CN-121990849-A - Method for preventing and treating sclerotinia rot of colza by antagonizing trichoderma strain

CN121990849ACN 121990849 ACN121990849 ACN 121990849ACN-121990849-A

Abstract

The invention discloses a method for preventing and controlling sclerotinia rot of rape by antagonizing trichoderma strains, which relates to the technical field of agricultural disease prevention and control, and comprises the specific steps of S100, screening and mutagenizing strains, namely separating trichoderma strains from rape rhizosphere soil in different environments, initially screening out strains with antagonism to sclerotinia by a plate-facing method, carrying out chemical mutagenesis treatment on the initially screened strains, and carrying out facing culture with sclerotinia again, screening out high-yield antagonism substances with larger inhibition zones, and mutagenized strains with strong stress resistance, and culturing to obtain trichoderma fermentation liquor.

Inventors

LAN BO
LI YUNPENG
LI BAOJIA
SUN QINGHUA
YANG ZIYU
DENG XING
YIN CHANGFA
YANG YINGQING

Assignees

江西省农业科学院植物保护研究所

Dates

Publication Date: 20260508
Application Date: 20260119

Claims (8)

1. A method for preventing and controlling sclerotinia rot of colza by antagonizing trichoderma strains is characterized by comprising the following specific steps: S100, separating trichoderma strains from rape rhizosphere soil in different environments, primarily screening out strains with antagonism to sclerotinia by a plate counter method, carrying out chemical mutagenesis treatment on the primarily screened strains, and carrying out counter culture with the sclerotinia again, screening out high-yield antagonistic substances with larger inhibition zone and mutagenized strains with strong stress resistance, and culturing to obtain trichoderma fermentation liquor; S200, extracting active substances, namely centrifuging trichoderma fermentation liquor to obtain supernatant, precipitating by ammonium sulfate, centrifuging, collecting precipitate, dissolving by using sterile water, dialyzing, and removing micromolecular impurities to obtain trichoderma antagonistic active substance concentrate; S300, preparing a biological fertilizer carrier, namely baking attapulgite to activate pores, treating earthworm cast charcoal with acetic acid solution, washing, mixing with alginic acid oligosaccharide, bacillus mucilaginosus powder and arbuscular mycorrhizal fungi spore agent, stirring to form a mixed material, adding trichoderma antagonistic active substance concentrate, uniformly stirring, spraying and mixing with chitosan oligosaccharide grafted sodium alginate aqueous solution to form a gel coating layer, granulating after adjusting the water content, curing, and coating with polyglutamic acid solution to prepare the biological fertilizer carrier; s400, seed dressing treatment, namely mixing and dressing rape seeds and a bio-fertilizer carrier according to the mass ratio of 1:4 before sowing rape, and airing after seed dressing to enable the surfaces of the seeds to be attached with the bio-fertilizer carrier; S500, applying in a growth critical period, namely diluting the biological fertilizer carrier for 30-50 times in a rape seedling period, applying the biological fertilizer carrier to the root system of a rape plant in a stripe mode, wherein the application amount per mu is 20-30kg, and applying the biological fertilizer carrier after diluting the biological fertilizer carrier for 20-40 times in the initial flowering period of the rape, wherein the application amount per mu is 15-25kg.
2. The method for antagonizing trichoderma strain to prevent and treat sclerotinia rot of rape according to claim 1, wherein in the screening and mutagenesis of the S100 strain, the trichoderma strain is isolated from rhizosphere soil of rape under different environments including a high temperature environment, a low temperature environment and a drought environment, wherein the high temperature environment is 42 ℃ to 45 ℃ for one week, the low temperature environment is-6 ℃ to-8 ℃ for one week, and the drought environment is 3% -4% of the soil moisture content for two weeks.
3. A method for preventing and treating sclerotinia rot of a trichoderma antagonistic strain according to claim 1, wherein in the screening and mutagenesis of the S100 strain, the strain having antagonism to sclerotinia is initially screened out by a plate-facing method, specifically, a sclerotinia sclerotiorum cake with a diameter of 5mm is inoculated in the center of a plate of a PDA solid medium with a diameter of 9cm, 4 trichoderma strain cakes with a diameter of 5mm are symmetrically inoculated at a position 2cm away from the edge of the sclerotinia sclerotiorum cake, a plate without the trichoderma strain is used as a control, each treatment is repeated 3 times, the culture is carried out in a constant temperature incubator at 25 ℃ for 5-7 days, the colony diameter of the sclerotinia sclerotiorum is measured, the inhibition rate of each trichoderma strain to the sclerotinia sclerotiorum is calculated, and the trichoderma strain with the inhibition rate of not less than 30% is screened out as the initially screened strain.
4. The method for antagonizing trichoderma strain to prevent and treat sclerotinia rot of rape according to claim 1, wherein in the step of screening and mutagenesis of the S100 strain, the primary screening strain is subjected to chemical mutagenesis treatment, the primary screening strain is inoculated into PDA liquid culture medium, the bacterial suspension is prepared by shaking culture for 24 hours under the conditions of 25 ℃ and 180r/min, ethyl methyl sulfonate solution with the final concentration of 0.1% -0.5% is added into the bacterial suspension, the light-shielding treatment is carried out for 30min to 120min under the condition of 25 ℃, and the reaction is stopped by adding an equal volume of 2.5% sodium thiosulfate solution after the mutagenesis.
5. The method for preventing and treating sclerotinia rot of trichoderma antagonistic strains according to claim 1, wherein the specific step of obtaining trichoderma antagonistic active substance concentrate in S200 active substance extraction is that the trichoderma antagonistic active substance concentrate is obtained by centrifuging at a rotation speed of 6000-8000r/min for 10-15min through a centrifuge tube in an environment of 2-6 ℃, discarding precipitate of trichoderma fermentation broth, collecting supernatant, adding ammonium sulfate, centrifuging for 15-20min at a temperature of 2-6 ℃ and 8000-10000r/min through a centrifuge tube, discarding supernatant, collecting precipitate, dissolving the precipitate with sterile water, dialyzing through a dialysis bag, and removing small molecular impurities.
6. The method for preventing and controlling sclerotinia rot of colza by antagonizing trichoderma strains according to claim 1, wherein in the preparation of the S300 bio-fertilizer carrier, 30-40 parts by mass of attapulgite with the grain diameter of 50-100 nanometers, 20-30 parts by mass of earthworm feces biochar, 10-15 parts by mass of alginic acid oligosaccharide, 2-4 parts by mass of bacillus mucilaginosus powder with the effective viable count of more than or equal to 5 multiplied by 10 10 CFU/g and 1-2 parts by mass of arbuscular mycorrhizal fungus spore agent with the spore content of more than or equal to 1000 are weighed according to the mass part, the attapulgite is baked and activated to form pores, the earthworm feces biochar is soaked in 5% acetic acid solution for 1 hour and then is washed to be neutral by deionized water, and the attapulgite, the bacillus mucilaginosus powder and the arbuscular mycorrhizal fungus spore agent are mixed and stirred to form a mixed material.
7. The method for preventing and controlling sclerotinia rot of colza by antagonizing trichoderma strains according to claim 1, wherein in the preparation of the S300 bio-fertilizer carrier, the volume mass ratio of the trichoderma antagonizing active substance concentrated solution to the mixed material is 1:15, the chitosan oligosaccharide grafted sodium alginate is prepared into an aqueous solution with the mass fraction of 2%, and the mass fraction of the polyglutamic acid solution is 0.5%.
8. The method for antagonizing trichoderma strain to prevent and treat sclerotinia rot of rape according to claim 1, wherein in the S500 growth critical period application, the bio-fertilizer carrier is applied to the root system of rape plant by a strip application mode, wherein the strip application mode is that ditches are formed around the root system of rape plant, the width of the ditches is 3-5cm, the depth is 5-8cm, the ditches are 5-10cm away from the root system of rape plant, the diluted bio-fertilizer carrier is applied into the ditches, and the ditches are covered with soil.

Description

Method for preventing and treating sclerotinia rot of colza by antagonizing trichoderma strain Technical Field The invention relates to the technical field of agricultural disease control, in particular to a method for controlling sclerotinia rot of colza by antagonizing trichoderma strains. Background Rape is one of important oil crops in China, the planting area is wide, the yield is stable, and the method has important significance for guaranteeing the safety of edible oil in China, however, during the growth process of rape, the rape sclerotinia is often affected by various diseases, the sclerotinia is a fungal disease caused by sclerotinia, the yield and the quality of the rape are seriously affected, the sclerotinia not only can cause the leaf and the stalk of the rape plant to be withered and yellow, but also can cause the rape seed to be not full, and the yield is greatly reduced, so that how to effectively prevent the rape sclerotinia and improve the yield and the quality of the rape becomes a problem to be solved urgently in the current agricultural field. In the traditional screening technology of trichoderma strains, although strains with a certain antagonism to sclerotinia sclerotiorum can be separated, the strains often have the defects of unstable antibacterial effect and poor stress resistance, particularly under different environmental conditions, the antagonism of the strains can be obviously changed, so that the control effect is greatly reduced, in addition, the traditional screening method often lacks more defects, the screening of strains with high-efficiency antagonism from a large number of strains is inconvenient, in the aspect of preparation of the biological fertilizer carrier, the carrier material is obviously insufficient in selection and proportion, so that the carrier is unstable in performance, a good growth environment cannot be provided for trichoderma, antagonism of trichoderma and nutrition of the biological fertilizer are difficult to fully exert, and meanwhile, the traditional carrier preparation method omits interaction between the carrier and microorganisms and influence of the carrier on growth and reproduction of microorganisms, so that improvement of biological control effect is limited. Thus, a method for preventing and treating sclerotinia rot of colza by antagonizing trichoderma strains is developed. Disclosure of Invention The invention aims to make up the defects of the prior art and provides a method for preventing and controlling sclerotinia rot of rape by antagonizing trichoderma strains, which can maintain high-efficiency antagonism and strong stress resistance by obtaining the trichoderma strains under different environmental conditions, greatly enhance the stability of the trichoderma strains on disease prevention and control, ensure that the trichoderma strains are prevented and controlled under different environmental conditions in the rape growing process, can effectively resist the invasion of sclerotinia sclerotiorum, reduce the dependence on specific environmental conditions, and prepare a biological fertilizer carrier by extracting antagonistic active substances thereof, and be applied to the planting process of rape, thereby effectively inhibiting the growth and propagation of sclerotinia sclerotiorum, and obviously reducing the morbidity and severity of sclerotinia sclerotiorum. The invention provides a method for preventing and treating sclerotinia rot of colza by antagonizing trichoderma strains, which comprises the following specific steps: S100, separating trichoderma strains from rape rhizosphere soil in different environments, primarily screening out strains with antagonism to sclerotinia by a plate counter method, carrying out chemical mutagenesis treatment on the primarily screened strains, and carrying out counter culture with the sclerotinia again, screening out high-yield antagonistic substances with larger inhibition zone and mutagenized strains with strong stress resistance, and culturing to obtain trichoderma fermentation liquor; S200, extracting active substances, namely centrifuging trichoderma fermentation liquor to obtain supernatant, precipitating by ammonium sulfate, centrifuging, collecting precipitate, dissolving by using sterile water, dialyzing, and removing micromolecular impurities to obtain trichoderma antagonistic active substance concentrate; S300, preparing a biological fertilizer carrier, namely baking attapulgite to activate pores, treating earthworm cast charcoal with acetic acid solution, washing, mixing with alginic acid oligosaccharide, bacillus mucilaginosus powder and arbuscular mycorrhizal fungi spore agent, stirring to form a mixed material, adding trichoderma antagonistic active substance concentrate, uniformly stirring, spraying and mixing with chitosan oligosaccharide grafted sodium alginate aqueous solution to form a gel coating layer, granulating after adjusting the water content, curing, and coating