Integrated Metabarcoding and Culturomic-Based Microbiome Profiling of Rice Phyllosphere Reveal Diverse and Functional Bacterial Communities for Blast Disease Suppression

Abstract

Phyllosphere—the harsh foliar plant part exposed to vagaries of environmental and climatic variables is a unique habitat for microbial communities. In the present work, we profiled the phyllosphere microbiome of the rice plants using 16S rRNA gene amplicon sequencing (hereafter termed metabarcoding) and the conventional microbiological methods (culturomics) to decipher the microbiome assemblage, composition, and their functions such as antibiosis and defense induction against rice blast disease. The blast susceptible rice genotype (PRR78) harbored far more diverse bacterial species (294 species) than the resistant genotype (Pusa1602) that showed 193 species. Our metabarcoding of bacterial communities in phyllomicrobiome revealed the predominance of the phylum, Proteobacteria, and its members Pantoea, Enterobacter, Pseudomonas, and Erwinia on the phyllosphere of both rice genotypes. The microbiological culturomic validation of metabarcoding-taxonomic annotation further confirmed the prevalence of 31 bacterial isolates representing 11 genera and 16 species with the maximum abundance of Pantoea. The phyllomicrobiome-associated bacterial members displayed antifungal activity on rice blast fungus, Magnaporthe oryzae, by volatile and non-volatile metabolites. Upon phyllobacterization of rice cultivar PB1, the bacterial species such as Enterobacter sacchari, Microbacterium testaceum, Pantoea ananatis, Pantoea dispersa, Pantoea vagans, Pseudomonas oryzihabitans, Rhizobium sp., and Sphingomonas sp. elicited a defense response and contributed to the suppression of blast disease. qRT-PCR-based gene expression analysis indicated over expression of defense-associated genes such as OsCEBiP, OsCERK1, and phytohormone-associated genes such as OsPAD4, OsEDS1, OsPR1.1, OsNPR1, OsPDF2.2, and OsFMO in phyllobacterized rice seedlings. The phyllosphere bacterial species showing blast suppressive activity on rice were found non-plant pathogenic in tobacco infiltration assay. Our comparative microbiome interrogation of the rice phyllosphere culminated in the isolation and identification of agriculturally significant bacterial communities for blast disease management in rice farming through phyllomicrobiome engineering in the future.

Rice leaf endophytic Microbacterium testaceum: Antifungal actinobacterium confers immunocompetence against rice blast disease

“…The endophytic M. testaceum isolated from rice leaf was found clustered with a potato leaf endophytic M. testaceum StLB037 ( Morohoshi et al, 2009 ). Before the present study, M. testaceum was reported in diverse habitats like water ( Epova et al, 2022 ), soil ( Yang et al, 2014 ), tannery effluents ( Elahi et al, 2019 ), phyllosphere ( Sahu et al, 2021a , b ), plant endosphere ( Kumar et al, 2021b ), and sometimes as an opportunistic pathogen in clinical samples ( Leal et al, 2016 ). We further studied the endophytism of the bacterium in rice plants using gfp-tagged M. testaceum D18::gfp that could be visualized in the root, stem as well as leaf tissues.…”

“…Along with Bacillus amyloliquefaciens , Microbacterium reduced Fusarium verticillioides and the mycotoxin fumonisin B1 and B2 in maize grains ( Sartori et al, 2012 ). Before the present study, leaf epiphytic Microbacterium testaceum was reported to elicit defense responses leading to blast disease suppression upon phyllo bacterization of rice cultivar Pusa Basmati-1 ( Sahu et al, 2021b ). However, published literature on the biocontrol potential of M. testaceum against blast disease is scanty.…”

“…Pelleted bacterial cells were washed with sterile saline and used for extracting genomic DNA. CTAB-method with slight modification was used to isolate bacterial genomic DNA ( Sheoran et al, 2015 ; Munjal et al, 2016 ; Eke et al, 2019 ; Sahu et al, 2021b ). Before PCR amplification, the integrity, purity, and yield of genomic DNA were tested spectrophotometrically (Biophotometer, Eppendorf, Hamburg, Germany) and electrophoretically.…”

New Insights on Endophytic Microbacterium-Assisted Blast Disease Suppression and Growth Promotion in Rice: Revelation by Polyphasic Functional Characterization and Transcriptomics

“…To meet future rice production challenges, there is an urgent need to develop strategies acceptable to all rice farming stakeholders. The plant microbiome, the phytobiome, presents a novel opportunity to harness pathogen-competing and crop-cooperating microbiota [ 12 ]. Rice is a tropical plant colonized by diverse microorganisms such as epiphytes and endophytes [ 13 , 14 ].…”

Structured Framework and Genome Analysis of Magnaporthe grisea Inciting Pearl Millet Blast Disease Reveals Versatile Metabolic Pathways, Protein Families, and Virulence Factors

“…From the year 2000 onwards, pearl millet blast disease was observed widespread across India [ 8 , 14 ]. Therefore, to prevent inadvertent dissemination and minimize the losses, the deployment of blast-resistant genes and the application of agrochemicals are widely practiced [ 8 , 14 , 15 , 16 ]. However, these strategies appear inadequate in blast-endemic areas because of variable host resistance, the emergence of new pathotypes, and the toxic nature of fungicides.…”