Isothermal layer bacillus R6 for preventing and treating pine wood nematode disease and application of isothermal layer bacillus R6

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to bacillus isothermal layer R6 for preventing and treating pine wood nematode disease and application thereof. The bacillus isothermal layer R6 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO. 32275. The strain has the activity of efficiently inhibiting and killing the pine wood nematodes, the fatality rate of fermentation liquor of the strain on the pine wood nematodes can reach 90% or above when OD600 is 0.5-2, and the disease index of pine tree seedlings infected with the pine wood nematode disease can be effectively reduced.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a stratospheric bacillus R6 for preventing and treating pine wood nematode disease and an application thereof. Background Art

[0002] Pine wilt disease (PWD), also known as pine wilt, is a devastating forest disease. The main pathogen of the disease is the pine wood nematode (Bursaphelenchus xylophilus), which belongs to the phylum Nematoda, class Nematoda, order Aphelenchus, family Aphelenchus, and genus Aphelenchus. It can parasitize pine trees and cause them to become infected. Pine wood nematodes have the characteristics of a wide host range, strong adaptability, fast spread, strong ability to expand ecological niches, and fast infection of host plant tissues, posing a serious threat to forest ecosystems worldwide. Pine wood nematodes can infect a variety of coniferous species, mainly including Pinus sylvestris, Pinus thunbergii, Pinus massoniana and other pine species, and can cause large-scale pine wilt, which will not only reduce the economic value of pine resources, but also cause serious damage to pine forest resources, leading to irreversible ecological and economic losses. Pine wood nematode disease is not only a plant disease, but also a multi-level and multi-dimensional ecological destructive disease, which urgently requires in-depth research and prevention and control.

[0003] The prevention and control measures of pine wood nematode disease mainly include physical control, chemical control and biological control. Among them, physical control often uses felling, burning, crushing and other methods to deal with diseased pine trees. This control method is simple and effective, but it requires a lot of manpower and material resources, the cost of prevention and control is high, and there are problems such as incomplete prevention and control. Chemical control often uses trunk injection, fumigation, root application and drone spraying of chemicals. This method acts quickly and has obvious effects. It can effectively reduce the incidence of pine wood nematode disease, but it is also easy to cause environmental pollution and increase the risk of pine wood nematode resistance. Biological control technology using biocontrol microorganisms can interfere with nematode behavior patterns or directly kill nematodes by killing with toxins, interfering with nematode behavior patterns, mobilizing fungi to prey on nematodes, competing for nutrition and digesting nematode surface tissues, blocking pine wood nematodes from invading trees, thereby preventing the occurrence of pine wood nematode disease. Biological control technology is environmentally friendly, efficient, highly specific and does not kill beneficial organisms. It protects ecological balance and is an important way to achieve green prevention and control of pine wood nematode disease. The discovery of biocontrol microorganisms with high inhibitory and killing activity against pine wood nematodes and the development and application of biocontrol microbial preparations for pine wood nematode disease have attracted widespread attention.

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

[0005] The purpose of the present invention is to provide a stratosphere bacillus R6 for preventing and controlling pine wood nematode disease and its application. The stratosphere bacillus R6 is separated from pine tree tissue, has stable and efficient activity in inhibiting and killing pine wood nematodes, and has a mortality rate of more than 90% for pine wood nematodes, which can effectively reduce the disease condition of pine tree seedlings.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to a Bacillus stratosphericus R6 for preventing and treating pine wood nematode disease, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.32275.

[0008] The stratosphere bacillus R6 is isolated from black pine and has a high lethality rate to pine wood nematodes, which can reach more than 90%. The 16S rRNA gene sequence is shown in SEQ ID No.1.

[0009] The stratosphere bacillus R6 was inoculated on a lysing broth (Luria-Bertani, LB) solid culture medium (ingredients: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, 10 g / L agar powder) for culture. After constant temperature culture at 30°C for 24 hours, round, uneven edges, rough, flat, opaque white colonies were formed.

[0010] The stratosphere bacillus R6 can produce obvious hydrolysis circles on protein solid culture medium, can decompose milky white lecithin into colorless dissolved phosphorus to form transparent circles on organic phosphorus solid culture medium, and can form orange halo on siderophore detection solid culture medium. Therefore, strain R6 can secrete protease, decompose organic phosphorus and produce siderophore, but strain R6 does not show transparent circles on solid culture medium of fat, cellulose, starch, inorganic phosphorus and nitrogen. Therefore, strain R6 cannot secrete lipase, cellulase and amylase, cannot decompose inorganic phosphorus and does not have nitrogen fixation ability.

[0011] Another aspect of the present invention also relates to a microbial preparation, which comprises: the above-mentioned stratosphere Bacillus R6.

[0012] The microbial preparation contains stratosphere bacillus R6, has good ability to kill pine wood nematodes, and can be used for the prevention and treatment of pine wood nematode disease.

[0013] Preferably, the microbial preparation comprises: a fermentation product of the stratosphere Bacillus R6 and / or a metabolite of the stratosphere Bacillus R6.

[0014] Preferably, the microbial preparation includes but is not limited to: a solid microbial preparation or a liquid microbial preparation.

[0015] Preferably, the viable count of the solid microbial preparation is 3.3×10 7 CFU / g and above.

[0016] Preferably, the viable count of the liquid microbial preparation is 3.3×10 7 CFU / mL and above.

[0017] Preferably, the solid microbial preparation further comprises a solid carrier, and the solid carrier includes but is not limited to one or more of talc, lignite, montmorillonite, alginate, silica, quartz powder, vermiculite, kaolin, light calcium carbonate, and diatomaceous earth.

[0018] Preferably, the solid bacterial agent further comprises one or more of an antioxidant, a binder and a water retaining agent.

[0019] Preferably, the antioxidant includes but is not limited to one or more of vitamin C, vitamin E and citric acid.

[0020] Preferably, the binder includes but is not limited to maltose and / or dextrin.

[0021] Preferably, the water retaining agent includes but is not limited to at least one of a water-absorbing resin and mannitol.

[0022] Preferably, the microbial preparation further comprises an auxiliary agent.

[0023] Preferably, the auxiliary agent includes at least one of a dispersant, a stabilizer, a disintegrant, a solvent or a wetting agent. In a specific embodiment, the auxiliary agent includes one or more of trehalose, glycerol, buffer salt, yeast extract, white carbon black, sodium chloride or amino acid.

[0024] Preferably, the microbial preparation further comprises an agriculturally effective amount of fertilizers and / or pesticides.

[0025] Preferably, the dosage form of the microbial agent includes at least one of wettable powder, water dispersible granules, water suspension or dispersible oil suspension.

[0026] Another aspect of the present invention also relates to a method for preventing and controlling pine wood nematode disease, comprising applying the stratosphere Bacillus R6 or the microbial preparation to a control object.

[0027] The method for controlling pine wood nematodes is to apply microbial preparations to pine trees, which belongs to a method for biological control of pine wood nematode disease and has the advantages of being economical, green, environmentally friendly, and highly efficient. Experiments on controlling pine wood nematode disease with stratosphere Bacillus R6 show that stratosphere Bacillus R6 has a good control effect on pine wood nematode disease.

[0028] Preferably, the bacterial solution containing the stratosphere bacillus R6 or the liquid containing the microbial preparation is sprayed on the control object. The control of pine wood nematode disease can be achieved only by spraying, which reduces the difficulty of operation.

[0029] Preferably, the control targets include: at least one of black pine, mongolica pine, red pine, larch or masson pine.

[0030] Preferably, the control targets include: Pinus sylvestris var. mongolica and / or Pinus thunbergii.

[0031] Another aspect of the present invention also relates to the use of the above-mentioned Stratospheric Bacillus R6 or the above-mentioned microbial preparation in preventing and treating pine wood nematode disease.

[0032] Another aspect of the present invention also relates to a growth inhibitor of pine wood nematodes, comprising the above-mentioned stratosphere Bacillus R6 or the above-mentioned microbial preparation.

[0033] Biological sample preservation information: Bacillus stratosphericus R6, the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with the deposit number CGMCC NO.32275. Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Postal Code 100101.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The Bacillus stratosphericus R6 provided by the present invention is isolated from black pine tissue and has high efficiency in inhibiting and killing pine wood nematodes. The fermentation liquid thereof is OD 600 =0.5-2, the mortality rate to pine wood nematodes reaches more than 90%, and can effectively reduce the disease index of pine seedlings infected with pine wood nematodes.

[0036] (2) The microbial preparation provided by the present invention, including the bacterial body of strain R6, its fermentation products or its metabolites, can be compounded with commonly used foliar fertilizers or pesticides to prevent and control plant diseases, especially pine wood nematode disease.

[0037] (3) The method for preventing and controlling pine wood nematode disease provided by the present invention is simple to operate and has a good prevention and control effect. It only requires applying stratosphere Bacillus R6 or a microbial preparation to the control object. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 The colony morphology of Bacillus stratosporus R6 is shown (scale bar: 1 mm);

[0040] Figure 2 For the identification of physiological and biochemical characteristics of Bacillus stratosporus R6 (A is fat medium, B is protein medium, C is cellulose medium, D is starch medium, E is organic phosphorus medium, F is inorganic phosphorus medium, G is nitrogen medium, and H is siderophore detection medium);

[0041] Figure 3 The results of methylene blue staining of pine wood nematodes treated with the fermentation liquid and the supernatant of the fermentation liquid of Stratosporum Bacillus R6;

[0042] Figure 4 The activity of the fermentation liquid and the supernatant of the fermentation liquid of Bacillus stratosporus R6 against pine wood nematodes;

[0043] Figure 5 The effect of concentration and treatment time of the fermentation liquid of Stratosporum Bacillus R6 on its activity against pine wood nematodes;

[0044] Figure 6 The results of methylene blue solution staining of pine wood nematodes treated with different concentrations of stratosphere Bacillus R6 fermentation liquid;

[0045] Figure 7 The results of the greenhouse disease prevention experiment on black pine seedlings (A is the water treatment group, B is the pine wood nematode treatment group, C is the stratospheric Bacillus R6 fermentation liquid and pine wood nematode treatment group, scale: 3 cm);

[0046] Figure 8 It is the disease index of black pine seedling greenhouse disease prevention experiment;

[0047] Fig. 9 The disease prevention effect of Stratospheric Bacillus R6 against pine wood nematode disease in greenhouse. DETAILED DESCRIPTION

[0048] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0049] Example 1 Identification of strain R6

[0050] (1) 16S rRNA gene sequencing analysis of strain R6

[0051] The genomic DNA of strain R6 was extracted and used as a template to PCR amplify the 16S rRNA gene fragment using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction system: template DNA 1μL, primer 27F 1μL, primer 1492R 1μL, 2×Taq Mixture 12.5μL, ddH2O 9.5μL, PCR reaction program: 98℃ pre-denaturation 5min, 98℃ denaturation 30s, 55℃ annealing 30s, 72℃ extension 2min, 30 cycles, 72℃ 5min. The amplified product was detected by 1% agarose gel electrophoresis, purified and sequenced, and the sequencing results are shown in SEQ ID No.1.

[0052] SEQ ID No.1:

[0053]

[0054] The obtained sequence was searched by BLAST in the National Center for Biological Information (NCBI) of the United States. The highest similarity with the 16SrRNA gene fragment sequence of strain R6 was Bacillus stratosphericus, with a homology of up to 99.86%. Therefore, the R6 strain was identified as stratosphere Bacillus and named as stratosphere Bacillus stratosphericus R6. The strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCCNO.32275.

[0055] (2) Morphological observation of strain R6

[0056] Strain R6 was inoculated into LB solid medium and cultured at 30°C for 24 h. Round, uneven-edged, rough-surfaced, flat, opaque white colonies were observed (see Figure 1 ).

[0057] (3) Physiological and biochemical reaction test of strain R6

[0058] The physiological and biochemical reaction test of strain R6 mainly refers to the "Common Bacteria System Identification Manual". This strain can produce obvious hydrolysis circles on protein solid culture medium, colorless transparent circles on organic phosphorus solid culture medium, and orange haloes around the colonies of microorganisms on the solid culture medium for siderophore detection. Therefore, strain R6 can secrete protease, decompose organic phosphorus and produce siderophore. Strain R6 does not produce transparent circles on solid plates of fat, cellulose, starch, inorganic phosphorus and nitrogen. Therefore, strain R6 cannot secrete lipase, cellulase and amylase, cannot decompose inorganic phosphorus, and does not have the ability to fix nitrogen (see Figure 2 ). The test results of strain R6 for hydrogen sulfide, catalase and IAA were all positive. The physiological and biochemical test results of strain R6 are shown in Table 1.

[0059] Table 1 Test results of physiological and biochemical characteristics of strain R6

[0060]

[0061] Example 2 Determination of the activity of stratosphere Bacillus R6 in killing pine wood nematodes

[0062] (1) Preparation of pine wood nematode suspension:

[0063] On the Botrytis cinerea plate with pine wood nematodes, sterile water is slowly injected until the fungal hyphae are completely covered, and then the system is left to stand for 10 minutes to separate the nematodes from the hyphae. Use a 5mL pipette to repeatedly suck and spit out the suspension to make the pine wood nematodes evenly suspended in sterile water. Subsequently, the suspension containing pine wood nematodes is transferred to a 15mL centrifuge tube and allowed to stand for 15-20min. After the nematodes naturally settle, the supernatant is carefully removed, leaving only about 1.5mL of pine wood nematode suspension. Add 400μL of streptomycin sulfate solution (concentration of 5g / L) to the worm suspension, and then adjust the total volume to 10mL with sterile water. After gently shaking the centrifuge tube, let it stand again for 15-20min, then remove the supernatant, leaving only about 1.5mL of nematode suspension, and repeat this process 3 times. After washing, gently shake the centrifuge tube to mix the nematode suspension, draw 20 μL of the suspension, and count the number of pine wood nematodes under a stereo microscope. Adjust the concentration of the suspension according to the statistical results so that 1 μL of the suspension contains 4-6 pine wood nematodes.

[0064] (2) Preparation of strain R6:

[0065] Preparation of fermentation broth of strain R6: The stratosphere Bacillus strain R6 stored in a -80℃ ultra-low temperature refrigerator was activated on LB solid medium by the three-zone streak method, incubated at 30℃ for 24h, and a single colony grown on the LB plate was picked and inoculated into LB liquid medium, which was placed in a shaker at 30℃, 200rpm, and shaken for 24h. The concentration of the bacterial suspension was adjusted to OD 600 =0.5, 1, 1.5, 2.

[0066] Preparation of the supernatant of the fermentation broth of strain R6: centrifuge the fermentation broth of strain R6 at 13000 rpm for 3 min, and the obtained supernatant is the supernatant of the fermentation broth of strain R6.

[0067] (3) Detection of pine wood nematode killing activity:

[0068] Take a 96-well plate, add 40 μL of the sample to be tested (fermentation liquid or fermentation liquid supernatant) of strain R6 and 20 μL of pine wood nematode suspension to each well, set 8 replicates for each treatment, and culture at 25°C for 36 hours. Add 200 μL of sterile water to each well to wash the nematodes, wash off the bacterial fermentation liquid or supernatant on the surface of the nematode body, and repeat the washing of the nematodes three times; then, add 4 μL of methylene blue solution (2 mg / mL), stain for 4 hours, wash off the dye with 200 μL of sterile water, and observe the morphology of the nematodes under a stereo microscope. The dead nematode body is stiff and dyed blue (see Figure 3 ), the bodies of the living nematodes were curled and not stained, and the corrected mortality of pine wood nematodes was calculated according to the following formula:

[0069]

[0070] The results showed that the fermentation liquid of Bacillus stratosporus R6 had stable high nematode activity. After 36 hours of co-culture, the corrected mortality rate of pine wood nematodes reached more than 90%. The supernatant of the fermentation liquid of strain R6 had almost no nematode killing activity (see Figure 3 , 4 ).

[0071] Set different fermentation broth concentrations (OD 600 The optimal nematode killing concentration and treatment time of the stratosphere Bacillus R6 fermentation liquid were determined by adjusting the co-culture time (12h, 24h, 36h and 48h) and the co-culture time (12h, 24h, 36h and 48h) of the pine wood nematode suspension and the fermentation liquid. The results showed that when the pine wood nematode suspension was co-cultured with the strain fermentation liquid for 48h and the fermentation liquid concentration was OD 600 = 0.5 to 2 can make the corrected mortality of pine wood nematodes reach more than 90% (see Figure 5 , 6 ), which is equivalent to a bacterial count of 3.3×10 7 CFU / mL~2×10 8 CFU / mL can make the corrected mortality of pine wood nematodes reach more than 90%, so strain R6 has the potential to be used as a highly effective biological control strain for killing pine wood nematodes.

[0072] Example 3 Greenhouse disease prevention experiment of black pine seedlings

[0073] Soak the black pine seeds in 7% potassium permanganate solution for 2 hours to sterilize the seeds on the surface. Subsequently, use sterile water to repeatedly wash the seed surface until the eluent is colorless to ensure that the potassium permanganate is completely removed. Wrap the sterilized seeds in moist sterile gauze, place them in a culture dish, and culture them at a constant temperature of 30°C for germination. The successfully germinated seeds are sown in sterilized soil (soil: vermiculite volume ratio is 3:1), and the culture conditions are set to a temperature of 23±2°C, a light cycle of 16 hours of light / 8 hours of darkness, and a relative humidity of 60%-80%, and the culture is continued for 30 days. After emergence, water once every 2 days. When the black pine seedlings grow to a plant height of about 7-9 cm and the number of needles reaches 30-50, they are selected as test subjects for disease prevention effect evaluation.

[0074] Use a syringe needle to make a wound in the phloem of the seedling trunk (about 2-3 cm from the soil surface), and install a 1 ml syringe needle at the wound. Use a small wooden stick about 10 cm long and a sealing film to fix the needle, and bandage the wound to ensure sealing. Then, inject 100 μL (concentration of 30 heads / μL) of pine wood nematode suspension into the seedling through the syringe needle, and spray 5 mL, OD 600= 2 of R6 fermentation liquid. 21 days after inoculation with nematodes, according to the published disease grade standard (reference "Functional analysis of Ludwig's Enterobacter in killing pine wood nematodes", Chinese Journal of Biological Control, 20230103, page 7, lines 5 to 11), the disease index of each treatment group was statistically analyzed, and the disease prevention effect was calculated according to the relevant formula. The calculation formula of disease index and disease prevention effect is as follows:

[0075]

[0076] The black pine seedlings inoculated with pine wood nematode suspension were used as the positive control, the black pine seedlings inoculated with pine wood nematode suspension and fermentation liquid of strain R6 were used as the treatment group, and the blank control was the black pine seedlings treated with sterile water. The results of the greenhouse disease prevention experiment showed that (see Figure 7 , 8 ), the disease index of the positive control group was 81.5; the disease symptoms of the treatment group were significantly alleviated compared with the positive control group, and the disease index was only 32.1, indicating that the fermentation liquid of strain R6 can significantly reduce the disease index of pine wilt disease in black pine seedlings under greenhouse conditions, effectively prevent and control the occurrence of pine wilt disease, and the disease prevention effect is 60.7% (see Fig. 9 ).

[0077] In summary, under greenhouse conditions, the stratosphere Bacillus R6 of the present invention can effectively prevent and control pine wood nematode disease.

[0078] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A strain of Bacillus stratosphericus R6 for controlling pine wood nematode disease, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC NO.32275.

2. A microbial preparation, characterized in that: The microbial preparation comprises: the stratosphere Bacillus R6 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation comprises: a fermentation product of the stratosphere bacillus R6 and / or a metabolite of the stratosphere bacillus R6.

4. The microbial preparation according to claim 2, characterized in that The microbial preparation includes: a solid microbial preparation or a liquid microbial preparation.

5. The microbial preparation according to claim 4, characterized in that The viable count of the solid microbial preparation is 3.3×10 7 CFU / g or above; And / or, the viable count of the liquid microbial preparation is 3.3×10 7 CFU / mL and above.

6. A method for preventing and controlling pine wood nematode disease, characterized in that: The stratosphere bacillus R6 according to claim 1 or the microbial preparation according to any one of claims 2 to 5 is applied to the control object.

7. The method for preventing and controlling pine wood nematode disease according to claim 6, characterized in that: The bacterial liquid containing the stratosphere bacillus R6 or the medicinal liquid containing the microbial preparation is sprayed on the control object.

8. The method for preventing and controlling pine wood nematode disease according to claim 6, characterized in that: The control targets include: at least one of black pine, mongolica pine, red pine, larch or masson pine.

9. Use of the stratospheric bacillus R6 according to claim 1 or the microbial preparation according to any one of claims 2 to 5 in preventing and controlling pine wood nematode disease.

10. A growth inhibitor of pine wood nematodes, characterized in that: The invention comprises the stratosphere bacillus R6 according to claim 1 or the microbial preparation according to any one of claims 2 to 5.

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