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Abstract. Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim ...
Antonie van Leeuwenhoek 81: 365–371, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria Valeria Bianciotto & Paola Bonfante∗ Dipartimento di Biologia Vegetale & Centro Studio sulla Micologia del Terreno del CNR, Viale Mattioli 25 10125 Torino Italy (∗ Author for correspondence; E-mail: [email protected]) Key words: cell surface molecules, endobacteria, mycorrhizal fungi, nif operon, rhizosphere, ribosomal genes, plant-growth promoting rhizobacteria

Abstract Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains. The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi. Abbreviations: AM – arbuscular mycorrhiza; BLOs – bacteria-like organisms; EPS – extracellular polysaccharides; PGPRs – plant-growth promoting rhizobacteria Introduction Mycorrhizal symbiosis, a mutualistic plant-fungus association, is an essential feature of the biology and ecology of most terrestrial plants, since it influences their growth, water and nutrient absorption, as well as protecting them from root diseases (Smith & Read 1997). Among mycorrhizal fungi, the Arbuscular Mycorrhizal (AM) ones are ancient Zygomycetes, which are known to have colonized the roots of the first land plants and are nowadays associated with about 80% of plant species. Due to their widespread presence in agricultural and natural soils, they represent an important microbial community of the rhizosphere (Martin et al. 2000). In this dynamic environment where bacteria, viruses, fungi, protozoa and microarthropods develop, and take advantage of organic matter released by the root (Weller & Thomashow 1994), AM fungi reside as spores, hyphae and propagules, and occupy the rhizoplane of the host plant producing a pre-symbiotic mycelium (Bianciotto & Bonfante 1999). This intricate network of extraradical hyphae plays a crucial role in mycorrhizal functioning, since

it acts as a bridge between the soil and roots. After the formation of specialized fungal structures, the appressoria, root colonization proceeds with the growth of intraradical hyphae which cross the epidermal layer, and eventually with the formation of intracellular, highly branched structures termed arbuscules which are exclusively located in cortical cells. During this ordered succession of events, AM fungi interact with bacteria which occupy certain specific fungal niches, i.e. spores, extraradical hyphae, intraradical mycelia. The interactions between AM fungi and bacteria have been the subject of great interest, which dates back to the pioneering work by Linderman (1988). It has become clear that rhizosphere microbial changes occur when mycorrhizas form, and that beneficial effects are the result of additive or synergistic interactions among all rhizosphere microbes (Linderman 1992). Therefore, a better understanding of such relationships has considerable ecological consequences, in addition to implications in sustainable agriculture. The first aim of this paper is to describe briefly the interactions between AM fungi and bacteria associated with the extraradical phase of

366 the symbiotic fungi paying particular attention to the molecular mechanisms which allow the physical contact between the microbes. The second part is devoted to the description of a rather unique example of symbiosis between bacteria and fungi: the endocellular bacteria which live inside certain AM isolates. They represent a bacterial community which topologically belongs to the rhizosphere, but in the mean time occupies an exclusive niche, i.e. the cytoplasm of spores and hyphae.

PGPR strains (Bianciotto et al. 1996a) would support this hypothesis. Bacterial attachment should be a general trait in the interactions between rhizosphere bacteria and mycorrhizal fungi: it was first hypothesised by Garbaye (1994) for explaining the beneficial effect of some mycorrhizal helper bacteria and then demonstrated by Nurmiaho-Lassila et al. (1997) during a microscopical investigation on Pinus sylvestris mycorrizospheres.

Soil bacteria are associated to mycorrhizal fungi

The molecular bases of PGPR attachment onto mycorrhizal fungi

Soil microbial communities change spatially and temporarily in the plant rhizosphere following the presence of mycorrhizal fungi, which induce alteration in the quality and quantity of root exudation, eventually leading to changes in microbe composition (Linderman 1992). The development of molecular methods to investigate microbial communities is rapidly changing information concerning the kind of microbes associated to AM roots. As an example, Marschner et al. (2001) used 16S rDNA profiles and multivariate analysis to demonstrate the change in the bacterial community of maize roots following the establishment of the mycorrhizal association. Similar data have also been reported for ectomycorrhizal roots from forestry soils by isolating bacterial colonies (for example: Timonen et al. 1998; Poole et al. 2001) or by detecting them with in situ techniques (Mogge et al. 2000). Bacteria have also been monitored during sporophore production, which often accomplishes the symbiotic ectomycorrhizal phase. For example, fluorescent Pseudomonas, Moraxella and Corynebacterium strains have been isolated from truffle ascomata (Bedini et al. 1999; Gazzanelli et al. 1999) as well as from other edible fungi, such as Cantharellus (Danell et al. 1993). Among the variety of soil bacteria taxa, increasing attention has been given to the presence of plantgrowth promoting rhizobacteria (PGPRs), due to their synergistic interactions with mycorrhizal fungi, in particular AM fungi (Barea 1997). It is known that colonization of sugar cane by PGPRs is increased when AM fungi are present in mixed inocula (Boddey et al. 1991). A possible explanation given by these authors is that AM fungi may act as a vehicle to spread PGPR to neighbouring rhizospheres. The observation of bacterial adhesion to AM fungal spores and hyphal structures, demonstrated in vitro for a variety of

Little is known on the molecular bases of PGPR attachment onto mycorrhizal fungi. By contrast, a large set of experiments has demonstrated the role of several surface components in the physical interactions between beneficial or detrimental rhizosphere bacteria and the plant root. Bacterial attachment generally proceeds through two consecutive steps (Vande Broek & Vanderleyden 1995). In the first step, the bacteria adhere loosely as single cells, whereas in the second step the bacteria become more firmly attached to the plant root and additional free bacteria are entrapped, resulting in the formation of large bacterial clusters at the attachment site. These two consecutive steps have also been described in the colonisation of inert solid substrates, where bacteria eventually assemble into complex clusters termed biofilms (Costerton et al. 1995). Appendages such as pili, fimbriae and flagella are involved in the initial attachment of bacteria to solid surfaces and their role has been demonstrated for a number of pathogenic and beneficial plant/microbe interactions (Vande Broek & Vanderleyden 1995). In particular, the adhesive protein richadesin is considered responsible for the first attachment step of R. leguminosarum to plant cells (Smit et al. 1992). In A. brasilense, a component located on the polar flagellum is involved in adhesion as mutants lacking polar flagella are strongly impaired in adsorption to the root (Croes et al. 1993), while Azoarcus, a nitrogen-fixing organism adheres to the root surface of rice seedlings thanks to the presence of type IV pili (Dorr et al. 1998). During the second step, extracellular polysaccharides are responsible for the firm anchoring of bacteria to the plant surface, and various polymers have been identified in different plant/bacterial combinations: the surface characteristics of A. brasilense relevant to cell aggregation and attachment to plant

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Figure 1. Transmission electron micrographs showing the attachment site of a bacterium (wild type strain sp7 A. brasilense) to the plant cell wall. Some extracellular material is visible around the bacterium (arrow). (W) plant cell wall, (E) epidermal cell, (B) bacterium. Bar 0.15 µm.

roots have been reviewed recently by Burdman et al. (2000). To understand the role of different surface components in the attachment of PGPRs to AM fungal structures and mycorrhizal roots, mutants of A. brasilense and R. leguminosarum strains impaired in the production of extracellular polysaccharides (EPS) and Pseudomonas fluorescens strains with an increased production of EPS have been tested (Table 1). Their attachment to transformed mycorrhizal carrot roots (Figure 1) as well as to in vitro growing AM hyphae has been tested in in vitro assays (Bianciotto et al. 2001a, b). The EPS reduced strains of A. brasilense and R. leguminosarum were shown to be strongly impaired in the attachment whereas both the mucoid strains (EPS+ ) of P. fluorescens formed a dense and patchy bacterial layer on the roots and fungal structures. The results demonstrate that EPS are involved in the anchoring of bacteria to both the surfaces (roots and fungal hyphae) and to the formation of root- and fungal associated biofilms, which can provide them the ability to survive in harsher environmental conditions. Finally, since EPS play a general role in the protection of bacteria against desiccation (Ophir & Gutnick 1994), this trait could be important in the production of inocula consisting of PGPRs and mycorrhizal fungi (Bianciotto et al. 2001a, b).

AM fungi and their intracellular bacteria Bacteria are often associated to eukaryotic cells to establish endocellular symbioses (Douglas 1994). However, the Mycota kingdom offers very limited examples of such symbioses: while bacteria forming a tri-partite associations with fungi and ants are extracellular (Currie 2001), Geosyphon piriforme surely contains both N-fixing cyanobacteria and Bacteria-like Organisms – BLOs- (Schubler & Kluge 2000). The widespread AM fungi are therefore unique in hosting bacteria in their cytoplasm as a rather common event. Intracellular structures very similar to bacteria and BLOs were first described in the 1970s (Mosse 1970; Scannerini & Bonfante 1991 for a review). Ultrastructural observations clearly revealed their presence in many field-collected fungal isolates. Further investigation on these BLOs, including the demonstration of their prokaryotic nature, was long hampered because of their inability to grow on plate. Only a combination of morphological observations (electron and confocal microscopy) and molecular analyses allowed us to identify BLOs as true bacteria and to start unravelling their symbiotic relationship with AM fungi (Bianciotto et al. 1996b). Isolate BEG 34 of Gigaspora margarita contains a large number of BLOs which can be easily detected by staining with fluorescent dyes specific for bacteria and capable of distinguishing between live and dead

368 Table 1. Adhesion of bacteria to root and fungal surfaces Species

Strain

Phenotype#

Adhesion class∗ Root Fungus

A. brasilense

Sp7 7030 AB7001(Sp7exoC::Tn5) AB7002(Sp7exoB1::Tn5) 7030::Tn5-23 7030::Tn5-101

WT WT EPSr EPSr EPS− EPS−

4 3 2 2 1 1

4 3 2 2 1 1

R. leguminosarum

B556 A507 A517

WT EPSr EPSr

3 1 1

3 1 1

P. fluorescens

CHA0 CHA211 CHA213M

WT EPS+ EPS+

2 4 4

2 4 4

∗ Four classes of bacterial attachment were defined: class 1, no attachment; class 2, a

few bacteria attached; class 3, bacteria evenly spread on the surface; class 4, dense coat of attached bacteria (from Bianciotto et al. 2001a with the permission of European Journal of Histochemistry). # WT: Wild type; EPSr : EPS reduced; EPS− : EPS deficient.

bacteria. About 2 150 000 live bacteria were counted in a single spore, which is a large structure of about 260–400 µm. Ultrastructural observations performed on high-pressure freezing/freeze-substituted samples revealed a large number of rod-shaped BLOs in the vacuoles of germinating spores (Figure 2), often associated with the abundant protein bodies (Bonfante et al. 1994). On the basis of the 16S rDNA sequences, the bacterial endosymbionts living in the fungus G. margarita (BEG 34) were first identified as belonging to the genus Burkholderia (Bianciotto et al. 1996b). To determine whether bacteria are also harboured by other members of Gigasporaceae, eleven fungal isolates collected from different geographic areas and belonging to six species were analysed by morphological and molecular approaches. A fluorescent dye was used to visualise the bacteria inside the spores of G. margarita, G. rosea, G. gigantea, G. decipiens, Scutellospora persica and S. castanea. 16S ribosomal genes from the sporal DNA were amplified by PCR using universal eubacterial primers and primers specific for the endobacteria identified in G. margarita BEG 34 (Bianciotto et al. 1996b, 2000). With the exception of the four G. rosea isolates, all other fungi harboured bacteria in their cytoplasm and gave an amplified fragment of the expected size with the universal primers (Bianciotto et al. 2000). Seven out of eight isolates (belonging to five different spe-

cies) could be also amplified with the specific primers. These specific primers were used as probes for in situ hybridization on G. margarita spores, where they successfully identified bacteria. The 16S rDNA amplified from isolates of Scutellospora persica, S.castanea and G. margarita was sequenced and aligned with the closest bacterial sequences available in databases. With neighbour-joining analysis a strongly supported branch containing all endosymbiotic bacteria so far sequenced in Gigasporaceae could be identified close to the genus Burkholderia, as well as to the genus Ralstonia and Pandorea. The tree shows that the endobacteria represent a new bacterial taxon (Bianciotto et al. 2002). Irrespectively of their final name Cendidetus glomeribacter pipesporerum, the results demonstrate that endobacteria are widespread in Gigasporaceae, and suggest that they represent a stable cytoplasmic component. Preliminary results showing that bacteria move along the fungal generations, following a vertical transmission mechanism (V. Bianciotto and G. Becard, unpublished) provide a first experimental confirmation of the statement. Associations between endosymbiotic bacteria and the Homoptera, Blattaria and Coleoptera are common. One of the best known is that between Buchnera and the aphids, where both partners are obligate and cannot survive in the absence of the symbiosis (Moran & Baumann 2000). The complete genome sequence

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Figure 2. Transmission electron micrograph of the cytoplasm of a G. margarita BEG 34 spore. Endobacteria are contained in vacuoles or free in the cytoplasm. Lipid drops are also visible in the sporal cytoplasm. (B) bacteria, (V) vacuole (L) Lipids. Bar: 0,4 µm.

of Buchnera has been recently reported (Shigenobu et al. 2000) and confirms that Buchnera is viable only in its limited niche, the bacteriocyte. The genome sequence also shows how Buchnera possesses genes for the biosynthesis of amino acids essential for the hosts, while it lacks genes involved in the biosynthesis of cell surface components or in defence (Shigenobu et al. 2000). The functional significance of AM fungal endobacteria is not clear; many attempts to cultivate them have been unsuccessful, suggesting that they are unculturable microbes. Using the Buchnera-aphid system as an analogy, we are investigating whether a genomic approach is more feasible. The finding that a genomic library developed from G. margarita spores is also representative of the bacterial genome (van Buuren et al. 1999) helped us to identify some of its genomic features. Among the bacterial genes so far identified (D. Minerdi, unpublished results), the most interesting genes are those involved in nutrient uptake (i.e. a putative phosphate transporter operon, pst) and a gene involved in colonisation events by bacterial cells (vac) (Ruiz Lozano & Bonfante 1999, 2000). A DNA region containing putative nitrogenase coding genes (nif operon) was also found (Minerdi et al. 2001). Analysis revealed three Open Reading Frames (ORFs) encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of component I and with component II

(NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the endobacterial nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. RT-PCR experiments with primers designed on endobacterial nifD and nifK genes and performed on total RNA extracted from germinated spores demonstrate the gene expression during this step of the fungal life cycle. In conclusion, the approach based on the sequencing of large DNA inserts is providing information on the potential physiological traits of a bacterial population, which has so far remained hidden inside its fungal hosts.

Conclusions In conclusion, the analysis of the multiple interactions established by AM fungi with bacterial cells offers new keys for understanding the complexity of AM symbiosis. The association between mycorrhizal fungi and some soil bacteria defines new parameters in the design of mixed inocula, while the identification of fungal strains which contain endosymbiotic bacteria with important genetic traits (for example potential nitrogen- fixing capacities) opens up new strategies for the rational use of AM fungi.

370 Acknowledgements We wish to thank Maria Teresa Della Beffa for the reference list. The research illustrated in this review has been funded by the EU GENOMYCA project, QLK5CT-2000-01319, by the Italian National Council of Research, and by the National Project Produzione Agricola nella Difesa dell’Ambiente (PANDA).

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