Deciphering Core-Phyllomicrobiome of Rice Genotypes Grown In Contrastingmountain And Island Agroclimatic Zones: Implications For Phyllomicrobiome Engineering Against Blast Disease
Authors: Sahu Kuleshwar Prasad , Aundy Kumar , Krishnan Sakthivel , Reddy Bhaskar , Kumar Mukesh , Patel Asharani , Sheoran Neelam , Subbaiyan Gopalakrishnan , Ganesan Prakash , Rathour Rajeev , Kumar Gautam Raj 
Published: September 2021

Abstract
Background The fundamental role and contributions of phyllosphere habitat in shaping plant functional ecology are poorly investigated, and often underestimated. Phyllosphere -the harsh and dynamic foliar-photosynthetic-habitat is continuously exposed to vagaries of changing weather events during the entire plant life. With its adapted microbiota, the phyllosphere-niche brings microbial diversity to the plant-holobiont pool and potentially modulates a multitude of plant and agronomic traits. The phyllomicrobiome structure and the consequent ecological functions are vulnerable to a host of biotic (Genotypes) and abiotic-factors (Environment) which is further compounded by agronomic-transactions on domesticated agricultural crops. However, the ecological forces driving the phyllomicrobiome assemblage and functions are among the most under-studied aspects of plant biology. Despite the reports on the occurrence of diverse prokaryotic phyla such as Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria on phyllosphere habitat, the functional characterization leading to their utilization for agricultural sustainability is not yet adequately explored.Currently, the metagenomic-Next-Generation-Sequencing (mNGS) technique scanning the conserved V3-V4 region of ribosomal RNA gene is a widely adopted strategy for microbiome-investigations. However, the structural and functional validation of mNGS annotations by microbiological methods is not integrated into the microbiome exploration-programs. In the present study, we combined the high throughput mNGS approach with conventional microbiological methods to decipher the core-functional-phyllomicrobiome of contrasting rice genotypes varying in their response to blast disease grown in contrasting agroclimatic zones in India. We, further, scanned the rice phyllosphere by electron microscopy to show the microbial communities on leaf. Magnaporthe oryzae-the phyllosphere pathogen inciting necrotic lesion on cereal crops is managed by the deployment of ‘non-durable’blast resistance genes and ‘toxic’ fungicidal molecules. Nowadays, there is a growing consensus for devising an alternative strategy for mitigating blast owing to a recent ban on the use of most commonly used fungicidal molecule, tricyclazole. In the present work, we further identified phyllosphere- core-functional microbial groups leading tothe proposal of phyllomicrobiome assisted rice blast management strategy.Multi-pronged activities of phyllomicrobiome against Magnaporthe oryzae (antifungal activity), rice innate immunity (defense elicitation), and rice blast disease (disease suppression) have beenelaborated for effective management of blast by phyllomicrobiome re-engineering. ResultsRice phyllomicrobiome of tropical “Island-Zone” displayed marginally more bacterial community diversity than that of temperate ‘Mountain-Zone’. Principal coordinate analysis based on Bray Curtis and ANoSIM method indicated nearly converging-phyllomicrobiome profiles on two contrasting rice genotypes grown in the same agroclimatic zone. However, the rice genotype grown in the contrasting Mountain-zone and Island-zone displayed diverse-phyllomicrobiome profiles indicating a strong influence of environmental factors rather than the genotype on phyllomicrobiome structure and assembly. The predominance of Phyla such as Proteobacteria, Actinobacteria, and Firmicutes was observed on the rice phyllosphere irrespective of the genotypes and environmental conditions. The core-microbiome analysis showed multi-microbiota-core consisting of Acidovorax, Arthrobacter, Bacillus, Clavibacter, Clostridium, Cronobacter, Curtobacterium, Deinococcus, Erwinia, Exiguobacterium, Hymenobacter, Kineococcus, Klebsiella, Methylobacterium, Methylocella, Microbacterium, Nocardioides, Pantoea, Pedobacter, Pseudomonas, Salmonella, Serratia, Sphingomonas and Streptomyceson phyllosphere of rice genotypes grown in contrasting agroclimaticzones. The linear discriminant analysis (LDA) effect size (LEfSe) method revealed ten and two distinct bacterial genera in blast-resistant and -susceptible genotypes, respectively. The analysis further indicated 15 and 16 climate-zone specific bacterial genera for Mountain and Island zone, respectively. SparCC based network analysis of phyllomicrobiome showed hundreds of complex intra-microbial cooperative or competitive interactions on the rice genotypes and agroclimatic zones. Our microbiological validation of mNGS data further confirmed the presence of resident Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, andSphingomonason the rice phyllosphere.Strikingly,the two contrastingagroclimatic zones displayed genetically identical bacterial isolates on the phyllosphere that could be attributed to the spatio-temporal transmission of core-phyllomicrobiome, perhaps, aided by rice seeds. A total of 59 distinct bacterial isolates were obtained, identified, and evaluated for their functional attributes on Magnaporthe oryzae and rice plant. The phyllomicrobiome associated core-bacterial communities showed secreted-metabolite and volatile-compound mediated antifungal activity on M. oryzae.Uponphyllobacterization (a term coined for spraying of bacterial cells on the phyllosphere), the core bacterial species such as Acinetobacter baumannii, Aureimonas sp., Pantoea ananatis, P. eucrina,andPseudomonas putidaelicited plant defense and contributed significantly to blast disease suppression. Transcriptional analysis by qPCR indicated induction of rice innate immunity associated genes such as OsPR1.1, OsNPR1, OsPDF2.2, OsFMO, OsPAD4, OsCEBiP, andOsCERK1in phyllobacterized rice seedlings. ConclusionsThe rice genotypes growing in a particular agroclimatic zone showed a convergent phyllomicrobiome assemblage and composition. Conversely, diverging phyllomicrobiome assembly was observed on rice genotype cultivated in the contrasting agroclimatic zones.Agroclimatic zones and the associated climatic-factors rather than plant-genotypesper se appeared to drive phyllomicrobiome structure and composition on the rice genotypes. Our integrated mNGS method and microbiological validation divulged Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, andSphingomonasas core phyllomicrobiome of rice. Genetically identical bacterial communities belonging to Pantoea intercepted on the phyllosphere of rice grown in the two contrasting agroclimatic zones are suggestive of spatio-temporal transmission of phyllomicrobiome aided by seed. The core-microbiome mediated phyllobacterization showed potential for blast disease suppression by direct-antibiosis and defense elicitation. The identification of phyllosphere adapted functional core-bacterial communities in our study and their co-occurrence dynamics presents an opportunity to devise novel strategies for rice blast management through phyllomicrobiome reengineering in the future.