Bacillus thuringiensis and application method thereof

Abstract

The fermentation liquor prepared from the bacillus thuringiensis has a prevention and treatment effect on cucumber root-knot nematode, and the bacillus thuringiensis is simple to culture, convenient to store, low in cost and simple to use, and has a very wide application prospect in nematode prevention and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Bacillus thuringiensis for preventing and controlling cucumber root-knot nematodes and an application method thereof. Background Art

[0002] my country is located in the temperate zone, with a wide variety of plants. At present, more than 100 crops, including food crops, oil crops, cash crops, fruit and vegetables, tropical crops, feed, flowers, medicinal materials, forage and forests, have been affected by nematode diseases and have been damaged to varying degrees. According to foreign reports, about 30% of the root rot of crops is related to nematodes. Many nematodes are very harmful to crops. For example, soybean cyst nematode disease can reduce production by 50% to 80% when the disease is severe, and even worse, it can lead to total crop failure. When peanut root-knot nematodes are severely infected, the yield reduction can reach more than 70%, and some fields will also experience total crop failure.

[0003] Cucumber root-knot nematodes are one of the most important diseases of cucumbers, which generally leads to a 20% reduction in yield. In severe cases, the reduction can reach more than 50%, or even a total loss. Cucumber root-knot nematodes mainly harm the roots of cucumbers, causing poor development of the cucumber root system, shrinkage and deformity of the main root and lateral roots, and the formation of nodules of varying sizes, shaped like chicken feet. The nodules sometimes grow in series, making the roots swollen and rough. The initial nodules are white, smooth and soft, and then turn yellow-brown to dark brown, with a rough or even cracked surface, and rot in severe cases.

[0004] The existing control methods for cucumber root knot nematode disease mostly use chemical control, which has the characteristics of good effect and convenient operation, but the long-term use of large amounts of chemical agents will cause environmental pollution. Therefore, it is necessary to develop a new control method to solve the above problems of the existing methods. Summary of the invention

[0005] The object of the present invention is to provide a Bacillus thuringiensis and its application, wherein the Bacillus thuringiensis ( Bacillus thuringiensis )SC fermentation liquid has the effect of preventing and controlling nematodes.

[0006] The technical solution of the present invention is: A Bacillus thuringiensis, characterized in that: the Bacillus thuringiensis strain is named ( Bacillus thuringiensis )SC, deposited in the Wuhan University Collection Center on April 9, 2024, with the accession number CCTCCNO: M 2024651.

[0007] The colonies formed by the Bacillus thuringiensis strain SC on the LB plate are round, with irregular edges, milky white to slightly yellow, dark and opaque in the center, and light and translucent at the edges.

[0008] The 16S rDNA sequence of the Bacillus thuringiensis strain is shown in the sequence listing SEQ ID No. 1.

[0009] The application method of Bacillus thuringiensis comprises the following steps: (1) The strain SC was fermented in LB medium at 28-30°C and 155-165 r / min for 48 h to obtain a fermentation broth.

[0010] (2) Dilute the fermentation broth to 1.0×10 6 -1.0×10 8 cfu / mL before use.

[0011] The beneficial effects of the present invention are: The fermented liquid of the bacillus thuringiensis has a preventive and therapeutic effect on cucumber root-knot nematodes, and the bacillus thuringiensis is simple to culture, easy to store, low in cost, and easy to use, and has a very broad application prospect in nematode control. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Bacillus thuringiensis ( Bacillus thuringiensis ) Front side of colony; Figure 2 Bacillus thuringiensis ( Bacillus thuringiensis ) The reverse side of the colony. DETAILED DESCRIPTION

[0013] Soil was taken from a cucumber field in Jingzhou City, Hubei Province, which was not damaged by nematodes, and brought back to the laboratory for storage and standby. Weigh 10 g of soil sample, add it to 90 mL of sterilized water, shake and culture at 160 r / min for 10 min, let it stand for 30 min, take 1 mL of supernatant, add it to a test tube containing 9 mL of sterile water, shake and mix, and make a sample suspension. Take 1 mL of sample suspension and add it to a test tube containing 9 mL of sterile water. Perform 10-fold serial dilutions in sequence. Take 50 μL of the 5th, 6th, and 7th dilutions, respectively, spread them on LB plates, and culture them at 28 ℃ for 24 h. According to the characteristics of morphology, size, color, etc., a total of 51 single colonies were picked, transferred to LB plates for culture, and stored for standby. The colonies formed by strain SC on LB plates are round, with irregular edges, milky white to slightly yellow, dark and opaque in the center, and light and translucent at the edges. See the attached instructions for details. Figure 1 and 2 .

[0014] The strain SC was inoculated into LB medium, cultured at 28-30°C and 155-165 r / min for 12 h, and centrifuged at 10,000 rpm for 10 min. DNA was extracted from the obtained bacteria using the kit FastPure Bacteria DNA Isolation Mini Kit-BOX 2 from Nanjing Novezan Biotechnology Co., Ltd.

[0015] The amplification primers were bacterial specific gene 16sDNA, the upstream primer was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), the downstream primer was 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the PCR reaction system was as follows: 94 ℃ 4 min (pre-denaturation), 94 ℃ 30 s, 59 ℃ 30 s, 72 ℃ 90 s, 32 cycles, 72 ℃ 5 min. The amplified PCR stock solution was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing, and the sequence was obtained: After comparison, SC was temporarily identified as Bacillus thuringiensis ( Bacillus thuringiensis ), deposited in the Wuhan University Collection Center on April 9, 2024, with the accession number CCTCC NO: M 2024651.

[0016] Control effect Healthy farmland soil was collected from the Yangtze University Experimental Base, air-dried, sieved, sterilized at 121 °C for 60 min, and placed in 500 g flower pots (10 cm × 10 cm × 10 cm). Root-knot nematode egg suspension was added to the sterilized soil and mixed thoroughly to make the egg content in the soil 1500 / pot. Cucumber seeds were treated with 4% sodium hypochlorite for 2 min, rinsed three times with sterile water, and germinated until the seeds turned white. After growing 2 to 3 true leaves, they were transplanted into pots, with 1 plant per pot.

[0017] To prepare fermentation broth, strain SC was inoculated into LB medium for activation (28°C, 155-165 r / min shaking culture for 24 h). After activation, the activated solution was inoculated into LB medium at a ratio of 5% for fermentation (28°C, 155-165 r / min shaking culture for 24 h).

[0018] The treatment group used root irrigation to inject 100 ml of fermentation liquid into each pot, and the control group injected an equal amount of sterile water. The experiment was designed to be repeated 4 times. Soil sampling was carried out 30 days later, and 100 g of soil samples were collected. The nematodes in the soil were separated using the modified Baermann funnel method. The nematode liquid was collected in a centrifuge tube. After standing for 2 hours, 2 / 3 of the supernatant was aspirated, and a certain amount of 100°C boiling water was added to the supernatant to kill the nematodes. The collected nematode liquid was dropped on a cell counting plate, and then observed and counted under a microscope to calculate the insect population reduction rate and insect population control effect. The formula is: P=(N CK -N t ) / N CK ×100%;EC=(P t -P CK ) / (1-P CK )×100%. Where EC is the corrected control effect, P is the insect population reduction rate, N t is the number of live insects after treatment, EC is the corrected control efficacy (%), P t is the insect population reduction rate of the treatment group, P CK The control group's insect population reduction rate was calculated. At the same time, the height and fresh weight of cucumber plants were counted, the number of root knots per plant was investigated, and the control effect was calculated using the formula: Control effect: Control effect = (number of root knots per plant in CK - number of root knots per plant in treatment) / number of root knots per plant in CK × 100%.

[0019] The results are shown in Tables 1 and 2. After 30 days of fermentation, the number of root-knot nematodes in the soil was significantly lower than that of the control, and the corrected control efficiency reached 69.99%. At the same time, the plant height and root length of the plants treated with the fermentation solution for 30 days were significantly higher than those of the control, and the number of cucumber root nodes treated with the fermentation solution was significantly lower than that of the control, and the control effect reached 53.05%.

[0020] Table 1 Effects of SC fermentation liquid on the number of nematodes in soil

[0021] Table 2 Effects of SC fermentation liquid on cucumber plants and its control effect

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus thuringiensis, characterized in that: The Bacillus thuringiensis strain is named as ( Bacillus thuringiensis )SC, deposited in the Wuhan University Collection Center on March 27, 2024, with the accession number CCTCC NO: M2024651.

2. The Bacillus thuringiensis according to claim 1, characterized in that: The colonies formed by the Bacillus thuringiensis strain SC on the LB plate are round, with irregular edges, milky white to slightly yellow, dark and opaque in the center, and light and translucent at the edges.

3. The Bacillus thuringiensis according to claim 1, characterized in that: The application method of Bacillus thuringiensis SC comprises the following steps: (1) Fermenting the strain SC in LB medium at 28-30°C and 155-165 rpm for 48 h to obtain a fermentation broth; (2) Dilute the fermentation broth to 1.0×10 6 -1.0×10 8 cfu / mL before use.

Images