Deciphering core phyllomicrobiome assemblage on rice genotypes grown in contrasting agroclimatic zones: implications for phyllomicrobiome engineering against blast disease
Authors: Sahu Kuleshwar Prasad , Kumar A. , Sakthivel K. , Reddy Bhaskar , Kumar Mukesh , Patel Asharani , Sheoran Neelam , Gopalakrishnan Subbaiyan , Prakash Ganesan , Rathour Rajeev , Gautam R. K. 
Published: May 2022

Abstract
Background
With its adapted microbial diversity, the phyllosphere contributes microbial metagenome to the plant holobiont and modulates a host of ecological functions. Phyllosphere microbiome (hereafter termed 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. However, the ecological forces driving the phyllomicrobiome assemblage and functions are among the most understudied aspects of plant biology. Despite the reports on the occurrence of diverse prokaryotic phyla such as Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria in phyllosphere habitat, the functional characterization leading to their utilization for agricultural sustainability is not yet explored.
Currently, the metabarcoding by Next-Generation-Sequencing (mNGS) technique is a widely practised strategy for microbiome investigations. However, the validation of mNGS annotations by culturomics methods is not integrated with the microbiome exploration program. In the present study, we combined the mNGS with culturomics to decipher the core functional phyllomicrobiome of rice genotypes varying for blast disease resistance planted in two agroclimatic zones in India. There is a growing consensus among the various stakeholder of rice farming for an ecofriendly method of disease management. Here, we proposed phyllomicrobiome assisted rice blast management as a novel strategy for rice farming in the future.

Results
The tropical "Island Zone" displayed marginally more bacterial diversity than that of the temperate ‘Mountain Zone’ on the phyllosphere. Principal coordinate analysis indicated converging phyllomicrobiome profiles on rice genotypes sharing the same agroclimatic zone. Interestingly, the rice genotype grown in the contrasting zones displayed divergent phyllomicrobiomes suggestive of the role of environment on phyllomicrobiome assembly. The predominance of phyla such as Proteobacteria, Actinobacteria, and Firmicutes was observed in the phyllosphere irrespective of the genotypes and climatic zones. The core-microbiome analysis revealed an association 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 Streptomyces on phyllosphere. The linear discriminant analysis (LDA) effect size (LEfSe) method revealed distinct bacterial genera in blast-resistant and susceptible genotypes, as well as mountain and island climate zones. SparCC based network analysis of phyllomicrobiome showed complex intra-microbial cooperative or competitive interactions on the rice genotypes. The culturomic validation of mNGS data confirmed the occurrence of Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, and Sphingomonas in the phyllosphere. Strikingly, the contrasting agroclimatic zones showed genetically identical bacterial isolates suggestive of vertical microbiome transmission. The core-phyllobacterial communities showed secreted and volatile compound mediated antifungal activity on M. oryzae. Upon phyllobacterization (a term coined for spraying bacterial cells on the phyllosphere), Acinetobacter, Aureimonas, Pantoea, and Pseudomonas conferred immunocompetence against blast disease. Transcriptional analysis revealed activation of defense genes such as OsPR1.1, OsNPR1, OsPDF2.2, OsFMO, OsPAD4, OsCEBiP, and OsCERK1 in phyllobacterized rice seedlings.

Conclusions
PCoA indicated the key role of agro-climatic zones to drive phyllomicrobiome assembly on the rice genotypes. The mNGS and culturomic methods showed Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, and Sphingomonas as core phyllomicrobiome of rice. Genetically identical Pantoea intercepted on the phyllosphere from the well-separated agroclimatic zones is suggestive of vertical transmission of phyllomicrobiome. The phyllobacterization showed potential for blast disease suppression by direct antibiosis and defense elicitation. Identification of functional core-bacterial communities on the phyllosphere and their co-occurrence dynamics presents an opportunity to devise novel strategies for rice blast management through phyllomicrobiome reengineering in the future.

Graphical abstract