Bacillus tequilensis strain HN-1 and application thereof

Abstract

The invention discloses a bacillus tequilensis strain HN-1 and application thereof, and belongs to the technical field of biological control of plant parasitic nematodes. The strain HN-1 is identified as bacillus tequilensis, the preservation date is July 28, 2025, the strain HN-1 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20251718, and the preservation number is CCTCC NO: M 20251718. The invention discovers that the bacillus tequilensis strain HN-1 has good insecticidal activity on meloidogyne incognita and pine wood nematode for the first time; the fermentation liquor and fermentation metabolites of the strain HN-1 can be used as a liquid fungicide, or the fermentation liquor is used for preparing a solid fungicide, and the fungicide can be used for preventing and treating meloidogyne incognita and pine wood nematodes in fields.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant parasitic nematodes, specifically relating to a Bacillus tekirae strain HN-1 and its application. Background Technology

[0002] Root-knot nematodes ( Meloidogyne spp. Root-knot nematodes can infect more than 2,000 plant species, causing crop yield reductions of 10% to 75%, making them a significant pest and disease threatening agricultural production. Currently, chemical agents remain the primary means of controlling root-knot nematodes, but they generally suffer from high toxicity, environmental pollution, and drug resistance. Existing biological control agents mostly rely on single or mixed strains, exhibiting drawbacks such as a single control mechanism, weak colonization ability, and unstable efficacy. (Pine wood nematode...) Bursaphelenchus xylophilus Pine wilt nematode is a tiny plant parasitic nematode belonging to the genus Pine Wood Nematode. It is extremely harmful to coniferous trees such as pine trees and is the pathogen that causes pine wilt disease (pine wilt disease). Pine wilt nematode is mainly spread naturally by vector insects such as the pine sawyer beetle. Current control measures mainly focus on controlling the number of vector insects to control the spread of pine wilt nematode.

[0003] Bacillus tekirulatus ( Bacillus tequilensis Bacillus tektii is a Gram-positive bacterium widely found in nature. It has the characteristics of simple nutritional requirements, strong resistance, and the ability to produce a variety of antibacterial substances. Studies have shown that Bacillus tektii has a significant inhibitory effect on plant pathogens such as Fusarium graminearum (e.g., CN 104403962 A). However, there are no reports on its application in the control of root-knot nematodes and pine wood nematodes. Summary of the Invention

[0004] The first objective of this invention is to provide a Bacillus tektii strain HN-1, the second objective is to provide the application of the Bacillus tektii strain HN-1 in the control of plant parasitic nematodes, and the third objective is to provide a plant parasitic nematode control agent prepared from the Bacillus tektii strain HN-1.

[0005] The first objective of this invention is achieved as follows: the Bacillus tekirae strain HN-1 is deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M 20251718, and strain HN-1 has activity against plant parasitic nematodes.

[0006] The second objective of this invention is achieved by the application of the Bacillus tekirii strain HN-1 in the control of plant parasitic nematodes, namely, the southern root-knot nematode or the pine wood nematode.

[0007] The third objective of this invention is achieved by providing a plant parasitic nematode control agent prepared from the Bacillus tektina strain HN-1, wherein the agent is a liquid agent prepared by using the fermentation broth or fermentation metabolites of strain HN-1, or a solid agent prepared by mixing the fermentation broth of strain HN-1 with an agriculturally acceptable carrier.

[0008] The Bacillus tergenta ( Bacillus tequilensis The strain HN-1 was isolated from tomato root soil collected in Yuxi City, Yunnan Province. This invention is the first to discover that the *Bacillus tektina* strain HN-1 exhibits highly effective killing activity against root-knot nematodes and pine wood nematodes. Compared to existing technologies, it has the following technical advantages: 1) The Bacillus subtilis strain HN-1 has significant nematicidal activity against the second instar larvae of the southern root-knot nematode and the pine wood nematode, which is superior to Bacillus subtilis. 2) The bacterial agent prepared by the Bacillus tekiria strain HN-1 is non-toxic and harmless to humans and animals and does not pollute the environment; 3) The fermentation conditions for preparing the bacterial agent by the Bacillus tekirae strain HN-1 are mild and the carrier sources are widely available, making it suitable for large-scale industrial production and reducing production costs. Attached Figure Description

[0009] Figure 1 Microscopic morphology of strain HN-1; Figure 2 Phylogenetic tree of strain HN-1; Figure 3 The images below the dotted lines show the root status of the negative control group, the HN-1 treatment group, and the positive control group in Example 9. Two plants from each group were photographed. The images below the dotted lines are magnified views of the corresponding photos above the dotted lines. Detailed Implementation

[0010] The present invention will be described in more detail below with reference to the test embodiments. However, the present invention is not limited thereto. Any modifications or improvements based on the present invention should be covered within the protection scope of the present invention.

[0011] The Bacillus tergentii described in this invention ( Bacillus tequilensis Strain HN-1, deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M 20251718, exhibits activity against plant parasitic nematodes.

[0012] The present invention relates to the application of Bacillus tekirii strain HN-1 in the control of plant parasitic nematodes, wherein the plant parasitic nematodes are southern root-knot nematodes or pine wood nematodes.

[0013] The present invention relates to a plant parasitic nematode control agent prepared from Bacillus tekirii strain HN-1. The agent is prepared by using the fermentation broth or fermentation metabolites of strain HN-1 as a liquid agent, or by mixing the fermentation broth of strain HN-1 with an agriculturally acceptable carrier to prepare a solid agent.

[0014] Furthermore, the preparation methods of the fermentation broth and fermentation metabolites are as follows: Strain strain HN-1 was inoculated into liquid medium No. 4 and cultured at 30℃ and 180 rpm for 12-48 h to obtain seed culture; the seed culture was inoculated into Modified Schaeffer's liquid medium at a volume ratio of 10% and cultured at 37℃ for 48-72 h to obtain fermentation broth; Centrifuge the fermentation broth at 5000 rpm for 20 minutes and collect the supernatant, which is the fermentation metabolite.

[0015] Furthermore, the formula of the No. 4 liquid culture medium, by mass fraction, is: peptone 0.5%, MgSO4 0.012%, glucose 0.5%, KH2PO4 0.01%, pH 7.0~7.2.

[0016] Furthermore, the Modified Schaeffer's liquid culture medium formulation, by mass fraction, contains: 0.8% peptone, 0.1% yeast extract, 0.12% MgSO4·7H2O, 0.34% KCl, 0.1% glucose, and pH 7.0~7.2.

[0017] Furthermore, the carrier includes, but is not limited to, starch or diatomaceous earth.

[0018] Furthermore, the application method of the liquid bacterial agent is as follows: (1) For the southern root-knot nematode, apply liquid inoculant to the roots before transplanting and / or apply solid inoculant in holes or liquid inoculant to the roots at the early stage of the southern root-knot nematode infestation. When dipping the roots, the concentration of live bacteria in the liquid inoculant should not be less than 2.0 × 10⁻⁶. 8 CFU / mL, the concentration of live bacteria in the liquid inoculant during root irrigation is 1.0 × 10⁻⁶. 8 ~3.0×10 8 cfu / mL, 300mL per plant for root irrigation; (2) For pine wood nematode, non-woven fabric strips soaked in liquid fungal agent were used to fill the borer holes of pine sawyer beetle. The concentration of live bacteria in the liquid fungal agent was 3.0 × 10⁻⁶. 8 cfu / mL.

[0019] Example 1: Isolation, purification and identification of Bacillus tekiria strain HN-1 I. Isolation and purification of strain HN-1 Strain HN-1 was isolated from tomato root soil collected in Yuxi City, Yunnan Province in October 2024. The specific isolation steps are as follows: 1. Prepare liquid medium No. 4 and solid medium No. 4: The formula for liquid medium No. 4, by mass fraction, is: peptone 0.5%, MgSO4 0.012%, glucose 0.5%, KH2PO4 0.01%, pH 7.0~7.2 (the same below); Based on the formula for liquid medium No. 4, prepare solid medium No. 4 according to the ratio of 15g agar per 1L of medium; 2. Weigh 10g of the obtained tomato root soil and add 10 times the volume of PBS buffer. Rotate the mixture at 4℃ for 24h to obtain a soil suspension. Place the soil suspension in an 80℃ water bath for 25min to remove the influence of other bacteria and retain as many Bacillus spores as possible in the soil sample. Precipitate the soil suspension after water bath treatment, take the supernatant and dilute it 1000 times, 10000 times and 100000 times with No. 4 liquid culture medium to obtain dilutions of different concentrations. 3. Take 30 mL of each of the different concentrations of dilution as samples, and culture and isolate them using the MISS CELL instrument (which has functions such as automatic culture and sorting of single strains). Then, in conjunction with streak culture on solid medium No. 4 and verification of the effect of killing southern root-knot nematodes, a Bacillus strain with the activity of killing southern root-knot nematodes was screened out and named HN-1.

[0020] II. Identification of strain HN-1 1. Morphological characteristics This strain forms white, round, smooth colonies on solid medium No. 4; the bacteria are rod-shaped, Gram-positive, and capable of producing spores. See [link to article]. Figure 1 ; 2. Physiological and biochemical characteristics Strain HN-1 possesses oxidase, arginine dihydrolase, gelatin hydrolase and amylase activities, and can utilize a variety of carbon sources to produce acid; 3. Molecular biological identification The strain HN-1 was subjected to bidirectional sequencing using 27F / 1492R (using universal primers selected according to known techniques), and a 1302 bp product was amplified by PCR. Its nucleotide sequence is shown in SEQ ID No. 1. A phylogenetic tree was constructed (see...). Figure 2 The strain HN-1 was identified as Bacillus tekirae ( Bacillus tequilensis ).

[0021] Example 2: Preparation of seed culture, fermentation broth, and fermentation metabolites of strain HN-1 1. Seed culture preparation: Strain HN-1 was inoculated into liquid medium No. 4 and cultured at 30℃ and 180 rpm for 48 h to obtain seed culture; 2. Fermentation broth preparation: The seed culture was inoculated into Modified Schaeffer's liquid medium at a volume ratio of 10% and cultured at 37℃ with shaking for 48 hours to obtain the fermentation broth of strain HN-1, in which the viable cell count was 3.0 × 10⁻⁶. 8 cfu / mL; The formula of Modified Schaeffer's liquid medium, by mass fraction: peptone 0.8%, yeast extract 0.1%, MgSO4·7H2O 0.12%, KCl 0.34%, glucose 0.1%, pH 7.0~7.2; 3. Preparation of fermentation metabolites: The fermentation broth of strain HN-1 was centrifuged at 5000 rpm for 20 min, and the collected supernatant was the fermentation metabolite of strain HN-1.

[0022] Example 3 Fermentation broth for strain HN-1 was prepared according to the method described in Example 2. Parameters were adjusted to ensure that the viable cell count in the fermentation broth of strain HN-1 obtained in step 2 was 3.6 × 10⁻⁶. 8 cfu / mL, used as a liquid bacterial agent for the control of plant parasitic nematodes.

[0023] Example 4 The fermentation broth of strain HN-1 prepared in Example 3 was centrifuged at 5000 rpm for 20 min, and the collected supernatant was the fermentation metabolite of strain HN-1, which was used as a liquid bacterial agent for controlling plant parasitic nematodes.

[0024] Example 5 The fermentation broth of strain HN-1 obtained in Example 3 was mixed with soluble starch at a mass ratio of 1:5 to prepare a solid bacterial agent for controlling plant parasitic nematodes.

[0025] Example 6: Nematode-killing activity test of fermentation broth of strain HN-1 1. Experiment on killing southern root-knot nematodes 1-1. Fermentation broth of strain HN-1: The fermentation broth of strain HN-1 obtained in step 2 of Example 2 was used, with a viable cell count of 3.0 × 10⁻⁶. 8 cfu / mL; Control group solution: Liquid culture medium No. 4 was used as the control group solution; 1-2. Preparation of test solution for second-instar larvae of Southern root-knot nematodes 1-2-1. Take the roots of tomatoes infected with Southern Root-knot Nematode, wash them, and then pick out the nematode eggs from the roots and place them in a petri dish containing purified water for incubation. After 3 days of incubation, observe the second instar larvae under a microscope. Collect the culture medium as the test solution for the second instar larvae, in which the concentration of the second instar larvae is 1~2 larvae / μL. 1-3. The experiment used the 96-well plate method. 1-3-1, Experimental Group: Take 20 μL of the fermentation broth of strain HN-1 and 80 μL of the second instar larval test solution (i.e., the fermentation broth of strain HN-1 was diluted 5 times) and co-culture for 48 h, then calculate the mortality rate. Take 10 μL of the fermentation broth of strain HN-1 and 90 μL of the second instar larval test solution (i.e., the fermentation broth of strain HN-1 was diluted 10 times) and co-culture for 48 h, then calculate the mortality rate. 1-3-2, Control Group: Replace the fermentation broth of strain HN-1 in the experimental group with an equal amount of control solution, and follow the same steps as 1-3-1 in this embodiment. 1-3-3. The corrected mortality rate of the experimental group was calculated using the following formula: Corrected mortality rate = (experimental group mortality rate - control group mortality rate) / 1 - control group mortality rate × 100% 1-4. Test Results As shown in Table 1, the corrected mortality rates of second-instar larvae for the fermentation broth of strain HN-1 diluted 5 times and 10 times were 94.57% and 83%, respectively.

[0026] 2. Tests on the killing of pine wilt nematodes 2-1. The fermentation broth of strain HN-1 and the control group test solution were the same as those used in the Southern Root-knot Nematode Killing Test in this example; 2-2 Preparation of pine wood nematode test solution Cultured in Botrytis cinerea ( Botrytis cinerea Pine wood nematodes were collected in 1.5 mL centrifuge tubes using the Bellman method. The culture medium of live pine wood nematodes was enriched by short-term centrifugation at 4400 rpm for 30 s, which is the pine wood nematode test solution with a concentration of 1-2 pine wood nematodes / μL. 2-3. The experiment used the 96-well plate method. 2-3-1, Experimental Group: 20 μL of the fermentation broth of strain HN-1 was co-cultured with 80 μL of pine wood nematode test solution (i.e., the fermentation broth of strain HN-1 was diluted 5 times) for 48 h, and the mortality rate was calculated. Take 10 μL of the fermentation broth of strain HN-1 and 90 μL of pine wood nematode test solution (i.e., the fermentation broth of strain HN-1 was diluted 10 times) and co-culture for 48 h, then calculate the mortality rate. 2-3-2, Control Group: Replace the fermentation broth of strain HN-1 in the experimental group with an equal amount of control solution, and follow the same steps as in step 2-3-1 of this embodiment. 2-3-3. The method for calculating the corrected mortality rate of the experimental group is the same as that for the Southern Root-knot Nematode Killing Experiment in this example; 2-4. Test Results As shown in Table 1, the corrected mortality rates of strain HN-1 fermentation broth diluted 5 times and 10 times for pine wood nematode were 93.21% and 82.32%, respectively.

[0027] Table 1 Results of the nematicide test of fermentation broth of strain HN-1 after 48 hours

[0028] Example 7 Nematode Activity Test of Bacillus subtilis Fermentation Broth 1. Experiment on killing southern root-knot nematodes 1-1. The Bacillus subtilis fermentation broth was prepared according to the method in Example 2. Bacillus subtilis was used instead of strain HN-1 for inoculation to prepare seed liquid and Bacillus subtilis fermentation broth, wherein the viable cell count was 3.2 × 10⁻⁶. 8 cfu / mL; the Bacillus subtilis used for inoculation was obtained from the strain bank of Yunnan University.

[0029] The control group solution was the same as the southern root-knot nematode killing test in Example 6; 1-2. Preparation of test solution for second-instar larvae of Southern root-knot nematodes is the same as in the Southern root-knot nematode killing test in Example 6. 1-3. The experiment used the 96-well plate method. 1-3-1, Experimental Group: Take 20 μL of Bacillus subtilis fermentation broth and 80 μL of second-instar larval test solution (i.e., Bacillus subtilis fermentation broth diluted 5 times) and co-culture for 48 h, then calculate the mortality rate. Take 10 μL of Bacillus subtilis fermentation broth and 90 μL of second-instar larval test solution (i.e., Bacillus subtilis fermentation broth diluted 10 times) and co-culture for 48 h, then calculate the mortality rate. 1-3-2, Control Group: Replace the Bacillus subtilis fermentation broth in the experimental group with an equal amount of the control group test solution, and follow the same steps as 1-3-1 in this embodiment. 1-3-3. The corrected mortality rate of the experimental group was calculated using the same method as in the Southern Root-knot Nematode Killing Experiment in Example 6; 1-4. Test Results As shown in Table 2, the corrected mortality rates of second-instar larvae for Bacillus subtilis fermentation broth diluted 5 times and 10 times were 87.13% and 79.78%, respectively. Combined with Example 6, it can be seen that the corrected mortality rate of second-instar larvae for strain HN-1 fermentation broth diluted 5 times and 10 times was significantly better than that for Bacillus subtilis fermentation broth. P <0.05); This indicates that strain HN-1 has significant killing activity against southern root-knot nematodes, and its effect is better than that of Bacillus subtilis. Good killing effect against southern root-knot nematodes can be obtained through the fermentation broth of strain HN-1.

[0030] 2. Tests on the killing of pine wilt nematodes 2-1. The Bacillus subtilis fermentation broth and control group test solution were the same as those used in the Southern Root-knot Nematode Killing Test in this example; 2-2. Preparation of pine wilt nematode test solution is the same as in the pine wilt nematode killing test in Example 6; 2-3. The experiment used the 96-well plate method. 2-3-1, Experimental Group: Take 20 μL of Bacillus subtilis fermentation broth and 80 μL of pine wood nematode test solution (i.e., Bacillus subtilis fermentation broth diluted 5 times) and co-culture for 48 h, then calculate the mortality rate. Take 10 μL of Bacillus subtilis fermentation broth and 90 μL of pine wood nematode test solution (i.e., Bacillus subtilis fermentation broth diluted 10 times) and co-culture for 48 h, then calculate the mortality rate. 2-3-2, Control Group: Replace the Bacillus subtilis fermentation broth in the experimental group with an equal amount of the control group test solution, and follow the same steps as in step 2-3-1 of this embodiment. 2-3-3. The method for calculating the corrected mortality rate of the experimental group is the same as that for the Southern Root-knot Nematode Killing Experiment in this example; 2-4. Test Results As shown in Table 2, the corrected mortality rates of Bacillus subtilis fermentation broth diluted 5 times and 10 times for pine wilt disease were 90.31% and 85.51%, respectively. Combined with Example 6, it can be seen that the corrected mortality rate of pine wilt disease by strain HN-1 fermentation broth is comparable to that of Bacillus subtilis fermentation broth, and the effect of strain HN-1 fermentation broth diluted 5 times is better. This indicates that strain HN-1 has significant insecticidal activity against pine wilt disease, and a good pine wilt disease killing effect can be obtained by using strain HN-1 fermentation broth.

[0031] Table 2 Results of the nematicide test of Bacillus subtilis fermentation broth over 48 hours

[0032] Example 8: Nematode-killing activity test of fermentation metabolites of strain HN-1 1. Experiment on killing southern root-knot nematodes 1-1. Fermentation metabolites of strain HN-1: The fermentation metabolites of strain HN-1 obtained in step 3 of Example 2 were used; the control group test solution was selected from liquid culture medium No. 4. 1-2. Preparation of test solution for second-instar larvae of Southern root-knot nematodes is the same as in the Southern root-knot nematode killing test in Example 6. 1-3. The experiment used the 96-well plate method. 1-3-1, Experimental Group: 20 μL of fermentation metabolite of strain HN-1 was co-cultured with 80 μL of second-instar larval test solution (i.e., the fermentation metabolite of strain HN-1 was diluted 5 times) for 48 h, and the mortality rate was calculated. 10 μL of fermentation metabolite of strain HN-1 was co-cultured with 90 μL of second-instar larval test solution (i.e., the fermentation metabolite of strain HN-1 was diluted 10 times) for 48 h, and the mortality rate was calculated. 1-3-2, Control Group: Replace the fermentation metabolites of strain HN-1 in the experimental group with an equal amount of control solution, and follow the same steps as 1-3-1 in this embodiment. 1-3-3. The method for calculating the corrected mortality rate of the experimental group is the same as that used in the pine wilt test in Example 6; 1-4. Test Results As shown in Table 3, the fermentation metabolites of strain HN-1 diluted 5 times and 10 times had corrected mortality rates of 93.78% and 81.26% for second-instar larvae, respectively, which were close to the effects of the fermentation broth of strain HN-1. This indicates that the fermentation metabolites of strain HN-1 can achieve a good effect in killing southern root-knot nematodes.

[0033] 2. Tests on the killing of pine wilt nematodes 2-1. The fermentation metabolites of strain HN-1 and the control group test solution were the same as those used in the Southern Root-knot Nematode Killing Test in this example. 2-2. Preparation of pine wilt nematode test solution is the same as in the pine wilt nematode killing test in Example 6; 2-3. The experiment used the 96-well plate method. 2-3-1, Experimental Group: 20 μL of fermentation metabolites of strain HN-1 were co-cultured with 80 μL of pine wood nematode test solution (i.e., the fermentation metabolites of strain HN-1 were diluted 5 times) for 48 h, and the mortality rate was calculated. 10 μL of fermentation metabolite of strain HN-1 was co-cultured with 90 μL of pine wood nematode test solution (i.e., the fermentation metabolite of strain HN-1 was diluted 10 times) for 48 h, and the mortality rate was calculated. 2-3-2, Control Group: Replace the fermentation metabolites of strain HN-1 in the experimental group with an equal amount of control solution, and follow the same steps as in step 2-3-1 of this embodiment. 2-3-3. The method for calculating the corrected mortality rate of the experimental group is the same as that for the Southern Root-knot Nematode Killing Experiment in this example; 2-4. Test Results As shown in Table 3, the corrected mortality rates of pine wilt nematodes were 89% and 83.82% for the fermentation metabolites of strain HN-1 diluted 5 times and 10 times, respectively, which were close to the effects of the fermentation metabolites of strain HN-1. This indicates that a good pine wilt nematode killing effect can be obtained by using the fermentation metabolites of strain HN-1.

[0034] Table 3 Results of the nematicide test of fermentation metabolites of strain HN-1 after 48 hours

[0035] Example 9: Experiment on the application of liquid bacterial agent of strain HN-1 in potted tomatoes to control southern root-knot nematodes. 1. Prepare transplanting pots and nutrient soil. Divide the plants into three groups based on the number of pots: a negative control group (CK group), a strain HN-1 treatment group, and a positive control group. Each group has 8 replicates, with one tomato seedling planted in each pot. The negative control group was treated with water. The strain HN-1 treatment group was treated with the liquid inoculant prepared in Example 3 (hereinafter referred to as inoculant A), with a viable count of 3.6 × 10⁻⁶ cells / year. 8 CFU / mL; the positive control group used the existing biocontrol agent for root-knot nematodes, Paecilomyces lilacinus (hereinafter referred to as Agent B), with a viable count of 2.0 × 10⁻⁶. 8 cfu / mL; 2. Referring to the experiment on killing southern root-knot nematodes in Example 6, prepare an inoculation solution of second-instar larvae of southern root-knot nematodes, wherein the concentration of second-instar larvae is 1500~1600 larvae / mL; 3. Experimental process 3-1. Take tomato seedlings for transplanting, including: 3-1-1. Tomato seedlings in the negative control group were dipped in clean water at the roots and then transplanted into pots normally, and watered with 100mL of clean water per plant. 3-1-2. Tomato seedlings in the HN-1 treatment group were dipped in inoculant A for 20 minutes. After transplanting, inoculant A was diluted to 2.0 × 10⁻⁶. 8 Water at a rate of 100 mL per plant (cfu / mL). 3-1-3. Tomato seedlings in the positive control group were dipped in inoculant B for 35 minutes (the number of viable bacteria after root dipping was equivalent to that in 3-1-2). After transplanting, inoculant B was applied at a rate of 100 mL per plant. 3-2. After transplanting, inject 1 mL of inoculum solution of second-instar larvae of southern root-knot nematodes into the root of each tomato seedling in each experimental group. 3-3. After the above treatment is completed, water twice a week. Collect tomato roots 40 days after transplanting, clean them, and observe the condition of the root knots. 4. Test Results: Numerous root knots formed in the negative control group, while the number of root knots was significantly reduced in the positive control group. The number of root knots in the HN-1 treatment group was also significantly reduced, and the effect of the HN-1 treatment group was comparable to that of the positive control group, with some plants exhibiting even fewer root knots (see reference). Figure 3 This indicates that the liquid bacterial agent prepared from strain HN-1 can be used for field control of root-knot nematodes in southern China and can achieve relatively ideal control results.

Claims

1. A strain of Bacillus tekirae, HN-1, is deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M 20251718. Strain HN-1 has activity against plant parasitic nematodes.

2. The application of the Bacillus tekirii strain HN-1 according to claim 1 in the control of plant parasitic nematodes, characterized in that, The plant parasitic nematode is either the southern root-knot nematode or the pine wood nematode.

3. A fungal agent for controlling plant parasitic nematodes prepared from the Bacillus tektii strain HN-1 according to claim 1, characterized in that, The microbial agent is a liquid microbial agent made from the fermentation broth or fermentation metabolites of strain HN-1, or a solid microbial agent made by mixing the fermentation broth of strain HN-1 with an agriculturally acceptable carrier.

4. The fungal agent for controlling plant parasitic nematodes according to claim 3, characterized in that, The preparation methods of the fermentation broth and fermentation metabolites are as follows: Strain strain HN-1 was inoculated into liquid medium No. 4 and cultured at 30℃ and 180 rpm for 12-48 h to obtain seed culture; the seed culture was inoculated into Modified Schaeffer's liquid medium at a volume ratio of 10% and cultured at 37℃ for 48-72 h to obtain fermentation broth; Centrifuge the fermentation broth at 5000 rpm for 20 minutes and collect the supernatant, which is the fermentation metabolite.

5. The fungal agent for controlling plant parasitic nematodes according to claim 4, characterized in that, The formula for liquid culture medium No. 4, by mass fraction, is: peptone 0.5%, MgSO4 0.012%, glucose 0.5%, KH2PO4 0.01%, pH 7.0~7.

2.

6. The fungal agent for controlling plant parasitic nematodes according to claim 4, characterized in that, The Modified Schaeffer's liquid culture medium formulation, by mass fraction, is: 0.8% peptone, 0.1% yeast extract, 0.12% MgSO4·7H2O, 0.34% KCl, 0.1% glucose, pH 7.0~7.

2.

7. The fungal agent for controlling plant parasitic nematodes according to claim 3, characterized in that, The carrier includes, but is not limited to, starch or diatomaceous earth.

8. The fungal agent for controlling plant parasitic nematodes according to claim 3, characterized in that, The application method of the liquid bacterial agent: (1) For the southern root-knot nematode, apply liquid inoculant to the roots before transplanting and / or apply solid inoculant in holes or liquid inoculant to the roots at the early stage of the southern root-knot nematode infestation. When dipping the roots, the concentration of live bacteria in the liquid inoculant should not be less than 2.0 × 10⁻⁶. 8 CFU / mL, the concentration of live bacteria in the liquid inoculant during root irrigation is 1.0 × 10⁻⁶. 8 ~3.0×10 8 cfu / mL, 300mL per plant for root irrigation; (2) For pine wood nematode, non-woven fabric strips soaked in liquid fungal agent were used to fill the borer holes of pine sawyer beetle. The concentration of live bacteria in the liquid fungal agent was 3.0 × 10⁻⁶. 8 cfu / mL.

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