Bacillus velezensis 30833 and its application

Abstract

The present invention discloses a Bacillus velezensis strain 30833, which is deposited in the General Microbiology Center of the China National Committee for the Collection of Microorganisms, with a deposit number of CGMCC No. 24597. The present invention provides a microbial agent containing strain 30833, and uses thereof. The strain 30833 of the present invention has a high preventive effect, with a preventive effect of 85.72%-91.95% against wilt diseases such as tomato wilt; secondly, it has a broad prevention and control spectrum, with 30833 having a high preventive effect against wilt diseases such as tomato wilt, watermelon wilt, and cucumber wilt caused by Fusarium oxysporum; in addition, the strain 30833 of the present invention also has the advantages of strong specialization, long-lasting efficacy, low resistance to drug resistance, and environmental friendliness.

Description

Technical Field

[0001] The invention belongs to the field of biocontrol microorganisms, and particularly relates to Bacillus velezensis, and also relates to a microbial agent produced by utilizing the bacterium, and uses thereof. Background Art

[0002] Fusarium oxysporum is a soil-borne pathogenic fungus with a worldwide distribution. It has a very wide host range and can cause wilt diseases in more than 100 plant species, including melons, Solanaceae, bananas, cotton, legumes, and flowers. Continuous cropping for many years will lead to increasingly severe diseases, causing huge losses to the production of economic crops such as melons.

[0003] At present, production mainly relies on soil fumigation, grafting technology and chemical agents to control wilt disease of vegetable plants. However, these control methods have the following problems: (1) Soil fumigation not only destroys the balance of soil microbial flora, but also causes serious soil and ecological environment pollution. (2) Although the use of grafting technology to control watermelon and cucumber wilt has a certain effect, it directly affects the quality and taste of the fruit, and this method is time-consuming and labor-intensive. (3) The use of chemical pesticides for control is prone to drug resistance and environmental pollution. The use of biocontrol microorganisms for biological control of plant diseases has received more and more attention due to its advantages such as high prevention efficiency, long-lasting drug effect, strong specialization, low resistance to drug resistance and environmental friendliness.

[0004] At present, the biocontrol microorganisms used to prevent and control wilt caused by Fusarium oxysporum mainly include Trichoderma fungi such as Trichoderma harzianum (Zhang Heqing et al. Sichuan Agricultural Science and Technology. 2020 (05): 35-37), and Bacillus bacteria such as Bacillus polymyxa, Bacillus megaterium, Bacillus subtilis, and Bacillus amyloliquefaciens. Their control efficiency against wilt is generally 60%-79%. Since the types of Fusarium oxysporum present in different ecological environments and different crop conditions are different, it is necessary to continuously screen strains that can prevent and control Fusarium oxysporum under corresponding environmental conditions in order to solve the problem in a targeted manner. Bacillus is a dominant species in the natural environment. Because it can produce a variety of antibacterial active substances (Li Baoqing et al. Chinese Agricultural Science. 2010, 43 (017): 3547-3554), and can form stress-resistant and heat-resistant spores, it is beneficial to product development and shelf life extension. It has a good safety evaluation and is easy to scale up production. Therefore, it has become an important resource for the development of microbial fungicides.

[0005] Fusaric acid (5-butyl-2-pyridinecarboxylic acid) is a non-specific toxin secreted by Fusarium oxysporum. It can increase the permeability of host plant cell membranes, reduce the content of mitochondrial reactive oxygen species in host cells, hinder ATP synthesis, cause plant metabolic disorders, and cause plant wilt and death (Zhang Jiacheng et al. Grassland Science. 2021, 38(08): 1513-1524). Fusaric acid can also inhibit the growth and metabolism of various microorganisms, including beneficial microorganisms (Bacon CW et al. Journal of applied microbiology. 2006, 100(1): 185-194; Raza W, et al. Plant Pathology. 2015, 64(5): 1041-1052). Therefore, the Fusarium oxysporum, which is present in large quantities in the rhizosphere of plants, inhibits the growth and colonization of rhizosphere biocontrol bacteria by producing fusaric acid (Chen Qiaohuan et al. Chinese Journal of Experimental Traditional Chinese Medicine. 2021, 27(11):7), especially the growth and colonization of Bacillus (Guo Qinggang et al. Acta Phytophylacica Sinica. 2013, 40(1):45-50), thereby reducing the biocontrol effect of biocontrol bacteria. Therefore, screening Bacillus that are both tolerant to fusaric acid and have strong antagonistic activity against Fusarium oxysporum will have the potential to become biocontrol bacteria for controlling crop wilt, and thus be used as an effective means to control plant wilt caused by Fusarium oxysporum. Summary of the Invention

[0006] In order to solve the problem of plant wilt caused by Fusarium oxysporum, the present invention aims to provide a Bacillus velezensis 30833.

[0007] Another object of the present invention is to provide a microbial agent containing the above-mentioned Bacillus Velez 30833.

[0008] The third object of the present invention is to provide uses of the above-mentioned Bacillus Velez 30833 and its bacterial agent.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] The invention discloses a Bacillus velezensis strain 30833, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 24597.

[0011] The present invention also provides the use of the Bacillus velezensis strain 30833 in preventing and treating crop wilt diseases such as tomato wilt, watermelon wilt or cucumber wilt.

[0012] The present invention also provides a microbial agent, which contains the above-mentioned Bacillus Velez subtilis strain 30833.

[0013] The present invention also provides the use of the microbial agent in preventing and treating crop wilt diseases such as tomato wilt, watermelon wilt or cucumber wilt.

[0014] The pathogen of tomato wilt is Fusarium oxysporumf.sp.lycopersici Snyder et Hansen.

[0015] The pathogen of watermelon wilt disease is Fusarium oxysporum f.sp. Hiveum (EF Smith) Wollen.

[0016] The pathogen of cucumber wilt is Fusarium oxysporum (Schl.) F. sp cucumerinum Owen.

[0017] A genetically engineered bacterium, wherein the starting strain of the genetically engineered bacterium is the above-mentioned Bacillus velezensis strain 30833, and the genetically engineered bacterium has the function of preventing and treating tomato wilt, watermelon wilt or cucumber wilt.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects: (1) High prevention efficiency. The prevention efficiency against tomato wilt is 91.95%, the prevention efficiency against watermelon wilt is 88.92%, and the prevention efficiency against cucumber wilt is 85.72%. (2) Broad prevention and control spectrum. It has high prevention efficiency against tomato wilt, watermelon wilt, cucumber wilt and other wilt diseases caused by Fusarium oxysporum. (3) The Bacillus Velezii of the present invention is a strain screened after high-temperature treatment at 80°C. It is resistant to high temperatures and can form stress-resistant and heat-resistant spores, which is beneficial to product development and shelf life extension. (4) The strain 30833 of the present invention also has the advantages of strong specialization, long-lasting efficacy, not easy to develop drug resistance and environmental friendliness.

[0019] Biological preservation: The Bacillus velezensis strain 30833 of the present invention is a sample collected by the inventor from healthy tomato plants and their soil in a diseased greenhouse in Handan, Hebei in August 2013, and was obtained by screening in an 80°C water bath and under the selection pressure of wilt pathogenic bacteria. It was deposited in the General Microbiology Center of the China Culture Collection Administration on March 28, 2022. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No. 24597. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis pattern of PCR amplification of the 16S rDNA gene of the strain 30833 of the present invention is shown.

[0021] Figure 2 This is the phylogenetic tree of strain 30833 constructed based on the 16S rDNA gene sequence.

[0022] Figure 3 The electrophoresis pattern of the gyrB gene of the strain 30833 of the present invention is shown in FIG.

[0023] Figure 4 This is the phylogenetic tree of strain 30833 constructed based on the gyrB gene sequence.

[0024] Figure 5 This is a bar chart comparing tomato yields treated with strain 30833. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific embodiments.

[0026] Example 1 Screening of biocontrol microorganisms that are resistant to fusaric acid and have antagonistic effects on Fusarium oxysporum

[0027] Proceed as follows:

[0028] (1) In the area where cucumber, tomato and watermelon wilt disease occurred, a total of 48 soil samples were collected from the rhizosphere of healthy plants. Each soil sample weighed 1 g and was added to a conical flask containing 10 mL of distilled water. The soil samples were shaken at 30°C and 180 rpm for 30 minutes, then taken out and allowed to stand at room temperature for 30 minutes. Then 1 mL of the supernatant was taken and placed at 80°C for 15 minutes, and cooled at room temperature to obtain soil suspensions of Bacillus in the form of spores.

[0029] (2) Dilute the soil suspension obtained in step (1) 100-fold with distilled water; take 100 μL of the diluted soil suspension and evenly spread it on LB medium containing 20 μg / mL fusaric acid (LB medium prepared according to conventional methods), and culture it at 37°C for 12 hours. Select the vigorously growing colonies, which are the fusaric acid-tolerant Bacillus.

[0030] (3) Antibacterial test: The target bacteria were Fusarium oxysporum f.sp.lycopersici FQ143, Fusarium oxysporum f.sp.Niveum XG0010 and Fusarium oxysporum f.sp.cucumerinum FOC 1-2-11, the pathogens of tomato, watermelon and cucumber wilt, respectively (all three pathogens were obtained from the Plant Disease Biological Control Laboratory, Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences). The pathogens were inoculated on PDA plates in advance and cultured at a constant temperature of 25°C until the mycelium on the plate covered the entire culture dish. Then, a 6 mm diameter bacterial block was punched out with a hole punch and inoculated on the center of a new pre-poured PDA plate with a diameter of 9 cm. The above-screened bacteria resistant to fusaric acid were inoculated at a distance of 2.5 cm and cultured in an incubator at 25° C. for 5 days. Bacteria resistant to fusaric acid and with an inhibition zone of more than 0.5 cm in three antibacterial activity tests were selected.

[0031] Results: 68 biocontrol strains were selected from the 500 endophytic microorganisms tolerant to fusaric acid, which had antibacterial effects (inhibition zone > 0.5 cm) on three pathogens (Fusarium oxysporum cucumber-specific type, watermelon-specific type and tomato-specific type) and were tolerant to fusaric acid.

[0032] Example 2 Preparation of biocontrol bacteria fermentation liquid of the present invention

[0033] The 68 fusaric acid-tolerant biocontrol bacteria obtained in Example 1 were inoculated into 5 mL of liquid LB medium, shaken and cultured at 180 rpm and 37°C for 12 h, and then transferred to 200 mL of LB liquid medium at a ratio of 1%, shaken and cultured at 180 rpm and 37°C for 48 h to obtain fermentation broths of 68 strains, and the bacterial cell concentration (CFU / mL) in the fermentation broth was calculated by the plate count method.

[0034] Example 3 Screening test of biocontrol strains with preventive effect on cucumber wilt

[0035] Proceed as follows:

[0036] (1) Cucumber (Zhongnong No. 6, highly susceptible to cucumber wilt) seed germination and seedling cultivation: Soak the cucumber seeds in 55°C warm water for 30 minutes, rinse with clean water, and then soak in clean water for 8 hours. After soaking, wrap them with wet gauze, germinate them in a 25°C incubator overnight, and then grow them in vermiculite.

[0037] (2) Application of biocontrol bacteria fermentation liquid: When the cucumbers grew two leaves and one heart, the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2) was applied, with 3 mL of the bacterial liquid applied to the roots of each seedling.

[0038] (3) Preparation of pathogen FOC 1-2-11 (cucumber-specific type) bacterial solution and soil mixing: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus blocks and inoculate them into PDB liquid culture medium. Cultivate at 25°C and 180 rpm for 5 days. Count the spores after filtering through sterile gauze. Mix 1 L of pathogen bacterial solution with 20 kg of sterile soil in a soil mixer to a final concentration of 5.0 × 10 5 Spore / Ksutsu.

[0039] (4) Transplanting: Transplant the seedlings 3 days after the transplantation. During the transplanting process, align the roots of the cucumber seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Then transplant them into small flower pots, and set up three replicates for each treatment.

[0040] (5) Second application of biocontrol bacteria: Three days after transplanting, apply the second application of biocontrol bacteria (the biocontrol bacteria fermentation liquid prepared in Example 2) with the same usage and dosage as the first application of biocontrol bacteria. Water and observe on time, and investigate the disease condition after 10 days.

[0041] Grading standards for cucumber wilt at the seedling stage (refer to the standards reported by Zhou Hongmei, Mao Aijun, etc.).

[0042] Disease index = ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value) × 100 (the same below).

[0043] Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100 (the same below).

[0044] (6) Select strains with a biocontrol effect of more than 60% for rescreening, and finally select 5 strains of biocontrol bacteria with high control effect.

[0045] The results showed that the strain named 30833 had a primary screening control effect of 84.16% against cucumber wilt, and a secondary screening control effect of 85.72%, indicating that the strain 30833 has a high and stable control effect against cucumber wilt.

[0046] Example 4 Screening test of biocontrol bacteria with preventive effect on tomato wilt

[0047] Proceed as follows:

[0048] (1) Germination and seedling cultivation of tomato seeds (Nami, highly susceptible to tomato wilt): Rinse the tomato seeds with clean water, wrap them with wet gauze after cleaning, germinate them in a 25℃ incubator overnight, and then grow them in vermiculite.

[0049] (2) Application of biocontrol bacteria fermentation liquid: When the tomato has two leaves and one heart, apply the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2). Dilute 150 mL of the fermentation liquid by half and pour it into the seedling tray.

[0050] (3) Preparation of pathogen FQ 143 (tomato-specific type) bacterial suspension: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus pieces, inoculate them into PDB medium, and shake at 25°C and 180 rpm for 5 days. After filtering through sterile gauze, count the spores at a concentration of 4 × 10 7 spores / ml.

[0051] (4) Transplanting: Transplant the seedlings 3 days after the transplantation. During the transplanting process, align the roots of the tomato seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Soak the pathogen solution below the roots for 60 minutes, and then transplant them into flower pots. Set up three replicates for each treatment.

[0052] Water and observe on time, checking the condition for the first time after about 7 days and the second time after two weeks.

[0053] Grading standards for tomato wilt at the seedling stage (refer to the standards reported by Bai Minzhan, Zheng Guibin, etc.).

[0054] The results showed that one of the strains, named 30833, had a 7-day control effect on tomato Fusarium wilt of 89.96% and a 15-day control effect of 91.95%, indicating that the 30833 strain had a high control effect on tomato Fusarium wilt.

[0055] Example 5 Screening test of biocontrol strains with preventive effect on watermelon wilt

[0056] Proceed as follows:

[0057] (1) Germination and seedling cultivation of watermelon seeds (Zaojia 84-24, highly susceptible to watermelon wilt): Soak the watermelon seeds in 55°C warm water for 30 minutes, stirring constantly, rinse with clean water, and then soak in clean water for 8 hours. After soaking, wrap them with wet gauze, germinate them in a 25°C incubator overnight, and then grow them in vermiculite.

[0058] (2) Application of biocontrol bacteria fermentation liquid: When the watermelon has two leaves and one heart, the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2) is applied, and 3 mL of the bacterial liquid is applied to the root of each seedling.

[0059] (3) Preparation of pathogen XG0010 (watermelon-specific type) bacterial solution and soil mixing: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus blocks, inoculate them into PDB medium, and shake at 25°C and 180 rpm for 5 days. Count the spores after filtering through sterile gauze. Mix 1 L of bacterial solution with 20 kg of sterile soil in a soil mixer to a final concentration of 5.0 × 10 5 Spore / gram soil.

[0060] (4) Transplanting: Transplant the seedlings 3 days after the sterilization. During the transplanting process, align the roots of the watermelon seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Then transplant them into flower pots. Set three replicates for each treatment.

[0061] (4) Apply the second biocontrol bacteria: Apply the second biocontrol bacteria (the biocontrol bacteria fermentation liquid prepared in Example 2) three days after transplanting the seedlings. The usage and dosage are the same as the first application of biocontrol bacteria. Water and observe on time. Investigate the disease condition after 10 days.

[0062] Grading standards for watermelon wilt at the seedling stage (refer to the standards reported by Yan Wen, Wang Xiqing, etc. for disease investigation).

[0063] The results showed that the strain named 30833 had a 7-day control effect on watermelon wilt of 84.72% and a 15-day control effect of 88.92%, indicating that the strain 30833 had a high control effect on watermelon wilt.

[0064] Example 6 Classification and Identification of Strain 30833 Selected by the Present Invention

[0065] Proceed as follows:

[0066] The genome of strain 30833 was extracted using a modified CTAB method. Sequences were amplified using primers targeting the 16S rDNA and gyrB genes, respectively. The universal primers for the 16S rDNA sequence were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. The universal primers for the gyrB gene were gyrB-F: 5'-TTGRCGGHRGYGGHTATAAAGT-3' and gyrB-R: 5'-TCCDCCSTCAGARTCWCCCTC-3'. PCR products were electrophoresed on 1% agarose gels and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The resulting sequences are listed in the sequence listings (SEQ ID No. 1) and (SEQ ID No. 2). The resulting sequences were subjected to homology analysis and multiple sequence alignment in the NCBI GenBank database. A phylogenetic tree was constructed based on the alignment using MEGA software.

[0067] Results The 16S rDNA fragment of 30833 obtained by PCR amplification was a DNA fragment of 1515 bp in size (see Figure 2 ), the gyrB gene of strain 30833 obtained by PCR amplification was a DNA fragment of 980 bp in size. The 16S rDNA gene sequence of strain 30833 was 99.93% similar to that of Bacillus velez. The gyrB gene sequence of strain 30833 was 98.47% similar to that of Bacillus velez. In addition, from the phylogenetic tree (see Figure 2 and Figure 4) It can also be seen that the 30833 strain belongs to the genus Bacillus Velezinsis in terms of classification, and is different from any known Bacillus Velezinsis strain, and is a new Bacillus Velezinsis strain.

[0068] Example 7 Indoor test on the efficacy of the fermentation broth, bacterial suspension and supernatant of the present invention against tomato wilt

[0069] Proceed as follows:

[0070] (1) Tomato seed germination and seedling cultivation: Rinse the tomato seeds with clean water, wrap them with wet gauze after cleaning, germinate them in a 25℃ incubator overnight, and then grow them in vermiculite.

[0071] (2) Preparation of 30833 biocontrol bacteria fermentation broth, cells, and supernatant: 30833 biocontrol bacteria were inoculated into 5 mL of liquid LB medium and cultured with shaking at 180 rpm and 37°C for 12 h. The culture was then transferred to 300 mL of liquid LB medium at a 1% ratio and cultured with shaking at 180 rpm and 37°C for 48 h to obtain a fermentation broth. The cell concentration (CFU / mL) in the fermentation broth was calculated using the plate count method. 150 mL of the biocontrol bacteria fermentation broth was centrifuged, the cells were resuspended, and the supernatant and the cells were each fixed to 150 mL.

[0072] (3) Application of 30833 fermentation liquid, bacteria and supernatant: When the tomato has two leaves and one heart, dilute 150 ml of fermentation liquid, bacteria and supernatant by half and pour it into the seedling tray to facilitate the full colonization of the 30833 strain or the absorption of active ingredients such as antibacterial substances by the roots.

[0073] (4) Preparation of pathogenic bacteria liquid: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum bacteria, inoculate into PDB medium, shake at 25°C and 180 rpm for 5 days, filter through sterile gauze, and count the spores. The concentration is 4.5×10 7 spores / ml.

[0074] (5) Transplanting seedlings: Transplant seedlings 3 days after the inoculation of antibacterial fermentation liquid, bacterial suspension and sterile supernatant. During the transplanting process, align the roots of the tomato seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wound treatment. Soak the pathogen liquid below the roots for 60 minutes, and then transplant them into flower pots.

[0075] (6) Water and observe on time, check the condition after about 10 days, and check the condition again after about 20 days.

[0076] The results showed that the control efficacy of 30833 fermentation liquid, bacterial suspension and sterile supernatant against tomato wilt was 84.68%, 89.91% and 26.58% respectively, indicating that the strain has a high control efficacy against tomato wilt.

[0077] Table 1 Control effects of 30833 bacterial suspension, sterile supernatant and fermentation liquid on tomato Fusarium wilt

[0078]

[0079]

[0080] Note: Data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level using Duncan's new multiple range test.

[0081] Example 8 Field control effect and yield increase test of strain 30833 of the present invention on tomato wilt

[0082] Proceed as follows:

[0083] The field efficacy of 30833 against tomato Fusarium wilt was evaluated in greenhouse plots in Dingxing County, Baoding City, Hebei Province, where Fusarium wilt is prevalent. The tomato variety was Provence. Before transplanting, 20 mL of the biocontrol agent's fermented liquid was applied to each seedling hole. Transplantation was carried out 12 hours after treatment. After transplanting, 30833 fermented liquid (100 million CFU / mL) was drip-irrigated at a rate of 500 L / mu (approximately 100 million CFU / mL). Each treatment consisted of five rows with four replicates. A water treatment served as a control. The incidence of Fusarium wilt was assessed during the flowering and fruiting stages of the tomatoes. Yield measurements were taken at the end of the growing season to compare tomato yield and red fruit percentage in the 30833-treated and untreated plots.

[0084] The grading standards for tomato wilt at maturity are the same as above.

[0085] The calculation formulas for disease index and prevention and control effect are the same as above.

[0086] Results (see Figure 5 The disease index for tomato wilt in the treatment area with strain 30833 of the present invention was 13.28, while the disease index in the control area was 52.56, indicating a field efficacy of 74.73% against tomato wilt. Treatment with strain 30833 increased tomato yield by 23.87%, and promoted the transition of green to red fruit, with the red fruit rate increasing by 38.02%. This demonstrates that strain 30833 of the present invention is effective in controlling tomato wilt in the field, significantly increasing tomato yield and the red fruit rate.

Claims

1. A Bacillus velezinoffii ( Bacillus velezensis )30833, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit number being CGMCC No.24597.

2. The Bacillus Velezii of claim 1 ( Bacillus velezensis )30833 is used in the prevention and treatment of tomato wilt, watermelon wilt or cucumber wilt; wherein the pathogen of tomato wilt is Fusarium oxysporum tomato-specific type; the pathogen of watermelon wilt is Fusarium oxysporum watermelon-specific type; the pathogen of cucumber wilt is Fusarium oxysporum cucumber-specific type.

3. A microbial agent, characterized in that: The microbial agent contains the Bacillus Velez 30833 according to claim 1.

4. Use of the microbial agent according to claim 3 in preventing and controlling tomato wilt, watermelon wilt or cucumber wilt; wherein the pathogen of tomato wilt is Fusarium oxysporum tomato-specific; the pathogen of watermelon wilt is Fusarium oxysporum watermelon-specific; and the pathogen of cucumber wilt is Fusarium oxysporum cucumber-specific.

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