Bacillus tequilensis R78 for preventing and treating pine wood nematode disease and application of bacillus tequilensis R78

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to bacillus tequilensis R78 for preventing and treating pine wood nematode diseases and application of the bacillus tequilensis R78. The bacillus tequilensis R78 for preventing and treating the pine wood nematode disease is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32106, and the preservation time is September 29, 2024. The bacillus tequilensis R78 can be used for preventing and treating the pine wood nematode disease. The bacillus tequilensis R78 is separated from pine trees, and fermentation liquor of the bacillus tequilensis R78 has stable and efficient pine wood nematode inhibiting and killing capacity. When OD600 is equal to 1.5, the fatality rate of fermentation liquor of the strain R78 on pine wood nematode can reach 95% or above, the disease index of pine tree infected with pine wood nematode disease can be remarkably reduced, and the prevention and treatment effect can reach 68.6%.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and more specifically, to a strain of Bacillus tekiria R78 for the prevention and control of pine wilt disease and its application. Background Technology

[0002] Pine wilt disease (PWD) is caused by the pine wood nematode (Pinus thunbergii). Bursaphelenchus xylophilus Pine wilt is a devastating forest disease caused by pine wilt nematodes, also known as pine blight or pine rot. Pine nematodes belong to the order Lyraeidae (…). Aphelenchoidea ), Slippery Blade Family ( Aphelenchoididae ), Umbrella Slippery Blade Genus ( Bursaphelenchus The nematode is slender, worm-shaped, and invisible to the naked eye. It can infect various pine species, including Scots pine, Masson pine, Chinese pine, sago palm, and black pine. After infection with pine wilt nematode, the vascular tissue of the pine tree is damaged, hindering the transport of water and nutrients, reducing transpiration, decreasing photosynthetic capacity, and reducing or even stopping resin secretion. The pine tree withers rapidly, eventually leading to the death of the entire tree. This disease is characterized by its wide host range, rapid spread, strong adaptability, and rapid infection of host plant tissues. It can cause the death of an entire pine tree in as little as 40 days after infection, and once pine trees are infected, it can easily cause large-scale mortality.

[0003] Control methods for pine wilt disease mainly include physical, chemical, and biological control. Physical control primarily involves clearing diseased and dead pine trees through methods such as felling, fumigation, and burning. However, physical control has drawbacks such as high manpower and material resources, high control costs, and the risk of spread. Chemical control mainly involves applying chemical agents through trunk injection, fumigation, and spraying to directly kill pine wilt nematodes or their vector insects. This is currently the most commonly used control method, but chemical control poses risks such as environmental pollution and the development of drug resistance, which may threaten ecological and environmental safety. Biological control, on the other hand, is a green, pollution-free, and environmentally friendly technology and is one of the important means of controlling pine wilt disease. These biocontrol microorganisms can resist pine wilt nematode infection through mechanisms such as secreting nematicidal substances, competing for nutrients and space, interfering with the physiological activities of nematodes, and activating the pine tree's own immune defense. Therefore, screening for biocontrol microorganisms with high efficacy in controlling pine wilt disease and developing and applying biocontrol microbial preparations for the prevention of pine wilt disease have attracted widespread attention.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus tektii R78 for the prevention and control of pine wilt disease and its application. The Bacillus tektii R78 and its fermentation broth have the ability to inhibit and kill pine wilt nematodes and prevent and control pine wilt disease.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: One aspect of the present invention relates to a strain of Bacillus tekirae for controlling pine wilt disease. Bacillus tequilensis R78, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32106, on September 29, 2024.

[0007] The aforementioned Bacillus tergentii ( Bacillus tequilensis R78, isolated from pine trees, is a type of Bacillus tekirae R78 and its fermentation broth. It has a stable and efficient ability to inhibit and kill pine wilt nematodes. This invention has been verified through greenhouse disease prevention experiments. Bacillus tekirae R78 can effectively reduce the disease index of pine trees infected with pine wilt nematodes without affecting the normal growth of pine trees.

[0008] The present invention relates to a microbial preparation comprising the aforementioned Bacillus tekiria R78.

[0009] The microbial preparation contains Bacillus tekiria R78, which has the function of preventing and controlling pine wilt disease. This microbial preparation also has the function of preventing and controlling pine wilt disease. Using it for the prevention and control of pine wilt disease not only has a good control effect, but also does not affect the growth of pine trees.

[0010] Furthermore, the microbial preparation includes the fermentation broth of Bacillus tekirulae R78.

[0011] This invention demonstrates that the fermentation broth of Bacillus tekirae R78 has good nematicidal ability through experiments measuring the nematicidal ability of the fermentation broth and fermentation supernatant.

[0012] This invention does not specifically limit the dosage form of the microbial preparation, and any dosage form conventional in the art can be selected. In some specific embodiments, the dosage form of the microbial preparation includes, but is not limited to, solid dosage forms or liquid dosage forms.

[0013] Furthermore, when the microbial preparation is a solid dosage form, the amount of *Bacillus tekiria* R78 contained is 4.8 × 10⁻⁶. 8 cfu·g -1 ~6.4×10 8 cfu·g -1 .

[0014] Furthermore, when the microbial preparation is a liquid preparation, the amount of *Bacillus tekiria* R78 contained is 4.8 × 10⁻⁶. 8 cfu·mL-1 ~6.4×10 8 cfu·mL -1 .

[0015] When the amount of Bacillus tektella R78 in a microbial preparation reaches a certain range, it can kill nematodes in a relatively short time and achieve a good control effect on pine wilt disease.

[0016] Furthermore, the microbial preparation further includes excipients. This invention does not specifically limit the excipients; conventional excipients in the art can be used to implement the technical solutions of this invention.

[0017] When necessary, the microbial preparations provided by this invention can also be combined with other conventional foliar fertilizers and / or pesticides in the art to promote the growth of pine trees, improve the disease resistance of pine trees, and reduce the susceptibility of pine trees to diseases.

[0018] Another aspect of the invention relates to a method for preventing and controlling pine wilt disease by applying the aforementioned Bacillus tektella R78 or the aforementioned microbial preparation to pine trees.

[0019] The method for preventing and controlling pine wilt disease described above is a biological control method that targets the pathogen of pine wilt disease and has advantages such as low cost, environmental friendliness, and good control effect.

[0020] Furthermore, the pine trees are sprayed with a fermentation broth containing the aforementioned Bacillus tekirae R78 or a medicinal solution containing the aforementioned microbial preparation.

[0021] Furthermore, the pine tree includes at least one of the following: black pine, Scots pine, red pine, larch, or Masson pine.

[0022] Another aspect of the present invention relates to the use of the aforementioned Bacillus tekiria R78 or the aforementioned microbial preparation in the prevention and control of pine wilt disease.

[0023] Another aspect of the invention relates to a growth inhibitor of pine wood nematode, comprising the aforementioned Bacillus tekirae R78 or the aforementioned microbial preparation.

[0024] The aforementioned pine wood nematode growth inhibitor has high infectivity to pine wood nematodes and can significantly inhibit their growth.

[0025] Biological sample preservation information: Bacillus tekirae ( Bacillus tequilensisR78, this strain was deposited on September 29, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32106. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Bacillus terrestris strain for preventing and controlling pine wilt disease provided by this invention ( Bacillus tequilensis R78, isolated from pine trees, has a stable and highly effective nematode-killing ability in its fermentation broth. This invention has been verified through greenhouse disease prevention experiments that Bacillus tekirae R78 can effectively reduce the disease index of pine wilt disease in pine trees without affecting the normal growth of pine trees.

[0027] (2) The microbial preparation provided by the present invention contains Bacillus tekiria R78, which has the function of preventing and controlling pine wilt disease. This microbial preparation also has the function of preventing and controlling pine wilt disease. Using it for the prevention and control of pine wilt disease not only has a good prevention and control effect, but also does not affect the growth of pine trees.

[0028] (3) The method for preventing and controlling pine wilt disease provided by the present invention belongs to biological control methods. It carries out targeted prevention and control of the pathogens of pine wilt disease and has the advantages of low cost, environmental friendliness and good control effect. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 Colony morphology of Bacillus tekiria R78 (scale bar: 1 mm); Figure 2 Methylene blue staining results of *Bacillus tegmentatus* R78 treatment of *Pinus wiltii* (A: water treatment group, B: fermentation broth treatment group of strain R78, C: fermentation broth supernatant treatment group of strain R78). Figure 3 The activity of Bacillus tekirae R78 fermentation broth and fermentation broth supernatant in killing pine wood nematodes; Figure 4 The effects of the concentration and treatment time of Bacillus tekiria R78 fermentation broth on its activity against pine wilt nematodes; Figure 5 Results of a greenhouse disease control experiment for black pine seedlings (A: pine wilt nematode treatment group; B: fermentation broth of strain R78 and pine wilt nematode treatment group; C: water treatment group; scale bar: 3 cm). Figure 6 The disease index of pine wilt disease in greenhouses using Bacillus tegmentata R78 fermentation broth; Figure 7 The effect of Bacillus tektii R78 fermentation broth on the prevention and control of pine wilt disease in greenhouses. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0032] Example 1 Identification of strain R78 (1) Morphological identification of strain R78 Strain R78 was inoculated onto LB solid medium and cultured at 30℃ for 24 hours. The colonies were milky yellow, nearly round, with irregular edges, and a rough, wrinkled surface. Figure 1 ).

[0033] (2) Sequencing analysis of 16S rRNA gene of strain R78 Genomic DNA was extracted from strain R78. PCR amplification was performed on the extracted genomic DNA using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The amplification system was 25 μL, specifically: 1 μL template DNA, 1 μL universal primer 27F, 1 μL universal primer 1492R, 12.5 μL 2×TaqMixture, and 9.5 μL ddH2O. The PCR reaction program was: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 2 min, repeated 30 times, with a final extension at 72℃ for 5 min. The amplified products were detected by 10 g / L agarose gel electrophoresis and then sequenced. The sequencing results are shown in SEQ ID No. 1. SEQ ID No. 1:

[0034] The 16S rRNA gene sequencing results of strain R78 were subjected to BLAST alignment at the National Center for Biotechnology Information (NCBI). The results showed that the strain with the highest similarity to strain R78 was... Bacillus tequilensis With a homology of 99.92%, strain R78 can be identified as *Bacillus tekirae*. Biological sample preservation information: *Bacillus tekirae* (… Bacillus tequilensis Strain R78 was deposited on September 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 32106. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0035] (3) Detection of physiological and biochemical properties of strain R78 The physiological and biochemical identification of Bacillus tekirae R78 was mainly based on the "Handbook of Systematic Identification of Common Bacteria". Strain R78 produces distinct clear zones on lipase and starch media, and distinct hydrolysis zones on protein solid media, indicating that this strain has the ability to secrete lipase, amylase, and protease. This strain can also produce catalase, but cannot produce hydrogen sulfide, and the negative result of the methyl red test indicates that this strain cannot produce acid by breaking down glucose. The identification results are shown in Table 1.

[0036] Table 1. Identification results of physiological and biochemical characteristics of strain R78

[0037] Example 2: Assay of the nematicidal activity of Bacillus tekiria R78 Preparation of pine wood nematode suspension: Botrytis cinerea plates infested with pine wood nematodes were washed with sterile deionized water to ensure the nematodes remained suspended. The nematode suspension was transferred to a 15 mL centrifuge tube and allowed to stand for 30 min. The supernatant was removed, and 400 μL of streptomycin sulfate (5 g / L) was added to the remaining 1 mL of suspension. The volume was adjusted to 10 mL, and the suspension was washed with sterile deionized water. The suspension was allowed to stand for 15-20 min, and the supernatant was removed. The nematodes were washed three times with sterile deionized water to remove the streptomycin sulfate from their bodies. Finally, 5 mL of pine wood nematode suspension was left in the 15 mL centrifuge tube. After shaking and mixing, 20 μL of the suspension was taken out and the number of pine wood nematodes was counted under a stereomicroscope. The 20 μL suspension was adjusted to contain approximately 50 pine wood nematodes.

[0038] Culture medium preparation: a. LB solid medium: 5g yeast extract, 10g sodium chloride, 10g tryptone, 1L distilled water, 20g agar powder, pH 7.0; b. LB liquid medium: 5g yeast extract, 10g sodium chloride, 10g tryptone, 1L distilled water, pH 7.0.

[0039] Preparation of bacterial culture: a. Fermentation broth preparation: Strain R78, stored at -80℃, was activated on LB solid medium using the three-zone streak method. It was then incubated in a 30℃ inverted incubator for 24 hours. Single colonies growing on the LB medium were picked and inoculated into LB liquid medium. The medium was incubated at 30℃ and 200 rpm for 24 hours. The bacterial culture was then adjusted to OD using LB liquid medium. 600 =2.0.

[0040] b. Preparation of supernatant for strain R78: Centrifuge the adjusted fermentation broth of strain R78 at 13000 rpm for 3 min. The resulting supernatant is the supernatant for strain R78.

[0041] Add 40 μL of fermentation broth (or supernatant) of strain R78 and 20 μL of pine wood nematode suspension to each well of a 96-well plate. For the blank control, add 40 μL of LB medium and 20 μL of pine wood nematode suspension. Each treatment has six replicates. Seal the edges of the 96-well cell culture plate with sealing film. Incubate in the dark at 25°C for 36 h. Then, wash the nematode surface with sterile water to remove the fermentation broth, repeating the washing three times. Stain the nematodes with 2 μL of 2 mg / mL methylene blue. After 4 h, wash the nematode surface with sterile water, repeating the washing three times. Observe the staining of the methylene blue-treated nematodes under a stereomicroscope. If the nematode is stiff and stained blue, it is a dead nematode; if the nematode can swim and its body is curved, it is a live nematode. Calculate the corrected mortality rate by calculating the number of dead nematodes using the following formula.

[0042]

[0043]

[0044] The results of the fermentation broth and fermentation supernatant of strain R78 in inhibiting pine wood nematode were analyzed, and the corrected mortality rate was calculated. Figure 2 The results showed that the fermentation broth of strain R78 could effectively inhibit and kill pine wood nematodes, with a corrected mortality rate of 96.5%; however, the corrected mortality rate of the fermentation supernatant of strain R78 in inhibiting and killing pine wood nematodes was only 10.8%, significantly lower than that of the fermentation broth of strain R78. Figure 3 In summary, the fermentation broth of strain R78 exhibits good inhibitory and killing ability against pine wood nematodes.

[0045] Example 3 Evaluation of the nematicidal activity of fermentation broth of strain R78 Preparation of fermentation broth with different bacterial counts of strain R78: Strain R78, stored at -80 ℃, was activated on LB solid medium using the three-zone streak method. After incubation at 30 ℃ for 24 h, single colonies grown on LB medium were picked and inoculated into LB liquid medium. The medium was then incubated at 30 ℃ and 200 rpm for 24 h. The concentration of the fermentation broth was adjusted using LB medium to OD values. 600= 0.5 (bacterial count 1.6 × 10⁻⁵) 8 cfu·mL -1 ), 1.0 (bacterial count 3.2×10), 8 cfu·mL -1 ), 1.5 (bacterial content 4.8×10), 8 cfu·mL -1 ) and 2.0 (bacterial count 6.4×10) 8 cfu·mL -1 The control group was LB medium, and the fermentation broth at the above concentrations was co-cultured with pine wood nematode suspension for 12, 24, 36, and 48 hours, respectively.

[0046] The results showed that the bactericidal effect of fermentation broth with different bacterial counts of strain R78 varied at different treatment times. Figure 4 The bactericidal effect showed an increasing trend with the increase of bacterial content in the fermentation broth and treatment time. When the bacterial content of strain R78 in the fermentation broth was 4.8 × 10⁻⁶, the bactericidal effect was significantly improved. 8 cfu·mL -1 ~6.4×10 8 cfu·mL -1 Furthermore, when the treatment time for pine wilt nematodes was 36 hours and 48 hours, the corrected mortality rate of pine wilt nematodes could reach over 90%; among them, the inoculation amount was 4.8 × 10⁻⁶. 8 cfu· mL -1 The treatment time was 36 hours, during which the corrected mortality rate of pine wilt nematode was highest, reaching 96.5%. In summary, the concentration of the fermentation broth of strain R78 within the OD range... 600 =1.5~2.0 (4.8×10 8 cfu·mL -1 ~6.4×10 8 cfu·mL -1 The nematode-killing effect is best when the treatment time is 36-48 hours.

[0047] Example 4: Greenhouse Disease Prevention Experiment for Black Pine Seedlings Soak black pine seeds in a 5% potassium permanganate solution for 1 hour. Then, wash the surface of the seeds with sterile water to remove the potassium permanganate until the eluent is clear and colorless. Wrap the seeds in moist, sterile gauze and place them in a petri dish. Incubate at 30°C in the dark, spraying with sterile water daily to maintain humidity until the seeds germinate to about 1.5cm. Sow the seeds in sterile soil (black soil:vermiculite volume ratio 3:1) under the following conditions: 23±2°C, 16h light / 8h dark cycle, relative humidity maintained at 60%-80%, watering every 2 days. After approximately 30 days of cultivation, when the black pine seedlings have grown to 7-9cm in height and have 30-50 needles, make an incision in the phloem of the seedling trunk (2-3cm from the root) using a syringe needle. Insert the needle of a 1mL syringe into the incision, secure the needle with a small wooden stick (approximately 10cm long) and sealing film, and bandage the wound. Add 100μL (30 nematodes / μL) of pine wilt nematode suspension using the syringe needle, and then spray the black pine seedlings with 5mL OD. 600 = 1.5% fermentation broth of strain R78 was used as a positive control, with black pine seedlings inoculated only with pine wilt nematode suspension and black pine seedlings inoculated only with sterile water as a blank control. 14 days after nematode inoculation ( Figure 5 Referring to the published disease severity standards for pine wilt disease (functional analysis of ganA in the killing of pine wilt by Enterobacter ludwig's bacillus, 20230103, Chinese Journal of Biological Control, see page 7, lines 5-11 for details), the disease index of each treatment was investigated and statistically analyzed, and the control effect was calculated. The formulas for calculating the disease index and control effect are as follows:

[0048] The results showed that the disease index of the positive control group was 74.9, while the disease index of the R78 strain treatment group was only 23.5, indicating a significant reduction in pine wilt disease symptoms. Figure 6 Under greenhouse conditions, the disease index of pine wilt disease in black pine seedlings can be significantly reduced, effectively reducing the occurrence of pine wilt disease, with a control effect of 68.6% (…). Figure 7 In summary, under greenhouse conditions, the Bacillus tektii R78 of this invention can effectively control pine wilt disease in black pine.

[0049] Although the present invention has been illustrated and described with specific embodiments, it should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and scope of the present invention; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention; therefore, this means that all such substitutions and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A strain of Bacillus tekiria, used to control pine wilt disease ( Bacillus tequilensis R78, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32106, on September 29, 2024.

2. A microbial preparation, characterized in that, The microbial preparation includes Bacillus tekirulae R78 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation includes the fermentation broth of Bacillus tekirulae R78.

4. The microbial preparation according to claim 2, characterized in that, The dosage forms of the microbial preparations include solid or liquid formulations.

5. The microbial preparation according to claim 4, characterized in that, When the microbial preparation is a solid dosage form, the amount of *Bacillus tergentii* R78 contained is 4.8 × 10⁻⁶. 8 cfu·g -1 ~6.4×10 8 cfu·g -1 ; And / or, when the microbial preparation is a liquid preparation, the amount of Bacillus tekirae R78 contained is 4.8 × 10⁻⁶. 8 cfu·mL -1 ~6.4×10 8 cfu·mL -1 .

6. A method for controlling pine wilt disease, characterized in that, Apply the Bacillus tekirae R78 of claim 1 or the microbial preparation of any one of claims 2 to 5 to pine trees.

7. The method for controlling pine wilt disease according to claim 6, characterized in that, Spray pine trees with a fermentation broth containing the aforementioned Bacillus tekirae R78 or a medicinal solution containing the aforementioned microbial preparation.

8. The method for controlling pine wilt disease according to claim 6, characterized in that, The pine trees include at least one of the following: black pine, Scots pine, red pine, larch, or Masson pine.

9. The use of Bacillus tekirulae R78 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 5 in the prevention and control of pine wilt disease.

10. A growth inhibitor for pine wood nematode, characterized in that, Includes Bacillus tekirae R78 as described in claim 1 or the microbial preparation as described in any one of claims 2 to 5.

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