Branches were collected from healthy kiwifruit plants in Changsha City, Hunan Province, China, where kiwifruit bacterial canker is the main disease during the bleeding period in the spring. The collected branches were washed with sterile water, followed by surface sterilization with 75% alcohol for 30 s, and sterile water washing three times. About 1 g of bark from the branch was ground into a slurry under aseptic conditions using an automatic sample grinding instrument (Shanghai Jingxin Industrial Development Co., Ltd., JXFSTPRP 24) by adding 9 mL of water and one gram of quartz sand. The slurry was diluted 10, 100, and 1000 times using sterile distilled water. A total of 100 μL of each dilution was added to lysogeny broth (LB) agar medium (agar 20 g, tryptone 10 g, NaCl 10 g, yeast extract 5 g in 1000 mL of sterile water at neutral pH) and incubated at 25°C for 72 h. Three replicates were conducted for each dilution concentration.

Based on the morphology of the cultures, six species of Bacillus subtilis were initially identified. These Bacillus isolates from kiwifruit were screened using pure culture (Cho et al., 2011) after morphological identification using a stereoscopic microscope to pick out purified Bacillus. Briefly, 100 μL of C48 pathogen (Psa) was spread evenly on a 20 mL plate (90 mm diameter) of King’s B (KB) solid medium (peptone 20 g, agar 15 g, glycerol 10 mL, K₂HPO₄ 1.5 g, and MgSO₄·7H₂O 1.5 g in 1000 mL sterile water at neutral pH). The bacterial isolate was then inoculated at six symmetrical sites 2.5 cm from the center of the KB solid plate. Plates inoculated only with the pathogen were used as controls. Each treatment was replicated three times. The plates were incubated in the dark at 25°C for 48 h and observed. The strongest antagonistic activity against the C48 pathogen was identified as JIN4 ( Supplementary Table 1 ).

After activation, JIN4 was inoculated onto KB solid medium plates and incubated for 24 h at 30°C to observe the growth of the colonies. A Gram Stain Kit (Morin et al., 2014) and a Spore Stain Kit (Beijing Solarbio Science & Technology Co., Ltd.) were used for routine Gram and budding staining of JIN4. The activated JIN4 was inoculated in a KB liquid medium (without agar; the others are the same as KB solid medium) and incubated at 30°C on a shaker at 160 r/min for 24 h. The culture medium was aspirated at an optical density of 0.5–0.8 and centrifuged. Then, the medium was discarded, and the bacterial sediment was collected at the bottom of the tube with 0.1 mol/L, pH = 7.0 phosphate-buffered saline (PBS), washed twice, where the supernatant was discarded, and a pre-cooled fixative (25% glutaraldehyde 10 mL, 0.2 mol/L PBS 50 mL, add water to 100 mL) was slowly added along the wall of the tube at 4°C and stored at 4°C for 12 h. After this, the fixative was decanted, and the sample was rinsed with 0.1 mol/L, pH = 7.0, phosphate buffer (PB) for 15 min three times. Then, the samples were fixed by soaking them in a 1% osmiridic solution for 2 h. The waste solution was discarded, and the samples were rinsed with PBS buffer for 15 min and rinsed three times. The dehydrated samples were soaked in six ethanol solutions of 30%, 50%, 70%, 80%, 90%, and 95% in descending order for 15 min and then treated with 100% ethanol twice for 20 min. A mixture of ethanol and isoamyl acetate (V/V=1:1) was used to soak the samples for 30 min, and then 100% isoamyl acetate was used to soak and treat the samples for 1 h. The samples were dried at the critical point of CO₂, plated with gold films, and observed using scanning electron microscopy, which was used for the collection of micrographs (Hick and Preece, 1990; Nakagawa et al., 2000; Saxeń, 2011; Novozhilov et al., 2022).

Strain JIN4 was further identified by amplifying 16S rDNA sequences using polymerase chain reaction (PCR) with the universal bacterial primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’-GGT TAC CTT GTT ACG ACT T-3’) (Zhang et al., 2014; Tang et al., 2020). The PCR amplification system (25 μL) contained 5 μL of DNA template, 12.5 μL of 2 × Es Taq MasterMix (CoWin Biosciences Co., Ltd., Beijing, China), 1 μL of each primer, and 5.5 μL of ddH₂O. PCR conditions were as follows: 94°C for 2 min, 35 cycles of 94°C for 30 s, 55.4°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 2 min. We also sequenced the gyrA gene of the JIN4 strain for molecular identification using the specific primers gyrA-F (5′-CAG TCA GGA AAT GCG TAC GTC CTT-3′) and gyrA-R (5′-CAA GGT AAT GCT CCA GGC ATT GCT-3′) (Borshchevskaya et al., 2013; Khademi et al., 2021). The PCR system was configured in the same manner as 16S rDNA. The PCR reaction was annealed at 55°C. The other PCR conditions were the same as those of 16S rDNA. The PCR products were purified and sequenced by Beijing Tsingke Biotech Co., Ltd.

The obtained 16S rDNA and gyrA sequences were compared with the sequences in the GenBank nucleic acid database of NCBI for BLAST homology analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with the reference sequences using Mega 7.0 software. Next, a phylogenetic tree was constructed using the neighbor-joining method bootstrap 1000 (Kuroha et al., 2009; Akter et al., 2022).

A single colony of JIN4 was inoculated in 50 mL of KB liquid medium and incubated at 28°C for 18 h at 160 r/min, with three replicates. The precipitate was collected by centrifugation at 4000 r/min for 3 min, washed twice with 0.01 mol/L PBS buffer (pH 7.2) to remove the medium, and stored at −80°C. The strains were stored on dry ice and sent to Biomarker Technologies (Beijing, China) for bacterial genome sequencing (Harris and Okoro, 2014).

The JIN4 genome was sequenced using PacBio RSII, a third-generation sequencing platform. PacBio sequencing uses SMRT chips as the sequencing vector and four-color fluorescent labeling of the four bases (dNTPs), and the type of bases entered can be determined based on the wavelengths and peaks of different base-pairing lights. Genome assembly was performed using Hifiasm (Cheng et al., 2021), and gene prediction was performed using Prodigal v2.6.3. Subsequent data analysis for mapping was performed using the Biomarker Cloud platform (https://www.biocloud.net).

Non-coding RNAs are RNA molecules that are transcribed from the genome but are not translated and expressed into proteins, such as mRNAs, ribosomal RNA (rRNA), transfer RNA (tRNA), and other non-coding RNAs, which are referred to as other-ncRNAs in this study. Predictive identification of non-coding RNAs was performed using Infernal v1.1.3 and tRNAscan-SE v2.0 (Nawrocki and Eddy, 2013) in combination with Rfam and the tRNAscan-SE database (Ioanna et al., 2017; Chan and Lowe, 2019). Clustered, regularly interspaced palindromic repeats (CRISPR) is a contiguous region of DNA in the genome that interferes with the replicative action of exogenous DNA and protects bacteria from the invasion of external DNA. We used CRT v1.2 (Bland et al., 2007) for CRISPR genome prediction.

Annotation analysis of the genome based on the gene ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Annotation of drug resistance genes based on the Comprehensive Antibiotic Resistance Database (CARD). The most widely used tool for the identification and analysis of biosynthetic gene clusters within bacterial and fungal genomes, antiSMARSH version 6.0.1 database online analysis (https://antismash.secondarymetabolites.org) (Blin et al., 2021), was used to analyze the predicted strains, the main secondary metabolites of JIN4, their gene cluster locations, and the use of the TBtools tool to map Circos to mark secondary metabolite gene cluster locations (Chen et al., 2020).

The following common physiological and biochemical characteristics (Tan et al., 2018; Zhu et al., 2020) of JIN4 were determined considering Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994) and The Manual for the Identification of Common Bacterial Systems, including the catalase test, oxidase, starch hydrolysis, methyl red (MR), Voges–Proskauer test (VP), gelatin liquefaction, nitrate reduction, H₂S production, citrate utilization, lecithinase test, malonate utilization, cellulolytic activity and fermentation utilization of sugars, containing glucose, D-galactose, D-arabinose, D-mannose, D-fructose, D-xylose, respectively (Ma et al., 2002; Wen et al., 2015; Moawia et al., 2016). Some biocontrol bacteria can also promote plant growth and development through the secretion of growth-promoting substances, such as indole-3-acetic acid (IAA) and siderophore, and the use of such growth-promoting bacteria can strengthen seedlings (Bach et al., 2016; Lau et al., 2020; Zhai et al., 2021). JIN4 was characterized for its functional substances and its ability to produce IAA (Maor et al., 2004; Nunes et al., 2023), phosphorus-dissolving ability (Nautiyala, 1999; Wang et al., 2020), and ammonia production capacity (Richard et al., 2018; Liu et al., 2019) was measured.

The antagonistic spectrum of JIN4 was obtained by testing the antagonism assay with eight other pathogens (including Colletotrichum gloeosporioides, Fusarium oxyporum f. sp. Nelumbicola, Fusarium oxysporium f. sp. Cucumerinum, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. Lini, Colletotrichum Linicolum, Phytophthora capsica, Phytophthora nicotianae) in face-to-face culture on potato dextrose agar (PDA) (potato 200 g, glucose 20 g, agar 15 g, sterile water to 1000 mL, pH neutral) medium (El-Hasan et al., 2018). PDA medium was used to activate these pathogens, 5 mm of the pathogenic cake was inoculated in the center of a 9 cm diameter PDA plate, and the biocontrol fungus JIN4 was spotted 2.5 cm from the center of the plate. The control plate was not inoculated with JIN4; each treatment was replicated three times. The plates were incubated at 28°C for 72 h. The inhibition effect was observed and measured using the cross-patch method.

The C48 pathogen (GenBank: MZ336040.1) used in this study was isolated from kiwifruit ulcer samples (Hunan Province, China) in our laboratory and preserved on KB agar medium (Mazumdar et al., 2007) slants at 4°C (Vicente et al., 2004). Before being used in the experiments, this pathogen was grown on KB agar plates at 25°C for two days in the dark.

A standoff culture of C48 was used to assess the inhibition capacity of the bacterium JIN4 in Petri dishes using the crossover method. The pathogenic bacteria JIN4 and C48 were scribed and activated in LB and KB liquid media and incubated at 160 r/min for 36 h at 25°C. A volume of 100 μL of C48 was inoculated and spread on a KB solid medium, and the surface was blown dry. A 6 mm diameter sterile paper pad was dipped in JIN4 and placed at four symmetrical spots. The diameter of the zone of inhibition, centered on the paper pad, was measured after 72 h of incubation at 25°C. The test was repeated in three sets.

On March 18, 2021, kiwifruit plots planted with hybrid kiwifruit varieties (Yongshun County, Xiangxi Tujia, and Miao Autonomous Prefecture, Hunan) were selected for consistent management levels. All kiwifruit plants were grouped (16 plants per group), and ulcer disease was counted in each group before application. The JIN4 bacteria solution diluted to Optical densit₆₀₀ (OD₆₀₀) = 0.2 and 500 times fine break (3% benziothiazolino, Xian Hytech Agrochemicals Co., Ltd.) was applied to each group of kiwifruit plants in 10 L batches, and the three groups were repeated. The disease incidence scoring of each group was investigated again 14 days after application, and the disease index, disease growth rate, and relative control efficiency were calculated. The kiwifruit bacterial canker field disease incidence scoring criteria are shown in Table 1 .

Bacterial suspensions of JIN4 and C48 with OD₆₀₀ = 0.2 were prepared using a KB culture medium. The test was conducted using potted seedlings of Cuiyu kiwifruit with a growth period of two years (3 pots per treatment, four plants per pot), and the pathogen C48 was inoculated 24 h earlier than the biocontrol bacterium JIN4 (experimental design, Table 2 ). Kiwifruit leaves were punctured with the tip of a 1mL sterile syringe prior to inoculation. A clean watering can was used to spray the prepared bacterial suspension spray evenly onto the leaves during inoculation until it began to drip water. Three leaves were taken for treatment 3 and treatment 4 on 1 d, and 3 formed leaves were taken for four groups of treatments on 2 d, 4 d, 6 d, 8 d, and 10 d and stored in a freezer at −80 °C after liquid nitrogen quick-freezing for the determination of PAL, POD, SOD, PPO, CAT, malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content.

The kiwifruit leaves were ground into fine powder under liquid nitrogen quick-freezing conditions and weighed into 0.1 g tubes, which were stored at −80°C for later use. 0.1 g of the aliquoted sample was added to 1 mL of 0.01 mol/L PBS buffer (pH = 7.2) and vortexed for 30 s. The supernatant was centrifuged at 4°C, 3500 r/min for 10 min, and the supernatant was used for POD, CAT, SOD, enzyme activity assay, and protein quantification. The sample was centrifuged at 10000 r/min for 10 min at 4°C. Subsequently, the supernatant was collected and used to determine the content of H₂O₂ and MDA. PAL and PPO crude enzyme solution extraction uses the extraction solution that comes with the kit.

The growth pattern of strain JIN4 was irregularly rounded at the edges, white, and with a dry, wrinkled surface, raised similar to a crater on the KB agar plates ( Figure 1A ). JIN4 for Gram-positive bacterium under microscopic observation ( Figure 1B ). A budding stain test revealed the presence of spore production in JIN4 ( Figure 1C ). Under scanning electron microscopy, JIN4 was found to have long rod-shaped cells with bluntly rounded ends, measuring 0.4–0.8 μm × 1–3 μm ( Figure 2A ), with sparse surrounding flagella and non-expanded sporangium ( Figure 2B ).

The 16S rDNA sequence and gyrA gene sequence were analyzed using the online BLAST in NCBI GenBank to further identify strain JIN4. The 16S rDNA sequence was found to exhibit 99% identity with the 16S rDNA sequence of B. velezensis, B. amyloliquefaciens, B. stercoris, B. vallismortis, and B. amyloliquefaciens. The phylogenetic tree based on the 16S rDNA sequences indicated that strain JIN4 (MZ277421.1) clustered with B. subtilis strain SBMP4 (NR 118383.1) ( Figure 3 ) (Serrao et al., 2024). The results from these tests suggested that strain JIN4 belonged to Bacillus. The 16S rRNA is highly conserved and may not be able to distinguish between closely related species. Using sequence differences in the gyrA gene proved to be a valuable tool for distinguishing closely related Bacillus species. The phylogenetic tree based on the gyrA sequences indicated that strain JIN4 clustered with B. velezensis strain H1 (OM523097.1) ( Figure 4 ). The JIN4 finally identified as B. velezensis.

The whole genome of B. velezensis strain JIN4 (GenBank: CP087137.1) has a genome size of 3,904,585 bp, 1 chromosome, and 46.57% of GC content. There are 3734 predicted genes, with an average gene length of 925 bp and a total gene length of 3,454,302 bp, accounting for 88.47% of the total genome length. Repetitive sequences had a predicted length of 4411 bp, representing 0.11% of the genome size. JIN4 was predicted to have nine each of 5S rRNA, 16S rRNA, and 23S rRNA. 5S rRNA contains 116 bp, 16S rRNA contains 1550 bp, and 23S rRNA contains 2930 bp ( Supplementary Table 2 ). Predictions on JIN4 are that there are 86 tRNAs with an average length of 77.1 bp. In addition to rRNA and tRNA, many other non-coding RNAs (ncRNAs) such as sRNA, snRNA, and miRNA, were also present in strain JIN4, with a total of 80 predicted, with an average length of 142.5 bp.

The CRISPR analysis of strain JIN4 predicts a total of 12 CRISPR regions, which are key components of the bacterial immune system. Bacteria use these gene clusters to detect and resist attacks from fragmented viruses and destroy their DNA, thereby achieving viral resistance ( Supplementary Table 3 ). The CARD annotation predicted resistance for JIN4 and identified the types of antibiotics tolerated ( Supplementary Table 4 ), providing an important reference for synergistic antibiotics in biocontrol applications. Three resistance genes were identified in JIN4, showing resistance to the following antibiotics: tetracycline, lincosamide, pleuromutilin, macrolide, oxazolidinone, streptogramin, and phenicol.

Of the 3734 genes predicted for Bacteroides JIN4, 67.65% were annotated based on the GO database ( Supplementary Figure 1 ), with 11 branches related to cellular components, 13 for molecular function, and 15 for biological processes. Nine branches contained more than 500 enriched genes, those involved in catalytic activity, metabolic processes, cellular processes, binding, single-organism processes, membranes, membrane parts, cells, and cell parts, comprising 1438, 1238, 1058, 1025, 963, 829, 785, 763, and 749, respectively.

According to the analysis of this strain in the KEGG database ( Supplementary Figure 2 ), 57.87% of the genes were annotated, and the genes were enriched in four metabolic pathways: metabolism, environmental information processing, genetic information processing, and cellular processes. Seven metabolic pathways were enriched with more than or equal to 50 genes, in descending order of ABC transporters, biosynthesis of amino acids, two-component systems, carbon metabolism, quorum sensing, purine metabolism, and ribosomes, which comprised 123, 120, 115, 99, 70, 62, and 56 gene annotation results. The synthesis of active compounds with resistance from secondary metabolites during growth is a disease control mechanism in B. biotrophicus. Using antiSMASH to predict the JIN4 genome ( Figure 5 ), nine secondary metabolite gene clusters were predicted. These were surfactin, butirosin (A/B), difficidin, bacillaene, fengycin, bacillibactin, bacilysin, macrolactin H, mersacidin, and various peptidoglycan and polyketide compounds.

The physiological and biochemical characteristics of strain JIN4 is listed in Table 3 . Strain JIN4 showed positive reactions in the catalase test, starch hydrolysis, VP, gelatin liquefaction, nitrate reduction, H₂S production, citrate utilization, lecithin enzyme activity, cellulolytic activity, fermentation utilization tests for glucose, D-mannose, D-fructose, and D-xylose. However, it was negative for the oxidase reaction, MR test, malonate utilization, D-galactose, and D-arabinose utilization. Based on these characteristics, JIN4 was found to be significantly similar to Bacillus subtilis (Manju and Shanmugam, 2013; Moawia et al., 2016; Kadiri et al., 2023). Qualitative detection of functional substances in JIN4 showed that strain JIN4 can produce IAA, phosphorus-dissolving, and ammonia production, promoting plant growth and development.

The ability of strain JIN4 to inhibit eight different pathogens was measured to determine the antifungal range of strain JIN4. The results showed that strain JIN4 inhibited the pathogens ( Figure 6 ). The strain showed different degrees of inhibition against various pathogens ( Table 4 ), with strain JIN4 showing the highest antagonistic activity against pepper blight pathogen, with 89.32% inhibition. The inhibition of the cucumber wilt pathogen was weak, but the inhibition ratio was still over 65%.

A standoff culture of C48 was used to assess the inhibition capacity of the JIN4 in Petri dishes using the crossover method. The results showed that the outer diameter of the zone of inhibition of Psa C48 by JIN4 after 72 h of incubation on KB plates was 26.45 ± 1.47 mm, and the inner JIN4 growth diameter was 9.70 ± 0.29 mm, with a difference of 16.75 mm ( Figure 7 ).

After 14 days of treatment, the disease growth rates of the CK group, the JIN4 group, and the 3% benziothiazolino were 27.33%, 10.87%, and 7.93%, respectively. The efficacy of JIN4 was 60.22%, and the efficacy of 3% benziothiazolino was 70.99% ( Table 5 ). JIN4 had a significant effect on the control of the kiwifruit bacterial canker. JIN4 is an endophytic bacterium isolated from kiwifruit in Hunan Province, China, which is better adapted to the environments of kiwifruit and Hunan Province. As an endophyte, JIN4 can effectively colonize kiwifruit plants, is not easily affected by the external environment, and forms a symbiotic relationship with the plants (Wicaksono et al., 2018; Ben Abdallah et al., 2016), which can allow symbiotic bacteria to play biological roles in the long term (Alsultan et al., 2019).

The treatment group CK did not affect the five enzyme activities, and two metabolites of kiwifruit leaves, and the change trends were the same. The other three treatment groups rapidly increased the kiwifruit leaf enzyme activities, and the enzyme activities were increased in descending order by treatment groups C48+JIN4, C48, and JIN4. Both the C48+JIN4 and C48 treatment groups were able to rapidly increase the H₂O₂ content and MDA content of kiwifruit leaves ( Figure 8 ). The results showed that JIN4 induced elevated activities of five antioxidant enzymes and elevated contents of two metabolites in kiwifruit, thereby increasing the resistance of kiwifruit plants to Psa infection.