CN105176894B - A kind of bacillus amyloliquefaciens and its microbial bacterial agent of prevention graw mold of tomato - Google Patents

Abstract

The invention belongs to Strategies of Agricultural Bio-control fields, specifically disclose a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain HMB27688 for preventing graw mold of tomato, and deposit number is CGMCC No.11457.The invention also discloses using the bacterium prepare microbial bacterial agent and they prevention graw mold of tomato on application.The present invention provides an efficient microorganism for prevention graw mold of tomato, and average preventive effect is and with strong points 80.0% or more；Secondly, microbial bacterial agent of the present invention belongs to environmentally friendly to people, animal safety；In addition, it is long using the present invention prevention graw mold of tomato lasting period, it is not likely to produce drug resistance；Preparation method of the present invention is simple, it is at low cost, using simple.

Description

A kind of Bacillus amyloliquefaciens and its microbial agent for preventing and treating gray mold of tomato

technical field

The invention belongs to the field of agricultural biological control, and in particular relates to a bacillus amyloliquefaciens for preventing and treating gray mold of tomato, a microbial agent containing the bacillus amyloliquefaciens, and also relates to their application in preventing and treating gray mold of tomato.

Background technique

Tomato gray mold is an important disease caused by Botrytis cinerea Pers. The disease mainly harms the fruit, causing the fruit to rot, and also harming the leaves, stems and flowers. The tomato yield can be reduced by 20% to 50%, and the loss is serious. The pathogenic bacteria have a wide range of hosts. In addition to infecting solanaceous vegetables, they can also infect crops such as melons, cabbage, kidney beans, lettuce, onions, and apples.

At present, the main control methods for tomato gray mold are: one is chemical control. Although chemical control has the advantages of quick effect and high efficiency, it is gradually eliminated due to the disadvantages of environmental pollution and natural enemies of pests caused by long-term use; the second is biological control, which has high efficacy, long duration and is not easy to control. The advantages of drug resistance and environmental friendliness are increasingly favored by people.

Bacillius sp is a kind of biocontrol bacteria that has received extensive attention. It has become an ideal biological control bacterium because of its wide distribution, easy isolation and culture, ability to produce spores with strong stress resistance, long storage period and convenient use. Biocontrol microorganisms. Because Bacillus can produce endophytic spores and has strong resistance to stress, compared with other types of biocontrol factors, it is more conducive to the production of bacterial agents, and survives, colonizes and reproduces in the dosage form processing environment. Therefore, screening for bacillus that has an inhibitory effect on pathogenic bacteria is one of the most effective methods for preventing and controlling related plant diseases. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is one of the bacillus used to prevent and control tomato gray mold. BA-KA3 (patent application number: 201410618581.5), SZ23 (patent application number: 201410683826.2), LH-1 (patent application number: 201410735508.6) and NCPSJ17 (patent application number: 201310112580.9), etc. Due to the diversity and synchronous evolution of pathogenic bacteria, it is necessary to find different new strains to control tomato botrytis.

Contents of the invention

The purpose of the invention is to provide a bacillus amyloliquefaciens strain for preventing and treating tomato gray mold, and the strain has the advantages of high efficiency and broad-spectrum bactericidal activity.

The second object of the present invention is to provide a microbial agent produced by the above-mentioned Bacillus amyloliquefaciens.

The third object of the present invention is to provide the preparation method of the above-mentioned microbial inoculum.

The fourth object of the present invention is to provide the use of the above-mentioned Bacillus amyloliquefaciens strain in preventing and treating Botrytis cinerea.

The fifth object of the present invention is to provide the use of the above-mentioned microbial agent in preventing and treating Botrytis cinerea.

The sixth object of the present invention is to provide an identification method for the above-mentioned Bacillus amyloliquefaciens strain.

The seventh object of the present invention is to provide an identification method for the above-mentioned microbial agent.

The present invention is realized through the following technical solutions:

A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HMB27688 has been preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms on September 29, 2015 (the preservation address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing ), the deposit number is CGMCC No.11457.

The microbial agent produced by the above-mentioned Bacillus amyloliquefaciens HMB27688 has an active ingredient of HMB27688 cells.

The above-mentioned microbial bacterial agent can be a liquid preparation.

The above-mentioned microbial bacterial agent, wherein the number of viable bacteria of HMB27688 is greater than 18.0×10 ⁸ cfu/mL.

The preparation method of above-mentioned microbial bacterial agent, comprises the steps:

(1) Strain activation: Activate the HMB27688 strain preserved at low temperature on LB plate medium, pick a single colony and culture it on LB slant medium at 25-35°C for 10-16 hours to obtain the activated strain;

(2) Preparation of seed liquid: scrape a ring with a sterile inoculation loop and inoculate the activated strain in step (1) into 100mL LB liquid medium, and cultivate it at 25-35°C with a shaker speed of 150-220rpm 10-16 hours to get the seed solution;

(3) Fermentation culture: according to the ratio of 1 to 3% by volume, the seed liquid of step (2) is inserted into the corn flour and soybean flour medium, at a temperature of 25 to 35°C and a shaking table speed of 150 to 220rpm Fermented and cultivated for 40 to 50 hours under certain conditions to obtain a fermented liquid;

(4) Detect the number of bacteria and spores in the fermentation broth, and stop the fermentation culture when the mature spores in the fermentation broth account for 90% of the total number of spores and bacteria; the obtained is the liquid preparation of the HMB27688 bacterial strain.

The LB plate medium, LB slant medium and LB liquid medium are all prepared according to conventional methods.

The LB plate medium or LB slant medium described in the above-mentioned preparation method step (1), its composition and weight ratio are: tryptone 8～12g, yeast extract 4～6g, sodium chloride 4～6g, Agar powder 12~18g, water 1000mL.

The LB liquid culture medium described in the step (2) of the above preparation method has components and weight ratios: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride, and 1000 mL of water.

The corn flour and soybean flour culture medium described in the step (3) of the above preparation method, its composition and weight percentage are: corn flour 1.0-3.0%, soybean flour 1.0-3.0%, NaCl 0.1-1.0%, MnSO ₄ . H ₂ O 0.5～1.0%, the rest is water; pH value is 7.2.

The preparation method of the corn flour and soybean flour medium includes mixing corn flour, soybean flour, NaCl and MnSO ₄ ·H ₂ O according to weight percentage, adding water, adjusting the pH and stirring evenly.

The application of the above bacillus amyloliquefaciens HMB27688 in the prevention and treatment of tomato gray mold.

The application of the above-mentioned microbial inoculant in the prevention and treatment of Botrytis cinerea.

The method of using the microbial agent of the present invention: dilute the microbial agent obtained above with water until the number of viable bacteria is 10 ⁷ cfu/mL, and spray the leaves before the onset of tomato gray mold.

Screening and isolation process of HMB27688 strain

The HMB27688 strain was isolated from the cotton field soil in Jingzhou City, Hubei Province by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. In April 2013, the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences collected soil samples from five points in the cotton fields of Jingzhou City, Hubei Province. After mixing, weigh 1g and put it into a 250mL sterile triangular flask, add 100mL of sterile water, and put it in a shaker. On the bed, shake at 170r/min for 30min, let it stand for 2h, take 10mL of supernatant and add it to a 50mL sterilized centrifuge tube, put it in a constant temperature water bath at 80°C for 30 minutes, then take 1mL and add 9mL of sterile water to form 10mL10 ^-3 times soil microorganisms Then dilute the soil suspension into 10 ^-4 , 10 ^-5 , 10 ^-6 times dilutions, take 200 μL of each concentration of microbial suspension and spread it on the LB medium plate, repeat 3 times for each concentration, at 30 ℃ Cultivate at constant temperature for 1d-3d to isolate and purify the bacteria. And with the tomato gray mold as the target, the biocontrol bacteria were screened by the plate confrontation method, the detached leaf method and the pot test method. Results A strain with obvious control effect on tomato gray mold was screened out and named as HMB27688.

Taxonomic identification of strain HMB27688:

(1) Identification of morphological characteristics

Bacteria were cultured on LB medium and were rod-shaped. After 10 hours of culture, spores were produced. The spores were mesenchymal, oval, and the cysts were not enlarged. On the nutrient agar plate, at the early stage of culture, the colonies are light milky white, pus-like, round, with neat edges, and the colonies are raised in the shape of steamed buns, with a moist surface; at the later stage of culture, the colonies are light yellow, with irregular edges, and the surface is dry and wrinkled; on the nutrient agar slant Straight line culture, in a straight line; static culture in liquid medium, the surface of the white bacterial film. These morphological characteristics are basically consistent with the morphological characteristics of Bacillus described in "Common Bacteria System Identification Manual" (edited by Dong Xiuzhu et al. Science Press. 2001), and it is preliminarily judged that strain HMB27688 belongs to Bacillus (Bacillus).

(2) Identification and classification by 16S rDNA sequence

Using the genomic DNA of HMB27688 as a template and using F27 and R1492 as primers to perform PCR amplification on 16S rDNA, the primer sequences are:

F27: 5'AGAGTTTGATCATGGCTCAG3'; (SEQ ID No: 1),

R1492: 5'GGCTACCTTGTTACGACTT3'; (SEQ ID No: 2);

The 16S rDNA amplification reaction system was 50 μL: 10×PCR Buffer (Mg ²⁺ ) 5 μL; dNTP Mixture (2.5 mM) 5 μL; Taq (5U/μL) 1 μL, F27 (10 μmol/L) 1 μL, R1492 (10 μmol/L) ) 1 μL; HMB27688 genomic DNA 50ng; ddH ₂ O to make up to 50 μL. The reaction conditions of PCR were 95°C for 5min; 30 cycles of 95°C for 30s, 55°C for 30s, 72°C for 1.5min, and 72°C for 10min. The resulting PCR amplification product was subjected to gel electrophoresis and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing to obtain the 16S rDNA sequence of HMB27688 (see SEQ ID No: 3). The 16S rDNA sequence of the obtained HMB27688 was compared in Genbank for homology, and the 16S rDNA homology of the bacterial strain HMB27688 and the genus Bacillus reached 99%; Together, it shows that HMB27688 belongs to the genus Bacillus.

(3) Identification and classification by gyrB gene sequence

Using HMB27688 genomic DNA as a template, using the degenerate primers gyrB-F and gyrB-R of the Bacillus gyrB gene as primers to perform PCR amplification to obtain PCR amplification products; wherein the sequences of the primers are:

gyrB-F: 5'TTGRCGGHRGYGGHTATAAAGT3'; (SEQ ID No: 4)

gyrB-R: 5'TCCDCCSTCAGARTCWCCCTC3'; (SEQ ID No: 5)

The PCR amplification reaction system of gyrB is 50 μL: 10×PCR Buffer (Mg ²⁺ ) 5 μL; dNTP Mixture (2.5mM) 5 μL; Taq (5U/μL) 1 μL; gyrB-F (10 μmol/L) 1 μL, gyrB-R (10 μmol/L) 1 μL; HMB27688 genomic DNA 50 ng; ddH ₂ O to make up to 50 μL. The reaction conditions of PCR were 95°C for 5min; 30 cycles of 95°C for 30s, 55°C for 45s, 72°C for 1min, and 72°C for 10min. The amplified product was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing to obtain the gyrB gene sequence of HMB27688 strain (see SEQ ID No: 6). The obtained gyrB gene sequence of the HMB27688 strain was homologously compared in Genbank, and a phylogenetic tree was constructed using MEGA software (Molecular Evolutionary Genetics Analysis, Molecular Evolutionary Genetics Analysis). As a result, it was found that HMB27688 had the highest homology with the gyrB gene sequence of Bacillus amyloliquefaciens; a phylogenetic tree was constructed (see Figure 2), and the HMB27688 strain was aggregated with Bacillus amyloliquefaciens, indicating that HMB27688 was Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), And is a new strain.

Based on the results of the above morphological characteristics, 16S rDNA and gyrB gene sequence homology comparative analysis, it can be known that HMB27688 belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and is different from the existing Bacillus amyloliquefaciens strains, and is a new strain.

The identification method of the above-mentioned Bacillus amyloliquefaciens bacterial strain HMB27688 comprises using the genomic DNA of the bacterial strain to be tested as a template, and carrying out PCR amplification with F27 and R1492 as primer pairs, if the obtained PCR amplification product is the nucleus shown in SEQ ID No:3 The nucleotide sequence is HMB27688 strain.

The identification method of the above-mentioned Bacillus amyloliquefaciens strain HMB27688 comprises using the genomic DNA of the bacterial strain to be tested as a template, and carrying out PCR amplification with gyrB-F and gyrB-R as primers, if the obtained PCR amplification product is SEQ ID No: 6 The nucleotide sequence shown is HMB27688 strain

The identification method of the above-mentioned microbial bacterial agent comprises using the genomic DNA extracted from the bacterial agent to be tested as a template, and carrying out PCR amplification with F27 and R1492 as primer pairs, if the obtained PCR amplification product is the nucleus shown in SEQ ID No:3 Nucleotide sequence, which is the HMB27688 microbial bacterial agent; or use the genomic DNA extracted from the bacterial agent to be tested as a template, and use gyrB-F and gyrB-R as primers for PCR amplification, if the obtained PCR amplification product is SEQ ID No : The nucleotide sequence shown in 6 is the HMB27688 microbial bacterial agent.

The present invention has the advantages and beneficial effects: (1) the present invention provides a highly efficient microorganism for preventing and treating tomato gray mold, and opens up a new control approach; (2) Bacillus amyloliquefaciens HMB27688 of the present invention is effective against tomato gray mold The drug effect is high, the average control effect is more than 80.0%, and the pertinence is strong; (3) the microbial bacterial agent of the present invention is safe to humans and animals, and belongs to the environment-friendly type; Produce drug resistance; (5) The preparation method of the present invention is simple, low in cost and easy to use.

Description of drawings

Fig. 1 is a phylogenetic tree diagram of the HMB27688 strain obtained according to the 16S rDNA sequence.

Fig. 2 is a phylogenetic tree diagram of the HMB27688 strain obtained according to the gyrB gene sequence.

Detailed ways

The following specific examples are used to further clearly explain the present invention, but do not limit the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods; the percentages in the following examples, unless otherwise specified, are all percentages by weight.

The preparation of embodiment 1HMB27688 microbial bacterial agent

Follow the steps below:

(1) Strain activation: The strain HMB27688 (Bacillus amyloliquefaciens strain HMB27688) stored at -80°C was deposited in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on September 29, 2015, and the preservation number is CGMCC No. 11457) was activated at 30°C on LB plate medium (its composition and weight ratio were: tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL), and single colony was picked on LB slant medium (its composition and weight ratio are: tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL) was cultivated at 30°C for 12 hours to obtain activated bacterial strains.

(2) Preparation of seed liquid: make LB liquid medium according to conventional methods (its composition and weight ratio are: tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL), and put it in a 250mL triangular flask Add 100mL of LB liquid medium, autoclave with moist heat, after the temperature drops to room temperature, insert an inoculation loop into each bottle of the strain activated in step (1), and carry out shaking culture under the condition of 30°C and shaker speed of 180rpm After 12 hours, the seed solution was obtained.

(3) Preparation of corn flour and soybean flour medium: add 1.5% corn flour, 2.0% soybean flour, 0.5% NaCl, and 0.6% MnSO ₄ H ₂ O to water according to weight percentage, stir and mix evenly to obtain corn flour and soybean Flour medium; divided into 500mL Erlenmeyer flasks, 200mL per bottle; sterilize the corn flour and soybean flour medium at 121°C for 30 minutes, then cool down to 30°C for later use.

(4) Fermentation culture: Inoculate 2 mL of the seed solution obtained in step (2) into 200 mL of each bottle of corn flour and soybean powder medium obtained in step (3); carry out fermentation and cultivation for 36 hours at 30° C. and a shaker speed of 180 rpm, and then Take a sample from the Erlenmeyer flask every 30 minutes for microscopic examination, count the number of spores and total thalline in the visual field, and calculate the spore rate (spore rate (%)=number of mature spores/(number of mature spores+number of thalline)× 100); when the spore rate reaches 90%, the fermentation culture is stopped; the co-fermentation culture is 48h, and the liquid preparation of Bacillus amyloliquefaciens HMB27688 is obtained.

Example 2 Antagonism test of bacillus amyloliquefaciens HMB27688 to Botrytis cinerea

(1) Test time and place: In early August 2013, it was carried out in the Plant Disease Biological Control Laboratory of the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences.

(2) Test method:

(1) Source of Botrytis cinerea for testing: Tomato Botrytis cinerea BC-1 strain was collected from tomato diseased fruit in Dongniudongzhuang Village, Rongcheng County, Baoding City, Hebei Province. It was isolated and purified by the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. The plant pathology department of the college identified it as Botrytis cinerea, and the pathogenicity test showed strong pathogenicity.

(2) Tablet confrontation experiment:

First the tomato Botrytis cinerea BC-1 is activated on the PDA plate, and after cultivating for 4 days, in the edge area of the colony, use a puncher ( ) punch holes to make the bacteria sheet, transfer the tomato Botrytis cinerea bacteria sheet to another PDA plate center, then connect the bacillus amyloliquefaciens HMB27688 activated in the embodiment 1 step (1) to 2.0 centimeters away from the indicator bacteria sheet place, set a blank control (do not spot the growth of Botrytis cinerea of tomato of HMB27688 strain). Cultivate at a constant temperature of 25°C. When the blank control is about to cover the entire petri dish, measure the control growth (colony radius) and treatment growth (inhibition growth radius after inoculation of HMB27688) of Botrytis cinerea. The antagonistic effect is represented by the inhibition rate. The calculation formula is:

Bacterial inhibition rate (%)=(control growth amount-treatment growth amount)/control growth amount×100

Result (see table 1) Bacillus amyloliquefaciens HMB27688 reaches 88.16% to the inhibitory rate of Botrytis cinerea of tomato; The transparent bacteriostatic band width is 11.5 millimeters; Illustrates that Bacillus amyloliquefaciens HMB27688 of the present invention has strong bacteriostasis to Botrytis cinerea of tomato, has the function of preventing and treating Botrytis cinerea. Biocontrol potential of Botrytis cinerea.

Antagonism test results of table 1 HMB27688 bacterial strains to Botrytis cinerea

strain name Control growth (mm) Treatment growth (mm) Bacteriostatic rate (%) HMB27688 38.0 4.5 88.16

Example 3 Contrast test of Bacillus amyloliquefaciens HMB27688 on control effect of Botrytis cinerea

(1) Test treatment:

(1) Microbial agent: the HMB27688 liquid preparation prepared in Example 1; diluted 50 times with water.

(2) Chemical agent: azoxystrobin suspension concentrate (250 g/L) (Syngenta Co., Ltd., UK); dilute 500 times with water.

(3) Blank control: clear water

(2) Test method: This test was carried out in the plant disease and biocontrol laboratory of the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. Cultivate tomato seedlings (tomato L-402, Liaoning horticultural seedling company, Liaoning Provincial Academy of Agricultural Sciences Horticultural Research Institute production) in 50 hole seedling trays, transplant in the flower pot of diameter 20 centimeters when growing 2 compound leaves, every Pot 1 seedling. When the plant grows to 7-8 compound leaves, select the same leaf age and uniform size leaves, first rinse the leaf surface with sterile water, dry the moisture with sterile filter paper, and then dilute the obtained water in Example 1 (suspension) Dip it in Bacillus amyloliquefaciens HMB27688 inoculum containing 10 ⁷ cfu/mL of bacteria, so that the leaf surface is fully covered with the drug, and then put it in a petri dish covered with double-layer sterile filter paper, and insert Botrytis cinerea BC at the same time -1 bacteria plate (diameter 6mm), moist culture. Leaves not treated with HMB27688 were used as blank control. After the blank control was fully developed, the diameter of the lesion was investigated, and the control effect was calculated.

Table 2 Contrast test results of Bacillus amyloliquefaciens HMB27688 on the control effect of Botrytis cinerea

deal with Lesion diameter (mm) Anti-efficacy (%) HMB27688 liquid preparation 4.0c 83.33 Chemicals 6.8b 71.67 blank control 24.0a --

Result (see table 2) detached leaf bioassay result shows, bacillus amyloliquefaciens HMB27688 of the present invention is 83.33% to the control effect of Botrytis cinerea, and the control effect of chemical agent is 71.67%, and HMB27688 is to the control effect of Botrytis cinerea of tomato It is obviously higher than that of chemical agents, which shows that Bacillus amyloliquefaciens HMB27688 and its microbial agents of the present invention have good control effects on tomato gray mold.

Example 4 Contrast test of Bacillus amyloliquefaciens HMB27688 on control effect of Botrytis cinerea

(1) Test treatment:

(1) Microbial agent: the HMB27688 liquid preparation prepared in Example 1; diluted 50 times with water.

(2) Chemical agent: azoxystrobin suspension concentrate (250 g/L) (Syngenta Co., Ltd., UK); dilute 500 times with water.

(3) Blank control: clear water

(2) Test method: In December 2013, it was carried out in the artificial climate chamber of the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences. Cultivate tomato seedlings (tomato L-402, produced by Liaoning Horticulture Seedling Company, Liaoning Academy of Agricultural Sciences Horticulture Research Institute) in the solar greenhouse. After the tomato grows 8 compound leaves, move them to the temperature-controlled and humidity-controlled cultivation room, and select plants of the same size. Tomato seedlings, the 50-fold water dilution of the HMB27688 liquid preparation prepared in Example 1 is evenly sprayed on the tomato seedlings, cultivated at 25°C for 24 hours, then insert the tomato cinerea bacterium plate (25 on the PDA plate) on each leaflet. Cultivate at ℃ for 3-5 days, with a diameter of 6mm), and keep moist. Set clean water and chemical agents as control, and investigate the incidence results after 3 days, measure the diameter (A, B) of each lesion with the cross measurement method, and calculate the lesion area and control effect.

Lesion area (mm ² )=π×A×B/4

Control effect (%)=(control lesion area-treatment lesion area)/contrast lesion area×100

Result (see Table 3) bioassay results in vivo showed that Bacillus amyloliquefaciens HMB27688 significantly inhibited the expansion of Botrytis cinerea on tomato leaves, and its effect of preventing and treating Botrytis cinerea was 86.70%, while the control effect of chemical agents was 73.02%. %; The control effect of HMB27688 of the present invention on tomato gray mold is significantly higher than that of chemical agents. It shows that Bacillus amyloliquefaciens HMB27688 and its microbial agent of the present invention have good control effect on tomato gray mold.

Table 3 Contrast test results of Bacillus amyloliquefaciens HMB27688 on the control effect of Botrytis cinerea

deal with Lesion area (mm ² ) Anti-efficacy (%) HMB27688 liquid preparation 15.02c 86.70 Chemicals 30.42b 73.02 blank control 112.76a --

Example 5 Contrast test of Bacillus amyloliquefaciens HMB27688 on control effect of Botrytis cinerea

(1) Test treatment:

(1) Microbial agent: the HMB27688 liquid preparation prepared in Example 1 was diluted 50 times with water.

(2) Chemical agent: azoxystrobin suspension concentrate (250 g/L) (Syngenta Co., Ltd., UK); dilute 500 times with water.

(3) Blank control: clear water

(2) Test method: From February to June 2014, it was carried out in Sunmaolong tomato greenhouse in Dadai Village, Mancheng County, Baoding City, Hebei Province. Botrytis cinerea occurred moderately on the tomato leaves in the whole shed at the beginning of the experiment, and the diseased leaves were removed before spraying. Three rows per plot, four replicates, arranged in random blocks. A knapsack electric sprayer was used to inoculate the bacterial agent of Bacillus amyloliquefaciens HMB27688 prepared in Example 1 (diluted with water, containing 10 ⁷ cfu/mL of bacterial cells); a control agent and a blank control were also set up. Spray again 7 days after the first spraying, and investigate the number of diseased leaves per plant in each treatment plot on the 7th day after the second spraying, and calculate the control effect.

Result (see table 4) after the Bacillus amyloliquefaciens HMB27688 inoculum was treated, the number of diseased leaves per plant of its tomato gray mold was significantly lower than that of the blank control and the chemical agent control, and its control effect reached 85.72%, indicating that the Bacillus amyloliquefaciens HMB27688 It has a good control effect on tomato gray mold.

Table 4 Contrast test results of Bacillus amyloliquefaciens HMB27688 on the control effect of Botrytis cinerea

deal with Number of diseased leaves per plant (pieces) Anti-efficacy (%) HMB27688 liquid preparation 0.67c 85.72 Chemicals 1.33b 71.52 blank control 4.67a ---

Claims (5)

1. A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HMB27688, which was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on September 29, 2015, with the preservation number CGMCCNo.11457. 2. utilize the microbial inoculum agent produced by Bacillus amyloliquefaciens HMB27688 described in claim 1, it is characterized in that its active ingredient is HMB27688 thalline. 3. according to the described microbial inoculum agent of claim 2, it is characterized in that described microbial inoculum agent is a liquid preparation. 4. the application of bacillus amyloliquefaciens HMB27688 described in claim 1 in the prevention and treatment of Botrytis cinerea. 5. the application of the microbial inoculant described in claim 2 or 3 in the prevention and treatment of Botrytis cinerea.