CN105483054A - Bacillus amyloliquefaciens WS-8 and application thereof - Google Patents

Abstract

The invention relates to a subspecies of Bacillus amyloliquefaciens WS-8 and its application, and discloses a subspecies of Bacillus amyloliquefaciens, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the number is CGMCC? No.11787, the deposit date is December 4, 2015. The invention also discloses the application of Bacillus amylobacter subspecies in the prevention and treatment of air-borne fungal diseases and soil-borne fungal diseases on the foliage of crops. The fermented liquid is diluted 50 times and sprayed once every 7-15 days, and the control effect on tomato leaf mold It can reach 72%, and it also has a good control effect on other air-borne and soil-borne fungal diseases; at the same time, the strain can also improve crop quality, effectively improve the leaf color of the plant, increase the content of chlorophyll, improve the disease resistance of the plant, and reduce pesticide residues , improve quality. The strain is convenient for artificial cultivation, has a fast propagation speed, is convenient for large-scale production, and has the potential of being developed into a biopesticide for preventing and controlling vegetable planting and post-harvest application.

Description

A kind of bacillus amyloliquefaciens WS-8 and its application

technical field

The invention relates to a strain of Bacillus amyloliquefaciens WS-8 and its application, belonging to the field of agricultural microorganisms and biotechnology, and has the functions of improving leaf color, increasing leaf chlorophyll content, improving plant system disease resistance, preventing and controlling airborne fungal diseases on crop leaves, Efficacy to improve quality.

Background technique

The pathogenic bacterium of leaf mold is Fulviafulva (Cooke) Cif. , which belongs to the subphylum Bacillus spp., and its synonym is Cladosporium fulvum Cooke. It is a common disease in facility tomato cultivation. It generally results in a 20-30% reduction in production, and can reach more than 50% in severe cases. At present, chemical pesticides are mainly used for prevention and control, but due to its complicated and complicated pathogenic differentiation, there are many physiological races; the control effect of chemical pesticides is getting worse and worse, and the amount of pesticides is increasing, resulting in pesticide residues in vegetables. Exceeding the standard, the planting environment is seriously polluted, hindering the sustainable development of agriculture. Due to its advantages of low residue and no pollution to the environment, and its important role in the sustainable development of agriculture, the protection of the agricultural ecological environment, and the guarantee of food safety, biopesticides have received more and more attention and love.

Bacillus spp . is a class of aerobic or facultative anaerobic, Gram-positive, spore-forming rod-shaped bacteria. The spores produced by Bacillus have strong ability to resist external environmental pressures such as drying, heat and ultraviolet radiation. At the same time, there are many strains with special functions in the Bacillus population, which can produce many kinds of active substances, such as polypeptides, cyclic peptides, Amino acid, protease and coumarin, etc., so it has a wide range of application values in the fields of industry, agriculture, medicine and food processing. Because the biocontrol bacillus has the ability to inhibit a variety of plant pathogenic bacteria, and is a non-pathogenic bacterium that exists widely in nature, it is harmless to humans and animals, and is friendly to the environment. Therefore, it is favored by plant protection and environmental protection experts at home and abroad. At present, biocontrol bacillus is widely used in the prevention and control of plant roots, branches, leaves and postharvest diseases of fruits and vegetables.

In the United States, three strains of Bacillus subtilis and one strain of B. amyloliquefaciens have obtained commercial production and application permits from the Environmental Protection Agency (EPA), and have entered large-scale promotion and application. Among them, the fungicide made by mixing Bacillus amyloliquefaciens FZB42 and Bacillus subtilis GB122 is named BioYield, which was developed by Gustafson Company and is used to prevent and control powdery mildew, downy mildew, blight, ash, etc. of vegetables, cherries, grapes, gourds and walnuts. Mildew and other diseases. Domestic fungicides As of December 2014, only one wettable powder of Bacillus amyloliquefaciens has been registered for the control of rice blast.

It is predicted that from 2014 to 2020, the global consumption and market value of pesticides and biopesticides will increase rapidly by more than 10%, and the Asia-Pacific region is expected to achieve the fastest growth. With the formulation of the "Thirteenth Five-Year Plan", the country pays more attention to the development of green agriculture and the protection of the ecological environment. The proportion of biopesticides used in vegetable cultivation will increase, providing a broader opportunity for the development of biopesticides. space. At present, most of the patents that have been authorized and published are Bacillus amyloliquefaciens for the control of soil-borne fungal diseases and post-harvest diseases of fruits. There are few related researches on the control of vegetable foliar fungal diseases, and there is no use of Bacillus amyloliquefaciens for the control of tomato leaf mold Related reports.

Contents of the invention

The object of the present invention is to provide a kind of bacillus amyloliquefaciens and its application, this bacterial strain is suitable for open field and greenhouse conditions, can improve the disease resistance of plant system, can control air-borne fungal disease and soil-borne fungal disease of plant foliage, especially Can control tomato leaf mold.

The present invention adopts following technical scheme:

The bacterial strain WS-8 described in the present invention is Bacillus amyloliquefaciens , the preservation number is CGMCCNo.11787, and the preservation date is December 4, 2015. The name of the preservation unit: China Microbiological Culture Preservation Management Committee General Microbiology Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101.

The Bacillus amyloliquefaciens of the present invention has simple culture conditions, and the colonies on the beef extract peptone medium plate are white, round, concave in the middle, smooth and opaque, and the size of the bacterial cells observed under an electron microscope is about 0.7 μm to 0.8 μm×1.8 μm ~3μm, mesozoic, oval; Gram-positive, facultative anaerobic bacteria; Physiological and biochemical indicators combined with 16SrDNA analysis, the strain was determined to be Bacillus amyloliquefaciens.

The optimal fermentation medium formula of Bacillus amyloliquefaciens WS-8 of the present invention is: starch 0.7%, bean cake powder 1.6%, sucrose 0.2%, yeast extract 0.08%, peptone 0.05%, ammonium sulfate 0.15%, dihydrogen phosphate Potassium 0.05%, magnesium sulfate 0.03%, manganese sulfate 0.01%, sodium chloride 0.02%, calcium carbonate 0.1%, pH7.3-7.5, the above are mass percentages.

The present invention also provides an understanding of the application of Bacillus amylobacter WS-8 in the prevention and treatment of crop foliage airborne fungal diseases and soil-borne fungal diseases. Specifically, the crop foliage airborne fungal diseases are: pear ring spot, pear black Spot disease, cucumber gray mold, tomato leaf mold, tomato gray mold, tomato early blight or tomato soot, the soil-borne fungal disease is blight, fusarium wilt, verticillium wilt or wilt.

Preferably, for the prevention and treatment of airborne fungal diseases on the foliage of crops, the fermentation broth of Bacillus amyloliquefaciens is diluted 10-100 times and then sprayed on the leaves, preferably 50 times diluted (the number of spores is 100 million/ml), Spray once every 7-15 days. When used to prevent and control soil-borne fungal diseases, the fermentation broth of Bacillus amyloliquefaciens is diluted 10-100 times and then the roots are irrigated, preferably 10 times diluted (the number of spores is 500 million/ml).

The present invention also provides the application of the Bacillus amyloliquefaciens WS-8 in improving the physiological and biochemical indexes and quality of crops, specifically for increasing the chlorophyll content, soluble protein content and ascorbic acid content in crop leaves. Preferably, the fermentation broth of Bacillus amyloliquefaciens WS-8 is diluted 10-100 times and then sprayed on crops, preferably 50 times diluted (the number of spores is 100 million/ml), and sprayed once every 10 days.

The Bacillus amyloliquefaciens WS-8 fermented liquid of the present invention is diluted 50 times and sprayed on the foliage, and the preventive effect to tomato leaf mold can reach 72%; Can effectively improve leaf color simultaneously, improve the content of chlorophyll in the blade; Improve The photosynthetic ability of leaves can promote the synthesis of soluble protein in photosynthetic products; increase the content of ascorbic acid in tomato and improve the quality of tomato. The control effects of foliar spraying on indoor airborne fungal diseases tomato gray mold, tomato early blight, cucumber gray mold, cucumber powdery mildew, and cucumber downy mildew were 70.2%, 71%, 70.8%, 72%, respectively. 71.1%; the control effect on ring spot and black spot of postharvest pears reached 64% and 69%; the control effect of root irrigation on tomato fusarium wilt and Verticillium wilt soil-borne fungal diseases in the shed reached 61.8% and 62% respectively. %, it also has a good inhibitory effect on food-borne Staphylococcus aureus. It can be seen that the high-efficiency application of the strain can effectively reduce the incidence of fungal diseases, reduce pesticide residues in products, and protect the ecological safety of the environment. Its potential market application prospect is broad.

Description of drawings

Fig. 1 is a phylogenetic tree of Bacillus amyloliquefaciens WS-8 of the present invention.

detailed description

The present invention will be further described below with reference to the drawings and specific embodiments, so that those skilled in the art can understand.

Embodiment 1: Isolation and identification of Bacillus amyloliquefaciens WS-8

The broad-spectrum antibacterial Bacillus amyloliquefaciens WS-8 of the present invention can be obtained by collecting diseased and healthy vegetable leaves in tomato and cucumber planting sheds in Dahe Town, Luquan, Shijiazhuang, and cultivating them in the laboratory according to the following conditions:

Isolation and purification of strain WS-8: Healthy samples and diseased leaves after removal of rot and mold were used, and 10 grams of leaves were put into phosphate buffer solution by gradient dilution method, shaken at room temperature 200r/min for 30min, and ultrasonically treated for 5min. The bacterial suspension was coated on a plate, and a single colony was picked and purified by streaking.

Screening of antagonistic strain WS-8: the purified single colony was used for primary screening and re-screening of resistant strains with tomato leaf mold and early blight as the target pathogenic strains; the WS-8 strain with good antagonistic effect was obtained.

The Bacillus amyloliquefaciens WS-8 described in the present invention is identified according to the phylogenetic analysis of its colony shape and individual morphological characteristics, physiological and biochemical characteristics and 16SrDNA gene sequence.

The individual morphology and colony characteristics of the strain are as follows: observed under the electron microscope, the size of the bacteria is about 0.7 μm-0.8 μm×1.8 μm-3 μm, and the spores are middle-grown and oval. The culture conditions are simple, and the colonies on the beef extract peptone medium plate are white, round, concave in the middle, smooth, opaque, and irregular at the edges.

Some physiological and biochemical identification results of the strain are as follows:

Table 1 Physiological and biochemical identification results of strain WS-8

.

The length of the 16SrDNA gene sequence of the strain is 1431bp. The phylogenetic tree (shown in Figure 1) was constructed using the sequence to determine the taxonomic status of the strain. The strain WS-8 is a subspecies of Bacillus amyloliquefaciens.

Embodiment 2: the antibacterial spectrum determination of bacillus amyloliquefaciens WS-8

The bacterial strain of the present invention, with air-borne fungal disease: pear ring spot, pear black spot, cucumber gray mold, tomato leaf mold, tomato gray mold, tomato early blight, tomato soot; soil-borne fungal disease : blight, fusarium wilt, verticillium wilt, splinter blight; food-borne bacteria: staphylococcus aureus, a variety of pathogenic strains were carried out to measure the antibacterial spectrum, the results are shown in Table 2.

Table 2 Antibacterial spectrum of Bacillus amyloliquefaciens subspecies WS-8

Note: The strength of antagonism is represented by "+''. "+": weak, the width of the inhibition zone is less than 1.5cm; "++'': medium strength, the width of the inhibition zone is between 1.5cm and 3.0cm;" +++": Very strong, with a width of the antibacterial zone greater than 3.0cm.

Embodiment 3: the cultivation and fermentation condition of bacillus amyloliquefaciens WS-8

Purify the strain with strong antibacterial effect, use beef extract peptone medium, and store it on a solid slant; pick a ring from the slant culture, inoculate it into the liquid medium of beef extract peptone and cultivate overnight at 37°C, as the strain seed bottle ready to use.

Fermentation culture conditions: fermentation medium: starch 0.7%, bean cake powder 1.6%, sucrose 0.2%, yeast extract 0.08%, peptone 0.05%, ammonium sulfate 0.15%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, manganese sulfate 0.01 %, sodium chloride 0.02%, calcium carbonate 0.1%, pH7.3-7.5, the above are mass percentages. Sterilize at 121°C for 20 minutes, after cooling, add the seed liquid to the fermentation medium with an inoculation amount of 1.5-2%, in a 600mL/3L Erlenmeyer flask, with a rotation speed of 180 rpm and a temperature of 30°C, and cultivate for 48 hours to obtain a fermentation liquid .

Embodiment 4: the application effect of Bacillus amyloliquefaciens WS-8 control tomato leaf mold

A pot experiment was adopted in the laboratory, and 5 treatments were set up: blank control (clear water treatment), 10-fold dilution of fermentation broth, 20-fold dilution of fermentation broth, 50-fold dilution of fermentation broth, and 100-fold dilution of fermentation broth; After the leaves are fully developed, the 5 treatments are sprayed with clear water and fermentation broth with different dilution ratios (the amount of spraying is based on spraying the front and back sides of the leaves wet and sprayed evenly), and the tomato leaf mold pathogen is inoculated 48 hours after spraying. 24 pots, set in three replicates. After the onset, the disease index and disease control effect of tomato leaf mold were counted (see Table 3), and the comprehensive application cost and control effect were calculated. The optimal concentration of the fermented liquid was 50 times diluted.

The classification criteria for tomato leaf mold disease are as follows:

Grade 1, no disease spots on the leaves;

Grade 2, there are lesion spots on the leaf edge and leaf tip, the lesion spots are rare, and the diameter of the lesion spot is D<0.6cm;

Grade 3, many lesions, but the diameter of the lesions is 0.6≤D<1.2cm; Grade 4, the lesions are many and continuous, and the diameter of the lesions is 1.2cm≤D<2.0cm; Grade 5, the lesions are contiguous, the disease Spot diameter D≥2.0cm.

Disease index calculation formula: disease index = 100 × ∑ (number of diseased plants at each level × representative value at each level) / (total number of investigated leaves × representative value at the highest level).

Table 3 Disease index and control effect of tomato treated with different dilution concentrations

.

Example 5: Field application effect of Bacillus amyloliquefaciens subspecies WS-8 in controlling tomato leaf mold

The application experiment of controlling tomato leaf mold in four consecutive crops was carried out in the tomato shed. The experiment set up 4 treatments: blank control, 50 times dilution of fermentation broth, chlorothalonil, and multi-antibiotics. Different treatments were separated by plastic films to Avoid the interaction between different treatments, and facilitate the statistics of experimental results. The blank control did not do any treatment; the fermented liquid spraying treatment was sprayed once every 10 days from the budding stage of tomato (the amount of spraying was based on spraying the front and back sides of the leaves wet and spraying evenly); chlorothalonil, multi-antibiotics According to the application experience of local vegetable farmers, spraying starts when tomato leaf mold shows signs of onset, and then sprays once every 7-10 days according to the disease. When the disease index of the blank control reached more than 50%, the disease index and disease prevention effect of other treatments of tomato leaf mold were counted (see Table 4).

Table 4 Disease index and disease control effect of fermented liquid and pesticide treatment tomato

.

Embodiment 6: the application effect of Bacillus amyloliquefaciens WS-8 to other air-borne and soil-borne fungal diseases

The control effect test of Bacillus amyloliquefaciens WS-8 on other air-borne and soil-borne fungal diseases was carried out respectively. For air-borne fungal diseases, 50 times diluted fermentation liquid was used for spraying, and for soil-borne fungal diseases, 10 times diluted fermentation liquid was used for irrigation. Roots were applied, and each plant was irrigated with 10ml. After 48 hours of application, each pathogenic bacteria was inoculated.

Table 5 Control effect of Bacillus amyloliquefaciens WS-8 on other airborne and soilborne fungal diseases

.

Example 7: Effect of Bacillus amyloliquefaciens WS-8 on physiological indexes and quality of tomato leaves

The comparative application experiment of fermentation broth and chemical agents was carried out in the tomato shed, and three treatments of 50-fold dilution of fermentation broth, chlorothalonil, and multi-antibiotics were set up, and the fermentation broth spraying treatment was sprayed once every 10 days from the beginning of the budding period of tomato According to the application experience of local vegetable farmers, chlorothalonil and polybacterial antibiotics are sprayed when tomato leaf mold shows signs of onset, and then sprayed at intervals of 7-10 days according to the disease until the disease is under control. After the fermentation broth was sprayed twice, the leaves at the second leaf position (the second leaf position counted from the growth point) were taken at intervals of 10 days to measure the content of chlorophyll and soluble protein; The ascorbic acid content of the tomatoes at the ear position was measured; the average results of 5 tests on tomato leaves and fruits are shown in Table 6. The tomatoes treated with fermentation broth were tested by the quality supervision department of Hebei Province, and the quality reached the standard of green vegetables.

Table 6 Physiological indicators of tomato leaves and vegetable quality in different treatments

.

SEQUENCELISTING

<110>Institute of Biology, Hebei Academy of Sciences

<120> A kind of Bacillus amyloliquefaciens WS-8 and its application

<160>1

<170>PatentInversion3.3

<210>1

<211>1431

<212>DNA

<213> Bacillus amyloliquefaciens WS-8

<400>1

TGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAA60

CACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGA120

TGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATG180

GACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGC240

CGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG300

GCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTG360

ATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAG420

GGCGGCACCTTGACGGTACCTAACCAGAAAAGCCACGGCTAACTACGTGCCAGCAGCCGCG480

GTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGT540

TTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGA600

ACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGT660

GGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAG720

CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA780

GTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG840

AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC900

ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAT960

CCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAG1020

CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGC1080

CAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT1140

GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAAC1200

AAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCG1260

CAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGC1320

GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACAC1380

CCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGTGACAAGAT1431

Claims (8)

1. a bacillus amyloliquefaciens WS-8, it has carried out preservation on December 4th, 2015 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCCNo.11787.

2. bacillus amyloliquefaciens according to claim 1, is characterized in that its substratum consists of: starch 0.7%, soybean cake powder 1.6%, sucrose 0.2%, yeast extract paste 0.08%, peptone 0.05%, ammonium sulfate 0.15%, potassium primary phosphate 0.05%, magnesium sulfate 0.03%, manganous sulfate 0.01%, sodium-chlor 0.02%, calcium carbonate 0.1%, pH7.3-7.5, is more than mass percent.

3. the application of the bacillus amyloliquefaciens described in claim 1 or 2 in control crop blade face gas borne fungus diseases and silborne fungal diseases.

4. application according to claim 3, it is characterized in that: described crop blade face gas borne fungus diseases is: black rot of pear, Pear black spot, gray mold of cucumber, leaf muld of tomato, graw mold of tomato, early blight of tomato or tomato sooty mold, and described silborne fungal diseases is damping-off, blight, verticillium or blight dis-ease.

5. application according to claim 4, is characterized in that: spray in blade face after doubly being diluted by the fermented liquid 10-100 of described bacillus amyloliquefaciens or fill with root.

6. the bacillus amyloliquefaciens described in claim 1 or 2 is improving the application in crop quality.

7. application according to claim 6, is characterized in that: described bacillus amyloliquefaciens is for improving the ascorbic acid content in crop leaf Determination of Chlorophyll content, soluble protein content and crop.

8. application according to claim 7, is characterized in that: spray in crop after doubly being diluted by the fermented liquid 10-100 of described bacillus amyloliquefaciens, preferably 50 times of dilutions.