CN106479934A - A kind of ash arrhizus bacteria antagonistic strain and its screening technique and application - Google Patents

Abstract

The invention belongs to the field of microbial technology and biological control, and in particular relates to a Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) BA-KA4 and its screening method and application. The present invention isolates the bacteria from the soil, and screens out the bacterium BA-KA4 with an inhibitory rate of 65% to Botrytis cinerea and stable inheritance by using the plate confrontation method. The strain BA‑KA4 was preliminarily identified as Bacillus amyloquefaciens by analyzing the culture characteristics, morphology, physiological and biochemical characteristics of the strain combined with the results of 16S rDNA sequence and gyr B gene sequence. Preserved strains, the registration number in the General Microbiology Center of the China Microbiological Strain Collection Management Committee is CGMCC No.12190, which has the best control effect on cucumber gray mold and has a long-lasting effect, and has a wide range of antibacterial spectrum.

Description

A kind of Botrytis cinerea antagonistic strain and its screening method and application

technical field

The invention belongs to the field of microbial technology and biological control, and specifically relates to a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BA-KA4 and a screening method and application thereof.

Background technique

Botrytis cinerea is a worldwide plant disease, which is caused by the fungus Botrytis cinerea, which infects various crops including cucumbers, tomatoes, grapes, etc. serious threat to the yield and quality of vegetables and fruits. At present, chemical pesticides such as pyrimethanil, azoxystrobin, carbendazim, dimethocarb, and saccharin are mainly used for prevention and control. The large-scale use of chemical pesticides has not only caused environmental pollution, but also caused botrytis cinerea to affect pyrimella. The resistance of amine, carbendazim, and diethofencarb to other agents, Wang Meiqin et al. have shown that the resistance of cinerea cinerea in Jinzhong area of Shanxi Province to Ditofencarb and Dimethocarb reached moderate resistance, and the resistance to carbendazim For high resistance. Research by Li Xinghong and others found that the resistance of tomato gray mold to pyrimethanil is very common in Beijing area, and the high antibacterial strains are the main ones. In production, new fungicides should be replaced to control tomato gray mold. Therefore, the development of safe, efficient, and non-resistance-inducing biological control methods is an important way to reduce the hazards of Botrytis cinerea, reduce environmental pollution, and ensure stable and high agricultural yields, and has increasingly become a research hotspot for Botrytis cinerea control.

At present, many strains with antagonistic effect on Botrytis cinerea have been screened out. Elad made Trichoderma harzianum wettable powder from Trichoderma harzianum T39 strain to control the occurrence of diseases such as greenhouse crops and grape gray mold. Dik et al. compared the control effects of Trichoderma harzianum T39, Aureobasidium pullulans, and Cryptococcus albicans on gray mold of cucumber and tomato, and found that the treatment of Trichoderma harzianum and Aureobasidium pullulans can reduce the mortality of stem lesions and plants 40%~100%, and its control effect is better than that of the fungicides, afendazim and chlorhein. Wei Yanmin et al. determined the antibacterial effect of 26 strains of actinomycetes on 5 kinds of pathogenic fungi, among which 5 strains had a stronger antibacterial effect on Botrytis cinerea, and the antibacterial activity was significantly higher than that of 50% carbendazim and other control chemicals. Han Siqin et al. isolated an actinomycete D2-4 from the soil of Changbai Mountain, and found that the fermentation liquid of this fungus had a strong inhibitory and control effect on tomato gray mold through plate antibacterial test, detached leaf test and pot test. . So far, there are more than 10 kinds of bacteria used in the research and application of Botrytis cinerea, mainly Bacillus, Pseudomonas, etc., which can control the occurrence and damage of botrytis in plants such as tomato, pepper, strawberry, apple and grape . The Bacillus licheniformis strains W 10, W 3 and Y 2-11-1 with strong antagonistic and broad antibacterial spectrum isolated by Tong Yunhui et al. have a control effect of 70% to 80% on tomato gray mold leaves and fruits, which is better than 50% Sukling 2000 times solution. Wang Wei et al found that the antagonistic bacterium x-75 has a good inhibitory effect on tomato Botrytis cinerea. Generally speaking, there are relatively few studies on the use of antagonistic bacteria to control Botrytis cinerea in my country. Wang Yingying et al. isolated a strain of Bacillus amyloliquefaciens, which is effective against plant pathogenic fungi such as Fusarium oxysporum and strawberry snakeeye. Strong inhibitory effect. For the first time, Wang Yiwen and others isolated a strain of Bacillus amyloliquefaciens from the surface of melon fruit, which has significant and broad-spectrum effects on postharvest pathogenic fungi such as Botrytis cinerea, Alternaria, Fusarium oxysporum, Aspergillus niger and Trichothecene pink. antagonism.

In this study, using the screening system of single-target primary screening and multi-target re-screening, Bacillus amyloliquefaciens BA-KA4, which has good antagonistic effect on Botrytis cinerea and a wide antibacterial spectrum, was screened out from soil samples, in order to prevent Botrytis cinerea. Provide material support for the biological control of disease.

Contents of the invention

The object of the present invention is to provide a kind of Bacillus amyloliquefaciens BA-KA4, which has the effect of inhibiting the growth of the crop gray mold pathogen, and has a wide antibacterial spectrum, especially has the ability to obviously inhibit the cucumber gray mold pathogen growth effect. On the other hand, the present invention also provides the screening method and application of the above bacterial strain.

In order to achieve the above object, the technical scheme adopted in the present invention is:

An antagonistic strain of Botrytis cinerea, which is Bacillus amyloliquefaciens BA-KA4, was preserved on March 8, 2016 in the General Microorganism Culture Collection Center of China Microbiology Culture Collection Management Committee, and the preservation number is: CGMCC No .12190.

Further, the said Botrytis cinerea antagonistic strain has the ability to inhibit Phytophthora capsici, Wheat scab, Wheat sheath blight, Tobacco Alternaria, Sclerotinia garlic, Botrytis cinerea, Tomato early blight, Pear Activity of black spot fungus growth.

A screening method for the above-mentioned Bacillus amyloliquefaciens BA-KA4, comprising the following steps:

(1) Separation, purification and preservation of bacteria in soil samples from different regions;

(2) Preliminary screening: use the plate confrontation method to screen antagonistic bacteria, use a puncher with a diameter of 5mm to punch holes on the edge of the cucumber gray mold, inoculate the bacteria block in the center of the PDA plate, and pick 4 different single colonies evenly. Connect it at a distance of 3cm from the indicator bacteria, and after culturing in the dark at 22°C for 5 days, observe the antibacterial effect, and select the strains with the antibacterial zone to enter the re-screening;

(3) Re-screening: use the cucumber gray mold as the indicator bacteria, and use the cross method to connect the strains entering the re-screening on the four corners 3cm away from the center of the plate, and move into the center of the plate at the same time. Botrytis cinerea with a diameter of 5mm For the agar block of the pathogen, after 5 days of dark culture at 22°C, observe the antibacterial results, measure the diameter of the inhibition zone, calculate the antibacterial rate, and screen out the strains with an inhibitory rate of more than 60%; carry out subculture, measure the genetic stability, and screen After cultivating 10 generations of strains with a bacteriostatic rate of more than 60%; select Phytophthora capsici, Wheat scab, Wheat sheath blight, Tobacco Alternaria, Garlic Sclerotinia, Tomato Botrytis cinerea, Tomato early blight, Pear Black spot bacteria were used as indicator bacteria, and the plate confrontation method was used to measure the bacteriostatic spectrum of the selected strains, observe the bacteriostatic effect, measure the diameter of the bacteriostatic zone, and calculate the bacteriostatic rate. A preferred strain is Bacillus amyloliquefaciens BA-KA4.

Further, the specific operation of step (1) is as follows: 120 soils collected from Yunnan, Heilongjiang, Xinjiang, and Hebei regions were used to separate the soil by using PB medium, and the soil was separated by dilution plate method, and cultivated in an incubator at 28°C for 3 days Finally, bacteria with different colony shapes were picked for 5 streak purifications, numbered, and stored in a -80°C refrigerator with 15% glycerol.

Further, the PB medium is: 5.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 15 g agar, pH 7.4, distilled water to 1 L, sterilized at 121 ° C for 20 min.

An application of the above-mentioned bacillus amyloliquefaciens BA-KA4 in preventing and treating crop gray mold diseases.

Further, the control method is to dilute Bacillus amyloliquefaciens BA-KA4 with sterile water to a bacterial suspension with a concentration of 1×10 ⁸ CFU/mL, sterilize at 121° C. for 20 minutes, and then spray the leaves on the crops.

The morphological characteristics of the Bacillus amyloliquefaciens CGMCC No. 12190 of the present invention are: no pigment is produced when grown on PB medium, the colonies are flat or round, milky white and opaque, the edges of the colonies are neat, and the surface has wrinkles. Gram-positive, microscopically observed as rod-shaped, producing spores, spores are oval or cylindrical. See Figure 3.

Physiological and biochemical identification of the strains was carried out referring to the "Berjamin Bacteria Identification Handbook" and "Common Bacteria Identification Handbook". The results are shown in Table 1. The antagonistic bacteria BA-KA4 was initially identified as Bacillus amyloliquefaciens.

Table 1 Physiological and biochemical identification results of antagonistic bacteria

Note: "+" means positive, "-" means negative

Bacillus amyloliquefaciens BA-KA4 of the present invention is effective against capsicum phytophthora, wheat scab, wheat sheath blight, tobacco red spot, garlic sclerotinia, tomato cinerea, tomato early blight, and pear black spot and many other pathogenic fungi have obvious inhibitory effects; and it is especially suitable for making agricultural preparations for preventing and controlling crop diseases caused by Botrytis cinerea infection.

Biological Sample Preservation Information

Bacillus amyloliquefaciens BA-KA4, classified as Bacillus amyloliquefaciens, was preserved on March 8, 2016 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, referred to as CGMCC, address: No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101, strain preservation number is CGMCC No.12190.

Description of drawings

Figure 1 Antagonistic effect of antagonistic bacteria BA-KA4 on cucumber Botrytis cinerea

Figure 2 Antibacterial spectrum of antagonistic bacteria BA-KA4

Figure 3 Colony morphology of antagonistic bacteria BA-KA4

Figure 4 Phylogenetic tree of antagonistic BA-KA4 based on 16S rDNA gene sequence

Fig. 5 Phylogenetic tree of antagonistic BA-KA4 based on gyrB gene sequence.

detailed description

Used test soil in the present invention is the soil sample collected from places such as Yunnan, Heilongjiang, Xinjiang, Hebei.

The pathogenic bacteria tested were Phytophthora capsici, Wheat scab, Wheat sheath blight, Tobacco Alternaria, Garlic Sclerotinia, Tomato Botrytis cinerea, Tomato early blight, and Pear black spot, which were isolated and preserved in our laboratory.

Cucumber seeds tested were purchased from Hebei Academy of Agricultural Sciences.

The test medium is: PDA medium (1 L): peeled potato 200.0 g, glucose 20.0 g, agar 15 g, distilled water to 1 L, sterilized at 115°C for 20 minutes; PB medium (1 L): beef extract 5.0 g, peptone 10.0 g, NaCl 5.0 g, agar 15 g, pH 7.4, distilled water to 1 L, sterilized at 121°C for 20 min.

Example 1 Isolation, Purification and Preservation of Bacteria in Soil

(1) 120 soils collected from Yunnan, Heilongjiang, Xinjiang, Hebei and other places were used to separate the soil by using PB medium and the dilution plate method. After culturing in an incubator at 28°C for 3 days, the colony with different morphology was picked. Bacteria were streaked and purified five times, numbered, and stored in a -80°C refrigerator with 15% glycerol.

(2) Use the plate confrontation method for primary screening of antagonistic bacteria. Use a puncher with a diameter of 5 mm to punch holes on the edge of the cucumber gray mold, inoculate the bacteria block in the center of the PDA plate, and pick 4 different single colonies evenly on the At a position 3cm away from the indicator bacteria, after culturing in the dark at 22°C for 5 days, the antibacterial effect was observed, and the strains with the antibacterial zone were selected for re-screening. 4,200 strains of bacteria isolated from 129 soil samples were subjected to plate confrontation tests with Botrytis cinerea, and 191 strains of bacteria with antagonistic effects on Botrytis cinerea were obtained through preliminary screening.

(3) Using Cucumber Botrytis cinerea as the indicator bacteria, use the cross method to spot the strains entering the primary screening on the four corners 3cm away from the center of the plate, and move the agar with a diameter of 5mm in the center of the plate with Botrytis cinerea at the same time After culturing in the dark at 22°C for 5 days, measure and record the diameter of the inhibition zone and calculate the inhibition rate, and screen out 10 strains with an inhibition rate of more than 60%. Inhibition rate (%) = (diameter of pathogenic bacteria colony in control group - diameter of pathogenic bacteria colony in treatment group) / diameter of pathogenic bacteria colony in control group × 100%. Carry out subcultivation, measure genetic stability, screen and cultivate the strains with bacteriostatic rate of more than 60% after 10 generations, and the bacteriostatic rates of the above 10 strains are all above 60% after 10 generations of culture; select Phytophthora capsici, Gibberella wheat Eight kinds of fungi, including germs, wheat sheath blight, tobacco red spot, garlic sclerotinia, tomato cinerea, tomato early blight, and pear black spot, were used as indicator bacteria for multi-target re-screening to observe the antibacterial effect and measure The diameter of the inhibition zone was used to calculate the inhibition rate, and the strain with the best antibacterial effect on the eight pathogenic fungi was selected, which was Bacillus amyloliquefaciens BA-KA4. The results are shown in Figure 1 and Figure 2, the bacteriostatic rate of BA-KA4 was the highest, 65%, and it was genetically stable. The antagonistic bacteria BA-KA4 had obvious inhibitory effects on 8 kinds of pathogenic fungi, and had a broad spectrum of inhibition, and the antagonistic ability to different pathogenic fungi was different. Among them, the inhibition rate of 5 kinds of pathogenic fungi reached more than 60%, respectively: the inhibition rate of pear black spot fungus was 75.9%, the inhibition rate of garlic Sclerotinia sclerotiorum was 65.9%, and the inhibition rate of tomato early blight was 62.9% , the inhibition rate of Botrytis cinerea was 61.8%, and the inhibition rate of Phytophthora capsici was 60.6%.

Example 2 Identification of antagonistic bacteria BA-KA4

(1) Culture characteristics and morphological characteristics of antagonistic bacteria

The bacterial strain BA-KA4, as shown in Figure 3, does not produce pigment when grown on PB medium, and the colonies are flat or round, milky white and opaque, with irregular edges and wrinkles on the surface. Gram-positive, microscopically observed as rod-shaped, producing spores, spores are oval or cylindrical.

(2) Physiological and biochemical characteristics of antagonistic bacteria

Refer to "Berjamin Bacterial Identification Manual" and "Common Bacterial System Identification Manual": VP test positive, methyl red test negative, indole test positive, gelatin liquefaction test positive, citrate utilization test positive, starch hydrolysis test positive , lipase negative, H ₂ S production test positive, nitrate reduction test positive, malonate utilization negative, tyrosine hydrolysis negative, cellulolysis positive.

(3) PCR amplification and sequence determination

(1) 16S rDNA sequence analysis and identification

The kit of Tiangen Company was used to extract bacterial genomic DNA, using this DNA as a template, with 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), sequence SEQ ID No.1 and 1492R (5'-GGYTACCTTGTTACGACTT-3'), The sequence SEQ ID No.2 is used as a primer to amplify the 16S rDNA of the bacterial strain. The PCR amplification conditions are: 95°C for 5 min; 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, 35 cycles; 72°C for 10 min. The 16S rDNA amplification product was sequenced in Jinweizhi Biotechnology Co., Ltd., and the sequence obtained by sequencing was compared with BLAST by NCBI database, and the phylogenetic analysis was carried out with the known sequence by MEGA5.2 software. Using the antagonistic bacteria BA-KA4 genomic DNA as a template, PCR amplification was carried out with primers 27F and 1492R, and the length of the 16S rDNA nucleotide sequence of the strain was measured to be 1394bp, as shown in the sequence SEQ ID No. Compared with the known 16S rDNA sequences, it was found that the antagonistic bacteria BA-KA4 strain was 100% similar to Bacillus methylotrophicus strain F29, Bacillus amyloloquefaciens strain CSM11, and Bacillus subtilis strain MB8, and a phylogenetic tree of 16S rDNA sequences was constructed (Figure 4). , it can be seen that the antagonistic bacteria BA-KA4 was identified as Bacillus amyloquefaciens.

(2) gyrB gene sequence analysis and identification

The kit of Tiangen Company was used to extract the bacterial genomic DNA, using this DNA as a template, the forward primer was UP1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3'), the sequence SEQ ID No.4, and the reverse primer was UP2r (5' -AGCAGGGTACGGAT GTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'), sequence SEQ ID No.5, amplified gyrB gene sequence. The PCR amplification conditions are: 95°C for 4min; 98°C for 10s, 62°C for 1min, 72°C for 2min, 30 cycles; 72°C for 8min. The amplified product of gyrB gene was sequenced in Jinweizhi Biotechnology Co., Ltd., and the sequence obtained by sequencing was compared with BLAST by NCBI database, and the phylogenetic analysis was carried out with the known sequence by MEGA5.2 software. The gyrB gene was amplified using the genomic DNA of the antagonistic bacteria BA-KA4 as a template, and the sequence length was about 1010bp, as shown in the sequence SEQ ID No.6. Compared with the 16S rDNA sequence of the model strain in NCBI, it was found that the antagonistic bacteria BA-KA4 The KA4 strain has the closest homology with Bacillus amyloquefaciens, with a similarity of 98%. A phylogenetic tree was constructed based on the gyrB gene sequence (Figure 5). The closest kinship.

Based on the culture characteristics, morphology, physiological and biochemical characteristics of the strain, and the analysis results of 16S rDNA sequence and gyrB gene sequence, it can be concluded that the antagonistic bacteria BA-KA4 strain is Bacillus amyloloquefaciens.

Example 3 Pot control effect test of Bacillus amyloliquefaciens BA-KA4 on cucumber gray mold

To prepare a seed bottle, pick a single colony of BA-KA4 and inoculate it into a 100 mL Erlenmeyer flask containing 20 mL of PB liquid medium, and culture it on a shaker at 28°C and 180 rpm for 24 hours. Draw 10 mL of the bacterial liquid from the seed bottle and inoculate it into a 500 mL Erlenmeyer flask containing 200 mL of PB liquid medium. After 48 hours of shaking culture at 28 °C and 180 rpm on a shaking table, dilute it with sterile water to 1×10 ⁸ CFU/mL, that is, For bacterial suspension. Take the same concentration of bacterial suspension and sterilize at 121°C for 20 minutes, which is the sterile fermentation broth. Azoxystrobin was diluted 1000 times with sterile water. Use a hole puncher to punch holes at the edge of Botrytis cinerea growing for 3 days, put the bacteria block into 0.9% normal saline, shake it at 22°C and 180 rpm for 30 minutes, and dilute it with sterile water to a concentration of 1×10 ⁶ per mL of spore suspension. Potted cucumber leaves were sprayed with bacterial suspension, sterile fermentation broth, and azoxystrobin for 6 hours, then inoculated with Botrytis cinerea spore suspension, and sterile water was used as a negative control. Moisturize in the dark for 48 hours, incubate at a constant temperature of 22°C, observe the disease on 5 days, 7 days, and 14 days, and calculate the disease index.

The experimental results are shown in Table 2. The control effect of the aseptic fermentation broth of Bacillus amyloliquefaciens BA-KA4 and azoxystrobin on cucumber botrytis can reach 100% in 5d and 7d, but the effect of azoxystrobin in 14d The control effect decreased, but the control effect of the sterile fermented liquid could still reach 97.5%. Compared with the treatment groups of azoxystrobin and bacterial suspension, the leaves treated with sterile fermentation broth were more dark green. Compared with the commonly used drug azoxystrobin, the short-term control effect is equivalent, but the metabolites of this strain last longer.

Table 2 Indoor control effect test of Bacillus amyloliquefaciens BA-KA4 on cucumber gray mold

In areas where cucumber botrytis is more common, the use of antagonistic microorganisms can reduce the use of chemical pesticides, solve the problem of pesticide residues, and delay the emergence of bacterial resistance.

Bacillus amyloliquefaciens is a bacterium with a high affinity with Bacillus subtilis, which can produce a series of metabolites during its own growth, which enable Bacillus amyloliquefaciens to have a wide range of inhibitory fungi and bacteria active. The control effect test of potted plants in this study showed that the control effect of the metabolites of Bacillus amyloliquefaciens BA-KA4 on cucumber gray mold could reach 100% at 5 days and 7 days, and still reach 97.5% after 14 days. Compared with the commonly used drug azoxystrobin, the short-term control effect is equivalent, but the metabolites of this strain last longer.

SEQUENCE LISTING

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Claims (7)

1. a Botrytis cinerea antagonistic strain, it is characterized in that, this bacterial strain is Bacillus amyloliquefaciens BA-KA4, on March 8th, 2016, preserved in China Microbiological Cultures Preservation Management Committee common microbial strains preservation center, The deposit number is: CGMCC No. 12190. 2. The Botrytis cinerea antagonistic strain according to claim 1, characterized in that it has the ability to inhibit Phytophthora capsici, Fusarium graminearum, Rhizoctonia cerealis, tobacco Growth characteristics of Alternaria alternata, Sclerotinia allii Saw, Botrytis cinerea, Alternaria solani, Alternaria alternate. 3. a screening method for Bacillus amyloliquefaciens BA-KA4 described in any one of claims 1-2, is characterized in that comprising the following steps: (1) Separation, purification and preservation of bacteria in soil samples from different regions; (2) Preliminary screening: use the plate confrontation method to screen antagonistic bacteria, use a puncher with a diameter of 5mm to punch holes at the edge of the activated Botrytis cinerea, pick the bacteria block and inoculate it in the center of the PDA plate, pick 4 different single The colony is evenly connected at a distance of 3cm from the indicator bacteria, and after 4-6 days of dark cultivation at 22°C, the antibacterial effect is observed, and the strains with the inhibition zone are selected for re-screening; (3) Re-screening: Using Botrytis cinerea as the indicator bacteria, use the cross method to connect the strains entering the re-screening on the four corners 2-3cm away from the center of the plate, and move the strains with Botrytis cinerea into the center of the plate at the same time. Agar block, cultured in the dark at 22°C for 5-7 days, observe the antibacterial results, measure the diameter of the inhibition zone, calculate the antibacterial rate, and screen the strains with an inhibition rate of more than 60%; measure the genetic stability, and screen and cultivate for 10 generations to inhibit the bacteria Strains with a rate of more than 60%; choose Phytophthora capsici, Wheat scab, Wheat sheath blight, Tobacco Alternaria, Garlic Sclerotinia, Tomato Botrytis cinerea, Tomato early blight, and Pear black spot bacteria as indicator bacteria , using the plate confrontation method to measure the antibacterial spectrum of the selected strains, observe the antibacterial effect, measure the diameter of the antibacterial zone, calculate the antibacterial rate, and screen out the strains with the best antibacterial effect on these eight kinds of pathogenic fungi, namely Bacillus amyloliquefaciens BA-KA4. 4. The screening method according to claim 3, characterized in that, the specific operation of step (1) is as follows: 120 soil samples collected from Yunnan, Heilongjiang, Xinjiang, and Hebei regions, using PB medium, using dilution coating Separation was carried out by the plate method, and after 3 days of culture in a 28°C incubator, bacteria with different colony shapes were picked for five times of streaking, purification, numbering, and storage in a -80°C refrigerator with 15% glycerol. 5. The screening method according to claim 4, wherein the PB medium is: beef extract 5.0 g, peptone 10.0 g, NaCl 5.0 g, agar 15 g, pH 7.4, distilled water to 1L, 115 Sterilize at ℃-121℃ for 20min-30min. 6. The application of the Bacillus amyloliquefaciens BA-KA4 described in any one of claims 1-5 in the prevention and treatment of botrytis cinerea. 7. The application according to claim 6, characterized in that the control method is to dilute Bacillus amyloliquefaciens BA-KA4 with sterile water to a bacterial suspension with a concentration of 1×10 ⁸ -1×10 ⁹ CFU/mL , After sterilizing at 115°C-121°C for 20 minutes, spray the leaves on the crops.