Whole-genome sequencing and biosynthetic gene cluster analysis of Bacillus subtilis BAGL as a potent antifungal biocontrol agent against phytopathogenic fungi.

Whole-genome sequencing and biosynthetic gene cluster analysis of Bacillus subtilis BAGL as a potent antifungal biocontrol agent against phytopathogenic fungi - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice Int Microbiol . 2026 Mar 13;29(4):549–564. doi: 10.1007/s10123-026-00802-7 Search in PMC Search in PubMed View in NLM Catalog Add to search Whole-genome sequencing and biosynthetic gene cluster analysis of Bacillus subtilis BAGL as a potent antifungal biocontrol agent against phytopathogenic fungi Beenish Amjad Beenish Amjad 1 Department of Microbiology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Iqbal Hall, Takatu Campus, Airport road, Quetta, Pakistan Find articles by Beenish Amjad 1 , Sana Sultan Sana Sultan 1 Department of Microbiology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Iqbal Hall, Takatu Campus, Airport road, Quetta, Pakistan Find articles by Sana Sultan 1 , Asma Abro Asma Abro 2 Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta, Pakistan Find articles by Asma Abro 2 , Bushra Tabassum Bushra Tabassum 3 School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan Find articles by Bushra Tabassum 3 , Muhammad Ilyas Muhammad Ilyas 4 Center of Omic Sciences, Islamia College, Peshawar, Pakistan Find articles by Muhammad Ilyas 4 , Nusrat Jahan Nusrat Jahan 2 Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta, Pakistan Find articles by Nusrat Jahan 2 , Samia Parveen Samia Parveen 1 Department of Microbiology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Iqbal Hall, Takatu Campus, Airport road, Quetta, Pakistan Find articles by Samia Parveen 1 , Anwar Khan Anwar Khan 1 Department of Microbiology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Iqbal Hall, Takatu Campus, Airport road, Quetta, Pakistan Find articles by Anwar Khan 1, ✉ Author information Article notes Copyright and License information 1 Department of Microbiology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Iqbal Hall, Takatu Campus, Airport road, Quetta, Pakistan 2 Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta, Pakistan 3 School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan 4 Center of Omic Sciences, Islamia College, Peshawar, Pakistan ✉ Corresponding author. Received 2025 Dec 31; Revised 2026 Feb 27; Accepted 2026 Mar 4; Issue date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13083322  PMID: 41820735 Abstract Bacillus subtilis BAGL, isolated from the rhizosphere, demonstrated significant antifungal activity against key phytopathogens, inhibiting R. solani by 73.7% , F. oxysporum by 55.15% , and (A) solani by 77% . Whole-genome sequencing revealed a genome size of 4,227,102 bp, GC content of 43.08%, and 4,494 predicted genes. The accession number (PRJNA1349010) for the whole genome was obtained from the NCBI GenBank. Phylogenetic analysis confirmed BAGL’s taxonomic position of BAGL within the B. subtilis clade. The genome harbored 17 biosynthetic gene clusters (BGCs) responsible for producing secondary metabolites with antimicrobial properties, such as surfactin, fengycin, bacillibactin, and subtilosin (A) Additionally, BAGL carries a diverse set of carbohydrate-active enzymes (CAZymes) that may contribute to its biocontrol efficacy by disrupting fungal cell wall. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed BAGL’s involvement in multiple metabolic pathways, including secondary metabolite biosynthesis and carbon metabolism. Gene Ontology (GO) analysis highlighted numerous catalytic and binding activities crucial for metabolic and cellular processes. The presence of these genetic features suggests that B. subtilis BAGL is an effective candidate for developing eco-friendly disease management strategies in agricultural settings. Future investigations into the active compounds produced by BAGL and in vivo trials are essential to determine their efficacy in real-world settings. Supplementary Information The online version contains supplementary material available at 10.1007/s10123-026-00802-7. Keywords: Whole-genome sequencing, Antifungal activity, Biosynthetic gene clusters, Genomic Annotation Introduction Global food security is critically dependent on sustainable agricultural productivity, which is continuously challenged by numerous factors, including the pervasive threat of phytopathogens. These pathogens are responsible for significant yield losses worldwide, impacting economies and livelihoods, with an estimated economic loss of more than US$220 billion annually and a reduction in global crop production of 10–15% (Singh et al. 2023 ; Mohammad-Razdari et al. 2022 ; Peng et al. 2021 ). In countries such as Pakistan, which rely on agriculture, such issues are especially severe, as the health of crops directly determines the financial stability of the country and the survival of its people in rural areas. Major staple crops, such as wheat, sugarcane, and maize, have experienced a drop in recent years, partly due to an attack by pathogens, including the widespread effects of fungal pathogens such as Rhizoctonia solani (causative agent of damping-off and root rot in a variety of crops), Fusarium oxysporum (causative agent of devastating wilt), and Alternaria solani (causative agent of early blight and leaf spots in vegetables). These have contributed to a significant loss of finances in Pakistan’s major agricultural sectors (Kumar et al. 2022 ; Riaz et al. 2020 ; Raza et al. 2021 ). Chemical fungicides are the dominant approach for controlling these plant pathogens. However, the prolonged use of these chemical fungicides has led to the development of resistance in various pathogens (Montesinos 2023 ) and has caused negative environmental impacts, increasing public concern and food safety issues. Consequently, the use of many chemical fungicides has been restricted or banned in several countries. Simultaneously, consumer preferences have shifted towards environmentally friendly, healthy, and sustainable agricultural products (Marian and Shimizu 2019 ). Consequently, biological control techniques, particularly those involving beneficial microbes, are gaining attention as eco-friendly and effective substitutes for traditional pesticides. Biological control relies on the use of microorganisms and their metabolic products to inhibit pathogen activity (Weng et al. 2022 ). Among microbial agents for biocontrol, Bacillus species are particularly promising because of their biosafety, broad-spectrum antimicrobial properties, and capacity to survive under diverse environmental conditions (Xu et al. 2020 ; Guillén-Navarro et al. 2023 ). These bacteria suppress pathogens through multiple mechanisms, including the production of lipopeptide antibiotics (e.g., iturin, fengycin, and surfactin) and hydrolytic enzymes (e.g., chitinase and cellulase), which foster antagonism, competition, and enhance plant resistance (Li et al. 2019 , 2022 ; Liu et al. 2019 ; Aziz et al. 2024 ). These capabilities make B. amyloliquefaciens and B. subtilis highly effective against a broad spectrum of plant pathogens, including Aspergillus spp., Fusarium spp., Curvularia spp., and Colletotrichum falcatum (Shahid et al. 2021 ). The advent of whole-genome sequencing and advanced bioinformatics has revolutionized the identification of biosynthetic gene clusters associated with antifungal activity and plant growth-promoting traits in beneficial Bacillus strains (Kopyltsov et al. 2022 ; Kravchenko et al. 2020 ). Although such genomic predictions are potent in determining possible candidates, experimental validation is essential to determine the biological relevance and functional implications of such clusters in pathogen suppression and plant defense induction (Durgadevi et al. 2021 ; Tian et al. 2021 ). Here, the isolation and intensive genomic profiling of new Bacillus subtilis strains, with an emphasis on their biosynthetic gene clusters (BGCs), provide a potent approach for the identification of novel antifungal agents. Although genomic studies have identified many BGCs in different Bacillus strains in different areas of the world, limited research has been conducted to combine such extensive genomic characterization and well-established functional validation with specific, high-frequency phytopathogens of crops in ecologically diverse regions such as Pakistan. Most research has focused on individual pathogens or has not provided sufficient genomic data to fully understand the molecular mechanisms underlying biocontrol activity. Despite significant advancements in the genomic characterization of Bacillus species and genomic predictions, a critical gap remains in our understanding of the biosynthetic potential of novel strains, particularly those from diverse ecological niches, such as rhizospheric soils in agricultural regions of Pakistan. Research often provides either genomic data without functional validation against pathogens or functional validation without detailed genomic insights, which hinders the development of effective region-specific biocontrol strategies. This study aimed to isolate and characterize a novel antifungal strain of Bacillus subtilis , BAGL, from rhizospheric soil in Pakistan. Using whole-genome sequencing and functional analyses, we identified biosynthetic gene clusters and mechanisms underlying the broad-spectrum activity of B. subtilis against key local phytopathogens, including Rhizoctonia solani , Fusarium oxysporum , and Alternaria solani . This integrated genomic and functional analysis fills a critical gap in understanding biocontrol in specific ecological niches while providing a framework for developing targeted, sustainable disease management solutions against major phytopathogens in Pakistan. Materials and methods Soil sampling and collection Soil samples were obtained from the rhizospheric zones of various healthy plants (wheat, potato, tomato, and maize) in the Quetta district of Pakistan. The samples were aseptically transported to the Applied Biotechnology Laboratory in sterile plastic bags and processed immediately. Bacteria were isolated as described by Zou et al. ( 2024 ) using the serial dilution method, and the serially diluted samples were spread onto Nutrient Agar (NA) plates. The plates were incubated at 32 °C for 72 h. The microbial colonies that emerged were further purified through multiple rounds of streaking to obtain isolated strains for further study. Phytopathogenic fungi Phytopathogenic fungi ( Alternaria solani , Rhizoctonia solani , and Fusarium oxysporum ) available in the Applied Biotechnology Lab from previous studies were grown according to routine protocols on potato dextrose agar (PDA) and kept at 25 °C. Evaluation of antifungal activity Bacteria isolated from the rhizospheres of different crops were screened for antifungal activity. Qualitative assay A qualitative assay was performed on all 54 bacterial isolates using the agar plug method described previously (Gupta et al. 2022 ) on potato dextrose agar (PDA) plates. Briefly, an approximately 5 mm plug of fungal pathogens was placed in the center of a PDA plate and incubated at 28 °C. After 3 days, bacterial isolates were spotted on the periphery of the Petri plates using a sterile wire loop and incubated for 7 d. The experiment was performed in triplicate. Quantitative assay To quantitatively evaluate the antifungal activity, a dual-culture assay was performed using the three bacterial isolates exhibiting antifungal activity. Only three bacterial isolates were selected for quantitative evaluation because they inhibited all the fungi tested. A 5 mm plug of fungal mycelium from phytopathogens such as A. solani , R. solani , and F. oxysporum was placed at the center of Potato Dextrose Agar (PDA) plates, as described by Azeem et al. ( 2022 ). The bacterial isolates were inoculated 3 cm away from the fungal plug as a loopful of bacterial culture or streak. The plates were incubated at 28 °C for 7 days. Fungal plug on PDA without bacteria was also kept as a negative control. Subsequently, the inhibition zones around the fungal plugs were measured, and the percentage of fungal growth inhibition was calculated using the following formula (Pasha et al. 2025 ), with the experiment performed in triplicate: Inhibition percentage =[100×(C-T)/C] Where. C = the radial growth of the fungus in the control plate (without bacteria), and. T = Radial growth on the treatment plate (with bacteria). Morphological characterization of Bacillus subtilis Bacterial isolates exhibiting ≥ 50% antifungal activity against all tested phytopathogenic fungi (BAGL) were examined both microscopically and macroscopically. Gram staining was used to determine cell shape, arrangement, and Gram reaction, classifying isolates as Gram-positive or Gram-negative. Colony characteristics, such as shape, edge, size, and pigmentation, were recorded on Nutrient Agar (NA) following established protocols (Lalaymia et al. 2022 ). Whole-Genome Sequencing Whole-genome sequencing was performed on only one bacterial isolate (BAGL) as it satisfied the inclusion criteria, that is, ≥ 50% of antifungal activity against all the tested phytopathogenic fungi. The BAGL isolate was incubated in Luria-Bertani (LB) broth at 28 °C with shaking at 180 rpm for 24 h. Genomic DNA from freshly cultured BAGL cells was isolated using the Thermo Scientific™ Genomic DNA Purification Kit (K0721; Thermo Fisher Scientific) according to the manufacturer’s protocol. Isolated genomic DNA was visualized on a 1% agarose gel (Hong et al. 2024 ). The extracted genomic DNA was stored for subsequent analysis. Whole-genome sequencing cell preparation and DNA processing were conducted following Microbes NG (Birmingham, UK) strain-submission procedures. Subsequent library preparation, DNA sequencing, and bioinformatics analysis were conducted by Microbes NG following in-house protocols. Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, USA) following the manufacturer’s instructions, with the following modifications: the input DNA was increased 2-fold, and the PCR elongation time was increased to 45 s. Libraries were sequenced using an Illumina NovaSeq 6000 (Illumina, San Diego, USA) with a 250 bp paired-end protocol. Reads were adapter trimmed using Trimmomatic version 0.30 (Bolger et al. 2014 ) with a sliding window quality cutoff of Q15, and FASTQC (Lokhande 2022 ) was used to assess the read quality. De novo assembly was performed on the samples using SPAdes version 3.7 (Bankevich et al. 2012 ), and the contigs were annotated using Prokka v1.14.6. The accuracy and completeness of the genome assembly were verified using QUAST (Sullivan et al. 2025 ). Phylogenetic profiling of Bacillus subtilis BAGL Phylogenetic analysis of the WGS of Bacillus subtilis strain (BAGL) was performed using AutoMLST 2.0, an automated multilocus sequence typing and genome-based taxonomic tool designed to infer evolutionary relationships from whole-genome data. The pipeline generated a phylogenetic tree in the Newick format, which was subsequently downloaded and imported into MEGA version 12 for visualization. In MEGA, the tree was rendered in a circular phylogram layout to provide a clearer representation of the clustering patterns and evolutionary distances among the Bacillus reference genomes. The use of established bioinformatics platforms ensured the accuracy, reproducibility, and clarity of phylogenetic output (Alanjary et al. 2019 ; Tamura et al. 2021 ). Annotation of Functional Elements in BAGL strain The predicted genes from the genome of strain BAGL were functionally annotated by comparison with several protein databases. The annotations were made by querying the NCBI (Nr) non-redundant protein database, SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Additionally, (COG), the Homologous Protein Cluster Database and Carbohydrate-Active Enzymes (CAZy) databases were used to further enhance functional predictions. For secondary metabolite analysis, gene clusters were predicted using antiSMASH ( Li et al. 2025 ), version 8-dev, with implemented subprograms including ClusterBlast, SubClusterBlast, KnownClusterBlast, and ActiveSiteFinder. Results Screening of antagonistic activity of bacterial isolates In this study, 54 bacteria were isolated from the rhizospheric region using the serial dilution technique on nutrient agar medium. Only seven bacteria showed antifungal activity against at least one tested phytopathogen; however, of the seven antifungal bacterial isolates, only three inhibited all the tested fungi. One bacterial isolate, BAGL, exhibited ≥ 50% antifungal activity against all tested fungi. Therefore, only the BAGL isolate was used for whole-genome sequencing. The effectiveness of the antifungal activity of BAGL was assessed against Alternaria solani , Fusarium oxysporum , and Rhizoctonia solani at 28 °C. The inhibition of fungal growth was measured on the 7th day post-inoculation (dpi). The control treatment included the addition of sterile water to the mixture. Figure 1 illustrates the broad-spectrum antifungal activity of BAGL, showing clear zones of inhibition against A. solani (Fig. 1 A), F. oxysporum (Fig. 1 B), and R. solani (Fig. 1 C). Fig. 1. Open in a new tab Broad-spectrum antifungal activity of Bacillus subtilis BAGL against major phytopathogens. The figure shows clear zones of inhibition, indicating the effective suppression of fungal growth by BAGL. (A) Inhibition of Alternaria solani . (B) Suppression of Fusarium oxysporum (C) Antifungal activity against Rhizoctonia solani Morphological Characterization of Bacillus subtilis BAGL The BAGL bacterial isolate was characterized using light microscopy and colony morphology, confirming it as Gram-positive B. subtilis strain. Microscopic observation after Gram staining showed that the cells had a purplish-blue rod-shaped structure, which is typical of the thick peptidoglycan layer of gram-positive bacteria. This staining pattern ensures that the cell wall is structurally intact, as is the case for Bacillus species. Macroscopically, when cultured on Nutrient Agar (NA), the colonies displayed distinctive morphological features consistent with B. subtilis . The colonies were milky white with irregular and wavy edges, which is a hallmark of this species. These colony characteristics, including shape, pigmentation, and edge morphology, further confirmed the identity of the isolate (Supplementary Figure S1 ). Whole genome sequencing of Bacillus strain BAGL The whole genome of Bacillus subtilis BAGL was sequenced using the Illumina NovaSeq 6000 (Illumina, San Diego, USA) with a 250 bp paired-end protocol, providing short-read data. After quality trimming with Trimmomatic and validation with FASTQC, the resulting sequence data were assembled with SPAdes version 3.7 and annotated via Prokka v1.14.6. The genome was composed of a circular chromosome with a total size of 4,227,102 bp and a GC content of 43.08%. A total of 4,494 genes were identified, including 4344 protein-coding genes (CDS) with a cumulative length of 3,714,453 bp, average CDS length of 855 bp, with the longest CDS being 7,683 bp and the shortest being 90 bp (Table 1 ). The non-coding RNA genes comprised 138 tRNA genes (total length of 5,981 bp, with an average length of 77.7 bp), and 12 rRNA genes were predicted, including three copies of 5 S rRNA, one partial copy of 16 S rRNA, and one partial copy of 23 S rRNA, one tmRNA (ssrA) gene while no pseudogenes were predicted using Prokka v1.14.6 (Fig. 2 ). Table 1. The complete genome overview of B. subtilis BAGL with others Items B. subtilis BAGL B. subtilis RS10 (Iqbal et al. 2021 ) B. subtilis KC14-1 (Li et al. 2025 ) B. subtilis MC4-2 (Shi et al. 2024 ) B. subtilis ATCC 13,952 (Li et al. 2015 ) B. subtilis XF-1 (Guo et al. 2015 ) B. amyloliquefaciens BS-3 (Dai et al. 2021 ) B. subtilis 168 (Kunst et al. 1997 ) Genomic size (bp) 4,227,102 4,457,201 3,908,079 4,076,630 3,876,276 4,061,186 3,870,130 4,215,606 G + C content % 43.7% 43.4 43.82 43.8% 45.8 43.8 46.9 43.5 Protein coding sequences 4344 4232 3895 4,207 3852 3,853 4161 4255 Average CDS size bp 855 8612 852 877 ---- 832 872 Number of tRNAs 138 86 86 85 72 77 92 86 Open in a new tab Fig. 2. Open in a new tab A genome map of whole genome sequence analysis of Bacillus subtilis BAGL Phylogenetic analysis of Bacillus subtilis BAGL Whole-genome phylogeny revealed that the BAGL isolate clustered firmly within the Bacillus subtilis lineage. In the circular phylogenetic tree, our sample, BAGL, was closely grouped with well-characterized strains, such as TS–GCF_000009045 ( Bacillus subtilis ), forming a highly supported node with strong bootstrap values. This cluster was clearly separated from other major Bacillus groups, including the B. pumilus , B. cereus , B. sonorensis , and B. amyloliquefaciens complexes, which all appeared as distinct branches. The consistently high support values across the topology confirmed the reliability of the inferred relationships and demonstrated that BAGL shared a recent common ancestor with members of the B. subtilis group. These results provide robust evidence for the taxonomic placement of strain BAGL in the Bacillus subtilis clade (Fig. 3 ). Fig. 3. Open in a new tab Whole-genome phylogenetic evolutionary tree of Bacillus subtilis BAGL. Accession number PRJNA1349010 indicates the current study Non redundant annotation Gene annotation was performed using PSI-BLAST against the NCBI NR database, resulting in the successful annotation of 24,855 genes. The predominant genus identified was Bacillus , with the majority of annotations corresponding to Bacillus , accounting for 3,395 genes, Bacillus subtilis for 2,620 genes, and Bacillus velezensis for 1,859 genes. Other notable species identified included Bacillus atrophaeus (1,160 genes), Bacillus halotolerance (774 genes), and Bacillus spizizenii (655). The top 20 species collectively accounted for the majority of the annotated genes, as shown in (Fig. 4 ). These findings suggest a significant representation of Bacillus species, which may reflect the microbial community structure or sample source. Fig. 4. Open in a new tab NR annotated genes in species of Bacillus subtilis (BAGL) Swiss-prot database annotation To enhance functional annotation, sequences were compared with the Swiss-Prot database, as shown in Fig. 5 . Among the 4,012 sequences analyzed, 3,918 unique hits were identified in Swiss-Prot. The bit score values ranged from 36.2 to 4998.0, indicating variable protein similarities. The five proteins with the highest bit scores were PPSB_BACC (Plipastatin synthase B,4998.0), DHBF_BACSU (Dimodular nonribosomal peptide synthase, 4626.0) WAPA_BACNB (tRNA(Glu)-specific nuclease WapA,3841.0) TMP_BPSPB (Probable tape measure protein,3531.0) and GLTA_BACSU (Glutamate synthase [NADPH] chain, large chain, 2980.0). Fig. 5. Open in a new tab Swiss Prot annotation of Bacillus subtilis BAGL strain KEGG pathway annotation To analyze the metabolic pathways, the KEGG database was used to identify the pathways in which Bacillus subtilis BAGL is involved. In 133 pathways, with a total of 4,317 genes. Most of these genes (2,321) were linked to metabolic processes, such as metabolic pathways, carbon metabolism, and biosynthesis of secondary metabolites (Fig. 6 ). The environmental information processing pathway contained 398 genes, genetic information processing contained 122 genes, cellular processes contained 56 genes, organismal systems contained 60 genes, and 1360 genes fell into the other category. The top pathways in each category are visually represented in a horizontal bar chart, with color-coded strips on the left denoting the functional categories. These findings highlight that the BAGL strain has a rich metabolic and regulatory gene pool, which may enable the production of various secondary metabolites. Fig. 6. Open in a new tab KEGG annotation genes of Bacillus subtilis BAGL strain Gene ontology annotation Gene Ontology (GO) analysis of Bacillus subtilis strain BAGL categorized annotated genes into the three major GO classes such as Biological Process, Cellular Component, and Molecular Function. The category of Biological Process showed high representation of biological process containing (850 genes), metabolic process containing (550 genes), and cellular process containing (550 genes), indicating vigorous metabolic and cellular activities. In the cellular component category, the top terms were cellular component (750 genes), intracellular anatomical structure (400 genes), and cytoplasm (370 genes). Molecular Function analysis revealed strong enrichment of molecular function (840 genes), catalytic activity (560 genes), and nucleotide binding (140 genes), emphasizing the strain’s active enzymatic and binding mechanisms. These results collectively describe the functional characteristics of the strain BAGL1 (Fig. 7 ). Fig. 7. Open in a new tab GO annotation and functional classification of Bacillus subtilis BAGL COG Annotation The Bacillus subtilis BAGL genome COG annotation indicated that the genes were broadly diversified, with a large share (1, 070 genes) belonging to the group of unidentified functions (S). This highlights the level of genetic factors whose contributions are yet to be understood, implying possible areas for further experimental characterization and functional genomics studies. The predominance of this category emphasizes the complexity of the genome and suggests that many genes may have novel or context-specific functions that are not yet reflected in the databases. In addition to the unknown functions, the genome exhibits strong representation of key metabolic and regulatory processes. Notably, genes involved in amino acid transport and metabolism (E, 353 genes) and carbohydrate transport and metabolism (G, 303 genes) suggest an active and versatile metabolic network essential for nutrient uptake and energy production. The presence of a substantial number of transcription-related genes (K, 343 genes) reflects the intricate regulatory mechanisms controlling gene expression. Additionally, the inorganic ion transport and metabolism category ( P = 252 genes) highlighted the importance of ion homeostasis and metal ion utilization in cellular physiology. The genome also contains rare categories represented by single genes, such as extracellular structures (W), chromatin structure and dynamics (B), and RNA processing and modification (A), which may correspond to specialized or unique functions in B. subtilis BAGL cells. The distribution pattern of these COG categories is visually summarized in Fig. 8 , which provides an overview of the functional landscape of bacterial genomes. Fig. 8. Open in a new tab COG functional classification diagram for Bacillus subtilis BAGL Strain Carbohydrate active enzyme CAzy Genome annotation identified the presence of six major groups of carbohydrate-active enzymes (CAzymes), as shown in Fig. 9 , including glycoside hydrolases (GH), glycosyltransferases (GT), carbohydrate-binding modules (CBM), polysaccharide lyases (PL), carbohydrate esterases (CE), and auxiliary redox enzymes (AA).The gene counts for each group were 887 for GHs, 290 for GTs, 40 for CBMs, 85 for PL, 120 for CE, and 69 for AA. Most gene sets comprised the GH and GT families, respectively. Further analysis revealed the presence of glucanases from the GH16 family and CBM3 modules, which are involved in β-glucan degradation. Enzymes for xylan degradation, including GH30-8 and GH11, were also detected, suggesting the potential for hemicellulose metabolism in the guts of termites. Additionally, enzymes associated with peptidoglycan, such as CBM50 and GT28, may play a role in modifying the cell wall. The presence of GH23 family lysozyme genes further suggests enzymatic capabilities for the degradation of dextran and Xylan. Fig. 9. Open in a new tab CAZy classification diagram for Bacillus subtilis BAGL strain Biosynthetic gene cluster of BAGL via antiSMASH The antiSMASH web server was used to predict natural product biosynthetic gene clusters within the genome of Bacillus subtilis BAGL. A total of 17 biosynthetic gene clusters were identified (Table 2 ),primarily involved in the production of secondary metabolites via nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) pathways. PKS clusters were further categorized into type 1 (T1PKS), type2 (T2PKS), and type 3 ( T3PKS). Among the identified cluster several were linked to the synthesis of terpenes (Regions 1.1,2.2,14.1), T3PKS (Region 2.1, NRPS (Regions 3.1, 4.1, 5.1, 18.1) NRPS-betalactone (Region 6.1), sactipeptide (Region 15.1), lanthipeptides (Region 16.2,and sidrophore production such as NRP metallophore clusters (Region 16.3) were found. Notably, multiple clusters showed high sequence similarity (> 90%) to known biosynthetic pathways, such as subtilosin A, pulcherriminic acid, fengycin, bacillibactin, and surfactin, highlighting their potential for producing metabolites. Although antiSMASH predictions may not cover all possible natural products, these results indicate that Bacillus subtilis BAGL possesses a rich and diverse set of biosynthetic pathways distributed across its genome. Furthermore, the genes associated with plant growth promotion is mentioned in Table 3 . Table 2. Biosynthetic gene cluster encoding secondary metabolites in strain BAGL Region Type From To Similarity Most similar known cluster 1.1 terpene 199,665 220,468 N/A 2.1 T3PKS 18,697 59,794 LOW 1- carbapen-2-em-3-carboxylic acid 2.2 Terpene precursor 236,240 257,130 3.1 NRPS 1 14,038 4.1 NRPS 1 10,377 5.1 NRPS 1 9,649 6.1 NRPS 1 27,573 Medium fengycin 14.1 terpene 15,505 37,403 15.1 sactipeptide 113,499 135,110 High subtilosin A 15.2 other 137,857 179,275 High bacilysin 15.3 RiPP-like 384,909 397,640 N/A 16.1 CDPS 118,870 139,616 high pulcherriminic acid 16.2 lanthipeptide-class-i 260,993 287,218 High subtilin 16.3 NRP-metallophore, NRPS, terpene-precursor 486,640 552,799 High bacillibactin 18.1 NRPS 196,019 222,591 Low surfactin 19.1 transAT-PKS, NRPS 1 64,662 21.1 anthipeptide-class-i 50,708 77,003 Open in a new tab Table 3. Genes associated with plant growth promotion Trait Gene involved Function / Role Nitrogen Assimilation Asparagine synthetase [glutamine-hydrolyzing]3 Nitrogen metabolism; supports plant nitrogen nutrition. Iron Acquisition Sirohydrochlorin ferrochelatase Iron acquisition, essential for chlorophyll production and plant growth. Phosphate solubilization Phosphoserine phosphatase RsbP Phosphate solubilization, enhancing phosphorus availability to plants. Phytohormone Synthesis (IAA) trpA, trpB, trpC, trpS IAA production; regulates root development and plant growth. Amino Acid Synthesis IlvB, ilvH, alsD Amino acid biosynthesis, essential for protein synthesis and stress response Potassium Transport KtrA, KtrB Potassium uptake, essential for nutrient balance and plant growth. Sodium, Lithium, Rubidium Transport Antiporter (Na+/K+/Li+/Rb+/H+ antiporter) Transport of sodium, potassium, lithium, and rubidium; contributes to osmotic regulation and stress tolerance. Sulfur Metabolism Cysteine desulfurase SufS Sulfur metabolism; involved in cysteine synthesis and stress tolerance. Biocontrol/Antifungal Activity Subtilisin amylosacchariticus Antifungal protease activity, protecting plants from pathogens. Colonization & Biofilm Formation FlgB, FlgC, tasA, tapA, sinR Biofilm formation, root colonization, and plant-microbe interactions. Open in a new tab Discussion This study focused on the isolation, antifungal potential, and whole-genome sequencing of Bacillus subtilis BAGL. Using whole-genome sequencing and functional analyses, we identified the biosynthetic gene clusters and molecular mechanisms responsible for the broad-spectrum activity against key phytopathogens, including Rhizoctonia solani , Fusarium oxysporum , and Alternaria solani . The observed broad-spectrum antifungal activity, with inhibition rates of up to 77% against (A) solani , 73.7% against R. solani , and 55.15% against F. oxysporum , is consistent with the known biocontrol capabilities of Bacillus species in suppressing multiple plant pathogens through the production of antimicrobial metabolites (Lim et al. 2018 ; König et al. 2024 ). The antifungal activity of BAGL suggests that it plays a crucial role in promoting plant growth by combating pathogen threats, similar to the biocontrol potential of (B) subtilis F62 and B. amyloliquefaciens Trb7, both of which are effective in controlling fungal diseases in crops (König et al. 2024 ; Balamurugan et al. 2024 ). The genomic investigation of B. subtilis BAGL entailed whole-genome sequencing to identify the biosynthetic gene clusters that produce antimicrobial compounds. This extensive genetic characterization indicated the existence of various clusters of genes that encode secondary metabolites, including lipopeptides, polyketides, and other bioactive molecules with antifungal properties. The Genomic mapping in this study revealed that BAGL possesses genes involved in antifungal activity, which likely enable it to antagonize phytopathogens. Genomic analysis of Bacillus subtilis BAGL not only confirmed its biocontrol potential but also provided detailed insights into its genetic architecture, supporting its functional versatility. Whole-genome sequencing data revealed a genome size of approximately 4.23 Mb with a GC content of 43.08%, consistent with that of typical B. subtilis strains. The identification of 4,494 predicted genes indicates a complex genome capable of encoding a wide array of enzymes, secondary metabolites, and regulatory proteins that contribute to the adaptability and efficacy of biocontrol agents. These genetic features suggest that BAGL possesses the molecular tools necessary to interact with plant pathogens, promote plant growth, and survive under diverse environmental conditions. Furthermore, the genomic features of BAGL are consistent with the overall genetic variation observed in the Bacillus genus (which is metabolically flexible and ecologically adaptable). This genetic variation among strains facilitates a broad spectrum of functional capabilities, including antimicrobial compound production, biofilm formation, and plant hormone and stress tolerance. These characteristics are essential for good biocontrol agents, as they help suppress pathogens and promote healthy crop growth sustainably. The comparative genomic context indicates how BAGL fits into this context and, therefore, its potential as a strong candidate for agricultural biotechnology and pest management of integrated strategies (Li et al. 2023 ). The BAGL strain contained genes such as trpA, trpB, trpC, and trpS, which regulate IAA production, a plant growth hormone that promotes plant growth. Essential genes, such as ilvB, ilvH, and alsD, are involved in amino acid synthesis. The decomposition of trace elements in soil also contributes to the growth of plants. Genes, including phoA, phoD, phoP, phoR, and ktrA, related to phosphorus and potassium proteins, were found in the BAGL strain. These findings suggest that B. subtilis BAGL enhances plant growth, similar to other plant growth-promoting rhizobacteria. Bacteria must adhere to plant roots or colonize plant surfaces to benefit plants, and their successful establishment is crucial for plant growth. These bacteria use flagella (FlgB and FlgC) to move toward plant surfaces and produce enzymes, proteins, and other compounds that interact with plants. The formation of biofilms is related to bacterial colonization, with various proteins, such as tasA, tapA, and sinR, being important. Phylogenetic analysis revealed that B. subtilis BAGL was closely related to other well-documented B. subtilis strains, supporting its taxonomic classification as a member of the B. subtilis clade. This observation corroborates previous findings that utilized whole-genome phylogenies to classify Bacillus strains, confirming the genetic characterization of BAGL strains. Thus, the close phylogenetic clustering of BAGL with well-documented B. subtilis strains robustly validates its taxonomic classification and genetic identity in accordance with established Bacillus taxonomies (Sraphet and Javadi 2025 ). The genome of Bacillus subtilis BAGL encompasses multiple biosynthetic gene clusters (BGCs) that are responsible for producing secondary metabolites with antimicrobial properties. In the case of the strain BAGL, 17 BCGs were identified based on AntiSMASH, and 10 BCGs were found in the genome of the biocontrol agent Bacillus subtilis MC4-2 (Shi et al. 2024 ). AntiSMASH identified 17 BGCs, including those responsible for the synthesis of surfactin, fengycin, bacillibactin, and subtilosin A, which are well-established antibacterial and antifungal agents (Abdel-Moghies et al. 2024 ). Surfactin is a cyclic lipopeptide synthesized by a nonribosomal peptide synthetase (NRPS) gene cluster. It is an antimicrobial biosurfactant with antibacterial and antifungal properties due to its ability to disrupt membranes and is a wide-spectrum antimicrobial agent. Bacillus subtilis strains have been demonstrated to express surfactin biosynthetic genes, which are conserved and play a role in antimicrobial defense (Li et al. 2015 , 2022 ). Fengycin is a lipopeptide with potent antifungal activity against filamentous fungi. The fengycin cluster of genes accommodates nonribosomal peptide synthetase (NRPS) enzymes that prepare fengycin homologs that interfere with fungal membranes. Differences between fengycin clusters in Bacillus subtilis strains determine their antifungal activity, and distinct genomic formats have been reported (Su et al. 2020 ). Bacillibactin is a catecholic siderophore that is produced by an NRPS gene cluster, which is essential in iron absorption and antimicrobial activities. Its small genotype cluster is highly preserved in Bacillus species (Chakraborty et al. 2022 ). Subtilosin A is a ribosomally expressed, post-translationally processed peptide that exhibits antibacterial activity with membrane-disruptive and quorum-sensing destabilizing effects. All these BGCs allow Bacillus subtilis BAGL to induce a repertoire of secondary metabolites with antibacterial and antifungal potential; thus, this strain is an attractive source of naturally occurring antimicrobial agents. These metabolites have been reported to have good antimicrobial activity against other Bacillus subtilis strains and species throughout their biocontrol benefits and possible medicinal uses (Kaspar et al. 2019 ). The detection of multiple nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) clusters in the BAGL genome highlights its potential for synthesizing a broad range of bioactive compounds, further validating its suitability for agricultural biocontrol applications. Genomic data also revealed that BAGL possesses a diverse array of carbohydrate-active enzymes (CAZymes), including xylanases, chitosanase peptidoglycanases and glucanases. This suggests that BAGL may compromise the structural integrity of fungal cell walls, thereby enhancing its ability to combat pathogens, as mentioned by Zhang et al. ( 2022 ). The presence of these CAZymes in B. subtilis BAGL indicates its potential role in the production of antimicrobial compounds and physical breakdown of fungal pathogens. Genome sequencing of B. subtilis strains has consistently revealed diverse CAZymes that enable plant polysaccharide degradation, highlighting their importance in agriculture and industry (Sraphet and Javadi 2025 ). KEGG analysis demonstrated that BAGL plays a role in multiple metabolic pathways, including secondary metabolite production and carbon metabolism, which improves the adaptability of a strain and its functional plasticity, contributing to its use in biocontrol and the production of new antimicrobial agents. These results were substantiated through Gene Ontology (GO) analysis, which indicated that many catalytic and binding activities are required in the process of metabolism and cellular processes. In addition to conducting in vitro antifungal tests and whole-genome sequencing, we identified biosynthetic gene clusters (BGCs) associated with antifungal activity. Gao et al. ( 2018 ) successfully isolated the strain B. subtilis VD18R19 from vanilla roots and identified six gene clusters responsible for the synthesis of antimicrobial secondary metabolites, including surfactin, plipastatin, bacillibactin, bacilysin, bacillaene, subtilosin A, and other antibacterial compounds. Similarly, (Wang XiaoYu et al. 2011 ) identified eight NRPS/PKS gene clusters that encode surfactin, bacillomycin L, fengycin, bacillibactin, bacilysin/anticapsin, macrolactin, bacillaene, difficidin, and other antibacterial substances from the entire genome of B. subtilis Bs-916. Qi et al. ( 2019 ) discovered seven gene clusters in B. subtilis BS-6 through whole-genome sequencing, which encode bioantibiotic synthesis-related substances, such as subtilin, subtilosin A, surfactin, bacillibactin, bacillaene, mycosubtilin, and rhizocticin. Despite all being B. subtilis strains, genome-wide analysis indicated that the gene clusters encoded different substances, likely due to the diverse origins of these strains. Conclusion In this study, we successfully isolated and characterized Bacillus subtilis BAGL, which demonstrated significant broad-spectrum antifungal activity against key phytopathogenic fungi, Rhizoctonia solani , Fusarium oxysporum , and Alternaria solani . Whole-genome sequencing and comprehensive genomic analysis revealed a well-annotated genome containing 17 biosynthetic gene clusters responsible for producing diverse secondary metabolites with potent antimicrobial properties, including surfactin, fengycin, bacillibactin, and subtilosin (A) The detection of an extensive array of carbohydrate-active enzymes further substantiates BAGL’s capacity to degrade fungal cell walls, thereby enhancing its biocontrol efficacy against phytopathogenic fungi in vivo. Phylogenetic analysis established the taxonomic placement of BAGL within the Bacillus subtilis clade, demonstrating its alignment with well-characterized strains of this species. Functional annotations using the KEGG, GO, and COG databases underscored the metabolic versatility and enzymatic potential of this strain, supporting its adaptability and effectiveness as a biocontrol agent in agriculture. Collectively, these integrated genomic and functional findings identify (B) subtilis BAGL as a promising candidate for eco-friendly and sustainable disease management strategies in agriculture, particularly in response to phytopathogenic challenges prevalent in Pakistan. Future studies focusing on the isolation and characterization of active compounds and in vivo efficacy trials will be essential to validate these findings and translate them into practical applications. Supplementary Information Below is the link to the electronic supplementary material. Supplementary Material 1. (76.8KB, odt) Acknowledgements The sequencing of the bacterial strains was supported by GetGenome and The Sainsbury Laboratory, Norwich, UK, with support from the Gatsby Charitable Foundation and the Biotechnology and Biological Sciences Research Council (BBSRC). Authors’ contributions This manuscript is part of the MS. thesis of BA under the supervision of AK, AA, and NJ. BA and SS completed the research; MI, BT, and AK conceptualized the study; MI and AA developed the bioinformatics tools and software; SP and NJ curated the data and performed the statistics; and BT and AK wrote, reviewed, and edited the manuscript. Funding No funding was received for this study. Data availability The datasets presented in this study can be found in the NCBI Sequence Database ( [https://www.ncbi.nlm.nih.gov/sra/PRJNA1349010](https:/www.ncbi.nlm.nih.gov/sra/PRJNA1349010) ) under the accession no. PRJNA1349010. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Abdel-Moghies AH, El-Sehrawy MH, Zakaria AE, Fahmy SM (2024) In vivo application of potent probiotics for enhancing potato growth and controlling Ralstonia solanacearum and Fusarium oxysporum infections. Antonie Van Leeuwenhoek 117(1):33 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Alanjary M, Steinke K, Ziemert N (2019) AutoMLST: an automated web server for generating multi-locus species trees highlighting natural product potential. Nucleic Acids Res 47(W1):W276–W282 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Azeem S, Agha SI, Jamil N, Tabassum B, Ahmed S, Raheem A, Jahan N, Ali N, Khan A (2022) Characterization and survival of broad-spectrum biocontrol agents against phytopathogenic fungi. Rev Argent Microbiol 54(3):233–242 [ DOI ] [ PubMed ] [ Google Scholar ] Aziz S, Jamshed SA, Mukhtar T, Irshad G, Ijaz SS, Raja MU (2024) Evaluation of Bacillus spp. as biocontrol agents against chili leaf spot caused by Xanthomonas vesicatoria . J Plant Dis Prot 131(3):987–997 [ Google Scholar ] Balamurugan A, Ashajyothi M, Velmurugan S, Charishma K, Sakthivel K, Muthamilan M, Kumar A (2024) Bacillus amyloliquefaciens: a versatile antimicrobial Firmicute for the suppression of wilt caused by Ralstonia pseudosolanacearum in tomato. Biocontrol Sci Technol 34(4):336–354 [ Google Scholar ] Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Chakraborty K, Kizhakkekalam VK, Joy M, Chakraborty RD (2022) Bacillibactin class of siderophore antibiotics from a marine symbiotic Bacillus as promising antibacterial agents. Appl Microbiol Biotechnol 106(1):329–340 [ DOI ] [ PubMed ] [ Google Scholar ] Dai LM, Li LL, Liu YX, Shi YP, Cai ZY (2021) Whole genome sequencing and genomics analysis of Bacillus amyloliquefaciens BS-3 with biocontrol activity. Microbiol China 48:2073–2088 [ Google Scholar ] Durgadevi D, Harish S, Manikandan R, Prabhukarthikeyan S, Alice D, Raguchander T (2021) Proteomic profiling of defense/resistant genes induced during the tripartite interaction of Oryza sativa , Rhizoctonia solani AG1-1A, and Bacillus subtilis against rice sheath blight. Physiol Mol Plant Pathol 115:101669 [ Google Scholar ] Gao S, Liu A, Sang L, Sun S, Gou Y, Wang Z (2018) Whole genome sequencing and comparative genomics analysis of Bacillus subtilis VD18R19 with biocontrol activity against pepper Phytophtora rot disease. Chin J Trop Crops 39:2021–2027 [ Google Scholar ] Guillén-Navarro K, López-Gutiérrez T, García-Fajardo V, Gómez-Cornelio S, Zarza E, De la Rosa-García S, Chan-Bacab M (2023) Broad-spectrum antifungal, biosurfactants and bioemulsifier activity of Bacillus subtilis subsp. spizizenii—A potential biocontrol and bioremediation agent in agriculture. Plants 12(6):1374 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Guo S, Li X, He P, Ho H, Wu Y, He Y (2015) Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants. J Ind Microbiol Biotechnol 42(6):925–937 [ DOI ] [ PubMed ] [ Google Scholar ] Gupta S, Pandey S, Sharma S (2022) Decoding the plant growth promotion and antagonistic potential of bacterial endophytes from Ocimum sanctum Linn. against root rot pathogen Fusarium oxysporum in Pisum sativum . Front Plant Sci 13:813686 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Hong B-Y, Driscoll M, Gratalo D, Jarvie T, Weinstock GM (2024) Improved DNA extraction and amplification strategy for 16S rRNA gene amplicon-based microbiome studies. Int J Mol Sci 25(5):2966 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Iqbal S, Ullah N, Janjua HA (2021) In vitro evaluation and genome mining of Bacillus subtilis strain RS10 reveals its biocontrol and plant growth-promoting potential. Agriculture 11(12):1273 [ Google Scholar ] Kaspar F, Neubauer P, Gimpel M (2019) Bioactive secondary metabolites from Bacillus subtilis : a comprehensive review. J Nat Prod 82(7):2038–2053 [ DOI ] [ PubMed ] [ Google Scholar ] König F, Sandri MR, Russi A, Granada CE, Schwambach J (2024) Biocontrol of tomato pathogens by Bacillus subtilis F62 and its synergistic action in plant growth promotion with Rhizobium sp. L5. Biocontrol Sci Technol 34(6):551–565 [ Google Scholar ] Kopyltsov SV, Milovanov AV, Elisiutikova AV, Savenkova DS, Petrova KO, Kwon DV, Koshchaev AG (2022) Draft Genome Sequence of Bacillus velezensis Strain Krd-20 with Antifungal Activity, a Biotechnologically Important Isolate from Wheat Root Zone. Microbiol Resource Announcements 11(10):e00656–e00622 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Kravchenko S, Poshvina D, Vasilchenko A, Vasilchenko A (2020) Draft genome sequence of the multiple antibiotic producer Bacillus velezensis X-BIO-1. Microbiol resource announcements 9(50):01319–01320. 10.1128/mra [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Kumar L, Chhogyel N, Gopalakrishnan T, Hasan MK, Jayasinghe SL, Kariyawasam CS, Kogo BK, Ratnayake S (2022) Climate change and future of agri-food production. Future foods. Elsevier, pp 49–79 Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G et al (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis . Nature 390:249–256 [ DOI ] [ PubMed ] [ Google Scholar ] Lalaymia I, Naveau F, Arguelles Arias A, Ongena M, Picaud T, Declerck S, Calonne-Salmon M (2022) Screening and efficacy evaluation of antagonistic fungi against Phytophthora infestans and combination with arbuscular mycorrhizal fungi for biocontrol of late blight in potato. Front Agron 4:948309 [ Google Scholar ] Li E, Yang H, Wang X, Wan L, Pan H, Zhu D (2015) Whole-genome sequencing and analysis of inosine-producing strain Bacillus subtilis ATCC 13952 [ PubMed ] Li Y, Héloir MC, Zhang X, Geissler M, Trouvelot S, Jacquens L, Henkel M, Su X, Fang X, Wang Q (2019) Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation. Mol Plant Pathol 20(8):1037–1050 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Li S, Xiao Q, Yang H, Huang J, Li Y (2022) Characterization of a new Bacillus velezensis as a powerful biocontrol agent against tomato gray mold. Pestic Biochem Physiol 187:105199 [ DOI ] [ PubMed ] [ Google Scholar ] Li L, Wang R, Liang X, Gai Y, Jiao C, Wang M (2023) Characterization of a Bacillus velezensis with antibacterial activity and its inhibitory effect on gray mold germ. Agronomy 13(6):1553 [ Google Scholar ] Li X, Chen Y, Yang S, Zhou Y, Yang C (2025) Whole genome-sequence analysis of Bacillus subtilis strain KC14-1 with broad-spectrum antifungal activity. BMC Genomics 26(1):319 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Lim SB, Junqueira ACM, Uchida A, Purbojati RW, Houghton JN, Chénard C, Wong A, Kolundžija S, Clare ME, Kushwaha KK (2018) Genome sequence of Bacillus velezensis SGAir0473, isolated from tropical air collected in Singapore. Genome Announcements 6(27):00642 – 00618. 10.1128/genomeA.00642-18 [ DOI ] [ PMC free article ] [ PubMed ] Liu Y, Lu J, Sun J, Lu F, Bie X, Lu Z (2019) Membrane disruption and DNA binding of Fusarium graminearum cell induced by C16-Fengycin A produced by Bacillus amyloliquefaciens . Food Control 102:206–213 [ Google Scholar ] Lokhande HA (2022) Bioinformatics analysis of miRNA sequencing data. MicroRNA Profiling: Methods and Protocols. Springer, pp 225–237 [ DOI ] [ PubMed ] Marian M, Shimizu M (2019) Improving performance of microbial biocontrol agents against plant diseases. J Gen Plant Pathol 85(5):329–336 [ Google Scholar ] Mohammad-Razdari A, Rousseau D, Bakhshipour A, Taylor S, Poveda J, Kiani H (2022) Recent advances in E-monitoring of plant diseases. Biosens Bioelectron 201:113953 [ DOI ] [ PubMed ] [ Google Scholar ] Montesinos E (2023) Functional peptides for plant disease control. Annu Rev Phytopathol 61(1):301–324 [ DOI ] [ PubMed ] [ Google Scholar ] Pasha R, Sultan S, Tabassum B, Waris M, Rehmat Z, Jahan N, Parveen S, Ahmed SS, Khan Malghani MG, Khan A (2025) Biocontrol potential of Bacillus subtilis WL2. 3 in mitigating Phytophthora infestans infection in potatoes. Int Microbiol :1–16 [ DOI ] [ PubMed ] Peng Y, Li SJ, Yan J, Tang Y, Cheng JP, Gao AJ, Yao X, Ruan JJ, Xu BL (2021) Research progress on phytopathogenic fungi and their role as biocontrol agents. Front Microbiol 12:670135 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Qi JM, Sun SS, Zhang DX, Xu ZW, Xu YP (2019) Identification and biocontrol activity analysis of Bacillus sp. BS-6 based on genome-wide data. Biotechnol Bull 35:111–118 [ Google Scholar ] Raza W, Ghazanfar MU, Sullivan L, Cooke DE, Cooke LR (2021) Mating type and aggressiveness of Phytophthora infestans (Mont.) de Bary in potato-growing areas of Punjab, Pakistan, 2017–2018 and identification of genotype 13_A2 in 2019–2020. Potato Res 64(1):115–129 [ Google Scholar ] Riaz M, Akhtar N, Khan SN, Shakeel M, Tahir A (2020) Neocosmospora rubicola: An unrecorded pathogen from Pakistan causing potato stem rot. Sarhad J Agric 36:906–912 [ Google Scholar ] Shahid I, Han J, Hanooq S, Malik KA, Borchers CH, Mehnaz S (2021) Profiling of metabolites of Bacillus spp. and their application in sustainable plant growth promotion and biocontrol. Front Sustainable Food Syst 5:605195 [ Google Scholar ] Shi C, Zeng S, Gao X, Hussain M, He M, Niu X, Wei C, Yang R, Lan M, Xie Y (2024) Complete Genome Sequence Analysis of Bacillus subtilis MC4-2 Strain That against Tobacco Black Shank Disease. International Journal of Genomics 2024 (1):8846747 [ DOI ] [ PMC free article ] [ PubMed ] Singh BK, Delgado-Baquerizo M, Egidi E, Guirado E, Leach JE, Liu H, Trivedi P (2023) Climate change impacts on plant pathogens, food security and paths forward. Nat Rev Microbiol 21(10):640–656 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Sraphet S, Javadi B (2025) Exploring genetic diversity and genomic insights of Bacillus subtilis isolates from cassava rhizosphere using molecular barcoding and whole genome sequencing. Sci Rep 15(1):22708 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Su Z, Chen X, Liu X, Guo Q, Li S, Lu X, Zhang X, Wang P, Dong L, Zhao W (2020) Genome mining and UHPLC–QTOF–MS/MS to identify the potential antimicrobial compounds and determine the specificity of biosynthetic gene clusters in Bacillus subtilis NCD-2. BMC Genomics 21(1):767 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Sullivan J, De Panis D, Galeone V, Mazzoni CJ (2025) Genome Evaluation Pipeline (GEP): A fully-automated quality control tool for parallel evaluation of genome assemblies. Bioinformatics Advances:vbaf147 [ DOI ] [ PMC free article ] [ PubMed ] Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38(7):3022–3027 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Tian D, Song X, Li C, Zhou W, Qin L, Wei L, Di W, Huang S, Li B, Huang Q (2021) Antifungal mechanism of Bacillus amyloliquefaciens strain GKT04 against Fusarium wilt revealed using genomic and transcriptomic analyses. Microbiologyopen 10(3):e1192 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Wang XiaoYu WX, Luo ChuPing LC, Chen ZhiYi CZ, Liu YongFeng LY, Liu YouZhou LY (2011) Nie YaFeng NY, Yu JunJie YJ, Yin XiaoLe YX The complete genome sequence of the gram-positive bacterium Bacillus subtils Bs-916 Weng W, Yan J, Zhou M, Yao X, Gao A, Ma C, Cheng J, Ruan J (2022) Roles of arbuscular mycorrhizal fungi as a biocontrol agent in the control of plant diseases. Microorganisms 10(7):1266 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Xu P, Xie S, Liu W, Jin P, Wei D, Yaseen DG, Wang Y, Miao W (2020) Comparative genomics analysis provides new strategies for bacteriostatic ability of Bacillus velezensis HAB-2. Front Microbiol 11:594079 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Zhang S, Li C, Si J, Han Z, Chen D (2022) Action mechanisms of effectors in plant-pathogen interaction. Int J Mol Sci 23(12):6758 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Zou Q, Zhang Y, Niu X, Yang H, Chu M, Wang N, Bao H, Zhan F, Yang R, Lou K (2024) Antifungal activity of Rhizosphere Bacillus isolated from Ziziphus jujuba against Alternaria alternata . Microorganisms 12(11):2189 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials Supplementary Material 1. (76.8KB, odt) Data Availability Statement The datasets presented in this study can be found in the NCBI Sequence Database ( [https://www.ncbi.nlm.nih.gov/sra/PRJNA1349010](https:/www.ncbi.nlm.nih.gov/sra/PRJNA1349010) ) under the accession no. PRJNA1349010. 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