CN105441485B - Method for simultaneously preparing leaf and root agricultural biocontrol preparation and application thereof - Google Patents
Method for simultaneously preparing leaf and root agricultural biocontrol preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for simultaneously preparing leaf and root agricultural biocontrol agents and application thereof, the product is obtained by liquid fermentation of paecilomyces lilacinus YT08 with the preservation number of CGMCC No.10026, fermentation liquor is subjected to solid-liquid separation after the fermentation is finished, the solid part containing thalli is directly used as the root agricultural biocontrol agent, and the liquid part is the leaf agricultural biocontrol agent. The preparation method is simple, the production process is easy to control, the method is suitable for large-scale production in factories, and the leaf application and root application agricultural biocontrol agents respectively have strong inhibition effects on phytopathogens and root-knot nematodes.
Description
Technical Field
The invention relates to a method for simultaneously preparing leaf and root agricultural biocontrol agents and application of the leaf and root agricultural biocontrol agents in biological control of crop pathogenic bacteria and root-knot nematodes, and belongs to the technical field of biology.
Background
Plant nematode disease is one of four main pathogenic organisms planted in crops, vegetables, fruit trees, forests, flowers and medicinal materials, and the plant nematode disease only has second damage to fungi and exceeds damage to bacteria and viruses. Plant parasitic nematodes cause about 1000 hundred million dollars of worldwide agricultural production loss every year, the most important global agricultural harmful nematodes are root-knot nematodes and cyst nematodes, the more serious nematodes in China include root-knot nematodes, soybean cyst nematodes, wheat grain nematodes, sweet potato stem nematodes, rice aphelenchoides besseyi nematodes, chestnut nematodes, pine wood nematodes and the like, and the nematode disease loss of the crop planting industry in China only reaches 600 million yuan. Plant parasitic nematodes are usually controlled by chemical nematicides, cultivation techniques, cultivation of resistant varieties and biological pesticides, but no specific chemical pesticide is available at present, and the use of nematicides is greatly limited due to potential environmental problems relating to human and animal health.
The paecilomyces lilacinus can be widely parasitized on nematode eggs, has high insecticidal effect, is considered to be the root-knot nematode biocontrol fungus with the greatest application prospect, has the characteristics of high efficacy, wide parasitism, easy culture and the like, and particularly has obvious efficacy on controlling plant pathogenic nematodes. As a biological preparation, the paecilomyces lilacinus has the characteristics of safety, high efficiency, long lasting period and the like in the nematicidal process, has the nematicidal activity no worse than that of a chemical nematicide and has the effect of promoting plant growth. The Paecilomyces lilacinus has nematicidal effect, can parasitize insects of hemiptera, homoptera, isoptera, coleoptera, lepidoptera and the like, and has antagonistic effect on plant pathogenic bacteria. The paecilomyces lilacinus secondary metabolite also has certain physiological effects, such as the production of similar indoleacetic acid products, the promotion of plant root systems and plant growth, the promotion of seed germination and growth, the production of multiple functional enzymes and a certain chemical pesticide degradation effect.
The chitosan oligosaccharide is a new generation of marine organism source pesticide, has the characteristics of no toxicity, no environmental pollution, double biological regulation functions of pesticide effect and fertilizer effect, can induce and activate the immune system of plants, and improves the antiviral ability of the plants. Chitosan oligosaccharide is a series of products with certain polymerization degree and structure, which are degraded by an enzyme method by using chitosan as a raw material. The chitosan oligosaccharide can be used as a plant growth regulator, can stimulate the immune system reaction of plants, and enhances the defense capability of the plants against plant diseases and insect pests. The basic research of chitosan oligosaccharide as a plant immune activation factor starts in the 60's of the 20 th century, and the chitosan oligosaccharide is reported to have better prevention and control effects on plant diseases such as tobacco mosaic virus, rice blast, sheath blight, sclerotinia sclerotiorum pathogenic bacteria and the like in succession, meanwhile, the research finds that the chitosan oligosaccharide has better induction and activation effects on various functional enzymes in plant bodies and can induce the synthesis of plant protection essence, and in addition, chitosan has certain insecticidal activity on pests such as lepidoptera, homoptera and the like. The biocontrol effect of the chitosan oligosaccharide has good control effect on the control effect of tobacco, rice, tomatoes, cotton, wheat, strawberries, rapes, cucumbers, soybeans, hot peppers, pawpaw and other crops. Therefore, the chitosan oligosaccharide has broad-spectrum antibacterial activity and is nontoxic to human and livestock, and the development of the chitosan oligosaccharide biogenic pesticide is an effective way for reducing the application amount of chemical pesticides.
Disclosure of Invention
Aiming at the technical background and the defects thereof, the invention provides a method for simultaneously preparing leaf and root agricultural biocontrol agents and application thereof in biological control of crop pathogenic bacteria and root-knot nematodes. The paecilomyces lilacinus YT08 is used as an original strain, and the leaf agricultural biocontrol preparation and the root agricultural biocontrol preparation are simultaneously produced and prepared by a liquid fermentation method, wherein the leaf agricultural biocontrol preparation has a strong inhibition effect on plant pathogenic fungi, and the root agricultural biocontrol preparation has a strong killing effect on root-knot nematodes.
The object of the invention can be achieved by the following measures:
1. the paecilomyces lilacinus has the characteristics of high chitosanase yield and is deposited under the name: YT08, depository: china general microbiological culture Collection center, preservation date: 26 days 11 months 2014, the preservation number is CGMCC No.10026, and the classification and the naming are as follows: paecilomyces lilacinus (A)Paecilomyces lilacinus）。
2. Strain activation
Activation medium (g/L): 3-5 parts of peptone, 1-3 parts of yeast powder, 10-20 parts of glucose, 2-5 parts of powdered chitosan, 1-3 parts of monopotassium phosphate, 1-2 parts of magnesium sulfate, 1-3 parts of disodium hydrogen phosphate and pH 7.0. The activated medium is 100ml dispensed into 500ml Erlenmeyer flasks and sterilized at 121 ℃ for 20 min. Selecting 1 ring from the strain preserved on the test tube slant, inoculating the strain in an activation culture medium, and culturing at 30-32 ℃ and 180rpm for 30-36 h until the strain is turbid to prepare an activation seed solution.
3. Liquid fermentation
(1) Shaking culture
With the preferred medium (g/L): 18-30 parts of glycerol, 25-40 parts of soybean peptone and MnSO41-3 parts of powdered chitosan (the deacetylation degree is more than 90%), 10-15 parts of crab shell powder, 2-5 parts of NaCl, and CaCl2 1～3，MgSO4 1～3，FeSO40.5-1.5, pH 5-6.5, subpackaging 200ml fermentation medium in each 500ml Erlenmeyer flask, and sterilizing at 121 ℃ for 20 min. According to the ratio of 0.8-1.5%v/v) inoculating the activated seed culture solution, then putting the seed culture solution into a rotary shaking table at 180rpm for culture, controlling the temperature to be 30-34 ℃, and fermenting for 5-8 d.
(2) Cultivation in fermenter
With the preferred medium (g/L): 18-30 parts of glycerol, 25-40 parts of soybean peptone and MnSO41-3 parts of powdered chitosan (the deacetylation degree is more than 90%), 10-15 parts of crab shell powder, 2-5 parts of NaCl, and CaCl2 1～3，MgSO4 1～3，FeSO40.5 to 1.5. The method comprises the steps of filling 70% of the mixture into a fermentation medium, performing steam sterilization at 118 ℃ for 20min, inoculating activated strains according to the proportion of 0.8-1.5% (v/v) under the condition of flame ring protection, controlling the pH value in the fermentation process to be kept at 5-6.5 all the time by adjusting an acid-base peristaltic pump, adjusting the culture temperature to be 30-34 ℃ by using a heating device of a fermentation tank, adjusting the dissolved oxygen parameter to be kept at more than 10% all the time by adjusting the amount of sterile air entering the tank and the stirring speed, and performing fermentation for 4-7 d.
4. Preparation of a product: centrifuging the fermentation liquor of the paecilomyces lilacinus YT08 for 5min at 10000r/min, and respectively collecting thallus precipitate and fermentation supernatant, wherein the fermentation supernatant can be directly used as a leaf agricultural biocontrol agent after being collected and filled, and meanwhile, the thallus precipitate containing part of undegraded chitosan and the like can be used as a root agricultural biocontrol agent. The prepared root application agricultural biological preparation is weighed to determine the thallus content in unit fermentation liquor volume, and meanwhile, the leaf application agricultural biological control preparation is used for respectively determining the activity of chitosan enzyme and the content of chitosan oligosaccharide by a DNS method.
The chitosanase assay used was: diluting leaves with agricultural biocontrol agent to a proper gradient, adding 0.2mL of water to 1mL, adding 1mL of colloidal chitosan substrate solution (1% mass fraction), carrying out heat preservation reaction in a water bath at 40 ℃ for 20min, adding 3mL of DNS reagent, carrying out boiling water bath for 5min, carrying out volume fixing to 25mL, and centrifuging 4mL of reaction sample liquid at 5000rpm for 10 min. The OD value was measured at 520nm using the control as a blank, and the enzyme activity was calculated from the DNS standard curve. Blank control group: 0.2mL of the leaf agricultural biocontrol agent diluted to the same gradient is added with water to 1mL, inactivated in a boiling water bath for 5min, added with the same volume of chitosan solution and DNS reagent, and treated by the same method as the sample group. Calculating enzyme activity: the umol number of glucosamine produced by hydrolysis in unit volume (mL) and unit time (min) is taken as a definition unit of enzyme activity.
The standard curve used was made: accurately weighing 0.13g of glucosamine hydrochloride dried at 100 ℃ to constant weight, adding distilled water to dissolve the glucosamine hydrochloride and fixing the volume to 100mL, and preparing a solution with the concentration of 6 umol/mL. And respectively adding 1mL of standard glucosamine solution, 1mL of ultrapure water and 3mL of DNS color developing solution into each 25mL colorimetric tube according to different concentration gradients, reacting for 5min in a boiling water bath, measuring the OD value at 520mn after the volume is up to 25mL, and drawing a standard curve.
The method for measuring the content of the chitosan oligosaccharide comprises the following steps: diluting the agricultural biocontrol preparation for leaves to a proper gradient within the accurate detection limit of a spectrophotometric method, respectively adding 1mL, 1mL of ultrapure water and 3mL of DNS color developing solution into each 25mL colorimetric tube, reacting for 5min in a boiling water bath, measuring an OD value at 520mn after the volume is reduced to 25mL, determining the amount of chitosan oligosaccharide according to a standard curve, and calculating the total amount of the chitosan oligosaccharide of the agricultural biocontrol preparation for leaves in unit volume.
The beneficial effects of the invention are as follows:
1. The paecilomyces lilacinus YT08 CGMCC No.10026 is a filamentous fungus with strong control effect on crop root-knot nematodes, can be colonized at the roots of plants after being applied to soil, generates a large amount of hydrolytic proteases such as chitosanase, chitinase and the like through the characteristics of the paecilomyces lilacinus YT08, and enters the root-knot nematodes as infection factors to achieve the effect of killing the root-knot nematodes, so that the fermented paecilomyces lilacinus YT08 can be used as an agricultural biocontrol agent for root application.
2. The fermentation culture medium is added with excessive powdered chitosan and crab shell powder, except that part of the chitosan and the crab shell powder are converted into chitosan oligosaccharide, part of undegraded chitosan and chitin are used as auxiliary factors for biological control of root-knot nematodes, and in addition, the chitosan and the chitin can also inhibit various plant pathogenic bacteria and are collected with thalli for use to form a synergistic effect of biological control, and the synergistic effect and the paecilomyces lilacinus YT08 are used as agricultural biocontrol agents applied to roots.
3. By utilizing the characteristic of high yield chitosan enzyme of paecilomyces lilacinus YT08, chitosan powder is added in the process of thallus fermentation to induce and generate chitosan enzyme, and the generated chitosan enzyme degrades chitosan in a culture medium to convert the chitosan into low molecular weight chitosan oligosaccharide with high physiological activity, wherein the chitosan oligosaccharide exists in a fermentation liquid in a dissolving mode and has an inhibiting effect on various plant pathogenic bacteria, so that the chitosan oligosaccharide can be used as a biological pesticide and is used for preventing and controlling the harm of the plant pathogenic bacteria on the leaf surfaces after being sprayed on the leaf surfaces.
4. The paecilomyces lilacinus YT08 can also generate a plurality of secondary metabolites during the fermentation culture process, is dissolved in water, can promote the growth of crops, and has a certain synergistic effect on the inhibition of plant pathogenic bacteria, so that the components and the generated chitosan oligosaccharide form a synergistic effect, and can be used for preventing and treating the harm of the pathogenic bacteria on the plant leaf surfaces after being sprayed on the leaf surfaces.
The invention has the following outstanding advantages:
1. By utilizing the characteristics of paecilomyces lilacinus YT08 CGMCC No.10026 for high yield of chitosanase and part of chitinase, powdery chitosan and crab shell powder are added in the fermentation production process, the agricultural biocontrol preparation for root application and leaves is simultaneously prepared in a fermentation process, the equipment investment is saved, the production field is reduced, and the production cost is reduced.
2. The paecilomyces lilacinus YT08 is easy to culture, the operation method is simple and efficient, and the paecilomyces lilacinus YT08 has an inhibiting effect on most pathogenic bacteria, so that the paecilomyces lilacinus YT08 is not easy to be infected with bacteria in the culture process.
3. The agricultural biocontrol preparation for root application and leaves prepared by the invention has strong inhibition effects on root-knot nematodes and phytopathogens respectively, and has wide market prospects.
Detailed Description
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative and not limiting of the scope of the invention.
EXAMPLE 1 Shake flask culture with leaf and root application of agricultural biocontrol agent
Selecting 1 ring from the strain preserved on the test tube slant, inoculating in an activation culture medium, and culturing at 30 deg.C and 180rpm for 32h until the strain is turbid to obtain an activation seed solution. Activation medium (g/L): peptone 3, yeast powder 1, glucose 10, powdered chitosan 2, potassium dihydrogen phosphate 1, magnesium sulfate 1, disodium hydrogen phosphate 1, and pH 7.0. The activated medium is 100ml dispensed into 500ml Erlenmeyer flasks and sterilized at 121 ℃ for 20 min.
With the preferred medium (g/L): glycerol 18, soy peptone 25, MnSO41, 20 parts of powdered chitosan (deacetylation degree of more than 90%), 10 parts of crab shell powder, NaCl 2 and CaCl2 1，MgSO4 1，FeSO40.5, pH 5, 200ml fermentation medium per 500ml Erlenmeyer flask, and sterilizing at 121 deg.C for 20 min. Inoculating the activated seed culture solution according to the inoculation amount of 0.8% (v/v), culturing in a rotary shaker at 180rpm, controlling the temperature at 30 deg.C, and fermenting for 6 d.
Centrifuging the fermentation liquor of the paecilomyces lilacinus YT08 for 5min at 10000r/min, and respectively collecting thallus precipitate and fermentation supernatant, wherein the fermentation supernatant can be directly used as a leaf agricultural biocontrol agent after being collected and filled, and meanwhile, the thallus precipitate containing part of undegraded chitosan is used as a root application agricultural biocontrol agent. In each liter of fermentation liquor after the fermentation is ended, the prepared root application agricultural biocontrol agent is weighed to determine that the wet weight is 142g, the activity of the chitosan enzyme in the leaf application agricultural biocontrol agent is determined to be 75.1U/mL, and the content of chitosan oligosaccharide is 7.2g by a DNS method.
example 2 cultivation in fermenter (10L) with preparation of leaf and root applied agricultural biocontrol formulations
Activation medium (g/L): peptone 5, yeast powder 3, glucose 20, powdered chitosan 5, potassium dihydrogen phosphate 3, magnesium sulfate 2, disodium hydrogen phosphate 3, and pH 7.0. The activated medium is 100ml dispensed into 500ml Erlenmeyer flasks and sterilized at 121 ℃ for 20 min. Selecting 1 ring from the strain preserved on the test tube slant, inoculating in an activation culture medium, and culturing at 32 deg.C and 180rpm for 36h until the strain is turbid to obtain an activation seed solution.
With the preferred medium (g/L): glycerol 30, soy peptone 40, MnSO43, 30 percent of chitosan powder (deacetylation degree of more than 90 percent), 15 percent of crab shell powder, 5 percent of NaCl, CaCl2 3，MgSO4 3，FeSO41.5. Adding 70% of the culture medium into fermentation medium at a liquid loading amount of 7L, steam sterilizing at 118 deg.C for 20min, and keeping the content of 1.5% under flame ring protection(v/v) inoculating activated strain, controlling pH to be always maintained at pH 6.5 by adjusting acid-base peristaltic pump, adjusting culture temperature to be 34 deg.C by using heating device of fermentation tank, adjusting dissolved oxygen parameter to be always maintained at above 10% by adjusting sterile air amount entering the tank and stirring speed, and fermenting for 7 d.
Centrifuging the fermentation liquor of the paecilomyces lilacinus YT08 for 5min at 10000r/min, and respectively collecting thallus precipitate and fermentation supernatant, wherein the fermentation supernatant can be directly used as a leaf agricultural biocontrol agent after being collected and filled, and meanwhile, the thallus precipitate containing part of undegraded chitosan is used as a root application agricultural biocontrol agent. In each liter of fermentation liquor after the fermentation is ended, the prepared root application agricultural biocontrol agent is weighed to determine the wet weight to be 203g, the activity of the chitosan enzyme in the leaf application agricultural biocontrol agent is determined to be 93.5U/mL, and the content of chitosan oligosaccharide is determined to be 8.4g by a DNS method.
example 3 cultivation in fermenter (50L) with preparation of leaf and root applied agricultural biocontrol formulations
Activation medium (g/L): peptone 4, yeast powder 2, glucose 15, powdered chitosan 3, potassium dihydrogen phosphate 2, magnesium sulfate 2, disodium hydrogen phosphate 2, and pH 7.0. The activated medium is 100ml dispensed into 500ml Erlenmeyer flasks and sterilized at 121 ℃ for 20 min. Selecting 1 ring from the strain preserved on the test tube slant, inoculating in an activation culture medium, and culturing at 30 deg.C and 180rpm for 34 h until the strain is turbid to obtain an activation seed solution.
With the preferred medium (g/L): glycerol 20, soy peptone 30, MnSO42, 25 percent of powdered chitosan (deacetylation degree of more than 90 percent), 12 percent of crab shell powder, 4 percent of NaCl, CaCl2 2，MgSO4 2，FeSO41. The fermentation medium is filled in 70% of the fermentation medium according to the proportion, the liquid filling amount is 35L, after steam sterilization at 118 ℃ for 20min, activated strains are inoculated according to the proportion of 1% (v/v) under the condition of flame ring protection, the pH value in the fermentation process is always maintained at pH 6 by adjusting an acid-base peristaltic pump, the culture temperature is adjusted to 32 ℃ by using a heating device of a fermentation tank, and the dissolved oxygen parameter is always maintained at more than 10% by adjusting the amount of sterile air entering the tank and the stirring speed, and the fermentation is carried out for 5 d.
Centrifuging the fermentation liquor of the paecilomyces lilacinus YT08 for 5min at 10000r/min, and respectively collecting thallus precipitate and fermentation supernatant, wherein the fermentation supernatant can be directly used as a leaf agricultural biocontrol agent after being collected and filled, and meanwhile, the thallus precipitate containing part of undegraded chitosan is used as a root application agricultural biocontrol agent. In each liter of fermentation liquor after the fermentation is ended, the prepared root application agricultural biocontrol agent is weighed to determine the wet weight of 185g, the activity of the chitosan enzyme in the leaf application agricultural biocontrol agent is determined to be 91.8U/mL, and the content of chitosan oligosaccharide is determined to be 8.6g by a DNS method.
Example 4 evaluation of Using Effect of root-applied agricultural biocontrol agent
The root applied agricultural biocontrol formulation prepared in example 1 was named PL-1, the root applied agricultural biocontrol formulation prepared in example 2 was named PL-2, and the root applied agricultural biocontrol formulation prepared in example 3 was named PL-3.
The culture medium is prepared by mixing disease-free soil, organic fertilizer and sand according to a ratio of 9:1:3, and the used culture medium has no nematode existence after detection. Tomato seedlings of about 12cm in length were picked and planted in 30X 30cm pots, 1 for each pot, for a total of 60 plants. 200 tomato root-knot nematodes of 2 years are planted in the culture medium of the tomato seedlings of 60 pots, wherein 3g of agricultural biocontrol agents are planted in 30 pots respectively, and the agricultural biocontrol agents marked as PL-1, PL-2 and PL-3 respectively account for 10 pots; another 30 pots were given 3g of wet distillers grains as a blank. And after 60 days of routine management, calculating the prevention and treatment effect.
The tomato root-knot nematode used was obtained by the following route: collecting root systems of root-knot nematode disease tomato plants in a greenhouse, firstly washing the root systems with water, cutting the root systems into small sections, adding the small sections into a sodium hypochlorite solution, sealing the openings, shaking the openings for 3 minutes, firstly sieving the root systems with a 200-mesh sieve, washing the root systems with distilled water, then sieving the root systems with a 500-mesh sieve, repeatedly washing the root systems with the distilled water, and collecting the root systems in a small sterile beaker. And (3) incubating the collected root-knot nematode eggs for 3 days at 26 ℃ to obtain 2-instar larvae of the tomato root-knot nematodes.
The severity of the disease is classified into 5 grades according to the number of roots born: the grade of the root knot is 0, the grade of the root knot accounts for 1-25% of the whole root system is 1, the grade of the root knot accounts for 26-50% of the whole root system is 2, the grade of the root knot accounts for 51-75% of the whole root system is 3, and the grade of the root knot accounts for 76-100% of the whole root system is 4. The calculation formula of the disease index and the prevention and treatment effect is as follows:
The pot culture test results are shown in Table 1, the control effects of the agricultural biocontrol agents applied to PL-1, PL-2 and PL-3 roots for controlling the tomato root-knot nematodes are 85.6%, 85.9% and 86.3%, the control effects of the three test agent groups are basically the same, and the root weight growth rate, the plant height growth rate and the overground part weight growth rate of the tomatoes in the treated group are obviously higher than those in the control group.
TABLE 1 prevention and control of tomato root-knot nematode by applying agricultural biocontrol agent to roots
Example 5 evaluation of inhibitory Effect of leaf agricultural biocontrol agent on plant pathogenic bacteria
The leaf agricultural biocontrol agent prepared in example 1 was named LBC-1, the leaf agricultural biocontrol agent prepared in example 2 was named LBC-2, and the leaf agricultural biocontrol agent prepared in example 3 was named LBC-3.
The inhibition effect of the leaf agricultural biocontrol agent on plant pathogenic bacteria is determined by adopting a hypha growth rate inhibition method. Adding the leaf agricultural biocontrol agent into sterilized and melted solid PDA culture medium, adding 5mL of the PDA culture medium per 100mL of the PDA culture medium, using 5mL of sterile water as a blank control, uniformly mixing and pouring the mixture into a flat plate. Firstly, respectively activating three plant pathogenic bacteria (apple anthracnose bacteria, watermelon fusarium wilt bacteria and tomato botrytis cinerea) by using a solid PDA culture medium until colonies with the diameter of more than 1cm are formed, then preparing fungus cakes according to concentric circles on the edges of the activated colonies of the agricultural pathogenic bacteria to be tested by using a puncher with the diameter of 0.5cm, inoculating the fungus cakes on a solid PDA flat plate added with a leaf agricultural biocontrol agent, setting 3 times for each treatment, culturing at the constant temperature of 28 ℃ for 5 days, and measuring the diameters of the colonies by using a cross method.
Bacteriostatic rate (%) = (control net growth amount-treated net growth amount)/control net growth amount 100%
Net growth = mean of triplicates (cm) -0.5cm
The evaluation of the effect of the leaf agricultural biocontrol agent is shown in table 2. As can be seen from Table 2, the prepared three leaf agricultural biocontrol agents have strong inhibition effects on apple anthracnose, watermelon fusarium wilt and tomato botrytis cinerea, and the leaf agricultural biocontrol agents prepared under different culture conditions have slightly different inhibition effects on plant pathogenic bacteria.
TABLE 2 evaluation of inhibitory Effect of leaf agricultural biocontrol agents on plant pathogenic bacteria
In conclusion, the invention has the advantages that the leaf agricultural biocontrol preparation and the root agricultural biocontrol preparation respectively have high inhibition rate on plant pathogenic bacteria and root-knot nematodes, the preparation method is simple, the control of process conditions is relatively easy, the preparation method is suitable for large-scale industrial production, and the preparation method has wide application prospect in the aspects of controlling plant pathogenic bacteria and root-knot nematode diseases of economic crops.
Claims (2)
1. a method for simultaneously preparing leaf application and root application agricultural biocontrol agents is characterized in that: is obtained by liquid fermentation of a strain with the preservation number of CGMCC No.10026, has strong inhibition effects on plant pathogenic bacteria and root-knot nematode respectively, and the preparation method comprises the following steps:
(1) Activating strains: selecting 1 ring from the strain preserved on the test tube slant, inoculating the strain in an activation culture medium, and culturing at 30-32 ℃ and 180rpm for 30-36 h until the strain is turbid to prepare an activation seed solution; the activation medium (g/L): 3-5 parts of peptone, 1-3 parts of yeast powder, 10-20 parts of glucose, 2-5 parts of powdered chitosan, 1-3 parts of monopotassium phosphate, 1-2 parts of magnesium sulfate, 1-3 parts of disodium hydrogen phosphate and pH 7.0;
(2) Shake flask culture liquid fermentation: subpackaging 200ml of preferable fermentation medium in per 500ml Erlenmeyer flask, sterilizing at 121 deg.C for 20min, inoculating activated seed culture solution according to the inoculation amount of 0.8-1.5% (v/v), and inoculatingCulturing in a rotary shaking table at 180rpm, controlling the temperature to be 30-34 ℃, and fermenting for 5-8 d; the preferred fermentation medium (g/L): 18-30 parts of glycerol, 25-40 parts of soybean peptone and MnSO41-3 parts of powdered chitosan 20-30 parts of crab shell powder 10-15 parts of NaCl 2-5 parts of CaCl2 1～3，MgSO41～3，FeSO4 0.5～1.5；
(3) Fermenting the culture liquid of the fermentation tank: loading 70% of the mixture into an optimized fermentation medium, sterilizing the mixture for 20min by steam at 118 ℃, inoculating activated strains according to the proportion of 0.8-1.5% (v/v), controlling the fermentation pH to be 5-6.5, adjusting the temperature to be 30-34 ℃, maintaining the dissolved oxygen at more than 10%, and fermenting for 4-7 d; the preferred fermentation medium (g/L): 18-30 parts of glycerol, 25-40 parts of soybean peptone and MnSO41-3 parts of powdered chitosan 20-30 parts of crab shell powder 10-15 parts of NaCl 2-5 parts of CaCl2 1～3，MgSO4 1～3，FeSO4 0.5～1.5；
(4) Preparation of a product: centrifuging the strain fermentation liquor of CGMCC No.10026 for 5min at 10000r/min, and respectively collecting thallus precipitate and fermentation supernatant, wherein the fermentation supernatant can be directly used as a leaf agricultural biocontrol agent after being collected and filled, and meanwhile, the thallus precipitate containing part of undegraded chitosan and the like is the root application agricultural biocontrol agent.
2. The method for simultaneously preparing a foliar and root application agricultural biocontrol agent according to claim 1 wherein the inducing substrates used are powdered chitosan and crab shell powder having a degree of deacetylation of 90% or more.
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| WO2014086753A3 (en) * | 2012-12-03 | 2014-08-14 | Bayer Cropscience Ag | Composition comprising biological control agents |
| WO2015069938A1 (en) * | 2013-11-06 | 2015-05-14 | The Texas A & M University System | Fungal endophytes for improved crop yields and protection from pests |
| CN104818216A (en) * | 2015-01-20 | 2015-08-05 | 鲁东大学 | Paecilomyces lilacinus for prevention and control of meloidogyne diseases of tomato and grape |
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| WO2014086753A3 (en) * | 2012-12-03 | 2014-08-14 | Bayer Cropscience Ag | Composition comprising biological control agents |
| WO2015069938A1 (en) * | 2013-11-06 | 2015-05-14 | The Texas A & M University System | Fungal endophytes for improved crop yields and protection from pests |
| CN104818216A (en) * | 2015-01-20 | 2015-08-05 | 鲁东大学 | Paecilomyces lilacinus for prevention and control of meloidogyne diseases of tomato and grape |
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