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

Kuleshwar Prasad Sahu,Mukesh Kumar,Aundy Kumar,Bhaskar Reddy,Asharani Patel,Narayanasamy Prabhakaran,Neelam Sheoran,Sahil Mehta,Pierre Eke

doi:10.3389/fmicb.2021.780458

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.

Highlights

Microbial communities have an evolutionary association with plant populations where they function as metaorganisms in the natural environment
Phyllomicrobiome profiling of blast-susceptible (PRR78) and blast-resistant rice (Pusa1602) genotypes planted in blastendemic locations was conducted using integrated 16S rRNA gene amplicon sequencing and microbiological methods
The metabarcoding-assisted taxonomic profiling of the phyllosphere microbiome of two rice genotypes revealed the abundance of bacterial phyla, Proteobacteria (70.7–91.0%), and class Gamma Proteobacteria on both the genotypes (70.4–91.0%)

Summary

Introduction

Microbial communities have an evolutionary association with plant populations where they function as metaorganisms in the natural environment. The microbial communities associated with the plants are called plant microbiome and microbial metagenome that often confer functional flexibility to the plant genome (Sessitsch et al, 2012). The plant microbiomes are presumed to modulate a variety of plant functions. The ecological role of the phyllosphere microbial communities on plant functional ecology is among the most understudied and underrated aspects in plant biology. The epiphytic phyllosphere microbiomes and their natural functions are increasingly investigated in crops like rice, wheat, maize, and soybean (Andrews and Harris, 2000; Bertani et al, 2016; Compant et al, 2019; Sahu et al, 2020). It is further reported that plantassociated bacteria are prolific for the secretion of primary and secondary metabolites and volatiles for plant growth, developmental regulation, and defense against stresses (Munjal et al, 2016; Rascovan et al, 2016; Sheoran et al, 2016; Gómez Expósito et al, 2017; Eke et al, 2019; Ashajyothi et al, 2020; Vandana et al, 2021)

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Conclusion