Bacillus aryabhattai LZ01 and application thereof in preparing preparation for preventing and treating rice brown planthopper

Abstract

The application discloses bacillus aryabhattai LZ01 and application of the bacillus aryabhattai LZ01 in preparation of a preparation for preventing and treating brown planthopper, and provides the bacillus aryabhattai LZ01. After the bacillus aryabhattai LZ01 is used for treating a rice cultivation substrate, the resistance of a rice variety to the brown planthopper can be improved, the damage of the brown planthopper to the rice can be alleviated, and the bacillus aryabhattai LZ01 has a good application prospect.

Description

(I) Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a strain of Bacillus argentea that enhances rice's resistance to brown planthoppers and its applications. (II) Background Technology

[0002] Bacillus species possess strong resistance, rapid colonization ability, strong nutrient competition, and the ability to induce plant resistance. Numerous reports have documented their applications in plant disease control and growth promotion. For example, *Bacillus subtilis* can stably survive and reproduce on soil and plant root and leaf surfaces, secreting antibiotics and hormones that stimulate plant growth, thus promoting disease resistance in host plants and enabling them to fight pathogens and diseases. Domestic and international reports indicate that some Bacillus species have shown good control effects against bacterial wilt of tomatoes, scab of wheat, wilt of watermelons, bacterial wilt of tobacco, wilt of cotton, and sheath blight of rice, promoting increased yields and income. *Bacillus thuringiensis* produces insecticidal proteins and is widely used in plant pest control, showing good control effects against lepidopteran larvae such as cabbage caterpillars, rice leaf rollers, inchworms, pine caterpillars, tobacco budworms, corn borers, cotton bollworms, rice leaf rollers, bagworms, and cutworms.

[0003] Bacillus aryabhattai is widely distributed, found in soil, animal intestines, and plant rhizosphere. This bacterium is mainly used for substance degradation, natural product synthesis, promoting plant growth, and enhancing plant stress resistance. Bacillus aryabhattai has strong environmental adaptability, easily surviving and colonizing in farmland soils and plant habitats, and is environmentally friendly, safe, and non-toxic. There are also some research reports and patents related to its application in agriculture. Patents CN201610036163.4, CN202111203354, CN202111159072.7, and CN202210026480 describe several strains of Bacillus aryabhattai. These strains can produce auxin IAA, siderophores, ammonia, cellulase, protease, etc., and have the ability to dissolve organophosphorus compounds. They have a significant growth-promoting effect on plants such as tomatoes and potatoes, and some can effectively inhibit the growth of potato scab pathogens. They have application value in the biological control of potato scab and the improvement of saline-alkali soil. Patents CN201310559441.0, CN201310559441.0, CN201510296912.2, CN201910375594.7, CN201910836765.1, CN201910836765.1, and CN202210230797 provide information on *A.* Bacillus species exhibit inhibitory activity against pathogens such as *Pseudomonas aeruginosa* (the pathogen causing pine needle brown spot), *Pinus pineus* (the pathogen causing pine twig blight), *Poplar rot* (the pathogen causing poplar canker), *Apple ring rot* (the pathogen causing apple ring rot), *Tomato gray mold* (the pathogen causing tomato gray spot), *Tobacco red spot* (the pathogen causing tobacco red rot), *Watermelon soft rot* (the pathogen causing tobacco black shank), *Rhizopus oryzae* (the pathogen causing rice blast), *Fusarium wilt* (the pathogen causing rice scab), *Microsporum* (the pathogen causing potato scab), and aflatoxin. Additionally, there are patents related to *Bacillus oryzae* that can degrade cypermethrin (CN201611136508.X) and polyethylene plastics (CN202111159072.7). Reports also indicate that *Bacillus oryzae* can degrade sugarcane bagasse and produce glucose and fructose. Patent CN109706102A discloses *Bacillus oryzae* MB35-5 (CGMCC). (No. 17204) possesses silicon-degrading capabilities, can reproduce and grow in soil, and produces metabolic products such as organic acids and polysaccharides, degrading potassium minerals. Patents CN202010759957 and CN202310729816 disclose *Bacillus aureus*, which respectively demonstrates good control efficacy against crop root-knot nematodes and pine wilt disease. This shows that *Bacillus aureus* strains are highly diverse, possessing multiple functions and different application values.

[0004] Research and applications of Bacillus aureus are still not in-depth or widespread enough, especially the screening and application of Bacillus aureus strains with specific functions.

[0005] Rice is one of my country's most important food crops, with an annual output of over 210 billion kilograms, accounting for more than 40% of the total grain output. However, rice is subject to various external environmental stresses at every stage of its growth and development. It has been reported that hundreds of insect species can feed on rice. The brown planthopper (Nilaparvata lugens Stal.) is a monophagous pest of rice, belonging to the family Hemiptera (Delphacidae). Brown planthoppers suck sap from the phloem of rice plants using their stylets and also transmit plant viruses. Mild infestations of brown planthoppers reduce the vitality of rice plants and decrease yield. Large infestations cause the base of the rice plants to blacken, rot, and smell foul, leading to clumps of dead stalks and lodging within a short period, resulting in no harvest. Traditional control of brown planthoppers relies mainly on chemical pesticides. The extensive use of pesticides not only increases production costs and damages the ecological environment but also increases the pests' resistance by hundreds of times, drastically reducing the effectiveness of pesticides. The application of pesticides also eliminates the natural enemies of brown planthoppers in rice paddies, making it extremely easy for brown planthoppers to "re-emerge". Relying on effective pesticides can no longer achieve the goal of sustainable management of rice planthoppers (Lou Yonggen and Cheng Jiaan, 2011).

[0006] Currently, there are no research reports or patent applications on the use of Bacillus aureus to improve the control of brown planthoppers in rice. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Bacillus argentea LZ01 that significantly improves rice resistance to brown planthoppers and its application in the preparation of agents for controlling brown planthoppers in rice, thereby solving the problems of strong drug resistance, easy variation in pathogenicity, and serious environmental impact of chemical pesticides in the control of brown planthoppers in rice production.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a strain of Bacillus aryabhattai LZ01, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20241485, deposit date July 4, 2024, address Wuhan University, Wuhan, China.

[0010] The present invention also provides the application of the aforementioned Bacillus oryzae LZ01 in the preparation of a formulation for controlling brown planthoppers in rice.

[0011] Furthermore, the preparation is made from the bacterial culture of Bacillus oryzae LZ01 expanded culture and rice nutrient solution.

[0012] Furthermore, the rice nutrient solution refers to Yoshida rice nutrient solution (Beijing Kulaibo Technology Co., Ltd.). The bacterial cell concentration in the preparation is 0.5–1.0 × 10⁻⁶. 8 CFU / mL.

[0013] Furthermore, the bacterial culture was prepared as follows: *Bacillus aureus* LZ01 was inoculated onto LB agar plates and incubated at 37°C for 24 hours for activation; single colonies of the activated bacteria were picked and inoculated into LB liquid medium and incubated at 37°C for 24 hours to obtain the seed culture; the seed culture was then inoculated into LB medium at a volume concentration of 1–3% and incubated at 37°C until OD... 600 The bacterial solution was obtained by setting the concentration to 0.6–0.8, wherein the bacterial content was ≥1×10⁻⁶. 8 CFU / mL.

[0014] Furthermore, the application involves adding the preparation to a sterilized cultivation substrate and then sowing rice seeds to obtain brown planthopper-resistant rice plants, effectively improving the resistance of rice plants to brown planthoppers.

[0015] Furthermore, the cultivation substrate 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] This invention provides a novel strain, Bacillus argentea LZ01. After treating rice cultivation substrate with this strain, it can improve the resistance / tolerance of susceptible rice varieties to brown planthoppers, effectively control the damage caused by brown planthoppers to rice, and has good application prospects. (iv) Description of the attached drawings

[0019] Figure 1 Photograph of bacterial colonies of strain LZ01 on a PDA plate.

[0020] Figure 2 Photographs of colonies of strain LZ01 on LB agar plates, where a represents the reverse side and b represents the front side.

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

[0022] Figure 4 Example 3: Selective insect inoculation test results after applying different microbial agents to sandy substrate; A is the result on the day of inoculation; B1 and B2 are the results of two replicates 14 days after inoculation; C is the statistical analysis of resistance level of three replicates.

[0023] Figure 5Example 3: Selective insect inoculation test results after applying different microbial agents to soil matrix; A: before insect inoculation; B: 7 days after insect inoculation; C: 10 days after insect inoculation; D: 13 days after insect inoculation; E: Arthrobacters p. colony morphology on LB agar plates; F: resistance grade statistics 13 days after insect inoculation. (V) Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

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

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

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

[0028] Strain LZ01 was isolated from fly ash treatment wastewater from a thermal power plant in Jiaxing, Zhejiang Province. Fly ash washing solutions in the environmental protection industry are rich in inorganic salts and contain very few microorganisms, which have a strong ability to utilize minerals for growth.

[0029] 1. Initial screening

[0030] PDA medium: Spread 100 μL of fly ash water onto PDA medium plates and incubate at 28°C for 24 h.

[0031] LB medium: Spread 100 μL of fly ash water onto LB medium plates and incubate at 37°C for 24 h.

[0032] Colonies grew on both of the above-mentioned culture media during the initial screening. The colonies were larger on PDA plates and smaller on LB plates. They were later identified as the same bacterium.

[0033] 2. Expand cultivation and domestication

[0034] (1) Inoculate the single colony screened in step 1 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 at 30°C for 24 h.

[0035] (2) Take 100 μL of the bacterial culture that became turbid after 24 h of culture 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). Incubate at 30 °C for 24 h.

[0036] (3) Inoculate 100 μL of the bacterial culture that became turbid after 24 h of culture in step (2) 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 incubate at 30 °C for 72 h.

[0037] (4) After culturing for 72 hours in step (3), the bacterial culture was diluted 1000 times with sterile deionized water, and 100 μL was spread on a PDA medium plate and incubated at 30℃. Figure 1 Observe for colony growth. After colony growth, perform streak plating purification, preserve, and send for testing. Select the grown colonies and inoculate them again into 100 mL of fly ash water containing 0.001 mg / L glucose and 0.001 mg / L yeast extract. Incubate at 30°C for 72 h, then spread on a PDA and observe for colony growth. If growth occurs, it proves that this strain has been domesticated and can grow in fly ash water with very low nutrient content. Finally, one strain was obtained, named strain LZ01.

[0038] 3. Identification of strain LZ01

[0039] (1) Colony morphology

[0040] The selected strain LZ01 was inoculated onto LB plates and incubated at 37°C for 24 hours. Colonies were observed to be opaque, round or oval in shape, milky white in color, and smooth in surface. Figure 2 ).

[0041] Dilute strain LZ01 with deionized water for 10 minutes. 6 After dilution, 100 μL of the culture was spread onto LB medium. After incubation at 37°C for 24 h, a single colony was picked and inoculated into 4 mL of liquid LB medium, and incubated at 37°C for 16 h. 500 μL of the culture was taken and an equal volume of 50% sterile glycerol was added. The strain was stored at -80°C for identification as a pure culture strain.

[0042] (2) 16S rDNA

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

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

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

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

[0047] The amplified PCR product of approximately 1.4 kb was recovered, purified, and sequenced after 1.5% agarose gel electrophoresis (Hangzhou Shangya Biotechnology Co., Ltd.). The nucleotide sequence is shown in SEQ ID NO.1. Based on the obtained 16S rDNA sequence, a Blast search for homologous sequences was performed in GenBank. The similarity between strain LZ01 and Bacillus aryabhattai was over 99%.

[0048] The 16S rDNA sequence obtained by PCR amplification of strain LZ01 is 1458 bp in length.

[0049]

[0050] Based on the colony morphology and the 16S rDNA sequence of strain LZ01, strain LZ01 was identified as Bacillus aryabhattai and named Bacillus aryabhattai LZ01. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20241485 and deposit date July 4, 2024.

[0051] Example 2: Insect-resistant effect of Bacillus aureus LZ01 under greenhouse pot conditions tested by the occlusion method.

[0052] (1) Seed pretreatment: The seeds of the rice variety Taichung Native 1TN1, which is susceptible to insects, were soaked in tap water for 1 day and then germinated at 35℃ for 1 day until the rice seeds showed white sprouts.

[0053] (2) Formulation

[0054] Bacillus aryabhattai preparation: Bacillus aryabhattai LZ01 was inoculated onto LB agar plates and incubated at 37°C for 24 h for activation. A single colony of the activated Bacillus aryabhattai LZ01 was picked and added to 4 mL of LB liquid medium and incubated at 37°C for 24 h to obtain the seed culture. Then, 4 mL of the seed culture was inoculated into 200 mL of LB liquid medium and incubated at 37°C for 6 h until OD (Organic Depth) reached. 600 The value was 0.8, and the number of cells obtained was 1×10. 8 The bacterial suspension was prepared at CFU / mL. 200mL of the bacterial suspension was added to 800mL of Yoshida rice nutrient solution (purchased from Beijing Kulaibo Technology Co., Ltd.) to prepare a formulation (diluted 5 times, resulting in a viable count of 0.2 × 10⁻⁶). 8 (CFU / mL)

[0055] Blank preparation: Prepared by adding 200 mL of LB liquid culture medium to 800 mL of Yoshida rice nutrient solution.

[0056] (3) Sowing

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

[0058] In the experimental group, 0.5 mL of Bacillus oryzae preparation was added to each of the above flowerpots at a rate of 1 gram of dry sand. In the control group, an equal amount of blank preparation was added, and the mixture was stirred evenly. 13 seeds pretreated in step (1) were sown in each flowerpot. The pots were placed in a greenhouse (26±2℃). When the rice plants reached the three-leaf stage after 16 days, the weaker plants were removed, leaving 10 uniformly growing plants. 10 second-instar brown planthopper nymphs were introduced into each plant, and a 40.0×7.5cm cover was placed over each cover, meaning 100 nymphs were introduced into each cover. The growth of the rice was observed. On the 7th day, when the pest level of the control group rice reached 7-9, photos were taken. Figure 3 ), and record the experimental group level.

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

[0060] On day 7, the experimental group had an average resistance level of 5, while the control group had a resistance level of 9, with all members dying.

[0061] Example 3: Selective test to evaluate the insect-resistant effect of Bacillus aureus LZ01 under greenhouse pot conditions.

[0062] 1. Sandy soil matrix

[0063] (1) Seed pretreatment: The seeds of the insect-susceptible rice variety TN1 were soaked in tap water for 1 day and then germinated at 35℃ for 1 day until the rice seeds showed white sprouts.

[0064] (2) Formulation

[0065] Experimental formulation: Bacillus oryzae LZ01 formulation, same as in Example 2.

[0066] Control formulation: Replace Bacillus argentis LZ01 in the bacterial agent of Example 2 with Bacillus subtilis (BPH-S33, a symbiotic Bacillus isolated from brown planthopper (refer to Acta Entomologica Sinica, 2023, 66(10): 1289-1301), and perform the same other operations.

[0067] Blank preparation: Same as in Example 2.

[0068] (3) Sowing

[0069] Sterilized dry sand was packed into turnover boxes (30.0 cm long × 17.0 cm wide × 8.0 cm high). Each turnover box was divided into three compartments: blank control group (CK), control group (GN), and experimental group (AS). The experiment was repeated three times.

[0070] Before sowing, each compartment of each turnover box was thoroughly watered with tap water and 0.5 mL of the preparation was added per gram of soil. The blank control group was given the blank preparation, the control group was given the control preparation, and the experimental group was given the experimental preparation. Different treatments were applied to three compartments in each turnover box. 35 seeds pretreated in step (1) were sown in each compartment. The boxes were placed in a light incubator at 28±2℃ with 16h / 8h light / dark conditions. After 16 days, 30 rice plants with basically uniform growth were retained and inoculated with 2nd instar nymphs of brown planthoppers at a rate of 10 nymphs per plant. After inoculation, each turnover box was covered with a cover measuring length × width × height (40.0 × 30.0 × 30.0 cm). 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] See results Figure 4 On day 14, the average resistance level of the experimental group treated with Bacillus aureus LZ01 preparation for irrigating sandy soil was level 5, while the resistance levels of the CK and GN groups were levels 7-9.

[0072] 2. Soil substrate

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

[0074] (2) Formulation

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

[0076] The control formulation (AR) was prepared by replacing Bacillus aryabhattai LZ01 with Arthrobacter.sp (a symbiotic bacterium isolated from the intestine of brown planthopper, numbered BPH-S14, see Acta Entomologica Sinica, 2023, 66(10): 1289-1301), with other procedures being the same as those for the formulation in Example 2.

[0077] (3) Sowing

[0078] Replace the sand in step 1 with soil, which was collected from the experimental field of the China National Rice Research Institute in Fuyang. After drying, the soil was crushed and passed 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 1]. Figure 5 Seven days after inoculation, the AR group showed signs of leaf drop and ecchymosis in the rice seedlings. By day 10, seedlings had reached level 7, and by day 13, they were mostly at level 9. The experimental group treated with Bacillus LZ01 soil irrigated with the Bacillus preparation had an average resistance level of 6.1 by day 13, compared to 7.0 for the control group (CK) and 8.6 for the AR group.

Claims

1. Bacillus aryabhattai LZ01, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20241485, deposited on July 4, 2024, Wuhan University, Wuhan, China.

2. The use of Bacillus oryzae LZ01 as described in claim 1 in the preparation of a formulation for controlling brown planthoppers in rice.

3. The application as described in claim 2, characterized in that, The preparation is made from the bacterial culture of Bacillus oryzae LZ01 expanded culture and rice nutrient solution.

4. The application as described in claim 3, characterized in that, The rice nutrient solution mentioned is Yoshida rice nutrient solution.

5. The application as described in claim 3, characterized in that, The bacterial cell concentration in the preparation is 0.5–1 × 10⁻⁶. 8 CFU / mL.

6. The application as described in claim 3, characterized in that, The bacterial culture was prepared as follows: Bacillus aureus LZ01 was inoculated into LB agar plates and incubated at 37°C for 24 hours to activate it; single colonies of the activated bacteria were picked and inoculated into LB liquid medium and incubated at 37°C for 24 hours to obtain the seed culture. The seed culture was inoculated into LB medium at a volume concentration of 1-3% and cultured at 37°C until the OD600 reached 0.6-0.8 to obtain the bacterial culture.

7. The application as described in claim 2, characterized in that, The application involves adding the preparation to a sterilized cultivation substrate before sowing rice seeds, which effectively improves the resistance of rice plants to brown planthoppers.

8. The application as described in claim 7, characterized in that, The cultivation substrate includes sand and soil.

9. The application as described in 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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