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Article

Exploring the Genome of Bacillus mojavensis Bai2-32 Against Root Rot Disease in Lycium barbarum L.

by
Yuexia Sha
1,*,†,
Qingchao Zeng
2,†,
Yanan Zhao
2 and
Bo Yang
3
1
Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750011, China
2
Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China
3
Agriculture, Forestry and Animal Husbandry Technology Extension Service Center of Ningxia Agricultural Reclamation Bureau, Yinchuan 750000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(12), 2832; https://doi.org/10.3390/agronomy15122832
Submission received: 4 November 2025 / Revised: 3 December 2025 / Accepted: 8 December 2025 / Published: 9 December 2025
(This article belongs to the Section Pest and Disease Management)
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Abstract

The root rot of Lycium barbarum represents the most severe soil-borne disease that impedes its production. The management of this disease primarily relies on chemical agents, which pose risk to both the environment and human health. In this study, we isolated Bacillus strains as potential biological control agents. Bai2-32 exhibited the strongest antagonistic activity against all five Fusarium species and demonstrated broad-spectrum antifungal activities. Field experiments further displayed that Bai2-32 provided excellent biocontrol efficacy. To understand the possible genetic determinants for biocontrol traits, we performed genome sequencing. The genome of B. mojavensis Bai2-32 consists of a 4,055,438 bp circular chromosome with a GC content of 43.67%, containing 3986 protein-coding genes. Phylogenetic analysis of Bacillus strains, utilizing a single core-genome approach, clearly placed the strain Bai2-32 within the B. mojavensis clade. Predictive analysis revealed that the genome encoded lipopeptides such as surfactin and fengycin, in addition to several active metabolite synthesis gene clusters. The results further support the potential of B. mojavensis Bai2-32 for application in agricultural production and suggest that it may be a promising biocontrol agent for further studies.

1. Introduction

Lycium barbarum, a perennial deciduous shrub, has been recognized as a valuable traditional medicinal material in China. Its fruits are rich in bioactive compounds, including polysaccharides, betaine, and carotenoids, which demonstrate significant efficacy in antioxidation, immune modulation, hepatoprotection, and vision enhancement. Furthermore, it constitutes an economically important crop in northwestern China, being widely cultivated in arid and semi-arid regions such as the Gansu, Ningxia, Xinjiang, and Qinghai provinces. This cultivation plays a crucial role in increasing local agricultural income and enhancing ecological sustainability [1,2]. However, the expansion of cultivation areas and the persistent practice of monocropping have led to the emergence of root rot in L. barbarum as the most severe soil-borne disease hindering industrial development. Yield losses in affected fields often exceed 35%, with severe infections resulting in complete plant mortality [3]. The pathogens responsible for root rot in L. barbarum are predominantly identified as Fusarium spp., including F. oxysporum, F. solani, F. culmorum, F. tricinctum, and F. equiseti [4]. Additionally, Rhizoctonia solani and Fusarium redolens can cause the disease either independently or through mixed infections [5]. The diversity of pathogens complicated disease prevention and management.
The management of L. barbarum root rot disease primarily relies on chemical agents. However, prolonged application of chemical agents enhances pathogen resistance and environmental pollution. Consequently, biological control has emerged as a significant research focus for the prevention and management of L. barbarum root rot. Previous investigations have demonstrated that microorganisms isolated from the soil and tissues of L. barbarum can effectively inhibit root rot pathogens. Among bacterial biocontrol strains, biological preparations made from spore-forming Bacillus are preferred due to their long-term viability, which facilitates the development of commercial products. B. amyloliquefaciens HSB1 achieved complete control efficacy against F. oxysporum by reshaping the rhizosphere bacterial community structure and suppressing pathogen hyphal growth [6]. B. velezensis K-9 inhibited up to 44.90% of the infection caused by Streptomyces scabies, the causative agent of potato scab. In 2021 and 2022, the potato yield for the B. velezensis K-9-treated plants was 12.44% and 12.65% higher than that for the control plants [7]. Additionally, B. subtilis J10-8 disrupted the pathogen’s hyphal structure via bacteriolytic action, achieving inhibition rates of up to 85% against F. solani [8]. Fungal biocontrol agents, including Metarhizium sp., Sinorhizobium sp., and dark septate endophytic fungi, have also demonstrated potential by competing for nutrients and secreting antimicrobial substances [9]. Furthermore, Alternaria, which can adapt to diverse lifestyles, have been reported to possess biocontrol potential as well [10]. Despite significant advancements in the exploration and application of biocontrol strains, most existing strains target only a single pathogen. Furthermore, their ability to colonize and the stability of their control efficacy under complex field conditions remain limited.
The present study aimed to identify highly effective Bacillus strains for biocontrol intended to prevent and manage root rot disease in L. barbarum. B. mojavensis Bai2-32 were identified through morphological analysis, physiological and biochemical profiling, and genome sequencing. The effectiveness of Bai2-32 in preventing L. barbarum root rot was later evaluated in pot and field experiments. Meanwhile, whole genome sequencing techniques were utilized to examine the gene clusters related to antimicrobial substance synthesis in Bai2-32, thus clarifying their possible biocontrol mechanisms. In the present study, the strain Bai2-32 was classified as B. mojavensis and its potential ability as a biocontrol agent for the management of L. barbarum root rot was evaluated. This study provided a foundation for further studies of functions and facilitated genetic engineering of B. mojavensis Bai2-32 to promote agricultural and industrial applications.

2. Materials and Methods

2.1. Strains and Growth Conditions

The strain Bai2-32 was routinely cultured at 37 °C in Luria–Bertani (LB) broth or on solid LB medium enhanced with 1.5% agar. The pathogenic fungi F. oxysporum 295 and F. verticillium 173 were obtained from the Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences. Additionally, F. moniliforme N19, F. solani N18, and F. oxysporum f. sp. niveum were isolated from the affected L. barbarum and stored at our laboratory. Meanwhile, Exserohilum turcicum, Alternaria alternata, Trichothecium roseum, Verticillium dahliae, Botrytis cinerea, Magnaporthe grisea, Colletotrichum orbiculare, and Sclerotinia fructigena were provided by the College of Plant Protection, China Agricultural University. The fungal strain was kept in paraffin at 4 °C at the Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry Sciences. The pathogens were propagated on potato dextrose agar (PDA) plates in a culture chamber at 25 °C for 7 days prior to use.
For preparing the spore suspension, firstly, Fusarium strains were inoculated onto PDA medium and incubated in the dark at 25 °C in a incubator for approximately 7 d. Once the mycelia completely covered the Petri dish, a small volume of sterile water was added, and the agar surface was gently scraped with a sterile brush to obtain the spore suspension. The concentration of the suspension was adjusted to 1 × 106 CFU/mL using a hemocytometer. For inoculum preparation, a single colony of Bai2-32, cultivated on LB medium plates, was transferred to 5 mL of LB broth and incubated at 37 °C on a shaker at 200 rpm for 12 h. Subsequently, 1 mL of the overnight culture was added to 100 mL of liquid LB medium, followed by incubation with shaking at 37 °C and 200 rpm for 48 h to obtain the fermentation broth of Bai2-32. The optical density was adjusted to approximately 1.0 at 600 nm, corresponding to approximately 1.0 × 108 CFU/mL.

2.2. Isolation of Bacterial Strains

More than 100 rhizosphere soil samples of L. barbarum were collected from Hongsipu District of Wuzhong City, Ningxia province. Subsequently, 10 g of soil was weighed and transferred into 100 mL of sterile water, followed by shaking for 1 h to ensure adequate mixing. Serial dilutions of 10−3, 10−4, and 10−5 were prepared, and 200 µL of each dilution was spread onto LB plates, which were then incubated at 37 °C for 48 h. Single colonies exhibiting distinct morphological characteristics were selected and purified. The purified strains were preserved in 40% glycerol at –80 °C for long-term storage.

2.3. Plate Antagonism Assay

Each tested plant pathogen mycelial plug (1 cm) was placed at the center of PDA medium plates. Bacterial strains under investigation were streaked (2 cm) using sterile toothpicks to transfer single colonies, positioned 2 cm away from both sides of the plug [11]. Plates without bacterial streaking served as the control. Meanwhile, 2 µL of the Bai2-32 strain culture was applied to the PDA plate after 1 day of incubation, positioned 2.5 cm from the pathogen disc. All plates were incubated in darkness at 28 °C, with four replicates for each treatment. After 5 days, the diameters of pathogen colonies and the inhibition zones were measured.

2.4. Strain Identification Using 16S rDNA Gene

The morphological characterization of the tested strain followed the methodology outlined in reference [12]. The physiological and biochemical characteristics of Bacillus species were evaluated using HBI Bacillus biochemical identification strips (HBIG14, Qingdao Hi-Tech Industrial Development Zone Haibo Biotechnology Co., Ltd., Xi’an, China). Species and genus identification was performed through bacterial 16S rDNA sequence analysis first. Genomic DNA was extracted, serving as the template for sequencing. The primer sequences are detailed as follows: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR conditions were as follows: an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s, concluding with a final extension at 72 °C for 10 min. The PCR products were purified and sequenced by Beijing Biomarker Biotechnology Co., Ltd., Beijing, China. The resultant sequences were compared against the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov, accessed on 26 September 2025) using the Basic Local Alignment Search Tool (BLAST) to identify the closest matches. Multiple sequence alignment and phylogenetic analysis were conducted by constructing a clustering phylogenetic tree using the neighbor-joining (NJ) method in MEGA 7.0 software [13].

2.5. The Control Efficacy of Bai2-32 in Greenhouse Conditions

After 30 days of growth of L. barbarum seedlings in a 25 °C chamber, root irrigation was conducted once using the fermentation broth of Bai2-32 (1 × 108 CFU/mL), with 20 mL applied to each plant. Twenty-four hours following root irrigation, a mixed spore suspension of four Fusarium species was inoculated at 20 mL per plant. The incidence of root rot was assessed after 10 days, and the disease index was subsequently determined. Each treatment was replicated four times. Each treatment include 10 L. barbarum seedlings. Meanwhile, more than 100 seeds of each treatment were used for the Astragalus membranaceus F. greenhouse experiment at 25 °C. The treatment for A. membranaceus is the same as mentioned above. Each experiment was replicated four times.
The investigation method adhered to the Guidelines for Field Efficacy Trials of Pesticides. The criteria for grading root rot assessment were established as follows: Grade 0: no visible symptoms; Grade 1: lesion area covering 1–5% of the root surface area; Grade 2: 6–10%; Grade 3: 11–20%; Grade 4: 21–40%; Grade 5: 41–60%; Grade 6: 61–80%; Grade 7: more than 80%.
The disease index was calculated using the following formula: Disease index = [∑(number of diseased roots in each grade × relative grade value)]/[total number of investigated roots × 7] × 100.

2.6. The Control Efficacy of Bai2-32 in Field Conditions

The test strain was activated for 48 h and subsequently inoculated into 1000 mL of seed culture medium composed of beef extract (3 g/L), peptone (10 g/L), and sodium chloride (5 g/L), maintaining a pH of 7.0. Following incubation, the mixture was incubated with shaking at 37 °C and 200 rpm for 12 h to obtain the seed solution. The seed solution was subsequently transferred into a fermenter at an inoculation rate of 6% for fermentation at 37 °C and 200 rpm, resulting in the fermentation broth of Bai2-32 after 48 h. Field efficacy trials for root rot management utilizing Bai2-32 were conducted on 5-year-old L. barbarum plants in Hongsipu District, Wuzhong City. Root rot was already present in the experimental field prior to the initial irrigation with the fermentation broth. Field experiments were conducted in 2023 in the L. barbarum fields of the same district as mentioned above. The variety of L. barbarum variety was Ningqi No. 1. Irrigation with the broth was performed three consecutively times on 24 July, 30 July, and 7 August 2022. On 5 September 2022, L. barbarum plants were excavated along with their root systems to evaluate the extent of root rot damage and to calculate the disease index. For the disease area, we randomly selected a 60 m2 plot for each treatment covering 20 plants, with four replicate plots for each treatment, and 500 mL of bacterial solution was applied per plot during each irrigation. Each plot was separated by 5 to 15 m. For healthy areas, the plot design was the same as mentioned above. Each experiment was repeated three times independently.

2.7. Bacterial DNA Extraction and Genome Sequencing

A solitary colony of B. mojavensis Bai2-32 was cultured overnight in LB broth at 37 °C. The extraction of genomic DNA was performed utilizing modified CTAB protocols, and then the DNA was subsequently dissolved in 100 μL of a tris-ethylenediaminetetraacetic acid buffer before being stored at –20 °C for later use. The NanoDrop ONE (Thermo Fisher Scientific, Waltham, MA, USA) instrument was used to evaluate both the quality and quantity of the DNA. For sequencing, both the Illumina (San Diego, CA, USA) NovaSeq 6000 and Oxford Nanopore GridION platforms facilitated the process, with support from Wuhan Benagen Technology Solutions Co., Ltd. (Wuhan, China) In the case of Illumina sequencing, 1 μg of the DNA sample underwent fragmentation through an ultrasonication technique, followed by size selection and end-repair procedures. Each fragment was then ligated with an adapter specific to Illumina. The resulting product was quantified, indexed, and sequenced on the NovaSeq 6000 platform. For the long-read sequencing approach, the genomic DNA was purified and directly utilized to construct a library with a ligation sequencing kit (SQK-LSK109), based on the manufacturer’s specifications. Barcoding of the DNA library was executed in accordance with the standard ONT protocol using the native barcoding expansion 1–12 kit. This library was then loaded into R9.4.1 flow cells and subjected to sequencing on a PromethION sequencer. The company used scripts to eliminate low-quality reads. High-quality sequences from both short-reads and long-reads were assembled into a cohesive sequence using Unicycler v.0.4.9 with default settings [14]. To predict protein-coding genes, Prokka v1.12 was employed, followed by functional annotation of these genes utilizing BLAST against the COG, Kyoto Encyclopedia of Genes and Genomes (KEGG), and InterPro databases [15,16]. Visualization of gene locations, GC skew, and GC content of the finalized annotated genome sequence was conducted using CIRCOS [17]. Additionally, the secondary metabolite gene clusters were examined using the antiSMASH v4.0.0rc1 program [18].

2.8. Phylogenomic Analysis Based on Genome Sequence

To create a phylogenetic tree for B. mojavensis Bai2-32 along with other Bacillus genomes, we procured genome sequences of Bacillus from the NCBI database, which included Paenibacillus polymyxa M1 as an outgroup. For the purposes of establishing the maximum likelihood (ML) phylogenetic tree among Bacillus species, we employed a sequence of single-copy core proteins that are shared across the Bacillus genomes. The extraction of single-copy core genes was performed from the PGAP table utilizing custom Perl scripts. Following this, multiple alignments of the amino acid sequences were created using MAFFT v7.310, and Gblocks was employed to select conserved blocks from these alignments [19,20]. The RAxML v8.2.10 software facilitated the construction of the maximum likelihood tree using the PROTGAMMALGX model, which included 100 bootstrap replicates [21]. The resultant tree was generated with the use of MEGA7 [13]. Furthermore, average nucleotide identity (ANI) values for the genome sequences were calculated utilizing JSpecies software with MUMmer alignment [22,23]. A heat map was produced using the R 3.6.0 package to validate the findings.

2.9. Pan-Genome Analysis

In order to pinpoint both core and specific genes in strains of B. mojavensis, we performed a pan-genome analysis with the PGAP software following genome annotation via Prokka [15]. The protein similarity method within the Pan-Genome Analysis Pipeline (PGAP) was employed to identify a collection of core orthologs from 30 strains of B. mojavensis. These core orthologs were subsequently grouped based on a minimum of 50% protein sequence similarity and 50% overlap with the longest sequence, maintaining an e-value threshold of 1 × 10−5 [24]. To extract the core genome along with specific genomes from the pan-genome dataset, we utilized a custom Perl script [21]. Functional annotation of the B. mojavensis pan-genomes was carried out using the COG database. We visualized the core genome and the pan-genomes with PanGP v1.0.1 software, producing distribution plots of the total and conserved genes identified through progressive sampling of ‘n’ genomes, adhering to the default settings [25].

2.10. Statistical Analysis

All experiments were performed with at least three independent biological replicates. Data are presented as mean ± SEM (standard error of the mean). Statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, Boston, MA, USA). Significance testing was performed through the t-test.

3. Results

3.1. The Strain Bai2-32 Showed Strong Inhibition Against Root rot in L. barbarum

Over 10,000 bacterial isolates were derived from around 100 samples of rhizosphere soil. Firstly, the plate confrontation method was employed to identity antagonistic strains capable of inhibiting the mycelial growth of F. oxysporum 295. The findings indicated that nearly 1700 bacterial strains exhibited antibacterial properties, accounting for 14.61% of the entire isolate collection. Next, we chose the strains with the inhibition rate exceeding 50% for further analysis, resulting in approximately 400 strains. Additionally, we assessed the inhibitory capacity of these strains against the remaining Fusarium pathogens. Ultimately, 12 biocontrol strains were identified that inhibited all five Fusarium strains, with Bai2-32 selected for subsequent investigation.
The strain Bai2-32 demonstrated an ability to inhibit the growth of Fusarium on the plates. The inhibition zones observed for F. oxysporum 295, F. solani N18, and F. verticillium 173 were greater compared to those for F. moniliforme N19 and F. oxysporum f. sp. niveum (Figure 1a). These findings indicated that Bai2-32 displayed a robust inhibitory effect on 295, N18, and 173, while showing less pronounced inhibition of N19 and F. oxysporum f. sp. niveum. Additionally, the antagonistic potential of strain Bai2-32 was evaluated against various other fungal pathogens. The results indicated that Bai2-32 effectively suppressed mycelium growth of these fungal pathogens, as evidenced by a clear inhibition zone. The strains displayed a broad spectrum of antagonistic activities (Figure 1b). The biological control capacity of strain Bai2-32 against root rot was assessed in both greenhouse conditions and field settings (Figure 1c). The disease index for L. barbarum plants treated with Bai2-32 was lower than that of the control plants. Specifically, the control plants had a disease index of 41.67, while L. barbarum plants treated with Bai2-32 exhibited a disease index of only 11.01, with a reduction of 73.58%. Meanwhile, Bai2-32 also demonstrated significant disease prevention against root rot in A. membranaceus under greenhouse conditions, with the disease index in the Bai2-32-treated plants (10.09%) showing a marked decrease compared to the control group (56.35%) (Figure 1d). The strain also had plant-growth-promoting ability; it significantly promoted the growth of plant height and root length (Figure 1e). A field experiment was also conducted to evaluate the control efficiency of Bai2-32. The disease index of L. barbarum plants for treatment with Bai2-32 measured 48.89, which showed a significant difference compared to the control group.
In conclusion, we isolated the biocontrol strain Bai2-32, demonstrating broad-spectrum antagonistic activities, plant-growth-promoting ability, and a beneficial microbe with potential for biotechnological application.

3.2. The Morphology of Strain Bai2-32

The morphological characteristics of Bai2-32 are illustrated in Figure 2. The single colonies cultivated on LB solid medium appeared large and opaque, exhibiting a slight yellow pigmentation at later stages, with a rough surface and multiple wrinkled edges. The physiological and biochemical reactions of strain Bai2-32 were examined, with positive results for citrate utilization, D-xylose, gelatin liquefaction, nitrate reduction, and starch hydrolysis, as shown in Table S1. In contrast, negative reactions were recorded for the V-P test, propionate utilization, L-arabinose, and D-mannitol. These features are considered characteristic morphological traits of Bacillus.

3.3. The General Genome Features of Bai2-32

This complete genome sequence of strain Bai2-32 was presented in this study, revealing a total genomic length of 4,055,438 bp and a GC content of 43.67%. It is estimated that the strain comprises 3986 protein-coding genes, among which 2794 have been assigned potential function, while 1192 are predicted to encode hypothetical proteins. The average length of these protein-coding genes is 887 bp, representing 87.18% of the overall genomic sequence. Furthermore, 86 tRNA and 30 rRNA genes were annotated in the genome sequence (Table 1 and Figure 2).
The classification function assigned the protein-coding genes to various COG functional groups. The results indicated that the highest number of genes (297) are involved in amino acid transport and metabolism. Additionally, 237 and 225 genes are involved in transcription and carbohydrate transport and metabolism. Meanwhile, a group of 45 genes are involved in secondary metabolites (Figure 2). As expected, surfactin and fengycin gene clusters were found in the genome sequence of Bai2-32. The entire secondary metabolism is displayed in Table 2.

3.4. Phylogenetic Analysis of Bai2-32

The sequencing results revealed that the 16S rDNA sequence length of this strain was 1253 bp, with the GenBank accession number PP762065. The obtained sequence exhibited over 99% similarity to Bacillus spp., as revealed by a BLAST search. Initial molecular identification, informed by 16S rDNA sequences, classified Bai2-32 within B. mojavensis (Figure 3). In recent years, the construction of phylogenetic trees based on core genome analysis has advanced towards a more standardized classification of bacteria. This research established a phylogenetic tree through core genome analysis to elucidate the evolutionary connections of Bai2-32. A phylogenetic tree representing Bacillus genomes was generated through the alignment of 715 single-copy core genes common to all genomes, employing the ML approach, and was rooted in P. polymyxa M1. Strain Bai2-32 clustered alongside other B. mojavensis strains, forming a sister group with B. mojavensis UCMB5075. The nodes in the phylogenetic tree received high bootstrap values (Figure 3). Furthermore, genomic relatedness for strain Bai2-32 was evaluated using ANI. The ANI comparisons for Bai2-32 relative to all B. mojavensis strains surpassed 95%, exceeding the established threshold. Strain Bai2-32 demonstrated a substantial nucleotide identity of 98.38% with UCMB5075, which serves as the type strain for the B. mojavensis species. Additionally, clustering analysis based on ANI values across strains was carried out to explore the evolutionary relationships among Bacillus strains, revealing that B. mojavensis strains tended to cluster together. However, certain inconsistencies arose when this data was compared to the phylogenetic tree derived from core genome sequences. Overall, strain Bai2-32 was classified as B. mojavensis based on morphological, physiological–biochemical, and molecular biological identification.

3.5. Pan-Genome Analysis of B. mojavensis

Our investigation determined that the pan-genome for the B. mojavensis strains examined comprises 7127 gene families. This study indicated that the core genome consists of 3061 genes, while the accessory genome contains 1940 genes, along with 2126 unique genes (Figure 4a). Interestingly, both B. mojavensis RO-H-1 and BD-627 exhibited the lowest specific genes, each having 24 unique genes. Conversely, B. mojavensis B-41811 showed the highest unique genes, totaling 236 (Figure 4a). The selected strains share a core genome that constitutes approximately 74.50% to 79.22% of the total genome repertoire. The sizes of both the core and pan-genome were estimated based on the selected genomic data. The pan-genome plot reveals that the curve for the pan-genome trend does not plateau and seems to expand with the incorporation of additional genomes into the analysis. Furthermore, it has been noted that the pan-genome curves for B. mojavensis align well with the mathematical functions of Heaps law, y = 625.05x0.54 + 3179.75, where y represents the size of the pan-genome and x indicates the count of sequenced genomes. Consequently, it is concluded that the pan-genome is an “open” pan-genome (Figure 4b). This open pan-genome offers considerable potential for discovering new genes as more B. mojavensis strains undergo sequencing. After the inclusion of the 30th genome, the analysis of the core genome demonstrated asymptotic behavior in relation to core gene counts. To enhance our understanding of the functional variation among B. mojavensis strains, COG annotations were assigned to the core, dispensable, and unique genes (Figure 4c). The core genome mainly comprises genes involved in amino acid (E), carbohydrate (G), and coenzyme (H) transport and metabolism, energy production and conversion (C), and transcription (K). On the other hand, the specific genome exhibits an increase in genes related to intracellular trafficking, secretion, and vesicular transport (U) and secondary metabolites related to biosynthesis, transport and metabolism (Q) (Figure 4c).

3.6. Antibiotic Gene Clusters in the Genome Sequence of Bai2-32

The genome sequence of B. mojavensis Bai2-32 contains 11 secondary metabolite biosynthetic gene clusters, including those for surfactin and fengycin. Among these, six gene clusters are annotated as known clusters, which include surfactin, zwittermicin A, fengycin, bacillibactin, subtilosin A, and bacilysin. Notably, surfactin and zwittermicin A exhibited 78% and 18% similarity to the known gene clusters, respectively, while the remaining clusters displayed 100% similarity. The ability of Bai2–32 to prevent and control root rot disease in L. barbarum may be linked to these antimicrobial gene clusters.

4. Discussion

There are lots of biocontrol strains used to control plant disease, such as Bacillus, Pseudomonas, and Streptomyces [26,27,28]. However, Bacillus species are characterized by several advantages, including a broad antibacterial spectrum, high stress tolerance, the ability to secrete multiple antimicrobial substances, rapid reproduction, and extensive natural distribution. These traits render them increasingly significant in the biological control of plant diseases [29,30]. B. mojavensis is commonly found in soil and animal intestines and is recognized as a safe biological resource [31]. Studies have demonstrated that the B. mojavensis strain Bai2-32 exhibits significant antibacterial activity against Fusarium, and field experiments have confirmed that this strain effectively prevents root rot disease in L. barbarum. Importantly, Bai2-32 exhibited broad-spectrum inhibitory activity, especially for all the plant pathogens of root rot (Figure 1). Previous reports indicate that B. mojavensis MTC-8 and B. mojavensis PS17 demonstrate potent antagonistic effects against various pathogens, including Magnaporthe oryzae, R. solani, Ustilaginoidea virens, Fusarium spp., A. alternata, V. dahliade, Epicoccum nigrum, Ascochyta pisi, Sclerotinia sclerotiorum, and Bipolaria maydis [32,33]. B. mojavensis represents a promising candidate for biological control microorganisms that could potentially be developed into commercial products in the future. Further in-depth research is necessary to elucidate the mechanisms of biocontrol for the development and application of biopesticides. Although B. mojavensis BmB4 was not found to exhibit any activity against phytopathogens, its extract demonstrated significant inhibitory effects against most pathogens. This strain is capable of producing various classes of lipopeptides, such as surfactin and fengycin, which possess broad-spectrum antibacterial activity [34]. Furthermore, B. mojavensis YL-RY0310 inhibited the growth of Penicillium expanum, and the author confirmed that lipopeptides could effectively inhibit the growth of P. expanum through DNA amplification analysis and the Oxford cup agar diffusion test [35]. The colonization process, particularly biofilm formation and motility, plays a crucial role in the efficacy of biocontrol bacteria. Notably, the antifungal activity and colonization ability of B. mojavensis D50 improved following medium optimization and metal ion adjustments [36]. We also need to find some solutions to help Bai2-32 to improve the colonization to inhibit the plant disease. Microbial whole genome sequencing has emerged as a vital and efficient detection technology in the field of biotechnology, being extensively employed for the analysis and prediction of various microbial functional genes. This technology facilitates the prediction of microbial secondary metabolite types and aids in elucidating their antimicrobial mechanisms. Ghazala et al. demonstrated that the isolated B. mojavensis 14 strain produced multiple lipopeptides belonging to the fengycin and surfactin families, which were identified as the principal active substances inhibiting potato dry rot [37]. The genome sequence of B. mojavensis Bai2-32 contains gene clusters for surfactin and fengycin (Figure 2 and Table 2). Future research should focus on determining the functions of these antibiotic gene clusters using gene-knockout technology. We need to check that the antibiotic gene cluster inhibited all five plant pathogens.
As technology advances, sequencing outcomes indicate that the plant microbiome is crucial for plant health and productivity. Pathogen infections lead to decreased community evenness and a more intense microbial network. Notably, infected leaves hosted microbes that possessed beneficial genomic traits and functions related to iron competition and potential antifungal properties [38]. The genus Bacillus emerged as the most abundant and essential microbial group associated with potential suppression of Verticillium wilt disease, as demonstrated through field, greenhouse, and laboratory experiments involving the smoke tree. Incorporating B. subtilis bioagents into the soil resulted in a reduced disease index and altered the structure of the soil microbiota [39]. Additionally, a microbial product made from B. subtilis and Trichoderma harzianum was effective in suppressing common scab disease and enhancing yield by boosting the relative abundance of beneficial bacteria [40]. Concurrently, bio-organic fertilizer that included B. amyloliquefaciens W19 demonstrated disease suppression, which was linked to its effects on the resident soil microbial communities, particularly increasing Pseduomonas spp. [41]. Numerous studies focus on the interactions among different species. Findings indicated that the biosynthesis pathways for branched-chain amino acids are involved in the syntrophic interaction between the inoculant B. velezensis SQR9 and the beneficial indigenous Pseudomonas stutzeri. This elucidates the mechanism of how synergistic interactions between biocontrol and indigenous strains enhance plant health [42]. The delivery of LtaE by Lysobacter enzymogenes, utilizing T4SS, triggers the production of the antifungal antibiotic 2,3-DAPG by interacting with the transcriptional repressor PhlF in Pseudomonas protegens [43,44]. Altering the plant microbiome is increasingly recognized as a sustainable environmental strategy for disease suppression and enhanced production [45]. The SynCom, which comprises Pantoea agglomerans, Acidovorax wautersii, and Burkholderia pyrrocinia, demonstrated a more pronounced effect in inhibiting the formation of rice false smut ball and enhancing resistance to M. oryzae [46]. There is limited understanding regarding the biocontrol strain B. mojavensis Bai2-32 and its role in regulating the plant microbiome to combat the disease, as well as its interactions with native microorganisms. Further experiments are necessary to investigate this.
The species within the genus of Bacillus exhibit significant taxonomic diversity, leading to ongoing confusion regarding the taxonomic status of certain Bacillus strains among researchers [47]. For instance, the current taxonomy of several strains within the B. pumilus group appears to be inaccurate based on ANI results [16]. The increasing availability of sequenced microbial genomes presents an excellent opportunity to reassess methodologies for understanding bacterial phylogeny [48]. Phylogenomic analysis reveals that the B. amyloliquefaciens group encompasses three species: B. siamensis, B. velezensis, and B. amyloliquefaciens [49]. In this study, we conducted phylogenetic analysis utilizing the single-copy core genome and ANI analysis, resulting in the classification of strain Bai2-32 as B. mojavensis (Figure 2). Comparative analyses were performed on four Bacillus strains that exhibit inhibitory effects against Verticillium wilt of cotton, revealing subtle differences in their genome sequences [11]. Notably, the core genome of B. velezensis is enriched in secondary metabolism genes when compared to B. siamensis and B. amyloliquefaciens, particularly regarding the fengycin gene clusters identified through comparative genomic analysis [49]. Additionally, it is essential to elucidate the mechanism underlying the biocontrol properties of these strains.

5. Conclusions

In summary, we isolated Bai2-32, which exhibited significant inhibitory effects on the root rot of L. barbarum. It was identified as B. mojavensis based on genomic sequencing. Predictive analysis indicated that the genome encodes lipopeptides, including surfactin and fengycin. These lipopeptides may play a crucial role in the biological control mechanisms of B. mojavensis Bai2-32. This study provided a valuable microbial resource for agricultural application. Further comprehensive research is required to elucidate the mechanisms by which the strain Bai2-32 acts as a biocontrol agent against plant disease.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15122832/s1. Table S1. Physiological and biochemical reactions of bacterial isolate Bai2-32. Figure S1. Genome map of B. mojavensis Bai2-32. Each circle has a different genome information, and circles from outside to inside: (1) scale marks (unit: Mb), (2, 3) protein-coding genes on the forward and reverse strands, respectively (color-coded by the functional categories), (4, 5) rRNA (blue) and tRNA (red) on the forward and reverse strands, respectively, (6) GC content (positive: red; negative: blue), and (7) GC skew (above average: aquamarine; below average: orange).

Author Contributions

Y.S. and Q.Z. wrote the draft of the manuscript, Q.Z. finalized the manuscript, Y.Z. and B.Y. prepared figures and tables, all authors proofread the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by the Ningxia Natural Science Foundation Project (2024AAC02062), the Ningxia Key Research and Development Program Project (2022BBF02031), the National Key R&D Program of China (2022YFD1401900), and the Science and Technology Research Project of Ningxia Academy of Agriculture and Forestry Sciences (NKYG-24-20).

Data Availability Statement

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBSJDY000000000. The version described in this paper is version JBSJDY010000000.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Strain Bai2-32 could serve as potential biocontrol agents against root rot. (a) The antagonistic activities of Bai2-32 against Fusarium on PDA plate. (b) Antagonistic activity of Bai2-32 against pathogenic fungi on PDA plate. (c) The biocontrol effect of Bai2-32 toward L. barbarum root rot in greenhouse experiment and field experiment. (d) The disease index of Bai2-32 and control in greenhouse experiments of A. membranaceus root rot. (e) The plant-growth-promoting ability of Bai2-32 for plant height and root length. Error bars indicate ± SD of three replicates. The statistical analysis was performed using GraphPad Prism 8 software through a t-test. ***, p < 0.001; *, p < 0.05.
Figure 1. Strain Bai2-32 could serve as potential biocontrol agents against root rot. (a) The antagonistic activities of Bai2-32 against Fusarium on PDA plate. (b) Antagonistic activity of Bai2-32 against pathogenic fungi on PDA plate. (c) The biocontrol effect of Bai2-32 toward L. barbarum root rot in greenhouse experiment and field experiment. (d) The disease index of Bai2-32 and control in greenhouse experiments of A. membranaceus root rot. (e) The plant-growth-promoting ability of Bai2-32 for plant height and root length. Error bars indicate ± SD of three replicates. The statistical analysis was performed using GraphPad Prism 8 software through a t-test. ***, p < 0.001; *, p < 0.05.
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Figure 2. Morphological and genomic characteristics of strain Bai2-32. (a) Colony appearance on LB medium. The picture was taken after 12 h of cultivation. (b) Gene clusters of surfactin and fengycin in the genome of Bai2-32. (c) COG functional categories of Bai2-32. The COG categories are as follows: energy production and conversion (C); cell cycle control, cell division, and chromosome partitioning (D); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); translation, ribosomal structure, and biogenesis (J); transcription (K); replication, recombination, and repair (L); cell wall, membrane, and envelope biogenesis (M); cell motility (N); posttranslational modification, protein turnover, and chaperones (O); inorganic transport and metabolism (P); secondary metabolites biosynthesis, transport, and catabolism (Q); general function prediction only (R); function unknown (S); signal transduction mechanisms (T); intracellular trafficking, secretion, and vesicular transport (U); and defense mechanisms (V).
Figure 2. Morphological and genomic characteristics of strain Bai2-32. (a) Colony appearance on LB medium. The picture was taken after 12 h of cultivation. (b) Gene clusters of surfactin and fengycin in the genome of Bai2-32. (c) COG functional categories of Bai2-32. The COG categories are as follows: energy production and conversion (C); cell cycle control, cell division, and chromosome partitioning (D); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); translation, ribosomal structure, and biogenesis (J); transcription (K); replication, recombination, and repair (L); cell wall, membrane, and envelope biogenesis (M); cell motility (N); posttranslational modification, protein turnover, and chaperones (O); inorganic transport and metabolism (P); secondary metabolites biosynthesis, transport, and catabolism (Q); general function prediction only (R); function unknown (S); signal transduction mechanisms (T); intracellular trafficking, secretion, and vesicular transport (U); and defense mechanisms (V).
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Figure 3. Phylogenetic examination of B. mojavensis Bai2-32. (a) A Phylogenetic assessment of Bai2-32 was performed utilizing 16S rDNA gene sequences. The phylogenetic tree was constructed using the NJ method. (b) The ML tree depicting various Bacillus strains was constructed based on 715 single-copy core genes via RAxML 8.2.10. P. polymyxa M1 served as the out-group. Bootstrap values, derived from 100 replications, are displayed at the nodes. (c) A heat map illustrating the ANI values among different Bacillus strains is provided. The numbers indicate the ANI values.
Figure 3. Phylogenetic examination of B. mojavensis Bai2-32. (a) A Phylogenetic assessment of Bai2-32 was performed utilizing 16S rDNA gene sequences. The phylogenetic tree was constructed using the NJ method. (b) The ML tree depicting various Bacillus strains was constructed based on 715 single-copy core genes via RAxML 8.2.10. P. polymyxa M1 served as the out-group. Bootstrap values, derived from 100 replications, are displayed at the nodes. (c) A heat map illustrating the ANI values among different Bacillus strains is provided. The numbers indicate the ANI values.
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Figure 4. Pan-genomes of B. mojavensis strains. (a) The quantity of unique genes associated with each B. mojavensis strain. The core genomes, which are shared among all strains, are represented in the inner circle. Unique genes for each strain can be found in the respective outer circles. The number indicated below each strain’s name corresponds to the CDS of that strain. (b) Graph representing the pan-genome and core genome of B. mojavensis. Blue dots illustrate the pan-genome size for each comparison among B. mojavensis genomes, whereas green dots depict the core genome size for the same comparisons. The median values are connected to illustrate the correlation between the number of genomes. (c) COG distribution of core and specific genes found across all 30 analyzed B. mojavensis strains. The COG categories are as follows: energy production and conversion (C); cell cycle control, cell division, and chromosome partitioning (D); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); translation, ribosomal structure, and biogenesis (J); transcription (K); replication, recombination, and repair (L); cell wall, membrane, and envelope biogenesis (M); cell motility (N); posttranslational modification, protein turnover, and chaperones (O); inorganic transport and metabolism (P); secondary metabolite biosynthesis, transport, and catabolism (Q); general function prediction only (R); function unknown (S); signal transduction mechanisms (T); intracellular trafficking, secretion, and vesicular transport (U); and defense mechanisms (V).
Figure 4. Pan-genomes of B. mojavensis strains. (a) The quantity of unique genes associated with each B. mojavensis strain. The core genomes, which are shared among all strains, are represented in the inner circle. Unique genes for each strain can be found in the respective outer circles. The number indicated below each strain’s name corresponds to the CDS of that strain. (b) Graph representing the pan-genome and core genome of B. mojavensis. Blue dots illustrate the pan-genome size for each comparison among B. mojavensis genomes, whereas green dots depict the core genome size for the same comparisons. The median values are connected to illustrate the correlation between the number of genomes. (c) COG distribution of core and specific genes found across all 30 analyzed B. mojavensis strains. The COG categories are as follows: energy production and conversion (C); cell cycle control, cell division, and chromosome partitioning (D); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); translation, ribosomal structure, and biogenesis (J); transcription (K); replication, recombination, and repair (L); cell wall, membrane, and envelope biogenesis (M); cell motility (N); posttranslational modification, protein turnover, and chaperones (O); inorganic transport and metabolism (P); secondary metabolite biosynthesis, transport, and catabolism (Q); general function prediction only (R); function unknown (S); signal transduction mechanisms (T); intracellular trafficking, secretion, and vesicular transport (U); and defense mechanisms (V).
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Table 1. The general genomic features of B. mojavensis Bai2-32.
Table 1. The general genomic features of B. mojavensis Bai2-32.
Bai2-32
Genome size (bp)4,055,438
GC content (%)43.67
Protein-coding genes3986
Gene length (bp)3,535,611
Gene average length (bp)887
Gene length/genome87.18
GC content in gene region (%)44.46
tRNA number86
rRNA number30
Table 2. The antibiotic gene clusters in the genome sequence of B. mojavensis Bai2-32.
Table 2. The antibiotic gene clusters in the genome sequence of B. mojavensis Bai2-32.
RegionTypeStrat RegionEnd RegionMost Similar Known ClusterSimilarity
Region 1NRPS352,981416,624surfactin78%
Region 2NRPS, T1PKS699,608780,185zwittermicin A18%
Region 3terpene1,192,6211,213,139--
Region 4lanthipeptide-class-ii1,871,4081,894,485--
Region 5NRPS, betalactone1,940,0952,017,749fengycin100%
Region 6terpene2,089,3702,111,268--
Region 7T3PKS2,158,4542,199,551--
Region 8NRPS3,068,3013,115,440bacillibactin100%
Region 9CDPS3,398,9203,419,666--
Region 10sactipeptide3,660,4783,682,090subtilosin A100%
Region 11other3,685,0693,726,487bacilysin100%
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Sha, Y.; Zeng, Q.; Zhao, Y.; Yang, B. Exploring the Genome of Bacillus mojavensis Bai2-32 Against Root Rot Disease in Lycium barbarum L. Agronomy 2025, 15, 2832. https://doi.org/10.3390/agronomy15122832

AMA Style

Sha Y, Zeng Q, Zhao Y, Yang B. Exploring the Genome of Bacillus mojavensis Bai2-32 Against Root Rot Disease in Lycium barbarum L. Agronomy. 2025; 15(12):2832. https://doi.org/10.3390/agronomy15122832

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Sha, Yuexia, Qingchao Zeng, Yanan Zhao, and Bo Yang. 2025. "Exploring the Genome of Bacillus mojavensis Bai2-32 Against Root Rot Disease in Lycium barbarum L." Agronomy 15, no. 12: 2832. https://doi.org/10.3390/agronomy15122832

APA Style

Sha, Y., Zeng, Q., Zhao, Y., & Yang, B. (2025). Exploring the Genome of Bacillus mojavensis Bai2-32 Against Root Rot Disease in Lycium barbarum L. Agronomy, 15(12), 2832. https://doi.org/10.3390/agronomy15122832

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