Résultat de la recherche
Plant growth stage, fertiliser management and bio-inoculation of arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria affect the rhizobacterial community structure in rain-fed wheat fields
The goal of this study was first to assess the dynamics of the bacterial community during a growing season in three Indian rain-fed wheat fields which differ mainly through their fertilizer management and yield and then to study the effects of PGPR/AMF bio-inoculations on the bacterial community structure and wheat growth. The bacterial community structure of the rhizosphere soil (RS) and the rhizoplane/endorhizosphere (RE) was determined by PCR-denaturing gradient gel electrophoresis. Seed treatments consisted of consortia of two PGPR strains alone or combined with AMF or AMF alone. The PGPR strains were Pseudomonas spp. which included some or all of the following plant growth promoting properties: phosphate solubilisation and production of indole-3-acetic acid, siderophores, 1-aminocyclopropane-1-carboxylate deaminase and diacetyl-phloroglucinol. The mycorrhizal inoculum was an indigenous AMF consortium isolated from the field with the lowest level of fertilization and yield. Variation partitioning analysis of the DGGE data indicated a predominant effect of the wheat growth stage (30.4% of the variance, P=0.001) over the type of field (9.0%, P=0.027) on the bacterial community structure in the RE. The impact of plant age in the RS was less than in the RE and the bacterial community structure of the field with the highest input of fertilization was very different from the low input fields. The bio-inoculants induced a significant modification in the bacterial community structure. In the RS, the bacterial consortia explained 28.3% (P=0.001) and the presence of AMF 10.6% (P=0.02) of the variance and the same trend was observed in the RE. Plant yield or grain quality was either increased or remained unaffected. For example, protein content was significantly higher in the treated plants' grain compared to the control plants; maximum values were obtained when the PGPR were co-inoculated with the AMF. The percentage of root colonization by AMF was significantly higher in the treatments containing a mycorrhizal inoculum than in the untreated control and remained unaffected by the PGPR treatments. In conclusion, the wheat rhizobacterial community structure is highly dynamic and influenced by different factors such as the plant's age, the fertilizer input and the type of bio-inoculant. In addition, there is a distance-related effect of the root on the bacterial community. Finally, a combined bio-inoculation of diacetyl-phloroglucinol producing PGPR strains and AMF can synergistically improve the nutritional quality of the grain without negatively affecting mycorrhizal growth.
Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns
Technical developments in molecular biology have found extensive applications in the field of microbial ecology. Among these techniques, fingerprinting methods such as denaturing gel electrophoresis (DGE, including the three options: DGGE, TGGE and TTGE) has been applied to environmental samples over this last decade. Microbial ecologists took advantage of this technique, originally developed for the detection of single mutations, for the analysis of whole bacterial communities. However, until recently, the results of these high quality fingerprinting patterns were restricted to a visual interpretation, neglecting the analytical potential of the method in terms of statistical significance and ecological interpretation. A brief recall is presented here about the principles and limitations of DGE fingerprinting analysis, with an emphasis on the need of standardization of the whole analytical process. The main content focuses on statistical strategies for analysing the gel patterns, from single band examination to the analysis of whole fingerprinting profiles. Applying statistical method make the DGE fingerprinting technique a promising tool. Numerous samples can be analysed simultaneously, permitting the monitoring of microbial communities or simply bacterial groups for which occurrence and relative frequency are affected by any environmental parameter. As previously applied in the fields of plant and animal ecology, the use of statistics provides a significant advantage for the non-ambiguous interpretation of the spatial and temporal functioning of microbial communities.
Bacteria Associated with Spores of the Arbuscular Mycorrhizal Fungi Glomus geosporum and Glomus constrictum
Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.
Bacterial community associated with the rhizosphere of wheat: interactions with arbuscular mycorrhizal fungi and selection of plant growth promoting rhizobacteria for the increase of wheat growth and soil health in Indian marginal rainfed fields
The objective of this thesis was to improve our knowledge on the interactions between wheat, rhizobacteria and arbuscular mycorrhizal fungi (AMF) in the mycorrhizosphere in order to define criteria for the selection of plant growth promoting bacteria (PGPR) strains in view of a PGPR/AMF dual inoculation in Indian wheat fields. First, microcosm systems were set-up to obtain mycorrhizosphere, AMF-free rhizosphere and root-free hyphosphere zones in order to examine the effects of AMF on the rhizobacterial community in the wheat mycorrhizosphere. The results showed that the bacterial community structure was more influenced by the type of rhizospheric fraction, the plant age and the plant specie than by the presence of AMF. However, the bacterial community was affected indirectly by AMF via a modification in the soil pH. In addition, there was a strong increase in the proportion of phosphate solubilizing bacteria in AMF related zones probably resulting from soluble phosphorus depletion in consequence to AMF phosphorus uptake. Secondly, spores of the arbuscular mycorrhizal fungi Glomus geosporum and G. constrictum were harvested from single spore derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants to determine if specific bacterial populations were associated with AMF spores. The bacterial communities associated with the spores were more influenced by the AMF than by the host plant. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated to taxonomic groups known to degrade biopolymers. These bacteria were probably feeding on the spore's outer hyaline layer. The third part of the study examined how PGPR strains directly affected AMF growth in the hyphosphere. An in vitro device, consisting of a two-compartmental Petri plate system using Ri T-DNA transformed clover roots permitting the separation of the hyphosphere from the mycorrhizosphere, was designed and tested. Even though the PGPR strains tested were all DAPG producers, their effects on the AMF development varied from inhibition to improvement of the hyphal biomass or spore production. For the fourth part of the study, we had to ensure that before applying the selected PGPR strains P. jessenii R62 and P. synxantha R81 in the fields, they were able to colonize the rhizosphere in situ. They were marked with the green fluorescent protein before testing them in greenhouse pot experiments. R62gfp had colonized the root at a later stage than R81gfp, explaining why the PGP effect of R62gfp was delayed. Both gfp strains were located in the upper part of the root but R81gfp was also detected near the root elongation zone. The fifth part was undertaken in the fields to confirm the positive interactions between AMF and the PGPR strains R62 and R81 and to assess the changes in the wheat bacterial rhizospheric community with respect to field conditions, plant age and PGPR/AMF bio-inoculation. As compared to the bacterial community of the rhizoplane/endorrhizosphere, the bacterial community of the root-adhering rhizospheric soil was more influenced by the field conditions such as an increase in fertilizer input. The bacterial community structure was also dependent on the plant's growth stage. In addition, the type of PGPR consortium had a greater impact on the bacterial community structure than the mycorrhizal colonization. The treatment composed of R62/R81 and an indigenous AMF consortium had not only a positive but also in some aspects, a synergistic effect on plant development.