A Lysinibacillus pakistanensis Sneb2537, a preparation capable of preventing and treating root-knot nematode disease, and its preparation method and application

Abstract

The present invention provides a Lysinibacillus pakistanensis Sneb2537 and a preparation capable of preventing and controlling root-knot nematode disease, as well as a preparation method and application thereof, belonging to the technical field of plant disease control. The present invention screens out Lysinibacillus pakistanensis Sneb2537 with high nematode-resistant activity from the soil, and the fermentation broth obtained by fermenting this strain can be directly used in each growth period of plants, and has a promoting effect on the prevention and control of root-knot nematode disease and the growth of plants. When the strain and the fermentation broth of the present invention are used as medicaments for preventing and controlling root-knot nematode disease, they have the characteristics of simple operation, high efficiency, safety, environmental protection, and not being prone to generating resistance, effectively reducing the damage of chemical medicaments to the environment and the threat to humans.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease control, and particularly relates to a Lysinibacillus pakistanensis Sneb2537, a preparation capable of preventing and treating root-knot nematode disease, a preparation method thereof, and an application thereof. Background Art

[0002] Root-knot nematodes (Meloidogyne spp.) are the most harmful plant parasitic nematodes in the world. They can infect more than 5,500 plants such as food crops and vegetables, and have a wide distribution range and strong destructive ability, bringing huge economic losses to agricultural production. According to statistics, there are 105 species of nematodes reported in the genus Meloidogyne in the world, and among them, 4 species are the most common: Meloidogyne incognita, Meloidogyne javanica, Meloidogyne hapla, and Meloidogyne arenaria. Due to the occurrence of root-knot nematode disease, the yield of general crops is reduced by 10% - 20%. When the disease is severe, the loss can reach 80%, and even the crops may fail completely. Every year, the losses of garden seedlings and vegetable planting industries due to the damage of root-knot nematodes reach 3 billion yuan.

[0003] Root-knot nematodes belong to sedentary endoparasitic nematodes. Under suitable temperature and humidity conditions, their reproduction speed is extremely fast. They can complete a life cycle in one month, and can carry out 6 infection cycles in one year in open field cultivation. Root-knot nematodes are more active in greenhouse cultivation, and can reproduce up to 10 generations in one year at most. After root-knot nematodes infect host plants, a large number of root knots are formed on their roots. The root knots are oval and yellowish-white at the initial stage of the disease, and turn brown in color at the later stage. The roots become rough and even rot. Since the conductive tissue of the roots is damaged and the normal functions of the roots are affected, the growth of the plants is slowed down, the leaves turn yellow, and even the whole plant may die. In addition to directly causing harm, the wounds caused by the infection of Meloidogyne incognita can also trigger the synergistic invasion of other pathogenic bacteria, such as Fusarium and Verticillium. The combined infection of multiple pathogenic bacteria shows various symptoms on the host and forms a result of 1 + 1 > 2, greatly aggravating the harm.

[0004] At present, the control of plant parasitic nematodes still mainly relies on chemical control. However, chemical control has problems such as high cost, chemical residues, phytotoxicity caused by improper application, the generation of drug resistance in nematodes after long-term application, and serious problems of life safety for humans and livestock and soil environmental pollution. An efficient and environmentally friendly control method for root-knot nematode disease is still an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a Lysinibacillus pakistanensis Sneb2537 and a preparation capable of preventing and controlling root-knot nematode disease, as well as its preparation method and application. The present invention adopts a biological control method, targeting plant root-knot nematodes, and screening Lysinibacillus pakistanensis Sneb2537 with high-efficiency anti-nematode activity from the soil. This strain has the characteristics of simple operation, high efficiency, safety, environmental protection, and is not prone to generating resistance. The fermentation broth fermented by the Lysinibacillus pakistanensis Sneb2537 strain obtained by the present invention can effectively reduce the harm of root-knot nematode disease to plants and promote plant growth.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a Lysinibacillus pakistanensis Sneb2537, which was deposited in the China General Microbiological Culture Collection Center on August 12, 2024, with the deposit number CGMCC NO. 31568.

[0008] The present invention provides a preparation capable of preventing and controlling root-knot nematode disease, and the preparation includes the Lysinibacillus pakistanensis Sneb2537 and / or the metabolite of the Lysinibacillus pakistanensis Sneb2537.

[0009] Preferably, the preparation includes a fermentation broth containing bacteria or a metabolite extract without bacteria.

[0010] Preferably, the viable bacteria concentration of Lysinibacillus pakistanensis Sneb2537 in the fermentation broth is 1.0×10 8 ~1.0×10 9 CFU / mL.

[0011] The present invention provides a preparation method of the preparation. The strain is activated, and the activated strain is inoculated into a liquid medium for fermentation to obtain a preparation capable of preventing and controlling root-knot nematode disease.

[0012] Preferably, the temperature of the fermentation is 25 - 35 °C, and the time of the fermentation is 1 - 3 d.

[0013] The present invention also provides the application of the Lysinibacillus pakistanensis Sneb2537, the preparation, or the preparation obtained according to the preparation method in preventing and controlling root-knot nematode disease.

[0014] Preferably, the control of root-knot nematode disease is to kill root-knot nematodes and / or inhibit the hatching of root-knot nematode eggs.

[0015] The present invention also provides the application of Lysinibacillus pakistanensis Sneb2537, the preparation or the preparation obtained according to the preparation method as a seed soaking and coating agent in promoting plant growth.

[0016] The present invention also provides the application of Lysinibacillus pakistanensis Sneb2537, the preparation or the preparation obtained according to the preparation method as a fertilizer in promoting plant growth.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) In the present invention, a strain is isolated from the soil, which is identified as Lysinibacillus pakistanensis and named Sneb2537. The fermentation broth obtained by fermenting the Lysinibacillus pakistanensis Sneb2537 strain obtained by the present invention has certain contact killing activity against the second-stage larvae of root-knot nematodes and can inhibit the hatching of root-knot nematode eggs.

[0019] (2) When the fermentation broth or fermentation filtrate is used in various growth periods of plants, it can effectively control root-knot nematode disease and promote plant growth. When the fermentation broth or fermentation filtrate is used for seed soaking and coating treatment, it can promote seed germination and has no inhibitory effect on plant growth; when the fermentation broth or fermentation filtrate is used for seedlings, it can resist root-knot nematode disease and promote the growth of plants.

[0020] (3) The strain, fermentation broth and fermentation filtrate of the present invention can be used as agents for controlling root-knot nematode disease, and have the characteristics of simple operation, high efficiency, safety, environmental protection and difficult to generate resistance, effectively reducing the damage of chemical agents to the environment and the threat to people. Description of the Drawings

[0021] Figure 1 To construct a phylogenetic tree of Lysinibacillus pakistanensis Sneb2537 strain based on 16S rDNA sequence.

[0022] Biological Deposit Description

[0023] Lysinibacillus pakistanensis Sneb2537, with the Latin name Lysinibacillus pakistanensis, this strain is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: August 12, 2024, deposit number: CGMCC NO. 31568. Detailed implementation manners

[0024] The present invention provides a Lysinibacillus pakistanensis Sneb2537, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 12, 2024, with the deposit number CGMCC NO. 31568.

[0025] The present invention provides a preparation capable of preventing and treating root-knot nematode disease, and the preparation comprises the Lysinibacillus pakistanensis Sneb2537 and / or the metabolite of the Lysinibacillus pakistanensis Sneb2537.

[0026] In the present invention, the preparation comprises a fermentation broth containing bacteria or a metabolite extract without bacteria; the viable bacteria concentration of Lysinibacillus pakistanensis Sneb2537 in the fermentation broth is 1.0×10 8 ~1.0×10 9 CFU / mL; the metabolite extract without bacteria is the fermentation filtrate obtained after centrifuging the fermentation broth of Lysinibacillus pakistanensis Sneb2537; the speed of centrifugation is 6000 - 10000 r / min, preferably 7000 - 9000 r / min, more preferably 8000 r / min, and the time of centrifugation is 2 - 10 min, preferably 4 - 8 min, more preferably 5 min.

[0027] The present invention provides a preparation method of the preparation. The strain is activated, and the activated strain is inoculated into a liquid medium for fermentation to obtain a preparation capable of preventing and treating root-knot nematode disease.

[0028] In the present invention, the activation method is to streak Lysinibacillus pakistanensis Sneb2537 on an LB solid medium and place it in an incubator at 25 - 30°C, preferably 26 - 39°C, more preferably 28°C; culture for 10 - 14 h, preferably 11 - 13 h, more preferably 12 h until single colonies grow, thus obtaining the activated strain. Pick a well-grown single colony and inoculate it into a 250 mL Erlenmeyer flask containing 120 - 170 mL of LB liquid medium. The amount of LB liquid medium is preferably 130 - 160 mL, more preferably 150 mL, and ferment at 25 - 37°C, preferably 27 - 30°C, more preferably 28°C for 1 - 3 d, preferably 2 d to obtain a preparation capable of preventing and treating root-knot nematode disease.

[0029] The present invention also provides the application of the Bacillus lysine var. pakistanensis Sneb2537, the preparation or the preparation obtained according to the preparation method in the prevention and control of root-knot nematode disease.

[0030] In the present invention, the prevention and control of root-knot nematode disease is to kill root-knot nematodes and / or inhibit the hatching of root-knot nematode eggs; the root-knot nematode is Meloidogyne incognita.

[0031] The present invention also provides the application of the Bacillus lysine var. pakistanensis Sneb2537, the preparation or the preparation obtained according to the preparation method as a seed soaking and coating agent in promoting plant growth.

[0032] In the present invention, when used as a seed soaking and coating agent, the Bacillus lysine var. pakistanensis Sneb2537 is prepared into a fermentation broth with a cell concentration of 1×10 8 CFU / mL or a metabolite extract without cells. The soaking time is 1 - 5 min, preferably 2 - 4 min, and more preferably 3 min; the plant is cucumber, and the cucumber variety is Zhongnong No. 6.

[0033] The present invention also provides the application of the Bacillus lysine var. pakistanensis Sneb2537, the preparation or the preparation obtained according to the preparation method as a fertilizer in promoting plant growth.

[0034] In the present invention, when used as a fertilizer, the Bacillus lysine var. pakistanensis Sneb2537 is prepared into a fermentation broth with a cell concentration of 1×10 8 CFU / mL or a metabolite extract without cells. The application amount is 5 - 15 mL / plant, preferably 7 - 12 mL / plant, and more preferably 10 mL / plant; the plant is cucumber, and the cucumber variety is Zhongnong No. 6.

[0035] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Preparation of LB solid medium: Dissolve 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium chloride, 1.0 g of glucose, and 20 g of agar in 1 L of distilled water, adjust the pH to 7.0 ± 0.2, and sterilize by moist heat at 121 °C for 30 min.

[0037] Preparation of LB liquid medium: Dissolve 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium chloride, and 1.0 g of glucose in 1 L of distilled water, adjust the pH to 7.0 ± 0.2, and sterilize by moist heat at 121 °C for 30 min.

[0038] Preparation of 30% glycerol: Mix 60 mL of glycerol with 140 mL of deionized water in a 200 mL blue-mouthed bottle, and sterilize it by moist heat at 121 °C for 30 min.

[0039] The LB solid medium, LB liquid medium, and 30% glycerol used in the subsequent examples were all prepared according to the above method.

[0040] Example 1 Obtaining of Lysinibacillus pakistanensis Sneb2537 Strain

[0041] Sampling was carried out from the cucumber field soil at No. 15-4, South Street, Hushitai, Dadong District, Shenyang City, Liaoning Province (longitude 123°51684, latitude 41°92332). Weigh 5 g of soil sample and add it to a 250 mL Erlenmeyer flask containing 45 mL of sterile water. After gradient dilution, take 25 μL of soil suspensions with dilution factors of 10 -2 、10 -3 、10 -4 respectively and spread them on the LB solid medium. Incubate at 37 °C for 12 h. Through the plate dilution method, the strains were isolated and purified. Prepare a mixture of the strain fermentation broth and 30% glycerol, and store it frozen at -80 °C in the refrigerator.

[0042] Example 2 Identification of Lysinibacillus pakistanensis Sneb2537 Strain

[0043] Determine the taxonomic status of the strain through morphological, physiological and biochemical characteristics, and 16S rDNA sequence analysis.

[0044] Morphological observation: Use the streak plate method to inoculate the purified strain in Example 1 on the LB plate, and place it in an inverted 28 °C constant temperature incubator for 12 h to observe the colony morphology, color, smell, etc.

[0045] Physiological and biochemical characteristics: Break open the neck of the physiological and biochemical tube (purchased from Qingdao Haibo Biotechnology Co., Ltd.) in the laminar flow hood. Use a sterilized pipette tip to pick a fresh single colony and inoculate it into the tube. After completion, seal it with a sealing film and place it in a 37 °C constant temperature incubator. Operate according to the instructions of the physiological and biochemical kit (purchased from Qingdao Haibo Biotechnology Co., Ltd.), and add the corresponding reagents according to the requirements to observe the final results.

[0046] 16S rDNA sequence analysis: Refer to the DNA extraction method of Sutyak et al. (Sutyak K E, Wirawan R E, Aroutcheva A A and Chikindas M L. 2008. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens [J]. Journal of Applied Microbiology, 104, (1067 - 1074).) to extract bacterial DNA. Using genomic DNA as a template, the universal bacterial primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’, SEQ ID NO.1) and 1492R (5’-ACGGCTACCTTGTTACGACTT-3’, SEQ ID NO.2) were used for 16S rDNA gene amplification. The 25 μL PCR amplification system was calculated as 25 μL: 1 μL of each upstream and downstream primer, 2 μL of DNA template, 12.5 μL of 2×Taq MasterMix (Dye olus), and 8.5 μL of ddH2O. The PCR amplification conditions were: pre-denaturation at 94 °C for 1.5 min, denaturation at 94 °C for 0.5 min, annealing at 58 °C for 0.5 min, extension at 72 °C for 1 min, 30 cycles, extension at 72 °C for 5 min, and storage at 4 °C. After the PCR products were detected by 1.0% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared and analyzed with the BLAST database in NCBI. Using the MEGA7.0 software, a phylogenetic evolution tree was constructed by the Neighbor-joining method. Then, the sequence results of the strain were submitted to GenBank of NCBI to obtain the accession number. The phylogenetic evolution tree is shown in Figure 1 .

[0047] The results showed that when the sequence results of the strain were submitted to GenBank of NCBI, the strain with the highest homology with the accession number PQ157562 was Lysinibacillus pakistanensis, with the accession numbers MH934930.1 and PP257351.1. Therefore, this strain was identified as Lysinibacillus pakistanensis.

[0048] Example 3: Experiment on the contact toxicity of the fermentation broth of Lysinibacillus pakistanensis Sneb2537 strain against Meloidogyne incognita and the inhibition of Meloidogyne incognita egg hatching

[0049] Preparation of the fermentation broth of Lysinibacillus pakistanensis Sneb2537: Streak the Lysinibacillus pakistanensis Sneb2537 strain obtained in Example 1 on an LB solid medium, place it in an incubator at 28 °C, and incubate for 12 h until single colonies grow, thus obtaining the activated strain. Use an inoculation loop to pick up a loopful of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 150 mL of LB liquid medium, and ferment it in a shaker for 2 d. The temperature of the shaker is 28 °C, and the rotation speed of the shaker is 200 r / min to obtain the fermentation broth.

[0050] Metabolite extract without bacteria: Centrifuge the above fermentation broth at 8000 r / min for 5 min to obtain a metabolite extract without bacteria (fermentation filtrate of the strain).

[0051] Use the cucumber roots to propagate and obtain oocysts of Meloidogyne incognita. Make a part of them into an egg suspension, and hatch J2 by the shallow dish method for another part to prepare a nematode suspension.

[0052] Take 400 μL of the fermentation filtrate of Lysinibacillus pakistanensis Sneb2537 strain, use ddH2O and LB medium treatment as the control group, add 100 μL of the nematode suspension (about 30 J2 nematodes), confront for 12 h and 24 h, observe the death of nematodes and record the number of dead nematodes, calculate the mortality rate and corrected mortality rate, and the results are shown in Table 1.

[0053] Mortality rate = (number of dead nematodes / total number of nematodes) × 100%.

[0054] Corrected mortality rate = (mortality rate of treated nematodes - mortality rate of control nematodes) / (1 - mortality rate of control nematodes) × 100%.

[0055] Table 1 Contact toxicity of Lysinibacillus pakistanensis Sneb2537 against Meloidogyne incognita

[0056]

[0057] Take 400 μL of the fermentation filtrate of Lysinibacillus pakistanensis Sneb2537 strain, use ddH2O and LB medium treatment as the control, add 100 μL of the egg suspension (about 300 eggs), culture for 24 h and 48 h, observe the number of hatched nematodes, calculate the egg hatching rate and relative inhibition rate, and the results are shown in Table 2.

[0058] Egg hatching rate = (number of hatched nematodes / number of test eggs) × 100%.

[0059] Relative inhibition rate = [(number of hatched nematodes in the control - number of hatched nematodes in the treatment) / number of hatched nematodes in the control] × 100%.

[0060] Table 2 Effects of Lysinibacillus pakistanensis Sneb2537 on the hatching rate of Meloidogyne incognita eggs

[0061]

[0062] As can be seen from Table 1 and Table 2, the corrected mortality rate of the fermentation filtrate of Lysinibacillus pakistanensis Sneb2537 against the second-stage larvae of Meloidogyne incognita can reach 64.83% at 12 h and 93.72% at 24 h; the relative inhibition rate of the fermentation filtrate of Lysinibacillus pakistanensis Sneb2537 against the hatching of Meloidogyne incognita eggs can reach 87.62% at 24 h and 89.22% at 48 h. The results show that Lysinibacillus pakistanensis Sneb2537 can effectively contact and kill the second-stage larvae of Meloidogyne incognita and inhibit the hatching of its eggs.

[0063] Example 4 Seed coating and germination test of Lysinibacillus pakistanensis Sneb2537

[0064] Prepare the fermentation broth of Lysinibacillus pakistanensis Sneb2537 according to the method of Example 3 for standby. Select the cucumber variety Zhongnong No. 6; disinfect the seeds by the warm water soaking method, soak them at 55 °C for 30 min for standby.

[0065] Soak the seeds in the strain fermentation broth for 3 min, and then place them in a sterile petri dish (d = 90 mm) lined with sterilized filter paper. Each treatment has 3 replicates, and each replicate has 10 seeds. Treat them with an equal amount of sterile water (ddH2O) and LB liquid medium as the control groups respectively. Place the petri dishes in a constant temperature and humidity incubator with a controlled temperature of 25 °C for cultivation. Start counting the number of germinated seeds after 24 h, count once a day. After culturing for 7 d, calculate the germination index and germination rate, measure the shoot length and root length, and calculate the vigor index. The results are shown in Table 3.

[0066] Germination rate = (Number of germinated seeds / Total number of tested seeds) × 100%

[0067] Germination index = Σ (Number of germinated seeds at different days / Corresponding germination days)

[0068] Seed vigor index = Seedling length × Germination index.

[0069] Table 3 Effects of coating cucumber seeds with Lysinibacillus pakistanensis Sneb2537 on their germination and growth

[0070]

[0071] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level by Duncan's test.

[0072] As can be seen from Table 3, after treatment with the fermentation broth of Lysinibacillus pakistanensis Sneb2537, the germination rate and germination index of cucumber seeds increased significantly, and there was no significant difference in root length and seed vigor compared with the control group, indicating that the fermentation broth of Lysinibacillus pakistanensis Sneb2537 can promote seed germination when used for soaking and coating cucumber seeds, and has no inhibitory effect on the growth of cucumber plants.

[0073] Example 5 Experiment on the control effect of the fermentation broth of Lysinibacillus pakistanensis Sneb2537 in potted plants

[0074] Prepare the fermentation broth of Lysinibacillus pakistanensis Sneb2537 and the nematode suspension respectively according to the method of Example 3 for standby. Select the cucumber variety Zhongnong No. 6, disinfect the seeds by the hot water soak method, soak them at 55 °C for 30 min, and culture them until the two-leaf and one-heart stage for standby.

[0075] Use ddH2O for root irrigation and inoculate with Meloidogyne incognita J2, use LB liquid medium for root irrigation and inoculate with J2, and use the medicament (1.8% abamectin emulsion, purchased from Yantai Oberth Biochemical Co., Ltd.) for root irrigation and inoculate with J2 as the control groups, and use the fermentation broth of Lysinibacillus pakistanensis Sneb2537 for root irrigation and inoculate with J2 as the treatment group. Add 2 mL of the second-instar larva suspension (about 1000 J2) to each seedling, and irrigate 10 mL of the fermentation broth of Lysinibacillus pakistanensis Sneb2537 (concentration about 1×10 8 CFU / mL) to each seedling in the treatment group, while irrigate the same amount of ddH2O or LB liquid medium or medicament (1.8% abamectin emulsion) to each seedling in the control groups. There are 5 replicates for each treatment, 1 seedling for each replicate, and they are placed completely randomly. After culturing for 30 d, investigate the number of root knots and calculate the root knot reduction rate, and use the root knot reduction rate to represent the relative control effect. The results are shown in Table 4.

[0076] Root knot reduction rate = [(number of root knots in the control - number of root knots in the treatment) / number of root knots in the control]×100%.

[0077] Table 4 Control effect of root irrigation with the fermentation broth of Lysinibacillus pakistanensis Sneb2537 on cucumber root-knot nematode disease

[0078]

[0079] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P<0.05 level by Duncan's test.

[0080] As can be seen from Table 4, in the pot experiment under room temperature conditions, after the cucumber seedlings were treated by irrigation with the fermentation broth of Lysinibacillus pakistanensis Sneb2537, the number of root knots in the cucumber roots was significantly lower than that in the control group of LB liquid medium, and there was no significant difference compared with the control group treated with avermectin. The reduction rate of root knots was 66.17%. Therefore, in the pot experiment, Lysinibacillus pakistanensis Sneb2537 can effectively control cucumber root-knot nematode disease.

[0081] Example 6 Effect of the fermentation broth of Lysinibacillus pakistanensis Sneb2537 on the growth of cucumber plants

[0082] The fermentation broth of Lysinibacillus pakistanensis Sneb2537 was prepared respectively according to the method of Example 3 and reserved. The cucumber variety Zhongnong No. 6 was selected, and the seeds were disinfected by the warm water soaking method, soaked at 55 °C for 30 min, and cultured until the two-leaf and one-heart stage and reserved.

[0083] Irrigation with ddH2O and irrigation with LB liquid medium were used as control groups respectively, and 10 mL of ddH2O or LB liquid medium was irrigated to each seedling. Irrigation with the strain of Lysinibacillus pakistanensis Sneb2537 was used as the treatment group, and 10 mL of the fermentation broth of Lysinibacillus pakistanensis Sneb2537 (concentration about 1×10 8 CFU / mL) was irrigated to each seedling. There were 3 replicates for each treatment, 3 seedlings for each replicate, and they were placed completely randomly. After culturing for 30 d, the plant height, root length, fresh root weight, and fresh plant weight of the cucumber plants were investigated.

[0084] Table 5 Effect of irrigation treatment with the fermentation broth of Lysinibacillus pakistanensis Sneb2537 on the growth of cucumber plants

[0085]

[0086] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P<0.05 level by Duncan's test.

[0087] As can be seen from Table 5, in the pot experiment under room temperature conditions, after the cucumber seedlings were treated by irrigation with the fermentation broth of Lysinibacillus pakistanensis Sneb2537, compared with the control group of LB liquid medium, the plant height, fresh plant weight, and fresh root weight all increased significantly, and there was no significant difference in root length compared with the control group of LB liquid medium. Therefore, in the pot experiment, Lysinibacillus pakistanensis Sneb2537 can effectively promote the growth of cucumber plants.

[0088] Example 7 Field control efficacy experiment of Lysinibacillus pakistanensis Sneb2537 strain

[0089] Prepare the fermentation broth of Lysinibacillus pakistanensis Sneb2537 according to the method of Example 3 respectively, and set aside. Select the cucumber variety Zhongnong No. 6. Disinfect the seeds by the warm water soaking method, soak them at 55 °C for 30 min, cultivate until the two-leaf and one-heart stage, and transplant them to the field with root-knot nematode disease.

[0090] At the time of transplanting, use ddH2O irrigation, LB liquid medium irrigation, and medicament irrigation as the control groups respectively. Irrigate 10 mL of ddH2O or LB liquid medium or medicament (1.8% abamectin emulsion) to each seedling. Use the irrigation of Lysinibacillus pakistanensis Sneb2537 as the treatment group, and irrigate 10 mL of the strain fermentation broth (concentration about 1×10 8 CFU / mL) to each seedling. After culturing for 30 days, measure the physiological indexes of cucumber plants such as plant height, root length, root fresh weight, and plant fresh weight, investigate the root-knot index and calculate the control effect.

[0091] Root-knot index grading standard: Grade 0, no root knots; Grade 1, there are some small root knots on the fibrous roots; Grade 2, there are some obvious small root knots on the fibrous roots, and no root knots on the main roots; Grade 3, there are some obvious large root knots on the fibrous roots, and no root knots on the main roots; Grade 4, most of the fibrous roots are large root knots, and no root knots on the main roots; Grade 5, half of the roots have root knots, and there are also root knots on the main roots; Grade 6, a large number of root knots appear on the main roots; Grade 7, most of the main roots have root knots; Grade 8, all main roots have root knots; Grade 9, all roots have root knots; Grade 10, the root knots are very serious and there are no fibrous roots.

[0092] Root-knot index = Σ[(number of diseased plants at each level × representative value at each level) / (total number of plants investigated × representative value of the highest level)]×100%

[0093] Control effect = [(control root-knot index - treatment root-knot index) / control root-knot index]×100%.

[0094] Table 6 Control effect of Lysinibacillus pakistanensis Sneb2537 irrigation on cucumber root-knot nematode disease

[0095]

[0096]

[0097] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P<0.05 level by Duncan's test.

[0098] As can be seen from Table 6, in the field experiment, after the cucumber seedlings were treated by irrigating the roots with the fermentation broth of Lysinibacillus pakistanensis Sneb2537, the control effect was 55.64%, which was significantly higher than that of the LB medium control group and the avermectin treatment control group; compared with the LB medium control group and the avermectin treatment control group, the root length, root fresh weight, plant fresh weight, and plant height were all significantly increased. Therefore, in the field experiment, Lysinibacillus pakistanensis Sneb2537 can effectively control cucumber root-knot nematode disease and promote the growth of plants.

[0099] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Lysinibacillus pakistanensis ( Lysinibacillus pakistanensis ), characterized in that The Lysinibacillus pakistanensis Sneb2537 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 12, 2024, with the deposit number of CGMCC NO. 31568.

2. A preparation capable of preventing and controlling root-knot nematode disease, characterized in that, The preparation includes the Lysinibacillus pakistanensis Sneb2537 described in claim 1.

3. The preparation according to claim 2, characterized in that, The preparation includes a fermentation broth containing bacterial cells.

4. The preparation according to claim 3, characterized in that, The viable cell concentration of Lysinibacillus pakistanensis Sneb2537 in the fermentation broth is 1.0×10 8 ~1.0×10 9 CFU / mL.

5. The preparation method of the preparation according to any one of claims 2 to 4, characterized in that, Activating the strain described in claim 1 and inoculating the activated strain into a liquid medium for fermentation can obtain a preparation capable of preventing and controlling root-knot nematode disease.

6. The preparation method according to claim 5, characterized in that, The temperature of the fermentation is 25 - 35 °C, and the time of the fermentation is 1 - 3 days.

7. Use of the Lysinibacillus pakistanensis Sneb2537 described in claim 1, the preparation described in any one of claims 2 - 4, or the preparation obtained by the preparation method described in any one of claims 5 - 6 in preventing and controlling root-knot nematode disease; The root-knot nematode is Meloidogyne incognita.

8. The application according to claim 7, characterized in that, The prevention and control of root-knot nematode disease is to kill root-knot nematodes and / or inhibit the hatching of root-knot nematode eggs.

9. Use of the Lysinibacillus pakistanensis Sneb2537 described in claim 1, the preparation described in any one of claims 2 - 4, or the preparation obtained by the preparation method described in any one of claims 5 - 6 as a seed soaking and coating agent in promoting plant growth; The promotion of plant growth is to promote the germination of cucumber seeds.

10. Use of the Lysinibacillus pakistanensis Sneb2537 described in claim 1, the preparation described in any one of claims 2 - 4, or the preparation obtained by the preparation method described in any one of claims 5 - 6 as a fertilizer in promoting plant growth; The promotion of plant growth is to increase the root length, root fresh weight, plant fresh weight, and plant height of cucumber plants.

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