Biogeography of symbiotic and other endophytic bacteria isolated from

RESEARCH ARTICLE

Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China Li Li1,2, Hanna Sinkko2, Leone Montonen2, Gehong Wei1, Kristina Lindstro¨m2 & Leena A. Ra¨sa¨nen2 1

College of Life Sciences, State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling, Shaanxi, China; and Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland

2

Correspondence: Gehong Wei, College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China. Tel.: +86 29 87091175; fax: +86 29 87091175; e-mail: [email protected] Received 21 December 2010; revised 16 August 2011; accepted 26 August 2011. Final version published online 17 October 2011. DOI: 10.1111/j.1574-6941.2011.01198.x

MICROBIOLOGY ECOLOGY

Editor: Philippe Lemanceau Keywords rhizobia; endophytic bacteria; nodulation; plant growth promoting activity; amplified fragment length polymorphism; biogeography.

Abstract A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.

Introduction Glycyrrhiza, ‘sweet root’, originating from Greek ‘glykos rhiza’, is a leguminous plant genus that contains about 20 species native mainly to Eurasia (Lewis et al., 2005). In China, roots and rhizomes of three Glycyrrhiza species, Glycyrrhiza uralensis Fish., Glycyrrhiza glabra L. and Glycyrrhiza inflata BAT, are used for medical purposes and in food. In general, G. glabra (European liquorice) is a Mediterranean species with an eastern boundary of Iran, Iraq, Central Asia and the northwestern part of China. Glycyrrhiza uralensis (Chinese liquorice) is found further east, growing in Central Asia, Mongolia and China. Glycyrrhiza inflata is found mainly in the Chinese province Xinjiang (Kondo et al., 2007; Hayashi & Sudo, 2009; Zimnitskaya, 2009). Glycyrrhiza uralensis and G. glabra are halophytic ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

perennial shrubs that grow to a height of 0.5–1.5 m. They have deep fusiform root systems, which in arid areas might be several meters long (Kushiev et al., 2005). In China, Glycyrrhiza species grow on dry grassy plains and sunny mountainsides in the northwestern provinces, where G. uralensis and G. glabra favour slightly alkaline and sandy soils. Both in China and Europe, liquorice is a top-selling herb and is applied in sweets and food industry for its sweet taste. In China, G. uralensis, ‘the king of herbs’, is one of the most used herb species in traditional medicine. Glycyrrhiza uralensis produces glycyrrhizin, a triterpenoid compound linked to several medical and other favourable properties, e.g. expectorant and antipyretic activities as well as antibacterial and antiviral effects (Fiore et al., 2008; Hayashi & Sudo, 2009). While there exist a vast FEMS Microbiol Ecol 79 (2012) 46–68

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Endophytic bacteria isolated from Glycyrrhiza nodules

number of pharmaceutical studies on different compounds produced by Glycyrrhiza spp. (e.g. 1500 reports on glycyrrhizin), much less is known about the biology and ecology of the plant itself. Roots and rhizomes of Glycyrrhiza have been collected from the wild. In China, collection of wild Glycyrrhiza plants was restricted in 1984 because it promoted desertification, and cultivation of G. uralensis was initiated in China in the early 1990s (Yamamoto & Tani, 2005). Glycyrrhiza can also be utilized in the rehabilitation of saline soils. In Uzbekistan, cultivation of G. glabra for 4 years in water-logged saline soils kept the water table below the critical level, decreased the soil and water salinity and successively increased the productivity of wheat and cotton crops compared to that of the local regional average (Kushiev et al., 2005). Glycyrrhiza spp. are able to fix N2 from the atmosphere in symbiosis with rhizobia. In this paper, rhizobia are defined as bacteria capable of inducing symbiotic N2-fixing nodules in legume roots or stems. Rhizobia spend part of their life cycle as saprophytes in the soil. Although Glycyrrhiza plants are widely cultivated, e.g. in China and southern Europe, their N2-fixing capacity has not been utilized, presumably due to lack of knowledge about their symbionts. The type strain of the species Mesorhizobium tianshanense, A-1BS, was isolated from Glycyrrhiza pallidiflora in Xinjiang, China (Chen et al., 1995), but otherwise there is no information about which rhizobial species induce effective nodules on the most common liquorice species, G. uralensis and G. glabra. Endophytic bacteria are defined as bacteria detected inside surface-sterilized plant parts or extracted from the inside of plant parts and having no visible harmful effects on plants. This definition includes internal colonizers with apparently neutral behaviour as well as symbionts (Hallman et al., 1997). In this paper, rhizobia are considered endophytic bacteria. Endophytic bacteria which are not able to form nodules on legumes are termed nonrhizobia. The host plant provides endophytes with a supply of nutrients and shelter from most abiotic stresses. In return for providing ‘bread and home’, plants may receive benefits from microbial associations by the enhancement of plant growth or reduction of plant stress, e.g. through the ability of bacterial ACC deaminase to modulate the level of ethylene produced by plants under stress (Lodewyckx et al., 2002; Hardoim et al., 2008). The purpose of this study was to explore the diversity of rhizobia that nodulate wild Glycyrrhiza plants in China. In previous studies all strains not producing typical rhizobial colonies were considered contaminants and were discarded from further investigations. This also resulted in concomitant loss of non-rhizobial endophytic bacteria in the root nodules. However, they might also FEMS Microbiol Ecol 79 (2012) 46–68

possess certain advantages, for instance assisting the symbiotic interaction between rhizobia and the host plant. Our strategy was to isolate all the rhizobia and non-rhizobial endophytes that inhabit root nodules. We also tested our non-rhizobial strains isolated from Glycyrrhiza for plant growth promoting (PGP) activities in cultures. Our third aim was to discover and identify rhizobial species that can be used as effective rhizobial inoculants in Glycyrrhiza cultivation in nitrogen-deficient soils or for rehabilitation of degraded soils. Biogeography is the study of the distribution of biodiversity over space and time (which species, how many, where and why; Hughes Martiny et al., 2006; Ramette & Tiedje, 2007a). Very little is known about the biogeography of rhizobia isolated from nodules of particular host plants. By applying multivariate analyses, we wanted to find out whether the variation of amplified fragment length polymorphism (AFLP) genomic fingerprint patterns of the bacterial strains obtained from Glycyrrhiza nodules exhibit specific biogeographical patterns. For the distancebased discriminant analysis (db-DA), our hypothesis was that AFLP patterns would be more similar in strains originating either from one geographical region or from the same Glycyrrhiza species. To understand mechanisms that drive the colonization of Glycyrrhiza nodules by particular bacterial species, we used the canonical correspondence analysis (CCA) to explore possible relationships between bacteria and environmental variables. We assumed that the more similar the AFLP patterns of strains were, the more similar the environmental requirements of the strains. Finally, we used variance partitioning to determine how much of the total variation in AFLP patterns is explained by the original Glycyrrhiza hosts and/or the geographic locations of the sampling sites.

Materials and methods Sampling

A total of 159 distinct bacterial strains, listed in Tables 1 and 2, were isolated from individual root nodules collected in June and August between 2001 and 2007 from G. uralensis, G. glabra, Glycyrrhiza squamulosa, Glycyrrhiza eurycarpa, G. inflata and a few taxonomically unidentified Glycyrrhiza plants growing on 40 sites in China. Twenty-seven sampling sites were located in the Xinjiang province in northwestern China. Because the Tianshan Mountains separate northern Xinjiang and southern Xinjiang, the sampling sites were further distinguished between them: 12 sites in the north and 15 sites in the south (Supporting Information, Fig. S1, Table S1). The rest of the sampling sites were situated relatively closely to each other in northern central China, in the Shaanxi ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

48

(six sites), Gansu (four sites) and Ningxia provinces (three sites) (Fig. S1, Table S1). The distance between the closest sites in Xinjiang and central China was about 2000 km. The sampling sites were located between latitudes 34° and 48° (N) and longitudes 75° and 109° (E), and at elevations between 290 and 2510 m above sea level. These data were obtained using the global positioning system. The climate of these regions is arid or semiarid, characterized by low rainfall, a long dry season and high evaporation, leading to high alkalization and salinization of soils. More detailed geographic and meteorological characteristics of each sampling sites are described in Table S1. Annual rainfall and annual mean temperature for each site were estimated from historical average data provided by the official regional websites. Soils of sampled sites were generally poor in organic matter and not fertilized. One plant species (several specimens) was sampled from each site. In Xinjiang, G. uralensis nodules were collected from 20 of 27 sites and G. glabra nodules were obtained from four sites. Nodules of G. squamulosa, G. eurycarpa and G. inflata were sampled only from a single site each. In central China, nodules from G. uralensis were obtained from six sites and those from unidentified Glycyrrhiza plants from seven sites (Table S1). Isolation of bacteria

Healthy, intact nodules were excised from the roots and placed in preservation vials containing a desiccant and brought to the laboratory. Nodules were surface-sterilized first with 95% ethanol for 5 min, then with 0.1% (w/v) mercuric chloride for 2–4 min and rinsed five times with sterile water according to Vincent (1970). Sterilized nodules were crushed, suspended in a small volume of sterile water, streaked out on yeast mannitol (YEM) agar (Vincent, 1970) and grown at 28 °C for 3–5 days. Single colonies were picked and checked for purity by repeated streaking and microscopic examination. When necessary, mixed bacterial cultures were purified by diluting cultures grown in YEM or tryptone-yeast extract (TY; Beringer, 1974) broth with Tween buffer (0.05 M phosphate, pH 6.6, 0.1% Tween 80) to separate different bacteria. The first dilution was shaken for at least 30 min, and dilutions of 10 5 and 10 6 were plated onto YEM agar containing 0.025 g kg 1 Congo red (Lindstro¨m et al., 1985) and grown for 3–5 days at 30 °C. All strains were maintained and stored on YEM agar and in 20% (v/w) glycerol at 70 °C. DNA extraction

DNA was isolated from rhizobial cells grown in TY broth for 2–3 days at 30 °C using mostly a hexadecylª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

L. Li et al.

trimethyl ammonium bromide (CTAB) procedure suitable for polysaccharide producing bacteria. This procedure, described in more detail in Appendix S1, was largely based on the CTAB method of Wilson (1994). Salt-extraction (Aljanabi & Martinez, 1997) with some modifications (Appendix S1) was applied when the CTAB method was unsuccessful. In both methods, DNA was extracted from a cell pellet of approximately 50 lL. The extracted DNA was dissolved in 30–50 lL of sterile ultra-pure water. AFLP fingerprinting

The AFLP procedure was largely based on the procedures described by Vos et al. (1995) and Dresler-Nurmi et al. (2000). The 25-lL restriction-ligation mixture contained 100–300 ng template DNA, 5 U EcoRI (Promega Corporation, Madison, WI) and Tru1l (MseI; MBI Fermentas), 10 pmol EcoRI, and 20 pmol Tru1l adapters and 1 U T4 DNA ligase in 19 ligase buffer (MBI Fermentas). This mixture was incubated at 37 °C and stored at 20 °C until use. Two primers with two selective bases (bold) were used in the PCR described in Appendix S1: EcoRIac (5′-gac tgc gta cca att cac-3′), and Tru1l-gc (5′-gat gag tcc tga agc-3′). The EcoRI-ac primer was fluorescently labelled at the 5′-end with blue 6-carboxyfluorescein (6FAM; TAG, Copenhagen A/S, Denmark). PCR products were checked, adjusted to a larger volume (50 lL) and purified with MicroSpin S-400 HR columns (Amersham Biosciences) according to the manufacturer’s instructions, and stored at 20 °C until use. Purified AFLP-PCR samples were analyzed by ABI Prism® 310 DNA Genetic Analyzer (PE Biosystems), the capillary of which was filled with denaturating polymer POP-6. A 3-lL volume of purified PCR product and 0.5 lL of internal red standard, GENESCAN™-500 TAMRA™ (Applied Biosystems, Carlsbad, CA) were added to 12 lL of deionized formamide. Samples were denatured for 2 min at 98 °C, chilled on ice and transferred to a sample tray. Samples were injected at 15 kV for 10 s into a 49-cm capillary and electrophoresed for 60 min at 50 °C. AFLP data were examined and processed using ABI GENESCAN analysis software (PE Biosystems) and transformed and analyzed using the BIONUMERICS software, version 5.0 (Applied Maths, Kortrijk, Belgium). Fragments between 35 and 500 bp were used for comparison. Fingerprints were normalized and checked manually. Fingerprint similarity values were calculated using the Dice coefficient with a position tolerance of 1.0% and optimization tolerance 0.5%. A dendrogram was constructed using the unweighted pair group method with arithmetic mean. FEMS Microbiol Ecol 79 (2012) 46–68

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Table 1. List of rhizobial strains isolated from root nodules of Glycyrrhiza spp. growing in Gansu, Ningxia, Shaanxi and Xinjiang in central and northwestern China Strain*

Geographic origin

Host plant

AFLP subgroup

NWXJ98 NWXJ100 NWXJ114 NWXJ90 NWXJ102 NWXJ111 NWXJ79 NWXJ97 NWXJ58 NWXJ06 NWXJ09 NWXJ35 NWXJ44 NWXJ80 NWXJ91 NWSX24 NWSX25 NWSX19 NWSX20 NWXJ52 NWXJ22-2 NWXJ36-2 NWXJ25 NWXJ30 NWXJ27-2 NWXJ43-2 NWXJ28 NWXJ32 NWGS07 NWNX05 NWGS10 NWGS11 NWSX23 NWSX26 NWSX08 NWSX11 NWGS03 NWSX06 NWGS01 NWGS09 NWSX09 NWSX10 NWGS02 NWSX01 NWSX02 NWSX05 NWSX15 NWXJ07 NWXJ12 NWXJ02 NWXJ05 NWXJ08 NWXJ14 NWXJ04 NWXJ15

Xinjiang Baicheng Xinjiang Baicheng Xinjiang Wuqia Xinjiang Baicheng Xinjiang Baicheng Xinjiang Tuofengxia Xinjiang Maigaiti Xinjiang Baicheng Xinjiang Habahe Xinjiang Bachu Xinjiang Bachu Xinjiang Chahan Xinjiang Haermodun Xinjiang Maigaiti Xinjiang Baicheng Shaanxi Yangling Shaanxi Yangling Shaanxi Yanan Shaanxi Yanan Xinjiang Beitun Xinjiang Ziniquan Xinjiang Chahan Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Hejing Xinjiang Ziniquan Xinjiang Ziniquan Gansu Chongxing Ningxia Zhongwei Gansu Chongxing Gansu Chongxing Shaanxi Yangling Shaanxi Yangling Shaanxi Yulin Shaanxi Yulin Gansu Huanxian Shaanxi Ziwuling Gansu Huanxian Gansu Chongxing Shaanxi Yulin Shaanxi Yulin Gansu Huanxian Shaanxi Huanglong Shaanxi Huanglong Shaanxi Ziwuling Shaanxi Yanan Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu

G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. squamulosa G. uralensis G. uralensis G. uralensis G. uralensis G. glabra G. uralensis G. squamulosa G. uralensis G. uralensis G. uralensis Glycyrrhiza sp. Glycyrrhiza sp. G. uralensis G. glabra G. glabra G. glabra G. glabra G. glabra G. uralensis G. glabra G. glabra Glycyrrhiza sp. Glycyrrhiza sp. Glycyrrhiza sp. Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis

1a 1a 1a 1a 1a 1a 1a 1a 1a 1b 1b 1b 1c 2 2 3a 3a 3a 3a 3a 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3b 3c 3c 3c 3c 3c 3c 3c 3c 3d 3d 3d 3d 3d 3d 3d 4a 4a 4a 4a 4a 4a 4a 4a

FEMS Microbiol Ecol 79 (2012) 46–68

Closest species and strain (accession no.), max identity Rhizobium daejeonense L61T (AY341343) 97%

Rhizobium daejeonense L61T (AY341343) 97% Rhizobium daejeonense L61T (AY341343) 99%

Rhizobium cellulosilyticum ALA10B2T (DQ855276) 98% Rhizobium cellulosilyticum ALA10B2T (DQ855276) 97% Phyllobacterium sp. STM370 (AJ968698) 96% Mesorhizobium mediterraneum LMG17148T (AM181745) 97%

Mesorhizobium tianshanense A-1BST (AF041447) 99%

Mesorhizobium tianshanense A-1BST (AF041447) 97%

Mesorhizobium tianshanense A-1BST (AF041447) 97% Mesorhizobium mediterraneum LMG17148T (AM181745) 97%

Mesorhizobium mediterraneum LMG17148T (AM181745) 99%

Rhizobium gallicum R602spT (U86343) 99%

Rhizobium gallicum R602spT (U86343) 96%

Mesorhizobium sp. Mad-1 (EF364378) 96%


ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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L. Li et al.

Table 1. Continued Strain*

Geographic origin

Host plant

AFLP subgroup

NWXJ16 NWXJ17 NWXJ18 NWXJ19 NWXJ01 NWXJ42 NWXJ87 NWGS06 NWSX04 NWSX13 NWSX14 NWXJ77 NWXJ78 NWXJ86 NWXJ115 NWXJ116 NWXJ81 NWXJ85 NWXJ82 NWXJ84 NWXJ83 NWXJ71 NWXJ73 NWXJ106 NWXJ107 NWGS05 NWSX22 NWXJ54 NWXJ03 NWXJ20 NWXJ21 NWXJ22 NWXJ27 NWXJ29 NWXJ37 NWXJ36 NWXJ34 NWXJ38 NWXJ40 NWXJ33 NWXJ41 NWXJ31 NWXJ26 NWXJ43 NWNX01 NWNX02 NWXJ10 NWXJ75 NWXJ76 NWSX03 NWXJ23 NWGS04 NWXJ92 NWXJ112 NWXJ88 NWXJ104

Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Bachu Xinjiang Hejing Xinjiang Maigaiti Gansu Minqin Shaanxi Huanglong Shaanxi Fuxian Shaanxi Fuxian Xinjiang Maigaiti Xinjiang Maigaiti Xinjiang Maigaiti Xinjiang Yuepuhu Xinjiang Yuepuhu Xinjiang Xiamale Xinjiang Maigaiti Xinjiang Xiamale Xinjiang Xiamale Xinjiang Xiamale Xinjiang12tuan Xinjiang12tuan Xinjiang Tada Xinjiang Tada Gansu Lingtai Shaanxi Yangling Xinjiang Gongliu Xinjiang Bachu Xinjiang Bachu Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Chahan Xinjiang Chahan Xinjiang Chahan Xinjiang Chahan Xinjiang Chahan Xinjiang Chahan Xinjiang Chahan Xinjiang Ziniquan Xinjiang Ziniquan Xinjiang Hejing Ningxia Yinchuan Ningxia Yinchuan Xinjiang Bachu Xinjiang Xiamale Xinjiang Xiamale Shaanxi Huanglong Xinjiang Ziniquan Gansu Huanxian Xinjiang Baicheng Xinjiang Tuofengxia Xinjiang Maigaiti Xinjiang Baicheng

G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. squamulosa Glycyrrhiza sp. G. uralensis Glycyrrhiza sp. Glycyrrhiza sp. G. squamulosa G. squamulosa G. squamulosa G. inflata G. inflata G. eurycarpa G. squamulosa G. eurycarpa G. eurycarpa G. eurycarpa G. glabra G. glabra G. glabra G. glabra Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis G. uralensis G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. glabra G. uralensis G. uralensis G. uralensis G. uralensis G. eurycarpa G. eurycarpa G. uralensis G. glabra G. uralensis G. uralensis G. uralensis G. squamulosa G. uralensis

4a 4a 4a 4a 4a 4a 4b 4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4c 4e 4e 4e 4f 4f 4f 4g 5a 5b 5d 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 5e 6a 6a 6a 6a 6a 6a 6a 6a 6a 6a 6a 6a


Closest species and strain (accession no.), max identity


Sinorhizobium meliloti LMG6133T (X67222) 97%

Sinorhizobium meliloti LMG6133T (X67222) 99% Sinorhizobium meliloti LMG6133T (X67222) 97%

Phyllobacterium ifriqiyense STM370T (AY785325) 97% Rhizobium giardinii H152T (U86344) 99% Rhizobium leguminosarum Rtl E12 (U73209) 97% Mesorhizobium amorphae ACCC19665T (AF041442) 94% Unidentified Rhizobium galegae LMG6214T (X67226) 97%

Mesorhizobium tianshanense ST-2 (AY225401) 99%

Mesorhizobium tianshanense ST-2 (AY225401) 99% Mesorhizobium tianshanense ST-2 (AY225401) 97% Agrobacterium tumefaciens LMG140T (AM181758) 96%

Agrobacterium tumefaciens LMG140T (AM181758) 97% Agrobacterium tumefaciens LMG140T (AM181758) 96%


51


Table 1. Continued Strain*

Geographic origin

Host plant

AFLP subgroup

NWXJ110 NWXJ109 NWXJ53 NWXJ108 NWNX03 NWXJ55

Xinjiang Tuofengxia Xinjiang Tuofengshang Xinjiang Beitun Xinjiang Tuofengshang Ningxia Yinchuan Xinjiang Gongliu

G. G. G. G. G. G.

6a 6b 6c 6c 6d 6e

uralensis uralensis uralensis uralensis uralensis uralensis

Closest species and strain (accession no.), max identity

Agrobacterium tumefaciens LMG140T (AM181758) 96% Unidentified Phyllobacterium bourgognense STM201T (AY785320) 97%

*Partial 16S rDNA was sequenced for strains written in bold.

Identification of bacterial strains

Based on the AFLP results, representative strains of each AFLP subgroup were subjected to partial sequencing of the 16S rRNA gene (1000 bp). Sequencing was done directly from PCR products that were amplified (Appendix S1) using primers fD1 (5′-aga gtt tga tcc tgg ctc ag3′) and rD1 (5′-aag gag gtg atc cag cc-3′) (Weisburg et al., 1991) by the Institute of Biotechnology, University of Helsinki. The most similar sequences were searched for and nucleotide alignments were constructed by SILVA comprehensive ribosomal RNA databases (http://www.arbsilva.de/aligner/) and manually corrected in ARB software (Ludwig et al., 2004). Results were confirmed by comparing sequences against a nucleotide database (BLAST). Nodulation tests

Thirty strains representing prevalent AFLP subgroups were tested for their nodulation ability on G. uralensis and G. glabra. Seeds were surface-sterilized by immersing them in concentrated H2SO4 solution for 1 h, rinsed with sterile water and germinated on 1% water agar for 2–3 days as described by Ra¨sa¨nen et al. (2001). Plants were grown in duplicate glass jars filled with a sterilized mixture of washed sand, vermiculite and ceramic Lecagravel (Ra¨sa¨nen et al., 2001). Five seedlings were transferred to each glass jar and inoculated on either the same or the following day with 10 mL of bacterial cultures grown in YEM broth to the early stationary phase and diluted with sterile water at 106 CFU mL 1. Non-inoculated plants were used as controls. The seedlings were grown in a growth cabinet with a 18 h light period (at 20 °C for 1 h, 24 °C for 16 h and 20 °C for 1 h) and with a 6-h dark period at 16 °C, and watered alternatively with sterilized quarter strength Jensen’s medium (Vincent, 1970) or water. At harvest, after 45 and 50 days, the colour and appearance of shoots were recorded. Roots were rinsed with tap water and the nodulation status of roots was checked. Nodulation results were recorded as positive (nodules were found) or negative (nodules were not found). Nodules were considered to be N2-fixing or FEMS Microbiol Ecol 79 (2012) 46–68

effective if plants were green and healthy, and ineffective if plants were yellow with a similar appearance to uninoculated plants. PGP activity tests

Indoleacetic acid (IAA) production was assayed from bacterial cultures grown in King’s B broth (King et al., 1954) for 3–7 days at 30 °C using a colorimetric method (Egamberdieva & Kucharova, 2009). After centrifugation, bacterial supernatant was mixed with the Salkowski reagent (1 : 1 v/v). A pink colour indicating indole production was recorded after 30 min. Results were confirmed by measuring the absorbance at 530 nm. Phosphate-solubilizing bacteria were screened on the solid Pikovskya’s medium supplemented with Ca3(PO4)2 (5 g L 1) and Bromophenol Blue (0.025 g L 1) (Srividya et al., 2009), and incubated for 5–7 days at 30 °C. Production of acid changed the indicator colour from blue to yellow. The formation of a clear halo around the bacterial colonies indicated a positive reaction. Siderophore production was assayed with chrome azurol S (CAS) agar (Alexander & Zuberer, 1991). After incubation for 5–7 days, siderophore-producing strains had an orange, purple or purplish-red halo around the colonies. Protease activity was assayed with YEM agar containing 5% skimmed milk. After incubation for 5–7 days, a clear halo around the bacterial colonies due to hydrolysis of milk casein indicated a positive reaction. Lipase activity was assayed with modified Sierra lipolysis agar (Sierra, 1957) supplemented with beef extract (3 g L 1) and ferrous citrate (0.2 g L 1). After autoclaving, 10 mL of Tween 80 and 50 mL of Victoria Blue B solution (0.1 g per 150 mL) was added to the medium. After incubation for 5 days, white calcium precipitates around the bacterial colonies indicated a positive reaction. Cellulase activity was assayed with GYM Streptomyces medium (DSMZ; http://www.dsmz.de/microorganisms/media_list.php; medium 65) but without CaCO3 and supplemented with carboxymethyl cellulose (5 g L 1; ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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L. Li et al.

Table 2. List of non-nodulating endophytic bacteria isolated from root nodules of Glycyrrhiza spp. growing in Xinjiang, Gansu, Ningxia and Shaanxi, with data about their nodulation and plant growth promoting activities

Strain*,†,‡ NWXJ80

†

NWXJ91 NWXJ11† NWNX04 NWGS12 NWXJ31-2 NWSX17

NWXJ93 NWXJ99† NWXJ49 NWXJ51 NWXJ95 NWXJ48 NWXJ47 NWXJ56 NWXJ57 NWXJ65†

NWXJ66 NWXJ113† NWXJ62 NWXJ63 NWXJ61

Geographic origin

Host plant

Xinjiang Maigaiti

G. squamulosa

2

Xinjiang Baicheng Xinjiang Bachu Ningxia Tongxin Gansu Chongxing Xinjiang Ziniquan Shaanxi Yanan

G. uralensis

Xinjiang Baicheng Xinjiang Baicheng Xinjiang Beitun Xinjiang Beitun Xinjiang Baicheng Xinjiang Beitun Xinjiang Baishidun Xinjiang Habahe Xinjiang Habahe Xinjiang 128tuan Xinjiang 128tuan Xinjiang Tuofengxia Xinjiang Heshuo Xinjiang Kelamayi Xinjiang Heshuo

AFLP subgroup

Closest species and strain (accession no.) max identity

Exo-enzymes††

IAA§

P solubilizing¶

Siderophore production**

+

+

++

+

2

+

+

++

+

G. uralensis

4d

+

Glycyrrhiza sp. Glycyrrhiza sp. G. glabra

4d

+++

4d

+

++

++

4d

+

+

++

Phyllobacterium sp. STM370 (AJ968698) 96%

Lipase

Cellulase

ng ++

+ +

+ +

+

ng

ng

Glycyrrhiza sp.

4d

G. uralensis

4d

+

++

+

G. uralensis

4d

+

++

+

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

G. uralensis

4d

ng

G. uralensis

4d

+

G. uralensis

4d


Paenibacillus amylolyticus NRS-290T (D85396) 95%

Protease‡‡

ng

+ +

+ ++

+

++

+ +

+

+

+ +

+ Paenibacillus graminis RSA19T (AJ223987) 93%

ng

+

+ ng

+ +

Paenibacillus sp.

+

ng

+


53


Table 2. Continued Exo-enzymes††

Host plant

AFLP subgroup

G. uralensis

4d

+

G. uralensis

4d

+

G. uralensis

4d

+

G. uralensis

4d

+

G. uralensis G. uralensis

4d 4d

Glycyrrhiza sp. G. uralensis

4d

+

++

4d

++

++

G. uralensis

4d

Paenibacillus sp.

Glycyrrhiza sp.

4d

Paenibacillus pasadenensis SAFN-007T (AY167820) 99%

Xinjiang Xiamale Xinjiang Xiamale Xinjiang Xiamale

G. eurycarpa G. eurycarpa G. eurycarpa

NWXJ45†

Xinjiang Aletai

G. uralensis

5c

NWXJ96

Xinjiang Baicheng Xinjiang Buerjin

G. uralensis

5c

G. uralensis

5f

Paenibacillus polymyxa EJS-3 (DQ120522) 99%

NWNX01

Ningxia Yinchuan

G. uralensis

6a

Agrobacterium tumefaciens LMG140T (AM181758) 96%

NWNX02

Ningxia Yinchuan Xinjiang Bachu Xinjiang Xiamale

G. uralensis

Strain*,†,‡ NWXJ69 NWXJ70 NWXJ67 NWXJ68 NWXJ74 NWXJ13 NWSX18 NWSX21 NWXJ64 NWGS08

NWXJ82 NWXJ84‡ NWXJ83

NWXJ46†

NWXJ10 NWXJ75

Geographic origin


Xinjiang Tekesi Xinjiang Zeketai Xinjiang Yishao Xinjiang Yishao Xinjiang Alaer Xinjiang Bachu Shaanxi Yanan Shaanxi Yangling Xinjiang 128tuan Gansu Chongxing

Paenibacillus sp.

IAA§

nd nd

P solubilizing¶

nd nd


nd nd

Protease‡‡

nd nd

Lipase

Cellulase

+

nd nd

nd nd +

ng

+

+ ++

++

4e

+

+

+

4e

+++

++

+

+

++

+

4e

Phyllobacterium ifriqiyense STM370T (AY785325) 97% Pantoea agglomerans SP1 (AF199029) 96%

+

+

+

+

++

+

+

++

++

+

+

+

++

+

++

+

6a

++

+

++

+

G. uralensis

6a

++

+

++

+

G. eurycarpa

6a

+

+

++

+


++

+

+


54

L. Li et al.

Table 2. Continued

Strain*,†,‡ NWXJ76 NWSX03 NWXJ23 NWGS04 NWXJ92† NWXJ112 NWXJ88

Geographic origin Xinjiang Xiamale Shaanxi Huanglong Xinjiang Ziniquan Gansu Huanxian Xinjiang Baicheng Xinjiang Tuofengxia Xinjiang Maigaiti

Host plant

AFLP subgroup


Exo-enzymes††

IAA§

P solubilizing¶


Protease‡‡

Lipase

Cellulase

G. eurycarpa G. uralensis

6a

++

+

++

+

6a

++

+

++

+

G. glabra

6a

+++

+

++

+

G. uralensis

6a

+

+

++

+

G. uralensis

6a

++

+

++

+

G. uralensis

6a

+

+

++

+

G. squamulosa

6a

+

++

++

+

+

+

++

+

Agrobacterium tumefaciens LMG140T (AM181758) 97% Agrobacterium tumefaciens LMG140T (AM181758) 96%

NWXJ104

Xinjiang Baicheng

G. uralensis

6a

NWXJ110

Xinjiang Tuofengxia Xinjiang Tuofengshang Xinjiang Beitun

G. uralensis

6a

+

+

++

+

G. uralensis

6b

+

+

++

+

G. uralensis

6c

+

+

++

+

G. uralensis

6c

+

+

++

+

G. uralensis

6d

Unidentified

+

+

++

+

G. uralensis

6e

P. bourgognense STM201T (AY785320) 97%

+

+

+

+

G. uralensis

6f

+

++

++

+

G. uralensis

6f

+

++

++

+

G. uralensis

6f

+

++

++

+

++

+

++

+

+

+

++

+

NWXJ109 NWXJ53

NWXJ108 NWNX03 NWXJ55†

NWXJ89 NWXJ103 NWXJ101

Xinjiang Tuofengshang Ningxia Yinchuan Xinjiang Gongliu Xinjiang Baicheng Xinjiang Baicheng Xinjiang Baicheng

NWXJ59

Xinjiang Habahe

G. uralensis

7a

NWSX12

Shaanxi Fuxian Gansu Chongxing

Glycyrrhiza sp. Glycyrrhiza sp.

7b

Xinjiang Ziniquan

G. glabra

NWGS13

NWXJ24


Agrobacterium tumefaciens LMG140T (AM181758) 96%

Erwinia cypripedii ATCC29267T (U80201) 95% Enterobacter cloacae 766 (AM778415) 98% Unidentified Rhodobacter changlensis JA139T (AM399030) 99% Unidentified

+

++

+

+

+

+

+

++

++

+

+


55


Table 2. Continued

Geographic origin

Host plant

NWSX16

Shaanxi Yanan

Glycyrrhiza sp.

NWSX07

Shaanxi Yulin

G. uralensis

Strain*,†,‡

AFLP subgroup

Closest species and strain (accession no.) max identity Microbacterium oxydans DSM20578T (Y17227) 97% Unidentified

Exo-enzymes††

IAA§

P solubilizing¶


++

++

+

++

Protease‡‡

Lipase

Cellulase

ng, Not growing in a medium used; nd, not done. *Partial 16S rDNA was sequenced for strains written in bold. † Strain was not able to nodulate the plant tested, G. uralensis. ‡ In plant tests only strain NWXJ84 (subgroup 4e) induced ineffective, small and white nodules on G. glabra but no nodules on G. uralensis. § IAA = production of the plant hormone IAA. Pink color indicated indole production. Values in parentheses refer to the absorbance at 530 nm: no production (< 0.1); + weak (0.1–0.2); ++ moderate (0.2–0.3); +++ good (> 0.3). ¶ Solubilisation of phosphate: no solubilisation; + production of acid (change in colour from blue to yellow); ++ solubilisation (a clear halo around the colony). **Siderophore production: no production; + weak; ++ moderate or strong. †† Plate assay. ‡‡ Protease production: no production; + weak halo around the colony; ++ clear halo around the colony.

Sigma) in place of glucose. After incubation for 5 days, plates were stained with Congo red solution and destained with NaCl according to Teather & Wood (1982). A clear or lightly coloured halo around the colonies indicated a positive reaction. Statistical analyses

Nonparametric multivariate methods were used to analyze data, as the environmental variables and AFLP fingerprint data obtained from bacterial strains were not normally distributed. Permutations were used to calculate the significance. P-values were considered significant when P 0.05. The nonparametric discriminant analysis based on distances (db-DA) (Anderson & Robinson, 2003) was performed to find out whether the AFLP fingerprint patterns discriminate between geographic sampling regions and/or host plant species. Strains were divided in advance (a priori) into groups within each category (sampling region and host plant). The db-DA was performed in the R environment (R Development Core Team, 2007) using the package BIODIVERSITYR (Kindt & Coe, 2005) and its function ‘CAPdiscrim’. The function ‘permutations’, with 9999 permutations, was used to test the null hypothesis that strains were distributed randomly over a priori groups. The homogeneity of multivariate group dispersions (variances) of a priori groups (Anderson, 2006) was tested using the package VEGAN (Oksanen et al., 2008) and its function ‘betadisper’. The function ‘permutest’, with 9999 permutations, was used to compare pairwise multivariate dispersions of the groups, the null hypothesis FEMS Microbiol Ecol 79 (2012) 46–68

being that there were no differences in dispersion between groups. For db-DA and the test for homogeneity of multivariate dispersions, the matrix of similarities (transformed to dissimilarities) of bacterial AFLP fingerprint data was obtained using the Dice coefficient. A constrained ordination method, CCA, was used to determine whether environmental variables of sampling sites could explain the distribution of particular bacterial groups, species (AFLP subgroups) or strains. CCA can model the unimodal relationships, such as those between AFLP patterns representing strains and environmental variables. CCA is also well suited for absence–presence data, such as ours. The response of the distribution of strains to environmental variables was first explored by fitting environmental variables on the AFLP ordination based on canonical analysis of principal coordinates (CAP; Anderson & Gribble, 1998) in the R environment (R Development Core Team, 2007) using the VEGAN package (Oksanen et al., 2008) and its functions ‘capscale’ and ‘ordisurf’. CCA was performed using the R package VEGAN (Oksanen et al., 2008) and its function ‘cca’. The function ‘permutest’ with 9999 random permutations with a reduced model was used to test the null hypothesis that the distribution of AFLP subgroups (species) or strains was unrelated to environmental variables. Variance partitioning (Borcard et al., 1992; Legendre & Legendre, 1998) was used to determine how much of the variation of AFLP patterns (strains) was explained by either the host plant species or the geographic location of the sampling site. First, a series of distance-based redundancy analysis (db-RDA; Anderson, 2001; McArdle & Anderson, 2001) was ran according to Legendre & ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

56

Legendre (1998) using the program DISTML (Anderson, 2004) with 9999 permutations of raw data. RDA runs were used to partition the variation to pure proportions of host plant and geographic location and to the shared proportion of both host plant and geographic location. For db-RDA, the matrix of similarities (transformed to dissimilarities) of AFLP fingerprint data was obtained using the Dice coefficient. Geographic coordinates (latitude and longitude, WGS 84) of the sampling sites were projected in the geographical information system and converted to metric coordinate units (projection China Gauss-Kruger Beijing 1954 zone 17) (MAPINFO PROFESSIONAL version 10.0; Pitney Bowes Software Inc. 2009).

Results Distribution of Glycyrrhiza isolates

Originally, 167 bacterial strains (Fig. S2) were isolated from surface-sterilized root nodules from five wild Glycyrrhiza species growing on 40 sites in four provinces in northwestern and central China. When identical strains were excluded, 159 distinct strains were identified from Glycyrrhiza nodules (Tables 1 and 2). Strains were considered to be identical when in the dendrogram of AFLP fingerprints, the similarity value of two or more strains was 100% and they originated from the same sampling site. Most of the sampling sites (27) were located in Xinjiang in northwestern China (Table S1) and most of the bacterial strains (115) were also derived from this province (Table 3). Furthermore, 37 strains were obtained from 12 sites in northern Xinjiang and 78 strains from 15 sites in southern Xinjiang. The rest of the strains came from 13 sites in the provinces Shaanxi (26 strains), Gansu (13 strains) and Ningxia (five strains) in central China (Table S1, Table 3). Almost 60% of the bacteria were isolated from G. uralensis. Of these, 22 strains originated from northern Xinjiang (11 sites), 49 strains from southern Xinjiang (9 sites) and 24 strains from Central China (6 sites). The distribution of strains among other host plants, which were sampled only in Xinjiang, was: 28 strains from G. glabra, eight strains from G. squamulosa, six strains from G. eurycarpa and two strains from G. inflata. Twenty strains were obtained from nodules of unidentified Glycyrrhiza plants growing on seven sites in central China (Table 3, Table S1). Diversity and identity of Glycyrrhiza isolates

In the AFLP analysis the strains were divided into seven genomic groups at the similarity level of 50%. Four single strains remained unclustered (Fig. S2). The seven groups ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

L. Li et al.

were further divided into subgroups at 60% similarity level (Fig. S2). Sequencing of partial 16S rRNA gene (1000 bp) from representative strains revealed that each AFLP subgroup consisted of strains belonging to the same species or to a distinct group of closely related strains (Tables 1 and 2). AFLP group 1 consisted of three subgroups (a, b, c), from which the largest subgroup 1a (nine strains) was identified as Rhizobium daejeonense. Subgroups 1b and 1c, altogether four strains, were identified as Rhizobium cellulosilyticum. AFLP group 2 contained two Phyllobacterium sp. strains. The majority of strains in AFLP group 3 belonged to the genus Mesorhizobium. Subgroups 3a and 3c with 13 strains represented Mesorhizobium mediterraneum and subgroup 3b with 12 strains M. tianshanense. Subgroup 3d with seven strains was related to Rhizobium gallicum (Table 1, Fig. S2). AFLP group 4 contained several different rhizobial species. Subgroups 4a and 4b (altogether 15 strains) belonged to the genus Mesorhizobium, termed Mesorhizobium group A. This group was especially interesting because the sequenced strains of subgroup 4a were not closely related to any recognized species, showing only 96 –98% identity to Mesorhizobium sp. strain Mad-1 (Table 1) isolated from Ononis tridenta in Spain (Rincoń et al., 2008). Subgroup 4c with 11 strains was related to Sinorhizobium meliloti. The small subgroups 4e and 4f with three strains each were related to Phyllobacterium ifriqiyense and Rhizobium giardinii, respectively. The only representative of subgroup 4g was identified as Rhizobium leguminosarum (Table 1, Fig. S2). AFLP group 5 contained the second largest Mesorhizobium subgroup, 5e (16 strains). The closest strain in the nucleotide database was M. tianshanense strain ST-2 (99% identity, Table 1) obtained from a chickpea nodule in Portugal (Laranjo et al., 2004). However, the taxonomy of this subgroup remains unresolved because other close strains in the database with equal identity belonged to the species M. mediterraneum, M. gobiense and M. tianshanense. Obviously, these closely related species cannot be distinguished reliably using the 16S rRNA gene alone for identification. Subgroup 5e was named Mesorhizobium group B. The single representatives of subgroups 5a and 5d were distantly related to Mesorhizobium amorphae and Rhizobium galegae, respectively. The 15 strains in subgroups 6a and 6c belonged to Agrobacterium tumefaciens. The only representative of subgroup 6b was also included in A. tumefaciens because strain NWXJ109 was situated between subgroups 6a and 6c in the dendrogram of AFLP fingerprints (Fig. S2). The one strain in subgroup 6e was Phyllobacterium bourgognense (Table 1, Fig. S2). FEMS Microbiol Ecol 79 (2012) 46–68

57


Table 3. Numbers of strains isolated from Glycyrrhiza nodules and their distribution among bacterial species and host plants growing in northern Xinjiang, southern Xinjiang and central China. Ninety-five strains were isolated from Glycyrrhiza uralensis (Gu), 28 strains from Glycyrrhiza glabra (Gg), 16 strains from Glycyrrhiza eurycarpa (Ge), Glycyrrhiza inflata (Gi) and Glycyrrhiza squamulosa (Gs), and 20 strains from taxonomically unidentified Glycyrrhiza species (Gsp)

Bacterial class Alphaproteobacteria M.mediterraneum M.tianshanense Mesorhizobium group A Mesorhizobium group B Rhizobium daejeonense Rhizobium cellulosilyticum Rhizobium gallicum Sinorhizobium meliloti Agrobacterium tumefaciens Phyllobacterium spp. Rhizobium giardinii Other species† Total Gammaproteobacteria Pantoea agglomerans Erwinia cypripedii Enterobacter cloacae Total Gram-positive bacteria Paenibacillus spp. Microbacterium oxydans Total Unidentified strains‡ Total All strains per region

AFLP subgroup

Northern Xinjiang

Central China*

Southern Xinjiang

Gu

Gg

Gu

Gg

Ge Gi Gs

Gu

Gsp

Strain no. per species

3a, 3c 3b 4a, 4b 5e 1a 1b, 1c 3d 4c 6a, b, c 2, 4e, 6e 4f 5a, 4g, 5d

1 0 0 0 1 0 0 0 1 1 0 1 5

0 6 0 6 0 0 0 0 1 0 0 0 13

0 1 14 3 7 3 0 0 7 1 0 0 36

0 1 0 7 0 1 0 0 0 0 3 1 13

0 0 1 0 1 0 0 7 3 4 0 0 16

9 0 0 0 0 0 6 1 4 0 0 0 20

3 4 0 0 0 0 1 3 0 0 0 2 13

13 12 15 16 9 4 7 11 16 6 3 4 116

5c 6f 7a

1 0 1 2

0 0 0 0

1 3 0 4

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

2 3 1 6

15 0 15 0 22

1 0 1 1 15 37

9 0 9 0 49

0 0 0 0 13

0 0 0 0 16 78

1 0 1 3 24

5 1 6 1 20 44

31 1 32 5 159

4d, 5f

5b, 6d, 7b

*Provinces Shaanxi, Gansu and Ningxia. Mesorhizobium amorphae (5a), Rhizobium leguminosarum (4g), Rhizobium galegae (5d), and Rhodobacter changlensis. ‡ AFLP subgroups 5b, 6d and 7b with single strains, and strains NWXJ24 and NWSX07. †

Except for the single strain in subgroup 5f identified as Paenibacillus polymyxa, 30 Paenibacillus strains were found mainly in subgroup 4d. Compared to other subgroups this subgroup was genetically more homogeneous, although its strains could represent several species (e.g. Paenibacillus pasadenensis, Paenibacillus amylolyticus, Paenibacillus graminis-like sp.). The rest of the identified endophytic bacteria represented a mixed collection of species having one to three strains: Pantoea agglomerans (subgroub 5c), Erwinia cypripedii (subgroup 6f), Enterobacter cloacae (subgroup 7a) and unclustered Rhodobacter changlensis and Microbacterium oxydans (Table 2, Fig. S2). Five strains remained unidentified because PCR of the 16S rRNA gene was not successful [single representatives of subgroups 5b (NWSX22), 6d (NWNX03) and 7b (NWSX12) and two unclustered strains (NWXJ24 and NWSX07)]. In summary, many rhizobial and non-rhizobial species were identified among Glycyrrhiza isolates. In total, 73% FEMS Microbiol Ecol 79 (2012) 46–68

of the strains were Alphaproteobacteria, with 57 strains representing Mesorhizobium, 25 Rhizobium, 11 Sinorhizobium (syn. Ensifer) and six Phyllobacterium. The rest of the 16 Alphaproteobacteria strains belonged mainly to the genus Agrobacterium (Table 3). Twenty percent of all the strains were Gram-positive bacteria, most of which belonged to the genus Paenibacillus within the Firmicutes (low G+C group). Six strains were members of the Gammaproteobacteria, representing the genera Erwinia, Pantoea and Enterobacter (Table 3). Symbiotic properties of Glycyrrhiza isolates

Our work revealed that mesorhizobia are the most important rhizobial symbionts for Glycyrrhiza plants growing in China. Fifty-eight percent of all rhizobial strains identified were mesorhizobia, comprising M. mediterraneum (AFLP subgroups 3a, 3c), M. tianshanense ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

58

L. Li et al.

(3b), Mesorhizobium group A (4a) and Mesorhizobium group B (5e) (Table 3). Nearly all mesorhizobial strains tested (14/15) induced N2-fixing nodules on G. uralensis and/or G. glabra (Table 4). Only the M. mediterraneum strain NWSX23 (3c) originally isolated from G. uralensis induced ineffective nodules on both test plants. The origin of a Mesorhizobium strain, i.e. host plant and isolation site, did not influence nodulation phenotype, and strains isolated from G. uralensis were capable of forming effective nodules on G. glabra and vice versa. Only one M. mediterraneum strain, strain NWSX20 (3a) isolated from an unidentified Glycyrrhiza plant species in central China, produced effective nodules on G. uralensis but ineffective ones on G. glabra (Table 4). The nodulation and N2-fixing capacity of the other rhizobial species, altogether 42 strains, was irregular. Four of 10 species, namely S. meliloti, R. gallicum, R. galegae and R. leguminosarum, had strains which were able to produce effective, N2-fixing nodules on G. uralensis and/or

G. glabra (Table 4). Effective R. galegae and R. leguminosarum were not typical symbionts of Glycyrrhiza because they possessed only a single representative each among the Glycyrrhiza isolates (Table 3). Sinorhizobium meliloti and R. gallicum also included ineffective (S. meliloti NWGS06) and non-nodulating strains (R. gallicum NWSX15), both of which were originally isolated from unidentified Glycyrrhiza species (Table 4). A few strains – M. mediterraneum NWSX20, S. meliloti NWXJ116 and R. leguminosarum NWXJ107 – showed stricter host specificity, producing effective nodules on G. uralensis but ineffective ones on G. glabra (Table 4). Strains belonging to R. giardinii and R. cellulosilyticum as well as Phyllobacterium strain NWXJ84 from subgroup 4e induced only ineffective nodules on both test plants (R. giardinii NWXJ73) or on G. glabra (R. cellulosilyticum NWXJ44, strain NWXJ84). The sequenced representative of subgroup 4e, strain NWXJ83, showed 97% identity to the P. ifriqiyense strain STM 370T (Table 2) obtained

Table 4. Nodulation and nitrogen fixation of Glycyrrhiza uralensis and Glycyrrhiza glabra inoculated with rhizobial strains isolated from Glycyrrhiza. Most strains originated in Xinjiang in northwestern China. Eight strains equipped with an asterisk (*) were obtained from the three provinces located in central China. Plants were grown in growth cabinet for 7 weeks in two replicate jars containing sterilized mixture of sand, vermiculate and Leca-gravel

Strain

Host plant

Closest species

AFLP group

NWXJ58 NWXJ114 NWXJ44 NWSX20* NWSX24* NWXJ32 NWXJ36-2 NWXJ43-2 NWGS10* NWGS03* NWSX23* NWSX10* NWSX15* NWXJ02 NWXJ14 NWXJ19 NWXJ81 NWXJ116 NWGS06* NWXJ73 NWXJ107 NWXJ54 NWXJ21 NWXJ31 NWXJ36 NWXJ43

G. uralensis G. uralensis G. uralensis Glycyrrhiza sp. G. uralensis G. glabra G. glabra G. uralensis Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis Glycyrrhiza sp. G. uralensis G. uralensis G. uralensis G. eurycarpa G. inflata Glycyrrhiza sp. G. glabra G. glabra G. uralensis G. glabra G. glabra G. glabra G. uralensis

R. daejeonense R. daejeonense R. cellulosilyticum M. mediterraneum M. mediterraneum M. tianshanense M. tianshanense M. tianshanense M. tianshanense M. mediterraneum M. mediterraneum R. gallicum R. gallicum Mesorhizobium sp. Mesorhizobium sp. Mesorhizobium sp. S. meliloti S. meliloti S. meliloti R. giardinii R. leguminosarum R. galegae Mesorhizobium sp. Mesorhizobium sp. Mesorhizobium sp. Mesorhizobium sp.

1a 1a 1c 3a 3a 3b 3b 3b 3b 3c 3c 3d 3d 4a 4a 4a 4c 4c 4c 4f 4g 5d 5e 5e 5e 5e

A A A

B B B B

G. uralensis

G. glabra

Nod

Fix†

+ + + + + + + + +

+ + + + + + +

+ + + + + + + + + + + + +

+ + + + + +

+ + + + + +

Nod

Fix

+ + + + + + + + + +

+ + + + + +

+ + + + + + + + + + + + +

+ + + + +

+ + + + +

Nod, nodulation; Fix, nitrogen fixation. This capability was deduced from the colour of plants. Green colour indicated effective nodules and yellow indicated ineffective ones.

†



59


from Lathyrus numidicus nodule (Mantelin et al., 2006). Strains belonging to R. daejeonense, Phyllobacterium sp. and P. bourgognense did not nodulate at all. This was also the case for the endophytic species A. tumefaciens, Pantoea sp. and Paenibacillus spp. (Tables 2 and 4). Altogether, of 115 strains belonging the Rhizobiales order, 57 rhizobial strains were effective Mesorhizobium species, 20 strains belonged to less effective species (S. meliloti, R. gallicum, R. galegae and R. leguminosarum) and nine strains to ineffective species. When the nodulation data were compared with data about bacterial species isolated from Glycyrrhiza nodules on each site, it appeared that G. uralensis plants growing in Xinjiang were seldom nodulated by rhizobial species (4/20 sites) that had formed N2-fixing nodules in our plants tests (Table 4, Table S1). In contrast to strains originating in Xinjiang, strains obtained from central China generally belonged to effective rhizobial species (Table S1). Paenibacillus sp. was frequently isolated from G. uralensis sampled in Xinjiang, especially from those sampled in northern Xinjiang (Table 3, Table S1). The above points raised the question: can environmental conditions explain the differences in bacterial populations between central China and Xinjiang?

(a)

(b)

Discrimination of strains within the sampling regions and host plants

We used db-DA to test the hypothesis that bacterial AFLP fingerprint patterns representing strains were more similar to each other when the strains originated from one geographical region or from one host plant species. Besides the ability to discriminate strains between different sampling regions or host plants, DA also has a classification function which reveals how big a percentage of the strains is classified according to the a priori hypothesis. DA was capable of discriminating bacteria between different Glycyrrhiza species and geographical regions but could not explain the mechanisms behind the divergence of bacteria. When bacterial strains were a priori divided into three groups according to the major sampling regions, i.e. northern Xinjiang, southern Xinjiang and central China, db-DA showed that 70% of all strains belonged to their a priori classes. Although there was some regional heterogeneity and overlap, strains obtained from central China were well separated from the Xinjiang strains along axis 1 (Fig. 1a). The DA method assumes that the dispersions (variances) of observations are approximately similar between the a priori groups to be analyzed. Strains of Paenibacillus sp., which were genetically rather homogeneous, constitute a big portion (40%) of the northern Xinjiang isolates FEMS Microbiol Ecol 79 (2012) 46–68

Fig. 1. Distance-based nonparametric discriminant analysis (DA) of a priori classified 159 bacterial strains obtained from root nodules of different Glycyrrhiza plant species growing in China. Coloured symbols indicate individual bacterial strains. Small numbers with letters refer to the major AFLP subgroups. Discrimination of strains based on (a) three geographical sampling regions and (b) four host plant species. In both cases, the P-value 0.0001 was based on 9999 permutations. The AFLP subgroup codes indicate bacterial species as follows: 1a, Rhizobium daejeonense; 1b and 1c, Rhizobium cellulosilyticum; 2, 4e and 6e, Phyllobacterium spp.; 3a and 3c, Mesorhizobium mediterraneum; 3b, Mesorhizobium tianshanense; 3d, Rhizobium gallicum; 4a, Mesorhizobium group A; 4c, Sinorhizobium meliloti; 4d, Paenibacillus spp.; 4f, Rhizobium giardinii; 4g, Rhizobium leguminosarum; 5e, Mesorhizobium group B; 6a, Agrobacterium tumefaciens; 6f, Erwinia cypripedii.

(Table 3, Fig. S2). Therefore, the composition of different AFLP patterns (strains) from northern Xinjiang was less diversified in comparison with those of the other ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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geographical groups. Thus, the dispersion (variance) in the northern Xinjiang group was greater than that of the other regional groups (Fig. S3) and the group dispersions between northern and southern Xinjiang and between northern Xinjiang and central China were not similar (P-values for pairwise comparisons were 0.0001 and 0.0056; Table S2). This argues slightly against the assumption of DA of similar group dispersions. Along axis 2, most strains originating in southern Xinjiang also differed from those obtained from the north. Paenibacillus strains were an exception, as their southern variants were not distinguishable from their northern variants (Fig. 1a). The abundance of Paenibacillus strains in northern Xinjiang possible strengthened the discrimination of AFLP patterns between northern and southern Xinjiang. Rhizobial AFLP subgroups (species) obtained from central China were clearly separated from those originating in Xinjiang. Only subgroups 3b (M. tianshanense) and 4c (S. meliloti) consisted of two geographically distinct populations (Fig. 1a). Strains representing A. tumefaciens (6a) were scattered near the origin of the ordination (Fig. 1a), indicating that the occurrence of A. tumefaciens was unrelated to the geography. When strains were a priori classified in four groups according to their host species, i.e. G. uralensis, G. glabra, G. eurycarpa + G. inflata + G. squamulosa and the unidentified Glycyrrhiza sp., db-DA revealed that 82% of all bacterial strains belonged to their a priori classes. Strains isolated from G. glabra were clearly separated from those obtained from G. uralensis and unidentified Glycyrrhiza plants (Fig. 1b). Furthermore, most strains isolated from G. uralensis differed from those obtained from the pooled three Glycyrrhiza species and unidentified Glycyrrhiza plants along axis 2 (Fig. 1b). The multivariate group dispersion of bacterial AFLP patterns was approximately the same in each plant group analyzed a priori. The dispersion was greater than in other plant groups only in the G. glabra group (Fig. S3) and the dispersions were different between G. glabra and G. uralensis (P-value 0.005; Table S2). Seventy percent of G. glabra isolates were represented by only two Mesorhizobium species (Table 3) and the strains, originating from three sampling sites (Table 1), were also genetically rather homogeneous (Fig. S2). Therefore, AFLP patterns obtained from the small number of G. glabra isolates showed less heterogeneity than those derived, for example, from numerous diversified G. uralensis isolates. This might partly explain why the G. glabra isolates separated so clearly from other bacteria in db-DA (Fig. 1b). Rhizobial AFLP subgroups (species) were typical for the particular host plant species (Fig. 1a). Strains belonging to subgroups 4a (Mesorhizobium group A) and 1a ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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(R. daejeonense) were characteristic for G. uralensis, whereas subgroups 3b (M. tianshanense), 5e (Mesorhizobium group B) and 4f (R. giardinii) were characteristic for G. glabra (Fig. 1b). In both DA plots, strains representing A. tumefaciens were scattered near the origin of the ordination (Fig. 1a and b), indicating that the variation in their AFLP patterns was not related to either the geography or the Glycyrrhiza hosts. Relationships between environmental factors and bacteria

CCA was applied to explore possible relationships between changes in environmental variables and variations in bacterial strains colonizing Glycyrrhiza nodules. CCA used the variation in the environmental matrix to explain variation in the bacterial AFLP fingerprint matrix. Our assumption was that the greater the similarity of the AFLP patterns in the strains, the more similar the environmental requirements of the strains. According to CCA all environmental variables used accounted for 8.4% of the total variation in the bacterial AFLP patterns. Although the variables used explained only a small part of the variation in AFLP patterns, CCA gave some clues to the factors that might be associated to bacteria isolated from Glycyrrhiza nodules. The CCA biplot indicated that latitude showed the strongest correlation to the variation in AFLP patterns, followed by altitude, longitude, annual temperature and annual rainfall. Among geographical variables, a scattered group of rhizobial strains obtained from central China was related to the increasing longitude (Fig. 2a and b). The sampling sites of central China are located east of Xinjiang. Paenibacillus strains (cluster P; Fig. 2a), isolated almost exclusively from G. uralensis, correlated to the increasing latitude, corresponding to sampling sites in northern Xinjiang (Fig. 2). Strains that belonged to AFLP subgroups 4a (Mesorhizobium group A) and 1a (R. daejeonense) and isolated from G. uralensis in southern Xinjiang were associated with the increasing altitude (cluster MRGU; Fig. 2a). The geographic location of sampling sites did not explain the distribution of the other strains obtained from Xinjiang. Paenibacillus strains were related to the decreasing rainfall. Thus, the drought could be a reason for the phenomenon that G. uralensis nodules in Xinjiang were commonly colonized by Paenibacillus spp. Strains belonging to subgroup 6a (A. tumefaciens) as well as most strains of subgroup 4c (S. meliloti) were associated with the increasing annual temperature and rainfall (Fig. 2a). In other words, these bacteria may favour warm and moist conditions. Strains belonging to two mesorhizobial subgroups 3b (M. tianshanense) and 5e (Mesorhizobium group B) were FEMS Microbiol Ecol 79 (2012) 46–68

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(a)

(b)

Fig. 2. CCA showing associations between environmental variables and AFLP patterns of 159 bacterial strains isolated from Glycyrrhiza nodules in China. Strains are displayed as points and variables as vectors. The arrows point to the direction of the most rapid change in the environmental variable. The lengths of the arrows are proportional to the correlation between ordination and environmental variable. Coloured symbols indicating individual bacterial strains were printed according to (a) bacterial genera, (b) geographical sampling regions and (c) Glycyrrhiza host plants. P-value 0.0001 was based on 9999 permutations. Clusters indicate: P (Paenibacillus), M (Mesorhizobium) and MRGU (Mesorhizobium and Rhizobium from Glycyrrhiza uralensis). AFLP subgroup codes indicate bacterial species as follows: 1a, Rhizobium daejeonense; 3b, Mesorhizobium tianshanense; 4a, Mesorhizobium group A; 4d, Paenibacillus spp.; 5e, Mesorhizobium group B.

not clearly associated with any environmental variables considered (cluster M, Fig. 2a). Although the strains originated from different host plants (G. glabra, G. uralensis, unidentified Glycyrrhiza spp.) as well as from different geographical regions (Xinjiang, central China) (Fig. 2b and c), CCA suggested that some unknown factor not included in this study united the strains in the Mesorhizobium cluster. Influence of host plant species and geographical location on bacterial variation

(c)

We used partitioning of the variance to determine how big a proportion of the variation in bacterial AFLP patterns was explained by the geographic location and host plant species. This test showed that the host Glycyrrhiza species of the strains accounted for 9.9% (pure proportion of host species) of the total variation in the AFLP patterns. The geographic location of the sampling sites, when latitude and longitude coordinates were converted into metric coordinate units, accounted for 6.9% (pure spatial proportion) of the total variation. The shared proportion, indicating the variation in AFLP patterns explained by both the host plant species and geographic location, was 2.4%. This leaves 85.7% of the total variation in bacterial AFLP patterns unexplained. It is possible that the dependence between AFLP patterns and geographic distances was not totally linear, which could have affected the results of partitioning the variance. However, these results were parallel with those obtained from CCA, indicating that the geographical location of sampling sites has a minor effect on which bacteria colonize Glycyrrhiza nodules. PGP activities among Glycyrrhiza isolates

Six different PGP activities were tested in vitro for 65 strains isolated from Glycyrrhiza nodules. Nearly all



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strains representing Alphaproteobacteria (Agrobacterium sp., Phyllobacterium spp.) and Gammaproteobacteria (Pantoea sp., Erwinia sp., E. cloacae) were able to produce cellulose and siderophores, i.e. molecules with a high affinity for iron, and at least a small amount of the auxin plant hormone indole-3-acetic acid (IAA) (Table 2). Only seven of 16 Agrobacterium strains and two strains, E. cloacae NWXJ59 and Microbacterium sp. NWSX16, showed a clear IAA production (Table 2). Only six strains representing Agrobacterium sp., Paenibacillus sp., Pantoea sp. and Erwinia sp. were able to solubilize phosphate, having a clear halo around the colony. However, except for the three P. ifriqiyense strains and R. changlensis strain NWGS13, all other Alpha- and Gammaproteobacteria tested produced organic acids (Table 2), indicating that they might be potential P-solubilizers (Vassilev et al., 2006). Besides the R. changlensis strain NWGS13 and unidentified strain NWXJ24, neither Alpha- or Gammaproteobacteria showed protease and lipase activity. Protease activity was typical for the Gram-positive Paenibacillus spp. (61%), followed by the production of cellulase (57%) and siderophores (38%), and small amount of IAA (44%). Similarly to other bacteria, phosphate solubilization, the clear production of IAA and lipase production were also rare among Paenibacillus strains (Table 2).

Discussion To avoid discarding putative novel symbiotic species and to obtain additional information on non-rhizobial endophytes colonizing Glycyrrhiza nodules, our strategy was to isolate all bacterial strains that inhabited the root nodules of the Glycyrrhiza plants sampled. Our work resulted in 159 distinct bacterial isolates, which represented mainly Alpha- and Gammaproteobacteria genera and Gram-positive Firmicutes. After testing for symbiotic properties (nodulation, N2 fixation) we divided the bacterial species into three categories (Fig. 3): (1) true symbionts, (2) sporadic symbionts and (3) non-nodulating species. The true symbionts, i.e. species that were common among Glycyrrhiza isolates and that, with few exceptions, were able to form N2-fixing nodules on the test plants G. uralensis and G. glabra, were all taxonomically Mesorhizobium sp. (Fig. 3). Four of five mesorhizobial AFLP subgroups represented the closely related lineage of M. mediterraneum, M. gobiense and M. tianshanense. The strains of the subgroup 4a were unrelated to any previously described Mesorhizobium species. Sporadic symbionts, i.e. species within which there was a large variation between effective, ineffective and nonnodulating strains, usually had only a single or a few representatives among Glycyrrhiza isolates. Based on the ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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nodulation phenotype, sporadic symbionts were further divided into two groups, those species with some effective strains and those with only ineffective strains. The first group included species for which N2 fixation ability was an exceptional characteristic (S. meliloti, R. gallicum) or which contained only a few but effective strains (R. galegae, R. leguminosarum). The species in the latter group with R. giardinii, R. cellulosilyticum and P. ifriqiyense-like strain NWXJ84 induced only ineffective nodules on G. uralensis and/or G. glabra. The non-nodulating species included the endophytic species Paenibacillus spp., A. tumefaciens, R. daejeonense, P. bourgognense, Phyllobacterium sp. and Pantoea sp. (Fig. 3). The type strains of R. cellulosilyticum and R. daejeonense isolated from environmental samples were capable of inducing ineffective nodules on alfalfa and carried some symbiotic genes (nodD or nifH) (Quan et al., 2005; Garcıá-Fraile et al., 2007). Phyllobacterium spp., identified first from leaf nodules, have also been detected in root nodules (e.g. Mantelin et al., 2006). Besides Phyllobacterium trifolii, which nodulated Trifolium and Lupinus species (Valverde et al., 2005), they did not renodulate their original hosts (Zakhia et al., 2006; Mahdhi et al., 2007; Rincoń et al., 2008; Bromfield et al., 2010), even though nodulation genes (nodD, nodC) were detected in a few strains (Valverde et al., 2005; Rincoń et al., 2008). Although the above-mentioned Rhizobium and Phyllobacterium species belong taxonomically to the Rhizobiaceae and Phyllobacteriaceae families in the Rhizobiales order and may carry some nodulation genes, their nodulation seems to be coincidental. Besides, none of them has been shown to induce N2-fixing nodules on any legumes. Among bacteria that induced ineffective nodules in our work, R. giardinii formed effective nodules, for example on Phaseolus vulgaris (Amarger et al., 1997), and therefore belong to the rhizobia. The ineffective R. cellulosilyticum and strain NWXJ84 representing P. ifriqiyense-like species as well as R. daejeonense, P. bourgognense and other Phyllobacterium spp., which did not nodulate Glycyrrhiza in our plant tests, could be considered transitional forms between nodulating rhizobia and non-nodulating endophytes. Glycyrrhiza glabra and G. inflata grow mainly in Xinjiang in the northwest, whereas G. uralensis grows in all parts of northern China (Kondo et al., 2007). In our study the geographic distribution of the Glycyrrhiza species sampled was similar. Based on genetic markers, Kondo et al. (2007) concluded that G. glabra is more closely related to G. inflata than to G. uralensis, G. glabra and G. inflata being equidistant to G. uralensis. However, the genetic difference between them is not large because G. uralensis can hybridize with G. glabra and G. inflata (Hayashi et al., 2003; Kondo et al., 2007). This close FEMS Microbiol Ecol 79 (2012) 46–68


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Fig. 3. Grouping of nodule-associated endophytic bacteria isolated from Glycyrrhiza spp. by their ability to form a N2-fixing symbiosis with Glycyrrhiza uralensis and Glycyrrhiza glabra when isolates were reinoculated on the host plants.

genetic relationship might explain why in our work most nodulating strains were capable of forming nodules on both G. uralensis and G. glabra. Endophytic bacteria identified from Glycyrrhiza nodules, namely Agrobacterium sp., Paenibacillus spp., Erwinia cypripedii, Pantoea agglomerans, E. cloacae, R. changlensis and Microbacterium oxydans, have been also previously detected in internal parts of roots, stems or nodules of various plants (Lodewyckx et al., 2002; Zakhia et al., 2006; Muresu et al., 2008). Even though inoculation of legumes with Agrobacterium alone hardly ever produces nodules (Mhamdi et al., 2005; Wang et al., 2006), our work as well those of others (e.g. de Lajudie et al., 1999; Bala et al., 2003; Mhamdi et al., 2005; Wang et al., 2006) indicated that Agrobacterium species frequently inhabit leguminous nodules. Only the phylogenetically different Agrobacterium strain IRBG74 was shown to induce effective nodules on tropical Sesbania plants (Cummings et al., 2009). In our work Paenibacillus spp. represented another big group of non-nodulating bacteria detected among Glycyrrhiza nodule isolates. Paenibacillus strains as well those of the closely related Bacillus genus have also been isolated from the nodules on other legumes (Bai et al., 2002; Zakhia et al., 2006; Muresu et al., 2008; ValvFEMS Microbiol Ecol 79 (2012) 46–68

erde et al., 2010). Thus, Gram-positive bacteria can also represent nodule dwellers even though they are usually considered laboratory contaminants. Of the PGP activities tested, the cellulase production activity was common among all Glycyrrhiza isolates tested. Hydrolytic bacterial cellulase might be needed when endophytic bacteria specifically penetrate plant roots or stems (Lodewyckx et al., 2002). It is very likely that endophytic bacteria obtained from the nodules are capable of colonizing the internals of roots and stems (Wang et al., 2006). In legumes with indeterminate nodules, such as Glycyrrhiza (Sprent, 2001), rhizobia enter the host plant through root hair invasion. It is still unknown whether non-nodulating endophytes can enter the nodules as free passengers during the infection by symbiotic rhizobia. Bacteria that produce siderophores can inhibit plant pathogens by competing with them for iron in the rhizosphere. Some plants can also bind, transport and exploit bacterial iron-siderophore complexes (Lodewyckx et al., 2002). Besides cellulase, protease produced by many Bacillus and Paenibacillus strains was proposed to degrade fungal cell walls, thereby inhibiting plant pathogens (Lodewyckx et al., 2002). Remarkable IAA and phosphate solubilizing ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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activities were not common among Glycyrrhiza isolates. In fact, many PGP activities are considered to be strainspecific rather than species-specific. The biosynthesis of IAA is widespread among plant-associated bacteria, including plant pathogens. Presumably, PGP bacteria use IAA as a part of their colonization strategy, involving phytostimulation and circumvention of plant defence mechanisms. Moreover, IAA might act as a signal molecule in bacteria–bacteria communication (Spaepen et al., 2007). Perhaps some of these functions explain why the production of IAA was common among our Agrobacterium strains. Recently, Muresu et al. (2008) demonstrated that the same root nodules of some Mediterranean legumes harboured prevailingly non-culturable rhizobia as well as a variety of culturable non-rhizobial endophytes. In our study, when only non-nodulating endophytes, such as Paenibacillus spp., were recovered from Glycyrrhiza nodules, the identity of the nodule-inducing organism remained unknown. As only intact, healthy nodules were collected from Glycyrrhiza roots, the reason for not getting rhizobia could either be that the original rhizobium or rhizobia died during the isolation or were not culturable under laboratory conditions. In many cases, the distinguishing of bacterial strains on the basis of 16S rRNA gene techniques is too coarse to detect biogeographic patterns of bacterial divergence (Cho & Tiedje, 2000; Papke & Ward, 2004; Hughes Martiny et al., 2006; Ramette & Tiedje, 2007a). AFLP, a whole-genome fingerprinting method, has been subjected to multivariate analyses in plant ecology (e.g. Muchugi et al., 2008) but has been used hardly at all for bacterial ecology. Only Ko¨lliker et al. (2005) included AFLP markers in multivariate analysis of grass pathogenic Xanthomonas isolates. Our work, among others (e.g. Portier et al., 2006), indicated that AFLP is a good method to identify strains belonging to the same genomic species or groups. In addition, AFLP has enough genetic resolution power for multivariate analyses to display biogeographic patterns of Glycyrrhiza isolates. A prerequisite of successful analyses and reasonable results would still be that a heterogeneous bacterial collection in particular consists mainly of approximately similar-sized AFLP groups and/or subgroups, which contain many strains. Plants can dramatically modify their soil environment through the rhizosphere effect, which is largely induced by rhizodeposition (Jones et al., 2009). Above-ground plants shape soil microbial communities (Garbeva et al., 2006; Bremer et al., 2009) through root exudates, this effect even being plant species-specific (Haichar et al., 2008; Micallef et al., 2009). In the case of legumes, there is in addition an effect of plant species due to the exchange of specific signal molecules between the host ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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plant and rhizobium preceding nodule formation (Dresler-Nurmi et al., 2009). Based on these assumptions, we postulated that the host plant species determine which endophytic bacteria are able to colonize Glycyrrhiza nodules. Among the multivariate analyses used, DA classified most bacterial strains into four a priori Glycyrrhiza classes, with 82% of all strains showing host specificity. This was a bigger proportion than the proportion of rhizobial strains from all strains analyzed (54%), suggesting that in addition to effective and ineffective rhizobia, other endophytic bacteria might also show a preference for a particular Glycyrrhiza species. The variance partitioning test, however, showed that the host Glycyrrhiza species explained only a small portion (9.9%) of the total variation in bacterial AFLP patterns. These apparently contradictory results are due to the fact that DA is only capable of discriminating strains between different Glycyrrhiza species. In other words, DA distinguishes strains that are more similar within a plant group than between plant groups but is not capable of explaining the mechanisms behind the divergence of bacteria. Recent studies dispute the first part of the paradigm ‘Everything is everywhere, but the environment selects’ presented by Baas-Becking in 1934 to explain microbial distribution (Hughes Martiny et al., 2006). Although many bacterial species, as defined by their 16S rRNA gene phylogeny, appear to be globally distributed, bacteria have shown site-specificity and endemism at the genotype or strain level (Cho & Tiedje, 2000; Ko¨lliker et al., 2005; Ramette & Tiedje, 2007a, b). Site-specificity or endemism was also detected among rhizobia isolated from leguminous nodules when ITS or repetitive element PCR (repPCR) fingerprinting were used for population analysis (Bala et al., 2003; Vinuesa et al., 2005). In this work, rhizobial strains showed regional endemism. Several rhizobial AFLP subgroups and subpopulations of M. tianshanense and S. meliloti sampled from different sites were unique either to central China or Xinjiang. However, the variance partitioning test displayed that the geographic distance explained a small proportion (6.9%) of the variation in bacterial AFLP patterns. The CCA results paralleled this. Only a few strains were clearly associated with the geographic location of their sampling sites. In addition, geographic variables (latitude, longitude and altitude) and climatic variables (annual site temperature and rainfall) of the sampling sites explained only 8.9% of the total variation in AFLP patterns. It is still controversial whether the geographic distance as such, affects bacterial community structure (Bissett et al., 2010; Chu et al., 2010). In our work the long distance between Xinjiang and central China (2000 km) and the isolated location of Xinjiang, which is surrounded by FEMS Microbiol Ecol 79 (2012) 46–68

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high mountains, explained only a small portion of the genetic variation of the bacteria. A reason for this could be that different soil bacteria have different dwelling or dispersion strategies. The study of Bissett et al. (2010) suggested that geographic distance has no influence on spore-forming taxa (e.g. Bacillaceae and Clostridiaceae), but does influence dispersal- or colonization-limited taxa such as Rhizobiaceae, Bradyrhizobiaceae and Xanthomonadaceae. Nemergut et al. (2011) demonstrated that relatively few taxa are cosmopolitan and the vast majority are restricted to individual assemblages. Moreover, the most abundant taxa appear also to be the most widely distributed. The above-mentioned phenomena were also observed in our study. Most rhizobial groups originated from one region, whereas a portion of the spore-forming Paenibacillus strains did not show clear region-specificity. The Paenibacillus population was also genetically more homogeneous than other bacterial groups, suggesting a close relationship of easily dispersing bacteria. The distribution of the A. tumefaciens strains differed from that of the rhizobial ones, being unrelated to the geographic origin or Glycyrrhiza host. Agrobacterium tumefaciens is very commonly identified among nodule isolates regardless of geographic sampling sites and host plant (e.g. de Lajudie et al., 1999; Bala et al., 2003; Mhamdi et al., 2005; Wang et al., 2006). Perhaps A. tumefaciens is a representative of cosmopolitan bacteria. Our results parallel the studies of Fierer & Jackson (2006) and Chu et al. (2010), which showed that the diversity of soil microbial populations is unrelated to variables predicting plant and animal diversity, such as annual site temperature and potential evapotranspiration, latitude and plant diversity. Only in the case of Paenibacillus strains, could the drought explain the phenomenon that G. uralensis nodules in Xinjiang were commonly colonized by Paenibacillus spp. Xinjiang has a typical continental climate with a low annual rainfall and high evaporation and a wide temperature range (Chen et al., 1995). Due to very resistant spores, Paenibacillus spp. are capable of surviving in dry environments. Edaphic factors such as soil pH, soil moisture, carbon and nutrient availability, and vegetation type (Fierer & Jackson, 2006; Chu et al., 2010) might be important factors in explaining variation of nodule isolates. The variance partitioning together with CCA indicated that the host plant species and geographic location as well as climatic variables measured explained only a small part of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more than the geography. However, DA showed that the AFLP fingerprint patterns, representing endophytic bacteria isolated from Glycyrrhiza nodules, showed host specificity FEMS Microbiol Ecol 79 (2012) 46–68

and regional endemism. Our work involved strains isolated from closely related species of the Glycyrrhiza genus, most rhizobial species being able to cross-nodulate different Glycyrrhiza species. This may explain why only a small portion of the total genetic variation of bacteria was accounted for host plant species. A study involving specifically rhizobial strains obtained from different leguminous genera combined with several strains per each sampling site and with several environmental variables, including soil related ones, would provide more information about the role of host plant in driving the colonization of leguminous nodules by particular bacterial species.

Conclusions Our work revealed that a wide variety of rhizobial and non-rhizobial endophytic bacteria can colonize Glycyrrhiza nodules. Glycyrrhiza plants are promiscuous in terms of nodulation because, besides mesorhizobia, the nodules harboured several other rhizobial genera. The classification of rhizobial species into true and sporadic symbionts is of practical importance. When looking for effective and stable inoculant strains for G. uralensis and G. glabra, our results suggest that these are to be found among the true symbiotic mesorhizobia. To our knowledge this study is the first in which multivariate analyses were applied to resolve biogeographical patterns of endophytic bacteria, including those of rhizobia obtained from leguminous nodules. DA, CCA and partitioning of the variance with essential explorative and additional tests, for example the one examining the homogeneity of multivariate group dispersions, were appropriate methods to show when variation in bacterial AFLP fingerprint patterns exhibited specific biogeographical patterns, thereby providing additional useful knowledge related to the large collection of bacteria.

Acknowledgements We thank Aneta Dresler-Nurmi and Petri Penttinen (University of Helsinki) for fruitful discussions and reading the manuscript and Jukka Rintala (Finnish Game and Fisheries Research Institute, RKTL) for projecting geographical coordinates in geographical information system and converting them to metric units. This work was funded by the National Science Foundation of China (31070444, 30970003, 31125007), the 973 project of China (2010CB126502), the Academy of Finland and the University of Helsinki. This work was shared with Northwest A & F University in China and University of Helsinki in Finland. ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Procedures used for the extraction of DNA, selective AFLP-PCR and 16S rDNA-PCR. Fig. S1. Geographic locations of the three sampling regions, northern Xinjiang, southern Xinjiang and central China, where Glycyrrhiza nodules were collected. Fig. S2. Dendrogram based on the AFLP fingerprinting analysis showing the genetic diversity among endophytic bacteria isolated from the root nodules of Glycyrrhiza spp. growing in China. Fig. S3. Box plots visualising the homogeneity of multivariate group dispersions obtained from bacterial AFLP fingerprint data when the matrix of similarities (transformed to dissimilarities) was obtained by using the Dice coefficient. Table S1. Environmental parameters, host plant and bacterial species identified from each sampling site. Table S2. Pairwise comparison of multivariate dispersions of a priori groups. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
