A strain of Bacillus belye GSSN and its application in the control of pine wilt disease

Abstract

This invention discloses a strain of Bacillus velezensis GSSN and its application in the control of pine wilt disease, belonging to the field of biotechnology. This invention discloses a Bacillus velezensis GSSN, which was deposited on May 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24849. It also discloses the application of aseptic fermentation broth of Bacillus velezensis GSSN and microbial agents containing Bacillus velezensis GSSN and / or its metabolites in the control of pine wilt disease by inhibiting and killing pine wilt disease and antagonizing pine wilt-associated fungi. The Bacillus velezensis GSSN and its metabolites disclosed in this invention have significant antagonistic effects against pine wilt disease and its associated fungi, providing a new type of biocontrol bacteria for the control of pine wilt disease and having important significance for the control of plant nematodes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Bacillus belye GSSN and its application in the control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a devastating disease of pine trees caused by the pine wood nematode (Bursaphelenchus xylophilus (Steiner & Bubrer) Nickle). It is a complex disease system with a variety of contributing factors, including the pine nematode, the host pine tree, vector insects, fungi, bacteria, human activities, and the environment. Pine wilt disease spreads rapidly, with early-stage infection sites often hidden and exhibiting latent infection, making it extremely difficult to control and eradicate to date. Since its discovery in Nanjing, China in 1982, pine wilt disease has spread to 731 counties in 19 provinces, causing the death of over 1 billion pine trees and resulting in direct and indirect economic and ecological losses amounting to hundreds of billions of yuan (National Forestry and Grassland Administration Announcement No. 6, 2022).

[0003] A crucial fungi-eating stage is present in the life cycle of the pine wood nematode. During this stage, the nematode sustains its growth and population reproduction by consuming various fungi. Currently, many fungi are known to promote the growth and reproduction of pine wood nematodes, while a small number are detrimental. Studies have shown that co-inoculating the symbiotic fungus *Sporothrix* sp. 1 with DAA extracted from the xylem into nematode-infested wood segments can induce the nematode to produce a large number of offspring with a significant female-to-male ratio and rapid development. In addition, the pine wood nematode can also produce ascarosides, substances that promote fungal growth and sporulation, thereby increasing the nematode population, raising the number of vector insects carrying the nematode, and exacerbating disease occurrence.

[0004] Currently, China's approach to pine wilt disease primarily focuses on prevention, addressing both the pathogen and its vectors through integrated physical, chemical, and biological control methods. However, none of these methods have completely controlled or eliminated the affected areas. In recent years, with increasing environmental awareness, biological control has gained growing importance. Currently, biological control of pine wilt disease can be broadly categorized as follows:

[0005] (1) Targeting vector insects: The pine sawyer beetle is the main vector of pine wood nematode. Control of the pine sawyer beetle involves both releasing pheromones to attract and kill it, and releasing its natural enemies to capture and kill it. However, a current problem is that releasing pheromones cannot account for the remaining number of beetles in the forest after control. Currently, the use of pathogenic fungi and insect natural enemies for pine wood nematode control is more common. Pathogenic fungi include Metarhizium anisopliae and Beauveria bassiana. Parasitic natural enemies mainly include Dastarcus helophoroides, Scleroderma guani, and Scleroderma sichuanensis. However, these methods are greatly affected by biotic and abiotic factors in the forest, and the control effect is not stable or certain.

[0006] (2) Targeting pathogenic nematodes: Currently, the most widely used biological fungicides for controlling pine wilt disease are trunk injection agents such as abamectin and emamectin benzoate, which can provide protection for 2-3 years with a single injection. However, these trunk injection agents are expensive, and the entire injection process is done manually, so the cost remains high, and they are usually only used in important areas or on ancient and famous trees. At present, China has also begun to carry out resistance breeding of pine trees against pine wilt disease, but this is a long-term project that cannot be achieved in the short term.

[0007] Therefore, researchers in many countries are working hard to find new ways to control plant nematodes. Developing highly effective and low-toxicity biological control agents is now urgent, and utilizing beneficial microorganisms to control nematodes has become an important approach to this development.

[0008] Bacillus spp. are rod-shaped, spore-producing, Gram-positive bacilli that are either aerobic or facultative anaerobic. They are widely found not only in soil, deep sea, intestines, and plants, but also in extreme environments such as hot springs and Antarctica. Bacillus can produce spores even under adverse environmental conditions, and these spores can be converted into powders without killing the bacteria, a significant advantage over other biocontrol bacteria. Since their discovery, Bacillus has been the subject of extensive research. Bacillus can produce a variety of secondary metabolites, and their strong activity against various plant pathogens (bacteria, fungi, nematodes, viruses, etc.) makes them important research subjects. Furthermore, Bacillus can produce various proteinaceous substances, such as chitinases and extracellular proteases. These substances can dissolve the fungal cell by acting on corresponding substrates on the fungal cell wall, causing cell dissociation and ablation, thereby achieving the purpose of disease control.

[0009] Bacillus velezensis is an aerobic, Gram-positive bacterium. As a novel biocontrol bacillus, Bacillus velezensis is resistant to high temperatures and ultraviolet radiation and produces stable substances. It has been reported that strains of this species can inhibit the growth of many pathogens, including bacteria, fungi, and nematodes. Studies have found that B. velezensis 504 has a significant antagonistic effect against the plant pathogen Xanthomonas. Currently, in vivo and in vitro experiments have confirmed that the antagonistic activity of B. velezensis FJAT-46737 is related to the secretion of lipopeptide substances, and it has good biocontrol potential against bacterial wilt. In one crop, B. velezensis can be used simultaneously to control pests and fungal plant diseases. Furthermore, B. velezensis also has a good antagonistic effect against nematodes. It has been reported that the lipopeptides of B. velezensis Bv-25 have strong antagonistic activity against root-knot nematodes (Meloidogyne spp.), and that its volatile organic compounds have a high repellent and paralyzing effect on root-knot nematodes. However, there are currently no records of research on the use of Bacillus belye against pine wilt disease and its antagonistic associated fungi for the control of pine wilt disease. Summary of the Invention

[0010] The purpose of this invention is to provide a strain of Bacillus belye GSSN and its application in the control of pine wilt disease, thus providing a new type of biocontrol bacteria for the prevention and control of pine wilt disease.

[0011] To achieve the above objectives, the present invention provides the following solution:

[0012] This invention provides a Bacillus belye GSSN, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24849, located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, on May 9, 2022.

[0013] This strain was isolated from the body surface of the bark beetle of Qinghai spruce. Its fermentation products have a strong antagonistic effect on pine wilt nematode and its associated fungi, thus it can effectively prevent and control pine wilt disease caused by pine wilt nematode.

[0014] The present invention also provides a plant disease control agent, characterized in that it comprises the Bacillus belye GSSN, its fermentation broth and / or its metabolites.

[0015] The fermentation culture conditions for the Bacillus belyssus GSSN are as follows:

[0016] (1) Culture medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000ml deionized water, sterilized at 121℃ for 30min, pH 7.2~7.4.

[0017] (2) Process conditions: pH 7.2-7.4, inoculum amount 2% (v / v), temperature 30℃, rotation speed 180-200r / min, and constant temperature and dark shaking culture for 72h.

[0018] The fermentation broth obtained by the above fermentation process was centrifuged at 10,000 r / min for 10 min at 4℃, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain sterile fermentation broth.

[0019] The present invention also provides a method for the prevention and control of plant diseases, wherein the plant disease control agent is applied to plants to suppress pathogenic nematodes and antagonize the growth of fungi associated with pathogenic nematodes.

[0020] Furthermore, the pathogenic nematode includes the pine wood nematode.

[0021] Furthermore, the plant disease mentioned is pine wilt disease.

[0022] Furthermore, the pathogenic nematode-associated fungi include Cytospora sp.1, Diplodia sapinea, Graphilbum xianjuensis, Ophiostoma album, Ophiostoma ips, Ophiostoma massoniana, Ophiostoma taizhouense, Pestalotiopsis sp.1, Phialocephala sp.1, Pseudocosmospora sp.1, Sporothrix macroconidia, Sporothrix zhejiangensis, and Xenoacremonium sp.1.

[0023] The present invention also provides the application of the Bacillus vesicularis GSSN or the plant disease control agent in the control of pine wilt disease.

[0024] The present invention also provides the application of the Bacillus vesiculosus GSSN or the plant disease control agent in the suppression and killing of pine wilt nematodes.

[0025] The present invention also provides the application of the Bacillus vesiculosus GSSN or the plant disease control agent in antagonizing fungi associated with pine wilt nematodes.

[0026] Furthermore, the fungi associated with the pine wood nematode include Cytospora sp.1, Diplodia sapinea, Graphilbum xianjuensis, Ophiostoma album, Ophiostoma ips, Ophiostoma massoniana, Ophiostoma taizhouense, Pestalotiopsis sp.1, Phialocephala sp.1, Pseudocosmospora sp.1, Sporothrix macroconidia, Sporothrix zhejiangensis, and Xenoacremonium sp.1.

[0027] The present invention discloses the following technical effects:

[0028] The *Bacillus belyceae* GSSN strain provided by this invention exhibits significant antagonistic effects against pine wilt disease, is environmentally friendly and pollution-free, and is of great significance for the control of plant nematodes. This strain also shows significant antagonistic effects against pine wilt disease-associated fungi, is environmentally friendly and pollution-free, and is important for controlling pine wilt disease by inhibiting the growth of associated fungi and controlling the pine wilt disease population.

[0029] The Bacillus GSSN culture provided by this invention is simple and easy to preserve, making it suitable for industrial production and showing good prospects for development and application. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The morphological characteristics of strain GSSN are shown in Figure 1, where a represents the colony morphology of strain GSSN and b represents the cell morphology of strain GSSN.

[0032] Figure 2 Phylogenetic analysis based on 16S rDNA gene sequences;

[0033] Figure 3 Phylogenetic analysis based on the gyrA gene sequence;

[0034] Figure 4 The morphological characteristics of pine wood nematodes after treatment with the fermentation broth of strain GSSN are shown in the figures. a is the control, b is the exudation of body fluid in nematodes, c is the breakage of nematodes, and d is the dissolution of substances inside the body cavity of nematodes.

[0035] Figure 5 The study investigated the antagonistic effect of sterile fermentation broth from strain GSSN on fungi associated with pine wood nematode, where A was Cytosporasp., B was D. sapinea, C was G. xianjuensis, D was O. album, E was O. ips, F was O. massoniana, G was O. taizhouense, H was Pestalotiopsis sp.1, I was Phialocephala sp., J was Pseudocosmosporasp., K was S. macroconidia, L was S. zhejiangensis, and M was Xenoacremonium sp.

[0036] Figure 6 The image shows the antagonistic effect of lipopeptides from strain GSSN on fungi associated with pine wood nematode. A-1 to k-1 are, in order: Bo. Cinerea, Cytospora sp.1, D. sapinea, G. xianjuensis, O. album, O. ips, O. taizhouense, Pestalotiopsis sp.1, Pseudocosmospora sp.1, S. macroconidia, and Xenoacremonium sp.1.

[0037] Figure 7 The image shows the antagonistic effect of GSSN protein substances on fungi associated with pine wood nematode. A-2 to k-2 are Bo. Cinerea, Cytospora sp.1, D. sapinea, G. xianjuensis, O. album, O. ips, O. taizhouense, Pestalotiopsis sp.1, Pseudocosmospora sp.1, S. macroconidia, and Xenoacremonium sp.1, respectively. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1: Isolation and Identification of Bacillus belyssus GSSN

[0044] 1. Isolation of strains

[0045] The bacterial strain GSSN was isolated from the body surface of the bark beetle that invades Qinghai spruce. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24849, located at No. 3, No. 1 Beichen Road, Chaoyang District, Beijing.

[0046] 2. Identification of strains

[0047] Morphological characteristics of GSSN: When strain GSSN was cultured on LB solid medium, the colonies were round with irregular edges, milky white in color, wrinkled surface, raised edges, and sunken center, and were opaque. The cells were rod-shaped. Figure 1 ).

[0048] Physiological and biochemical identification: Bacterial GSSN was transferred to IF-B inoculum using a sterilized cotton swab, and the turbidity was adjusted to 90%–98%. It was then transferred to a GenIII identification plate and incubated at 30°C for 24 hours. Identification was performed using the Biolog microbial identification system. The results showed that strain GSSN could utilize 54 carbon sources and was sensitive to 11 chemical substances (Table 1).

[0049] Table 1. Physiological and biochemical indicators of strain GSSN

[0050]

[0051]

[0052] Note: "+" indicates that it can be used, "-" indicates that it cannot be used, and "B" indicates a weak reaction.

[0053] Based on the morphological and physiological-biochemical characteristics of the strain, and with reference to the "Handbook for Identification of Common Bacteria", strain GSSN was determined to belong to the genus Bacillus.

[0054] Identification of GSSN strains using molecular biological methods:

[0055] The 16S rDNA and gyr A sequences were determined to be 1471 bp and 947 bp, respectively, as shown in SEQ ID No: 1 and No: 2. Both sequences were submitted to the GenBank database, with accession numbers ON797328 and ON033177, respectively.

[0056] The 16S rDNA sequences were aligned using BLAST, and highly homologous strain sequences were found in GenBank. The sequences with the highest similarity to the GSSNs of *Bacillus amyloliquefaciens*, *Bacillus velezensis*, and *Bacillus siamensis* were found, all with homology exceeding 99%. Using MEGA 7.0 software, 1000 similarity replicates were performed using the neighbor-joining method, and a phylogenetic tree was constructed. Figure 2 The results showed that strain GSSN also clustered with these three bacteria, with a node support rate higher than 95%. We further constructed a phylogenetic tree of gyrA and found that strain GSSN was associated with Bacillus velezensis (B. velezensis FZB24). T ) clustered on one branch, with a node support rate of 100 ( Figure 3Based on comprehensive morphological characteristics, physiological and biochemical index detection, and phylogenetic analysis, we identified the strain GSSN as *Bacillus velezensis*. This strain has been deposited at the China General Microbiological Culture Collection Center on May 9, 2022, at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing.

[0057] Example 2: Antagonistic effect of Bacillus belysin GSSN sterile fermentation broth on pine wood nematode.

[0058] Preparation of sterile fermentation broth: The strain was inoculated into 100 mL / 250 mL LB liquid medium and cultured at 30 °C and 180 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into 100 mL / 250 mL LB liquid medium at a 2% inoculation rate and cultured at 30 °C and 200 rpm for 48 h. The fermentation broth was then centrifuged at 4 °C and 10,000 rpm for 10 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the sterile fermentation broth of *Bacillus belyssiensis* GSSN was obtained and stored at 4 °C for later use.

[0059] Preparation of nematode solution: Botrytis cinerea, preserved in our laboratory, was inoculated onto PDA plates and cultured at 25°C for 7 days. Then, pine wood nematodes, preserved in our laboratory, were inoculated onto plates contaminated with Botrytis cinerea and cultured in the dark at 25°C for 7 days. The pine wood nematodes were then collected using the Bellman funnel method, and surface sterilized with 0.1% sodium hypochlorite solution. They were subsequently counted under a microscope to obtain a nematode solution (approximately 160 nematodes per 20 μL).

[0060] Assay method: The sterile fermentation broth prepared in Example 2 and the nematode broth were tested at ratios of 1:1 and 1:3, respectively (treatment group). The sterile fermentation broth was replaced with LB liquid medium and sterile water at the same ratio as the control group. The mixture was placed in a dark constant temperature incubator at 25°C. The mortality rate of nematodes was calculated at 12h, 24h and 36h, respectively. Each treatment was repeated 3 times.

[0061] Mortality determination of pine wood nematodes: Take 20 μl of nematode suspension onto a glass slide, add the same volume of sterile water, and incubate at room temperature for 30 minutes. Then observe under a 10x objective lens. If the nematodes are motionless and exhibit a large "C" or "J" shape, and gentle tapping of their bodies does not elicit any response, the nematodes are considered dead. If the nematodes move in an "S" shape, curled, or spiral motion, they are considered alive. Calculate the mortality rate and corrected mortality rate of pine wood nematodes using the following formula:

[0062] Mortality rate (%) = (Number of dead nematodes / Total number of nematodes) × 100%

[0063] Corrected mortality (%) = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%

[0064] The results of the antagonistic activities of the sterile fermentation broth at different fermentation times against Bursaphelenchus xylophilus are shown in Table 2 - 3.

[0065] Table 2 Antagonistic activities of the sterile fermentation broth at different fermentation times against Bursaphelenchus xylophilus (1:1)

[0066]

[0067] Note: The data in the table are the average values of 3 replicates. Values with the same letter in the same column indicate no significant difference at the P < 0.05 level (DMRT method). Judgment of activity level; "-" indicates NA ≤ 10%; "+" indicates 10% < NA < 30%; "++" indicates 30% < NA < 50%; "+++" indicates 50% < NA < 80%; "++++" indicates NA > 80%. NA represents the corrected mortality rate of nematodes, the same below.

[0068] Table 3 Antagonistic activities of the fermentation broth at different fermentation times against Bursaphelenchus xylophilus (1:3)

[0069]

[0070]

[0071] The antagonistic activities of the sterile fermentation broth of bacterium GSSN at different fermentation times against nematodes were determined by the dipping method. The results showed that the sterile fermentation broth had a strong antagonistic effect on Bursaphelenchus xylophilus. The mortality rates of the control groups LB and sterile water were both below 10%, and there was no significant difference (Table 2, 3). In the 1:1 test, the antagonistic activities of the sterile fermentation broth at 48 h were significantly higher than those of other sterile fermentation broths at the same time at 12 h and 24 h. At 36 h, all the tested nematodes died. In the 1:3 test, similar test results were also obtained, but the corrected mortality rates of Bursaphelenchus xylophilus in the sterile fermentation broths at 24 h, 36 h, and 48 h were all lower than those in the corresponding 1:1 test, and the tested nematodes in the sterile fermentation broth at 24 h did not all die until 48 h. This indicates that the concentration of antibacterial substances in the fermentation broth also has a certain effect on the antagonistic efficiency. In the antagonistic tests of the sterile fermentation broths at three different fermentation times, phenomena such as the ablation, fracture of the body wall of Bursaphelenchus xylophilus, and the dissolution of substances in the body cavity were all observed ( Figure 4 ).

[0072] Example 3 Antagonistic effects of the sterile fermentation broth, protein substances, and lipopeptide substances of Bacillus velezensis GSSN against the associated fungi of Bursaphelenchus xylophilus

[0073] 1. Antagonistic effect of the sterile fermentation broth of Bacillus velezensis GSSN against the associated fungi of Bursaphelenchus xylophilus

[0074] The associated fungi used in the pine wilt nematode feeding experiment in this embodiment were provided by the Forest Pathology Laboratory of the Institute of Forest Ecology, Environment and Nature Conservation, Chinese Academy of Forestry, as shown in Table 4.

[0075] Table 4. Information on associated fungi used in the pine wood nematode feeding experiment.

[0076]

[0077] Preparation of sterile fermentation broth: The strain was inoculated into 100 mL / 250 mL LB liquid medium and cultured at 30 °C and 180 rpm for 24 h to obtain seed culture. The seed culture was then inoculated into 100 mL / 250 mL LB liquid medium at a 2% inoculation rate and cultured at 30 °C and 200 rpm for 48 h. The fermentation broth was then centrifuged at 4 °C and 10,000 rpm for 10 min, and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the sterile fermentation broth of *Bacillus belyssiensis* GSSN was obtained and stored at 4 °C for later use.

[0078] PDA-treated plates were prepared by mixing the sterile fermentation broth of strain GSSN with PDA medium cooled to 50°C at a ratio of 1:5 and then pouring the mixture into 90 mm petri dishes. PDA-treated plates (treatment group) were prepared by replacing the sterile fermentation broth with the same volume of sterile water as the control group.

[0079] Culture of associated fungi: The associated fungi of pine wood nematode in Table 4 were inoculated into PDA plates, inverted and cultured in a dark incubator at 25℃ for 7 days. The associated fungi in Table 4 were prepared into mycelial cakes using a sterile punch with a diameter of 7 mm for later use.

[0080] Aseptic fermentation broth antibacterial activity assay: The mycelial growth rate method was used to analyze the antagonistic effect of the aseptic fermentation broth of strain GSSN on associated fungi. Aseptic inoculation needles were used to pick up fungal pellets and inoculate them into the center of PDA-treated plates (treatment group and control group), which were then incubated at 25℃ in the dark for 7 days. The colony diameter was measured using the cross-crossing method, and the inhibition rate was calculated. Each treatment was repeated three times. The inhibition rate was calculated using the following formula:

[0081] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100%

[0082] 2. Antagonistic effect of Bacillus belysin GSSN lipopeptides on fungi associated with pine wood nematode.

[0083] Preparation of lipopeptides: The GSSN seed culture (obtained in the sterile fermentation broth preparation step) was inoculated into LB liquid medium at a 2% inoculation rate and cultured at 30℃ and 180 rpm for 48 h. After centrifugation at 4℃ and 10000 rpm for 10 min, the bacterial cells were discarded, and the supernatant was collected. The pH of the sterile fermentation broth was adjusted to 2 with 6 mol / mL hydrochloric acid for acidification precipitation, and the mixture was allowed to stand at 4℃ for 24 h. The mixture was then centrifuged again at 4℃ and 10000 rpm for 10 min, and the supernatant was collected. The precipitate was dissolved in anhydrous methanol at a ratio of 1:10 (solid:liquid). The solution was placed on a shaker and incubated at 30℃ and 120 rpm for 2 h. Afterward, the mixture was centrifuged at 4℃ and 10000 rpm, and the supernatant and precipitate were collected separately. This step was repeated for extraction. The resulting supernatant was rotary evaporated at 40℃ until approximately 1 / 6 of the liquid remained in the flask. The solution was collected and stored at -20℃ for later use.

[0084] Preparation of conidium suspension: Pine wood nematode-associated fungi were inoculated into MEA medium (Table 4) and incubated in a 25℃ dark incubator for 10 days. On a clean bench, an appropriate amount of sterile water was added to the culture dish. The surface of the colonies was gently scraped with a sterile inoculation needle, and the mixture was allowed to stand for 10 minutes. Finally, the culture dish was gently shaken, and the mycelia were filtered out using sterile absorbent cotton to obtain the conidium suspension. The concentration of the conidium suspension was adjusted to 1×10⁻⁶ with sterile water. 6 Units / mL are available for use.

[0085] Antibacterial activity assay of lipopeptides: The antibacterial activity was determined using the agar well diffusion method. 200 μl of fungal conidial suspension was spread onto PDA medium, and after drying, 100 μl of the prepared lipopeptide was injected into each well using a 7 mm diameter sterile punch. The mixture was incubated at 25°C in the dark for 5–10 days, with daily observation and recording of antibacterial activity. The diameter of the inhibition zone was measured using the cross-hatching method. Controls were prepared by adding the same volume of sterile water and anhydrous methanol, and each treatment was repeated three times. The antagonistic effects of the lipopeptide extract from strain GSSN against the fungus associated with pine wood nematode are shown in Table 5.

[0086] Table 5. Antagonistic effects of GSSN lipopeptide extracts from strain GSSN on fungi associated with pine wood nematode.

[0087]

[0088] Note: "++++" indicates an inhibition zone diameter greater than 22 mm, indicating strong antibacterial activity; "+++" indicates an inhibition zone diameter of 17–21 mm, indicating relatively strong antibacterial activity; "++" indicates an inhibition zone diameter of 12–16 mm, indicating moderate antibacterial activity; "+" indicates an inhibition zone diameter of 7–11 mm, indicating weak antibacterial activity.

[0089] 3. Antagonistic effect of Bacillus belysin GSSN protein on fungi associated with pine wood nematode.

[0090] Preparation of protein substances: The GSSN strain seed culture was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 48 h. After centrifugation at 4℃ and 10000 rpm for 10 min, 1.1 L of fermentation supernatant was obtained. The supernatant was transferred to a 2 L beaker, and ammonium sulfate powder was slowly added in batches, not exceeding 10 g each time. Addition was repeated only after the previous addition of ammonium sulfate had completely dissolved, until the solution reached a saturation concentration of 30%. The mixture was stirred continuously with a glass rod during this process. The solution was allowed to stand at 4℃ for 24 h, and then centrifuged at 4℃ and 10000 rpm for 10 min. The supernatant and protein precipitate were collected separately. The protein precipitate was dissolved in 1×PBS solution (pH 7.4) to obtain a protein solution. The obtained supernatant was transferred to a clean beaker, and ammonium sulfate powder was added continuously until the solution reached a saturation of 60%. The solution was allowed to stand at 4℃ for 24 h, and then centrifuged at 4℃ and 10000 rpm for 10 min. The protein precipitate and supernatant were collected separately. Dissolve the protein precipitate in 1×PBS solution with pH 7.4, combine it with the protein solution obtained in the first step, and store it at -20°C for later use.

[0091] Antibacterial activity assay of protein extracts: Antibacterial activity was determined using the agar well diffusion method. 200 μl of fungal conidial suspension was spread onto PDA medium, dried, and then perforated using a 7 mm diameter sterile punch. 100 μl of the prepared protein extract was injected into each well, and the mixture was incubated at 25°C in the dark for 5–10 days. Antibacterial activity was observed and recorded daily, and the diameter of the inhibition zone was measured using the cross-sectional method. Controls were prepared by adding the same volume of sterile water and 1×PBS solution, with each treatment repeated three times. The antagonistic effects of the GSSN strain protein extract on the fungus associated with pine wood nematode are shown in Table 6.

[0092] Table 6. Antagonistic effects of bacterial GSSN protein extracts on fungi associated with pine wood nematode.

[0093]

[0094] Note: "++++" indicates an inhibition zone diameter greater than 22 mm, indicating strong antibacterial activity; "+++" indicates an inhibition zone diameter of 17–21 mm, indicating relatively strong antibacterial activity; "++" indicates an inhibition zone diameter of 12–16 mm, indicating moderate antibacterial activity; "+" indicates an inhibition zone diameter of 7–11 mm, indicating weak antibacterial activity.

[0095] The results show that:

[0096] The antifungal activity of the aseptic fermentation broth of strain GSSN against associated fungi was analyzed using the growth rate method. The results showed that the aseptic fermentation broth of strain GSSN significantly inhibited the growth of associated fungi, with inhibition rates exceeding 60%. Figure 5 The inhibitory activity against fungi Cytospora sp. 1, G. xianjuensis, O. ips, O. taizhouense, Pestalotiopsis sp. 1, and Phialocephala sp. 1 all exceeded 90%. It also exhibited strong antifungal activity against the dominant fungi O. ips and O. taizhouensis, with inhibition rates of 97.86% and 94.02%, respectively.

[0097] The antagonistic effects of lipopeptides on fungi associated with pine wood nematode were analyzed using the inhibition zone method. Results showed that lipopeptides exhibited significant antifungal activity against all tested fungi, with inhibition zones appearing, while methanol solution showed no inhibitory effect on the associated fungi (Table 5). Analysis revealed that the lipopeptide extracts showed the strongest antifungal activity against the pathogen *D. sapinea* and the dominant fungus *O. taizhouense*, with highly significant differences. The inhibition zone diameters were 23.6 mm and 25 mm, respectively, and the inhibitory activity intensity was "++++". It also showed strong inhibitory activity against the dominant fungus *O. ips*, with an inhibition zone diameter of 20.6 mm and an inhibitory activity intensity of "+++". It also showed strong inhibitory activity against the fungi *Bo. cinerea* and *Pestalotiopsis* sp. 1, which have high reproductive efficiency against pine wood nematode, with inhibition zone diameters of 21.8 mm and 18.5 mm, respectively, and the inhibitory activity intensity was "+++". Among these fungi, the inhibition efficiency against S. macroconidia was the lowest, with an inhibition zone diameter of 11.3 mm and an antibacterial activity intensity of only "++".

[0098] The antifungal activity of protein extracts against associated fungi was analyzed using the inhibition zone method. The results showed that the protein extracts inhibited all associated fungi of *Pinus wiltii* (Table 6). The strongest inhibitory effect was observed against strain *Cytospora* sp. 1, with a highly significant inhibition zone diameter of 23.1 mm and an inhibitory activity intensity of "++++". This was followed by strain *B. cinerea*, with an inhibition zone diameter of 19 mm and an activity intensity of "+++". Inhibitory effects were also observed against the dominant fungi *O. taizhouense* and *O. ips*, with inhibition zone diameters of 14.5 mm and 17.4 mm, respectively, and inhibitory activity intensities of "++" and "+++", respectively. Furthermore, significant antifungal activity was observed against other fungi, with inhibitory activity intensities all above "++".

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for controlling pine wilt disease, characterized in that, Apply plant disease control agents containing Bacillus velezensis GSSN or Bacillus velezensis GSSN and / or its sterile fermentation broth to plants to suppress pine wilt nematodes and antagonize the growth of fungi associated with pine wilt nematodes. The GSSN preservation number of the Bacillus belyssus is CGMCC No. 24849; The fungi associated with the pine wood nematode include Cytospora sp. 1, Diplodia sapinea, Graphilbumxianjuensis, Ophiostoma album, Ophiostoma ips, Ophiostoma massoniana, Ophiostomataizhouense, Pestalotiopsis sp. 1, Phialocephala sp. 1, Pseudocosmospora sp. 1, Sporothrix macroconidia, Sporothrix zhejiangensis, and Xenoacremonium sp.

1.

2. The application of a plant disease control agent containing Bacillus velezensis GSSN or Bacillus velezensis GSSN and / or its sterile fermentation broth in the control of pine wilt disease; The GSSN preservation number of the Bacillus belyssus is CGMCC No. 24849; The plant disease control agent containing Bacillus vesicularis GSSN or its sterile fermentation broth controls pine wilt disease by inhibiting and killing pine wilt nematodes and antagonizing the growth of fungi associated with pine wilt nematodes.

3. Application of a plant disease control agent containing Bacillus velezensis GSSN or Bacillus velezensis GSSN and / or its sterile fermentation broth in antagonizing fungi associated with pine wilt nematode; The GSSN preservation number of the Bacillus belyssus is CGMCC No. 24849; The fungi associated with the pine wood nematode include Cytospora sp. 1, Diplodia sapinea, Graphilbumxianjuensis, Ophiostoma album, Ophiostoma ips, Ophiostoma massoniana, Ophiostomataizhouense, Pestalotiopsis sp. 1, Phialocephala sp. 1, Pseudocosmospora sp. 1, Sporothrix macroconidia, Sporothrix zhejiangensis, and Xenoacremonium sp. 1.

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