Bacillus subtilis, microbial agent thereof and application of bacillus subtilis in prevention and treatment of cucumber bacterial angular leaf spot

Abstract

The invention provides bacillus subtilis, a microbial agent of the bacillus subtilis and application of the bacillus subtilis to prevention and treatment of cucumber bacterial angular leaf spot. The bacillus subtilis is a bacillus subtilis strain A45, and the preservation number is CGMCC No.33289. The invention further provides a preparation method of the bacillus subtilis. The microbial agent of the bacterial strain can be used for efficiently inhibiting the growth of the Pseudomonas amygdali pv.lachrymans (Pseudomonas amygdali pv.lachrymans) of the cucumber bacterial angular leaf spot pathogen, and can be used for efficiently inhibiting the growth of the Pseudomonas amygdali pv.lachrymans. The strain code not only can be used for synthesizing known gene clusters of antibacterial substances such as abeleventin, bacitene, tripeptide tripeptide, catechol siderophore, pexamic acid, subtilosin A, lysobacin and the like, but also can be used for synthesizing a new gene cluster of surfactin, 1-carbapaen-2-em-3-carboxylic acid and tylosin A as well as a gene cluster synthesized by two brand-new terpenoids. The microbial agent disclosed by the invention not only can prevent cucumber angular leaf spot, but also has a remarkable promoting effect on cucumber growth.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of plant diseases, and particularly relates to a strain of Bacillus subtilis, its microbial agent and its application in controlling cucumber bacterial angular leaf spot disease. Background Art

[0002] Cucumber bacterial angular leaf spot disease is one of the common and serious diseases in cucumber production. Cucumber angular leaf spot disease can occur and cause damage from the seedling stage to the adult plant stage. The disease onset time is short, and it spreads quickly, often causing serious economic losses. With the increase in the protected cultivation area of cucumbers, and the fact that the protected cultivation environment is conducive to the occurrence and prevalence of bacterial diseases, the angular leaf spot disease shows an increasing trend year by year. Currently, it has become one of the main bacterial diseases in protected cucumber production.

[0003] This disease is mainly caused by Pseudomonas amygdalipv. lachrymans (Pal). The original name of this pathogen was Pseudomonas syringae pv. lachrymans (Smith & Bryan) Yong, Dye & Wilkie (also known as Pseudomonas syringae pv. lachrymans). The pathogen mainly overwinters on seeds and can survive for more than two years. It can also overwinter in the soil with diseased residues. It can invade through stomata, lenticels, hydathodes and wounds and spread by dripping and splashing through rainwater, watering, water droplets on the shed roof, dew and guttation at the leaf margin. Currently, chemical pesticides are mainly relied on for prevention and control. When preventing, preparations containing zhongshengmycin, kasugamycin or copper quinolate can be selected; when controlling, multiple applications of pesticides are required, and pesticides with different action mechanisms should be selected for rotation, such as 40% thiazole zinc suspension, 3% cymoxanil microemulsion, 5% allicin microemulsion, 77% copper hydroxide wettable powder, 30% succinic acid copper wettable powder, 20% thiodiazole copper suspension, etc. Bacillus subtilis, Paenibacillus polymyxa, Bacillus thuringiensis, Bacillus licheniformis, Bacillus velezensis, etc. have been widely used in the prevention and control of plant diseases. However, there is still relatively little research on the biological control of cucumber bacterial angular leaf spot. Raupach G S and Kloepper J W (2000) found that the plant growth-promoting rhizobacteria (PGPR) Bacillus pumilus INR7, Curtobacterium flaccumfaciens ME1 and Bacillus subtilis GB03, when used alone or in combination to treat cucumber seeds, could all reduce the disease index of cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans to varying degrees, with the highest control effect being 57.37% (Plant disease, 2000, 84(10): 1073-1075). Li Baoju et al. from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, invented and announced that when spraying at the stage of two true leaves and one heart of cucumber, 1×10 85 mL of the Bacillus velezensis ZF145 bacterial suspension at CFU / mL can achieve a pot control effect of 71.10% against cucumber bacterial angular leaf spot caused by Pseudomonas amygdalipv. lachrymans (Pal) (CN112746046B). Summary of the Invention

[0004] The object of the present invention is to provide a strain of Bacillus subtilis, its microbial agent, and its application in controlling cucumber bacterial angular leaf spot.

[0005] The present invention is achieved as follows:

[0006] The present invention provides a strain of Bacillus subtilis strain A45 with a preservation number of CGMCC No. 33289.

[0007] The present invention provides the application of Bacillus subtilis in controlling plant diseases or in preparing a microbial agent for controlling plant diseases, where the plant disease is cucumber bacterial angular leaf spot caused by Pseudomonas amygdalipv. lachrymans.

[0008] The present invention also provides a microbial agent, which contains Bacillus subtilis with a preservation number of CGMCC No. 33289 or contains the Bacillus subtilis A45 and its metabolites.

[0009] The microbial agent provided by the present invention is prepared by the following method:

[0010] (1) Inoculate the freshly activated A45 slant strain into a shake flask seed medium and culture it on a shaker at 28 - 32°C and 180 - 200 rpm for 12 - 16 h;

[0011] (2) Inoculate the cultured shake flask seeds into a seed tank at an inoculation amount of 0.3 - 0.5%, culture at 28 - 32°C, with an aeration rate of 0.5 - 1.0 v.v.m, for 5 - 8 h;

[0012] (3) Then transfer the seeds in the seed tank to a fermentation tank at an inoculation amount of 3.0 - 6.5%, culture at 28 - 32°C, with an aeration rate of 0.6 - 1.2 v.v.m, ferment for 20 - 30 h, and use the spore formation rate greater than 95% as the discharging standard. After fermentation is completed, the fermentation broth is obtained;

[0013] (4) Use the fermentation broth to prepare a liquid microbial agent or a solid soluble microbial agent;

[0014] Among them, the shake flask seeds use PCB medium, and the medium compositions used in the seed tank and fermentation tank are (g / L): corn steep liquor 80 - 100, soybean meal 5 - 10, starch 40 - 60, sodium chloride 1.5 - 3.0, manganese sulfate 0.1 - 0.2, pH 7.0 ± 0.2, sterilized at 121 °C for 30 min.

[0015] For the microbial inoculant of the present invention, in the step (3), the discharging standard is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%.

[0016] For the microbial inoculant of the present invention, in the step (4), the steps for preparing the liquid inoculant are as follows: adding xanthan gum with a final concentration of 0.1 - 0.3%, or gellan gum with a final concentration of 0.02 - 0.05%, or polyvinylpyrrolidone K90 (PVP K90) with a final concentration of 0.3 - 0.5% to the fermentation broth obtained in the step (3), mixing evenly and then aseptically filling to obtain the liquid inoculant; in the step (4), the steps for preparing the solid soluble inoculant are as follows: spray-drying the fermentation broth obtained in the step (3) and then adding soluble carriers such as Jia Yifen and dextrin to obtain the soluble solid inoculant.

[0017] For the application of the Bacillus subtilis of the present invention in preventing and controlling plant diseases, when transplanting, dip the roots with the microbial inoculant, dilute the liquid inoculant 100 - 200 times for use, and dilute the soluble solid inoculant 300 - 400 times for use.

[0018] Advantages of the present invention:

[0019] 1. The Bacillus subtilis A45 microbial inoculant provided by the present invention can efficiently inhibit the growth of Pseudomonas amygdali pv. lachrymans, the causative agent of cucumber bacterial angular leaf spot.

[0020] 2. The mechanism by which the Bacillus subtilis A45 microbial inoculant provided by the present invention efficiently inhibits the growth of Pseudomonas amygdali pv. lachrymans, the causative agent of cucumber bacterial angular leaf spot, is that in addition to the known gene clusters encoding the synthesis of antibacterial substances such as fengycin, bacilysin, subtilosin A, catechol-type siderophore, putrebactin, subtilosin A and lysobacterin, this strain also synthesizes a new gene cluster for surfactin, 1-carbapen-2-em-3-carboxylic acid, tylosin A, and two brand-new terpene compound synthesis gene clusters, providing technical support and theoretical basis for the prevention and control of cucumber angular leaf spot.

[0021] 3. The Bacillus subtilis A45 microbial inoculant of the present invention can not only prevent cucumber angular leaf spot caused by Pseudomonas amygdali pv. lachrymans, but also significantly promote the growth of cucumbers.

[0022] Biological Deposit Description

[0023] Bacillus subtilis A45 was isolated from soil samples collected from cucumber bacterial angular leaf spot-prone plots in Quzhou County, Hebei Province. It was deposited at the China General Microbiological Culture Collection Center on January 3, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33289. Description of the Drawings

[0024] Figure 1 It is a diagram showing the antagonistic effect of the PCB fermentation supernatant of the Bacillus isolate A45 on Pseudomonas amygdali pv. lachrymans in Example 1.

[0025] Figure 2 It is the spore formation stage during fermentation, with a spore formation rate > 95%.

[0026] Figure 3 It is the spore shedding stage during fermentation, with a spore shedding rate > 90%. Detailed Description of the Invention

[0027] 1. Isolation, Screening and Identification of Strains

[0028] Isolation and Screening of Strains

[0029] The plate dilution method was used to isolate Bacillus strains from cucumber rhizosphere soil samples collected from severely angular leaf spot-infected areas, and further screen for antagonistic bacteria against Pseudomonas amygdali pv. lachrymans. A total of 65 antagonistic bacteria (A1 - A65) were finally obtained. The inhibitory effect of the PCB fermentation broth supernatant of each isolate on cucumber bacterial angular leaf spot was determined by the cylinder-plate method. Among them, A45 had the largest inhibition zone, reaching 30.0 mm.

[0030] Strain Identification

[0031] First, the preliminary identification of A45 was carried out based on the phylogenetic analysis of the 16S rRNA gene sequence, and then whole-genome sequencing was performed to determine its taxonomic status at the species level according to the dDDH and ANI values.

[0032] Example 1 Isolation and Screening of Strains

[0033] PCB Medium (g / L): Peptone 5.0, Yeast Extract Powder 2.5, Glucose 1.0, pH: 7.4 ± 0.2, sterilized at 121 °C for 30 min. Adding 15 g / L of agar to the PCB medium gives the PCA medium.

[0034] Soil samples were collected from high-incidence cucumber bacterial angular leaf spot plots in Quzhou County, Hebei Province. 10 g of soil samples were added to a triangular flask containing 10 - 25 glass beads and 90 mL of sterile water, and the soil samples were shaken to disperse them to prepare a soil suspension. After boiling the soil suspension in a water bath for 5 min, it was diluted into three gradients of 10-4, 10-5, and 10-6. 0.2 mL of the diluted soil suspension was pipetted onto the surface of a PCA plate medium, spread evenly, and left standing for 5 min. The culture dishes were inverted for cultivation, the separation and cultivation temperature was 30 °C, and the growth of soil-separated microbial colonies was observed within 24 - 48 h of cultivation time. Well-grown single colonies were picked and transferred to a PCA slant medium and cultivated at 30 °C for 24 h.

[0035] Using an inoculation loop, 1 loop of fresh culture of the soil-separated strain was inoculated into a 500 mL triangular flask containing 100 mL of PCB medium. After culturing at 30 °C and 180 - 200 rpm for 24 h, the supernatant was taken by aseptic operation after centrifugation, and the antagonistic effect of the supernatant of the fermentation broth of each strain against Pseudomonas amygdali pv. lachrymans was determined by the cylinder-plate method. A total of 65 isolates with inhibition zones were screened. Among them, 18 isolates had inhibition zones directly greater than 20 mm (Table 1), and the inhibition zone of A45 was the largest, reaching 30 mm ( Figure 1 as shown).

[0036] Table 1 Sizes of inhibition zones of fermentation supernatants of antagonistic strains screened

[0037]

[0038] Example 2 Strain identification

[0039] Extraction of colony PCR template: 30.0 μL of cell lysate was added to a PCR tube, and a single colony was picked into the PCR tube and lysed at 100 °C for 10 min. Centrifuged at 10000 rpm for 5 min, and the supernatant was taken as the template for PCR amplification.

[0040] The composition of the PCR amplification reaction system is shown in Table 2; the PCR amplification program was: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, 30 cycles, and post-extension at 72 °C for 5 min. After electrophoresis detection of the PCR products, Sanger sequencing was performed.

[0041] Table 2 PCR reaction system

[0042]

[0043] The sequencing results were aligned with the sequences of all type strains in the Blast and GenBank nucleic acid databases, and it was found that the 16S rRNA gene sequences of A45 were exactly the same as those of the type strains ATCC6051 and str168 of Bacillus subtilis.

[0044] The measured 16S rRNA gene sequence of A45 is shown in SEQ ID No.1.

[0045] Furthermore, the whole genome shotgun (WGS) strategy was adopted to construct libraries with different insert fragments. Using the second-generation sequencing technology (Next-Generation Sequencing, NGS), based on the Illumina NovaSeq sequencing platform, and at the same time using the third-generation single molecule sequencing technology, based on the Oxford Nanopore ONT sequencing platform, the whole genome sequencing of these libraries was carried out respectively. The genome size of A45 is 4215636 bp, the GC content is 43.51%, circular chromosome, and no plasmid. The A45 genome was submitted to the Type Strain Genome Server (https: / / tygs.dsmz.de / ) for genome alignment, and it was found that the dDDH value of this strain and Bacillus subtilis ATCC6051T was 100%; submitting the genome sequences of the two to https: / / www.ezbiocloud.net / tools / ani for calculation found that the ANI value was also 100%. The above results indicate that the taxonomic status of A45 is Bacillus subtilis.

[0046] 2. Analysis of genes encoding secondary metabolite synthesis

[0047] Use antiSMASH (https: / / antismash.secondarymetabolites.org) to search for genes encoding secondary metabolites from the determined A45 genome sequence and analyze the mechanism of A45 against the pathogen Pseudomonas amygdali pv. lachrymans of cucumber angular leaf spot.

[0048] Example 3 Known gene clusters encoding the synthesis of known antibacterial active products

[0049] It was found that A45 encodes multiple antibacterial-related active gene clusters. The secondary metabolite-encoding genes identical to known gene sequences include the gene clusters for Fengycin, Bacillaene, Sublancin, Bacillibactin, Pulcherriminic acid, subtilosin A, and Bacilysin.

[0050] Example 4 Antibacterial active products encoded by the newly synthesized gene cluster

[0051] It was found that the similarity between the Surfactin synthesis gene cluster encoded by A45 and known genes is up to 82%, the similarity between the 1-carbapen-2-em-3-carboxylic acid synthesis gene cluster and known genes is up to 16%, and the similarity between the thailanstatin A synthesis gene cluster and known genes is up to 10%. In addition, A45 also encodes two brand-new terpene compound synthesis gene clusters (see Table 3).

[0052] Table 3 Secondary metabolite synthesis gene clusters encoded by the A45 genome discovered by antiSMASH analysis and their sequence similarities with known genes

[0053]

[0054] 3. Fermentation method and preparation of microbial agents

[0055] Inoculate the freshly activated A45 slant culture into a shake flask seed medium (PCB medium), and culture it on a shaker at 28 - 32°C and 180 - 200 rpm for 12 - 16 h. Inoculate the cultured shake flask seeds into a seed tank at an inoculation amount of 0.3 - 0.5%, culture at 28 - 32°C, with an aeration rate of 0.5 - 1.0 v.v.m for 5 - 8 h; then transfer the seeds in the seed tank to a fermentation tank at an inoculation amount of 3.0 - 6.5%, culture at 28 - 32°C, with an aeration rate of 0.6 - 1.2 v.v.m, and ferment for 20 - 30 h. Use the condition that the spore formation rate is greater than 95% as the standard for discharging the tank. The preferred condition is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%.

[0056] Among them, the shake flask seeds use PCB medium, and the media used for the seed tank and fermentation tank have the following composition (g / L): corn steep liquor 80 - 100, soybean meal 5 - 10, starch 40 - 60, sodium chloride 1.5 - 3.0, manganese sulfate 0.1 - 0.2, pH 7.0 ± 0.2, sterilized at 121°C for 30 min.

[0057] After fermentation, xanthan gum with a final concentration of 0.1 - 0.3%, or gellan gum with a final concentration of 0.02 - 0.05%, or polyvinylpyrrolidone K90 (PVP K90) with a final concentration of 0.3 - 0.5% is added to the fermentation broth. After thorough mixing, it is aseptically filled to obtain a liquid microbial agent. After spray-drying the fermentation broth, soluble carriers such as filler powder and dextrin are added to reach an appropriate number of bacteria, obtaining a soluble solid microbial agent (powder).

[0058] Example 5 Preparation of Liquid Microbial Agent

[0059] (1) The preserved A45 strain is inoculated onto a PCA slant and cultured at 28 °C for 20 h for activation. Three loops of the activated A45 slant strain are inoculated into a 3 L Erlenmeyer flask containing 500 mL of seed medium (PCB medium).

[0060] It is cultured on a shaker at 28 °C and 200 rpm for 12 h to obtain a shake flask seed solution.

[0061] (2) The cultured shake flask seed is inoculated into a 1.5-ton seed tank containing 0.8 tons of fermentation medium at an inoculation amount of 0.3%. It is cultured at 28 °C, with an aeration rate of 0.8 v.v.m and a stirring speed of 200 rpm for 6.5 h to obtain the seed tank seed.

[0062] (3) The cultured seed tank seed is transferred to a 20-ton fermentation tank containing 14 tons of fermentation medium at an inoculation amount of 5%. It is fermented at 28 °C, with an aeration rate of 0.9 v.v.m and a stirring speed of 160 rpm for 24 h to obtain the fermentation broth. At the end of fermentation, the spore formation rate is 100% ( Figure 2 as shown), and the spore shedding rate is 90% ( Figure 3 as shown).

[0063] (4) 0.2% (m / v) xanthan gum is added to the fermentation broth and stirred until completely dissolved to obtain a liquid microbial agent. Samples are taken, diluted and spread on PCA plates, and cultured at 28 °C for 20 h for counting. The effective viable count of the liquid microbial agent is 2.8×

[0064] 10 10 CFU / mL.

[0065] Among them, the media used in the seed tank and fermentation tank are (g / L): corn steep liquor 100, soybean meal 7.5, starch 45, sodium chloride 2.0, manganese sulfate 0.1, pH 7.2, sterilized at 121 °C for 30 min.

[0066] After filling the liquid microbial agent and storing it at room temperature, the survival rates are measured at 3 months, 6 months, and 12 months, which are 100%, 98.6%, and 95.8% respectively.

[0067] Example 6 Preparation of Soluble Solid Microbial Agent

[0068] The fermentation liquid obtained in step (3) of Example 5 was spray-dried at an air inlet of 180°C and an air outlet of 90°C to obtain a solid bacterial agent powder. The effective bacterial content of the powder was 3.1×10 11 CFU / g.

[0069] Add Jiayi powder or dextrin in the ratio of 1 part of solid bacterial agent powder to 2 parts of soluble carrier, mix thoroughly to obtain a soluble solid bacterial agent with an effective bacterial content of 100 billion / g.

[0070] 4. Usage and dosage

[0071] A45 microbial agent is used to prevent and treat bacterial angular spot of cucumber caused by Pseudomonas amygdalipv. lachrymans. It can be applied from transplanting to maturity, and is best used before and in the early stages of the disease. Liquid agents can be diluted 150-250 times, and the best effect is achieved by diluting 200 times for spraying. Solid agents can be diluted 600-1000 times, and the best effect is achieved by diluting 800 times for spraying. If dipping the roots during transplanting, dilute the liquid agent 100-200 times, and the solid agent 300-400 times.

[0072] Example 7A45 Liquid bacterial agent spray control effect on cucumber bacterial angular spot disease

[0073] When the potted cucumbers grew to two leaves and one heart, 1×10 8 CFU / mL cucumber bacterial angular leaf spot pathogen Pseudomonas amygdali pv. lachrymans suspension, sprayed 24 hours later, after the leaf surface was dried, Bacillus subtilis A45 liquid agent was diluted 200 times and sprayed on the cucumber plants. The clear water control was only inoculated with pathogenic bacteria without spraying A45 agent. After inoculation, the cucumber seedlings were placed in a moisturizing cabinet with a relative humidity of 80±5% and a temperature of 26-28℃ for moisturizing culture for 24 hours, and then transferred to a normal seedling greenhouse for culture, and repeated 3 times. 4 days after inoculation, the clear water control was fully diseased, and the disease index and control effect were calculated.

[0074] Bacterial angular leaf spot of cucumber: graded according to the surface area of leaves occupied by angular leaf spot.

[0075] Level 0: no lesions;

[0076] Level 1: 0%-5%;

[0077] Level 3: 5%-25%;

[0078] Level 5: 25%-50%;

[0079] Level 7: 50%-75%;

[0080] Level 9: Above 75%.

[0081] Disease index = 100 × Σ (number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × representative value of the highest level of disease)

[0082] Control effect (%) = 100 × (control disease index - treated disease index) / control disease index.

[0083] Table 4 Control effect of spraying A45 liquid bactericide on cucumber bacterial angular leaf spot in pot experiments

[0084] Treatment Usage concentration and application method Disease index Control effect (%) A45 liquid bacterium agent Dilute 200 times and spray 5.52±0.25 80.74±1.33 Control Spray with clear water 28.72±1.33 ---

[0085] Example 8 Control effect and growth promotion effect of dipping roots with A45 solid bactericide on transplanted cucumber bacterial angular leaf spot

[0086] Select cucumber seedlings with 3 true leaves. Before transplantation, the control group was not treated with anything, the model group dipped the roots with a bacterial suspension of Pseudomonas amygdali pv. lachrymans at a concentration of 1×10 8 CFU / mL, and the experimental group dipped the roots with a composite bacterial suspension of Pseudomonas amygdali pv. lachrymans at a concentration of 1×10 8 CFU / mL and 1 / 300 of A45 solid bactericide. Twenty days after transplantation, the disease index and control effect were calculated, the length of the cucumber vines was measured, the number of leaves was counted, and the growth promotion effect of A45 on cucumbers was observed.

[0087] The method for grading angular leaf spot, calculating the disease index and control effect was the same as in Example 7.

[0088] Table 5 Control effect of dipping roots with A45 solid bactericide on transplanted cucumber bacterial angular leaf spot

[0089]

[0090] Comparative Example 1: Comparative experiment of different fermentation media

[0091] (1) Inoculate the freshly activated A45 slant strain into the shake flask seed medium (PCB medium), and culture it on a shaker at 28°C and 200 rpm for 12 h.

[0092] (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation amount of 0.5%, culture at 28°C, and the aeration rate is 0.7 v.v.m for 10 h;

[0093] (3) Then transfer the seeds in the seed tank to the fermentation tank at an inoculation amount of 5.5%, culture at 28°C, and the aeration rate is 0.8 v.v.m for 30 h.

[0094] Among them, the seed media for the seed tank and the fermentation media for the fermentation tank are respectively the following 3 types:

[0095] Medium 1 (g / L): Corn steep liquor 100, soybean meal 7.5, starch 45, sodium chloride 2.0, manganese sulfate 0.1, pH 7.2, sterilized at 121 °C for 30 min.

[0096] Medium 2 (g / L): Corn steep liquor 90, soybean meal 10.0, starch 55, sodium chloride 3.0, manganese sulfate 0.2, pH 7.2, sterilized at 121 °C for 30 min.

[0097] Medium 3 (g / L): Peptone 25.0, soybean meal 20.0, sucrose 50.0, sodium chloride 0.8, manganese sulfate 0.4, pH 7.2, sterilized at 121 °C for 30 min.

[0098] After fermentation, 10000 rpm, 4 °C centrifugation for 6 min was performed on the fermentation broth, and then the centrifuged supernatant was filtered through a 0.22 μm microporous filter membrane. The antibacterial effect of the filtrate against the pathogen of cucumber bacterial angular leaf spot, Pseudomonas amygdali pv. lachrymans (Pal), was determined by the Oxford cup method. The results are shown in Table 6. The larger the diameter of the antibacterial circle, the better the antibacterial effect.

[0099] Table 6 Antibacterial effects of fermentation broths obtained by fermenting different fermentation media under the same conditions against Pseudomonas amygdali pv. lachrymans (Pal)

[0100] Culture medium Diameter of inhibition zone (mm) 1 38 2 36 3 14

[0101] Comparative Example 2:

[0102] (1) Inoculate the freshly activated A45 slant strain into the shake flask seed medium (PCB medium), and culture it on a shaker at 30 °C and

[0103] 200 rpm for 12 h.

[0104] (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation amount of 0.5%, culture at 30 °C, and the aeration rate is 0.8 v.v.m for 8 h;

[0105] (3) Then transfer the seeds in the seed tank to the fermentation tank at an inoculation amount of 5.0%, ferment at 30 °C and an aeration rate of 1.0 v.v.m for 25 - 30 h, and discharge the tank under the following three different conditions. The first: The spore formation rate is 100% and the spore shedding rate is 95%; the second: The spore formation rate is 96% and no shedding; the third: The spore formation rate is 85% and no shedding.

[0106] Among them, the seed tank medium and the fermenter medium are both: corn steep liquor 100, soybean meal 7.5, starch 45, sodium chloride 2.0, manganese sulfate 0.1, pH 7.2, sterilized at 121°C for 30 min.

[0107] After fermentation, count the number, and then count again after storing at room temperature for 6 months. The results are shown in Table 7.

[0108] Table 7 Effects of different fermentation end-point criteria on the survival rate of A45 fermentation broth during storage at room temperature

[0109] Standard for fermentation end point (discharging the tank) Survival rate after storing at room temperature for 6 months (%) Sporulation rate 100% and spore shedding rate 95% 99.6±0.25 Sporulation rate 96%, not shed 89.7±2.28 Sporulation rate 85%, not shed 65.7±3.62

Claims

1. A strain of Bacillus subtilis strain A45, with the preservation number of CGMCC No. 33289.

2. The application of the Bacillus subtilis described in claim 1 in the prevention and control of plant diseases or in the preparation of a microbial agent for preventing and controlling plant diseases, wherein the plant disease is cucumber bacterial angular leaf spot caused by Pseudomonas amygdali pv. lachrymans.

3. A microbial inoculant, characterized in that, Containing Bacillus subtilis A45 with the preservation number of CGMCC No. 33289 or containing the Bacillus subtilis A45 and its metabolites.

4. The microbial inoculant according to claim 3, characterized in that Prepared by the following method: (1) Inoculate the freshly activated A45 slant strain into a shake flask seed medium, and culture it on a shaker at 28 - 32 °C and 180 - 200 rpm for 12 - 16 h; (2) Inoculate the cultured shake flask seeds into a seed tank at an inoculation amount of 0.3 - 0.5%, at 28 - 32 °C, with an aeration rate of 0.5 - 1.0 v.v.m, and culture for 5 - 8 h; (3) Then transfer the seeds in the seed tank to a fermentation tank at an inoculation amount of 3.0 - 6.5%, at 28 - 32 °C, with an aeration rate of 0.6 - 1.2 v.v.m, and ferment for 20 - 30 h. When the spore formation rate is greater than 95% as the discharge standard from the tank, the fermentation is completed to obtain the fermentation broth; (4) Use the fermentation broth to prepare a liquid microbial agent or a solid soluble microbial agent; Among them, the shake flask seeds use PCB medium, and the media used in the seed tank and fermentation tank are composed of (g / L): corn steep liquor 80 - 100, soybean meal 5 - 10, starch 40 - 60, sodium chloride 1.5 - 3.0, manganese sulfate 0.1 - 0.2, pH 7.0 ± 0.2, sterilized at 121 °C for 30 min.

5. The microbial inoculum according to claim 4, characterized in that, In the step (3), the discharge standard from the tank is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%.

6. The microbial inoculant according to claim 4, wherein In the step (4), the steps for preparing the liquid microbial agent are: adding xanthan gum with a final concentration of 0.1 - 0.3%, or gellan gum with a final concentration of 0.02 - 0.05%, or polyvinylpyrrolidone K90 with a final concentration of 0.3 - 0.5% to the fermentation broth obtained in the step (3), mixing evenly and then aseptically filling to obtain the liquid microbial agent; in the step (4), the steps for preparing the solid soluble microbial agent are: spray - drying the fermentation broth obtained in the step (3) and then adding soluble carriers such as calcium superphosphate and dextrin to obtain the soluble solid microbial agent.

7. Use of the Bacillus subtilis according to claim 2 in the prevention and control of plant diseases, characterized in that, When transplanting, dip the roots with the microbial agent described in claim 4. The liquid microbial agent is diluted 100 - 200 times for use, and the soluble solid microbial agent is diluted 300 - 400 times for use.

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