Bacillus aryabhattai LZ01 and application of bacillus aryabhattai LZ01 in preparation of preparation for preventing and treating rice brown planthopper

Abstract

The invention discloses bacillus aryabhattai LZ01 and application thereof in preparing a preparation for preventing and treating rice brown planthopper, and provides the bacillus aryabhattai LZ01, after a rice culture medium is treated by the strain, the resistance / tolerance of insect-susceptible rice varieties to the brown planthopper can be improved, the damage of the brown planthopper to rice is relieved, and the bacillus aryabhattai LZ01 has a good application prospect.

Description

(I) Technical field

[0001] The invention relates to the field of agricultural microorganisms, and in particular to a strain of Bacillus aggradii capable of improving the resistance of rice to brown planthoppers and an application thereof. (II) Background technology

[0002] Bacillus has strong stress resistance, rapid colonization, strong nutritional competition, and the ability to induce plant resistance. There are many reports on its application in plant disease control and growth promotion. For example, Bacillus subtilis can survive and reproduce stably in the soil and on the surface of plant roots and leaves, and can secrete antibiotics and hormones that stimulate plant growth, prompting parasitic plants to develop disease resistance and fight against pathogenic diseases. Some Bacillus species reported at home and abroad have good control effects on tomato bacterial wilt, wheat fusarium wilt, watermelon wilt, tobacco bacterial wilt, cotton wilt, rice sheath blight, etc., and promote increased production and income. Bacillus thuringiensis can produce proteins with insecticidal activity, which are widely used in plant pest control and have good control effects on lepidopteran pest larvae, such as cabbage worms, rice sheathworms, loopers, pine caterpillars, tobacco budworms, corn borers, cotton bollworms, rice leaf rollers, moths, cutworms, etc.

[0003] Bacillus aryabhattai is widely distributed and has been found in soil, animal intestines, plant rhizosphere, etc. The bacteria are mainly used in substance degradation, natural product synthesis, plant growth promotion, and improving plant stress resistance. Bacillus aryabhattai has strong environmental adaptability, is easy to survive and colonize in habitats such as farmland soil and plants, and has the characteristics of being environmentally friendly, safe and non-toxic. There are also some research reports and patents in agriculture and other aspects. Patents CN201610036163.4, CN202111203354, CN202111159072.7, and CN202210026480 describe several strains of Bacillus aryabhattai. These strains can produce IAA, siderophore, ammonia, cellulase, protease, etc., have the ability to dissolve organophosphorus compounds, and have a significant growth-promoting effect on plants such as tomatoes and potatoes. Some can effectively inhibit the growth of potato scab pathogens, and have application value in the biological control of potato scab and the improvement of saline-alkali land; Patents CN201310559441.0, CN201310559441.0, CN201510296912.2, CN201910375594.7, CN201910836765.1CN201910836765.1, CN202210230797 provide Aspergillus niger Bacillus has the activity of inhibiting pathogens, such as pine needle brown spot pathogen pine needle disc spore fungus, pine dieback pathogen, poplar rot pathogen, apple ring rot pathogen, tomato gray mold pathogen, tobacco red star pathogen, watermelon soft rot pathogen, tobacco black shank disease, rice blast, wilt, potato scab, microsporus, aflatoxin, etc.; in addition, there are patents related to Bacillus argyi that can degrade cypermethrin (CN201611136508.X) and polyethylene plastic (CN202111159072.7). There are also reports showing that Bacillus argyi can degrade sugarcane bagasse and produce glucose and fructose; Bacillus argyi MB35-5 (CGMCC No.: 17204) has the function of dissolving silicon, can reproduce and grow in the soil, and produce metabolites such as organic acids and polysaccharides, degrading mineral potassium; the Bacillus arguta provided by patents CN202010759957 and CN202310729816 have good preventive effects on crop root-knot nematodes and the function of preventing and controlling pine wood nematode disease. It can be seen that Bacillus arguta strains are rich in diversity and have multiple functions and different application values.

[0004] The related research and application of Bacillus arginense are still not deep and extensive enough, especially the screening and application of Bacillus arginense strains with specific functions are rare.

[0005] Rice is one of the most important food crops in my country, with a total annual rice output of more than 210 billion kilograms, accounting for more than 40% of the total grain output. However, rice is subject to various external environmental factors at all stages of growth and development. According to reports, hundreds of insect species have been found to feed on rice. Brown planthopper (Nilaparvata lugens Stal.) is a monophagous rice pest belonging to the family Hemiptera: Delphacidae. Brown planthoppers suck the sap from the phloem of rice through their mouth needles and spread plant viruses. Mild damage by brown planthoppers leads to reduced growth vitality and yield of rice plants. When the number of insects is large, the base of the rice will turn black, rot and become smelly. In a short period of time, the rice will clump and die in pieces, and no grain will be harvested. Traditional brown planthopper control is mainly based on chemical pesticides. The large-scale use of pesticides not only increases production costs and damages the ecological environment, but also increases the resistance of pests by hundreds of times, and the efficacy of the pesticides drops sharply. The application of pesticides also eliminated the natural enemies of brown planthoppers in rice fields, which can easily lead to the "re-emergence" of brown planthoppers. Relying on special pesticides can no longer achieve the goal of sustainable control of rice planthoppers (Lou Yonggen and Cheng Jiaan, 2011).

[0006] At present, there are no research reports or patent applications on the use of Bacillus aeruginosa to improve the control of rice against brown planthoppers. (III) Summary of the invention

[0007] The present invention aims to provide a strain of Bacillus agni LZ01 which can significantly improve the resistance of rice to brown planthoppers and its application in preparing a preparation for controlling rice brown planthoppers, so as to solve the problems of strong drug resistance, easy variation of harmfulness and serious environmental impact of chemical pesticides in the control of brown planthoppers in rice production.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a strain of Bacillus aryabhattai LZ01, which is deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20241485, a deposit date of July 4, 2024, and an address of Wuhan University, Wuhan, China.

[0010] The present invention also provides an application of the Bacillus arifugeii LZ01 in preparing a preparation for controlling rice brown planthoppers.

[0011] Furthermore, the preparation is prepared by mixing the bacterial solution of expanded culture of Bacillus aggrad LZ01 and rice nutrient solution.

[0012] Furthermore, the rice nutrient solution refers to Yoshida rice nutrient solution (Beijing Coolaibo Technology Co., Ltd.). The bacterial concentration in the preparation is 0.5-1.0×10 8 CFU / mL.

[0013] Further, the bacterial solution is prepared as follows: Bacillus aggregatus LZ01 is inoculated into LB plate culture medium and cultured at 37°C for 24 hours for activation; a single colony after activation is picked and inoculated into LB liquid culture medium and cultured at 37°C for 24 hours to obtain seed solution; the seed solution is inoculated into LB culture medium at an inoculum concentration of 1 to 3% by volume and cultured at 37°C until OD 600 is 0.6 to 0.8, and the bacterial solution is obtained, wherein the bacterial content is ≥ 1×10 8 CFU / mL.

[0014] Furthermore, the application is to add the preparation into a sterilized cultivation substrate, and then sow rice seeds to obtain rice plants resistant to brown planthoppers, thereby effectively improving the resistance of rice plants to brown planthoppers.

[0015] Furthermore, the cultivation medium includes sand or soil.

[0016] Furthermore, the dosage of the preparation is 0.5-1.0 mL / g based on the weight of the cultivation substrate.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] The invention provides a new strain, Bacillus arifugeii LZ01, which can improve the resistance / tolerance of susceptible rice varieties to brown planthoppers after treating rice cultivation substrates, effectively prevent and control the damage of brown planthoppers to rice, and has good application prospects. (IV) Description of the drawings

[0019] Figure 1 , Colony photos of strain LZ01 on PDA plate.

[0020] Figure 2 , Colony photos of strain LZ01 on LB medium plate, a represents the reverse side, b represents the front side.

[0021] Figure 3 , Example 2: Insect resistance of rice after applying strain LZ01 in sandy soil matrix; A represents the 7-day covering state; B represents the covering removal state; C represents the insect resistance level of the experimental group and the blank group.

[0022] Figure 4 , Example 3: Test results of selective inoculation after applying different bacterial agents in sandy soil matrix; A is the day after inoculation; B1 and B2 are the results of two repetitions 14 days after inoculation; C is the resistance level statistics of three repetitions.

[0023] Figure 5, Example 3 Test results of selective insect inoculation after applying different bacterial agents in the soil matrix; A is before insect inoculation; B is 7 days after insect inoculation; C is 10 days after insect inoculation; D is 13 days after insect inoculation; E is the colony morphology of Arthrobacter sp. on the LB plate; F is the resistance level statistics 13 days after insect inoculation. (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] LB medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1 L distilled water, pH 7.0.

[0026] PDA medium (1 L): 200 g potato, 20 g glucose, 15 g agar, 1 L distilled water, natural pH.

[0027] Example 1. Screening and identification of Bacillus aryabhattai LZ01

[0028] The strain LZ01 was isolated from the fly ash treatment wastewater of the Jiaxing thermal power plant in Zhejiang. The fly ash washing liquid in the environmental protection industry is rich in inorganic salts and contains very few microorganisms, which have a strong ability to grow using minerals.

[0029] 1. Initial screening

[0030] PDA medium: 100 μL of fly ash water was spread on a PDA medium plate and cultured in a 28°C incubator for 24 h.

[0031] LB medium: 100 μL of fly ash water was spread on an LB medium plate and cultured in a 37°C incubator for 24 h.

[0032] Colonies grew on both of the above culture media in the initial screening. On the PDA plate, the colonies were larger. On the LB plate, the colonies were smaller. They were later identified as the same bacteria.

[0033] 2. Expand cultivation and domestication

[0034] (1) The single colony screened in step 1 was inoculated into a flask containing 100 mL of fly ash water containing 0.1 mg / L glucose (carbon source) and 0.1 mg / L yeast extract (nitrogen source), and cultured at 30°C for 24 h.

[0035] (2) Take 100 μL of the bacterial solution that becomes turbid after culturing for 24 h in step (1) and inoculate it into a flask containing 100 mL of fly ash water containing 0.1 mg / L glucose (carbon source) and 0.1 mg / L yeast extract (nitrogen source), and incubate it at 30°C for 24 h.

[0036] (3) 100 μL of the turbid bacterial solution obtained in step (2) after culturing for 24 h was inoculated into a flask containing 100 mL of fly ash water containing 0.01 mg / L glucose (carbon source) and 0.01 mg / L yeast extract (nitrogen source), and the mixture was incubated at 30°C for 72 h.

[0037] (4) After 72 hours of culture in step (3), the bacterial solution was diluted 1000 times with sterile deionized water, and 100 μL was spread on a PDA medium plate and cultured at 30°C ( Figure 1 ). Observe whether there are colonies growing. After the colonies grow, purify them by streaking, store them, and send them for testing. Select the colonies that grow and inoculate them again into 100mL of fly ash water containing 0.001mg / L glucose and 0.001mg / L yeast extract. After culturing at 30℃ for 72h, apply PDA and observe whether there are colonies growing. If there is growth, it proves that this strain has been domesticated and can grow in fly ash water with extremely low (nutrient) feed amounts. Finally, a strain was obtained, recorded as strain LZ01.

[0038] 3. Identification of strain LZ01

[0039] (1) Colony morphology

[0040] The screened strain LZ01 was inoculated onto an LB plate and cultured at 37°C for 24 h. The colonies were observed to be opaque, round or oval, milky white, and smooth ( Figure 2 ).

[0041] The strain LZ01 was diluted with deionized water to 10 6 After the culture was doubled, 100 μL was spread on LB medium. After culturing at 37°C for 24 hours, a single colony was picked and inoculated into 4 mL of liquid LB medium and cultured at 37°C for 16 hours. 500 μL of culture solution was added with an equal volume of 50% sterilized glycerol and the strain was stored in a -80°C refrigerator for identification as a pure culture strain.

[0042] (2) 16S rDNA

[0043] Take 2 mL of the culture solution from step (1) and centrifuge at 8000 rpm for 10 minutes to precipitate the bacteria. Use a bacterial genomic DNA extraction kit to extract genomic DNA. Use the genomic DNA as a template and use 16S rDNA universal primers 27f and 1492r to perform PCR amplification of 16S rDNA.

[0044] 27f: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0045] 1492r: 5'-GGTTACCTTGTTACGACTT-3'.

[0046] PCR reaction conditions: 94°C pre-denaturation for 30 s; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 60 s, 35 cycles.

[0047] The amplified PCR product of about 1.4 kb was purified and sequenced after 1.5% agarose gel electrophoresis (Hangzhou Shangya Biotechnology Co., Ltd.), and the nucleotide sequence is shown in SEQ ID NO. 1. Based on the obtained 16SrDNA sequence, the homologous sequence was searched in GenBank by Blast, and the similarity between strain LZ01 and Bacillus aryabhattai was more than 99%.

[0048] The 16SrDNA sequence obtained by PCR amplification of strain LZ01 was 1458 bp in length.

[0049]

[0050] Based on the colony morphological characteristics and the 16SrDNA sequence of strain LZ01, strain LZ01 was identified as Bacillus aryabhattai, named Bacillus aryabhattai LZ01, and deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M20241485 and the deposit date of July 4, 2024.

[0051] Example 2: Testing the insect-resistant effect of Bacillus agni LZ01 in greenhouse potted plants by cage method

[0052] (1) Seed pretreatment: Seeds of the insect-susceptible rice variety Taichung Native 1TN1 were soaked in tap water for 1 day and then germinated at 35°C for 1 day until the rice seeds turned white.

[0053] (2) Preparation

[0054] Preparation of Bacillus aryabhattai LZ01: Inoculate Bacillus aryabhattai LZ01 on LB plate and culture at 37℃ for 24h for activation; pick a single colony of Bacillus aryabhattai LZ01 after activation and add it to 4mL LB liquid culture medium, culture at 37℃ for 24h to obtain seed liquid. Then inoculate 4mL seed liquid into 200mL LB liquid culture medium and culture at 37℃ for 6h until OD 600 The cell number was 1×10 8 CFU / mL of bacterial solution. Take 200mL of bacterial solution and add it to 800mL of Yoshida rice nutrient solution (purchased from Beijing Coolbo Technology Co., Ltd.) to make a preparation (dilute 5 times, that is, the number of viable bacteria is 0.2×10 8 CFU / mL).

[0055] Blank preparation: 200 mL of LB liquid medium was added to 800 mL of Yoshida rice nutrient solution.

[0056] (3) Seeding

[0057] Sterilized dry sand was placed in small flower pots (10.0 cm in height × 7.5 cm in diameter) and soaked with tap water. The small flower pots were divided into an experimental group (AS) and a control group (CK), with 3 replicates in each group.

[0058] In each of the above flower pots in the experimental group, 0.5 mL of Bacillus aeruginosa preparation was added per gram of dry sand weight, and in the control group, an equal amount of blank preparation was added, and the mixture was stirred evenly. Thirteen seeds pretreated in step (1) were sown in each flower pot. The seeds were placed in a greenhouse (26±2°C). After 16 days, when the rice had grown to three leaves and one heart, the weakly growing rice plants were cut off, and 10 rice plants with uniform growth were retained. Ten 2-year-old brown planthopper nymphs were inoculated per plant, and a 40.0×7.5 cm cage was placed, i.e., 100 nymphs were inoculated in each cage. The growth of the rice was observed. When the rice pest level in the control group reached level 7-9 on the 7th day, photos were taken ( Figure 3 ), and record the experimental group level.

[0059] Level: The level of damage to rice seedlings by brown planthoppers is evaluated according to the group standard T / ZNZ 023-2020 "Technical Procedures for Identification of Rice Resistance to Brown Planthoppers" of the Zhejiang Provincial Agricultural Product Quality and Safety Society. Level 1: "Extremely slight damage"; Level 3: "Partial yellowing of the first and second leaves of most plants"; Level 5: "Obvious yellowing, nearly half of the plants are wilting or dying"; Level 7: "More than half of the plants are dying"; Level 9: "All plants are dying".

[0060] On the 7th day, the average resistance level of the experimental group was 5, while all deaths in the control group were at level 9.

[0061] Example 3: Selective test to test the insect-resistant effect of Bacillus aeruginosa LZ01 in greenhouse potted plants

[0062] 1. Sandy soil matrix

[0063] (1) Seed pretreatment: Seeds of the insect-susceptible rice variety TN1 were soaked in tap water for one day and then germinated at 35°C for one day until the rice seeds turned white.

[0064] (2) Preparation

[0065] Experimental preparation: The preparation of Bacillus arnoldii LZ01 is the same as that in Example 2.

[0066] Control preparation: The Bacillus axothrix LZ01 in the bacterial agent of Example 2 was replaced with Bacillus subtilis (isolated from the symbiotic Bacillus BPH-S33 in the brown planthopper (refer to Acta Entomologica Sinica, 2023, 66(10): 1289-1301), and the other operations were the same.

[0067] Blank preparation: same as Example 2.

[0068] (3) Seeding

[0069] The sterilized dry sand was placed in a turnover box (length 30.0×width 17.0×height 8.0 cm), and each turnover box was divided into three compartments, namely the blank control group (CK), the control group (GN), and the experimental group (AS). Repeat 3 times.

[0070] Before sowing, each grid of each group of turnover boxes was irrigated with tap water and 0.5 mL of the preparation was added per gram of soil. The blank control group was added with the blank preparation, the control group was added with the control preparation, and the experimental group was added with the experimental preparation. Three grids of each turnover box were added with different treatments. 35 seeds pretreated in step (1) were sown in each grid. Placed in a light incubator at 28±2°C, 16h / 8h light / dark. After 16 days, 30 rice plants with basically the same growth were retained and inoculated with 2nd instar nymphs of brown planthoppers at a rate of 10 heads / plant. After inoculation, each turnover box was covered with a cage of length×width×height (40.0×30.0×30.0cm). The distribution of brown planthoppers on the rice plants was observed after 2 days. The resistance level of the rice plants was recorded and photographed on the 10th day.

[0071] Results Figure 4 On the 14th day, the average resistance level of the experimental group treated with Bacillus agglutinosa LZ01 preparation for irrigation of sand was 5, while that of the CK group and GN group was 7-9.

[0072] 2. Soil matrix

[0073] (1) Seed pretreatment: same as step 1.

[0074] (2) Preparation

[0075] The experimental preparation (AS) and blank preparation (CK) were prepared in the same manner as in step 1.

[0076] The control preparation (AR) is a preparation in which Bacillus aryabhattai LZ01 is replaced with Arthrobacter.sp (a symbiotic bacteria isolated from the intestine of brown planthopper, numbered BPH-S14, reference: Acta Entomologica Sinica, 2023, 66(10): 1289-1301), and other operations are the same as those of the preparation in Example 2.

[0077] (3) Seeding

[0078] The sand in step 1 was replaced with soil collected from the experimental field of the Chinese Academy of Rice Research in Fuyang. After drying, the soil was crushed with a hammer and sieved through a 20-mesh sieve to remove impurities.

[0079] The experimental conditions and methods of step 1 were used, and the results are shown in Figure 5 , 7 days after inoculation, the AR group showed dryness of rice seedlings, 7-level seedlings appeared on the 10th day, and basically 9-level on the 13th day. The experimental group treated with Bacillus LZ01 preparation for soil irrigation had an average resistance level of 6.1 on the 13th day, the control group CK was 7.0, and the AR group was 8.6.

Claims

1. Bacillus aryabhattai LZ01, deposited in China Center for Type Culture Collection, CCTCC NO: M20241485, deposit date July 4, 2024, address: Wuhan University, Wuhan, China.

2. Use of the Bacillus arifugeyi LZ01 according to claim 1 in preparing a preparation for controlling rice brown planthopper.

3. The use according to claim 2, characterized in that The preparation is prepared by mixing bacterial liquid of expanded culture of Bacillus aggradi LZ01 and rice nutrient solution.

4. The use according to claim 3, characterized in that The rice nutrient solution is Yoshida rice nutrient solution.

5. The use according to claim 3, characterized in that The bacterial concentration in the preparation is 0.5 to 1×10 8 CFU / mL.

6. The use according to claim 3, characterized in that The bacterial solution is prepared as follows: Bacillus aggregatus LZ01 is inoculated into LB plate culture medium and cultured at 37°C for 24 hours for activation; a single colony after activation is picked and inoculated into LB liquid culture medium and cultured at 37°C for 24 hours to obtain seed solution; The seed liquid is inoculated into LB medium at an inoculum volume concentration of 1-3%, and cultured at 37° C. until OD600 is 0.6-0.8 to obtain the bacterial liquid.

7. The use according to claim 2, characterized in that The application is to add the preparation into the sterilized cultivation substrate and then sow rice seeds, so as to effectively improve the resistance of rice plants to brown planthoppers.

8. The use according to claim 7, characterized in that The cultivation medium includes sand and soil.

9. The use according to claim 7, characterized in that The dosage of the preparation is 0.5-1.0 mL / g based on the weight of the cultivation substrate.

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