Bacillus altitudinis JZBQ5 and application thereof in plant disease prevention and growth promotion

Abstract

The invention provides bacillus altitudinis JZBQ5 and application of the bacillus altitudinis JZBQ5 in plant disease prevention and growth promotion. The preservation number of the bacillus altitudinis JZBQ5 is CGMCC (China General Microbiological Culture Collection Center) The bacillus altitudinis JZBQ5 can improve the disease resistance of strawberries, and has a good control effect on powdery mildew and anthracnose of strawberries. Meanwhile, growth of cucumbers and wheat is promoted, and plant diseases such as cucumber root rot and wheat stem rot are effectively prevented and controlled. The strain provides a new strain resource for biological control of crop diseases such as strawberry powdery mildew, and a theoretical basis is provided for a safe and efficient green biocontrol preparation.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of Bacillus subtilis JZBQ5 and its application in plant disease prevention and growth promotion. Background Technology

[0002] With the rapid development of facility agriculture and large-scale planting, the frequency and severity of crop diseases have increased year by year, seriously restricting the high-quality and efficient development of agricultural production. Among them, strawberry powdery mildew is caused by obligate parasites (… Podosphaera aphanis Caused by [unspecified pathogen], this pathogen cannot survive and reproduce independently on in vitro culture media, making traditional antagonistic screening methods ineffective and resulting in a severe shortage of biocontrol microbial resources. Strawberry powdery mildew is particularly severe in protected cultivation, significantly reducing strawberry yield and quality, causing huge economic losses. Simultaneously, fungal diseases such as strawberry anthracnose, cucumber root rot, and wheat stem rot are also prevalent, posing serious threats to the production of their respective crops. Currently, crop disease control still mainly relies on chemical pesticides, but long-term and excessive use has led to increasingly prominent problems such as enhanced pathogen resistance, excessive pesticide residues in agricultural products, and environmental pollution, which does not meet the needs of green agricultural development. Biological control, with its environmentally friendly, residue-free, and sustainable advantages, has become an important direction to replace chemical control. Bacillus, due to its strong resistance, ease of cultivation, and diverse functions, has become a research hotspot in biocontrol microorganisms.

[0003] Highland Bacillus ( Bacillus altitudinis As an important member of the Bacillus genus, *Bacillus oryzae* has been shown in studies to have certain control effects on cotton verticillium wilt and tobacco black shank. However, to date, no literature has reported that *Bacillus oryzae* has a good control effect on strawberry powdery mildew, and biocontrol strains targeting strawberry powdery mildew remain scarce. Therefore, screening for *Bacillus oryzae* strains that have control effects on strawberry powdery mildew and other diseases, and can also promote crop growth, is of great significance for enriching biocontrol strain resources and promoting the development of green biocontrol agents. Summary of the Invention

[0004] To address the scarcity of biocontrol resources for strawberry powdery mildew and the narrow and limited disease control spectrum of existing microbial strains, this invention provides a strain of Bacillus hygroscopicus JZBQ5. This strain can effectively control various crop diseases, including strawberry powdery mildew, while also promoting the growth of cucumbers and wheat, providing a new solution for green crop production.

[0005] To achieve the above objectives, the present invention adopts the following technical solution; The Bacillus hygroscopicus provided by this invention ( Bacillus altitudinis JZBQ5, its taxonomic name is clearly defined as Bacillus hygroscopicus. Bacillus altitudinisIt was deposited on August 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.35673.

[0006] This invention provides a fermentation agent, comprising a fermentation broth or bacterial suspension obtained by fermenting and culturing the aforementioned Bacillus hygroscopicus JZBQ5, or a dry powder agent obtained by spray drying the fermentation broth.

[0007] The aforementioned Bacillus cereus JZBQ5 was inoculated into a liquid culture medium for fermentation to obtain a fermentation broth. The liquid culture medium may contain nutrients such as carbon sources, nitrogen sources, and inorganic salts. Carbon sources may include glucose, sucrose, and starch, while nitrogen sources may include yeast extract, tryptone, and soybean meal.

[0008] The fermentation conditions include a temperature of 25–37°C, a pH of 6.5–7.5, and controlling the dissolved oxygen level during fermentation to ensure efficient cell proliferation.

[0009] This invention provides the application of the aforementioned Bacillus cereus JZBQ5 in the prevention and control of plant diseases, wherein the plant fungal disease is caused by the strawberry anthracnose fungus (… Colletotrichum gloeosporioides Strawberry root rot pathogen ( Fusarium proliferatum ), cucumber wilt pathogen ( Fusarium oxysporum f. sp. cucumerinum), cucumber root rot fungus ( Fusarium solani ), Fusarium wilt of watermelon ( Fusarium oxysporum f. sp. niveum), *Gynostemma pentaphyllum* (f. sp. niveum), *Gynostemma pentaphyllum* leaf spot pathogen ( Alternaria tenuissima ), Jujube fruit shrinkage pathogen ( Alternaria alternata ), Jujube anthracnose bacteria ( Colletotrichum gloeosporiode ), Jujube black skin fungus ( Colletotrichum nymphaeae ); Wheat stem rot fungus ( Fusarium pseudograminearum ), wheat root rot pathogen ( Bipolaris sorokiniana ), wheat scab ( Fusarium graminearum ), the pathogen of Verticillium wilt in Cotinus coggygria ( Verticillium dahliae ), Euonymus anthracnose bacteria ( Colletotrichum siamense ), Oomycetes arachnidosis fungus ( Cladobotryumvarium ) and shiitake mushroom green mold ( Trichoderma atroviride Diseases caused by one or more of the following.

[0010] Specifically, the applications include the control of strawberry powdery mildew, the control of strawberry anthracnose, the control of cucumber root rot, and the control of wheat stem base rot.

[0011] This invention provides the application of the aforementioned Bacillus subtilis JZBQ5 in promoting plant growth, including its application in promoting cucumber growth and its application in promoting wheat growth.

[0012] The *Bacillus hygroscopicus* JZBQ5 strain provided by this invention possesses multiple functions. It can effectively control various diseases in strawberries, cucumbers, and wheat, promote crop growth, and enhance the disease resistance of strawberries. This strain has significant theoretical research value and application prospects. It can serve as a high-quality experimental material for studying the interaction mechanisms between biocontrol Bacillus and different crops. It is also an excellent strain for the development of green biofertilizers and biofungicides, providing new strain resources for the integrated management of crop diseases and important theoretical basis for the development of safe and efficient green biocontrol agents. Attached Figure Description

[0013] Figure 1 The morphological characteristics of Bacillus alpineus of the present invention are shown.

[0014] Figure 2 The present invention presents the basic characteristics and gene clusters of Bacillus altissima, wherein A represents plate confrontation, B represents a phylogenetic tree, and C represents gene cluster analysis.

[0015] Figure 3 Phylogenetic trees for strain JZBQ5 are shown, where A is a phylogenetic tree constructed based on the 16S rRNA gene and B is a phylogenetic tree constructed based on the gyrB gene.

[0016] Figure 4 The control effect of strain JZBQ5 on strawberry powdery mildew is shown in Figure A, where A represents the control effect on strawberry powdery mildew in potted plants and leaves, and B represents the incidence rate of strawberry powdery mildew.

[0017] Figure 5 The study investigated the effects of strain JZBQ5 on the MAPK signaling pathway in strawberry leaves. In this study, A represents the analysis of the MAPK signaling pathway, B represents the transcriptional analysis of important genes in the MAPK pathway, and C represents the verification of the transcriptional analysis of important genes in the MAPK pathway.

[0018] Figure 6 The study investigated the effects of strain JZBQ5 on the ethylene signaling pathway in strawberry leaves. In this study, A represents the analysis of the ethylene signaling pathway, and B represents the verification of the transcriptional expression of important genes in the ethylene pathway.

[0019] Figure 7 The ex vivo control efficacy of strain JZBQ5 against strawberry anthracnose is shown in the figure. In this figure, A represents an ex vivo leaf, and B represents the area of ​​leaf infected with anthracnose.

[0020] Figure 8 The study aimed to assess the control effect of strain JZBQ5 on cucumber root rot. In the study, A represents potted cucumber plants with root rot, B represents cucumber seedlings in the control group, C represents cucumber seedlings in the pathogen group, and D represents the group treated with strain JZBQ5.

[0021] Figure 9 The study evaluated the growth-promoting and yield-increasing effects of strain JZBQ5 on cucumbers in pots and in the field. In this study, A represents the growth indicators of potted cucumber seedlings and cucumbers, while B represents the application effect and growth and yield indicators in the field.

[0022] Figure 10 The control efficacy of strain JZBQ5 against wheat stem base rot is shown in Figure 1. A represents the control efficacy against wheat stem base rot in potted plants, B represents the disease index of wheat in potted plants, and FP represents the control efficacy against *Fusarium graminearum* inoculation. Fusarium pseudograminearum ) processing group.

[0023] Figure 11 The effect of strain JZBQ5 on wheat growth promotion is evaluated. In this figure, A is the wheat growth promotion control and B is the wheat growth index.

[0024] Figure 12 The inhibition rates of 305 pesticide molecules against strain JZBQ5 were calculated.

[0025] Preservation of biological materials Accession number: CGMCC No. 35673 Name: Bacillus hygroscopicus JZBQ5 Classification and nomenclature: Bacillus hygroscopicus Bacillus altitudinis , Survival status: Survival Preservation period: August 18, 2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Detailed Implementation

[0026] Example 1: Isolation and Identification of Bacillus hygroscopicus JZBQ5 I. Isolation and purification of Bacillus hygroscopicus JZBQ5 In August 2024, rhizosphere soil samples were collected from healthy cucumber plants at a cucumber planting base in Daxing District, Beijing (39°42′N, 116°34′E). Collection method: Vigorous cucumber plants were selected, dug up entirely with roots, and the non-rhizosphere soil was shaken off. The soil attached to the roots (≤2mm thickness) was collected, placed in sterile sampling bags, and stored at 4℃. The samples were then brought back to the laboratory for processing within 24 hours.

[0027] Soil suspension preparation: Weigh 10g of rhizosphere soil sample and add it to an Erlenmeyer flask containing 90mL of sterile physiological saline. Add sterile glass beads, shake at 28℃ and 180r / min for 30min, let stand for 10min, and take the supernatant as a 10⁻¹ dilution.

[0028] Serial dilution: The 10⁻¹ diluent was serially diluted 10-fold using sterile physiological saline to obtain 10⁻², 10⁻³, and 10⁻¹ solutions, respectively. 4 10⁻ 5 Soil suspension of a certain concentration.

[0029] Spread culture: Take 10⁻³, 10⁻ 4 10⁻ 5 0.1 mL of each of the three concentrations of suspension was evenly spread on LB solid medium plates, with three replicates for each concentration, and incubated at 30°C for 24-48 h.

[0030] Purification culture: Select single colonies with different morphologies (colony color, size, edge characteristics, etc.) from the plate, inoculate them into fresh LB solid medium, and purify them by streak culture at 30℃ for 24 hours. Repeat the streak 3 times until a pure strain with uniform colony morphology is obtained.

[0031] Strain preservation: The purified strain was inoculated into LB liquid medium and cultured at 30℃ and 200r / min for 16h with shaking. 800μL of the bacterial solution was mixed with an equal volume of 50% glycerol and stored at -80℃ for later use.

[0032] II. Preliminary screening of antibacterial activity of strains (plate confrontation test) Tested pathogens: Vegetable and fruit pathogens: Strawberry anthracnose fungus ( Colletotrichum gloeosporioides Strawberry root rot pathogen ( Fusarium proliferatum ), cucumber wilt pathogen ( Fusarium oxysporum f. sp. cucumerinum), cucumber root rot fungus ( Fusarium solani ), Fusarium wilt of watermelon ( Fusarium oxysporum f. sp. niveum), *Gynostemma pentaphyllum* (f. sp. niveum), *Gynostemma pentaphyllum* leaf spot pathogen ( Alternaria tenuissima Fruit tree pathogens: Jujube fruit shrinkage pathogen ( Alternaria alternata ), Jujube anthracnose bacteria ( Colletotrichum gloeosporiode ), Jujube black skin fungus ( Colletotrichum nymphaeae ); Field crop pathogens: Wheat stem rot fungus ( Fusarium pseudograminearum ), wheat root rot pathogen ( Bipolaris sorokiniana ), wheat scab ( Fusarium graminearum ); Pathogenic fungi of garden and landscaping plants: Verticillium wilt of Cotinus coggygria ( Verticillium dahliae ), Euonymus anthracnose bacteria ( Colletotrichum siamense ); Edible fungal pathogens: Oomycetes oothecae (Arachnidus oothecae) Cladobotryumvarium ), Shiitake mushroom green mold ( Trichoderma atrovirideAll of these were isolated from our laboratory, identified using conventional identification methods, and preserved for screening antibacterial activity.

[0033] Culture media: LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0-7.2, solid medium supplemented with 18 g / L agar) was used for bacterial isolation, purification and culture; PDA medium (200 g / L potato, 20 g / L glucose, 18 g / L agar) was used for pathogen culture and plate confrontation test.

[0034] Pathogen culture: Fusarium oxysporum and Anthrax sicca were inoculated into PDA medium and cultured at 25°C for 7 days to activate the pathogens.

[0035] Preparation of fungal cakes: Using a sterile punch with a diameter of 6 mm, fungal cakes are punched from the edge of the activated pathogen colony and set aside for later use.

[0036] Confrontation culture: Take an LB solid medium plate and inoculate the center of the plate with a pathogenic fungal disc; symmetrically around the fungal disc (2.5 cm from the edge of the plate), inoculate 2 μL of LB liquid culture of strain JZBQ5 (1×10⁻⁶). 8 (CFU / mL), with plates uninoculated with JZBQ5 strain serving as blank controls. Each treatment was performed in triplicate and incubated at 25°C for 6 days.

[0037] Inhibition rate calculation: Measure the diameter of pathogen colonies and calculate the inhibition rate. The formula is: Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0038] The results showed that a strain with strong antibacterial activity against plant pathogenic fungi such as Fusarium and Anthracnose was screened from the rhizosphere soil of cucumbers in Daxing, Beijing, and named JZBQ5.

[0039] This strain forms grayish-white colonies on LB agar plates with regular edges, smooth surfaces, opaqueness, non-stickiness, and low mobility. It stains purple with Gram stain, indicating that it is a Gram-positive bacterium. Figure 1 ).

[0040] like Figure 2 The plate confrontation experiment showed that strain JZBQ5 had an inhibition rate of more than 55% against Fusarium oxysporum and Anthrax sicca.

[0041] Table 1. Inhibition rate of JZBQ5 strain against various plant pathogenic fungi indicator bacteria Antibacterial rate (%) Strawberry anthracnose fungus ( ) 58.6 ± 4.2 Strawberry root rot pathogen ( ) 49.3 ± 3.8 Cucumber root rot pathogen ( ) 63.7 ± 5.1 Fusarium wilt of cucumber (f. sp. cucumerinum) 60.8 ± 5.1 Fusarium wilt of watermelon (f. sp. niveum) 55.2 ± 4.5 Kale leaf spot pathogen ( ) 45.8 ± 3.3 Wheat stem rot fungus () 67.4 ± 5.3 Wheat root rot fungus ( ) 52.9 ± 4.0 Fusarium head blight of wheat ( ) 61.5 ± 4.8 The anthracnose bacterium of jujube ( ) 48.7 ± 3.6 Jujube fruit shrinkage pathogen ( ) 56.3 ± 4.3 Jujube black skin fungus ( ) 54.9 ± 5.0 Anthracnose fungus of Euonymus alatus ( ) 51.8 ± 3.9 The pathogen causing Verticillium wilt in Cotinus coggygria ( ) 44.5 ± 3.2 Shiitake mushroom green mold pathogen ( ) 58.2 ± 5.4 Oomycetes oothecae (spider web fungus) 59.7 ± 4.6 III. Identification of strain JZBQ5 In the molecular identification experiment of the target strain, 16S rRNA gene-specific PCR amplification was first performed using the universal bacterial primer pair 27F (forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (reverse primer: 5'-GGTTACCTTGTTACGACTT-3'). The reaction program was as follows: 95℃ pre-denaturation for 5 min, followed by 30 cycles (94℃ denaturation for 1 min, 56℃ annealing for 2 min, and 72℃ extension for 2 min), a final extension at 72℃ for 10 min, and finally, incubation at 4℃. The product was stored; then, targeting the gyrB gene, PCR amplification was performed using specific primer pairs gyrB-F (forward primer: 5'-GAAGTCATCATGACCGTTTCGCAYGCNGGNGGNAARTTYGA-3') and gyrB-R (reverse primer: 5'-AGCAGGGTACGGATGTGCGGAGCCRTCNACRTCNGCRTNGTCAT-3'). The program was 95℃ pre-denaturation for 5 min, followed by 30 cycles (94℃ denaturation for 1 min, and optimized de-denaturation at 55~62℃). The gyrB gene amplification product was subjected to bidirectional sequencing using Seq-gyrB-F (forward sequencing primer: 5'-GAAGTCATCATGACCGTTTCGCA-3') and Seq-gyrB-R (reverse sequencing primer: 5'-AGCAGGGTACGGATGTGCGGAGCC-3'). The experiment was performed by Sangon Biotech (Shanghai) Co., Ltd. The obtained sequencing peaks were then assembled, proofread, and... Low-quality bases were removed to obtain the complete coding region sequence of the gyrB gene. Finally, the 16S rRNA gene and gyrB gene reference sequences of homologous strains were downloaded from the GenBank database and imported into Mega11 software along with the experimentally obtained target sequence. Multiple sequence alignment was performed using the ClustalW algorithm, and a phylogenetic tree was constructed using the Neighbor-Joining method. The reliability of the phylogenetic tree branches was verified using the Bootstrap method (repeated 1000 times). The phylogenetic position of the target strain was determined by combining sequence homology analysis and the phylogenetic tree topology. Figure 3 The 16S rRNA gene of strain JZBQ5 is shown in SEQ ID NO. 1, and the gyrB gene is shown in SEQ ID NO. 2.

[0042] Based on the phylogenetic tree constructed using the 16S rRNA gene, strain JZBQ5 clearly clustered with […]. Bacillus altitudinis Branches, with known strains B. altitudinisThe homology of 41KF2b (NR 042337.1) is 98%, indicating a very close phylogenetic relationship. Furthermore, based on the gyrB gene, a phylogenetic tree was constructed, showing that JZBQ5 and... B. altitudinis T12 (PV554264.1) forms the same small branch, which is further clarified as B. altitudinis .

[0043] 2.3.1 Whole-genome sequencing and phylogenetic analysis Genomic DNA extraction: Genomic DNA was extracted from strain JZBQ5 using a genomic DNA extraction kit following the instructions. The purity and integrity of the DNA were then assessed by agarose gel electrophoresis and Nanodrop to ensure that the DNA met sequencing requirements.

[0044] Genome sequencing: Qualified genomic DNA samples were sent to Beijing Novogene Bioinformatics Technology Co., Ltd., where whole genome sequencing was performed using the Illumina HiSeq sequencing platform to obtain high-quality genomic sequence data.

[0045] Phylogenetic tree construction: The genome sequences obtained from sequencing were assembled and annotated. The genome sequences of the Bacillus hygroscopicus model strain and related biocontrol strains (GR-8, FD48, GLB197, etc.) were selected as references, and the average nucleotide identity (ANI) between strain JZBQ5 and the reference strain was calculated. Based on 11 conserved genes of strain JZBQ5 (16S_RimM, rpoA, PyrG, gyrB, gyrA, EF-Tu, rpsS_bact, L27, ruvB, rpsB_bact, infA, the spliced ​​sequences of the 11 conserved genes in the JZBQ5 genome are shown in SEQ ID NO. 3), a phylogenetic tree was constructed using AntoMLST with maximum likelihood method. The bootstrap value was set to 1000 replicates to verify the reliability of evolutionary branches.

[0046] pan-genome analysis The genome sequences of Bacillus subtilis strains GR-8, FD48, and GLB197 were compared with the genome sequence of strain JZBQ5. Pan-genome analysis was performed using Anvi'o software to count the functional proteins among strains and clarify the specificity of strain JZBQ5.

[0047] Analysis of gene clusters for the biosynthesis of secondary metabolites The AntiSMASH 8.0 online analysis tool (https: / / antismash.secondarymetabolites.org / ) was used to predict the secondary metabolite biosynthesis gene clusters of strain JZBQ5, and the type, number and integrity of the gene clusters were analyzed.

[0048] 2.4 Preservation of microbial strains The identified strain JZBQ5 was inoculated onto LB solid slant medium and cultured at 30°C for 24 hours before being sent to the China General Microbiological Culture Collection Center (CGMCC) for preservation. The preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 35673.

[0049] Phylogenetic analysis showed that this strain shared an average nucleotide identity (ANI) of 98.8% with *Bacillus glomeratus* strain GR-8, and was located on the same evolutionary branch as GR-8 in the phylogenetic tree with a confidence level of 99%. Combined with morphological characteristics, strain JZBQ5 was confirmed as *Bacillus glomeratus*. Pan-genome analysis revealed that the functional protein differences between *Bacillus glomeratus* JZBQ5 and reference strains GR-8, FD48, and GLB197 exceeded 5%, demonstrating the specificity of this strain at the functional gene level and providing a molecular basis for its unique biocontrol function. AntiSMASH analysis showed that the genome of strain JZBQ5 contained 11 secondary metabolite biosynthesis gene clusters, covering various types including siderophores, terpenoids, and non-ribosomal peptides. Among them, a complete lichenysin biosynthesis gene cluster was included. Lichenysin, as a lipopeptide with surface and antibacterial activity, is presumably one of the important functional substances for the disease-preventing effect of strain JZBQ5.

[0050] Example 2: Control and resistance mechanism of Bacillus hygroscopicus JZBQ5 against strawberry powdery mildew 1. Experimental materials Preparation of bacterial suspension: Strain JZBQ5 was inoculated into LB liquid medium and cultured at 30℃ with shaking at 200 rpm for 24 h. After centrifugation and concentration, the concentration was adjusted to 10 with sterile physiological saline. 8 CFU / mL, for later use.

[0051] Strawberry plants used in the test: Strawberry plants with uniform growth were selected from the Changping strawberry planting base in Beijing. These included infected strawberry plants with uniform powdery mildew infection, disease grade 3 (leaves covered with obvious white powdery mycelium), and healthy strawberry plants without powdery mildew symptoms and in good growth condition. All plants were transplanted with soil to 20cm diameter flowerpots (substrate: garden soil: humus: perlite = 3:2:1) for later use.

[0052] 2. Test Methods 2.1 Experimental Design and Processing The transplanted strawberry plants were placed in an artificial climate chamber for one week to acclimatize, and then divided into three groups of 10 plants each. The main treatments were as follows: Control group (CK): Select healthy strawberry plants and spray each plant with an equal amount of sterile water. The amount of water sprayed should be enough to moisten the leaves without dripping. Powdery mildew treatment group (SPM): Selected infected strawberry plants, without fungicide treatment, only sprayed with an equal amount of sterile water at the same time point; Inoculant and powdery mildew treatment group (JZBQ5): Select susceptible strawberry plants and spray each plant evenly with 20 mL of a 10% concentration. 8 The appropriate amount of JZBQ5 bacterial suspension (CFU / mL) should be sprayed until the leaves are moist but not dripping wet.

[0053] All treatment groups were cultured under the same artificial climate conditions, with normal watering management during the culture period to avoid cross-contamination.

[0054] 2.2 Survey on the effectiveness of prevention and control After 14 days of cultivation, the disease was characterized by the appearance of a distinct white powdery substance on the leaf surface, as calculated below: Incidence rate (%) = (Number of diseased leaves / Total number of leaves surveyed) × 100%; Prevention and control effect (%) = (SPM incidence rate - JZBQ5 incidence rate) / SPM incidence rate × 100%.

[0055] 2.3 RNA-seq transcriptome analysis 2.3.1 Sample Collection and RNA Extraction After 14 days of cultivation, three strawberry plants were randomly selected from each group, and the top functional leaves (3rd-4th leaves) were collected and immediately frozen in liquid nitrogen at -80℃ for later use. Total RNA was extracted from the leaves using an RNA extraction kit (Aikerui Biotechnology Co., Ltd.), and the purity and integrity of the RNA were detected by agarose gel electrophoresis and Nanodrop to ensure that it met the sequencing requirements.

[0056] 2.3.2 Transcriptome sequencing and differential gene analysis Qualified RNA samples were sent to Beijing Biomarker Biotechnology Co., Ltd., where RNA-seq sequencing was performed using the Illumina NovaSeq platform. After filtering, assembling, and annotating the sequencing data, differentially expressed genes (DEGs) in the JZBQ5 group were analyzed, with the SPM group as a control. The focus was on changes in the expression of signaling pathways related to plant disease resistance (such as the MAPK signaling pathway and the ethylene signaling pathway) and defense genes.

[0057] 2.4 qPCR Validation Genes with significantly different disease resistance characteristics identified in the transcriptome analysis were selected for qPCR validation. Actin The gene was used as an internal reference gene. RNA was reverse transcribed into cDNA using a reverse transcription kit, and amplification was performed according to the qPCR kit instructions. A 2... ⁻ΔΔCt The method calculates the relative expression level of genes.

[0058] 3. Experimental Results like Figure 4 As shown, compared with the SPM group, the white powdery mycelium on strawberry leaves in the JZBQ5 group was significantly reduced, and the incidence rate was reduced by 77.05% compared with the SPM group, significantly alleviating the severity of strawberry powdery mildew, indicating that Bacillus oryzae JZBQ5 has excellent control effect on strawberry powdery mildew. GO functional enrichment and KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in plant disease resistance-related pathways, among which the MAPK signaling pathway was significantly activated, and the transcription levels of several key genes were greatly increased; in particular, the overall ethylene signaling genes underwent significant changes, participating in the regulation of plant defense responses (…). Figure 5 , Figure 6 qPCR validation showed that it could increase the defense protein level by more than 15 times. PR1 Transcription, RTE1 , ETR , MKK9 , MPK6 , EIN3 The transcriptional expression of genes such as [list of genes] was significantly upregulated. These results suggest that the JZBQ5 strain may enhance strawberry resistance to powdery mildew by activating the MAPK pathway.

[0059] Example 3: The control efficacy of Bacillus hygroscopicus JZBQ5 against strawberry anthracnose 1. Experimental materials JZBQ5 bacterial culture: Strain JZBQ5 was inoculated into LB liquid medium and cultured at 30℃ with shaking at 200 rpm for 24 h. After centrifugation and concentration, the concentration was adjusted to 10 with sterile physiological saline. 8 CFU / mL, for later use.

[0060] Siamese anthrax ( Colletotrichum siamense Spore suspension: *Bacillus thaliana* was inoculated onto PDA medium and incubated at 28°C for 7 days to activate the spores. The colony surface was rinsed with sterile saline, and mycelia were removed by filtration through double-layer sterile gauze. After counting with a hemocytometer, the spore concentration was adjusted to 10⁻⁶. 6 Spores / mL, for later use.

[0061] Select healthy, disease-free, and uniformly growing mature strawberry plants of the variety "Hongyan". Collect mature functional leaves (4th-6th leaves) from the middle and lower parts of the plant. The leaves should be undamaged and free from deformities to ensure the uniformity of the experimental materials.

[0062] 2. Test Methods Strawberry leaves were collected and rinsed with sterile water to remove surface dust. They were then immersed in a 75% ethanol solution for 10 seconds, quickly removed, rinsed three times with sterile water, and drained. 20 μL of sterile water was dropped onto the center of each leaf surface as the control group (CK). Alternatively, 20 μL of 10% ethanol solution was dropped onto the center of each leaf surface. 6 A spore suspension of *Anthracis sicca* spores at a concentration of spores / mL was used as the pathogen group (CS); 20 μL of a 10% concentration was added dropwise to the center of the leaf surface. 6 After incubating a suspension of *Anthrax sicca* spores (spores / mL) at 28°C and 80% relative humidity in the dark for 24 hours, 20 μL of a 10% concentration was added dropwise at the same location. 8 JZBQ5 bacterial suspension at CFU / mL. Leaves were placed in sterile petri dishes lined with two layers of moist sterile filter paper, and the lesion area was measured after 7 days. Control efficacy (%) = (average lesion area of ​​control group - average lesion area of ​​treatment group) / average lesion area of ​​control group × 100%.

[0063] 3. Experimental Results After 7 days of cultivation, obvious lesions appeared on the leaves of the CS group. The lesions were black with clear edges, and the average lesion area was 35.2 mm². The lesion area in the JZBQ5 group was significantly reduced, with an average lesion area of ​​only 5.68 mm². Calculations showed that Bacillus thuringiensis JZBQ5 achieved a control effect of 83.8% against strawberry anthracnose. Figure 7 This indicates that the strain can effectively inhibit the infection and spread of *Anthracnose sicca*, and has excellent control effects against strawberry anthracnose.

[0064] Example 4: Control effect of Bacillus hygroscopicus JZBQ5 on cucumber root rot 1. Experimental materials The JZBQ5 strain was inoculated into LB liquid medium and cultured at 30℃ with shaking at 200 rpm for 24 h. After centrifugation and concentration, the concentration was adjusted to 10 with sterile physiological saline. 8 CFU / mL, for later use; Fusarium solani (… Fusarium solani Inoculate onto PDA medium and incubate at 28°C for 7 days. Rinse the colony surface with sterile water, filter through double-layer gauze to remove mycelia, and obtain a spore suspension. Adjust the concentration to 10. 6 Spores / mL, for later use.

[0065] Healthy seeds of the cucumber variety "Jinyuan Cucumber" were selected, disinfected by soaking in 55℃ warm water for 15 minutes, and then sown in a seedling substrate (garden soil: humus: perlite = 3:2:1). The seeds were cultivated under the conditions of 25℃ temperature, 60% relative humidity, 12h light / 12h dark. When the seedlings grew to the 4-leaf and 1-heart stage, seedlings with uniform growth and no diseases or pests were selected as test materials.

[0066] 2. Test Methods The cucumber seedlings were transplanted into pots filled with seedling substrate, one seedling per pot. After transplanting, they were allowed to acclimatize for 3 days and then divided into 3 groups of 10 seedlings each. The control group (CK) was watered with 22 mL of sterile water; the control group (CK) was watered with 2 mL of a 10% sterile water solution. 6 A spore suspension of *Fusarium solani* spores at a concentration of 10 spores / mL, with 2mL of sterile water added, constitutes the pathogen group (FS); 2mL of a 10 spore concentration is applied to the cucumber rhizosphere soil as a top dressing. 6 A suspension of Fusarium spores per mL was prepared for solanaceous Fusarium spores, and 20 mL of a 10% concentration was simultaneously applied as irrigation. 8 The treatment group (JZBQ5) consisted of a JZBQ5 bacterial suspension at CFU / mL. Two weeks after inoculation, the incidence of root rot in cucumber plants in each group was investigated, and the disease severity was graded according to the cucumber root rot grading standard.

[0067] Disease Index (DI) = ∑(Number of diseased plants at each level × Disease level value) / (Total number of plants surveyed × Highest level value) × 100; Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0068] 3. Experimental Results Two weeks after inoculation with *Fusarium solani*, cucumber plants in the FS group generally showed symptoms of root rot, with brown lesions appearing on the roots. Some plants experienced root rot and death, with an incidence rate of 90% and a disease index of 76.8. In the JZBQ5 group, the symptoms were significantly reduced, with only a few plants showing scattered lesions on the roots without rot, and the disease index dropping to 13.0. Calculations showed that *Bacillus hygroscopicus* JZBQ5 achieved an 83% control effect against cucumber root rot caused by *Fusarium solani*. Figure 8 This indicates that the strain can effectively inhibit pathogen infection of cucumber roots, demonstrating excellent biocontrol potential.

[0069] Example 5: Effect of Bacillus hygroscopicus JZBQ5 on cucumber growth promotion and yield increase 1. Experimental materials Liquid inoculum: Inoculate JZBQ5 strain into LB liquid medium, incubate at 30℃ with shaking at 200 rpm for 24 h, centrifuge to concentrate, and adjust the concentration to 10 with sterile physiological saline. 8 CFU / mL, for later use; Dry powder inoculant: The fermented bacterial broth is adsorbed by light calcium carbonate and prepared into a powder using a spray drying process. It should be diluted with water to a concentration of 10% before use. 8 CFU / mL, for later use.

[0070] Pot experiment: Healthy seeds of cucumber variety “Jingyan 118” were selected, disinfected by soaking in 55℃ warm water for 15 minutes, and sown in seedling substrate (garden soil: humus: perlite = 3:2:1). The seeds were cultivated under the conditions of 22℃ temperature, 65% relative humidity, 14h light / 10h dark. When the seedlings grew to the 2-leaf and 1-heart stage, seedlings with uniform growth and no diseases or pests were selected as test materials. Field trial: Cucumber fields in a vegetable planting base in Zhuozhou City, Hebei Province were selected, and the cucumber variety "Jingyan 118" was selected. The seedlings were cultivated to the 3-leaf and 1-heart stage using conventional seedling raising methods before transplanting.

[0071] 2. Test Methods 2.1 Potted plant growth promotion experiment The cucumber seedlings were transplanted into pots filled with seedling substrate and allowed to acclimatize for 2 days. They were then divided into a control group (CK) and a treatment group (JZBQ5), with 12 seedlings in each group. Each seedling in the control group was watered with 20 mL of sterile water, while each seedling in the treatment group was watered with 20 mL of a 10% sterile water solution. 8 JZBQ5 bacterial suspension at CFU / mL. During the cultivation period, watering was normal and the substrate moisture was kept consistent. After 20 days of cultivation, key growth indicators of the two groups of cucumber seedlings were measured.

[0072] 2.2 Field trials to promote growth and increase yield The field trial for promoting growth and increasing yield included a control group (CK) and a treatment group (JZBQ5). Each group consisted of three plots of 20 m² each, with 1 m wide isolation rows between plots, arranged in a randomized block design. The control group received routine field management after cucumber transplanting (watering, fertilization, pest and disease control, but no application of fungicides). The treatment group received 20 mL of a 10% concentration fungicide per plant 7 days after transplanting (seedling stage). 8 A diluted bacterial solution of JZBQ5 dry powder (CFU / mL) was applied as a second irrigation treatment at the same dosage after a 7-day interval. Other field management practices remained the same as the control group. Twenty days after the second irrigation, 10 cucumber plants were randomly selected from each plot to measure plant height and stem diameter at 5 cm from the base. Once the cucumbers entered their peak fruiting period, the cucumber yield of each plot was measured for one week, and the yield increase rate was calculated.

[0073] 3. Experimental Results Pot experiments showed that strain JZBQ5 significantly promoted the growth and development of cucumber seedlings in terms of plant height and root length, and significantly enhanced the overall growth of the plants, resulting in robust seedling stems, well-developed root systems, and superior growth compared to the control group. In field trials, this strain significantly improved cucumber plant height and stem diameter, promoting vegetative growth. Field yield measurements showed an 11.94% increase compared to the control group, demonstrating a stable field growth-promoting and yield-increasing effect. Figure 9 ).

[0074] Example 6: Control effect of Bacillus hygroscopicus JZBQ5 on wheat stem base rot 1. Experimental materials The JZBQ5 strain was inoculated into LB liquid medium and cultured at 30℃ with shaking at 200 rpm for 24 h. After centrifugation and concentration, the concentration was adjusted to 10 with sterile physiological saline. 8 CFU / mL, for later use; Fusarium graminearum ( Fusarium pseudograminearum Inoculate onto PDA medium and incubate at 28°C for 7 days. Rinse the colony surface with sterile water, filter through double-layer gauze to remove mycelia, and obtain a spore suspension. Adjust the concentration to 10. 6 Spores / mL, for later use. Select wheat seedlings at the three-leaf stage with good uniform growth, and choose plants with uniform growth and no pests or diseases as test materials, for later use.

[0075] 2. Test Methods Wheat seedlings were transplanted into pots containing a seedling substrate (garden soil: humus: perlite = 3:2:1), one seedling per pot. After transplanting, the seedlings were allowed to acclimatize for 3 days and then divided into 3 groups, with 10 seedlings in each group: the control group (CK) was watered with an equal volume of sterile water; the control group was watered with 2 mL of a 10% sterile water solution first. 6 A suspension of *Fusarium graminearum* spores at a concentration of 10 / mL was used to irrigate an equal volume of sterile water after 24 hours; this was the pathogen group (FP). Alternatively, 2 mL of a 10% concentration of *Fusarium graminearum* spores was first irrigated into the wheat rhizosphere soil. 6 A spore suspension of *Fusarium graminearum* with a concentration of 10 spores / mL was applied to the soil, and 20 mL of the suspension was applied to each pot after 24 hours. 8 The treatment group (JZBQ5) consisted of JZBQ5 bacterial suspension at CFU / mL. All treatment groups were cultured at 22℃, 65% relative humidity, and a 14h / 10h light / dark cycle, with normal watering management to maintain consistent substrate moisture during cultivation. Two weeks after treatment, the disease severity was graded according to the wheat stem rot grading standard, and the control effect was assessed using the disease index method. Disease Index (DI) = ∑(Number of diseased plants at each level × Disease grade value) / (Total number of plants surveyed × Highest grade value) × 100; Control effect (%) = (Disease index of pathogen group - Disease index of treatment group) / Disease index of pathogen group × 100%.

[0076] 3. Experimental Results Wheat plants in the FP group generally showed symptoms of stem base rot, with brown lesions appearing at the base of the stem. In some plants, the lesions encircled the stem circumference, and the leaves turned yellow and wilted, resulting in weakened growth. In the JZBQ5 group, the symptoms were significantly reduced, with only a few plants showing a small number of light brown lesions at the base of the stem, without any lesions encircling the stem circumference. The plants showed good growth. Calculations showed that the disease index in the FP group was significantly higher than that in the JZBQ5 group, and the control efficiency of *Bacillus hygroscopicus* JZBQ5 against wheat stem base rot caused by *Fusarium graminearum* reached 72%. Figure 10 This indicates that the strain can effectively inhibit the infection of the wheat stem base by pathogens, demonstrating good biocontrol potential.

[0077] Example 7: Growth-promoting effect of Bacillus hygroscopicus JZBQ5 on wheat seedling growth 1. Experimental materials The JZBQ5 strain was inoculated into LB liquid medium and cultured at 30℃ with shaking at 200 rpm for 24 h. After centrifugation and concentration, the concentration was adjusted to 10 with sterile physiological saline. 8 CFU / mL was used as the bacterial solution for seed coating. Plump, uniform, undamaged, and disease-free wheat seeds were selected and prepared for use. The seedling substrate consisted of a 2:1 mixture of sandy loam and humus, sterilized by high-pressure steam at 121℃ for 30 minutes, and then filled into 15cm diameter flowerpots.

[0078] 2. Test Methods Wheat seeds were soaked in a 1% sodium hypochlorite solution for 10 minutes (stirring during soaking), rinsed 5 times with sterile water, and drained. They were then divided into two groups (50 seeds per group): the control group was soaked in an equal volume of sterile water for 30 minutes and air-dried; the treatment group was soaked in 10% sodium hypochlorite solution for 10 minutes and air-dried. 8 Soak the seeds in CFU / mL JZBQ5 bacterial solution for 30 min (stirring to ensure uniform contact) and air dry to complete the coating. Sow the seeds of both groups into flowerpots containing sterilized substrate and culture them under the conditions of 22℃, 65% relative humidity, 14h light / 10h dark. During the period, water normally to keep the substrate moisture consistent. After 14 days of culture, measure the plant height (vertical height from substrate surface to growth point) and root length (length from root base to taproot tip) with a tape measure, calculate the average value and perform significance analysis.

[0079] 3. Experimental Results After 14 days of cultivation, the wheat seedlings were growing well and free from pests and diseases. The test results showed ( Figure 11The average plant height of wheat seedlings in the control group was 24.6±3.3 cm, and the average root length was 14.4±4.5 cm. In the JZBQ5 seed-coated group, the average plant height was 27.6±3.2 cm, and the average root length was 17.1±3.5 cm. Compared with the control group, the plant height in the treated group increased significantly by 11.99%, and the root length increased significantly by 16.25%, indicating that Bacillus hygroscopicus JZBQ5 seed-coating treatment can effectively promote the growth and development of wheat seedlings and significantly improve plant height and root length.

[0080] Example 8: Adaptability test of Bacillus hygroscopicus JZBQ5 to chemical pesticides 1. Experimental materials The tested strain was *Bacillus hygroscopicus* JZBQ5, which was inoculated into LB liquid medium and cultured at 30°C with shaking at 200 rpm for 24 h. The concentration was then adjusted to 10. 6 CFU / mL was prepared as the bacterial solution for testing and kept on standby. The test pesticides were 305 pesticide standards provided by MCE Company, dissolved in appropriate solvents to prepare a 10mM concentration and kept on standby. The experimental equipment included 96-well cell culture plates and an ELISA reader, which were sterilized before use.

[0081] 2. Test Methods Compatibility was tested using a 96-well plate in vitro culture method: 200 μL of 10 [unspecified ingredient] was added to each well. 6 JZBQ5 LB bacterial suspension (CFU / mL) was used in the experimental group, with 2 μL of 10 mM pesticide standards added. The control group received 2 μL of sterile solvent. Each group had three replicate wells. The 96-well plate was incubated at 30℃ for 24 hours, and the OD of each well was measured using a microplate reader. 600 Value. The antibacterial rate is calculated using the formula: Antibacterial rate = (OD value of control group) / (OD value of control group). 600 -Experimental group OD 600 (value) / control group OD 600 The value is multiplied by 100%, and the effect of pesticides on the growth of bacterial strains is evaluated by the inhibition rate.

[0082] 3. Experimental Results After 24 hours of cultivation, no contamination was observed in the bacterial solutions at any of the wells, and the test results were stable and reliable. Of the 305 pesticides tested, 257 pesticides showed an inhibition rate ≤50% against the strain, indicating a weak or no effect on its growth. These included fungicides such as pyraclostrobin, azoxystrobin, and triadimefon, as well as various insecticides and plant growth regulators. Only 48 pesticides showed an inhibition rate greater than 50% against strain JZBQ5, significantly inhibiting its growth. The results indicate that ( Figure 12 Bacillus hygroscopicus JZBQ5 exhibits good adaptability to most chemical pesticides and has the potential to be used in conjunction with most pesticides.

[0083] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. In summary, according to the principles of the invention, this application is intended to include any modifications, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A strain of Bacillus hygroscopicus JZBQ5, characterized in that, Its taxonomic name is Bacillus hygroscopicus. Bacillus altitudinis This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35673.

2. A microbial inoculant, characterized in that, The active ingredient of the bacterial agent is *Bacillus hygroscopicus* JZBQ5 as described in claim 1, and the formulation of the bacterial agent is fermentation broth, bacterial suspension, or dry powder, wherein the viable count of *Bacillus hygroscopicus* JZBQ5 is not less than 10. 8 CFU / mL or 10 8 CFU / g.

3. The application of Bacillus cereus JZBQ5 as described in claim 1 in the prevention and control of plant fungal diseases and / or diseases caused by pathogens of edible fungi, characterized in that, The plant fungal disease mentioned is caused by strawberry anthracnose fungus (… Colletotrichum gloeosporioides Strawberry root rot pathogen ( Fusarium proliferatum ), cucumber wilt pathogen ( Fusarium oxysporum f. sp. cucumerinum), cucumber root rot fungus ( Fusarium solani ), Fusarium wilt of watermelon ( Fusarium oxysporum f.sp. niveum), the fungus causing leaf spot of Chinese kale ( Alternaria tenuissima ), Jujube fruit shrinkage pathogen ( Alternaria alternata ), Jujube anthracnose bacteria ( Colletotrichum gloeosporiode ), Jujube black skin fungus ( Colletotrichum nymphaeae ); Wheat stem rot fungus ( Fusarium pseudograminearum ), wheat root rot pathogen ( Bipolaris sorokiniana ), wheat scab ( Fusarium graminearum ), the pathogen of Verticillium wilt in Cotinus coggygria ( Verticillium dahliae ), Euonymus anthracnose bacteria ( Colletotrichum siamense One or more of the diseases caused by the edible fungus, the pathogen of which is *Oomycetes ootheca* (a spider web pathogen). Cladobotryumvarium ) and shiitake mushroom green mold ( Trichoderma atroviride One or two of them.

4. The application according to claim 3, characterized in that, The plant fungal diseases mentioned are one or more of the following: strawberry powdery mildew, strawberry anthracnose, cucumber root rot, and wheat stem base rot.

5. The application of Bacillus subtilis JZBQ5 as described in claim 1 in promoting plant growth, characterized in that, The plant in question is cucumber and / or wheat.

6. The application of the microbial inoculant according to claim 2 in the prevention and control of plant fungal diseases and / or diseases caused by pathogens of edible fungi, characterized in that, The plant fungal disease mentioned is caused by strawberry anthracnose fungus (… Colletotrichum gloeosporioides Strawberry root rot pathogen ( Fusarium proliferatum ), cucumber wilt pathogen ( Fusarium oxysporum f. sp. cucumerinum), cucumber root rot fungus ( Fusarium solani ), Fusarium wilt of watermelon ( Fusarium oxysporum f. sp. niveum), *Gynostemma pentaphyllum* (f. sp. niveum), *Gynostemma pentaphyllum* leaf spot pathogen ( Alternaria tenuissima ), Jujube fruit shrinkage pathogen ( Alternaria alternata ), Jujube anthracnose bacteria ( Colletotrichum gloeosporiode ), Jujube black skin fungus ( Colletotrichum nymphaeae ); Wheat stem rot fungus ( Fusarium pseudograminearum ), wheat root rot pathogen ( Bipolaris sorokiniana ), wheat scab ( Fusarium graminearum ), the pathogen of Verticillium wilt in Cotinus coggygria ( Verticillium dahliae ), Euonymus anthracnose bacteria ( Colletotrichum siamense One or more of the diseases caused by the edible fungus, the pathogen of which is *Oomycetes ootheca* (a spider web pathogen). Cladobotryumvarium ) and shiitake mushroom green mold ( Trichoderma atroviride One or two of them.

7. The application according to claim 5, characterized in that, The plant fungal diseases mentioned are one or more of the following: strawberry powdery mildew, strawberry anthracnose, cucumber root rot, and wheat stem base rot.

8. The application of the microbial inoculant according to claim 2 in promoting plant growth, characterized in that, The plant in question is cucumber and / or wheat.

9. A method for controlling plant diseases and promoting growth, characterized in that, The microbial agent described in claim 2 is applied to the plant or its growing environment by spraying, watering or seed dressing; 1) When used to control strawberry powdery mildew and anthracnose, spray the fungicide at a ratio of 1:100-1:300 and apply it every 7-10 days from the strawberry seedling stage to the flowering stage. 2) When used to prevent and control cucumber root rot and promote cucumber growth, dilute the inoculant at a ratio of 1:200-1:400 by irrigation and apply it once a week after cucumber transplanting for 2-3 consecutive times. 3) When used to prevent and control wheat stem base rot and promote wheat growth, seed dressing or seedling irrigation can be used. When dressing seeds, the dosage of the inoculant is 10-20 mL of original inoculant solution per 100 g of seeds. When irrigating seedlings, dilute the inoculant at a ratio of 1:300-1:500 and apply it along the roots.

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