CN111349589A - Bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus roxburghii and application thereof - Google Patents
Bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus roxburghii and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus formosanus and application thereof, and belongs to the technical field of crop disease prevention and treatment. The strain is bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) GZ5, which has been registered and preserved in the China general microbiological culture Collection center on 21.11.2019 with the preservation number of CGMCC No. 18987. The strain GZ5 is obtained from healthy tomato rhizosphere soil in Guangxi county, Fujian province, is harmoniously compatible with soil ecology, and is non-toxic and non-pathogenic. The bacillus amyloliquefaciens GZ5 strain is obtained by screening through a plate confrontation test, can effectively inhibit hypha growth and spore germination of rhizoctonia solani, and is in contact with the environmentGood compatibility and good development and application prospect.
Description
Technical Field
The invention relates to bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus formosanus and application thereof, belonging to the technical field of crop disease prevention and treatment.
Background
Anoectochilus roxburghii is a rare Chinese medicinal material in China, can be used as a medicine for fresh or dry whole herbs, and is a name of 'Yaowang' in folk. Because the wild anoectochilus formosanus has strict requirements on ecological environment, poor adaptability, low reproduction rate and slow growth, and because of the unique nutritive value and medicinal value, the wild anoectochilus formosanus is artificially over-mined to become an endangered variety which is listed as a provincial wild medicinal material protection variety by Fujian province. At present, the artificial cultivation of anoectochilus roxburghii is mainly carried out, but the disease problem is highlighted day by day in the cultivation process. Wherein the microorganism is Fusarium oxysporum (F.), (Fusarium oxysporum) The caused stem rot is one of the main diseases in the cultivation of the anoectochilus formosanus and can cause damage to the anoectochilus formosanus in different growth periods. When the disease occurs, the base of the plant stem appears yellow brown water stain-like disease spots which rapidly develop to a circle around the stem, and the disease part is dry and rotten. The disease development of the stem rot is rapid, seedlings rapidly fall down and die suddenly, and the damage rate is up to 90% when the disease is serious, thereby bringing great economic loss to planters.
The research on the comprehensive control of the stem rot of the anoectochilus formosanus has less reports, the chemical control is the main control method for the disease in the current production, and the research of shouasong and the like shows that the tebuconazole missible oil has better inhibition effect on the stem rot of the anoectochilus formosanus, and the inhibition toxicity of the tebuconazole missible oil is 92.50 times that of carbendazim. Besides, the commonly used bactericides include chlorothalonil, mancozeb, thiophanate-methyl and the like. The chemical bactericide plays a certain role in preventing and treating the stem rot of the anoectochilus formosanus, but with the massive and unscientific use of the chemical bactericide, many defects are increasingly prominent, such as the generation of drug resistance of pathogenic bacteria, the damage to the environment, the ecological system, pesticide residue and the like. In recent years, with the deepening of the understanding of people on ecological agriculture, the exploration of effective and environment-compatible prevention and treatment measures has attracted the general attention of scholars at home and abroad. Biological control is an important development concept in current agricultural practice, has the characteristics of no harm to human and livestock, no pollution, no residue, reproducibility, difficulty in causing pests to have drug resistance, easiness in industrialization, low cost and the like, can reduce the use of pesticides to the maximum extent, reduces the environmental pollution of the pesticides, has very unique advantages, accords with the current tendency of agricultural sustainable development in China, and has extremely wide development prospect.
Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The bacillus subtilis is a gram-positive bacterium with extremely high affinity with bacillus subtilis, is widely distributed in nature, is easy to separate and culture, has broad-spectrum antibacterial activity, does not pollute the environment, is nontoxic to people and livestock, and has strong stress resistance and good stability. It can produce many kinds of high-efficiency active metabolite, effectively inhibit the activity of fungi and other bacteria, promote plant growth and induce organism to generate resistance, and control the growth of plant pathogenic bacteria and the invasion of organism in many aspects. At present, the bacterium is widely applied to the disease control of flowers, fruits and various crops. Qiu light and other researches show that the bacillus amyloliquefaciens can be fast colonized on the root circumference of the strawberry, so that soil-borne diseases can be prevented and controlled, the disease resistance of the strawberry can be improved, the quality of the strawberry can be improved, and the yield can be increased; the researches of Ganberg and the like show that the bacillus amyloliquefaciens has obvious promotion effects on the germination rate of sesame seeds, the plant height, the root length, the freshness and the dry weight of sesame seedlings and has control effects on sesame stem blight. Research shows that the bacillus amyloliquefaciens can produce plant growth hormone IAA, stimulate the growth of crops and induce defense systems of the crops to generate resistance, so that the effect of inhibiting plant pathogenic bacteria and preventing plant diseases is achieved, the growth of plant roots can be promoted, and the yield of the crops is increased.
Until now, related reports have been made on chemical control of stem rot of anoectochilus formosanus, but few reports have been made on research on biological control of stem rot of anoectochilus formosanus. The bacillus amyloliquefaciens GZ5 with the biocontrol effect is obtained by early separation in the laboratory, and is used as the biocontrol bacterium to study the inhibition effect of the strain on the growth of the hyphae of the pathogenic bacteria fusarium oxysporum of the stem rot of the anoectochilus roxburghii and the inhibition effect of the strain fermentation liquid and the sterile filtrate thereof on the hyphae growth and the spore germination of the pathogenic bacteria of the stem rot of the anoectochilus roxburghii, so that practical basis is provided for the biological control of the stem rot of the anoectochilus roxburghii and the deep development and application of the biocon.
Disclosure of Invention
The invention aims to: aiming at the problems that the existing roxburgh anoectochilus terminal bud stem rot seriously occurs, the area is enlarged year by year, the chemical prevention and control effect is not ideal, and the environmental pollution is caused, the bacillus amyloliquefaciens for preventing and controlling the roxburgh anoectochilus terminal bud stem rot and the application thereof are provided.
The invention is realized by the following technical scheme:
the bacillus amyloliquefaciens for preventing and treating the stem rot of anoectochilus formosanus is identified as bacillus amyloliquefaciens by morphological observation, culture characteristic observation and molecular biological researchBacillus amyloliquefaciens) GZ5, which was deposited in the general microbiological culture Collection center of China general microbiological culture Collection Committee in 2019 at month 11 and 21, and has the following addresses: the collection number of the strain is CGMCC No.18987, No. 3 of Xilu No.1 of Beijing, Chaoyang, and institute of microbiology of Chinese academy of sciences.
The strain GZ5 is streaked and inoculated in an LB culture medium (l 0g of tryptone, 5 g of yeast powder, 5 g of sodium chloride and 15 g of agar powder, distilled water is added to the volume to be 1000 mL, the solution is dissolved, the pH value is adjusted to be 7.0-7.2, and autoclaving is carried out for standby), and the bacterial colony characteristics and morphological characteristics are observed after the strain is cultured for 48 hours at 37 ℃. Colony characteristics: the bacterial colony of the strain is round, milky white, opaque, neat in edge, convex in center, wet in surface and slightly sticky. Morphological characteristics: the strain GZ5 has gram-positive stain, short rod-shaped thallus, blunt ends, endogenous spores, non-expanded spore cysts and oval spores, and is mesogenic to subterminal.
The biocontrol microbial inoculum prepared from the bacillus amyloliquefaciens strain is prepared by the following steps:
the strain is inoculated in LB liquid culture medium at 37 ℃ and 180 r/min for 24h to prepare seed liquid, and 10mL of the seed liquid is inoculated in the fermentation culture medium. The formula of the optimized fermentation medium is as follows: 10g of tryptone, 5 g of yeast powder, 5 g of sodium chloride and 15 g of agar powder are added with distilled water to reach the constant volume of 1000 mL, and the mixture is dissolved and the pH value is adjusted to be 7.0-7.2. Tong (Chinese character of 'tong')Through exploration of culture conditions, the optimal fermentation conditions are as follows: inoculating 5% (v/v), bottling 100 mL/250 mL, fermenting culture medium pH 7.0-7.2, culturing at 37 deg.C and 180 r/min for 48 hr to obtain a solution with a concentration of about 10%8CFU/ml strain GZ5 fermentation broth. And centrifuging the fermentation liquor at 4 ℃ and 8000 r/min for 20 min, and filtering by using a 0.22 mu m bacterial filter to obtain GZ5 sterile fermentation liquor, namely the biocontrol microbial inoculum.
The application of the bacillus amyloliquefaciens GZ5 in preventing and treating the stem rot of anoectochilus roxburghii, in particular to a method for inhibiting hypha growth and spore germination of the stem rot of the anoectochilus roxburghii, which comprises the following steps:
inhibition effect of GZ5 living body on hypha growth of pathogenic bacteria of stem rot of anoectochilus formosanus
Taking the PDA and LB culture medium for standby, melting in a microwave oven, and mixing in equal proportion. Inoculating fusarium oxysporum hypha blocks (D =7 mm) in the center of a PDA and LB equal-proportion mixed culture dish, dipping GZ5 bacterial liquid on a plate by using an inoculating ring at a position 2.5cm away from the opposite two sides of the hypha blocks for streaking, taking the fusarium oxysporum hypha blocks with the same size as a control, repeating for 3 times, placing in a constant-temperature incubator at 28 ℃ for culture, and counting the bacteriostasis rate.
Inhibition effect of strain GZ5 fermentation liquor on hypha growth of pathogenic bacteria of stem rot of anoectochilus formosanus
Placing fusarium oxysporum hypha blocks (D =7 mm) in the center of a PDA flat plate, placing sterilization filter paper sheets with the diameter of 5 mm at positions of 2.5cm away from each other at the upper and lower opposite positions of the hypha blocks, dropwise adding 10mL of strain GZ5 fermentation liquor on each filter paper sheet, repeating for 3 times by taking the fusarium oxysporum hypha blocks with the same size as a control, placing at 28 ℃ for constant-temperature culture, and counting the bacteriostasis rate.
Inhibition effect of strain GZ5 fermentation liquor on spore germination of pathogenic bacteria of stem rot of anoectochilus roxburghii
Measuring the germination rate of spores by a concave slide spore germination method, namely dripping strain GZ5 fermentation liquor with the concentration of 1 × 10 on a concave slide in turn according to the volume ratio of 1: 1: 15Fusarium oxysporum spore suspension per mL, 1 wt.% glucose aqueous solution, and LB liquid medium instead of fermentation liquid in the control group. 28 ℃ moisture-preserving culture, namely, putting the concave glass into a large-size culture dish which is fully paved with wet filter paper for useAnd sealing the preservative film, observing the germination condition of fusarium oxysporum spores under an optical microscope after 24 hours of treatment, wherein the germination standard is that the length of a bud tube reaches half of the diameter of the spores, and if the fusarium oxysporum does not germinate or germinate in a small quantity after 24 hours, observing once every 2 hours at least for 5 visual fields, and observing the germination condition of 30-40 spores in each visual field.
Inhibition effect of strain GZ5 on growth of pathogenic bacteria hypha of stem rot of anoectochilus roxburghii
2 mL of the strain GZ5 sterile fermentation liquid is added into the melted 20 mL of PDA culture medium which is cooled to about 45 ℃ to be mixed and poured into a flat plate, and the antibacterial flat plate of the GZ5 sterile fermentation liquid is prepared. Fusarium oxysporum hypha blocks with the diameter of 7mm are inoculated in the center of the plate. Adding 2 mL of LB liquid culture medium into 20 mL of melted PDA culture medium cooled to about 45 ℃, mixing and pouring into a flat plate, inoculating a fusarium oxysporum hypha block with the diameter of 7mm in the center of the flat plate as a control, repeating for 3 times, placing the flat plate in an incubator at 28 ℃, culturing, measuring the growth diameter of a bacterial colony by a cross method, and counting the bacteriostasis rate.
Inhibition effect of strain GZ5 on spore germination of pathogenic bacteria of stem rot of anoectochilus roxburghii by sterile fermentation broth
Measuring the germination inhibition rate of the spores by adopting a concave slide spore germination method, and sequentially dripping sterile fermentation liquor of strain GZ5 on a concave slide according to the volume ratio of 1: 1: 1, wherein the concentration is 1 × 105Fusarium oxysporum spore suspension per mL, 1% glucose aqueous solution, and LB liquid medium instead of sterile broth were added as controls, and each treatment was repeated 3 times. And (3) carrying out moisture-preserving culture at 28 ℃, observing the germination condition of the fusarium oxysporum spores under an optical microscope after 24 hours of treatment, wherein the germination standard is that the length of a bud tube reaches half of the diameter of the spores, and if the fusarium oxysporum spores do not germinate or germinate in small quantity after 24 hours, observing once every 2 hours at least for 5 visual fields, and observing 30-40 spore germination conditions in each visual field.
The invention has the beneficial effects that:
1. the screened bacillus amyloliquefaciens GZ5 has strong inhibiting effect and can obviously inhibit the hypha growth and the spore germination of the stemona of anoectochilus roxburghii. The strain is separated from the tomato rhizosphere soil, is harmonious and compatible with soil ecology, is a biological preparation, has no series of problems caused by the use of chemical pesticides, can reduce agricultural pollution, can effectively prevent and treat the stem rot of the anoectochilus formosanus, and has good development and application prospects.
2. The bacillus amyloliquefaciens strain has simple culture conditions, is easy to store and industrial production, and is an ideal biocontrol bacterium.
Drawings
FIG. 1 shows the inhibition effect of GZ5 living body on the hypha growth of pathogenic bacteria of the stem rot of Anoectochilus roxburghii.
FIG. 2 shows the inhibition effect of fermentation liquor of strain GZ5 on the hypha growth of pathogenic bacteria of stem rot of Anoectochilus roxburghii.
FIG. 3 shows the inhibition effect of fermentation liquor of strain GZ5 on spore germination of pathogenic bacteria of stem rot of Anoectochilus roxburghii.
FIG. 4 shows the inhibition effect of the strain GZ5 on the growth of the hypha of pathogenic bacteria of the stem rot of Anoectochilus roxburghii.
FIG. 5 shows the inhibition effect of the strain GZ5 on the spore germination of pathogenic bacteria of stem rot of Anoectochilus roxburghii.
FIG. 6 is an electron microscope observation of the strain GZ5 after the live body treatment of pathogenic bacteria of stem rot of Anoectochilus roxburghii.
Detailed Description
For a better understanding of the present invention, reference is made to the following examples and accompanying drawings which are set forth to illustrate, but are not to be construed as the limit of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
EXAMPLE identification of the isolation of antagonistic bacteria
l, isolation of the Strain
And lightly shoveling away surface soil around the roots of the healthy tomato plants, taking a soil sample with the depth of 5-10 cm by using a soil sampler, pouring the soil sample into a sterilized and standby self-sealing bag, recording in real time, and taking the self-sealing bag back to the laboratory for separation. Randomly weighing 10g of each part of soil, placing the weighed soil into a 250mL triangular flask filled with 90 mL of sterile water and a small amount of glass beads, fixing a sealing film at the bottle mouth, placing the bottle mouth on a 200 r/min shaking table, shaking for 30 min, standing for 5 min, respectively sucking 1 mL of soil sample suspension by using a liquid transfer gun, adding the soil sample suspension into a test tube filled with 9 mL of sterile water, and fully mixing to obtain the soil sample10-2The soil sample diluent is prepared by the same method in turn by 10-3、10-4、10-5、10-6And (5) diluting the soil sample. And uniformly coating 100 ul of the prepared soil sample diluent with each gradient multiple in an LB flat plate respectively, sealing a sealing film, and inversely placing the flat plate in a biochemical incubator at 37 ℃ for culturing for 2-4 d. Observing the color, shape, transparency and other characteristics of the bacterial colony, re-purifying the bacterial colony of different types, transferring the obtained pure culture to LB inclined plane, and storing in 4 deg.C refrigerator for further use.
Screening the fusarium oxysporum antagonistic strain by adopting a plate confronting method. Taking the PDA and LB culture medium for standby, melting in a microwave oven, mixing in equal proportion and pouring. A punch with the diameter of 7mm is used for punching a bacterial cake with the same diameter on the edge of an activated fusarium oxysporum colony, then the bacterial cake is inoculated in the center of a PDA and LB proportional mixing culture dish, meanwhile, an inoculating ring is used for dipping activated antagonistic bacterial liquid to be detected on an LB culture medium at the position 2.5cm away from the two opposite sides of the bacterial cake and streaking is carried out, only fusarium oxysporum bacterial cake with the same size is inoculated in the center of a flat plate to serve as a control, each treatment is repeated for 3 times, the culture is carried out in a constant temperature incubator at the temperature of 28 ℃, and the size of a bacteriostatic zone is measured when a control group grows to the edge of the flat plate.
Observation of morphological characteristics
And (3) streaking and inoculating an antagonistic strain GZ5 with the most obvious bacteriostatic zone to an LB culture medium (l 0g of tryptone, 5 g of yeast powder, 5 g of sodium chloride and 15 g of agar powder, adding distilled water to a constant volume of 1000 mL, dissolving and adjusting the pH to 7.0-7.2, and performing autoclaving for later use), and performing colony characteristic and morphological characteristic observation after culturing for 48 hours at 37 ℃. Colony characteristics: the bacterial colony of the strain is round, milky white, opaque, neat in edge, convex in center, wet in surface and slightly sticky. Morphological characteristics: the strain GZ5 is gram-positive, the thallus is rod-shaped, the two ends are blunt, the spore is generated in the thallus, the spore sac is not expanded, the spore is oval, and the thallus is mesogenic to subterminal. Preliminarily judging whether the strain GZ5 belongs to the genus Bacillus according to morphological characteristics (Bacillus sp)。
16S rDNA sequence analysis
After extracting the genome DNA of the strain GZ5 by the genome extraction kit, carrying out PCR amplification on the 16S rDNA by adopting universal primers L1: 5-AGTCGTAACAACGTAGCCGT-3 'and L2: 5-GTGCCAAGGCATCCACC-3'. And recovering PCR amplification products, connecting, transforming and identifying, and then sending the positive clone to the biological engineering (Shanghai) Limited company for sequencing, wherein the total length of the sequence is 1000 bp (please see the sequence). The obtained sequence is submitted to a GenBank database for BLAST analysis and alignment, and the sequence is found to have more than 99 percent of sequence homology with the bacillus amyloliquefaciens.
The sequence is as follows:
GTGCAAGGGCGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGATTTATTTCGAAGCAACGCGAAGATCTTACCAGTTTTGACATCCTCTGATATCCTAGAGATAGACGTCCCT。
identifying Strain GZ5 as Bacillus amyloliquefaciens combining morphological and cultural characteristics: (Bacillus amyloliquefaciens)。
Example two: inhibition effect of GZ5 living body on hypha growth of pathogenic bacteria of stem rot of anoectochilus formosanus
Taking the PDA and LB culture medium for standby, melting in a microwave oven, and mixing in equal proportion. Inoculating fusarium oxysporum hypha blocks (D =7 mm) in the center of a PDA and LB equal-proportion mixed culture dish, dipping GZ5 bacterial liquid on a plate by using an inoculating ring at a position 2.5cm away from the opposite two sides of the hypha blocks for streaking, taking the fusarium oxysporum hypha blocks with the same size as a control, repeating for 3 times, placing in a constant-temperature incubator at 28 ℃ for culture, and counting the bacteriostasis rate. As shown in FIG. 1, Fusarium oxysporum mycelia grew vigorously on the medium without GZ5 living body, while mycelia growth was significantly inhibited on the medium with GZ5 living body, and the inhibition rate of mycelia growth was 63.75% after 6 d of culture.
Example three: inhibition effect of strain GZ5 fermentation liquor on hypha growth of pathogenic bacteria of stem rot of anoectochilus formosanus
Placing fusarium oxysporum hypha blocks (D =7 mm) in the center of a PDA flat plate, placing sterilization filter paper sheets with the diameter of 5 mm at positions of 2.5cm away from each other at the upper and lower opposite positions of the hypha blocks, dropwise adding 10mL of strain GZ5 fermentation liquor on each filter paper sheet, taking the fusarium oxysporum hypha blocks only connected with the same size as a control, repeating for 3 times, placing at the constant temperature of 28 ℃ for culture, and counting the bacteriostasis rate. The result is shown in figure 2, the strain GZ5 fermentation liquor has obvious inhibition effect on the growth of fusarium oxysporum hyphae, and the hypha growth inhibition rate is 62.58% after 6 d culture.
Example four: inhibition effect of strain GZ5 fermentation liquor on spore germination of pathogenic bacteria of stem rot of anoectochilus roxburghii
Measuring the germination rate of spores by a concave slide spore germination method, namely dripping strain GZ5 fermentation liquor with the concentration of 1 × 10 on a concave slide in turn according to the volume ratio of 1: 1: 15Fusarium oxysporum spore suspension per mL, 1 wt.% glucose aqueous solution, and LB liquid medium instead of fermentation liquid in the control group. And (3) carrying out 28 ℃ moisturizing culture, namely placing the concave glass into a large-size culture dish fully paved with wet filter paper, sealing the concave glass with a preservative film, observing the germination condition of fusarium oxysporum spores under an optical microscope after 24 hours, wherein the germination standard is that the length of a bud tube reaches half of the diameter of the spores, observing the spores once every 2 hours if the spores are not germinated or the germination quantity is small after 24 hours, observing at least 5 visual fields for each treatment, and observing the germination condition of 30-40 spores for each visual field. The result is shown in fig. 3, after the fermentation broth GZ5 is treated for 24 hours, the spore germination rate is 11.1%, and the spore germination inhibition rate is 78.83%, which indicates that the fermentation broth GZ5 significantly inhibits the spore germination of fusarium oxysporum.
Example five: inhibition effect of strain GZ5 on growth of pathogenic bacteria hypha of stem rot of anoectochilus roxburghii
2 mL of the strain GZ5 sterile fermentation liquid is added into melted 20 mL of PDA culture medium cooled to about 45 ℃ to be mixed and poured into a flat plate, and the antibacterial flat plate of the GZ5 sterile fermentation liquid is prepared. Fusarium oxysporum hypha blocks with the diameter of 7mm are inoculated in the center of the plate. Adding 2 mL of LB liquid culture medium into 20 mL of melted PDA culture medium cooled to about 45 ℃, mixing and pouring into a flat plate, inoculating a fusarium oxysporum hypha block with the diameter of 7mm in the center of the flat plate as a control, repeating for 3 times, placing the flat plate in an incubator at 28 ℃, culturing, measuring the growth diameter of a bacterial colony by a cross method, and counting the bacteriostasis rate. As shown in FIG. 4, in the medium containing the GZ5 sterile fermentation broth, Fusarium oxysporum filaments grew slowly and the mycelia grew sparsely, and the amount of the mycelia decreased significantly, and the colony growth diameter was 22 mm after 6 days of culture, and the rate of inhibition of mycelia growth was 63.33%.
Example six: inhibition effect of strain GZ5 on spore germination of pathogenic bacteria of stem rot of anoectochilus roxburghii by sterile fermentation broth
Measuring the germination inhibition rate of the spores by adopting a concave slide spore germination method, and sequentially dripping strain GZ5 sterile fermentation liquor with the concentration of 1 × 10 on a concave slide according to the volume ratio of 1: 1: 15Fusarium oxysporum spore suspension per mL, 1 wt.% aqueous glucose solution, control added LB liquid medium instead of sterile broth, each treatment was repeated 3 times. Carrying out moisture-preserving culture at 28 ℃, observing the germination condition of fusarium oxysporum spores under an optical microscope after processing for 24 hours, wherein the germination standard is that the length of a bud tube reaches half of the diameter of the spores, and observing the spores once every 2 hours if the spores are not germinated or the germination quantity is small after 24 hours. At least 5 fields of view are observed in each treatment, and 30-40 spore germination conditions are observed in each field of view. The results are shown in fig. 5, the spore germination rate is 26.73% and the spore germination inhibition rate is 54.59% after the fermentation broth is treated for 24h, which indicates that the strain GZ5 has a certain inhibition effect on the germination of fusarium oxysporum spores.
Example seven: electron microscope observation of Anoectochilus roxburghii stem rot pathogenic bacteria treated by GZ5 living body
After treating fusarium oxysporum according to the method of example two, a small amount of hyphae at the edge of the inhibited colony was picked up in the experimental group, hyphae at the edge of the colony was picked up in the control group, and the morphology of the hyphae was observed under an electron microscope. As shown in FIG. 6, the control hyphae grew vigorously, were long, uniform and full, while the experimental hyphae malformed, shriveled and shriveled. It can be seen that the GZ5 living body treatment can cause obvious change of the shape of fusarium oxysporum hyphae.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> institute of plant protection of academy of agricultural sciences of Fujian province
<120> bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus roxburghii and application thereof
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cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180
ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
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tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420
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gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660
tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720
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cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840
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Claims (5)
1. A bacillus amyloliquefaciens strain for preventing and treating stem rot of anoectochilus formosanus is characterized in that the strain is amyloliquefaciens
Bacillus (A), (B)Bacillus amyloliquefaciens) GZ5, which has been deposited in the China general microbiological culture Collection center on 21.11.2019 with the preservation number of CGMCC 18987.
2. A biocontrol bacterial agent prepared from the strain of Bacillus amyloliquefaciens according to claim I.
3. The biocontrol microbial inoculum of claim 2 wherein said biocontrol microbial inoculum is fermented from said bacillus amyloliquefaciens strain GZ 5.
4. The biocontrol microbial inoculum of claim 3 characterized in that: inoculating the strain into an LB liquid culture medium at 37 ℃ and 180 r/min, culturing for 24h to prepare a seed solution, inoculating 10mL of the seed solution into a fermentation culture medium, wherein the fermentation conditions are as follows: inoculating 5% (v/v), bottling 100 mL/250 mL, fermenting culture medium pH 7.0-7.2, culturing at 37 deg.C and 180 r/min for 48 hr to obtain 10% (v/v) extract8CFU/ml strain GZ5 fermentation broth; centrifuging the fermentation liquor at 4 ℃ and 8000 r/min for 20 min, and filtering by using a 0.22 mu m bacterial filter to obtain an aseptic fermentation liquor, namely the biocontrol microbial inoculum; the formula of the fermentation medium is as follows: 10g of tryptone, 5 g of yeast powder, 5 g of sodium chloride and 15 g of agar powder are added with distilled water to reach the constant volume of 1000 mL, and the mixture is dissolved and the pH value is adjusted to be 7.0-7.2.
5. The use of a bacillus amyloliquefaciens strain according to claim 1 for controlling stem rot of anoectochilus roxburghii.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373083A (en) * | 2021-05-19 | 2021-09-10 | 福建省农业科学院植物保护研究所 | Biocontrol microbial inoculum for preventing and treating phytophthora blight of taro and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368747A (en) * | 2015-12-02 | 2016-03-02 | 中国农业科学院麻类研究所 | Bacillus amyloliquefaciens strain and application thereof |
| CN106906167A (en) * | 2017-04-26 | 2017-06-30 | 北京农学院 | One plant of biological and ecological methods to prevent plant disease, pests, and erosion bacillus amyloliquefaciens and its application |
| CN108690821A (en) * | 2017-12-07 | 2018-10-23 | 中国热带农业科学院环境与植物保护研究所 | One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
| CN109554319A (en) * | 2019-01-31 | 2019-04-02 | 中国科学院微生物研究所 | A kind of Bacillus strain and its application |
| CN110172429A (en) * | 2019-06-13 | 2019-08-27 | 江苏牧之歌生态农业科技有限公司 | A kind of Biocontrol Bacillus that improves is to the culture medium and cultural method of plant pathogenic fungi bacteriostatic activity |
| CN110734871A (en) * | 2019-08-30 | 2020-01-31 | 山东蔚蓝生物科技有限公司 | kinds of bacillus amyloliquefaciens and application thereof in agricultural production |
-
2020
- 2020-04-29 CN CN202010353836.5A patent/CN111349589B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368747A (en) * | 2015-12-02 | 2016-03-02 | 中国农业科学院麻类研究所 | Bacillus amyloliquefaciens strain and application thereof |
| CN106906167A (en) * | 2017-04-26 | 2017-06-30 | 北京农学院 | One plant of biological and ecological methods to prevent plant disease, pests, and erosion bacillus amyloliquefaciens and its application |
| CN108690821A (en) * | 2017-12-07 | 2018-10-23 | 中国热带农业科学院环境与植物保护研究所 | One plant height imitates bacillus amyloliquefaciens and its microbial inoculum and application |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
| CN109554319A (en) * | 2019-01-31 | 2019-04-02 | 中国科学院微生物研究所 | A kind of Bacillus strain and its application |
| CN110172429A (en) * | 2019-06-13 | 2019-08-27 | 江苏牧之歌生态农业科技有限公司 | A kind of Biocontrol Bacillus that improves is to the culture medium and cultural method of plant pathogenic fungi bacteriostatic activity |
| CN110734871A (en) * | 2019-08-30 | 2020-01-31 | 山东蔚蓝生物科技有限公司 | kinds of bacillus amyloliquefaciens and application thereof in agricultural production |
Non-Patent Citations (3)
| Title |
|---|
| SHIWANI GULERIA等: "Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum", 《INT J FOOD MICROBIOL》 * |
| 张莹等: "解淀粉芽孢杆菌L-H15的促生与抗病特性研究", 《农业机械学报》 * |
| 李玉洋等: "尖孢镰刀菌拮抗菌SN06的分离、鉴定及其对棉花枯萎病的防治效果", 《山东农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113373083A (en) * | 2021-05-19 | 2021-09-10 | 福建省农业科学院植物保护研究所 | Biocontrol microbial inoculum for preventing and treating phytophthora blight of taro and application thereof |
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