CN113373083A - Biocontrol microbial inoculum for preventing and treating phytophthora blight of taro and application thereof - Google Patents
Biocontrol microbial inoculum for preventing and treating phytophthora blight of taro and application thereof Download PDFInfo
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 33
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- 238000009629 microbiological culture Methods 0.000 claims abstract description 7
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Abstract
The invention discloses a biocontrol microbial inoculum for preventing and treating taro epidemic disease and application thereof, belonging to the technical field of crop disease prevention and treatment. The bacillus amyloliquefaciens 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 biocontrol microbial inoculum comprises bacillus amyloliquefaciens GZ5 fermentation liquor or bacillus amyloliquefaciens GZ5 sterile fermentation liquor. The prepared biocontrol microbial inoculum for preventing and treating the phytophthora blight can effectively inhibit the growth of phytophthora blight.
Description
Technical Field
The invention belongs to the technical field of crop disease control, and particularly relates to a biocontrol microbial inoculum for controlling taro epidemic disease and application thereof.
Background
The taro is native to Indian eastMinistry and Malaysia and other tropical areas have a long history of cultivation in China, and are mainly planted in south China, southwest China and Yangtze river watersheds. In recent years, with the adjustment of agricultural industrialization, the planting area of the taro is continuously enlarged. Prepared from Phytophthora taro (A)Phytophthora colocasiaeRacib) is one of the most common and most important diseases causing the most serious loss in taro cultivation, and occurs and prevails in each taro production area. The disease mainly damages leaves and petioles, and also damages corms in serious cases, generally, the disease incidence is 20% -35%, a large number of leaves are rotten and plants are withered frequently in the outbreak period, the yield and the quality of the taros are seriously influenced, the yield loss is up to 30% -40% in serious cases, and some fields are even completely harvested.
Until now, the prevention and treatment of the taro epidemic disease mainly depends on the cultivation of disease-resistant varieties and the mass use of chemical pesticides. The cultivation of disease-resistant varieties is the most successful measure for preventing and treating epidemic diseases, but the toxic composition of the epidemic diseases has the characteristic of variability, so that the resistance of the disease-resistant varieties cultivated by using the main resistance genes is lost, and the screening and cultivation of new varieties with the epidemic disease resistance in a short time are very difficult. Although chemical pesticides play an important role in the development of agriculture, long-term use of chemical pesticides can have many negative effects. The use of chemical pesticides not only lacks long-term effectiveness, but also has the problems of ecological environment destruction, poisoning of people and livestock and beneficial organisms, great exceeding of the residual quantity of chemical substances in food and feed, influence on the living quality of people and livestock and the like, and the production and use of some high-toxicity and high-residue chemical pesticides are forbidden in many countries and regions. In the times that people pay more and more attention to their living environment and advocate green life, the exploration of effective biocontrol measures undoubtedly has great theoretical and practical significance.
Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) Widely distributed in nature, easy to separate and culture, broad-spectrum antibacterial activity, no environmental pollution, no toxicity to human and livestock, strong stress resistance and good stability. It can produce various high-efficiency active metabolites, effectively inhibit the activity of fungi and other bacteria, promote plant growth and induceThe organism can generate resistance by itself, and the growth of plant pathogenic bacteria and the invasion effect on the organism can be controlled in various aspects. At present, the bacterium is widely applied to disease control of various crops such as fruits, vegetables, flowers and the like, but the research on the disease control of taro is rarely reported. The bacillus amyloliquefaciens GZ5 with the biocontrol effect is obtained by separation in the laboratory, the growth inhibition effect of the biocontrol microbial inoculum on phytophthora taro hyphae is researched, and practical basis is provided for biological control of phytophthora taro and deep development and application of the biocontrol microbial inoculum.
Disclosure of Invention
The invention aims to: aiming at the problems that the taro epidemic disease is seriously generated, the area is enlarged year by year, the chemical control effect is not ideal, and the environmental pollution is caused at the same time, the biocontrol microbial inoculum for preventing the taro epidemic disease and the application thereof are provided.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biocontrol microbial inoculum for preventing and treating phytophthora blight comprises bacillus amyloliquefaciens GZ5 fermentation liquor or bacillus amyloliquefaciens GZ5 sterile fermentation liquor.
The Bacillus amyloliquefaciens GZ5 is Bacillus amyloliquefaciens (A), (B) and (C)Bacillus 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 preparation method of the bacillus amyloliquefaciens GZ5 fermentation liquid comprises the following steps:
(1) inoculating a bacillus amyloliquefaciens GZ5 strain into an LB liquid culture medium, culturing at 30 ℃ at 200 r/min for 24 h to prepare a seed solution;
(2) and (2) inoculating 10 mL of the seed solution prepared in the step (1) into a fermentation culture medium, and performing fermentation culture to obtain a fermentation liquor of the bacillus amyloliquefaciens GZ 5.
The formula of the fermentation medium in the step (2) is as follows: 10 g of tryptone, 5g of yeast powder and 5g of sodium chloride, adding distilled water to a constant volume of 1000 mL, dissolving and adjusting the pH value to 7.0-7.2; the fermentation culture conditions are as follows: inoculation amount of 3% (v/v), packagingThe bottle amount is 100 mL/250 mL, the culture is carried out for 48 hours at 30 ℃ and 200 r/min, and the prepared concentration is 108 CFU/ml fermentation broth of Bacillus amyloliquefaciens GZ 5.
The preparation method of the bacillus amyloliquefaciens GZ5 sterile fermentation liquid comprises the following steps: and centrifuging the fermentation liquor of the bacillus amyloliquefaciens GZ5 at 4 ℃ and 8000 r/min for 20 min, and filtering by using a 0.22 mu m bacterial filter to obtain the bacillus amyloliquefaciens GZ5 sterile fermentation liquor.
The application mode of the biocontrol microbial inoculum for preventing and treating the taro epidemic disease comprises the following steps: irrigating 100ml of biocontrol microbial inoculum at the young seedling stage or the adult plant stage of the taros, and continuously using for 2-3 times at intervals of 2 days each time.
The application of the biocontrol microbial inoculum for preventing and treating the taro epidemic disease in preventing and treating the taro epidemic disease.
The invention has the beneficial effects that:
1. the screened bacillus amyloliquefaciens GZ5 has strong inhibiting effect and can obviously inhibit the growth of phytophthora taro. Because the bacillus amyloliquefaciens GZ5 is separated from the tomato rhizosphere soil and is harmoniously compatible with soil ecology, the biocontrol microbial inoculum prepared by the fermentation liquor has no a series of problems caused by the use of chemical pesticides, reduces agricultural pollution, can effectively prevent and treat the taro epidemic disease, 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 colony morphology of strain GZ5 on LB medium.
FIG. 2 shows the inhibition effect 1 of the strain GZ5 on the growth of phytophthora tarda hyphae.
FIG. 3 shows the inhibition of the growth of phytophthora tarda hyphae by the strain GZ5 2.
FIG. 4 shows the inhibition effect of strain GZ5 on the growth of phytophthora tarda hyphae in the sterile fermentation broth.
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
The strain of the biocontrol microbial inoculum for preventing and treating the taro blight is bacillus amyloliquefaciensBacillus 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 separation and identification method of the strain is the same as that of the embodiment 1 in the application number of 202010353837X, the invention name of which is a strain of bacillus amyloliquefaciens and the application thereof in preventing and treating potato late blight.
Example two inhibition of Phytophthora tara hypha growth by Strain GZ5
1. Activated culture of strain GZ 5: taking out the separated and stored Bacillus amyloliquefaciens GZ5 from a refrigerator at-80 deg.C, and standing at room temperature
After the upper layer is dissolved, the strain ring is dipped in GZ5 bacterial liquid and streaked on an LB plate, and the strain is cultured in a constant temperature incubator at 30 ℃ for 24 hours for later use.
Preparation of a rye culture medium: soaking rye 60 g in deionized water for 36 h, mashing, soaking in 55-60 deg.C water bath for 1-2 h, filtering with four layers of gauze, collecting supernatant with distilled water to 1000 mL, adding agar powder 13.5g, and autoclaving at 121 deg.C for 30 min.
And (3) determining the inhibition effect of the strain GZ5 on the growth of phytophthora tarda hyphae: a plate opposing culture method is adopted, phytophthora taro hypha blocks with consistent fungus age and diameter of 7mm are inoculated in the center of a mixed culture dish of a rye culture medium and an LB culture medium in equal proportion, GZ5 bacterial liquid is inoculated at the position 2.5 cm away from the two opposite sides of the hypha blocks, or GZ5 bacterial liquid is dipped by an inoculating loop and streaked, phytophthora taro which is not inoculated with GZ5 is used as a control, the steps are repeated for 3 times, and the bacterial zone size is measured after the mixture is placed in a constant-temperature incubator at 28 ℃ for culture for 3 days, so that the strain GZ5 has good antagonistic action on the growth of the phytophthora taro hypha (figures 2 and 3).
EXAMPLE three preparation of Strain GZ5 sterile fermentation broth
The preparation of the strain GZ5 sterile fermentation liquid comprises the following steps:
(1) inoculating a bacillus amyloliquefaciens GZ5 strain into an LB liquid culture medium (l0 g of tryptone, 5g of yeast powder, 5g of sodium chloride, 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) at 30 ℃ and 200 r/min for culturing for 24 hours to prepare a seed solution;
(2) inoculating 10 mL of seed liquid into a fermentation medium, and performing fermentation culture to obtain a strain GZ5 fermentation liquid;
(3) and centrifuging the fermentation liquor of the strain GZ5 for 20 min at 4 ℃ at 8000 r/min, and filtering by using a 0.22 mu m bacterial filter to obtain the strain GZ5 sterile fermentation liquor.
The formula of the fermentation medium in the step (2) is as follows: 10 g of tryptone, 5g of yeast powder and 5g of sodium chloride, adding distilled water to a constant volume of 1000 mL, dissolving and adjusting the pH value to 7.0-7.2; the fermentation conditions were: inoculating 3% (v/v), bottling 100 mL/250 mL, fermenting culture medium pH 7.0-7.2, culturing at 30 deg.C and 200 r/min for 48 hr to obtain 10% (v/v) extract8 CFU/ml fermentation broth of strain GZ 5.
Example inhibition of four Strain GZ5 bacteria fermentation broth on Phytophthora taro hypha growth
Diluting the strain GZ5 sterile fermentation liquid prepared in the third embodiment by 100 times, adding 2 mL of the diluted sterile fermentation liquid into melted 20 mL of rye culture medium cooled to 45 ℃, uniformly mixing, pouring into a flat plate to prepare a bacteriostatic flat plate of the strain GZ5 sterile fermentation liquid, and inoculating phytophthora tarda mycelium blocks with the diameter of 7mm in the center of the flat plate. Adding 2 mL of sterile water into the melted 20 mL of rye culture medium cooled to about 45 ℃, uniformly mixing and pouring into a flat plate, and inoculating a phytophthora taro hypha block with the diameter of 7mm in the center of the flat plate as a control. Each treatment was repeated 3 times, incubated at 28 ℃ in an incubator, and the hyphal growth diameter was measured by the cross method after 3 days. The result shows that the GZ5 sterile fermentation broth has obvious inhibition effect on the growth of phytophthora taro hyphae, and the bacteriostasis rate is 60.1% (figure 4).
Example five-strain GZ5 fermentation liquor potted plant control effect test
Selecting 3-4-leaf-size taro variety (June red) seedlings with epidemic disease, and irrigating roots of each seedling with 100ml of strain GZ5 fermentation liquor (10) prepared in the third embodiment8CFU/ml). Irrigating roots after 24 h and inoculating phytophthora taroZoospore suspension (10)3spores/mL), 50mL per strain. After 2 days of inoculation, the root is irrigated once by 100ml of strain GZ5 fermentation liquor (10)8CFU/ml). A600-time liquid irrigation treatment of 75% chlorothalonil wettable powder (Shandong Lvfeng pesticide Co., Ltd.) is used as a pesticide control, and a fermentation medium and a plant treated by clear water are used as a blank control. Each treatment was 10 seedlings, 3 replicates per treatment. And investigating the morbidity every day after the disease is developed, and counting the morbidity and disease index until the clear water control morbidity reaches more than 80 percent.
Grading standard of taro epidemic disease:
level 0: no disease spots;
and (l) grade: the area of the lesion spots accounts for less than 5% of the area of the whole leaf;
and 3, level: the area of the lesion spots accounts for 6 to 10 percent of the area of the whole leaf;
and 5, stage: the area of the lesion spots accounts for 11 to 25 percent of the area of the whole leaf;
and 7, stage: the area of the lesion spots accounts for 26-50% of the area of the whole leaf;
and 9, stage: the area of the lesion spots accounts for more than 50 percent of the area of the whole leaf.
Disease index = [ Σpathological stage number × representative number)/(most serious pathological stage number × total number of leaves) ] × 100
The control effect = [ (blank control disease index-treatment disease index)/blank control disease index ] × 100%
The experimental results are shown in table 1, and the results show that the fermentation liquor of the tested bacillus amyloliquefaciens GZ5 can obviously reduce the disease index of the phytophthora blight, the control effect is as high as 64.25 percent and is only slightly lower than 70.42 percent of the control effect of 600 times of chemical pesticide 75 percent chlorothalonil wettable powder. In addition, the results in the table also show that the disease index of the fermentation medium treatment is slightly lower than that of the clear water treatment. The greenhouse pot experiment result shows that the bacillus amyloliquefaciens GZ5 can effectively prevent and treat the phytophthora blight and has good application potential.
TABLE 1 prevention and treatment effect of Bacillus amyloliquefaciens GZ5 fermentation liquid on taro blight
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.
Claims (7)
1. A biocontrol microbial inoculum for preventing and treating taro epidemic disease is characterized in that: the biocontrol microbial inoculum comprises bacillus amyloliquefaciens GZ5 fermentation liquor or bacillus amyloliquefaciens GZ5 sterile fermentation liquor.
2. The biocontrol microbial inoculum for preventing and treating the phytophthora blight of the taro as claimed in claim 1, which is characterized in that: the bacillus amyloliquefaciens GZ5 is bacillus amyloliquefaciens (A)Bacillus 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.
3. The biocontrol microbial inoculum for preventing and treating the phytophthora blight of the taro as claimed in claim 1, which is characterized in that: the preparation method of the bacillus amyloliquefaciens GZ5 fermentation liquid comprises the following steps:
(1) inoculating a bacillus amyloliquefaciens GZ5 strain into an LB liquid culture medium, culturing at 30 ℃ at 200 r/min for 24 h to prepare a seed solution;
(2) and (2) inoculating 10 mL of the seed solution prepared in the step (1) into a fermentation culture medium, and performing fermentation culture to obtain a fermentation liquor of the bacillus amyloliquefaciens GZ 5.
4. The biocontrol microbial inoculum for preventing and treating the phytophthora blight of taro as claimed in claim 3, which is characterized in that: the formula of the fermentation medium in the step (2) is as follows: 10 g of tryptone, 5g of yeast powder and 5g of sodium chloride, adding distilled water to a constant volume of 1000 mL, dissolving and adjusting the pH value to 7.0-7.2; the fermentation culture conditions are as follows: inoculating 3% (v/v), bottling 100 mL/250 mL, culturing at 30 deg.C for 48 hr to obtain 10% (v/v) solution8 CFU/ml fermentation broth of Bacillus amyloliquefaciens GZ 5.
5. The biocontrol microbial inoculum for preventing and treating the phytophthora blight of the taro as claimed in claim 1, which is characterized in that: the preparation method of the bacillus amyloliquefaciens GZ5 sterile fermentation liquid comprises the following steps: and centrifuging the fermentation liquor of the bacillus amyloliquefaciens GZ5 at 4 ℃ and 8000 r/min for 20 min, and filtering by using a 0.22 mu m bacterial filter to obtain the bacillus amyloliquefaciens GZ5 sterile fermentation liquor.
6. The use mode of the biocontrol microbial inoculum for preventing and treating the taro epidemic disease as the claim 1 is characterized in that: irrigating 100ml of biocontrol microbial inoculum at the young seedling stage or the adult plant stage of the taros, and continuously using for 2-3 times at intervals of 2 days each time.
7. The use of the biocontrol bacterial agent for preventing and treating phytophthora blight of taro as claimed in claim 1.
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