Bacillus velezensis strain JZ01, microbial inoculum containing bacillus velezensis strain JZ01 and application of bacillus velezensis strain JZ01

Abstract

The invention relates to a bacillus velezensis strain JZ01, a fungicide containing the same and application of the bacillus velezensis strain JZ01. The bacillus velezensis strain JZ01 can be used for efficiently antagonizing cucumber brown spot pathogen cercospora, and effectively killing root-knot nematode. Furthermore, the invention also develops a synergist of the bacterial strain, and the research finds that the trigonelline can be used as the synergist to significantly increase the effect of the trigonelline on resistance to cephalosporium, while the ganoderic acid A can enhance the effect of the trigonelline on killing root-knot nematode, so that the trigonelline can be used for developing a more efficient compound bacterial agent. Besides, the bacillus velezensis strain and the fungicide containing the bacillus velezensis strain are microbial preparations and have the advantages of being safe and environmentally friendly, and therefore the bacillus velezensis strain and the fungicide containing the bacillus velezensis strain have high industrial value and application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms and biological pesticides, and particularly relates to a strain of Bacillus velezensis JZ01, a microbial agent containing the same and application thereof. BACKGROUND

[0002] Cucumber brown spot caused by Cercospora is a very harmful disease in cucumber planting agriculture. When the disease is serious, it will not only cause cucumber yield reduction and quality decline, but also may lead to absolute yield loss. At present, the chemical method is mainly used to prevent and control this kind of brown spot caused by Cercospora, such as spraying 75% chlorothalonil wettable powder, 50% thiram and other chemical agents. However, long-term use of chemical agents not only promotes the increasing resistance of Cercospora, but also aggravates environmental pollution, and further causes a series of food safety problems. Therefore, it is urgent to explore safe, efficient and environmentally friendly biological control means. At present, there are few studies on biological control of cucumber brown spot caused by Cercospora, and relevant reports are also very limited.

[0003] Cucumber root-knot nematode disease is also a big problem in cucumber production, which is mainly caused by Meloidogyne. Root-knot nematodes invade cucumber lateral roots and fibrous roots, forming yellowish to yellowish-brown tuberculate root knots, in which there are parasitic white root-knot nematodes; after the development of the disease, the plant is dwarfed, the number of cucumbers is reduced and the cucumbers are small, the leaves are yellow and yellow, and finally withered and dead, which seriously affects yield and quality. Root-knot nematodes overwinter in soil with root tissue, survive for 1-3 years, and are transmitted by infested soil, diseased roots and irrigation water; 2nd instar larvae invade root tips in spring to cause infection, and the cycle of generations continues to harm. Among the current control methods, agricultural measures such as pest-free soil seedling raising and crop rotation have limited effect; chemical agents such as 10% clonitralid, 3% mila, and avermectin have effects, but they can cause environmental pollution and pesticide residues, and destroy soil microecology. Although microbial control has been explored, there are still problems such as insufficient control effect. Therefore, it is urgent to develop new biological control methods that are efficient, safe and environmentally friendly.

[0004] Bacillus velezensis is one of the most potential bacteria for biological control of plant diseases and pests, and has been reported to play a role in many plant diseases, but there is no report on its use for preventing and controlling the above-mentioned pathogens and plant diseases caused by them. Bacillus velezensis SUMMARY

[0005] OBJECTIVE ​To address the problems of strong drug resistance, significant environmental impact, and food safety concerns associated with existing chemical control methods for cucumber brown spot disease caused by Cercospora, this invention provides a strain of Bacillus belyceta JZ01, a fungal agent containing it, and their applications. The Bacillus belyceta JZ01 strain or its fungal agent exhibits highly effective antagonism against Cercospora and kills root-knot nematodes, playing a crucial role in the biological control of crops.

[0006] Furthermore, through research on the endogenous metabolites of the *Bacillus belyssae* strain, this invention discovered that its active ingredient, trigonelline, itself has an antagonistic effect against *Cercospora glomerata*, the pathogen causing cucumber brown spot disease. Further verification revealed that trigonelline can act as a synergist to significantly enhance the antagonistic effect of the *Bacillus belyssae* strain against *Cercospora glomerata*. Moreover, its active ingredient, ganoderic acid A, can kill root-knot nematodes, especially second-instar larvae. Further verification revealed that ganoderic acid A can act as a synergist to significantly enhance the effect of the *Bacillus belyssae* strain in killing root-knot nematodes, especially second-instar larvae.

[0007] Solution To achieve the objectives of this invention, the following technical solution is provided.

[0008] In a first aspect, the present invention provides a strain of Bacillus belyssus ( Bacillus velezensis strain JZ01, wherein Bacillus belyssus ( Bacillus velezensis Strain JZ01 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2025, with accession number CGMCCNO.35305.

[0009] The *Bacillus belye* of this invention was isolated from the sediment at the mouth of a river. Through comparative experiments, the inventors discovered that the isolated *Bacillus belye* (… Bacillus velezensis The strain JZ01 exhibits strong antagonistic activity against Cercospora, demonstrating a strong control effect against cucumber brown spot disease caused by Cercospora and effectively inhibiting the pathogen of cucumber brown spot disease; furthermore, the Bacillus belyssus of this invention ( Bacillus velezensis Strain JZ01 also has the effect of killing root-knot nematodes, especially the second instar larvae of root-knot nematodes. Therefore, it can be used to effectively prevent and control cucumber root-knot nematode disease caused by root-knot nematodes.

[0010] The Bacillus belesiensis of the present invention ( Bacillus velezensis Strain JZ01 was identified as *Bacillus belesiensis* through morphological identification, physiological and biochemical characterization, and 16S rRNA sequencing. Bacillus velezensis) having a 16S rRNA gene sequence of SEQ ID NO: 1.

[0011] The Bacillus velezensis (Bacillus velezensis) Bacillus velezensis The colony state of the Bacillus velezensis (Bacillus velezensis)

[0012] In a second aspect, the present application provides a microbial agent comprising at least one selected from the group consisting of: viable cells, freeze-dried cells, inactivated cells of the Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) strain JZ01 as described in the first aspect above; and optionally, the strain culture is a fermentation broth, a fermentation supernatant of the strain and / or a concentrated or dried product of the foregoing.

[0013] The dosage form of the microbial agent can be a conventional dosage form in the art, for example, a liquid or solid preparation, and the dosage form can be adjusted according to the application requirements, and is preferably a liquid preparation or a powder.

[0014] In preferred embodiments, the microbial agent further comprises a synergistic agent.

[0015] Optionally, the synergistic agent is an active metabolite of the Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) strain JZ01, and preferably, the synergistic agent is trigonelline and / or ganoderic acid A.

[0016] In some preferred embodiments, the synergistic agent is ganoderic acid A, and the content of the synergistic agent in the microbial agent is 1%-5%, preferably 3%.

[0017] In other preferred embodiments, the synergistic agent is trigonelline, and the content of the synergistic agent in the microbial agent is 0.1%-0.5%.

[0018] In a third aspect, the present application provides use of the Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) strain JZ01 as described in the first aspect above or the microbial agent as described in the second aspect above in the preparation of a biological pesticide.

[0019] In feasible embodiments, the biological pesticide is used for any one or more of the following purposes: (1) for antagonizing Cercospora, preferably Cercospora so Cercospora kikuchii ; (2) for preventing and treating cucumber brown spot caused by Cercospora, preferably Cercospora so Cercospora kikuchii ; (3) for killing root-knot nematodes, preferably cucumber root-knot nematodes; (4) for preventing and treating cucumber root-knot nematode disease caused by root-knot nematodes, preferably cucumber root-knot nematodes.

[0020] In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Bacillus velezensis In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Cercospora kikuchii Cercospora kikuchii In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica).

[0021] In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Cercospora kikuchii Cercospora kikuchii In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Bacillus velezensis

[0022] In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Bacillus velezensis In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica).

[0023] In a fourth aspect, the present application provides (1) live bacteria, lyophilized bacteria, inactivated bacteria of Bacillus velezensis (preferably Bacillus velezensis strain JZ01) as described in the first aspect above, and / or (2) the use of trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica (preferably Cercospora sochanica) and / or for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Bacillus velezensis

[0024] Beneficial effects (1) The Bacillus velezensis (preferably Bacillus velezensis strain JZ01) of the present application has a strong antagonistic effect on Cercospora sochanica (preferably Cercospora sochanica), and can be used for preventing and treating cucumber brown spot disease caused by Cercospora sochanica (preferably Cercospora sochanica). Bacillus velezensis ​​​​Strain JZ01 has excellent antagonistic effect on Cercospora and high killing effect on root-knot nematode; specifically, the inhibition rate of strain JZ01 on Cercospora beticola reaches 82.96%; the 16h killing rate of strain JZ01 on root-knot nematode reaches 81.6%, and the 20h killing rate reaches 100%.

[0025] (2) The study found that the metabolites of Bacillus velezensis strain JZ01, trigonelline and ganoderic acid A, can be used as synergists to significantly improve the efficacy of the strain JZ01 in resisting Cercospora and killing root-knot nematode, so that the control effect of the strain JZ01 exceeds the level of existing chemical fungicides, and therefore, the strain JZ01 can be used for developing a high-efficiency compound microbial agent. Bacillus velezensis The synergists, ganoderic acid A and trigonelline, are extracted from the metabolites of Bacillus velezensis strain JZ01, and no exogenous substances are added, so that the compound microbial agent containing the synergists is relatively safe and environmentally friendly. Bacillus velezensis

[0026] In summary, whether it is the Bacillus velezensis strain JZ01 itself or the compound microbial agent further containing the synergists ganoderic acid A and trigonelline, the strain JZ01 has the characteristics of high efficiency, safety and environmental protection, and therefore has high industrial value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] One or more embodiments are illustrated by way of example in the figures that form a part of this disclosure and which are shown by way of example, and not limitation, as being implemented in one or more specific modes of doing work in certain embodiments. The preferred implementations of the present application have significant related arts, and any implementation described herein of the preferred implementations of the present application should be within the skill of a designer of a microbial agent.

[0028] Figure 1 is the colony state of the strain JZ01 recorded in embodiment 1 of the present application after dilution plate culture for 3 days. -4 is the colony state of the strain JZ01 recorded in embodiment 1 of the present application after dilution plate culture for 3 days.

[0029] Figure 2 is the plate confrontation experiment result of the strains JZ01 and JZ02 recorded in embodiment 1 of the present application.

[0030] Figure 3 is the colony morphology of the single colony of the strain JZ01 recorded in embodiment 1 of the present application at different culture time periods.

[0031] Figure 4 is the colony morphology of the strain JZ01 recorded in embodiment 1 of the present application under a microscope. DETAILED DESCRIPTION

[0032] ​In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall into the scope of the present application. Unless otherwise explicitly indicated, in the entire specification and claims, the term "comprise" or its variants such as "contain" or "include" should be understood as including the stated elements or components, and not excluding other elements or components.

[0033] In addition, in order to better illustrate the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0034] Embodiment 1: Screening and identification of strain JZ01 of the present application This embodiment provides experiments related to the isolation, purification, anti-aspergillus tail function screening and strain identification of strain JZ01 of the present application.

[0035] 1. Isolation and purification of strain JZ01

[0036] The strain JZ01 involved in the present application is isolated from river mud collected at the estuary of Dagu River in Qingdao, and the specific isolation method is as follows: (1) River mud collection: five-point sampling method is used to collect river mud at five random representative sampling orientations at the estuary of Dagu River, 2-3 kg is collected at each orientation to ensure that the sample amount meets the subsequent experimental analysis requirements; after collection, the samples are placed in sampling bags, and the sampling time, location and collector are marked on the sampling bags, and then placed in a foam box containing biological ice bags and transported back to the laboratory.

[0037] (2) Preparation of sample suspension: accurately weigh 10 g of river mud sample and put it into a triangular flask containing 90 mL of sterile water, and 25-30 glass beads are added in advance in the triangular flask; after placing, it is placed in a 30℃ constant temperature shaking incubator, and the shaking rate is set to 150 r / min, and uniformly shaken for 30 min to make the river mud particles fully dispersed and uniformly mixed in the sterile water, thereby preparing the river mud sample suspension.

[0038] (3) Sample suspension gradient dilution: 1 mL of mixed sample suspension was taken from the flask with a 1 mL sterile pipette, added to a test tube containing 9 mL of sterile water and mixed well, then 1 mL of the diluted sample suspension was re-pipetted from the test tube and added to another large test tube containing 9 mL of sterile water, mixed well; in turn, 10 -1 ~10 -6 Different dilutions of river mud solution.

[0039] (4) Coating: the bottom of three plates of beef extract peptone agar medium was written with 10 -4 , 10 -5 , 10 -6 three dilutions with a marker pen, then a pipette was taken and 10 -4 , 10 -5 , 10 -6 mL of soil dilution was pipetted onto the prepared plate medium with sterile operation; a sterile coating rod was used to evenly coat the medium surface, and it was placed at room temperature for 5-10 min to allow the bacterial solution to be absorbed on the medium.

[0040] Beef extract peptone agar medium formula: beef extract 3.0 g, peptone 10.0 g, NaCl 5.0 g, agar 15-25 g, water 1000 ml, pH 7.4-7.6.

[0041] (5) Culture: the coated beef extract peptone agar medium plate was sealed and inverted in a 30°C incubator for constant temperature culture for 2-3 days, and the growth of colonies of different concentrations on the plate was observed at regular intervals.

[0042] (6) Single bacteria picking: a sterile inoculation loop was used to pick a small amount of single colonies of different colony morphologies that grew after culture on the beef extract peptone agar medium plate, and inverted in a 30°C incubator for constant temperature culture for 16-24 h, and the appearance of the single colonies was observed again to see if it was consistent, and staining was observed to check if it was a single microorganism; if it was a single microorganism, it proved that the purification was complete, if there were mixed bacteria, it needed to be separated and purified again until a pure culture was obtained.

[0043] The results are shown in Figure 1 , 10 -4 dilution plate had a better colony distribution, moderate number, no spread growth, and different colony morphologies of strains were clearly visible; through single bacteria picking plate streaking, 16 strains of pure culture were obtained, which were numbered as JM01-JM08, JX01-JX05, and JZ01-JZ03 according to the single bacteria picking time.

[0044] 2. Screening of strains antagonistic to Cercospora The present application takes the pathogenic fungus of cucumber brown spot Cercospora kikuchii (the pathogenic fungus is isolated from cucumber leaves of cucumber brown spot by itself, and verified by Koch's postulates, and identified as Aspergillus by strain identification Cercospora kikuchii ) as an indicator strain, and the antagonistic function of 16 strains of Aspergillus is explored by plate confrontation culture, and the specific method is as follows: (1) Activation of the test strain: the 16 strains isolated above are streaked on beef extract peptone agar medium, and cultured at 30°C for about 18h; no contamination is observed, i.e. successful activation.

[0045] (2) Activation of the indicator pathogenic fungus: the Aspergillus plate stored in the 4°C refrigerator is inoculated on modified potato dextrose agar (PDA) medium under sterile operation, and cultured at 28°C for more than 50h, and the pathogenic fungus is grown to 3 / 4 of the plate, i.e. successful activation.

[0046] The formula of modified potato dextrose agar (PDA) medium is as follows: potato 200g, glucose 20g, cucumber leaves 30~50g, agar 15~20g, distilled water 1000mL, pH natural.

[0047] (3) Plate confrontation culture: 0.3~0.5cm 2 in size of activated Aspergillus is inoculated in the center of the modified PDA medium plate, and the activated test strain is inoculated on both sides 2cm away from the center, and the two sides without inoculation are used as controls, and each treatment is repeated 3 plates; after the treatment is completed, it is cultured at 28°C, and the inhibition effect of the test strain on the pathogenic fungus is observed every day, and after the pathogenic fungus on the control sides grows to the edge of the plate, the size of the lesion of each treatment group is measured, and the inhibition rate is calculated according to the following formula: inhibition rate = [(control colony radius-treatment colony radius)] / control colony radius x 100%.

[0048] The results are shown in Table 1.

[0049] Table 1, inhibition effect of different strains on cucumber brown spot pathogen ; Note: "-" indicates no antagonistic effect; "+" indicates 0

[0050] Result analysis: Among the 16 strains to be tested, JM03, JM05, JM06, JM07, JX01, JX05, JZ01 and JZ02 showed inhibitory effect on the cucumber brown spot pathogen (see Table 1); further analysis found that strains JZ01 and JZ02 had an inhibitory effect on the cucumber brown spot pathogen of more than 80%, 82.96% and 80.74% respectively (see Table 1, and the results of the confrontation experiment thereof are shown in Figure 2 ), which was significantly better than the other six strains. The results preliminarily indicated that strains JZ01 and JZ02 both had the function of efficiently inhibiting the cucumber brown spot pathogen and had the potential to be developed into related microbial pesticides.

[0051] 3. Screening of strains with root-knot nematode killing function Through the above antagonistic research of the aspergillus, it was determined that strains JM03, JM05, JM06, JM07, JX01, JX05, JZ01 and JZ02 could inhibit the cucumber brown spot pathogen aspergillus on the plate. In order to further screen strains with root-knot nematode killing activity from the above 8 strains, the root-knot nematode killing function of the above 8 strains was detected, and the specific method steps were as follows: (1) Preparation of different test strain bacterial liquid: The 8 strains to be tested were activated by plate streaking in advance, and the method was the same as that in part 2, (1) of Example 1; after activation, a small piece of culture medium with bacterial turf was taken with a sterile spatula and inoculated into liquid beef extract peptone medium, and then cultured in a constant temperature shaking incubator at 30°C and 150 r / min for 18 h, thereby different test strain bacterial liquid was prepared.

[0052] (2) Preparation of root-knot nematode suspension: The suspension of the second instar larvae of the root-knot nematode (which was obtained by self-separation and culture from the root system soil of cucumber plants and was identified as the second instar larvae of the root-knot nematode by the identification method recognized in the art) was loaded into a centrifuge tube and enriched in a centrifuge, and then resuspended with sterile water to prepare a root-knot nematode second instar larvae suspension with a concentration of about 500 pieces / 100 μL.

[0053] (3) Killing activity detection: The test strain bacterial liquid and the root-knot nematode second instar larvae suspension (shaken) were mixed in a 96-well plate at a volume ratio of 1:1, each being 100 μL; the liquid beef extract peptone medium was used as a blank control; the mixture was incubated at room temperature for 24 h; after the treatment was completed, the root-knot nematode death was directly observed under a low-power microscope, and the dead root-knot nematode was defined as the standard of the dead root-knot nematode, and the root-knot nematode mortality of ≥5% was defined as "having root-knot nematode killing activity", and the root-knot nematode mortality of <5% was defined as "having no root-knot nematode killing activity".

[0054] The results are shown in Table 2 below.

[0055] Table 2, different strains of nematode root-killing effect statistics ; Note: "+" represents "root-knot nematode activity", "-" represents "no root-knot nematode activity".

[0056] Table 2 shows that among the 8 strains detected, 3 strains have the function of killing second instar larvae of root-knot nematodes, which are JM03, JM05 and JZ01, and the remaining 5 strains have no root-knot nematode activity. Therefore, strains JM03, JM05 and JZ01 have antagonistic activity of Cercospora and root-knot nematode activity, and therefore have multifunctionality.

[0057] 4, Screening of high-efficiency root-knot nematode-killing strain JZ01 In order to further compare the root-knot nematode-killing activity of strains JM03, JM05 and JZ01, we screened strains with better root-knot nematode-killing activity, and prepared the bacterial liquid of the three strains by the above method, and prepared 6.0x10 9 CFU·mL -1 Standard bacterial liquid. The same method was used to detect the killing activity of different test strains of standard bacterial liquid on second instar larvae of root-knot nematodes. The concentration of the standard bacterial liquid applied was 3.0x10 9 CFU·mL -1 After treatment, observe the death of root-knot nematodes every 4h, count the number of dead root-knot nematodes and calculate the mortality according to the following formula: mortality (%) = number of dead root-knot nematodes in each group / total number of root-knot nematodes in each group x 100.

[0058] The results are shown in Table 3 below.

[0059] Table 3, the killing effect of three strains of the same concentration on root-knot nematodes ; Result analysis: JZ01 with a concentration of 3.0x10 9 CFU·mL -1 Dead root-knot nematodes were found after 4h of treatment, and JM05 and JM05 with the same concentration were found to have dead root-knot nematodes after 8h and 12h of treatment, respectively (see Table 3); among them, strain JZ01 had a root-knot nematode mortality of 100% after 20h of treatment (see Table 3), and the effect of killing root-knot nematodes was significantly better than the other two strains. The above results fully demonstrate that under the same strain concentration, JZ01 shows higher root-knot nematode-killing activity. Therefore, strain JZ01 is selected for the following strain identification and efficacy verification.

[0060] 5, Strain JZ01 strain identification (1) Morphological identification The colony morphology of strain JZ01 cultured for different time periods (16h, 32h, 48h, respectively) is shown in FIG. 1, which shows that when strain JZ01 is cultured on beef extract protein peptone agar medium, the single colony is round, oval to irregular in shape in the early stage (16h), the surface is smooth, wet and sticky, and opaque; in the middle stage (32h), the single colony morphology is consistent with that in the early stage, the surface is smooth, wet and sticky, milky white, and the edge begins to appear spreading radial growth, and the middle part is slightly convex; in the late stage (48h), the surface is dry, and the edge appears jagged radial growth and dry. Figure 3

[0061] The bacterial body and spore morphology of strain JZ01 are shown in FIG. 2, which shows that by Gram staining, it is found that the bacterial body of strain JZ01 shows purple, which is judged to be a Gram-positive bacterium, and the bacterial body is rod-shaped; in the later stage, it is found that the strain produces spores, and the spores are mesosporous, so we judge that it is a spore-forming bacterium. Figure 4

[0062] (2) Physiological and biochemical identification Referring to the "Common Bacteria System Identification Manual", the partial physiological and biochemical characteristics of strain JZ01 are tested (the detection method is a conventional detection method in the art), and the results are shown in Table 4.

[0063] Table 4, physiological and biochemical characteristics of strain JZ01 ; Table 4 (continued), physiological and biochemical characteristics of strain JZ01 ; Note: "+" represents a positive reaction, and "-" represents a negative reaction.

[0064] According to the physiological and biochemical characteristics of strain JZ01, it is preliminarily confirmed that it is a bacterium of the genus Bacillus, which is consistent with the morphological identification.

[0065] (3) Molecular biology identification Strain JZ01 was sent to the Institute of Microbiology, Chinese Academy of Sciences for 16S rRNA gene sequencing, which showed that the full length of 16S rRNA gene was 1413 bp, and the specific sequence was SEQ ID NO: 1; BLAST comparison was performed on GenBank, and it was found that the base similarity reached 99.79% with PP086812.1; combined with morphological identification and physiological and biochemical identification, strain JZ01 was identified as Bacillus velezensis (Bacillus velezensis). Bacillus velezensis Bacillus velezensis The strain has been preserved in the China General Microbiological Culture Collection Center on July 21, 2025, and the preservation number is CGMCC NO. 35305. ​​​

[0066] 16S rRNA gene sequence determination results are as follows:

[0067] Example 2: Preparation of high-content Bacillus velezensis JZ01 inoculant This example provides methods and processes related to the preparation of Bacillus velezensis JZ01 mother liquor, mother powder, and inoculant.

[0068] 1. Obtaining Bacillus velezensis JZ01 mother liquor The present application optimizes the fermentation formula and process suitable for Bacillus velezensis JZ01 through experiments, and prepares Bacillus velezensis JZ01 mother liquor suitable for production through industrial fermentation method. The specific operation steps are as follows: (1) Preparation of shake flask seed liquid a. Streaking on beef extract peptone agar medium, incubating at 30-32°C for about 18-20 hours; observing for contamination. If contaminated, continue to pick single colonies for streaking according to colony morphology until the colonies are single.

[0069] b. After observing no contamination, pick three single colonies and inoculate into beef extract peptone liquid medium, incubate at 30-32°C, 90-120 r / min for 18-20 hours.

[0070] c. After incubation, observe by microscopy for contamination and consistency of colony growth. Select the best one from the three bottles without contamination for standby.

[0071] d. Prepare beef extract peptone liquid medium according to 0.5-1% of seed tank feed; select the best one according to 0.1-0.5% inoculation amount and inoculate into the seed liquid medium, incubate at 30-32°C, 90-120 r / min for 18-20 hours to obtain the shake flask seed liquid.

[0072] (2) Primary seed fermentation a. Feeding: Feed seed fermentation medium into the tank according to 60-70% of tank volume, and additionally add 1-2% water for consumption.

[0073] The formula of the seed fermentation medium (according to mass ratio) is as follows: glucose 1.95-2.15%, corn starch 1.9-2.1%, yeast extract powder 1.4-1.6%, peptone 0.15-0.25%, magnesium sulfate 0.02-0.05%, potassium dihydrogen phosphate 0.04-0.05%, beef extract 0.1-0.3%, and the rest is water.

[0074] b. Sterilization and cooling: sterilize according to the conventional sterilization requirements, maintain 121±1°C, 0.05MPa high temperature and high pressure for 30-40 minutes; after sterilization, cool to 32±1°C.

[0075] c. Inoculation and fermentation: the seed liquid of the shake flask is inoculated into the sterilized seed tank, and the inoculation amount is 0.5-1 v / v% of the material liquid. After inoculation, the fermentation is carried out according to the following process.

[0076] Fermentation process: the temperature is maintained at 32±1℃; 0-18h: the ventilation ratio is 1:0.5 (the ventilation gas is sterile air filtered by three stages), and the rotating speed is 60r / min. When OD600=0.8-0.9, the fermentation is completed, and the seed is ready for transfer.

[0077] (3) Secondary expansion fermentation a. Feeding and sterilization and temperature reduction are the same as the primary seed fermentation.

[0078] b. Seed transfer: the seed fermented in the primary fermentation is directly pumped into the matched fermentation tank, and the inoculation amount is 9.5-10.5%. After inoculation, the fermentation is carried out according to the following process.

[0079] Fermentation process: the temperature is maintained at 32±1℃; 0-6h: the ventilation ratio is 1:0.5, and the rotating speed is 60r / min; 6-25h: the ventilation ratio is 1:1, and the rotating speed is 100r / min; 26-42h: the ventilation ratio is 1:1.2, and the rotating speed is 120r / min. The above ventilation gas is sterile air filtered by three stages.

[0080] When the spore rate is ≥85% under microscopic examination, the fermentation is completed, and the bacillus velezensis JZ01 mother liquor is obtained, the effective viable bacterial count is 100-150 billion / mL, and the spore rate is ≥85%.

[0081] 2. Preparation of bacillus velezensis JZ01 microbial agent On the basis of obtaining the bacillus velezensis JZ01 mother liquor, the mother powder is obtained by the spray drying method, and then the bacillus velezensis JZ01 microbial agent (powder) is obtained by formula processing. The specific method steps are as follows: (1) Preparation of mother powder: take the bacillus velezensis JZ01 mother liquor prepared in the early stage, add 3%-5% (w / v, i.e. mass volume ratio in g / mL) of full water soluble benefit powder (ordinary commercial product) in proportion, fully stir and mix to ensure that the benefit powder is completely dissolved and uniformly compatible with the bacterial mother liquor to form a stable spray precursor liquid; The above precursor liquid is introduced into the spray drying equipment, and the equipment operation parameters are set and stabilized: the inlet temperature is 160-170℃ (to ensure that the precursor liquid is quickly atomized and preliminarily dehydrated), and the outlet temperature is 60-65℃ (to avoid the inactivation of bacteria caused by too high outlet temperature, and to ensure the effective removal of water), and the equipment is started for spray drying. After the drying is completed, a powdery product is collected, which is the B. velezensis JZ01 mother powder. The water content of the mother powder is detected by a moisture meter to ensure that it is stably controlled at 8% to 12% (w / w, i.e., mass percentage); the number of live bacteria in the mother powder is detected by a plate counting method (or other live bacteria detection method) to reach a quality standard of 80 billion to 120 billion CFU / g (colony forming unit / gram), and the mother powder is qualified for use in subsequent experiments or storage only after the quality standard is met.

[0082] (2) Preparation of the raw powder: According to the content of B. velezensis JZ01 in the mother powder, the raw powder containing 20 billion / g of B. velezensis JZ01 is prepared by adding the benefit powder in a weight ratio of 1: (3-5) of the mother powder to the benefit powder.

[0083] (3) Preparation of the finished product: On the basis of the B. velezensis JZ01 raw powder, we compound the product according to the following formula: 1.3-1.5wt% glucose + 0.3-0.4wt% amino acid powder + 2.8-5.2wt% sodium dodecyl sulfate + 75-80wt% raw powder + 12.9-20.6wt% benefit powder, to obtain the B. velezensis JZ01 microbial agent, in which the content of B. velezensis JZ01 is about 150-160 billion CFU / g.

[0084] Example 3: Screening of the B. velezensis JZ01 microbial agent synergist This example provides experiments related to the screening of the B. velezensis JZ01 microbial agent synergist.

[0085] 1. Antagonism of JZ01 strain fermentation supernatant against Cercospora and nematode-killing function determination (1) Strain activation: streaking on beef extract peptone agar medium, 32°C culture for about 18h, and observing that the plate is not contaminated to indicate successful activation.

[0086] (2) Preparation of fermentation supernatant: using a loop to inoculate beef extract peptone liquid medium, 32°C, 100r / min culture for 18-20h to obtain fermentation broth; after centrifugation, the upper liquid is filtered by a 0.22μm bacterial filter to obtain the fermentation supernatant.

[0087] (3) Detection of antagonism against Cercospora: taking PDA medium cooled to 45°C-50°C after melting, adding JZ01 strain fermentation supernatant at a volume ratio of 1:19, quickly and gently inverting to mix (avoiding the generation of too many bubbles to affect the flatness of the plate), immediately pouring the mixed liquid into a sterile culture dish, and naturally cooling to complete solidification in a clean bench to prepare a sample plate containing the fermentation supernatant. The beef extract peptone liquid medium was used as a blank control. The same operation as the sample group was performed to prepare the control plate to ensure that all experimental conditions were identical except for the presence of fermentation supernatant. This eliminated unrelated variable interference. The activated C. herbarum was selected, and a sterile forceps or punch was used to cut a virus block with a size of 0.3-0.5 cm 2 The virus block was inoculated into the central position of the sample plate and the control plate (when inoculating, the virus block was gently pressed to make one corner of the virus block tightly adhere to the culture medium to avoid falling off). Three biological replicates were set for each treatment group (sample group and control group) to ensure the reliability of the experimental results; All inoculated plates were placed in a constant temperature incubator at 28-30°C in the dark. The growth state of the virus in the two groups of plates was observed and recorded every day from the first day of culture (such as colony diameter, mycelium density, and lesion degree). The inhibitory effect of bacterial fermentation supernatant on the target pathogenic bacteria was analyzed by comparing the growth differences between the sample group and the control group. When the pathogenic bacteria in the control group grew over the plate, the size of the lesion in the experimental group was measured, and the inhibition rate was calculated. The inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter x 100. The results are shown in Table 5.

[0088] (4) Root-knot nematode killing activity detection: The second instar larvae of root-knot nematode suspension were pre-warmed at room temperature, and a pipette was used to gently blow and suck 5-10 times to fully shake, ensuring uniformity of the second instar larvae of root-knot nematode. At the same time, it was confirmed that the fermentation supernatant had no precipitate and no bacterial contamination, and was ready for use; In the target reaction wells of a sterile 96-well cell culture plate (or enzyme-labeled plate), 100 μL of the second instar larvae of root-knot nematode suspension was added, followed by 100 μL of fermentation supernatant, and the two were mixed at a volume ratio of 1:1 (the total reaction volume in each well was 200 μL). After adding the sample, the pipette was gently blown and sucked 3-5 times to ensure that the supernatant and the second instar larvae of root-knot nematode suspension were in uniform contact, and by fixing the initial sample amount of the second instar larvae of root-knot nematode suspension, the number of second instar larvae of root-knot nematode in each well was about 200; The blank control group was operated according to the same system as above: 100 μL of the second instar larvae of root-knot nematode suspension and 100 μL of beef extract peptone liquid medium (instead of bacterial fermentation supernatant) were added to the corresponding wells, and they were also fully mixed to ensure that, except for the variable "fermentation supernatant", the reaction volume, the second instar larvae of root-knot nematode suspension, and the operation environment of the control group and the sample group were completely consistent, excluding the influence of the culture medium itself on the activity of the second instar larvae of root-knot nematode; Seal the 96-well plate with sterile sealing film (to prevent changes in concentration due to liquid evaporation during incubation, or contamination by external microorganisms) and place it in a room temperature (recommended to control the room temperature fluctuation range of 23-25°C, to avoid temperature changes affecting the activity of the second instar juveniles of root-knot nematodes) environment for 24 hours of incubation; After treatment, directly observe the death of root-knot nematodes under a low-power microscope, calculate the mortality rate, and take the rigid and immobile root-knot nematodes as the standard for dead root-knot nematodes. The results are shown in Table 6.

[0089] Table 5, Inhibition effect of fermentation supernatant of strain JZ01 on the pathogen of cucumber brown spot disease ; Table 6, Killing effect of fermentation supernatant of strain JZ01 on root-knot nematodes ; The results show that the fermentation supernatant of strain JZ01 has a significant inhibitory effect on the growth of the pathogen of cucumber brown spot disease, with an inhibition rate of 62.96% (see Table 5). The fermentation supernatant of strain JZ01 starts to work after 12 hours of treatment on root-knot nematodes, and reaches 56.5% after 32 hours of treatment (see Table 6). The results show that the metabolic products of strain JZ01 also have anti-Aspergillus and root-knot nematode killing functions.

[0090] 2, Identification of metabolic active products of strain JZ01 and detection of their anti-Aspergillus and root-knot nematode killing effects In the previous experiment, we have determined that the metabolic products produced by strain JZ01 have anti-Aspergillus and root-knot nematode killing effects. In order to find out the specific main metabolic substances that play a role, whether they can be used as a synergist of Bacillus velezensis JZ01 inoculant, and play a synergistic effect in the field application, we detected the metabolic components in the fermentation broth of JZ01 strain by LC-MS. A total of 238 kinds of metabolites were detected, and the main types and quantities are shown in Table 7.

[0091] Table 7, Types and quantities of metabolic active products of strain JZ01 ; According to the detection results, most of the water-soluble active products were selected, and deionized water was used as the solvent, with a concentration of 2%. At the same time, deionized water was used as a control to detect the anti-Aspergillus and root-knot nematode killing activities of various active products. The methods used are the same as those described in 1 above.

[0092] The results of anti-cercospora activity detection are shown in Table 8, which shows that among the active substances detected, three active substances can show inhibition effect on cucumber brown spot pathogen Cercospora beticola on the plate at a final concentration of 0.1%, which are Trigonelline, 4-Oxohexanoic acid and Phe-Tyr, and the inhibition rates are 48.52%, 12.59% and 22.59% respectively.

[0093] Table 8, Inhibition effect of different active substances on cucumber brown spot pathogen ; The results of nematode-killing activity detection are shown in Table 9, which shows that among the active substances detected, two active substances have a killing effect on root-knot nematodes of more than 10% at a final concentration of 1%, which are Cystathionine and Ganoderic acid A, and the killing effects are 17.5% and 48.5% respectively.

[0094] Table 9, Killing effect of different active substances on root-knot nematodes ; The above results show that among the many JZ01 metabolic active substances, Trigonelline has the best antagonistic effect on cucumber brown spot pathogen Cercospora beticola, and Ganoderic acid A has the best killing effect on second instar larvae of root-knot nematodes. Therefore, Trigonelline and Ganoderic acid A have the potential to be developed into strain JZ01 fungicide synergist.

[0095] 3, Field test of Ganoderic acid A improving the control effect of JZ01 fungicide on cucumber root-knot nematode disease In order to clarify the synergistic effect of Ganoderic acid A (purchased from Chengdu Maidesheng Technology Co., Ltd.) on Bacillus velezensis JZ01 fungicide, we carried out field test. The test site is selected in the vegetable planting base of Shouguang City, Weifang City, Shandong Province. The cucumber planted in this area is harmed by root-knot nematodes all year round, and the variety is Shouyan 101. The specific test arrangement is as follows: (1) Test agents

[0096] ① JZ01 fungicide: the viable bacterial count is 5.0 billion CFU / gram, which is prepared by Shandong Bin'nan Technology Co., Ltd.

[0097] ② 10% thiadiazolidine granules: provided by the test site, which is a chemical root-knot nematode-killing agent commonly used in previous years.

[0098] ③ JZ01 fungicide + 1% Ganoderic acid A: according to the mass ratio, Ganoderic acid A is added to JZ01 fungicide, and the final content of Ganoderic acid A is 1%.

[0099] (4) JZ01 microbial agent + 3% ganoderic acid A: according to the mass ratio, add ganoderic acid A in JZ01 microbial agent, and the final content of ganoderic acid A is 3%.

[0100] (5) JZ01 microbial agent + 5% ganoderic acid A: according to the mass ratio, add ganoderic acid A in JZ01 microbial agent, and the final content of ganoderic acid A is 5%.

[0101] (6) 3% ganoderic acid A: inactivate JZ01 microbial agent at high temperature, and add ganoderic acid A according to the mass ratio, and the final content of ganoderic acid A is 3%.

[0102] (2) Test method After the transplanting of cucumbers is completed, irrigation is carried out, and the dilution is 1000 times, 2.5 kg per mu. The second time is applied after 15-20 days of the first application, and the whole test process is applied twice. The use of thiazolylphosphine is according to the normal farmer's use method. The treatment without applying any nematicide is used as a blank control; each designed test area is more than 0.5 mu.

[0103] (3) Investigation and statistical method After the last drip irrigation is completed for 30 days, on-site investigation is carried out. The disease classification of cucumber root-knot nematode of each treatment is counted, and the control effect is calculated; according to the 5-point sampling method, 20 plants of each treatment are randomly investigated, and the repetition is 3 times.

[0104] The root-knot nematode disease classification standard is as follows: 0 level: no galls on root system; 1 level: a small amount of galls on root system; 3 level: two-thirds of root system covered with small galls; 5 level: root system covered with galls and secondary galls; 7 level: root system forms root nodule.

[0105] The disease index and control effect are calculated according to the following formula. Disease index = [∑ (number of each disease level x representative value of each disease level) / (total number of plants x representative value of highest disease level)] x 100. Control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area x 100.

[0106] (4) Test results The results are shown in Table 10: when JZ01 microbial agent is used alone, the control effect on root-knot nematode reaches 64.71%; compared with 10% thiazolylphosphine granules, there is no significant difference; it is proved that JZ01 microbial agent can effectively replace 10% thiazolylphosphine granules, and there is no chemical residue.

[0107] Table 10, control effect of each nematicide on cucumber root-knot nematode disease ; Note: the same letter after the same column data in the table indicates that there is no significant difference, and different letters indicate that there is a significant difference.

[0108] Result analysis: On the basis of JZ01 microbial agent, the addition of 1%, 3% and 5% ganoderma acid A, the control effect is 67.50%, 75.42% and 76.25% respectively; Compared with the former, it increased by 2.79%, 10.71% and 11.54% respectively; It shows that the addition of ganoderma acid A can significantly improve the control effect of JZ01 microbial agent. Among them, the effect of adding 3% and 5% ganoderma acid A is the best, which is significantly higher than that of 10% thiazolylphosphine granules (65.83%); And when the content of ganoderma acid A is 3% and 5%, there is no significant difference between the two, so the appropriate amount of ganoderma acid A in JZ01 microbial agent is 3%.

[0109] 4. Field test of trigonelline improving the control effect of JZ01 microbial agent on cucumber brown spot In order to clarify the synergistic effect of trigonelline (purchased from Wuhan Guanying Biological Technology Co., Ltd.) on bacillus velezensis JZ01 microbial agent, we carried out field test. The test site is located in the vegetable planting base of Xian County, Liaocheng City, Shandong Province. The cucumber variety is Cuiyu No. 3, which is planted in this area all year round and is affected by cucumber brown spot. The specific test arrangement is as follows: (1) Test agent

[0110] ① JZ01 microbial agent: the viable bacterial content is 5.0 billion CFU / gram, which is prepared by Shandong Bin'agricultural Technology Co., Ltd.

[0111] ② 5% chlorothalonil wettable powder: provided by the test site, which is a chemical fungicide commonly used in previous years.

[0112] ③ JZ01 microbial agent + 0.1% trigonelline: according to the mass ratio, add trigonelline in JZ01 microbial agent, and the final content of trigonelline is 0.1%.

[0113] ④ JZ01 microbial agent + 0.3% trigonelline: according to the mass ratio, add trigonelline in JZ01 microbial agent, and the final content of trigonelline is 0.3%.

[0114] ⑤ JZ01 microbial agent + 0.5% trigonelline: according to the mass ratio, add trigonelline in JZ01 microbial agent, and the final content of trigonelline is 0.5%.

[0115] ⑥ 0.3% trigonelline: inactivate JZ01 microbial agent by high temperature, add trigonelline according to the mass ratio, and the final content of trigonelline is 0.3%.

[0116] (2) Test method The first leaf spraying was carried out when the cucumber just entered the fruiting stage, the dilution ratio was 500 times, the dosage was 150g / acre, and the second spraying was carried out after 15 days. Chlorothalonil is used according to the normal method of farmers. The treatment without any disease control agent is blank control; Each design test area is more than 0.5 mu.

[0117] (3) Investigation and statistical method Field investigation was carried out 5d, 10d, 15d after the last spraying was completed. The disease classification of cucumber brown spot of each treatment was counted, and the control effect was calculated; according to the 5-point sampling method, 20 plants were randomly investigated for each treatment, and the test was repeated 3 times.

[0118] The brown spot classification standard: 0 level: no disease spot; 1 level: the disease spot area accounts for less than 5% of the whole leaf area; 3 level: the disease spot area accounts for 6%~10% of the whole leaf area; 5 level: the disease spot area accounts for 11%~25% of the whole leaf area; 7 level: the disease spot area accounts for 26%~50% of the whole leaf area; 9 level: the disease spot area accounts for more than 51% of the whole leaf area.

[0119] The disease index and control effect were calculated according to the following formula.

[0120] Disease index = [∑ (number of each disease level × representative value of each disease level) / (total number of plants × representative value of the highest disease level)] × 100. Control effect (%) = (disease index of the control area - disease index of the treatment area) / disease index of the control area × 100.

[0121] (4) Test results Table 11, control effect of each pesticide on cucumber brown spot ; Note: The same letter after the same column data in the table indicates that there is no significant difference, and different letters indicate that there is significant difference.

[0122] The results are shown in Table 11; from Table 11, it can be seen that when JZ01 fungicide is used alone, the control effect on cucumber brown spot is 69.75%, which is lower than 75.86% of 5% quinoxyfen wettable powder. However, when 0.1%, 0.3% and 0.5% of trigonelline is added respectively, the control effects are 81.73%, 81.81% and 82.07% respectively; which are increased by 11.98%, 12.06% and 12.32% respectively compared with the former; and are significantly better than the control effect (75.86%) of 5% quinoxyfen wettable powder; which indicates that the addition of trigonelline can significantly improve the control effect of JZ01 fungicide. Since the addition amount of trigonelline is 0.1%, 0.3% and 0.5%, there is no significant difference in the control effect among the three, and it is preliminarily determined that the suitable addition amount of trigonelline in JZ01 fungicide is 0.1%.

[0123] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A strain of Bacillus velezensis strain JZ01, characterized in that, The taxonomic name of the bacillus velezensis strain JZ01 is Bacillus velezensis , which was preserved in the China General Microbiological Culture Collection Center on July 21, 2025, and the preservation number is CGMCC NO.35305.

2. A microbial agent comprising at least one selected from the group consisting of viable bacteria, freeze-dried bacteria, inactivated bacteria, and strain culture of Bacillus velezensis strain JZ01 according to claim 1.

3. The bacterial agent of claim 2, wherein The strain culture is fermentation broth, fermentation supernatant, and / or concentrated or dried product of the foregoing of the strain; And / or, the microbial agent is a liquid preparation or a powder.

4. The bacterial agent of claim 2 or 3, characterized in that, The microbial agent further comprises a synergist, which is trigonelline and / or ganoderic acid A.

5. The bacterial agent of claim 4, characterized in that, The synergist is ganoderic acid A, which is present in the microbial agent at a content of 1%-5%.

6. The bacterial agent of claim 4, wherein The synergist is trigonelline, which is present in the microbial agent at a content of 0.1%-0.5%.

7. Use of Bacillus velezensis strain JZ01 according to claim 1 or the microbial agent according to any one of claims 2-6 in the preparation of a biopesticide.

8. Use according to claim 7, characterized in that, The biopesticide is used for any one or more of the following purposes: (1) for antagonizing Cercospora sochanica; (2) for preventing and treating Cercospora sochanica-induced cucumber brown spot; (3) for killing root-knot nematode; (4) for preventing and treating root-knot nematode-induced cucumber root-knot nematode disease.

9. Use of viable bacteria, freeze-dried bacteria, inactivated bacteria, and strain culture of Bacillus velezensis strain JZ01 according to claim 1 and / or trigonelline in the preparation of a pesticide for antagonizing Cercospora sochanica and / or for preventing and treating Cercospora sochanica-induced cucumber brown spot.

10. Use of viable bacteria, freeze-dried bacteria, inactivated bacteria, and strain culture of Bacillus velezensis strain JZ01 according to claim 1 and / or ganoderic acid A in the preparation of a pesticide for killing root-knot nematode and / or for preventing and treating root-knot nematode-induced cucumber root-knot nematode disease.

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