Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens.

BACKGROUND: Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles. RESULTS: Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin. CONCLUSION: The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Neolamarckia cadamba hosts a putative novel deltapartitivirus: a revelation by transcriptome data-mining.

Neolamarckia cadamba (Roxb.) Bosser is a medicinally important, fast-growing, timber-yielding tree species. In the present study, the virome of N. cadamba was explored using the publicly available N. cadamba transcriptome datasets and a putative novel virus, tentatively named as cadamba cryptic virus 1 (CdbCV1), was identified. CdbCV1 contained two genome segments, each coding for a single protein. CdbCV1 RNA1 (1564 nt) encoded for an RNA dependent RNA polymerase (RdRp) protein while CdbCV1 RNA2 (1492 nt) encoded for a coat protein (CP). Phylogenetic and sequence similarity analyses revealed the relatedness of CdbCV1 to pepper cryptic virus 1 and pittosporum cryptic virus 1. Based on the species demarcation criteria, genome organization and phylogeny, CdbCV1 can be regarded a new member of the genus Deltapartitivirus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00845-8.

Hydroxamate siderophores secreted by plant endophytic Pseudomonas putida elicit defense against blast disease in rice incited by Magnaporthe oryzae.

Our study focuses on hydroxamate-type siderophores from Pseudomonas putida BP25, known for chelating ferric iron and aiding microbial growth in iron-deficient environments. Confirmed through CAS-agar and tetrazolium tests, a purified siderophore extract was obtained via ion-exchange chromatography. Applying varying concentrations of this siderophore to rice seedlings demonstrated concentration-dependent effects on shoot and root phenotypes. Prophylactic application on rice leaves significantly reduced blast severity (68.7%-97.0%), surpassing curative application (47.5%-86.87%). Additionally, the siderophore treatment elevated peroxidase, polyphenol oxidase, and total phenols in rice plants. Defense-related genes linked to salicylic acid (OsPR1.1, OsNPR1, and OsPDF2.2), and other pathways (Oshox24, OsCLE, and OsGLP3-3, OsEIN2.4, and OsCSE) promoting blast suppression showed upregulation. However, the OsACS6 gene associated with ethylene-induced internodal elongation was significantly downregulated. Overall, our findings propose that the siderophore from P. putida BP25 induces defense gene transcription, offering potential for sustainable rice production via bio-formulation.

Enhancing defense against rice blast disease: Unveiling the role of leaf endophytic firmicutes in antifungal antibiosis and induced systemic resistance.

Rice remains the primary staple for more than half of the world's population, yet its cultivation faces numerous challenges, including both biotic and abiotic stresses. One significant obstacle is the prevalence of rice blast disease, which substantially diminishes productivity and increases cultivation costs due to frequent fungicide applications. Consequently, the presence of fungicide residues in rice raises concerns about compliance with international maximum residue limits (MRLs). While host resistance has proven effective, it often remains vulnerable to new variants of the Magnaporthe oryzae pathogen. Therefore, there is a critical need to explore innovative management strategies that can complement or enhance existing methods. An unexplored avenue involves harnessing endophytic bacterial communities. To this end, the present study investigates the potential of eleven endophytic Bacillus spp. in suppressing Pyricularia oryzae, promoting plant growth, and eliciting a defense response through phyllobacterization. The results indicate that the secreted metabolome and volatilome of seven tested isolates demonstrate inhibitory effects against P.oryzae, ranging from a minimum of 40% to a maximum of 70%. Bacillus siamensis L34, B. amyloliquefaciens RA37, B. velezensis L12, and B. subtilis B18 produce antifungal antibiotics targeting P.oryzae. Additionally, B. subtilis S4 and B. subtilis S6 emerge as excellent inducers of systemic resistance against blast disease, as evidenced by elevated activity of biochemical defense enzymes such as peroxidase, polyphenol oxidase, and total phenol content. However, a balance between primary metabolic activity (e.g., chlorophyll content, chlorophyll fluorescence, and photosynthetic rate) and defense activity is observed. Furthermore, specific endophytic Bacillus spp. significantly stimulates defense-related genes, including OsPAD4, OsFMO1, and OsEDS1. These findings underscore the multifaceted potential of endophytic Bacillus in managing blast disease through antibiosis and induced systemic resistance. In conclusion, this study highlights the promising role of endophytic Bacillus spp. as a viable option for blast disease management. Their ability to inhibit the pathogen and induce systemic resistance makes them a valuable addition to the existing strategies. However, it is crucial to consider the trade-off between primary metabolic activity and defense response when implementing these bacteria-based approaches.

New Insights on Endophytic Microbacterium-Assisted Blast Disease Suppression and Growth Promotion in Rice: Revelation by Polyphasic Functional Characterization and Transcriptomics.

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.

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.

Endophytic bacteria of desert cactus (Euphorbia trigonas Mill) confer drought tolerance and induce growth promotion in tomato (Solanum lycopersicum L.).

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.