CN108293480B - Method for preventing and treating tomato gray mold by using biocontrol microbial inoculum - Google Patents
Method for preventing and treating tomato gray mold by using biocontrol microbial inoculum Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention belongs to the technical field of biology, and discloses a method for preventing and treating tomato gray mold by using a biocontrol microbial inoculum, which comprises the following steps: adding the biocontrol microbial inoculum into water with the weight of 50-100 times, stirring uniformly, activating at 25-30 ℃ for 3-5h, then spraying 10-20ml of biocontrol microbial inoculum on tomatoes according to the amount of each plant, and repeatedly spraying once after 1 week. The invention adopts the biocontrol agent to carry out field control on the tomato gray mold, completely has no a series of problems caused by chemical control, and has good control effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preventing and treating tomato gray mold by using a biocontrol microbial inoculum.
Background
Tomato gray mold is one of important diseases in tomato production, and poses a great threat to tomato production in protected areas. The tomato is generally reduced in yield by more than 30% due to the fact that the flowers, the fruits, the leaves and the stems can be damaged, particularly the green fruits are damaged the most, and the yield and the quality of the tomatoes are greatly influenced. At present, the method occurs in various regions in China and becomes a restrictive obstacle for tomato facility cultivation. At present, because the resistance source of the gray mold is not found, disease-resistant breeding is difficult to carry out, and the control of the gray mold of the tomato still depends on chemical agents for control. However, long-term and continuous medication causes pathogenic bacteria to gradually generate drug resistance, which affects the control effect, causes environmental pollution and pesticide pollution of fruits. Therefore, the search and screening of microbial strains with strong inhibition on botrytis cinerea is the main direction for preventing and treating botrytis cinerea. The biological pesticide is used for replacing chemical pesticide to prevent and treat plant diseases, although the generation of drug-resistant pathogenic strains can be avoided, the biological pesticide has the defects of unclear prevention and treatment mechanism, slower prevention effect, unstable effect and the like.
The biological pesticide is mainly a biological control agent, is used as a novel control measure, has the characteristics of safety, effectiveness and durability compared with other methods, and particularly avoids a series of problems caused by chemical control. The biocontrol microbial inoculum control method is to kill gray mold by adopting microorganisms, is popularized and used in agriculture at present, and is characterized in that 3 hundred million CFU/gram Trichoderma harzianum 300 sprays are added with water and sprayed once every 5 to 7 days before or at the early stage of disease attack, so that the gray mold can be controlled to a certain degree. The document 'screening research of chemical agents and biocontrol strains for preventing and controlling the tomato gray mold, Anzhenfu, 2016 years of northeast agricultural science' tests the control effect of the tomato gray mold in the field by adopting the chemical agents and the biocontrol strains; wherein, the control effect of the chemical agent on the tomato gray mold is between 60 and 70 percent; the control range of the biocontrol strain fermentation liquor on the tomato gray mold is 45.73-61.09%, and the control effect is trichoderma in the highest degree.
In recent years, biological control methods for gray mold have become a focus of research, and a large number of antagonistic microorganisms have been screened and utilized for gray mold control. A single strain often has the defects of low control effect, slow pesticide effect and the like, and a plurality of biopesticide companies begin to research composite biocontrol preparations at present. The compound biocontrol preparation generally refers to a preparation comprising more than two microorganisms, and the selection of strains in the compound biocontrol preparation is more critical and difficult; if the selection is careless, the strains can play antagonistic roles instead. The development of a method for preventing and treating tomato gray mold by using the composite biocontrol microbial inoculum is particularly urgent.
Disclosure of Invention
In order to overcome the defects that the chemical control method in the prior art is easy to generate antagonism and pollution and the control effect of a single biocontrol microbial inoculum is poor, the invention provides a method for controlling the tomato gray mold by using the biocontrol microbial inoculum.
The invention is realized by the following technical scheme:
the method for preventing and treating the tomato gray mold by using the biocontrol microbial inoculum comprises the following steps:
adding the biocontrol microbial inoculum into water with the weight of 50-100 times, stirring uniformly, activating at 25-30 ℃ for 3-5h, then spraying 10-20ml of biocontrol microbial inoculum on tomatoes according to the amount of each plant, and repeatedly spraying once after 1 week.
Preferably, the first and second electrodes are formed of a metal,
the tomatoes are in the early stage of the gray mold.
In particular, the amount of the solvent to be used,
the biocontrol microbial inoculum is prepared by the following process: uniformly mixing the trichoderma koningii-myceliophthora yeast mixed fermentation liquor and the pseudomonas stutzeri culture solution according to the volume ratio of 3:2, and then carrying out vacuum freeze drying to obtain the bacterial powder.
Preferably, the first and second electrodes are formed of a metal,
the pseudomonas stutzeri is ATCC 17588; the Trichoderma koningii is ATCC 66766; the filamentous yeast is ATCC 201110.
In particular, the amount of the solvent to be used,
the preparation method of the trichoderma koningii-trichosporon mixed fermentation liquor comprises the following steps:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr to obtain Trichosporon sinensis seed liquid;
2) inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
3) inoculating the trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5-7%, and continuously culturing for 18 hours at 30 ℃ to obtain the trichoderma koningii-filamentous spore yeast mixed fermentation liquid.
Preferably, the first and second electrodes are formed of a metal,
the formula of the fermentation medium is as follows: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate.
In particular, the amount of the solvent to be used,
the preparation method of the Pseudomonas stutzeri culture solution comprises the following steps:
performing streak culture on pseudomonas stutzeri on an LB (lysogeny broth) plate to obtain a single colony; selecting single colonies, respectively inoculating to LB liquid culture medium, culturing, and shake culturing at 28 deg.C and 200r/min for 24 hr to obtain seed solution; then inoculating the strain into an amplification culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 28 ℃ to obtain the pseudomonas stutzeri culture solution.
Preferably, the first and second electrodes are formed of a metal,
the formula of the amplification culture medium is as follows: 12g/L of corn starch, 10g/L of yeast extract, 5g/L of sodium chloride, 2g/L of urea, 0.1g/L of monopotassium phosphate, 0.1g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate and 0.01g/L of manganese sulfate.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the invention adopts the biological control microbial preparation to control the gray mold of the tomato, has no series of problems caused by chemical control, is beneficial to the pollution-free production of crops, and farmers can avoid or reduce other measures for controlling the gray mold and improving the stress resistance and promoting the growth, thereby not only reducing the burden of the farmers for planting, but also being beneficial to the export of the crops, improving the product quality and increasing the added value of the products.
The trichoderma koningii and the trichoderma koningii have no antagonistic action and can symbiose with each other, the trichoderma koningii can secrete cellulase, and the cellulose in the straw powder is decomposed into a carbon source which can be utilized by the trichoderma koningii, so that the trichoderma koningii grows by utilizing the carbon source, and the yeast is inoculated after 6 hours; the saccharomycetes can promote the growth of trichoderma koningii in turn, greatly improve the enzyme yield of the trichoderma, and further improve the ability of resisting botrytis cinerea;
the pseudomonas stutzeri can secrete antibacterial substances, can secrete various active enzymes and phytostimulants, promotes the growth of plant roots and plants, enhances the stress resistance, enables the plants to generate antagonistic action on the gray mold, and can inhibit the gray mold through the competition of nutrition and space; the inventor finds in experiments that pseudomonas stutzeri and the saccharomycete-trichoderma koningii have good synergistic effect and can greatly improve the capability of killing gray mold, probably because the pseudomonas stutzeri and the saccharomycete-trichoderma koningii have symbiotic relation, metabolites of pseudomonas stutzeri provide necessary growth promoting factors for the propagation of the saccharomycete so as to promote the rapid propagation and generate a large amount of antagonistic substances, and then the gray mold can not grow normally under the action of the substances; the inventors will further study the specific action mechanism in subsequent experiments. In addition, the test is only researched on the antagonism of botrytis cinerea, and the control effect on other soil-borne pathogenic bacteria is also to be further researched.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The method for preventing and treating the tomato gray mold by using the biocontrol microbial inoculum comprises the following steps:
adding the biocontrol microbial inoculum into 100 times of water by weight, stirring uniformly, activating at 30 ℃ for 3h, spraying 20ml of biocontrol microbial inoculum to tomatoes according to the amount of each plant, and repeatedly spraying once after 1 week.
The tomatoes are in the early stage of gray mold attack;
the biocontrol microbial inoculum is prepared by the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 2 multiplied by 10)7CFU/ml) into a fermentation medium, culturing at 30 deg.C for 6 hr, and inoculating 5-7% of the filamentous yeast seed solution (with an inoculation density of 0.5 × 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on pseudomonas stutzeri on an LB (lysogeny broth) plate to obtain a single colony; selecting single colonies, respectively inoculating to LB liquid culture medium, culturing, and shake culturing at 28 deg.C and 200r/min for 24 hr to obtain seed solution; then according to the inoculation amount of 10% (inoculation density is 5X 10)7CFU/ml) is inoculated into an enlarged culture medium and cultured for 24 hours at 28 ℃ to obtain a Pseudomonas stutzeri culture solution; the formula of the amplification culture medium is as follows: 12g/L of corn starch, 10g/L of yeast extract, 5g/L of sodium chloride, 2g/L of urea, 0.1g/L of monopotassium phosphate, 0.1g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate and 0.01g/L of manganese sulfate.
Uniformly mixing the trichoderma koningii-myceliophthora yeast mixed fermentation liquor and the pseudomonas stutzeri culture solution according to the volume ratio of 3:2, and then carrying out vacuum freeze drying to obtain the bacterial powder.
The pseudomonas stutzeri is ATCC 17588; the Trichoderma koningii is ATCC 66766; the filamentous yeast is ATCC 201110.
Example 2
The method for preventing and treating the tomato gray mold by using the biocontrol microbial inoculum comprises the following steps:
adding the biocontrol microbial inoculum into 50 times of water by weight, stirring uniformly, activating at 28 ℃ for 5h, then spraying 10ml of biocontrol microbial inoculum to tomatoes according to the amount of each plant, and repeating the spraying once after 1 week.
The tomatoes are in the early stage of gray mold attack;
the biocontrol microbial inoculum is prepared by the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 2 multiplied by 10)7CFU/ml) into a fermentation medium, culturing at 30 deg.C for 6 hr, and inoculating 5-7% of the filamentous yeast seed solution (with an inoculation density of 0.5 × 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on pseudomonas stutzeri on an LB (lysogeny broth) plate to obtain a single colony; selecting single colonies, respectively inoculating to LB liquid culture medium, culturing, and shake culturing at 28 deg.C and 200r/min for 24 hr to obtain seed solution; then according to the inoculation amount of 10% (inoculation density is 5X 10)7CFU/ml) is inoculated into an enlarged culture medium and cultured for 24 hours at 28 ℃ to obtain a Pseudomonas stutzeri culture solution; the formula of the amplification culture medium is as follows: 12g/L of corn starch, 10g/L of yeast extract, 5g/L of sodium chloride, 2g/L of urea, 0.1g/L of monopotassium phosphate, 0.1g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate and 0.01g/L of manganese sulfate.
Uniformly mixing the trichoderma koningii-myceliophthora yeast mixed fermentation liquor and the pseudomonas stutzeri culture solution according to the volume ratio of 3:2, and then carrying out vacuum freeze drying to obtain the bacterial powder.
The pseudomonas stutzeri is ATCC 17588; the Trichoderma koningii is ATCC 66766; the filamentous yeast is ATCC 201110.
Example 3
And (3) field control of tomato botrytis cinerea:
the selected variety of the tomatoes is Yukang No. 4, and the tomatoes are planted in rows in a greenhouse, and the row spacing of the rows is controlled to be 40 multiplied by 50 cm. The planting time of the tomato seedlings is 1 month and 7 days, the biocontrol microbial inoculum is sprayed at the early stage of the gray mold disease in 2 months and 3 days, and the biocontrol microbial inoculum is sprayed again in 2 months and 10 days, wherein the biocontrol microbial inoculum is sprayed according to the amount of 20ml per plant.
Grouping: experimental groups: example 1; control group 1: the procedure of example 1 was otherwise the same as in the case of the fermentation broth of Trichoderma koningii-myceliophthora; control group 2: the same procedure as in example 1 was repeated except that a culture solution of Pseudomonas stutzeri was used; control group 3: trichoderma koningii and myceliophthora serissa were fermented separately, and Trichoderma koningii fermentation broth, myceliophthora serissa fermentation broth, and Pseudomonas stutzeri culture broth were mixed to prepare bacterial powder, and the rest was the same as in example 1.
Spraying 50 plants each time, and setting three times of repeated tests; after the second spraying for 7 days, the disease index and the control effect of the botrytis cinerea are detected (according to the GB/T17980.28-2000 standard), and the field control effect is shown in the table 1:
TABLE 1
| Group of | Index of disease condition | The control effect is% |
| Blank control group | 23.9 | -- |
| Control group 1 | 8.6 | 64.02 |
| Control group 2 | 11.3 | 52.72 |
| Control group 3 | 6.9 | 71.13 |
| Experimental group | 4.5 | 81.17 |
And (4) conclusion: as can be seen from table 1 above, the experimental group and the control groups 1 to 3 can control the tomato gray mold, wherein the control effect of the experimental group is the best, and can reach more than 80%, while the control group 2 is the lowest, and is only 52.72%, thus the selection of the strain has a great influence on the control effect; although the control group 3 also adopts three strains, the difference from the biological control bacteria agent of the experimental group is mainly that the fermentation mode is different, the control effects of the two strains are obviously different, and the experimental group is improved by 10 percent compared with the control group 3, probably because the trichoderma koningii is inoculated to generate the cellulase, and the straw powder cellulose is decomposed into the carbon source which can be utilized by the trichosporon, so that the trichosporon grows by utilizing the carbon source, and the trichosporon promotes the growth of the trichoderma koningii in turn, and the synergistic effect of the two strains is obvious.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (5)
1. The method for preventing and treating the tomato gray mold by using the biocontrol microbial inoculum comprises the following steps:
adding the biocontrol microbial inoculum into water with the weight of 50-100 times, uniformly stirring, activating at 25-30 ℃ for 3-5h, then spraying 10-20ml of biocontrol microbial inoculum on tomatoes according to the amount of each plant, and repeatedly spraying once after 1 week;
the biocontrol microbial inoculum is prepared by the following process: uniformly mixing the trichoderma koningii-myceliophthora yeast mixed fermentation liquor and the pseudomonas stutzeri culture solution according to the volume ratio of 3:2, and then carrying out vacuum freeze drying to obtain bacterial powder;
the pseudomonas stutzeri is ATCC 17588; the Trichoderma koningii is ATCC 66766; the filamentous yeast is ATCC 201110;
the preparation method of the trichoderma koningii-trichosporon mixed fermentation liquor comprises the following steps:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr to obtain Trichosporon sinensis seed liquid;
2) inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
3) inoculating the trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5-7%, and continuously culturing for 18 hours at 30 ℃ to obtain the trichoderma koningii-filamentous spore yeast mixed fermentation liquid.
2. The method of claim 1, wherein the tomatoes are at an early stage of gray mold development.
3. The method of claim 1, wherein the fermentation medium is formulated as: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate.
4. The method according to claim 1, wherein the preparation method of the culture solution of Pseudomonas schleri comprises the following steps:
performing streak culture on pseudomonas stutzeri on an LB (lysogeny broth) plate to obtain a single colony; selecting single colonies, respectively inoculating to LB liquid culture medium, culturing, and shake culturing at 28 deg.C and 200r/min for 24 hr to obtain seed solution; then inoculating the strain into an amplification culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 28 ℃ to obtain the pseudomonas stutzeri culture solution.
5. The method of claim 4, wherein the formulation of the amplification medium is: 12g/L of corn starch, 10g/L of yeast extract, 5g/L of sodium chloride, 2g/L of urea, 0.1g/L of monopotassium phosphate, 0.1g/L of dipotassium phosphate, 0.01g/L of ferrous sulfate and 0.01g/L of manganese sulfate.
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