CN117106663A - A strain of Bacillus siamese that resists rice smut and its application - Google Patents

Abstract

The invention provides a strain of Bacillus siamese that antagonizes rice smut disease and its application. A strain of Bacillus siamensis N288 that antagonizes rice smut disease has a deposit number of CGMCCNO.26570. The present invention can produce microbial inoculants through biocontrol bacteria, which has a good control effect on rice smut, thereby reducing pesticide investment and environmental pollution, and is of great significance in protecting the sustainable development of farmland ecology.

Description

A Bacillus siamese strain resistant to rice smut and its application

Technical field

The invention belongs to the technical fields of microbiology and plant disease biocontrol, and specifically relates to a strain of Bacillus siamensis N288 that antagonizes the rice smut fungus and its application.

Background technique

Rice is the most important food crop in my country and even the world. It provides food for nearly half of the world's population. However, the occurrence of various diseases every year leads to a large reduction in rice yield. Rice smut disease (Ustilaginoidea virens) is one of the main diseases affecting rice yield. Rice smut occurs in various rice growing areas in my country. This disease mainly occurs in the ear of rice. The pathogen infects the ear of rice and forms rice balls. Rice smut pathogens not only cause reduced rice yields and poor rice quality, but also produce toxins that endanger human health.

Current prevention and control strategies for rice smut disease mainly include breeding of excellent disease-resistant varieties, chemical control and biological control, among which chemical control is the main measure. However, long-term use of chemical pesticides can easily cause pathogenic bacteria to develop drug resistance, resulting in reduced control effectiveness. At the same time, long-term application of chemical pesticides will also cause pollution to the ecological environment and cause a series of problems such as food safety. In view of the above problems, the use of beneficial microorganisms to prevent and control rice smut disease has gradually attracted the attention of agricultural workers.

Compared with other methods, biological control is safe, effective, and environmentally friendly. Biocontrol preparations are effective in disease control, have no residue, do not pollute the environment, and are harmless to humans and animals. Biocontrol bacteria can secrete antibacterial substances to inhibit the growth of pathogenic bacteria, and can also produce certain substances to promote plant growth, thereby improving plant immunity and reducing the occurrence of diseases.

Therefore, in order to reduce the occurrence of rice smut disease in farmland, it is necessary to screen biocontrol microorganisms for this disease and fully explore and utilize beneficial microorganisms in the rhizosphere. This will help reduce the input of chemical fertilizers and pesticides, reduce environmental pollution, and achieve sustainability. develop.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a Bacillus siamese strain for preventing and treating rice smut disease. The present invention collects rice rhizosphere soil from a rice test field in Liuhe District, Nanjing City, Jiangsu Province, and isolates 1,883 bacterial strains. After screening, a strain N288 with extremely strong antagonistic effect on rice smut fungus was obtained. Based on the 16S rDNA gene sequence analysis of its colony and cell morphological characteristics, it was identified as Bacillus siamensis. And it will be deposited in 2023 to the China General Microbial Culture Collection and Management Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number: CGMCC NO.26570. The present invention studies the control effect of the antagonistic bacteria on rice smut, and provides scientific basis for the biological control of rice smut and the further development and utilization of biocontrol fungicides.

The invention provides a Bacillus siamensis N288 strain for preventing and treating rice smut disease, and its deposit number is: CGMCC NO.26570.

The present invention also provides a microbial agent containing the above-mentioned Bacillus siamensis N288.

The invention also provides a method for preparing a microbial agent of Bacillus siamensis N288. The Bacillus siamensis strain N288 is inoculated into a liquid LB culture medium, placed in a constant temperature shaker at 30°C, and cultured with shaking at 180-200r/min for 24 hours. .

The present invention also provides the effect of the above-mentioned Bacillus siamensis N288 strain or inoculant in preparing and controlling crop diseases.

Preferably, the pathogenic bacteria of the soil-borne diseases include rice Aspergillus oryzae, rice blast fungus, rice sheath blight, wheat scab, gray mold, Sclerotinia sclerotiorum, and banana Fusarium wilt.

Preferably, the crops are rice, wheat, rape, and banana.

The present invention isolates and screens the bacterial strain Bacillus siamese N288, which is extremely resistant to rice smut disease, from the rhizosphere of healthy plants in rice smut-occurring plots. The B. siamese strain N288 is also resistant to rice blast fungus and rice blast fungus. Sheath blight, wheat scab, Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium wilt of banana have strong antagonistic effects. The results of the pot experiment showed that the Bacillus siamese strain N288 has a significant control effect on rice smut disease. Bacillus siamese N288 of the present invention is of great significance to the restoration of continuous cropping soil and the prevention and control of crop diseases.

Description of drawings

The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. In the attached picture:

Figure 1 shows the plate antagonistic effect of strain N288 of the present invention on rice smut fungus; A in the figure is Ustilaginoidea virens, and B is strain N288.

Figure 2 shows the colony morphology of strain N288 of the present invention.

Figure 3 is a phylogenetic tree of the 16S rDNA gene sequence of the antagonistic strain N288 constructed in the present invention.

Figure 4 shows the plant height and root length of rice in the pot experiment in Example 3 of the present invention.

Figure 5 shows the content of rice sorghum pathogens in the rice rhizosphere in the pot experiment in Example 3 of the present invention.

Figure 6 shows the confrontation culture method used in Example 4 of the present invention to determine the antagonistic effect of Bacillus siamese strain N288 on different pathogenic bacteria.

Biological sample preservation information

N288, classified as Bacillus sp., is deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The deposit date is February 20, 2023. The deposit number is CGMCCNO.26570. The deposit address is Beichen West, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, No. 3, Road 1.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified. Quantitative experiments in the following examples were repeated three times.

Example 1 Isolation and purification of rice smut antagonistic bacterium N288

The inventor of the present application collected the rhizosphere soil of healthy rice from the rice smut-infected fields in Liuhe District, Nanjing City, Jiangsu Province, sealed it in a sterile ziplock bag, brought it back to the laboratory, and temporarily stored it at 4°C for separation. Antagonistic bacteria.

The target pathogen tested was Ustilaginoidea virens, which was provided by the Plant Protection Institute of the Jiangsu Academy of Agricultural Sciences. The bacterial culture medium was LB, and the fungal culture medium was PDA.

Bacteria were isolated using the soil dilution method, cultured in continuous streaks until pure, mixed in 40% glycerol at a volume ratio of 1:1, and stored in a -80°C refrigerator for later use. The antagonistic effect of bacteria on rice sorghum pathogen was determined using plate confrontation method. For initial screening, rice smut bacteria are inoculated on PDA plates for activation. After sufficient mycelium has grown, use a 5mm diameter hole punch to prepare a mushroom cake in the center of a new PDA plate, and place the purified bacteria 3cm away from the edge of the mushroom cake. For bacteria, 4 spots were picked on each plate and cultured in the dark in a biochemical incubator at 28°C. At the same time, a control group was set up to inoculate only rice smut bacteria. When the pathogenic bacterial colonies grew to a certain size, strains with antagonistic effects were saved for re-screening. The inhibition zone method was used to conduct rescreening experiments.

1883 bacterial strains were isolated and purified from the rice rhizosphere soil. Through preliminary screening using the plate confrontation method, 249 antagonistic strains with inhibition zone diameters greater than 3 mm were obtained, accounting for 13.2% of all strains. The inhibition zone method was used to continue re-screening the antagonistic bacteria, and 7 strains with inhibition zone diameters greater than 16 mm were obtained (Table 1). The results of the plate antagonistic effect of strain N288 of the present invention on rice smut fungus are shown in Figure 1.

Table 1 Inhibitory effects of bacteria on rice smut fungus

Example 2 Identification of rice smut antagonistic bacterium N288

The selected antagonistic strain N288 was identified using a method combining colony morphology and molecular biology. PCR reaction system (50 μL system): 2 μL of each primer, 25 μL of 2x Mix, add ddH ₂ O to make up to 50 μL. The primer is 27F, a universal primer selected for general bacterial identification. The most commonly used universal primer is 27F/1492R. (27F: 5'-AGAGTTTGATCCTGGCTCAG-3') and (1492R: 5'-GGTTACCTTGTTACGACTT-3'). Reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 55 s, annealing at 50°C for 50 s, extension at 72°C for 1 min, extension at 72°C for 10 min, and storage at 16°C after 35 cycles. After the PCR product was detected on 1.5% agarose gel electrophoresis, it was sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. The sequencing results are shown in SEQ ID NO.1. The measured sequences were compared by BLAST through the Eziocloud database, and MEGA software was used to conduct phylogenetic analysis of the isolated bacteria.

On the LB medium plate, the colonies of strain N288 were milky white, with uneven edges, wrinkles on the surface, and a certain degree of adhesion. The colony morphology of strain N288 is shown in Figure 2.

The sequencing results of strain N288 were compared on the Ezbiocloud website and showed the highest similarity with the model strain Bacillus siamensisKCTC 13613 ^T , reaching 99.23%, and was identified as Bacillus siamensis (see Figure 3 for the phylogenetic tree). The strain will be deposited in 2023 to the China General Microbial Culture Collection and Management Center, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCCNO.26570.

Example 3 Pot experiment on the prevention and treatment of rice smut with the antagonistic bacterium Bacillus siamese N288

The rice variety tested was Liangyoupeijiu, and the strain tested was the rice smut antagonistic bacterium Bacillus siamese N288 in Example 2, deposit number: CGMCC NO.26570.

Preparation of Bacillus siamese inoculant: Inoculate Bacillus siamese strain N288 into liquid LB culture medium, place it in a constant temperature shaker at 30°C, and culture it with shaking at 200r/min for 24 hours. Use sterile water to adjust the concentration of the cultured fermentation broth to a viable bacterial count of 1×10 ⁸ cfu/ml.

Preparation of rice smut pathogenic bacteria suspension: Inoculate the preserved rice smut pathogenic bacteria into the liquid PDA culture medium with an inoculation needle, place it on a constant temperature shaker at 28°C, and culture it with shaking at 140r/min for 5-7 days. Dilute with sterile water to a spore suspension with a concentration of 1×10 ⁶ /ml.

Select healthy rice seedlings of the same size for planting, wash away the floating soil with clean water, and wash them again with 75% alcohol. Put 5kg of sterilized soil into a 10L plastic bucket, plant 6 rice seedlings evenly, and water regularly.

Before rice heading, the Bacillus siamese inoculant was poured into the rice rhizosphere. Two days later, the rice smut pathogenic bacteria suspension was poured into the rice rhizosphere to set up a control group.

After the rice matures, the rice rhizosphere soil is collected, soil microbial DNA is extracted, and qPCR is performed to quantify the pathogenic bacteria content in the rice rhizosphere. And measure rice plant height and root length.

Bacillus siamese inoculant can promote rice growth to a certain extent (Figure 4), and can inhibit the rice sorghum pathogen in the rice rhizosphere (Figure 5).

Example 4 Determination of the antibacterial spectrum of the rice smut antagonistic bacterium Bacillus siamese strain N288

The pathogenic microorganisms of plant fungal diseases with a relatively common incidence were selected as experimental materials, and the antagonistic spectrum of Bacillus N288 was determined using the plate confrontation method. Among them, the pathogenic fungi are: rice blast fungus (Magnaportheoryzae), rice sheath blight (Rhizoctonia solani Kühn), wheat scab (Fusariumgraminearum), gray mold (Botrytis cinerea), rapeseed sclerotiorum (Sclerotiniasclerotiorum), banana Fusarium oxysporum f.sp.cubense. As shown in Figure 6, Bacillus siamese N288 not only has a strong inhibitory effect on rice smut disease caused by Chlorosomes oryzae, but also has a strong effect on rice blast pathogens and banana fusarium wilt pathogens. In addition, it has a strong inhibitory effect on rice smut. Fusarium blight pathogens, wheat scab pathogens, gray mold pathogens and rapeseed sclerotinia pathogens also have certain inhibitory effects, which shows that Bacillus N288 has potential preventive effects on various diseases.

Claims (9)

1. A strain of Bacillus siamensis N288, its deposit number is CGMCCNO.26570. 2. The microbial agent of Bacillus siamensis N288 according to claim 1. 3. The preparation method of the microbial inoculant according to claim 2, characterized in that the Bacillus siamensis N288 strain is inoculated into LB liquid culture medium and placed in a constant temperature shaker at 30°C, 180-200r /min shaking culture for 24 hours. 4. Application of the Bacillus siamensis N288 strain according to claim 1 in the preparation and prevention of plant fungal diseases. 5. Application according to claim 4, characterized in that the pathogenic bacteria of the plant diseases are selected from the group consisting of rice Aspergillus oryzae, rice blast fungus, rice sheath blight, wheat scab, gray mold, and rapeseed fungus. Sclerotinia sclerotiorum or Fusarium wilt of banana. 6. The application according to claim 5, characterized in that the plant is rice, wheat, rapeseed, or banana. 7. Application of the inoculant according to claim 2 in the preparation and prevention of plant fungal diseases. 8. Application according to claim 7, characterized in that the pathogenic bacteria of the plant diseases are selected from the group consisting of rice Aspergillus oryzae, rice blast fungus, rice sheath blight, wheat scab, gray mold, and rapeseed fungus. Sclerotinia sclerotiorum or Fusarium wilt of banana. 9. The application according to claim 8, characterized in that the plant is rice, wheat, rapeseed, or banana.