Bacillus velezensis strain and application thereof

Abstract

The invention belongs to the field of development and utilization of microbial resources and germplasm resources, and particularly relates to a bacillus velezensis strain and application thereof, the classification name of the bacillus velezensis strain is bacillus velezensis ZJU-117, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2025294, and the preservation date is February 25, 2025. The bacillus velezensis strain provided by the invention has a prevention and treatment effect up to 40% on wheat stem rot, and can simultaneously prevent and treat plant pathogenic bacterial diseases such as carrot soft rot, bacterial wilt, rice bacterial leaf streak, wheat bacterial leaf streak, rice bacterial leaf blight, tomato bacterial leaf spot and the like; the composition has a wide prevention and treatment range and a good prevention and treatment effect.

Description

Technical Field

[0001] The invention belongs to the field of development and utilization of microbial germplasm resources, and specifically relates to a Bacillus velez strain and application thereof. Background Art

[0002] Wheat is one of the three major food crops in the world and the most important food crop in my country. With population growth and economic development, the demand for wheat continues to grow. Wheat stem rot is a soil-borne disease that is mainly caused by Fusarium graminearum infecting the base of wheat stems and can cause harm throughout the entire growth period. In recent years, due to factors such as the extensive return of straw to the field and the continuous accumulation of fungal sources, the scope of the disease has continued to expand and has caused serious impacts. This not only seriously threatens the stable yield and harvest of wheat, but also poses a serious threat to the quality and safety of wheat. Wheat stem rot can cause browning and easy breakage at the base of the stem, and eventually lead to white ears of wheat plants, unfull grains or even seedless grains, which greatly affects the yield of wheat. In addition, after being infected by pathogens, a series of secondary metabolites mainly composed of trichothecenes will be produced in the plant body. Ingestion of wheat containing such toxins will pose a threat to the life and health of humans and other mammals.

[0003] At present, the prevention and control of wheat stem rot mainly focuses on "one turning, one mixing and one spraying", adopting a comprehensive prevention and control technology that combines chemical seed mixing, disease-resistant varieties and cultivation measures. However, due to the lack of disease-resistant varieties, chemical control is still the most common prevention and control method for wheat stem rot. Although chemical control can control the occurrence of diseases and pests to a certain extent, it also has disadvantages such as environmental pollution, increased drug resistance, damage to non-target organisms and short-term effects.

[0004] As an alternative to chemical fungicides, biological control is gradually becoming an important and active research field in the prevention and control of plant diseases, especially soil-borne diseases. More and more biocontrol strains, especially Bacillus, are being discovered as biocontrol agents to control wheat stem rot. The biocontrol mechanism of Bacillus usually includes the following three aspects: (1) producing secondary metabolites to fight pathogens and pests; (2) improving the host plant microbial community and promoting host nutrition and growth; (3) stimulating plant host defense and producing systemic resistance (induced systemic resistance, ISR). At present, there are few reports on the use of Bacillus as a biocontrol agent to simultaneously control wheat stem rot, carrot soft rot, bacterial wilt, rice bacterial leaf streak, wheat bacterial leaf streak, rice white leaf blight and tomato bacterial leaf spot. Summary of the invention

[0005] In view of the demand for biological fungicides in the prior art, the present invention provides a Bacillus Velezii strain and its application, and the specific technical scheme is as follows: In a first aspect, the present invention provides a strain of Bacillus Velezii, which is classified as Bacillus Velezii. Bacillus velezensis ZJU-117, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCCNO: M 2025294, and the preservation date is February 25, 2025.

[0006] In a second aspect, the present invention provides a biocontrol agent, wherein the active ingredient of the biocontrol agent is the above-mentioned Bacillus Velezii strain.

[0007] Furthermore, in the biocontrol agent, the concentration of Bacillus Velezii is 1×10 7 ~1×10 8 cfu / mL.

[0008] In a third aspect, the present invention provides a method for preparing the above-mentioned biocontrol agent, comprising the following steps: (1) inoculating the Bacillus Velezii into LB liquid culture medium to obtain seed liquid; (2) The obtained seed solution was inoculated into the culture medium for expansion and the concentration of Bacillus Velezii in the fermentation broth was adjusted to 1×10 7 ~1×10 8 cfu / mL, and the biocontrol agent is obtained.

[0009] Furthermore, the inoculation amount of the seed liquid is 1% of the total volume of the seed liquid after inoculation.

[0010] In a fourth aspect, the present invention provides a biofungicide, comprising the above-mentioned biocontrol agent and Jinggangmycin; the concentration of the biocontrol agent is 1×10 7 ~1×10 8 cfu / mL; the volume ratio of the biocontrol agent to Jinggangmycin is 50~500mL:1.5g.

[0011] In the process of applying the biocontrol agent prepared by Bacillus Velezii to prevent and control wheat base stem rot, it was found that the biocontrol agent has a good control effect on wheat base stem rot indoors, and the control effect can reach more than 65%. In the field, the biocontrol agent is used in combination with Jinggangmycin. Compared with the use of Jinggangmycin alone, the control effect can be increased by up to 35%, indicating that the biocontrol agent and biological fungicide provided by the present application have good application prospects in biological control of diseases.

[0012] In a fifth aspect, the present invention provides the use of the above-mentioned strain, or the above-mentioned biocontrol agent, or the above-mentioned biofungicide in antagonizing Fusarium graminearum.

[0013] In a sixth aspect, the present invention provides the use of the above-mentioned strain, or the above-mentioned biocontrol agent, or the above-mentioned biological fungicide in controlling wheat basal rot disease.

[0014] Further, the application method is: during the green-reverting stage of wheat, evenly spray the strain, biocontrol agent or biological fungicide on the root base of wheat.

[0015] Further, the wheat varieties are Tianhe 6 and / or Jimai 22.

[0016] In a seventh aspect, the present invention provides the use of the above-mentioned strain, or the above-mentioned biocontrol agent, or the above-mentioned biological fungicide in controlling carrot soft rot, bacterial wilt, rice bacterial leaf streak, rice bacterial blight, wheat bacterial leaf streak and / or tomato bacterial leaf spot.

[0017] In this application, it is found that the ZJU-117 strain not only has an antagonistic effect against Fusarium pseudograminearum, but also has an antagonistic effect against the pathogens of various diseases occurring on different crops, indicating that the ZJU-117 strain and the biocontrol agent and biological fungicide prepared therefrom have a certain control effect on these diseases.

[0018] Further, the pathogen of carrot soft rot is Pectobacterium carotovorum subsp. carotovorum Pcc ; the pathogen of bacterial wilt is Ralstonia solanacearum GMI1000; the pathogen of rice bacterial leaf streak is Xanthomonas oryzae pv. oryzicola RS105; the pathogens of rice bacterial blight are Xanthomonas oryzae pv. oryzae PXO99A and / or Xanthomonas oryzae pv. oryzae GX-1; the pathogen of wheat bacterial leaf streak is Xanthomonas translucens pv. undulosa NXtc01; the pathogen of tomato bacterial leaf spot is Pseudomonas syringae pv. tomato DC3000.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention screened a strain of Bacillus velezensis, which has a control effect of up to 40% on wheat basal rot disease, and can simultaneously control plant pathogenic bacterial diseases such as carrot soft rot, bacterial wilt, rice bacterial leaf streak, wheat bacterial leaf streak, rice bacterial blight and tomato bacterial leaf spot, with a wide control range and good control effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the colony morphology diagram of the biocontrol agent ZJU-117 in Example 1 and the antagonistic effect diagram against Fusarium pseudograminearum; wherein, A is the colony morphology diagram of the biocontrol agent ZJU-117 on the culture medium, and B is the antagonistic effect diagram of the biocontrol agent ZJU-117 against Fusarium pseudograminearum.

[0021] Figure 2Graph showing the determination results of EC50 for the inhibition of the mycelial growth of Fusarium pseudograminearum by strain ZJU-117 in Example 3; among them, A is a diagram showing the state when the mycelia of Fusarium pseudograminearum cover the blank control plate, B is a diagram showing the size state of the colonies of Fusarium pseudograminearum in the biocontrol agent plates at each gradient, C is a fitting curve of the correlation between the bacterial content in the plate and the inhibition rate, the horizontal axis is the concentration of strain ZJU-117 Log 10 CFU / mL, and the vertical axis is the diameter (cm) of the colonies of Fusarium pseudograminearum, and R 2 represents the degree of fitting between the estimated value of the trend line and the corresponding actual data.

[0022] Figure 3 Diagram showing the inhibitory effect of the sterile supernatant of the biocontrol agent prepared from the biocontrol strain ZJU-117 on the spores of Fusarium pseudograminearum in Example 3.

[0023] Figure 4 Diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against different strains in Example 4; among them, A is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Pectobacterium carotovorum subsp. carotovorum pcc ; B is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Ralstonia solanacearum GMI1000; C is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Xanthomonas oryzae pv. oryzicola RS105; D is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Xanthomonas oryzae pv. oryzae PXO99A; E is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Xanthomonas oryzae pv. oryzae GX-1; F is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Xanthomonas translucens pv. undulosa NXtc01; G is a diagram showing the antagonistic effect of the biocontrol agent ZJU-117 against Pseudomonas syringae pv. tomato DC3000; H is a histogram showing the diameter of the inhibition zone of the biocontrol agent ZJU-117 against the above 7 plant pathogenic bacteria.

[0024] Figure 5 Diagram showing the control effect of the biocontrol strain ZJU-117 on wheat basal rot in the field of Tianhe 6 and Jimai 22 in Example 5; among them, A is a diagram showing the control effect of the biocontrol strain ZJU-117 on wheat basal rot in the field of Tianhe 6; B is a diagram showing the control effect of the biocontrol strain ZJU-117 on wheat basal rot in the field of Jimai 22.

[0025] Figure 6 Diagram showing the control effect of the biocontrol strain ZJU-117 on potted wheat basal rot of Jimai 22 indoors in Example 6. Detailed implementation methods

[0026] To enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and only represents a part of the embodiments of the present invention, rather than all of the embodiments.

[0027] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in this field and can be obtained through commercial channels. The experimental methods without detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0029] EC50, the half-maximal effective concentration, generally refers to the concentration of a drug or compound required to produce half of the maximum effect on an organism. In the following embodiments, it specifically refers to the strain concentration when the inhibition rate of strain ZJU-117 on the mycelium of Fusarium pseudograminearum reaches 50%.

[0030] The following embodiments relate to the following media and culture solutions: R 2 Formula of Medium R: yeast extract 0.5 g / L, peptone 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 0.04 g / L, sodium pyruvate 0.3 g / L, agar 15 g / L; Formula of 10% TSA Medium: tryptone 1.5 g / L, soy peptone 0.5 g / L, sodium chloride 0.5 g / L, agar 15 g / L.

[0031] Formula of LB Medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, adjust the pH to 7.2.

[0032] Formula of LBGM Medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, 50% glycerol 20 ml / L, 1.51×10 -2 g / L MnSO 4 , adjust the pH to 7.2.

[0033] Formula of WA Solid Plate: peptone 5 g / L, glucose 10 g / L, beef extract 3 g / L, sodium chloride 5 g / L, agar powder 15 g / L, pH = 7.0.

[0034] PDA medium formulation: 200 g / L of potato, 20 g / L of glucose, 10 g / L of agar powder, natural pH.

[0035] CMC liquid medium formulation: 15 g / L of sodium carboxymethyl cellulose, 1 g / L of yeast extract, 1 g / L of ammonium nitrate, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate heptahydrate.

[0036] NA medium: 5 g / L of tryptone, 3 g / L of beef extract, 1 g / L of yeast extract, 10 g / L of sucrose, 12 g / L of agar powder, pH = 7.0. Sterilize at 121 °C for 20 minutes. Omit agar powder for liquid medium.

[0037] KB medium: 20 g / L of peptone, K 2 HPO 4 1.5 g / L, 15 g / L of glycerol, 12 g / L of agar powder, pH = 7.2. Sterilize at 121 °C for 20 minutes. Omit agar powder for liquid medium. 1 mol / L of MgSO 4 ·7H 2 O is sterilized separately and prepared immediately before use, with a ratio of 5 mL / L.

[0038] BG medium: 10 g / L of peptone, 1 g / L of yeast extract, 1 g / L of casein amino acids, 5 g / L of glucose, 12 g / L of agar powder, pH = 7.0. Sterilize at 121 °C for 20 minutes. Omit agar powder for liquid medium.

[0039] Example 1 Obtaining of ZJU-117 Strain 1. Isolation of culturable Bacillus Cut 1 g of diseased tissue with perithecia from the diseased part of the wheat straw and place it in a sterilized mortar for grinding. Add the residue obtained after grinding into a sterilized 50 mL centrifuge tube, add 10 mL of sterile water, and resuspend it with a vortex oscillator. Mix the sample thoroughly under the conditions of a shaker at 30 min and 180 rpm; Pipette 100 μL of the suspension into a 1.5 mL sterile EP tube, and add 900 μL of sterile water to prepare a 10 -1 dilution; Dilute according to the same steps to obtain dilutions with concentration gradients of 10 -2 、10 -3 、10 -4 、10 -5 Concentration gradient dilutions. Pipette 100 μL of the dilution under each gradient and spread it evenly on R 2A, on TSA plates with 10% TSA for separating bacteria (to inhibit the growth of fungi, carbendazim or tebuconazole with a final concentration of 5 ppm can be added to the plates). Place the plates in an incubator at 30 °C and take out the plates after 1 d, 3 d, and 5 d respectively to observe whether colonies grow. Pick single colonies of Bacillus with different morphological sizes and colors and place them on new R 2 A, TSA plates for purification. Pick a purified strain the size of a toothpick tip and culture it overnight in LB medium to obtain the corresponding seed solution. Take 900 μL of the seed solution and add 50% glycerol in a 1:1 ratio, and store it at -80 °C to complete the preservation of the strain. Finally, classify and number according to the sampling location of the sample.

[0040] 2. Determination of the antagonistic activity of strains against Fusarium pseudograminearum on plates Use the plate confrontation culture method to determine the antagonistic activity of Paenibacillus strains against Fusarium pseudograminearum. First, pick Paenibacillus colonies the size of a toothpick tip from the activated bacterial plates and draw two parallel lines on the left and right sides (3 cm from the center of the plate) of the WA solid plate, and culture in an incubator at 30 °C for 24 h. Inoculate Fusarium pseudograminearum stored at 4 °C onto a PDA plate for activation. After the fungus fills the plate, use a sterilized punch to evenly punch circular discs with a diameter of 6 mm from the outer edge of the colony, with the mycelium side facing down, and inoculate the discs in the center of the WA plate, and place it in an incubator at 25 °C for culture. Use the WA plate with Fusarium pseudograminearum inoculated in the center as a blank control. When the edge of the control colony is about to expand to the entire plate, measure the antagonistic distance of Paenibacillus and take pictures for recording. Each treatment is repeated 3 times.

[0041] Method for measuring the antagonistic distance on plates: Measure the vertical distances from the left and right sides and the middle part of the Fusarium pseudograminearum colony to the Paenibacillus strain at the edge of the WA plate (measure the antagonistic distances on both sides, a total of 6 values), and calculate the average antagonistic distance according to the results.

[0042] Evaluate the antagonistic activity of Bacillus strains against Fusarium pseudograminearum according to the size of the antagonistic distance: 0 cm, no antagonistic effect; 0 - 0.5 cm, slight antagonistic effect; 0.5 - 1.5 cm, medium antagonistic effect; > 1.5 cm, strong antagonistic effect. Screen out the Bacillus strain ZJU-117 with strong antagonistic effect, and the detection results are as shown in Figure 1 B in. As can be seen from Figure 1 B in, the strain ZJU-117 has a strong inhibitory effect on the growth of Fusarium pseudograminearum colonies, with an antagonistic distance of 1.82 cm and a significant antagonistic effect.

[0043] 3. Morphological observation and taxonomic confirmation of strain ZJU-117 Bacillus velezensis ZJU-117 is Gram-positive, with rod-shaped and large oval cells, often in pairs, not forming endospores but forming cysts and capsular mucus, and moving by peritrichous flagella. After culturing on LB medium for 24 h, colonies of 1.5 mm can be formed, as shown in A of Figure 1 , showing milky white, round colonies, with a smooth and slightly convex surface and a viscous texture. Its 16S rDNA sequence is as shown in SEQ ID NO.1. The comparison result in NCBI shows that it is Bacillus velezensis ( Bacillus velezensis ).

[0044] SEQ ID NO.1:

[0045] Example 2 Preparation of Biocontrol Bactericide Inoculate the strain ZJU-117 into LB liquid medium, and culture it with shaking at 30 °C and 180 rpm for 20 - 24 hours. Then take samples in a laminar flow hood to measure the OD value at 600 nm, and use the uninoculated culture solution as a blank control during the measurement process; When the culture reaches an OD 600 between 0.5 and 0.8, add the seed bacterium solution to a 500 mL glass bottle containing fermentation culture solution (LBGM medium) at a volume ratio of 1:1000, and culture it with shaking at 30 °C and 180 rpm for 72 h to obtain the fermentation broth of the strain ZJU-117. The pH value of the fermentation broth is stable at 7.2. Dilute it in gradients, spread it on plates for detection, and adjust the concentration of the bacterial suspension to 1×10 7 ~10 8 cfu / mL to obtain the biocontrol bactericide.

[0046] Example 3 Antagonism of the Strain ZJU-117 Against Fusarium pseudograminearum 1. EC50 of the Strain ZJU-117 in Inhibiting the Mycelial Growth of Fusarium pseudograminearum Add the gradient-diluted strain ZJU-117 to the WA medium cooled to 40 - 50 °C, so that the final concentration of ZJU-117 is 10 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL in turn to prepare WA plates containing the biocontrol bacterium ZJU-117. Use the WA plate without the biocontrol bacterium as a blank control.

[0047] Inoculate a disc of Fusarium pseudograminearum in the center of the plate, and culture it at 25 °C until the mycelium covers the blank control plate (as shown in A in Figure 2 ). Then count the data and analyze it, measure the size of the colonies in the plates containing different gradients of the biocontrol bacterium (as shown in A in Figure 2 ), and calculate the median effect concentration (EC50).

[0048] The results are shown in B in Figure 2 . As the bacterial content in the plate increases, the inhibition rate of the mycelium increases accordingly. The statistical results show that there is a linear relationship between the bacterial content in the plate and the inhibition rate: y = -0.6771x + 4.8305 (R 2 = 0.9028) (1) where x is the concentration of the strain ZJU-117 (Log10 CFU / mL), Y is the colony diameter of *Fusarium pseudograminearum* (cm), and R 2 represents the degree of fit between the estimated value of the trend line and the corresponding actual data.

[0049] Calculated by formula (1), when the amount of ZJU-117 bacteria in the plate is about 10 3 CFU / mL, it can inhibit the mycelial growth of 50% *Fusarium pseudograminearum*, that is, the EC50 of ZJU-117 against *Fusarium pseudograminearum* = 10 3 CFU / mL.

[0050] 2. Activity of the cell-free supernatant of the biocontrol agent ZJU-117 in inhibiting the mycelial growth and spore germination of *Fusarium pseudograminearum* The fermentation broth of the biocontrol bacterium ZJU-117 strain obtained in Example 2 was centrifuged at 10,000 rpm for 20 min, and the cell-free supernatant was taken. The cell-free supernatant was filtered through a bacterial filter (0.22 μm) and used later.

[0051] Five fungal discs were punched out at the edge of the colony of *Fusarium pseudograminearum* with a 1 cm puncher and added to a conical flask containing 100 mL of CMC culture medium. The mixture was cultured at 25°C and 180 rpm for 5 days; the crude spore liquid obtained by shaking culture was filtered through three layers of sterilized filter paper to remove the mycelium. After centrifugation at 6,000 rpm for 10 min, the CMC medium was removed to obtain the spore precipitate of *Fusarium pseudograminearum*. The spore precipitate was resuspended with the same volume of ddH 2 O, and after pipetting and mixing evenly, 10 μL was taken for counting under a hemocytometer, and the spore concentration was recorded (the concentration is preferably 10 6 ), and it was stored in a 4°C refrigerator for later use.

[0052] The inhibitory effect of the supernatant of the biocontrol agent on the spores of *Fusarium pseudograminearum* was tested by the agar punching method: the obtained spore liquid of *Fusarium pseudograminearum* was added to the PDA medium melted to 37°C - 40°C at a final concentration of 10 4 cfu / mL (to prevent bacterial contamination, kanamycin with a final concentration of 50 ppm was added to the melted medium), and after thorough mixing, the mixed PDA medium was poured into a petri dish with a diameter of 6 cm to obtain a PDA plate containing spores; a puncher with a pore diameter of 8 mm was used to punch a hole in the center of the plate. After sealing the bottom with the melted PDA medium and thoroughly drying, 300 μL of the cell-free supernatant of the biocontrol agent was added. After culturing at 25°C for 36 h, the diameter of the inhibition zone was measured and photographed (as Figure 3 shown), indicating that the cell-free supernatant can effectively inhibit the mycelial growth.

[0053] Example 4 Determination of the plate antagonistic activity of ZJU-117 strain against other plant pathogens The Bacillus ZJU-117, negative control Bacillus 9-71 and Pectobacterium carotovorum used in the experiment were cultured at 30 °C and 180 rpm, and the other 6 bacterial strains were cultured at 28 °C and 180 rpm until the OD600 of the bacterial solution reached 1.0 (the concentration was about 10 8 ). Then, the 7 pathogenic bacteria were diluted at a ratio of 1:2000 and added to the culture medium to make bacterial plates.

[0054] Among them, ZJU-117, 9-71 and Pectobacterium carotovorum Pcc used LB medium; Xanthomonas oryzae pv. oryzicola RS105, Xanthomonas oryzae pv. oryzae GX-1, Xanthomonas oryzae pv. oryzae PXO99A and Nxtc01 used NA medium; Pseudomonas syringae pv. tomato DC3000 used KB medium; Ralstonia solanacearum GMI1000 used BG medium.

[0055] 2 μL of the bacterial solutions of ZJU-117 and the negative control with OD600 adjusted to 1.0 were directly spotted onto the plates. The thiazole zinc used was a 30% thiazole zinc suspension agent with the active ingredient, which was diluted with water to a final concentration of 50 ppm, 100 ppm, 200 ppm, and 400 ppm. First, 2 μL was taken and spotted onto filter paper, and after air-drying, it was pasted onto the plate. Diameter = diameter of the inhibition zone - diameter of the colony. The antagonistic activity of the ZJU-117 strain against the pathogenic bacteria causing carrot soft rot, bacterial wilt, bacterial leaf streak of rice, bacterial leaf streak of wheat, bacterial blight of rice was detected, with different concentrations of thiazole zinc as the control. The detection results are shown in Figure 4 A - G in.

[0056] From Figure 4 A - G in, it can be seen that the ZJU-117 strain has good antagonistic effects against the pathogenic bacteria causing carrot soft rot, bacterial wilt, bacterial leaf streak of rice, bacterial leaf streak of wheat, bacterial blight of rice and bacterial leaf spot of tomato.

[0057] Example 5 Field control effect and indoor control evaluation of the ZJU-117 strain against wheat basal rot 1. Evaluation of the field control effect of ZJU-117 against wheat basal rot The field control effect experiment was carried out in Caiyuan Village, Chuwang Town, Neihuang County, Anyang City, Henan Province and Zhouzhuang Village, Jiaomiao Town, Qihe County, Dezhou City, Shandong Province from February to May 2023. The wheat grew well in the field, and the soil water and fertilizer conditions were moderate. The tested wheat varieties were Tianhe No. 6 and Jimai 22, and the areas of the test plots were 25 m 2 and 30 m 2, three experimental plots (three replicates) were randomly arranged in the field for each treatment. When the wheat was in the green-reverting stage in the field, the biocontrol agent was sprayed at the base of the roots (500 mL + 5 L of water), and Jinggangmycin and water were also sprayed simultaneously.

[0058] The application concentration of the biocontrol agent ZJU-117 was 10 7 ~10 8 cfu / mL, and the application dose was 6.67 L / 667 m 2 , and the application dose of Jinggangmycin was 33 mL / 667 m 2 .

[0059] After the disease situation of wheat stem base rot in the field was stable and the wheat entered the milk-ripe to wax-ripe stage, disease investigations were carried out on all plot treatments. During the investigation, five-point sampling was carried out in each treatment area of the experimental field, with 20 plants at each point, a total of 100 plants. The leaf sheaths were peeled off to record the disease grade, and the disease index and relative disease index were calculated. The disease conditions of some sampled wheat ears were as shown in Figure 5 A and B in. Combining the experimental results of the two places, statistical analysis and evaluation were carried out on the biocontrol strain ZJU-117 in the field experiment (as shown in Table 1 and Table 2).

[0060] According to the proportion of diseased plants showing brown discoloration at the base of the stem (or white ear symptoms caused by stalk rot) in the total number of plants, the severity of the disease was divided into 6 grades (Grade 0: disease-free; Grade 1: brown discoloration at the base of the stem to the first node or the first leaf; Grade 2: brown discoloration at the base of the stem to the second node or the second leaf; Grade 3: brown discoloration at the base of the stem to the third node or the third leaf; Grade 4: brown discoloration at the base of the stem above the third node but without forming a withered white ear or the fourth leaf; Grade 5: withered white ear or not heading due to disease).

[0061] According to the above statistical results, the disease index of each treatment was calculated, and it was calculated according to formula (2): Calculation formula: ) / )×100 (2) In formula (2): DI is the disease index; i is the disease grade (0 to n); X i is the number of units at level i; S i is the representative value of the severity at level i; S max is the highest value of the severity level.

[0062] Table 1 Evaluation of the control effect of wheat stem base rot in the field for each treatment group (Anyang, Henan)

[0063] Table 2 Evaluation of the control effect of wheat stem base rot in the field for each treatment group (Dezhou, Shandong)

[0064] 2. Evaluation of the Control Effect of Bacillus velezensis ZJU-117 against Wheat Basal Rot in the Laboratory In the above experiment, conidia of Fusarium pseudograminearum at a concentration of 1×10 5 cfu / mL were mixed with nutrient soil, and wheat seeds soaked in the fermentation broth at a concentration of 1×10 7 cfu / mL for 24 h were sown on the same day, with seeds soaked in water as the control. Three days after inoculation, the roots were irrigated with the fermentation broth at a concentration of 1×10 7 cfu / ml. As shown in Figure 6 Table 3, the fermentation broth of Bacillus velezensis ZJU-117 could significantly reduce the disease index at the diseased sites after inoculation. The average disease index of the control was 70.54±10.59, while the average disease index of the fermentation broth of the biocontrol bacteria was 24.30±7.67, and the control effect exceeded 60%, indicating certain biocontrol potential.

[0065] Table 3. Evaluation of the Effect of Different Treatments on Inhibiting the Toxin Production of Wheat Basal Rot in the Laboratory

[0066] As shown in Table 1 and Table 2, the combined application of the biocontrol agent ZJU-117 and jinggangmycin maintained a field control effect against wheat basal rot above 40%. As shown in Table 3, the biocontrol agent ZJU-117 could significantly inhibit the occurrence of wheat basal rot under laboratory conditions, with an inhibition effect of up to 65%, showing good application prospects.

Claims

1. A Bacillus Velez strain, characterized in that Bacillus velez Bacillus velezensis ZJU-117, the preservation unit is China Center for Type Culture Collection, the preservation number is CCTCC NO: M2025294, and the preservation date is February 25, 2025.

2. A biocontrol agent, characterized in that: The active ingredient of the biocontrol agent is the Bacillus Velezii strain described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that In the biocontrol agent, the concentration of Bacillus Velez is 1×10 7 ~1×10 8 cfu / mL.

4. The method for preparing the biocontrol agent according to claim 2 or 3, characterized in that: The following steps are involved: (1) inoculating the Bacillus Velezii into LB liquid culture medium to obtain seed liquid; (2) The obtained seed solution was inoculated into the culture medium for expansion and the concentration of Bacillus Velezii in the fermentation broth was adjusted to 1×10 7 ~1×10 8 cfu / mL, and the biocontrol agent is obtained.

5. The preparation method according to claim 4, characterized in that: The inoculation amount of the seed solution is 1% of the total volume of the seed solution after inoculation.

6. A biocide, characterized in that: The method comprises the biocontrol agent as claimed in claim 2 and Jinggangmycin; wherein the concentration of the biocontrol agent is 1×10 7 ~1×10 8 cfu / mL; the ratio of the volume of the biocontrol agent to the effective ingredient of Jinggangmycin is 50~500mL:1.5g.

7. Use of the strain according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, or the biofungicide according to claim 5 in antagonizing Fusarium graminearum.

8. Use of the strain according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, or the biofungicide according to claim 5 in preventing and treating wheat stem base rot.

9. Use of the strain according to claim 1, or the biocontrol agent according to any one of claims 2 to 3, or the biofungicide according to claim 5 in preventing and controlling carrot soft rot, bacterial wilt, rice bacterial leaf streak, wheat bacterial leaf streak, rice bacterial leaf blight and / or tomato bacterial leaf spot.

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