Genetic and functional characteristics of rice leaf endophytic actinobacterial member, Microbacterium are described. Morphotyping, multilocus sequence analysis and transmission electron microscopy indicated the species identity of the endophytic bacterium, OsEnb-ALM-D18, as Microbacterium testaceum. The endophytic Microbacterium showed probiotic solubilization of plant nutrients/minerals, produced hydrolytic enzyme/phytohormones, and showed endophytism in rice seedlings. Further, the endophytic colonization by M. testaceum OsEnb-ALM-D18 was confirmed using reporter gene coding for green fluorescence protein. Microbacterium OsEnb-ALM-D18 showed volatilome-mediated antibiosis (95.5% mycelial inhibition) on Magnaporthe oryzae. Chemical profiling of M. testaceum OsEnb-ALM-D18 volatilome revealed the abundance of 9-Octadecenoic acid, Hexadecanoic acid, 4-Methyl-2-pentanol, and 2,5-Dihydro-thiophene. Upon endobacterization of rice seedlings, M. testaceum altered shoot and root phenotype suggestive of activated defense. Over 80.0% blast disease severity reduction was observed on the susceptible rice cultivar Pusa Basmati-1 upon foliar spray with M. testaceum. qPCR-based gene expression analysis showed induction of OsCERK1, OsPAD4, OsNPR1.3, and OsFMO1 suggestive of endophytic immunocompetence against blast disease. Moreover, M. testaceum OsEnb-ALM-D18 conferred immunocompetence, and antifungal antibiosis can be the future integrated blast management strategy.
Aims
To characterize the functional role of extracellular polysaccharides and lipopolysaccharide (LPS) extracted from endophytic Pseudomonas putida BP25 (PpBP25) against rice blast.
Methods and Results
We profiled the transcriptome of endobacterized rice seedlings using RNA-seq. Fluorescence imaging of interaction between Magnaporthe:: gfp and P. putida:: mCherry was performed on rice phylloplane using confocal laser scanning microscopy (CLSM). Microbial polysaccharides, exopolysaccharide (EPS), and LPS extracted from PpBP25 were characterized using Fourier-transform infrared-spectroscopic analysis (FTIR). Biochemical assays and gene expression analysis were conducted on EPS- and LPS-treated rice seedlings. A detached-leaf assay was designed to test the blasticidal-effect of bacterial-endophyte, EPS, and LPS on rice phylloplane. PpBP25 elicited defense in rice with a consequently altered seedling phenotype. Rice cultivar, Pusa Basmati-1, colonized by PpBP25 showed an altered transcriptome profile displaying a total of 110-downregulated and 68-upregulated genes (P < 0.005) representing growth/development and defense pathways, respectively. CLSM of PpBP25 bacterized phylloplane showed reduced conidial-germination and mycelial-biomass of Magnaporthe oryzae. To decipher the elicitor role of polysaccharides, we purified and characterized EPS and LPS using FTIR. Rice treated with the EPS and LPS showed root-growth inhibition the phenotype of MAMP-triggered immunity. While the EPS showed blast suppressive activity at 1–20 mg mL−1 (79.80%–86.87% reduction over control), the LPS exhibited 78.0%–79.8% reduction at 20–200 μg mL−1on rice. Polysaccharides treated seedling showed elevated activities of peroxidase and polyphenol-oxidase activities, and total-phenols content. Treated plantlets showed up regulation of OsPR1.1,OsPR3, OsGLP3-3,OsZFP179, and Oshox24 as well as downregulation of OsACS6.
Conclusions
We showed that P. putida Bp25 and its cell wall-associated polysaccharides could elicit defense against rice blast
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