Application of bacillus coagulans strain Rhs-L31 in prevention and treatment of pine wood nematode disease

Abstract

The invention provides application of a bacillus subtilis strain Rhs-L31 in prevention and treatment of pine wood nematode disease, and relates to an agricultural environment microbial technology. In particular relates to a bacillus friaricola strain Rhs-L31 which is separated from rhizosphere soil of Chinese pines and application of the bacillus friaricola strain Rhs-L31 in prevention and treatment of pine wood nematode diseases. The strain is preserved in the China Center for Type Culture Collection in Wuhan University on December 25, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) 20242847. The in-vitro poisoning activity of the strain Rhs-L31 on pine wood nematodes can reach 100% within 24 hours; a fermentation filtrate and a zymophyte suspension of the strain Rhs-L31 have a remarkable inhibition effect on the propagation of the pine wood nematodes; the prevention and treatment effect of a zymophyte suspension of the strain Rhs-L31 on the pine wood nematode disease of the Chinese pine potted seedlings can reach 50%-72.5%; the bacillus subtilis strain Rhs-L31 disclosed by the invention has a good application prospect in the field of pollution-free prevention and treatment of pine wood nematode diseases.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural environmental microorganisms, and in particular to application of a cold-resistant Bacillus strain Rhs-L31 in preventing and controlling pine wood nematode disease. Background Art

[0002] Pine wilt nematode (PWN) is considered one of the most dangerous and destructive forest pests in the world. Once a pine tree is infected with PWN, it will die in as little as 40 days, and a large pine forest will be destroyed within 3 to 5 years, causing extremely serious losses to forest resources. Due to its wide range of adaptability, many host species, fast spread, high mortality rate of infected pine trees, and difficulty in prevention and control, pine wilt disease is distributed in Japan, China, South Korea, the United States, Canada, Mexico, Portugal, and Spain, posing a serious threat to pine forests in various countries around the world, seriously affecting the ecological balance of forests and the sustainable development of the forestry industry. In my country, pine wilt disease is listed as the number one forest plant quarantine object in China. In summary, the severity and widespread distribution of pine wilt disease pose a huge threat to pine forests around the world, especially in Asia. Its rapid spread and high mortality rate make this disease an important issue in forestry.

[0003] For the prevention and control of pine wood nematode disease, traditional prevention and control methods include physical prevention and chemical prevention, but since both methods have their own limitations, research on environmentally friendly biological control has received more and more attention. With the increasing awareness of ecological protection, biological control strategies have become increasingly important in the prevention and control of pine wood nematode disease. At present, biological control of pine wood nematode disease is mainly divided into two directions: targeting vector insects and targeting pathogenic nematodes. Targeting vector insects: As the main transmitter of pine wood nematodes, the prevention and control measures include the use of biological pheromones for trapping and killing, and releasing its natural enemies for biological control, such as parasitic natural enemies such as flower beetles and tube-legged beetles. However, these methods are easily affected by other biological and non-biological factors in the forest, resulting in unstable or unpredictable control effects; targeting pathogenic nematodes: At present, the main biological nematicides used to control pine wood nematodes include avermectin and emamectin benzoate, which are applied by trunk injection and can be effective for 2 to 3 years. However, this method of application is labor-intensive and has high cost of agents, resulting in expensive overall control costs. Therefore, the development of microbial agents with high lethality to pine wood nematodes in nature has become a hot area for the development of new nematicides. Cold-resistant Bacillus has the functions of cold resistance and resistance to plant pathogens, can grow in a wide range of temperature and pH values, and has a good inhibitory effect on a variety of plant pathogens. For example, the inhibition rate of banana wilt pathogens is as high as 98%. The potential application value of this strain is to prepare biocontrol bacteria to replace or reduce the use of chemical pesticides. In view of this, the present invention is specially proposed. Summary of the invention

[0004] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides a cold-resistant Bacillus strain Rhs-L31, which solves the deficiencies in the existing prevention and control technology for pine wood nematode disease.

[0005] (II) Technical solution Another object of the present invention is to provide the results of in vitro toxicity activity of the cold-resistant Bacillus strain Rhs-L31 against pine wood nematodes.

[0006] Another object of the present invention is to provide the results of the inhibition of reproduction of pine wood nematodes by the cold-resistant Bacillus strain Rhs-L31.

[0007] Another object of the present invention is to provide the application of cold-resistant Bacillus strain Rhs-L31 in the prevention and treatment of pine wood nematode disease in potted pine seedlings To achieve the above objectives, the present invention is implemented through the following technical solutions: The cold-resistant Bacillus strain Rhs-L31 provided by the present invention has been deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number being CCTCC NO: M 20242847.

[0008] Furthermore, the cold-resistant Bacillus strain Rhs-L31 was fermented in LB liquid medium at 25°C, 150rpm, and dark culture for 4 days to obtain the fermentation suspension (1×) and fermentation filtrate (without bacteria, 1×) of the strain, and diluted with sterile water to prepare 5-fold (0.2×) and 10-fold (0.1×) fermentation suspension and fermentation filtrate, respectively. Then, each fermentation suspension and fermentation filtrate were co-incubated with pine wood nematodes for 24 hours, and the number of surviving and dead nematodes was counted. The in vitro toxicity of Rhs-L31 against pine wood nematodes was analyzed by the corrected mortality rate of pine wood nematodes.

[0009] Furthermore, the cold-resistant Bacillus strain Rhs-L31 was fermented in LB liquid medium at 25°C, 150rpm, and dark culture for 4 days to obtain the fermentation suspension and fermentation filtrate (without bacteria). Botrytis cinerea was inoculated on a PDA medium plate. After 7 days of culture, 50µL of a suspension containing 600 pine wood nematodes was dripped into the center of the Botrytis cinerea colony. At the same time, 100µL of Rhs-L31 fermentation filtrate or fermentation suspension was also dripped into the inoculation site. The same amount of LB liquid medium was added as a control. The concentration of the fermentation suspension was 7.02×107CFU / mL. Then it was placed in a constant temperature incubator for 7 days, and the number of surviving nematodes and eggs was counted. The effect of Rhs-L31 on the reproduction of pine wood nematodes was analyzed by the reproduction coefficient and egg laying amount of pine wood nematodes.

[0010] Furthermore, based on the artificial inoculation system of pine wilt disease, the trunk punch injection method was used to analyze the preventive effect (first inject Rhs-L31 suspension, then inoculate pine wood) and therapeutic effect (first inoculate pine wood nematodes, then inject Rhs-L31 suspension) of the cold-resistant Bacillus Rhs-L31 suspension on pine wilt disease. The concentration of the fermented bacterial suspension was 7.02×107CFU / mL. The incidence and severity of the disease in potted pine seedlings were statistically analyzed, the disease index and biocontrol effect of each treatment were calculated, and the density of pine nematodes in pine trees was determined.

[0011] (III) Beneficial effects The present invention provides an application of a cold-resistant Bacillus strain Rhs-L31 in preventing and treating pine wood nematode disease. The application has the following beneficial effects: 1. The present invention provides a cold-resistant Bacillus strain Rhs-L31, which has significant toxic activity against pine wood nematodes.

[0012] 2. The present invention provides a cold-resistant Bacillus strain Rhs-L31, and determines the effects of the fermentation bacterial suspension and fermentation filtrate of the cold-resistant Bacillus Rhs-L31 on the reproduction of pine wood nematodes. The reproduction coefficient and egg laying amount of pine wood nematodes are used as detection indicators. The results show that the strain has a significant inhibitory effect on the reproduction of pine wood nematodes.

[0013] 3. The present invention provides a cold-resistant Bacillus strain Rhs-L31, and evaluates the preventive and therapeutic effects of cold-resistant Bacillus Rhs-L31 on pine wood nematode disease in potted pine seedlings. It was found that the preventive effect was more significant, with the prevention efficiency of pine wood nematode disease reaching up to 72.5%, and significantly reducing the nematode density in the pine tree, indicating that the strain has great potential in the biological prevention and control of pine wood nematode disease, laying an important foundation for the later development of bacterial agents, forest application and commercialization of Rhs-L31, and providing a new biocontrol bacteria resource for controlling pine wood nematode disease using biological control technology.

[0014] 4. The present invention provides a cold-resistant Bacillus strain Rhs-L31. The application of this microbial product helps to reduce or replace the use of other chemical pesticides in the prevention and control of pine wood nematode disease, is beneficial to the green prevention and control of pine wood nematode disease, and is beneficial to maintaining the sustainable development of forestry ecosystems. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Live pine wood nematodes (A) and dead pine wood nematodes (B).

[0016] Figure 2 To analyze the toxic activity of cold-resistant Bacillus strain Rhs-L31 against pine wood nematodes.

[0017] Figure 3 Colony morphology (A) and spore staining (B) of the cold-resistant Bacillus strain Rhs-L31.

[0018] Figure 4 This is the effect of the cold-resistant Bacillus strain Rhs-L31 on the reproduction of pine wood nematode.

[0019] Figure 5 This is the growth status of Pinus tabulaeformis seedlings in different treatment groups with the cold-resistant Bacillus strain Rhs-L31.

[0020] Figure 6 The results of potted control of pine wilt disease by the strong cold-resistant Bacillus strain Rhs-L31. The incidence of pine wilt disease in different treatments (A); the disease index of pine wilt disease in different treatments (B); the preventive and therapeutic effects of strain Rhs-L31 on pine wilt disease (C); the density of pine wilt disease in pine wilt disease in different treatments (D). DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Isolation of biocontrol strains and determination of toxicity activity A strain of cold-resistant Bacillus (Peribacilllus frigoritolerans) Rhs-L31 for pine wood nematode disease was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO: M20242847. The biocontrol strain Rhs-L31 was isolated from rhizosphere soil microorganisms of Pinus tabulaeformis. The co-incubation method was used to find that the strain Rhs-L31 showed significant toxicity to pine wood nematodes, and then the effect on the reproduction of pine wood nematodes was determined. The isolation of the biocontrol strain Rhs-L31 and the determination of its toxicity to pine wood nematodes are as follows: (1) Isolation of biocontrol strains The rhizosphere soil of Pinus tabulaeformis was collected from the roots of 20-year-old healthy Pinus tabulaeformis in Huanshan Park, Chengguan Town, Ningshan County, Shaanxi Province (N33°18′25″, E108°18′39″) in September 2022. The specific collection process is as follows: Four 20-year-old healthy Pinus tabulaeformis trees were randomly selected, and a rhizosphere soil sampling point was set 15 cm away from the base of the trunk as the center. The soil 10 to 20 cm away from the surface soil was dug with a shovel, and the fine roots of Pinus tabulaeformis in the soil (diameter <2 mm) were collected. The large particles of soil were shaken off and put into a plastic bag. The root tissue samples brought back were taken out of the ice box, and the soil on the fine roots was shaken off vigorously and collected in a plastic bag. This is the rhizosphere soil of Pinus tabulaeformis. Take 2.5 g of each of the 4 rhizosphere soil samples collected, mix them thoroughly, and transfer them to a sterile glass conical flask (250 mL) containing 90 mL of sterile water. After vigorous shaking and mixing for 15 minutes, let it stand for 5 minutes. The upper solution is the 10-1 g / mL soil suspension. Then use the 10-1 g / mL soil suspension to prepare soil dilution solutions with concentrations of 10-2 g / mL, 10-3 g / mL and 10-4 g / mL, respectively, and set them aside.

[0023] 30 µL of soil suspension was taken and dropped onto the LBA plate, then evenly spread with a sterile applicator, and 3 plates were spread for each concentration (the concentrations used were 10-2g / mL, 10-3g / mL, and 10-4g / mL), the culture dish was sealed with a sealing film, and then placed in a 25°C incubator for constant temperature culture for 30 days. The single colonies grown on the culture medium were observed every day, and the single colonies with different colony morphology that appeared on the plate every day were inoculated on the newly configured plate with a sterile toothpick for purification and preservation until there were no more colonies on the plate.

[0024] The obtained strains were stored in 30% glycerol and placed in a low-temperature environment of -80°C.

[0025] The LBA culture medium is prepared by: 10 g of tryptone, 3 g of yeast extract, 10 g of NaCl, 15-20 g of agar, 1000 mL of water, and pH 7.4-7.6.

[0026] (2) Determination of the toxicity of biocontrol bacteria against pine wood nematodes LB liquid medium was used to carry out liquid fermentation culture of the isolated strains of rhizosphere soil microorganisms of Pinus tabulaeformis. The culture conditions were 25°C, 150rpm, and dark culture for 4 days. The fermentation suspension (1×) and fermentation filtrate (without bacteria, 1×) of the strain were obtained, and diluted with sterile water to prepare fermentation suspension and fermentation filtrate diluted 5 times (0.2×) and 10 times (0.1×), respectively.

[0027] Inoculate Botrytis cinerea on PDA medium and culture for 7 days until the colonies cover the entire medium plate. Inoculate pine wood nematodes on the Botrytis cinerea colonies in an ultra-clean workbench and continue to culture at 25°C without darkness for 7 days until an oily sheen appears on the surface of the medium. Then rinse the pine wood nematodes from the culture dish with sterile water and collect them by centrifugation to form a pine wood nematode suspension.

[0028] 480 μL of each fermentation bacterial suspension (1×, 0.2× and 0.1×) and fermentation filtrate (1×, 0.2× and 0.1×) were added to sterile centrifuge tubes, and then 20 μL of pine wood nematode suspension (mixed larvae and adults, a total of 200 heads) was added to each centrifuge tube, and placed in a constant temperature incubator at 25°C in the dark for 24 hours. After the incubation, the number of surviving and dead nematodes under each treatment was detected by combining safranin staining and morphological observation. Dead nematodes were stained red by safranin and appeared in the shape of "C" or "J", and had no response to physical stimulation; while surviving nematodes were not colored and remained active (see Figure 1 ). Through nematicidal activity screening, it was found that the fermentation suspension and fermentation filtrate (without bacteria) of a bacterium Rhs-L31 had significant in vitro toxicity against pine wood nematodes. The corrected mortality of pine wood nematodes reached 100% when treated with the original solution; after treatment with the fermentation suspension and fermentation filtrate diluted 5 times, the corrected mortality of pine wood nematodes remained at 76.90% and 72.60% ( Figure 2 ).

[0029] The mortality rate and corrected mortality rate of pine wood nematodes in rhizosphere soil of Pinus tabulaeformis were calculated according to the following formula.

[0030] Mortality rate (%) = Corrected mortality rate (%) = Wherein, Nd is the number of nematodes killed in the treatment group; Nt is the total number of nematodes; d is the nematode mortality rate in the treatment group (%); c is the nematode mortality rate in the control group (%).

[0031] Example 2: Identification of biocontrol strain Rhs-L31 A single colony of the biocontrol strain Rhs-L31 was selected and inoculated on LBA medium. The colony morphology was as follows: Figure 3 A. The Schaeffer-Fulton spore staining kit was used to detect whether the strain Rhs-L31 produced spores. It was found that the strain Rhs-L31 could produce a large number of spores, such as Figure 3 As shown in B.

[0032] The DNA of strain Rhs-L31 was extracted by CATB method, and the 16SrDNA sequence of strain Rhs-L31 was amplified and sequenced by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') of 16SrDNA. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] The PCR amplification reaction system of the 16SrDNA sequence of strain Rhs-L31 was 25 μL, including: 12.5 μL 2×ESTaqMasterMix, 1 μL template DNA, 1 μL 27F primer, 1 μL 1492R primer and 9.5 μL sterile distilled water. PCR amplification was carried out in a PCR thermal cycler, and the specific program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles; 72℃ final extension for 10 min. After the PCR was completed, agarose gel electrophoresis was used to detect the length of the amplified fragment, and the product of successful PCR amplification was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. According to the comparison of the 16SrDNA sequence of strain Rhs-L31 in the NCBI database, it was identified as Peribacilllus frigoritolerans. The strain was deposited in the China Center for Type Culture Collection on December 18, 2024, with the accession number CCTCC NO: M 20242847.

[0034] Sequencing results of the 16SrDNA amplified fragment of strain Rhs-L31: Example 3: Detection of the effect of the biocontrol strain Rhs-L31 on the reproduction of pine wood nematodes The cold-resistant Bacillus strain Rhs-L31 was fermented in LB liquid medium at 25℃, 150rpm, and dark culture for 4 days to obtain the fermentation suspension and fermentation filtrate (without bacteria). Botrytis cinerea was inoculated on a PDA medium plate. After 7 days of culture, 50µL of suspension containing 600 pine wood nematodes was dripped into the center of the Botrytis cinerea colony. At the same time, 100µL of Rhs-L31 fermentation filtrate (T1 group) or fermentation suspension (T2 group) was also dripped into the inoculation site. The same amount of LB liquid medium was added as the control (CK group). The concentration of the fermentation suspension was 7.02×107CFU / mL. Then, it was placed in a constant temperature incubator for 7 days. First, the pine wood nematodes were rinsed from each culture dish with sterile water. Each culture dish was rinsed with 5mL of sterile water in small amounts and multiple times, and then the pine wood nematodes were collected by centrifugation.

[0035] Combining the methods of safranin staining and morphological observation, the number of surviving pine wood nematodes and the number of eggs under each treatment were counted under an optical microscope. By comparing with the analysis of the CK group, the effect of Rhs-L31 on the reproduction of pine wood nematodes was clarified. The experimental results showed that the fermentation filtrate and fermentation bacterial suspension of strain Rhs-L31 had a significant inhibitory effect on the reproduction of pine wood nematodes. The reproduction coefficient and egg production of the CK group were 59 and 1302, respectively, while the reproduction coefficients of the T1 and T2 groups were only 4.1 and 1.32, and no new eggs were formed ( Figure 4 ). This shows that both the fermentation filtrate and the fermentation bacterial suspension of Rhs-L31 can significantly inhibit the reproduction of pine wood nematodes.

[0036] The reproduction coefficient of pine wood nematodes on each PDA plate was calculated according to the following formula: Reproduction coefficient = Wherein, Pf is the number of surviving nematodes in each plate after 7 days of cultivation, and Pi is the initial inoculum number of nematodes in each plate.

[0037] Example 4: Application of strain Rhs-L31 in pine wood nematode disease Five-year-old healthy Pinus tabulaeformis seedlings planted in greenhouse pots at Northwest Agriculture and Forestry University were used as the research objects. Five different treatment groups were designed, including LB liquid culture medium treatment (CK group), pine wood nematode treatment (PWN group), Rhs-L31 fermented bacterial suspension treatment (Rhs-L31 group), injection of Rhs-L31 fermented bacterial suspension first and then inoculation of pine wood nematodes (preventive group: Rhs-L31→PWN), and inoculation of pine wood nematodes first and then injection of Rhs-L31 fermented bacterial suspension (treatment group: PWN→Rhs-L31). All Pinus tabulaeformis seedlings were cultured and observed in the greenhouse of Northwest Agriculture and Forestry University. Three replicates were set for each treatment, and each replicate contained three Pinus tabulaeformis seedlings. The concentration of Rhs-L31 bacterial suspension was 7.02×107CFU / mL.

[0038] Before treatment, wet sterile cotton was used to clean the branches and trunks of healthy Chinese pine seedlings. Then, two small holes were drilled on the stems of each Chinese pine seedling using a handheld electric drill. Each hole was 4.5 mm in diameter and 5 mm in depth, with a spacing of 10 cm between the two holes. For the CK group, each hole on each Chinese pine seedling was covered with sterile cotton and 5 ml of LB medium was added to it. Each well was sealed with plastic film to prevent rapid evaporation of water, and the cotton and plastic film were removed after 7 days. For the PWN group, each well was inoculated with 50 µL of PWN suspension (a mixture of larvae and adults) containing 3000 heads. The inoculation holes of the PWN group, the prevention group, and the treatment group were also sealed with plastic film, and the plastic film was removed after 7 days of treatment. The Rhs-L31 group was treated in the same way as the CK group, except that 5 ml of Rhs-L31 strain fermentation suspension was used instead of LB. The initial treatment in the Rhs-L31→PWN prevention group was the same as that in the Rhs-L31 group. After 7 days of cultivation in the greenhouse, the cotton and plastic film covering the holes were removed, and 50 μL of pine wood nematode suspension (containing 3000 PWN) was inoculated in each hole of each Chinese pine seedling. The PWN→Rhs-L31 group was first inoculated with pine wood nematodes, and then the fermented suspension of the Rhs-L31 strain was applied 7 days later.

[0039] The disease conditions of the pine seedlings in each treatment group were observed and recorded at 7, 14, 21, 28 and 35 days after inoculation with PWN, and the incidence rate was calculated. The pine seedlings were divided into 5 sections on average, and the severity of the disease in each section was counted. The severity of the disease (S) was divided into 5 levels (S1-S5). S1: All needles were in a healthy green state; S2: Yellow needles appeared, but the number of yellow needles did not exceed one-quarter; S3: Dead needles appeared, and the number of yellow or dead needles was between one-quarter and one-half; S4: The number of yellow or dead needles was between one-half and two-thirds, and the pine wood segment showed slight wilting; S5: The number of yellow or dead needles exceeded two-thirds, and the pine seedling segment was obviously dehydrated and wilted. The representative values ​​of each disease severity were 0, 1, 2, 3, and 4, respectively. The disease index of pine wilt disease under each treatment was calculated, and the preventive effect of Rhs-L31 preventive treatment (Rhs-L31→PWN group) and therapeutic treatment (PWN→Rhs-L31 group) on pine wilt disease was calculated.

[0040] 35 days after inoculation with PWN, the whole pine seedlings in the PWN group were in a wilting and dead state. Each pine seedling in the PWN group, Rhs-L31→PWN group, and PWN→Rhs-L31 group was collected, and the roots, stems, and leaves of each pine seedling were cut into small pieces, mixed evenly, and weighed. Subsequently, the pine wood nematodes in all tissues of each seedling were separated by the Baermann funnel method, and the number of pine wood nematodes (Number of nematodes, NN) was counted under a microscope. The density of pine wood nematodes in pine seedlings under different treatments was expressed as the number of nematodes per gram of fresh weight (FW) of pine (NN g-1FW).

[0041] The incidence rate, disease index, control effect and pine wood nematode density were calculated using the following formula: Incidence (%) = ×100% Disease index (%) = ×100% Control effect (%) = ×100% Density of pine wood nematodes (NN g -1 FW) = The pot experiment showed that the strain Rhs-L31 had no toxicity to pine seedlings. The growth conditions of pine seedlings treated with Rhs-L31 alone were basically the same as those of the control group, and both remained in a healthy state. 35 days after inoculation with pine wood nematodes, all the needles of the Chinese pine seedlings in the PWN group lost their green leaves and dried up, and the pine seedlings were in a state of significant wilting. The incidence of the disease in the Rhs-L31→PWN group (prevention group) was significantly lighter than that in the PWN group, and the number of green or dried needles of each pine seedling was less than half. The WN→Rhs-L31 group (treatment group) also had a certain effect on alleviating the occurrence of pine wood nematode disease, but the number of dead needles was significantly more than that in the prevention group ( Figure 5 ).

[0042] By analyzing the incidence and disease index of pine seedlings in each treatment group at 7, 14, 21, 28 and 35 days after inoculation with PWN, it was found that the pine wilt disease appeared on the 5-year-old pine seedlings at 14 days after inoculation with PWN. As the disease progressed, the symptoms became more severe. At 35 days, all pine seedlings were infected with the disease, and the disease index was as high as 100 ( Figure 6 AB). The pine seedlings in the prevention group (Rhs-L31→PWN) and treatment group (PWN→Rhs-L31) of strain Rhs-L31 also showed mild symptoms on day 14, but the disease was milder, especially in the Rhs-L31→PWN group. As the disease progressed, the incidence rate continued to increase. At day 35, the incidence rate of the Rhs-L31→PWN group was 83.3%, while the incidence rate of the PWN→Rhs-L31 group was 100% ( Figure 6 A). Although there was no significant difference in the incidence between the Rhs-L31→PWN group and the PWN→Rhs-L31 group, the disease index of the Rhs-L31→PWN group (47.91) was significantly lower than that of the PWN→Rhs-L31 group (81.25) and the PWN group (100) ( Figure 6 B). By calculating the control effect of Rhs-L31→PWN group and PWN→Rhs-L31 group on pine wood nematode disease, it was found that the control effect of Rhs-L31→PWN group on pine wood nematode disease was between 50% and 72.5%, while the control effect of PWN→Rhs-L31 group was lower than that of Rhs-L31→PWN group, between 15.6% and 54.2% ( Figure 6 C), therefore, in the prevention and control of pine wood nematode disease, strain Rhs-L31 has a better preventive and protective effect.

[0043] At 35 days after inoculation with PWN, it was found that all treatments with strain Rhs-L31 could significantly reduce the density of PWN in pine seedlings. Compared with the PWN group, the density of pine wood nematodes in the Rhs-L31→PWN group was significantly reduced by 43.16%, and the density of pine wood nematodes in the PWN→Rhs-L31 group was significantly reduced by 21.32%, indicating that strain Rhs-L31 can effectively inhibit the reproduction of pine wood nematodes in Chinese pine seedlings, and the inhibitory effect of the prevention group (Rhs-L31→PWN group) was more obvious than that of the treatment group (PWN→Rhs-L31 group) (Figure 8D).

[0044] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A cold-resistant Bacillus strain Rhs-L31, characterized in that: The cold-resistant Bacillus strain Rhs-L31 was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO: M 20242847.

2. The cold-resistant Bacillus strain Rhs-L31 according to claim 1, characterized in that: The cold-resistant Bacillus Rhs-L31 is used in preventing and controlling pine wood nematode disease.

3. The in vitro toxicity activity of a cold-resistant Bacillus strain Rhs-L31 according to claim 1, characterized in that: The components of the fermentation medium were 10 g of tryptone, 3 g of yeast extract, 10 g of sodium chloride, and 1 L of water. The fermentation conditions were 25°C, 150 rpm, and dark culture for 4 days. The fermentation bacterial suspension and fermentation filtrate (without bacteria) had significant in vitro toxic activity against pine wood nematodes. After 24 hours of co-incubation, the corrected mortality rates of pine wood nematodes reached 100%. After 5-fold dilution, the corrected mortality rates of pine wood nematodes were 76.90% and 72.60%, respectively.

4. The method for determining the effect of a cold-resistant Bacillus strain Rhs-L31 on the reproduction of pine wood nematodes according to claim 1, characterized in that: The fermentation filtrate or fermentation bacterial suspension of the Rhs-L31 strain has a significant inhibitory effect on the reproduction of pine wood nematodes, significantly reducing its reproduction coefficient and egg-laying amount. The concentration of the fermentation bacterial suspension is 7.02×107CFU / mL.

5. The preventive effect of the cold-resistant Bacillus Rhs-L31 fermentation bacterial suspension on pine wood nematode disease in potted Chinese pine seedlings according to claim 4 is characterized in that: The control effect on pine wood nematode disease can reach 50% to 72.5%, and significantly reduces the density of pine wood nematodes in pine trees.

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