CN116515689A - Bacillus bailii B125 for preventing and treating strawberry leaf blight and application thereof - Google Patents
Bacillus bailii B125 for preventing and treating strawberry leaf blight and application thereof Download PDFInfo
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- CN116515689A CN116515689A CN202310414287.1A CN202310414287A CN116515689A CN 116515689 A CN116515689 A CN 116515689A CN 202310414287 A CN202310414287 A CN 202310414287A CN 116515689 A CN116515689 A CN 116515689A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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Abstract
The invention relates to bacillus subtilis B125 for preventing and treating strawberry leaf blight and application thereof. The bacillus belicus (Bacillus velezensis) B125 is preserved in China general microbiological culture Collection center (CGMCC) with a strain preservation number of CGMCC No.26583 in 2023 and 2 months and 20 days. The invention provides a culture method of bacillus belicus B125. The invention also provides application of the bacillus belicus B125 in preparing a microbial agent for preventing and treating strawberry leaf blight. The bacillus belicus (Bacillus velezensis) B125 is obtained by first separation, has obvious inhibition effect on strawberry leaf blight bacteria, can be used for preventing and treating the strawberry leaf blight bacteria and preparing a microbial inoculum for preventing and treating the strawberry leaf blight bacteria, and has wide application prospect.
Description
Technical Field
The invention relates to bacillus subtilis B125 for preventing and treating strawberry leaf blight and application thereof, and belongs to the technical field of microorganisms.
Background
Strawberry (Fragaria ananassa) is a perennial herb of the genus strawberry of the family rosaceae, and is popular with more and more consumers because of its rich nutrition and high economic value. In recent years, the strawberry industry in China develops rapidly, the cultivation area expands year by year, but as the cultivation area expands continuously and soil continues to grow for years, the strawberry leaf diseases are serious, and a great challenge is brought to strawberry production.
The strawberry leaf blight is a leaf disease which is difficult to control in the current strawberry planting area, once the strawberry leaf blight is generated, photosynthesis and nutrient utilization of plants are seriously affected, and finally, the quality and yield of strawberries are reduced. If the prevention and the control are not carried out in time, the yield is reduced, and if the yield is increased, the yield is increased. It is reported that the pathogenic bacteria causing strawberry leaf blight at present mainly include Pestalotiopsis (Pestalotiopssspp.).
At present, the method for preventing and treating the leaf blight of the strawberries still takes chemical prevention and treatment as the main method, but more and more traditional chemical pesticides are gradually forbidden due to the non-negligible defects of high toxicity, high residue, environmental pollution, harm to people and livestock and the like. Biological control is environment-friendly, and pathogenic bacteria are not easy to generate drug resistance. The biocontrol microbial agent has simple production process, can promote the growth of crops and has wide application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides bacillus belgium B125 for preventing and treating strawberry leaf blight and application thereof. The bacillus belicus (Bacillus velezensis) B125 strain has remarkable antagonism on pathogenic bacteria causing strawberry leaf blight, is high in safety, free of hemolysis, harmless to crop growth and capable of promoting plant growth.
The technical scheme of the invention is as follows:
in a first aspect, the present invention provides bacillus belicus (Bacillus velezensis) B125 for controlling strawberry leaf blight, deposited in the China general microbiological culture Collection center, address: the collection number of the strain is CGMCC No.26583 of the institute of microbiological study, national academy of sciences, no. 3, north Chen West Lu, 1, of the Chaoyang area of Beijing city.
In a second aspect, the invention also provides a culture method of bacillus beljalis B125, which specifically comprises the following steps:
(1) Inoculating bacillus belicus B125 into an LB liquid culture medium, and culturing for 12-16 hours at 36-38 ℃ to obtain seed liquid;
(2) Inoculating the seed liquid prepared in the step (1) into an LB liquid culture medium according to the volume percentage of 5-10%, and performing expansion culture for 20-24 h at 36-38 ℃ to prepare bacillus belicus B125 bacterial liquid.
In a third aspect, the invention also provides application of the bacillus beijerinckii B125 in preparing a microbial agent for preventing and treating strawberry leaf blight.
Preferably according to the invention, said application comprises the steps of:
1) Inoculating bacillus belicus B125 into an LB liquid culture medium, and culturing for 12-16 hours at 36-38 ℃ to obtain seed liquid;
2) Inoculating 10-15% of the seed liquid prepared in the step 1) into a solid fermentation culture medium according to the volume percentage, culturing for 72-84 hours at 36-38 ℃, and then drying for 5-7 hours at 60-70 ℃ to obtain bacillus beijerinus B125 solid fermentation product;
3) Crushing the bacillus belicus B125 solid fermentation product prepared in the step 2), and sieving the crushed bacillus belicus B125 solid fermentation product with a 75-85-mesh sieve to obtain the microbial agent for preventing and treating strawberry leaf blight.
According to the present invention, preferably, in the step 1), the seed liquid concentration is not less than 3.0X10 8 CFU/mL。
According to a preferred embodiment of the present invention, in step 2), the formula of the solid fermentation medium is: 60 to 65 percent of bran, 20 to 25 percent of straw powder, 4 to 6 percent of glucose, 2 to 3 percent of peptone, 1 to 1.5 percent of monopotassium phosphate, pH control of 7.0 to 7.5 and water content control of 50 to 60 percent.
According to the invention, in the step 3), the spore content of bacillus beliensis B125 in the microbial agent for preventing and treating strawberry leaf blight is not less than 1.0x10 11 The spore rate is not lower than 95 percent per gram.
The components of the invention are all commercially available products, and the prior art can be adopted in the non-exhaustive place.
The invention has the beneficial effects that:
1. the bacillus belicus (Bacillus velezensis) B125 is obtained by first separation, has obvious inhibition effect on strawberry leaf blight bacteria, can be used for preventing and treating the strawberry leaf blight bacteria and preparing a microbial inoculum for preventing and treating the strawberry leaf blight bacteria, and has wide application prospect.
2. The microbial agent for preventing and treating the strawberry leaf blight provided by the invention has an obvious inhibition effect on strawberry leaf blight bacteria, has no hemolysis phenomenon, is harmless to crops, has high safety, can promote plant growth, is suitable for industrial production, and has high application value.
Description of the drawings:
FIG. 1 is a diagram showing the identification of Bacillus belicus (Bacillus velezensis) B125;
in the figure: a is a morphological diagram of a strain B125 on a culture medium; b is a gram of strain B125; c is a spore staining chart of the strain B125.
FIG. 2 is a 16S rDNA phylogenetic tree of Bacillus beleiensis (Bacillus velezensis) B125.
FIG. 3 shows antagonism of leaf blight of strawberry by Bacillus belicus B125.
FIG. 4 shows the effect of microbial agents containing Bacillus belicus B125 on controlling strawberry leaf blight.
FIG. 5 shows a B.bailii B125 cultured on a blood plate.
FIG. 6 shows a cabbage after use of a microbial agent containing Bacillus belicus B125.
Detailed Description
The invention is further clearly illustrated by the following examples, which are not to be construed as limiting the invention in any way, in order to make the objects, technical solutions and advantages of the invention more apparent. The experimental methods in the following examples are conventional methods unless otherwise specified; the percentages in the examples below are by weight unless otherwise indicated.
Bacillus belicus (Bacillus velezensis) B125 was deposited at the China general microbiological culture Collection center, address: the collection number of the strain is CGMCC No.26583 of the institute of microbiological study, national academy of sciences, no. 3, north Chen West Lu, 1, of the Chaoyang area of Beijing city.
Example 1
1. Screening and isolation of strains
Healthy strawberry plant rhizosphere soil is selected from a strawberry planting base, a sterilization sampling shovel is used for collecting soil with a good growth condition and a strawberry rhizosphere range of 5-20 cm in the same area, a sample is put into a sterilization bag, brought back to a laboratory and stored in a refrigerator at 4 ℃. When separating, weighing 10g of soil sample, placing into a triangular flask filled with sterile water and glass beads, shaking for 30min, standing for 10min after the soil sample is uniformly dispersed to form bacterial suspension with concentration of 0.1g/ml, and diluting the bacterial suspension to obtain the bacterial suspension with concentration of 10 -2 ，10 -3 ，10 -4 ，10 -5 ，10 -6 ，10 -7 And g/ml bacterial suspension, respectively taking 100 mu L of bacterial suspension, uniformly coating the bacterial suspension on LB solid culture medium, culturing the bacterial suspension in a constant temperature incubator at 37 ℃ for 24 hours, picking single bacterial colonies similar to bacillus, and storing the single bacterial colonies in the culture medium at 4 ℃.
Then, taking strawberry leaf blight bacteria (Neopetalotopsisclovispora) as screening strains, adopting a flat plate counter culture method, taking strawberry leaf blight bacteria cakes with the diameter of 5mm after activation, inoculating the strawberry leaf blight bacteria cakes to the center of a PDA solid culture medium, inoculating bacillus at a position which is 2.5cm away from the strawberry leaf blight bacteria cakes, taking non-inoculated bacillus as a control, repeating each treatment for 3 times, culturing for 7d and 7d at a constant temperature of 28 ℃, calculating the bacteriostasis rate, screening the strain with the optimal inhibition effect, and obtaining the strain B125.
Antibacterial ratio (%) = (diameter of control pathogenic bacteria-diameter of counter-cultured pathogenic bacteria)/diameter of control pathogenic bacteria×100%.
2. Identification of strains
The colony of the strain B125 on the LB medium is milky white, opaque, irregular in edge and easy to pick (FIG. 1A). Gram staining and spore staining were performed to find that the bacterial cells were gram positive bacteria (FIG. 1B). The bacterial cells were short-stalk-shaped, and short-stalk spores were produced (FIG. 1C). The physiological and biochemical identification results of the bacteria show that the strain has positive gelatin liquefaction test, positive lecithin enzyme activity test, positive starch hydrolysis test, negative indole test, positive V-P test, positive motility test, positive contact enzyme test, positive anaerobic growth test, positive citrate test, positive nitrate reduction test and negative methyl red test.
DNA from the screened strain B125 was extracted using a BioTeke bacterial genome extraction kit, specific extraction method was referred to the instructions of the kit, PCR amplification was performed using the 16S rRNA gene of the B125 strain with the 27F and 1492R primer pair, the amplified product was ligated to pMD19-T vector (TaKaRa, china), and sequenced on a Applied Biosystems 3730xl DNA sequencer.
The nucleotide sequence of the PCR primer is as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'，
1492R：5'-GGTTACCTTGTTACGACTT-3'；
the PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 90s;30 cycles, extension at 72℃for 5min, storage at 4 ℃.
Sequencing to obtain fragment length 1292bp, and the sequence is shown as SEQ ID NO. 1. Standard strain sequences were obtained from NCBI database, and 16S rDNA sequences of the isolated strain and the reference strain were analyzed using MEGA-7 to construct phylogenetic tree of the isolated strain and the reference strain as shown in fig. 2. As can be seen from FIG. 2, the 16S rDNA gene sequence of the bacillus strain B125 provided by the invention has extremely high similarity with the 16S rDNA gene sequence of Bacillus velezensis, so that the B125 is determined to belong to Bacillus velezensis, and the bacillus strain is identified as bacillus beliensis (Bacillus velezensis) and named as bacillus beliensis (Bacillus velezensis) B125.
Example 2
The culturing method of bacillus belgium B125 specifically comprises the following steps:
(1) Inoculating bacillus belicus B125 into LB liquid culture medium, and culturing at 37deg.C for 14 hr to obtain seed solution;
(2) And (3) inoculating 10% of the seed liquid prepared in the step (1) into an LB liquid culture medium according to the volume percentage, and performing expansion culture for 24 hours at 37 ℃ to prepare bacillus bailii B125 bacterial liquid.
Example 3
Antagonism of bacillus belicus B125 against strawberry leaf blight.
Taking out the strawberry leaf blight bacteria (Neopentalotopsisclovispora) from a strain tube, inoculating the strain tube onto a PDA solid culture medium, standing at 28 ℃ for 7 days, taking out a bacterial cake with the diameter of 5mm from the cultured strawberry leaf blight bacteria (Neopentalotopsisclovispora) by using a puncher, placing the bacterial cake with the diameter of 5mm in the center of the PDA solid culture medium, inoculating Bacillus berensis B125 bacterial liquid around 2.5cm away from the bacterial cake, and inoculating Bacillus berensis B125 bacterial liquid (CK) in a control group. Then, the plate was cultured at 28℃for 7 days, the diameter of the strawberry leaf blight fungus was measured, and the plate inhibition ratio of the strain B125 against the strawberry leaf blight fungus was calculated.
The antagonism effect of bacillus belicus B125 against strawberry leaf blight is shown in fig. 3, and the inhibition ratio is shown in table 1.
TABLE 1
From fig. 3 and table 1, it can be seen that bacillus belgium B125 has a strong inhibition effect on strawberry leaf blight bacteria, has an obvious inhibition zone, and the inhibition rate of bacillus belgium B125 on strawberry leaf blight bacteria reaches 75.44%. The bacillus belgium B125 can be used for preventing and controlling strawberry leaf blight.
Example 4
A preparation method of a microbial agent containing bacillus bailii B125 comprises the following steps:
1) Inoculating Bacillus bailii B125 into LB liquid medium, culturing at 37deg.C for 14 hr to obtain strain with concentration of 5.0X10 8 Seed solution of CFU/mL;
2) Inoculating the seed liquid prepared in the step 1) into a solid fermentation culture medium according to the volume percentage of 10%, culturing for 80 hours at 37 ℃, and then drying for 6 hours at 65 ℃ to obtain bacillus belicus B125 solid fermentation product;
the formula of the solid fermentation culture medium is as follows: 60-65% of bran, 20-25% of straw powder, 4-6% of glucose, 2-3% of peptone, 1-1.5% of monopotassium phosphate, 7.0-7.5% of pH and 50-60% of water content;
3) Crushing the bacillus belicus B125 solid fermentation product prepared in the step 2), and sieving the crushed bacillus belicus B125 solid fermentation product with a 80-mesh sieve to obtain the microbial agent containing bacillus belicus B125, wherein the specific indexes of the microbial agent are shown in table 2.
TABLE 2
Example 5
The effect of the microbial agent containing bacillus belicus B125 on preventing and controlling strawberry leaf blight is measured.
Firstly, selecting strawberry leaves with similar sizes, sterilizing and cleaning for standby. Preparation of Bacillus bailii B125 at a concentration of 1X 10 according to the number of viable bacteria of microbial agent powder containing Bacillus bailii B125 prepared in example 4 6 CFU/mL、1×10 7 CFU/mL、1×10 8 The CFU/mL bacterial liquid is used for stabbing leaves by a puncture method, 20 mu L of bacterial liquids with different concentrations are respectively inoculated at the wound positions of the leaves, 20 mu L of spore suspension of pathogenic bacteria strawberry leaf blight (Neopralotiopsis sclerotium) is inoculated after air drying, and clear water control treatment and pesticide tebuconazole (diluted according to the use instructions) treatment are arranged. Finally, the treated leaf blade is placed in a culture dish with filter paper for moisturizing culture, and the disease condition and the size of the disease spots on the strawberry leaf blade are observed and measured after 3d, and the results are shown in fig. 4 and table 3.
Control effect= (control lesion diameter-treatment lesion diameter)/control lesion diameter x 100%.
TABLE 3 Table 3
As shown in FIG. 4 and Table 3, the microbial agent containing Bacillus bailii B125 provided by the invention has an obvious inhibition effect on strawberry leaf blight.
Example 6
Safety assay of bacillus belicus B125.
1. Bacillus bailii B125 was streaked onto blood plates and cultured for 24 hours as shown in FIG. 5.
As can be seen from FIG. 5, no hemolytic ring appeared on the blood plate, indicating that Bacillus bailii B125 blood plate was not hemolytic, and was a safe microbial preparation product.
2. Bacillus bailii B125 was prepared at a concentration of 1X 10 9 And transplanting young cabbages with similar sizes into pots, diluting 1g of the microbial inoculum for 500 times, irrigating the cabbages, and measuring the growth index of the cabbages after 30 days by using clear water as a control, wherein the results are shown in fig. 6 and table 4.
TABLE 4 Table 4
As can be seen from fig. 6 and table 4, the microbial agent containing bacillus belicus B125 is harmless to crops and can also effectively promote plant growth.
In conclusion, the bacillus beljalis B125 is obtained by separating from healthy strawberry rhizosphere, has a very good inhibition effect on strawberry leaf blight bacteria, further verifies the good biocontrol effect of the bacillus beljalis B125 on strawberry leaf blight bacteria through an indoor in-vitro leaf test, has high safety, has industrial production conditions, has no hemolysis phenomenon, is harmless to crops, and can promote plant growth. Therefore, bacillus beleiensis B125 is a biocontrol strain with development and application values, can be used for preventing and controlling strawberry leaf blight bacteria and preparing a microbial inoculum for preventing and controlling the strawberry leaf blight bacteria, and has high application value and wide application prospect.
Claims (7)
1. Bacillus belicus (Bacillus velezensis) B125 for preventing and treating strawberry leaf blight was deposited in China general microbiological culture Collection center, address: the collection number of the strain is CGMCC No.26583 of the institute of microbiological study, national academy of sciences, no. 3, north Chen West Lu, 1, of the Chaoyang area of Beijing city.
2. The method for culturing bacillus belgium B125 according to claim 1, which comprises the steps of:
(1) Inoculating bacillus belicus B125 into an LB liquid culture medium, and culturing for 12-16 hours at 36-38 ℃ to obtain seed liquid;
(2) Inoculating the seed liquid prepared in the step (1) into an LB liquid culture medium according to the volume percentage of 5-10%, and performing expansion culture for 20-24 h at 36-38 ℃ to prepare bacillus belicus B125 bacterial liquid.
3. The use of bacillus beljalis B125 according to claim 1 for the preparation of a microbial agent for controlling leaf blight of strawberries.
4. A use according to claim 3, comprising the steps of:
1) Inoculating bacillus belicus B125 into an LB liquid culture medium, and culturing for 12-16 hours at 36-38 ℃ to obtain seed liquid;
2) Inoculating 10-15% of the seed liquid prepared in the step 1) into a solid fermentation culture medium according to the volume percentage, culturing for 72-84 hours at 36-38 ℃, and then drying for 5-7 hours at 60-70 ℃ to obtain bacillus beijerinus B125 solid fermentation product;
3) Crushing the bacillus belicus B125 solid fermentation product prepared in the step 2), and sieving the crushed bacillus belicus B125 solid fermentation product with a 75-85-mesh sieve to obtain the microbial agent for preventing and treating strawberry leaf blight.
5. The use according to claim 4, wherein in step 1), theThe seed liquid concentration is not lower than 3.0X10 8 CFU/mL。
6. The use according to claim 4, wherein in step 2) the solid fermentation medium is formulated as follows: 60 to 65 percent of bran, 20 to 25 percent of straw powder, 4 to 6 percent of glucose, 2 to 3 percent of peptone, 1 to 1.5 percent of monopotassium phosphate, pH control of 7.0 to 7.5 and water content control of 50 to 60 percent.
7. The use according to claim 4, wherein in step 3), the spore content of Bacillus bailii B125 in the microbial agent for controlling strawberry leaf blight is not less than 1.0X10% 11 The spore rate is not lower than 95 percent per gram.
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