Bacillus cereus for preventing and treating root-knot nematode and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a bacillus cereus for preventing and treating root-knot nematode and application thereof. The bacillus cereus is named as bacillus cereus ZWL-B-24, is preserved in Guangdong Microbial Culture Collection Center (GDMCC) with an address of 5th floor, building 59, Guangdong Academy of Microbiology, 100, Middle Junli Street, Guangzhou, and a preservation number of GDMCC No: 63680, and a preservation time of July 24, 2023. The bacillus cereus obtained in the application is derived from sweet potato soil, has a strong soil colonization capacity, and due to a long-term coexistence with sweet potato root systems, mutual symbiosis, a series of similar survival mechanisms are formed, and the bacillus cereus is applied to the prevention and treatment of sweet potato root-knot nematode disease, and is more safe and reliable for the growth, edible and processing products of sweet potato.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a strain of Bacillus cereus for controlling root-knot nematodes and its application. Background Technology

[0002] Root-knot nematodes are a class of highly specific soil-borne pathogens with a wide range of hosts and simple transmission routes. The southern root-knot nematode (Meloidogyne incognita), also known as the sweet potato root-knot nematode, is one of the most important root-knot nematodes damaging crops.

[0003] Root-knot nematodes infect the roots of crops, forming nodules that hinder the absorption of water and nutrients. Furthermore, the wounds created by these nematodes promote the infection of other plant pathogens. In the early stages, this results in stunted growth and weakened immunity; in later stages, it leads to reduced yields, crop failure, or even death, severely jeopardizing agricultural development.

[0004] Root-knot nematodes parasitize the phloem of plant roots. Due to their unique body structure, they exhibit strong resistance to pesticides. Furthermore, the lack of resistant crop varieties and high multiple cropping index mean that current control of root-knot nematodes primarily relies on chemical methods. While chemical control can achieve significant results, long-term use not only results in high toxicity and environmental pollution but also increases nematode resistance, leading to ever-increasing pesticide dosages. Therefore, biological methods are currently the main research direction for the prevention and control of plant root-knot nematodes.

[0005] Bacillus cereus is widely distributed in soil, air, water, and plants. It shares a high degree of similarity with Bacillus thuringiensis in its 16S rRNA and biological characteristics. Bacillus cereus is classified into virulent and non-virulent strains. Non-virulent strains are commonly used as plant growth promoters, microbial additives in animal feed, and biocides. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a strain of Bacillus cereus that produces a large number of spores, has strong soil colonization ability, high pathogenicity to root-knot nematodes, is non-toxic to humans, has good stability of fermentation broth, and is suitable for industrial production for the control of root-knot nematodes. This strain is named Bacillus cereus ZWL-B-24, providing a new resource and direction for biological control to replace chemical control, and can be developed and utilized as a biological pesticide in the future.

[0007] This invention utilizes Bacillus cereus, screened from sweet potato soil, for the control of sweet potato root-knot nematodes. Due to its long-term coexistence and mutualistic relationship with sweet potato roots, it has formed a series of survival mechanisms similar to those of sweet potato. Therefore, its application in the control of sweet potato root-knot nematode disease is safer and more reliable for the growth, consumption, and processing of sweet potatoes.

[0008] Another objective of this invention is to provide a biological agent prepared based on the above-mentioned Bacillus cereus ZWL-B-24.

[0009] Another objective of this invention is to provide an application of the above-mentioned biological agents in the prevention and control of root-knot nematode diseases, particularly in the prevention and control of root-knot nematode diseases in southern regions.

[0010] Another objective of this invention is to provide the application of the above-mentioned Bacillus cereus ZWL-B-24 in the prevention and control of root-knot nematode diseases, especially in the prevention and control of root-knot nematode diseases in southern regions.

[0011] Another objective of this invention is to provide the application of the above-mentioned Bacillus cereus ZWL-B-24 in a simple and efficient fermentation process in a culture medium containing sweet potato.

[0012] The objective of this invention is achieved through the following solution:

[0013] A strain of Bacillus cereus, named Bacillus cereus ZWL-B-24, is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63680, and deposited on July 24, 2023.

[0014] When the aforementioned Bacillus cereus is cultured on LB medium at 34°C for 24 hours, the colonies are round or nearly round with irregular edges, white and opaque, slightly raised, and have a waxy surface.

[0015] The aforementioned Bacillus cereus is a Gram-positive bacterium. Under a microscope, the bacterial cells appear as oval rod-shaped cells arranged in short or long chains. It produces spores, which are oval and located centrally or near the terminal end. At the end of the spore growth phase, rhomboid parasporal crystals are produced at the terminal end of the Bacillus cereus spores.

[0016] A biological agent prepared based on the above-mentioned Bacillus cereus.

[0017] The biological agent is prepared by liquid fermentation of the Bacillus cereus, preferably including the following steps: inoculating Bacillus cereus into a fermentation medium and culturing it to obtain the biological agent.

[0018] The fermentation medium consists of: 35-50 g / L soybean meal powder, 8-12 g / L corn starch, 10-15 g / L glucose, 1-3 g / L sodium chloride, 1-3 g / L magnesium sulfate, and 1-3 g / L potassium dihydrogen phosphate.

[0019] The culture refers to culturing at 30-32℃, with an aeration rate of 0.5-1.5 vvm and a rotation speed of 300-600 rpm for 28-36 hours.

[0020] The present invention also provides the application of the above-mentioned biological agents in the prevention and control of root-knot nematode diseases, especially in the prevention and control of root-knot nematode diseases in southern regions.

[0021] The present invention also provides the application of the above-mentioned Bacillus cereus in the prevention and control of root-knot nematode diseases, especially in the prevention and control of root-knot nematode diseases in southern China.

[0022] The present invention also provides the application of the above-mentioned biological agents in the inhibition of root-knot nematodes, particularly in the inhibition of southern root-knot nematodes.

[0023] The present invention also provides the application of the above-mentioned Bacillus cereus in the inhibition of root-knot nematodes, particularly in the inhibition of southern root-knot nematodes.

[0024] This invention also provides the application of the aforementioned Bacillus cereus ZWL-B-24 in a simple and efficient fermentation process in a medium containing sweet potato. The Bacillus cereus ZWL-B-24 of this invention exhibits excellent and efficient fermentation activity in a medium prepared from sweet potato. Furthermore, the sweet potato-containing medium described in this invention has simple components, is easy to prepare, and is readily applicable in practical applications.

[0025] In this invention, the culture medium contains sweet potato.

[0026] Furthermore, the culture medium contains 100-300 g / L of sweet potato. Even further, the culture medium contains 200 g / L of sweet potato.

[0027] Furthermore, the culture medium also contains 10-30 g / L of soybean meal powder. Even further, the culture medium also contains 20 g / L of soybean meal powder.

[0028] Furthermore, the culture medium comprises: 10-30 g / L soybean meal powder and 100-300 g / L sweet potato. Even further, the culture medium comprises: 20 g / L soybean meal powder and 200 g / L sweet potato.

[0029] Furthermore, the culture medium can be prepared with water or using sweet potato starch waste liquid. The *Bacillus cereus* ZWL-B-24 of this invention also exhibits excellent and efficient fermentation activity in the culture medium prepared using sweet potato starch waste liquid.

[0030] Furthermore, the culture medium can be inoculated and fermented after simple high-temperature sterilization. High-temperature sterilization can be carried out at 100°C.

[0031] Furthermore, the fermentation refers to culturing at 26-36℃ and a rotation speed of 100-250rpm for 36-72h.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. The Bacillus cereus obtained in this invention originates from sweet potato soil and has a strong soil colonization ability. Due to its long-term coexistence and mutual benefit with sweet potato roots, it has formed a series of survival mechanisms similar to those of sweet potato. Applying it to the prevention and control of sweet potato root-knot nematode disease is safer and more reliable for the growth, consumption and processing of sweet potatoes.

[0034] 2. Both Bacillus cereus and its biological agents of the present invention have strong inhibitory and toxic effects on southern root-knot nematodes, and have excellent biocontrol effects against southern root-knot nematodes. Moreover, the biological source is environmentally friendly and non-toxic, and has little impact on the ecological environment.

[0035] 3. The Bacillus cereus obtained by this invention has low requirements for culture conditions, is easy to culture, and has good prospects for development and application. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a single colony image of Bacillus cereus ZWL-B-24 on LB medium.

[0038] Figure 2 Gram staining and basic fuchsin staining of Bacillus cereus ZWL-B-24.

[0039] Figure 3 Phylogenetic tree of Bacillus cereus ZWL-B-24 based on 16S rDNA gene sequence.

[0040] Figure 4 Phylogenetic tree of Bacillus cereus ZWL-B-24 based on gmk gene sequence.

[0041] Figure 5Phylogenetic tree of Bacillus cereus ZWL-B-24 based on the ccpA gene sequence.

[0042] Figure 6 and Figure 7 This image shows the toxicity effect of Bacillus cereus ZWL-B-24 on nematodes. Among them, Figure 6 For CK water; Figure 7 Dilute the fermentation broth of Bacillus cereus ZWL-B-24 by 100 times.

[0043] Figure 8 This image shows the inhibitory effect of the biological agent Bacillus cereus ZWL-B-24 on nematodes. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0045] Example 1: Isolation, purification and preservation of Bacillus cereus ZWL-B-24

[0046] (1) Composition of LB solid medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 15-17g agar, 1000mL water.

[0047] (2) Isolation and purification of Bacillus cereus: Soil samples used for strain isolation were collected from the Lufeng Sweet Potato Industrial Park in Guangdong Province. 10g of soil sample was weighed and added to 90mL of sterile water containing 0.1% Tween under aseptic conditions. The mixture was then incubated at 28°C for 1 hour. The culture solution was serially diluted with sterile water containing 0.1% Tween to different dilution ratios, spread on LB agar, and incubated at 34°C for 2 days to isolate various bacteria from the soil. Bacteria with significant differences in size, shape, color, texture, and surface structure were selected and purified using the streak plate method to obtain single colonies for later use.

[0048] (3) Determination of the physicochemical properties of Bacillus cereus ZWL-B-24

[0049] The Bacillus cereus ZWL-B-24 sample isolated in this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center, and the center provides the analysis and testing report. Detailed results are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0054] The test methods used are all standard test methods, listed below:

[0055] a. NA medium: 3g beef extract, 5g peptone, 2.5g glucose, 15g agar powder, 1000mL distilled water, pH 7.0.

[0056] b. Catalase test: Prepare 3-10% hydrogen peroxide, pick a small loop of freshly cultured test bacteria and smear it on a glass slide that has been dotted with hydrogen peroxide solution; if bubbles are produced, it is positive, otherwise it is negative.

[0057] c. Oxidase test: Purchase oxidase test strips, pick freshly cultured bacterial growth to be tested, and smear it on the test strip; if the bacterial growth turns red within 10 seconds, it is positive; if it turns red within 10-60 seconds, it is a delayed reaction; if no red appears or if it turns red after more than 60 seconds, it is negative.

[0058] dVP test: The culture medium formula is 5g peptone, 5g glucose, 5g dipotassium hydrogen phosphate, 1000mL water, pH 7.0-7.2. Dispense 4-5mL into each tube and sterilize at 115℃ for 30min. Inoculate the test bacteria into the culture medium and incubate at 37℃ with shaking for 24h. Mix an equal volume of the culture medium with 40% sodium hydroxide, add a small amount of creatine. If the culture medium turns red after 10min, it is positive; otherwise, it is negative.

[0059] e. Acid production test: The sugars and alcohols to be tested include ribose, xylose, galactose, glucose, fructose, mannose, mannitol, sucrose, arabinose, and glycerol; the culture medium formula is 1.0 g diammonium hydrogen phosphate, 0.2 g potassium chloride, 0.2 g magnesium sulfate, 0.2 g yeast extract, 10.0 g sugar or alcohol, 3 mL 1% bromothymol blue aqueous solution, 1000 mL distilled water, pH 7.0-7.2. Dispense into test tubes and sterilize at 112℃ for 30 min; pick freshly cultured... For bacterial testing, four test tubes were inoculated by puncture. Two of these tubes were sealed with sterile petroleum jelly (approximately 0.5-1.0 cm thick) to isolate them from air, serving as closed tubes. The other two tubes were left unsealed with petroleum jelly, serving as open tubes. Uninoculated closed and open tubes were used as controls. The tubes were incubated at 37°C for 1, 2, 3, 7, and 14 days. The results were observed after the culture medium turned yellow. A positive result was indicated by the culture medium turning yellow, while a negative result was indicated by the culture medium remaining dark. Among these, only open tubes produced acid and turned yellow, indicating oxidative acid production. Both open and closed tubes produced acid and turned yellow, indicating fermentative acid production.

[0060] f. Starch hydrolysis test: While preparing NA solid culture medium, add soluble starch to make the final concentration 0.2%, sterilize at 121℃ for 20 min, and pour into plates for later use; pick freshly cultured test bacteria and inoculate them into the above plates, incubate at 37℃ for 2-5 days, after the formation of obvious colonies, drop iodine solution onto the plate. If the plate turns blue-black and there is a transparent ring around the colony that does not change color, it indicates starch hydrolysis and is positive; if it remains blue-black, it is negative.

[0061] g. Citrate utilization test: The culture medium formula is 1g sodium chloride, 0.2g magnesium sulfate hexahydrate, 0.5g ammonium dihydrogen phosphate, 2g sodium citrate, 1000mL distilled water, and 20mL 0.04% phenol red solution. Sterilize at 121℃ for 20min and pour into plates for later use. Pick freshly cultured test bacteria and streak them on the above plates. Incubate at 37℃ for 3-7 days. If the culture medium is alkaline (the indicator turns blue or pink), it is positive; otherwise, it is negative.

[0062] h. Indole acid production test: Prepare a 1% tryptone aqueous solution, pH 7.2-7.6, dispense into test tubes, and sterilize at 115℃ for 30 min; separately prepare the following reagents—8g of p-dimethylaminobenzaldehyde, 760mL of 95% ethanol, and 160mL of concentrated hydrochloric acid; pick the bacteria to be tested and culture them in the above culture medium with shaking for 1d, 2d, 4d, and 7d. Then, slowly add 3-5mL of the above reagents along the wall of the test tube to the surface of the culture medium. If a red color appears at the liquid interface, it is positive; otherwise, it is negative.

[0063] i. H2S Production Test: The culture medium formula is 10g peptone, 5g sodium chloride, 10g beef extract, 0.5g cysteine, and 1000mL distilled water, pH 7.0-7.4. Dispense the medium into test tubes and sterilize at 112℃ for 30min. Cut ordinary filter paper into strips 0.5-1cm wide, the length depending on the height of the test tube and culture medium. Soak the paper strips in 5-10% lead acetate solution, then dry them in an oven and sterilize them in petri dishes. Inoculate freshly cultured test bacteria into the above culture medium. Then, using sterile forceps, pick up a strip of lead acetate paper, plug it tightly with cotton, and suspend it in the tube. Incubate at 37℃, observing after 3d, 7d, and 14d. A blackening of the paper strip indicates a positive result; no color change indicates a negative result.

[0064] (3) The purified strain was subjected to 16S rDNA sequence analysis. Nucleotide homology was compared with the registered sequences in GenBank using the Blast program. The 16S rDNA sequence showed 100% homology with Bacillus cereus, Bacillus wiedmannii, and Bacillus proteolyticus, 99.93% homology with Bacillus fungorum, and 99.78% homology with Bacillus thuringiensis. This confirmed its classification and name as Bacillus cereus. It was named Bacillus cereus ZWL-B-24, and the sequence fragment (SEQ ID NO.1) is as follows:

[0065]

[0066]

[0067] Bacillus cereus ZWL-B-24 was cultured on LB medium at 34°C for 24 hours. The colonies were round or nearly round with irregular edges, white and opaque, slightly raised, and had a waxy surface (as shown in the image). Figure 1 (As shown).

[0068] The preservation center's test report states that this strain is a Gram-positive rod-shaped bacterium. Under a microscope, the bacteria appear as oval rods, arranged in short or long chains, and can produce spores. The spores are oval, centrally or proximally arranged. In the late stage of spore growth, rhomboid parasporal crystals are produced at the ends of the bacterium. Gram staining and basic fuchsin staining images are shown below. Figure 2 .

[0069] (4) The purified strain was sequenced to obtain the gmk gene sequence, which is 519 bp. The sequence fragment (SEQ ID NO.2) is as follows:

[0070]

[0071]

[0072] (5) The ccpA gene sequence obtained by sequencing the purified strain is 886 bp, and the sequence fragment (SEQ ID NO.3) is as follows:

[0073]

[0074] (6) Figures 3-5As shown, a phylogenetic tree of Bacillus cereus ZWL-B-24 was constructed using MEGA software based on the 16S rDNA, gmk, and ccpA gene sequences using the neighbor-joining method. The tree was calculated with 1000 replicates, and nodes showing a Bootstrap value greater than 50% were indicated. The superscript "T" indicates the type strain. The 16S rDNA, gmk, and ccpA gene sequences of Bacillus cereus ZWL-B-24 showed 100%, 100%, and 99.04% homology with Bacillus cereus, respectively. Its morphological characteristics and physiological life processes were most similar to those of Bacillus cereus, and it was identified as Bacillus cereus.

[0075] (7) Preservation of Bacillus cereus ZWL-B-24: The strain was deposited on July 24, 2023 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63680.

[0076] Example 2: Biological preparation of Bacillus cereus ZWL-B-24

[0077] The biological agent of Bacillus cereus ZWL-B-24 is prepared by liquid fermentation of the aforementioned Bacillus cereus, preferably including the following steps: inoculating Bacillus cereus into a fermentation medium and culturing it to obtain the biological agent.

[0078] The specific steps are as follows:

[0079] (1) Composition of the culture medium:

[0080] The seed culture medium consists of: 10g peptone, 5g yeast extract, 10g sodium chloride, and 1000mL water.

[0081] The fermentation medium consists of: 35-50 g / L soybean meal, 8-12 g / L corn starch, 10-15 g / L glucose, 1-3 g / L sodium chloride, 1-3 g / L magnesium sulfate, and 1-3 g / L potassium dihydrogen phosphate.

[0082] (2) Seed activation: Bacillus cereus ZWL-B-24 was inoculated on LB solid medium and cultured at 34℃ for 24h to obtain activated seeds;

[0083] (3) Seed culture: From the activated seeds obtained in step (2), pick a single colony and inoculate it into the seed culture medium. Shake at 180-200 rpm and 30-32℃ for 16-18 hours to obtain seed liquid.

[0084] (4) Fermentation culture: The seed culture obtained in step (3) is inoculated into the fermentation medium at an inoculum rate of 1-5%, and cultured at 30-32℃, an aeration rate of 0.5-1.5 vvm, and a rotation speed of 300-600 rpm for 28-36 h to obtain the fermentation broth. The viable bacterial content of the Bacillus cereus biological agent obtained by fermentation is 40-50 × 10⁻⁶. 8 cfu / mL.

[0085] Example 3: Toxicity test of Bacillus cereus ZWL-B-24 against Southern root-knot nematodes

[0086] The fermentation broth of the *Bacillus cereus* strain ZWL-B-24 of the present invention (Example 2) was diluted 100-fold and 200-fold, respectively. Water (CK) was used as a blank control. Equal volumes of each solution were added to containers, and 100 *Southern root-knot nematodes* were added to each group. After incubation at room temperature for 18 hours, nematode mortality was observed. The results are shown in Table 2. Figures 6-7 The experimental results showed that in the CK water group, 14 nematodes died and 86 survived; after 100-fold dilution of the fermentation broth of the Bacillus cereus strain ZWL-B-24 of this invention, 93 nematodes died and 7 survived, with a corrected mortality rate of 91%; as shown in Table 2 and... Figures 6-7 It is evident that the Bacillus cereus ZWL-B-24 of this invention has a significant inhibitory effect on southern root-knot nematodes, exhibiting excellent biocontrol efficacy. At a 100-fold dilution, it can kill 91% of the nematodes within 18 hours.

[0087] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100

[0088] Table 2

[0089] Initial number of nematodes Number of surviving nematodes Adjusted mortality rate (%) CK 100 86 / Fermentation broth diluted 100 times 100 7 91 Fermentation broth diluted 200 times 100 21 75

[0090] Example 4: Experiment on the control of southern root-knot nematodes by Bacillus cereus ZWL-B-24 under potted conditions

[0091] (1) Groups (fermentation culture medium is Example 2): ① Water control group, CK; ② Bacillus cereus ZWL-B-24 fermentation culture medium diluted 100 times; ③ Bacillus cereus ZWL-B-24 fermentation culture medium diluted 200 times;

[0092] Take coconut coir soil infested with nematodes (approximately 3000 southern root-knot nematodes per pot of equal volume) and transplant it into crops (tomato seedlings). After transplanting, each group is watered with 80 mL of the solution. During the observation period, watering is done regularly and in measured amounts to ensure crop survival. 30 days after treatment, the crop roots are cleaned and the number of root knots is counted. The results are shown in Table 3. Figure 8Experimental results showed that the crop roots treated with the Bacillus cereus ZWL-B-24 fermentation broth of this invention had only a small number of root knots, while the CK group produced a large number of root knots. This indicates that the Bacillus cereus ZWL-B-24 fermentation broth of this invention has a strong inhibitory effect on root-knot nematodes, with a biocontrol effect of 82.2%.

[0093] Control efficacy (%) = [(Number of root knots in CK group - Number of root knots in treatment group) / Number of root knots in CK group] × 100

[0094] Table 3

[0095] deal with Number of root nodes Prevention and control efficacy (%) CK 79 / Fermentation broth diluted 100 times 14 82.2 Fermentation broth diluted 200 times 29 63.2

[0096] (2) Groups: ① Water control group, CK; ② Bacillus cereus ZWL-B-24 fermentation broth diluted 100 times; ③ Bacillus cereus ZWL-B-24 fermentation broth diluted 200 times.

[0097] Take coconut coir soil infested with nematodes (each pot contains approximately 4000 southern root-knot nematodes in an equal volume of soil) and transplant sweet potato seedlings (1 seedling / pot, 10 pots per treatment, 4 replicates). After transplanting, each group's solution is applied, 1000 mL per seedling as the initial watering. 15 days later, a second application is made (500 mL of each treatment solution is applied to each pot). 45 days after the second application, a third application is made (1000 mL of each treatment solution is applied to each pot). During the observation period, watering is done regularly and in measured amounts to ensure normal crop growth for a total of 120 days. After the sweet potatoes mature, they are cleaned and the sweet potatoes and nematode infection status of each treatment are recorded. The average results of the 4 groups are shown in Table 4. As shown in the table, sweet potatoes treated with the fermentation broth of Bacillus cereus ZWL-B-24 of this invention showed only minor nematode damage, while the infection rate in the control group (CK) reached 76.3%, demonstrating that Bacillus cereus ZWL-B-24 of this invention has a significant effect on controlling southern root-knot nematodes. Furthermore, the yield of sweet potatoes treated with the fermentation broth of Bacillus cereus ZWL-B-24 of this invention was significantly increased.

[0098] Infection rate (%) = (Number of infected individuals / Total number of individuals) × 100

[0099] Control efficacy (%) = [(Infection rate of CK group - Infection rate of treatment group) / Infection rate of CK group] × 100

[0100] Table 4

[0101]

[0102] Example 5: High-efficiency fermentation of Bacillus cereus ZWL-B-24

[0103] (1) Composition of culture medium: 10-30g soybean meal powder, 100-300g sweet potato, 1000mL water.

[0104] (2) Preparation of culture medium: Crush or mash sweet potatoes, add water (or sweet potato starch waste liquid) and stir thoroughly to obtain sweet potato fermentation slurry. Then add soybean meal powder and stir evenly. After high-temperature sterilization, the culture medium is obtained. High-temperature sterilization can be carried out at 100℃.

[0105] (3) Seed cultures of Bacillus cereus ZWL-B-24 of the present invention, Bacillus amyloliquefaciens subsp. plantarum ZFH-3 (see CN201510103023.X), and Bacillus cereus BCJB01 (see CN201510081973.7) were inoculated into the above-mentioned culture medium at an inoculation rate of 3%, with 20g of soybean meal powder used in each culture. The cultures were cultured at 34°C and 200rpm for 36-72h. The fermentation medium of Example 2 was used for comparison. The colony counts of the obtained fermentation broth are shown in Table 5. The results show that Bacillus cereus ZWL-B-24 of the present invention can achieve high-efficiency fermentation in a medium containing sweet potato, and has significantly outstanding fermentation activity compared with other Bacillus cereus BCJB01 strains and other Bacillus amyloliquefaciens ZFH-3 strains.

[0106] Table 5

[0107]

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of Bacillus cereus, characterized in that, It was named Bacillus cereus ( Bacillus cereus ZWL-B-24 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63680, and deposited on July 24, 2023.

2. A biological agent, characterized in that, It was prepared based on the Bacillus cereus strain described in claim 1.

3. The biological agent according to claim 2, characterized in that, The preparation is carried out by the following steps: Bacillus cereus is inoculated into a fermentation medium and cultured to obtain a biological agent.

4. The biological agent according to claim 3, characterized in that: The fermentation medium consists of: 35-50 g / L soybean meal powder, 8-12 g / L corn starch, 10-15 g / L glucose, 1-3 g / L sodium chloride, 1-3 g / L magnesium sulfate, and 1-3 g / L potassium dihydrogen phosphate. The culture is carried out at 30-32℃, with an aeration rate of 0.5-1.5 vvm and a rotation speed of 300-600 rpm for 28-36 hours.

5. The use of Bacillus cereus as described in claim 1 or the biological agent as described in any one of claims 2-4 in the prevention and control of root-knot nematode disease in southern China.

6. The use of Bacillus cereus as described in claim 1 or the biological agent as described in any one of claims 2-4 in the inhibition of southern root-knot nematodes.

7. The application of Bacillus cereus as described in claim 1 in simple fermentation in a culture medium containing sweet potato.

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