III. Ecological and evolutionary implications of MHB 23
IV. The question of MHB specificity 27
V. Mechanisms of the MHB effect 28
VI. Role of MHB in mycorrhizal functions 31
VII. Conclusions and research priorities 32
In natural conditions, mycorrhizal fungi are surrounded by complex microbial communities, which modulate the mycorrhizal symbiosis. Here, the focus is on the so-called ‘mycorrhiza helper bacteria’ (MHB). This concept is revisited, and the distinction is made between the helper bacteria, which assist mycorrhiza formation, and those that interact positively with the functioning of the symbiosis. After considering some examples of MHB from the literature, the ecological and evolutionary implications of the relationships of MHB with mycorrhizal fungi are discussed. The question of the specificity of the MHB effect is addressed, and an assessment is made of progress in understanding the mechanisms of the MHB effect, which has been made possible through the development of genomics. Finally, clear evidence is presented suggesting that some MHB promote the functioning of the mycorrhizal symbiosis. This is illustrated for three critical functions of practical significance: nutrient mobilization from soil minerals, fixation of atmospheric nitrogen, and protection of plants against root pathogens. The review concludes with discussion of future research priorities regarding the potentially very fruitful concept of MHB.
The concept of ‘mycorrhiza helper bacteria’ (MHB), since it was introduced and discussed in a previous Tansley Review (‘Helper bacteria: a new dimension to the mycorrhizal symbiosis’; Garbaye, 1994), has given rise to novel research on diverse plant–fungus model systems which has produced a good crop of new results. It is thus timely to revisit this particular field of research at the interface of plant science, mycology, bacteriology and rhizosphere ecology, which is more generally related to the major research topic of fungal–bacterial interactions in ecosystems (de Boer et al., 2005; Artursson et al., 2006). The main issue here is not so much the generality of MHB (this will be summarized in the next section of this review) but, more than that, the role of MHB in the mycorrhizal symbiosis from evolutionary, functional and ecological points of view, in relation to other thriving research fields such as the taxonomy, phylogenetics and functional diversity of mycorrhizal fungi.
II. Evidence for the occurrence of MHB
The majority of plants from terrestrial ecosystems interact with symbiotic mycorrhizal fungi (for a review, see Smith & Read, 1997). There are different types of mycorrhizal symbiosis, the arbuscular mycorrhizal one being the most common and the most frequent all over the world. The arbuscular mycorrhizal symbiosis is formed by obligately symbiotic fungi from the Glomeromycota phylum. Colonization of plant roots by arbuscular mycorrhizal fungi is achieved via spores and mycelia originating from infected roots. The hyphae enter the root tissues and develop inter- and intracellularly, forming running hyphae, coils and arbuscules. In contrast to arbuscular mycorrhizal symbiosis, a relatively small number of plants live in symbiosis with ectomycorrhizal fungi, mainly forest trees in temperate and boreal ecosystems. Ectomycorrhizal symbioses are formed by a large number of fungal species, mainly Basidiomycetes, but also Ascomycetes. Spores produced by the sporocarps of ectomycorrhizal fungi germinate and colonize first, second and further orders of lateral roots which are called fine roots or short roots. The fungus forms a mantle which encloses the root. Some hyphae extend to the surrounding soil; others pass between the epidermal and cortical cells and form the so-called Hartig net, the site for nutritional exchange between the fungal and plant cells.
The MHB concept is generic. It depends neither on the type of the mycorrhizal symbiosis nor on the taxonomy of the MHB strains. To date, many bacterial strains have been reported to be able to promote either arbuscular or ectomycorrhizal symbioses (Table 1; for a review, see Garbaye, 1994; Barea et al., 2002; Johansson et al., 2004; Artursson et al., 2006; Duponnois, 2006). In the case of the arbuscular mycorrhizal fungi, many examples of MHB have been described in the literature since the first mention by Mosse (1962) in the genus Glomus. Of the ectomycorrhizal fungi, to date only Basidiomycetes have been reported to positively interact with MHB. Nevertheless, in the case of the ectomycorrhizal Ascomycete Tuber melanosporum, Mamoun & Olivier (1992) reported an indirect helper effect of soil pseudomonads on the T. melanosporum symbiosis. This could result from a protective effect of the pseudomonads against soil-borne competitors in the T. melanosporum mycorrhizosphere. The MHB strains that have been identified to date belong to many bacterial groups and genera, as illustrated in Table 1, such as gram-negative Proteobacteria (Agrobacterium, Azospirillum, Azotobacter, Burkholderia, Bradyrhizobium, Enterobacter, Pseudomonas, Klebsiella and Rhizobium), gram-positive Firmicutes (Bacillus, Brevibacillus, and Paenibacillus) and gram-positive Actinomycetes (Rhodococcus, Streptomyces, and Arthrobacter). Further research will be needed to determine if MHB also exist in noneasily culturable bacterial groups that are known to inhabit the mycorrhizophere such as the Acidobacterium group (Burke et al., 2006). Many plant models have been used to study the MHB effect, including herbaceous and woody plant species, mainly from temperate ecosystems (Table 1). Only a few studies have focused on tropical plant species.
Table 1. Examples from the literature of mycorrhiza helper bacteria (MHB) with significant effects on mycorrhiza formation
III. Ecological and evolutionary implications of MHB
Although MHB have been found every time they have been looked for, under very different environmental conditions and in various plant–mycorrhizal fungus symbioses, one should ask whether there exist specific niches for MHB. The MHB isolates described in the literature have been isolated from diverse environments (Table 1). Some of these environments were related to the mycorrhizal fungi, such as mycorrhizas, mycorrhizospheres, fruiting bodies of ectomycorrhizal fungi and spores of arbuscular mycorrhizal fungi. Others, such as rhizospheres, rhizobial nodules and galls, did not appear to be directly related to the fungal environment. Garbaye & Bowen (1989) demonstrated that the majority of the bacterial isolates from the mantle of the Pinus radiate–Rhizopogon luteolus ectomycorrhizas were able to promote the ectomycorrhizal symbiosis. Similarly, Founoune et al. (2002b) revealed a strong relationship between the mycorrhizosphere origin of fluorescent pseudomonads and their helper effect on the Acacia holosericea–Pisolithus sp. ectomycorrhizal symbiosis. By contrast, Frey-Klett et al. (2005) observed, in the mycorrhizosphere, a counter-selection of the bacterial isolates able to repress the establishment of the Douglas-fir (Pseudotsuga menziesii)–Laccaria bicolor symbiosis. Further research is needed to determine whether MHB preferentially live in association with mycorrhizal fungi rather than in other soil compartments, or whether mycorrhizal fungi select MHB around their hyphae. Such a close association between fungal hyphae and helper bacterial isolates was previously reported in the case of the fungal plant pathogen Stagnospora nodorum: disease helper bacteria were successfully isolated from field and laboratory cultures of the fungus which failed to be decontaminated in vitro, and these bacteria were proved to promote fungal pathogenicity (Dewey et al., 1999; Newton & Toth, 1999).
If the presence of helper bacteria is advantageous to the fungi, we might also expect a positive effect of the fungus on the populations of MHB. This aspect has received little attention to date, with most studies on MHB having been dedicated to analysis of the impact of the MHB on fungal behaviour or to determining the mechanisms of the helper effect. We have demonstrated that, in glasshouse conditions, the survival in the soil of the MHB Pseudomonas fluorescens strain BBc6R8 is significantly improved by the presence of the ectomycorrhizal strain L. bicolor S238N from which it was isolated, in the prescence or absensce of Douglas-fir roots (unpublished results). Interestingly, the survival of strain BBc6R8 is not improved by the presence of nonmycorrhizal Douglas-fir roots (Frey-Klett et al., 1997), suggesting that this bacterial strain depends more on the presence of the fungus than on that of the roots. Similarly, the arbuscular mycorrhizal fungus Glomus mosseae improved the long-term survival of the MHB strain P. fluorescens 92rk in the rhizosphere of tomato (Lycopersicon esculentum) plants (Gamalero et al., 2004).
Although a close association with mycorrhizal fungi cannot be considered the rule for all the MHB studied to date, one should address the question of the physical relationship that exists between the mycorrhizal fungal cells and the cells of the MHB strains originating from the hyphosphere of the mycorrhizal fungi. The P. fluorescens strain BBc6R8 was shown to attach to the hyphae of different ectomycorrhizal fungi (Sen et al., 1996). We revealed recently that this bacterial strain is also able to develop biofilm-like structures on L. bicolor hyphae in vitro (Fig. 1; unpublished results). This observation is in accordance with a previous hypothesis which proposed that, after inoculation, the population of BBc6R8 decreases in the soil but concentrates in target niches such as the fungal cell wall (Frey-Klett et al., 1999). Bacterial colonization of arbuscular mycorrhizal hyphae (Toljander et al., 2006) and biofilm formation on ectomycorrhizal hyphae (Nurmiaho-Lassila et al., 1997; Sarand et al., 1998) have already been reported. However, the MHB status of these bacteria has not yet been described. Further studies are needed in order to determine whether MHB form biofilms randomly on the surfaces of fungal hyphae or in specific locations where key fungal metabolites, such as trehalose, are exuded.
When associated with plant roots, mycorrhizal fungi receive up to 30% of the total carbon fixed and frequently transform it into trehalose, a dissacharide that has been proposed to behave as a carbon sink (Lopez et al., 2007; Wiemken, 2007). Interestingly, several studies have highlighted the possible role of trehalose in the interactions between bacteria and mycorrhizal fungi. Trehalose was reported to be responsible for the selection of specific bacterial communities in the mycorrhizospheres of tree roots in forest nurseries and plantations (Frey et al., 1997; Izumi et al., 2006b; Uroz et al., 2007). Moreover, it was demonstrated to be involved in the growth-promoting effect of a MHB strain of Pseudomonas monteilii on the ectomycorrhizal fungus Pisolithus albus in a two-compartment plate assay (Duponnois & Kisa, 2006): P. monteilii significantly increased fungal growth when cultivated on a minimal medium amended with trehalose, while none of the other seven organic compounds tested produced the same effect. Finally, we recently observed that the biofilm-forming MHB strain P. fluorescens BBc6R8 is chemoattracted by trehalose as well as by the mycelium of L. bicolor (unpublished results), which was proved to accumulate trehalose in its hyphae (Martin et al., 1984). Therefore, we can now hypothesize that fungal metabolites, such as trehalose, secreted from fungal cells, can facilitate the colonization of the hyphae and the formation of MHB biofilms on them. However, whether the biofilm development of MHB on the fungal hyphae is required for the expression of the MHB effect remains to be determined: are there specific metabolites that MHB synthesize only when structured in biofilms and that would mediate the MHB effect? It is well known that the virulence of the human bacterial pathogen Pseudomonas aeruginosa relies on the production of different factors such as antibiotics. Their synthesis is triggered by quorum-sensing signals which are critical for the development of mature bacterial biofilms on the eucaryotic host (Whiteley et al., 1999). Riedlinger et al. (2006) recently identified a novel antibiotic, termed auxofuran, to be responsible for the growth promotion of the ectomycorrhizal fungus Amanita muscaria by the MHB strain Streptomyces sp. AcH 505. Interestingly, these authors also demonstrated a stimulating effect of the ectomycorrhizal fungus on auxofuran synthesis by the bacteria, which seems to be related to acidic pH. Whether the formation of AcH 505 biofilms on the fungal hyphae is required for the synthesis of auxofuran and for the mycorrhiza helper effect is intriguing and warrants further experiments.
In addition to the ability of MHB to form biofilms on the surface of fungal hyphae, one should ask if they can also colonize fungi intracellularly. Such a hypothesis was suggested by Frey-Klett et al. (1999) to account for the helper effect of low population densities of the MHB strain P. fluorescens BBc6R8. At that time, the presence of endobacteria had already been detected in some species of arbuscular mycorrhizal fungi and in the related fungus Geosiphon pyriforme (Schüßler et al., 1994; Perotto & Bonfante, 1997). Endobacteria have now been reported in other fungi (Lumini et al., 2006), such as the Zygomycete plant pathogen Rhizopus sp. (Partida-Martinez & Hertweck, 2005), the ectomycorrhizal Basidiomycete L. bicolor (Bertaux et al., 2003, 2005), and the edible white-rot fungus Pleurotus ostreatus (R. Yara, GSF, Germany, pers. comm.). Among the bacteria that intracellularly colonize arbuscular and ectomycorrhizal fungi, one should ask whether there are any MHB. Because most of these bacteria are unculturable, it is very difficult to answer such a question. Nevertheless, there is evidence that some of these endobacteria are at least beneficial to their mycorrhizal fungal host. By comparing lines of Gigaspora margarita harbouring endosymbiotic Candidatus Glomeribacter gigasporarum with lines that have been cured, Lumini et al. (2007) proved very recently that the presence of endosymbiotic bacteria strongly improves the presymbiotic growth of the fungus, as shown by increased hyphal elongation and branching following treatment with root exudates. Under optimal glasshouse conditions, no significant effect of the presence of the endosymbiotic bacteria on the intensity of mycorrhiza formation in pot experiments was observed. We might expect that, because of their positive impact on the presymbiotic growth of the fungus, these endobacteria could behave as true MHB when the environmental soil conditions are not favourable to fungal survival. Bruléet al. (2001) previously made the same suggestion in the case of the MHB effect of P. fluorescens BBc6R8 on the ectomycorrhizal Douglas-fir–L. bicolor symbiosis. Interestingly, this symbiosis is also significantly promoted by a Paenibacillus sp. isolate, purified from a contaminated fermentor culture of L. bicolor and suspected to come from within the fungal cells (Bertaux et al., 2003). This Paenibacillus sp. isolate was recently shown to significantly promote the growth of L. bicolor in vitro (Deveau et al., 2007).
If fungus-beneficial endobacteria can inhabit the mycorrhizal fungal mycelium, the existence and the evolution of such an intimate relationship between the bacteria and their fungal hosts are in themselves intriguing. Interestingly, the two mycorrhizal fungi G. margarita and L. bicolor illustrate two different evolutionary processes of the bacterial colonization of fungal cells. Gigaspora margarita is an example of long-lasting coevolution between the fungus and its endobacteria, as suggested by the strict vertical transmission of Candidatus Glomeribacter gigasporarum through fungal spore generations (Bianciotto et al., 2004), by the small genome size of the endobacteria, and by the difficulties in cultivating these bacteria in vitro (Jargeat et al., 2004). Arbuscular mycorrhizal fungi have been suspected to play a crucial role in facilitating the colonization of the land by plants during the Ordovician period (Redecker et al., 2000). Therefore, we may speculate whether the endobacteria found in some arbuscular fungi could have behaved as ancient MHB and could have contributed to the success of the early colonization of terrestrial plants. In contrast to arbuscular mycorrhizal fungi, L. bicolor harbours fluctuating endobacterial communities that appear to be environmentally acquired (Bertaux et al., 2005). Whether these endobacteria result from the bacterial biofilms that colonize the hyphae extracellularly remains to be determined. Our hypothesis is that the intracellular colonization of L. bicolor by soil bacteria would confer on the fungal host the ability to adapt to changing environments, especially during its presymbiotic life in the soil. More generally, the transient but frequent intracellular colonization of eukaryotes, such as ectomycorrhizal fungi, by bacteria could increase the occurrence of horizontal gene transfer between bacterial and eukaryotic genomes, as suggested by Brown (2003). Gene transfer has been reported to occur between fungi, and this may have a strong impact on the virulence of plant-pathogenic fungal isolates (Friesen et al., 2006). The suspected cases of fungus–fungus gene transfer events are restricted to selfish genetic elements, plasmids, introns and transposons (Rosewich & Kistler, 2000). Although it has rarely been reported, it is likely that horizontal transfer of genetic material occurs between bacteria and fungi. It has been suggested that antibiotic biosynthetic genes are horizontally transferred from gram-positive bacteria to fungi (Buades & Moya, 1996). Additional support for horizontal transfer from bacteria to fungi has been found in studies on hydrolytic enzyme-encoding genes (Li et al., 1997; Liu et al., 1997). Further evidence for bacterium–fungus gene transfer is now emerging from the analyses of fungal genome sequences which reveal the presence of bacterial genes (F. Martin, INRA France, pers. comm.). The mechanisms of horizontal gene transfer from bacteria to fungi are unknown. Because fungi feed by absorption, the ‘you are what you eat’ hypothesis, which was proposed to account for horizontal gene transfer in the case of other eukaryotic cells (Doolittle, 1998), is unlikely to account for gene transfer in fungi (Richards et al., 2006). Therefore, more attention should be paid to fungal endobacteria and their possible involvement in horizontal gene transfer in the future. The ectomycorrhizal fungus L. bicolor, for which the genome sequence is now available and which harbours intracellular bacteria potentially beneficial to fungus biology, is a good candidate model for such studies.
IV. The question of MHB specificity
An important property of the MHB is their fungus specificity (for pathogenic as well as mycorrhizal fungi). The MHB often enhance mycorrhiza formation by some fungi but inhibit the establishment of symbiosis by others (Garbaye & Duponnois, 1992). Most fungus-specificity investigations have involved single isolates of ectomycorrhizal fungal species (Duponnois & Garbaye, 1991; Garbaye & Duponnois, 1992; Duponnois et al., 1993). When several isolates of a single ectomycorrhizal species have been used for MHB analyses, diversity in the responses to a MHB among the fungal isolates has become apparent. Whereas Duponnois & Garbaye (1991) observed a significant promotion by P. fluorescens BBc6 of ectomycorrhiza formation between an American Laccaria laccata isolate and Douglas-fir, using the same MHB strain and an Australian L. laccata strain, Dunstan et al. (1998) recorded significantly inhibited ectomycorrhiza formation. This ‘fungal isolate specificity’ may reflect the genetic distance between isolates of different origin of this taxonomically challenging species (Kropp & Mueller, 1999), the recent acquisition of a resistance mechanism in the American isolate, or the loss of a resistance mechanism in the Australian isolate against antagonistic substances produced by P. fluorescens BBc6.
Although the MHB strain Streptomyces sp. AcH 505 promotes the growth of A. muscaria and Suillus bovinus, it inhibits the growth of Hebeloma cylindrosporum. Antibiosis seems to be the major mechanism underlying the antagonism between AcH 505 and H. cylindrosporum. Indeed, whereas H. cylindrosporum is sensitive to the dominant antibiotic WS-5995 B produced by AcH 505 (Keller et al., 2006), A. muscaria is tolerant to this antibiotic (Riedlinger et al., 2006). The same applies to the few isolates of Heterobasidion investigated to date (Lehr et al., 2007), explaining the ‘fungus specificity’ of AcH 505. Interestingly, by exudating organic acids or protons, A. muscaria is able to inhibit the production of WS-5995 B by Streptomyces sp. AcH 505 (Riedlinger et al., 2006). More generally, other mycorrhizal fungi produce organic acids (van Hees et al., 2006), suggesting that they may influence the fungus–MHB interactions by modulating the spectrum of bacterial antibiotic production.
MHB could also reduce concentrations of antifungal metabolites in the mycorrhizosphere by direct antagonism against microbes that are harmful to mycorrhizal fungi. The fungus-specific MHB might act in such a way, inhibiting microbial strains that act against them. It will be interesting to see which factors other than water-soluble antibiotics are responsible for fungus specificity. By growing the MHB and ectomycorrhizal fungi separately in two compartments communicating only through the atmosphere, Garbaye & Duponnois (1992) revealed that fungus specificity factors include gaseous compounds. Using solid-phase microextraction and gas chromatography–mass spectrometry, Barbieri et al. (2005) were able to analyse 65 volatiles produced by Staphylococcus pasteuri, which is strongly antagonistic towards Tuber borchii mycelium in cocultures without liquid contact. Similar methods could perhaps be used to clarify the gaseous fungus specificity factors indicated by the study of Garbaye & Duponnois (1992).
Because of their selectivity, Duponnois et al. (1993) suggested that MHB could become an alternative to soil fumigation; for example, they could be simultaneously used for controlled mycorrhization and for antagonism against competitive symbiotic and/or phytopathogenic fungi. In the light of ‘fungal isolate specificity’, however, our data on the phytopathogen Heterobasidion annosum and Streptomyces sp. AcH 505 are of concern (Lehr et al., 2007). We observed that, while 11 H. annosum strains tested were suppressed by AcH 505, root infection with one fungal isolate was promoted by the MHB strain. This suggests that some MHB behave as helpers of both symbiotic and pathogenic fungi. Such a hypothesis is consistent with the recent observation of the helper effect of the MHB P. fluorescens BBc6R8 on the wheat pathogen Gaeumannomyces graminis (A. Sarniguet, INRA, France, pers. comm.).
V. Mechanisms of the MHB effect
MHB promote the establishment of symbiosis by stimulating mycelial extension; increasing root–fungus contacts and colonization; and reducing the impact of adverse environmental conditions on the mycelium of the mycorrhizal fungi. For example, spore germination and mycelial growth may be enhanced by MHB through the production of growth factors, through the detoxification of antagonistic substances, or through the inhibition of competitors and antagonists. In the following, we revisit the major hypotheses explaining the helper effect in Garbaye (1994), covering some of the topics in Figs 2 and 3, and provide evidence for a multitude of MHB mechanisms.
1. Spore germination
As early as 1962, Mosse showed that some MHB and their culture filtrates were able to stimulate arbuscular mycorrhizal fungal spore germination of G. mosseae. Direct contact between the spores and bacteria was necessary for the induction of spore germination in Glomus clarum (Xavier & Germida, 2003), indicating a ligand–receptor interaction between the two microbes. These spore germination stimulatory bacteria were accompanied by other bacterial isolates producing antagonistic volatiles, suggesting the presence of a complex bacterial consortium on the G. clarum spore surface that regulates germination. In contrast, volatile compounds produced by different species of Streptomyces were proved to promote the germination of G. mosseae spores (Tylka et al., 1991). Working with the Paenibacillus validus–Glomus intraradices interaction, Hildebrandt et al. (2002) showed that the otherwise obligately symbiotic G. intraradices could grow and sporulate in fungus–bacterium cocultures. A specific carbon source, raffinose, was detected in bacterial cultures and mycelial growth was supported by this sugar (Hildebrandt et al., 2006).
2. Mycelial growth
Fungus–bacterium cocultures are easily produced and thus have often been used to screen for potential MHB strains for mycorrhiza inoculation experiments. A significant correlation has been shown to exist between increases in mycelial biomass and promotion of mycorrhiza establishment (Garbaye & Bowen, 1989; Gryndler & Vósatka, 1996; Founoune et al., 2002a). The MHB P. monteilii produces currently unknown gaseous compounds that increase the growth rate of P. albus when the bacteria are grown on tryptic soy broth agar or on a minimal medium with trehalose (Duponnois & Kisa, 2006). The arbuscular mycorrhizal colonization of roots with Glomus fistulosum and the growth rate of the hyphae in the soil substrate were significantly higher when the fungus was coinoculated with Pseudomonas putida or with the low-molecular-weight fraction of the bacterial culture supernatant (Vósatka & Gryndler, 1999), indicating that the effective substances were in this fraction.
The bacterial secondary metabolite responsible for growth promotion of A. muscaria by Streptomyces sp. AcH 505 has been isolated from bacterial suspension cultures, and its structure has been elucidated (Keller et al., 2006; Riedlinger et al., 2006). This novel compound, 5,6,7-trihydro-7-hydroxy-3-prolylbenzofuran-4-1, was classified as auxofuran because of its auxin-reminiscent structure. The strain AcH 505 exuded auxofuran, with a concentration range of between 10 nm and 1 µm being observed in the culture media, which was optimal for growth promotion of ectomycorrhizal fungi (Riedlinger et al., 2006; our unpublished results). The expression of the A. muscaria gene acetoacyl co A synthetase (AmAacs) was up-regulated by auxofuran treatment, indicating an activation of sterol biosynthesis by this substance (Riedlinger et al., 2006).
Coculture with MHB may influence the interacting fungal hyphae in various ways, depending on the MHB–mycorrhizal fungus pair. Comparing the impact of different MHB on the growth and morphology of L. bicolor mycelium, we have demonstrated that, of the tested strains, P. fluorescens BBc6R8 is the only MHB that simultaneously enhances significantly the growth, the branching angle and the branching density of the mycelium, as well as the number of apices (Deveau et al., 2007). In the case of the MHB Streptomyces sp. AcH 505, although it promotes mycelial extension of A. muscaria, it sharply reduces hyphal biomass/colony area ratio as a result of a reduction in mycelial density (Schrey et al., in press). Moreover, it reduces the thickness of the fungal hyphae (Maier, 2003). We recently analysed the structural background of this hyphal thinning: bacterial inoculation leads to a change in the organization of the actin cytoskeleton in A. muscaria (Schrey et al., in press). As expected in the light of these results, the A. muscaria–Streptomyces sp. AcH 505 interaction is accompanied by altered fungal gene expression levels. Fungal genes related to signal transduction pathways, cell stress and cell growth, metabolism and cell structure were found to be up-regulated in mycelium cocultured with AcH 505 (Schrey et al., 2005; Tarkka et al., 2006). In the case of the P. fluorescens BBc6R8–L. bicolor S238N pair, a gene profiling approach has also recently revealed that morphological modifications of the fungal mycelium induced by this MHB strain are coupled to pleiotropic alterations of the fungal transcriptome. The latter are partly specific to the interaction with the strain BBc6R8 and suggest that the strain BBc6R8 induced a shift from the saprotrophic stage of the fungus to the infectious stage (Deveau et al., 2007). The expression level of the A. muscaria cyclophilin gene AmCyp40, which encodes a prolyl isomerase that is involved in cell growth and the cell stress response, is 9-fold up-regulated in A. muscaria–Streptomyces sp. AcH 505 coculture (Schrey et al., 2005). To analyse the specificity of A. muscaria–MHB Streptomyces interactions, the modifications of the expression of the cyclophilin gene AmCyp40 were monitored in the presence of culture supernatants of AcH 505 and of another MHB, Streptomyces setonii AcH 1003. Inoculation with culture supernatants of both MHB led to a large increase in AmCyp40 expression, but the application of the culture supernatant of Streptomyces sp. AcH 504, a bacterium that is not a MHB, did not lead to altered AmCyp40 expression (Schrey et al., 2005). Later we observed that the antibiotic WS-5995 B produced by AcH 505 induces AmCyp40 expression (Riedlinger et al., 2006), but it is currently unknown which factor(s) from AcH 1003 is responsible for the induction of AmCyp40.
Certain fungus–plant combinations may produce an observable MHB effect only when fungal growth is inhibited by the soil substrate. Pseudomonas fluorescens BBc6R8 had a significant positive influence on fungal biomass only when the nursery soil was autoclaved before the bacteria and fungal inoculum were added (Bruléet al., 2001). The authors suggested that toxic metabolites are released by autoclaving, inhibiting mycelial development. The bacterium would then detoxify the soil, restoring soil conduciveness. In accordance with the data obtained by Bruléet al. (2001) with an ectomycorrhizal model, a Bacillus sp. strain had a stronger positive effect on the intensity of root cortex colonization and arbuscule formation by G. intraradices when the plants were subjected to drought stress than in a normally watered soil (Vivas et al., 2003). MHB may also release plants from stress caused by heavy metal pollution. It was recently demonstrated that bacteria isolated from heavy metal-contaminated soils had a strong positive impact on spore germination and on presymbiotic fungal growth under toxic concentrations of heavy metals: bacterial inoculation not only reduced damage to G. mossae hyphae but even resulted in increased mycelial growth and mycorrhiza formation (Vivas et al., 2005). Also, under natural conditions soil solution commonly contains substances that inhibit mycelial growth, and at least some MHB are able to detoxify these molecules. For instance, Duponnois & Garbaye (1990) showed that MHB reduced the concentrations of phenolic antagonistic substances produced by mycorrhizal fungi.
4. Increased branching of the root system and facilitated colonization
Stimulation of lateral root formation is a frequently observed characteristic of MHB (Duponnois, 1992; reviewed in Garbaye, 1994; Poole et al., 2001; Schrey et al., 2005), which essentially leads to an increase in potential points at which plant and fungus can interact. As well as increasing the number of lateral roots, the Bacillus strain isolated by Bending et al. (2002) increased the formation of only first-order ectomycorrhiza roots, and Burkholderia and Rhodococcus strains isolated by Poole et al. (2001) increased the formation of only second-order ectomycorrhiza roots in Scots pine (Pinus sylvestris). The mechanisms underlying these spatially organized root ramifications did not relate to bacterial colonization patterns (Poole et al., 2001). Phytohormones, including auxins and ethylene, have been implicated in producing morphological changes in roots during mycorrhiza formation (Kaska et al., 1999), including the formation of lateral roots and dichotomous branching of short roots (Barker & Tagu, 2000). However, no link has yet been established between stimulated root branching and auxin or ethylene production by MHB. Two non-indol-3-acetic acid (IAA)-producing strains isolated from Lactarius rufus mycorrhizas, Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, were found to promote dichotomous root branching in Scots pine seedlings (Aspray et al., 2006a). This suggests that other growth factors are produced by EJP67 and EJP73, such as ethylene which has been implicated in dichotomous root branching (Kaska et al., 1999), or that the two bacteria modulate hormone production or transport in Scots pine.
Plants produce chemotropic signals to direct mycelial growth towards the fine roots. In both arbuscular mycorrhizas and ectomycorrhizas it has been shown that these substances include flavonoids (Lagrange et al., 2001; Akiyama et al., 2002). MHB could indirectly facilitate root colonization by mycorrhizal fungi, by inducing the release of plant flavonoids. Xie et al. (1995) demonstrated that the nodulation (Nod) factors produced by a MHB Bradyrhizobium japonicum strain stimulated the production of flavonoids by soybean (Glycine max) seedlings and mycorrhiza formation. They also showed that exogenously applied flavonoids and Nod factors similarly promoted mycorrhiza formation. They hypothesized that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization.
A direct contact between MHB and plant roots may be required for the promotion of mycorrhizal symbiosis, as demonstrated by Aspray et al. (2006b) for the MHB Paenibacillus sp. strain EJP73. This recent study indicates that the strain EJP73 exudes substances related to the MHB effect only when in contact with the roots; that these substances are attached to the bacterial cell wall; and/or that these substances are short-lived and therefore have to be produced continuously (Aspray et al., 2006b). MHB effectors that facilitate root colonization could be plant cell wall-digesting enzymes, which would enhance the penetration and the spreading of the fungus within the root tissues (Mosse, 1962), or suppressors of the plant defence response (Lehr et al., 2007). The impact of MHB on plant gene expression levels can now be examined on a larger scale because the genome sequences of many plants are available or their annotation is in progress, and large databases of expressed plant genes exist.
VI. The role of MHB in mycorrhizal functions
In Fig. 3, we propose a simplified representation of the main interactions in the mycorrhizosphere during the functioning of the already-established symbiosis. As in Fig. 2, the bacterium has been placed in the centre of the figure in order to emphazise its specific helper activities. Three categories of interactions warrant special attention because of their practical importance in plant production and their potential applications in agriculture, horticulture and forestry: nutrient mobilization from soil minerals, fixation of atmospheric nitrogen, and protection of plants against root pathogens.
Through weathering and solubilization processes, soil minerals derived from the parent rocks are often the only or the dominant source of all plant nutrients (except nitrogen) in natural ecosystems that are not influenced by humans. Soil and rhizosphere microbes, including fungi as well as bacteria, are known to contribute to mineral weathering by secreting protons and complexing agents such as low-molecular-weight organic anions or siderophores. However, the influence of the whole mycorrhizal complex, comprising the root, the symbiotic fungus and other associated microorganisms, on mineral weathering has been investigated only recently. In situ evidence exists that ectomycorrhizas and their extramatrical mycelium can locally solubilize minerals (Landeweert et al., 2001), but the roles of the different partners of the ectomycorrhizal complex are poorly understood. Frey-Klett et al. (2005) showed that, in a forest nursery, the proportion of phosphorus-solubilizing and siderophore-producing fluorescent pseudomonads was much higher in Douglas-fir–L. bicolor ectomycorrhizas than in the surrounding root-free soil. Similarly, Calvaruso et al. (2007) observed that the oak (Quercus sessiliflora)–Scleroderma citrinum mycorrhizosphere significantly structured the culturable bacterial and fungal communities, in the organomineral horizon of a forest stand, by selecting very efficient strains for phosphorus mobilization. Further experiments revealed a higher proportion of culturable bacteria able to release iron from a mineral, biotite, in oak–S. citrinum ectomycorrhizas than in the bulk soil (Uroz et al., 2007). Using a nonculturable approach, F. Rineau (pers. comm.) showed that the capacity of beech (Fagus sylvatica)–Xerocomus sp. ectomycorrhizas to complex iron and to modify the pH of the rhizosphere was significantly reduced when the samples were pretreated with rifampicin and chloramphenicol, suggesting that mycorrhiza-associated bacteria contributed to the measured activities. In the case of arbuscular mycorrhizas, Toro et al. (1997) demonstrated experimentally that two MHB strains of Enterobacter sp. and B. subtilis, when inoculated together with G. intraradices into onion, enhanced phosphorus uptake from rock phosphate; this is consistent with the recent demonstration by Jayasinghearachchi & Seneviratne (2005) that the solubilization of rock phosphate is enhanced by formation of mixed biofilms between phosphate-solubilizing saprotrophic fungi and a Bradyrhizobium elkanii strain. Taken together, these recent findings strongly suggest that ectomycorrhiza-associated bacteria complement the roles of the external mycelium by mobilizing nutrients from minerals.
Nitrogen is another element essential to plant nutrition. It is absorbed by the roots as nitrate and ammonium derived from soil organic matter through decomposition by saprobes. In the case of acidic and/or cold soil ecosystems, where nitrogen is sequestrated in high carbon:nitrogen and recalcitrant humic compounds, nitrogen is a limiting factor for plant nutrition (Read, 1991). This is why special attention has been paid to potential nitrogen fixation by bacteria associated with ectomycorrhizas, the dominant mycorrhizal type of trees in mostly nitrogen-poor temperate and boreal forests. In boreal forests, the total nitrogen input attributable to biological fixation has been estimated to be approx. 20 kg ha−1 yr−1 by DeLuca et al. (2002). Recently, Paul et al. (2007) showed that nitrogen fixation by tuberculate ectomycorrhizas formed by Suillus tomentosus on Pinus contorta in British Columbia was approx. 10% of the average activity reported for Frankia nodules on Alnus rubra. As early as 1987, Li demonstrated effective nitrogenase activity of bacteria associated with Douglas-fir tuberculate ectomycorrhizas in Oregon (Li, 1987). In 1992, Li et al. found that some of these bacteria belonged to the Bacillus group. More recently, Rózycki et al. (1994) isolated diazotrophic (acetylene-reducing) Pseudomonas and Bacillus spp. from P. sylvestris and Q. robur ectomycorrhizas in Poland and Paul (2002) isolated the nitrogen-fixing bacterium Paenibacillus amylolyticus from the inner tissue of the S. tomentosus tuberculate ectomycorrhizas of lodgepole pine (Pinus contorta) in British Columbia. This presence of nitrogen-fixing bacteria in these diverse ectomycorrhizal types clearly supports their potential for improving plant nutrition. Using molecular methods, Timonen & Hurek (2006) and Izumi et al. (2006a) found DNA sequences of the nifH gene (encoding nitrogenase) in P. sylvestris–S. bovinus and Pinus nigra–Suillus variegates ectomycorrhizas, respectively. The genomic nifH signature (DNA) was present both in a productive and in a nonproductive P. nigra stand, while transcripts (mRNA) were found only in the nonproductive stand (Izumi et al., 2006a). Comparison of the DNA and mRNA sequences revealed that the nifH gene was not always transcribed, and that the actively transcribing bacteria were different from those detected using the DNA-based approach. The dominant active bacteria belonged to Methylocella spp. and Burkholderia spp. All these results suggest that diazotrophic bacteria embedded in ectomycorrhizal tissues contribute to nitrogen input in forest ecosystems by directly providing nitrogen of atmospheric origin to the two partners of the symbiosis, thus bypassing the organic matter decomposition pathway. These bacteria can therefore be assigned to the MHB category, because they are suspected to positively interact with the functioning of the mycorrhizal symbiosis. Nevertheless, direct confirmation of nitrogen fixation and the quantification of the net nitrogen input remain to be performed. Understanding the regulation of the expression of nitrogenase genes is another challenge: in the association between Pleurotus ostreatus (a nonmycorrhizal mushroom) and Bradyrhizobium sp., nitrogen fixation only occurred in the bacterial biofilm associated with the fungal mycelium (Jayasinghearachchi & Seneviratne, 2004).
Finally, mycorrhiza-associated bacteria also contribute, together with the fungal symbiont, to protection against root pathogens. In vitro antagonism against phytopathogens by mycorrhiza-associated bacteria has been frequently observed (Schelkle & Peterson, 1996; Becker et al., 1999; Maier et al., 2004). Moreover, Frey-Klett et al. (2005) revealed a significantly higher proportion of fluorescent pseudomonads inhibiting the growth of seven root-pathogenic fungi belonging to the genera Rhizoctonia, Fusarium, Phytophthora and Heterobasidion in the Douglas-fir–L. bicolor ectomycorrhizas than in the surrounding bulk soil. Concerning arbuscular endomycorrhizas, Citernesi et al. (1996) and Linderman (1994) reviewed a number of similar examples, and speculated about the fact that arbuscular mycorrhizal fungi are relatively tolerant to bacterial antagonists that inhibit fungal root pathogens. They concluded that, having coevolved with plants, they would be adapted to be compatible and function in concert with such antagonists. More recently, Budi et al. (1999) and Selim et al. (2005) focused on a strain of Paenibacillus isolated from the rhizosphere of sorghum that proved to be compatible with arbuscular mycorrhiza development but antagonistic towards soilborne fungal pathogens. This strain was found to produce small peptides which were responsible for the antagonistic effect against pathogens but were harmless to the symbiotic fungi. All these findings suggest that MHB could have evolved selective mechanisms of interaction with their microbial surroundings, having neutral or positive effects on their host mycorrhizal associations but negative effects on the root pathogens that might threaten their very habitat. Whether this hypothesis represents a general feature of MHB remains an open question because some MHB can also behave as helpers of both symbiotic and pathogenic fungi, as discussed in Section IV, ‘The question of MHB specificity’.
VII. Conclusions and research priorities
Before concluding, we feel it necessary to clarify a point of semantics about the very definition of ‘mycorrhiza helper bacteria’. The term ‘mycorrhization helper bacteria’ was first coined by Duponnois & Garbaye (1991) to refer only to bacteria that promote the establishment of the root–fungus symbiosis, while either ‘mycorrhiza’ or ‘mycorrhization’ has been used with the same intended meaning by later authors. We think that the use of the word ‘mycorrhiza’ is misleading because it suggests that the bacterium helps the functioning of the already-established symbiosis. Instead, in most instances we would prefer the use of the word ‘mycorrhization’, which strictly relates to the process of mycorrhiza formation in the applied context of mycorrhizal inoculation (a technique commonly known as ‘controlled mycorrhization’). Therefore, for the sake of the intelligibility of future papers, we propose the following working definitions: while the abbreviation MHB will be used for both meanings, ‘mycorrhization’ will strictly refer to the bacteria that help mycorrhiza formation, and ‘mycorrhiza’ will refer to those that interact positively with the functioning of the symbiotic organ, keeping in mind that these two functional categories might be represented by different or overlapping groups of microorganisms.
When revisiting the concept of MHB proposed 13 yr ago, we must ask the question: what is new? The answer clearly is: a lot! Evidence for mycorrhization helper bacteria exists in diverse mycorrhizal systems. The development of nucleic acid-based methods has yielded new insights: in the taxonomy of MHB; into the interactive mechanisms that are important in the helper effect; and in the detection of endofungal bacteria which might prove to form a three-partner symbiosis within the mycorrhizal association. In addition, the appearance of a new research field, dealing with the mycorrhiza helper bacteria, meaning those that contribute to mycorrhizal functions after the establishment of the root–fungus symbiosis, has been facilitated by studies of mycorrhization helper bacteria. Thus, the MHB concept has proved to be a fruitful one.
However, knowledge of biological interactions in the mycorrhizosphere is still rudimentary, and a lot of substantial research is needed to understand them fully. We suggest five research priorities to achieve this goal.
1Searches for MHB in a wider range of mycorrhizal systems should be carried out in order to better explore the question of their specificity. The combination of molecular approaches (Izumi et al., 2006c) may allow the identification and functional characterization of mycorrhiza-associated bacteria, but culture-based methods remain necessary to demonstrate MHB effects and to study the mechanisms.
2Identification of marker traits and genes specific for MHB functions should be performed (as initiated by Schrey et al., 2005 and Deveau et al., 2007), both in fungi and in bacteria. This should provide short cuts when looking for helpers in new mycorrhizal systems (Frey-Klett & Garbaye, 2005). On the fungal side of the system, this task will be greatly facilitated by the recent genome sequencing of two model species of mycorrhizal fungi, G. intraradices and L. bicolor. On the bacterial side, a rapidly increasing number of genomes are being sequenced, including species inhabiting the rhizospheres and mycorrhizospheres of plants.
3Imaging techniques should be used to specifically localize bacterial cells and their activities related to the helper effect, while addressing the question of metabolic adjustments during fungal–bacterial cometabolism. This approach is likely to significantly improve our understanding of helper mechanisms.
4The contribution of mycorrhiza-associated bacteria (the mycorrhiza helpers) to mycorrhizal functions such as nutrient uptake, protection of the roots against phytopathogens and provision of the plant with growth factors should be investigated. This would undoubtedly provide a new dimension to the physiology, ecology and evolutionary biology of mycorrhizal symbioses.
5Last but not least, the principles and practices of controlled mycorrhization in agriculture, horticulture and forestry should be revisited: helper bacteria may improve the efficiency of fungal inocula with a low extra cost, because bacteria are easier to grow in commercial quantities than most mycorrhizal fungi. This means that more MHB work should be dedicated to model mycorrhizal fungi that are of obvious commercial interest as well as being of use as research laboratory models. Such fungi include G. intraradices (whose genome has been sequenced) for arbuscular mycorrhizas and Pisolithus spp. and L. bicolor (the genome of the latter having been sequenced) for ectomycorrhizas. In addition, growing concern about the pollution of soils, and the resulting trend towards reducing the input of chemicals in plant production, should foster more environmentally friendly practices such as controlled mycorrhization or microbial bioremediation, for instance by using mycorrhizal fungi as carriers of depolluting bacteria (Sarand et al., 1998). This converging of scientific and practical interests, supported by the development of genomics, may represent a unique opportunity to place MHB at the forefront of future mycorrhiza research and to boost the more general field of fungal–bacterial interactions in ecosystems.