Biocontrol bacterium and application thereof in prevention and treatment of soil-borne diseases

Abstract

The invention relates to a novel biocontrol bacterium and application thereof in prevention and treatment of soil-borne diseases of crops. The biocontrol bacterium GDMCC No: 62599 disclosed by the invention can be used for effectively preventing and treating soil-borne diseases of crops and has a remarkable growth promoting effect on the crops; especially in the aspect of planting and application of tomatoes, hot peppers, broccoli and watermelons, the composition not only has remarkable prevention and treatment effects on tomato bacterial wilt, pepper phytophthora blight, broccoli black rot and watermelon fusarium wilt, but also can remarkably promote the growth of the crops and improve the yield, and also has the efficacy of enhancing the salt tolerance of the crops; furthermore, compared with a chemical agent in the prior art, the biocontrol bacterium disclosed by the invention is non-toxic to people and livestock, free from environmental pollution and residue, and beneficial to sustainable development of an ecological environment.

Description

Technical Field

[0001] The invention belongs to the field of plant protection, and relates to Bacillus velez for preventing and controlling soil-borne plant diseases, promoting growth and improving salt tolerance of crops and applications thereof. Background Art

[0002] Plant soil-borne diseases refer to diseases caused by pathogens infecting plants through soil or pathogens left in the soil with plant diseased residues. They are highly hidden and contagious and are called the "cancer" of plants. Common and destructive soil-borne diseases include blight, bacterial wilt, black rot, soft rot, and wilt. Once these diseases occur, they will lead to reduced crop yields or even crop failure. At present, the prevention and control of soil-borne diseases is mainly based on chemical prevention and control. Although it has the characteristics of low cost and quick effect, the long-term use of chemical pesticides will lead to problems such as increased resistance of pathogens, drug residues, and soil pollution.

[0003] The use of environmentally beneficial microorganisms to control the occurrence of soil-borne diseases and to induce and improve crop resistance has been a hot topic in recent years. In particular, these microorganisms are non-polluting to the environment, which has aroused the interest of scientists in using biological control methods to control diseases and cope with adverse environments. Bacillus velezensis ), can produce a series of metabolites during its own growth process, which enable Bacillus velez to have a wide range of fungal and bacterial inhibitory activities, or induce plant resistance. It can affect many metabolic processes of plants in different ways or pathways, increase plant root length, change plant root morphology, and promote the root system's absorption of water and nutrients. Bacillus velez can improve the disease resistance of plants and resist the infection of pathogens.

[0004] Bacillus Velez is widely distributed in nature and can inhibit a variety of fungi and bacteria. Therefore, it has broad application prospects in biological control and can appropriately replace traditional chemical pesticides. At the same time, with the improvement of molecular research technology, the gene functions of Bacillus Velez are better understood, increasing the feasibility of application. And the optimization of fermentation conditions has also made the large-scale application of Bacillus Velez possible. Summary of the invention

[0005] The purpose of the invention is to address the shortcomings of the existing prevention and control of soil-borne diseases and to provide a green and environmentally friendly Bacillus Velezii BR11 that can improve broad-spectrum plant disease prevention, growth promotion, and salt tolerance.

[0006] Another object of the present invention is to provide the application of the Bacillus Velez BR11.

[0007] The purpose of the present invention can be achieved through the following technical solutions: The biocontrol bacterium BR11 is Bacillus velezinsis ( Bacillus velezensis ). The biocontrol bacterium BR11, which is used to control soil-borne diseases of various crops, promote growth, and improve crop stress resistance, is classified and named Bacillus Velezii ( Bacillus velezensis ), deposited in Guangdong Provincial Microbiological Culture Collection Center on June 21, 2022, with the culture collection number GDMCCNo: 62599.

[0008] The Bacillus Velezii BR11 is preferably used in preventing and controlling tomato bacterial wilt, pepper blight, broccoli black rot, and watermelon wilt, promoting the growth of the aboveground or underground parts of tomatoes, peppers, broccoli, and watermelons and improving their quality, and improving plant stress resistance.

[0009] The bacterial agent prepared by the Bacillus Velezii BR11 of the present invention.

[0010] The bacterial agent of the present invention is preferably prepared by the following method: the Velezella BR11 described in claim 1 is cultured in LB culture medium at 28°C and 180 rpm for 12-16 h, then centrifuged at 6000 rpm for 10 min to obtain bacterial cells, and diluted with sterilized water to prepare a bacterial agent, wherein the total concentration of live bacteria in the finished bacterial agent is 1×10 9 -1×10 10 CFU / mL.

[0011] The microbial agent is treated by rhizosphere irrigation or foliar spraying.

[0012] The present invention uses a microbial agent that is non-toxic to the environment to prevent and treat tomato bacterial wilt, pepper blight, broccoli black rot, and watermelon wilt, providing a green and environmentally friendly Bacillus Velezii agent. It solves the problems of poor effects or residues of other methods such as chemical agents, protects the environment, and promotes sustainable agricultural development.

[0013] The Velez Bacillus BR11 agent can be stored at room temperature for 6 months to 1 year. The greenhouse disease prevention test results of plants treated with the agent showed that the prevention effect on bacterial wilt was as high as 65.28%, the prevention effect on pepper blight was as high as 56.21%, the prevention effect on broccoli black rot was more than 53.33%, and the prevention effect on watermelon wilt was more than 60.37%; the field disease prevention and growth promotion test results of plants treated with the agent showed that it had significant disease prevention and control effects on the above soil-borne diseases, and could significantly promote crop growth, increase crop yields, and improve the adaptability of crops to adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 , Bacillus velezensis BR11 based on 16S r DNA (A), ikB (B) Phylogenetic tree of Bacillus constructed from gene sequences Figure 2 Antagonistic effects of Bacillus Velezii BR11 (A) and lipopeptide bold substance (B) on pathogenic bacteria Figure 3 , Bacillus Velez BR11 treatment on crop disease prevention effect Figure 4 , Velez Bacillus BR11 treatment on crop growth promotion effect DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0016] Example 1 Molecular Identification of Bacillus velez BR11

[0017] The BR11 strain is Bacillus Velezii. It was isolated from the soil of a tomato field in Shandong Province and obtained by plate confrontation and enzyme activity screening. The 16S r DNA, ikB Gene sequence analysis identified the strain as Bacillus velezinsis ( Bacillus velezensis ).

[0018] The Bacillus velez Bacillus velezensis ) was deposited in Guangdong Microbiological Culture Collection Center on June 21, 2022. The deposit address is: Guangdong Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province. The strain deposit number is GDMCC No: 62599. \ Bacillus Velez Bacillus velezensis ) 16S rDNA of BR11 strain, ikB PCR amplification and sequencing of genes.

[0019] The strain was inoculated into LB medium, cultured at 28°C and 180 rpm for 12h, and then centrifuged at 6000 rpm for 10 min to obtain the bacteria. After the bacterial gene DNA was extracted using a bacterial genome kit (Sapsen), the 16S rDNA was amplified using forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). After the amplification of the 16S rDNA fragment was observed by gel electrophoresis, the PCR stock solution was sent for sequencing. The PCR conditions were: 95 °C pre-denaturation for 4 min, 94 °C denaturation for 1 min, 54 °C annealing for 1 min, 72 °C extension for 1 min, 35 cycles, 72 °C extension for 10 min, and 4 °C storage. The length of the 16S rDNA partial gene sequence is about 1500 bp, and the result is shown in the sequence table SEQ ID No. 1.

[0020] The forward primer gyrB-F (5'-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCR TCNGTCAT-3') and the reverse primer gyrB-R (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGN AARTTYGA-3') were used to ikB Gene amplification, PCR reaction conditions are the same as above. ikB The length of the partial gene sequence is about 1500 bp, and the result is shown in the sequence table SEQID No.2.

[0021] The 16S r DNA, ikB The sequences were compared in the GenBank database, the corresponding gene sequences of related strains were downloaded, and phylogenetic trees were constructed based on sequence similarity using MAGE 11. r DNA, ikB The position of the sequence in the phylogenetic tree ( Figure 1 ) are all related to Bacillus velezinsis ( Bacillus velezensis ) gathered into one branch.

[0022] Example 2 Preparation of Bacillus Velez BR11 Agent

[0023] A preparation of a Bacillus Velez BR11 bacterial agent with significant biological control effect on soil-borne diseases and growth promotion, stress resistance and microecological improvement, comprising a microbial component: Bacillus Velez BR11 (preservation number is GDMCC No: 62599).

[0024] The preparation method of Bacillus Velez BR11 bacterial agent is as follows: Bacillus Velezii BR11 was cultured in LB medium at 28°C and 180 rpm for 12-16 h, and then centrifuged at 6000 rpm for 10 min to obtain the bacterial cells, which were diluted with sterile water to prepare a bacterial agent. The total concentration of live bacteria in the finished bacterial agent was 1×10 9 -1×10 10 CFU / mL.

[0025] Example 3 Determination of the antagonistic activity of live Bacillus velez BR11 and lipopeptide extracts against various pathogens

[0026] Bacillus velez Bacillus velezensis ) The live bacteria and lipopeptide extracts of BR11 strain have inhibitory activity against pathogenic oomycetes, fungi and bacteria.

[0027] Bacillus velez Bacillus velezensis ) The common fermentation method of BR11 is: culture in LB culture medium at 28°C and 180 rpm with shaking for 12 hours.

[0028] Bacillus velez Bacillus velezensis )The extraction method of BR11 lipopeptides is as follows: BR11 common fermentation broth is inoculated into Landy culture broth at 1% (v / v) inoculation volume and cultured at 28℃ 180 rpm for 48h. The bacterial suspension is centrifuged at 10000 rpm for 20 min to obtain the supernatant, and the lipopeptides in the supernatant are extracted by acid precipitation. The extraction method is as follows: the supernatant is adjusted to pH 2.0, allowed to stand for 5-8h, and then centrifuged to obtain the precipitate, which is then redissolved with methanol, adjusted to pH 7.0, allowed to stand overnight, centrifuged at 10000 rpm for 20 min, the supernatant is rotary distilled, and the methanol is redissolved to obtain the BR11 lipopeptide crude extract.

[0029] The plate confrontation method was used to determine the antagonistic activity of live bacteria and lipopeptide extracts of BR11 strain against pathogenic oomycetes and fungi. Pathogenic oomycetes and fungi include: Fusarium solani, Rhizoctonia solani, Rhizoctonia solani, root rot fungi, cucumber wilt, watermelon wilt, Fusarium oxysporum, and Phytophthora. The experimental method is: take an 8mm pathogen cake and inoculate it in the center of the PDA plate, and spot 10μL BR11 bacterial solution (OD 600 =1) and crude lipopeptide extracts. The results showed that BR11 lipopeptide extracts had significant antagonistic effects on Fusarium solani, Rhizoctonia solani, Rhizoctonia solani, root rot pathogens, cucumber wilt, watermelon wilt, and Fusarium oxysporum. Figure 2 shown.

[0030] Example 4 Determination of biocontrol potential of Bacillus velez BR11 strain

[0031] The biocontrol potential of BR11 was determined and evaluated with the following scoring criteria: 1 point for IAA production, 1 point for nitrogen fixation NFb, 1 point for JNFb, 1 point for phosphate solubilization NPA, 1 point for OPA, 1 point for ACC deaminase, and 1 point for siderophore activity. The highest score was 7 points. The results are shown in Table 1: The method for determining the IAA production capacity is based on Sawar and Kremer (1992): 16 g of dimethylaminobenzaldehyde is dissolved in 1520 ml of 95% alcohol and 320 ml of concentrated hydrochloric acid to prepare Ehrlich's reagent. 1% tryptone aqueous solution (pH = 7.0-7.5) is used as the culture medium. After the bacteria are cultured at 28°C and 200 rpm for 48 hours, 3-5 ml of Ehrlich's reagent is added, and the liquid surface is shaken and observed to see if it turns red, indicating that IAA is produced. At the same time, the strains that have been tested in the laboratory to produce IAA and those that do not produce IAA are used as positive and negative controls, respectively.

[0032] Determination of bacterial nitrogen fixation ability: Pick a single colony of BR11 and culture it in 2 ml LB culture medium for 18 h. Pipette 10 μl of the bacterial solution onto the NFb plate. The strain that can grow in the nitrogen fixation medium (NFb) and make the culture medium turn blue is considered to have nitrogen fixation activity and recorded as 1. If it does not change color, it is recorded as 0.

[0033] Determination of bacterial phosphate solubilization ability: Pick a single colony of BR11 and culture it in 2 ml LB culture medium for 18 h, then pipette 10 μl of the bacterial solution onto the plates (OPA organic phosphorus medium and NPA inorganic phosphorus medium) and culture them at 28℃-30℃ for 96 h. Observation method: The presence or absence of transparent circles on the NPA plates is recorded as 1, and the absence is recorded as 0; the presence or absence of transparent circles on the OPA plates is recorded as 1, and the absence is recorded as 0.

[0034] Determination of bacterial ACC deaminase activity: The strain was inoculated on ADF solid medium, and the strain that could grow on the medium with ACC as the only nitrogen source after 5 passages was selected as the ACC deaminase-positive strain.

[0035] The siderophore-producing activity was tested according to the method of Shin et al. (2001). Solution A: (1) 121 mg of solid chrome azuroin S was weighed and dissolved in 100 mL of double distilled water; (2) 20 mL of trivalent iron ion solution (1 mM FeCl3·6H2O as solute, 10 mM hydrochloric acid as solvent); (3) 145.8 mg of hexadecyltrimethylammonium bromide was dissolved in 80 mL of deionized water. The three solutions were mixed and the volume was adjusted to 200 mL, the pH was adjusted to neutral, and sterilized at 121°C for 20 min. Solution B: 60.48 g of piperazine-1,4-diethanesulfonic acid was added to 1800 mL of WA culture medium, the pH of the culture medium was adjusted to 6.8 with 50% (W / V) NaOH solution, and sterilized at 121°C for 20 min. After sterilization, mix liquid A and liquid B evenly and pour into plates. Inoculate the bacteria and culture at 28℃ for 1-3 days to observe the presence of yellow transparent circles. If there is a yellow transparent circle, it is marked as 1 for siderophore activity and 0 for no siderophore activity.

[0036] Table 1: Results of bacterial activity tests

[0037] Example 5 Bacillus Velez BR11 can control major soil-borne diseases of tomatoes, peppers, broccoli and watermelons In the greenhouse growth promotion experiment of tomatoes, after the tomato seedlings were grown in plug trays, they were transplanted to 43×19×15 cm disposable 10L nutrient soil, with 8 plants per pot. After transplanting, each plant in the treatment group was watered with 200-fold dilution, and the bacterial concentration was 5.0×10 7 CFU / mL of the inoculant, 40 mL of water was poured into each plant in the control group, and 48 plants were poured into each treatment. 36 hours after transplanting, 4×10 5 CFU / mL concentration of bacterial wilt pathogen 40mL. Each treatment of 48 plants. After 3 days of normal watering, the growth conditions are 28℃, photoperiod 12h / 12h. After 30 days of treatment, the severity of the disease was investigated and graded and counted. The effect of BR11 on tomato bacterial wilt disease prevention was 65.28% compared with the control group. Figure 3 shown.

[0038] In the field disease prevention test of peppers, broccoli, and watermelons, peppers, broccoli, and watermelons were transplanted to the field after they grew to three leaves and one heart. The treatment area was one acre for the control group and one acre for the treatment group. There were 4,000 pepper plants per acre, with a row spacing of 50 cm and a spacing of 30 cm. In the pepper continuous cropping field, the diseases that usually occurred were sporadic viral diseases and pepper blight. The broccoli plant spacing was 45 cm, and 4,500 plants per acre were in the broccoli continuous cropping field. Broccoli black rot occurred every year. The watermelon plant spacing was 30 cm, and 600 plants per acre were in the watermelon continuous cropping field. Watermelon wilt occurred every year. The disease occurred naturally in the field. When transplanting, the roots were irrigated with 2L of bacterial agent diluted per acre, and the control group was irrigated with the same amount of water. After the peppers had grown for 60 days, the incidence of pepper blight was investigated and graded and counted. The disease prevention effect of BR11 on pepper blight was 56.21% compared with the control group. Figure 3 After 45 days of growth, the incidence of broccoli black rot was investigated and statistically classified. The prevention effect of BR11 on broccoli black rot was 53.33%. Figure 3 20 days after watermelon transplanting, the incidence of watermelon wilt was investigated and graded and counted. The control effect of BR11 on watermelon wilt was 60.37%. Figure 3 shown.

[0039] Example 6 Bacillus Velez BR11 can promote the growth of tomatoes, peppers, broccoli, etc. In the greenhouse growth promotion experiment of tomatoes, after the tomato seedlings were grown in plug trays, they were transplanted to 7.7×4.8×11 cm disposable medium with 250 g of nutrient soil. The treatment group was watered with 200 times dilution and the bacterial concentration was 5.0×10 7 CFU / mL of inoculant was added to 20mL, and the control group was watered with 20mL of water. Each treatment had 24 plants and the control had 48 plants. After 3 days, normal watering was applied. The growth conditions were all 28℃ and the photoperiod was 12h / 12h. The growth promotion effect was counted after 20 days of treatment. Each treatment had 24 seedlings. The amount of watering should be reduced before statistics, but the seedlings should not wilt due to lack of water. The plants in each treatment group were removed from the soil (note to keep the fibrous roots intact as much as possible). Use a soft brush to brush off the soil at the roots of the tomatoes, then rinse the roots in water until the soil is completely washed off, and dry them for later use. Cut the tomato seedlings from the base of the stem, measure the aboveground length, underground length, aboveground fresh weight, underground fresh weight, and stem thickness to calculate the growth promotion effect. Place the cut underground part in an aluminum box according to the treatment, wrap the aboveground part with newspaper according to the treatment, and then place it in a water-proof constant temperature incubator and dry it at 80℃ to constant weight. Take it out and measure the aboveground dry weight and underground dry weight.

[0040] Calculation formula:

[0041] In the field growth promotion experiment of pepper, broccoli and watermelon, pepper, broccoli and watermelon were transplanted to the field after they had three leaves and one heart. During transplanting, the root was irrigated at a dosage of 2L per mu. The growth promotion effect was calculated after 30 days of growth for pepper, 45 days for broccoli and 20 days for watermelon. The yield was recorded and calculated at harvest time.

[0042] BR11 promoted tomato plant height by 48.93%, stem diameter by 16.52%, and biomass by 25.32%. BR11 promoted pepper plant height by 36.53%, stem diameter by 25.35%, and yield by 21.33%. BR11 promoted broccoli plant height by 13.53%, leaf number by 2, and broccoli yield by 43.52%. BR11 promoted watermelon plant height and stem length by 40.53%, leaf number by 7, and watermelon yield by 30.28%. Growth-promoting effects such as Figure 4 shown.

[0043] Example 7 Bacillus Velezii BR11 improves crop salt tolerance Preparation of salt soil: Screening test using 2g·kg -1 Saline soil. First, air dry the test soil to constant weight, 2g·kg -1 The salt soil was prepared by dissolving 0.4 g of sodium chloride in 50 ml of water and mixing it with 200 g of soil.

[0044] In the greenhouse salt tolerance and growth promotion experiment, cucumbers were transplanted to 250 g of nutrient soil in a disposable medium of 7.7×4.8×11 cm after they grew to two leaves and one heart, and tomatoes were transplanted to 3 leaves and one heart after they grew to three leaves and one heart. The watering concentration was 5.0×10 7 CFU / mL bacterial solution 20mL, normal watering after 3 days, growth conditions are 28℃, photoperiod 12h / 12h. The growth promotion effect was counted after 20 days of treatment. 24 seedlings were treated in each treatment. The amount of watering should be reduced before statistics, but the seedlings should not wilt due to lack of water. Remove the plants in each treatment group from the soil (pay attention to keep the fibrous roots intact as much as possible). Use a soft brush to brush off the soil at the roots of the tomatoes, then rinse the roots in water until the soil is completely washed, dry the water for later use. Cut the tomato seedlings from the base of the stem, measure the aboveground length, underground length, aboveground fresh weight, underground fresh weight, and stem thickness to calculate the growth promotion effect. Place the cut underground part in an aluminum box according to the treatment, wrap the aboveground part with newspaper according to the treatment, and then place it in a water-proof constant temperature incubator and dry it at 80℃ to constant weight. Take it out and measure the aboveground dry weight and underground dry weight.

[0045] Calculation formula: Under greenhouse conditions, 20 days after cucumber transplantation, the BR11 strain increased the cucumber biomass by 42.25%, as shown in Table 2. Under greenhouse conditions, 20 days after tomato transplantation, the BR11 strain increased the tomato biomass by 48.38%, as shown in Table 3.

[0046] Table 2 Cucumber biomass increase results deal with Plant height (cm) Stem diameter (mm) Fresh weight (g) Dry weight (g) Biomass increase (%) BR11 15.73±0.42a 4.19±0.15a 6.78±0.21a 1.01±0.03a 42.25 CK 8.23±0.25b 2.3±0.03b 4.28±0.08b 0.71±0.02b No salt 9.43±0.21c 3.69±0.21c 5.74±0.1c 0.83±0.04c Note: The values ​​are mean ± standard error. Different letters indicate significant differences among treatments at the significance level of P=0.05.

[0047] Table 3 Tomato biomass increase results deal with Plant height (cm) Stem diameter (mm) Fresh weight (g) Dry weight (g) Biomass increase (%) BR11 18.73±0.42a 4.19±0.15a 7.69±0.28a 0.92±0.04a 48.38 CK 11.27±0.12b 3.27±0.07b 4.88±0.04b 0.62±0.03b No salt 16.43±0.21c 3.69±0.21c 5.7±0.08c 0.77±0.03c Note: The values ​​are mean ± standard error. Different letters indicate significant differences among treatments at the significance level of P=0.05.

[0048] In summary, the biocontrol bacteria BR11 of the present invention can effectively prevent and control soil-borne diseases of crops and has a significant growth-promoting effect on crops; especially in the application of tomato, pepper, broccoli and watermelon planting, it not only has significant control effects on tomato bacterial wilt, pepper blight, broccoli black rot and watermelon wilt, but also can promote the growth of these crops and increase yield, and at the same time has the effect of improving the salt tolerance of crops.

[0049] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0050] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

[0051] Sequence Listing: SEQ ID No.1 SEQ ID No.2

Claims

1. A biocontrol bacterium, characterized in that: The biocontrol bacteria is Bacillus velez acillus velezensis ), deposited in Guangdong Microbiological Culture Collection Center, and its culture collection number is GDMCC No: 62599.

2. A fungal drug preparation, characterized in that: The fungicide preparation comprises the biocontrol bacteria as claimed in claim 1 and an adjuvant acceptable in pesticides.

3. Use of the biocontrol bacteria as claimed in claim 1 in preventing and controlling soil-borne diseases of crops, in promoting crop growth, or in the cultivation of tomatoes, peppers, broccoli, and watermelons.

4. Use of the fungicide preparation as claimed in claim 2 in preventing and controlling soil-borne diseases of crops, in promoting growth and stress resistance of crops, or in the cultivation of tomatoes, peppers, broccoli and watermelons.

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