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Mycorrhizal fungi are an ubiquitous component of most ecosystems throughout the world and play an important role in soil processes (Smith & Read, 1997). As the fungal symbiosis modifies root functions, its microbial communities differ from those of the uninfected rhizosphere and of the surrounding soil (Katznelson et al., 1962; Ames et al., 1984; Garbaye & Bowen, 1987, 1989; Garbaye, 1991). This microbial compartment is commonly named ‘mycorrhizosphere’ (Linderman, 1988). The microorganisms associated with the mycorrhiza may complement mycorrhizal activities (i.e. N2-fixing bacteria, phosphate-solubilizing bacteria) (Secilia & Bagyaraj, 1987; Toro et al., 1996). However, more specific relationships between the microbial populations of the mycorrhizosphere and the fungal symbionts can affect the establishment of the mycorrhizal symbiosis (Garbaye & Bowen, 1987). Some bacteria can have a negative or positive effect on mycorrhiza formation (Bowen & Theodorou, 1979; Garbaye & Bowen, 1989).
Mycorrhiza Helper Bacteria (MHB) which have been previously defined as telluric bacteria promoting the development of mycorrhizal symbiosis (Garbaye, 1994), have been isolated from different plant–fungal combinations such as AM fungi (herbaceous plants) (Meyer & Linderman, 1986; Paula et al., 1992; Staley et al., 1992; von Alten et al., 1993; Requena et al., 1997) or ectomycorrhizal fungi (trees) (Dunstan et al., 1998; Duponnois & Garbaye, 1991a; Rózycki et al., 1994). The location and survival of MHBs have been assessed (Frey-Klett et al., 1997) as well as their selectivity and specificity towards the fungal symbiont from which they have been isolated (Garbaye & Duponnois, 1992; Duponnois et al., 1993).
However, the effect of MHBs on ectomycorrhizal symbiosis has only been investigated with a few northern hemisphere tree species (Pseudotsuga menziesii, Quercus robur) (Duponnois & Garbaye, 1991a,b; Garbaye et al., 1992) and a limited number of fungi, principally Laccaria bicolor (Duponnois & Garbaye, 1991a,b; Frey-Klett et al., 1997; Frey et al., 1997). To our knowledge, only one fast growing tree species has been studied (Eucalyptus diversicolor) to determine the impact of MHBs on its ectomycorrhizal status (Dunstan et al., 1998).
As Australian Acacias are frequently used in agroforestry plantations to rehabilitate degraded soils in sahelian areas, it is very important to identify factors which could optimize the effects of controlled mycorrhization. This study was undertaken with A. holosericea, an Australian acacia frequently planted in dry tropical areas and an ectomycorrhizal fungus, Pisolithus sp. strain COI 007, previously screened for its stimulating effect on A. holosericea plant growth (Duponnois et al., 2000). The aims of this study were to explore the interactions between the establishment of this ectomycorrhizal symbiosis and two bacterial strains belonging to the fluorescent pseudomonad group, isolated from a soil highly colonized by ectomycorrhizal fungi.
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P. alba (COI007) dramatically increased plant growth: shoot biomass was increased by 448%. This beneficial effect of the ectomycorrhizal symbiosis has previously been demonstrated with another Pisolithus isolate COI024 collected from an A. holosericea plantation in Senegal (+142% in shoot biomass) (Duponnois et al., 2000). This positive effect was increased with HR13 and HR26 coinoculated with COI007 (shoot biomass enhanced, respectively, by 46% and 23.2% above that measured in the control without bacteria). The percentage ectomycorrhizal infection was also significantly promoted by 158.2% and 31.3%, respectively. Both bacterial strains can be considered as MHB. After 3 months the bacterial inoculants could no longer be detected in the soil or rhizosphere plate counts (a PCR-DGGE analysis would confirm the results). A decline in MHBs after 3 months culture has been already described with BBc6, a P. fluorescens isolate, in the ectomycorrhizal association between Douglas fir and Laccaria bicolor (Frey-Klett et al., 1997). This result suggests that the survival of these two bacterial inoculants is probably dependent on host plant (HR13 and HR26 were present in the mycorrhizosphere of 3-month-old A. mangium) and on soil conditions (general soil conditions differ under the two Acacia stands). Moreover, the sterilized soil was rapidly recolonized by other bacteria. After 3 month culture, the bacterial community composition could be different from the native soil in which HR13 and HR26 were isolated and could include bacteria that are antagonistic to the inoculant strains.
Sequence analysis of both Pseudomonas isolates showed that HR13 and HR26 were authentic species of Pseudomonas which includes three subgroups, P. aeruginosa, P. fluorescens and the P. syringae subgroup (Palleroni, 1984). The Pseudomonas putida group (HR26) was included in the P. fluorescens subgroup (Anzai et al., 2000). To our knowledge, these species of Pseudomonas have not previously been studied for their effects on ectomycorrhizal establishment.
Greater percentage ectomycorrhizal infection was correlated with the total number of nodules per root systems. It is thought that nodule formation and functioning are dependant on mycorrhizal inoculation (Cornet & Diem, 1982). The main explanation is that the improvement of P uptake by the host plant resulting from mycorrhizal symbiosis enhances nodulation and N2 fixation (Cornet & Diem, 1982).
The MHB effect was accompanied by an increase in the ergosterol content in the soil. This shows that the bacterium was acting on fungal growth in the soil and consequently could enhance the effective mycelial surface areas provided by fungal hyphae to explore greater volumes of soil and to overcome mineral nutrient and water depletion zones near active root surface. This supports the hypothesis of Frey-Klett et al. (1997) who suggested that the main mechanism involved in the MHB effect concerned the bacterial influence on the fungal growth. Moreover, it has previously been demonstrated that the ability of the bacteria to stimulate mycelial growth was strongly correlated with their effect on the ectomycorrhizal establishment (Garbaye, 1994).
The cocultures of the bacteria and the fungus showed that the radial growth was stimulated by HR13 and fungal biomass (expressed as ergosterol) by HR26. This suggests that different mechanisms may be involved in the MHB effect. Moreover, increased respiration (expressed by the CO2 production) demonstrates some increase in metabolism in the presence of both bacteria.
Another hypothesis to explain the MHB effect has been suggested by Garbaye (1994). The MHBs could improve the receptivity of the root to the fungus before the first mycorrhizas are formed. It has been demonstrated recently that hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation of root hairs and consequently, is involved in establishment of mycorrhizal association (Bèguiristain & Lapeyrie, 1997; Ditengou et al., 2000). In our study, both bacterial isolates stimulated phenolic production by the fungus. Further investigations must be done to clarify this bacterial effect on the phenol compounds and, in particular, on hypaphorine production.
In conclusion, it appears that some bacteria can help the establishment of ectomycorrhizal symbiosis in tropical conditions with an Australian Acacia. The main mechanisms involved in this phenomena concern the interactions between the MHB and the fungal symbiont. Further research must be undertaken to identify the compounds responsible for the promotion of the fungal growth and the signal molecules implied in the recognition processes between the host and the fungus. As the bacterial inoculants disappeared after 3 month culture, the bacterial effect must be transient and could involve some changes in the recolonizing indigenous microbial community (included Rhizobium in only the coinoculation treatments). In future experiments, a sequential harvesting could determine these interactions between the inoculant bacteria and the other microbial components of the system (Rhizobium, indigenous microbial communities, arbuscular mycorrhizal fungi).
From a practical point of view, the use of MHBs could facilitate the introduction of controlled mycorrhization in nursery and forestry practices through the soudano-sahelian areas.