Bacillus velezensis strain jz01, bacterial agent containing same, and application thereof

Abstract

The present application relates to a bacillus velezensis strain JZ01, a microbial agent containing the same and application thereof. The bacillus velezensis strain JZ01 can efficiently antagonize the pathogen of cucumber brown spot and effectively kill root-knot nematodes. Further, the present application also develops a synergist of the strain. It is found that trigonelline can be used as a synergist to significantly increase the efficacy of the strain against the pathogen of cucumber brown spot, and ganoderic acid A can enhance the efficacy of the strain in killing root-knot nematodes, so that a more efficient compound microbial agent can be developed. In addition, the bacillus velezensis strain and the microbial agent containing the same are microbial agents, which are safe and environmentally friendly, and therefore have high industrial value and application prospect.

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 serious 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 soil with worms, diseased roots and irrigation water; 2nd instar larvae invade root tips to cause infection in spring, and the cycle of generations continues to harm. Among the current control methods, agricultural measures such as pest-free soil seedling and crop rotation have limited effect; chemical agents such as 10% clonitralid, 3% milaer and abamectin 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 in biological control of plant diseases and pests, which has been reported to play a role in many plant diseases, but there is no report on its use in preventing and controlling the above-mentioned pathogens and plant diseases caused by them. Bacillus velezensis SUMMARY

[0005] OBJECTIVE

[0006] ​In order to solve the problems of strong drug resistance, great influence on the environment, food safety and the like in the chemical control of cucumber brown spot caused by Cercospora in the prior art, the present application provides a bacillus velezensis strain JZ01, a microbial agent containing the same and application thereof. The bacillus velezensis strain JZ01 or the microbial agent containing the same has the effects of highly antagonizing Cercospora and killing root-knot nematodes, and can play a key role in the biological control of crops.

[0007] Further, the present application finds, through the study of the endogenous metabolites of the bacillus velezensis strain, that the active ingredient trigonelline itself has the effect of antagonizing the pathogen Cercospora of cucumber brown spot, and further finds that the trigonelline can be used as a synergist to significantly improve the effect of the bacillus velezensis strain in antagonizing the pathogen Cercospora of cucumber brown spot. In addition, the active ingredient ganoderic acid A itself can kill root-knot nematodes, especially second instar larvae of root-knot nematodes, and further finds that the ganoderic acid A can be used as a synergist to significantly improve the effect of the bacillus velezensis strain in killing root-knot nematodes, especially second instar larvae of root-knot nematodes.

[0008] Solution

[0009] In order to achieve the object of the present application, the present application provides the following technical solutions.

[0010] In a first aspect, the present application provides a bacillus velezensis (Bacillus velezensis) strain JZ01, wherein the bacillus velezensis (Bacillus velezensis) strain JZ01 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2025, and the preservation number is CGMCC NO. 35305. Bacillus velezensis Bacillus velezensis The bacillus velezensis of the present application is isolated from the silt at the estuary of a river, and the inventors find, through confrontation experiments, that the isolated bacillus velezensis (Bacillus velezensis) strain JZ01 has a strong antagonistic effect on Cercospora and a strong control effect on cucumber brown spot caused by Cercospora, and has the function of highly inhibiting the pathogen of cucumber brown spot. Further, the bacillus velezensis (Bacillus velezensis) strain JZ01 of the present application also has the effect of killing root-knot nematodes, especially second instar larvae of root-knot nematodes, and thus can be used to effectively control cucumber root-knot nematode disease caused by root-knot nematodes.

[0011] The bacillus velezensis (Bacillus velezensis) strain JZ01 of the present application is identified as bacillus velezensis (Bacillus velezensis) through morphological identification, physiological and biochemical characteristic identification and 16S rRNA sequencing. Bacillus velezensis Bacillus velezensis The bacillus velezensis (Bacillus velezensis) strain JZ01 of the present application is identified as bacillus velezensis (Bacillus velezensis) through morphological identification, physiological and biochemical characteristic identification and 16S rRNA sequencing.

[0012] The bacillus velezensis (Bacillus velezensis) strain JZ01 of the present application is identified as bacillus velezensis (Bacillus velezensis) through morphological identification, physiological and biochemical characteristic identification and 16S rRNA sequencing. Bacillus velezensis Bacillus velezensis ​​​) having a 16S rRNA gene sequence of SEQ ID NO: 1.

[0013] The Bacillus velezensis (Bacillus velezensis) Bacillus velezensis The colony state of the Bacillus velezensis (Bacillus velezensis)

[0014] 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 / or a culture of the strain, optionally, the culture of the strain is a fermentation broth, a fermentation supernatant and / or a concentrated or dried product of the foregoing.

[0015] The microbial agent can be in a form of a conventional dosage form in the art, for example, a liquid or a solid preparation, and the dosage form can be selected according to the application requirement, preferably, a liquid preparation or a powder.

[0016] In preferred embodiments, the microbial agent further comprises a synergistic agent.

[0017] Optionally, the synergistic agent is an active metabolite of the Bacillus velezensis (Bacillus velezensis) Bacillus velezensis strain JZ01, preferably, the synergistic agent is trigonelline and / or ganoderic acid A.

[0018] 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%.

[0019] 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%.

[0020] 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.

[0021] In feasible embodiments, the biological pesticide is used for any one or more of the following purposes:

[0022] (1) for antagonizing Cercospora, preferably Cercospora sochii; Cercospora kikuchii

[0023] (2) for preventing and treating cucumber brown spot caused by Cercospora, preferably Cercospora sochii; Cercospora kikuchii

[0024] (3) for killing root-knot nematodes, preferably Meloidogyne arenaria;

[0025] ​​(4) Used to prevent and control cucumber root-knot nematode disease caused by root-knot nematodes, preferably cucumber root-knot nematodes.

[0026] Fourthly, the present invention provides (1) Bacillus belyesensis as described in the first aspect above ( Bacillus velezensis (1) Live, lyophilized, inactivated, or cultured strain JZ01 (optionally, the cultured strain is the fermentation broth, fermentation supernatant, and / or the aforementioned concentrated or dried product), and / or (2) trigonelline in the preparation of a solution for antagonizing Cercospora (preferably Cercospora). Cercospora kikuchii ) and / or for the prevention and control of diseases caused by Cercospora (preferably Cercospora) Cercospora kikuchii Its use in pesticides to treat cucumber brown spot disease caused by )

[0027] Fifthly, the present invention provides an antagonistic agent against Cercospora (preferably Cercospora). Cercospora kikuchii ) and / or control of pathogens caused by Cercospora (preferably Cercospora) Cercospora kikuchii A method for treating cucumber brown spot disease caused by (1) Bacillus belesiensis as described in the first aspect above, the method comprising: applying an effective amount of (1) Bacillus belesiensis as described in the first aspect above to the plant in need or its growing medium (e.g., soil). Bacillus velezensis (1) Live bacteria, freeze-dried bacteria, inactivated bacteria, strain culture of strain JZ01 (optionally, the strain culture is the fermentation broth, fermentation supernatant and / or the aforementioned concentrated or dried product of the strain), and / or (2) trigonelline.

[0028] In a sixth aspect, the present invention provides (1) Bacillus belye as described in the first aspect above ( Bacillus velezensis (1) Live, freeze-dried, inactivated, or cultured strain JZ01 (optionally, the cultured strain is the fermentation broth, fermentation supernatant, and / or the aforementioned concentrated or dried product), and / or (2) the use of ganoderic acid A in the preparation of pesticides for killing root-knot nematodes (preferably cucumber root-knot nematodes, such as second-instar larvae of root-knot nematodes) and / or for controlling cucumber root-knot nematode disease caused by root-knot nematodes.

[0029] In a seventh aspect, the present invention provides a method for killing root-knot nematodes (preferably cucumber root-knot nematodes, such as second-instar larvae of root-knot nematodes) and / or preventing cucumber root-knot nematode disease caused by root-knot nematodes, the method comprising: applying an effective amount of (1) Bacillus belesiensis as described in the first aspect above to the plant in need or its growing medium (e.g., soil). Bacillus velezensis (1) Live bacteria, freeze-dried bacteria, inactivated bacteria, strain culture of strain JZ01 (optionally, the strain culture is the fermentation broth, fermentation supernatant and / or the aforementioned concentrated or dried product of the strain), and / or (2) ganoderic acid A.

[0030] Beneficial effects

[0031] (1) The Bacillus belye of the present invention ( Bacillus velezensis Strain JZ01 exhibits excellent antagonistic activity against Cercospora and highly effective killing of root-knot nematodes. Specifically, its inhibition rate against Cercospora, the pathogen causing cucumber brown spot disease, reaches 82.96%; the killing rate against root-knot nematodes is as high as 81.6% after 16 hours and 100% after 20 hours.

[0032] (2) Research has found that the aforementioned Bacillus belyssus ( Bacillus velezensis The metabolites trigonelline and ganoderic acid A of strain JZ01 can act as synergists to significantly enhance its efficacy against Cercospora and root-knot nematodes, making its control efficacy significantly surpass that of existing chemical fungicides. Therefore, it can be used to develop highly effective compound fungicides. Since the synergists ganoderic acid A and trigonelline are derived from Bacillus belye (… Bacillus velezensis The compound bacterial agent is extracted from the metabolites of strain JZ01 and does not contain any exogenous substances. Therefore, the compound bacterial agent containing it is relatively safe and environmentally friendly.

[0033] In summary, both the Bacillus vesiculosus strain JZ01 itself and the compound bacterial agent containing synergists ganoderic acid A and trigonelline possess high efficiency, safety, and environmental friendliness, thus exhibiting high industrial value and application prospects. Attached Figure Description

[0034] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the implementation. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0035] Figure 1 It is the 10 described in Embodiment 1 of this invention. -4 Colony status after 3 days of incubation on dilution plates.

[0036] Figure 2 These are the results of the plate confrontation experiment of strains JZ01 and JZ02 described in Example 1 of this invention.

[0037] Figure 3 These are the colony morphologies of single colonies of strain JZ01 described in Example 1 of this invention at different time periods during culture.

[0038] Figure 4 The microscopic morphology of strain JZ01 is described in Example 1 of this invention. Detailed Implementation

[0039] 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 clearly 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.

[0040] 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.

[0041] Embodiment 1: Screening and identification of the strain JZ01 of the present application

[0042] This embodiment provides experiments related to the isolation and purification of the strain JZ01 of the present application, the screening of the anti-aspergillus and nematode-killing functions, and the identification of the strain.

[0043] 1. Isolation and purification of the strain JZ01

[0044] 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:

[0045] (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.

[0046] (2) Preparation of sample suspension: accurately weigh 10 g of river mud sample and place it in a triangular flask containing 90 mL of sterile water, and 25-30 glass beads are added in advance; after placing it in a 30℃ constant temperature shaking incubator, set the shaking rate to 150 r / min, and uniformly shake 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.

[0047] (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.

[0048] (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.

[0049] 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.

[0050] (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 in the plate was observed at regular intervals.

[0051] (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.

[0052] The results are shown in Figure 1 , 10 -4 dilution plate, the colony distribution was better, the number was moderate, there was 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.

[0053] 2. Screening of strains antagonistic to Cercospora

[0054] The present application takes the pathogenic fungus of cucumber brown spot disease as the indicator strain, and the specific method is as follows: Cercospora kikuchii The pathogenic fungus is isolated from cucumber leaves of cucumber brown spot disease, and is verified by Koch's postulates, and is identified as Aspergillus by strain identification Cercospora kikuchii ) for inquiry of antagonistic function of 16 strains of Aspergillus, and the specific method is as follows:

[0055] (1) Activation of the tested strain: the above-mentioned 16 strains were streaked on beef extract peptone agar medium, and cultured at 30°C for about 18h; no contamination was observed, and the activation was successful.

[0056] (2) Activation of the indicator pathogenic fungus: the stored Aspergillus plate was taken out from the 4°C refrigerator, and inoculated on modified potato dextrose agar (PDA) medium under sterile operation, and cultured at 28°C for more than 50h, and the pathogenic fungus was grown to 3 / 4 of the plate, and the activation was successful.

[0057] 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, and pH is natural.

[0058] (3) Plate confrontation culture: 0.3~0.5cm 2 of the activated Aspergillus was inoculated in the center of the modified PDA medium plate, and the activated tested strain was inoculated on both sides 2cm away from the center, and the un-inoculated two sides were used as controls, and each treatment was repeated for 3 plates; after the treatment was completed, the inhibition effect of the tested strain on the pathogenic fungus was observed every day, and after the pathogenic fungus on the control two sides grew to the edge of the plate, the size of the disease spot of each treatment group was measured, and the inhibition rate was calculated according to the following formula: inhibition rate = [(control colony radius-treatment colony radius)] / control colony radius x 100%.

[0059] The results are shown in the following table 1.

[0060] Table 1, inhibition effect of different strains on cucumber brown spot disease pathogen

[0061] ;

[0062] Note: "-" indicates no antagonistic effect; "+" indicates 0

[0063] 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.

[0064] 3. Screening of strains with root-knot nematode killing function

[0065] 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:

[0066] (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.

[0067] (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.

[0068] (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".

[0069] The results are shown in Table 2 below.

[0070] Table 2, different strains of nematode root-killing effect statistics

[0071] ;

[0072] Note: "+" represents "root-killing activity", "-" represents "no root-killing activity".

[0073] 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 tail fungus and root-knot nematode activity, and therefore have multifunctionality.

[0074] 4. Screening of high-efficiency root-knot nematode-killing strain JZ01

[0075] In order to further compare the root-knot nematode-killing activity of strains JM03, JM05 and JZ01, and screen strains with better root-knot nematode-killing activity, we 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, every 4h, the death of root-knot nematodes was observed by microscope, the number of dead root-knot nematodes was counted and the mortality was calculated according to the following formula, taking the rigid and immobile root-knot nematodes as the dead root-knot nematodes: mortality (%) = number of dead root-knot nematodes in each group / total number of root-knot nematodes in each group x 100.

[0076] The results are shown in Table 3 below.

[0077] Table 3, the killing effect statistics of three strains of root-knot nematodes at the same concentration

[0078] ;

[0079] Result analysis: the concentration of 3.0x10 9 CFU·mL -1JZ01 was found to have dead root-knot nematodes after 4h of treatment, and JM05 and JM05 were found to have dead root-knot nematodes after 8h and 12h of treatment, respectively, at the same concentration (see Table 3); among them, the strain JZ01 reached 100% root-knot nematode mortality after 20h of treatment (see Table 3), and the root-knot nematode killing effect 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.

[0080] 5. Strain identification of strain JZ01

[0081] (1) Morphological identification

[0082] The colony morphology of strain JZ01 cultured for different time periods (16h, 32h, 48h) is shown in Figure 3 , which shows that after culturing on beef extract peptone agar medium, the single colony of strain JZ01 is round, oval to irregular in shape in the early stage (16h), with smooth and moist surface, sticky, and opaque; the single colony morphology is consistent with the early stage in the middle stage (32h), with smooth and moist surface, sticky, milky white, and the edge begins to appear spreading radial growth, with a slightly raised middle part; in the late stage (48h), the surface is dry, and the edge appears jagged radial growth and dry.

[0083] The bacterial body and spore morphology of strain JZ01 are shown in Figure 4 , which shows that through Gram staining, the bacterial body of strain JZ01 shows purple, which is judged as Gram-positive bacteria, and the bacterial body is rod-shaped; through simple staining in the late stage, it is found that the strain produces spores, and the spores are mesosporangium, so we judge that it is a spore-forming bacteria.

[0084] (2) Physiological and biochemical identification

[0085] Referring to the "Common Bacteria System Identification Manual", the partial physiological and biochemical characteristics of strain JZ01 were tested (the detection method was the conventional detection method in the art), and the results are shown in Table 4.

[0086] Table 4, physiological and biochemical characteristics of strain JZ01

[0087] ;

[0088] Table 4 (continued), physiological and biochemical characteristics of strain JZ01

[0089] ;

[0090] Note: "+" represents positive reaction, "-" represents negative reaction.

[0091] According to the physiological and biochemical characteristics of the strain JZ01, it is preliminarily confirmed that it is a bacterium of Bacillus, which is consistent with the morphological identification.

[0092] (3) Molecular biology identification

[0093] The 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 as shown in 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, the 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.

[0094] The results of 16S rRNA gene sequence determination are as follows:

[0095] ​

[0096] Example 2: Preparation of high-content Bacillus velezensis JZ01 inoculant

[0097] This example provides methods and processes related to the preparation of Bacillus velezensis JZ01 mother liquor, mother powder, and inoculant.

[0098] 1. Obtaining Bacillus velezensis JZ01 mother liquor

[0099] 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:

[0100] (1) Preparation of shake flask seed liquid

[0101] a. Streaking on beef extract peptone agar medium, incubating at 30~32℃ for about 18~20h; observing for contamination. If contaminated, continue to pick single colonies and streak according to colony morphology until the colonies are single.

[0102] b. After observing no contamination, pick three single colonies and inoculate into beef extract peptone liquid medium, incubate at 30~32℃, 90~120r / min for 18~20h.

[0103] c. After incubation, observe by microscopy for contamination and consistency of colony growth state. Select the best one from the three bottles without contamination for standby.

[0104] 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℃, 90~120r / min for 18~20h, to obtain the shake flask seed liquid.

[0105] (2) Primary seed fermentation

[0106] a. Feeding: feed seed fermentation medium into the tank according to 60~70% of tank volume, and additionally add 1~2% water for consumption.

[0107] 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.

[0108] b. Sterilization and temperature reduction: according to the conventional sterilization requirement, maintain 121±1℃, 0.05MPa high temperature and high pressure sterilization for 30-40min; after sterilization, reduce the temperature to 32±1℃.

[0109] c. Inoculation and fermentation: inoculate the seed tank after sterilization with the seed liquid in the shake flask, and the inoculation amount is 0.5-1v / v% of the material liquid; after inoculation, carry out fermentation according to the following process.

[0110] Fermentation process: maintain the temperature 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.

[0111] (3) Secondary expansion fermentation

[0112] a. The feeding and sterilization and temperature reduction are the same as the primary seed fermentation.

[0113] b. Seed transfer: directly pump the seed after the primary fermentation into the matched fermentation tank, and the inoculation amount is 9.5-10.5%; after inoculation, carry out fermentation according to the following process.

[0114] Fermentation process: maintain the temperature 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 ventilation gas is sterile air filtered by three stages.

[0115] When the spore rate under microscope is ≥85%, 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%.

[0116] 2. Preparation of bacillus velezensis JZ01 microbial agent

[0117] 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, and the specific method steps are as follows:

[0118] (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 total water soluble benefit powder (ordinary commercial product) in proportion, fully stir and mix, ensure that the benefit powder is completely dissolved and uniformly compatible with the bacterial mother liquor, and form a stable spray powder precursor liquid;

[0119] The precursor liquid is introduced into a spray drying device, and the operation parameters of the device are set and stabilized: the inlet temperature is 160-170°C (to ensure rapid atomization and preliminary dehydration of the precursor liquid), and the outlet temperature is 60-65°C (to avoid inactivation of the bacterial body caused by high outlet temperature, while ensuring effective removal of water); the device is started for spray drying;

[0120] After drying, the 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 8-12% (w / w, i.e., mass percentage); the number of viable bacteria in the mother powder is determined by plate counting method (or other viable bacteria detection method), which needs to reach 80-120 billion CFU / g (colony forming unit / gram) quality standard, and only after passing the quality standard can it be used for subsequent experiments or storage.

[0121] (2) Preparation of raw powder: according to the content of B. velezensis JZ01 in the mother powder, add the benefit powder to prepare B. velezensis JZ01 raw powder with a content of 20 billion / g, according to the weight ratio of mother powder: benefit powder of 1:(3-5).

[0122] (3) Preparation of finished product: on the basis of B. velezensis JZ01 raw powder, we compound 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 B. velezensis JZ01 inoculant, in which the content of B. velezensis JZ01 is about 150-160 billion CFU / g.

[0123] Example 3: Screening of B. velezensis JZ01 inoculant synergist

[0124] This example provides related experiments for screening of B. velezensis JZ01 inoculant synergist.

[0125] 1. Antagonism of JZ01 strain fermentation supernatant to Cercospora and nematode killing function determination

[0126] (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 be considered as successful activation.

[0127] (2) Preparation of fermentation supernatant: use 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 fermentation supernatant.

[0128] (3) Antagonistic activity of Cercospora detection: Take the PDA medium cooled to 45~50°C after melting, add JZ01 strain fermentation supernatant according to the volume ratio of 1:19, mix gently (avoid generating too many bubbles to affect the flatness of the plate), immediately pour the mixed liquid into a sterile culture dish, and place it in a clean bench to cool naturally to complete solidification, and prepare a sample plate containing fermentation supernatant;

[0129] Take beef extract protein peptone liquid medium as a blank control, prepare a control plate according to the same operation as the sample group, and ensure that all experimental conditions are consistent except for "whether containing fermentation supernatant" to exclude irrelevant variable interference;

[0130] Select activated Cercospora, and use sterile forceps or puncher to cut virus blocks with a size of 0.3~0.5 cm 2 (ensure that the size of the block is uniform and the edge is complete), and inoculate them into the central position of the sample plate and the control plate (when inoculating, gently press the block to make one corner of it tightly adhere to the medium to avoid falling off). Each treatment group (sample group, control group) has 3 biological replicates to ensure the reliability of the experimental results;

[0131] Put all inoculated plates into a constant temperature incubator at 28~30°C in the dark, and observe and record the growth state of the virus in the two groups of plates (such as colony diameter, mycelium density, and lesion degree) every day from the first day of culture. By comparing the growth differences of the sample group and the control group, the inhibitory effect of bacterial fermentation supernatant on the target pathogen is analyzed. When the pathogen in the control group covers the plate, measure the size of the lesion in the experimental group and calculate the inhibition rate. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter x 100. The results are shown in Table 5.

[0132] (4) Root-knot nematode killing activity detection: The second instar larva suspension of root-knot nematode was pre-warmed at room temperature, and a pipette was used to blow and suck 5~10 times to fully shake, ensuring that the second instar larvae of root-knot nematode were uniform; At the same time, confirm that the fermentation supernatant has no precipitate and no bacterial contamination, and prepare for use;

[0133] In the target reaction well of a sterile 96-well cell culture plate (or enzyme-labeled plate), first add 100 μL of well-shaken root-knot nematode second instar larva suspension, then add 100 μL of fermentation supernatant, and mix them thoroughly according to the volume ratio of 1:1 (the total reaction volume in each well is 200 μL). After adding the sample, use a pipette to gently blow and suck 3~5 times to ensure that the supernatant and the root-knot nematode second instar larva suspension are in uniform contact, and by fixing the initial sample amount of the root-knot nematode second instar larva suspension, the number of root-knot nematode second instar larvae in each well is about 200;

[0134] The blank control group was operated according to the same system: 100 μL of second instar nematode suspension and 100 μL of beef extract peptone liquid medium (instead of bacterial fermentation supernatant) were added to the corresponding holes, and they were mixed thoroughly to ensure that the reaction volume, second instar nematode suspension, and operation environment of the control group and sample group were completely consistent, excluding the influence of the culture itself on the activity of the second instar nematode.

[0135] The 96-well plate with completed sample addition was sealed with sterile sealing film (to prevent liquid evaporation during incubation, which would cause changes in concentration, or contamination by external microorganisms) and placed in a room temperature (recommended control room temperature fluctuation range: 23-25°C, to avoid temperature changes affecting the activity of the second instar nematode) environment for 24 h of incubation.

[0136] After treatment, direct microscopic examination was performed under a low-power microscope to observe the death of the nematodes and calculate the mortality rate, with the standard for dead nematodes being rigid and immobile nematodes. The results are shown in Table 6.

[0137] Table 5, Inhibition effect of fermentation supernatant of strain JZ01 on the pathogen of cucumber brown spot disease

[0138] ;

[0139] Table 6, Killing effect of fermentation supernatant of strain JZ01 on nematodes

[0140] ;

[0141] 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 begins to take effect after 12 h of treatment, and reaches 56.5% after 32 h of treatment (see Table 6). This result shows that the metabolic products of strain JZ01 also have anti-Aspergillus and nematocidal functions.

[0142] 2. Identification of metabolic active products of strain JZ01 and detection of their anti-Aspergillus and nematocidal effects

[0143] In the previous experiment, we determined that the metabolic products produced by strain JZ01 have anti-Aspergillus and nematocidal 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 field application, we detected the metabolic components in the fermentation broth of JZ01 strain using LC-MS. A total of 238 kinds of metabolites were detected, and the main types and quantities are shown in Table 7.

[0144] Table 7, Types and quantities of metabolic active products of strain JZ01

[0145] ;

[0146] According to the detection results, the pure product of most water-soluble active products was selected, deionized water was used as the solvent, and the concentration was adjusted to 2%; at the same time, deionized water was used as a control to detect the anti-Aspergillus caespiossus and nematode-killing activity of various active products. The method used is the same as that described in 1 above.

[0147] The anti-Aspergillus caespiossus activity detection results are shown in Table 8, which shows that among the active substances detected, three active substances can exhibit inhibition effect on the pathogen of cucumber brown spot disease Aspergillus caespiossus 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.

[0148] Table 8, Inhibition effect of different active substances on cucumber brown spot pathogen

[0149] ;

[0150] The nematode-killing activity detection results 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.

[0151] Table 9, Killing effect of different active substances on root-knot nematodes

[0152] ;

[0153] The above results show that among the many JZ01 metabolic active products, Trigonelline has the best antagonistic effect on the pathogen of cucumber brown spot disease Aspergillus caespiossus, and Ganoderic acid A has the best killing effect on the second instar larvae of root-knot nematodes. Therefore, Trigonelline and Ganoderic acid A have the potential to develop into strain JZ01 fungicide synergist.

[0154] 3. Field test of Ganoderic acid A improving the control effect of JZ01 fungicide on cucumber root-knot nematode disease

[0155] In order to clarify the synergistic effect of Ganoderic acid A (purchased from Chengdu Maidesheng Technology Co., Ltd.) on Bacillus velezensis JZ01 fungicide, we conducted a field test. The test site was 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:

[0156] (1) Test agent

[0157] ① JZ01 microbial agent: the viable bacterial count is 5.0 billion CFU / gram, prepared by Shandong Bin'ang Technology Co., Ltd.

[0158] ② 10% fosthiazate granules: provided by the test site, a chemical nematicide commonly used in previous years.

[0159] ③ JZ01 microbial agent + 1% ganoderic acid A: ganoderic acid A is added to the JZ01 microbial agent according to the mass ratio, and the final content of ganoderic acid A is 1%.

[0160] ④ JZ01 microbial agent + 3% ganoderic acid A: ganoderic acid A is added to the JZ01 microbial agent according to the mass ratio, and the final content of ganoderic acid A is 3%.

[0161] ⑤ JZ01 microbial agent + 5% ganoderic acid A: ganoderic acid A is added to the JZ01 microbial agent according to the mass ratio, and the final content of ganoderic acid A is 5%.

[0162] ⑥ 3% ganoderic acid A: the JZ01 microbial agent is inactivated at high temperature, and ganoderic acid A is added according to the mass ratio, and the final content of ganoderic acid A is 3%.

[0163] (2) Test method

[0164] After the cucumber transplanting and planting are completed, irrigation and application are carried out, 1000 times dilution, 2.5 kg / acre, the second application is carried out 15-20 days after the first application, and two applications are carried out throughout the test process. Fosthiazate is used according to the normal farmer's use method. The treatment without applying any nematicide is used as the blank control; each designed test area is more than 0.5 acres.

[0165] (3) Investigation and statistical method

[0166] After 30 days of the last drip irrigation, on-site investigation is carried out. The disease classification of cucumber root-knot nematodes in each treatment is counted, and the control effect is calculated; according to the 5-point sampling method, 20 plants in each treatment are randomly investigated, and the investigation is repeated 3 times.

[0167] Root-knot nematode disease classification standard: 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 small galls and secondary galls; 7 level: root system forms root nodule.

[0168] The disease index and control effect are calculated according to the following formula. Disease index = [∑(number of plants of each disease level × representative value of each disease level) / (total number of plants × representative value of highest disease level)] × 100. Control effect (%) = (disease index of control area - disease index of treatment area) / disease index of control area × 100.

[0169] (4) Test results

[0170] 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% fosetyl-Al granules, there is no significant difference; it is shown that JZ01 microbial agent can effectively replace 10% fosetyl-Al granules, and there is no chemical residue.

[0171] Table 10, control effect of each agent on cucumber root-knot nematode disease

[0172] ;

[0173] Note: in the same column of the table, the same letter after the data indicates that there is no significant difference, and different letters indicate that there is a significant difference.

[0174] Result analysis: on the basis of JZ01 microbial agent, the addition of 1%, 3% and 5% ganoderic acid A has a control effect of 67.50%, 75.42% and 76.25% respectively; compared with the former, it is increased by 2.79%, 10.71% and 11.54% respectively; it is shown that the addition of ganoderic acid A can significantly improve the control effect of JZ01 microbial agent. Among them, the effect is the best when the amount of ganoderic acid A is 3% and 5%, which is significantly higher than the control effect (65.83%) of the control chemical pesticide 10% fosetyl-Al granules; and when the content of ganoderic acid A is 3% and 5%, there is no significant difference between the two control effects, so it is determined that the appropriate amount of ganoderic acid A added in JZ01 microbial agent is 3%.

[0175] 4, Field test of trigonelline improving the control effect of JZ01 microbial agent on cucumber brown spot

[0176] 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 selected in the vegetable planting base of Xian County, Liaocheng City, Shandong Province. The cucumber planted in this area is harmed by cucumber brown spot all the year round, and the variety is Cuiyu No. 3. The specific test arrangement is as follows:

[0177] (1) Test agents

[0178] ① JZ01 microbial agent: the viable bacterial content is 5.0 billion CFU / gram, which is prepared by Shandong Bin'agro Technology Co., Ltd.

[0179] ② 5% chlorothalonil wettable powder: provided by the test site, which is a chemical fungicide commonly used in previous years.

[0180] 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%.

[0181] 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%.

[0182] ⑤JZ01 fungicide + 0.5% trigonelline: add trigonelline to JZ01 fungicide according to mass ratio, and the final content of trigonelline is 0.5%.

[0183] ⑥0.3% trigonelline: inactivate JZ01 fungicide at high temperature, and add trigonelline according to mass ratio, and the final content of trigonelline is 0.3%.

[0184] (2) Test method

[0185] Spray the first time when the cucumber just enters the fruiting stage, the dilution ratio is 500 times, the dosage is 150 g / mu, and the second time is sprayed after 15 days. The use of chlorothalonil is according to the normal farmer's use method. The treatment without applying any disease prevention agent is blank control; each designed test area is more than 0.5 mu.

[0186] (3) Investigation and statistical method

[0187] After the last spraying is completed for 5d, 10d, and 15d, on-site investigation is carried out. The disease classification of cucumber brown spot of each treatment is counted, and the control effect is calculated; according to the 5-point sampling method, 20 plants are randomly investigated for each treatment, and the investigation is repeated for 3 times.

[0188] The brown spot disease classification standard is as follows: 0 level: no disease spot; 1 level: the disease spot area accounts for less than 5% of the entire leaf area; 3 level: the disease spot area accounts for 6%~10% of the entire leaf area; 5 level: the disease spot area accounts for 11%~25% of the entire leaf area; 7 level: the disease spot area accounts for 26%~50% of the entire leaf area; and 9 level: the disease spot area accounts for more than 51% of the entire leaf area.

[0189] The disease index and control effect are calculated according to the following formula.

[0190] Disease index = [∑ (number of each disease level plants x representative value of each disease level) / (total number of plants x representative value of the highest disease level)] x 100. Control effect (%) = (disease index of the control area - disease index of the treatment area) / disease index of the control area x 100.

[0191] (4) Test results

[0192] Table 11, control effect of each pesticide on cucumber brown spot

[0193] ;

[0194] Note: the same letter after the same column data in the table means that there is no significant difference, and different letters mean that there is a significant difference.

[0195] 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% 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; it is indicated 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 effects of the three, and it is preliminarily determined that the suitable addition amount of trigonelline in JZ01 fungicide is 0.1%.

[0196] Finally, it should be pointed out that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; 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 belyssus JZ01, characterized in that, The taxonomic name of the Bacillus belysium strain JZ01 is: Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2025, with accession number CGMCC NO.35305.

2. An agent comprising at least one selected from the following: live bacteria, freeze-dried bacteria, inactivated bacteria, or bacterial cultures of Bacillus berberis strain JZ01 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The strain culture is the fermentation broth, fermentation supernatant and / or the concentrated or dried product of the fermentation broth and fermentation supernatant of the strain. And / or, the microbial agent is a liquid preparation or a powder.

4. The microbial agent according to claim 2 or 3, characterized in that, The microbial agent also contains a synergist, which is trigonelline and / or ganoderic acid A.

5. The microbial agent according to claim 4, characterized in that, The synergist is ganoderic acid A, and its content in the fungal agent is 1%-5%.

6. The microbial agent according to claim 4, characterized in that, The synergist is trigonelline, and its content in the bacterial agent is 0.1%-0.5%.

7. The use of the Bacillus belyssus strain JZ01 as described in claim 1 or the bacterial agent as described in any one of claims 2-6 in the preparation of biological pesticides; in, The biopesticide is used for any one or more of the following purposes: (1) Used to antagonize Cercospora Cercospora kikuchii ; (2) Used to prevent and control diseases caused by Cercospora Cercospora kikuchii This leads to cucumber brown spot disease; (3) Used to kill root-knot nematodes; (4) Used to prevent and control cucumber root-knot nematode disease caused by root-knot nematodes.

8. The live, lyophilized, inactivated, or cultured *Bacillus belyssus* strain JZ01 as described in claim 1, or the live, lyophilized, inactivated, or cultured *Bacillus belyssus* strain JZ01 as described in claim 1, and trigonelline in the preparation of a treatment for antagonizing *Cercospora*. Cercospora kikuchii And / or used to prevent and control diseases caused by Cercospora Cercospora kikuchii The use of pesticides that cause cucumber brown spot disease.

9. The use of live bacteria, freeze-dried bacteria, inactivated bacterial cells, and strain cultures of Bacillus vesiculosus strain JZ01 as described in claim 1, or the use of live bacteria, freeze-dried bacteria, inactivated bacterial cells, strain cultures, and ganoderic acid A of Bacillus vesiculosus strain JZ01 as described in claim 1 in the preparation of pesticides for killing root-knot nematodes and / or for controlling cucumber root-knot nematode disease caused by root-knot nematodes.

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