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Adaxial, abaxial phylloplane (leaf), and spermoplane (seed) are proximal yet contrasting habitats for a microbiota that needs to be adequately explored. Here, we proposed novel methods to decipher the adaxial/abaxial-phylloplane and spermoplane-microbiomes. Comparison of 22 meta barcoded-NGS datasets (size of total data set-1980.48 Mb) enabled us to fine-map the microbiome of the rice foliar niche, which encompasses the lower, middle, top leaf as well panicle. Here, the total- and the cultivable-microbiome profiling revealed 157 genera representing ten phyla and 87 genera from 4 bacterial phyla, respectively, with a predominance of Proteobacteria and Actinobacteria. Interestingly, more bacterial communities (124-genera) preferred the abaxial than the adaxial phylloplane (104-genera) and spermoplane (67-genera) for colonization. The microbiome profiles were nearly identical on the aromatic (125-genera) and non-aromatic rice (116-genera) with high representation of Pantoea, Methylobacterium, Curtobacterium, Sphingopyxis, and Microbacterium. The culturomics investigation confirmed the abundance of Pantoea, Chryseobacterium, Pseudomonas, Acinetobacter, Sphingobacterium, and Exiguobacterium. One hundred bacterial isolates characterized and identified by polyphasic-taxonomic tools revealed the dominance of Acinetobacter, Chryseobacterium, Enterobacter, Massilia, Pantoea, Pseudomonas, and Stenotrophomonas on adaxial/abaxial-phylloplane and spermoplane. The study culminated in identifying hitherto unexplored bacterial communities on the adaxial/abaxial phylloplane and spermoplane of rice that can be harnessed for microbiome-assisted rice cultivation in the future., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Plant growth-promoting endophytic microbes have drawn the attention of researchers owing to their ability to confer fitness benefits in many plant species. Here, we report agriculturally beneficial traits of rice-leaf-adapted endophytic Microbacterium testaceum . Our polyphasic taxonomic investigations revealed its identity as M. testaceum . The bacterium displayed typical endophytism in rice leaves, indicated by the green fluorescence of GFP-tagged M. testaceum in confocal laser scanning microscopy. Furthermore, the bacterium showed mineral solubilization and production of IAA, ammonia, and hydrolytic enzymes. Tobacco leaf infiltration assay confirmed its non-pathogenic nature on plants. The bacterium showed antifungal activity on Magnaporthe oryzae , as exemplified by secreted and volatile organic metabolome-mediated mycelial growth inhibition. GC-MS analysis of the volatilome of M. testaceum indicated the abundance of antimicrobial compounds. Bacterization of rice seedlings showed phenotypic traits of MAMP-triggered immunity (MTI), over-expression of OsNPR1 and OsCERK , and the consequent blast suppressive activity. Strikingly, M. testaceum induced the transcriptional tradeoff between physiological growth and host defense pathways as indicated by up- and downregulated DEGs. Coupled with its plant probiotic features and the defense elicitation activity, the present study paves the way for developing Microbacterium testaceum- mediated bioformulation for sustainably managing rice blast disease.

Genetic and functional characteristics of rice leaf endophytic actinobacterial member, Microbacterium are described. Morphotyping, multilocus sequence analysis and transmission electron microscopy indicated the species identity of the endophytic bacterium, OsEnb-ALM-D18, as Microbacterium testaceum . The endophytic Microbacterium showed probiotic solubilization of plant nutrients/minerals, produced hydrolytic enzyme/phytohormones, and showed endophytism in rice seedlings. Further, the endophytic colonization by M. testaceum OsEnb-ALM-D18 was confirmed using reporter gene coding for green fluorescence protein. Microbacterium OsEnb-ALM-D18 showed volatilome-mediated antibiosis (95.5% mycelial inhibition) on Magnaporthe oryzae . Chemical profiling of M. testaceum OsEnb-ALM-D18 volatilome revealed the abundance of 9-Octadecenoic acid, Hexadecanoic acid, 4-Methyl-2-pentanol, and 2,5-Dihydro-thiophene. Upon endobacterization of rice seedlings, M. testaceum altered shoot and root phenotype suggestive of activated defense. Over 80.0% blast disease severity reduction was observed on the susceptible rice cultivar Pusa Basmati-1 upon foliar spray with M. testaceum . qPCR-based gene expression analysis showed induction of Os CERK1, Os PAD4, Os NPR1.3, and Os FMO1 suggestive of endophytic immunocompetence against blast disease. Moreover, M. testaceum OsEnb-ALM-D18 conferred immunocompetence, and antifungal antibiosis can be the future integrated blast management strategy., Competing Interests: Author PN was employed by T-Stanes & Company Ltd., Coimbatore, India. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Patel, Sahu, Mehta, Balamurugan, Kumar, Sheoran, Kumar, Krishnappa, Ashajyothi, Kundu, Goyal, Narayanasamy and Kumar.)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KV declared a shared affiliation with the authors, to the handling editor at the time of the review., (Copyright © 2021 Sahu, Patel, Kumar, Sheoran, Mehta, Reddy, Eke, Prabhakaran and Kumar.)

Weeds, pests, and pathogens are among the pre-harvest constraints in rice farming across rice-growing countries. For weed management, manual weeding and herbicides are widely practiced. Among the herbicides, glyphosate [N-(phosphonomethyl) glycine] is a broad-spectrum systemic chemical extensively used in agriculture. Being a competitive structural analog to phosphoenolpyruvate, it selectively inhibits the conserved 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme required for the biosynthesis of aromatic amino acids and essential metabolites in eukaryotes and prokaryotes. In the present study, we investigated the antifungal and defense elicitor activity of glyphosate against Magnaporthe oryzae on transgenic-rice overexpressing a glyphosate-resistance OsEPSPS gene (T173I + P177S; TIPS OsmEPSPS) for blast disease management. The glyphosate foliar spray on OsmEPSPS transgenic rice lines showed both prophylactic and curative suppression of blast disease comparable to a blasticide, tricyclazole. The glyphosate displayed direct antifungal activity on Magnaporthe oryzae as well as enhanced the levels of antioxidant enzymes and photosynthetic pigments in rice. However, the genes associated with phytohormones-mediated defense (OsPAD4, OsNPR1.3, and OsFMO) and innate immunity pathway (OsCEBiP and OsCERK1) were found repressed upon glyphosate spray. Altogether, the current study is the first report highlighting the overexpression of a crop-specific TIPS mutation in conjugation with glyphosate application showing potential for blast disease management in rice cultivation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have deciphered the leaf endophytic-microbiome of aromatic (cv. Pusa Basmati-1) and non-aromatic (cv. BPT-5204) rice-genotypes grown in the mountain and plateau-zones of India by both metagenomic NGS (mNGS) and conventional microbiological methods. Microbiome analysis by sequencing V3-V4 region of ribosomal gene revealed marginally more bacterial operational taxonomic units (OTU) in the mountain zone at Palampur and Almora than plateau zone at Hazaribagh. Interestingly, the rice leaf endophytic microbiomes in mountain zone were found clustered separately from that of plateau-zone. The Bray-Curtis dissimilarity indices indicated influence of geographical location as compared to genotype per se for shaping rice endophytic microbiome composition. Bacterial phyla, Proteobacteria followed by Bacteroidetes, Firmicutes, and Actinobacteria were found abundant in all three locations. The core-microbiome analysis devulged association of Acidovorax; Acinetobacter; Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium; Aureimonas; Bradyrhizobium; Burkholderia-Caballeronia-Paraburkholderia; Enterobacter; Pantoea; Pseudomonas; Sphingomonas; and Stenotrophomonas with the leaf endosphere. The phyllosphere and spermosphere microbiota appears to have contributed to endophytic microbiota of rice leaf. SparCC network analysis of the endophytic-microbiome showed complex cooperative and competitive intra-microbial interactions among the microbial communities. Microbiological validation of mNGS data further confirmed the presence of core and transient genera such as Acidovorax, Alcaligenes, Bacillus, Chryseobacterium, Comamonas, Curtobacterium, Delftia, Microbacterium, Ochrobactrum, Pantoea, Pseudomonas, Rhizobium, Rhodococcus, Sphingobacterium, Staphylococcus, Stenotrophomonas, and Xanthomonas in the rice genotypes. We isolated, characterized and identified core-endophytic microbial communities of rice leaf for developing microbiome assisted crop management by microbiome reengineering in future., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Dark brown necrotic lesions caused by Magnaporthe oryzae on rice foliage is a contrasting microhabitat for leaf-colonizing microbiome as compared with the surrounding healthy chlorophyll-rich tissues. We explored culturable bacterial communities of blast lesions by adopting microbiological tools for isolating effective biocontrol bacterial strains against M. oryzae. 16S rRNA gene sequencing-based molecular identification revealed a total of 17 bacterial species belonging to Achromobacter (2), Comamonas (1), Curtobacterium (1), Enterobacter (1), Leclercia (2), Microbacterium (1), Pantoea (3), Sphingobacterium (1), and Stenotrophomonas (5) found colonizing the lesion. Over 50% of the bacterial isolates were able to suppress the mycelial growth of M. oryzae either by secretory or volatile metabolites. Volatiles released by Achromobacter sp., Curtobacterium luteum, Microbacterium oleivorans, Pantoea ananatis, Stenotrophomonas maltophilia, and Stenotrophomonas sp., and were found to be fungicidal while others showed fungistatic action. In planta pathogen challenged evaluation trial revealed the biocontrol potential of Stenotrophomonas sp. and Microbacterium oleivorans that showed over 60% blast severity suppression on the rice leaf. The lesion-associated bacterial isolates were found to trigger expression of defense genes such as OsCEBiP, OsCERK1, OsEDS1, and OsPAD4 indicating their capability to elicit innate defense in rice against blast disease. The investigation culminated in the identification of potential biocontrol agents for the management of rice blast disease.

Endophytic bacteria isolated from cactus were characterized and assessed for their capability to induce drought tolerance and growth promotion in tomato. A total of 191-bacteria representing 13-genera and 18-species were isolated from wild cactus, Euphorbia trigonas. Bacillus (58), Lysinibacillus (36), Enterobacter (29), Stenotrophomonas (18), Lelliottia (12) and Pseudomonas (12) were the most represented genera. 16S rDNA sequence (>1400-bp) comparison placed the bacterial isolates with Bacillus xiamenensis; Bacillus megaterium; Bacillus cereus; Bacillus amyloliquefaciens; Bacillus velezensis; Brevibacillus brevis; Lysinibacillus fusiformis; Enterobacter cloacae; Lelliottia nimipressuralis; Proteus penneri; Sphingobacterium multivorum; Klebsiella pneumoniae; Pseudomonas putida; Pseudomonas aeruginosa; Stenotrophomonas maltophilia; Citrobacter freundii; Chryseobacterium indologenes and Paracoccus sp. Bacillus xiamenensis was identified for the first time as plant endophyte. Upon bacterization, the endophytes triggered germination and growth promotion in tomato as indicated by 118 % and 52 % more root-biomass under drought-free and drought-induced conditions, respectively. Bacillus amyloliquefaciens CBa_RA37 and B. megaterium RR10 displayed broad spectrum endophytism in tomato. Bacterization of tomato with cactus endophyte showed altered oxidative status, stomatal and photosystem II functioning, internal leaf temperature and relative water content suggestive of physiological de-stressing from moisture stress. Activity of oxidative stress enzymes such as guaiacol peroxidase and catalase was also indicative of endophyte assisted de-stressing of tomato. Re-irrigation on 20-days of drought infliction showed 86.9% recovery of B. amyloliquefaciens CBa_RA37 primed tomato when non-primed plantlets succumbed. The cactus endophytic bacterial strain B. amyloliquefaciens CBa_RA37 showed promise for low-cost, efficient and environmentally friendly bio-inoculant technology to mitigate drought in arid zones of Asian and African continents., (Copyright © 2019 Elsevier GmbH. All rights reserved.)