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. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04881-4.


Introduction
Pearl millet [Cenchurus americanus (L.) R. Br.] is the sixth most predominant cereal crop cultivated both in arid and semi-arid tropical regions worldwide, grown for food and forage.India, being the largest producer of millets, accounts for 36% of the world's millet production [1].Pearl millet is grown on an area of 7.65 million hectares with an average production of 10.86 million metric tonnes during 2020-21 [2], and its standalone contribution was 40.51% of the total millet production from India.However, pearl millet is susceptible to several diseases [3][4][5], and blast disease induced by Magnaporthe grisea is a global threat with high genetic variability and increasing virulent races [6][7][8].Since host plant resistance is vulnerable to new pathotypes and chemical pesticides are under the limelight, there is a need to develop an effective, alternative, environment-friendly sustainable disease management strategy.
Exploring the invisible, complex microbial community that includes a diverse and complex mixture of different species of microorganisms on and within plant spheres, which have subtle and relatively constant partner relationships can be a potential option for green technologies in plant disease management [9].Plant microbiome, having evolutionary significance regulates the physiological functions of plant metabolism [10][11][12][13][14][15].Plant microbiome members, unlike other microbiota, develop an intimate relationship with plant metabolism and influence it in harmony to alleviate various biotic and abiotic stress conditions.Among all, the firmicutes are well known for their abundance on plants, and the genus Bacillus has a special place with extensive studies on agroecology, including nutrient cycling [16][17][18], disease suppression [19][20][21], plant growth regulation [22,23], diversity enhancement [24,25] and phytoremediation [26,27].With this relevance, the pearl millet microbiome is under exploration of late, and associated microbes from the root, leaf, and stem regions of pearl millet are attributed in many studies to its drought stress tolerance ability [28].Pearl millet leaf and root-originated Bacillus subtilis, Bacillus pumilus strains have shown their ability to enhance the shoot and root dry mass under phosphorous deficit conditions and are known to produce siderophores and Indole Acetic Acid, which benefit from withstanding adverse conditions [29].Previously, pearl millet endophytic Bacillus amyloliquefaciens EPP90 was found to have multiple stress tolerance mechanisms and plant growth promotion activity [30].However, little is known about the pearl millet microbiome's relation to foliar blast disease.Especially, the compact structure of the pearl millet spike and its exposure to extreme weather events challenge the spermosphere microbes to thrive on and withstand the stress.However, the spermosphere microbial functionality and influence on pearl millet growth and blat disease are less studied.In this study, two pearl millet panicle-associated Bacillus strains, PBs 12 and PBl 36, were identified and characterized for their plant growth promotion and broad-spectrum antifungal activity against pearl millet blast-causing Magnaporthe grisea as well as a few crop and tree pathogens.With the increasing public and government criticism on large-scale dependency of pesticides for plant disease management, this study will help in developing a suitable bioformulation for crop protection.

Plant material and growth condition
The pearl millet genotype 7042S, which was raised in the experimental field at the University of Mysore, Manasagangotri (12.30°N, 76.64° E), Karnataka, India, was used in the present study.Panicle samples for isolation of spermosphere-associated microbes were collected at the physiological maturity stage (110-120 days) of the pearl millet in sterilized centrifuge tubes (50 ml), and the samples were immediately processed for panicle-associated microbe isolation under aseptic conditions in the laboratory at the Institution of Excellence, Vijnana Bhavana, University of Mysore, Manasagangotri.

Isolation of pearl millet panicle-associated bacteria and morphotyping
Pearl millet panicle-associated epiphytic bacteria were isolated, according to Eyre et al. [31].Briefly matured panicles from pearl millet plants (n = 12) were cut into two equal parts under aseptic conditions and transferred to the 500-ml glass bottles containing 300 ml of sterile 1X phosphate buffer saline with 0.1% Tween (PBST) at pH 7.2 and placed on an orbital shaker (180 rpm) for 30 min.The washed buffer alone (300 ml) was collected in new 500 ml bottles, and the washing step was repeated twice with 100 ml of PBST buffer.Pellets were harvested from panicle-washed buffer-microbe suspension and resuspended in 10 ml of PBS (PH-7.2) after centrifugation at 6000 rpm for 15-20 min.Serial dilutions made with sterile distilled water and 1 ml of 10 -4 , 10 -5 , 10-6, and 10 -7 dilutions were poured onto nutrient agar (NA) media amended with 5% 2,3,5-triphenyl tetrazolium chloride (n = 3 replications for each dilution).Culture morphotyping was recorded 48 h after incubation at 28 °C.Wellseparated, individual, distinct colonies were selected based on colony morphology and re-streaked onto fresh NA plates.Among several morphotypes on the NA surface, the two distinct colonies that displayed the most abundance were named PBs 12 and PBl 36, preserved at -80 °C for further characterization.

Biochemical analysis of the pearl millet panicle-associated bacterial isolates PBs 12 and PBl 36
The pearl millet panicle-associated bacterial isolates PBs 12 and PBl 36 with distinct morphology were tested for their gram staining, gram-reaction (3% KOH test), siderophore production (CAS assay), citrate, potassium utilization, chitinase and lipase activity, starch utilization, ammonia production, and cellulase activity, respectively.Single colonies of 24-h-old culture were inoculated in the specific medium for all the biochemical tests.All the tests were conducted, and the observations were recorded as per the standard protocols [32,33].

DNA extraction and 16S rRNA sequencing
Genomic DNA was extracted from two representative isolates (PBs 12 and PBl 36) according to Chen and Kuo [34], and the qualitative and quantitative analysis of genomic DNA was performed using Nanodrop 2000 (Thermofisher, USA).Amplification of 16S rRNA was performed by Thermocycler (Eppendorf, Germany) using primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3').PCR samples with genomic DNA were subjected to initial denaturation at 96 °C for 5 min, 35 cycles of denaturation at 95 °C for 60 s, annealing at 58 °C for 60 s, and extension at 72 °C for 60 s, followed by a final extension at 72 °C for 10 min.A 1% agarose gel electrophoresis with ethidium bromide was used to separate the PCR amplicons, and they could be seen on a QuantityOne UV transilluminator (BioRad, USA).Obtained amplicons of the 16S rRNA gene (~ 1500 bp) were used for bi-directional Sanger sequencing (Eurofins Scientific, India).The nucleotide sequences were assembled (> 1400 bp; DNA Baser assembler v5), trimmed using CLC sequence viewer 8.0.0 (https:// clc-seque nce-viewer.softw are.infor mer.com/8.0/), and curated sequences were compared with NCBI GenBank sequences (https:// blast.ncbi.nlm.nih.gov/) based on the BLAST search tool (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).The curated sequences were finally submitted to NCBI GenBank via the submission portal BankIt (https:// submit.ncbi.nlm.nih.gov/ subs/ genba nk/) to generate accession numbers.The maximum likelihood method with the Tamura-Nei model [35] and 1000 bootstrap replications were used for the phylogenetic analysis.Similarity percentage was considered for species-level grouping of the isolates, wherein Bacillus thuringiensis species was considered an outgroup.

Growth promotion activity of pearl millet panicle associated Bacillus strains PBs 12 and PBl 36
The effect of pearl millet panicle associated with two Bacillus strains, PBs 12 and PBl 36, on seed germination, enhancing seedling growth parameters including shoot and root length, and seedling vigour index, on pearl millet cv.7042S, was investigated by a seed priming assay [36].Bacillus strains PBs 12 and PBl 36 were grown on NA media, and a 24-h-old culture was used for the preparation of a 1.0 OD (1 × 10 8 cfu/mL) bacterial suspension.Pearl millet cv.7042S seeds were soaked in bacterial suspension overnight and later allowed to grow for 7 days under climate-controlled greenhouse conditions at 28 °C with 90% relative humidity.Similar treatments with sterile water alone served as controls, and four replications were maintained for each treatment separately.The growth parameters, including germination percentage, root, and shoot length, were recorded, whereas seedling vigour was calculated using the given formula [37].
Data analysis was performed using the raw data from all the recorded parameters separately for statistical ANOVA analysis, applying the Completely Randomized Design (CRD) through the WASP 2.0 (Web Agri Stat Package) software, which can be accessed at http:// www.Dual-culture confrontation assays were carried out on potato dextrose agar medium as described by Dennis and Webster [38].The following 6 fungal plant pathogens, comprising Magnaporthe grisea (foliar blast of pearl millet), Sclerotium rolfsii (stem rot of tomato), Fusarium solani (root rot and wilt in malabar neem), Ganoderma spp. 1 and 2 (wood rots in Tectona grandis and Pongamia pinnata), and Alternaria alternata (leaf blight of Tectona grandis) were used for in vitro screening with Bacillus strains PBs 12 and PBl 36, respectively.Bacterial cultures were grown on NA media, and a 24-h-old culture was used for the dual-culture assay.Mycelial discs of 0.5 mm from 7-day-old fungal cultures placed on PDA plates and loops of bacterial culture streaked on the opposite side of the same Petri plate were perpendicular to each bacterial isolate, whereas the fungal disc alone in the media served as the control.Three replications were maintained for all the treatments with each fungus, and inoculated plates were incubated at 26 ± 1 °C for 7 days.When the control plates attained full mycelial growth, the radial growth of the fungal pathogens was calculated, and percent inhibition over control was estimated by using the given formula [39].
The inhibition percentage values were further analyzed by statistical ANOVA, applying the Completely Randomized Design (CRD) through the WASP 2.0 (Web Agri Stat Package).The mean values have been grouped based on Critical Difference (CD) to indicate the significance at probability (p) = 0.05%.The experiment was repeated twice to confirm the reproducibility of the results.(100 -µg mL −1 ) was grown on Nutrient agar media [NA, gL −1 Peptone 5.0; Beef Extract 3.0; NaCl 5.0; Agar 15.0; pH 7.0 ± 0.2] and incubated at 28 °C for 48 h.The fresh culture was washed with 0.5% NaCl used for DNA extraction using the CTAB method.DNA quality check was performed using Nanodrop and agarose gel electrophoresis (0.8% Agarose Gel) respectively.

Library preparation and QC
The samples that passed quality control were selected for further processing in the library preparation step.To summarize, we used 100 nanograms of DNA to create libraries with unique identifiers using the Truseq Nano library preparation kit from Illumina (#20015964).The final libraries were assessed for concentration using a Qubit 4.0 fluorometer from Thermofisher (#Q33238) and a DNA HS assay kit (#Q32851), following the provided instructions.We determined the size and base length of the fragmented DNA libraries by running them on an Agilent Tapestation 4150 system with highly sensitive D1000 screen tapes (#5067-5582) according to the manufacturer's protocol.

Genome assembly, molecular phylogenetic analysis and annotation
The paired-end files were imported for analysis, and a De novo genome assembly was performed using CLC Genomics Workbench version 12.0.The resulting assembled genome served as the basis for additional scaffolding performed on the Medusa server (http:// combo.dbe.unifi.it/ medusa/).The final scaffold files of both PBs 12 and PBl 36 draft genomes were utilized in subsequent steps, including molecular phylogeny and functional annotation analysis.To establish comparative genomic relationships, the OrthoANI algorithm was employed to calculate average nucleotide identity (ANI), and a UPGMA tree was generated by aligning whole genomes.This analysis included reference sequences for Bacillus subtilis (NCIB3610, NBRC 13719, DSM  [40]. The assembled genome was subsequently annotated using the Pathosystems Resource Integration Centre (PATRIC) platform (https:// www.patri cbrc.org/).PAT-RIC facilitated the extraction of information related to sequence length, N50, G + C content, a circular view of the genome, pathway summaries, and cellular subsystems.Additionally, a comprehensive analysis was conducted to identify biosynthetic gene clusters responsible for producing secondary metabolites in the Bacillus strains PBs 12 and PBl 36.This analysis was carried out using the antiSMASH server (Antibiotics and Secondary Metabolite Analysis Shell) version 4.1.0(http:// antis mash.secon darym etabo lites.org) as detailed by Medema et al. [41] and Blin et al. [42].Finally, the whole genome data of Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 was submitted to the NCBI GenBank through the genome submission process under Biosample IDs: SAMN31359864 and SAMN31577901.

Morphological and biochemical characterization of two pearl millet panicle microbiome associated Bacillus strains PBs 12 and PBl 36
The pearl millet panicle-associated microbiota was dominated by the genera Bacillus, comprising Bacillus licheniformis, Bacillus paralicheniformis, Bacillus subtilis, Bacillus megaterium, and Bacillus aryabhattai, apart from other important bacterial species like Pantoea stewartii and Sphingomonas sanguinis.Among all, two isolates were observed as the most abundant Bacillus species that displayed the highest antifungal activity.The two representative isolates were named PBs 12 and PBl 36 and considered for further characterization.
The colony morphology of Bacillus sp.PBs 12 was observed as round to irregular pale creamy white colonies with a shiny surface and diffused irregular margins, while Bacillus sp.PBl 36 produced irregular pale white/ash-colored colonies with spreading margins and rough surface (Table 1).The biochemical analysis revealed that the bacterial isolates are gram-positive, rod-shaped and did not produce fluorescence on King's B media.They also lack chitinase and lipase activity and can grow at 37°C within 24 h.These bacterial isolates varied in their citrate utilization ability, starch hydrolysis, siderophore production, and ammonia production.Bacillus subtilis PBs 12 tested positive for siderophore production, able to utilize citrate but lacking lipase and chitinase activity.Both PBs 12 and PBl 36 were found to have cellulase activity and were negative for indole production and nitrate utilization tests (Supplementary Table 1).
The 16S ribosomal RNA sequence (1416 bp) of representative Bacillus isolates, PBs 12 and PBl 36 were checked for low-quality bases, and the contigs were end-trimmed and curated using a CLC sequence viewer.The BLAST analysis in the NCBI Nucleotide BLAST database using the 16S r RNA-curated sequence of PBl 36 revealed its close similarity with the wheat seed endophyte, Bacillus licheniformis strain PZ-54 from New Delhi, India (MT184872.1),with a 99.44% identity.Similarly, PBs 12 revealed its close similarity with Bacillus subtilis strain soil G2B (MT184872.1)from Gujarat, India, with 98.31% identity.Curated sequences of 16S r RNA from both PBs 12 and PBl36 were submitted to the NCBI GenBank (Table 1).The accession numbers allotted were as follows for Bacillus subtilis PBs 12 (accession no.: OL674150) and Bacillus licheniformis PBl 36 (accession no.: OL674149).Phylogenetic analysis using 16S ribosomal RNA sequence based on the maximum likelihood tree construction method revealed that isolate PBs 12 clustered with Bacillus subtilis strains and PBl 36 closely clustered with the other Bacillus licheniformis strains.Both Bacillus licheniformis PBl 36 and Bacillus subtilis PBs 12 were distant from other Bacillus species such as Bacillus megaterium, Bacillus altitudinis, Bacillus mycoides and Bacillus thuringiensis (Fig. 1).

Effect of pearl millet panicle-associated Bacillus strains PBs 12 and PBl 36 on pearl millet cv. 7042S seedling growth
Bacillus strains PBs 12 and PBl 36 were used for seed priming to test their efficacy on plant growth promotion.Germination percentage (%), seedling vigour index, seedling shoot, and root length were estimated to find the growth promotion potential.Seed priming with PBs 12 enhanced the seedling vigour index up to 1186.55 with the enhanced seedling shoot (6.87 cm) and root length (5.62 cm), while PBl 36 showed maximum plant growth promotion activity with the highest seedling vigour index (1324.8),shoot length (7.38 cm), and root length (6.42 cm).In sterile water-treated mock plants, seedling shoots and root length were 5.01 and 5.56 cm, respectively, and seedling vigour index was recorded as 1004.15(Table 2), indicating both the Bacillus isolates have a considerable plant-growth-promoting function.

Antifungal activity of pearl millet panicle associated Bacillus strains PBs 12 and PBl 36
Several Bacillus species are well known for their antimicrobial activity.The strains PBs 12 and PBl 36 were tested against taxonomically diverse plant pathogens and found effective in the suppression of their mycelial growth.Dual-culture confrontation assays against diverse fungal plant pathogens comprising Magnaporthe grisea (foliar blast of pearl millet), Sclerotium rolfsii (stem rot of tomato), Fusarium solani (root rot and wilt in malabar neem), Ganoderma spp. 1 and 2 (wood rots in Tectona grandis and Pongamia pinnata) and Alternaria alternata (leaf blight of Tectona grandis) revealed that the tested Bacillus strains were able to effectively inhibit the mycelial growth up to 25 to 78% (Table 3).Bacillus subtilis PBs 12 (60.4-78.70%) showed strong antifungal activity and inhibited > 60% of mycelial growth in all test pathogens, with a minimum inhibition percentage of 60.40 against Magnaporthe grisea and a maximum of 78.70% against Alternaria alternata followed by Bacillus licheniformis PBl 36, which recorded the lowest inhibition percentage (25%) against Ganoderma spp.and the maximum (69.5%) against Athelia rolfsii.However, both the isolates have proven to have strong antifungal activity against taxonomically diverse plant pathogens from both crops and trees (Fig. 2).

Whole genome sequencing, assembly, and annotation
The    2).

Functional genome annotation of Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36
Functional annotation of the assembled genome was performed using PATRIC, the bioinformatics resource centre (BRC) approach (https:// www.bv-brc.org/).A   14), amino acid metabolism ( 13), metabolism of cofactors and vitamins (12).Two pathways were found related to signal transduction, one with the immune system and others related to various metabolic activities (Table 6).The pathway summary for all the pathway classes is presented along with the information on the number of genes annotated in Supplementary

Identification of secondary metabolite coding gene clusters Mining of secondary metabolite gene clusters from the whole genome sequences of Bacillus strains PBs 12 and PBl 36
The antifungal activity exhibited by Bacillus strains PBs 12 and PBl 36 was tested by prediction of secondary

Discussion
Microbes provide a wide range of services and benefits to plants, and in return, they receive reduced carbon and other metabolites [43] for their survival.These microbial communities have been associated with almost all terrestrial plants since their earliest evolution to assist early land plants faced with challenges such as access to nutrients, novel and often stressful conditions, and pathogens, but little is known about their complete functional role [44].In the present study, pearl millet panicle-associated Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 were found to have good plant growth promotion and significant antifungal potential.The two strains were also observed to be the most abundant microbiome member species of pearl millet indicating their versatile roles.Bacillus species are one of the most abundant species on plants due to their ability to survive for long periods under harsh environmental conditions.Previously, few studies focused on the pearl millet (leaves, stems, and roots) associated microbiota and its relation to plant growth and stress responses, especially concerning drought and salinity [16,[45][46][47][48].Still, the pearl millet panicle microbiome is under-explored for its potential against foliar diseases including blast, a fungal disease caused by Magnaporthe grisea which is a known threat for breeding and cultivation.In this scenario, pearl millet panicle-associated Bacillus subtilis PBs 12 was observed as one of the most abundant species on panicles, which produced round to irregular, flat, non-fluid-type, creamy white colonies with irregular borders.The PBs 12 is a gram-positive, endospore-forming bacterium able to utilize citrate, cellulose, and starch.It is also able to produce siderophores, which are known for their elicitor activity to induce plant defense and enhance plant growth promotion [49,50].The second prominent isolate, Bacillus paralicheniformis PBl 36 produced creamy white rough lichen-like wrinkled colonies with an irregular margin and found motile, gram-positive bacteria with cellulase and starch hydrolysis activity.The morpho-biochemical characters were identical to those of previous characterization studies [51][52][53].In 16S rRNA-based phylogenetic  [54,55].However, up to the genus level, it will provide a considerable identity for most of the bacteria.After initial characterization, both the strains PBs 12 and PBl 36 were used for further studies.Bacillus species are well known for their plant growth promotion potential by positively regulating the plant metabolism for enhanced growth.To test this behavior, seed priming assays were conducted on pearl millet cv.7042S with both PBs 12 and PBl 36.The significant shoot and root growth increase along with seedling vigour index in 7-day-old seedlings by PBs 12 and PBl 36 gave hints on their ability to promote plant growth, which can be considered as mutually beneficial plant-microbe interaction.There are several reports on the plant growth promotion activity of Bacillus subtilis [22,26,56] and Bacillus paralicheniformis [57][58][59].The direct effects of microbe-induced plant growth are mainly derived either from their capacity to improve the nutritional status of plants or from the production of phytohormones [60].Several growth-promoting traits, such as indole-3 acetic acid production, zeatin synthesis, phosphate solubilization, and siderophore production, are exhibited by Bacillus sp.[59].Unlike Bacillus paralicheniformis, Bacillus subtilis is one of the most widely used and studied PGPR (Plant Growth Promoting Rhizobacteria) and a highly promising candidate for agricultural applications [61,62].Identification of native pearl millet strains with plant growth promotion abilities will help sustain pearl millet production in near future.
Further, in dual culture direct inhibition assays with different potential fungal plant pathogens (Sclerotium rolfsii; Magnaporthe grisea; Fusarium solani; Alternaria alternata; Ganoderma sp. 1 and 2) of Basidiomycota and Ascomycota, including the most economically important pearl millet blast pathogen Magnaporthe grisea, the direct inhibition of mycelial growth of all the test pathogens by PBs 12 (60.40-78.70%)and PBl 36 (25.36-69.54%)confirmed their broad-spectrum antifungal activity.Here, PBs-12 has shown a comparatively high inhibitory effect against all pathogens.In our recent study, the leaf endophytic Bacillus sp.showed great antifungal activity and induced systemic resistance against rice blast disease caused by Magnaporthe oryzae [24].However, the metabolites that induced such a phenomenon are yet to be explored.The information behind this significant antifungal antibiosis of Bacillus sp. can aid in the identification of beneficial bioproducts such as elicitor compounds to stimulate plant immunity against blast disease [63][64][65].The ability of bacteria to  secrete secondary metabolites such as antibiotics, bacterial organic volatile compounds, cell-wall-degrading enzymes, hormones, plant defense-modulating elicitor compounds, and antioxidants is indeed attributed to their antimicrobial potential [26,[66][67][68][69]. To further understand and explore these traits, whole genome sequencing has been performed for both Bacillus strains PBs12 and PBl36, and secondary metabolite biosynthetic potential has been studied.
In the present investigation, we have reported the genomes of pearl millet panicle-associated Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36.Bacillus subtilis PBs 12 has a 4020447 bp circular chromosome that consists of 4171 genes with 43.68% G + C      [76].This creates difficulty in the correct identification of Bacillus paralicheniformis using a 16S rRNA-based approach.Further, in PATRIC-based functional annotation, a total of 136 (PBs 12) and 134 (PBl 36) pathway classes were found, where the majority of genes were found to encode the biosynthesis of secondary metabolites (23) followed by xenobiotic biodegradation and metabolism (21), carbohydrate metabolism (15), lipid metabolism (14), amino acid metabolism (13), and metabolism of cofactors and vitamins (12).Two pathways were found to relate to signal transduction, one with the immune system and the other with various metabolic activities.Interestingly, the ability to code ABC-type Fe3 + -siderophore transport system, periplasmic iron-binding component which was evident from PBs 12 genome backed its siderophore production in vitro and indirectly siderophore mediated growth promotion in pearl millet that was observed in seed priming assays [77].Similarly, trans zeatin ribose (ZR) synthesis by zeatin biosynthesis pathway found in PBl 36 further backed cytokinin mediated growth promotion observed in pearl millet [78].Due to their ability to synthesize several enzymes and antimicrobial compounds, B. subtilis and B. paralicheniformis have been used for decades in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals, and several other consumer products [79][80][81][82].Also, they are significant contributors in agriculture, industry, biomaterials, and medicine [82][83][84][85].Previous discoveries of novel metabolites secreted by Bacillus sp.such as Bacitracin, Fengycin, Lichenysin and Lantipeptide, siderophores, and cyclic lipopeptides, are evident from genome research studies that help identify biosynthesis pathways and genes involved in the production of various secondary metabolites [83,[85][86][87].Identification of key cellular pathways for secondary metabolite biosynthesis helps in the selection of efficient biopesticides and biostimulants for crop protection.In the prediction of secondary metabolite coding gene clusters, a total of 14 genomic regions in PBs 12 and 15 in PBl 36 identified to be involved in coding for epipeptides, Ripp (Ribosomally synthesized and post-translationally modified Peptides), sactipeptide, Ripp recognition element (RRE), lanthipeptide, Type III polyketide synthases (PKSs), and NRPS (Non-Ribosomal Peptide Synthetase).Several antibiotic coding gene clusters for thailanstatin A, bacilysin, subtilosin, subtilin, bacillibactin, fengycin, surfactin are present in PBs 12 and compounds bacillibactin E/F, geobacillin, lichenysin, butirosin A/B, schizokinen, fengycin, bacitracin are evident in PBl 36 genome.The presence of four antimicrobial Peptide genes, spaS (375 bp), spoVG (226 bp), bacA (498 bp) and srfAA (201 bp) in PBs 12 and PBl 36 also further confirmed their potential in the secretion of antimicrobial cyclic lipopeptides such as subtilin, bacylisin and surfactin.The ability of these antimicrobial compounds in plant disease control is enormous, and they are known to initiate a distinct pattern of defense responses in plants.In present study the observed antifungal activity of both the isolates against taxonomically different plant pathogens is mainly attributed to the antimicrobial cyclic lipopeptides production ability observed and validated from their genomes.This is evident when Bacillus subtilis PBs 12, shown the presence of subtilin, bacylisin, and surfactin biosynthesis genes, also significantly inhibited all the test plant pathogens, irrespective of their taxonomic position and host plant in dual culture confrontation assays.Previously, mycosubtilin, a cyclic lipopeptide produced from Bacillus subtilis shown to induce local resistance against a necrotrophic pathogen, Botrytis cinerea [76].Iturin is another strong antifungal compound secreted by several Bacillus species and is well known for its potential to trigger immune responses in various hosts and also for its use in plant disease management, including tomato grey mould, bacterial diseases of cucurbits, tomato late blight, and pepper anthracnose [88][89][90].The enhanced levels of subtilin produced by Bacillus amyloliquefaciens in wheat spikes are directly linked to the biological control of Fusarium head blight disease caused by Fusarium graminearum [91].
Further mining of the Minimum Information about a Biosynthetic Gene Cluster (MiBiG) from known gene clusters revealed that PBs 12 and PBl 36 are involved in the biosynthesis of a total of 25458 and 33000 secondary metabolite products from each genome.The list of MiBiG products ranged from alkaloids, polyketides, polysaccharides, terpenes, anti-coumarins, siderophores, lipo/glycopeptides, antibiotics, etc. Bacillus, being an important genus, has been in scientific research and demonstrated its unique abilities for agriculture [92].B. subtilis has revolutionized the industry of bioinoculants, especially biofertilizers [93][94][95] and biopesticides [96][97][98].B. paralicheniformis is also known for its nematicidal [99], antifungal [59], antibacterial [100], and growth promotion [59] activities.With this, PBs 12 and PBl 36 isolates inevitably show antifungal potential, but they are unique in that they stand against pearl millet blast disease and other important tree diseases (Malabar neem wilt, Acacia, and Pongamia root rot).This study provided more insights into the genomes of PBs 12 and PBl 36 to further understand their genetic potential.The available genetic information strongly suggests that Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl 36 have a huge potential to initiate studies on the development of bio-fungicide formulations for both agricultural and forestry pathogens.

Conclusion
The study showed that Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 have the potential to enhance the growth of pearl millet and protect it from the Magnaporthe grisea that causes pearl millet blast disease.Also, they have antagonism against taxonomically diverse crop and tree pathogens.This research revealed the genome potential of PBs 12 and PBl 36 which can naturally produce several antimicrobial compounds like siderophores, polysaccharides, antibiotics, and lipopeptides.The findings open up opportunities for further investigation into the pearl millet microbiome, its impact on crop growth and health, and the possibility of developing a biological disease management strategy for crop protection.
Seed vigour index = Germination percentage (%) × Seedling length (mm) ccari.res.in/ wasp2.0/index.php.The mean values have been grouped based on Critical Difference (CD) to indicate the significance at probability (p) = 0.05% Dual-culture confrontation assay of Bacillus strains PBs 12 and PBl 36 with taxonomically diverse fungal plant pathogens

Fig. 1
Fig. 1 16S ribosomal RNA based phylogenetic analysis of pearl millet panicle associated Bacillus subtilis PBs 12 and Bacillus licheniformis PBl 36 Maximum-likelihood tree constructed based on Tamura-Nei model (1993) for Bacillus strains ( Bacillus subtilis PBs 12 and Bacillus licheniformis PBl 36) derived from MUSCLE alignment of 16S ribosomal RNA partial sequences [Boot strap values are based on 1000 repetitions and Bacillus thuringiensis strain IAM12077 considered as an outgroup]

Table 1
The identity of pearl millet panicle microbiome member species Bacillus subtilis and Bacillus paralicheniformis based on morphology and 16S r RNA gene sequencing

Table 2
Seed priming with pearl millet panicle associated Bacillus strains PBs 12 and PBl 36 to test their plant growth promotion activity Data sharing the same letter designation in each column, indicate no significant difference between them at a significance level of p = 0.05 CD (Critical Difference): Smallest difference between treatments means likely to indicate true change 24 h old bacterial cultures of PBs12 and PBl 36 used to prepare 1 OD (1 × 10 8 cfu/ml) cell suspension.Pearl millet cv.7042S seeds (n = 100) were soaked overnight in cell suspensions and incubated for 7 days at 27 o C temperature and 98% relative humidity.Data recoded and interpreted 7 days after incubation Seedling vigour index was calculated using the formula: Seedlingvigourindex * = [(Seedlinglength) * (Germinationpercentage)]chromosome, which consists of 4606 genes with 45.83% G + C content (Table4).A circular genome view (Fig.3) of both PBs12 and PBl 36 is presented, where contigs/ chromosomes, CDS, non-CDS, AMR (antimicrobial resistance) genes, VF (virulence factor) genes, transporters, drug targets, GC content, and GC skew are depicted from the outer to the inner ring with different colour codes.

Molecular phylogenetic analysis of Bacillus strains PBs12 and PBl36 using whole genome sequences, with a focus on average nucleotide identity The
comparative genome average nucleotide identity (ANI) was calculated using the OrthoANI algorithm for phylogenetic analysis.The average nucleotide identity analysis was performed between whole genome sequences of Bacillus subtilis (PBs 12, NCIB3610, NBRC 13719, DSM 10, KCTC 3135, 168); Bacillus paralicheniformis (PBl 36, A4-3, PRO 109, MDJK 30, Bac 84, ATCC 14580); and other Bacillus species (B.halotolerans, B. velezensis, B. amyloliquefaciens, B. pumilus, B. thuringiensis) as out groups (Table5).The strain Bacillus subtilis PBs 12 closely clustered with Bacillus subtilis strains (ATCC12633, NCIB3610, NBRC 13719, DSM 10, KCTC 3135, 168) and Bacillus paralicheniformis PBl 36 with A4-3 and PRO 109 and showed 98.70% and 99% average nucleotide identity, respectively.Among all, the Bacillus subtilis PBs12 showed close association with the strain NBRC 13719 of Japan, with the highest (98.75%) identity.Similarly, PBs 12 shared 76.18 to 87.01% identity with B. halotolerans, B. velezensis, and B. amyloliquefaciens, which were distant from Bacillus subtilis strains in the phylogenetic tree (Fig.4).On the other hand, Bacillus paralicheniformis PBs 12 was very similar to the strain A4-3 from South Korea, sharing 99.25% identity.It was also similar to B. amyloliquefaciens, B. pumilus, and B. thuringiensis, but not to other Bacillus paralicheniformis strains.The comparative ANI analysis for each genome combination has been given to show a distinct relationship between strains (Supplementary Table

Table 3
Ability of pearl millet panicle associated Bacillus licheniformis PBl 36 and Bacillus subtilis PBs 12 on mycelial growth inhibition of taxonomically diverse plant pathogens in Invitro dual culture assays Data sharing the same letter designation in each column, indicate no significant difference between them at a significance level of p = 0.05 CD (Critical Difference): Smallest difference between treatments means likely to indicate true changeMycelial growth was recorded once the mock plate attained full petri plate coverage of each inoculated fungal species here Athelia rolfsii (3 days); Magnaporthe grisea (10 days); Fusarium oxysporum (6 days); Alternaria sp.(6 days); Ganoderma spp. 1 (3 days); Ganoderma spp. 2 (4 days).Mycelial growth inhibition was calculated using the formula: [(Mycelial growth in mock plate-Mycelial growth in treated plate)/ Mycelial growth in mock plate]*100

Table 3 .
To further investigate the biocontrol potential of PBs 12 and PBl 36, the pathways classes related to secondary metabolite biosynthesis, antifungal/antimicrobial compounds secretion, plant disease resistance priming and biodegradation of heavy metals were analyzed.The focus on selected pathways such as biosynthesis of polyketides and non-ribosomal peptides, biosynthesis of secondary metabolites, signal transduction and xenobiotics biodegradation revealed the ability of the PBs 12 and PBl 36 genome for several antimicrobial compounds (Table7).Under the pathway class, biosynthesis of polyketides and non-ribosomal peptides found in both PBs 12 and PBl 36, while polyketide sugar unit biosynthesis was observed only in PBs 12.In the second class, biosynthesis of secondary metabolite pathways involved in caffeine metabolism, several antibiotics, isoquinoline alkaloid biosynthesis, terpenoid backbone biosynthesis, phenylpropanoid biosynthesis etc.The phosphatidylinositol signaling system and mTOR signaling pathway were found under the signal transduction class.Similarly, both PBs 12 (atrazine, naphthalene, tetrachloroethane, anthracene, benzoate, caprolactam, hexachlorocyclohexane, geraniol, styrene) and PBl 36 (1,4-Dichlorobenzene, benzoate, biphenyl, hexachlorocyclohexane, geraniol, styrene, toluene) showed xenobiotics biodegradation related metabolism indicating their bioremediation potential (Table7).Finally, the PBs 12 and PBl 36 showed 289 subsystems distributed in 11 subsystem superclasses (Supplementary Table4) with a total of 1,799 and 1,850 genes in each genome.Among which 35 subsystems are directly involved in stress response, defense and virulence with 136 and 124 genes.

Table 4
Genome statistics of pearl millet associated Bacillus subtilis and Bacillus paralicheniformis Fig. 3 Circular genome view of pearl millet associated Bacillus sp. a Bacillis subtilis PBs 12; b) Bacillus licheniformis PBl 36 metabolite coding gene cluster analysis.In total 14 genomic regions in PBs 12 and 15 in PBl 36 identified to be involved in coding for epipeptides, Ripp (Ribosomally synthesized and Post-translationally modified Peptides), sactipeptide, Ripp recognition element (RRE), lanthipeptide, Type III polyketide synthases (PKSs),

Table 6
Summary of pathway classes in pearl millet adapted Bacillus subtilis and Bacillus paralicheniformis genomes

Table 7
Pathways involved in secondary metabolite biosynthesis and bioremediation in Bacillus strains PBS12 and PBl36

Table 8
Summary of secondary metabolite gene clusters mined by AntiSmash2.0 in Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36