A bacillus firmus strain rhs-l1 and application thereof in prevention and treatment of pine wood nematode disease

Abstract

The application belongs to the field of agricultural environmental microorganism technology, and particularly relates to a strong Bacillus firmus strain Rhs-L1 isolated from the rhizosphere soil of Pinus tabulaeformis and application of the strain in the prevention and treatment of pine wood nematode disease. The strain was preserved in the China Center Type Culture Collection of Wuhan University on April 18, 2024, and the preservation number was CCTCC M 2024708. The fermentation filtrate and fermentation bacterial suspension of the strain Rhs-L1 have significant inhibitory effects on the reproduction of pine wood nematode; the prevention and treatment effect of the fermentation bacterial suspension of the strain Rhs-L1 on the pine wood nematode disease of Pinus tabulaeformis pot seedlings can reach 81.0%; the strong Bacillus firmus strain Rhs-L1 of the application has a good application prospect in the nuisance-free prevention and treatment field of pine wood nematode disease.

Description

Technical Field

[0001] This invention relates to the field of agricultural environmental microbiology, specifically to a strong Bacillus strain Rhs-L1 and its application in the control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a highly dangerous forest disease affecting pine trees. It is characterized by its wide transmission routes, rapid spread, and difficulty in control. Infected pine trees typically die within 40 days, earning it the nickname "cancer of the pine." The pine wilt nematode has been listed as a global quarantine pest. This disease is a complex system comprised of factors such as the pine wilt nematode, vector insects, host plants, environmental factors, and human behavior, making its control extremely challenging. Due to rapid economic development and accelerated globalization, pine wilt disease has spread rapidly throughout the world via trade. This large-scale expansion has immediately drawn the attention of many countries, leading to the pine wilt nematode being listed as a quarantine pest in over 40 countries. Among the most severely affected countries are the United States, Canada, Japan, China, and South Korea, seriously impacting local ecological landscapes and ecological security.

[0003] Currently, control measures for pine wilt disease mainly include felling and burning or fumigating infected trees, controlling the vector insect longhorn beetle, injecting pesticides (mainly chemical nematicides such as aldicarb and abamectin) through trunk drilling, and directly killing the pine wilt nematodes. At present, pine wilt disease control relies primarily on chemical control, which was once popular due to its rapid effect and ease of application. However, the extensive use of chemical pesticides has led to problems such as pesticide residues and pathogen resistance in forest ecosystems, which have attracted great attention from relevant departments and researchers. To protect the forestry ecological environment, countries worldwide are gradually restricting and phasing out the use of highly toxic and persistent chemical pesticides, shifting their focus to the development and research of environmentally friendly biological pesticides or biocontrol agents. Some progress has been made in the control of pine wilt disease, providing resources for green control of the disease and contributing to the sustainable development of forest ecosystems.

[0004] Biological control based on microbial resources is an important approach to the green and pollution-free control of plant diseases. Bacillus, widely distributed in natural environments such as soil, rhizosphere, and within plants, can produce stress-resistant spores and diverse antimicrobial metabolites, effectively colonizing plants and protecting them from pathogen infection. It is considered an environmentally friendly biocontrol agent with broad application prospects. The functions and activities of Bacillus in different habitats vary. Bacillus discovered in habitats related to host plants has great potential for the biological control of plant diseases. There are successful cases of using Bacillus for plant disease control in production. However, there are currently no reports on the application of *Bacillus thuringiensis* strains to the control of pine wilt disease. Therefore, studying the toxic activity of *Bacillus thuringiensis*, a soil microorganism in the rhizosphere of *Pinus tabuliformis*, against pine wilt nematodes and evaluating its potted plant control efficacy against pine wilt nematodes is of great significance, providing a promising new approach for the biological control of pine wilt disease. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing control technologies for pine wilt disease by providing a biocontrol-strengthening Bacillus strain Rhs-L1 for controlling pine wilt disease.

[0006] Another objective of this invention is to provide results on the in vitro toxicity of Bacillus thuringiensis strain Rhs-L1 against pine wood nematodes and its effect on reproduction.

[0007] Another objective of this invention is to provide an experimental model for determining the effect of Bacillus thuringiensis strain Rhs-L1 on the reproduction of pine wood nematode.

[0008] Another objective of this invention is to provide the application of the Bacillus thuringiensis strain Rhs-L1 in the prevention and control of pine wilt disease in potted Pinus tabuliformis seedlings.

[0009] The technical solution provided by this invention is as follows:

[0010] This invention provides a Bacillus thuringiensis strain Rhs-L1, which was deposited at the China Center for Type Culture Collection on April 18, 2024, with accession number CCTCC M2024708.

[0011] Furthermore, the 16S rRNA sequence of the Bacillus thuringiensis strain Rhs-L1 is shown in SEQ ID NO:1.

[0012] Furthermore, the *Bacillus thuringiensis* strain Rhs-L1 was isolated from the rhizosphere soil of *Pinus tabuliformis*.

[0013] The present invention also provides a bacterial agent containing the above-mentioned Bacillus thuringiensis strain Rhs-L1.

[0014] Furthermore, the bacterial agent is a fermentation suspension of the *Bacillus thuringiensis* strain Rhs-L1, and the concentration of the fermentation suspension is 1×10⁻⁶. 6 CFU / mL.

[0015] The present invention also provides the above-mentioned microbial agent for the prevention and control of pine wilt disease in plants, wherein the plant is Pinus tabuliformis.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] (1) The present invention screened a biocontrol strain Bacillus thymophilus Rhs-L1, which has significant toxic activity against pine wood nematode;

[0018] (2) The effect of the biocontrol strain Bacillus thymotherum Rhs-L1 on the reproduction was determined. The reproduction coefficient and oviposition of pine wood nematode in the fermentation broth were used as the detection indicators. It was found that the strain had a significant inhibitory effect on the reproduction of pine wood nematode, and the experimental model of the effect of the biocontrol strain on the reproduction of pine wood nematode was clarified.

[0019] (3) The efficacy of biocontrol strains against pine wilt disease in potted Pinus tabuliformis seedlings was studied. The efficacy against pine wilt disease can reach 81.0%, which has good application prospects in the pollution-free control of pine wilt disease. It also lays the foundation for the development of Rhs-L1 microbial agents, forest trials and its commercialization and industrial production.

[0020] (4) Applying this microbial product does not have a series of problems caused by the use of chemical pesticides. It can eliminate or reduce the amount of other chemical pesticides used to control root rot of Sichuan pepper. It is not only conducive to the green control of pine wilt disease, but also can make up for the problems of pesticide residue, drug resistance and re-outbreak of harmful organisms caused by the use of chemical pesticides, which is conducive to maintaining the sustainable development of forest ecosystem. Attached Figure Description

[0021] Figure 1 A schematic diagram of pine wood nematodes (A) alive and (B) dead;

[0022] Figure 2 Schematic diagram of colony morphology (A) and spore staining (B) of Bacillus thuringiensis strain Rhs-L1;

[0023] Figure 3 Figure showing the effect of Bacillus thuringiensis strain Rhs-L1 on the reproduction of pine wood nematode;

[0024] Figure 4 A schematic diagram of an experimental model showing the effect of biocontrol strains on the reproduction of pine wood nematode;

[0025] Figure 5A schematic diagram of the test analysis of the control efficacy of Bacillus thuringiensis strain Rhs-L1 against pine wilt disease in potted Pinus tabuliformis seedlings.

[0026] Figure 6 The growth effects of different treatment groups of Pinus tabuliformis seedlings with Bacillus thuringiensis strain Rhs-L1 are shown in the figure.

[0027] Figure 7 The diagram shows the control efficacy of Bacillus subtilis strain Rhs-L1 on potted Pinus tabuliformis seedlings. The effects of different treatments on the infection rate of Pinus tabuliformis seedlings are shown in Figure 1 (A), the effects of different treatments on the disease index of Pinus tabuliformis seedlings are shown in Figure 2 (B), the effects of different treatments on the control effect of different treatments on Pinus tabuliformis seedlings are shown in Figure 3 (C), and the effects of different treatments on the density of Pinus tabuliformis nematodes in Pinus tabuliformis seedlings are shown in Figure 4 (D). Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0029] Example 1: Isolation and Virulence Determination of Biocontrol Strains

[0030] A biocontrol strain of Bacillus firmus, Rhs-L1, for pine wilt disease was deposited at the China Center for Type Culture Collection (CCTCC) on April 18, 2024, with accession number CCTCC M 2024708. This Bacillus firmus strain Rhs-L1 was isolated from rhizosphere soil microorganisms of Pinus tabuliformis. A co-incubation method revealed that strain Rhs-L1 exhibited significant toxic activity against pine wilt nematodes. Subsequently, its effect on pine wilt nematode reproduction was determined. The methods for isolation, toxic activity determination, and determination and identification of the reproductive effect of the biocontrol strain Rhs-L1 are as follows:

[0031] (1) Isolation of biocontrol strains

[0032] Rhizosphere soil samples were collected from the Pinus tabuliformis forest in Huanshan Park, Chengguan Town, Ningshan County, Shaanxi Province. The specific collection process was as follows: Four healthy 20-year-old Pinus tabuliformis trees were randomly selected. Rhizosphere soil sampling points were set 15cm away from the base of the trunk. Soil samples were dug up from 10-20cm below the surface soil with a shovel. Fine Pinus tabuliformis roots (diameter <2mm) were collected from the soil. Large soil particles were shaken off and placed in a plastic bag. The root tissue samples were taken out of the ice box and the soil on the fine roots was shaken off and collected in a plastic bag. This is the Pinus tabuliformis rhizosphere soil.

[0033] Take 2.5g of each of the four collected rhizosphere soil samples, mix thoroughly, and transfer to a sterile glass Erlenmeyer flask (250mL) containing 90mL of sterile water. Vigorously shake to mix for 15min, then let stand for 5min. The supernatant solution is the 10... -1 A soil suspension of g / mL was then used with 10 g / mL soil solution. -1 Soil suspensions of g / mL were prepared to a concentration of 10 g / mL. -2 g / mL, 10 -3 g / mL and 10 -4 Prepare a soil dilution solution at a concentration of g / mL for later use.

[0034] Take 30 μL of soil suspension and drop it onto a plate. Then, spread it evenly using a sterile spreader. Spread 3 plates for each concentration (the concentration used is 10). -2 g / mL, 10 -3 g / mL and 10 -4 The culture dish was sealed with sealing film (g / mL) and then placed in an incubator at 25℃ for 30 days. Single colonies growing on the culture medium were observed daily, and newly formed single colonies with different morphologies were inoculated onto newly prepared plates using sterile toothpicks for purification and preservation until no more colonies were added to the plates.

[0035] The obtained strain was preserved in 30% glycerol and kept at -80°C.

[0036] The LBA medium is prepared as follows: 10g tryptone, 3g yeast extract, 10g NaCl, 15-20g agar, 1000mL water, pH 7.4-7.6.

[0037] (2) Determination of the toxic activity of biocontrol bacteria against pine wood nematode

[0038] The co-incubation method was used to detect the toxic activity of all microbial strains isolated from the rhizosphere soil of Pinus tabuliformis against pine wood nematodes.

[0039] Determination of the toxic activity of the biocontrol strain against pine wood nematode: Botrytis cinerea was inoculated onto PDA medium and cultured for 7 days until the Botrytis cinerea covered the entire medium plate. Pine wood nematodes were then inoculated onto the Botrytis cinerea mycelium in a clean bench and cultured at 25°C in the dark for 7 days. Once the surface of the medium became glossy, the pine wood nematodes were isolated using the Bellman funnel method. After centrifugation and enrichment, a pine wood nematode suspension was obtained. 480 μL of the bacterial fermentation filtrate (concentration 1×10⁻⁶) was added to a sterile centrifuge tube. 620 μL of pine wood nematode suspension (approximately 200 nematodes) and 20 μL of safranin (CFU / mL) were incubated in a constant temperature shaking incubator at 25°C and 150 rpm for 12 hours. The survival of the pine wood nematodes was assessed using a combination of safranin staining and morphological observation. Dead pine wood nematodes stained red and formed "C" or "J" shapes, showing no response to physical stimuli. Surviving pine wood nematodes did not stain and exhibited a bent, active state. The images of dead and surviving pine wood nematodes are shown below. Figure 1 Strains exhibiting significant toxic activity against pine wilt nematode (corrected mortality rate ≥80%) were screened, resulting in 42 biocontrol strains in the initial screening. The following are the identification, reproductive effects, and pot control efficacy results of strain Rhs-L1.

[0040] Example 2: Identification of biocontrol strain Rhs-L1

[0041] Single colonies of the biocontrol strain Rhs-L1 were picked and inoculated onto LBA medium. The colony morphology was as follows: Figure 2 As shown in Figure A. The Schaeffer-Fulton spore staining kit was used to detect whether strain Rhs-L1 produced spores. It was found that strain Rhs-L1 could produce a large number of spores, such as... Figure 2 As shown in B.

[0042] DNA was extracted from strain Rhs-L1 using the CATB method. The 16S rDNA of strain Rhs-L1 was then amplified and sequenced. Primer synthesis and sequencing were performed by Shanghai Sangon Biotech Co., Ltd. Based on the alignment of the 16S rDNA sequence of strain Rhs-L1 with the NCBI database, it was identified as *Bacillus firmus*. The primers used for PCR amplification of the 16S rDNA of strain Rhs-L1 were: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3').

[0043] The PCR amplification reaction system for 16S rDNA of strain Rhs-L1 was 25 μL, including 12.5 μL 2×ES TaqMasterMix, 1.0 μL template DNA, 1.0 μL 27F primers, 1.0 μL 1492R primers, and 9.5 μL distilled water. PCR amplification was performed in a thermal cycler: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 45 s, for 30 cycles; and a final extension at 72℃ for 10 min. After PCR, the fragment size was verified by gel electrophoresis, and the PCR products were sent to a sequencing company for sequencing.

[0044] Sequencing results of the 16S rDNA amplified fragment of strain Rhs-L1:

[0045]

[0046] Example 3: Determination of the effect of biocontrol strain Rhs-L1 on the reproduction of pine wood nematode

[0047] (1) Effects of biocontrol strains on the reproduction of pine wood nematode:

[0048] Effects of biocontrol strains on the reproduction of pine wood nematodes: The reproduction effect of strain Rhs-L1 was investigated on PDA plates covered with *Botrytis cinerea*. The number of pine wood nematodes and eggs on the plates was measured after 7 days of incubation. Three equal aliquots of pine wood nematode suspension (approximately 200 nematodes per aliquot) were evenly inoculated onto PDA plates completely covered with *Botrytis cinerea* colonies. Each aliquot was placed on the circumference of the colony, 2 cm from the center. Subsequently, 0.5 mL of fermentation filtrate or fermentation suspension of strain Rhs-L1 was added to the inoculation site. All petri dishes were incubated in the dark at 25°C for 7 days. After incubation, pine wood nematodes and eggs on each PDA plate were collected using the Bellman funnel method and observed under a microscope to count the number of live pine wood nematodes and eggs. The inhibitory effect on the reproduction of pine wood nematodes is shown in the figure below. Figure 3 The concentration of the fermentation filtrate or fermentation suspension of strain Rhs-L1 was 1×10⁻⁶. 6 CFU / mL.

[0049] (2) Experimental model of the effect of biocontrol strains on the reproduction of pine wood nematode

[0050] This experiment designed a novel experimental model to evaluate the impact of biocontrol strains on the reproduction of pine wood nematode. This model has several key advantages, including simplicity, low cost, and intuitiveness. A schematic diagram of the model can be found below. Figure 4 .

[0051] In summary, the biocontrol strain Rhs-L1 was identified as Bacillus firmus, which was deposited at the China Center for Type Culture Collection on April 18, 2024, with accession number CCTCC M 2024708. This strain has significant effects on the in vitro toxicity and reproduction of pine wood nematode.

[0052] Example 4: Application of strain Rhs-L1 in pine wilt disease

[0053] Two-year-old healthy Pinus tabuliformis seedlings cultivated in greenhouses at Northwest A&F University were used as the research subjects. Strains were treated and pine wilt nematodes were inoculated. At the beginning of treatment, the surface of the treated areas on healthy branches was cleaned with damp, sterile cotton. Five different treatment groups were designed: inoculated only with LB liquid medium (CK group), inoculated only with pine wilt nematode suspension (Bx group), inoculated only with strain fermentation suspension (Rhs-L1 group), inoculated first with Rhs-L1 strain fermentation suspension and then inoculated with pine wilt nematodes 7 days later (Rhs-L1→Bx group), and inoculated first with pine wilt nematodes and then inoculated with Rhs-L1 strain fermentation suspension 7 days later (Bx→Rhs-L1 group). All treated Pinus tabuliformis seedlings were cultivated in the greenhouses of Northwest A&F University. Two small holes were drilled 10 cm apart on the stem of each Pinus tabuliformis seedling using a hand-held electric drill. The drill bit diameter was 4.5 mm, and the depth of each hole was 5 mm. In the CK group, each well of each Pinus tabuliformis seedling was covered with sterile cotton, and then 5 mL of LB medium was added to the sterile cotton. Each well was sealed with plastic film to prevent rapid moisture evaporation, and the cotton and plastic film were removed after 7 days. For the Pinus tabuliformis seedlings requiring pine wilt disease treatment, only 3000 pine wilt nematodes were inoculated per well, for a total of 6000 pine wilt nematodes per seedling; the concentration of the bacterial strain fermentation suspension was the same. The inoculation wells of the Bx group were also sealed with plastic film, and the plastic film was removed after 7 days of treatment. The Rhs-L1 group was treated the same as the CK group, except that 5 mL of Rhs-L1 bacterial strain fermentation suspension was used instead of LB. The initial treatment of each seedling in the Rhs-L1→Bx group was the same as the Rhs-L1 group, and the seedlings were cultured in a greenhouse for 7 days, after which the cotton and plastic film covering the wells were removed. Subsequently, as with the Bx group, each Pinus tabuliformis seedling was inoculated with Bx. The Bx→Rhs-L1 group, like the Bx group, was first inoculated with pine wood nematodes, and then, like the Rhs-L1 group, was treated with NI strain fermentation suspension. See the schematic diagram below. Figure 5 The concentration of the fermentation filtrate or fermentation suspension of strain Rhs-L1 was 1×10⁻⁶. 6 CFU / mL.

[0054] Disease incidence was observed and recorded in each treatment group of Pinus tabuliformis seedlings at 7, 14, 21, 28 and 35 days after treatment. The seedlings were divided into 5 segments, and the severity of each segment was graded. The results were statistically analyzed and the disease index and control effect were calculated.

[0055] Disease severity (S) is divided into 5 levels, from S1 to S5, with representative values ​​of 0, 1, 2, 3, and 4 respectively. S1: All needles show no symptoms and are in a healthy, green state; S2: Yellowing needles appear, but the number of yellowed needles does not exceed one-quarter; S3: One-quarter to one-half of the needles turn yellow; S4: More than half but no more than two-thirds of the needles turn yellow, and the pine seedlings show slight wilting; S5: More than two-thirds of the needles turn completely yellow, accompanied by significant wilting of the pine seedlings.

[0056] After 35 days of treatment, the density of pine wood nematodes in each Pinus tabuliformis seedling in the Bx group, Rhs-L1→Bx group, and Bx→Rhs-L1 group was calculated. Roots, stems, and leaves from each seedling were collected, cut into small pieces, mixed thoroughly, and weighed. Subsequently, the Bellman funnel method was used to isolate pine wood nematodes from all tissues of each seedling, and the number of pine wood nematodes (NN) was counted under a microscope. The density of pine wood nematodes in Pinus tabuliformis seedlings under different treatments was expressed as the number of nematodes per gram of fresh weight (FW) of Pinus tabuliformis (NN·g). -1 FW).

[0057]

[0058] like Figure 6 As shown, 35 days after inoculation with pine wilt nematodes, the Pinus tabuliformis seedlings in group Bx showed severe disease, with all needles drooping and turning reddish-brown, exhibiting an overall wilted state. The Pinus tabuliformis seedlings treated with the appropriate bacterial strain showed less disease compared to group Bx. Specifically, the Pinus tabuliformis seedlings inoculated with the Rhs-L1 strain fermentation suspension for 7 days before inoculation with pine wilt nematodes showed even less disease than those inoculated with the Rhs-L1 strain fermentation suspension for 7 days before inoculation.

[0059] like Figure 7 As shown, after treatment with Rhs-L1 strain, *Pinus tabuliformis* seedlings (Rhs-L1→Bx and Bx→Rhs-L1 groups) began to show signs of disease after 14 days, with infection rates ranging from 15.0% to 40.0%. By day 35, the infection rate of *Pinus tabuliformis* seedlings in all treatment groups reached 100%. Figure 7 A). Compared with *Pinus tabuliformis* seedlings pretreated with *Bacillus thuringiensis* strain Rhs-L1 before inoculation with pine wilt disease (Rhs-L1→Bx group), the disease index of *Pinus tabuliformis* seedlings treated with *Bacillus thuringiensis* Rhs-L1 fermentation suspension (Rhs-L1→Bx and Bx→Rhs-L1 groups) was significantly lower than that of *Pinus tabuliformis* seedlings inoculated only with pine wilt disease (Bx group), with a significant reduction of 0.1–4.3 times, respectively. Figure 7 B), its control effect was significantly increased by 1.0 to 3.2 times, with a control effect of 11.8 to 81.0% ( Figure 7 C). At 35 days, the density of pine wilt nematodes in *Pinus tabuliformis* seedlings treated with *Bacillus thuringiensis* Rhs-L1 fermentation suspension (Rhs-L1→Bx and Bx→Rhs-L1 groups) was significantly lower than that in *Pinus tabuliformis* seedlings inoculated only with pine wilt nematodes (Bx group). Furthermore, compared to *Pinus tabuliformis* seedlings treated with Rhs-L1 strain after pine wilt nematode inoculation (Bx→Rhs-L1 group), pretreatment with Rhs-L1 strain before pine wilt nematode inoculation significantly improved the pine wilt nematode inoculation. The density of pine wood nematodes in *Pinus tabuliformis* seedlings (Rhs-L1→Bx group) was significantly reduced by 0.8 times, indicating that the endophytic fungus Rhs-L1 in *Pinus tabuliformis* can effectively inhibit the reproduction of pine wood nematodes in the seedlings. Furthermore, the inhibitory effect of Rhs-L1 strain pretreated seedlings before pine wood nematode inoculation (Rhs-L1→Bx group) was significantly greater than that of Rhs-L1 strain treated seedlings after pine wood nematode inoculation (Bx→Rhs-L1 group). Figure 7 D).

[0060] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A strain of Bacillus firmus, Rhs-L1, characterized in that, The Bacillus thymophilus strain Rhs-L1 was deposited at the China Center for Type Culture Collection on April 18, 2024, with accession number CCTCC M2024708.

2. The robust Bacillus strain Rhs-L1 according to claim 1, characterized in that: The 16S rRNA sequence of the Bacillus thuringiensis strain Rhs-L1 is shown in SEQ ID NO:

1.

3. The robust Bacillus strain Rhs-L1 according to claim 1 or 2, characterized in that: The robust Bacillus strain Rhs-L1 was isolated from the rhizosphere soil of Pinus tabuliformis.

4. A microbial agent, characterized in that, Contains the Rhs-L1 strain of Bacillus fibrosum as described in any one of claims 1-3.

5. The microbial agent according to claim 4, characterized in that, The bacterial agent is the Bacillus thuringiensis strain Rhs-L1. The fermentation suspension has a concentration of 1×10⁻⁶. 6 CFU / mL.

6. The application of the fungal agent as described in claim 4 or 5 in the control of pine wilt disease, characterized in that, The plant in question is a Chinese pine.

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