion of fine root (diameter ≤ 0.2 mm) length to total root length (PFR) was c.2 mg g-1. Lower than this level, TRL continuously increased with the increasing shoot P concentration, and higher than this level, TRL maintained at c. 75 m pot-1. However, SRL and PFR showed a unimodal pattern. Rhizosphere acidification was positively correlative with shoot P concentration when which was lower than c. 5 mg g-1. There were two critical levels of shoot P concentration for acid phosphatase activity in rhizosphere, which reached up to the maximum at c. 2 mg g-1, and then continuously decreased with the increasing shoot P concentration until to c.4 mg g-1. Finally, acid phosphatase activity did not change significantly at high shoot P status. P deficiency induced citrate accumulation in rhizosphere of wheat, while it suppressed malate accumulation synchronously. In conclusion, the critical levels of shoot P concentration were specific for adaptations of soil phosphorus acquisition, and different among them. Thus, wheat did not regulate adaptations of soil phosphorus acquisition synchronously with increasing shoot P concentration.

Are hotspots of plant and of microbial organic phosphorus mineralization in the rhizosphere spatially separated? Marie Spohn*1, Michal Cormann1, Yakov Kuzyakov2, Michael Schloter3, Nicole Simone Treichel3, Doreen Fischer3 1 Department of Soil Ecology, Bayreuth Center of Ecology and Environmental Research (BayCEER), University Bayreuth, Germany, 2Department of Soil Science of Temperate Ecosystems, Georg-August-University Göttingen, Germany, 3Helmholtz Zentrum München, Research Unit Environmental Genomics, Neuherberg, Germany In two rhizobox experiments, we tested the hypothesis that hotpots of microbial and of root organic phosphorus mineralization in the rhizosphere are spatially separated. For this purpose, we applied soil zymography, which is a novel method that allows for imaging and analyzing the in situ distribution of enzyme activity in soil. In the first experiment with Lupinus albus L. we found that acid phosphatase activity (produced by plants and microorganisms) was closely associated with roots, while alkaline phosphatase activity (produced exclusively by microorganisms) was more widely distributed in the rhizosphere. This led to a 2.5-times larger area of alkaline than of acid phosphatase activity. This finding indicates that microorganisms also mineralized organic phosphorus in 33 Nutrient Acquisition 1

Monday 22 June – Parallel session areas of the rhizosphere, in which only little organic phosphorus mineralization by the roots occurred. In the second experiment, we analyzed the distribution of acid and alkaline phosphatase activity in the rhizosphere of barley (Hordeum vulgare L.) and the abundance of various bacterial groups by combining soil zymography with fluorescence-in situ-hybridization (FISH). Again acid phosphatase activity was strongly associated with the roots and was highest at the root tips. The number of bacterial cells – mostly beta-proteobacteria, firmicutes and gammaproteobacteria – was larger at the root surface several cm away from the root tip than at the root tip. Taken together, our results showed that acid phosphatase activity was highest at the root tips, where the number of bacterial cells was significantly lower than at the older root zones. The two experiments indicate that hotspots of plant and of microbial organic phosphorus mineralization in the rhizosphere are spatially separated. This spatial niche differentiation might alleviate direct competition between plants and microorganisms for phosphorus.

Using Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy to study phosphorus dynamics in the rhizosphere Dalton Abdala*1, Donald Sparks2, Paul Northrup3 1 Brazilian Synchrotron Light Laboratory, Brazil, 2University of Delaware, United States, 3National Synchrotron Light Source, United States Phosphorus (P) is a major plant nutrient that usually represents a limiting factor in terms of soil fertility and plant nutrition. This is because its low availability, particularly in tropical soils, directly impacts its utilization by plants, leading to limited plant growth and crop yields. Understanding the P chemistry in the rhizosphere is ultimately important to devise strategies that will allow plant growers to overcome the limited uptake and utilization of P by plants. Rhizosphere science has benefited from developments made to analytical techniques, particularly on in situ techniques. This has led to a more comprehensive understanding of the reactions taking place in that region, i. e., reactions controlling the uptake and release of heavy metals and plant nutrients in the rhizosphere. Further advances will, however, be facilitated upon the utilization of more sophisticated in situ techniques, i. e., techniques relying on high flux, intensity and highly collimated beam, such as those based on synchrotron radiation. In this sense, EXAFS is a powerful technique for in situ studies and has been used to address the fate and transformation of plant nutrients and heavy metals at the soil/plant interface. I myself am the first to ever employ this technique to address P surface complexation at mineral/water interfaces and our published papers on P-EXAFS highlight the leap in information quality that P-EXAFS analysis can provide at the molecular/atomic scale, required to understand more fundamental aspects of the rhizosphere chemistry.

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Monday 22 June – Parallel session Phosphorus EXAFS studies will certainly advance the current understanding of P dynamics in the rhizosphere and thus of P uptake by plants. In my talk, I will be presenting the advances that we have made on mechanistic aspects of P chemistry in soils via EXAFS spectroscopy and how this technique can help us shed some light on the intricate processes in the rootzone.

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Monday 22 June – Parallel session Root Endophytes Convenors: Kari Saikkonen, MTT Agrifood Research, Finland, and Leo van Overbeek, Wageningen University, the Netherlands

Convenor talk Endophytic fungi and nutrient cycling Kari Saikkonen* Natural Resources Institute Finland, Finland Few recent studies reveal how fungal endophytes associated with plant foliage can affect litter decomposition rates, stimulate soil carbon sequestration and alter the flux of greenhouse gases (CO2 and N2O) from soil to the atmosphere. This suggests that fungal endophytes may affect belowground food webs and processes they may control. Here I briefly review the current knowledge of how the foliar endophytic fungi and nutrient cycling interact, and aim to highlight general rules and gaps in knowledge. Current endophyte literature suggests that endophytes can modulate the decomposition of plant litter and thus nutrient cycling at least in three ways: (1) by acting as saprotrophs in abscised plant parts, (2) by affecting the amount and/or quality of the plant litter, and (3) by affecting the abundance, richness and composition of decomposer organisms. Yet, we are only beginning to understand how the endophyte-mediated aboveground and belowground processes link with each other.

The endofungal bacterium Rhizobium radiobacter RrF4 promotes disease resistance and plant growth independent of its fungal host Piriformospora indica Karl-Heinz Kogel*1, Jafargholi Imani1, Ibrahim Alabid1, Huijuan Guo1, Peter Kämpfer2, Martin Hardt3, Jochen Blom4, Michael Rothballer5, Anton Hartmann5, Stefanie Glaeser2 1 Justus Liebig University, Department of Phytopathology, Germany, 2Justus Liebig University, Department of Applied Microbiology, Germany, 3Justus Liebig University, Biochemical Research Centre Seltersberg, Germany, 4Justus Liebig University, Department of Bioinformatics and Systems Biology, Germany, 5Helmholtz Zentrum München, Germany The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) forms an intimate mutualistic tripartite symbiosis with the beneficial endophytic Sebacinalean fungus Piriformospora indica (Basidiomycota) and a broad range of host plants. While attempts to cure P. indica from RrF4 repeatedly failed, the bacterium could be isolated nonetheless from the fungus and multiplied in pure culture. Here we report on RrF4’s genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic Agrobacterium tumefaciens C58, except vibrant differences in both the tumor-inducing (pTi) and the assessor (pAt) plasmids, which can explain the loss of RrF4’s pathogenicity. Similar to its fungal host P. indica, RrF4 colonizes roots of monocotyledonous and dicotyledonous 36 Root Endophytes

Monday 22 June – Parallel session plants without substantial host preference. RrF4 forms aggregates of attached cells and dense biofilms at the root surface of maturation zones and accumulate at lateral root protrusions, the potential entry side to the inner root tissue. RrF4-colonized plants have increased biomass and enhanced systemic resistance against bacterial leaf pathogens. RrF4mediated resistance to Pseudomonas syringae pv. tomato DC3000 was compromised in Arabidopsis mutants indicative of the induced systemic resistance (ISR) pathway, a phenomenon that earlier also was observed with P. indica-mediated resistance. Consistent with this, RrF4- and P. indica-induced pattern of defence gene expression in barley roots were similar. In clear contrast to P. indica, but similar to endophytic plant growth promoting rhizobacteria (PGPR), RrF4 colonized not only the root outer cortex but spread beyond the endodermis into the stele. Together our data show that RrF4 exhibit virtually the same mechanistic beneficial activity on plants as its host fungus P. indica, but shows differences in the root colonization pattern.

Detection of spatial dynamics of PAC requires long observation periods Sophie Stroheker*, Valentin Queloz, Thomas N. Sieber ETH Zurich, Institute of Integrative Biology, Switzerland Dark septate endophytes of the Phialocephala fortinii s.l. – Acephala applanata species complex (PAC) are abundant root colonizers of coniferous and ericaceous plants in boreal and temperate forest ecosystems. So far, 21 morphologically indistinguishable but reproductively isolated cryptic species (CSPs) were characterized. PAC species live sympatrically and form complex communities in roots. PAC community structure was spatially stable for three years in a previous experiment, suggesting inability of PAC to disperse in soil and/or mutual inhibition of neighboring PAC genotypes. Now, we repeated the analysis ten years later. The community structures were determined in 2004 and 2014 in a 40/50-yr-old Norway-spruce plantation. Norway-spruce roots were excavated at the same 16 grid points in both years to determine the PAC community using microsatellite genotyping. The majority of the PAC isolates belonged to CSP1, 2, 3, 4 and 5 in both years. CSP12 and Acephala applanata were found in 2014 only. CSPs were the same at only two grid points in both years. CSP1 and CSP3 were the most frequent species in both years. However, CSP3 was more frequent (47%) than CSP1 (26%) in 2004 whereas in 2014 the situation was opposite. In conclusion, significantly more than three years are required to detect shifts in the PAC community structure. The observed shifts suggest that PAC are able to disperse in soil and to colonize other roots, though very slowly. In a next step, the PAC communities in various sterile and non-sterile soil substrates enclosed in ingrowth bags (50 micrometers mesh width) buried at the same grid points will be determined using sterile Norway-spruce seedlings as bait und by next generation sequencing of DNA extracts to confirm dispersal of PAC in soil.

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Monday 22 June – Parallel session Biogeographical diversity of root associated microbial communities in arctic and alpine tundra plants Manoj Kumar*1, Riitta Nissinen2, Jan Dirk Van Elsas1, Angela Sessitsch3, Livio Antonielli3 1 University of Groningen, the Netherlands, 2University of Jyväskylä, Finland, 3AIT Austrian Institute of Technology GmbH, Austria We are interested in bio-geographical diversity and functioning of plant associated microbial communities in the Arctic and Alpine tundra plants. In this study we addressed community composition of soil, rhizosphere and root endophytic bacterial and fungal communities associated with two arcto-alpine plant species, Oxyria digyna and Saxifraga oppositifolia in three climatic regions. The samples were collected from Kilpisjärvi, Finland (sub-arctic), NyAlesund, Svalbard (high arctic) and Innsbruck, Austria (alpine). Terminal restriction fragment analysis (T-RFLP) and massive parallel sequencing were used to address the influence of location, climate and plant species on structure of soil and plant microbial communities. Community fingerprinting analysis of bulk and rhizosphere soils revealed that soil bacterial communities were primarily shaped by climate, geographical location and sampling sites. Within the sampling sites the influence of individual plant species on rhizosphere community composition was clear, suggesting that plant species select their own rhizosphere bacterial community from available local soil microflora. A similar trend was detectable in the community fingerprinting and sequence data of fungal communities of bulk soil and rhizosphere soil communities from different climatic zones. In contrast, the community structures of root endophytic fungi were primarily influenced by plant species, and only secondarily by climatic zones, soil properties or geographical distance. Sequence analysis of soil and rhizosphere fungal communities indicated an increase in phylum Zygomycota and decrease in phylum Chytridiomycota along the latitude from Austria, Kilpisjärvi to NyAlesund, while no such trend was observed in other phyla. In addition, we observed that O. digyna and S. oppositifolia had several plant species specific root endophytic fungal OTUs present across all the regions. These host plant specific fungal species were absent in both bulk and rhizosphere soils.

Metagenomic mining of the endophytic plant microbiome Victor J Carrion*1, Manoeli Lupatini1, Ruth Gomez-Exposito1, Eiko E. Kuramae1, Thierry K.S. Janssens2, Mattias de Hollander1, Jos M. Raaijmakers1 1 Netherlands Institute of Ecology (NIOO-KNAW), Netherlands, 2MicroLifeSolutions, Netherlands The plant microbiome represents an enormous untapped resource for discovering novel microorganisms, traits, genes and bioactive compounds. Endophytes are “microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects”. Endophytic bacteria can promote plant growth, protect against pathogens by producing a range of natural products or activating the plant immune system. Here we focus on the diversity and metabolic potential of the endophytic community of sugar beet plants grown in soils that are naturally suppressive to specific fungal diseases. 16S-rRNA amplicon sequencing showed that the overall structures of the endophytic bacterial communities are very similar for plants grown in suppressive and nonsuppressive (conducive) soils. However, 38 Root Endophytes

Monday 22 June – Parallel session substantial variations were observed in the relative abundance of certain bacterial groups, with Azospira, Chitinophagaceae, Devosia and Flavobacterium being more abundant in the roots of sugar beet plants grown in disease suppressive soil. In vitro experiments with Chitinophaga and Devosia isolates obtained from the endophytic community showed a minor inhibitory activity against R. solani. In planta experiments are currently conducted to resolve if these or other endophytic bacterial genera play a role in the natural protection of plants against fungal infections. Metagenome sequencing of the endophytic community is ongoing to understand the functional potential of the microorganisms that live inside the plant root tissue.

Factors affecting the occurrence of fungal root endophytes and their interaction with plants Jose G. Maciá-Vicente*, Sevda Haghi Kia, Kyriaki Glynou Goethe University Frankfurt/Institute of Ecology, Evolution and Diversity, Germany The importance of fungal endophytes in the functioning of natural ecosystems is commonly assumed given their high prevalence and ubiquity in plants. However, clear effects on plant performance can only be attributed to specific fungi in a few cases, and hence we still lack a general framework of understanding of the ecological significance of these symbionts. We aim at defining functional groups of fungi according to their interaction with plants by combining information on their genetic and biochemical diversity, their ecology, and their direct effect on host's development. For that purpose, we have assembled a broad collection of root-endophytic strains originating from diverse plants and ecological contexts. Most of them come from an extensive sampling of Microthlaspi perfoliatum, a Brassicaceae that lacks mycorrhizal associations. In this we have identified a clear latitudinal gradient defining the composition of its associated endophytic assemblages, which consist of a combination of fungal generalists and others whose presence is determined by specific soil or climatic conditions. These results suggest working hypotheses on the potential function of certain fungal groups that we aim to address by characterizing several of their traits. We have sequenced the ribosomal DNA region of all strains, and have obtained the profiles of secondary metabolites production for most of them. Additionally, we have tested a subset of 128 strains representing most fungal taxa in laboratory inoculation assays of three host plants: M. perfoliatum, arabidopsis and barley. Our results indicate a slight plant-parasitic behavior by most fungi under the assayed conditions, whose outcome is dependent on the strain-host combination. We are currently trying to determine whether the detrimental effect on plant's development is balanced under certain environmental conditions by yet-unknown benefits driven by the endophyte. We intend to combine the above data to gain insight into the evolutionary processes leading to plant-fungal symbioses.

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Monday 22 June – Parallel session Actinobacterial endophytes enhance nitrogen use efficiency with legume crops Christopher Franco*1, Hoang Xuyen Le1, Ross Ballard2 1 Flinders University, Australia*, 2South Australian Research & Development Institute, Australia Selected actinobacterial endophytes improve lucerne plant growth by enhancing nodulation and symbiotic nitrogen fixation by Sinorhizobium meliloti RRI 128. The effect of two Streptomyces strains, LuP30 and LuP47B, were examined with and without the presence of S.meliloti on lucerne at three levels of NH4NO3 supplied: 3, 25 and 50 mg.kg-1 soil under greenhouse conditions. Both strains, when added as the sole inoculant to lucerne seed, improved plant dry weight, in comparison to the untreated control, at 25 and 50 mg N supply 8 weeks after sowing. Plant growth with the actinobacteria alone was similar to that of plants inoculated with the rhizobial inoculant alone. Co-inoculation of Streptomyces spp. LuP30 or LuP47B with S. meliloti significantly increased plant dry matter by up to 35% at 3 mgN.kg-1 and 46% at 25 mgN.kg-1. Co-inoculation of the rhizobium and the actinobacteria significantly increased the number of nodules during the early growth phase (4 weeks) compared with S.meliloti alone at 25 mg N.kg-1. Significant increases in the nitrogen content of lucerne plants co-inoculated with S.meliloti and either Streptomyces sp. LuP30 or LuP47B were observed, when compared to plants treated with the rhizobium alone. Using 15N-labeled NH4NO3 it was determined that the actinobacterial partners were responsible for the significant increases in symbiotic nitrogen fixation of 50 to 70% compared to lucerne plants inoculated only with S.meliloti. These two actinobacteria also showed significant improvement of plant growth when added with S.meliloti to Lucerne plants challenged with pathogenic Rhizoctonia in soil, compared to the untreated control. Actinobacterial endophytes, such as the Streptomyces species described here, have the potential to be applied to legume crops to enhance their symbiosis with rhizobia and significantly improve nitrogen use efficiency, and also to protect the crop against fungal root pathogens.

Bacterial root endophytes of plants growing on oil sands reclamation covers in Alberta, Canada Eduardo Kovalski Mitter*, Renato de Freitas, James Germida University of Saskatchewan, Department of Soil Science, Canada Root endophytes play a key role in supporting plant health and growth in both managed and natural ecosystems. The potential use of bacterial endophytes to assist plant growth on oil sands reclamation covers requires an understanding of the diversity and metabolic potential of these endophytes. This study assessed the diversity of bacterial endophytes and rhizosphere bacteria associated with annual barley (Hordeum vulgare L.) and sweet clover (Melilotus albus) growing on a reclaimed landscape at one of the oil sands mining sites in the Athabasca Region. Plants and peat-mineral soil samples were collected at different locations along two transects at two distinct cover management areas and analyzed for nutrient and hydrocarbons contents. Root-associated microbial communities were assessed by culture40 Root Endophytes

Monday 22 June – Parallel session dependent and culture-independent techniques including denaturing gel gradient electrophoresis (DGGE) and Illumina MiSeq. Available nutrients and total hydrocarbon contents varied at distinct locations. Of 614 isolated bacterial endophytes, 100 randomly selected isolates were identified by 16S rRNA sequencing in which the class Gammaproteobacteria, commonly found in agricultural crops, was predominant amongst these isolates. Rhizosphere and endophytic bacterial community structure analyzed by culture independent techniques varied depending on slope positions and cover types. Selected bacterial endophyte bands from DGGE gel closely matched growth promoting bacteria and potential hydrocarbon degraders, suggesting they may assist plant growth on reclamation covers. Our results suggest that plants growing on oil sands reclamation covers host a wide range of bacterial endophytes, which potentially could be used to assist plant establishment and growth in these areas.

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Monday 22 June – Parallel session Metabolomics Convenors: Nicole van Dam, German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Germany, and Harro Bouwmeester, Wageningen University, the Netherlands

Convenor talk The use of metabolomics to shine light in the black box of the rhizosphere Harro Bouwmeester*1, René Kuijken2, Desalegn Etalo3, Robert Hall2 1 Wageningen University, Netherlands, 2Wageningen UR, Netherlands, 3Netherlands Institute for Ecological Research, Netherlands There is increasing evidence that the rhizosphere microbiome has more effect on the functioning of plants than was previously anticipated. The composition of the rhizosphere microbial community is in turn likely to be strongly affected by the host plant, although the experimental evidence to support this is still scarce. The full details of the mechanisms by which plants shape their rhizosphere microbiome are hence still largely unknown. One of these mechanisms is the chemical communication between plants and their rhizosphere micro-organisms. Examples are the secretion of flavonoids and strigolactones for communication with rhizobial and AM fungal symbionts, respectively. But plants secrete a wealth of molecules from their roots into the medium in which they grow. These molecules range from components that are simply being food for micro-organisms, to signalling molecules or antibiotics that, together, must play a role in shaping the subterranean microbial community. There is scattered evidence that there is genetic variation in the amount and composition of the metabolites secreted by plants into their rhizosphere and that environmental conditions can affect exudation, as well as (consequently?) the microbial community. One of the tools to shed further light on this communication between plants and the rhizosphere organisms is metabolomics, an analytical –omics approach in which hundreds to thousands of metabolites can be detected in a single sample using advanced LC- and GCMass Spectrometry. Statistics is subsequently used to infer relationships between metabolite profiles and biological processes. The principle of metabolomics will be introduced and the use of metabolomics to analyse plant root exudates, the possible pitfalls as well as the potential uses will be discussed.

Metabolomics meets natural variation - integrative analysis of Arabidopsis thaliana exudates Susann Mönchgesang*1, Nadine Strehmel1, Stephan Schmidt1, Christoph Böttcher2, Franziska Taruttis3, Erik Müller1, Lore Westphal1, Steffen Neumann1, Dierk Scheel1 1 Leibniz Institute of Plant Biochemistry, Germany, 2Current address: Julius Kühn Institute, Germany, 3Current address: University of Regensburg, Germany Several metabolomics studies have focused on the aboveground part of plants. Interactions belowground are still poorly understood. Naturally occurring genetic diversity within a species, referred to as natural variation, might influence exudation and consequently the 42 Metabolomics

Monday 22 June – Parallel session chemical composition of the rhizosphere. Especially secondary metabolites are essential for communication at the plant-root-interface. The major challenge in metabolomics remains in compound identification. 19 parental lines of the Arabidopsis MAGIC collection were grown in a hydroponic system, and their exudates were subjected to UPLC/ESI-QTOF-MS analysis. Targeted and nontargeted metabolite profiling were performed, and observations were analyzed in the context of the 19 sequenced genomes. The study uncovered distinct metabolite profiles of root exudates. Hierarchical clustering revealed relationships between selected accessions in exudates, which could only be partly projected to the root profile and/or genetic distance. A user-friendly tool for pre-screening the association between genetic and metabolite patterns was developed. Due to the high variability, the absence of a metabolic feature in at least one accession was used as a stringent measure. These patterns of absence over 19 accessions were compared to nonsense mutations in enzyme-encoding genes from AraCyc. Out of 1950 features in the exudate (-) screen, 1352 were absent in at least one accession. In total, 27 features were matched to two to eighteen stop codons with occurrences in one, two or three accessions. Further filter criteria were applied to select targets for tandem mass spectrometry for in-depth metabolite identification. Integrating knowledge from other omics resources will support metabolite identification and even unravel novel biological functions. This study will shed light on communication in the rhizosphere and its natural variation in the model species Arabidopsis thaliana.

Identification of small molecules secreted by roots in response to shoot applied biotic stresses Hassan Massalha*1, Elisa Korenblum1, Jedrzej Szymanski1, Saul Burdman2, Noam Alkan3, Murad Ghanim3, Asaph Aharoni1 1 Weizmann Institute of Science, Israel, 2Hebrew University of Jerusalem, Israel, 3Agricultural Research Organization, Israel Plants undergo sensitization as a result of biotic and abiotic stresses. Induced resistance is expressed not only locally at the site of induction but also systemically in all plant tissues, even in those spatially separated from the inducer. This phenomenon is known as Induced Systemic Resistance (ISR). ISR can be developed as a result of a wide range of inducers, including pathogenic infection, attack of insect herbivores, root colonization by specific beneficial microbes, chemical treatments and more. In this study, we aim to understand how plants process foliar biotic stress cues and transmit them belowground in order to adjust the rhizosphere environment. Therefore, exudates and root tissue were collected from tomato plants growing in hydroponic cultures, whose foliage had been infected by fungi, bacteria and insect. Metabolome and transcriptome data is currently being generated and analyzed in order to characterize either common or unique small molecules secreted from roots under different foliar biotic stresses.

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Monday 22 June – Parallel session Convenor talk Ecometabolomics in the rhizosphere: So what? Nicole van Dam* German Institute for Integrative Biodiversity Research (iDiv) Halle-JenaLeipzig, Germany By nature, soil communities are extremely diverse. As a consequence, the rhizosphere is populated with a countless number of organisms, such as nematodes, fungi, bacteria and arthropod herbivores. Each of these organisms, alone and in combination, may interact with the plant. As there is no daylight and sound waves do not carry very far in the soil matrix, chemical communication is likely to be the norm belowground. Using state-of-the-art metabolomic analyses and specific experimental set-ups we can determine the chemical compounds excreted by plants and/or the interacting organisms in the rhizosphere. However, seen the sheer numbers of interactors present in a natural rhizosphere community, it is hard to determine who exactly is ‘talking’, who is ‘listening’ and what are the consequences for the performance of each species involved in the chemical communication. This knowledge is of absolute importance, if we truly want to achieve a better understanding of the ecological role of plant-environment interactions in the context of soil biodiversity. With this presentation, I would like to invite you to a discussion on how we can achieve this. What kind of set-ups and experiments will bring us beyond the ‘one plant-one organism’ interactions? Which technologies and approaches are needed to achieve true rhizosphere eco-metabolomics?

44 Metabolomics

Monday 22 June – Parallel session Signaling Convenors: Christoph Keel, University of Lausanne, Switzerland, and Vittorio Venturi, International Centre for Genetic Engineering and Biotechnology, Italy

Phloroglucinol functions as an intercellular chemical messenger with broad transcriptional effects in Pseudomonas protegens Pf-5 Joyce Loper*1, Jennifer Clifford1, Alex Buchanan2, Qing Yan2, Jeff Chang2 1 USDA-ARS at Oregon State University, United States, 2Oregon State University, United States Bacteria can be both highly communicative and highly competitive in the rhizosphere and antibiotics play a role in both of these processes. Among the large spectrum of antibiotics produced by the rhizosphere bacterium Pseudomonas protegens Pf-5, two—pyoluteorin and 2,4-diacetylphloroglucinol (DAPG)— function in intracellular and intercellular communication, both as autoinducers of their own production. Here, we demonstrate that phloroglucinol, an intermediate in DAPG biosynthesis, can serve as an intercellular signal influencing both the production of pyoluteorin and inhibition of Pythium ultimum, a phytopathogenic oomycete sensitive to pyoluteorin. The analysis of RNAseq data sets showed that phloroglucinol influences the transcription of pyoluteorin biosynthesis genes in a concentration-dependent manner via a mechanism involving the transcriptional regulator PltR. Furthermore, phloroglucinol had broad effects on the transcriptome of Pf-5, significantly altering the transcription of 289 genes by at least two fold. The 130 genes most highly regulated by phloroglucinol (≥4 fold) fall into 14 role categories, reflecting the influence of phloroglucinol on diverse aspects of bacterial physiology and highlighting interactions between primary and secondary metabolic pathways. The effects of nanomolar versus micromolar concentrations of phloroglucinol differed both quantitatively and qualitatively, influencing the expression of distinct sets of genes or having opposite effects on transcript abundance of certain genes. Therefore, our results support the concept of hormesis, a phenomenon associated with signaling molecules that elicit distinct responses at different concentrations. P. protegens controls the relative production of phloroglucinol vs. DAPG via PhlG and PhlA, two enzymes in the DAPG biosynthesis pathway. Just as concentration mediates the inhibitory versus signaling functions of an antibiotic through the process of hormesis, the relative concentrations of PG versus DAPG mediate the distinct roles of the pathway in the communication or competition critical to the bacterial lifestyle in the rhizosphere.

Effect of maize diversification on its cooperation with the model PGPR Pseudomonas fluorescens F113 Claire Prigent-Combaret*, Vincent Walker, Daniel Muller, Yvan Moënne-Loccoz Microbial Ecology UMR5557, France The efficacy of cooperation between cereals and Plant Growth-Promoting Rhizobacteria (PGPR) differs according to the genotypes of the two partners, but the functional traits responsible for these differences are poorly understood. We hypothesize that taking into account the genetic structure of cereals resulting from crop diversification will help 45 Signaling

Monday 22 June – Parallel session understand differences in their association with PGPR partners. We addressed this hypothesis with maize (a plant whose dissemination and diversification resulted in the formation of 5 main genetic groups) and Pseudomonas fluorescens F113, a type of Pseudomonas PGPR found worldwide in association to cereal roots. We determined whether 15 maize lines representative of maize diversity differ or not regarding the composition of their root exudate profiles, the ability of their exudates to trigger expression of plant-beneficial genes of the model PGPR F113, as well as their ability to respond to F113 inoculation. An experimental system was set up to grow maize lines in axenic conditions and collect root exudates. The chemical composition of root exudates was compared using HPLC-based metabolic profiling and their impact on the expression of two F113 plant-beneficial genes (such as acdS encoding the enzymatic activity 1-aminocyclopropane-1-carboxylate deaminase and phlABCD involved in the biosynthesis of the antimicrobial compound 2,4- diacetylphloroglucinol) analyzed. We showed that maize lines differ from each other regarding their root exudate composition and their ability to induce the expression of plant- beneficial genes in F113. These differences do not relate to the genetic relationships between maize lines, suggesting that the establishment of efficient cooperation with PGPR was not influenced by the evolutionary history of maize diversification.

Convenor talk A sensor protein controlling host-specific expression of an insect pathogenicity factor in plant-beneficial rhizosphere pseudomonads Peter Kupferschmied1, Maria Péchy-Tarr1, Nicola Imperiali1, Monika Maurhofer2, Christoph Keel*1 1 University of Lausanne, Switzerland, 2ETH Zurich, Switzerland Pathogens and pest insects that attack belowground parts of agricultural crop plants are very difficult to control by conventional methods. The application of microbial control agents is a promising alternative. We discovered that phylogenetically defined groups of root-colonizing pseudomonads, specified by Pseudomonas protegens and Pseudomonas chlororaphis, exhibit potent entomopathogenic activities in addition to their known plant-beneficial activities, which include the suppression of soil-borne fungal and oomycete pathogens, the promotion of plant growth and the induction of systemic plant defenses. These bacteria colonize larvae of pest insects, particularly Lepidoptera and Diptera, with ease. Upon ingestion by pest larvae, they colonize their gut, invade and proliferate in their blood system, and ultimately kill the insects. We try to unravel molecular mechanisms that allow these fascinating pseudomonads to distinguish between plant and insect environments and to adapt their activities specifically to the two contrasting hosts. Insect pathogenicity of the pseudomonads depends to an important part on the production of a large insecticidal protein, which we termed the Fit toxin. Remarkably, expression of the potent insecticidal toxin is switched on only in the insect but not on plant roots. We dissected a sophisticated regulatory system which assures the host-dependent tight control of Fit toxin production. A hybrid sensor histidine kinase that shares a sensing domain with a histidine kinase regulating carbon uptake in Proteobacteria emerged as the key element of host sensing. Our data indicate that inhibition of the sensor during root colonization is the underlying mechanism by which the bacteria discriminate

46 Signaling

Monday 22 June – Parallel session between the plant and insect hosts. We anticipate that additional factors relevant for insect interaction are also controlled in a host-dependent manner.

Convenor talk Interspecies and interkingdom signaling in plant associated bacteria Vittorio Venturi* ICGEB, Italy It is believed that most bacteria live in constant association or in the vicinity of many different bacterial species. In addition, most bacteria are now believed to produce and respond to chemical signals in a cell-density dependent manner in a process known as quorum sensing (QS). QS results in a synchronous response of bacterial populations which confers them a form of multicellularity and enables them to adapt and survive to challenging environments. Most bacterial QS studies thus far have involved mono-species (in fact mono-strain) set up which are rather distant from what occurs in nature. It is our major interest to investigate chemical signaling in interspecies bacterial communities and the possible role of chemical signals in plant-bacteria interactions. We are using endophytes which enter the plant via the rhizosphere to study bacterial interspecies interactions. The other emerging aspect of communication in plant associated bacteria is the presence of interkingdom signaling between plant and bacteria. If and how do bacteria and plant communicate still remains at large unknown. As plant associated bacteria have been living many million of years alongside plants it is most probable that signaling mechanisms between them have evolved. Studying this aspect, we have found a new family of bacterial regulators (which are closely associated to the quorum LuxR-family proteins) which bind low molecular compound(s) produced by plants and regulate gene expression. The role of this protein has been studied in a plant beneficial rhizosphere bacteria and a plant pathogenic bacteria showing that it plays crucial roles. This sub-family of LuxR family regulators is very common in plant-associated bacteria thus constituting a major form of interkingdom signaling.

47 Signaling

Tuesday 23 June – Parallel session Nutrient Acquisition 2 Convenors: Hans Lambers, the University of Western Australia, Australia, and Jianbo Shen, China Agricultural University, China

Root exudate effects on zinc availability to plants Ellis Hoffland*1, Sjoerd Van der Zee1, Andreas Duffner1, Hans Meeussen2 1 Wageningen University, Netherlands, 2Nuclear Research & Consultancy Group, Netherlands Zinc is an essential micronutrient, limiting crop production on ±30% of the world’s agricultural soils. The availability of Zn to plants depends on concentrations and distribution of Zn species in the rhizosphere rather than on the total Zn content of soils. The concentration and distribution of Zn species in the soil solution vary significantly between bulk and rhizosphere soil due to root exudation, microbial activity, Zn uptake and pH effects. To understand and predict Zn bioavailability we combined multisurface chemical speciation and transport models into a new Orchestra-based tool that describes interactions between root exudates and a nutrient that binds to the soil solid phase. Interaction of Zn, Fe and citrate with goethite and soil organic matter, degradation of citrate, and diffusion of Zn towards and citrate away from the root surface are considered in the model. During the conference, we present results on quantitative effects of root citrate exudation on bioavailability of Zn in various soils, varying in pH, soil organic matter or metal(hydr)oxides. The model output contributes to our understanding of success or failure of plant species or genotypes to mobilize growth-liming nutrients from various soils through root exudates.

Iron deficiency-induced root exudation of coumarins in Prunus rootstocks grown at high pH Ana Alvarez-Fernandez*, Yolanda Gogorcena, Javier Abadia, Anunciacion Abadia Aula Dei Experimental Station-CSIC, Spain Plant Fe deficiency is a problem in calcareous soils, due to the low solubility of Fe(III) hydroxide. Peach trees are among the fruit crop species most sensitive to Fe-deficiency. Prunus rootstocks differ widely in their susceptibility to Fe deficiency. Phenolics root secretion is associated to Fe deficiency tolerance in different plant species. Recent studies have shown that the fluorescent coumarin scopoletin along with other coumarins are exudated by the roots of the model species Arabidopsis and tomato in response to Fe deficiency. However, no information on Fe deficiency-induced root phenolics secretion has been reported for any fruit tree species so far. In the present work, two Prunus rootstocks (GF677 and Cadaman) with different tolerance to lime-induced chlorosis were grown for two weeks in half-strength Hoagland solution buffered at 7.5 pH, including either 0 (Fe-deficient plants) or 90 μM Fe(III)– EDDHA (Fe-sufficient plants). Iron deficiency induced the root secretion of fluorescent compounds in both genotypes, and leaf chlorosis was less intense in the Fe-efficient rootstock (GF677) than in the Fe-inefficient one (Cadaman). Root extracts and exudates were analyzed by liquid chromatography coupled to mass spectrometry. Several glycosides of 48 Nutrient Acquisition 2

Tuesday 23 June – Parallel session different coumarins accumulated in GF677 roots in response to Fe deficiency. Also, several coumarins and coumarin derivatives were secreted in response to Fe deficiency by GF677 roots. The concentration of these compounds in root extracts and exudates of Fe-deficient Cadaman plants were lower than those from GF 677 roots. Phenolics removal from growth medium of Fe-deficient plants, by circulating the nutrient solution through a C18 resin column, led to a more advanced leaf chlorosis in GF677 but not in Cadaman. This supports that the root secretion of phenolics by Fe-efficient Prunus rootstocks could play an important role in Fe deficiency tolerance at high pH.

Analysis of a rice mutant with altered root system architecture in a nutrientdependent manner Toru Fujiwara*, Yoshihiro Ohomori, Ryohei Yoshinaga, Nobuhiro Tanaka University of Tokyo/Graduate School of Agricultural Life Sciences, Japan Root system architecture (RSA) is an important factor of nutrient uptake from soil. In general, root elongation/root branching is enhanced under mild deficiency of nutrients including nitrogen, phosphorus and sulfur, while deficiency of other nutrients such as boron does not enhance root development. RSA is affected by nutrient status and different nutrients have been shown to have different impacts on RSA. Regulation of RSA is important for adaptation to various nutrient conditions in soil. Several genes have been shown to be important for nutrient dependent RSA changes in Arabidopsis thaliana, but in rice, little is known about the molecular mechanisms of the nutrient-dependent modulation of RSA. In the course of our screening of Taichung65 N-methyl-N-nitrosourea mutagenized rice mutant population, we found a mutant with root growth defects at the stage of seedlings grown in deionized water culture but not strongly in normal hydroponic culture. It turned out that in this mutant, root growth was strongly impaired under low nutrients, but not when normal nutrients were supplied. Shoot growth is also affected under the low nutrients condition but not as strongly as root growth. We then investigated which essential nutrient deficiency is causing this phenotype and found that respective deficiencies of N, P and Ca affect the root growth of the mutant. Composition of nutrients in the mutant was not strongly affected, suggesting that the mutant is not defective in nutrient uptake or translocation but mechanisms in sensing nutrient condition and/or regulating root development accordingly. The gene responsible for the phenotype is located around 25 Mb on chromosome 2 and mutations were found in several genes in the mapped region including a gene possibly involved in phytohoromone metabolism. Confirmation of the gene responsible for the phenotype is in progress.

49 Nutrient Acquisition 2

Tuesday 23 June – Parallel session Focusing or broadening the spectrum of arbuscular mycorrhizal fungal symbionts - What is better for plant nutrition and biomass production? Hannes Gamper*, Muhammad Ali, Renata Slavíková, Emmanuel Frossard ETH Zurich, Agricultural Sciences, Switzerland Whether single or multiple, native or foreign arbuscular mycorrhizal fungi (AMF) make plants grow better or improve their mineral nutrition is still a largely unanswered question. Here, we report on the outcomes of a fully cross-factorial pot experiment, involving a wild type and an isogenic mycorrhizal symbiosis-defective mutant of Lotus japonicus, still able to form symbioses with N2-fixing bacteria. Plants were raised in either slightly alkaline, slightly acidic, or a 1:1 (w/w) mixture of these soils. Prior to transplantation into pots, plants were either preinoculated or not with a strain of Funneliformis caledonius, Acaulospora scrobiculata, or the mixture of the two AMF and transplanted into native or gamma-irradiated soils after washing the roots free of soil. Similarly, plants were also pre-inoculated with the native AMF assemblages of the pure or mixed study soils. Plants inoculated with F. caledonius grew better in the alkaline soil and those with A. scrobiculata grew better in the acidic soil, compared to the inoculation treatment with the other fungus and soil of the opposite pH. Co-inoculation of both AMF strains yielded plants of comparable size to those inoculated with F. caledonius, that were bigger than those inoculated with A. scrobiculata. In the mixed soil, the plants inoculated with F. caledonius and the mixture of the two AMF grew equally well but better than the plants inoculated with A. scrobiculata. We can thus conclude that mycorrhizal symbiotic functioning is dependent on a matching between soil pH and AMF identity, which seems to make mixtures of strains more effective than single-strain inoculants in novel soil environments. Ongoing analyses on fungus-mediated 15N and 33P uptake, fungal root colonization, and P availability in soil, are expected to provide insights into the drivers of differential growth responses to AMF inoculation in living field soil with an indigenous AMF assemblage.

Maize root and rhizoshere responses to nutrient patches under progressing topsoil drought - trade-offs and constraints Eric Visser*1, Christian Fritz2, Dina in 't Zandt2, Chantal Le Marié3, Andreas Hund3, Lidia Campos4, Blanca San Segundo4, Philippe Hinsinger5, Claude Doussan6, Annette Bérard6 1 Radboud University Nijmegen, Institute for Water and Wetland Research, Netherlands, 2Radboud University Nijmegen, Netherlands, 3Swiss Federal Institute of Technology Zurich, Switzerland, 4Center for Research in Agricultural Genomics (CRAG), Spain, 5INRA UMR Eco&Sols, France, 6INRA UMR EMMAH (Environnement Méditerranéen et Modélisation des Agro-Hydrosystèmes), France Drought spells during the growing season of crops are expected to become more frequent due to clime change, particularly in the Mediterranean area. In the framework of the EU FP7 programme EURoot, twenty consortium members joined forces and are currently identifying crop traits that are advantageous for yield in conditions of progressing top soil drought. This presentation will show how maize plants respond to such drought conditions when challenged with patches of nutrients in the soil. These patches proved to be disadvantageous

50 Nutrient Acquisition 2

Tuesday 23 June – Parallel session when shallowly applied, causing drought to affect nutrient availability and accumulated root biomass in the top soil layer. Deep patches were initially negatively affecting plant growth in irrigated conditions, but then appeared to have an at least partially mitigating effect when top soil drought occurred later in the season. These experiments were performed in large mesocosms. Plants were grown outside under a rain shelter at a density similar to the field. The large containers enabled us to track root development non-destructively via minirhizotron tubes, and to collect rhizosphere and root samples to determine the soil metabolic profiles and mycorrhizal colonization as affected by nutrients and drought. Strong effects were found of soil water content and nutrient conditions on these rhizosphere processes, with a local rather than systemic character. The project in general shows the importance of involving multiple disciplines when tackling more complex environmental interactions on plant-rhizosphere responses.

Mineral nutrition of campos rupestres plant species on contrasting nutrientimpoverished soil types Rafael Oliveira*1, Hugo Galvão1, Mariana de Campos2, Cleiton Eller1, Stuart Pearse2, Hans Lambers2 1 University of Campinas, Brazil, 2University of Western Australia, Australia Nutrient availability plays a pivotal role in structuring plant communities. In ancient and nutrient-impoverished landscapes, plant communities tend to be phosphorus- (P) limited and species-rich, with nonmycorhizal species with root specializations that increase P acquisition being prominent. In Brazil, the campos rupestres occur over the Brazilian shield, and are characterized by acidic nutrient-impoverished soils, which are particularly low in phosphorus (P). Despite recognition of the campos rupestres as a global biodiversity hotspot, little is known about the diversity of P-acquisition strategies and other aspects of plant mineral nutrition in this region. To explore nutrient-acquisition strategies and assess aspects of plant P nutrition, we measured leaf P and nitrogen (N) concentrations, characterized root morphology and determined the percentage arbuscular mycorrhizal (AM) colonization of 50 dominant species in six communities, representing a gradient of soil P availability. Leaf manganese (Mn) concentration was measured as a proxy for carboxylate-releasing strategies. Communities on the most P-impoverished soils had the highest proportion of nonmycorrhizal (NM) species, the lowest percentage of mycorrhizal colonization, and the greatest diversity of root specializations. The large spectrum of leaf P concentration and variation in root morphologies show high functional diversity for nutritional strategies. Higher leaf Mn concentrations were observed in NM compared with AM species, indicating that carboxylate-releasing P-mobilizing strategies are likely to be present in NM species. The soils of the campos rupestres are similar to the most P-impoverished soils in the world. The prevalence of NM strategies indicates a strong global functional convergence in plant mineral nutrition strategies among severely P-impoverished ecosystems.

51 Nutrient Acquisition 2

Tuesday 23 June – Parallel session Uptake of asparagine and inorganic nitrogen in inoculated and non-inoculated white clover plants Weronika Graj*1, Sandra Jämtgård2, Torgny Näsholm2, Jim Rasmussen1, Mogens Nicolaisen1, Inge Fomsgaard1 1 Aarhus University, Denmark, 2Swedish University of Agricultural Sciences, Sweden White clover (Trifolium repens), a forage legume, is a valuable species. It can fix N directly from the air via symbiosis with Rhizobium, which is reflected in generally high N content in the plant. All stored N-compounds when released to the surrounding plants after clover decay, can act as a good source of nutrients. Moreover, when sown with grasses, clover increases significantly forage quality due to its high N content. In spite of evidences that white clover is economically attractive due to its role in the soil N flow, there has been only little attention paid to amino acid uptake by Trifolium species with no focus on white clover. Amino acids are highly abundant in soil, thus they also play an important role in the total N dynamics. Understanding the processes that govern N fluxes, particularly N uptake under white clover cultivation, is of major importance with respect to both environmental concerns and the quality of crop products. The first objective of this project was to determine the general white clover potential for amino acid uptake in hydroponic experiment where the labelled L-Asparagine-13C4,15N2 was used. Analysis of the results showed recovery of labelled L-Asparagine-13C4,15N2 as well as metabolites from the plant material confirming its uptake of the amino acid. The next step is to conduct two other experiments with aim to (1) assess how the uptake of 13C4 15N2-Asn changes when amino acid is supplied together with the NH4NO3 as the source of inorganic nitrogen (IN), (2) elucidate how 13C415N2-Asn acquisition by white clover changes when both the IN source in the medium is present and the plant is inoculated with Rhizobia. It is planned to finalize hydroponic experiments within the next two months, therefore all the results from three different hydroponic setups will be presented.

Image based modelling of nutrient uptake by root hairs Keith Daly*, Sam Keyes, Shakil Masum, Tiina Roose University of Southampton, United Kingdom Root hairs provide a significant contribution to the uptake of low mobility nutrients such as phosphate. However, in order to fully understand their and quantify their role non-invasive measurements of the root and soil structure are essential. Three dimensional imaging of plant roots in-situ using X-ray computed tomography is a rapidly growing field which provides resolutions as low as 500 nm. Using this technique combined with image based modelling we can image and quantify the role of root hairs in nutrient uptake. The root and root hairs are imaged in-situ and the soil, water, air and root material are segmented from the image to provide a computational representation of the geometry which can be used for numerical simulation. Previous modelling of this type has been constrained as finite computational resources limit the maximum domain size on which models can be run and, hence, limit the timescale over which the model is valid. We have overcome this by combing rigorous averaging and upscaling techniques with targeted simulations which capture the bulk soil properties and root hair – soil interactions in the rhizosphere. Specifically the bulk 52 Nutrient Acquisition 2

Tuesday 23 June – Parallel session soil properties are found from a representative volume of far from the root. The bulk soil is patched onto another representative volume containing the root structure and rhizosphere soil. The size of each of these volumes is increased until the uptake properties converge. This approach allows us to determine the size of the rhizosphere and model an effectively infinite volume of soil about the root. This increases the timescale over which the model is valid to longer than the root hair lifetime.

53 Nutrient Acquisition 2

Tuesday 23 June – Parallel session Engineering the Rhizosphere Convenors: Yves Dessaux, CNRS, France, and Paul Schulze Lefert, Max Planck Institute for Plant Breeding Research, Köln, Germany

Convenor talk by Paul Schulze Lefert Abstract to be announced

Biogeography of microbial phosphorus solubilization and applications to improve crop yields Peter Baas*1, Colin Bell2, Lauren Mancini2, Melanie Lee2, Richard Conant2, Matthew Wallenstein2 1 Colorado State University, United States, 2Colorado State University, USA Although phosphorus is an essential nutrient for plant growth our understanding of the controls of the biogeography of microbial communities capable of liberating phosphorus from stabilized pools via solubilization is very limited. The importance of plant-microbial interactions in governing this ecosystem property is even less clearly understood. Further, current agricultural practices result in the majority of applied phosphorus fertilizer to either be immediately made unavailable by chemical-physical interactions with soil surfaces or result in run-off and associated water pollution. Could the inoculation of a highly productive microbial community improve plant phosphorus uptake? In the current study, we investigated phosphorus solubilization rates of cultures grown in selective media from soils originating from all over the United States. We selected the most productive culture and prepared it for a greenhouse inoculation experiment. We compared plant productivity (common vegetables, herbs and turf) in greenhouse experiments among plants inoculated with the selected culture against traditional fertilizer based approaches in addition to elucidating any synergistic effects. We used agricultural soil with a legacy of fertilization (turf) or mixed 1:1 with potting soil (tomato, jalapeno and basil). Our results suggest that cultures developed from soils high in total phosphorus have a greater ability to solubilize and are dominated by Pseudomonas and Enterobacter species. Turf productivity showed an interactive synergistic effect when applying both turf starter (traditional management) and our culture inoculum. Initial results suggest a similar pattern when the traditional Miracle Grow was combined with the inoculum for jalapeno, tomato and basil. In conclusion, our results suggest a paramount opportunity exist to use a selection approach to develop microbial communities capable of improving soil phosphorus solubilization. Development and application of this research could have major implications for the understanding of the controls of microbial biogeography in addition to management applications to improve crop phosphorus use efficiency.

54 Engineering the Rhizosphere

Tuesday 23 June – Parallel session Helping the quorum-quenching biocontrol agents using biostimulation Denis Faure*1, Jérémy Cigna2, Anthony Kwasiborski1, Pauline Dewaegeneire3, Amélie Beury3 1 CNRS, France, 2CNRS/SIPRE, France, 3SIPRE, France Degradation of quorum-sensing (QS) signals N-acylhomoserine lactones (AHL) by soil bacteria is proposed as a beneficial trait for protecting crops, such as potato plants, against the worldwide pathogen Pectobacterium. Our previous studies revealed three quorum- quenching (QQ) activities (amidase; lactonase and reductase) in Rhodococcus erythropolis, which therefore appeared as one of the most effective QQ-bacteria. As a consequence, native and introduced R. eythropolis populations may act as plant-protective agents. The question arises how to stimulate the rhizosphere colonization by R.erythropolis. We characterized an AHL-degrading, biocontrol R. erythropolis isolate R138, which was introduced in the potato rhizosphere. We also identified gamma-heptalactone (GHL) and gamma-caprolactone (GCL) as biostimulating agents that efficiently promote the growth of the QQ-bacterium R. erythropolis. In greenhouses (hydropony and soil cultures), different combinations of GHL- and R138treatments were compared in root colonization by AHL-degrading bacteria using a cultivation-based approach (percentage of QQ-bacteria), rrs-pyrosequencing (total bacterial community) and qPCR of the qsdA gene that encodes an AHL-lactonase in R. erythropolis. The higher densities of AHL-degrading R. erythropolis population in rhizosphere were observed when GHL- and R138-treatments were associated. Under this condition, the introduced R. erythropolis displaced the native R. erythropolis population. Chemical analyses revealed that GHL and GCL, and their by-products, gamma-hydroxyheptanoic acid and gammahydroxycaproic acid, rapidly disappeared from the rhizosphere and did not accumulate in plant tissues. In field assays, we monitored the introduced R. erythropolis and Pectobacterium pathogen using appropriate strain-selective primers which were developed from their complete genome. We also evaluated plant-protection against the introduced and native Pectobacterium populations. Upon biostimulation, an efficent colonization of the potato rhizosphere by R. erythropolis was observed, and as well as a decrease of plant symptom level in some assays. This study highlights biostimulation as a potential approach for improving root colonization by beneficial bacteria.

55 Engineering the Rhizosphere

Tuesday 23 June – Parallel session Water Relations Convenors: Claude Doussan, INRA, France, and Doris Vetterlein, UFZ, Germany

Convenor talk Root age distribution: How does it matter in plant processes? A focus on water uptake Doris Vetterlein*1, Claude Doussan*2 1 UFZ, Germany, 2INRA - UMR 1114 EMMAH, France As root grows from apex, a gradient in age arises between distal and proximal sections of the root. During the root system growth process, young branch roots emerge from the axis and other younger roots (e.g. adventitious roots) may reach same domain of soil as older roots. In relation with the root developmental pattern of the species and environmental conditions, this will result in various distributions of root ages and root types with depth. It is known that a number of root functions will vary with root type and root tissue age (e.g. respiration, exudation, ion uptake, root hydraulic conductance, mucilage release, soil compaction, hydrophobicity, …) and so will the resulting rhizosphere properties. The impact of the distribution of root age with depth, and related functions, on the overall functioning of the root system is fundamental for an integration of processes on the root system scale. In this presentation, we give a quick review of age distribution within root systems in relation with variation of root functions, considering root type and plant species (monocot/dicot, perennial/annuals) which may exhibit different patterns. We will also point to deficit in data and identify some knowledge gaps, while trying to define an integrated descriptor of root age at the root system level. We will exemplify then the possible effect of root aging and function, in relation with root system developmental pattern, with a functional root architecture model of water uptake. We will focus on 2 contrasted root architectures resulting in a more or less sharp gradient in root age along the soil profile. The implications of these root distributions, for water uptake, in relation with variation in root hydraulic conductance or other processes (cortex decrepitude for example) will be examined at the profile and whole root system levels. These behaviors will be compared to an integrated root system age index.

The importance of mucilage for water uptake of maize roots Mutez A. Ahmed*1, Mohsen Zarebanadkouki2, Andrea Carminati2 1 University of Göttingen, Germany, 2Göttingen University, Germany As plant roots take up water and the soil dries, water depletion is expected to occur in the rhizosphere. However, by exuding mucilage, plants maintain the rhizosphere wetter and possibly more conductive than the bulk soil. Indeed, our recent measurements with a root pressure probe technique demonstrated that mucilage exudation facilitates the water flow to roots in dry soils. However, the generalization of this finding to large root systems is 56 Water Relations

Tuesday 23 June – Parallel session not trivial. Mucilage is primarily produced at the root tips and it is not clear how long mucilage persists at the root surface before being degraded. The role of mucilage in facilitating water uptake is therefore closely related to the ability of the young distal root segments to take up and transport water. The question is: how active are the young distal root segments in water uptake? We used neutron radiography to trace the transport of deuterated water (D2O) into maize roots. Maize plants were grown in sandy soils for five weeks. Then we injected D2O in different locations along the roots. The transport of D2O was simulated using a convection– diffusion numerical model. By fitting the observed D2O transport we quantified the diffusion coefficient and water uptake along the roots. The maize root architecture consisted of a primary root, 5-6 seminal roots, 4-5 crown roots and many lateral roots. The results show that the distal parts of the seminal roots were not active in root water uptake. In contrast, we found that the distal parts of crown roots were very active in taking up water and transporting it to the shoot. Interestingly, we also found that the crown roots exuded a large quantity of mucilage. Based on these observations, it is very tempting to conclude that mucilage exudation is a trait that favors the acquisition of water from dry soils.

Development of root-soil contact and gap formation of Vicia faba in drying soil Nicolai Koebernick*, Steffen Schlüter, Sebastian Blaser, Doris Vetterlein Helmholtz Centre for Environmental Research - UFZ, Germany Root-soil contact is a prerequisite for the uptake of water and nutrients from the soil matrix. Root shrinkage and subsequent loss of root-soil contact in drying soil has been shown repeatedly, but only for Lupinus albus the dynamics of root shrinkage and its relationship with plant water status was investigated. We studied the development of root-soil contact of taproot and lateral roots of Vicia faba L. during a drying period using X-ray CT. Plants (N=4) were grown in cylinders filled with a sandy soil. They were placed in a climate chamber and after two weeks under well-watered conditions a drying period was initiated. During the drying period, samples were repeatedly scanned with an X-ray CT scanner to visualize root-soil contact. Soil matric potential, transpiration rate, and stomatal conductance were measured daily. At the end of the drying period, plants were irrigated and CT scanned three hours after re-watering. Root-soil contact of taproots did not exceed 60 %, even before there was substantial shrinkage of roots, while lateral roots had an initial root-soil contact close to 90 %. Transpiration rate and stomatal conductance decreased at soil matric potentials between -5 and -10 kPa. Roots started to shrink at matric potentials below -30 kPa. Lateral roots had a higher relative shrinkage (up to 42 %) than taproots (up to 23 %), but they retained higher root-soil contact. Root-soil contact of the taproot decreased to 16.5 – 27.9 %, lateral roots had a minimum root-soil contact of 47.3 – 74.9 %. Three hours after re-watering, root shrinkage was completely reversed and root-soil contact was close to initial values. This study confirms previous findings with lupin roots, in that roots shrink after transpiration rate 57 Water Relations

Tuesday 23 June – Parallel session decreases. In contrast to lupin, lateral roots of fava beans shrink more than taproots, but they still retain partial contact to soil.

58 Water Relations

Tuesday 23 June – Parallel session Food-web Interactions Convenors: Mark Bradford, Yale School of Forestry & Environmental Studies, USA, and Katarina Hedlund, Lund University, Sweden

Convenor talk Structure and soil food webs: are there really root versus detritus-based compartments? Mark Bradford* Yale University, United States Classical soil food-web theory identifies distinct energy channels (or compartments) through which resources flow belowground. Channels are initially divided based on their basal resource, either roots or detrital inputs. The latter channel is then further sub-divided into bacterial- and fungal-based compartments, which are considered to differ markedly in their response to disturbance and influences on carbon and nutrient cycling rates and storage. However, recent conceptualizations blur these distinctions and their functional significance. Instead, labile root inputs such as exudates are suggested to fuel more than half of the activity of belowground food webs, and to provide the primary substrate for formation of stable soil carbon. I consider how the recent consideration of root inputs as a dominant food source for both symbiotic and free-living soil microbes necessitates a re-evaluation of the connectedness and functional significance of presumed energy channels in soil food webs.

Convenor talk Intensive agriculture influence soil food webs, biodiversity and ecosystem functions Katarina Hedlund* Lund University, Department of Biology, Sweden Intensification of agricultural has profound effects on soil food webs and the rates and levels of ecosystem functions produced. Food web theory predicts relations between diversity stability and functions that influence the rates of C and N processed in the food web. Agricultural food webs from 4 European countries provide evidence on how diversity and food web structure is influenced by intensive agriculture and climate change factors. Results from a European study will show examples on how diversity and functions of food webs are influences by intensification and climate change. This has implications on multiple functions from soils.

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Tuesday 23 June – Parallel session Composition of soil biodiversity networks, functional changes in nutrient dynamics, and consequences for vegetation succession Elly Morrien*, Emilia Hannula NIOO-KNAW, Netherlands The majority of current theory on plant community ecology has been based on vegetation succession at abandoned arable land. This shows how habitat filtering and competition for limiting resources structures plant community composition. Currently, there is consensus that plant community development is the result of those factors, as well as of interactions with belowground biota. Here, we address the question how soil community diversity and structure affect nitrogen and carbon cycling during secondary succession. In 2011, we visited 9 grassland sites, categorized as recent, mid-term, long-term abandoned ex-arable fields. Bacteria and fungi were identified by pyrosequencing, while archaea were identified using TRFLP. The protists, micro-fauna, nematodes, enchytraeids and earthworms were extracted and morphologically identified until species (taxon) level. In total, around 15 000 species were identified from the soils. We created a Spearman-rank correlation matrix based on abundance data of species which we visualized in a network. In 2012, intact soil cores with comparable plant vegetation were collected from the same sampling points. Stable isotope probing of the cores was performed using dual labelled 15N ammonium nitrate (15NH4 15NO3) and 13C was fed to the plants in the form of 13CO2. The soil food web structure was resolved by identifying the microbes using phospholipid markers and identifying soil fauna by morphology into similar aggregated groups as for the network analysis, both combined with isotopic measurements. With the combination of the visualized network and a functional study on living soil cores, we provide evidence that the conversion of soil food web structure appears to be more important than a quantitative change in biodiversity. Moreover, we show that structural changes in the food web topology also leads to functional changes in the soil food web which can act as a driving force during land use change after human disturbance.

Food web structure affects mycorrhization and seedling survival Sina Adl1, Felicity Crotty*2, Melanie Wilson3 1 University of Saskatchewan, Canada, 2Aberystwyth University, United Kingdom, 3Dalhousie University, Canada We tested several hypotheses to show that grazing by microarthropods and nematodes on mycorrhizae can have both beneficial and detrimental consequences on plant growth and survival. We established microcosms with Eastern White Pine seedlings (Pinus strobus) and manipulated the food web by adding and removing consumer trophic levels. The experiments were conducted under typical field abundances with all co-occurring species extracted from the same forest soil. Seedlings that did not receive mycorrhizae, or that were exposed to excessive grazing on mycorrhizae, grew poorly and seedling survival was poor compared to other treatments. Excessive grazing on mycorrhizae occurred when predators on the fungivores were removed. In these cases, seedlings died due to poor or no mycorrhization during the first 90 days of growth. Multivariate analysis of the data showed that survival and seedling biomass correlated most, and strongly, with the level of mycorrhization by treatment and by individual seedling. We further measured mycorrhizae 60 Food-web Interactions

Tuesday 23 June – Parallel session consumption rates (µm h-1) by obtaining functional response curves of the fungivorous nematodes on mycorrhizae using a dual-labelled fluorescence technique. We also measured grazing rates of the microarthropods on mycorrhizae, and of predatory microarthropods on the fungivorous consumers, using 13C and 15N stable isotope ratio mass spectrometry. We modified the technique to measure as low as one individual microarthropod instead of 1 mg bulked dry mass. By measuring 13C and 15N accumulation in body mass, in excreted pellets, and in moulted cuticles, we calculated assimilation efficiencies for all fungivores and their predators. Our results showed trophic cascades occur in the fungivore energy pathway, and we have obtained detailed measurements of the rates of ingestion, assimilation and transfer to higher trophic level. Changes in soil community structure that affect the amount of grazing on mycorrhizae affects seedling survival and growth, causing mortality when fungi are overgrazed.

Detritus-based trophic cascades mediate the affect of plant-based cascades on N cycling in old-field systems Robert Buchkowski*, Oswald Schmitz Yale University, United States Nitrogen (N) cycling is a fundamental process central to numerous ecosystem functions and services. Accumulating evidence suggests that species within detritus-based and plant-based food chains can play an instrumental role in regulating this process. However, the effects of each food chain are usually examined in isolation of each other, so it remains uncertain how interactions between these food chains impact N cycling. We experimentally manipulated the species composition of detritus-based (isopods and spiders) and plant-based (grasshoppers and spiders) food chains individually and in combination within mesocosms in an old-field ecosystem in Northeastern Connecticut. We tested (1) their relative impact on nitrogen cycling and (2) whether interactions between them moderated the net impact on N cycling. We found that spiders in plant-based food chains exerted the only significant positive effect on nitrogen cycling increasing N mineralization by 42 ng-N×gdry weight-1×day-1 and soil N by 112 ng-N×gdry weight-1. Detritus-based food chains had no effect on nitrogen cycling in isolation; but when combined with plant-based food chains mitigated the positive effects of plant-based species. A possible mechanism explaining this mitigation was the reduction in grasshopper survival by 20% in treatments with isopods and spiders. Our results suggest that detritus-based species ultimately limited rates of N cycling by eroding the enhancing effects of plant-based species through antagonistic interactions.

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Tuesday 23 June – Parallel session Evaluation of entomopathogenic nematode soil food webs in Swiss soils reveals major differences between agricultural and natural habitats Raquel Campos-Herrera*1, Xavier Chiriboga1, Geoffrey Jaffuel1, Ruben Blanco-Perez1, Anna-Sofia Hug2, Reto Giulio Meuli2, Ted Turlings1 1 University of Neuchâtel, Switzerland, 2Agroscope Institute for Sustainability Sciences ISS, Switzerland Entomopathogenic nematodes (EPNs) naturally occur in natural and agricultural soils and contribute to the control of soil dwelling insect pests. We hypothesized that EPN prevalence and equilibrium are negatively affected by agricultural practices and this should be reflected in their presence in agricultural soils as compared to natural areas. Organisms associated with EPN, such as free-living nematodes (FLNs) that compete for insect cadavers, ectoparasitic bacteria that limit nematode movement, and nematophagous fungi (NF) should also be affected. By combining traditional and molecular methods we explored EPN soil food web in different Swiss sites categorized as agricultural (wheat and other annual crops) and natural areas (forest and grassland). In spring 2013-2014, soil samples were recovered from each geo-referenced locality (Swiss Soil Monitoring Network NABO-project, n = 30; NRP68nematode project, n = 10). Higher numbers of EPN were recorded in natural areas (P = 0.072), but this did not translate in higher EPN activity, richness or diversity. The FLN Acrobeloides-group was also higher in natural areas (P = 0.002). In contrast, NF were present in higher numbers in agricultural areas (P = 0.002), suggesting that they may be a key factor in limiting the native EPN biocontrol potential. The sampled Swiss natural areas were distributed among higher altitudes (>800 masl), with higher organic matter (OM, >5%) and pH slightly >5 (P < 0.01) than the agricultural areas (500 masl, OM >2%, and pH ≈6.5). These differences might also modulate the EPN soil food web assemblage. A new survey planned for spring-2015 will provide additional measurements to confirm these patterns, in order to determine critical soil properties and biotic factors that shape the EPN assemblages in Swiss soils. This knowledge will provide insights into how soils can be modified to increase biological control of soil-borne pests in order to enhance crops health.

Grasses do not feed soil microbes to obtain nutrients Søren Christensen*1, Marie Dam2 1 Copenhagen University, Dept. Biology, Sect. Terrestrial Ecology, Denmark, 2Copenhagen University, Dept. Biology; Sect. Terrestrial Ecology, Denmark It is a well-established idea that release of organic carbon from roots aids the plant in accessing nutrients for growth. The proposed mechanism behind this is that the extra activity of the soil decomposer community mediated by this carbon leads to increased release of plant nutrients otherwise bound in dead organic matter. Part of the literature supports this idea, part does not. We performed a study where the grass cover in a global change study was defoliated two times; early in the growing season during active growth and late in the season after grass growth ceased. We hypothesized that defoliated plants would have an increased demand for nutrients, and therefore release more carbon into the rhizosphere, in the early growth phase, and even more so at elevated CO2 with reduced nutrient availability 62 Food-web Interactions

Tuesday 23 June – Parallel session in the plant-soil system. The defoliation late in the season increased microbial growth rate as well as protozoan numbers in the rhizosphere, both indicating an increased microbial production due to the defoliation. The defoliation effect on soil biota was most marked at elevated CO2. However, a similar defoliation during active plant growth early in the season reduced microbial biomass and nematode populations in the rhizosphere which suggests that microbial production was reduced due to defoliation. Moreover, the defoliation effect was not affected by CO2. Our results therefore do not suggest that root exudation is a mechanism for plants to obtain nutrients from soil in these grasses. We suggest that the contrasting results from the literature are at least partly due to differences in the growth phase of the plants: Late in the season at low nutrient demand defoliated plants release organic carbon; early season during active growth plants withhold their resources and do not release organic carbon from the roots when defoliated.

Multitrophic interactions in the rhizosphere: Key roles of protists Stefan Geisen*1, Michael Bonkowski2, Wim van der Putten1 1 NIOO-KNAW, Netherlands, 2University of Cologne, Germany Plant roots host an enormous diversity of associated organisms. Among them are bacterivorous protists that impact plant performance as major drivers of nutrient cycling, while other protists act as devastating plant pathogens. Species-specific functional differences remain, however, elusive and even the community structure of soil protists is largely unknown. To increase the knowledge on soil protists I am using a multi-methodological approach including cultivation, microcosm experiments and environmental sequencing. Using a combination of cultivation- and sequence-based methods I showed that the protist community structure in soils is more complex than previously assumed. In functional studies on cultivated taxa I revealed that protists directly interact with a range of other organisms, e.g. as predators of fungi and nematodes. The potential importance of these novel trophic nodes in soils was underpinned by an environmental sequencing approaches revealing ubiquitous presence of the respective protists in soils, and further uncovered a range of animal parasites. Now I aim at identifying (protist) pathogens as drivers of intracontinental plant range expansion. I cultivated three plants that expanded their ranges in the past 200 years and closely related native species in soils from Slovenia and the Netherlands. I sterilized roots, cultivated root-endoparasitic eukaryotic microbes and am now evaluating species- and location-specific pathogenicity in microcosm experiments. Preliminary results indicate that plant species host different plant pathogens, which likely play a role in determining success of plant range-expansion.

63 Food-web Interactions

Tuesday 23 June – Parallel session Rhizosphere Microbiome 2 Convenors: Rodrigo Mendes, Brazilian Agricultural Research Corporation, Embrapa Environment, Brazil, and Phil Poole, University of Oxford, UK

Microbes From Inner Space: III. The impact of geography and biotic stress on the root endophytic and rhizospheric microbiomes of Trifolium repens Richard Johnson*1, Rhys Jones1, Paul Maclean1, Jana Monk1, Katharine Adam2, Alison Popay1, Nigel Bell1, Damien Fleetwood3 1 AgResearch Ltd, New Zealand, 2AgReseach Ltd, New Zealand, 3Biotelliga Ltd, New Zealand Trifolium repens (white clover) is the key legume component of New Zealand pastoral agriculture. Although the benefits from symbiosis with rhizobia are well known, the identity and potential benefits of non-rhizobia endophytic and rhizospheric microbes associated with white clover remain unknown. We have performed MiSeq rRNA amplicon sequencing from sterilised T. repens roots and rhizosphere soil from ten different New Zealand locations. Further to this we have compared the microbiome of T. repens growing in these soils free of invertebrate pests or challenged by Meloidogyne hapla nematodes and Costelytra zelandica scarab larvae. By characterising the microbiome of T. repens roots growing in these soils we will identify geographic and biotic factors influencing the taxonomic make-up of these communities, including whether T. repens “recruits” different microbes in response to nematode versus insect pests. These studies will complement parallel experiments to determine the biological soil suppression potential against invertebrate pests (Bell et al, this volume), and the potential for beneficial microbes to be carried endophytically in white clover seed (Monk et al, this volume).

Dynamics of plant-internal oxygen and analysis of rhizoplane methanotrophic communities in Phragmites australis Anna Fausser*1, Michael Hoppert2, Paul Walther3, Marian Kazda1 1 University of Ulm, Institute for Systematic Botany and Ecology, Germany, 2GeorgAugust Universität Göttingen, Institute of Microbiology & Genetics, Germany, 3University of Ulm, Central Electron Microscopy Unit, Germany Plants in wetland ecosystems are adapted to anaerobic sediments by supplying air to submerged organs and into the rhizosphere. Around the roots, phytotoxic compounds are oxidised and oxygen-dependent bacteria can grow. Dynamics of oxygen were monitored in the pith cavities of rhizomes of Phragmites australis (Cav.) Trin. ex Steud. in a constructed wetland (Slavošovice, Czech Republic) over several days. The internal oxygen concentration was measured every five minutes using an optical detection system (Fa. PreSens). The presence of methane-oxidising bacteria in the rhizoplane of P. australis was tested, as oxygen is a rare resource in the rhizosphere. Therefore roots samples were fixed chemically for analysis in transmission electron microscopy and immunolabelling. 64 Rhizosphere Microbiome 2

Tuesday 23 June – Parallel session Oxygen concentrations in the rhizomes showed distinct diurnal dynamics. Soon after sunrise rhizome oxygen concentrations increased steeply to almost ambient levels around 19-20 % during midday. In the evening, oxygen concentrations declined exponentially over night to minimum values between 6 to 9 %. This indicates for high oxygen consumption in the rhizosphere. Pressurised ventilation mechanisms ceased during night while submerged organs and root-associated microbes consumed restricted internal oxygen reserves. Distinctly lower slopes of oxygen decline during late night hours implicate a down regulation of biological and biogeochemical processes in the rhizosphere, roots and rhizomes. In the dense root-associated biofilms of P. australis we detected up to 37 % of potential methaneoxidising bacteria. These extensive microbial biofilms in the rhizoplane of wetland plants are presumably the key oxygen consumer. This confirms that methane oxidation is a common and important microbial process related to oxygen-releasing roots of wetland macrophytes. These findings accentuate the importance of oxygen supply to the rhizosphere by aerenchymatic macrophytes for the degradation of organic compounds and methane oxidation.

Nitrogen cycling and rhizosphere microbiome of summer active perennial grasses in Australian soils Vadakattu Gupta*1, Marcus Hicks1, Kroker Stasia1, Bill Davoren1, Phil Ward1, David Ferris2, Rick Llewellyn1 1 CSIRO, Australia, 2DAFWA, Australia In the lower rainfall mixed farming regions of southern Australia perennial pasture species are recommended for economic and environmental sustainability. Perennial grasses grown during summer in South Australia and Western Australia provide dense rhizosphere environments (considered N-limited) with significant inputs of C through rhizodeposition providing a C-rich environment for soil bacteria esp. N cycling microbial communities. We investigated rhizosphere bacterial communities (taxonomic and functional) associated with summer active perennial grasses such as Megathyrsus maximus Jacq. cv. Petrie and Gatton, Panicum coloratum L. cv. Bambatsi (Bambatsi panic), Chloris gayana Kunth cv. Katambora and Digitaria eriantha Steud. cv. Premier. Samples were collected from field experiments on sandy-textured soils at Karoonda, South Australia and Moora, Western Australia were analysed for the abundance (qPCR) and composition of total bacteria (16S amplicon sequencing), N2-fixing bacteria (nifH-TRFLP) and the amount of non-symbiotic 15N2 fixation. Genetic diversity of bacteria was higher in the grass rhizosphere compared to that in the barley rhizosphere. Proteobacteria were the dominant species in both grass and barley rhizospheres, although there were significant differences in Phyla level composition. For example, Proteobacteria, Actinobacteria and Firmicutes were higher in the Bambasti Panic rhizosphere whereas Actinobacteria and Gemmatimonadetes were higher than Barley rhizosphere. Populations of diazotrophic bacteria in the grass rhizosphere soils were similar to populations in the roots but the diversity of diazotrophic bacteria was significantly higher in the rhizosphere than the roots. Different grass species promoted the abundance of specific members of the nifH community, suggesting plant-based selection from the soil microbial community. Diazotrophic N2 fixation estimates for the grass roots ranged between 0.92 and 2.35 mg15N/kg root/day. 65 Rhizosphere Microbiome 2

Tuesday 23 June – Parallel session Our results suggest that a greater rhizosphere bacterial diversity under perennial grasses coupled with higher carbon inputs and greater volume of rhizosphere soil result in significant changes in the N cycling processes and soil C turnover.

The microbiome of the seagrass Zostera marina responds to a temperature shift in a mesocosm experiment Jenna Lang*, Laura Reynolds, Jonathan Eisen, Jay Stachowicz University of California, United States Despite their success on land, only ~60 of the >250,000 angiosperm species live fully submerged in the ocean. These species (all called seagrasses) represent three independent invasions of the marine environment. Life under the sea is extreme. Seagrasses are bathed in salt water, sunk below meters of light-scattering water, and rooted in toxic, sulfuric, wavebattered sediments. Among the three seagrass lineages, convergent evolution has led to similar morphological and physiological adaptations to the marine habitat. Have the microbes associated with seagrass roots and leaves undergone a similar convergence? What role do they play in seagrass adaptation to the marine environment? The Seagrass Microbiome Project (seagrassmicrobiome.org) uses evolutionary and ecological approaches to tackle these questions. Here, we present a 16S rDNA PCR survey of microbes associated with seagrass roots. This was conducted in conjunction with a mesocosm common garden experiment, in which 7 genotypes of the seagrass Zostera marina were subjected to a month-long period of warming. Previous work has shown that these 7 genotypes differ significantly with respect to shoot production, biomass, and nutrient uptake rates. During the experiment, all plant genotypes responded positively to the increased temperature, but differed in their ability to tolerate the shift back to ambient. There were no significant differences in microbial community composition between the Zostera marina genotypes, despite differential plant genotype performance during the experiment. However, specific microbial taxa were correlated with plant traits and responded to the warming treatment. The most abundant rhizosphere taxa are involved in sulfur oxidation and nitrogen fixation, and thus are prime candidates for seagrass symbionts. These include members of the genera Arcobacter, Desulfobulbus, Sulfurimonas, Winogradskyella, and Thalassospira. We’ll discuss ongoing efforts to cultivate these taxa for future experimental work.

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Tuesday 23 June – Parallel session Human Pathogens in the Rhizosphere Convenors: Jeri Barak, University of Wisconsin-Madison, USA, and Gabriele Berg, Graz University of Technology, Austria

Convenor talk Environmentally-dependent regulation of root colonization factors in Salmonella enterica Jeri Barak* University of Wisconsin-Madison, United States Plants are now recognized as a significant secondary host in the life cycle of enteric human pathogens, such as Salmonella enterica. S. enterica is exposed to numerous environmental stresses during colonization of its hosts; stresses common in plant niches include plant defense responses and fluctuations in temperature, humidity, and nutrient availability. Despite these obstacles, S. enterica is ubiquitous in the plant environment, persisting for months on roots and in the rhizosphere during crop production. This adaptation has led to increased incidence of human disease from consumption of contaminated fresh produce. A common route for S. enterica into production fields is via water used for irrigation or pesticide application from which Salmonella is commonly isolated. Thus, we predict that the bacterium’s ability to transition from a motile lifestyle to a sessile lifestyle (on plants) would impact its success on plants. In support of this idea, several S. enterica attachment factors contribute to colonization of roots: proteinaceous curli, O-antigen capsule, and cellulose. In vitro CsgD-activated transcription of adrA initiates cellulose production. The diguanylate cyclase AdrA synthesizes the signaling molecule cyclic dimeric GMP (c-di-GMP) which binds the PilZ domain of the cellulose synthase BcsA, activating production of cellulose. Interestingly, an adrA mutant is not defective in root colonization. The lack of an in planta phenotype for an adrA mutant could be explained by an additional diguanylate cyclase activating BcsA during root colonization. However, this putative activation does not appear to occur in vitro as an adrA mutant is devoid of cellulose production in the typical biofilm formation assays. We hypothesize that a subset of c-di-GMP metabolizing proteins are functionally active in the rhizosphere. A putative diguanylate cyclase involved in the in planta activation of BcsA would demonstrate both environment-specific activity as well as potential functional redundancy with AdrA.

67 Human Pathogens in the Rhizosphere

Tuesday 23 June – Parallel session Convenor talk The microbial network and role of pathogens in vegetables and their impact on human health Gabriele Berg*1, Armin Erlacher1, Martin Grube2, Massimiliano Cardinale2, Kornelia Smalla3, Robert Krause4 1 Graz University of Technology, Austria, 2University of Graz, Austria, 3Julius KühnInstitut – Federal Research Centre for Cultivated Plants (JKI), Germany, 4Medical University of Graz, Austria The highly diverse microbiomes of vegetables roots are reservoirs for opportunistic and emerging pathogens. In recent years, an increased consumption, larger scale production and more efficient distribution of vegetables together with an increased number of immunocompromised individuals resulted in an enhanced number of documented outbreaks of human infections associated with the consumption of vegetables. The occurrence of potential pathogens in vegetable microbiomes, the impact of farming and processing practices, and plant and human health issues will be presented. Lettuce (Lactuca sativa L.) and rucola (syn. arugula, Eruca sativa Mill.) will be used as example to explain the microbial networks in the rhizosphere as well as the possibility to use this knowledge to avoid outbreaks of human pathogens and to promote biocontrol approaches. In addition, the potentially positive aspects of the vegetables microbiome for the gut microbiota and human health will be discussed.

Molecular basis to attachment of Escherichia coli O157:H7 to plant hosts Nicola Holden*1, Ashleigh Holmes1, Yannick Rossez1, Louise Birse1, Henriette Lodberg Pedersen2, William Willats2 1 The James Hutton Institute, United Kingdom, 2University of Copenhagen, Denmark Enterohaemorrhagic Escherichia coli (EHEC) are able to actively interact with plants and use them as secondary hosts. Proliferation occurs under permissive conditions, for example when the bacteria colonise susceptible hosts or reside in favourable niches such as the rhizosphere. Specific molecular interactions underpin the associations, using the same general mechanisms that are recognised in well-known plant-associated bacteria. Attachment is normally a prerequisite to successful colonisation of a eukaryotic host, regardless of species or even kingdom. Adherence is mediated by surface-associated bacterial factors such as fimbriae/pili and flagella. Specific interactions occur when adherence factors recognise and bind to receptor target. Characterisation of the interactions between bacteria and animal hosts has shown that adhesins (normally supported on fimbrial structures) target glycoproteins that decorate host cell surfaces. Thus, in order to characterise adherence interactions of E. coli O157:H7 with plant hosts, we have used a high throughput screening approach of plant cell wall glycans. This was coupled with additional screening and functional assays to identify bacterial adherence factors in E. coli O157:H7 Sakai that enable functional binding in planta. The work has focused on plant roots because these pathogens colonise roots in substantially higher numbers that the phyllosphere.

68 Human Pathogens in the Rhizosphere

Tuesday 23 June – Parallel session Several adherence factors in E. coli O157:H7 Sakai were found to bind to plant roots, including Escherichia coli common pili (ECP). ECP interacted predominantly with long chain arabinans present in pectin, and there was some indication of interaction with certain arabinogalactoproteins (AGP). The ECP receptor was found to be enriched in certain plant tissues and species, which may in turn influence distribution of the pathogen. This work has relevance to food safety and risk analysis, but also adds to fundamental knowledge about plant-microbe interactions.

Plant root colonization by entero-aggregative Escherichia coli Leo van Overbeek* WUR Plant Research International, Netherlands The causative agent responsible for the outbreak of hemorrhagic uremic syndrom (HUS) among consumers of fenugreek sprouts in Hamburg, 2011, was caused by an enteroaggregative E. coli O104:H4 (EHEC) strain. Most likely, the pathogen was already present on, or inside fenugreek seeds and from there it was able to contaminate sprouts. This is a remarkable phenomenon because the pathogen must have survived long-term storage and shipping on, or inside seeds. It was therefore our purpose to investigate whether this specific group of E. coli were able to survive on, or in seeds and to colonize plants emerging from contaminated seeds. Work currently in progress in our laboratory is to explore the possibilities of entero-aggregative E. coli to systemically spread through plants with eventual risks on contamination of reproductive organs and developing seeds. We, therefore, used a mildly virulent entero-aggregative strain of E. coli, belonging to E. coli O104, and studied possibilities of outgrowth from pea seeds to emerging plants. We demonstrated that the bacteria on seed were able to spread to shoots and roots of emerging plants, with higher colony forming units present in root versus shoot tissue. Later in the development of these plants, until flowering and pod and seed production stages, E. coli O104 CFUs were only observed in root tissue and rhizosphere soil, but not in shoots, leaves and reproductive organs. As low numbers of contaminants may already result in disease outbreak, we will further explore the possibility of shoot and seed contamination of pea plants in greenhouse and field studies. We tentatively concluded that pea seed contamination with E. coli O104 will result in contamination and efficient colonization of roots and rhizosphere soil, but not in contamination of shoot and reproductive organs.

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Tuesday 23 June – Parallel session Below and Above Ground Interactions Convenors: Alison Bennet, The James Hutton Institute, UK, and Susanne Wurst, University of Berlin, Germany

Convenor talk Genotypic and species variation predict above-belowground interactions Alison Bennett*1, Ali Karley1, Niall Millar2, Matthew Emslie-Smith3 1 The James Hutton Institute, United Kingdom, 2University of Dundee, United Kingdom, 3University of St. Andrews, United Kingdom While host plant genotype has long been recognized as an important factor in plantherbivore and above-belowground interactions, the role of herbivore genotype has largely been ignored despite our wide knowledge of the variation in genetic background and even endosymbionts present in herbivores. In a series of studies using potato we have been exploring the role of herbivore genotype, host plant genotype, and the interactions between them in above-belowground interactions. We have manipulated soil and soil symbiont communities, plant host and aphid genotype, and recently we have found an interaction between all three factors that influences plant resource allocation, aphid growth rates, soil symbiont associations, and aphid parasitism rates. In particular we found that AM fungi promoted direct over indirect defense in potato, and that interactions between plant host and aphid genotype influence the rate of AM fungal colonization. We clearly show that insect and plant genotype influence the outcome of above-belowground interactions.

Convenor talk Root herbivore effects on grassland plants depend on root herbivore history of the soil and land use intensity Ilja Sonnemann, Susanne Wurst* Freie Universität Berlin, Germany Root herbivores are known to affect plant performance. However, the root herbivore net effect may be highly context dependent, as their influence is partly indirect, via alterations of plant interactions with other organisms. In a greenhouse experiment, we determined root herbivore effects on a plant community, as influenced by different soil biota communities. A grassland plant community was grown either with or without Agriotes spp. larvae (Coleoptera: Elateridae), in a first phase with two different soil biota communities, and in a second phase with these communities and their soil substrates that had either been Agriotes trained or not. We found that Agriotes training of the soil substrate determined plant community productivity, and root colonization by arbuscular mycorrhizal fungi (AMF) in the model plant species Plantago lanceolata, while actual root herbivory had no effects on these parameters. In a field experiment we determined root herbivore effects on plant interactions with aboveand belowground organisms at different land use intensities. Local P. lanceolata plants were 70 Below and Above Ground Interactions

Tuesday 23 June – Parallel session established with and without Agriotes larvae in their root system in grasslands of different management intensity, to determine potential root herbivore impacts on plant interactions with AMF and invertebrate shoot herbivores. We found that root herbivore impacts on AMF root colonization and leaf damage by shoot herbivores depended on cattle grazing intensity and mowing frequency, respectively. Overall, we showed (I) indirect effects of root herbivores on plant performance via soil organisms and (ii) impacts of land use intensity on root herbivore effects on plant interactions with above- and belowground organisms.

Do shoot and root strategies for resource use are coordinated along a secondary succession gradient undergoing frequent disturbance? Amandine Erktan*1, Diane Bouchet2, Catherine Roumet3, Alexia Stokes1, François Pailler1, François Munoz4 1 INRA UMR AMAP, France, 2UMR AMAP, France, 3CNRS (CEFE), France, 4Université Montpellier UMR AMAP, France Replacement of species, with contrasting ecological strategies for resource use, occurs along successional gradients and with disturbance. Studying the variations of plant ecological strategies provides insightful information to better understand plant community dynamics and functioning. Shoot and root plant functional parameters (morphological, phenological or physiological characteristics of plant communities) can be used as proxies’ for ecological strategies. Our objective is to assess linkages between shoot and root strategies in plant communities along a secondary succession gradient and to test whether disturbance modifies these relationships. We analyzed 24 plant communities forming a 64 year long successional gradient (from 5 to 69 year old) along roadsides. A width of 1 m next to the road is periodically mown, resulting in frequent disturbance. We measured variations in the community functional structure using six above- and below- ground functional parameters characterizing plant morphology and chemistry. Results revealed parallel variations in shoot and root functional parameters along the successional gradient. For instance, earlysuccessional plant communities showed thin organs - both leaves and roots - with high surface area/mass, usually associated with an efficient resources use - both above (light) and belowground (nutrients). Along succession, both leaves and roots become coarser with a decrease in surface area/mass, suggesting a coordinated change in both above and belowground compartments toward more conservative strategies. Conversely, variations in shoot and root functional parameters in response to frequent disturbance (mowing) were independent. For instance, while leaves became thinner with higher surface area/mass when periodically mowed, root morphological parameters remained the same. This suggests that mowing tend to favor leaf traits associated with acquisitive strategies and that roots act as a buffer compartment, with low sensitivity and a relative independence to disturbance of the aboveground compartment.

71 Below and Above Ground Interactions

Tuesday 23 June – Parallel session Plant-soil feedback effects on plant chemistry and performance of aboveground herbivores Martine Kos*, Martijn Bezemer Netherlands Institute of Ecology (NIOO-KNAW), Netherlands Plants can influence the (a)biotic conditions of the soil they grow in, and this, in turn, can change the performance of plants that grow later in the soil. This process is named plant–soil feedback. Plant–soil feedback studies have typically focus on effects on plant biomass, but plant–soil feedback effects can also change aboveground plant–insect interactions via soilmediated changes in plant chemistry. To what extent these effects depend on the identity of the plant species that conditioned the soil, and how they are influenced by soil nutrient availability is unknown. We performed a series of greenhouse experiments to examine how plant–soil feedback effects of a range of plant species influence the growth and primary (amino acid) and secondary (pyrrolizidine alkaloids) chemistry of ragwort (Jacobaea vulgaris) and the performance of specialist and generalist aphids, and whether fertilisation influences these plant–soil feedback effects on aboveground plant-aphid interactions. We observed that different plant species, via species-specific effects on soil fungal communities, exerted different plant-soil effects on J. vulgaris biomass, amino acid concentrations in phloem exudates and aphid performance. Furthermore, we observed a strong interaction between soil conditioning and fertilisation on amino acid and pyrrolizidine alkaloids concentrations and on aphid performance, with opposite effects of soil conditioning at the two fertilisation levels. We provide novel evidence that plant–soil feedback effects on plant chemistry and aphid performance depend greatly on the plant species that conditioned the soil, and that fertilisation can alter the plant–soil feedback effects on aboveground plant-insect interactions, even directionally.

Unravelling mechanisms linking plant community composition and soil-disease suppression Ellen Latz*1, Nico Eisenhauer2, Björn C. Rall3, Stefan Scheu4, Alexandre Jousset5 1 German Centre for Integrative Biodiversity Research (iDiv) Halle-JenaLeipzig, Germany, 2German Centre for Integrative Biodiversity Research (iDiv) HalleJena-Leipzig & University of Leipzig, Germany, 3German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig & University of Jena, Germany, 4University of Göttingen, Germany, 5University of Utrecht, Netherlands Soil-borne plant diseases cause dramatic yield losses, and adverse impacts of applied chemical pesticides on the environment and human health foster the need for alternative disease control. Pathogen antagonistic microbes primarily drive natural disease suppression in soil. Accordingly, research so far mostly focused on microbial agents, but plant disease suppression is most likely dependent on a complex set of rhizosphere properties. Via measuring plant influences on several biotic as well as abiotic soil properties and analysing their direct as well as indirect influence on soil disease suppressive ability in a structural equation model, we were able to reveal that pathogen suppressiveness is indeed interactively shaped by a complex set of factors which are essentially driven by previous plant community composition. Our results indicate that among an important indirect role of plant 72 Below and Above Ground Interactions

Tuesday 23 June – Parallel session communities in shaping soil-disease suppression (e.g. via changes in root biomass, soil pH and the abundance of important bacterial biocontrol agents), plants interactively directly affect the soils suppressive potential. This study represents an important first step in understanding the complexity of mechanisms linking plant community composition and disease suppression. We propose that an interactive synergy of direct as well as indirect effects of plants therefore indeed have to be taken into account in order to predict and manipulate plant-disease suppression.

The root of rhizobacteria-induced systemic resistance Corné Pieterse*, Christos Zamioudis, Roeland Berendsen, Peter Bakker Utrecht University, Plant-Microbe Interactions, Netherlands Plants nurture a large community of plant growth-promoting rhizobacteria (PGPR) that provide them with essential services, such as enhanced mineral uptake, nitrogen fixation, growth promotion, and protection from pathogens. These plant microbiota are predominantly hosted by the root system. Selected PGPR promote plant health by stimulating the plant’s immune system, a phenomenon called induced systemic resistance (ISR). The molecular mechanisms underpinning ISR have been intensively studied in the interaction between the model plant Arabidopsis thaliana (Arabidopsis) and the PGPR strain Pseudomonas fluorescens WCS417 (WCS417). WCS417-mediated ISR is effective against a broad variety of pathogens and even insect herbivores. WCS417-ISR requires functional jasmonic acid and ethylene signaling pathways and its expression in foliar tissues is controlled by the transcriptional regulators NPR1 and MYC2. Large scale gene expression analyses revealed that the establishment of WCS417-ISR in foliar tissues is not associated with major changes in gene expression. Instead, ISR-expressing leaves are primed for accelerated defense gene expression, which only becomes apparent after pathogen attack. This phenomenon is known as priming and provides a cost-effective mechanism of protection against pathogens and pests. In contrast to leaves, roots reprogram the expression of a large set of genes in response to colonization by WCS417. The root-specific R2R3-type MYB transcription factor MYB72 emerged as an important early regulator in the onset of rhizobacteria-ISR. Recently, we demonstrated that MYB72 is essential for both ISR and the production of phenolic compounds that are excreted in the rhizosphere to mobilize iron. Our recent work highlights the role of rhizobacterial volatiles in this processs and provides a thus far unidentified mechanistic link between the ability of rhizobacteria to stimulate systemic immunity and iron uptake mechanisms in host plants.

Organic amendment modulates the nematode-induced aboveground defense responses of plant genotypes differing in phytohormonal signaling pathways Zhenggao Xiao*, Manqiang Liu, Xiaoyun Chen, Linhui Jiang, Huixin Li, Feng Hu Nanjing Agricultural University, China The defense responses triggered by belowground herbivores that would increase aboveground plant systemic resistance are mediated by phytohormones. But it remains 73 Below and Above Ground Interactions

Tuesday 23 June – Parallel session indefinite how these defense response changes are mediated by endogenous jasmonic acid (JA), particularly the interaction with salicylic acid (SA). Moreover, the extent of plant systemic defense may be mediated by the biotic and abiotic factors in rhizosphere. To resolve this scientific issue, we investigated whether root-feeding nematodes (Meloidogyne incognita)induced foliar resistance differs in soil amendment type (inorganic vs organic) with three isogenic tomato genotypes of distinct jasmonic acid synthesis. The phytohormonal changes in leaves were analyzed by liquid Chromatograph Mass Spectrometer. Foliar crucial regulated gene-expressions of JA/SA pathway and related resistance were dissected by Real-time Quantitative PCR Detecting System. We found that the number of root galls of the three genotypes was less under organic amendment than under inorganic amendment. Regardless of amendment type, there were significantly fewer galls on 35S (JA over-expressing genotype) than on Wt (wild-type), particularly than on spr2 (JA-deficient genotype). Moreover, there were antagonistic interactions between the SA and JA signaling pathways and defense responses. In the Wt and 35S genotypes, root-knot nematodes increased the foliar JA concentration under organic than under inorganic amendment, thus up-regulating the protease inhibitors (PIN1, PIN2) gene expression and down-regulating the PAL gene expression. Compared with organic amendment, the nematode induced SA accumulation and SlWRKY70 transcript levels up-regulation further triggered the suppression of JA-induced defense response (i.e. PIN1, PIN2) under inorganic amendment. Our results suggested that root herbivore would modulate the interactions of aboveground phytohormonal signaling pathways, conferring various resistances in response to divergent environment of rhizosphere. It provides a great potential of adopting organic amendment to increase plant defenses to herbivores.

The microbial landscape in soils - Biogeography of soil microorganisms in the German Biodiversity Exploratories Ellen Kandeler* University of Hohenheim, Germany In the framework of an initiative to advance biodiversity research in Germany, three exemplary large-scale and long-term research sites were established to understand the role of land-use and management for biodiversity and to understand the role of biodiversity for ecosystem processes across Germany (www.biodiversity-exploratories.de). This platform is used to investigate the effects of land-use intensity on single trophic levels, interacting organisms, as well as foodweb structures. We identified general relationships between abiotic soil properties and soil biota across spatial scales, ecosystems (forest and grassland) and different land-use intensities. Whereas abiotic soil properties and climatic conditions mainly explained differences in microbial community structure and function on the regional scale, on the local and plot scale differences were related to the land-use intensity level. In 2011, we tested whether microbial biogeography of a grassland plot (10 m x 10 m) changes over the growing season. We sampled intensively the plot six times within one season to cover the different stages of substrate release and nutrient demands of plant communities. Microbial community spatial structure (based on PLFA data) was positively correlated with the local environment in spring and autumn, while the density and diversity of plants had an additional effect in the summer period. Spatial relationships among plant and microbial communities 74 Below and Above Ground Interactions

Tuesday 23 June – Parallel session were detected only in the early summer and autumn periods when aboveground biomass increase was most rapid and its influence on soil microbial communities was greatest due to increased demand by plants for nutrients. Individual properties exhibited varying degrees of spatial structure over the season. Finer phylogenetic resolution of microbial groups using next generation sequencing helped determine the importance of plant species density, composition, and growth stage in shaping microbial community composition and spatial patterns.

75 Below and Above Ground Interactions

Tuesday 23 June – Parallel session Root Turnover Convenors: Luke McCormack, Chinese Academy of Sciences, China, and Alexandra Weigelt, University of Leipzig, Germany

Convenor talk What are we measuring and what does it mean when reporting root turnover and decomposition in terrestrial ecosystems? Luke McCormack*1, Alexandra Weigelt2 1 Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, China, 2University of Leipzig, Germany Root turnover represents the combined processes of birth, death, and subsequent replacement of a root population. Collectively, root turnover accounts for a large, but highly variable portion of net primary productivity in terrestrial ecosystems and mediates the capacity for soil nutrient and water uptake by a plant. Additionally, the loss and subsequent decomposition of roots also accounts for a large portion of soil respiration and soil nutrient cycling. Meaningful interpretation of these ecosystem processes and their appropriate representation in terrestrial biosphere models therefore requires accurate and consistent characterization of root turnover and its component parts. However, difficulties measuring rhizosphere processes coupled with a high degree of functional and structural diversity among and within root systems have made it difficult to precisely and consistently measure root turnover and may confound our understanding of decomposition processes. This has then severely limited comparisons of root dynamics across species and sites. While measuring root and rhizosphere processes is likely to remain logistically challenging for some time into the future, there are several simple steps that can be taken today to significantly improve the consistency and accuracy of efforts to measure and model fine-root processes. In this open-ended presentation we will discuss use of the orderbased and functional classifications as opposed to traditional classifications of fine roots (i.e. all roots 1.5 includes microbial biomass, basal respiration and SOM turnover rate. Increased SOM turnover rate is connected with better SOM quality in rhizosphere soil as evidenced by the increase in polysaccharides portion in rhizosphere SOM revealed by 13C-MAS-NMR spectroscopy. C:N:P stoichiometry in microbial biomass might also affect the SOM susceptibility to decomposition; C:N ratio in rhizosphere microbial biomass was higher than that in bulk soil, while C:P displayed the opposite tendency. 176 Rhizosphere and Climate Change

Monday 22 June – Poster session The most drastic differences between top and deep rhizosphere were found in basal respiration and SOM turnover rates. Rf for SOM turnover in top soil was about 1.5, while in the deep soil horizon it was as high as 6. Thus, deep rhizosphere was found to be the more pronounced hot spot of biological activity than top one.

177 Rhizosphere and Climate Change

Monday 22 June – Poster session Metabolomics 100

Genomic, metabolomic and functional characterization of beneficial Burkholderia species from natural disease suppressive soils Victor J Carrion*1, Desalegn W. Etalo2, Viviane Cordovez2, Kazuki Fujiwara3, Irene de Bruijn2, Victor de Jager2, Jos M. Raaijmakers2 1 Netherlands Institute of Ecology (NIOO-KNAW), Netherlands, 2Netherlands Institute of Ecology (NIOO-KNAW), Netherlands, 3National Agriculture and food Research Organization, Japan

Disease-suppressive soils are ecosystems in which crop plants suffer less from specific diseases than expected owing to the activities of antagonistic rhizosphere microorganisms. For most disease-suppressive soils, however, the beneficial microbes and underlying mechanisms involved in pathogen control are largely unknown. In previous studies, we identified key bacterial taxa involved in suppression of the fungal root pathogen Rhizoctonia solani by PhyloChip-based metagenomics of the rhizosphere microbiome of sugar beet seedlings. Members of the Proteobacteria, Firmicutes, and Actinobacteria were found to be consistently associated with disease suppression. Here we focus on the β-Proteobacteria, specifically on the Burkholderia genus. We isolated approximately 50 Burkholderia strains from the rhizosphere of sugar beet seedlings grown in a soil suppressive to R. solani. Based on MLST sequencing, these isolates were classified as B. caledonica, B. graminis, B. hospita, B. pyrrocinia and B. terricola. These five Burkholderia species showed different activities. B. graminis inhibited R. solani via volatile organic compounds (VOCs), whereas B. caledonica, hospita, pyrrocinia and terricola showed antifungal, antioomycete activity and antibacterial activity. Strains of all five species exhibited in planta activity against R. solani. Comparative VOCs profiling of all five species revealed that sulphur-containing compounds were unique for B. graminis. By comparative genomics of the five fully sequenced species/strains, three unique gene clusters were identified in the B. graminis genome that are most likely involved in the biosynthesis of these sulphur-containing VOCs. Comparative genomics also revealed the presence of four non-ribosomal peptide synthetase gene clusters in B. caledonica, hospita, pyrrocinia and terricola that may contribute to the observed broad-spectrum antimicrobial activities. Mutagenesis, cloning, heterologous expression and chemical analyses are ongoing to resolve the functions of these novel gene clusters identified in the genomes of the five Burkholderia species from disease suppressive soil.

178 Metabolomics

Monday 22 June – Poster session 101

Mining rhizobacteria-induced metabolome reprograming in plants Desalegn Etalo*1, Judith van de Mortel2, Je Seung Jeon1, Ric de Vos3, Henk Gude4, Thierry Janssen5, Jos Raaijmakers1 1 Department of Microbial Ecology, Netherlands Institute of Ecology, NIOOKNAW, Netherlands, 2HAS University of Applied Sciences, Netherlands, 3Plant Research International, Wageningen University and Research Centre, Netherlands, 4Flower Bulbs, Applied Plant Research, Wageningen University & Research Centre, Netherlands, 5MicroLifeSoilutions, Netherlands

The colonization of plant roots, stem, leaves or fruits by beneficial microorganisms can protect plants from diseases, promote growth and enhance yield/biomass. Application of the beneficial rhizobacterium Pseudomonas fluorescens strain SS101 to roots of Arabidopsis thaliana resulted in a distinct shift in the plant transcriptome as compared to non-treated plants, with approximately 1150 plant genes differentially regulated. Gene set enrichment analysis (GSEA) revealed that genes involved in sulfur metabolism were among the most overrepresented gene clusters that were up-regulated in plants treated with the beneficial rhizobacterium. Furthermore, the upregulated gene category included also genes involved in sugar metabolism and auxin biosynthesis. In line with the transcriptome data, the metabolome analyses showed that sulfur containing metabolites were among the metabolites that accumulated in Arabidopsis plants treated with the rhizobacterium. Based on the observed transcriptome and metabolome changes associated to sulfur metabolism in Arabidopsis, experiments are ongoing to boost the production of specific compounds, including sulforaphane, in other cruciferous plants like broccoli. Furthermore, root, bulb and sprout samples obtained from two daffodil cultivars (Carlton, Geranium) treated with rhizobacterial strain SS101 were subjected to untargeted metabolomics. Our analyses of these bulb crops indicated that specific groups of metabolites like spermidine-conjugates were induced only in the sprout of both cultivars that were treated with the rhizobacteria. Collectively, these results show that specific rhizobacteria can (re)program the plant metabolome.

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Discriminating disease suppressive soils for cereal pathogen Rhizoctonia solani AG-8 using metabolomics Helen Hayden*, Simone Rochfort, Vilnis Ezernieks, Pauline Mele Department of Environment & Primary Industries, Australia

Metabolomic analyses were used to investigate possible functional mechanisms for disease suppression in soils known to be suppressive to the fungus Rhizoctonia solani AG-8, which infects cereal crops. Disease suppression refers to a lack of disease manifestation even in the presence of the pathogen, host plant and favourable environmental conditions. Currently the only way to identify fields with disease suppression of R. solani AG-8 is by doing glasshousebased pot trials. Soil samples were collected from two adjacent fields, one known to have a high disease suppression and the other low disease suppression resulting in infected cereal crops. Samples were collected at different times throughout the cropping cycle over two years and analysed for their metabolite profiles. Differentiation of the high and low suppression fields was carried out using multivariate analyses of liquid chromatography mass spectrometry (LC-MS) data, acquired in both the positive and negative ionisation modes, and 179 Metabolomics

Monday 22 June – Poster session nuclear magnetic resonance (NMR) data. Several peaks were significantly more abundant in the high suppression soil for the positive and negative ionisation modes. Potential LC-MS biomarkers for the high suppression soil were identified and corroborated by analysing two years of soil samples. The structure of these LC-MS biomarkers was elucidated using accurate mass data and MS fragmentation spectrum information. Analyses of the NMR loadings plots identified soils with high suppression have a greater abundance of polyols and terpenes. Two dimension NMR identified sugar biomarkers in the high suppression soils. Metabolite biomarkers with high abundance in disease suppressive soils were shown to match standards of macrocarpals, which are phloroglucinol containing compounds. Suppression of Rhizoctonia solani AG-8 may be occurring through an antibiotic mode of action as shown for soils suppressive to take all disease of cereals (Gaeumannomyces graminis var. tritici) or through microbial effects upon the plant-pathogen interaction.

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Effects of microbial signaling molecules on the growth and secondary metabolism of Arabidopsis thaliana Katharina Sklorz*, Michael Bonkowski University of Cologne, Zoological Institute, Germany

All plants are simultaneously colonized by a multitude of microorganisms of very different taxonomic affiliation. It is still uncertain how plants orchestrate the complex interaction with the surrounding microbiome, but it is clear that specific signaling molecules must exist that mediate the communication between plant species and their root microbes. We investigated the role of bacterial autoinducers (N-Acyl-Homoserine Lactones, AHL´s), which are employed in quorum sensing systems in various gram negative bacteria, on root growth and the secondary metabolism of Arabidopsis thaliana. We will discuss the observed patterns in the light of plant defense and interkingdom communication.

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Presence of a growth-promoting endophyte affects the composition of plant secondary metabolites and root exudates in Arabidopsis thaliana Katja Witzel*1, Nadine Strehmel2, Susanne Baldermann1, Susanne Neugart1, Dierk Scheel2, Monika Schreiner1, Rita Grosch1, Silke Ruppel1 1 Leibniz Institute of Vegetable and Ornamental Crops, Germany, 2Leibniz Institute of Plant Biochemistry, Germany

We have isolated the strain Kosakonia radicincitans (DSM 16656) from the phyllosphere of winter wheat under temperate conditions. Growth promotion of root and shoot, along with increased yield, was conferred by inoculation of different crop and model plant species. Endophytic plant growth promoting bacteria are discussed to impact significantly plant physiology, but regulatory pathways and biochemical alterations are still unclear. Therefore, transcriptome and metabolome investigations were conducted under controlled conditions to gain a better understanding. A global transcriptome analysis of Arabidopsis thaliana plants inoculation with Kosakonia radicincitans identified an over-representation of genes involved in secondary plant metabolism. Profiling the glucosinolate, carotenoid and phenylpropanoid 180 Metabolomics

Monday 22 June – Poster session content of leaves and roots revealed a specific response to endophytic colonization. As some secondary plant metabolites are known to act as signaling molecules in the rhizosphere, such as flavonoids and coumarins, root exudates of control and inoculated plants were collected and analyzed. More than 50 primary and secondary metabolism compounds were differentially enriched when plants were colonized by K. radicincitans. The results and possible implications of these analyses on plant-endophyte interactions are discussed.

181 Metabolomics

Monday 22 June – Poster session Root Endophytes

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Consortia of ACC deaminase-producing bacteria (both endophytic and rhizospheric) isolated from avocado plants mitigate salt stress of wheat plants Patricio Barra*1, Nitza Inostroza1, Maria de la Luz Mora1, David Crowley 2, Milko Jorquera1 1 Universidad de La Frontera, Chile, 2University of California Riverside, USA

Plants growing under salinity stress conditions increase ethylene production, which triggers inhibition of root elongation. Bacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) cleave the ethylene precursor, 1-aminocyclopropane-1-carboxylate, thus decreasing ethylene levels and consequently their detrimental effects. Some bacterial strains also produce phytohormone indol acetic acid (IAA), which increases cell elongation. Therefore, we hypothesize that ACCD- and IAA-producing bacteria (both endophytic and rhizospheric bacteria) isolated from avocado plants are able to mitigate salt stress effects of wheat plants; and we also hypothesize than bacterial location (both endophytic and rhizospheric), as well as, the levels of ACCD and IAA production by bacteria have different effects on salt stress response of wheat plants. Twelve bacterial strains were isolated from avocado plants, and four bacterial consortia were formulated, each composed of three strains, as follows: 1) Endophytic bacteria with higher ACCD and IAA production; 2) Endophytic bacteria with lower ACCD and IAA production and 3) Rhizobacteria with higher ACCD and IAA production; 4) Rhizobacteria with lower ACCD and IAA production. Wheat seeds were inoculated with the bacterial consortia, and then seeds were grown under salt stress conditions. Length, dry weight and superoxide dismutase (SOD) activity of wheat shoot and roots were determined. The results showed that at lower levels of bacterial IAA and ACCD production, the endophytic bacteria were more efficient than rhizobacteria consortia mitigating salt stress effects. Between rhizobacteria consortia, only those with higher production were able to promote the growth of stressed plants. Both endophytic bacteria and rhizobacteria consortia with higher production were able to increase SOD activity. Finally, bacterial strains isolated from avocado plants mitigate plant stress and therefore have the potential to be used as commercial inoculum of avocado plants.

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Stenotrophomonas rhizophila SPA P69: deep insights into an endophytic stress protecting agent Gabriele Berg*, Henry Müller Graz University of Technology, Austria

Stenotrophomonas rhizophila is able to promote plant growth of many crops and to protect roots against biotic and a-biotic stresses. We studied mechanisms associated with osmotic stress using transcriptomic and microscopic approaches. In response to salt or root extracts, the transcriptome of S. rhizophila SPA P69 (syn. DSM14405T) changed drastically. We found a notably similar response for several functional gene groups responsible for general stress 182 Root Endophytes

Monday 22 June – Poster session protection, energy production, and cell motility. However, unique changes in the transcriptome were also observed: the negative regulation of flagella-coding genes together with the up-regulation of the genes responsible for biofilm formation and alginate biosynthesis were identified as a single mechanism of S. rhizophila against salt shock. However, production and excretion of glucosylglycerol (GG) were found as a remarkable mechanism for the stress protection of this strain. For S. rhizophila treated with root exudates, the shift from the planktonic lifestyle to a sessile one was measured as expressed in the down-regulation of flagellar-driven motility. These findings fit well with the observed positive regulation of host colonization genes and microscopic images that show different colonization patterns of oilseed rape roots. Spermidine, described as a plant growth regulator, was also newly identified as a protector against stress. In addition to both the changes in life style and energy metabolism, phytohormons, and osmoprotectants were also found to play a key role in stress protection. Risk assessment studies reveal no health risks. This is mainly because SPA P69 is unable to growth at the human body temperature, 37°C due to the absence of heat shock genes and a temperature-regulated suicide mechanism. Taken together SPA P69 is a promising endophytic stress protecting agent (SPA) ready for commercial applications.

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Better efficiency of biofertilizers in combination with N2-fixing endophytes in low quality sandy soil Borbala Biro*, Zita Szalai, Tamás Kocsis, Zsolt Kotroczó Corvinus University of Budapest, Hungary

Biofertilizer and/or soilconditioner products are containing very often the various types of the Nitrogen-fixing and P-mobilising microorganisms, as only one or 2-3 types of species components. Question arises if single or combined products are more efficient and what is the main driving force for the efficiency in a low quality sandy soil? Tomato Solanum lycopersicon Mill. ’Mobil’ was used in a field experiment, inoculated with Trichoderma harzianum T-22 and TDM, Hungarian Trichoderma inoculums, including also of Azotobacter and Azospirillum N2-fixers in one product. There were patentkali and calcinit fertilizers applied at 1200 kg/ha doses also to the soil. Inoculation treatment was performed twice during the vegetation, at the sowing and at the time of the plantation of tomato seedlings. Growth of tomato, shoot and root biomass was assessed and general soil characterization, including physical-chemical parameters, the MPN counts of some beneficial physiological groups in tomato rhizosphere, outside and inside the plants. Results were evaluated by statistical probes. The beneficial effect was realised with the combined tomato inoculation in comparison with the only Trichoderma strain application. Effect of Trichoderma TDM inoculation was improved with free-living and associative Nitrogen-fixers and thus provided better nutrition. The relatively high P-content (430 mg/kg) was found in the slightly humous sandy soil, where the N2-fixing microbes might able to improve the N,P, K ratio for the more efficient tomato growth. Plant microbe interaction is largely dependent on the soil nutrient-status and the balance among main nutritive elements. In this process the multifunctional inoculums might providing greater plant-growth promotion beyond the single strain effects. 183 Root Endophytes

Monday 22 June – Poster session 110

A survey of bacterial root endophytes associated with vegetation at a bitumen impacted site Natalie Blain*1, Bobbi Helgason2, James Germida1 1 University of Saskatchewan, Canada, 2Agriculture and Agri-Food Canada, Canada

Bacterial root endophytes can help alleviate plant stress and promote plant growth, and the potential use of these endophytes to assist land reclamation is receiving increased interest. Bitumen is a heavy oil that influences plant growth due to its hydrocarbon composition. The Bitumount provincial historical site is considered the location of two of the world’s first oil sands (bitumen) extraction plants. Operations at Bitumount began in 1923 and ceased completely in 1958. Through natural reclamation, vegetation has re-colonized the area including hardened bitumen. The Bitumount site offers a unique opportunity to study plantmicrobe associations that have co-adapted to this stressed environment. Sampling locations were established in June 2014, and plant and soil samples collected. A total of 6 different plant species were identified and sampled based on their abundance at each sampling location. They included: Bromus inermis, Equisetum spp., Agropyron trachycaulum, Poa pratensis, Fabaceae spp. and Fragaria virginiana. Soil samples were analyzed for hydrocarbon content, which ranged from 330 to 24,700 mg kg-1 throughout the site. Culture dependent and independent methods were used to characterize bacterial root endophytes. The number of culturable root endophytes varied significantly (p shoots ³ roots. The ratio of nifH-gene to 16S rDNA-gene was higher at naturally saline test site (0.6) compare to the test site with anthropogenic salinity (0.4). Most of isolated diazotrophs were identified to the phylum Proteobacteria and Actinobacteria.

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Reaction of perennial ryegrass (Lolium perenne L.)/Glomus spp. symbiotic association on infection by Fusarium poae and Rhizoctonia solani Malgorzata Jeske*, Dariusz Panka, Marcin Juda, Karol Lisiecki, Katarzyna Koczwara, Monika Młynska UTP University of Science and Technology, Poland

Perennial ryegrass is one of the most important grasses in Poland, used for pasture and for turf. It is often attacked by numerous pathogens, which can decrease yield and its quality. Use of fungicides for plants protection against diseases is often limited. The other way of protection of perennial ryegrass can be use of arbuscular mycorrhizal fungi (AMF) of the Glomus genus. These fungi belong to the most commonly occurring soil microorganisms of the world and are associated with at last 80% of plants of the Earth. Arbuscular mycorrhizal fungi increase the root absorptive area and hence the plant nutrition. Additionally, AMF increase the tolerance of plants to heavy metals, water stresses, as well as pathogenic 191 Root Endophytes

Monday 22 June – Poster session fungi. The exact mechanism of higher resistance of AMF infected plant is yet not fully understood, but it can be assumed that PR (Pathogenesis-related) proteins have a significant role. Detailed experiments in controlled conditions were conducted to determine the impact of AMF on induction of specific defense mechanism, including production of PR proteins: chitinases and β-1,3-glucanases in perennial ryegrass. Severity of plant’s infection was estimated 2, 4, 6, and 8 days after inoculation with F. poae and R. solani. The level of chitinases and glucanases in plants was determined 0, 2, 4, 6 and 8 days after infection with use of Abeles et al. (1970) method with modifications. The specific activity of chitinases and glucanases in the extracts was expressed as moles of reducing sugars released in one minute of incubation per one milligram of total protein in the extract. Occurrence of Glomus spp. affected the amount of chitinases and β-1,3-glucanases in perennial ryegrass. The amount of enzymes was also dependent on the time after infection. Results indicate the role of PR proteins in resistance of plants exposed to infection.

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Genetic analysis of root endophytic Pseudomonas putida bp25 and chemoprofiling of its antimicrobial volatile organic compounds Aundy Kumar*1, Neelam Sheoran1, Agisha Valiya Nadakkakath2, Vibhuti Munjal1, Aditi Kundu3, Kesavan Subaharan4, Vibina Venugopal4, Suseelabhai Rajamma2, Santhosh Eapen2 1 Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, India, 2Division of Crop Protection, ICAR-Indian Institute of Spices Research, India, 3Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, India, 4Division of Crop Protection, ICAR-Central Plantation Crops Research Institute, India

Black pepper (Piper nigrum) associated bacterium BP25 was isolated from root endosphere of apparently healthy cultivar, Panniyur-5 that protected black pepper plants against Phytophthora capsici and Radopholus similis -the major production constraints. The bacterium was genetically characterized and mechanisms of its antagonistic action against major plant pathogens elucidated. The polyphasic phenotypic and genotypic analysis revealed its identity as a strain of Pseudomonas putida (PpBP25). Multi Locus Sequence Typing revealed that the bacterium shared gene sequences with several other isolates representing diverse habitats. Tissue localization assays exploiting green fluorescence protein expression clearly indicated that PpBP25::gfp endophytically colonized not only its host -black pepper, but also other plants such as edible ginger (Zingiber officinale) and a model plant, Arabidopsis thaliana. Coupled with PpBP25 colonies enumerated from internal plant tissues four weeks post inoculation indicated its stable establishment and persistence in the plant system. Strikingly, the bacterium inhibited broad range of economically significant plant pathogens representing diverse taxonomic groups such as Phytophthora capsici; Pythium myriotylum; Giberella moniliformis; Rhizoctonia solani; Athelia rolfsii; Colletotrichum gloeosporioides; Magnaporthe oryzae; Ralstonia solanacearum; Xanthomonas axonopodis pv. punicae; Xanthomonas oryzae pv. oryzae; and plant parasitic nematode, Radopholus similis by its volatile organic compounds (VOCs). Gas Chromatography/Mass Spectrometry (GC/MS) based chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1,-3Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25 192 Root Endophytes

Monday 22 June – Poster session metabolites. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence of many aromatic compounds such as Dimethyl trisulfide; Dimethyl disulphide; Pyrazine, 2,5dimethyl-; Pyrazine, methyl-; Pyrazine, 2-ethyl-5-methyl-; Pyrazine, 2-Ethyl-3,6-dimethyl; Heptamethyl-2-nonene; β-Naphthol; Octadecyl vinyl ether; Tetradecane, 2,6,10-trimethyl; Cyclobutene, 2-propenylidene-; Heptamethyl-1-nonene; 1,8-Nonadien-3-ol; Octadecanal,2bromo; Isoamyl alcohol; and 1-Undecene. The work paved way for profiling and consequent identification of several broad spectrum anti-oomycetes, antifungal, antibacterial and nematicidal VOCs in black pepper root endophytic bacterium for next generation plant disease management.

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Evaluation of ACC deaminase-producing endophytic bacteria isolated from organic products by mung bean assay Hiroaki Matsuoka*1, Yoshinari Ohwaki1, Junko Terakado-Tonooka2, Fukuyo Tanaka Tanaka1 1 National Agriculture and Food Research Organization Agricultural Research Center, Japan, 2Saga University, Japan

Bacterial endophytes have been associated with the growth promotion of various crops. In particular, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing bacteria have received widespread attention because of its ability to improve the stress tolerance of plants by lowering ethylene level under environmental stress. We supposed that organic crops which are exposed to frequent attacks of pathogen and pest may establish the beneficial interaction with bacteria with such property. In this study, therefore, we isolate and evaluate ACC deaminase-producing bacteria from organic crops in order to find new candidates of biocontrol agents in practical use for cultivated crops. Bacterial strains were isolated from the fruit or root of organic crops including carrot, aroid, turnip, sweet pepper, and apple by using Dworkin–Foster minimal-salt medium with ACC as a sole nitrogen source. The isolated strains were further selected from amplifying the ACC deaminase synthase (acdS) gene by colony PCR. Moreover, the strains possessing acdS genes were examined for enzyme activity by measuring the production of α-ketobutyrate from ACC ,and bioassay with growth-response of mung bean in the presence of ACC under gnotobiotic condition. Twenty-four strains were found to have acdS genes in all the 80 strains isolated on the medium containing ACC. Of those, 10 strains were observed ACC deaminase activity by enzyme assay. Among them, 4 strains were shown to promote the shoot elongation of mung bean in the bioassay. Interestingly, the ACC deaminase activity in the isolated strains showed different trends with growth response of mung bean. Our data indicate that selected bacteria in organic crops produce ACC deaminase and have potential for plant growth promotion. We will also report the results of inoculation of selected 3 strains into cultivated crop.

193 Root Endophytes

Monday 22 June – Poster session 125

Influence of plant host species on the root associated microbial community and functional redundancy of the root microbiome under phosphate starvation Tatiana Mucyn*1, N. W. Breakfield2, G. Castrillo1, S. H. Paredes1, S. M. Yourstone1, C. Hunter1, J. L. Dangle1 1 University of North Carolina, Chapel Hill, NC, USA, 2NewLeaf Symbiotics, St Louis, MO, USA

Plants harbor a specific and complex microbiota at the surface of the root (rhizosphere) and within the root (endophytic compartment) which influences plant health and productivity. Various factors such as soil type, abiotic/biotic stress, host developmental stage, and host species shape the root microbiome. The extent to which the host selects its root microbial community remains poorly understood. To determine the influence of plant host species on the assembling of both the rhizospheric and endophytic microbiome, we are comparing the well-studied bacterial root bacterial microbiome of Arabidopsis thaliana Col-0, with those of two monocot species Brachypodium distachyon Bd21 and Setaria viridis A10-1 from plants grown in the same wild soil, using high-throughput bacterial 16S rDNA, as well as fungal internal transcribed spacer 2 profiling. We are characterizing both core and species-specific root microbiomes and are exploring the hypothesis that plants more closely related to each other share more similar microbial communities. The Dangl laboratory has isolated ~600 bacterial strains from the rhizoplane and endophytic compartments of Arabidopsis thaliana and is evaluating the effect of these isolates on plant health under various nutrient starvation stresses. This bacterial collection is currently being tested on Setaria viridis focusing on the identification of isolates that rescue phosphate starvation stress to establish to whether the core microbiome may also present functional conservation across species. We are in parallel establishing a collection of bacterial strains isolated from the root of Setaria viridis to refine our characterization of both the core and potential species-specific microbiome.

126

ACC deaminase-expressing endophyte increases plant tolerance to FD phytoplasma infection Elisa Gamalero1, Elisa Bona1, Giorgia Novello*1, Cristina Marzachì2, Luciana Galetto2, Flavio Veratti2, Nadia Massa1, Bernard Glick3, Simone Cantamessa1, Giovanni D'Agostino4, Graziella Berta1 1 Università del Piemonte Orientale, DiSIT, Italy, 2CNR, Italy, 3University of Waterloo, Canada, 4Mybasol srl, Italy

Flavescence dorée (FD) is an epidemic yellows disease of grapevine caused by a phytoplasma (FDP), living in the phloem for which there is currently no cure. We have previously shown that treatment of plants with rhizobacteria leads to mitigation of phytoplasma-induced disease. The alleviation of a variety of disease symptoms by rhizobacteria may involve the reduction of the stress-related plant ethylene via the 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We tested whether the endophytic bacteria Pseudomonas migulae 8R6, able to synthesize ACC deaminase, can limit the damages induced by FDP. Since FD-infected grapevines show symptoms the year after infection or later, the FDP host plant periwinkle was used as model. The use of a mutant of strain 8R6, lacking the ability to produce ACC deaminase, may 194 Root Endophytes

Monday 22 June – Poster session demonstrate the involvement of ethylene in the development of FDP symptoms in the plant. The strain 8R6 colonized the internal tissue of periwinkle, thus confirming its endophytic aptitude and significantly reduced the number of symptomatic plants (53% vs. 93%). A lower number of symptomatic plants was also detected in plants inoculated with the 8R6 mutant compared to controls (73% vs 93%) thus suggesting that ACC deaminase is not the only mechanisms involved in the increased tolerance to FD infection. Quantification of phytoplasma inside the leaves was not affected by both bacterial strains. However, FD phytoplasma title was under the quantification threshold in 38% of plants inoculated with the strain 8R6. This value was more than what recorded for FD infected plants (14%). ACC deaminase activity seems to be involved, as FD phytoplasma population was under the quantification threshold in 8% of the plants inoculated with the mutant. In conclusion, ACC deaminase activity of P. migulae 8R6 might help the plant to regulate the level of the stressrelated hormone ethylene, potentially leading to improved tolerance to phytoplasma infection.

127

The roles of root colonizing bacterial endophytes on suppression of Fusarium oxysporum f. sp. cucumerinum and growth promotion of cucumber plants Hatice Ozaktan*, Ayşe Gul, Bi̇ rsen Çakir, Lalehan Yolageldi, Mustafa Akbaba, Sahika Akat Ege University, Turkey

The aim of this study was to find that endophytic bacterial (EB) isolates obtained from healthy cucumber plant tissues could promote the plant growth and marketable fruit yield and suppression of soilborne plant pathogen of cucumber plants caused by Fusarium oxysporum f. sp. cucumerinum (FOC). Cucumber plants with treated EB as seed coating and soil drenching (108–109 CFU ml-1). were transplanted to peat inoculated with FOC spore suspension (105 spore ml-1 ). According to growth chamber test results, 38% of tested EB strains exhibited the disease reduction between 30 to 60 %, comparing to only FOC inoculated plants. In the next step of this research, EB strains applied as seed coating and soil drenching inhibited the Fusarial wilt development at the rate of 49 to 52% compared to only FOC inoculated cucumber plants for two months growing period in soilless growing system. Moreover, EB treatments, without being any disease pressure, increased the total marketable fruit yield at the rate of 5 to 28% compared to non-treated cucumber plants for 2 months growing period in soilless cultivation. The population density of some EB strains in the root tissues was determined as approximately 103-105 CFU plant g-1 75 days after seed bacterization. It was determined that colonization rate of EB strains in the root tissues was higher than shoot tissues. So, the application of EB as seed coating and soil drenching was recorded as effective and practical techniques in terms of biological control, growth promotion of cucumber plants and root colonization of bacteria inside the plant tissues.

195 Root Endophytes

Monday 22 June – Poster session 128

Siderophore production and iron phosphate solubilization by root endophytic bacteria isolated from maize Vitoria Palhares*1, Ivanildo Marriel2, Eliane Gomes2, Ubiraci Lana2, Crisia Abreu3, Cássia Almeida1, Christiane Oliveira2 1 UniFEMM, Brazil, 2Embrapa Mayze and Sorghum, Brazil, 3Federal University of Minas Gerais, Brazil

Tropical soils generally exhibit acidic condition with predominant phosphate immobilized in insoluble forms of iron (P-Fe). Many soil microorganisms solubilize P from P-Fe by siderophores and organic acids production. The microorganisms that live in the rhizosphere and in the interior of plants (facultative endophytic) with potential for phosphate solubilization are important to enhance the production of the maize crop in order to reduce the use of soluble fertilizers. The objective of this work was to isolate efficient facultative endophytic bacteria solubilizing iron phosphates and evaluate their production of siderophores. Maize plants were harvested at flowering stage and 113 bacteria were isolated from roots (54.9%), leaves (20.4%) and sap (24.8%). Fifty-eighty selected strains, most root endophytic, were evaluated in liquid culture medium containing iron phosphate, after 9 days of incubation. Soluble phosphorus was determined using modified ammonium molybdate method. The production of siderophores was through inoculation of microorganisms in solid medium containing the indicator cromoazurol. Strains were identified based in the 16S rDNA gene. Maize plants had a high diversity of endophytic bacteria solubilizing iron phosphate and siderophore producing. One strain of Pantoea and two of Bacillus showed the highest P solubilization, releasing 68.7; 64.1 and 64.08 mgP.L-1, respectively. The siderophore produced by 65% of the evaluated microorganisms was the type carboxylate, and the most strains producing siderophores are efficient in solubilizing P associated with iron. The use these facultative endophytic solubilizing microorganisms as inoculant may be considered a promising strategy in environmental and economic terms for maize production.

129

Effect of GFP-labeled Paenibacillus polymyxa on growth of agricultural crops Kiran Preet Padda*, Akshit Puri, Christopher Chanway University of British Columbia, Canada

Paenibacillus polymyxa strain P2b-2R, previously isolated from internal stem tissue of a naturally regenerating pine seedling, fixes nitrogen in association with lodgepole pine seedlings and promotes their growth. This strain has been shown to colonize lodgepole pine seedling tissues endophytically using a green fluorescent protein (GFP)-labelled derivative of the wild type. We wanted to see if the GFP labelled derivative of P2b-2R would fix N and promote growth of agricultural crops such as corn and canola in ways similar to the wild type strain. We inoculated corn and canola seeds with wild type P2b-2R or the GFP labelled derivative and grew seedlings for 40 days in an N-limited soil mix. Seedlings were harvested 20, 30 and 40 days after inoculation and evaluated for biological nitrogen fixation and growth promotion.

196 Root Endophytes

Monday 22 June – Poster session Seedlings inoculated with the GFP labelled P2b-2R strain derived small amounts of N from the atmosphere (up to 17%) but were 40% taller and accumulated 70% more biomass than those treated with wild type P2b-2R. We conclude that GFP modification of strain P2b-2R resulted in a significant enhancement of the wild type’s growth promotion efficacy on corn and canola and facilitated some in situ biological nitrogen fixation with these two crop species.

130

Next generation sequencing analysis of soil and plant associated fungal assemblages in sub-Arctic sand dune ecosystem Anbu Poosakkannu*, Riitta Nissinen, Minna-Maarit Kytöviita University of Jyväskylä, Finland

Our research focusses on plant associated microbial communities in the sub-arctic inland primary successional site, located in an Aeolian sand dune area in subarctic Northern Fennoscandia (68° 29' N). One of the very few species capable of colonizing these Arctic sand dunes and enabling ecosystem restoration is the grass Deschampsia flexuosa. In the study site, the early successional stage is characterized by D. flexuosa growing as monoculture in the blow-out areas. Late successional stage is mountain birch forest vegetation with continuous ground cover vegetation composed of abundant D. flexuosa together with other plants and under the cover of mountain birch trees. This goal of this study was to assess the community composition of fungal assemblages in soil (bulk and rhizosphere) and in the internal tissues (endophytes) of D. flexuosa growing in the two different successional stages. Endophytic fungal studies in cold environment especially in arctic are very limited, although they can improve host nutrient acquisition and protect the plants from different stresses. Deschampsia flexuosa (leaf and root), rhizosphere and bulk soil samples were collected from four different blow-out areas between 150 and 2250 meters apart. We collected eight biological replicates (two samples per blow out area and successional stage). Soil and plant associated fungal community structure was studied using Next generation Ion torrent sequencing of partial internal transcribed region (ITS) amplicons. Mothur based bioinformatics analysis of ITS sequences revealed 99217 quality-filtered sequences which are abundant (OTUs with less than 10 sequences were removed) that were separated into 2781 species-level OTUs. Of fungal taxa, Ascomycota (67.1%), Basidiomycota (18.9 %), encompassed the largest proportion of OTUs. The phylum Ascomycota was the predominant phylum identified and present in all samples followed by Basidomycota, Zygomycota and Glomeromycota. Endophytic samples are dominated by phylum Ascomycota. The compositional variation was mainly accounted for by successional stages.

197 Root Endophytes

Monday 22 June – Poster session 131

Can an endophyte isolated from lodgepole pine trees reside inside agricultural crops and fix N? Akshit Puri*, Kiran Preet Padda, Christopher Chanway University of British Columbia, Canada

Several Paenibacillus strains that were able to fix nitrogen were isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus Polymyxa P2b-2R, was found to fix high amounts of nitrogen when reintroduced to the gymnosperms, lodgepole pine and western red cedar. We wanted to determine if Paenibacillus polymyxa P2b-2R could colonize, fix N and promote the growth of important agricultural crops such as corn and canola. We inoculated corn and canola seeds with P. polymyxa strain P2b-2R and grew seedlings for 40-60 days. Corn seedlings were harvested 10, 20 and 30 days after inoculation and canola seedlings were harvested 20, 40 and 60 days after inoculation for evaluation of endophytic and rhizospheric colonization. Seedlings were also evaluated for biological nitrogen fixation and growth promotion at these harvest intervals. P2b-2R colonized rhizosphere as well as inside tissues of root (endophytically). Corn and canola seedling growth was promoted significantly by inoculation with P2b-2R with an increase of up to 35% in height and 30% in biomass from the controls. P2b-2R also provided more than 20% of foliar nitrogen. These results suggest that Paenibacillus polymyxa P2b-2R has a broad range of plant hosts and is able to fix N and promote the growth of at least certain agricultural crops.

132

Maize roots endophytic bacteria and their potential as plant growth promoting and biological control agents João A. C. Vieira1, Natalia L. S Alves1, Christiane A. O. Paiva2, Vera Lúcia Santos*1 1 Laboratory of Applied Microbiology, Microbiology Department, Institute of Biological Science, Federal University of Minas Gerais, Brazil, 2Embrapa Milho e Sorgo Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Brazil

Studies have shown that many endophytic microorganisms, which lives asymptomatically within plant causing no signs of harm to the host, may work as plant-growth promoters and/or biocontrol agents which has made them valuable for agriculture for improving crop performance. In this work, endophytic bacteria were isolated from roots of Pioneer 30F35 Herculex hybrid corn crops, cultivated with and without phosphorus fertilization. After identification by partial sequencing of the 16S rDNA gene, a total of 80 bacteria was evaluated regarding solubilization of inorganic phosphate (CaPO4), antagonizing bacterial (Bacillus subtilis and Pantoea ananatis) and phytopathogenic fungal (Fusarium verticillioides and Coletotrichum graminicola) growth, mineralization of phytate and production of IAA (indole-3-acetic acid). There was an equal distribution of the isolates from Actinobacteria, Firmicutes and Proteobacteria clades, and only one isolate (Flavobacterium acidificum RT3B41) of Bacteroidetes phylum was found. The strains were grouped into 26 genera, which the 198 Root Endophytes

Monday 22 June – Poster session most frequent were Bacillus, Leuconostoc, Pseudomonas, Serratia and Enterobacter. From the total, 32 isolates were able to solubilize inorganic phosphate (dosages between 10 and 527 mg/l) and 45 showed production of IAA (4.5 to 111 μg/ml). In the plate antagonism test, 10 isolates inhibited the growth of B. subtilis, 6 of the gram negative pathogen P. ananatis, 11 of the fungus C. graminicola and 4 of F. verticillioides. In plate qualitative tests, 52 isolates also had the ability to mineralize phytate. This work demonstrates the enormous potential application of these isolates, which must still be confirmed by in vivo and field tests.

133

Development of a bacterial cell enrichment method for the analysis of the endophytic microbiota in sugarcane stems Stefan Schwab*1, Carlos dos Santos2, Daniel de Souza2, Dayana Rosa2, José Baldani1 1 Embrapa Agrobiologia, Brazil, 2Universidade Federal Rural do Rio de Janeiro, Brazil

Sugarcane is an important culture in Brazil, with high economic support and social relevance. Sugarcane plants are rich in endophytic bacteria, which can promote and modulate plant growth through diverse mechanisms, such as phytohormone production, antagonistic activity against phytopathogens, and improvement of nutrient utilization by the plant; however, most bacteria are yet uncultivable. Therefore, cultivation-independent strategies are essential to better understand their genetic diversity and functional characteristics. Metagenomic surveys of endophytic microbiota currently represent a challenge, due to the low number of bacterial cells in relation to the host plant. In this work a bacterial cell enrichment procedure was established, cultivation-independently, from the inner tissues of the base of sugarcane stems. Results showed that the enriched material contained bacterial cells that are colony-forming, and can be visualized by bright-field microscopy or Gram stain test. DNA content analysis of the enriched material revealed efficient elimination of plant DNA, and results of PCR and ARDRA showed that bacterial DNA is predominant. Preliminary results of 16S rDNA sequencing revealed the presence of alpha-, beta- and gamma-Proteobacteria. In order to obtain a deeper insight of the bacterial taxa, 16S rDNA sequencing of the enriched material on Illumina platform is being conducted, and results will be presented. The developed method may allow, in the future, accessing the sugarcane endophytic microbiome, and reveal bacterial genetic resources with agrobiotechnological applications.

134

Indirect stimulation of the vegetative growth of ‘Elkat’ strawberry plants by the bacterium Pantoea sp. (N52AD) Pawel Trzciński*, Lidia Sas Paszt, Edyta Derkowska, Michał Przybył The Research Institute of Horticulture, Poland

The most important problems of agriculture are: increasing the abundance of macro- and microelements in the soil, maintaining adequate moisture levels, and protection from diseases and pests. Excessive use of fertilizers (mineral and manure) and pesticides may reduce soil fertility and biodiversity, and contaminate surface and ground waters. One way of reducing the use of fertilizers and chemical plant protection products is to employ beneficial microorganisms which can decrease the number of pathogens in the soil, increase the availability of macro- and microelements to plants, and improve the resistance of plants to 199 Root Endophytes

Monday 22 June – Poster session stress caused by, for example, drought. Among the most common and universal microorganisms used are arbuscular mycorrhizal fungi. By colonizing the roots of the host plant, these fungi increase the uptake of water, macro- and microelements by the plant and protect it from soil-borne pathogens. The degree of the symbiosis is affected by some microorganisms, for example, mycorrhiza helper bacteria. Application of such bacteria can improve the colonization of plant roots by autochthonous mycorrhizal fungi or those present in commercial products, thus allowing plants to make a more efficient use of nutrients and consequently contributing to a reduction in the amounts of the fertilizers and pesticides used. The aim of this study was to evaluate the impact of the bacteria from the genus Pantoea (strain N52AD) on the degree of colonization of strawberry roots by mycorrhizal fungi and on the vegetative growth of strawberry plants. Frigo plantlets of the cultivar Elkat were planted in pots filled with the soil collected from an experimental field in Dąbrowice. In the experiments conducted in 2012-2013 in greenhouse conditions, the plants inoculated with the N52AD strain were colonized by arbuscular mycorrhizal fungi to a greater extent and produced a greater mass of leaves than the control plants (non-inoculated with N52AD strain).

135

Improvement of soil fertility and plant production in the Democratic Republic of Congo by implementation of arbuscular mycorrhizal fungi and Sebacinales Jolien Venneman*, Danny Vereecke, Geert Haesaert Department of Applied Biosciences, Faculty of Bioscience Engineering, University Ghent, Belgium

A growing world population is associated with an increased demand for food. This trend is most pronounced in developing countries where the availability of fertile arable land is becoming rather limited. An important reason is the typical physical and chemical properties of tropical soils, e.g. P-fixation and low cation exchange capacity (CEC), in addition to soil degradation caused by unsustainable agricultural practices. Since the access to mineral fertilizers is very restricted in tropical regions, the mentioned problems should be addressed through the application of an integrated soil fertility management. This includes the use of sustainable cultivation techniques in combination with adapted plant genetic material, micro-doses of chemical fertilizers, and maximal amounts of organic matter. In our research, we particularly focus on the implementation of growth promoting micro-organisms, which are isolated from soil and plant root samples collected in the Democratic Republic of Congo, region of Kisangani. Arbuscular mycorrhizal fungi and basidiomycetes belonging to the order Sebacinales are two groups of root endophytes that attracted our attention. To reveal the mechanisms behind the plant-fungus interactions we combine molecular diversity analysis with functional tests. In addition, we plan to set up onfarm based systems for mass production of inoculum in Kisangani in order to provide local farmers with the knowledge on how to maintain the beneficial indigenous micro-organisms.

200 Root Endophytes

Monday 22 June – Poster session 136

Monitoring and quantifying Bacillus mycoides in the potato rhizosphere Yanglei Yi*, Jan Spoelder, Marielle van den Esker, Oscar Kuipers University of Groningen, Netherlands

Bacillus mycoides is a rod-shaped soil bacterium belonging to the B. cereus species-group. On agar plates, a fungus-like shape forms resulting from cells linked end to end. B. mycoides is reported to have plant growth-promoting effects on sugar beet, cucurbits and tobacco. B. mycoides is believed to be an important bacterium for potato growth as well, due to its abundance in potato rhizosphere and endosphere without causing visible signs of infection. Unraveling mechanisms mediating plant host-endophytes recognition, establishment and colonization dynamics requires a reliable method to monitor these processes. Visualization of cells in the rhizo-/endo-sphere using fluorescent protein (FP) as a marker has been adopted by many researchers to study plant-microbe interactions. However, many laboratories struggle with incorporating foreign DNA into Gram-positive bacteria including some wild isolates of Bacillus. Here, we describe a method to transform the microbe by electroporation with a plasmid encoding a constitutive promoter FP, resulting in fluorescent cells. Combination of quantitative and qualitative data achieved by both molecular and microscopy methods are probably the best choice to monitor endo- and rhizo-bacteria, with the advantages of each technique complementing the drawbacks of the other.

201 Root Endophytes

Monday 22 June – Poster session Root Development

137

Architectural analysis of date palm root systems (Phoenix dactylifera L.) Mimoun Asma*1, Stokes Alexia2, Rey Hervé3, Lecoustre René3, Jourdan Christophe4, Bennaceur Malika1 1 Oran 1 University, Algeria, 2INRA, Montpellier, France, 3AMAP/CIRAD BIOS, France, 4CIRAD-Eco&Sols, France

Architectural traits were used to describe the structure and development of the date palm (Phoenix dactylifera L.), root system. To characterize root system architecture, two parallel experiments were conducted: one in a rhizotron whereby root growth was measured over time and one in a nursery bags from which roots were sampled regularly. These roots were sectioned and examined under a light microscope in order to determine how the anatomical structure of roots changes as they age. Double staining was performed on roots at different ages and from diverse root zones. The topology (arrangement of axes relative to each other) and typology (classification of different root axes based morphological and functional criteria) of root systems was measured in the first rhizotrons. Root diameter and root elongation were measured regularly on seedlings grown from seed (0 to 6 months). RhizoDigit software (©CIRAD) was used to digitize root observations and to compile data for statistical analyses. Results showed that during this developmental period, the root system architectural unit comprises seven root types having distinct characteristics and spread over three different topological orders (primary, secondary and tertiary). The establishment of different types of root axes evolves over time and depends on the root category. Root growth was variable depending on the root type and branching order of the diameter according to its class. There was an absence of both radical mortality and the arrest in growth of short secondary roots during this period. The anatomies of the radical and adventitious roots were typical of monocotyledonous roots. Radical anatomy also had a characteristic spatial and temporal development during the juvenile stage. We will use these results to develop three-dimensional models for use in agricultural management.

139

Root architecture phenotyping of different quinoa (Chenopodium quinoa Willd) accessions José Correa*1, Phil Pstrong2, Francisco Pinto2, Kurt Ruf1, Iván Matus1, Kerstin Nagel2, Fabio Fiorani2, Manuel Pinto1 1 Instituto de Investigaciones Agropecuarias, Chile, Chile, 2Forschungszentrum Jülich, Germany

Quinoa has received an increasing attention because of its nutritional value, adaptability to different environmental constraints, and its vast biodiversity. All these crop aspects are relevant for global food security. Root system architecture plays an important role in determining the ability of a plant to explore the soil and foraging for water and nutrients. These aspects linked to root architecture are not yet well characterized in quinoa accessions and genetically diverse panels. According to that, two pilot experiments were carried out to evaluate the applicability of a rhizotron system for root assessment in quinoa. Plants were grown under greenhouse conditions in rhizotrons at IBG2: Plant Sciences at 202 Root Development

Monday 22 June – Poster session Forschungszentrum Jülich GmbH, Germany. The first experiment focused to establish whether the visible root traits at the interface of the rhizoboxes correlate with root and shoot traits measured destructively and the second one to analyze the effect of water stress on root architectural parameters. To represent the diversity of quinoa, three Chilean wild ecotypes and three commercial varieties graciously supplied by Wageningen breeding program, were used. During the second experiment the water stress treatment was performed on the more vigorous ecotypes. The following traits were measured: length of primary, lateral and tertiary roots; root system depth, width and area; and root dry weight. Correlations and differences among ecotypes for all traits were found. In addition, the effect of water stress on root architecture was mainly manifested by a decrease of root system length, width, laterals, and area. In conclusion, there are relationships between traits measured in rhizotrons and traits of the plant/root system allowing analyzing the variation observed in the plant/root system and providing details about the effect of water stress on root development. These findings indicate that protocols for screening germplasm in pre-breeding scenarios could be developed specifically for quinoa.

141

First root allometric model for shifting cultivation systems in PDR Lao Iain McNicol1, Nicholas J Berry1, Thilde B Bruun2, Andreas de Neergaard*2, Casey Ryan1 1 University of Edinburgh, Scotland, 2University of Copenhagen, Denmark

Shifting cultivation remains an important land use across of Southeast Asia and other parts of the tropics. This practice creates complex mosaic landscapes with regrowing fallows of various age interspersed with active fields and patches of mature forest. Quantifying root carbon stocks in these secondary forests is limited by the availability of reliable allometric models for these systems, inhibiting the development of policies aimed at reducing the intensification of shifting cultivation systems (REDD+). We developed new allometric models for prediction of both tree stem and root biomass in shifting cultivation systems based on a destructive harvest of 150 trees from Luang Prabang Province, Laos PDR. This study is the first to develop allometric models of root biomass for shifting cultivation landscapes, which we hypothesised would be a major carbon pool given that resprouting, and associated high root biomass, is a common physiological trait. Tree height was less important for estimating root biomass with “diameter only” models performing best. Resprouting trees exhibited significantly greater root biomass compared to trees growing from seed. Our best-fit allometric models were subsequently applied to 12 nearby plots which span a chronosequence of fallows to calculate the impact of accounting for resprouting allometry on forest biomass. We found that root biomass stocks (Mg/ha) were around 58% (22 - 85%) higher after accounting for resprouting, resulting in an average 9.5% (4 - 13%) increase in total biomass stocks, thus demonstrating the need to correctly account for re-sprouting trees in shifting cultivation fallows. Our analysis suggests that using our models will substantially improve the accuracy of tropical estimates of tree biomass and its distribution among different pools in shifting cultivation fallows. We also find that models fit using non-linear regression provide equally good fits to the data compared to the traditional approach of logtransforming biomass data.

203 Root Development

Monday 22 June – Poster session 145

Root dynamics of two grapevine cultivars differing in their hydraulic behavior Jhonathan Ephrath*1, Brian Hoefgen2, Uri Hochberg2, Shimon Rachmilevitch2 1 Ben Gurion University of the Negev, Jacob Blaustien Inst. for Desert Research, Israel, 2Blaustein Institutes for Desert Research, French Associates Institute for Biotechnology and Agriculture of Drylands, Ben Gurion University of the Negev, Israel

The interaction between scion and rootstock in grafted grapevines is extensively investigated. Nonetheless, the effect of canopy on root morphology has not been widely tested in field conditions. We tested root morphology in response to two irrigation regimes for two hydraulically different cultivars Shiraz (SH) and Cabernet Sauvignon (CS) grafted onto the same rootstock, 140 Ruggeri. These two grapevine cultivars have different aboveground hydraulic behavior (SH near-anisohydric and CS near-isohydric), and it is assumed that these differences may also cause belowground differences. This research was conducted in a four year old experimental vineyard at Ramat Negev, Israel. The grapevines underwent different irrigation regimes (50% and 25% of crop evapotranspiration). Aboveground and belowground physiological parameters were measured for a period of 16 months. Transparent tubes were installed to a depth of 180 cm at distances of 25 and 75 cm from the trunk. Root photographs were taken during different developmental stages of the year using a minirhizotron. Our results indicated that scion hydraulic behavior affected root dynamics. Larger differences were seen in SH than compared to CS in response to the irrigation treatment. It seems that SH invested more growth directly below the trunk, while CS roots were more evenly distributed throughout the soil profile.

146

Modelling of root dynamics in split-root rhizoslides reveals strong selective root placement of maize in response to nitrogen Dina in 't Zandt*1, Chantal Le Marié2, Norbert Kirchgessner2, Eric J.W. Visser1, Andreas Hund2 1 Radboud University Nijmegen, Netherlands, 2Swiss Federal Institute of Technology Zurich, Switzerland

The plant’s root system is highly plastic, and can respond to environmental stimuli such as high nitrogen in patches. A root generally responds to a nitrogen patch by selective placement of new lateral roots within the patch to increase nitrogen uptake efficiency. This is a desirable trait in breeding programs, since it may decrease NO3 - leaching and N2O emission. Roots of maize (Zea mays L.) were grown without nitrogen in split-root rhizoslides, a system that enables non-destructive root measurements via direct imaging of the root system. Half of the root system was subjected to high nitrogen after 15 days, and root growth was traced for a subsequent 15 days and modelled. The elongation rates of crown axile roots on the nitrogen-treated side followed a logistic increase to a 5.3 cm d-1 maximum, 95% of which was reached within 4 days. On the untreated side, axile root elongation dropped linearly to 1.2 cm d-1 within 6.4 days and stayed constant thereafter. Twice as many lateral roots were formed on the crown axis on the nitrogen side compared to the untreated side. Furthermore, the elongation rates of laterals exposed to high nitrogen increased linearly with 204 Root Development

Monday 22 June – Poster session most of the roots reaching an asymptote approximately 8 days after start of the nitrogen treatment. The laterals without nitrogen did not elongate. Furthermore, it was shown that the crown root system had a greater influence on shoot performance than the seminal root system 15 days after nitrogen application.

147

Effect of phenanthrene exposure on apoplastic barriers formation in maize Pierre Leglize*, Joan Dupuy, Quentin Vincent, Ivan Zelko, Christian Mustin, Stéphanie Ouvrard, Thibault Sterckeman Université de Lorraine, France

Management of Polycyclic Aromatic Hydrocarbons (PAH) contaminated soils through phytoremediation process is promising in controlled conditions. However, in situ experiments were not so successful due to plant growth limitation induced by PAH toxicity. Improving revegetation of contaminated site appears therefore crucial in phytoremediation application but requires a better understanding of contaminant’s impact on plant functioning. PAH exposure affects plant physiology such as mineral nutrition. Because the nutrient uptake is linked with the maturation of exodermis and endodermis, we focused on (i) the effect of PAH on the suberin lamellae formation and (ii) the localization of phenanthrene in root. Maize plants were grown in contaminated sand (50 and 150 mg PHE kg-1 dry sand) for 10 or 20 days. Epi-fluorescence microscopic observation of root sections was used to assess PHE localization within the root and maturation of exodermis and endodermis by Fluorol yellow 088 detection of suberin. For 10 days of cultivation, suberization of exodermis and endodermis of maize exposed to PHE was more extensive and PHE were only observed within suberized exodermis. This could be related to an inducer effect of PHE on the deposition of hydrophobic compounds within exodermis, which may act as barrier against PHE penetration. However after 20 days of exposure, exodermis and endodermis of non-exposed roots were totally suberized whereas PHE-exposed roots where less suberized. After 20 days, PHE seemed to inhibit root maturation, which could be caused by its toxicity as it is related with the plant biomass reduction. Furthermore, PHE patches were located only within suberized exodermis and endodermis, which may therefore act as retention zone where the hydrophobic PHE accumulates during its radial transport.

148

Responses of root development and zinc uptake to heterogeneous soil water distribution in lupin and cucumber Huifang Ma*1, Michael Wassilios Evangelou1, Peter Vontobel2, Rainer Schulin1 1 ETH Zurich, Institute of Terrestrial Ecosystems, Switzerland, 2Paul Scherrer Institute, Switzerland

The distribution of moisture is typically heterogeneous in soil even at the scale of a root system, and plant root systems are known to adapt to such heterogeneity. In this study we investigated how root growth allocation of two common crop plants, white lupin (Lupinus albus) and cucumber (Cucumis sativus L.), responded to lateral heterogeneity in soil moisture 205 Root Development

Monday 22 June – Poster session over time with neutron radiography and how this affected zinc (Zn) uptake in a low-Zn sandy soil with patchy Zn distribution. Single plants were grown in Al-containers (inner size: 27 cm length × 27 cm height × 1.2 cm thickness). To one lateral section of each container 20 mg Zn/kg were added. Soil water heterogeneity was achieved by mixing coarse sand into the soil in one lateral section of the respective containers, either on the same (cis) side as or on the side opposite (trans) to the Zn-enrichment, while no sand was added to control containers. The same amount of water was added to all containers. As a result the soil water content decreased in the order: sections without sand in heterogeneous containers > control containers > sections with sand in heterogeneous containers. In lupin, root growth (primary root length, cluster root number and length, cluster root length/total root length, and root dry weight) was reduced in the heterogeneity treatments, with fewer roots being produced in sand than in no-sand sections, while Zn uptake was not affected. In cucumber, the heterogeneity treatment increased root growth (root length density and total dry weight) and shoot Zn uptake. Lupin roots responded already after 6 days to the heterogeneity treatment, cucumber roots only after 35 days.

149

Effect of drought on surface and deep root dynamics of teak (Tectona grandis) and rubber (Hevea brasiliensis) trees in mainland South East Asia Jean-Luc Maeght*1, Corentin Clément2, Santimaitree Gonkhanmdee3, Oloth Sengtaheuanghoung4, Alexia Stokes5, Alain Pierret6 1 UMR Iess Paris ; IRD, France, 2Iess Paris IRD Nafri, Lao People's Democratic Republic, 3Faculty of Agriculture, Thailand, 4NAFRI, Lao People's Democratic Republic, 5INRA UMR-AMAP, France, 6UMR Iess Paris ; IRD - NAFRI, Lao People's Democratic Republic

We present the main results of two experiments conducted in a teak (Tectona grandis) tree plantation in Northern Laos and a rubber (Hevea brasiliensis) tree plantation in North East Thailand. We aimed at documenting the response of fine roots to seasonal and induced drought and quantify the corresponding carbon stocks. Fine root growth of 20-year old teak trees and 18-year old rubber trees were monitored at monthly intervals over 2 and 3 years, respectively. Observations were made using root windows installed at 50 cm depth increments, to a depth of 4.5 m. A rainfall exclusion experiment was performed in the teak plantation. Destructive samples were used to quantify fine root-related carbon stocks. In the case of teak, fine roots were found to a depth of 12 m. In both species, root dynamics is linked to transient changes in water availability. This work shows that deep fine roots are organs of foremost functional importance and we demonstrate their instrumental role in deep-water extraction during critical dry periods. The presence of significant amounts of deep roots in the soil profile further indicates that they should be taken into account when quantifying long-term carbon storage.

206 Root Development

Monday 22 June – Poster session 150

The characteristics of root cell components of plants adapted to acidic soil Eriko Maejima*1, Toshihiro Watanabe1, Tadao Wagatsuma2, Mitsuru Osaki1 1 Research faculty of Agriculture, Hokkaido University, Japan, 2Faculty of Agriculture, Yamagata University, Japan

Melastoma malabathricum and Melaleuca cajuputi naturally grow in highly acidic soil in tropical and subtropical zones. They are both reported to be highly aluminum tolerant, but their tolerance mechanisms still remain to be explained. Melastoma malabathricum accumulates high concentration of aluminum, whereas Melaleuca cajuputi accumulates low concentration of aluminum. Plasma membrane and cell wall compositions, organic acids released from root tips, and/or phenolics in root cells have often been reported to affect aluminum tolerance and aluminum accumulation in roots. In this study, we focused on woody species in Melastomataceae and Myrtaceae and investigated their root cell components possibly responsible for aluminum tolerance and/or accumulation. Melastoma malabathricum (Melastomataceae), Tibouchina urvilleana (Melastomataceae), and Melaleuca cajuputi (Myrtaceae) were grown in hydroponic culture and transferred to an aluminum treatment solution (phosphorus-free nutrient solution containing 0 or 500 μM AlCl3) for 1 week. Aluminum concentration in roots and leaves, lipid and cell wall composition, and organic acid and phenolics concentrations in roots were determined. We also investigated rice (Oryza sativa L.), an aluminum-tolerant crop, for comparison. Aluminum concentration in Melastoma malabathricum and Tibouchina urvilleana was higher than that in Melaleuca cajuputi. Melastoma malabathricum and Melaleuca cajuputi contained much lower proportion of phospholipid in root cells than that in rice, suggesting that the lipid composition in the plasma membrane in root cells is involved in aluminum tolerance mechanisms in such highly tolerant plants. Moreover, all woody plants investigated contained the highest concentration of phenolics compared with that in rice. Although Melaleuca cajuputi contained highest concentration of phenolics in roots, the extracted phenolics did not have the capacity to chelate aluminum. In contrast, phenolics extracted from Melastoma malabathricum formed a stable chelate with aluminum. These results suggest that phenolics in roots are likely to be involved in the detoxification of aluminum in roots of Melastoma malabathricum.

151

Root system of crops under different soil management systems in sugarcane field reform Gustavo Mateus*1, Felipe Giglio Bernardoni2, Denizart Bolonhezi2, Rafael Müller3, Carlos Alexandre Costa Crusciol3, Humberto Sampaio Araújo2 1 São Paulo Agency of Agribusiness Technology, Brazil, 2São Paulo Agency of Agribusiness Technology - APTA, Brazil, 3Department of Crop Science, College of Agricultural Sciences - FCA, Sao Paulo State University - UNESP, Brazil

Sustainable agricultural production systems that improve the quality of soil and water and reduce the emission of greenhouse gases, are essential in modern agriculture. This work was developed in the São Paulo Agency of Agribusiness Tecnhology, Andradina, São Paulo, Brazil. The aim of this study was to evaluate the effect soil management systems and different 207 Root Development

Monday 22 June – Poster session rotation system in sugarcane field reform on the root dry matter and root length of rotated crops. The experimental design was randomized blocks in split plots with four replications. The plots treatments consisted of three soil management systems, (conventional tillage, minimum tillage and no-tillage). The split plot consisted of four comercial crops, corn, peanut, sorghum and soybeans, a choice of green manure (Crotalaria juncea + jack beans), and fallow system. Roots were sampled in the flowering plants. No significant interactions were found among the soil management systems and different crops rotation in sugarcane field reform, for all variables. This can be explained by the dry spell and high temperatures occurring during crop development, which allowed the species presented similar behavior for the root dry matter and root length. Thus the volume of soil explored by the roots was the similar to the species regardless of the tillage system. The yield of root dry matter was observed values of 2.053, 2.496 and 1.195 g m-3 in the layers 0-10, 10-20 and 20-40 cm, respectively. Regardless of soil management systems and rotated crops was found that 80.7% of the root system concentrated at between the layers 0-20 cm.

152

Rooting of jujube (Ziziphus jujube mill) Li variety cuttings using some root promoting micro-organism and plant growth regulators Nabil Omar* Agricultural Research Centre, ARC, Egypt

This study was conducted at the experimental nursery of the Horticulture Research Institute, Giza, Egypt during 2008 and 2009 seasons. Semi-hard wood cuttings were taken from mature 15 years old trees of jujube (Ziziphus Jujube Mill) Li. Roots treatments inoculated with some plant growth promoting rhizobacteria strains (PGPR) 1- Bacillus polymyxa, 2- Bacillus circulans, 3- Bacillus megaterium, 4- Bacillus pasteurii, 5- Pseudomonas fluorescens, 6- Yeast strain (Saccharomyces cerevisiae), and mixed inoculants from previous PGPR strains. In addition, indole butyric acid (1000 and 2000 ppm), naphthalene acetic acid (1000 and 2000 ppm) were also tested as compared with untreated (control). The obtained results showed that, the effect of Bacillus megaterium as PGPR resulted in the highest significant rooting percentage (60% and 50%). On the contrary, the lowest significant effect of treatments was found as a result of naphthalene acetic acid at 1000 ppm and control during the two seasons of the study. Histological studies revealed that the callus originated from the cambial and phlom parenchyma cells below the cork cells from these protrusions the adventitious roots were developed. In conclusion the use of PGPR offers an attractive way to replace chemical fertilizer and supplements most of the isolates result in a significant increase in plant height, root length, and dry matter production of shoot and root of plants.

208 Root Development

Monday 22 June – Poster session 153

Effect of Streptomyces sp. isolated from acidic cultures of minerals in root development in environments polluted with mercury Sara Liz Pacheco Huerta*, Jasmin Elena Hurtado Custodio, Patricia Sheen Cortavarria Universidad Peruana Cayetano Heredia, Peru

Two strains of Streptomyces sp. (E1 and K2) and one strain of Streptomyces variabilis (K1A) were isolated from acidic cultures of mineral ores from Peruvian highlands. Molecular and phenotypic identification of the isolate strains was performed. The isolates were also evaluated to determine their resistance to metals, growth at different concentrations of sodium chloride, growth to different pH and exposed to different temperatures. The Streptomyces sp. strains (E1 and K2) were able to grow in 100 ppm of mercury and Streptomyces variabilis (K1A) was able to grow in 50 ppm of mercury. The three strains were evaluated to determine their ability to contribute to the development of roots in Lactuca sativa and Medicago sativa when exposed to mercury. L. sativa showed regular root development in 10 ppm mercury. However, when treated with Streptomyces sp (E1 and K2) and Streptomyces variabilis (K1A), L.sativa showed a remarkable root development in length in up to 50 ppm of mercury. Moreover, M.sativa has the ability to tolerate concentrations of mercury up to 100 ppm allowing a significant root development. In presence of Streptomyces sp (E1 and K2) the root development was in up to 200 ppm of mercury. The results obtained in this study allowed us to identify the ability of these strains to contribute to the development of plants in contaminated areas and therefore allow the restoration of polluted environment.

154

Chili pepper growth and drought tolerance are regulated by Bacillus vallismortis strain EXTN-1 producing volatile organic compounds Kyungseok Park*, Sarnalee Dutta National Academy of Agricultural Science, South Korea

The plant growth-promoting rhizobacteria (PGPR), especially Pseudomonas spp. and Bacillus spp., colonize roots of monocots and dicots, and directly or indirectly promote plant growth and elicit induced systemic resistance, among which Bacillus vallismortis strain EXTN-1 plays a pivotal role in enhancing induced systemic resistance (ISR) against multiple pathogens in a variety of crops. Despite of accumulating evidence the role of EXTN-1, the precise underlying mechanism of volatile organic compounds (VOCs) emitted by EXTN-1 has not been conclusively studied. In this study, in order to elucidate the potential physiological role of VOCs from EXTN-1 we investigated the plant growth and development in response to VOCs. Any phenotypic changes were not observed in aerial parts of VOCs-exposed chili pepper plants. However, root architecture was significantly altered in which increased root biomass was obtained in VOCs-exposed plants. Interestingly, a combination treatment between EXTN1 and VOCs from EXTN-1 had a negative effect on pepper growth. In addition, root colonization rate in rhizosphere was higher in VOCs-exposed plants, relative to control plants. These results suggest that increased population density of Bacillus may positively modulate the VOCs-mediated plant growth promotion. Intriguingly, improved drought tolerance was exhibited in plants exposed to VOCs derived from EXTN-1. Our data clearly propose that

209 Root Development

Monday 22 June – Poster session VOCs from EXTN-1 contribute to augmentation of plant growth and that VOCs are normally required for abiotic stress tolerance.

155

Response of plant roots and mycorrhizal fungi to soil hypoxia Yasmine Piñuela Samaniego*1, Peter Železnik2, Hojka Kraigher2, Irena Maček1 1 University of Ljubljana, Biotechnical Faculty, Slovenia, 2Slovenian Forestry Institute, Slovenia

Root growth and colonization with plant symbiotic arbuscular mycorrhizal (AM) fungi was measured in barnyard grass seedlings exposed to geological CO2 and hypoxia. Barnyard grass (Echinochloa crus-galli (L.) Beauv.) is an annual C4 grass and a cosmopolitan weed. It has been shown to be tolerant to soil hypoxia and flooding. Our experiment was conducted in the area of natural CO2 springs (mofettes) in Slovenia, and was designed to follow the growth of Echinochloa in three different CO2 soil exposures: control ( 2 m) and acidic while the rendisol is shallow (YM-42> MY-29.

110

Plant nitrophily drives plant productivity and plant-microbial interactions Barbara Pivato*1, Hugues Busset2, Florence Deau2, Annick Matejicek2, Philippe Lemanceau2, Laurent Philippot2, Delphine Moreau2 1 UMR Agrécologie - INRA Dijon, France, 2INRA, UMR1347 Agroécologie, France

The interest for plant interactions, especially between crop and weed species, in agricultural situations has increased with the pressure to reduce the use of chemical inputs. Thus, a better knowledge is required in plant traits mediating the issue of plant competition and how this competition is impacted by the environment. Nitrophily is a plant trait referring to plant habitat, ranging from oligotrophic species mostly found in soils with low N content to nitrophilic species better adapted to soils with high N content. We hypothesize that nitrophily impacts (i) the issue of plant-plant interactions, through competition for N and light and (ii) plant associations-rhizospheric microbial community interactions. We can expect that microbial community from nitrophilic/oligotrophic roots association is (i) different from community associated to the same plants cultivated alone, and (ii) differently impacted according to the association type. To test these hypotheses, three nitrophilic and two oligotrophic graminaceous species (one crop plant and four weeds) were cultivated in mono- or bi-specific associations in a soil supplemented or not with N. The issue of the plant-plant interactions was assessed by checking their productivity through biomass measures. The impact of the different plant combinations on total bacterial communities was characterized by A-RISA (AutomatedRibosomal Intergenic Spacer Analysis) fingerprinting.

284 Root-Root Interactions

Tuesday 23 June – Poster session The biomass was significantly higher in nitrophilic than in oligotrophic species when grown in N supplemented soil. Bi-specific associations showed different root biomass compared to monocultures in both N conditions. Genetic structure of rhizospheric bacterial community differed according to the N level. In higher N availability, bacterial communities structures were very similar, whereas in N limiting conditions, the rhizosphere of bi-specific associations harbored different bacterial communities in comparison to monocultures. These results suggest that nitrophilic plant trait is a driver of plant productivity via plant-plant competition, and this affects rhizospheric microbial community in N limiting conditions.

111

From pots to plots: Hierarchical trait-based prediction of population biomass in a mesic grassland Thomas Schröder-Georgi*1, Christian Wirth2, Karin Nadrowski3, Sebastian T. Meyer4, Liesje Mommer5 1 University Leipzig, Biology, Germany, 2University of Leipzig, Institute of Biology / German Centre for Integrative Biodiversity Research (iDiv), Germany, 3University of Leipzig, Institute of Biology, Germany, 4Technische Universität München, Department of Ecology and Ecosystemmanagement, Germany, 5Wageningen University, Nature Conservation and Plant Ecology group, Netherlands

Plant functional traits are powerful tools when it comes to the prediction of plant performance and ecosystem functioning. For a long time ecologists focused on aboveground traits but widely ignored root traits because their sampling and measurement is expensive and labourintensive. A number of newer studies have shown that root traits link not only to belowground (e.g. N cycling) but also to aboveground functions (e.g. biomass production). So far it has never been tested, how the importance of root traits for plant performance depends on the hierarchical level (individual vs. population). We hypothesized that root traits are more important on the population level than on the individual level, due to an increase in process complexity. We used univariate and multiple regression analyses to test the importance of 35 root-, leafand stature traits for the prediction of individual and population biomass production of 59 European grassland species. We found that traits of all three clusters (root, leaf and stature) correlate with both individual and monoculture biomass. The most parsimonious multiple regression model significantly improved for both, individual and monoculture biomass, when root traits are included. However, whereas root traits improved the individual biomass model by only 3% they improved the monoculture biomass model by 28%. Root traits are important for both, individual and population performance but the significance of root traits increases with increasing hierarchical level.

285 Root-Root Interactions

Tuesday 23 June – Poster session 112

Research on rhizosphere soil phosphorus fractions and availability in a maizesoybean relay intercropping system Chun Song*1, Xia Xiao2, Lu Mao2, Min Xu2, Taiwen Yong3, Wenyu Yang3 1 Sichuan Agricultural University, China, 2Institute of Ecological and Environmental Sciences, College of Resources and Environment, Sichuan Agricultural University, China, China, 3Key Laboratory of Crop Ecophysiology and Farming System in Southwest China, College of Agronomy, Sichuan Agricultural University, China, China

Intercropping is a traditional and sustainable cultivation model based on ecology principle of plant-plant interaction and plant-soil feedback between different biological species. However, the knowledge of crop rhizosphere soil phosphorus forms transformation and availability under intercropping system is still limited. This research focused on the effect of root interaction on crop aboveground biomass, rhizosphere soil phosphorus availability in a maize-soybean relay intercropping system by treated maize and soybean root system with root separation by polyvinyl chloride (PVC) clapboards or without root separation in PVC boxes. Two seedlings of maize and four seeds of soybean were planted per PVC box, and the distances between maize and soybean plants were 60cm. Soluble chemical reagents of CO(NH2)2, KH2PO4 and K2SO4 were applied as N, P and K fertilizer. We collected plant and soil samples in maize silking and maturity stages, and soybean branching, early flowering and maturity stages separately, and then determined the crop aboveground biomass and rhizosphere soil phosphorus fractions, to clarify the mechanism of rhizosphere soil phosphorus fractions transformation and bioavailability in maize-soybean relay intercropping system. The plant biomass data showed that soybean aboveground biomass were significantly higher in treatments without root separation. The phosphorus fractionation results showed that treatments without root separation significantly improved the rhizosphere soil NaHCO3-Po and NaHCO3-Pi content in maize silking stage, whereas decreased NaHCO3-P o content in soybean branching stage, and the NaHCO3-Pi content in soybean early flowering stage. These results indicated that maize had stronger competition for phosphorus uptake than soybean during maize and soybean co-growth stage. Residue P content of maize rhizosohere soil in maturity stage considerably higher than soybean maturity stage, which indicated that soybean had higher soil phosphorus use efficiency. In a word, maize-soybean relay intercropping can improve soil phosphorus use efficiency by root interaction, so as to improve crop yield.

286 Root-Root Interactions

Tuesday 23 June – Poster session 113

Root priority effects: Are root-root interactions in grasslands significantly affected by who interacts with whom and who arrives first? Vicky Temperton*1, Philipp von Gillhaussen2, Marco Brendel3, Marc Faget4, Stephan Blossfeld3 1 Plant Sciences (IBG-2), Forschungszentrum Jülich & Leuphana University Lüneburg, Germany, 2Bayreuth University, Germany, 3Plant Sciences (IBG-2), Forschungszentrum Jülich, Germany, 4University of Louvain, Germany

Whilst we all know that the timing of interactions between plants has important implications for plant performance, we still know relatively little about how community assembly (either aboveground or belowground) is affected by who arrives first. In a large field experiment we tested the possible role of priority effects of by sowing either grasses, legumes or nonlegume forbs five weeks before the other two functional groups, at the same time as testing effects of sowing a low or a high diversity seed mixture. Intriguingly, the species richness of the mixture did not have much of an effect on aboveground biomass, whereas the arrival time of each functional group had a very significant effect on aboveground as well as belowground productivity. When legumes were sown before forbs and grasses, community root productivity was significantly lower during the first growing season than when grasses were sown first. A repeat measurement of root turnover using in-growth cores in 2014 (data pending) will show whether this pattern was stable and created a priority effect underground or not. Which species interact can also affect rhizosphere pH. In a controlled experiment combining maize with bean, we measured rhizosphere pH and found that if these two species were grown together variation in pH was lower than when each species was growing alone. We also tested whether there were species-specific differences in the extent to which grass species experienced nitrogen facilitation when interacting with the legume Trifolium repens and this also in presence of an invasive non-legume forb Senecio inaequidens. The grass Holcus lanatus benefitted the most from growing near the legume, but pots with Festuca pratensis in had highest root biomass and less pronounced facilitation effects. Overall our data show that plant root performance is clearly affected by the identity and functional traits of the neighbouring plants.

114

Characteristics of phenolic acids exuded by roots in wheat and faba bean intercropping Jingxiu Xiao*1, Yi Zheng2, Li Tang1 1 Yunnan Agricultural University, China, 2Southwest Forestry University,Yunnan Agricultural University, China

Phenolic acids play important role in rhizosphere process, and few works have been cited on the effects of intercropping on phenlic acids exudation. Wheat and faba bean is an important planting pattern in southwest of China due to its significant yield advantages and diseases resistance. Hydroponic culture was conducted and the exudates form the roots were collected and examined by HPLC to investigate the effects of wheat and faba bean intercropping (W//F) on the phenolic acids exudation. The results showed that the three 287 Root-Root Interactions

Tuesday 23 June – Poster session phenolic acids were identified in root exudates: ρ-hydroxybenzoic, vanillic and syringic acid in both mono and intercropping. Intercropping decreased total amounts of phenolic acids in root exudates by 39.0%-70.1% and 37.56 -57.79% respectively, in comparison with that of mono cropping wheat (MW) and faba bean (MF). The maximum amount of phenolic acids exuded by root was found on 35d after transplanting in both mono and intercropping treatments, and exudation rate tended to decrease with crop growth from35d to 85d. During whole crops growth stages, intercropping significant decreased exudation rate of ρhydroxybenzoic and syringic acid by 41.06%-100% and 37.89%-64.35% respectively in comparison with that of MW. In W//F, vanillic and ρ-hydroxybenzoic acids were not detected from root exudates on 35d and 55d after transplanting respectively, which were detected till to85d in mono cropped wheat and faba bean.

288 Root-Root Interactions

Tuesday 23 June – Poster session Root Turnover

115

Root decomposition along a grassland plant diversity gradient Hongmei Chen*1, Arthur Gessler2, Hans de Kroon3, Michael Scherer-Lorenzen4, Liesje Mommer5, Christian Wirth1, Alexandra Weigelt1 1 University of Leipzig, Germany, 2Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Switzerland, 3Radboud University, Netherlands, 4University of Freiburg, Germany, 5Wageningen University, Netherlands

Decomposition of plant litter is a key process for carbon cycling and nutrient availability in ecosystems. However, we know very little about decomposition of root compared to leaf litter, especially in the light of biodiversity-ecosystem functioning relationships. To elucidate how plant diversity affects root decomposition in grassland and to disentangle the effects of root litter quality from environmental factors (including both abiotic and biotic aspects), three decomposition experiments using a litter-bag approach were conducted at the Jena Experiment, Germany, on 80 plots differing in species richness (1, 2, 4, 8, 16 species): 1) decomposition of plot-specific roots in their origin plots to assess plant diversity effects; 2) decomposition of plot-specific roots in a common-garden plot to assess root litter quality effects; and 3) decomposition of standard roots (from Lolium perenne) in all plots to assess general environmental effects. The litter bags were installed in April 2014 and retrieved 1, 2 and 4 months afterwards to trace the decomposition progress. Mass loss was determined as a measure of decomposition rate. With increasing plant diversity, decomposition of plot-specific roots decreased both in their origin plots and in the common-garden plot, while decomposition of standard roots did not change along the plant diversity gradient. These patterns remained constant over time. Moreover, in the common-garden plot, there was a negative effect of the presence of grasses on root decomposition, which increased along the diversity gradient. Our results suggest: 1) that the overall effect of plant diversity on root decomposition is negative; 2) that this effect is primarily due to changing root litter quality rather than environmental factors; and 3) that grasses have a substantially higher negative effect on root litter quality and thus decomposition compared with other functional groups.

289 Root Turnover

Tuesday 23 June – Poster session 116

Fertilization and root presence of permanent grassland have no effect on microbial biomass and mineralization Christoph Knoblauch*1, Conor Watson2, Nicole Wrage-Mönnig3, Rolf Becker4, Clara Berendonk5, Florian Wichern2 1 Rhine-Waal University of Applied Sciences, Faculty of Life Sciences, Germany, 2Faculty of Life Sciences, Rhine-Waal University of Applied Sciences, Germany, 3Faculty of Agricultural and Environmental Sciences, University of Rostock, Germany, 4Faculty of Communication and Environment, Rhine-Waal University of Applied Sciences, KampLintfort, Germany, 5Chamber of Agriculture North Rhine-Westphalia, Kleve, Germany

The release of nitrogen (N) from soil organic matter (SOM) and plant residues is an important source of available N in permanent grassland. Added inorganic fertilizer can result in an alteration of root biomass and therefore also influence the input and turnover of SOM. However, there is a lack of knowledge about interactions of inorganic fertilization and mineralization in grassland soils. The objectives of the current study were to (i) measure the carbon (C) and N mineralization of grassland soils, (ii) to quantify microbial biomass (MB) and (iii) to monitor the effect of roots on C and N mineralization. We initiated a short-term incubation experiment with varying organic and inorganic fertilizer rates and the presence or absence of course roots and measured the effects on C and N mineralization and MB. Soil was taken from a fertilizer field trial on permanent grassland (Lolium perenne L.) and visible roots were removed manually. During incubation, we measured soil respiration at least once a week for a period of 35 days. Inorganic N and MB were determined at the beginning and end of the experiment. The presence of roots did not significantly affect the mineralization processes within the contrasting fertilizer rates. However, inorganic N tended to increase with a higher application rate of the two fertilizer types. Neither cumulative soil respiration nor MB was affected by fertilizer type or application rate. Yet, we determined a hysteresis effect of the pre-equalized water content on soil respiration. Especially at the beginning, water content concealed the importance of fertilizer rate in terms of soil respiration. In conclusion, the heterogeneity of the taken samples within each fertilizer treatment predominated over the amount and type of fertilizer or presence and absence of roots.

117

Long live the roots! Characterizing root lifespan in a grassland biodiversity experiment Natalie Oram*, Liesje Mommer, Frank Berendse, Jasper van Ruijven Nature Conservation and Plant Ecology, Wageningen University and Research Centre, Netherlands

Since the discovery of the positive plant diversity-productivity relationship in grasslands, the mechanisms underlying this relationship have become central research questions. It is wellknown that greater plant species diversity results in higher standing root biomass. However, whether this is the result of increased root production or decreased root mortality is largely unknown. Yet, understanding the dynamics of root biomass is crucial with respect to carbon and nutrient cycling. Increased root production could lead to enhanced organic matter inputs into the soil, whereas increased root lifespan may actually slow down these inputs, leading to different soil C and N dynamics in the long term. An increasingly positive plant-soil feedback, as well as changes in microclimate and nutrient uptake that parallel high plant diversity may 290 Root Turnover

Tuesday 23 June – Poster session lead to a more favourable environment for roots. We hypothesize that root lifespan will be positively correlated with species richness; greater root lifespan in diverse plots will contribute to the observed increase in standing root biomass. We test this hypothesis in a long term experimental grassland biodiversity experiment in Jena. We aim to quantify root lifespan in a novel and more precise way using repeated imaging of roots through minirhizotrons and quantification of root decomposition with a litterbag experiment. Root morphology and standing root biomass will also be measured. Imaging started in March, 2015, one year after the minirhizotrons were installed. Preliminary results will be presented.

118

Effect of nitrogen form, pH and plant species in the mobilization and acquisition of P from a recycled phosphorus fertilizer Ana Alejandra Robles Aguilar*1, Nicolai David Jablonowski2, Vicky M. Temperton2 1 Forschungszentrum Jülich GmbH, Germany, 2Forschungszentrum Jülich GmbH, Institute für Bio- und Geowissenschaften, Germany

World phosphorus resources are limited. Therefore recycling of phosphorus from waste materials is important, and struvite (MgNH4PO46H2O) is a common precipitate recovered from waste water treatments or during anaerobic digestion of manure. Our approach is to evaluate how mobilization of phosphorus may differ when two different species (narrow– leaved Lupin and Maize) are grown in an acidic or alkaline sand with phosphorus added as either struvite or as superphosphate. Nitrogen was applied as ammonium or nitrate as an important factor that could affect phosphorus availability by changing soil pH. The parameters to evaluate the mobilization of phosphorus from struvite were phosphorus uptake, phosphorus present in the soil and comparison of plant performance within the different treatments. Lupines are capable of symbiotically fixing atmospheric nitrogen, as well as to release phosphate-mobilizing carboxylates. These two traits make lupines good candidates forstudying nutrient mobilization in the rhizosphere. In order to observe if citrate (mimicking root exudates) was able to make the P from the struvite more available compared with water, our study also included a flushing experiment with citrate in columns filled with two different sands of acidic and alkaline pH mixed with struvite. We predict that lupine growing on alkaline sand will have better access to phosphorus in struvite (than on acidic sand) due to its ability to acidify the rhizosphere via exudation of carboxylates. Thus we expect lupine to have higher biomass when growing on alkaline sand with an ammonium supply.

119

Plant presence reduces root decomposition rate of non-legume species Sirgi Saar*1, Marina Semchenko1, Janna M Barel2, Gerlinde DeDeyn2 1 Tartu University, Estonia, 2Wageningen University, Netherlands

Litter nutrient concentrations are important traits for litter decomposition, which generally proceeds faster with increased nutrient concentrations in the litter. However, the presence of living roots can affect decomposition rates due to a priming effect by releasing root exudates which can give saprotrophic microbes an energy boost, enabling them to degrade the litter 291 Root Turnover

Tuesday 23 June – Poster session faster. To test whether and how plant presence affects decomposition of roots with different traits, we used dead roots of seven species (3 grasses, 3 legumes, 1 forb) as litter material and measured the litter mass loss after 8 weeks of incubation in soil with or without a white clover (Trifolium repens) plant. We expected that the decomposition rate would increase in the presence of a living plant, especially in the case of nitrogen-rich litter, because root exudates can provide a source of easily available carbon and stimulate microbial activity while subsequent nitrogen release from the litter could further enhance microbial activity and thus decomposition. On the contrary, we found that the decomposition rate of grass and forb roots decreased in the presence of living roots, while the decomposition rate of legume roots was not significantly affected by the presence of a living plant. Our results show that root decomposition rate can be slowed down in the presence of a living plant, but that this effect depends on the properties of the decomposing roots, with reduction being pronounced in root litter poor in N and P but not in the relatively nutrient-rich legume root litters.

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Consequences of plant kin recognition for microbial soil feedback and root decomposition Marina Semchenko*1, Anu Lepik2, Sirgi Saar2 1 University of Manchester, United Kingdom, 2Department of Botany, University of Tartu, Estonia

Recent studies have shown that plants are able to detect the genetic identity of their neighbours. Some species proliferate roots when grown next to unrelated individuals but apparently avoid direct competition with kin by reducing root growth. Neighbour recognition can be mediated by root exudates and involve complex changes in root growth and morphology. The consequences of such behaviour for ecosystem functioning are as yet unknown. In this study, we examined the consequences of kin recognition for microbial soil feedback and root decomposition. In the conditioning stage, plants of Deschampsia caespitosa were grown in groups of either siblings or unrelated individuals from the same population. In the feedback stage, new seedlings were planted into soil conditioned by either siblings or non-siblings. Conditioned soil included dead roots, allowing estimation of root litter decomposition during the feedback stage of the experiment. We found that microbial soil feedback for seedlings at early stages of development tended to be more negative on soil previously occupied by siblings. However, soil feedback did not differ between sibling and non-sibling soils at later stages of development. We also found evidence for significantly slower decomposition of root litter obtained from plants grown in groups of siblings compared with groups of unrelated individuals. Roots of plants that grew with siblings also had a significantly higher C:N ratio. This may explain slower root decomposition and indicate increased investment into pathogen defence at the expense of competitive ability when interacting with kin.

292 Root Turnover

Tuesday 23 June – Poster session 121

Phosphorus availability and its interaction with plant belowground carbon, nitrogen and phosphorus input into Oxisol Pierre Stevenel*1, Astrid Oberson1, Samuel Abiven2, Idupulapati M. Rao3, Emmanuel Frossard1 1 ETHZ - Institute of Agricultural Sciences, Switzerland, 2University of Zürich Department of Geography, Switzerland, 3International Center for Tropical Agriculture, Colombia

Phosphorus (P) deficiency is an issue for a large part of tropical soils worldwide. Low P availability in Oxisols is mainly due to strong P sorption and often limits plant growth. This feature affects the carbon (C) cycle by influencing its capture by plants, and the input of belowground C contained in roots and rhizodeposition. Moreover, low P availability influences nitrogen (N) input in soil by limiting symbiotic N2 fixation by legumes. Additionally, plants have developed strategies to cope with P deficiency, such as root system extension or exudation enhancement, which also determine belowground organic matter input. However, a quantitative understanding of combined root and rhizodeposition C and N inputs in response to P availability has not yet been studied. In this project, we want to investigate the role of P availability on the regulation of belowground C, N and P inputs, and its impact on the turnover of these inputs in soil nutrient pools over time. Experiments will be performed with two tropical plant species: the legume Stylosanthes guianensis and the grass Brachiaria decumbens. Plants will be grown in an Oxisol along a gradient of plant available P. A promising tri-isotopes (13C, 15N, 33P) labeling method will be used to quantify belowground C, N, P inputs. The isotopic composition and isotope recovery in shoots, roots and root free soil will be determined. Following this experiment and after removing the aboveground biomass, nutrient turnover will be studied in soils incubated during three months. The incorporation of plant belowground input of C, N and P into soil pools over time will be determined with the isotopic composition. This project will provide a better understanding of the influence of P availability on C and N inputs to soil but also strong foundations on the long term storage of those nutrients in soils.

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Root activity and dormancy in forest communities along an elevational gradient Yan Wang*1, Zhun Mao2, John Kim3, Christophe Jourdan4, Herve Ray4, Alexia Stokes3 1 Université Montpellier 2, France, 2c IRSTEA, UR EMGR, 2 Rue de la Papeterie, BP 76, 38402 Saint-Martin-d’Hères Cedex, France, France, 3INRA, UMR AMAP, Boulevard de la Lironde, 34398 Montpellier Cedex 5, France, France, 4CIRAD, UMR Eco&Sols – Ecologie Fonctionnelle & Biogéochimie des Sols & Agroécosystèmes (Montpellier SupAgroCIRAD-INRA-IRD), 2 Place Viala, 34060 Montpellier, France, France

Belowground processes in plant communities drive the sequestration of carbon into soil, but how these processes are modified by climate remains largely unexplained. Elevational gradients are unique tools for studying the responses of communities to climatic variability in situ.

293 Root Turnover

Tuesday 23 June – Poster session We installed rhizotrons in contrasting forest communities at 1400 m, 1700 m and 2000 m. We measured the number of growing roots and elongation every month over 4 years. Results showed that mean daily root elongation rate (RER) was driven by soil temperature between 0-8°C, outside which extreme soil temperatures perturbed growth. RER peaked in springtime, and a smaller peak was sometimes observed in autumn. Cumulated root length was not significantly different between altitudes, although the growing season was significantly shorter at 2000 m. However, when winter snow cover was > 6 months, root growth was severely limited the following year. Root longevity was dependent on altitude and the season in which roots were initiated; roots emitted in the autumn lived significantly longer than those initiated in springtime. Root diameter was a significant factor explaining much of the variability. Few differences in root demography between contrasting plant communities within each altitude were found, and were explained by air or soil temperature. We conclude that at high altitudes and when extreme abiotic events are not restrictive to carbon supply, more resources are invested belowground in a shorter period of time, revealing a plastic response to climatic variables within a community.

294 Root Turnover

Tuesday 23 June – Poster session BIOFECTOR

123

Single and tripartite biofectors for tomato (var. Mobil) upscaled from pots to plots Borbala Biro*, Zita Szalai, Anita Dudás, Tamás Gáspár, Heléna Wass-Matics, Zsolt Kotroczó Corvinus University of Budapest, Hungary

Living and non-living bioeffectors are beneficial treatments of organic and sustainable agricultural practices. It is a question if single or 2-3-types of microbes in one particular product is the most necessary or how to successfully upscaling the beneficial effects from pots to the fields? Tomato Solanum lycopersicon Mill. ’Mobil’ was used in the experiment. Bioeffector products, as BE1: Trichoderma harzianum T-22 and BE-4: Hungarian Trichoderma TDM, including Azotobacter and Azospirillum N2-fixers, BE2: Pseudomonas sp., BE3: Bacillus amyloliquefaciens Rhizovital 42 F1 were applied on the bases of the supplier’s recommendations. The plants were grown in 2.5 kg soil/pot in 4 replicates. TSP (0,62g), Rock-phosphate RP (1,59g), Patentkali (1,67g) and Calcinit (1,46g) were applied at the pots and 1200 kg/ha from both at the field. BE application was performed both at sawing and at the time of plantation. Growth of tomato, shoot and root biomass was assessed and general soil characterization, including MPN counts of bioeffectors in tomato rhizosphere. Results were evaluated by statistical probes. Upscaling was resulting the similar beneficial effect at Bacillus (BE3) bioeffector, with greater variability and less improvement at the field. Shoot biomass production was less variable parameter than the root or fruit biomass, more particularly also among the rather changeable field condition. Combined tripartite microbial inoculations seem to have better beneficial influence, then the only single bioeffector treatments in general. Effect of BE1 Trichoderma inoculation was improved for instance in combination with free-living and associative Nitrogen-fixers, due to the relatively high P-content (430 mg/kg) of the slightly humous sandy soil. Plant microbe interaction is largely dependent on the soil nutrient-status and the balance among nutritive elements.

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Soil application of seaweed extracts and micronutrients to improve the cold stress tolerance in maize during early growth Klara Bradacova*1, Nino Weber2, Markus Weinmann2, Günter Neumann2 1 University of Hohenheim, Institute of Crop Science (340h), Germany, 2University Hohenheim, Institute of Crop Science (340h), Germany

Low soil temperature in spring is a major constraint for cultivation of tropical crops in temperate climates, associated with impaired seedling development, inhibition of root growth and activity.

295 BIOFECTOR

Tuesday 23 June – Poster session In this study, we tested the potential of commercial bio-effector products such as seaweed extracts and rhizobacteria (strains of Bacillus and Pseudomonas) with plant growth promoting potential to improve the tolerance of maize to low root zone temperatures during early growth. Maize (v. Colisee) was cultivated in pots containing 2 kg of a fully fertilized silty-loam soil (pH 6.8). In order to control the root zone temperature (RZT), pots were installed in a cooling system. After germination at 22 °C, the cold stress phase (12-14 °C) started at 14 days after sowing to simulate a cold-period in spring. Bioeffectors were supplied close to the plants with a dosage of 109 CFU kg-1 soil for bacteria and 0.017 g kg-1 soil for seaweed extracts at 0, 2 and 4 weeks after sowing. In two independent experiments, positive BE effects on plant growth and particularly on root development at low RZT were exclusively detected for seaweed extracts with high Zn/Mn contents and similar growth promotions were induced by sole application of Zn and Mn in comparable amounts. This finding suggests that also the seaweed extracts were mainly acting via improved Zn and Mn supply to the plants. Assays of different oxidative stress markers showed that the beneficial effect of Zn/Mn treatments was associated with increased superoxide dismutase (SOD) acitivity in the root tissue, playing a key role in antioxidative defense mechanisms. In this context, Zn/Mn supplementations may effectively improve cold stress tolerance of maize by their function as enzymatic co-factors particularly for SOD. Accordingly, formation of necrotic leaves in cold-stressed plants was associated with a low Zn-nutritional status.

125

The combination of humic substances and inoculation with different microorganisms enhances the growth of maize (Zea mays L.) Vincenza Cozzolino*, Hiarhi Monda, Daniele Todisco, Davide Savy, Giovanni Vinci, Alessandro Piccolo University of Naples Federico II, Italy

Plant biostimulants, or bioeffectors (BEs), include diverse substances and rhizosphere microorganisms that enhance plant growth, contributing to improve fertilizer use efficiency, enhancing nutrient uptake, and increasing root health through competition with root pathogens. The well-known direct effect of humic substances (HS) on root growth has produced an expanding market for HS extracted from various composted green wastes, that represent a more sustainable source than those obtained from expensive fossil matrices, such as lignites or peats. The aim of this work was to evaluate the ability of these new biotechnological tools to promote the growth of maize plants in soil with low P availability. A pot trial was performed with the following BE treatments: 1. B0 (no inoculation), 2. B2, Pseudomonas sp plant growth promoting bacteria (Proradix), B3, Bacillus amyiloliquefaciens PGP (Rhizovital 42); MYC Funneliformis mosseae and Rhizophagus irregularis, commercial inoculum of arbuscular mycorrhizal fungi (Aegis); HA, humic acid (HA) from composted artichoke wastes. We evaluated the benefits of each BE alone or in combination. The P addition to soil comprised: P0 (no P addition), and CM (composted cow and buffalo manure at 50 mg P/kg soil). The impact of P fertilization, inoculation and HS were evaluated on biomass production and P and 296 BIOFECTOR

Tuesday 23 June – Poster session N content. The plants were grown in pots filled with 5 kg of substrate, prepared using an alkaline clay-loam soil and harvested 8 weeks after sowing. The benefits of the combined action of compost, microorganisms and HS were evident. Inoculation with the bioeffectors enhanced plant growth, as well as N and P content in shoots, especially in treatments where each microorganisms were mixed with HA. This new generation of biological products based on HS and selected beneficial microorganisms provides opportunities for an effective biological support of a sustainable crop production.

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Combinations of rhizosphere-competent fungal and bacterial isolates improve nutrient acquisition and growth in maize and tomato Joerg Geistlinger*1, Markus Weinmann2, Borbala Biro3, Alessandro Piccolo4, Shekhar Sharma5, Rainer Borriss6 1 Anhalt University of Applied Sciences, Germany, 2University of Hohenheim, Germany, 3Corvinus University of Budapest, Hungary, 4University of Naples Federico II, Italy, 5Agri-Food & Biosciences Institute, United Kingdom, 6ABiTep GmbH, Germany

BIOFECTOR (7th EU Framework Programme, grant agreement 312117) works on “an improved understanding and utilization of biological processes supporting soil fertility”, essentially on the soil microbiome and bioactive natural compounds interacting with the root system and/or mutual or symbiotic microbes, termed bio-effectors (“BEs”). Here, we report on the results of Work Package 2: BE combinations and synergisms. Ten partners from 5 EU countries agreed on a corporate standardized experimental design for greenhouse and field trials with tomato and maize as model crops. Local soils received basal fertilization (low in P) and were enriched with standard BEs (BE1= Trichoderma harzianum (TRIANUM-P™), BE2= Pseudomonas spec. (PRORADIX™), BE3= Bacillus amyloliquefaciens (RHIZOVITAL™)), combinations thereof and in combination with newly developed fungal and bacterial strains as well as extraction products from seaweed and compost. Main findings after 2 years of research were in maize: (1) combining standard BEs1-3 in a loamy top soil with low P availability has no positive effects, but growth-promotion was achieved by combining BE2 with seaweed extracts. (2) BEs1-3 have no effects on cold stress induced necrotic leaf areas but necrosis could be completely avoided by combining Zn/Mn micronutrients with seaweed extracts. (3) Composted cow manure with the combination of BE3 and humic acid extracted from compost yielded 65% more dry weight biomass. (4) Cultivar/strain-specific effects between different maize cultivars and Trichoderma strains were confirmed by qPCR. In tomato (1) the combination of Trichoderma with associated N-fixing bacteria had positive effects on yield, (2) the combination of Trichoderma and Gram+ bacteria (Bacilli) doubled yield, (3) early application of Piriformospora indica (Sebacinales) caused positive seedling development and (4) co-colonization of tomato roots by different Trichoderma strains was proven by molecular marker application. Currently new combinatorial BE-products are developed on the bases of Trichoderma, seaweed extracts rich in micronutrients, Bacilli and N-fixing bacteria.

297 BIOFECTOR

Tuesday 23 June – Poster session 127

Influence of bioeffectors application on maize growth and yield Martin Kulhanek*, Pavel Tlustoš, Jiří Balík, Zlata Holečková, Jindřich Černý Czech university of life sciences in Prague, Czech Republic

Phosphorus will become probably the limiting nutrient in near future. The sources of suitable rock phosphates for phosphorus fertilizer production are restricted. Therefore, the improvement of phosphorus acquisition from less available soil forms is needed. One of these ways is the bioeffectors (BEs) application. The aim of this study was to confirm, that the BEs have a positive influence on soil phosphorus mobilisation leading to better P uptake by plants and their higher yields and quality. In this study, the data from pot experiment were evaluated. Three BEs – Trianum (Trichoderma harzianum), RhizoVital (Bacillus amyloliquefaciens) and Proradix (Pseoudomonas sp.) in different combinations with rock phosphate and triple superphosphate were tested on maize plants. The soils from two sites (Humpolec and Lukavec) with low P level and diluted with sand (2:1 soil:sand) were used for the experiment. Plant height, shoot and root dry mass yield, and P plant contents were measured. The average plant height was significantly higher at all treatments fertilized with triple superphosphate in first two months of the experiment. Thereafter, at harvest after 15 weeks of growth, significant differences among plant heights disappeared. The above ground biomass dry weight was significantly higher at the treatments fertilized with triple superphosphate only at Lukavec site. The significant influence of BEs on plant height and above ground dry weight was not confirmed. The root dry weight was slightly, but not significantly higher at the treatments without P fertilization. On the contrary, the lowest root dry mass weight was measured at the triple superphosphate treatments. From the obtained data is possible to conclude, that used bio-effectors did not significantly influenced the plant height and root and shoot dry biomass amount weight as well.

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Effect of bio-effective microorganisms on early Maize root development and P uptake under low temperature Jonas Duus Stevens Lekfeldt*, Beatriz Gomez Muñoz, Jakob Magid, Lars Stoumann Jensen, Andreas de Neergaard University of Copenhagen, Denmark

Maize is a rapidly expanding crop in Northern Europe, but emergence and establishment can be challenged by low soil temperatures in early spring. Microbial inoculants have been demonstrated to stimulate root development, and may help overcoming initial water and nutrient stress, caused by poor root development. However, if soil nutrient levels are low, early investment of plant biomass in root development may not be a successful strategy from a nutrient acquisition point of view. We tested the ability of selected microorganisms with known root stimulation potential (Penicillium sp; Bacillus sp) and humic acids on maize root development in soils of low and medium P availability at 10 and 16 degrees. The soil used was sandy loam from a long-term depletion trial, one with very low P availability, as well as a moderate P treatment, fertilized with animal manure for the past 10 year. 25% sand was mixed into the soils to enable root re-isolation at harvest, and avoid water-logging. Plants were destructively harvested after 5 and 8 weeks, and analysed for shoot and root biomass, plant P in root, shoot and remaining in the seed. Root architecture parameters from 298 BIOFECTOR

Tuesday 23 June – Poster session entire isolated roots were assessed using WinRhizo software. Soil extractable P and pH was measured at planting and harvest. Results from the ongoing trial will be presented at the conference.

129

Microbial bio-effectors combined with manure proliferate plant growth of tomato: investigations on the modes of action Zhifang Li*1, Justus Riemann1, Angelika Lüthi1, Nino Weber1, Markus Weinmann1, Gheorghe Poşta2, Günter Neumann1 1 University Hohenheim, Institute of Crop Science (340h), Germany, 2Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Horticulture and Forestry, Romania

When tested under practice conditions, commercial bio-effector preparations induced strong growth improvements (80 % increase in plant height) of tomato seedlings grown on a substrate with composted cow-manure (45 %), garden soil (30 %), peat (15 %), and sand (10 %), but without mineral fertilizers. To study the modes of action how these products based on (Bacillus strains FZB42 and R41, Pseudomonas sp. DSMZ 13134 and Penicillium sp. PK 112) isolates could improve plant growth, pot experiments under controlled conditions were conducted. The question whether the plant growth-promoting effect was due to mobilization of sparingly available mineral nutrients from the humified manure was studied in comparative tests where the portion of manure was replaced by an increased share of peat in the substrate while adding mineral nutrients in easily available forms or not. The role of manure as a carbon source supporting the establishment and activity of the introduced microbial strains was tested through substitution by alternative carbon sources. Root morphological characteritics and colonization by arbuscular mycorrhizal (AM) fungi were assessed to detect if an improved spatial acquisition of mineral nutrients could explain the beneficial effects of the bio-effectors. Contrary to the strong effects observed under practice conditions, weak or no growth responses to the bio-effector treatments were found in plants under controlled conditions. However, the Pseudomonas treatment induced a significant increase in root hair length. The biomass production of tomato plants grown on the substrate with manure raised by 9 % in response to the combined application of the Penicillium preparation with an AM inoculum (Glomus intraradices). Plant growth-promotion by microbial bio-effectors, therefore, the combination of diverse modes of action seems to be involved, whose relative importance may vary depending on the environmental conditions.

299 BIOFECTOR

Tuesday 23 June – Poster session 130

The effect of biomass ashes and bioeffectors on soil solution composition Filip Mercl*, Václav Tejnecký, Petra Hubová, Pavel Tlustoš, Pavla Ochecová, Jiřina Száková Czech University of Life Sciences in Prague, Czech Republic

Biomass ash, the waste material coming from biomass combustion, is generated in large quantities worldwide. Mineral nutrients, bound in ash, are usually poorly plant-available, therefore there is an effort to enhance their availability using Bioeffectors (BEs). Objectives of this study was to i. assess the influence of biomass ash on soil solution composition and ii. investigate BEs ability to enhance availability of nutrients from ash in soil-plant system. In the pot experiment, plants of wheat (Triticum aestivum L.) and maize (Zea mays L.) were grown in BE inoculated mixture of soil and biomass ash under outdoor precipitationcontrolled conditions. Two different types of ash were used (straw and wood ash), and two different BEs were tested (BE3 - Rhizovital® and BE4 – Bacto_Prof). Soil solution was sampled using suction cups (MacroRhizons, Rhizosphere Research Products B.V., Netherlands) from the root zone of plants five times during the vegetation period. In samples of soil solution, concentrations of phosphorus (P) and major low-molecular-mass organic acid (LMMOA) anions (acetate, formate, lactate, oxalate, pyruvate) were determined. The yield of grain and their P content were also determined. Results showed different influence of ashes on soil solution composition. Application of straw ash significantly increased P concentrations in soil solution throughout the vegetation period. Both types of ashes did not differ in the influence on the grain yield, but straw ash significantly increased P content in wheat grains. We observed no significant influence of tested BEs on parameters listed above. Changes in relative and total amount of individual LMMOAs after ash addition were registered. BE4 significantly changed oxalate content in soil solution for one treatment (wheat + straw ash) in early stage of plant development (27 days after planting).

131

Humic, fulvic and water-soluble organic fractions from different compost structure-bioactivity relationship revealed by chemical-biological characterization Hiarhi Monda*, Vincenza Cozzolino, Riccardo Spaccini, Alessandro Piccolo University of Naples Federico II, Italy

The influence of humified organic matter on soil fertility has led to a growing interest in the use of humic substances (HS) as soil amendments, revealing their ability to positively affect biochemical processes underlying plant growth and nutrition. An ecologically based approach to farm management consist in the use of compost, recognized as a natural method for recycling organic wastes, and widely used as soil conditioner and fertilizer. In this study we combine the bright side of both technologies by comparing three different organic extracts, humic acids (HA), fulvic acids (FA) and water-soluble organic matter (WOM), from composts made out of different biomass wastes: tomato (C1), artichoke (CYN), and artichokefennel (CYN+F). We conducted bioactivity assay on maize seedling with the humic extracts, which were thoroughly characterized. All the extracts generally increased the whole plant

300 BIOFECTOR

Tuesday 23 June – Poster session biomass and positive effects were observed on root length and chlorophyll content. The molecular composition assessed by CPMAS-NMR spectroscopy and pyrolysis-GC-MS of all original composts appeared generally similar, whereas that of the HA and FA extracts varied somewhat as by hydrophobic and hydrophilic components. However, all HA and FA extracts were generally low in aromatic compounds, while they were abundant in biolabile compounds, such as carbohydrates and lipids. The hydrophobic/hydrophilic ratio seemed to play an important role in determining their bioactivity. Moreover, the biological assay pointed out that the larger the content of carbohydrates and sugar-like compounds, as well as nonlignin aromatic components, and the smaller the amount of phenols groups, the greater was the biological/chemical effects exerted by HS. These results confirm the role of humified organic matter in stimulating plant growth, especially when the content of potentially bioavailable hydrophilic components is predominant.

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Supplementation of silicon in maize (Zea mays) to improve P acquisition and AMF symbiosis efficiency Narges Moradtalab*1, Markus Weinmann2, Roghieh Hajiboland3, Günter Neumann2 1 University of Hohenheim, Institute of Crop Science (340h), Germany, 2University Hohenheim, Institute of Crop Science (340h), Germany, 3University of Tabriz, Iran

Phosphorus (P) deficiency is a major limiting factor for plant growth and development. Based on preliminary observations in strawberry (see: Moradtalab et al., this issue), we hypothesized that an optimum level of silicon (Si) supplementation improves arbuscular mycorrhizal (AM) colonization and P acquisition in maize. In this work, the effects of Si supplementation (as K2SiO3) and a commercial silicone fertiliser (Actisil, Yara, ) on AMF colonization, symbiosis efficiency and P availability were studied in maize (Zea mays L. cv. Colisee) plants grown in organic farming soil under greenhouse conditions with additional AM inoculation. Concentrations of 6, 100, 1000 and 10000 mg Si/kg soil (as K2SiO3 by adding to the soil) and 6 mg Si/kg soil of commercial Actisil were applied to the 3 L pots. Application of Actisil was performed either as start application or by weekly irrigation. No P fertilizer was added to the soil within first 4 weeks but after appearance of stronger deficiency symptoms, a moderate P fertilisation was performed with 50 mg P kg-1 soil. Si-supplied plants showed less P deficiency symptoms and growth stimulation especially at application rates of 6 and 100 mg kg-1 as compared with plants without Si and 1000 and 10000 mg/kg Si. Application of 10000 mg kg-1 Si exerted detrimental effects on plant growth and the plants died after 5 weeks application. Start application with Actisil was more effective than weekly fertigation. Our results indicated an optimal level of Si application at 6 mg Si/kg soil. Evaluation of AM colonisation is currently on the way.

301 BIOFECTOR

Tuesday 23 June – Poster session 133

Effects of silicon supplementation in drought-stressed strawberry plants in the presence or absence of arbuscular mycorrhizal fungi Narges Moradtalab*1, Roghieh Hajiboland2, Markus Weinmann3, Günter Neumann3 1 University of Hohenheim, Institute of Crop Science (340h), Germany, 2University of Tabriz, Iran, 3University Hohenheim, Institute of Crop Science (340h), Germany

Drought stress seriously affects plant growth and development. In this work the effects of silicone (Si) supplementation (3 mM, as Na2SiO3) were studied in strawberry (Fragaria × ananassa var. Parus) plants. Three levels of irrigation (field capacity, 0.75 field capacity, 0.35 field capacity) and two levels of inoculation with arbuscular mycorrhizal fungi (G. Clarum) (‒AMF, +AMF) were applied in a pot experiment and plants were cultivated for 6 weeks under growth chamber conditions. Plant dry matter production and protein content decreased in drought-stressed plants associated with significant reduction of net photosynthesis. In response to drought, soluble carbohydrates and proline were accumulated in the leaves up to 2 and 10 folds, respectively. Application of Si and AMF significantly increased shoot and root dry weight and relative water content not only in drought-stressed but also in well-watered plants. Both Si and AMF treatments significantly increased leaf photosynthesis rate, protein and relative water content and decreased leaf accumulation of osmolytes and malondialdehyde. Combined Si and AMF treatments exerted stronger effects on the improvement of physiological and biochemical parameters of drought-stressed plants than single applications, associated with significantly higher AMF root colonization of +Si treated plants. Our results suggest a beneficial effect of Si on yield, photosynthesis and water relation parameters as well as AMF responsiveness in strawberry plants.

134

Improved P acquisition in crops by inoculation with P-solubilising microorganisms - vision or reality? Guenter Neumann*1, Mira Kuhlmann2, Nicole Probst3, Mehdi Nkebiwe2 1 University of Hohenheim, Germany, 2University of Hohenheim, Institute of Crop Science (340h), Germany, [email protected], Germany

Mobilisation of sparingly-soluble phosphates via pH modifications of the growth medium and release of chelating metabolites are widespread mechanisms for P acquisition in many plant growth-promoting microorganisms (PGPMs) cultivated on artificial media and is promoted also as a measure to improve P acquisition in crops. However, within the BIOFECTOR-Project, more than ten experiments in eight countries with three different crops and nine P-solubilising microorganisms failed to show any crop benefit via acquisition of sparingly-soluble P sources. Therefore, this study aimed to characterise critical factors, determining microbial mobilisation of sparingly-soluble P sources in the rhizosphere. Maize was used as test crop with low adaptive potential for root-induced P-solubilisation. A calcareous Loess sub-soil, extremely low in available P (2-3 ppm) and organic matter (0.1%) but rich in acid-soluble Ca-P was supplied (1) with or (2) without rock phosphate fertilisation and (3) with soluble P as positive control. Plants inoculated with 6 different P-solubilising microorganisms (Pseudomonas sp. Poradix®, Pseudomonas sp., Paenibacillus mucilaginosus, 302 BIOFECTOR

Tuesday 23 June – Poster session Bacillus subtilis, Streptomyces spp., Penicillium sp.) were cultivated on this substrate, ensuring that plant P-acquisition in treatments (1 and (2) was only possible after microbial Ca-P solubilisation. However, all microbial inoculants failed to stimulate P-acquisition of the test plants and even exerted inhibitory effects on plant growth. Also simultaneous fertigation with a mixture of glucose and glycine had only marginal effects on crop performance, suggesting that limitation of root exudates as C and N source was not a critical factor. However, successful P acquisition and plant growth promotion was observed after diluting a similar growth substrate with 75 % (w/w) washed quartz sand, drastically lowering the pH buffering capacity and the CaCO3 concentration for unspecific binding of P-solubilising chelators. These findings suggest that the soil-buffering capacity may be a major constraint which limits the P-solubilising potential of many PGPMs.

135

Placement of Pseudomonas sp. PRORADIX around NH4+-based fertilizer depots in maize stimulates root exploitation of the fertilizer depot Peteh Mehdi Nkebiwe*1, Markus Weinmann2, Günter Neumann2, Torsten Müller3 1 University of Hohenheim, Plant Nutrition: Fertilisation and Soil Matter Dynamics (340i), Germany, 2University of Hohenheim, Plant Nutrition: Nutritional Crop Physiology (340 h), Germany, 3University of Hohenheim, Plant Nutrition: Fertilisation and Soil Matter Dynamics (340 i), Germany

Combining fertilizer placement and inoculation with plant growth-promoting rhizobacteria that stimulate root-growth and solubilize phosphates may be a strategy to improve root exploitation of fertilizer depots. Moreover, root colonization by such rhizobacteria may be enhanced in the depot zone with intense root development as a consequence of increased availability of root exudates induced by localized supply of root-attracting nutrients like ammonium. After confirming that the rhizobacteria strain Proradix®WP (Pseudomonas sp. DSMZ13134, Sourcon Padena, Germany) grows normally at high ammonium concentrations (50 mM) in the presence or absence of the nitrification inhibitor 3,4-Dimethylpyrazole phosphate, we investigated the effect of placing concentrated ammonium sulfate (64 mg N ml-1) with 3,4Dimethylpyrazole phosphate as a fertilizer depot in combination with PRORADIX as inoculant on growth-stimulation of maize roots (Zea mays L.), rhizosphere pH changes, root colonization by PRORADIX, nitrogen (N) and phosphorous (P) uptake in a rhizobox experiment on a calcareous Loess subsoil, pH 7.6 and in a field experiment on silty loam, pH 7.1. In the rhizobox experiment, increased root length densities around the NH 4+-depot were recorded 8 weeks after sowing, associated with intense root surface and rhizosphere acidification by 2 pH units and to higher N and P uptake per plant compared to the control variant with homogenous NO3 - fertilization. This may be attributed to improved spatial nutrient acquisition and to mobilization of acid-soluble calcium-phosphates, which are usually formed in neutral to alkaline carbonate-rich soils. Particularly in the depot zone, a high root colonization density of Pseudomonas was detected by culturing root extracts on a selective nutrient medium. In the field, root length density was doubled in the ammoniumdepot zone inoculated with PRORADIX at 11 weeks after sowing, as a first indication of improved root exploitation of the fertilizer depot, induced by simultaneous rhizobacteria inoculation under field conditions. 303 BIOFECTOR

Tuesday 23 June – Poster session 136

No matter whether dead or alive? An investigation of bio-effector application in tomato and maize experiments under phosphorus limited soil conditions Dinah Reinhardt*1, Marie Spohn2, Sven Marhan1, Namis Eltlbany 3, Kornelia Smalla3, Ellen Kandeler1 1 University of Hohenheim/Institute of Soil Science and Land Evaluation, Germany, 2University of Bayreuth/Department of Soil Ecology, Germany, 3Julius Kühn-Institute/Institute for Epidemiology and Pathogen Diagnostics, Germany

Bio-effectors are viable organisms and active natural compounds that are able to promote plant growth and health. This ability is enabled, for instance, by mobilizing sparingly soluble mineral nutrients like phosphorus. Particularly plant growth promoting rhizobacteria (PGPR), such as strains from genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate mobilizers but the functional mechanisms are still poorly understood. In the frame of the BioFector project we focus on the effects of phosphorus solubilizing rhizobacteria. We hypothesize: (I) Plant growth promoting effects of PGPR are based on an increasing phosphatase activity (especially acid phosphatase) and here we assume a higher activity in rhizosphere than in bulk soil; (II) PGPR colonization will be displayed in phosphatase activity hotspots. Two rhizobox experiments (tomato and maize) under phosphorus limited soil conditions were performed using Pseudomonas jessenii as the bioeffector. Experiments comprised five control treatments, including inoculation with native soil microorganisms and dead Pseudomonas jessenii cells, respectively. During plant cultivation a soil in situ zymography for determination of spatial and temporal phosphatase activity was conducted. Results showed a significant plant growth promoting effect, but contrary to our expectation, in treatments with living as well as dead Pseudomonas jessenii cells. In both treatments we observed yields that nearly corresponds to the optimally phosphorus fertilized plants; 79% in plant height and 83% in biomass, for example. First evaluation of zymography indicates a higher acid phosphatase activity in rhizosphere than in the bulk soil across all treatments. A particularly high activity was revealed in maize, especially on root tip surface. Additional analyses will identify whether the improved plant growth is based on an increasing nutrient availability and uptake. In addition, we will analyse the cell components of lysed P. jessenii to identify potential plant growth promoting compounds.

137

Max-Res TGA and FTIR evaluation of plant growth promoting rhizobacteria David Nelson, Eugene Carmichael, Graham McCollum, JR Rao, Gary Lyons, Shekhar Sharma* Agri-Food Biosciences Institute, United Kingdom

Analysis of bacterial biomass provides valuable information on the cell contents and structural differences in cell wall polysaccharides. The objective is to develop rapid methods for evaluating plant growth promoting rhizobacteria (PGPR) by Fourier Transform Infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) maximum resolution (Max-Res) protocol. Bacterial strains (BM- Bacillus mucilaginosus, FBZ42- B. amyloliquefaciens A, RLH – B. subtilis S, 4.2 – Burkhoweria sp, H2.6/RC2.5 – Rahnella aquatalis) were inoculated in potato dextrose 304 BIOFECTOR

Tuesday 23 June – Poster session broth for 4 days and incubated on a rotary shaker at 160 rpm for 48 h at 25oC. After removing the supernatants by centrifugation at 6000g for 10 min, the bacterial pellets were washed three times with distilled water and lyophilized. FTIR spectra of the samples were obtained using a diamond ATR. TGA was carried out at a heating rate of 20oC/min in nitrogen. TGA heating rates for maximum resolution (Max Res) analysis were programmed at 20oC / min when no weight loss was detected and a heating rate of 5oC / min on detection of weight loss. The six isolates can be distinguished from the differences in infrared functional groups and TGA composition data of the isolates. Max-Res analysis of the isolates showed compositional differences between the six isolates as indicated by differences in the weight loss steps.

138

Development of Quality Assurance protocols for biostimulant products Shekhar Sharma*, Eugene Carmichael, Eugene Conlon, Trevor Martin, Chris Selby Agri-Food Biosciences Institute, United Kingdom

Plant biostimulants are usually formulated using a range of components including macroalgae, animal or plant proteins, amino acids, minerals and plants extracts containing a wide range of trace compounds. The objective is to develop quality assurance (QA) protocols for evaluating physico-chemical characteristics of biostimulants. Six formulations (Rygex, Algavyt, Ryzoset, Manek, Ecoryg and Algavyt Zn/Mn – Agriges products) containing algal/plant extracts, humic and amino acids, lipids and inorganic components were assessed for particle size distribution, zeta potential and auxin activity. The particle size range of biostimulant products can be reduced by employing a wet mill and micro-fluidiser. The particle sizes of Rygex, Algavyt, Manek and Ecoryg were less than Rizoset and Algavyt Zn/Mn. Among the six products, Algavyt and Manek showed the highest zeta potential (ZP) values. The formulations were assessed for auxin activity using a mung bean rooting bioassay (cytokinin bioassay result not presented). Measurements of total bean roots per cutting and root length demonstrated the presence of significant auxin activities in some formulations (e.g. Rygex and Ecoryg). The gross differences in the particle size, ZP and composition of the products could be used for developing formulations to improve field performance and stability during transportation and storage.

139

Field performance of barley treated with two biostimulant products Trevor Martin, Fiona Clarke, Chris Selby, Eugene Conlon, JR Rao, Shekhar Sharma* Agri-Food Biosciences Institute, United Kingdom

Two types of arable fields were selected on the basis of their nutrient status: (a) standard input field – regularly applied with inorganic fertiliser to maintain the soil nutrient status for good plant growth and (b) low input field - not treated with fertiliser during the past ten years, with the exception of nutrient input grazing animal waste. The objective is to evaluate 305 BIOFECTOR

Tuesday 23 June – Poster session if certain biological extracts can influence the yield of spring barley in either a standard input soil or a soil of low nutritional value. Two formulations of biostimulants supplied by Bioatlantis, Ireland (A039F249 and D002G138) were assessed for biological activity and evaluated as foliar treatments. Spring barley was sown at both sites (standard nutrient input soil and low input soil) in randomised blocks. Treatment applications at three rates were applied during the growth of the crop. Mung bean bioassays was carried out to monitor growth hormone activities of the products to ascertain their biostimulant potential. Yields and 1000 grain weights were assessed at harvest. The bioassay of the products using mung bean root extension data showed that two products (A039F249 and D002G138) are biologically active. The application of D002G138 increased the number of mung bean roots, while both products at concentrations 1 and 5 gml-1 initiated greater root length. Field trials with spring barley at the two concentrations showed differing levels of chlorophyll between sites but no significant differences in either the yield of grain or the 1000 grain weight.

140

Response of cold and heat stressed Arabidopsis and wheat plants on the expression of salicylic acid and jasmonic acid Colin Fleming, Thomas Fleming, Colin McRoberts, Stewart Floyd, Shekhar Sharma* Agri-Food Biosciences Institute, United Kingdom

The impact of cold and heat stresses on the productivity of arable crops is expected to be a major factor worldwide in reducing yields by 10 to 30% . This study was aimed at developing tools to monitor key biotic and abiotic stress related plant hormones such as salicylic acid and jasmonic acid in wheat and Arabidopsis plants. The objective is to monitor salicylic acid (SA) and jasmonic acid (JA) expression in Arabidopsis and wheat plants in response to cold and heat stress. Heat and cold stresses (4oC and 38oC for 18 hrs) were applied to plants in growth cabinets. SA and JA levels were measured directly using Q-TOF-LC/MS. Total mRNA was extracted and cDNA produced before RT-PCR, targeting the PR1 SA pathway gene and MYC2 JA pathway gene. The conclusions are (a) heat and cold stress significantly affected the concentration of SA and JA in wheat and Arabidopsis plants (b) heat and cold stress changed expression levels of wheat genes in the SA and JA pathways and (c) these models can be used to monitor the effects of stress modifying materials on monocotyledons and dicotyledons.

306 BIOFECTOR

Tuesday 23 June – Poster session 141

Evaluation of anion exchange membrane for monitoring of phosphate solubilisation in soil types Chris Selby, Eugene Carmichael, Eugene Conlon, Shekhar Sharma*, JR Rao Agri-Food Biosciences Institute, United Kingdom

It is relatively easy to identify soil bacteria capable of solubilising phosphorous by plating them on microbiological media containing an insoluble phosphate (P) and observing rings of media clearing around active colonies. The objective is to estimate phosphorous solubilisation in soils using an anion exchange membrane (AEM) protocol. A method was evaluated to confirm if these organisms including AFBI P solubilisers and Porodix are equally active in agricultural soils. The method involved using sections of AEM (1.5 x 4 cm) to act as “artificial roots” by placing them in soils for a given period then recovering them to assay (molybdate reactive P) captured PO4 3- ions. The conclusions are (a) AEMs can be used to assess the availability of P to plants in agricultural soils and give a good correlation with estimates found using conventional chemical analyses of soils; (b) AEMs can be used to distinguish P availabilities between soils and effects of supplementation of soils with various P salts; (c) No significant increased P solubilization occurred with either the AFBI P solubilisers or Porodix regardless of P supplementation or soil type and (d) Future work will focus on why the bacterial inoculae do not appear to be solubilising P in soils. In the first instance this will involve providing additional supplements such as increased organic matter or organic compounds common in root exudates (eg glutamic and aspartic acids and their amines) to provide the bacteria with a more readily available energy source.

142

Evaluation of ryegrass extracts for plant bio-stimulant activities Chris Selby, Eugene Carmichael, Eugene Conlon, Shekhar Sharma* Agri-Food Biosciences Institute, United Kingdom

Plant fibres are generally regarded as being the major product of the bio-refining of grasses. However, this process also produces large volumes of aqueous extract that is potentially useful to the agricultural and horticultural industries. Here we compare the plant defence elicitor activity (French bean cell suspension cultures) and auxin-like activity (mung bean hypocotyl rooting test) of extracts produced from high pressure screw-pressing with those prepared by homogenising foliage in water for. The objective is to evaluate if ryegrass extracts have the potential to act as crop bio-stimulants by assessing their biological activity in a series of bioassays. Two growth hormone (auxin and cytokinin) bioassays were used to compare screw-pressed juice with extracts prepared by homogenising foliage with water. In both assays, all six extracts were equalised to the lowest sugar content sample, as assayed using the anthrone reagent, by dilution with water. Extracts of perennial ryegrass contained potent elicitors of plant defence that caused rapid and intense browning of cultured French bean cell suspensions. This is indicative of the 307 BIOFECTOR

Tuesday 23 June – Poster session synthesis of phenolic compounds, many of which have phytoalexin activity. Extracts produced by screw-pressing were marginally but significantly more active than those produced by homogenisation of tissues in water. This was illustrated by the significant interaction between extraction method and treatment concentration. Differences between ryegrass varieties were small and insignificant, particularly if attempts were made to equalise sample strengths using a measure such as sugar concentration. Root induction (a) and growth (b) in hypocotyl cuttings of mung bean indicate that the ryegrass extracts are rich in “auxin-like” or auxin cofactor activity. Studies are in progress to characterise the extract components responsible for the biological activities reported here.

143

Quantitative tracing of two Pseudomonas strains in the roots and rhizoplane of maize, as related to their plant growth-promoting effect in contrasting soils Carla Mosimann1, Thomas Oberhänsli1, Dominik Ziegler2, Dinah Reinhardt3, Ellen Kandeler3, Thomas Boller4, Paul Mäder1, Cécile Thonar*1 1 FiBL (Research Institute of Organic Agriculture), Switzerland, 2Mabritec AG, Switzerland, 3University of Hohenheim, Institute of Soil Science and Land Evaluation, Germany, 4Zürich-Basel Plant Science Center, University of Basel, Switzerland

Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition and can act as biocontrol agents by suppressing soil-borne diseases. Efficient strains can be formulated as microbial inoculants and their successful use for field application often requires a certain ability of the strains to survive in the soil where they are inoculated. In this respect, there is a need to create tools enabling the tracing of inoculated PGPR which can also serve to monitor their spread in space and time. In this poster we describe the development and application of a molecular method allowing the quantitative detection of two Pseudomonas strains (Pseudomonas fluorescens Pf153 and Pseudomonas sp. DSMZ 13134) contained in commercial formulations. The method is based on a Taqman qPCR assay targeting two polymorphic regions of the bacterial genome in order to ensure the specificity of the detection. Inoculation experiments with maize were conducted in three contrasted soils. Eight weeks after planting and inoculation, Pf153 could still be detected and its persistence in root and rhizoplane was shown to be the best in the organic soil (versus the two other conventional soils). On the other side, DSMZ 13134 could not be detected anymore after 8 weeks but its persistence after 4 weeks in root and rhizoplane samples was equally good in the three tested soils. Combined with the plant responses, our data indicate that the persistence of the two strains cannot clearly explain the measured plant biomass and nutrient acquisition (nitrogen and phosphorus) due to inoculation of the two strains. The discrepancy observed between the level of colonization by the strains and their effects on plant indicate that more research is needed to elucidate the mechanisms and conditions leading to successful application of PGPR. The developed tools will definitively contribute to this objective.

308 BIOFECTOR

Tuesday 23 June – Poster session 144

Metabolites Profiling of Zea mays inoculated with the Bacillus amyloliquefaciens as Bioeffector Vincenza Cozzolino, Giovanni Vinci*, Hiarhi Monda, Daniele Todisco, Alessandro Piccolo University of Naples Federico II, Italy

Bacillus amyloliquefaciens represents the largely studied plant growth-promoting rhizobacteria (PGPR) that competitively colonize plant roots and can act as either biofertilizers or antagonist biopesticides or both. PGPR enhance growth and yield of cereals through mechanisms that include production of growth stimulating phytohormones, solubilization and mobilization of phosphate and induction of plant systemic resistance to pathogens. For this reasons, PGPR represent biofertilizers well recognized as efficient tool for sustainable and safe agriculture. In this study, we investigated the effects of Bacillus amyiloliquefaciens when inoculated on plants of Zea mays under different P amendments. In order to improve our understanding we determined the metabolic profiling followed by multivariate analysis. Gas chromatography– mass spectrometry (GC-MS) was employed to screen potential differences among metabolic leaf extracts. A number of 56 primary polar metabolites, comprising amino acids, organic acids, sugars, phenolic acids, amino sugars, sugar alcohols, and 7 unknown compounds, were identified. The PCA showed a robust reproducibility among five replicates, revealed a significant separation between control and treatments with Bacillus a. Moreover, the different P amendments (no P addition, triple super phosphate, rock phosphate, composted cow manure, composted horse manure) showed a substantial difference in plants metabolic profiles. A significant variance from control was found for plants undergone to both Bacillus a. inoculation and compost treatment, whereby the difference in metabolic profile was accounted by a large abundance of sugars and organic acids metabolites. These results indicated that the different P amendments and the presence of Bacillus amiloliqefaciens strongly influenced the presence and distribution of primary metabolites. However, more detailed analyses of the metabolome (secondary metabolites) will better elucidate how Bacillus a. affect the metabolic pathways and consequently the performance of maize plant.

145

Bioeffectors promoting Bioeffectors - Seaweed extracts as prebiotics for plant growth-promoting rhizobacteria? Nino Weber*1, Markus Weinmann1, Peteh Mehdi Nkebiwe2, Kristin Dietel3, Sarah Symanczik4, Günter Neumann1, Uwe Ludewig1 1 University Hohenheim, Institute of Crop Science (340h), Germany, 2University Hohenheim, Institute of Crop Science (340i), Germany, 3ABiTEP GmbH, Germany, 4FiBL, Switzerland

A major constraint for a successful practical application of plant growth-promoting microorganisms is the limited reproducibility of the desired effects, depending on often unknown external factors. One important determinant for effective application of microbial bioeffectors is their rhizosphere competence and effective root colonization of the host plant, 309 BIOFECTOR

Tuesday 23 June – Poster session frequently promoted by repeated inoculations with high inoculum densities. However, under practical conditions, this approach is not always economic. Using the prebiotic potential of various natural compounds such as seaweed extracts to stimulate microbial growth may be an alternative strategy. Our in vitro studies with bacterial suspensions of low CFU showed highly prebiotic potential of selected seaweed extracts on strains of Bacillus amyloliquefaciens and Pseudomonas sp. “Proradix” which could not be attributed to carbon source effects as demonstrated by control treatments with glucose. In a pot experiment with maize, combinations of Proradix® and seaweed extracts led to significantly higher plant biomass compared to the untreated control, even on a well fertilized field soil. Based on these results, promising product combinations were tested in a field experiment with maize on a silty loam (pH 6.8) and fertilization according to farmers practice. For tracing of the bacterial strains in the field selective plating assays and RT-qPCR with specific primers were employed. Plant sampling at four weeks after sowing revealed no effect of the selected seaweed extracts (Superfifty, Algafect) either on root colonization of Proradix or on the total cell number of B. amyloliquefaciens. However, the proportion of Bacillus spores was increased by Algafect. No significant effects on biomass production and final grain yield of maize were observed. The results suggest that seaweed extracts may exert prebiotic effects on plant growth-promoting bacteria but the characterization of conditions determining positive interactions with the host plant still needs further investigation.

146

Strategies for bio-effector application to improve the growth and mineral nutrition of maize under field conditions Markus Weinmann*1, Mehdi Peteh Nkebiwe2, Nino Weber1, Klara Bradacova1, George Fora3, Torsten Müller2, Günter Neumann1 1 University Hohenheim, Institute of Crop Science (340h), Germany, 2University Hohenheim, Institute of Crop Science (340i), Germany, 3Banat’s University of Agricultural Sciences and Veterinary Medicine, Romania

For many bio-effectors, such as plant growth-promoting microorganisms, the ability to support the mineral nutrition of plants has been proven under controlled conditions. Mobilization of mineral nutrients, stimulation of root growth and activity, and beneficial interactions with other microorganisms are among the most noticeable modes of action. These properties could be effectively utilized for the development of more environmentally friendly and sustainable plant nutrition strategies that are less dependent on mineral fertilizers. A critical task is to meet the demand of high yielding crops for phosphate and other essential minerals that are sparingly supplied by the soil or from alternative fertilizers. However, the agricultural implementation of such approaches is still limited and this is likely due to a lack of appropriate application techniques ensuring the functioning of bio-effectors under adverse environmental conditions. In the present work, commercial preparations of bacterial (Bacillus strains FZB42 and R41, ABiTEP GmbH, Berlin, Germany; Pseudomonas sp. DSMZ 13134, Sourcon Padena GmbH & Co. KG, Tübingen, Germany) and fungal (Penicillium sp. PK 112, Bayer CropScience Biologics 310 BIOFECTOR

Tuesday 23 June – Poster session GmbH, Malchow/Poel, Germany) bio-effectors were tested with different application methods to improve the phosphorus nutrition of field grown maize. No significant improvements in growth or mineral nutritional status of the plants were found when the bio-effectors were applied by seed incrustation, broadcast distribution with soil incorporation, or spray application on top of the plants. These findings suggest that the application of these microorganisms as discrete measures is not decisive for the productivity of maize on fertile soils. Advanced application strategies, such as the combination of bioeffectors with fertilizer placement and/or the use of recycling fertilizers are proposed to provide viable alternatives to the current practice of mineral fertilization.

147

Microbial bio-effectors combined with manure proliferate plant growth of tomato: investigations on the modes of action Zhifang Li1, Justus Riemann1, Angelika Lüthi1, Nino Weber1, Markus Weinmann*1, Gheorghe Posta2, Günter Neumann1 1 University Hohenheim, Institute of Crop Science (340h), Germany, 2Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Horticulture and Forestry, Romania

When tested under practice conditions, commercial bio-effector preparations induced strong growth improvements (80 % increase in plant height) during the early development of tomato seedlings grown on a substrate prepared with composted cow manure (45 %), garden soil (30 %), peat (15 %), and sand (10 %), but without mineral fertilizers. To study the modes of action how these products based on bacterial (Bacillus strains FZB42 and R41, Pseudomonas sp. DSMZ 13134) and fungal (Penicillium sp. PK 112) isolates could improve plant growth, pot experiments under controlled conditions were conducted. The question whether the plant growth-promoting effect was due to mobilization of sparingly available mineral nutrients from the humified manure was studied in comparative tests where the portion of manure was replaced by an increased share of peat in the substrate while adding mineral nutrients in easily available forms or not. The role of manure as a carbon source supporting the establishment and activity of the introduced microbial strains was tested through substitution by alternative carbon sources. Root morphological characteritics and colonization by arbuscular mycorrhizal (AM) fungi were assessed to detect if an improved spatial acquisition of mineral nutrients could explain the beneficial effects of the bio-effectors. Contrary to the strong effects observed under practice conditions, weak or no growth responses to the bio-effector treatments were found in plants under controlled conditions. However, the Pseudomonas treatment induced a significant increase in root hair length. The biomass production of tomato plants grown on the substrate with manure raised by 9 % in response to the combined application of the Penicillium preparation with an AM inoculum (Glomus intraradices). In plant growth-promotion by microbial bio-effectors, therefore, the combination of diverse modes of action seems to be involved, whose relative importance may vary depending on the environmental conditions.

311 BIOFECTOR

Tuesday 23 June – Poster session Food-web Interactions

148

Carbon flow and enzyme activities during protozoan grazing in rhizosphere and detritusphere Sebastian Löppmann*1, Fionn Clissmann2, Anna Gunina1, Johanna Pausch1, Robert Koller3, Michael Bonkowski2, Yakov Kuzyakov1 1 University of Göttingen, Büsgen-Institute, Germany, 2University of Cologne, Institute of Zoology, Germany, 3Forschungszentrum Jülich, Institute of Bio- and Geosciences, Germany

The differences in complexity and accessibility of plant provided carbon (C) sources for the soil food web result in two major decomposition pathways based on 1) root and shoot litter, and 2) rhizodeposits (especially exudates). The amount and quality of substrates entering the soil mainly controls microbial processes in the rhizosphere and detritusphere. Furthermore, soil fauna has important functions in regulating microbial activity and enzymatic substrate utilization. A triple-labeling (13C, 14C and 15N) experiment was conducted focusing on the identification of C resources (rhizodeposited C by 14C and root litter by 13C) that fuel microbial-protozoan interactions in two soil hotspots: rhizosphere and detritusphere. Soil was taken from an arable field planted with maize, autoclaved and re-inoculated with a microbial community extracted from the same soil. The following treatments were established: 1) no addition of plant C, 2) addition of sterilized 13C /15N labeled maize root litter, representing detritusphere 3) 14CO2 pulse labeling of growing maize plants, representing rhizosphere. To determine the effects of faunal grazing on nutrients (by 15N) release each treatment was setup with and without amoeba. Enzyme kinetics was implemented as indicator for microbial activity and 13C flux to the microbial pool was determined by 13C-PLFA analysis. We analyzed14C, 13C and 15N in the microbial biomass, soil, plant, root and CO2. Additionally, microbial biomass was assigned by dsDNA extraction. Between 65-89% of soil released 14CO2 was respired by roots and microorganisms in the first 3 h after the 14CO2 pulse. First results showed higher 14C activity in the microbial biomass in presence of protozoa. The dsDNA-extracted microbial biomass was significantly higher in litter amended and planted soil compared to the unplanted control. Furthermore, the DNA contents were increased in the presence of protozoa. Clear effects of enzymatic affinity to the specific substrate were elucidated during protozoan grazing.

312 Food-web Interactions

Tuesday 23 June – Poster session 149

Litter decomposition and home-field advantage of range-expanding plant species Marta Manrubia Freixa*, Ciska Veen, Wim van der Putten NIOO-KNAW, Netherlands

The current climate warming enables many native plants to expand ranges to higher altitudes and latitudes. Plants develop in close interaction with soil organisms in a direct (e.g. via pathogenesis) and indirect way (e.g. via the detritus food web). During range shifts, these specific plant-soil interactions might become temporally disrupted since soil organisms have limited dispersal capacity. Consequently, range-expanding plants are expected to benefit from the release of soil-borne pathogens and suffer from losing positive interactions with specialized decomposer organisms. The present research aims to study local specialization by decomposers of range-shifting plant species along latitudinal gradients and determine whether specialization results in ‘home field advantage’. We tested the hypothesis that soil microbial communities from the native range have higher affinity with litter of range-expanding plants than communities in the new range. We collected senesced leaves and soil of two range-expanding plants (Centaurea stoebe and Lactuca serriola) and a native congener (Centaurea jacea) in the native and expansion range. We set up a 97-day microcosm incubation experiment for each plant species. Litter was reciprocally transplanted to all soils within and between ranges and we measured CO2 fluxes over the incubation period. Soil heterotrophic respiration response to litter addition was used as a proxy for decomposition activity. We found that soil and litter origin had the strongest effect on soil heterotrophic respiration for all 3 plant species, whereas no local specialization effect was observed in litter transplants within or between ranges. High within-range variability suggested that litter decomposition controls operate at a highly local scale.

150

A root-feeding insect in the shallow soil zone significantly alters yield, mortality, and size structure of Lolium perenne populations Tomonori Tsunoda*1, Naoki Kachi2, Jun-Ichirou Suzuki2 1 Tokyo Metropolitan University / Yokohama National University, Japan, 2Tokyo Metropolitan University, Japan

We evaluated the effects of vertical distributions of a larva of a root-feeding insect, Anomala cuprea, on the yield, mortality, and size structure of Lolium perenne populations. We conducted a growth experiment with a two-way factorial randomized block design with nine replications. Factors included plant density (36 plants per pot, and six plants per pot) and vertical distribution of A. cuprea. Pot soil was divided into three distinct vertical zones (top, middle and bottom) with a wire gauze, and each pot received a larval insect into one zone. There were two additional treatments, one with a larva but without a partition and one without an insect. Yield of L. perenne significantly decreased in the treatments with herbivory, with the largest decrease in the top feeding zone, i.e. the shallowest soil. Yield was also significantly reduced at low plant density, but was more affected by herbivory. Plant mortality occurred only when the herbivore was in the top feeding zone. Plant density did not significantly alter the number of dead plants. At low plant density, the shoot biomass of the 313 Food-web Interactions

Tuesday 23 June – Poster session three largest plants significantly decreased with a larval herbivore, but the three smallest plants not. Thus, the standard deviation in shoot biomass significantly decreased under herbivory. At high plant density, shoot biomass was not significantly affected by herbivory, irrespective of plant ranks. The standard deviation in the shoots was smaller at high density than at low density. In contrast with aboveground herbivory, which is known to increase size variance in plant populations, the root-feeding of an insect in shallow soil decreased the size variance in a sparse plant population.

151

Top-down and bottom-up control of generalist root-feeding nematodes in the rhizospheres of range-expanding plant species and native congeners Rutger Wilschut*, Stefan Geisen, Wim van der Putten NIOO-KNAW, Netherlands

As a consequence of climate warming many plant species are expanding their range to higher latitudes and altitudes, thereby leaving behind their rhizosphere soil organisms. It is unknown how new rhizosphere communities become assembled in the new range. Generally, rangeexpanding plant species are less negatively affected by soil communities in the new range than congeneric native plant species. This may be due to a lower amount of herbivores and pathogens that are able to attack the range expanding plants in the new range, either because of a lack of co-evolutionary history or by stronger direct or indirect defense mechanisms. We study how specific multitrophic interactions develop in the rhizospheres of rangeexpanding plant species. In a greenhouse experiment we exposed two range-expanding plant species and their congeneric natives to different generalist root-feeding nematodes and inoculated microbial communities that may contain antagonists of the nematodes. Subsequently, we examined if nematode population growth differed between rangeexpanding and native plant species and if top-down control of nematodes by the microbial communities was plant species-dependent. We tested the hypotheses that 1) if top-down control of root-feeding nematodes is plant-mediated, this will be stronger in native species than in range-expanders, as native plants have a shared evolutionary history with the local soil community and 2) range-expanding plants are better defended against generalist rootfeeding nematodes than congeneric natives, because of strong direct defense mechanisms. Our results suggest that these defense mechanisms are species-specific, rather than depending on range shift capacity.

314 Food-web Interactions

Tuesday 23 June – Poster session Engineering the Rhizosphere

152

Severe rhizosphere oxygenation optimising the carbon footprint of Paludiculture // (Sustainble biomass production on peat soils) Christian Fritz*1, Eric Visser2, Alfons Smolders3, Leon Lamers2, Florian Wichern1, Bikila Dullo4, Theo Elzenga5, Veronica Pancotto6, Ab Grootjans5 1 Rhine-Waal University of Applied Sciences, Germany, 2Radboud University, Netherlands, 3B-Ware Research Center, Netherlands, 4Addis Abeba University, Ethiopia, 5Rijksuniversiteit, Netherlands, 6CADIC, Argentina

Water-logging is a major threat to plant health and causes substantial methane emissions from crops and natural vegetation. Flood adapted plant species influence soil processes by releasing root exudates and degradable litter. This can increase methane production and nutrient availability. In contrast, little is known to what extent root derived oxygen caps methane and nutrient release. Our research tested which growth conditions are necessary to maintain oxygen release at sufficient rates to dominate rhizosphere processes. Root-methane-soil interactions were studied by comparing methane emissions, stock and oxygen availability in depth profiles below stands of cushion plants and paludicrops. We followed rhizosphere nutrient-availability along depth profiles in Ethopia, Argentina and the Netherlands. We investigated nutrient cycling by 15N field experiments and N:P ratios of plant organs. Cushion plants, Eriocaulon schimperi and Astelia pumila, formed extensive root systems up to 150 cm deep. Root biomass (3590 g.DW.m-²) dominated the belowground biomass of cushion plants but resulted in a higher nutrient demand. Roots of paludicrops, Typha spec. and Phragmites spec., were in general shallow (< 70 cm) and root proliferation was sensitive to vertical nutrient availability. In contrast, soils surrounding cushion plant roots were depleted in methane and plant-available nutrients. Main finding is that methane emissions were then cut to zero high root densities promoting soil oxygen (> 1.5 mg.l-1). High soil N:P ratios and low temperatures seemed major controls on root density and rhizosphere oxygenation. Increase in shallow root length was associated with higher 15N recycling Our study shows that roots of cushion plants and biomass crops can dominate biogeochemical processes at the ecosystem scale given certain growth conditions. Oxygen release from the dense root biomass was sufficient to cause a thorough soil oxidation. Soil nutrient ratios seem promising in reducing greenhouse gas emissions from water-logged fields by tuning the root system architecture.

315 Engineering the Rhizosphere

Tuesday 23 June – Poster session 153

Biocontrol practices alter rhizosphere community structure in pepper and suppress Phytophthora blight Lori Hoagland*, Dan Egel, Natasha Cerruti, Jyothi Thimapuram, Clayton Colling, Ketaki Bhide Purdue University, United States

Phytophthora blight has become one of the most serious threats to the vegetable industry in the Midwest U.S. Controlling Phytophthora capsici, the pathogen responsible for Phytophthora blight is difficult because it has a broad host range, spreads rapidly under ideal environmental conditions, and produces resilient spores that can survive indefinitely in soil. There is also insufficient resistance in crop varieties, and P. capsici is now resistant to many fungicides. Biocontrol practices that are thought to suppress soil-borne pathogens via changes in soil microbial communities could help control Phytophthora blight, though the mechanisms are not well understood limiting the effective deployment of these practices. In this study, we evaluated the potential for various cover crop species grown alone, or in combination with a biochar amendment made from hardwood forest species to suppress Phytophthora blight in two naturally infested field soils. Changes in the rhizosphere community structure of a subsequently planted pepper crop were quantified with semi- selective media and 16S sequencing performed by Illumina MiSeq and analyzed using QIMME. A wheat cover crop and biochar, alone and in combination, increased pepper root and shoot biomass, and increased the abundance of Pseudomonas fluorescens in the pepper rhizosphere in both soils. In the low organic matter soil, P. capsici abundance in the pepper rhizosphere was reduced most by treatments that received the wheat cover crop, whereas in the high organic matter soil P. capsici was reduced most by treatments that received biochar. These results indicate that including a wheat cover crop and applying biochar can improve pepper growth in soil infested with P. capsici, though the suppression mechanisms and most effective biocontrol strategy could vary given soil type. Results of on-going sequencing analyses will provide additional insights to the potential mechanisms regulating P. capsici suppression and enhanced pepper growth observed in this study.

154

Beneficial interaction between tomato plant and Flavobacterium strains isolated from tomato rhizosphere Seon-Woo Lee*1, Soo Yeon Choi1, Eun Joo Jung1, Ju Yeon Song2, Jihyun Kim2 1 Dong-A University, South Korea, 2Yonsei Unversity, South Korea

Plant and microbe interaction in rhizosphere may influence various plant functions such as plant growth, plant development and tolerance to stresses. Microbial community analysis of tomato rhizosphere from our study revealed that members of phylum Bacteroidetes were abundant in tomato rhizosphere compared to bulk soil. We hypothesized that those bacteria dominating tomato rhizosphere contribute to the plant function. A pair of specific primer targeting those bacteria in class Flavobacteria was designed and used to select bacterial isolates in a member of the class Flavobacteria by colony PCR of bacterial colonies derived from tomato plant rhizosphere. A number of bacteria were isolated and examined for phenotypes relevant to plant-beneficial interaction, such as biofilm formation, seedling growth promotion, and antimicrobial activities. Five strains were selected for further study because they showed plant-beneficial effects. One of the isolates, F. daejeonensis RCH33, 316 Engineering the Rhizosphere

Tuesday 23 June – Poster session showed the delayed occurrence of bacterial wilt in tomato plants, when RCH33 strain was treated in tomato rhizosphere prior to the bacterial wilt pathogen Ralstonia solanacearum inoculation by soil-soaking method. Genomes were analyzed for all five strains in Flavobacterium and revealed that they harbor genes for plant growth promotion, such as auxin biosynthesis and ethylene removal, and for secondary metabolites which may be involved in beneficial effect to plants. However, one of novel species of Flavobacterium TCH32 did not contain any known genes for beneficial interaction with plant, while plant growth promotion effect and the capability of biofilm formation were remarkable. We are under investigation of bacterial gene expression in planta for these Flavobacterium strains.

155

The right set of circumstances – Selecting microbial bio-effectors for applications in alternative fertilisation systems – the BIOFECTOR project Guenter Neumann* University of Hohenheim, Germany

Practical application of plant growth-promoting microorganisms (PGPMs) as so-called biofertilisers to improve growth and nutrient acquisition of crops is frequently biased by low reproducibility of the desired effects. Various environmental factors including biotic and abiotic stresses, competition with the indigenous microflora and genotypic differences in host compatibility may limit the rhizosphere competence, the survival and thus the efficiency of the microbial inoculants. BIOFECTOR is a collaborative project, located within the 7th EU Framework Programme with the aim to select suitable microbial partners for applications in various alternative fertilisation systems. It is expected that a strategic combination of microbial strains adapted to the culture conditions characteristic for the respective fertilisation systems will reduce the variability of host plant responses and thus the efficiency of the plant-microbial interaction. First promising results but also drawbacks and open questions are summarised with respect to PGPM use for improved nutrient acquisition from organic recycling fertilisers, in fertiliser placement strategies and mobilisation of sparingly soluble nutrients.

156

The engineered rhizosphere bacterium help plant sense a hazards chemical Choong-Min Ryu*, Hae-Ran Lee, Soohyun Lee KRIBB, South Korea

Synthetic biology is a new field of biological engineering that generates new biological modules and synthesizes novel pathways to reprogram organisms. It is valuable to the development of reporter plants to provide a rapid, low-cost, and in situ monitoring of environmental hazards and plant diseases. The reporter plants are also useful for the ecological risk assessment of industrial chemicals. We attempted to develop a reporter plant that senses hazardous aromatic compounds such as toluene. Bacterial two-component signal transduction system such as TodST of a rhizosphere bacteria Pseudomonas putida is useful to construct artificial genetic regulatory module for synthetic biology. For developing toluene sensing reporter plant, we exploited root-rhizosphere bacteria (rhizobacteria) interaction. First, we generated P. putida KT2440 to produce indole-3-acetic acid (IAA) depending on 317 Engineering the Rhizosphere

Tuesday 23 June – Poster session toluene concentrations through TodST system. Secondly, we manipulate Arabidopsis and tobacco plant to elicit RNAi of the magnesium chelatase (ChlH) gene induced by concentration dependent manner of IAA produced by P. putida under presence of toluene. Collectively, we provide new two-step chemical sensing system which turn over signals from chemical sensing rhizobacteria to plant indicator.

157

Plant-Sediment Microbial Fuel Cells: electron transfer mechanisms and their energy efficiencies David Strik*, Koen Wetser, Cees Buisman Wageningen University, Netherlands

Wetlands can be used to generated electricity by means of Plant-Sediment-Microbial Fuel Cells (PSMFC). In this since 2008 emerging technology, solar energy is converted into electricity. Basically the technology consists of: 1) an anode which is placed in the vicinity of plants roots to take-up electrons; 2) an electric circuit to harvest energy out of released electrons.; and 3) a cathode placed at an oxygen rich environment to deliver electrons for oxygen reduction. Experimental work supports that electrochemical active bacteria are responsible for catalysis of anodic oxidation and cathodic reduction reactions. The PSMFC provides a new solid electron acceptor and donor in the wetland. Plants and sediments both provide a wide pool of (potential) electron donors (e.g. exudates, lysates, sulfide) and electron acceptors (e.g. oxygen, metals, nitrate, sulfate, carbon dioxide). Electron donors can be converted and transferred via different direct and indirect mechanisms. E.g. micro-organisms can oxidise acetate and directly deliver electrons to the anode. Also, microbial or plant-based mediators can be used to transfer electrons from micro-organisms to the solid electrode. Based on thermodynamics and actual conditions, one can calculate the energetically most attractive route for electron transfer at different sites like root tips and root-turn over locations. Aim of this study was to determine (novel) theoretical pathways of electron transfer mechanisms in the Plant-Sediment Microbial Fuel Cells and reveal their energy efficiency. With this work we present the working principles of novel electron transfer pathways and provide in-sights on effective placement of anodes and cathodes. Past experimental results were linked to the outcome of the model. By further experimental elucidation of the proposed model, the actual processes and efficiencies in Plant-Sediment Microbial Fuel Cells can be quantified.

318 Engineering the Rhizosphere

Tuesday 23 June – Poster session 158

Rhizogenesis: Exploring the physical development of the emerging root:soil interface Jonathan Helliwell1, Craig Sturrock*1, Sacha Mooney 1, Anthony Millar2, Richard Whalley3 1 University of Nottingham, United Kingdom, 2John Innes Centre, United Kingdom, 3Rothamsted Research, United Kingdom

The rhizosphere is a distinct zone of soil directly influenced by a plant root, with all below ground resources passing through this dynamic zone prior to capture by plants. Therefore the physical nature of the interface between the rhizosphere and the bulk soil is crucial for plant development. It is well known that the soil microbial community play a significant role in the evolution of the rhizosphere and some studies have shown that it is structurally a different environment to the bulk soil. However how this evolution or genesis is influenced by the underlying soil physical properties and how this interacts with different plant species is less well understood. Examining the undisturbed rhizosphere has represented a major obstacle to research, due to its microscopic size and often fragile nature. Here we have employed X-ray Computed Tomography (CT) to successfully map the physical architecture of the developing rhizosphere in natural soils for the first time. We compared the temporal changes to the intact porous structure of the rhizosphere during the emergence of a developing root system, by assessing changes to the soil porous architecture across a range of soil textures and plant species. Our results indicate the physical zone of influence of a root at an early stage is more localised than previously thought (at the µm rather than mm scale). Soil porosity increases at the immediate root surface due to localised crack formation in both fine and coarse textured soils. As such the soil porous architecture at the root interface is enhanced and not compacted as previously considered. This 'rhizosphere structure' and associated dynamics have important consequences for several important root-soil processes including water uptake efficiency and gaseous exchange between individual aggregates and subsequently our efforts to model their behaviour.

159

Effects of crop rotation on pathogen-suppressive activity and shifts in antibiotic activity of soilborne actinomycete communities Patricia Vaz*1, Nora Altier2, Carlos Pérez3, Linda Kinkel4 1 INIA- Las Brujas, Uruguay, 2Sección Bioinsumos, INIA- Las Brujas, Uruguay, 3Facultad de Agronomía, Universidad de la República, Uruguay, 4Department of Plant Pathology, University of Minnesota, United States

Indigenous soil microbes have intrinsic potential to protect plants from pathogens, yet our understanding of the factors that determine the dynamics of antagonistic populations remains limited. Bacterial antibiotic production may change in the proximity of another organism, thus not only the ability of the bacterial community to produce antibiotics but also interactions with neighbors are likely to determine community antagonistic potential. We explored the relationships between crop rotation and edaphic characteristics with community antagonistic activity, and with induction of changes in inhibition among sympatric actinomycetes. Soil samples were taken from an experiment established 13 years earlier, with 10 treatments in a randomized block design (n = 3 blocks). Bacterial, actinomycete, and inhibitor densities were measured for all plots. Mean inhibition of actinomycetes against 319 Engineering the Rhizosphere

Tuesday 23 June – Poster session target organisms was also determined. Three plots were selected for isolation of a random collection of sympatric actinomycetes using a 1 cm diameter soil corer. Ten isolates from each community were tested for their abilities to induce changes in inhibition among all possible sympatric isolate pairs. Soils having different rotation histories had significantly different bacterial, actinomycete and inhibitor densities. Soil communities from longer rotations had significantly higher microbial densities than communities from shorter rotations. Moreover, communities with higher inhibitor densities had better inhibitors. Soil nitrate, organic matter and soluble potassium were significantly correlated with microbial community characteristics. Inhibition among sympatric isolates was both increased and decreased by the presence of a partner. However, no significant differences in the frequencies of such shifts were observed among communities. Our work suggests that agronomic practices that contribute to increasing total bacterial densities are likely to enhance the potential for microbial communities to suppress plant pathogens. In this study, longer rotations, with higher plant diversities over time, were most effective in achieving high bacterial densities and strong inhibitory populations in soil.

160

Managing replant disease of apple and sweet cherry with compost Tristan Watson*1, Louise Nelson1, Tom Forge2 1 The University of British Columbia - Okanagan Campus, Canada, 2Agriculture and Agri-Food Canada, Canada

Replant disease refers to the poor growth of fruit trees planted into soil previously used for tree-fruit production. The plant-parasitic nematode, Pratylenchus penetrans, and a diverse array of root-associated fungi have been implicated in replant disease worldwide. Recent restrictions on soil fumigants have generated interest in alternative management strategies, particularly those associated with promotion of beneficial microorganisms in the rhizosphere. This study aimed to investigate: (1) the impacts of amending soil with biocontrol agents and composts on plant growth and recovery of root pathogens, and (2) differences in N mineralization between two different composts. A greenhouse pot experiment was performed, consisting of apple and sweet cherry seedlings grown in old apple orchard soil amended with either a biocontrol agent, an agricultural or municipal waste compost, granular chemical fumigant (Basamid®), or nothing (untreated control). After 19 weeks growth, the abundance of P. penetrans was determined for each pot, and colonization by root-associated fungi assessed through isolation and molecular identification (ITS region). Differences in soil microbial communities were determined using Biolog EcoPlatesTM combined with real-time PCR analyses of total bacteria. Differential nutrient mineralization was assessed using freeliving nematode community indices of nutrient enrichment as well as analysis of NO3-N content. Fumigation and compost amendments decreased the abundance of P. penetrans and colonization by root-associated fungi, however only fumigation and agricultural waste compost improved root volume. Fumigation and compost amendments selected for distinct microbial communities, with agricultural waste compost supporting a greater abundance of total bacteria relative to non-compost amended treatments. Similarly, agricultural waste compost also supported a greater nematode enrichment index, lower channel index, and higher soil NO3-N content relative to municipal waste compost, suggesting enhanced Nmineralization through the bacterial decomposition pathway. Overall, agricultural waste 320 Engineering the Rhizosphere

Tuesday 23 June – Poster session compost shows potential as a non-fumigant option for control of replant disease impacting apple and sweet cherry.

321 Engineering the Rhizosphere

Wednesday 24 June – Poster session Symbiosis

1

Effect of Sinorhizobium sp. and phosphorus on growth, nodulation, and nitrogen fixation of fenugreek (Trigonella foenum-graecum L.) Abdolreza Akhgar*, Sara Larki Vali-e-Asr University of Rafsanjan, Iran

The Legume-rhizobia symbiosis is the basis of biological nitrogen fixation and improving soil fertility. Inoculation of legumes with rhizobia is an important practice to maximize biological N2 fixation capacity in legume crops. Phosphorus is also an essential element for Rhizobia to convert atmospheric nitrogen into an ammonium form usable by plants. In this study, a greenhouse experiment was carried out to investigate effects of simultaneous application of Sinorhizobium sp. isolates and phosphorus levels on growth, nodulation, and nitrogen fixation of fenugreek. The experiment studied four Sinorhizobium isolates combined with four phosphorus levels (0, 20, 40 and 60 mg kg-1soil). The Sinorhizobium isolates were isolated from root nodules of fenugreek grown in farms located in Khuzestan and Kerman Province. The greenhouse experiment showed that simultaneous application of Sinorhizobium and phosphorus significantly increased shoot dry weight and nodule number and significantly enhanced N, P, Ca, Mg, Fe and Mn uptake.

2

Plant Growth Promoting Rhizobacteria (PGPR) to benefit root architecture and nutrition of nursery crops Shelby Berg*, Stephen Mudge, Susanne Schmidt, Yun Kit Yeoh The University of Queensland, Australia

A healthy root system is fundamental for plant growth and development. Plant growth promoting rhizobacteria (PGPR) form complex symbiotic relationships with plants and benefit plant growth in the rhizosphere, on the root surface, within intercellular spaces and root cells. Here we investigated microbes for their ability to act as PGPR and colonise root systems of young maize and tomato plants in the presence of native soil biota. A number of putative PGPR isolates with strong phytate degrading and phosphorus solubilising activities were identified of which two isolates (an Enterobacter sp. and Burkholderia sp.) were selected for further testing. Tomato and maize seedlings inoculated with only Burkholderia or both Enterobacter and Burkholderia, cultivated in low P soil did not show improved plant growth. However Enterobacter inoculation led to increased shoot and root biomass, root surface area, branching and lateral root growth of both maize and tomato seedlings. Such changes in root architecture are desirable for nursery container production whereby roots inoculated with PGPR remain confined within the pot and do not proliferate from the base. These results also suggest that the Enterobacter strain, although originating from the sugarcane rhizosphere, has a wider host range. We are currently using genetically-engineered Enterobacter expressing green fluorescent protein (GFP) to study microbial colonisation of root systems and survival post-inoculation. The effects of Enterobacter inoculation on pre-existing rhizosphere microbial populations is also being analysed using bacterial community profiling technologies. We discuss the findings in context of formulating efficient PGPR inoculums to specifically target crops of interest for growth promotion. 322 Symbiosis

Wednesday 24 June – Poster session 3

Nutritional value of tomato and strawberry fruits is affected by plant inoculation with soil bacteria Elisa Bona*1, Elisa Gamalero2, Guido Lingua2, Simone Cantamessa2, Nadia Massa2, Valeria Todeschini2, Paola Manassero2, Giorgia Novello2, Andrea Copetta2, Giovanni D'Agostino3, Graziella Berta2 1 Università del Piemonte Orientale, Italy, 2Università del Piemonte Orientale, DiSIT, Italy, 3Mybasol, srl, Italy

Soil bacteria can stimulate plant growth; however, little information is available about the impact of these bacteria on the nutritional value of fruits. Therefore, we considered two economically relevant crops: tomato for the evidence of reduced risk of cancer and cardiovascular diseases and strawberry because they are an important source of antioxidants and anti-inflammatory phytonutrients. Tomato plants were inoculated with Pseudomonas sp. 19Fv1T, or Pseudomonas fluorescens C7 and grown in open field condition. Strawberry plants were inoculated with P. fluorescens Pf4 or Pseudomonas spp. 5Vm1K and grown in pot under glasshouse condition. Both plant species were subjected to reduced fertilization. The impact of the bacterial strains on the fruit nutritional value was assessed by measuring the concentration of soluble sugars, organic acids, vitamins, anthocyanin, lycopene and b-carotene together with nitrate and nitrite amount in fruits. The size and biomass of tomato fruits were unaffected by the bacterial strains. However, the sugar content was modulated by the microorganisms (i.e. fructose increase and glucose reduction). 19Fv1T induced an enhancement of malic, tartaric, ascorbic and glutamic acids, beta carotene and a reduction of oxalic acid in the fruits compared to controls. The strain C7 induced reduction of the glutamic, oxalic and ascorbic acid, and decrease of nitrite level in fruits compared to those produced by uninoculated plants. Both strawberry fruit size and yield were increased by the bacterial. The concentrations of sugars were differently affected according to the microorganism. Both bacteria enhanced the amount of ascorbic acid in fruits, but only Pf4 increased folic acid concentration. Fruit yield and quality can be improved by plant inoculation with soil bacteria; this can have a real world application, leading to economical, ecological and human health benefits in relation to the reduced chemical input and to the increased quality of the fruits.

4

Roots and nodules bacterial diversity of Scorpiurus muricatus in western Algeria Zoulikha Bouchiba*1, Zineb faiza Boukhatem1, Zohra Ighilhariz1, Mustapha MIsbah el Idrissi2, Abdelkader Bekki1 1 University of Oran 1 Ahmed Benbella, Algeria, Algeria, 2University Mohamed V Agdal,Rabat - Morocco, Morocco

Algeria is facing chronic forage deficit, in order to improve the production and contribute to increasing animal production for sustainable development, the use of spontaneous forage legumes of pastoral interest as Scorpiurus muricatus ssp. sulcatus seems interesting. The 323 Symbiosis

Wednesday 24 June – Poster session diversity of microorganisms associated with this plant is very little studied worldwide. The purpose of this study is to investigate the genotypic characteristics of microorganisms associated with this plant. Fifty nodular and root bacterial strains respectively were isolated from plants sampled in six regions in western Algeria. Nodular isolates are grouped into different clusters according to profiles obtained by repPCR by ERIC and BOX primers. NodC amplification allowed us to select renodulant strains. Nodular and root endophytes are studied by PCR-RFLP of DNAr16s and compared with references strains, a representative of each group is sequenced. The endophytes ability to solubilize phosphorus and IAA production is determined.

5

Biological Nitrogen Fixation Book Volume I and II Frans J. de Bruijn* INRA/CNRS Laboratory of Plant Microbe Interactions, France

Nitrogen is arguably the most important nutrient required by plants. However, the availability of nitrogen is limited in many soils and although the earth’s atmosphere consists of 78.1% nitrogen gas (N2) plants are unable to use this form of nitrogen. To compensate, modern agriculture has been highly reliant on industrial nitrogen fertilizers to achieve maximum crop productivity. However, a great deal of fossil fuel is required for the production and delivery of nitrogen fertilizer. Moreover carbon dioxide (CO2) which is released during fossil fuel combustion contributes to the greenhouse effect. Biological nitrogen fixation is one alternative to nitrogen fertilizer. It is carried out by prokaryotes using an enzyme complex called nitrogenase and results in atmospheric N2 being reduced into a form of nitrogen diazotrophic organisms and plants are able to use (ammonia). It is this process and its major players which will be discussed in the “Biological Nitrogen Fixation” Books. The best known and most extensively studied example of biological nitrogen fixation is the symbiotic interaction between nitrogen fixing “rhizobia” and legume plants. Here the rhizobia induce the formation of specialized structures (“nodules”) on the roots of the legume plant and fix nitrogen which is directly assimilated by the host plant;. It is this symbiotic interaction which will be highlighted in the Book. While legumes are important as major food and feed crops, cereals such as wheat, mays and rice) do not have this symbiotic nitrogen fixing interaction with rhizobia. It has thus been a “holy grail” to transfer the ability to fix nitrogen to the cereals and different timely approaches towards this goal are also discussed in the Books.

324 Symbiosis

Wednesday 24 June – Poster session 6

Phosphorus use efficiency in N2-fixing legume-rhizobia symbioses Jean Jacques Drevon*1, J. Abadie1, L. Amenc1, A. Bargaz2, O. Dommergue3, M. Lazali4, M. Zamanallah5 1 INRA Ecologie Fonctionnelle & Biogéochimie des Sols & Agroécosystèmes, France, 2Swedish University of Agricultural Sciences, Department of Biosystems and Technology, Sweden, 3Laboratoire des Symbioses Tropicales et Méditerranéennes, Campus International de Baillarguet, France, 4Université de Khemis Miliana, Algeria, 5CIMMYT, Southern Africa Regional Office, Zimbabwe

Low phophorus availability in about 40% of the world’s arable land limits crop yield, most particularly for leguminous crops when their growth depends upon symbiotic N2-fixation (SNF). Therefore, our work aims to increase the phosphorus use efficiency (PUE) for SNF, and its contribution to a more effective coupling between the P and N bio-geochemical cycles. Myo-inositol hexakisphosphate (phytate) constitutes the main source of organic P in soils, but is unavailable to plants. Phytases are the only phosphatases able to hydrolyse phytate efficiently into inorganic Pi, thus increasing P bio-availability for plants. In this work we demonstrate the existence of phytase activity in root-nodules, and show the expression of a purple acid phytase within nodule inner-cortex by in situ RT-PCR. Also histidine acid- and bpropeller- phytases (HAP and BPP) were found among legume-nodulating bacteria, i.e. rhizobia, with major expression in nodule infected-cells. Moreover, phosphoenol pyruvate phosphatase and trehalose 6P phosphatase were discovered in nodules with puzzling localization. The nodular expression of all these genes and the enzymatic activity of their products increased significantly under P-deficiency, and varied among recombinant inbred lines of Phaseolus vulgaris that are contrasting in their PUE for SNF. It is concluded that the differential expression of bacterial and plant phosphatase-genes in nodules offers a new understanding of the N2-fixing legume physiology and its specific P requirements. The potential of these phosphatase-genes for a virtuous circle of P and N soil-fertility will be addressed in relation with the interdisciplinary research strategy of the FABATROPIMED federative project of Agropolis Montpellier.

7

Diagnosis of common bean inoculation needs and selection of effective bacterial biofertilizers adapted to soil conditions of the Skhirate region in Morocco Imane El Attar*1, Jamal Aurag2, Imane Thami Alami3, Mohamed El Khadir3 1 Laboratory of Microbiology and Molecular Biology (LMBM), Faculty of Science, Mohammed V University, Morocco, 2Laboratory of Microbiology and Molecular Biology (LMBM, LMI), Faculty of Science, Mohammed V University, Morocco, 3National Institute for Agricultural Research (INRA), Morocco

The Skhirate region (North-West part of Morocco) provides about 20% of of common bean production in the country under an intensive production system based on the uncontrolled use of chemical fertilizers. In this context our research project aims to reduce the amount of fertilizers used during the cultivation of common bean causing negative environmental impacts. For this purpose, we plan to develop bacterial biofertilizers that can contribute efficiently to the nutrition of common bean and its growth by replacing, partially, the intakes of mineral fertilizers. This approach is based on the establishment of a sustainable 325 Symbiosis

Wednesday 24 June – Poster session agrobiological system for the nutrition of common bean using mostly symbiotic nitrogen fixation and inorganic phosphate solubilization. During the growing season of common bean, a general survey was carried out in the Skhirate region in order to diagnose the nodulation status of this legume and determine if there is a need for inoculation with bacterial inocula. Common bean nodulation was found to be very low or even absent in most of the visited fields, thus we decided to carry out additional surveys in other production areas in Morocco (Beni Mellal, Moulay Bouselham and Berkane). Nodules collected during these surveys have enabled the creation of a collection of 143 bacterial isolates which were subjected to a physiological characterization, especially their tolerance to salt and pH stresses and their potential plant growth promoting activities (PGP activities). The results of this characterization revealed a large number of isolates with interesting PGP activities and tolerance to both types of stress studied. The symbiotic properties of these isolates are being characterized under controlled conditions. The strains able to establish a symbiotic relationship with the host plant (Phaseolus vulgaris) will be identified by sequencing the 16S rRNA gene.

8

Does mycorrhizal phosphate uptake influence the root-associated microbiome? Nina Gerlach*, Martin Willmann, Eva Koebke, Alexander van Burgeler, Marcel Bucher University of Cologne, Botanical Institute, Cologne Biocenter, Cluster of Excellence on Plant Sciences (CEPLAS), Germany

The plant microbiome is a key determinant of plant growth and fitness. Arbuscular mycorrhizal fungi are part of the fungal microbiome of most terrestrial plants and function as an extension of the root system facilitating bi-directional exchange of soil-born nutrients and plant-derived carbon between both symbionts. Maize plants colonized by arbuscular mycorrhizal fungus Rhizophagus irregularis exhibited enhanced growth under nutrient-deficient conditions. Systemic metabolic alterations included anthocyanin and lipid metabolism likely in response to an improved P-status of mycorrhizal maize leaves. An overall increase in leaf C versus N metabolism highlighted changes in metabolic fluxes. A parallel induction of defense gene expression and accumulation of secondary metabolites suggested priming of mycorrhizal maize leaves. Phosphate uptake in plants is mediated by transporters of the Pht1 protein family. A pht1;6 transposon insertional maize mutant is strongly impaired in mycorrhizal phosphate uptake and thus exhibited reduced biomass accumulation in agricultural soil poorly fertilized with phosphate. Arbuscular mycorrhizal fungal colonization of isolated pht1;6 plants was strongly diminished. However, when cultivated together with wild type plants, fungal colonization of pht1;6 was restored but mutant roots exhibited increased arbuscule degeneration and formation of strongly septated stunted arbuscules. In consideration of the role of Pht1;6 in maize root and shoot physiology, our current interest is on the impact of Pht1;6 transporter activity on the root-associated fungal microflora 326 Symbiosis

Wednesday 24 June – Poster session including mycorrhizal but also other fungi. To this end, the fungal microbiome of pht1;6, heterozygous and wild type plants was analyzed by a PCR-based automated ribosomal intergenic spacer analysis. Trans-complementation assays highlighted the impact of mycorrhizal neighboring plants on the mutant root-associated microbiome. Moreover the taxonomic structure of fungal assemblages as a function of Pht1;6 will be resolved by a culture-independent molecular approach.

9

Endofungal bacteria in plant symbiotic fungi Anton Hartmann*1, Dan Li1, Michael Rothballer1, Jochen Blom2, Peter Kämpfer3, Stefanie P. Glaeser3, Karl-Heinz Kogel4 1 Helmholtz Zentrum München, German Research Center for Environmental Health, Department Environmental Sciences, Research Unit Microbe-Plant Interactions, Germany, 2Bioinformatics and Systems Biology, Justus Liebig University, Germany, 3Institute of Applied Microbiology, Research Centre for BioSystems, Land Use and Nutrition, Justus Liebig University, Germany, 4Institute of Phytopathology, Research Centre for BioSystems, Land Use and Nutrition, Justus Liebig University, Germany

Endofungal bacteria occur in some ectomycorrhizal as well as arbuscular mycorrhizal fungi; a mycorrhizal helper function was attributed to these bacteria. In the order of Sebacinales (Basidiomycota), also endofungal bacteria have been demonstrated. In many cases, the endofungal bacteria are not cultivated yet, but in the case of Piriformospora indica, the endofungal bacterium Rhizobium radiobacter F4 (RrF4) was cultured and its genome and physiology could be investigated. A better understanding of the role of R. radiobacter in this tripartite plant-fungus-bacterium symbiosis is of high relevance, because P. indica has a wide range of plant beneficial applications in agriculture. The genome of RrF4 has been sequenced by second generation pyro-sequencer (454 GS FLX Titanium). It is organized in a circular and a linear chromosome and two plasmids (pTiF4 and pAtF4) in the same manner as in R. radiobacter C58 (formerly Agrobacterium tumefaciens). The average amino acid identity (AAI) of RrF4 to biovar I strains (e.g. C58) is 99.8%. While RrF4 and C58 showed a high degree of similarity based on the circular and linear chromosomes, the plasmids were more diverse. In addition to some rearrangements between the two plasmids, RfF4 most importantly lacks – in contrast to C58 - the complete transferred DNA (T-DNA) region and some adjacent genes belonging to the nopalin catabolic region. Like in C58, strain RrF4 contains the structural gene traI for N-acylhomoserine lactone (AHL)synthesis and traR for the AHL-receptor traR. Detailed chemical analysis showed that RrF4 produces a range of oxo- and hydroxyl-AHLs with C8-, C10-, or C12 alkyl side chains. These autoinducer signaling molecules are able to induce systemic resistance in different plant hosts as well as having plant growth promoting effects. We therefore hypothesize that the endofungal bacterium RrF4 of P. indica contributes to its plant growth promoting action in the tripartite plant-fungal-bacterium symbiosis.

327 Symbiosis

Wednesday 24 June – Poster session 10

An investigation on the colonisation of bacterial endophytes in oilseed rape, grown in the presence of entomopathogenic nematodes Mary Jo Hurley*, Dina Brazil, Thomais Kakouli-Duarte Institute of Technology Carlow, Ireland

Pathogenic microorganisms and pests are a predominant threat in agriculture worldwide and control of these has relied heavily on chemical pesticides. The overuse of pesticides has been directly related to the intensification of agriculture over previous decades, but with substantial environmental costs. The overall aim of this project focuses on the biocontrol of economically important insects utilising a combination of entomopathogenic nematodes and bacterial endophytes, to help reduce pesticide input in the environment. Both entomopathogenic nematodes and endophytic bacteria have widespread applications as biocontrol agents, however the potential of synergism between these nematodes and endophytic bacteria has yet to be explored. Investigating the effects of various endophytic bacteria on nematode biology and behaviour will not only contribute to knowledge on these but also increase nematode efficiency and predictability within a dynamic environment. Results are presented here from experiments designed to (a) investigate the effects of bacterial endophytes on nematode virulence and (b) examine the potential of increased bacterial-plant colonisation in the presence of entomopathogenic nematodes. Nematode dose response experiments were carried out to determine negative or stimulatory effects of endophytes on nematode infectivity. In all bacterial treatments Heterorhabditis bacteriophora infected in lower numbers than Steinernema feltiae and Steinernema carpocapsae. Following bacterial exposure fewer S. feltiae (e-nema) infective juveniles were recovered, when compared to the control. Moreover, for all nematodes species fewer numbers were recovered from insects exposed to bacterial strain L228. Bacterial colonisation of oilseed rape was determined via bacterial plate counts and the polymerase chain reaction, using green fluorescent protein specific primers. To date gfp labelled bacteria have been isolated from root, rhizosphere and stem samples, in the presence and absence of nematodes. This work is currently on-going.

11

Symbiotically fixed N substitutes fertilizer N by enhancing C and N assimilation during reproductive stages of soybean Jian Jin*1, Yansheng Li2, Xiaobing Liu2, Guanghua Wang2, Zhenhua Yu2, Mathesius Ulrike3, Judong Liu2, Herbert Stephen4 1 Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, China, 2Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, China, 3Division of Plant Science, Research School of Biology, The Australian National University, Australia, 4Stockbridge School of Agriculture, University of Massachusetts, USA

Excessive fertilizer has been commonly applied in the soybean cropping system in fertile Mollisols in northeast China. It is necessary to understand the mechanisms of how reducing N fertilizer application impacts the plant N acquisition and remobilization, which is associated

328 Symbiosis

Wednesday 24 June – Poster session with photosynthetic C assimilation and seed yield. Thus, the aim of this study was to investigate the origin of plant N, i.e. N2 fixation-, fertilizer- and soil-derived N under two different N application levels, and the subsequent influence on C assimilation. A pot experiment was conducted with soybean grown in a Mollisol supplied with 5 (N5) or 100 (N100) mg N kg-1 soil. Nitrogen was applied as 19.83% of 15N atom excess in urea before sowing, and 13CO2 labeling was performed at the R5 (initial seed-filling) stage. Plants were harvested at R5 and R8 (full maturity) stages to determine the 15N and 13C abundance in plant tissues. Seed yield and N content were not affected by different N rates. The symbiotically fixed N accounted for 64% of seed N in N5, while fertilizer-derived N dominated seed N in N100, resulting in 58% of seed N. The proportion of soil-derived N in seed showed no difference between the two N treatments. The enhanced N2 fixation in N5 significantly increased the assimilation of N and C during the seed-filling period compared to N100. The nodule density (nodule number per unit root length) and the amount of photosynthetically fixed 13C in roots in N5 were greater than those in N100. These results indicate that prolonging N2 fixation to increase assimilated N during the seed-filling period is likely to meet N demand for maintaining soybean yield when fertilizer N supply is reduced. More allocation of photosynthetic C to roots and enhanced nodulation would greatly contribute to the alteration of N acquisition pattern.

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Selection of specific Rhizobium leguminosarum genotypes by different Fabeae legume hosts: A Pool-Seq mesocosm study Beatriz Jorrin*1, Amalia Soenens1, Juan Imperial2 1 Centro de Biotecnolgia y Genomica de Plantas. Universidad Politecnica de Madrid, Spain, 2Centro de Biotecnolgia y Genomica de Plantas. Universidad Politecnica de Madrid. Centro Superior de Investigaciones Cientificas, Spain

Rhizobium leguminosarum bv viciae can establish effective symbioses with members of the Fabeae legume tribe (Pisum, Lathryrus, Lens and Vicia). Previous studies have suggested that different Fabeae select specific genotypes of rhizobia from those available in soil. We extended these observations at the genomic level by applying a Pool-Seq analysis to isolates selected from a soil population by pea, lentil, fava and vetch plants, and showed that plantselected sub-populations differ at the single nucleotide polymorphism level. The nature and extent of plant-specific genotypic preference were further studied by performing controlled mesocosm assays. Sterilized seedlings were planted in soil:vermiculite 1:1, and 21-day roots were collected, sterilized superficially, homogenized and centrifuged at low speed. This supernatant was used as inoculant for the next cycle of selection in the same soil with new seedlings. After 5 cycles, roots were collected, and rhizobia (25 from each host) isolated from nodules. Each of the isolates was grown separately, all cultures from the same host were pooled, and their pooled DNA was isolated and subjected to Pool-Seq analysis. Sequence reads were aligned against the R. leguminosarum bv viciae 3841 reference genome and both coverage and polymorphism analyses were performed for specific regions. No genotypic selection by the host was observed in the rDNA region, whereas increased genotypic selection was very clear along the mesocosm experiment for the nod cluster, but only in the pea and vetch subpopulations. In contrast, no further polymorphism profile changes were observed in the nod region for the fava and lentil subpopulations after the first 329 Symbiosis

Wednesday 24 June – Poster session round of plant host selection. These results suggest the existence of differences in plant host selection of rhizobial genotypes along the mesocosm experiment for different hosts and genomic regions.

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Effect of different biochars on the establishment of the symbiosis between Rhizophagus irregularis and leek grown in peat-based substrate Vicky Levesque*1, Hani Antoun1, Martine Dorais2, Noura Ziadi2, Martin Trépanier3 1 Laval University, Canada, 2Agriculture and Agri-Food Canada, Canada, 3Premier Tech, Canada

Arbuscular mycorrhizae are affected by the use of peat-based substrates and inhibition of mycorrhization was observed with Allium species. We hypothesized that the use of biochar could alleviate the observed inhibition of mycorrhization of leek transplants grown in a soilless mix containing 75 to 85% sphagnum peat moss and inoculated with Rhizophagus irregularis. Since biochar physicochemical properties are influenced by feedstock nature and the pyrolysis temperature used, five different biochars were produced (maple bark 400˚C, 550˚C and 700˚C, pine chips 700˚C and willow chips 400˚C) and applied at three different rates (5, 10 and 15% in volume). The experiment was realized during nine weeks in a greenhouse with 5x3 factorial design for biochar types and rates. A control (0% of biochar) was also included for comparison purpose. Biochars were added to potting mix and inoculated with monoxenic culture of Rhizophagus irregularis DAOM 197198. Leek (Allium ampeloprasum var. Lancelot) was used as host plant. The effect of biochar amendment on leek root colonization by mycorrhizae, plant biomass and phosphorus uptake were evaluated at the seventh leaf stage. Results indicated that the type of biochar and the level of amendment have an effect on root colonization by mycorrhizal fungi. Indeed, in comparison to the control, the addition of 15% of maple biochar increased (ρ < 0.05) the percentage of root colonization (ranging from 23% to 46%). Nevertheless, the results have shown a reduction (ρ < 0.05) in shoot dry matter biomass and phosphorus uptake with maple biochar. Observed decreases could be attributed to the early stage of plant development. In conclusion, biochar could alleviate peat inhibition in soilless mix and improve mycorrhization, which will produce more robust leek transplants.

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Nitrogen transfer in soybean/maize intercropping system inoculated arbuscular mycorrhizal fungi and rhizobium Shumin Li*1, Aiyuan Zhang2, Lingbo Meng3, Xiaoguang Han2, Fei Wang2, Dejiang Wang2 1 Northeast Agricultural University, China, 2Resource and Environmental College, Northeast Agricultural University, China, 3Department of Life Science, Harbin University, China

The tripartite symbiosis between legumes, rhizobia and mycorrhizal fungi are generally considered to be beneficial for nitrogen (N) uptake of legumes, but the facilitation of the symbiosis in legume/non-legume intercropping system is not clear.Therefore, the aims of the research are 1) to certify if the dual inoculation can facilitate the N uptake and N transfer in 330 Symbiosis

Wednesday 24 June – Poster session maize/soybean intercropping system, 2) to calculate how much N will be transferred from soybean to maize. A pot experiment with different root separations (solid barrier, mesh(30µm) barrier and no barrier) was conducted and 15N isotopic tracing method was used to calculate how much N transferred from soybean to maize inoculated arbuscularmycorrhizal fungi (AMF) and rhizobium in soybean (Glycine max L.cv. Dongnong No.42)/maize (Zea mays L.cv.DongnongNo.48) intercropping system. In comparison with the inoculatingGlomus mosseae(G.m.), Rhizobium SH212 and no inoculation, both inoculating Glomus mosseaeand Rhizobium SH212 increased the N uptake of soybean by 28.73%, 39.62% and 93.07% in solid barrier system. N uptake of maize inoculated both Glomus mosseae and rhizobium was 1.20, 1.28 and 1.67 times greater than that ofinoculating Glomus mosseae, Rhizobium SH212 and no inoculationrespectively. In addition, the amount of N transferred from soybean to maize of dual inoculation with mesh barrier was 7.25 mg, 7.01 mg and 11.21 mg greater than that ofinoculating Glomus mosseae, Rhizobium SH212 and no inoculation, and similarly 6.40 mg, 7.58 mg and 12.46 mg increased in no barrier system. Inoculating both AMF and rhizobium in soybean/maize intercropping system could improve the N fixation efficiency of soybean, and promote N transfer from soybean to maize, which result in the improvement of yield advantage in legume/non-legume intercropping.

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Insect vectors efficiently convey complex endophytic communities across grapevine plants Sebastian Lòpez-Fernàndez, Valerio Mazzoni, Pier Luigi Bianchedi, Ilaria Pertot, Andrea Campisano* Fondazione Edmund Mach, Italy

Microbial endophytes colonize the inner tissues of plants. It is commonly held that most endophytes invade the host tissues through the roots or through discontinuities on the plant surface, including wounds and stomata. Microorganisms can also be transferred through root anastomoses, as it occurs for instance with some pathogenic mollicutes, such as the phytoplasmas. Some insects able to penetrate the plant surface are also vectors of phytoplasmas. Very little is known about the ability of such vectors to harbour and transfer other microorganisms. To unravel the ecological role of insects for endophytic microorganisms, we used freshly hatched nymphs of the sap-feeding leafhopper Scaphoideus titanus (vector) to transport microorganisms across grapevine plants. We used adult, greenhouse-grown (donor) plants with an established endophytic fauna, and micropropagated (acceptor) grapevines hosting no detectable bacteria. We used 454 pyrosequencing of the bacterial 16S rDNA gene to estimate the composition of bacterial endophytic communities in donor plants, vector insects and acceptor plants, and to track microbial communities along the insect-plant-microbe network. After contact with the vector, acceptor plants were colonized by a complex endophytic community dominated by Proteobacteria, highly similar to that present on donor plants. Interestingly, a similar bacterial community, but with a higher ratio of firmicutes, was found on S. titanus. Insects feeding only on acceptor plants transferred an entirely different bacterial community dominated by Actinobacteria, where the opportunistic human pathogen Mycobacterium abscessus played a major role. Despite the fact that insects dwelled mostly on 331 Symbiosis

Wednesday 24 June – Poster session plant stems, the bacterial communities in plant roots resembled more closely those inside and on insects, when compared with above-ground plants. We establish here for the first time the potential of insect vectors to transfer entire bacterial communities across plants. We also define the probiotic role of plants and microbial endophytes in establishing microbial communities in plant-feeding insects.

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Inoculation with selected strains of Azospirillum spp. replaces nitrogen in pearl millet (Pennisetum glaucum (L.)) in Brazilian cerrado soil Ivanildo Evodio Marriel*1, Izabelle Melo2, José A. Rodrigues1, Christiane A Oliveira1, Eliane A. Gomes1, Francisco A Souza1 1 Embrapa Maize and Sorghum, Brazil, 2UFSJ, Brazil

The cultivation of pearl millet has received increasing attention as an alternative crop for mulch formation in no-till systems and as forage for livestock production, mainly in the Brazilian Cerrado fields. There are evidences of the benefic contribution of the inoculation of diazotrophic bacteria to agronomic crops as a source of nitrogen (N), including pearl millet, but there is no strains recommended for this crop in the country. Here we report the effect of inoculation of Azospirillum strains on pearl millet (BRS 1501) growth and nitrogen fertilizer replacement. We evaluated 20 Azospirillum strains (CMS01 to 20) under three doses of N (0, 30 and 60 kg N.ha-1 on field condition at the Municipality of Sete Lagoas, MG State, Brazil. The crop was cultivated in plots at the field; the experimental design was randomized blocks with four replications. A basic fertilization was applied at sowing and consisted of 40 kg ha-1 of urea, 400 kg.ha-1 of superphosphate, 100 kg ha-1 of potassium chloride and 20 kg ha-1 FTE. At the flowering stage, plants were collected for determination of dry matter accumulation, concentration and content of macronutrients in the shoots. There were significant differences (p Pinus mugo> Rhododendron hirsutum. In roots, the enzyme activities of βglucosidase, N-acetyl-β-D-glucosaminidase, acid phosphatase, leucine aminopeptidase were higher in mycorrhizal root tips than non-mycorrhizal tips. Between ectomycorrhizal species, the activity of acid phosphatase, averaged for all species of mycorrhizas investigated, was higher in both tree species at the high elevation site, compared to Picea abies at the lower 340 Symbiosis

Wednesday 24 June – Poster session elevation. No clear relationship was shown between enzyme activity in the soil and that of the dominant ectomycorrhizal species, and also between the enzyme activity and rate of net nitrogen mineralization.

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Modelling resource allocation in the legume - rhizobium symbiosis Annet Westhoek*1, Neil Dalchau2, Lindsay A. Turnbull3 1 University of Oxford, United Kingdom, 2Computational Science Laboratory, Microsoft Research, United Kingdom, 3University of Oxford, Department of Plant Sciences, United Kingdom

The symbiosis between legumes and the nitrogen-fixing bacteria, rhizobia, is of major global importance, with symbiotic nitrogen fixation accounting for a third of the total nitrogen input in agricultural systems. Increasing demands for protein are only likely to increase reliance on legumes, which already form a key source of protein in human diets. Rhizobia provide legumes with nitrogen in return for carbon in the form of photosynthates. It is well established that investment by legumes in rhizobial symbionts depends both on the external nitrogen environment and on the rhizobial strain. However, we lack a quantitative understanding of how legumes allocate resources to symbionts of different nitrogen-fixing efficiency under a wide range of external nitrogen concentrations. In this study, we develop a model which investigates how a plant can maximise its fitness through optimising its resource allocation. The model simulates the growth of a plant infected with one or more rhizobial strains. We assume that growth is limited either by carbon or by nitrogen. The plant obtains carbon via investment in leaves while nitrogen is obtained by direct uptake via roots (which depends on the soil nitrogen concentration) and/or via nitrogen fixation in nodules (which depends on the nitrogen fixation rate of rhizobial strains). We then use the model to test alternative hypotheses about resource allocation strategies under different conditions. In particular we investigate whether the optimal allocation strategy depends on the assumptions made about the way inorganic nitrogen is supplied in soil. The model will be able to answer questions such as: 1) under what conditions investment in inefficient strains could be beneficial for legume growth; 2) and what would be optimal allocation for maximum growth?

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Symbiotic effectiveness and host range of indigenous Rhizobia isolates nodulating Sesbania sesban Wassie Haile Woldeyohannes*1, Elias Dogiso Dagne2 1 Hawassa University, Ethiopia, 2Bureau of Agriculture, Ethiopia

Exploitation of biological N-fixation as a cheap and sustainable source of N for plants requires identification of Rhizobia strain capable of inducing nodulation in plants and effective in fixing atmospheric nitrogen. Experiments were conducted to evaluate symbiotic 341 Symbiosis

Wednesday 24 June – Poster session effectiveness of forty indigenous Rhizobia isolates on Sesbania sesban and to determine their host-ranges. Each isolate was grown on YEMB for 3-5 days in the Laboratory and inoculated to pre-germinated Sesbania seeds in modified Leonard Jars. -Ve and +ve-N control treatments were also included. The experiment was laid out in CR design with three replications. Data on nodulation, plant growth and yield parameters; and plant tissue N content were collected and subjected to ANOVA. Cluster analysis of data was also done. The results revealed that all isolates except AC100e induced nodulation on S. sesban. The isolates varied significantly in their effects on nodulation and plant growth parameters, biomass yield and tissue N contents of S. sesban. Accordingly, 70% of the isolates produced significantly higher growth and biomass yield of the plant than that produced in –N control. But, only 25% of the isolates produced growth and biomass yield of the plant similar to that produced in +N treatment. Cluster analysis data revealed that the 40 isolates were grouped in to six clusters. Those isolates in cluster-VI were AC50b, AC51C, AC61a, AC61d and AC100c resulted in the highest yield and N content of S. sesban and were best. The results of cross inoculation study revealed that out of 10 isolates tested only 40 and 90% of the isolates induced nodulation and growth on soybean and haricot bean respectively. It is concluded that there is a high potential to isolate infective and effective Rhizobia strains capable of inducing nodulation and N-fixation in S. sesban, Soybean and haricot bean from indigenous sources in Ethiopia.

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Arbuscular mycorrhizal fungal hyphal exudates prime bacterium mediated phytate mineralization in hyphosphere Lin Zhang*1, Minggang Xu2, Yu Liu3, Fusuo Zhang1, Angela Hodge4, Gu Feng1 1 China Agricultural University, China, 2Chinese Academy of Agricultural Sciences, China, 3Zhejiang University, China, 4University of York, United Kingdom

Ecology and evolutionary biology seeks to understand how cooperative strategies evolve and are maintained in species networks. Here, we focus on the three-partner relationship between plants, arbuscular mycorrhizal fungi (AMF) and hyphosphere bacteria to ask if the interaction between AMF and bacteria can pay back an essential resource (in this case phosphorus) to their associated host plant by consuming plant derived carbon (C). A microcosm and two Petri plate experiments which separate the plant roots, AMF hyphae and bacteria were conducted to demonstrate the direct effects of hyphal exudates on the growth and activity of bacteria in organic phosphorus (P) mobilization and, the reciprocal impact of the bacteria on growth and activity of the AMF in P uptake and transfer to the plant. Results showed that AMF released substantial C to the environment, triggering bacterial growth and activity resulting in enhanced organic P mineralization and turnover. While, in return, bacteria enhanced AMF hyphal proliferation which in turn, resulted in enhanced capture of the available P released. Under low soil C:P conditions, the AM fungi-bacteria interaction improved aboveground plant P nutrition. Our results suggest a C-P tradeoff occurs in plantAMF-bacteria systems. AMF and bacteria share the photosynthate of the plant, and as reciprocation, the AMF -bacteria interaction repays the plant with P by jointly mobilizing soil organic P forms and P acquisition for the plant through division of labour between these two microbial groups.

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Wednesday 24 June – Poster session 33

Specificity and resilience of the arbuscular mycorrhizal fungal community in intensive agroecosystems Jiachao Zhou*, Xiaojing Wang, Yang Chen, Gu Feng China Agricultural University, China

The composition of arbuscular mycorrhizal (AM) fungi communities can have a large effect on the performance of their plant hosts. The dynamic changes of AMF in ecosystem is hotspot recently. It is generally believed that the growth period of plants influenced the AMF community composition obviously. In agroecosystem, whether AMF communities can keep stable and be helpful for crop growth under traditional fertilization and pesticide is yet unresolved. We have carried out indoor pot and field test, to study the effect od benomyl and N fertilization on AMF communities and crop development. Changes in the community were characterized by root colonization, cloning, sequencing, tRFLP and DNA copy. While we used biomass and shoot P to measure crop development. Benomyl inhibited AM fungi infection while N fertilization increased abundance of AMF, some new batchs of fungi emerged after nitrogen added AMF diversity after nitrogen added. Community composition changed at different development stages of crop growth. The study found that after using benomyl G. intraradices appeard, the reason might be that G. intraradices can produce rich exogenous hyphae network and has strong restorability, thus adapt to benomyl. In vegetative growth period, AMF abundance was larger than reproductive period and different groups of AMF respond to differennt period altered. In vegetative period, mycorrhizal colonization rate was positive correlation with shoot biomass, nitrogen and phosphorus absorption. During reproductive growth stage, mycelium density was positive correlation with phosphorus absorption. In addition, during reproductive period mycorrhizal colonization has linear negative correlation with nitrogen nutrient absorption. In maturation phase, mycelium density had certain relevance with corn biomass, grain yield and harvest index. AM fungi in this study differed greatly in their response to perturbation and can be helpful to plant growth. In agroecosystems, AMF communities was important to crop even if apply traditional fertilization and pesticide.

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Wednesday 24 June – Poster session Towards Application

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ToT (transfer of technology) needs for promoting farmers on IPNM in agricultural production system in Indian sub-continent Manas Mohan Adhikary*, Anandamoy Puste, Kalyan Jana Bidhan Chandra Krishi Viswavidyalaya (State Agricultural University), India

Modernizing agriculture is essential for meeting challenges of reducing hunger and poverty, increasing & sustaining productivity. Agriculture is the backbone of growth for developing countries like India, may achieve how well and how fast be able to manage a rural transformation (ToT) on integrated plant nutrient management (IPNM). Keeping this, field research on IPNM was emphasized during consecutive summer seasons at farmers’ field in different agro-zones of this sub-tropics. Improvised field demonstrations were undertaken on rice cv. Satabdi (IET 4786) in medium (S1) and medium-lowland situation (S2) to standardize production trend with IPNM (both organic and inorganic sources), which comparable with control and farmer’s practice [T1 - Control, T2 - Farmer’s practice, applied generally N:P 2O5:K2O @ 50:30:30 as inorganic sources, T3 - N:P 2O5:K2O @ 120:60:60 as 100% inorganic and T4 and T5 - 25% of N through FYM (farm yard manure) as organic, and 25% of N through green manuring + 75% of N:P2O5:K2O @ 120:60:60 kg ha-1, respectively as inorganic sources]. Results showed that grain yield of rice were significantly increased with IPNM in balanced form over farmers & control plot. Among organic sources, highest yield exhibited with green manuring along (25% N) with 75% of N, P 2O5 and K2O through fertilizers. Gaining yield of rice is almost double in respect to farmers’ practice of the zones (Cluster I to IV). It reveals that benefit-cost ratio had gone in favour of highest productivity of rice grain (3.58 t ha-1), using maximum and balanced use of plant nutrients (IPNM), which is more compatible with the nutrients available to the crop plants and this was economically viable (B-C ratio 2.28) to the rural farming communities, it may be concluded that ToT field-based technological intervention on IPNM is imperative for boosting up crop productivity and promoting livelihoods of the rural farming community.

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Combined use of Kosakonia radicincitans and Trichoderma harzianum in the northern and southern hemisphere: First results Beatrice Berger*1, Hernan Paillan Legüe2, Eduardo Donoso3, Silke Ruppel1 1 Leibniz Institute of Vegetable and Ornamental Crops, Germany, 2Universidad de Talca, Chile, 3Bio Insumos Nativa, Chile

Microorganisms originating from nature are promising candidates to keep high yield gain and quality standards of agricultural products. However, successful use of microorganisms in bio-economy presupposes elucidation and understanding of microbial behavior in various environmental niches. Our bilateral project, funded by the BMBF (Project Management Jülich), between Germany (IGZ, Grossbeeren) and Chile (University of Talca) aims to study mechanisms of plant-microbe interaction using the plant growth-promoting bacteria Kosakonia radicincitans (German partner) and the fungal antagonist Trichoderma harzianum strain Queule (Chilean partner) in interaction with various horticultural plants. In cooperation with our Chilean partners we investigate the microbial mechanisms on the plant when grown 344 Towards Application

Wednesday 24 June – Poster session under different nitrogen- and phosphor nutrition regimens, salinity stress under natural occurring soil conditions in the southern and northern hemisphere. In detail, we will measure changes in primary and secondary plant metabolism, moreover the nutrient up-take by the plant and we will evaluate the growth -promoting potential of bacteria and fungi as well as their colonization ability. Our results are intended to support the use of microbial products worldwide. Here we present first results on the combination of K. radicincitans and T. harzianum in greenhouse and field studies.

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Efficacy evaluation of seed coated plant – root associated plant growth promoting rhizobacteria in microcosm system Rita Choudhary*, Alok Adholeya The Energy and Resources Institute (TERI), India

Plant growth promoting Rhizobacteria (PGPR) are the important group of microorganisms which play a major role in stimulating plant growth through mobilizing nutrients in soils, producing numerous plant growth regulators, protecting plants from phytopathogens by controlling or inhibiting them and by improving soil fertility. This study was conducted with a view to isolate bacteria associated with roots of wheat, maize and soybean from different locations of Punjab, Rajasthan and Madhya Pradesh, India. A total of 130 bacterial isolates were screened biochemically for their plant growth promoting traits like phosphate solubilization, production of Indole Acetic acid (IAA), hydrogen cyanide (HCN) and siderophore. It was found that 58.5% of them showed IAA production, 22% showed phosphate solubilization, 48.3% siderophore production, 70% showed HCN production, whereas 9.7 % isolates showed all the plant growth promoting characteristics. Under polyhouse conditions, the efficacy of these biochemically characterized isolates was assessed. Formulations were prepared and an uniform 2300) from a number of sources including the ecto and endorhizosphere of native plants, cereals, legumes and included endophytes and rhizosphere colonising microorganisms. The first phase of the high-throughput in planta screening consisted of field soil with added R. solani in 50 ml tubes and test strains added as a suspension directly to seeds. The 4.3 % of strains that reduced disease symptoms were then screened in more rigorous disease bioassays and characterised for properties important for the commercial development for such products. The 43 effective strains represented a diversity of microbial genotypes that included fungi and 4 phyla of bacteria-Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. Further rigorous evaluation of minimum effective inoculum reduced the number to 6 strains for field trails. A range of inoculum delivery methods –from seed coating, drenches at different locations around the seed at sowing- and testing methods to account for the patchiness of the disease- resulted in the selection of three strains which were effective in a range of soil types in more than a single season. This holistic approach coupled with a team that understood each class of microorganism showed how inoculants can be developed to be effective in the rhizosphere.

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The effect of nitrogen fertilization on rooting patterns and nitrogen recovery of catch crops Dina in 't Zandt*1, Alison Arico2, David Lehnert2, Christian Fritz1, Florian Wichern2 1 Radboud University Nijmegen, Netherlands, 2Rhine-Waal University of Applied Sciences, Germany

In agriculture, nitrogen is typically applied in excess to increase crop yield. However, crops only take up 30-50% of the applied nitrogen leaving large amounts of nitrogen behind causing nitrate leaching and subsequent pollution of ground, surface and coastal waters. To reduce nitrate leaching, residual nitrogen can be immobilized in plant biomass by cultivating so called catch crops after harvesting the main crop. The objective of our study was to establish the relationship between root distribution and residual nitrogen immobilization by catch crops. In addition, we studied the effect of nitrogen addition to stimulate plant growth and nitrogen uptake. Since soil microorganisms compete with plants for nitrogen and immobilize nitrogen, microbial biomasses were also estimated. In a pot experiment, three catch crops, Raphanus sativus oleiformis L., Brassica rapa oleifera L. and Phacelia tanacetifolia Benth., were grown in a loamy sand soil at three fertilizer levels: 0 kg ha-1, 40 kg ha-1 and 80 kg nitrogen ha-1. Catch crops decreased nitrate concentrations up to tenfold compared to soils without catch crops. Interestingly, Brassica produced almost twice as many roots as Raphanus, but recovered comparable amounts of nitrate. Phacelia, on the other hand, captured a smaller fraction of the applied nitrogen, which was consistent with a decrease in root length and an increase in root diameter. For both Brassica and Raphanus, leftover nitrogen was comparable between all three nitrogen levels indicating that these plants respond to higher nitrogen availability by increasing their nitrogen uptake. Furthermore, nitrogen fertilization increased microbial carbon, but not nitrogen biomass, whereas catch crop cultivation increased only microbial nitrogen biomass. In conclusion, catch crops did not only contribute to reduced nitrogen losses by nitrogen uptake, but also by stimulating microbial nitrogen immobilization. 348 Towards Application

Wednesday 24 June – Poster session 44

Effects of microbial fertilizer on soil chemical and biological properties of soil cultivates with wheat, barley and corn plant in different climatic conditions Nurgül Kitir*, Meti̇ n Turan Yeditepe University, Turkey

This study was conducted to determine effectiveness of microbial fertilizers on soil chemical, biological properties and biodiversity of soil cultivated with wheat, barley and corn plant under different climatic conditions in Turkey. The Microbial Fertilizer was applicate in field conditions to wheat, barley and corn plant 30 L/ha. Each treatment were conducted three region that Kayseri, Urfa and Erzurum in Turkey. Plant and soil samples were taken at the end of the growing period each plant and each region. Some soil enzymes such as acid and alkaline phosphate, urease and dehydrogenase, amino acid exhausted from soil and plant roots, and macro and micronutrients elements of plant, soil microbial type and amount of the microorganism in the soil rhizospere were determined. The results obtained have shown that amino acids, plant and soil enzymes exhausted from root parts significantly affected the LifeBac NP microbial fertilizer. Results show that efficiency of LifeBac NP microbial fertilizer was affected to plant species and region climatic conditions. All plant species and region that studied LifeBac NP microbial fertilizer increased yield and yield parameters, and this increasing value has been very significantly as statical.

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Development of an atlas of fine roots of European tree species Tanja Mrak*, Jožica Gričar, Hojka Kraigher Slovenian Forestry Institute, Slovenia

Tree fine roots are an active component of belowground carbon cycle because of their fast turnover rates, and the exchange sites for nutrients and water. In this part of the rhizosphere, multiple interactions with various soil organisms occur, mycorrhizas being one of the central for the functioning of the forest ecosystems. Identification of tree fine roots is needed to elucidate the role of different tree species in belowground functional traits. Furthermore, tree root identification may serve in the field of cultural heritage protection as tree roots may cause structural damages to historical buildings. Since the molecular tools for identification of tree fine roots are costly and do not allow for quantification of species occurrence, the identification with anatomical-morphological approaches is a good alternative, especially for routine work. In our study ten temperate European tree species were investigated and for each species 3-5 individuals sampled. Roots of 5, 3 and 1 mm in diameter, as well as the most distal fine roots, were embedded into paraffin, longitudinal and radial sections prepared and studied with a light microscope. Morphology of fine roots was observed under the dissecting microscope and photographed. Compared to the stem wood of the same species, root wood differs in several characteristics, such as wedging growth rings, pattern of porosity, smaller size of vessels etc. Some characteristics not present in stem wood occur in fine roots – e.g. central channel in Abies alba Mill. roots. As the bark and primary tissues represents a high proportion of tissues in roots of smaller diameter, their anatomical characteristics can be used for identification purposes. The most important morphological characters for identification are the colour and texture of the bark, pattern of ramification and type of mycorrhiza. 349 Towards Application

Wednesday 24 June – Poster session 46

Analysis of secondary metabolites produced by different strains of Pseudomonas chlororaphis isolated from halophytes, mesophytes and xerophytes Salma Mukhtar*1, I Shahid1, Muhammad R2, Deeba Baig1, Rahman Saleem3, Kauser Malik1 1 Forman Christian College (A Chartered University), Pakistan, 2Pakistan Council of Science and Technology, Pakistan, 3School of Science and Engineering, LUMS, Pakistan

Several bacterial strains have been isolated from plants growing in diverse environments, such as halophytes, mesophytes and xerophytes (paragrass, sugarcane, cotton, cactus), of Pakistan. Among these, eight isolates were identified as strains of Pseudomonas chlororaphis subspecies chlororaphis and P. chlororaphis subspecies aurantiaca, based on 16SrRNA gene sequence. Four strains, RP4 (paragrass), ARS38 (cotton), FS2 (cactus) and PB-St2 (sugarcane), representing isolates from three different habitats, were selected for detailed study. These strains were screened for phosphate solubilization, indole acetic acid production, activity against plant pathogens, phenazine O (phzO) and pyrrolnitrin A (prnA) genes. All strains were positive for antifungal activity, indole acetic acid production, phzO and prnA genes. Secondary metabolites produced by these strains were analyzed and compared with each other by using MS technique. Phenazines, cyclic lipopeptides, homserine lactones, pyoverdin, pyrolnitrin and derivatives of lahorenoic acid have been detected in variable amount in these strains. Screening of these compounds against fungal pathogens is going on. These strains have great potential to be used as biocontrol agent due to phenazines and antibiotic production.

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Studies on the bio-ecological characteristics and control methods of melon necrotic spot nepovirus (MNSV) and its soil fungal vector, Olpidium spp. Jin-woo Park*1, Kyung-Seok Park2, Se-weon Lee3 1 Agricultural Microbiology Division, National Academy of Agricultural Science(NAAS), South Korea, 2National Academy of Agricultural Science(NAAS), South Korea, 3Technology Cooperation Bureau, South Korea

Melon necrotic spot nepovirus(MNSV) transmitted by seed and fungal vector Olpidium spp. in soil, is a most serious viral disease on melon. This study aimed to analyze the biological characteristics of MNSV and the impact of soil environment on the outbreak of a disease, as well as to select chemical control agents against Olpidium spp. which transmits MNSV. For the detection of MNSV in plant and Olpidium spp. in soil, this study used the real-time PCR, and genetic analysis of capsid protein gene of MNSV and resting spore ITS region of Olpidium were performed, respectively. Based on the result, it was found that among MNSV 128 isolates, the homogeny of 34 isolates whose pathogenicity is strong was more than 96% while 88~95% variations were confirmed over 84 isolates whose pathogenicity was weak. In addition, the genetically homology was found to be higher related to Spanish and Israeli strain than Japanese strain. Genetically analysis result showed that the Olpidium isolated from MNSV occurring areas was identified to be Olpidium blassicae. As a result of the analysis of the correlation between MNSV outbreak and in-plastic house environmental factors, it was found that MNSV occurrence was severe in soil with alkalinity of more than 7.5 while moisture in soil had no big impact on MNSV occurrence. The results of the study showed that the proper soil temperature for MNSV outbreak was 25°C. The result of survey on the density 350 Towards Application

Wednesday 24 June – Poster session of Olpidium spp. which transmits MNSV from the surface soil to 45cm on a 15cm unit by six regions was found to be high at the layer near the surface soil of the region with high Olpidium spp. From the seven kinds of agricultural chemicals selected for fungal vector control, the Benomyl and Chlorothalonil water-dispersible powders were found to have high control effect.

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The interactions between plant, microorganism and soil affect Fe acquisition in cucumber plants Youry Pii*1, Alexander Penn1, Concetta Eliana Gattullo2, Ignazio Allegretta2, Roberto Terzano2, Carmine Crecchio2, Tanja Mimmo1, Stefano Cesco1 1 Free University of Bolzano, Italy, 2University of Bari, Italy

Plants have evolved two different strategies (Strategy I and II) to cope with Fe shortage, based on the exudation of organic and inorganic compounds to favor its mobilization and the root uptake. The role of the soil biotic component in the nutritional processes in the rhiszophere needs to be elucidated, since plants inoculated with PGPR showed an increased content of nutrients and a stronger resistance to abiotic stresses. The aim of the present work is the evaluation of the physiological effects, induced by Azospirillum brasilense in a calcareous soil on cucumber plants. Plants were grown in hydroponic Fe deficient solution followed by a 7-day period of contact with the A. brasilense-inoculated calcareous soil. At sampling, biometrics measurements, quali-quantitative analyses of root exudates and analyses of the nutrients content in plant tissues were carried out. Variations in soil mineralogy were assessed by X-ray powder diffraction (XRPD). Our results showed that A. brasilense facilitates plant growth in calcareous soils due to an enhanced recovery from the micronutrient deficiency. A. brasilense increases most likely the Fe availability within the rhizosphere by a) affecting the solubilisation of Fe thanks to the siderophore release and b) up and down-regulating the exudation activity of plants with an effect also on its molecular complexity. Further studies are needed to better understand and highlight the interactions between these two mechanisms and microorganisms. In particular, the present study shed light for the first time on two AAs, namely Gly and Glu, which could be involved in the plant-microorganism-soil interaction for the retrieval of Fe within a calcareous soil. XRPD analysis revealed a slight decrease of calcite and an increase of smectite under Fe-deficiency conditions.

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Wednesday 24 June – Poster session 49

Growth and nutrient uptake of sugarcane inoculated with diazotrophs Veronica Reis*1, Valfredo Chaves2, Willian Pereira2 1 Embrapa Agrobiologia, Brazil, 2Universidade Federal Rural do Rio de Janeiro, Brazil

Sugarcane is a important crop for Brazilian economy by the production of sugar, ethanol and energy. Technologies that favor growth and better use of nutrients using diazotrophic plant growth promoting bacteria (PGPB) can reduce costs, environment impact and improve crop yield. The aim of this work was to evaluate the effects of inoculation with selected PGPB in the sugarcane varieties RB867515 and IACSP95-5000. Germination was measure during 40 days in a sterile substrate sand/vermiculite and also growth and biomass accumulation was measured in pots containing a mixture of sand/soil during 50 days. Treatments used: uninnoculated control and inoculation with G. diazotrophicus (Gd - strain PAL-5T); H. rubrisubalbicans (Hr - HCC103); H. seropedicae (Hs HRC54); A. amazonense (Aa - CBAMc) and B. tropica (Bt - PPe8T) applied in as a mixture or individually by immersion using a sett composed of a single stem node. The assays used a randomized block experimental design with 8 replications. In general inoculation with PGPR improved germination, biomass accumulation and nutrient uptake specially phosphorus and potassium but the response was dependent by the variety and strain used, showing that plant-bacteria interaction was modulated by plant and bacteria genotypes. the magnitude of inoculation has a different response in each variety but the single inoculation of Gd, Hr and Hs presented better results in the two sugarcane genotypes tested.

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Enhancing the effective use of rhizobium inoculants by legume growers in southeastern Australia Maarten Ryder*1, Judith Rathjen1, Matthew Denton1, Ross Ballard2 1 School of Agriculture, Food and Wine, University of Adelaide, Australia, 2South Australian Research and Development Institute, Australia

Legumes growers in Australia commonly use rhizobium inoculants. However farmers can be unsure whether or how frequently inoculants should be used on a particular field. A nodulation assessment guide has been made available (online at http://www.agwine.adelaide.edu.au/research/farming/legumes-nitrogen/legumeinoculation/) that guides grain legume growers in determining whether or not the inoculation of a crop has been successful. Preliminary surveys have been conducted in several farming regions of southeastern Australia to test this approach. Although nodulation levels have often been rated as adequate, poor nodulation of an inoculated crop should be investigated, and a troubleshooting guide is being developed to help solve problems with nodulation. Poor nodulation of an uninoculated crop implies that inoculation is advisable in future, to help improve nodulation and nitrogen fixation rates. Farmers are also often uncertain about any negative effects of mixing inoculant with other treatments at sowing, for example fertilizers, trace elements or pesticides. The compatibility of faba bean rhizobia (R. leguminosarum bv. viciae WSM1455) with a liquid zinc preparation used by farmers for improved plant nutrition 352 Towards Application

Wednesday 24 June – Poster session was tested. When mixed in proportions used by the farmer, the zinc sulphate preparation (final pH approx pH 3.2) was found to kill 80% of rhizobia within 10 minutes and no rhizobia were detected after 2 h of incubation. This result clearly shows an incompatibility between current farm practice and rhizobium survival, and has led to a change in method. Further data on the impact of Zn, low pH and water quality on survival of rhizobia at sowing time will be presented.

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Two volatile organic compounds trigger plant self-defense against a bacterial pathogen and a sucking insect in cucumber under open field conditions Choong-Min Ryu, Geun Cheol Song* KRIBB, South Korea

Systemic acquired resistance (SAR) is a plant self-defense mechanism against a broad-range of pathogens and insect pests. Among chemical SAR triggers, plant and bacterial volatiles are promising candidates for use in pest management, as these volatiles are highly effective, inexpensive, and can be employed at relatively low concentrations compared with agrochemicals. However, such volatiles have some drawbacks, including the high evaporation rate of these compounds after application in the open field, their negative effects on plant growth, and their inconsistent levels of effectiveness. Here, we demonstrate the effectiveness of volatile organic compound (VOC)-mediated induced resistance against both the bacterial angular leaf spot pathogen, Pseudononas syringae pv. lachrymans, and the sucking insect aphid, Myzus persicae, in the open field. Using the VOCs 3-pentanol and 2-butanone where fruit yields increased gave unexpectedly, a significant increase in the number of ladybird beetles, Coccinella septempunctata, a natural enemy of aphids. The defense-related gene CsLOX was induced by VOC treatment, indicating that triggering the oxylipin pathway in response to the emission of green leaf volatiles can recruit the natural enemy of aphids. These results demonstrate that VOCs may help prevent plant disease and insect damage by eliciting induced resistance, even in open fields.

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From seed to whole plant: seed defense priming by rhizobacteria and its determinant dipeptide Choong-Min Ryu, Geun Cheol Song*, Hye Khung Choi KRIBB, South Korea

Seed priming is a technique to be controlled hydration and drying of seeds resulting in more rapid germination when are re-imbibed. Defense priming that has been induced by pretreatment of certain beneficial microbes and natural/synthetic compounds can enhance defense responses more rapidly or aggressively to biotic- or abiotic stresses. In this study, we were newly developed a immerged technology of the two priming methods referred to as “seed defense priming (SDP)” that is by seed priming with its supernatant (secreted metabolites) from root-associated Bacillus spp. in order to induction of systemic resistance (ISR) seven after transplanting to field. Seed defense priming mediated by bacterial supernatants from the 1800 strains of Bacillus spp. isolated from various soil samples in South Korea were tested ISR against Pseudomonas syringae pv. lachrymans (PSL) in cucumber 353 Towards Application

Wednesday 24 June – Poster session seedlings. Symptom development on the SDP with strains PB69 and 1628 was reduced 40 and 28% respectively in vitro. Under field condition, pretreated pepper plants were assessed their disease severity by infiltration of Xanthomonas axonopodis pv. vesicatoria (Xav) at 20, 30, and 40 days post-transplanting (DAT). SDP by Bacillus spp. strains PB69 and 1628 elicited ISR as compared to control treatment at 20 and 30 but not 40 DAT. Cyclo (leu - pro) was isolated by various column chromatography and NMR spectra from culture filtrates of PB69. SDP by cyclo (leu - pro) (0.1 ppm) induced systemic resistance in cucumber plant. Our results indicate that seed defense priming triggered by bacterial supernatants can be de novo method to induced ISR even under field condition and cyclo (leu - pro) involves in the activator of plant defense reactions, leading to induced resistance against PSL in cucumber.

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Is thinking about a below-ground landscape useful for scaling up understanding to practice? Elizabeth Stockdale* Newcastle University, United Kingdom

Below-ground processes result from the interaction of soil habitats and their associated populations where the structure, composition and flows between these components are critical in defining the outcome and rate of the processes observed at the soil scale. Soil can be conceptualised as a series of linked habitats, including the rhizosphere, rather than a single habitat for soil organisms. Habitat types in soil: Resources (places): root, root surface (rhizoplane), rhizosphere, organic matter (litter to old humus), mineral surfaces. Pores (spaces): storage (Air/water filled), transmission (AIR filled) and residual (WATER filled). A multi-habitat (landscape) conceptualisation has been shown to provide a useful representation of soil faunal populations. However, application of landscape ecology approaches to below-ground ecology is not easy. Above-ground landscape ecology is moving away from a simple patch-matrix view of landscape and consequently connectivity is considered as an aggregate property of the structural configuration of the landscape elements. Habitat characteristics must be defined from across a range of scales and pattern prediction is complex and multifactorial – interaction between access to resources and refuge from predators. Habitat elements defined below-ground should clearly differ in quality; in the Table each has distinctive physical and chemical characteristics together with distinguishable communities of soil organisms. The context and connectivity of these elements are then key. The plant or plant community integrates across the diversity of below-ground ecosystem functioning via the roots and rhizosphere; in some way this role can be compared to that of the top predator in above ground systems.

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Wednesday 24 June – Poster session This conceptual model is applied to an analysis of the impacts of agricultural management on the size, activity and diversity of soil organisms and highlights the key role of the rhizosphere in mediating plant-soil interactions and their resilience.

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Innovative biochar (hydrothermal carbonization) as an additive to soil-substrate for sustainable plant production Maren Stollberg*1, Thorsten Kraska2, Ralf Pude2, Guido Dericks3, Ulrich Schurr1, Arnd Kuhn1 1 Forschungszentrum Jülich, Germany, 2Friedrich-Wilhelms Universität Bonn, Germany, 3Grenol GmbH, Germany

Just as natives peoples thousands of years ago started the story about “Terra preta”, an anthropogenic fertile tropical soil, we would like to imitate this phenomenon today using hydrothermal carbonization (HTC-biochar). The advantage of HTC-biochar is the possible use of all kinds of wet organic material as a parent material. We used digestate originating from biogas production which was converted to charcoal in a reactor at 200°C and 20 bar within 6 hours. The better-known pyrolysisbiochar instead is based on dry woody material only. In first pre-experiments we found a depression of Lactuca sativa var. crispa. growth after adding HTC-Biochar to field soil to increase carbon content. Therefore our aim was to remove any growth-reducing substances (e.g. aromatic compounds) by different extra treatments of the HTC-Biochar. The biochar treatments were (1) drying at 80°C, drying and washing with (2) water or (3) with 10% Ethanol solution. 5% HTC-Biochar (dry matter) was mixed with a silty loamy soil (field-soil, 40 soil-points, sieving at 2 mm) in pots and either Zea maize, Lactuca sativa var. crispa. or Brassica rapa subsp. pekinensis were grown on this substrate for 14-49 days. We performed a sequence of 3 - 4 harvests per species. Growth parameters such as dry matter, leaf area and minerals were measured. Zea maize showed after 14 days a negative effect on plant growth with untreated HTC-Biochar and also of the pyrolysis variant. The biomass of these variants was increased up to 20% compared to the control variant (pure soil) after 28 and 35 days. All HTC-Biochar treatments in comparison to untreated HTC-Biochar -as well as compared to pure soil- had negative effects on plant biomass. Therefore our chosen extra treatment of HTC-Biochar did not lead to an improvement in plant growth. Our assumption is that we lost mineral nutrients during these treatments.

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Wednesday 24 June – Poster session 55

Tracing of Pseudomonas inoculants in root and rhizosphere samples Carla Mosimann, Sarah Symanczik, Thomas Oberhänsli, Paul Mäder, Cécile Thonar* FiBL (Research Institute of Organic Agriculture), Switzerland

Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition and can act as biocontrol agents by suppressing soil-borne diseases. Efficient strains can be formulated as microbial inoculants and their successful use for field application often requires a certain ability of persistence in the soil where they are inoculated. In this respect, there is a need to create tools enabling the tracing of inoculated PGPR which can also serve to monitor their spread in space and time. Here we report the development and application of a molecular method allowing the quantitative detection of two Pseudomonas strains contained in commercial formulations. The method is based on a Taqman qPCR assay targeting two polymorphic regions of the bacterial genome in order to ensure the specificity of the detection. The assays have been used with several samples (root or rhizosphere DNA) originating from various pot and field experiments with maize as host plant. The first results achieved in pot experiments indicate that for one strain the survival is influenced by the soil management (organic versus conventional) and that in general the high abundance of native Pseudomonas strains will not prevent a reasonable persistence of the inoculated Pseudomonas strains. In field conditions, the method has shown that the strain survival was improved when inoculated in combination with compost amendments. More results using these detection tools will be presented and will highlight the set of conditions (e.g. soil, inoculation techniques and frequency, co-inoculation or combination with other amendments) associated with reasonable persistence of inoculated PGPR.

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Altered carbon and nitrogen cycling in soils following biochar application Tess van de Voorde*1, Simon Jeffery1, T. Martijn Bezemer2, Jan Willem Van Groenigen1, Liesje Mommer1 1 Wageningen University, Netherlands, 2NIOO-KNAW, Netherlands

Biochar, pyrolysed biomass, is being widely promoted as a means to improve soil quality, sequester carbon, and improve soil-based ecosystem services. However, large knowledge gaps remain and the majority of research has been performed in managed agricultural land or controlled pot experiments. We aimed to study the effects of biochar amendment under semi-natural conditions. To do so we utilise a field experiment set up in 2011 in a nature restoration area near Ede, The Netherlands. Biochar was produced from cuttings collected from a local semi-natural grassland and pyrolyzed at 400oC or 600oC. The field experiment consisted of 4 treatments in 6 replicate blocks, resulting in 24 plots in total. The four treatments are: incorporation of biochar produced at 400oC, biochar produced at 600oC, incorporation of the non-pyrolyzed cuttings from which the biochar was produced, and a control treatment in which no material was incorporated (Control). Biochar and residue were applied at a rate of 10 ton/ha and mixed through the top soil layer (~10 cm). 356 Towards Application

Wednesday 24 June – Poster session Three years later, in autumn 2014, we collected soil samples in all plots, which were used for a series of experiments to investigate the functioning of the soil microbial community, focussing especially on carbon (C) and nitrogen (N) cycling. We hypothesised that microbial communities exposed to biochar (1) differ in their ability to utilise a range of carbon substrates, and that (2) these communities will be better at decomposing more recalcitrant substrates. Overall, net N mineralisation measurements, a denitrification inhibition assay, substrate induced respiration experiments and a MicroRespTM assay (both using multiple Csubstrates), showed increased CO2 production in the soils that received biochar and hay residue, and reduced N2O production in the biochar amended soils, as compared to the noamendment control. We will link these findings to functional gene activities using GeoChip 5.0.

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Root traits and aboveground yield of silage maize varieties under field conditions Nick Van Eekeren*1, Natalie Oram2, Joachim Deru1 1 Louis Bolk Institute, Netherlands, 2Nature Conservation and Plant Ecology, Wageningen University and Research Centre, Netherlands

Drought tolerance of agricultural crops is critically important in the face of water shortages caused by climate change and competition for drinking water. Silage maize is a major fodder crop for the dairy industry on dry sandy soils in The Netherlands. Root architectural traits play a key role in improving drought tolerance of crops. Our objective was to study root architecture of silage maize in relation to aboveground production under field conditions. An experiment with 14 commercial maize varieties was carried out on a sandy soil in Loosbroek, The Netherlands. The experiment was managed according to common agricultural practice. At harvest, aboveground nitrogen content (g N / kg dry matter) and yield (kg N / ha) were measured along with other yield components (dry matter, starch, calculated feeding value). Root traits were quantified in two ways: 1) using ‘shovelomics’: a practical in-field measurement of brace and crown root number, angle, and branching; 2) harvested brace and crown roots were dried, weighed and scanned. Root length was determined with image software, discerning lateral from main roots. Aboveground, results show that nitrogen content differed between varieties and nitrogen yield did not. In root architecture, we found differences in crown root branching and root weight between varieties but not in root biomass or angle. Variety effects were also found in the proportion of lateral root length in total root length. Aboveground nitrogen yields were significantly positively correlated with root traits: total root length (R2 = 0.29), lateral root length (0.28) and crown root branching (0.32). There was no correlation between root traits and nitrogen content. Equivalent responses were found for the other yield components. Our results show that root traits that are quickly assessed in the field could be used in maize breeding to improve drought tolerance without affecting yield components.

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Wednesday 24 June – Poster session 58

Root properties of dike vegetation and their effects on concentrated flow erosion Wouter Vannoppen*1, Jean Poesen1, Sarah De Baets2, Matthias Vanmaercke1, Patrik Peeters3, Bart Vandevoorde4 1 KU Leuven, Belgium, 2University of Exeter, United Kingdom, 3Flanders Hydraulics Research, Belgium, 4Research Institute for Nature and Forest, Belgium

The predicted climate change and the associated sea level rise are major challenges for the near future. The Scheldt basin will be exposed to an increased risk of flooding due to wave overtopping. To safeguard the land from dike breakthrough both above-ground and belowground biomass are important to keep the erosion resistance of the dikes sufficiently high. Plant roots are more effective in reducing soil erosion by concentrated flow compared to plant shoots. Therefore, the main goal of this study is to determine the erosion-reducing potential of the belowground biomass of dike vegetation types. Root properties of five dike vegetation types were studied: i.e. species-rich grassland (SR), species-rich grassland dominated by Arrhenatherum elatius (SRAe), grassland dominated by Arrhenatherum elatius (Ae), grassland dominated by Arrhenatherum elatius with few nettles (AeN) and nettle- dominated vegetation (NDV). The erosion-reducing effect (RSD) was estimated using a Hill curve model linking RSD to root length density (RLD, km/m³) based on an analysis of a global database on the erosionreducing potential of plant roots. Results based on the measured RLD in the topsoil (0-5cm) indicate that erosion rates would be reduced by more than 80% compared to a bare soil for dike vegetation types without nettles (129 < RLD < 235 km/m³). Dike vegetation types with nettles (22 < RLD < 58 km/m³) were less effective in controlling erosion due to concentrated flow. The degree of overgrowth of grassland by nettles, which have thicker roots and therefore a lower RLD, explains this pattern. To maintain a high resistance of the topsoil against concentrated flow erosion it is important to avoid the overgrowth of grassland by nettles through an effective management of the dike vegetation. Preventing the enrichment of the topsoil by nutrients is therefore an important measure.

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How do plant roots affect rill and gully erosion? Wouter Vannoppen*1, Jean Poesen1, Matthias Vanmaercke1, Sarah De Baets2 1 KU Leuven, Belgium, 2University of Exeter, United Kingdom

An important ecosystem service of plant roots is their effectiveness in controlling concentrated flow erosion rates. However an extrapolation of model results from individual case studies to estimate the erosion-reducing potential of plant roots is not reliable as rootsoil interactions in different types of environments, with differences in both root and soil characteristics are not yet fully understood. The objectives of this study were therefore: i) to provide a state of the art on studies quantifying the erosion-reducing potential of plant roots in controlling concentrated flow erosion rates; and ii) to explore the overall trends in erosion reduction as a function of root density (RD, kg/m³) and root length density (RLD, km/m³), root system architecture and soil texture, based on a meta-analysis of published research results. We therefore compiled a dataset of measured relative soil detachment rates (RSD) for RD and RLD. The decreases in RSD as a function of RD and RLD could be best described by a Hill curve model. Taking into account root system architecture and soil texture improved the model accuracy, especially for root length density. Fibrous root systems are in general more 358 Towards Application

Wednesday 24 June – Poster session effective in controlling soil erosion by concentrated flow as they have a larger root-soil contact. As there was little variation in soil types among the collected data, the effect of soil texture on the erosion-reducing potential of plant roots could not be proved. The remaining unexplained variance is attributed to measuring errors and differences in experimental set ups (e.g. plant species tested, soil characteristics) that could not be explicitly accounted for. By taking these uncertainties into account we were able to establish relationships to assess the likely erosion-reducing effects of plant roots which are reliable irrespective of differences in root and soil characteristics.

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Mixtures of maize genotypes produce more biomass than monocultures when mycorrhizal Xinxin Wang*1, Ellis Hoffland1, Gu Feng2, Thomas Kuyper1 1 Wageningen University, Netherlands, 2China Agricultural University, China

Arbuscular mycorrhizal fungi can play a key role in enhancing plant productivity in multispecies natural ecosystems; however, their role in enhancing crop productivity and P uptake efficiency in single species genotype mixtures is hardly known. Therefore, we grew monocultures (one genotype) and mixtures of two genotypes of maize in low P soils in a greenhouse (with Funneliformis mosseae) and in a field experiment with an unidentified species mixture. We measured P uptake, hyphal length density and plant biomass. Genotype mixtures showed overyielding and enhanced P-uptake when mycorrhizal but not when nonmycorrhizal. The increase in relative yield total and P uptake was largely due to complementarity effects, and not to enhanced competitive ability of the larger genotype. Genotype mixing increased hyphal length density. The effects occurred both in the greenhouse and in the field. Our results suggest that genotype mixing increases the extent and activity of the mycorrhizal network. So mixing maize genotypes may be beneficial for enhancing productivity and P uptake efficiency.

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Wednesday 24 June – Poster session Rhizo Remediation and Fate of Pollutants

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On-site PAH removal and microbial diversity during six-years monitoring under plant-assisted attenuation Thierry Beguiristain*1, Amélia Bourceret1, Aurélie Cébron1, Noële Raoult2, Pierre Faure1, Emilie Tisserant3, Pascal Poupin1, Pascale Bauda1, Corinne Leyval1 1 LIEC UMR 7360 CNRS-Université de Lorraine, France, 2GISFI, France, 3IAM UMR1136, INRA, France

During the XXth century, the intensive coal and steel industries contributed to the chronic pollution of soils. Their dismantling left large areas of wasteland soils highly contaminated by recalcitrant organic compounds such as polycyclic aromatic hydrocarbons (PAH). We are interested in studying the potential for soil bioremediation using plants assisted rhizodegradation combining the potential of plant with those of rhizospheric microorganisms. Our objectives were to evaluate the fate of PAH and characterize the microbial diversity of an aged contaminated soil using on-site natural attenuation in experimental plots. A 24 plots devices with 6 different treatments in 4 replicates allowed the comparison between bare soil and different rhizospheric soils (alfalfa, naturally colonising vegetation) during 6 years monitoring. Soil characteristics and fate of pollutants were analysed together with microbial parameters. Our work is one of the first to characterize the microbial diversity in such aged PAH-contaminated soil. The impact of plant rhizosphere on both bacterial and fungal densities and diversities was assessed using qPCR and pyrosequencing. During this 6 year period of time an increase of pH and C/N was observed and a 50 % PAH removal was estimated. Plants had a limited impact on these soil parameters. On the contrary, plants favoured a higher density and originally increased the diversity of microorganisms. The bacterial community was dominated by Proteobacteria, Actinobacteria and Bacteroidetes and the fungal community was mainly represented by Ascomycota. Moreover some OTUs were foster by plants, such as members of Arthrobacter, Fusarium, Bionectria, Acremonium genera. Similarly, the plant rhizosphere seemed to favour the PAHdegrading bacteria belonging to the Actinobacteria as shown by qPCR targeting PAH-ring hydroxylating dioxygenases. Plants seemed to favour the PAH-degrading functional community but, in such aged contaminated soils, the main limiting factor to their activity remained the low PAH-bioavailability.

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Dark-septate endophytic fungi: a solution for trees on metal-contaminated sites? Damien Blaudez*1, Charlotte Berthelot1, Julie Foulon2, Michel Chalot2, Corinne Leyval1 1 Université de Lorraine, France, 2Université de Franche-Comté, France

Dark-septate endophytes (DSE) are conidial or sterile ascomycetous fungi that colonize living plant roots without causing apparent negative effects. DSE comprise a heterogeneous group

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Wednesday 24 June – Poster session of root associated endophytic fungi, characterized by melanized inter- and intra-cellular running hyphae and microsclerotia within the epidermis and/or the cortex of plant roots. High tolerance of DSE to metal pollution and their relatively high abundance in contaminated habitats suggest that DSE might have an important function for host survival in these extreme conditions. Fungal endophytes could affect heavy metal uptake of their host plants and increase plant metal tolerance. Therefore, in the context of phytoremediation assisted by symbiotic fungi, we have first isolated a set of DSE strains from poplar roots from metal-polluted sites. A wide-range screening of the strains was performed to select the best promising symbionts. Fungal isolates were identified as members of the Phialophora, Cadophora, Leptodontidium, and Exophiala genera. They were characterized for their plant growth promoting abilities, through different tests such as production of indol-3 acetic acid (AIA), release of volatile organic compounds or production of antifungal compounds. Metal tolerance of the fungal isolates was also studied under axenic conditions. For the most promising strains, an inoculation experiment was performed to monitor the effects of the fungi on the growth of poplar and birch on metal-contaminated soils. Moreover, we also investigated the interaction between DSE and endomycorrhizal fungi through dual inoculations of host plants.

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Degradation of iprodione, vinclozolin and propanil by an Arthrobacter spp. strain isolated from a pristine rhizospheric soil Marco Campos*1, Dimitrios Karpouzas2, Maria Cristina Diez3 1 Universidad de la Frontera, Chile, 2Department of Biochemistry and Biotechnology, University of Thessaly., Greece, 3Chemical Engineering Department, Universidad de La Frontera., Chile

Biological degradation constitutes the major processes controlling the dissipation of pesticides in soil. In this way, rhizodegradation in cooperation with pesticide-degrading bacteria could be a suitable tool to avoid point source contamination by iprodione, vinclozolin and propanil pesticides. We aimed to test the degradation of iprodione, vinclozolin and propanil by a rhizospheric bacteria strain isolated from a pristine grassland. Rhizospheric soil samples collected from ryegrass (Lolium perenne) grassland with and without previous exposure to pesticides were used for the bacterial isolation. After several enrichment in mineral soil medium (MSM) amended with iprodione, the isolation of an effective iprodione-degrading culture (C2.7) was reached from the pristine soil enrichments. Molecular fingerprinting revealed that C2.7 was composed of two strains identified via cloning as Arthrobacter spp. (C1) and Achromobacter spp. (C2). Degradation studies with the purified strains C1, C2 and their combination in minimal and rich media showed that C1 was the key iprodione-degrader, whereas C2 was only able to slowly co-metabolize iprodione. After that, C1 capacity to degrade pesticides of similar chemical structure to iprodione (vinclozolin, procymidone, propanil, diuron and isoproturon) was investigated. This strain completely degraded vinclozolin, a chemical analogue of iprodione belonging to the dicarboxamide family, in 20 days, and this was accompanied by bacterial growth and the 361 Rhizo Remediation and Fate of Pollutants

Wednesday 24 June – Poster session formation of 3,5-dichloraniline. In contrast, this only partially degraded propanil with the production of small amounts of 3,4-DCA, while no degradation of procymidone (the other member of the dicarboxamide fungicides family) diuron and isoproturon were observed. These results provided first evidence that the degradation of iprodione proceeds via cleavage of the carboxamide bond common in iprodione and vinclozoline. Additionally, degradation of propanil provide significant information about the degrading versatility of our Arthrobacter spp. strain for future application in rhizodegrading systems.

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Short-term effect of plants on the identity, abundance and activity of phenanthrene-degrading microorganisms slows down bioremediation in a contaminated soil François Thomas, Aurelie Cebron* LIEC, UMR7360 CNRS-Universite de Lorraine, France

Microbial degradation is a promising soil remediation strategy for polycyclic aromatic hydrocarbons (PAHs) frequently polluting post-industrial environments, but the effect of plants on PAH dissipation rates and overall microbial community diversity and activity, is still unclear. Here, we compared the short-term dynamics of pollution, microbial communities and PAH-degraders in bare or ryegrass-vegetated aged-contaminated soil, using phenanthrene as a model PAH. Actively growing roots were allowed to colonize phenanthrenerecontaminated soil for 2 to 10 days in compartmented microcosms. Samples were collected from rhizospheric soil directly adherent to roots, bulk soil from vegetated microcosms and bare soil from non-vegetated controls. Phenanthrene concentrations were significantly lower after 10 days in bare soil than in rhizospheric or bulk vegetated soils. Measurements of total dissolved organic carbon, organic acids and carbohydrates showed that root exudation provided labile substrates that might be preferentially consumed instead of phenanthrene, therefore impeding its dissipation. Although the abundance of 16S rRNA genes and transcripts increased throughout the time course for both Bacteria and Archaea, these communities were more active in rhizospheric than in bulk vegetated soil after 8 days. In contrast, while plants favored the abundance of PAH-degrading genes compared to bare soil, their transcription level was similar in all conditions except after 10 days where the Actinobacteria activity was enhanced in bulk vegetated soil. To specifically target metabolically active PAH-degraders, similar microcosms were spiked with 13C-labelled phenanthrene in a stable isotope probing experiment. After 10 days, 16S rRNA gene fingerprinting revealed that 13C-phenanthrene was metabolized by different bacterial taxa depending on the presence of plants. Metagenomic characterization of the 13C-DNA fractions is underway. Together with the analysis of variations in total and active microbial community composition over the 10 days, it will provide an unprecedented view of the effect of plants on the identity of PAH-degraders in aged-contaminated soils.

362 Rhizo Remediation and Fate of Pollutants

Wednesday 24 June – Poster session 66

Characterizing zinc tolerance genes in Suillus luteus, an ectomycorrhizal fungus with properties promising for use in phytostabilization applications Laura Coninx*1, Joske Ruytinx2, Michiel Op De Beeck3, Vangronsveld Jaco1, Colpaert Jan1 1 Universiteit Hasselt, Belgium, 2INRA, France, 3Lund University, Sweden

Pyrometallurgical industry and mining activities have led to the contamination of vast areas with heavy metals. In these areas, biodiversity of plants and micro-organisms is often greatly reduced. Surviving organisms are subjected to a high selection pressure for metal tolerance, often resulting in the evolution of metal tolerant ecotypes of plants, fungi and bacteria. Zn contamination in the northern part of Limburg (Belgium), has led to the evolution of Zn tolerant ecotypes of Suillus luteus (L.) Roussel, an ectomycorrhizal basidiomycete that forms symbiotic associations with Pinus sylvestris L. These Zn tolerant ecotypes thrive in heavily contaminated soils and in the meantime protect their hosts from metal toxicity. This protective feature combined with the fact that S. luteus is a pioneer species common to sandy soils in temperate climate regions make S. luteus a suitable candidate for use in phytostabilization applications. However, to fully exploit the potential of such applications, a better understanding of the Zn tolerance mechanism is crucial. Previous investigations have shown that the basis of the tolerance trait is a mechanism promoting Zn efflux. Yet, since Zn is an essential nutrient, many homeostatic pathways are expected to be involved in maintaining an optimal Zn concentration in all cell compartments. This makes it a challenging task to characterize the tolerance mechanism. Hence, we first focused on establishing which Zn homeostatic pathways are present in S. luteus. Here we report 7 Cation Diffusion Facilitator proteins and 4 Zrt- Irt- like Proteins that have been identified in the S. luteus genome. Further characterization of these proteins and their response to increased zinc concentrations in zinc tolerant and sensitive isolates may lead to a better characterization of the zinc tolerance mechanism in S. luteus.

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Bacterial resources for assisted phytostabilization of acid mine drainageaffected mountain stream bank Anna Corsini*1, Sarah Zecchin1, Milena Colombo1, Nicoletta Guerrieri2, Giorgio Lucchini1, Gian Attilio Sacchi1, Lucia Cavalca1 1 University of Milano, Italy, 2CNR-ISE, Italy

Several areas of the Italian Alps have an arsenic content that exceeds the Italian law limit (20 mg kg-1, D.Lgs 152/2006), due to mineralogy of bedrock and to mining activities. Rio Rosso mountain stream (Anzasca Valley, Piedmont), is affected by arsenic (1015 mg kg-1) and iron (22 g kg-1) contaminated sediments that leach from an abandoned gold mine. In view of a bacterial-assisted phytostabilization action, rhizospheric bacteria and root endophytes of liverwort, fern and willow inhabiting stream bank were isolated and characterized. A total of 240 colonies were isolated and screened for arsenic-related features and plant growpromoting traits. Most of rhizobacteria and endophytes were resistant up to 7500 mg L-1 arsenate (21 and 20 respectively) and up to 750 mg L-1 arsenite (17 and 24 respectively), regardless plant species. More than 80% of the isolates were capable to reduce 75 mg L-1 of arsenate, while arsenite oxidation rarely occurred (