CN113373091A - Biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight - Google Patents
Biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight Download PDFInfo
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Abstract
The invention provides a biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight, belonging to the technical field of plant protection. The biocontrol strain Bacillus thuringiensis FJ2B-25 has been preserved in China general microbiological culture Collection center (CGMCC) at 3.15.2021 with the preservation number of CGMCC No. 22014. Biocontrol strain Bacillus thuringiensis FJ2B-25 and biocontrol strain Bacillus thuringiensis FJ2B-25 for treating Rhizoctonia solani (Rhizoctonia solani) as pathogenic bacteria of rice sheath blightRhizoctonia solani) Has remarkable inhibiting effect. The strain has wide application prospect in the aspects of developing a biocontrol microbial inoculum for green prevention and control of rice sheath blight and separating a compound with bacteriostatic activity.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a biocontrol strain bacillus thuringiensis FJ2B-25 for controlling rice sheath blight.
Background
The rice is the planting surface in the worldThe crops with the largest accumulation are the first crops and the most main grain crops in China. However, the occurrence of rice diseases and pests causes the reduction of yield and quality, and one important disease is rice sheath blight disease. The disease is distributed in the whole world, has strong pathogenicity and very high prevalence frequency, thereby causing serious harm to the global rice production. The rice sheath blight disease is caused by Rhizoctonia solani (F.), (Rhizoctonia solani) Causing the fungus to be Dermatopsis citrulli of Basidiomycota in its sexual stage: (Thanatephorus cucumeris Donk). Rhizoctonia solani (A), (B), (C)Rhizoctonia solani) Does not produce asexual spores, exists in the form of hyphae or sclerotia and serves as a primary infection source of the next infection. It is particularly important that the sclerotia produced by the method can live in soil for a long time, so that the sclerotia becomes a stubborn soil inhabitant. Resistance of rice to sheath blight belongs to horizontal disease resistance, and it is difficult to find a variety that is highly resistant to production. At present, the effective means for preventing and treating the rice sheath blight still utilizes chemical pesticides to kill the pathogenic bacteria. Although effective, chemical control also has many negative effects, such as: toxic and side effects on human and livestock, soil environment pollution, resistance to pathogenic bacteria, pesticide residue generation and the like. These negative effects will become an obstacle to the effective implementation of green and environmentally friendly planting and sustainable development of agriculture. Therefore, it is urgent to develop a safe, effective, and environmentally friendly measure for preventing and treating sheath blight of rice. Previous researches find that the beneficial microorganisms are used for preventing and treating the banded sclerotial blight pathogenic bacteria, so that the method is a safe and effective way for replacing chemical pesticides. Some fungi, bacteria and actinomycetes having a control effect on rice sheath blight have been found, but these bacteria have limitations in use. In addition, the control of diseases by utilizing the microbiome is one of the hot spots of the current research, and more microbial resources are needed for artificially preparing the microbiome. Therefore, it is particularly important to screen more biocontrol bacteria for the microbial control of rice sheath blight.
Disclosure of Invention
The invention aims to provide a biocontrol strain bacillus thuringiensis FJ2B-25 separated and screened from paddy field soil for preventing and treating rice sheath blight, aiming at the problems of harm caused by chemical prevention and control of rice sheath blight, insufficient microbial pesticide and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight, wherein the Bacillus thuringiensis FJ2B-25 is classified and named as bacillus thuringiensis (Bacillus thuringiensis) (B)Bacillus thuringensis) The microbial inoculum is preserved in the China general microbiological culture Collection center, and the addresses are as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is 2021 year, 3 months and 15 days, and the preservation number is CGMCC No. 22014.
A biocontrol microbial inoculum for preventing and treating rice sheath blight comprises a biocontrol strain bacillus thuringiensis FJ2B-25 or a biocontrol strain bacillus thuringiensis FJ2B-25 sterile fermentation broth.
The preparation method of the aseptic fermentation liquor of the biocontrol strain bacillus thuringiensis FJ2B-25 comprises the following steps:
(1) inoculating biocontrol bacterium Bacillus thuringiensis FJ2B-25 into a sterile LB liquid culture medium, and performing activated shaking culture at 37 ℃ at 180 r/min for 12 h to obtain activated bacterium liquid;
(2) transferring 1 mu L of the activated bacterial liquid obtained in the step (1) into 50 mL of LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180 r/min for 24 h;
(3) and (3) collecting the bacterial liquid obtained by culturing in the step (2), centrifuging for 10 min at 8,000 r/min, and filtering the supernatant by using a sterile filter membrane of 0.22 mu m to remove thalli to obtain sterile fermentation liquid.
The LB liquid culture medium comprises the following components: 1% of Tryptone, 0.5% of Yeast extract, 0.5% of NaCl and the balance of distilled water.
The biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight is used for inhibiting rhizoctonia solani (F) (F2B-25)Rhizoctonia solani) The use of (1).
The biocontrol microbial inoculum for preventing and treating the rice sheath blight is applied to prevention and treatment of the rice sheath blight.
The invention has the beneficial effects that:
the screened biocontrol strain FJ2B-25 for preventing and treating rice sheath blight and the sterile fermentation liquid thereof for treating rhizoctonia solani (a pathogen of rice sheath blight)Rhizoctonia solani) Has remarkable inhibiting effect. The strain is separated and screened from rice rhizosphere soil, is harmonious and compatible with soil ecology, and is used as a biological preparation without a series of problems of toxic and side effects, drug residues and the like caused by the use of chemical pesticides; can obviously reduce agricultural pollution and improve soil environment while effectively preventing and treating the rice sheath blight. The strain has simple culture conditions, is easy to store and industrial production, and becomes an ideal biocontrol microbial inoculum.
Description of the drawings:
FIG. 1 colony morphology of bacterial strain FJ2B-25 and its gram stain. A: is a colony morphology map of bacterial strain FJ 2B-25; b: is a gram stain of bacterial strain FJ 2B-25.
FIG. 2 phylogenetic tree of the 16S rDNA sequence of bacterial strain FJ 2B-25.
FIG. 3 bacterial strain FJ2B-25 and Rhizoctonia solani (Rhizoctonia solani) which is a pathogen of rice sheath blightRhizoctonia solani) And (3) performing antagonistic culture on GD-118. A: bacterial strains and Rhizoctonia solani (Rhizoctonia solani) which is a pathogen of rice sheath blightRhizoctonia solani) A colony morphology map of GD-118 confrontation culture; b: bacterial strains and Rhizoctonia solani (Rhizoctonia solani) which is a pathogen of rice sheath blightRhizoctonia solani) Histogram of bacteriostatic bandwidth in GD-118 confrontation culture.
FIG. 4 shows the bacteria strain FJ2B-25 in the fermentation liquid for rhizoctonia solani, a pathogen of rice sheath blightRhizoctonia solani) Inhibition of the growth of GD-118. A: rhizoctonia solani (Rhizoctonia solani) pathogenic bacterium of rice sheath blightRhizoctonia solani) Colony morphology of GD-118 in PDA culture medium containing bacterial strain FJ2B-25 sterile fermentation liquid and sterile water as control; b: rhizoctonia solani (Rhizoctonia solani) pathogenic bacterium of rice sheath blightRhizoctonia solani) Statistical analysis of colony diameters of GD-118 in PDA medium containing bacterial strain FJ2B-25 sterile fermentation broth and sterile water as controls.
FIG. 5 FJ2B-25 Strain sterile fermentation broth of Rhizoctonia solani ((S. solani))Rhizoctonia solani) The effects of sclerotia germination and hyphal growth. A: FJ2B-25 strain sterile fermentation liquor and rhizoctonia solani after sterile water treatment (Rhizoctonia solani) Morphograms of sclerotium germination; b: FJ2B-25 strain sterile body fermentation liquor and rhizoctonia solani (F) after sterile water treatmentRhizoctonia solani) And (5) counting the hypha indexes of sclerotium germination.
Detailed Description
The present invention will be further described with reference to specific embodiments, illustrative examples and illustrations of the invention herein are provided to explain the present invention and are not intended to limit the invention.
Example 1 isolation and characterization of the strains
1. Soil microorganism separation by soil dilution coating plate method
(1) Collecting rice rhizosphere soil of a Huangshan test base of a rice institute of agricultural academy of sciences of Fujian province;
(2) adding 10 g soil sample into 90 mL sterile water, shaking for 20 min, adding 0.5 mL solution into 4.5 mL sterile water test tube, sequentially diluting by multiple times to obtain 10-1、10-2、10-3、10-4And 10-5Five sample solutions with different dilutions;
(3) diluting with 10 degree of dilution by a dilution coating plate method-3And 10-4Two concentrations were spread on LB solid medium (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, 1.5% Agar, the balance distilled water);
(4) and (3) performing constant-temperature culture at 37 ℃, starting to pick a single bacterial colony when the bacterial colony on the plate is formed, performing zone streaking culture on the plate respectively, performing constant-temperature culture at 37 ℃, observing whether the bacterial colony is single, and numbering after confirmation.
2. Preliminary screening of biocontrol strain of rice sheath blight
All bacterial strains isolated from the soil were separately treated with Rhizoctonia solani (a. solani)Rhizoctonia solani) Simultaneously inoculating to PDA plate for culturing at 29 deg.C for 2 d, and observing whether there is Rhizoctonia solani (B) (Rhizoctonia solani) Bacteria with inhibitory action on hypha growthThe strains are preliminarily judged as Rhizoctonia solani: (Rhizoctonia solani) The strains with the inhibiting effect are further screened by adopting a confrontation culture method, and each strain is subjected to three times of repeated screening. Finally, the bacterium FJ2B-25 was found to be Rhizoctonia solani (Rhizoctonia solani) Has obvious inhibition effect on hypha growth. Bacterial colonies of strain FJ2B-25 were pale yellow, circular or oval in shape on PDA plates, irregular in edges, opaque on the surface, and slightly raised from the middle (FIG. 1A).
3. Identification of bacterial Strain FJ2B-25
Identifying the cell morphology of the screened biocontrol bacterium FJ2B-25 by using a gram staining method, wherein the staining method comprises the following specific steps: fixing a bacterial liquid smear, sucking 10 mu L of bacterial liquid onto a glass slide by using a liquid transfer gun, and uniformly coating the bacterial liquid smear, wherein the bacterial liquid smear cannot be excessively thick during smearing; then adding crystal violet for dyeing for 1 min, and washing with distilled water; adding iodine solution to stain for 1 min, covering the slide, washing with distilled water, and absorbing water with filter paper; adding a destaining solution, shaking the glass slide for 20 s-30 s, washing with distilled water, and absorbing water by using filter paper; adding safranin, dyeing for 1 min, washing with distilled water, and drying with filter paper; finally, the oil lens is examined and photographed, and the result is shown in fig. 1B: the cells were straight rod-shaped and the strain was blue in gram stain, thus indicating that the strain was a gram-positive bacterium.
Extracting gDNA of the bacterial strain FJ2B-25 by using a bacterial genome DNA extraction kit of Tiangen Biochemical technology (Beijing) Co., Ltd; then, bacterial 16S rDNA gene universal primer 27-F (
5'-AGAGTTTGATCCTGGCTCAG-3') and 1492-R (5'-GGTTACCTTGTTACGACTT-3'), and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 40 s, extension at 72 ℃ for 40 s, 30 cycles, and extension at 72 ℃ for 10 min; the resulting PCR product was sequenced by Piano Biotechnology (Fuzhou) Inc., and the 16S rDNA sequence (SEQ ID NO. 1) of bacterial strain FJ2B-25 was determined to have the highest homology to Bacillus thuringiensis and 100% similarity when BLAST alignments were performed on the NCBI website. Performing sequence analysis by MEGA7 software, constructing phylogenetic tree, and showing that it is related to Bacillus thuringiensisRecently (fig. 2). The bacterial strain FJ2B-25 is judged to be bacillus thuringiensis (Bacillus thuringiensis) by combining the result of the last step of bacterial morphology identificationBacillus thuringensis). The strain is preserved in the China general microbiological culture Collection center, and the addresses are as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is 2021 year, 3 months and 15 days, and the preservation number is CGMCC No. 22014.
Example 2: the separated strain of the invention has the function of inhibiting the growth of rhizoctonia solani
The isolated FJ2B-25 bacterial strain was first activated in LB liquid medium (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, balance distilled water). (iii) Rhizoctonia solani, a pathogenic bacterium of sheath blight of rice, which is given by professor medal all over the country of southern China agricultural university ((Rhizoctonia solani) The GD-118 strain was inoculated on PDA for activation. Inoculating the pathogenic bacteria of rice sheath blight disease cultured in PDA culture medium for 2 days to a new PDA culture medium
One third of the diameter of the sample was inoculated with 10. mu.L of activated and homogenized FJ2B-25 strain solution at the other end of the same PDA plate, and 10. mu.L of sterilized water was added as a control, and the two solutions were symmetrical to the center point. And (3) after the bacterial liquid is dried, sealing and placing the bacterial liquid in a constant-temperature incubator at 29 ℃, observing the growth condition of hyphae every two hours, and measuring the diameter of a bacteriostatic circle. As shown in fig. 3: strain FJ2B-25 Rhizoctonia solani (F.solani) compared with control groupRhizoctonia solani) The average bacteriostatic bandwidth of the strain is 7.61 mm, the strain has obvious bacteriostatic bands, and the control group and the treatment group have significant differences, which shows that the strain FJ2B-25 has significant inhibitory effect.
Example 3: the separated bacterial strain fermentation product sterile filtrate of the invention (rhizoctonia solani: (Rhizoctonia solani) Effect of growth and sclerotium Germination
Firstly, inoculating the FJ2B-25 strain into a test tube filled with 3 mL of LB liquid culture medium (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, and the balance of distilled water); then carrying out shake culture at 37 ℃ and 180 r/min for 12 h; then taking 1 mL of bacterial liquid to inoculate into a shake flask filled with 50 mL of LB liquid culture medium, and carrying out shake culture at 37 ℃ and 180 r/min for 24 h; then loading into a 50 mL sterile centrifuge tube, centrifuging at 4 deg.C for 10 min at 8,000 r/min to remove thallus, filtering the supernatant with a 0.22 μm sterile filter membrane to remove thallus, and subpackaging in a sterile tube to obtain the sterile fermentation broth of FJ2B-25 strain.
Adding 1 mL of FJ2B-25 strain sterile fermentation broth into 9 mL of PDA solid culture medium which is sterilized, cooled to 45 ℃ and not condensed, uniformly mixing, and pouring; inoculating Rhizoctonia solani (Rhizoctonia solani) with diameter of 5 mm at the center of the plateRhizoctonia solani) GD-118 fungus cake, simultaneously adding 1 mL of sterile water into 9 mL of PDA culture medium which is sterilized, cooled to 45 ℃ and not condensed, uniformly mixing, and then pouring the mixture to serve as a control. Culturing at 29 deg.C, and measuring the growth diameter of hypha every four hours. Each treatment included three replicates and the entire experiment was repeated three times. The results show that: control group and Rhizoctonia solani (Rhizoctonia solani) treated with sterile filtrate of strain FJ2B-25Rhizoctonia solani) The growth diameters of (a) were 55.7 mm and 40.7 mm, respectively, with significant differences between the control and treatment groups. According to the formula of the bacteriostasis rate: bacteriostatic ratio (%)
The bacteriostasis rate of the strain FJ2B-25 is 29.59 percent, and the strain FJ2B-25 has the bacterial strain rhizoctonia solani (Rhizoctonia solani) for the pathogenic bacteria of rice sheath blight diseaseRhizoctonia solani) The growth of hyphae was significantly inhibited (FIG. 4).
Analysis of the inhibitory effect of the fermentation broth sterile filtrate of bacterial strain FJ2B-25 on sclerotium germination:
culturing Rhizoctonia solani (Rhizoctonia solani) of rice sheath blight disease pathogenic bacteria on PDA culture mediumRhizoctonia solani) GD-118, after the sclerotium grows out, the sclerotium which is basically the same in size and has no pollution is selected by a pair of tweezers, then the sclerotium is put into sterile water to be cleaned, grown hypha is removed, and the sclerotium is wiped clean by sterile filter paper and dried at room temperature. Pouring the FJ2B-25 strain sterile fermentation liquid into a sterile culture dish on a super clean bench, and soaking the pretreated sclerotium in the FJ2B-25 strain sterile fermentation liquidSoaking in sterile water of control group at room temperature for 30 min. Then taking out the sclerotium, and putting the sclerotium on filter paper for airing; then inoculating the mycelia on a PDA flat plate, uniformly inoculating four sclerotia on each plate, finally culturing at 29 ℃, taking out every half hour, observing the growth condition of mycelia on the sclerotia under a microscope, and counting the number of stages of the mycelia in germination. And (3) grading the germination hyphae: grade 0, the number of hyphae is 1; the germination number of grade 1 hyphae is 1-20; grade 2, the number of germination hyphae is 21-50; grade 3, the number of germination hyphae is 51-80; 4, 81-140 germinated hyphae; grade 5, the number of the germination hyphae is more than 141. Each treatment included three replicates and the entire experiment was repeated three times. Calculating the hypha germination index according to a germination hypha index formula: germination hypha index = ∑ (number of stages of germination hyphae at each stage × representative value at each stage)/(total number of investigation × representative value at highest stage) × 100. The results are shown in FIG. 5: control group and Rhizoctonia solani treated with the sterile fermentation broth of FJ2B-25 Strain ((R))Rhizoctonia solani) GD-118 sclerotium germination hypha indexes are 71.67% and 33.33%, respectively, and significant differences exist between the control group and the treatment group. It can be seen that the treatment of the fermentation broth of a sterile strain of strain FJ2B-25 resulted in Rhizoctonia solani: (Rhizoctonia solani) The hypha index of the sclerotium germination is obviously reduced. Simultaneously, the sterile fermentation liquor of the strain FJ2B-25 is rhizoctonia solani (F: (F))Rhizoctonia solani) The GD-118 sclerotium hypha has obvious bacteriostatic effect. As described above, the sterile fermentation broth of FJ2B-25 strain can inhibit Rhizoctonia solani (F.solani) ((R))Rhizoctonia solani) Germination and growth of sclerotia.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight
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| CN115851533A (en) * | 2022-12-05 | 2023-03-28 | 众乐(潍坊)生物科技有限公司 | Bacillus thuringiensis and application thereof in inhibition of livestock and poultry feed mould |
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