A functional study on the multilateral symbiosis of the fungal order Sebacinales with plant hosts and bacteria

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SharmaMonica-2008-06-30.pdf (2.68 MB)

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2008

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DOI:
http://dx.doi.org/10.22029/jlupub-10100

Abstract

The order Sebacinales is the most basal order of Hymenomycetes (Basidiomycetes) which contains an amazing diversity of mycorrhiza. Frequent detection of Sebacinales in ecological studies using various molecular techniques suggests their possible role in shaping plant communities. The root-colonizing mutualistic fungus Piriformospora indica is axenically cultivable and represent the model organism for symbiotic species of the order Sebacinales. In the present work, axenic cultures of seven isolates of Sebacina vermifera have been established and their phylogenetic relationship was characterized by sequencing and comparing nuclear genes coding for the internal transcribed spacer (ITS) region. Despite their sequence similarity for the large ribosomal subunit (nucLSU), they showed difference in the ITS region. Isolates were clustered into three different groups based on their ITS sequences. Morphological variation in terms of colony morphology and growth, hyphal and spore size and number of nuclei per hyphal cell and spore was also observed for the isolates. The isolates were further evaluated in barley for their biological potential with respect to growth promotion and ability to induce systemic resistance against the biotrophic leaf pathogen Blumeria graminis f. sp. hordei (powdery mildew). All isolates conferred beneficial effects (growth augmentation and induced resistance) on two cultivars of barley i.e., cvs. Ingrid and Golden Promise. Hence, the data support the working hypothesis that the mutualistic symbiosis of crop plants and Sebacinales has a great potential for sustainable agriculture.While culturing P. indica, initial signs of bacterial presence were observed several times after mechanical rupturing of fungal mycelium. Using universal primers, bacterial 16S-rRNA gene from the metagenomic DNA of P. indica and seven isolates of S. vermifera was amplified. Cloning, sequencing and phylogenetic analysis of 16S-rRNA gene sequences revealed the association of P. indica with Rhizobium radiobacter, while 3 isolates S. vermifera complex showed association with 3 different bacteria belonging to Paenibacillus sp., Acinetobacter sp. and Rhodococcus sp. R. radiobacter (PABac) was isolated from P. indica and its axenic culture was established. All bacterial sequences from P. indica and PABac were identical within the amplified region of the bacterial 16S-rRNA gene displaying the presence of a unique bacterial isolate. This notion was confirmed by detecting single dominant band in denaturing gradient gel electrophoresis (DGGE) analysis of 16S-rRNA gene. The Sebacinales associated bacteria were localized using fluorescence in situ hybridization (FISH). R. radiobacter could be detected close to and in the hyphal walls of P. indica. Paenibacillus sp. was localized within the cytosol of S. vermifera MAFF305838. Endocellular nature of Paenibacillus sp. was further confirmed by transmission electron microscopy (TEM).Vertical transmission of R. radiobacter was proved by single spore cultures of P. indica. Real time quantitative PCR showed an average of 0.035 ng of R. radiobacter DNA per 100 ng of P. indica DNA. Various attempts to cure P. indica from bacteria (by growing single spore cultures, young mycelium and regenerated hyphal protoplast on medium containing antibiotic for five generations) were not successful which indeed suggests towards strong association between P. indica - R. radiobacter. Initial biochemical characterization of R. radiobacter revealed its ability to produce auxin from tryptophan. Biological activities (growth augmentation and induction of systemic resistance against powdery mildew) of R. radiobacter in host plants (Hordeum vulgare and Arabidopsis thaliana) were demonstrated. In addition, using Arabidopsis mutants defective in various defence pathways clarified that R. radiobacter induces a type of resistance reminiscent of jasmonate-dependent induced systemic resistance (ISR) provided by non-pathogenic rhizobacteria. Thus, the Sebacinales-associated bacteria might be involved in the growth response of the host plant as well as in the evasion of the host defense response by the mutualistic fungi. Together, these findings suggest that Sebacinales species undergo complex symbioses involving bacteria and requires reconsideration of the role played by the fungus in its symbiotic interaction with the plant.

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