Deciphering Core-Phyllomicrobiome of Rice Genotypes Grown in Contrasting Mountain and Island Agroclimatic Zones: Implications for Microbiome Engineering Against Blast Disease

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 identied phyllosphere- core-functional microbial groups leading to the 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 been elaborated for effective management of blast by phyllomicrobiome re-engineering. respectively. The analysis further indicated 15 and 16 climate-zone specic 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 conrmed the presence of resident Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, and Sphingomonas on the rice phyllosphere. Strikingly, the two contrasting agroclimatic 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, identied, 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. Upon phyllobacterization (a term coined for spraying of bacterial cells on the core bacterial species such as Acinetobacter baumannii, Aureimonas sp., Pantoea ananatis, P. eucrina, and Pseudomonas putida elicited plant defense and contributed signicantly to blast disease suppression. Transcriptional analysis by qPCR indicated induction of rice innate immunity associated genes and in seedlings. structure and composition on the rice genotypes. Our integrated mNGS method and microbiological validation divulged Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, and Sphingomonas as 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 identication 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. is the ability of synthetic-microbiome to buffer against environmental perturbations. However, the development of such synthetic microbiomes is, often, hampered by our limited understanding of the core functional microbiome. Harnessing the potential of naturally occurring phyllomicrobiome for foliar disease and crop management has not been attempted till date. Since the phyllosphere microbiomes have been reported to play a pivotal role in growth, development, and defense against biotic and abiotic stress, proling the phyllomicrobiome for deciphering the functions assumes signicance. With this background, the current investigation was conducted to identify the core-phyllomicrobiome of rice and its potential to suppress blast disease. We, further, attempted to decipher the major driver(s) of phyllosphere microbiome composition using the integrated metagenomic Next Generation Sequencing (mNGS) approach and conventional microbiological methods. For this purpose, phyllomicrobiome samples were generated from two contrasting rice genotypes differing for a single resistance-gene, Pi2 conferring complete resistance to blast, thereby varying for their reaction to blast disease, grown in two-contrasting agro-climatic zones in India separated by more than 2800 Km. The agroclimatic zones represented Mountain-zone in the Himalayan region (Palampur) and Island-zone in Andaman Island in the Bay-of-Bengal, India (Port Blair). While the mountain-zone in Palampur is an endemic-location for blast disease, the island-zone in Port Blair is non-endemic. We identied the core-phyllomicrobiome of rice genotypes in the combined and comparative mNGS and microbiological data. The results indicated the association of complex microbial assemblages displaying diverse-functions on the rice phyllosphere for rice blast management. Our in-vitro screening of phyllomicrobiome against M.oryzae and in-planta evaluation trial against rice blast disease further conrmed the potential of functional-microbial groups for phyllomicrobiome assisted rice cultivation in the future. antifungal mediated antagonistic (11.8 %), Curtobacterium (9.3 %), Pseudomonas (8.7 %), andSphingomonas (8.6 %) on rice spermosphere. They also observed that the seed microbiome appeared to be highly stable and protected owing to their natural encapsulation in the seed coat that enables them to be inherited, known as vertical transmission. The core-phyllomicrobiome assemblage observed in our study seems to be less or unaffected by local climatic conditions of either hill ecosystem or coastal ecosystem and genotype differences. Therefore, it is concluded that the spermosphere of PRR 78 and Pusa 1602 harboured a core-phyllomicrobiome consisting of Acinetobacter, Arthrobacter, Bacillus, Curtobacterium, Enterobacter, Exiguobacterium, Kineococcus, Methylobacterium, Microbacterium, Paenibacillus, Pantoea, Pseudoalteromonas, Pseudomonas, Rhodococcus, and Sphingomonas. According to Eyre et al [69] an ideal core microbiome is dened as the microbiota shared between genotypes grown in geographical areas that do not share common environmental conditions. The genotypes, PRR 78, and Pusa 1602 grown in contrasting agroclimatic zones representing the lower-Himalayan region and coastal Island region showed the consistent presence of bacterial genera that are reported as core seed microbiome. Along with the recent shreds of evidence from rice seed microbiomes, it is further speculated that the rice seeds played a carrier of microbiome which enabled its spatio-temporal transmission across diverse geographical locations and seasons.

Molecular diversity analysis and identi cation of phyllomicrobiome associated bacterial species BOX-PCR DNA ngerprinting: Genomic DNA of each of the bacterial isolates was isolated by the CTAB method prescribed by Moore et al [34]. Isolated and puri ed genomic DNA was quantitated and quality checked electrophoretically and spectrophotometrically (NanoDrop 2000, ThermoScienti c, USA). Finally, the genomic DNA was reconstituted at 100 ng µl -1 and used as a template in PCR ampli cation. Box-PCR based DNA-ngerprinting was performed for diversity analysis as well to eliminate the duplicate isolates from the collection [39]. The BOX-PCR amplicon pro ling technique speci cally ampli es the noncoding conserved sequences in the bacterial genome and is considered a highly discriminatory DNA ngerprinting technique for bacteria [40,41]. Amplicons were resolved in 1.0 % agarose gel at 30 volts for 10-12 hours and image-captured (QuantityOne, BioRad Laboratories, USA). Isolates showing identical amplicon pro les were presumed to be duplicates and represented one BOX-Amplicon Group. One representative isolate from each BOX-Amplicon Group was eventually used in the downstream work.
16S rRNA gene sequencing: Ampli cation of 16S rRNA gene was performed using universal primers 27F (27F: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (1492R: 5'-GGTTACCTTGTTACGACTT-3') to amplify the 1465 bp region to establish bacterial identity [42,43]. Then, the PCR products resolved in 1.0 % agarose gel were excised from the agarose gel and eluted using a gel elution kit (Wizard® SV Gel and PCR Clean-Up System) according to the manufacturer's instructions (Promega Corporation, USA). The cycle-sequencing reaction was performed using 20-30 ng of the puri ed amplicon using the ABI PRISM BigDye Terminators v3.1 cycle sequencing kit (Applied Biosystems Foster City, CA, USA) according to the manufacturer's instruction. The puri ed product was sequenced bi-directionally to obtain maximum coverage of the spacer region. The sequences were end trimmed, edited, and contig assembled using DNAbaser (http://www.dnabaser.com/download/DNA-Baser-sequence-assembler/). The curated sequences were, further, subjected to Basic Local Alignment Search Tool analysis (NCBI nucleotide BLAST) to establish their identity by closest match (https://www.ncbi.nlm.nih.gov/nucleotide/). All curated 16S rRNA gene sequences of phyllosphere bacterial species were submitted to GenBank database and assigned accession numbers.

Functional screening of phyllosphere bacterial communities
In vitro antifungal activity on Magnaporthe oryzae: Volatile and secretory metabolite mediated antagonistic assay of bacterial isolates were conducted on M. oryzae (isolate 1637) by dual-culture confrontation method. The percent inhibition of mycelial growth over mock was estimated by adopting the methods described by Sheoran et al [42] and Munjal et al [43]. Additionally, the fungicidal or fungistatic nature of the bacterial volatiles on M. oryzae was also determined. Brie y, bacterial isolates found completely inhibiting the growth of M. oryzae were further allowed to reestablish mycelial-growth. Based on the regrowth of the mycelium, the bacterial volatile were either categorized as fungicidal or fungistatic.
The radial growth of the fungus was measured and percent inhibition of growth over control was calculated with the help of the following formula Where I = percent inhibition C = Colony diameter in control T = Colony diameter in treatment In planta blast suppressive activity: The bacterial isolates signi cantly antagonistic to blast fungus in vitro were selected for this assay. Blast susceptible rice genotype, Pusa Basmati-1, was allowed to germinate in bacterial cell suspension (2×10 7 CFU mL -1 ) for ve days. Upon germination, the transplants were, further, grown in a climate-controlled greenhouse set at temperature 28°C ±2 °C/ RH 90+10 % /Light/dark cycles 14/10 h. Seedlings were foliar sprayed with phyllosphere bacterial suspension (Phyllobacterization; 10 7 CFU mL -1 ) and challenged with a conidial-suspension of M. oryzae 1637 (2 × 10 5 conidia mL -1 ) three weeks post sowing according to the protocols of Rajashekara et al [44]. Blast disease index was determined seven days post-inoculation using a 0-5 disease rating-scale where 0= no evidence of infection; 1.0= brown specks smaller than 0.5 mm in diameter; 2.0= brown-specks of 0.5-1.0 mm in diameter; 3.0= roundish to elliptical lesions of about 1.0-3.0 mm in diameter; 4.0= typical spindle-shaped blast lesion, 3 mm or longer with little or no coalescence of the lesion; 5.0= same as 4.0 but half or more leaves killed by coalescence of lesions. Plants rated 0.0-2.0 were considered resistant, 3.0 as moderately susceptible, and 4.0-5.0 were considered susceptible [45]. The disease severity was calculated using the following formula.
Further, the percent reduction in disease severity as compared to control was estimated using the following formula.
Where RDS = Reduction in Disease Severity (%) C = Disease Severity in control T = Disease Severity in treatment.
Brie y, whole seedlings of Pusa Basmati-1 bacterized with 2×10 7 CFU mL -1 sampled at 24, 48, and 72 hours were immediately snap-frozen using liquidnitrogen (to arrest all the cellular activity) and then stored instantly at -80°C till further use. Total RNA was isolated using the SV Tool RNA Isolation System according to the manufacturer's instruction (Promega, Madison, USA). The quality and quantity of RNA were assessed spectrophotometrically (NanoDrop 2000, ThermoScienti c, USA) as well as by agarose gel electrophoresis. The experiment was repeated two times with three technical replications.

PCoA based Bray Curtis and ANoSIM
PCoA of metagenome reads of contrasting rice genotypes, PRR 78, and Pusa 1602 by Bray-Curtis and ANoSIM revealed converging and shared microbiome assemblage on rice genotypes when grown in the same agroclimatic-zone. The same genotype, either PRR 78 or Pusa 1602, showed diverging-microbiome composition when grown in another agroclimatic zone, either Mountain or Island-zone (Fig. 3).
Linear discriminant analysis (LDA) effect size (LEfSe) analysis The LDA-LEfSe score calculated at 2.0 signi cance level revealed microbial-biomarker pro les for rice genotypes and agroclimatic zones. The result showed a total of 10 biomarkers for Pusa 1602 and two for PRR 78. Klebsiella and Exiguobacterium were found to be a unique microbial biomarker for PRR 78 while Methylobacterium, Janibacter, Frankia, Macrococcus, Leptolyngbya, Shigella, Pseudacidovorax, Anoxybacillus, and Cellulosimicrobium were predicted to be a biomarker of Pusa 1602. For the geographical location, a total of 15 biomarkers for the mountain zone at Palampur and 16 for the Island zone for Port Blair Lysinibacillus, Alkaliphilus, Cylindrospermum, Enterococcus, Bi dobacterium, Arthrospira, Leptolyngbya, Candidatus-Aquiluna, Agromyces, Lactobacillus, Leifsonia, Clostridium, Streptomyces, Bacillus, and Curtobacterium were identi ed as a biomarker for island zone ( Supplementary Fig. 1).

SparCC network of variety and location
Network analysis showed the positive (cooperative) and negative (competitive) interactions within the phyllomicrobiome members on the phyllosphere. In agroclimatic zones and rice genotypes, as many as 68 bacterial genera were predicted to interacting among themselves showing positive and negative interactions on the phyllosphere (Supplementary Table 4; Supplementary Fig. 2). SparCC based network analysis of phyllomicrobiome showed 128 and 127 cooperative interactions on the rice genotypes and agro-climatic zones, respectively; as many as 104 and 108competitive interactions were also predicted on the genotypes and climatic zones.
Comparative mNGS analysis of contrasting rice genotypes The bacterial taxa can be considered as a member of "core microbiota" if it is "consistently" associated with all genotypes of a particular species. All other bacterial species may belong to "satellite microbiota" members. Comparative mNGS analysis of rice genotypes revealed the dominance of Proteobacteria, Firmicutes, and Actinobacteria on both the rice genotypes. A total of 11 phyla were found predominated in Pusa 1602 compared to PRR 78; they were Deinococcus-Thermus, Aqui cae, Gemmantimonadetes, Chloro exi, Acidobacteria, Planctomycetes, Verucomicrobia, Actinobacteria, Proteobacteria, Bacteroidetes, and Nitrospirae. On the other hand, only three phyla Firmicutes, Fusobacteria, and Cyanobacteria were found predominated in PRR 78 ( Supplementary Fig. 3). Phyllomicrobiome pro les of all taxonomic hierarchies are furnished in Supplementary Fig. 3. Phyllomicrobiome at genus level showed primarily Pantoea followed byCurtobacterium, Methylobacterium, Exiguobacterium, andBacillus on Pusa 1602; PRR 78 showed the dominance of Exiguobacterium followed by Pantoea, Sphingomonas, Curtobacterium, and Arthrobacter (Table 2

Core microbiome analysis
Core-microbiome at the genus level was analyzed for rice genotypes as well as for the agroclimatic zones. Core microbiome of blast susceptible genotype, PRR 78 was found consisting of 17 bacterial genera with a maximum prevalence of Pantoea, Klebsiella, and Exiguobacterium. Blast resistant genotype Pusa 1602 showed core microbiota composed of 19 genera with the maximum prevalence of Pantoea, Methylobacterium, and Exiguobacterium. For agroclimatic zones, the core phyllomicrobiome at the mountain zone was found comprising of 20 genera with the high representation of Pantoea, Microbacterium Exiguobacterium, and Arthrobacter. Similarly, the core phyllomicrobiome at the Island zone displayed 16 genera with the maximum prevalence of Pantoea, Methylobacterium, Exiguobacterium, Curtobacterium, and Bacillus.

Scanning Electron Microscopic imaging of phyllomicrobiome
The SEM imaging of rice phyllomicrobiome revealed the physical presence of bacterial cells aggregates of 5-8 cells, and unevenly distributed solitary bacterial-cells on the phyllosphere of rice genotypes. The Eukaryotic cells and hyphal fragments were also found scattered among the prokaryotic cells ( Fig.   6).
Further, comparative analysis of phyllomicrobiome of rice samples con rmed the consistent association of Acinetobacter, Curtobacterium, Enterobacter, Exiguobacterium, Pantoea, Pseudomonas, and Sphingomonas in Mountain and Island agroclimatic zones in both the mNGS and microbiological approaches (Data not shown). Bacterial genera such as Acinetobacter, Curtobacterium, Enterobacter, Exiguobacterium, Pantoea, Pseudomonas, andSphingomonas were consistently associated with both the genotypes in all samples (data not shown).  [59] proposed a concept of keystone microbial species which is central to the microbial community assemblage and the sustainability of the ecological niche. Adapted microbial communities developing an intimate association with that of plant species during their co-evolution are termed as core-microbiome (or core-microbiota) which is speculated to be vertically transmitted in successive generations of plants [60]. Nevertheless, microbiome composition on a plant niche is in uenced by plant genotype, habitat, ecosystem, as well as macro and micro-climatic conditions [61]. It is further reported that long-term seasonal patterns related to climatic variations serve a vital role in shaping the phyllosphere microbiome as compared to short-term weather uctuations during crop season [62].
The plant phyllosphere is one of the habitats for diverse microorganisms that are adapted to survive intra-day vagaries of weather as well as the nutrientdepleted niches. However, the major drivers of phyllosphere microbiome structure and composition are not adequately understood. Although speculated from the microbiome pro le of multiple genotypes, the core-phyllomicrobiome of rice is not elucidated thoroughly. We attempted to integrate both mNGS and microbiological strategies to characterize the core phyllomicrobiome of the rice genotype. For this, rst, we sequenced phyllosphere metagenome of two rice genotypes contrasting for their reaction to blast disease grown in two contrasting agroclimatic zones of India namely, the Mountain zone in the Himalayan Hill and the Island zone in the Andaman Island situated in the Bay-of-Bengal. Uniquely, the phyllomicrobiome in our study represented blast susceptible genotype PRR 78, and Pusa 1602 -the near-isogenic line of PRR 78, introgressed with Pi2 gene conferring complete resistance to blast disease. Most of the phyllomicrobiome studies, till now, focused mostly on pro ling of microbiome using mNGS methods alone. Furthermore, very few attempts have been made to exploit the phyllomicrobiome for crop production and protection. Therefore, the ultimate goal of our investigation was to decipher the functional corephyllomicrobiome of rice for exploiting phyllomicrobiome assisted rice cultivation with a focus on blast disease management. While the blast mitigation strategy by R-genes is threatened by new pathotypes, the fungicide is environmentally unsafe and is no longer accepted in trade [30,63]. Hence, there is a need for alternative approaches for blast disease management preferably through eco-friendly strategies.
The core-phyllomicrobiome assemblage observed in our study seems to be less or unaffected by local climatic conditions of either hill ecosystem or coastal ecosystem and genotype differences. Therefore, it is concluded that the spermosphere of PRR 78 and Pusa 1602 harboured a core-phyllomicrobiome consisting of Acinetobacter, Arthrobacter, Bacillus, Curtobacterium, Enterobacter, Exiguobacterium, Kineococcus, Methylobacterium, Microbacterium, Paenibacillus, Pantoea, Pseudoalteromonas, Pseudomonas, Rhodococcus, and Sphingomonas. According to Eyre et al [69] an ideal core microbiome is de ned as the microbiota shared between genotypes grown in geographical areas that do not share common environmental conditions. The genotypes, PRR 78, and Pusa 1602 grown in contrasting agroclimatic zones representing the lower-Himalayan region and coastal Island region showed the consistent presence of bacterial genera that are reported as core seed microbiome. Along with the recent shreds of evidence from rice seed microbiomes, it is further speculated that the rice seeds played a carrier of microbiome which enabled its spatio-temporal transmission across diverse geographical locations and seasons.
The impact of disease resistance conferring-gene (R-gene) introgression in cultivated crops on phyllomicrobiome composition and assemblage is recently reported [67]. The rice line IR24 introgressed with Xa4 gene conferring resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae showed a reduction in the abundance of Actinobacteria, but an increase in Proteobacteria and Firmicutes compared to IR24. Similarly, the rice line R711+SAox had a decrease in the abundance of Firmicutes and an increase in Proteobacteria. A signi cant in uence of plant genotype on rhizosphere and endosphere microbiome is also reported by several workers [71][72][73].
Bacterial communities identi ed on rice phyllomicrobiome by mNGS were further validated by culture-based microbiological methods which yielded 78 bacterial morphotypes. More number of morphotypes was isolated from 30 days old rice seedlings as compared to 15 days old seedlings suggestive of the expansion of microbial biomass on plant niches upon aging. These isolates were further characterized using BOX-AIR-PCR ngerprinting that resulted in 59 discrete isolates based on the amplicon pro le of the isolates. BOX-PCR is one of the frequently used molecular tools in bacterial typing and biogeography studies of microbial isolates [39,74]. The BOX-PCR ngerprinted 59 phyllosphere bacterial isolates represented 13 genera and 29 species based closest match of 16S rRNA gene sequence in multiple databases. Interestingly, as many as six bacterial morphotypes from mountain-zone and four from tropical island-zone were found sharing all BOX-PCR amplicons; they can be considered as genetically identical isolates. The most frequented bacterial species in the cultivated phyllomicrobiome belonged to Acinetobacter, Acidovorax, Curtobacterium, Enterobacter, Pantoea, Pseudomonas, and Sphingomonas. We observed genetically identical Pantoea ananatis in the phyllomicrobiome obtained from the two agroclimatic zones. Interception of genetically identical OsEp-Plm-15P9 and OsEp-AN-15A10 identi ed as Pantoea ananatisrepresenting contrasting and well-separated agroclimatic zones is indicative of vertical transmission of phyllomicrobiome. The evidence generated for vertical transmission of phyllomicrobiome may be attributed to rice seeds. Recently Charishma [75] reported high-frequency occurrence of Pantoea ananatis on rice spermosphere and phyllosphere of Pusa Basmati-1 and VLD85 by adopting dual mNGS and microbiological methods. Spermosphere microbiome seems to have spread to rice phyllomicrobiome pool during seedling emergence and further plant growth. Our data on seed transmission of phyllomicrobiome supported the observations of Kim et al [70]. Altogether, it may be concluded that rice spermosphere is among the primary sources of the core phyllomicrobiome, and the rice grown in contrasting geographical locations may have acquired the phyllomicrobiome from the spermosphere as well.
The core bacterial genera such as Acinetobacter (pale brown), Aeromonas (dark brown), Aureimonas(yellow), Curtobacterium (yellow; red), Exiguobacterium (yellow; orange), Methylobacterium (pink), Microbacterium (yellow), Micrococcus (yellow; red), Pantoea (yellow), and Sphingomonas (yellow) found consistently on phyllosphere are frequently reported pigment-producing species. Dark pigmentation is one of the adaptive traits of bacteria and other microbes in the phyllosphere [61,76]. The pigmentation of many Aeromonas species is attributed to the production of L-3, 4-dihydroxyphenylalanine (L-DOPA) based melanin [77]. Rice foliar niche is frequently cited habitat for pink-pigmented-facultative -methylotrophic (PPFM) bacteria and yellow-pigmented Pantoea that is tolerant of harmful -ray radiation as well as nutritional and moisture stress [76]. Recently, Carvalho and Castillo [78] reported the signi cant role of sunlight in shaping the microbiome of the phyllosphere. The intimate association of Pantoea ananatis with the phyllosphere of many plants including rice plants is reported [79,80]. Microbacterium testaceum is reported to degrade N-acyl-homoserine lactone on a potato leaf and is considered as an aggressive plant colonizer involved in natural biocontrol against plant pathogen [81]. Microbacterium has been reported in the rice phyllosphere and spermosphere [68,82,83]. The phyllomicrobiome data further revealed horizontal microbiome transmission from insects like Anopheles stephensi to rice as evident from the interception of Asaia-a mosquito-associated bacteria on phyllosphere samples from Andaman Island that is endemic for malaria [84].
Techniques such as uorescent in situ hybridization (FISH) and SEM are among the frequently used methods to visualize native microbial cells as well as to analyse the spatial distribution of microbial cells on phyllosphere [85,86]. Our SEM imaging indicated the physical presence of bacterial cells aggregates of 5-8 cells, and unevenly distributed solitary bacterial cells on the rice phyllosphere. The formation of aggregates or bio lms by bacterial communities is touted as one of the adaptive mechanisms on the nutrient-depleted harsh plant habitat like phyllosphere [10,87].
Phyllobacterized plants showed an elevated expression of defense genes such as OsCEBiP, OsCERK, OsPR1.1, OsNPR1,OsPDF2.2, OsFMO, andOsPAD4; signi cant induction of OsCEBiP, OsCERK1, andOsPAD4 was observed in rice seedlings sprayed with Pantoea or Aureimonas. OsCEBiP and OsCERK1 are known to interact with chitin to activate MAMP Triggered Immune (MTI) responses in plants [46]. OsCERK1 is a receptor-like kinase (RLK) believed to perceive fungal-chitin and bacterial-peptidoglycan [47]. OsPAD4 and OsEDS1 play an important role in jasmonic acid-mediated induced systemic resistance. The accumulation of rice phytoalexin mamilactone-A is reported to be modulated by the expression of the OsPAD4 gene and is known to govern blast resistance [48,49,98]. Marginal induction of OsNPR1, OsFMO, OsPDF2.2, and OsPR1.1 was observed in bacterized seedlings. OsNPR1 is the central regulator of salicylic acid (SA)-mediated defense signaling which is also responsible for the reallocation of energy and resources during the defense response [50].
Similarly, OsFMO1 is also an essential component for induced systemic acquired resistance [52,53]. OsPDF2.2 is a plant defensin responsible for the inhibition of the growth of fungi [51]. OsPR1.1 is an acidic pathogenesis-related protein, and a marker for salicylic acid-mediated SAR [54].
Black pepper endophyte, Pseudomonas putida BP25 has been recently reported to induce defense in rice plants against blast disease [94]. Similarly, Arabidopsis thaliana genes governing SA-mediated defense and growth promotion were found up-regulated by P. putida BP25 [99] and Bacillus megaterium BP17 [100]{Vibhuti, 2017 #61; Akamatsu, 2013 #44}. Species belonging to Microbacterium and Stenotrophomonas have also been recently reported to elicit defense against rice blast disease [101]. Patel et al [102] recently reported the antifungal and defense elicitation activity by BVC belongs to pyrazine against the rice blast disease.

Conclusion
A converging phyllomicrobiome assemblage was observed on rice genotypes grown in a particular agroclimatic zone. Conversely, rice genotype grown in contrasting agroclimatic zones displayed divergent phyllomicrobiome assemblage. Agroclimatic zone and the associated climatic factors rather than hostgenotype per se appears to drive phyllomicrobiome structure and composition on the rice genotypes. Our integrated approach revealed Acinetobacter, Aureimonas, Curtobacterium, Enterobacter, Exiguobacterium, Microbacterium, Pantoea, Pseudomonas, andSphingomonas as core phyllomicrobiome of rice. Genetically identical bacterial communities intercepted on the phyllosphere of rice grown in the contrasting agroclimatic zone are suggestive of spatiotemporal transmission of phyllomicrobiome aided by seed. The core microbiome mediated phyllobacterization showed potential for blast disease suppression which could be attributed to direct-antibiosis as well as indirect-elicitation of innate immunity in rice. The identi cation 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.   Microbiome Analyst [37] was utilized for the determination of core phyllomicrobiome    Extended error bar plots for the top 31 microbiota at the genus level (a) Extended error bar plots for the top microbiota at the Genus level for two genotypes.
(b) Extended error bar plots for the top microbiota at the Genus level for two climatic zones. Sorted by signi cance in ascending order, mean proportion and their differences for phyllosphere microbiota are shown i. Genus Exiguobacterium, Sphingomonas, Klebsiella, Pseudomonas, and Arthrobacter in PRR 78 were signi cantly higher in abundance than that in Pusa 1602 ii. Genus Methylobacterium, Cronobacter, Pantoea, Curtobacterium, and Clavibacter in Pusa 1602 were signi cantly higher in abundance than that in PRR 78 iii. Genus Pantoea, Arthrobacter, Exiguobacterium, Klebsiella, and Methylobacterium in the Mountain zone at Palampur were signi cantly higher in abundance than that in the Island zone at Port Blair iv. Genus Curtobacterium, Bacillus, Sphingomonas, Clavibacter, and Cronobacter in the Island zone at Port Blair were signi cantly higher in abundance than that in the Mountain zone at Palampur Figure 6 SEM images of rice phyllosphere with bacterial and fungal cells/mycelium on the surface Phyllosphere adapted bacterial isolates found promising for in vitro inhibition of Magnaporthe oryzae and in planta suppression of rice blast disease