Rice foliar-adapted Pantoea species: Promising microbial biostimulants enhancing rice resilience against foliar pathogens, Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae.

Foliar fungal blast and bacterial leaf blight have significant impacts on rice production, and their management through host resistance and agrochemicals has proven inadequate. To achieve their sustainable management, innovative approaches like leveraging the foliar microbiome, which collaborates with plants and competes against pathogens, are essential. In our study, we isolated three Pantoea strains (P. agglomerans Os-Ep-PPA-1b, P. vagans Os-Ep-PPA-3b, and P. deleyi Os-Ep-VPA-9a) from the rice phylloplane. These isolates exhibited antimicrobial action through their metabolome and volatilome, while also promoting rice growth. Our analysis, using Gas Chromatography-Mass Spectrometry (GC-MS), revealed the presence of various antimicrobial compounds such as esters and fatty acids produced by these Pantoea isolates. Inoculating rice seedlings with P. agglomerans and P. vagans led to increased root and shoot growth. Additionally, bacterized seedlings displayed enhanced immunocompetence, as evidenced by upregulated expressions of defense genes (OsEDS1, OsFLS2, OsPDF2.2, OsACO4, OsICS OsPR1a, OsNPR1.3, OsPAD4, OsCERK1.1), along with heightened activities of defense enzymes like Polyphenol Oxidase and Peroxidase. These plants also exhibited elevated levels of total phenols. In field trials, the Pantoea isolates contributed to improved plant growth, exemplified by increased flag-leaf length, panicle number, and grains per panicle, while simultaneously reducing the incidence of chaffy grains. Hypersensitivity assays performed on a model plant, tobacco, confirmed the non-pathogenic nature of these Pantoea isolates. In summary, our study underscores the potential of Pantoea bacteria in combatting rice foliar diseases. Coupled with their remarkable growth-promoting and biostimulant capabilities, these findings position Pantoea as promising agents for enhancing rice cultivation.

Fine-scale mapping of the microbiome on phylloplane and spermoplane of aromatic and non-aromatic rice genotypes.

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.

Cell wall polysaccharides of endophytic Pseudomonas putida elicit defense against rice blast disease.

AIMS: To characterize the functional role of extracellular polysaccharides and lipopolysaccharide (LPS) extracted from endophytic Pseudomonas putida BP25 (PpBP25) against rice blast. METHODS AND RESULTS: We profiled the transcriptome of endobacterized rice seedlings using RNA-seq. Fluorescence imaging of interaction between Magnaporthe:: gfp and P. putida:: mCherry was performed on rice phylloplane using confocal laser scanning microscopy (CLSM). Microbial polysaccharides, exopolysaccharide (EPS), and LPS extracted from PpBP25 were characterized using Fourier-transform infrared-spectroscopic analysis (FTIR). Biochemical assays and gene expression analysis were conducted on EPS- and LPS-treated rice seedlings. A detached-leaf assay was designed to test the blasticidal-effect of bacterial-endophyte, EPS, and LPS on rice phylloplane. PpBP25 elicited defense in rice with a consequently altered seedling phenotype. Rice cultivar, Pusa Basmati-1, colonized by PpBP25 showed an altered transcriptome profile displaying a total of 110-downregulated and 68-upregulated genes (P < 0.005) representing growth/development and defense pathways, respectively. CLSM of PpBP25 bacterized phylloplane showed reduced conidial-germination and mycelial-biomass of Magnaporthe oryzae. To decipher the elicitor role of polysaccharides, we purified and characterized EPS and LPS using FTIR. Rice treated with the EPS and LPS showed root-growth inhibition the phenotype of MAMP-triggered immunity. While the EPS showed blast suppressive activity at 1-20 mg mL-1 (79.80%-86.87% reduction over control), the LPS exhibited 78.0%-79.8% reduction at 20-200 µg mL-1on rice. Polysaccharides treated seedling showed elevated activities of peroxidase and polyphenol-oxidase activities, and total-phenols content. Treated plantlets showed up regulation of OsPR1.1,OsPR3, OsGLP3-3,OsZFP179, and Oshox24 as well as downregulation of OsACS6. CONCLUSIONS: We showed that P. putida Bp25 and its cell wall-associated polysaccharides could elicit defense against rice blast.

Rice leaf endophytic Microbacterium testaceum: Antifungal actinobacterium confers immunocompetence against 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 OsCERK1, OsPAD4, OsNPR1.3, and OsFMO1 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.

Deciphering core-microbiome of rice leaf endosphere: Revelation by metagenomic and microbiological analysis of aromatic and non-aromatic genotypes grown in three geographical zones.

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.