Tansley Review No. 76 Helper bacteria: a new dimension to the mycorrhizal symbiosis

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1 Neu-Phytol. (1994), 128, Tansley Review No. 76 Helper bacteria: a new dimension to the mycorrhizal symbiosis BY J. GARBAYE INRA, Centre de Recherches Forestieres de Nancv, Champenoux (France) (Accepted 25 May 1994) CONTENTS Summary 197 I. Introduction 197 II. Evidence for helper bacteria 198 III. Fungus-specificity of MHBs 200 IV. Mechanisms underlying the MHB effect Previous studies and data Effect of MHBs on the receptivity of the root Effect of MHBs on the root-fungus recognition Effect of MHBs on the funga] growth Modification of the rhizospheric soil by MHBs 6. Effect of MHBs on the germination of fungal propagules V. Ecological and evolutionary implications of MHBs VI. Practical applications of MHBs VII. Conclusions and perspectives Acknowledgements References SUMMARY The symbiotic establishment of mycorrhizal fungi on plant roots is affected m various ways by the other microorganisms of the rhizosphere, and more especially by bacteria. This review discusses the case of some of these bacteria which consistently promote mycorrhizal development, leadmg to the concept of ' mycorrhization' helper bacteria (MHBs). Examples of MHB evidence are given from the literature, with special reference to the Douglas fir (Pseudotsuga menzeisii Mirb. Eranco)-Lacron'o laccata Scop, ex Fr. ectomycorrhizal combination which has been more extensively studied. The fungal specificity of some MHBs and the various mechanisms underlying their effect are discussed, considering five hypotheses: effects on the receptivity of the root, effects on the root-fungus recognition, effects on the fungal growth, modification of the rhizospheric soil, and effects on the germination of the fungal propagule. MHBs are then considered for their ecological and evolutionary implications, and examples of practical applications in forest nurseries are given: when added to the fungal inoculum, MHBs can improve the success of ectomycorrhizal inoculation of planting stocks with fungi selected for their outstanding growth stimulation after outplanting. The conclusion points out a number of fundamental questions which remain unanswered about mycorrhization helper bacteria and suggests some investigation priorities in this new field of mycorrhiza research. Key words: Rhizosphere, mycorrhizas, bacteria. 1. INTRODUCTION Root exudates provide most of the low molecular weight, easily absorbed carbon compounds available for microorganisms in the soil; that is why the largest and most diversified microbial populations in soils are in the rhizosphere, bacteria being particularly abundant. On the other hand, the roots of most terrestrial plants are inhabited by symbiotic fungi forming specialized structures known as mycorrhizas. This mycorrhiza] symbiosis is actively studied for its beneficial effect on plant growth and for its potential use in agriculture and forestry. Understanding the ecology and physiology of the association and learning how to control its establishment and stability is therefore a key issue of plant sciences. From the germination of propagules in the soil to the achievement of the true symbiosis, the fungus has to grow in the soil and colonize the root surface

2 198 y. Garbaye before reaching receptive entry points. During this free-hving stage of its development, it interacts with the rhizospheric bacterial populations (Rambelli, 1973; Bowen & Theodorou, 1979; Bowen, 1980; De Oliveira & Garbaye, 1989; Garbaye, 1991a). For instance, Bowen & Tbeodorou (1979) showed under in vitro conditions that some soil and rhizosphere bacteria affected the growth of the ectomycorrhizal fungus Rhizopogon luteoliis along the root of young seedlings of Pinus radiata, and that this effect was either positi\ e or negative depending on the bacterial isolate. The latter example shows that most of these interactions are competitive but that some others can be beneficial to the mycorrhizal infection process. This review will analyze a range of results illustrating the fact - more particularly in the case of ectomycorrhizas, where literature is more abundant - and discuss the present trends in this new field of mycorrhiza research. The scope of the review is limited to positive effects of rhizobacteria on early myeorrhiza establishment: with a few exceptions, it does not encompass inhibitory interactions and the spread, stability and functioning of the symbiosis. For instance, the effect on plant growth is not discussed. ti. EVIDENCt; FOR HELPKK B,^CTERIA Ridge & Theodorou (1972) found that fumigation with methyl bromide enhanced infection of Finns radiata by Rhizopogon luteolus m one nursery soil but reduced it in another one; they concluded that this might have been related to different microorganisms recolonizing the soils. Similarh', Garbaye (1983) showed that some soils are less conducive to mycorrhiza formation by Hebeloma crustiliniforme with beech seedlings when fumigated with methyl bromide, while other soils show greater receptivity. Marx et al. (1982, 1984) reported that vegetative inoculum of Pisolithus tinctorius contaminated by fungi and bacteria was sometimes more efficient for mycorrhiza formation than non-contaminated inoculum when inoculating pine seedlings in fumigated nursery soils. These indirect results strongly suggest that interactions exist between soil microflora and mycorrhizal infection. Direct experimental evidence of this hypothesis was given by Garbaye & Bowen (1987). Three soils, with the same pedologieal origin but different management histories and different microbial communities, were steam-sterilized and separately reinoculated or not with the total microflora of one of the three soils. They were also inoculated under standardized conditions with one of the following ectomycorrhizal fungi: Paxillus involutus Batsch. ex Fr., Hebeloma crustuliniforme Quelet or Rhizopogon luteolus. Fr. and Nordh. Seedlings of Pinus radiata D. Don. were grown in pots filled with the prepared soils. The results clearly showed that, in a given soil characterized by its physico-chemical properties, n:iycorrhiza! establishmem was strongly affected by the microbial environment. They also revealed that enhancements of mycorrhiza] infection by soil microfloras outnumbered negative effects. Moreover, in a given soil, the three fungi tested reacted differently to each microflora, suggesting specific interactions. This meant that rhizospheric interactions with mycorrhizal fungi cannot be discussed only in terms of competition, but that synergism should be taken into account and may play an important role in the general ecologn' and distribution of mycorrhizal symbioses. However, at that stage, it was impossible to know if the recorded effects were due to individual microbial strains or to complex interactions within whole communities. Experiments controlling single isolates were necessary. With arbuscular endomycorrhizas, Meyer & Linderman (1986) showed that a rhizospheric strain of Pseudomonas putida enhanced mycorrhizal infection and grov\th of subterranean clover; with ectomycorrhizas, De Oliveira (1988) found that several bacterial isolates from forest soils stimulated mychorrhiza formation by Hebeloma crustuliniforme with beech seedlings under controlled conditions. Thus, it was well demonstrated that the 'helper' effect of the soil microflora recorded in the previous experiments can be due to individual bacterial strains. Garbaye & Bowen (1989) went a step further and postulated that true helper bacteria should be adapted to living in association w ith the fungus; therefore, if they were to be found in a soil, they were probably more frequent in the close \-icinity of the fungus, i.e. in the mantle in the case of ectomycorrhizas (bacteria are frequently seen embedded in the outer mantle: Foster & marks, 1966; Duponnois, 1992; Fig. la). Thus, these authors isolated bacteria from surface-sterilized ectomycorrhizas formed by Rhizopogon luteolus with the roots of Pinus radiata and tested them for their effect on mycorrhiza formation with the same symbiotic partners in a sterilized soil. They found about 10** colony-forming units g^^ (fresh weight) mycorrhiza. These were mostly fluorescent pseudomonads, and 80"o of them displayed a significant positive effect on mycorrhiza establishment, while only 20% were neutral of inhibitory. However, there was no clear relation between the taxonomy of the bacteria and their effect on mycorrhiza formation. Garbaye, Duponnois & Wahl (1990), Duponnois & Garbaye (1991 a) and Duponnois (1992) used the same approach with another symbiotic partnership: Douglas fir {Pseutotsuga menziesii Mirb. Franco) and the ectomycorrhizai fungus Laccaria laccata Scop, ex. Fr. They screened 47 strains (mostly fluorescent pseudomonads and sporulating bacilli, depending on

3 Helper bacteria in mycorrhizal formation 199 Figure 1. {a) Transmission electron micrography of the cross-section of the mantle of an ectomycorrhiza formed by Laccaria laccato with a Douglas fir seedling in a forest nursery; arrowheads show bacteria embedded in the outer mantle (on the left-side of the photograph): scale-bar, S jim. (b) and {c) Fluorescence light micrography (acridine orange staining) of Laccaria laccata mycelium in contact witb two isolates of rhizospbere bacteria adhering (6) or not (c) to the hyphae; scale-bar, 10/im.

4 200 y. Garbaye Table 1. Effect of four MHBs [isolated from sporocarps or mycorrhizas of Laccaria laccata associated with Douglas fir) ort mycorrhiza formation {per cent of mycorrhizal short roots) of Douglas fir seedlings inoculated with different ectomycorrhizal fungi Bacterial isolates Fungal isolates None MB3 SHBl BBc6 SBc5 Nursery experiment (Number of Laccaria laccata S-238 Laccaria bicolor D-101 Laccaria proxima Hebeloma cylindrosporum Paxillus irtvolutus Pot experiment in the glasshouse freedom for error, 12) Laccaria laccata S-238 Laccaria bicolor 993 Laccaria bicolor S-3 Laccaria bicolor A4B3 x A1B2 Thelephora terrestris degrees oi freedom for error, 12) '1 93' f (xenic conditions) (Number of degrees of ' S-\ ' ' '3-l- 30'4 + 47' In vitro experiment (aseptic synthesis in test-tubes) (Number of degrees of freedom for error, 33) Laccaria laccata S-238 Hebeloma cylindrosporum Paxillus inuolutus QBC ' Cenococcum geophilum u- Values followed by a sign are significantly higher ( + ) or lower ( ) than the one corresponding to the control treatment with no bacteria in the same line of the table (005 probability level); percentages of mycorrhizal short roots were transformed by arcsin square root prior to test of significance, : experiment not done. From Garbaye & Duponnois, ]992. the site) isolated from L. laccata mycorrhizas at developing in an atmosphere with normal CO., various sites and found that about 50,, of them very concentration (which is not the case when the whole significantly enhanced mycorrhiza formation. The plant and its rhizospheric microorganisms are grown most efficient isolates in this respect changed the together in the tube). mycorrhizal index (per cent of short roots mycor- In conclusion, 'mycorrhization' helper bacteria rhizal with L. laccata) from 67 "o in the control (MHB) are probably quite common: they were treatment up to 97% with the bacterium. In found every time they were looked for, under very addition, the same effect was consistently repro- different site conditions and in various plant-fungus ducible as well under axenic in vitro conditions combinations. Moreover, they seem to be closely (aseptic synthesis in test-tubes: Duponnois & associated with the mycorrhizal fungi in the sym- Garbaye, 1991 a, 6), as in glasshouse or nursery- biotic organs, experiments; this proves that the stimulation is an intrinsic property of each single isolate, indepenj,, r It 11 HI. FUNGUS-SPECIFICITY OF MHBS dently of any mteraction withm the microbial community of the rhizosphere. Incidentally, this Atnong the various cases of'mycorrhization'helper observation also validates the use of aseptic synthesis bacteria presented above, the one concerning as well representative of phenomena occurring under Laccaria laccata is particularly interesting because it natural conditions. However, it should be noted that provides evidence of a fungal selectivity of the MHB three precautions were taken to maximize the effect, as was indicated by the earlier results of similarity with in vivo conditions: the roots grew in Garbaye & Bowen (1987). a peat-vermicuhte substrate closer to natural soil Table 1 (from the results of Garbaye & Duponnois, (adsorbing properties of the humic colloids of the 1992) shows that, in a wide range of experimental peat) than the agar commonly used in this type of conditions, the MHB isolated from the Douglas test, the mineral nutrient solution did not contain hr-laccaria laccata S238 symbiotic combination sugar, and the top of the plant grew outside the tube consistently stimulated mycorrhiza formation by L. through a plastic seal preventing contamination. The laccata and L. bicolor (two very closely-related two latter precautions ensured that the root exudates species) with Douglas fir, had no effect on L. were only dependent on photosynthesis by the shoot proxima, and inhibited the symbiotic establishment

5 Helper bacteria in mycorrhizal formation 201 of fungi belonging to other genera. Thus, in this system, MHBs are clearly seleetive of the fungal species, and the term fungus-specific can be used. This finding suggests that MHBs are adapted to live in the elose vicinity of the mycorrhizal fungus, which is consistent with the observations made in Section II about the high frequency of helpers among the bacterial populations isolated from the mycorrhizas. In contrast, the same work and Garbaye, Churin & Duponnois (1992) have shown that the same bacterial isolates consistently promoted mycorrhiza formation with L. laccata and different host-plants (four conifers: Picea abies, Pmus nigra, Pinus sylvestris, Pseudotsuga menziesii and an angiosperm: Quercus robur). Therefore, in the system under study, the MHB effect is not plant-speeifie. IV. MECHANISMS UNDERLYING THE MHB EFFECT 1. Previous studies and data To date, little work has been specifically aimed at elucidating the mechanisms involved in the effect of helper bacteria on the mycorrhizal symbioses. The following discussion is supported by little experimental evidence and often refers to analogies with more advanced fields of the literature about the relationships between roots and rhizospheric microorganisms. Most available data concern the MHBs associated with the Douglas fir-laccaria laccata symbiotie combination which has been extensively studied and already mentioned above. In this system, it was shown in Section II that the MHB effect was reproducible under in vitro gnotobiotic conditions, independently of the other soil BACTERIUM ]. root-fungus recognition and attachment Figure 2. Simplified representation of the rhizosphere pointing out five possible ways by which a bacterium can promote mycorrhizal establishment. The numbers refer to the five hypotheses discussed in Section IV. microorganisms. This provides an experimental model for studying the mechanisms involved in the enhancement of mycorrhiza formation by individual bacterial strains. This simplified system has three partners; the plant, the symbiotic fungus and the bacterium. By growing them axenically alone, in pairs, or in threes, and by analyzing their biochemical interactions, it is thus theoretically possible to detect explanatory mechanisms. Figure 2 represents the main hypotheses to be discussed. 2. Effect of MHBs on the receptivity of the root According to this first hypothesis, the bacterium, proliferating in the rhizosphere before any in\'olvement of the symbiotic fungus, improves the reeeptivity of the root to mycorrhiza formation. Duponnois (1992) has shown that the MHBs associated with the Douglas fir-l. laccata symbiosis produced IAA - most of them even in tryptophanfree media - and that exogenous IAA stimulated short root initiation on Douglas fir seedlings. This may be one ofthe mechanisms involved m the helper effect: if the plant develops more short roots in the same volume of soil, the probability of encounter between a soil-borne fungal propagule and a short root increases. However, the number of bacterial isolates tested was too small to generalize from this observation. It is also worth noting that L. laccata itself produces IAA from tryptophan, making it difficult to assess the real contribution of the bacterium in the complete system, and that other root-growth regulating substances might be involved. Moreover, Nylund (1988) suggests that the role of IAA in ectomycorrhiza formation has probably been overestimated and that the receptivity of the root to fungal colonization is rather mainly regulated by the nitrogen and phosphorus status of the plant. Duponnois (1992) also hypothesized that MHBs could soften the cell walls and the middle lamella bet%veen the cells of the root cortex by producing specific enzymes and thus making fungal penetration easier. At least two out of four such enzyme activities (endoglucanase, cellobiose hydrolase, pectate lyase and xylanase) were detected in pure cultures of each of several MHBs from the Douglas fir-l, laccata system. This tends to support the hypothesis, as did the early work of Mosse (1962) which showed that some microorganisms (e.g. Pseudomonas sp.) producing cell wall-degrading enzymes promoted the establishment of arbuseuiar endomycorrhizas on clover roots under aseptic conditions. This author also showed that replacing the living bacterial cells by filtrates of bacterial cultures or by solutions of the enzymes had the same effect. It is thus clearly established that this type of mechanism can explain some cases of the MHB effect.

6 202. Garbaye 3. Effect oj MHBs on root-fungus recognition According to this second hypothesis, the bacterium interferes with the plant-fungus recognition mechanisms which are the first steps of the interactive process leading to the symbiosis. Anderson (1988) reviewed the different chemical elicitors and mediators, produced either by the plant or the fungus, involved in the mutual recognition: phenolic compounds, enzymes, lectin and polysaccharide or glycoprotein fibrils (permitting surface attachment), volatile and phytohormones. T'he cellwall molecular patterns of both partners also play a role. A bacterium can obviously interfere with this 'biochemical dialogue' between the two potentially symbiotic partners by breaking down or transforming the mediating substances, or contributing to the production of some key-compounds such as auxin and enzymes. The attachment of bacteria to either the root or the fungus, or both, can also modify the cell-wall properties or facilitate the establishment of the symbiosis by providing a mechanical link between the two partners (Duponnois, 1992, and Figs. 1 b, c). When studying the attachment problem, one should take into account the fact that the mycelium of ectomycorrhizal fungi can be either hydrophilic or hydrophobic (Unestam, 1991). Experimental results are lacking, but this second hypothesis is worth considering because it could easily explain the fungus-selectivity of some MHBs which was shown in Section III. 4. Effect of MHBs on the fungal growth According to this third hypothesis, the bacterium helps the growth of the fungus in its saprophytic. presymbiotic stage in the rhizospheric soil or on the root surface. Because of the relative simplicity of the experiments involved (co-culturing the fungus and the bacterium), this hypothesis has been more intensively tested than the previous ones. Cocultures of two microorganisms in a Petri dish or in any in vitro axenic system have been widely used for studying useful negative interactions, for instance detecting antibiosis or competitive inhibition for biological control. These techniques, generally using rich media, need some modifications when working on positive interactions - as with MHBs - which are more likely to be trophic, i.e. involving a metabolite produced by the bacterium and used by the fungus. In order to detect such effects, the medium in which the fungus is grown should be as poor as possible. However, it should be remembered, when interpreting experiments of this type, that artificial media will always be very different from the real conditions prevailing in the rhizosphere. Duponnois (1992) used such a test with a poor medium to compare a number of bacterial isolates ranging from mycorrhization inhibitors to mycor Effect on tnycorrhiza formation (%) Figure 3, Relationship between the effects of 20 bacterial strains isolated from the Douglas hr-haccaria laccata ectomycorrhizas and associated sporocarps on the radial growth of h. laccata in vitro and on the mycorrhizal establishment (mycorrhiza] index) of L. taciata with Douglas fir in glasshouse pot experiments. The effects are expressed as per cent of the value of the parameter in the control with no bacteria. The correlation coefficient (R = 0-9) is significant at the 0-01 probability level. rhization helpers toward Laccaria laccata, as determined previously in co-inoculation experiments with Douglas fir seedlings under controlled conditions. He found a highly significant correlation between the ability of the bacterial isolates to reduce or promote the mycelial growth of L. laccata and their effect on mycorrhiza formation (Fig. 3). Although a correlation does not prove any direct causal relationship, this result strongly suggests that the MHBs associated with the Douglas fir/l. laccata symbiotic combination mainly act by helping the fungus to grow and colonize the surface of the long roots and reach the infection-receptive short roots, or to survive and grow in the soil explored by the developing root system. In turn, the larger volume of soil occupied by the mycelium increases the probability of encounter with a root of the host-plant. This type of mechanism has been described by mathematical models in the case of some soil-borne diseases (Baker, Maurer & Maurer, 1967; Ricci & Messiaen, 1976). The experimental results of Garbaye & Bowen (1989) with another symbiotic combination {Pinus radiata and Rhizopogon luteolus) also showed that the ability of some bacteria isolated from R. luteolus ectomycorrhizas to limit or enhance mycorrhiza formation was mainly due to their negative or positive effect, respectively, on the growth of mycelium in the soil. These authors had designed a system of glass-boxes containing natural soil where it was easy to place precisely the standardized mycelial inoculum and a calibrated drop of bacterial sus-

7 Helper bacteria in mycorrhizal formation 203 Table 2. Effect of four MHBs (isolated from sporocarps or mycorrhizas of Laccaria laccata associated with Douglas fir) on the mycelia! growth in vitro of different fungal strains (mean radial growth of colonies in mm) zvith liquid contact Bacterial isolates Fungal isolates None MB3 SflBl BBc6 SBc5 Laccaria laccata S-238 Hebeloma cylindrosporum Faxillus involutus NAU Paxillus involutus QBC Cenococcum geophilum Thelephora terrestris D'5O 3' '00 0' '80- O'OO ' I Values followed by a sign are significantly higher ( + ) or lower ( ) than the one corresponding to the control treatment in the same line of the table (0-05 probability level). Number of degrees of freedom for error, 54. From Garbaye & Duponnois pension close to an emerging short root, and observe the development of the fungus and of the root during the following days. By comparing large numbers of inoculation points with or without bacteria, it was thus possible to separate the effects of the bacteria on mycelial growth from the inoculum, on rhizoplane colonization, and on establishment of the symbiosis (mantle formation). Moreover, looking back at the Douglas fir-laccaria laccata mycorrhizal combination, and to the bacteria isolated from and screened for their MHB effect in this system, it appears that the selectivity (fungusspecificity) pattern of these bacteria against other fungi is consistent with the pattern of their effects on the growth of the same fungi ifi vitro (Table 2 from Garbaye & Duponnois, 1992, to be compared with Table 1 in Section III). This point of view is supported by the key work of Bowen & Theodorou (1979). By measuring the mycelial growth of ectomycorrhizal fungi along roots of Pinus radiata in aseptic systems and introducing individual strains of rhizosphere bacteria, they found up to 70",, increase in growth of some fungi with one bacterium and a significant decrease with another. Furthermore, there was fungus specificity in the stimulation. Incidentally, the same study showed that two non-inhibitory pseudomonads could reduce the effect of a negative pseudomonad; this opens another possibility of MHBs: compe'tition with mycorrhization-inhibiting bacteria. In conclusion, all these results tend to indicate that some sort of trophic stimulation (i.e. involving nutritional relations) of tbe fungal growth by the bacterium is the main mechanism involved in the MHB effect, at least in the case of ectomycorrhizas. This interpretation could also partly explain why some MHBs are fungus-selective. In order to analyze these interactions in more detail, two sub-hypotheses within hypothesis 3 can be considered. Sub-hypothesis 3.1 involves the production by the bacterium of metabolites directly used by the fungus as nutrients or enhancing its anabolism (growth factor-type effect). Duponnois (1992), Duponnois & Garbaye (1990) and Garbaye & Duponnois (1992) have tested this sub-hypothesis with MHBs associated with Hebeloma crustttliniforme, Paxillus involutus and Laccaria laccata. They found that some organic acids (predominantly malic and citric) were excreted by the MHBs, and that these acids represented a carbon source as good as glucose for the fungal growth. This can indeed explain part of the MHB effect, but not the very selective specificity already discussed. More interestingly, the same authors also found, by growing the two organisms grown separately in two compartments communicating only through the atmosphere, that volatile compounds were also involved in the specificity (Garbaye & Duponnois, 1992; see Table 3 to be compared to Table 1 in Section HI and to Table 2). Attempts to identify these volatile substances are presently underway. Carbon dioxide could play a role, because it has been shown that, depending on its concentration, it differentially inhibits or stimulates the growth of different fungi (Boasson & Shaw^ 1979, 1981; Le Tacon, Skinner & Mosse, 1983; Straatsma, Van Griensven & Bruinsma, 1986; Imolehin & Grogan, 1980). However, a preliminan experiment was inconclusive (Duponnois, 1992). Ethylene could also be involved, as shown by Imolehin & Grogan (1980) with the root pathogen Sclerotinia minor, as well as ammonia, amines, alcohols, sulphur compounds or low-molecular weight fatty acids (Duponnois, 1992). However, all these experiments having been done in very artificial laboratory conditions, great caution should be taken in extrapolating to the rhizosphere. Nitrogen fixation should also be considered as a possible reason of some MHB effects: Rambelii (1973) showed that some diazotrophic bacteria isolated from ectomvcorrhizas stimulated the growth

8 204 J. Garbaye Table 3. Effect of four MHBs {isolated from sporocarps or mycorrhizas of Lacearia laccata associated with Douglas fir) on the mycelial growth {mean radial growth of 10 colonies in mm) of different fungal strains with no liquid contact after 8 d incubation, at 25 C Bacterial isolates Fungal isolates None MB3 SHBl BBc6 SBc5 Lacearia laccata S-238 Lacearia laccata S 1023 Lacearia laccata CHAM 3 Lacearia laccata 003 Lacearia hicolor CRBF 569 Laeearia hicolor CRBF 581 Lacearia bieolor Lacearia bieolor D-101 Laeearia bieolor Laeearia proxima Hebeloma cylindro.tporum Paxillus involutus QBC Cenococcum geophilum Thelephora terrestris Suillus bovinus Pisolithus tinetorius 115 0S '05 0'60 VOO l'5o '25 l'75-l '05 + 1'85-i '20-1'25- ]' ' '87- l'2o i MO M Values followed by a sign are significantly higher (+) or lower ( ) than the one corresponding to the control treatment in the same line of the table {unpaired Student's / test with 10 replicates, 0-05 probability level), n.d: experiment not done. Xunnber of degrees of freedom for error, 225. From Garbaye & Duponnois, of the fungus by releasing ammonium. This possibility is further supported by Li & Castellano (1984) and Li & Huang (1987) who demonstrated that acetylene-reducing bacteria are associated with ectomycorrhizas of Douglas fir and with the connected fungal sporocarps of Hymenogaster parksii, Tuber melanosporum, Suillus ponderosa, Heheloma crustuliniforme, Lacearia laccata and Rhizopogon vinicolor. More recently, Li, Massicote & Moore (1992) found that the tuberculate ectomycorrhizas formed by Rhizopogon vinicolor with Douglas fir contained nitrogen-fixing, spore-forming Bacillus sp.; -ft^ater extracts from the fungal tissues of the tuberculate mycorrhizal clusters enhanced the nitrogenase activity of the bacterium, suggesting there is a close nutritional relationship between the two microorganisms. However, in the four works cited, the bacterial isolates were not tested for their MHB properties. In contrast, Chanway & HoII (1991) found that a nitrogen-fixing Bacillus promoted the growth of Pinus contorta seedlings but did not interfere with ectendomycorrhiza formation with Wilcoxina mikolae. Sub-hypothesis 3.2 involves detoxification of the fungal environment by the bacterium: like most microorganisms, ectomycorrhiza] fungi grown in vitro excrete metabolites which accumulate in the nutrient medium and inhibit mycelial growth. If it is assumed that this self-intoxication phenomenon also occurs in the rhizosphere, MHBs could act by using or breaking down the fungal metabolites and detoxifying the fungal habitat. Such a mechanism has been shown by Duponnois & Garbaye (1990) to occur with the ectomycorrhizal fungi Paxillus involutus and Hebeloma crustuliniforme and a range of bacteria isolated from forest soils where these fungi commonly form mycorrhizas with beech (Fagus silvatica). The isolates which displayed an MHB effect toward these fungi also detoxified the liquid media in which the fungi were grown. The toxic fungal metabolites were dark polyphenols in the case of P. involutus and unidentified colourless substances in the case of H. crustuliniforme. 5. Modification of the rhizospheric soil by MHBs According to this fourth hypothesis, the metabolic activity of the bacterium multiplying in the rhizosphere modifies the physico-chemical properties of the soil in a way facilitating mycorrhizal infection. As the rhizospheric soil is the habitat of the root as well as of the fungus, both can be affected by these changes and we have to consider indirect mechanisms, more complex and difficult to approach experimentally than those discussed before. Results in this field are still rare, but several processes are likely to be involved, for instance changes of ph or the complexing of ions by siderophore-producing fluorescent pseudomonads, as suggested by the work of Olivier & Mamoun (1988) and Mamoun & Olivier (1989,

9 Helper bacteria in mycorrhizal formation , 1992) with Tuber melanosporum ectomycorrhizas. The potential role of siderophores is worth special attention in future research because Reid et al. (1984, 1986) and Szaniszlo et al. (1981) have sbown that chelating ligands, and more especially hydroxamate siderophore, were produced by ectomycorrhizal fungi and contributed to mineral uptake by the root. If some ectomycorrhizosphere bacteria as MHBs are able to complement this siderophore production, the whole root-fungus interactive equilibrium is likely to be modified. More generally, Nylund (1988) and Wallander & Nylund (1991) showed that the nutrient balance of the plant (mostly nitrogen and phosphorus) is a key factor in mycorrhizal development; therefore, rhizospheric bacteria could regulate such development by mobilizing mineral ions or by competing for their uptake. For instance, Azcon-Aguilar et al. (1986) reported that inoculation with phosphatesolubiltzing bacteria enhanced the arbuscular endomycorrhizal infection of soybean. However, this result is difficult to interpretate: under improved phosphorous nutrition of the plant, root cell membranes have a lower permeability thereby limiting the loss of root metabolites, which should rather limit mycorrhizal infection (Reid, 1990). 6. Effect of MHBs on the germination of fungal propagules According to this fifth and last hypothesis, the bacterium triggers or accelerates the germination of spores, sclerotia, or any other dormant propagules specialized in the conservation and dissemination of the fungus in the soil. In nature, this is often the first step leading to mycorrhiza formation. In the case of ectomycorrhizas, this possibility was completely neglected in the experiments reported above, where only 'artificial', non-dormant mycelial propagules were used from the beginning of the screening process. Fries (1987) reports several examples of stimulation of basidiospore germination by yeasts (mostly Rhodotorulla species) and bacteria, but the diftusible substances involved have not been identified. Although he acknowledges in the same review that data are too scarce for any ecological interpretation, the possibility that some MHBs might have an effect on spore germination should be investigated more fully. More recently, AH & Jackson (1989) sbowed that corynebacteria and Psueodomonas stutzeri stimulated germination of spores of basidiomycetes. However, the stimulations reported by these authors are weak if compared with those due to root exudades. The subject is better documented for arbuscular endomycorrhizas. For instance, Mosse (1962) and Azcon (1987) found that some rhizosphere bacteria and their culture filtrates enhanced the germination of chlamydospores of Glomus mossea. Mayo, Davis & Motta (1986) obtained similar results with Glomus versiforme and spore-associated bacteria, as well as Linderman & Paulitz (1990) with other models. However, as in the case of basidiospores of ectomycorrhizal fungi, the substances involved are not known. It is probable that most MHB effects recorded with arbuscular endomycorrhizas (e.g. Meyer & Linderman, 1986; Von Alten, Lindemann & Shonbeck. 1993) are partly due to such a mechantsm, because the fungal inoculum used contained many spores, which are known to play a major role in the establishment of arbuscular endomycorrhizas. In conclusion, it appears that none of the five proposed hypotheses can presently be discarded, given the scarcity of data. However, at least in the case of the ectomycorrhizas studied to date, the stimulation of the fungal growth by bacterial metabolic activity seems to be the primary cause of the helper effect of MHBs, as well as of their selectivity towards the fungus. V. ECOLOGICAL.\ND EVO Ll'TI O N AR Y IMPLICATIONS OF MHBS The very fact that some rhizospheric bacteria may help mycorrhizal symbioses to establish means that the fitness of a plant species or of an individual m a given ecosystem, partly conditioned by the fungal associates, is improved if MHBs are associated with the compatible mycorrlitzal fungi present tn the site. Therefore, the competitive success of both plants and fungi depends in part on MHBs, This adaptive mechanism is even more critical when MHB strains are fungus-selective as was demonstrated with L. laccaia. Chanway, Tukington & Holl (1991) discussed the ecological and evolutionary implications of specificity between plants and rhizosphere microorganisms. When dealing with the infectivity of mycorrhizal fungi, they concluded that, in contrast with pathogenic associations, there is no obvious selection pressure for specificity to evolve between both organisms because the root-inhabiting microbe is protected against competition ; at the same time, they acknowledge that cases of narrow host-specificity are frequent with ectomycorrhizas. This contradiction might be resolved if the infection process was mediated by some rhizospheric microorganisms such as MHBs. It is well documented that free-living rhizobacteria are sometimes plant-specific with mutual benefit (i.e. a selection pressure exists, due to the complex and highly competitive rhizospheric environment: Neal, Larson & Atkinson, 1973); if the same bacterium happens to be a fungus-specific mycorrhization helper with mutual-benefit interactions with the fungal symbiont, this could lead to the plant-specificity of the mycorrhizal association. However, this speculation cannot be supported until

10 206 y. Garbaye it is demonstrated that MHBs benefit more from the mycorrhizal association than from each partner individually. Moreover, the only mycorrhizal system where the fungus-specificity of MHBs has been demonstrated is the Douglas fir/l. laccata one, which gives no clue since L. laccata has a very broad host range. The succession pattern of ectomycorrhizal fungi during the aging of a forest stand is an intriguing phenomenon which has been well described (Mason, Wilson & Last, 1983) but incompletely understood. It is tempting to hyporhesize that the slow buildingup of populations of MHBs specific of late stage fungi is the factor delaying their symbiotic establishment. However, the common observation that these so-called 'late stage' fungi can initiate mycorrhizas with young tree seedlings in sterile conditions but not in non-sterile soil suggests that inhibiting bacteria, rather than helpers might be involved. More generally, the question arises whether MHBs are ordinary rhizobacteria occasionally acting as mycorrhization helpers when occurring by chance near a symbiotic fungus, or whether they are dependent on the fungus and follow it in all its developmental stages. Experimental evidence is lacking, but the fact that tbe sporocarps of some ectomycorrhizai fungi such as Laccaria, Tuber, Suillus, Hymenogaster and Cantharellus (Danell, Alstrom & Terstrom, 1993) are always inhabited by large bacterial populations tend to support the latter hypothesis. VI. PRACTICAL.-APPLICATIONS OF MHBS Many research programmes are carried out worldwide with the aim of using mycorrhizal symbioses to improve plant yield in agriculture and forestry. Some of them have already been followed by commercial applications (Garbaye, 1991 b). Although they differ in their approaches, depending on the plant and the agricultural context, all of them involve soil or substrate inoculation with spores or mycelium of the desired fungi which have been selected for their outstanding efficiency in promoting plant growth. Artificially introducing an alien microorganism into the soil is always a challenge because of the competition exerted by the resident microflora and of the specific physico-chetnical properties of the soil which determine the equilibrium of its microbial community. The inconsistent and deceptive results often recorded with PGPRs (plant growth-promoting rhizobacreria) and some free-living antagonists of root pathogens illustrate this difficulty. On the other hand, results are generally better with symbiotic microorganisms such as mycorrhizal fungi or nodule-forming and nitrogen-fixing bacteria; among the latter, rhizobia are now widely and routinely used for inoculating soybean and other legume crops. The reason for this success is probably that the natural habitat of the symbiotic tnicroorganisms is the root rather than the soil; therefore, the competition tends to be limited to other symbionts, except during the early pre-infection stage, when the potential symbiont is still free-living in the rhizosphere or colonizing the rhizoplane. However, even w-hen mycorrhizal inoculation can be mastered, economic considerations make it desirable to reduce the inoculum dose and to optimize the competitiveness of the introduced fungus to ensure a long-lasting symbiotic effect. This goal is commonly achieved by manipulating the soil properties (amendments, fertilization) or microfiora (killing the symbiotic competitors by fumigation). Introducing MHBs together with the desirable fungus can be an alternative to these techniques. Von Alten et al. (1993) have discussed the potential of MHBs in relation to arbuscular endomycorrhizas of field and horticultural crops, but the most advanced results so far concern forestry, with the Douglas fir/l. laccata symbiotic combitiation already presented above. The strain S238N of L. laccata has been shown consistently to improve the early growth of conifer plantations in Western Europe (Le Tacon et al., 1992) and commercial forest nurseries in France are beginning to produce and sell Douglas fir planting stocks inoculated and mycorrhizal with L. laccata S238N. This large-scale development of controlled mycorrhization provided tbe opportunity to tesr the effect of MHBs in routine nursery conditions. Experiments were carried out for 4 consecutive years in three nurseries producing 2-yr-old bare-root Douglas fir planting stocks in natural sandy soils. Four strains of MHBs, isolated and selected from the Douglas fir/l. laccata symbiotic combination as described before in section II, were used: these were two pseudomonads and two sporulating bacilli belonging to the subtilis group. The first set of experiments, done in soil fumigated with methyl bromide as routinely practised before the fungal inoculation, was aimed at testing the first property of the L. laccata MHBs, i.e. their stimulatory effect on mycorrhiza formation by L. laccata. This effect was consistently confirmed in all trials (Duponnois & Garbaye, 1992), increasing the mycorrhizal index of the plants (per cent of short roots mycorrhizal with L. laccata) from around 60% to over 90% in the soils most receptive to L. laccata inoculation, and from 25 % to 75 % in less receptive ones. Table 4 shows that the MHB inoculation makes it possible to reduce the dose of fungal inoculum by at least one half while ensuring the same mycorrhizal index. This result is important from an economic viewpoint. Tbe same series of experiments also demonstrated that the application of the four MHBs tested can be effective with doses as low as 10* living bacterial cells m~^, mixed into the

11 Helper bacteria in mycorrhizal formation 207 Table 4. Effect of three MHBs and the mycelium dose on the success of ectomycorrhizal inoculation of Douglas fir seedlings with Laccaria laccata S238N in a bare-root forest nursery L. laccata mycorrhizal index {%) Microbial treatment Low dose High dose Laccaria laccata alone L. laccata-\-mhb strain MB3 93* 90* L./flcca/a + MHB strain SHBl 92* 89* L. laccata+ MHB strain SBc5 84* 63 The inoculum, mixed into the soil prior to sowing, was beads of calcium alginate gel containing both microorganisms (only the fungus in the control). Two inoculation doses were applied, on a square metre basis: 1 g mycelium (dry weight) and 10" bacterial cells, or 0-5 g mycelium and " bacterial cells. Results are expressed as per cent of short roots mycorrhizal with L. laccata 6 months after sowing (mycorrhizal index). Values followed by an asterisk in a column are significantly different from the control (0-05 probabilit>- level). Number of degrees of freedom for error, 9. From Duponnois & Garbaye, top-soil at a depth of about 10 cm (Table 5). This corresponds to 10 bacteria cm"^, or an average distance of 4'6 mm between two bacteria. This density is surprisingly low when compared with those reported as minimum for PGPRs (10*^- 10* m""^), and has only been reported as effective by Brown (1972). Besides its economica] value, this result suggests that the introduced bacteria rapidly multiply and colonize the soil. The second set of experiments, done in nonfumigated soil, was aimed at testing the second property of L. laccata MHBs, i.e. their selectivity for L. laccata against other fungi. The practical purpose of these trials was to assess the possibility of replacing soil fumigation by bacterial inoculation as a mean of suppressing the competition exerted by undesirable resident symbionts (mostly Thelephora terrestris and an unidentified white mycorrhiza m the nurseries studied). All the tests were positive, confirming the validity of this alternative. Table 6 shows that the results (in terms of L. laccata mycorrhizal index) can even be better with the MHBs than with the fumigation. However, the best results are clearly obtained with the combination of the two treatments (MHB inoculation and fumigation), where the drastic suppressive effect of methyl bromide and the specific stimulation of L. laccata establishment by the MHB are added. Table 6 also shows that the two treatments produced effects at different rates: fumigation was effective only during the first year, before the soil was re-colonized by symbiotic competitors, while the MHB effect increased with time. This result is consistent with the effectiveness of low doses of bacterial inoculum already discussed, suggesting that the population of the introduced bacterium build up in the rhizosphere during the growth of the seedlings. Studies of the population dynamics of the introduced bacterial strain are under way. Other experiments tried the simultaneous inoculation of several MHB strains, with the purpose of detecting possible additive stimulatory effects due to different mechanisms (see above. Section IV). All tests were negative, suggesting that the various MHB strains tested either compete and exclude each other or help mycorrhizal development by the same mechanism. The former hypothesis is supported by recent results by H. Gryta (unpublished data) which show that strains of Bacillus subtilis successfully compete with a Pseudomonas ftuorescens and prevent the latter from colonizing the rhizosphere of Douglas fir in a nursery soil. In conclusion, MHBs could be an important aid toward controlled mycorrhization techniques in forestry practice, by extending their application to Table 5. Effect of four MHBs applied at three doses as a bacterial suspension on the success of ectomycorrhizal inoculation of Douglas fir seedlings with Laccaria laccata S23SN in a bare-root forest nursery L. laccata mycorrhizal index Microbial treatment 10" cfu m"' ' \0' cfu m'^ 10'" cfu m-^ Laccaria laccata alone L. laccata^mhb strain MB3 L. /flccaia+mhb strain SHBl L. laccata+mwb strain SBc5 L. laccata + MHh strain BBc * 77* 70* 76* 60 96* 92* 68 86* 60 89*,87* 59 Mm The fungal inoculum, mixed into the soil prior to sowing, was a peat-vermiculite mix thoroughly colonized by L. laccata mycelium. Results are expressed as per cent of short roots mycorrhizal with L. laccata 6 months after sowing (mycorrhizal index). Values followed by an asterisk in a column are significantly different from the control (0-05 probability level). Number of degrees of freedom for error, 12. From Duponnois & Garbaye, 1992.

12 208. Garbaye Table 6. Comparison of effects of soil fumigation with methyl bromide and of the MHB strain BBc6 on the percentage of short roots mycorrhizal by Lacearia laccata of 2 yr-old Douglas fir planting stocks inoculated with L. laccata mycelium in a bare-root forest nursery Soil treatment L. laeeata alone L. laeeata+ 'QEc() L. /afffl/a-h fumigation From Duponnois et at., Mycorrbization C^o) Year Year naturally poorly receptive nursery soils, saving fungal inoculum, improving the mycorrhiza] quality of the planting stocks and/or suppressing the need for soil disinfection in bare-root nurseries. The latter point is particularly important because methyl bromide, the most efficient and commonly used fumigant. is dangerous for man and will probably soon be banned in many countries in spite of its wide range of application against soil-borne insects, pathogens and weed seeds. The use of MHBs as an adjuvant to fungal inoculum should be simple and cheap: they are easy to grow rapidly in large quantities m liquid medium and can be entrapped in alginate gel together with the mycelium, as illustrated in Table 4. In anticipation of such an application to commercial inoculum, the whole process of selecting and using MHBs for controlled mycorrhization has been patented (Garbaye & Duponnois, 1991). Vlt. CONCLUSIONS AND PERSPECTIVES Among the wide variety of bacteria and other microorganisms living in the rhizosphere, it is probable that all types of interactions with mycorrhizal symbioses occur, from inhibition to stimulation and with various mechanisms involved. The term 'mycorrhization helper bacteria' was proposed here only for the sake of convenience and to make the discussion easier: it obviously hides a much more complex continuum in reality. Nevertheless, as shown by evidence of experiments and some applied results, the MHB concept has proved to be fecund and to work in practice: it gives a new dimension to mycorrhiza research by identifying a category of rhizosphere microorganisms which cannot be ignored w^hen studying mycorrhizal symbioses in their natural environment. MHBs are especially important whenever the competitiveness and population dynamics of mycorrhizal fungi are considered. Unfortunately, our description of the phenomenon is still fragmentary. Results are particularly scarce with arbuseuiar endomycorrhizas and nonexistent with other minor types as ericoid or orchidoid mycorrhizas. More research is needed before the concept of MHB can be generalized. However, based on the few data available, this tentative and provisional definition is suggested: MHBs are bacteria associated with mycorrhizal roots and mycorrhizal fungi which selectively promote the establishment of mycorrhizal symbioses. Many fundamental questions remain unanswered about MHBs: do they benefit from the presence of the fungus and do they tend to be specifically attached to it (in that case they w'ould be part of a three-partner symbiosis)? Which groups of bacteria are most commonly involved? Do they share common metabolic traits? What are the dominant mechanisms explaining the stimulation? Do they also play a role in the functioning of the mycorrhizas after the symbiosis is established? Are they the rule w'ith all types of mycorrhizal symbioses and in all environments? What are their population dynamics in the soil and the rhizosphere? How stable is the association? Do MHBs play a role in the fungal reproductive processes? Put another way, is it normal for plants and fungi to benefit from some sort of microbial intermediaries or 'match-makers' in forming the mycorrhizal symbioses? 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