Bacillus safensis and application thereof in prevention and control of pine wood nematode disease

Abstract

The invention discloses bacillus safensis and application thereof in prevention and control of pine wood nematode diseases, and belongs to the technical field of biocontrol of plant diseases. The invention discloses bacillus safensis GDWS1 which is separated from resistant masson pine tissues and has a relatively good inhibition effect on pine wood nematode diseases, and the preservation number of the bacillus safensis GDWS1 is GDMCC No: 66264. The strain GDWS1 has high lethal activity on pine wood nematodes, the correction death rate of the strain GDWS1 on the pine wood nematodes reaches 90% or above, and active substances capable of poisoning and killing the pine wood nematodes and good in stability can be generated; the requirements on culture conditions are low, and the ecological environment is friendly and pollution-free. A greenhouse control experiment is carried out by using pinus massoniana seedlings, the strain GDWS1 can effectively reduce the disease index of the pinus massoniana seedlings inoculated with pine wood nematodes, the control effect reaches 45.88%, and the strain has a good application prospect in prevention and control of pine wood nematode diseases, provides a new biocontrol bacterium type for biological prevention and control of pine wood nematode diseases, and has the potential of being developed into biopesticides.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease biocontrol, and particularly relates to a strain of Bacillus saffron and its application in preventing and controlling pine wood nematode disease. Background Art

[0002] Pine Wilt Disease (PWD), also known as pine wilt, is a serious quarantine disease caused by the pine wood nematode (Bursaphelenchus xylophilus), resulting in the mass mortality of pine trees. Pine wood nematodes can infect 106 host plant species, 60 of which have been confirmed to be naturally infected. As an important native tree species in my country's subtropical regions, Masson pine is renowned for its high resin yield, excellent wood properties, and rapid growth. It is an essential tree species for soil and water conservation, windbreak and sand fixation, and landscape design.

[0003] Pine wood nematode is limited by its own mobility and its transmission mainly relies on insect vectors. In nature, pine sawyer beetle (Monochamus alternatus) is the key vector for spreading pine wood nematode disease, and its population density indirectly affects the occurrence of pine wood nematode disease.

[0004] (1) Targeting insect vectors: Monochamus alternatus is the main vector of pine wood nematodes. Natural enemies of Monochamus alternatus, such as Scleroderma guani, Dastarcus helophoroides, Cleridae, and Scleroderma sichuanensis, are introduced. However, these methods are greatly affected by biological and non-biological factors in the forest, and the control effects are unstable and uncertain.

[0005] (2) Targeting pathogenic nematodes: Currently, tree trunk injection technology is used for prevention and control, and the agents include tea saponin solution, emamectin, and abamectin. After injection, the protection lasts for 2-3 years, but because injection requires a lot of manpower and material resources, the cost of prevention and control is too high, so it is only used in ancient trees in scenic areas. Therefore, searching for microorganisms and their metabolites that are highly lethal to pine wood nematodes in nature has become a research hotspot for the development of new nematicides.

[0006] Significant progress has been made in the use of microorganisms to control parasitic nematodes. For example, the metabolite avermectin from the actinomycete Streptomyces genus has been used to control pine wilt disease. Among fungi, the nematode-eating fungus Esteyavermicola is a natural enemy of pine wilt nematodes. Its crescent-shaped spores can adhere to and infect the nematodes, ultimately killing them. Among bacteria, widely commercialized nematode-killing bacterial strains primarily come from Pseudomonas fluorescens, Bacillus, Actinomycetes, and Pasteuria. These strains are primarily used to control crop nematodes. However, relatively few bacterial strains for controlling pine wilt disease have been put into production. Therefore, the development of new pine wilt biocontrol bacteria is urgently needed.

[0007] Compared to actinomycetes and fungi, bacteria reproduce quickly and are easy to culture. Plant endophytes are also superior to soil bacteria, overcoming the poor colonization problems previously encountered by biocontrol bacteria isolated from natural environments. Compared to epiphytic and rhizospheric bacteria in natural environments, they possess a stable habitat, are less susceptible to external influences, and are able to colonize and transmit pathogens within plants, inducing systemic resistance. Therefore, plant endophytic bacteria hold great promise for development and application in nematode disease control. In recent years, many researchers have attempted to isolate endophytic bacteria from Masson pine trees that can control plant diseases. Yuan Wenting first isolated 20 endophytic bacterial strains from Masson pine and, through plate confrontation assays, screened three strains with strong antagonistic effects against pathogens (Yuan Wenting, 2010).

[0008] Therefore, the development of highly effective, low-toxic biological control agents is urgent, and the use of beneficial microorganisms to control nematodes has become an important approach for developing these agents. However, there are currently no reports on the antagonistic effects of Bacillus saffron against pine wood nematodes. In light of this, the present invention was proposed. Summary of the Invention

[0009] In order to overcome the shortcomings and deficiencies of the prior art in preventing and treating pine wood nematode disease, the present invention aims to provide a strain of Bacillus saffron GDWS1.

[0010] Another object of the present invention is to provide a biological preparation comprising the above-mentioned Bacillus sabdariffa GDWS1.

[0011] Another object of the present invention is to provide an application of the above-mentioned Bacillus sabdariffa GDWS1 or biological preparation.

[0012] The Bacillus safensis GDWS1 of the present invention is a microorganism with nematicidal activity screened from healthy Masson pine plant tissues, provides a theoretical basis for the development of plant tissue microbial resources and the utilization of pine wood nematode-killing endophytes, and has important practical significance.

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] A strain of Bacillus safensis, named Bacillus safensis GDWS1, was isolated and purified from resistant Masson pine branch tissues.

[0015] The preservation information of the Bacillus safensis GDWS1: Preservation unit: Guangdong Microbial Culture Collection Center (GDMCC), preservation time is May 7, 2025, preservation address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, preservation number: GDMCC No: 66264.

[0016] The Bacillus safensis GDWS1 has the following morphological characteristics:

[0017] The colonies are usually white or light yellow, opaque, round, with irregular edges, a smooth and shiny surface, slightly sticky, and Gram-positive.

[0018] A biological preparation comprising the above-mentioned Bacillus saffron GDWS1, or at least one of its fermentation broth, fermentation broth supernatant, and fermentation filtrate.

[0019] Furthermore, the fermentation broth is prepared by liquid fermentation of the Bacillus sp. GDWS1, preferably comprising the following steps: inoculating the seed liquid of the Bacillus sp. GDWS1 into LB liquid culture medium for fermentation to obtain the fermentation broth.

[0020] Preferably, the inoculation amount of the seed solution is 1-2% (further 2%);

[0021] Preferably, the fermentation culture conditions are 26-32° C., 180-200 r / min shaking culture for more than 2 days in the absence of light; more preferably, 28±1° C., 180-200 r / min shaking culture for 2-7 days in the absence of light.

[0022] Preferably, the pH of the LB liquid culture medium is 6 to 8, further 7±0.5.

[0023] Preferably, the bottling volume of the LB liquid culture medium is 50-150 / 250 mL; further 100 / 250 mL.

[0024] Preferably, the seed liquid is obtained by first activating Bacillus sabdariffa GDWS1 and then picking a single colony and culturing it in LB liquid culture medium.

[0025] The culture conditions are as follows: 28-30° C., 180-200 r / min shaking culture for 8-15 h; further as 28° C., 180 r / min shaking culture for 8 h.

[0026] Furthermore, the fermentation broth supernatant is prepared by centrifuging the fermentation broth.

[0027] Preferably, the centrifugation conditions are 2-8° C., 8000-10000 rpm, and 8-10 min; more preferably, 4° C., 10000 rpm, and 10 min.

[0028] Furthermore, the fermentation filtrate is prepared by filtering the supernatant of the fermentation broth.

[0029] Preferably, the filtration is performed using a microporous filter membrane with a pore size of 0.22 μm.

[0030] The present invention also provides applications of the above-mentioned Bacillus sabdariffa GDWS1 or biological preparation, which are one or more combinations of the following applications:

[0031] (1) Application in the prevention and control of pine wood nematode disease;

[0032] (2) Application in the preparation of preparations for the prevention and control of pine wood nematode disease;

[0033] (3) Application in killing pine wood nematodes;

[0034] (4) Application in the preparation of preparations for killing pine wood nematodes.

[0035] The present invention has the following advantages and effects compared to the prior art:

[0036] (1) The present invention uses resistant Masson pine tissue as the isolation object to isolate a Bacillus sabdariffa that has a good inhibitory effect on pine wilt disease, named Bacillus sabdariffa GDWS1. The present invention provides a new type of biocontrol bacteria for the biological control of pine wilt disease and has the potential to be developed into a biological pesticide.

[0037] (2) The biological preparation provided by the present invention contains Bacillus sabdariffa GDWS1, which has high lethal activity against pine wood nematodes, wherein the corrected mortality rate against pine wood nematodes is more than 90%, and can be as high as 93.60%, and can produce active substances that kill pine wood nematodes, and the nematode-killing active substances have good stability; greenhouse control experiments were carried out using Pinus massoniana seedlings, and Bacillus sabdariffa GDWS1 can effectively reduce the disease index of Pinus massoniana seedlings inoculated with pine wood nematodes, with a control effect of 45.88%. This strain has good application prospects in the prevention and control of pine wood nematode disease.

[0038] (3) The biological preparation provided by the present invention has low requirements for culture conditions, and the strains contained therein are derived from resistant Masson pine tissues, which are friendly to the ecological environment and pollution-free. Therefore, the biological preparation has a good development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a diagram showing the growth status of strain GDWS1 on LB solid culture medium.

[0040] Figure 2 This is the Gram staining result (10x40) of strain GDWS1.

[0041] Figure 3 This is the phylogenetic tree result of strain GDWS1 constructed based on the 16S rDNA gene sequence.

[0042] Figure 4 This is an analysis of the nematicidal activity of the filtrate after fermentation of Bacillus saffron GDWS1 in different culture media.

[0043] Figure 5 This is an analysis of the nematicidal activity of the fermentation filtrates of Bacillus saffron GDWS1 at different culture times.

[0044] Figure 6 This is an analysis of the nematicidal activity of the filtrates fermented at different temperatures using Bacillus saffron GDWS1.

[0045] Figure 7 This is an analysis of the nematicidal activity results of fermentation filtrates of Bacillus saffron GDWS1 at different initial pH values.

[0046] Figure 8 This is an analysis of the nematicidal activity of the fermentation filtrates of Bacillus saffron GDWS1 at different rotation speeds.

[0047] Figure 9 This is an analysis of the nematicidal activity results of fermentation filtrates of Bacillus saffron GDWS1 with different liquid volumes.

[0048] Figure 10This is a diagram showing the effect of Bacillus saffron GDWS1 in preventing and controlling pine wood nematode disease; among them: a: CK1, first inoculate with pine wood nematode suspension and then with sterile water; b: CK2, no inoculation with pine wood nematodes and then with sterile water; c: first inoculate with pine wood nematode suspension and then apply GDWS1 fermentation liquid. DETAILED DESCRIPTION

[0049] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0050] The culture medium formulas involved in the examples are as follows:

[0051] LB medium: Weigh 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of NaCl, add 1000 mL of water and stir to mix. Then add 15.0 g of agar (LB liquid medium does not contain agar), dissolve thoroughly, and dispense into 250 mL Erlenmeyer flasks. Autoclave at 121°C for 15 min and dry for later use.

[0052] NB medium: 10.0 g of peptone, 3.0 g of beef extract, and 5.0 g of NaCl were dissolved in 1000 mL of ultrapure water by heating, divided into 250 mL Erlenmeyer flasks, and sterilized by autoclave at 121°C for 15 min. The mixture was then dried for later use.

[0053] NA medium: Weigh 10.0 g of peptone, 3.0 g of beef extract, 5.0 g of NaCl, and 15.0 g of agar, dissolve in distilled water, and dilute to 1000 mL. Pour the mixture into 500 mL conical flasks, autoclave at 121°C for 20 min, and set aside.

[0054] KB medium: Weigh 20.0 g of peptone, 10.0 g of glycerol, 1.5 g of K2HPO4, and 1.5 g of MgSO4·7H2O, add 1000 mL of water, stir and mix thoroughly, and after fully dissolving, divide the mixture into 250 mL Erlenmeyer flasks and sterilize them by autoclave at 121°C for 15 min. Dry and set aside.

[0055] YT medium: Weigh 10.0 g of peptone, 5.0 g of yeast extract powder, 1.0 g of glucose, and 5.0 g of NaCl, add 1000 mL of water, stir and mix thoroughly, dissolve thoroughly, dispense into 250 mL Erlenmeyer flasks, autoclave at 121°C for 15 min, and dry for later use.

[0056] SOB medium: Weigh 20.0 g of peptone, 5.0 g of yeast extract powder, 0.5 g of NaCl, 0.18 g of KCl, and 0.95 g of MgCl2·H2O, add 1000 mL of water, stir and mix thoroughly. Dissolve thoroughly and dispense into 250 mL Erlenmeyer flasks. Autoclave at 121°C for 15 min and dry for later use.

[0057] BYP medium: Weigh 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast extract powder, 5.0 g of glucose, and 5.0 g of NaCl, add 1000 mL of water, stir and mix thoroughly, dissolve thoroughly, dispense into 250 mL Erlenmeyer flasks, autoclave at 121°C for 15 min, and dry for later use.

[0058] Example 1 Isolation of strain GDWS1 and its activity against pine wood nematodes

[0059] 1. Isolation and Purification of Strain

[0060] (1) Resistant Pinus massoniana branches were collected from Fengkai County, Zhaoqing City, Guangdong Province. Sterile water was used to rinse the surface of the branches. After absorbing the water with sterile filter paper, the branches were cut into 2 cm segments and surface disinfected. The specific steps were as follows: first, soak them in 75% alcohol by volume for 30 seconds, rinse them once with sterile water, then disinfect them with 10% NaClO solution for 5 minutes, and rinse them four times with sterile water. The last rinse solution was applied to a NA plate to test whether the surface disinfection was thorough. If no colonies were generated on the culture dish, it was confirmed that the surface disinfection was thorough. Otherwise, re-isolation was required.

[0061] (2) Cut the branch material that has been surface disinfected as described above into small pieces under sterile conditions, place it in a sterile mortar, add sterilized quartz sand and grind it, then place it in a sterilized centrifuge tube filled with 10 mL of sterile water, and culture it on a shaker at 28°C and 180 r / min for 1 hour to form a uniform plant tissue solution. Dilute the plant tissue solution with sterile water to different concentration gradients, and take 100 μL of the dilution solution with different concentration gradients, spread it on a NA plate, and culture it at 28°C for 1-2 days. Observe the growth of the colony, and then pick a single colony based on its morphological characteristics and inoculate it on an LB plate for purification to obtain a single strain. Bacteria were preserved in a 30% glycerol suspension at -80°C.

[0062] 2. Preparation of Fermentation Filtrate

[0063] (1) The strain purified in step 1 was inoculated into 50 mL of LB liquid medium and cultured on a shaker at 28°C and 180 rpm for 8 h until the OD value of the bacterial solution reached 0.5 to obtain a seed solution;

[0064] (2) inoculating the seed solution obtained in step (1) into a flask containing fresh LB liquid medium at an inoculum size of 2% by volume and a liquid volume of 100 / 250 mL, and culturing the mixture on a shaker at 28° C. and 180 rpm for 2 days in the absence of light to obtain a fermentation broth;

[0065] (3) The fermentation broth obtained in step (2) was centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was taken and filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0066] 3. Antagonistic effect of the fermentation filtrate of the strain on pine wood nematodes

[0067] The nematicidal activity of the selected bacteria was determined by the immersion method. The specific method is as follows:

[0068] (1) Add 250 μL of sterile fermentation filtrate and 250 μL of pine wood nematode suspension (containing about 50 pine wood nematodes) to a 48-well plate, shake gently to mix, and place in a constant temperature incubator at 28°C. Observe the survival of the nematodes every 12 hours using an inverted microscope and calculate the corrected mortality rate.

[0069] (2) After 24 h of treatment, add an equal volume of sterile water to each well and mix well. Let it stand for 5-10 min, then aspirate an equal volume of supernatant. Repeat this three times to allow nematode recovery.

[0070] (3) After 24 hours of recovery, observe the nematodes. When the nematodes are rigid and do not respond to stimulation with the dissecting needle, the nematodes are considered dead. An equal amount of uninoculated LB liquid medium is used as a control. Each treatment is replicated three times. The mortality and corrected mortality of pine wood nematodes are calculated according to the following formula:

[0071] Mortality (%) = (number of dead nematodes / total number of nematodes) × 100%;

[0072] Corrected mortality rate (%) = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%;

[0073] After comparative screening, a strain with high lethal activity against pine wood nematodes was finally obtained, numbered GDWS1. The corrected mortality rate of its sterile fermentation filtrate against pine wood nematodes can reach 90.67%, and subsequent identification work is being carried out on it.

[0074] Example 2 Identification of strain GDWS1

[0075] 1. Morphological identification

[0076] The experimental methods in the Manual of Identification of Common Bacteria were referred to, and the colony morphology, Gram staining characteristics, and optical microscopy morphology of strain GDWS1 on LB solid medium were recorded.

[0077] 2. Physiological and biochemical identification

[0078] Referring to the physiological and biochemical indicators in the Manual of Identification of Common Bacteria Systems, the physiological and biochemical indicators such as catalase, starch hydrolysis, carbohydrate acid production, and citrate were measured for each strain.

[0079] 3.16S rDNA sequence analysis

[0080] Genomic DNA from strain GDWS1 was extracted and used as a template for 16S rDNA amplification using universal primers (upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The amplified product was sent to Guangzhou Aiki Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of strain GDWS1 was compared with known sequences in the GenBank database using Blast analysis to analyze homology.

[0081] Morphological identification results showed that strain GDWS1 grew best on LB solid medium. The colonies of strain GDWS1 were white or light yellow, round, opaque, with irregular edges, smooth and concave surface, and high viscosity ( Figure 1 ), Gram stain positive ( Figure 2 ).

[0082] The results of physiological and biochemical identification showed that strain GDWS1 was positive in the catalase test, carbohydrate acid production test, citrate test, casein hydrolysis test, and VP test, but negative in the starch hydrolysis test and phosphatidylcholine test. In the glucose oxidation and fermentation test, only the open tube produced acid and turned yellow, indicating that it was an oxidative type.

[0083] Using the genomic DNA of strain GDWS1 as a template, after PCR amplification, the isolated PCR product showed a fluorescent band of approximately 1000 bp by electrophoresis on a 1.5% agarose gel. After sequencing, the 16S rDNA sequence was determined as shown in SEQ ID No. 1.

[0084]

[0085] After Blast alignment, 1000 similarity calculations were performed using the neighbor-joining method using MEGA11 software to construct a phylogenetic tree. The strain was clustered in the same branch with the Bacillus safensis strain HNS-S11 with accession number PP087017.1 in GenBank, and the homology was 99.89% ( Figure 3 ).

[0086] Therefore, combined with the morphological characteristics, physiological and biochemical characteristics and 16S rDNA identification results of GDWS1, the strain GDWS1 isolated by the present invention with high lethal activity against pine wood nematodes is Bacillus safensis, named Bacillus safensis GDWS1, and the strain was deposited in the Guangdong Provincial Microbial Culture Collection on May 7, 2025, abbreviated as GDWS1, and the storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the storage number is GDMCC No: 66264.

[0087] Example 3 Effect of culture conditions on the production of active substances by strain GDWS1

[0088] 1. Culture medium selection

[0089] (1) The seed liquid of strain GDWS1 was inoculated into 100 / 250 mL of LB, NB, KB, YT, SOB, and BYP liquid culture media at a volume percentage of 2%, and cultured on a shaker at 28°C and 180 r / min in the absence of light for 48 h to obtain fermentation broth.

[0090] (2) The fermentation broth obtained in step (1) was placed in a 50 mL centrifuge tube, centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0091] (3) Referring to Example 1, the immersion method was used to determine the corrected mortality of pine wood nematodes by the sterile fermentation filtrate.

[0092] like Figure 4 As shown in the results, strain GDWS1 was able to grow and produce nematicidal substances in all six liquid culture media. However, the nematicidal activities of the sterile fermentation filtrates obtained from different culture media were different. Among them, the nematicidal activity of strain GDWS1 after culture in LB liquid medium was the highest, which was better than that of the other five liquid culture media.

[0093] 2. Selection of cultivation time

[0094] The seed liquid of strain GDWS1 was inoculated into LB liquid culture medium at an inoculum rate of 2% by volume and cultured on a shaker at 28°C and 180 r / min in the absence of light for 1 day, 2 days, 3 days, 5 days or 7 days to obtain fermentation liquid; sterile fermentation filtrate was prepared according to step 1 and its corrected mortality rate against pine wood nematodes was determined.

[0095] like Figure 5 As shown in the figure, with the increase of culture time, the nematicidal active substances of strain GDWS1 continued to accumulate, and a higher concentration of nematicidal active substances was produced on the second day of culture, with the corrected mortality rate of pine wood nematodes reaching 90.71%.

[0096] 3. Selection of culture temperature

[0097] The seed liquid of strain GDWS1 was inoculated into LB liquid culture medium at an inoculum rate of 2% by volume and cultured on a shaker at different temperatures (20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C or 36°C) and in the absence of light at 180 r / min for 2 days to obtain fermentation liquid; the sterile fermentation filtrate was prepared according to step 1 and its corrected mortality rate for pine wood nematodes was determined.

[0098] like Figure 6 As shown, the filtrate of the sterile fermentation of strain GDWS1 at 28°C had the best activity in killing pine wood nematodes, with a corrected mortality rate of 91.70% against pine wood nematodes.

[0099] 4. Selection of starting pH

[0100] The seed liquid of strain GDWS1 was inoculated into LB liquid culture medium with different pH values (5, 6, 7, 8, 9 or 10) at an inoculum rate of 2% by volume, and cultured on a shaker at 28°C and 180 r / min in the absence of light for 2 days to obtain fermentation liquid; sterile fermentation filtrate was prepared according to step 1 and its corrected mortality rate for pine wood nematodes was determined.

[0101] like Figure 7 As shown, when the initial pH was 7.0, the sterile fermentation filtrate of strain GDWS1 had the best activity in killing pine wood nematodes, with a corrected mortality rate of 92.40% against pine wood nematodes.

[0102] 5. Selection of different speeds

[0103] (1) The seed liquid of strain GDWS1 was inoculated into LB liquid culture medium with a pH of 7.0 at an inoculum rate of 2% by volume, and cultured on a shaker at 28°C in the absence of light at different speeds (100 r / min, 120 r / min, 140 r / min, 160 r / min, 180 r / min, 200 r / min, 240 r / min or 260 r / min) for 2 days to obtain a fermentation liquid; referring to step 1, a sterile fermentation filtrate was prepared and its corrected mortality rate against pine wood nematodes was determined.

[0104] like Figure 8 As shown, when the rotation speed is 200 rpm, the sterile fermentation filtrate of strain GDWS1 has the best activity in killing pine wood nematodes, with a corrected mortality rate of 93.50% against pine wood nematodes.

[0105] 6. Different liquid volume options

[0106] Use Erlenmeyer flasks to prepare liquid culture media of different specifications: 50 / 250mL, 100 / 250mL, 150 / 250mL, and 200 / 250mL. According to the optimal culture conditions screened out above, the seed liquid of strain GDWS1 was inoculated into the Erlenmeyer flasks of the above specifications of LB liquid culture medium (pH = 7) at an inoculum rate of 2% by volume. The culture was shaken on a shaker at 28°C and 200r / min for 2d without light to obtain fermentation liquid; refer to step 1 to prepare sterile fermentation filtrate and determine its corrected mortality rate for pine wood nematodes.

[0107] like Figure 9 As shown, when the liquid volume was 100 / 250 mL, the sterile fermentation filtrate of strain GDWS1 had the best activity in killing pine wood nematodes, with a corrected mortality rate of 93.60% against pine wood nematodes.

[0108] Example 4 Effect of strain GDWS1 on preventing and treating pine wood nematode disease

[0109] 1. Preparation of GDWS1 fermentation broth: The seed liquid of strain GDWS1 was inoculated into LB liquid culture medium at an inoculum volume percentage of 2%, and cultured on a shaker at 200 rpm at 28°C in the absence of light for 2 days to obtain GDWS1 fermentation broth.

[0110] 2. Divide 90 two-year-old Masson pine seedlings with the same growth potential and a height of 35 to 40 cm into three groups: CK1 group, CK2 group, and GDWS1 group, with 30 plants in each group.

[0111] 3. Inoculation method of young shoot epidermis scratching (Zhang Jiawen, 2018): Use a sterile surgical blade to make two longitudinal wounds about 0.5-1mm deep and 1.5cm long at the part where the needles were pulled out, about 3-5cm away from the top of the Masson pine seedling. Use sealing film to stick to the trunk at this scratched part to form a funnel shape, and inject pine wood nematode suspension into it. Regularly add sterile water to the funnel, bag it to keep it moist for 3 days to maintain the activity of the nematodes, and culture it in a greenhouse environment. Manage it according to the conventional water and fertilizer management methods for Masson pine.

[0112] 4. CK1 group: First inoculated with a pine nematode suspension and then with sterile water. 3000 pine nematodes were inoculated with 1 mL of pine nematode suspension per plant, and 30 mL of sterile water per plant. CK2 group: No pine nematodes were inoculated, then with sterile water. 30 mL of sterile water per plant. GDWS1 group: First inoculated with a pine nematode suspension and then with GDWS1 fermentation liquid. 3000 pine nematodes were inoculated with 1 mL of pine nematode suspension per plant, and 30 mL of GDWS1 fermentation liquid per plant. Seven days after inoculation with pine nematodes, the CK1 and CK2 groups were each inoculated with 30 mL of sterile water. The GDWS1 group was inoculated with 30 mL of GDWS1 fermentation liquid.

[0113] GDWS1 fermentation liquid was applied by root irrigation method, and the incidence of nematode inoculation was recorded 63 days later. Figure 10 Statistics on the incidence rate, disease grade, disease index, and control efficacy showed that after applying GDWS1 fermentation liquid, the disease index was 38.33, the incidence rate was 43.33%, and the control efficacy was 45.88%. The results are shown in Table 1.

[0114] Incidence rate (%) = number of diseased plants / total number of inoculated plants × 100%;

[0115] Disease index = ∑(number of plants at each disease level × representative value of disease level) / (total number of plants × representative value of the highest disease level)] × 100;

[0116] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0117] Table 1 Analysis of the effect of Bacillus saffron GDWS1 on the prevention and treatment of pine wood nematode disease

[0118]

[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Bacillus saffron, characterized in that: The name is Bacillus safensis GDWS1, and it was deposited on May 7, 2025 in the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 66264.

2. A biological agent, characterized in that: The biological preparation comprises at least one of the Bacillus sp. GDWS1, the fermentation broth of Bacillus sp. GDWS1, the supernatant of the fermentation broth of Bacillus sp. GDWS1 and the fermentation filtrate of Bacillus sp. GDWS1 as claimed in claim 1.

3. The biological agent according to claim 2, characterized in that: The fermentation liquid of Bacillus sabdariffa GDWS1 is prepared by liquid fermentation of the Bacillus sabdariffa GDWS1 described in claim 1.

4. The biological agent according to claim 3, characterized in that: The seed liquid of Bacillus sabdariffa GDWS1 was inoculated into LB liquid culture medium for fermentation to obtain a fermentation liquid.

5. The biological agent according to claim 4, characterized in that: The inoculation amount of the seed solution is 1-2%; The fermentation culture conditions are 26-32° C., 180-200 r / min shaking culture for more than 2 days under no light conditions; The pH of the LB liquid culture medium is 6-8.

6. The biological agent according to claim 4, characterized in that: The seed liquid is obtained by first activating Bacillus sabdariffa GDWS1 and then picking a single colony to culture in LB liquid culture medium.

7. The biological agent according to claim 6, characterized in that: The culture conditions are 28-30° C. and 180-200 r / min shaking culture for 8-15 hours.

8. The biological agent according to any one of claims 2 to 7, characterized in that: The supernatant of the fermentation broth of Bacillus sabdariffa GDWS1 is prepared by centrifuging the fermentation broth of Bacillus sabdariffa GDWS1; The fermentation filtrate of Bacillus sabdariffa GDWS1 is prepared by filtering the supernatant of the fermentation broth of Bacillus sabdariffa GDWS1.

9. The biological agent according to claim 8, characterized in that: The centrifugal conditions are 2-8°C, 8000-10000 rpm, and 8-10 min; The filtration is performed using a microporous filter membrane with a pore size of 0.22 μm.

10. Use of the Bacillus sabdariffa according to claim 1 or the biological agent according to any one of claims 2 to 9, characterized in that: One or more combinations of the following applications: (1) Application in the prevention and control of pine wood nematode disease; (2) Application in the preparation of preparations for the prevention and control of pine wood nematode disease; (3) Application in killing pine wood nematodes; (4) Application in the preparation of preparations for killing pine wood nematodes.

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