CN105802859B - Trichoderma pseudokoningii TG-102 and application thereof in prevention and control of Aspergillus flavus bacterial contamination - Google Patents
Trichoderma pseudokoningii TG-102 and application thereof in prevention and control of Aspergillus flavus bacterial contamination Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fertilizers, and particularly discloses a trichoderma koningii strain and application thereof in prevention and control of aflatoxin pollution of corn. The Trichoderma pseudokoningii strain disclosed by the invention is preserved by China center for type culture collection with the preservation number of CCTCC NO: M2016146. The Trichoderma pseudokoningii strain disclosed by the invention has an obvious effect of preventing and treating aflatoxin and toxin pollution infecting corns and peanuts, and has strong antibacterial and detoxifying activities. The invention also provides an organic fertilizer containing the strain, after the organic fertilizer is applied to soil, the strain can be rapidly propagated and survived for a long time at the rhizosphere of crops to form dominant flora, the application of the microbial organic fertilizer can prevent and control the pollution of aflatoxin to agricultural products, can reduce the residue of chemical pesticides, and has wide prospect in modern agricultural planting and agricultural product quality safety application.
Description
Technical Field
The invention relates to the technical field of microbial fertilizers, and particularly discloses a trichoderma pseudokoningii strain, a bacterium-containing organic fertilizer and application of the trichoderma pseudokoningii strain and the bacterium-containing organic fertilizer in prevention and control of aspergillus flavus bacterial pollution of corn.
Background
Corn is an important food crop in the world and also an important feed raw material. At present, mycotoxin is inevitably generated in the storage process of corn, and the problem of pollution of mycotoxin, particularly aflatoxin, to grains and feeds such as corn is a worldwide problem. Aflatoxins (AF) are secondary metabolites produced by fungi such as aspergillus flavus and aspergillus parasiticus, and are a class of mycotoxins with similar physicochemical properties and structures. Aflatoxin contamination causes severe losses to the production of corn, peanuts and other crops, and annual economic losses can reach billions of dollars. Aflatoxin is a strong carcinogenic and virulent mycotoxin which pollutes agricultural products widely, wherein B1 is the most common and the strongest toxicity, and human or animals can cause pathological changes and even death of organisms after eating the agricultural products polluted by the aflatoxin, thereby seriously threatening the health and life safety of consumers. Therefore, the prevention and control of aflatoxin pollution in agricultural products such as corn, peanut and the like are not slow.
The infection of the aspergillus flavus on the corn mainly occurs in the field growth period, and the toxin generation and pollution are mainly in the storage period, so that how to prevent and control the infection of the aspergillus flavus on the corn in the field is very important. At present, no ideal measures are available for controlling aspergillus flavus infection in the growth period of corn, and chemical control is mainly adopted. Because the chemical prevention and control cost is high, the environment is easily polluted, and the pathogens easily generate drug resistance and even drug resistance to the bactericide, the search for a prevention and control measure which is healthy to human bodies and friendly to the environment is imperative. Researches at home and abroad find that a plurality of microorganisms in nature can inhibit and degrade aflatoxin in corns or feeds in the growth period and the storage period, and the microorganisms comprise bacteria, saccharomycetes, moulds, actinomycetes, algae and the like. The microbial preparation or enzymes generated by the microbial preparation are used for inhibiting bacteria and reducing toxicity, so that the ecological environment is not damaged, the quality of agricultural products is not reduced, and the nutritional value of the product can be increased by some varieties.
The method is characterized in that aflatoxin and toxin are taken as research objects, endophytic bacteria are separated from healthy corn grains, antagonistic bacteria are screened by a living method, the antibacterial activity of the antagonistic bacteria is researched, and a new way is expected to be explored for solving the bottleneck problem of prevention and control of corn aflatoxin pollution.
Disclosure of Invention
The invention aims to provide trichoderma koningii TG-102 (Trichoderma koningi TG-102) with strong bacteriostatic and detoxifying activities.
The invention also aims to provide the application of the strain in preventing and controlling the pollution of aspergillus flavus to corn, the preparation of the microbial organic fertilizer for preventing and controlling the pollution of aspergillus flavus to corn by fermentation and the fermentation preparation method thereof.
The purpose of the invention is realized by the following technical scheme:
an antibacterial and detoxicating Trichoderma pseudokoningii TG-102 with preservation number of CCTCC NO: M2016146, which is preserved by China center for type culture Collection with preservation date of 2016, 3 and 28 days.
Selecting province healthy corn kernels such as Hebei, Shandong, Shanxi and Jilin, soaking the weighed corn kernels in a sterile triangular flask with 3% sodium hypochlorite solution for 10 minutes, finally washing with sterile water for 5 times, and draining off water. The corn kernel is clamped by tweezers, the kernel is averagely divided into 3 equal parts by sterile scissors and is placed in a slant culture medium, the kernel is cultured at the constant temperature of 28 ℃, the growth condition of bacterial colonies is observed every day, single bacterial colonies with the shape similar to trichoderma are picked and transferred to a slant plate for purification culture, and the mirror identification is preliminarily identified as the trichoderma and then numbered and stored on the slant.
Slant culture medium: 200g of potato, 15g of glucose, 20g of agar and 1000 ml of distilled water. Sterilizing at 121 deg.C for 20 min.
Morphological characteristics:
the diameter of a bacterial colony on a slant culture medium for 5 days at 20 ℃ is 9.0 cm, the growth speed is high, the bacterial colony is in a spider silk shape to a wool shape and is white, the surface is green due to spore production under scattered light, the back is colorless, and the bacterial colony is slightly green due to large spore production in the later period; the multi-branched tree-like conidiophores form rather loose flora; the main branches are 4-5 mu m wide, and the side branches are more, so that a pyramid is formed; the lateral phialides can reach 5 and are arranged in a quasi-wheel shape or are irregularly arranged along the small lateral branch; the lateral bottle stalk is slightly constricted at the base part, the middle part is expanded and gradually narrowed upwards to form the bottle stalk of 5-7 multiplied by 3-3.5 mu m; the terminal and atypical phialides are relatively long and slender, 13-18 x 2.5-3.5 mu m; the phialides are mostly grown on the carriers at a large angle and sometimes slightly bent to the top end; the phialides are spherical conidium heads, the conidium is nearly spherical or inverted egg-shaped, the wall is smooth and light green, and the grain size is 2.8-3.2 multiplied by 2.5-2.8 mu m. The results are shown in FIGS. 1 and 2.
The strain was identified as Trichoderma pseudokoningii TG-102 by the 16S rDNA method (the 16S rDNA is shown in SEQ ID N0: 1).
The invention also provides the application of the strain with the preservation number of CCTCC NO: M2016146 in preventing and controlling the pollution of aspergillus flavus to corn.
The invention also provides application of the strain with the preservation number of CCTCC NO: M2016146 in preparation of the microbial organic fertilizer for preventing and controlling aspergillus flavus pollution to corn.
The microbial organic fertilizer for preventing and controlling the pollution of aspergillus flavus to corn is prepared by fermenting a strain with a preservation number of CCTCC NO: M2016146.
The invention also provides a microbial organic fertilizer, wherein the content of the strain with the preservation number of CCTCC NO: M2016146 in the organic fertilizer is 1.5 multiplied by 108More than cfu/g, and the organic matter content is 38-45 percent.
The invention also provides a method for preparing the microbial organic fertilizer by fermenting the strain with the preservation number of CCTCC NO: M2016146, which comprises the following steps:
(1) slant culture: inoculating the strain with the preservation number of CCTCC NO: M2016146 into slant culture medium, and culturing at 25-28 deg.C for 5-7 days;
(2) liquid culture in eggplant bottles: inoculating the trichoderma pseudokoningii after slant culture into a liquid culture medium, and culturing for 3-5 days in an incubator at the temperature of 25-28 ℃.
(3) And (3) medium-sized fermentation tank amplification liquid culture: and (3) flushing the trichoderma pseudokoningii in the step (2) with sterile water, inoculating the trichoderma pseudokoningii into a medium-sized fermentation tank, and keeping the dissolved oxygen concentration of about 20% in the fermentation process at the temperature of 28-30 ℃, the stirring speed of 200-250r/min and the ventilation volume of 10-15L/min.
(4) Solid culture: inoculating the liquid strain obtained in the step (3) into a solid culture medium according to the inoculation amount of 10% of the volume ratio, transferring the solid culture medium into a culture room for culture after inoculation, controlling the temperature of the culture room at 28-31 ℃, the temperature of the culture medium at 25-29 ℃, controlling the relative humidity of air at 95-98%, controlling the thickness of the culture medium at 5-7cm, and culturing for 5-7 days.
(5) And (3) drying: after the solid fermentation is finished, the organic fertilizer is naturally dried until the water content is controlled to be 7-10%, and the Trichoderma pseudokoningii TG-102 solid culture can be obtained.
(6) Performing solid fermentation on a mixture of trichoderma pseudokoningii, pre-decomposed cassava residues and livestock and poultry manure, adjusting the water content, adding 20 kg of trichoderma pseudokoningii TG-102 solid culture into each ton of solid organic materials, performing turning after fermenting for two days, performing turning every day, and finishing the fermentation after 5-7 days; drying at 60 deg.C or below to make its water content less than 30%, and packaging.
The preferable technical scheme of the fermentation method of the microbial organic fertilizer provided by the invention is as follows:
the formula of the slant culture medium in the step (1) is as follows: 200g of potato, 15g of glucose, 20g of agar and 1000 ml of distilled water.
The formula of the liquid culture medium in the step (2) is as follows: wheat bran 2.2 wt%, glucose 1.0 wt%, magnesium sulfate 0.45 wt%, potassium dihydrogen phosphate 0.35 wt%, calcium chloride 0.3 wt%, and water in the balance, and has pH 6.5-6.8.
The liquid culture medium in step (2) is sterilized at 115-125 deg.C for 25-35 min, preferably at 121 deg.C under humid heat for 30 min.
The formula of the solid culture medium in the step (4) is as follows: the weight ratio of wheat bran to wheat straw powder is 8: 2, sterilizing at 121 ℃ for 30min with water content of 70 percent and pH of 6.5.
The invention also provides application of the microbial organic fertilizer in preventing and controlling aspergillus flavus on corn.
Compared with the prior art, the invention adopting the technical scheme has the following beneficial technical effects:
1. m2016146 Trichoderma pseudokoningii TG-102 with a preservation number of CCTCC NO is obtained by separating from Zhengshan rice and Zhengshan rice grain, and is found by indoor antagonism experiment, living body bacteriostasis and detoxification experiment and field control experiment to have strong bacteriostatic ability, good and stable bacteriostasis and detoxification effect, and easy culture.
2. The invention also provides an organic fertilizer containing the strain, after the organic fertilizer is applied to soil, the strain can be rapidly propagated and survived for a long time at the rhizosphere of crops to form dominant flora, the application of the microbial organic fertilizer can prevent and control the pollution of aflatoxin to agricultural products, can reduce the residue of chemical pesticides, and has wide prospect in modern agricultural planting and agricultural product quality safety application.
Strain preservation: the culture is preserved in China center for type culture Collection (CCTCC for short), and the preservation addresses are as follows: wuhan, Wuhan university. M2016146, the classification name of Trichoderma pseudokoningii TG-102, Latin name: trichoderma koningi TG-102. The preservation date is 2016, 3 and 28.
Drawings
FIG. 1 is a morphology of a slant plate culture of Trichoderma pseudokoningii TG-102.
FIG. 2 is a conidiophores and conidiophores of Trichoderma pseudokoningii TG-102.
FIG. 3 is a graph showing the inhibitory effect of Trichoderma pseudokoningii TG-102 on Aspergillus flavus on maize.
FIG. 4 is a graph of the inhibitory effect of Trichoderma pseudokoningii TG-102 metabolites on Aspergillus flavus on plates.
FIG. 5 is a graph showing the effect of Trichoderma pseudokoningii TG-102 on soil microflora.
Detailed Description
The invention is further described with reference to specific examples, but the methods and technical parameters involved in the schemes are not to be construed as limiting the invention.
Example 1
Isolation and identification of the Strain having the accession number CCTCC NO: M2016146
Selecting province healthy corn kernels such as Hebei, Shandong, Shanxi and Jilin, soaking the weighed corn kernels in a sterile triangular flask with 3% sodium hypochlorite solution for 10 minutes, finally washing with sterile water for 5 times, and draining off water. The corn kernel is clamped by tweezers, the kernel is averagely divided into 3 equal parts by sterile scissors and is placed in a slant culture medium, the kernel is cultured at the constant temperature of 28 ℃, the growth condition of bacterial colonies is observed every day, single bacterial colonies with the shape similar to trichoderma are picked and transferred to a slant plate for purification culture, and the mirror identification is preliminarily identified as the trichoderma and then numbered and stored on the slant.
Slant culture medium: 200g of potato, 15g of glucose, 20g of agar and 1000 ml of distilled water. Sterilizing at 121 deg.C for 20 min.
Morphological characteristics:
the diameter of a bacterial colony on a slant culture medium for 5 days at 20 ℃ is 9.0 cm, the growth speed is high, the bacterial colony is in a spider silk shape to a wool shape and is white, the surface is green due to spore production under scattered light, the back is colorless, and the bacterial colony is slightly green due to large spore production in the later period; the multi-branched tree-like conidiophores form rather loose flora; the main branches are 4-5 mu m wide, and the side branches are more, so that a pyramid is formed; the lateral phialides can reach 5 and are arranged in a quasi-wheel shape or are irregularly arranged along the small lateral branch; the lateral bottle stalk is slightly constricted at the base part, the middle part is expanded and gradually narrowed upwards to form the bottle stalk of 5-7 multiplied by 3-3.5 mu m; the terminal and atypical phialides are relatively long and slender, 13-18 x 2.5-3.5 mu m; the phialides are mostly grown on the carriers at a large angle and sometimes slightly bent to the top end; the phialides are spherical conidium heads, the conidium is nearly spherical or inverted egg-shaped, the wall is smooth and light green, and the grain size is 2.8-3.2 multiplied by 2.5-2.8 mu m. The results are shown in FIGS. 1 and 2.
The strain was identified as Trichoderma pseudokoningii TG-102 by the 16S rDNA method (the 16S rDNA is shown in SEQ ID N0: 1).
The strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2016146, and is classified and named as Trichoderma pseudokoningii TG-102 with the preservation date of 2016, 3 and 28 days.
Example 2
Experiment on inhibition effect of strain with preservation number of CCTCC NO: M2016146 on maize aspergillus flavus
(1) The inhibition effect of trichoderma pseudokoningii TG-102 on pathogenic bacteria is as follows: inoculating prepared Trichoderma pseudokoningii TG-102 cake and pathogenic bacteria cake at 3cm distance on the slant plate culture medium, culturing in opposite directions, and culturing in 25 deg.C dark culture box with the bacteria cake inoculated with each test bacteria as control. Each treatment was set to 3 replicates. And 3d, measuring the colony diameter, and calculating the inhibition rate of the test strain on the growth of pathogenic bacteria. In 350 strains tested, the strain TG-102 has the strongest inhibition effect on aspergillus flavus, and Table 1 indicates that the applied 10 strains have better inhibition rate on aspergillus flavus.
TABLE 1 inhibition ratio (%) of 10 strains of Trichoderma pseudokoningii TG-102 and the like to Aspergillus flavus of maize
Note: the experimental data in the table are the average of three replicates.
(2) Indoor living body biocontrol effect experiment of trichoderma pseudokoningii TG-102 strain
The trichoderma spores in the eggplant bottles are washed down by sterile water and inoculated into a medium-sized fermentation tank. The fermentation process maintains the dissolved oxygen concentration at about 20%, the temperature at 28-30 deg.C, the stirring speed at 200-250r/min, and the ventilation amount at 10-15L/min.
Inoculating the fermentation liquor into a solid culture medium according to the inoculation amount of 10% of the volume, and transferring the solid culture medium into a culture room for culture after the inoculation is finished. The temperature of the culture room is controlled at 28-31 deg.C, the temperature of the culture medium is controlled at 25-29 deg.C, the relative humidity of air is controlled at 90-95%, the thickness of the culture medium is 5-7cm, and the culture time is 5-7 days.
And after the culture is finished, naturally drying the culture in the air to obtain the zymophyte agent of the antagonistic strain.
The culture conditions of the incubator are as follows: the temperature is 28 ℃, the humidity is 70-80%, and the illumination is 12 h of darkness/12 h of illumination. The control was blank with 3 replicates per treatment. The control effect and the toxicity inhibition rate of the biocontrol bacteria on the aspergillus flavus are calculated and shown in the table 2 and the figure 3.
The result shows that the obtained biocontrol bacteria have good disease prevention effect on corn grains and have high aflatoxin pollution inhibition rate. The disease prevention effect of the antagonistic strain on the aspergillus flavus is respectively 44.2% -87.0%, wherein the control effect of the strain TG-102 on the aspergillus flavus is the highest and reaches 87.0%, and the control effects of the strain T12-1 and the strain T40-3 are respectively 69.5% and 68.8%.
The strains with better poison inhibition effect are TG-102, T21-2 and T40-3 respectively, the inhibition rates are 85.0%, 75.2% and 68.5% respectively, and the strain has further research value.
TABLE 2 preventive and therapeutic effects and toxicity inhibiting rates of antagonistic bacteria on Aspergillus flavus
Note: data are mean of triplicates
Example 3
Microbial organic fertilizer prepared by fermenting strain with preservation number of CCTCC NO: M2016146 and preparation method thereof
The microbial organic fertilizer for preventing and controlling the corn pollution caused by field aspergillus flavus is prepared by fermenting a strain with the preservation number of CCTCC NO: M2016146, wherein the content of the strain with the preservation number of CCTCC NO: M2016146 in the organic fertilizer is 1.5 multiplied by 108More than cfu/g, and the organic matter content is 38-45 percent.
The method for preparing the microbial organic fertilizer by fermenting the strain with the preservation number of CCTCC NO: M2016146 comprises the following steps:
(1) slant culture: the strain with the preservation number of CCTCC NO: M2016146 is inoculated in a slant culture medium and cultured for 6 days at 25-28 ℃.
The formula of the slant culture medium is as follows: 200g of potato, 15g of glucose, 20g of agar and 1000 ml of distilled water.
(2) Liquid culture in eggplant bottles: inoculating the trichoderma pseudokoningii after slant culture into a liquid culture medium, and culturing for 4 days in an incubator at the temperature of 25-28 ℃.
The formula of the liquid culture medium is as follows: 2.2% of wheat bran, 1.0% of glucose, 0.45% of magnesium sulfate, 0.35% of monopotassium phosphate, 0.3% of calcium chloride and the balance of water, wherein the pH value is 6.7.
The liquid medium was sterilized at 121 ℃ for 30min under moist heat.
(3) And (3) medium-sized fermentation tank amplification liquid culture: flushing the trichoderma pseudokoningii in the step (2) with sterile water, inoculating the trichoderma pseudokoningii into a medium-sized fermentation tank, and keeping the dissolved oxygen concentration of about 20% in the fermentation process at the temperature of 28-30 ℃, the stirring speed of 200-250r/min and the ventilation volume of 10-15L/min;
(4) solid culture: inoculating the liquid strain obtained in the step (3) into a solid culture medium according to the inoculation amount of 10% of the volume ratio, transferring the solid culture medium into a culture room for culture after inoculation, controlling the temperature of the culture room at 28-31 ℃, the temperature of the culture medium at 25-29 ℃, controlling the relative humidity of air at 95-98%, controlling the thickness of the culture medium at 5-7cm, and culturing for 6 days.
The formula of the solid culture medium is as follows: the weight ratio of wheat bran to wheat straw powder is 8: 2, sterilizing at 121 ℃ for 30min with water content of 70 percent and pH of 6.5.
(5) And (3) drying: after the solid fermentation is finished, the organic fertilizer is naturally dried until the water content is controlled to be 7-10%, and the Trichoderma pseudokoningii TG-102 solid culture can be obtained.
(6) Performing solid fermentation on the trichoderma pseudokoningii TG-102 and a mixture of pre-decomposed cassava residues and livestock and poultry manure, adjusting the water content, adding 20 kg of trichoderma pseudokoningii TG-102 solid culture into each ton of solid organic materials, turning the materials after fermenting for two days, turning the piles every day, and finishing the fermentation after 6 days; the content of Trichoderma pseudokoningii TG-102 in the solid fermentation microbial organic fertilizer reaches 5 multiplied by 108More than cfu/g, and the organic matter content is 38-45 percent. Drying at 60 deg.C or below to water content of 30% or below, and packagingAnd (6) packaging to obtain the finished product.
Example 4
Experiment on inhibition effect of microbial organic fertilizer prepared by fermenting strain with preservation number of CCTCC NO: M2016146 on corn aspergillus flavus
The field test plot is randomly designed and repeated for 3 times, and the area of each plot is 15m multiplied by 3 m. The biocontrol agent is added in an amount of 4 kilograms per mu (1.5 multiplied by 10)6Spore g-1) When the corn is sowed, a chemical control method (0.5 g of 70% thiophanate methyl wettable powder per plant) and a control are set along with fertilizer application, and the application method of the pesticide is the same as a biocontrol preparation. After harvesting maize, 5 spots of 10 plants per spot were randomly investigated for each treatment. The antagonistic bacteria toxicity inhibiting rate and the growth promoting effect on the corn are calculated, and the results are shown in table 3.
The result shows that the obtained biocontrol trichoderma has a certain growth promoting effect on the growth of corns and has a high aflatoxin pollution inhibition rate. The growth promoting effect of the better several antagonistic bacteria on the aspergillus flavus is 0.8-10.5%, wherein the growth promoting effect of the strain TG-102 is the highest and is 10.5%, and the growth promoting effects of the strains T95-2 and T40-3 are 5.5% and 4.5% respectively. The strains with better poison inhibiting effect are TG-102, T40-3 and T85-5 respectively, and the inhibition rates are 90.5%, 60.5% and 58.3% respectively.
TABLE 3 antagonistic bacteria inhibiting rate of Aspergillus flavus production and promoting corn growth
| Survey index | TG-102 | T95-2 | T85-5 | T12-1 | T21-2 | T40-3 | T100 | T22-1 |
| The growth promoting rate% | 10.5 | 5.5 | 0.0 | 2.5 | 2.7 | 4.5 | 0.0 | 0.8 |
| Toxicity inhibiting rate/%) | 90.5 | 50.5 | 58.3 | 50.8 | 53.2 | 60.5 | 23.5 | 45.0 |
Determination of bacteriostasis rate of Trichoderma pseudokoningii TG-102 metabolite
Preparation of biocontrol bacteria metabolite
Activating biocontrol bacterium trichoderma pseudokoningii TG-102 on a slant, putting a loop in a slant culture solution, loading the solution in 100mL/300 mL, carrying out shake culture at 30 ℃ and 160 r/min for 72h, and preparing the following treatment solutions: supernatant fluid: the culture was centrifuged at 12000 r/min for 15min, and the supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain a sterile supernatant.
Bacterial suspension: centrifuging the culture solution at 12000 r/min for 15min, discarding the supernatant, washing with sterile water for 3 times, centrifuging, and adding sterile water.
Crude protein extraction: the culture solution is separated at 12000 r/min at 4 ℃ for 15min, the precipitate is discarded, solid ammonium sulfate is added into the supernatant to 70 percent of saturation, the mixture is kept stand at 4 ℃ overnight, the mixture is centrifuged at 10000 r/min at 4 ℃ for 20min, the supernatant is discarded, the precipitate is suspended by 1/25 volume of 10 mmol/L phosphate buffer solution with the pH value of 7.0, and then the bacterial filter with the diameter of 0.22 mu m is used for filtering to remove the possibly existing bacteria.
Influence of biocontrol bacteria metabolite on growth of aspergillus flavus hyphae
The determination method comprises the following steps: taking Aspergillus flavus bacterial suspension of 1.5X 1065mL, placing the mixture into a triangular flask (100 mL/350 mL) with a slope, wherein the temperature is reduced to about 45 ℃, shaking for 2min, and uniformly pouring the mixture into a culture dish. Punching 3 holes at equal intervals around the inclined plane plate by using a puncher, respectively adding 3 mL of supernatant, filtrate, frozen supernatant, frozen filtrate and crude protein extract by using a pipette gun, culturing at 30 ℃ for 5 days, detecting the radius of each treatment inhibition zone when CK grows over the plate, and repeating each treatment for three times. The results are shown in Table 4 and FIG. 4.
The result shows that the biocontrol strain culture solution has 100 percent of inhibition effect on the growth of aspergillus flavus hyphae; the inhibition effect of the crude protein extract is also strong and reaches more than 95 percent; the biocontrol bacteria filtering liquid and the freeze-filtration bacteria inhibiting rate are both over 30 percent, wherein the TG-102 bacteria inhibiting rate is the strongest and respectively reaches 60.5 percent and 58.5 percent, and then T85-5 reaches 57.3 percent and 55.5 percent; the bacteriostatic rates of the biocontrol bacteria supernatant and the frozen supernatant are more than 45%, wherein the bacteriostatic rate of TG-102 is the strongest and respectively reaches 65.0% and 68.0%, and T85-5 is the strongest and respectively reaches 58.5% and 63.2%, which is shown in Table 4.
TABLE 4 inhibitory Effect of biocontrol bacteria metabolites on Aspergillus flavus hypha growth
Root colonization experiment of trichoderma pseudokoningii TG-102
The corn seeds are subjected to indoor germination acceleration and are selected to have consistent growth vigorThe germination seeds of (1) were sown 10 seeds per pot and repeated five times. Inoculating the strain after the corn seedlings emerge. The spore suspension concentration of Trichoderma pseudokoningii TG-102 is 1.5 × 106Per mL, the spore suspension was injected into the rhizosphere soil with a syringe, 15mL per hole, with clear water as a control. The first sample was collected 1 day after irrigation and every 7 days thereafter for 4 weeks. Accurately weighing 1 g of soil sample to be detected, placing the soil sample into a test tube filled with 9ml of sterile water, carrying out vortex oscillation for 3min to fully disperse microorganisms in the soil, and standing for 1min to obtain 10-1Diluent, use 10-3And 10-4Coating the diluted solution on a TSM culture medium, culturing for 72h at 28 ℃, and measuring Trichoderma pseudokoningii TG-102 in the rhizosphere soil of the corn plants, wherein the measured Trichoderma pseudokoningii TG-102 is used as an index of the rhizosphere colonization capacity of Trichoderma fungi. The rhizosphere soil of maize plants not inoculated with trichoderma was used as a control during counting. The results are shown in Table 5.
Trichoderma semi-selective medium (TSM medium): MgSO (MgSO)4(7H20) 0.2 g，K2HPO40.9 g， KCl0.15 g，NH4NO31.0 g, 3.0 g of glucose, 0.15 g of rose bengal, 0.3 g of 60% dixon wettable powder, 0.2g of PCNB0, 20g of agar and 950ml of distilled water. Sterilizing at 20 deg.C for 121 min.
TABLE 5 rhizosphere colonization ability of Trichoderma TG-102 in peanut plants (. times.10)x/g)
| Treatment of | 7 |
14 days | 28 days |
| TG-102 | 2×105 | 4×104 | 5×103 |
| TG-102- |
3×105 | 2.8×104 | 2×103 |
Note: the experimental data in the table are the average of three replicates.
Influence of biocontrol bacteria on corn rhizosphere microflora
The PCR-DGGE technology is mainly used for researching the diversity and population difference of microorganisms, and the method discloses DNA sequence genetic polymorphism based on a 16SrDNA gene sequence and is used for deducing the phylogenetic relationship of the microorganisms. The experiment researches the influence of the biocontrol strain TG-102 on the microbial diversity of the corn rhizosphere soil, and the result is shown in figure 5.
Those skilled in the art can implement the present invention in various embodiments without departing from the spirit and scope of the present invention, and the above description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention, and all equivalent variations made by the present specification and drawings are included in the scope of the present invention.
SEQUENCE LISTING
<110> Tangshan city livestock and poultry aquatic product quality monitoring center
<120> Trichoderma pseudokoningii TG-102 and application thereof in prevention and control of aflatoxin contamination of maize
<130>1
<160>1
<170>PatentIn version 3.3
<210>1
<211>574
<212>DNA
<213>Trichoderoma koningiiTG-102
<400>1
ccaaacccaa tgtgaacgtt accaaactgt tgcctcggcg ggatctctgc cccgggtgcg 60
tcgcagcccc ggaccaaggc gcccgccgga ggaccaacca aaactcttat tgtatacccc 120
ctcgcgggtt ttttttataa tctgagcctt ctcggcgcct ctcgtaggcg tttcgaaaat 180
gaatcaaaac tttcaacaac ggatctcttg gttctggcat cgatgaagaa cgcagcgaaa 240
tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga acgcacattg 300
cgcccgccag tattctggcg ggcatgcctg tccgagcgtc atttcaaccc tcgaacccct 360
ccggggggtc ggcgttgggg atcggccctg ccttggcggt ggccgtctcc gaaatacagt 420
ggcggtctcg ccgcagcctc tcctgcgcag tagtttgcac actcgcatcg ggagcgcggc 480
gcgtccacag ccgttaaaca cccaacttct gaaatgttga cctcggatca ggtaggaata 540
cccgctgaac ttaagcatat caataagcgg agga 574
Claims (10)
1. An antibacterial and detoxicating Trichoderma pseudokoningii TG-102 (Trichoderma koningi TG-102) is characterized in that the preservation number is CCTCC NO: M2016146, and the preservation date is 2016, 3 and 28 days.
2. The use of the strain having the accession number CCTCC NO: M2016146 of claim 1 for controlling Aspergillus flavus contamination of corn.
3. The use of the strain of claim 1, having a accession number of CCTCC NO: M2016146, in the preparation of a microbial organic fertilizer for preventing and controlling Aspergillus flavus contamination to corn.
4. A microbial organic fertilizer is characterized in that the content of a strain with the preservation number of CCTCC NO: M2016146 in the organic fertilizer is 1.5 multiplied by 108More than cfu/g, haveThe content of organic substances is 38-45%.
5. The fermentation method of the microbial organic fertilizer of claim 4, comprising the following steps:
(1) slant culture: inoculating the strain with the preservation number of CCTCC NO: M2016146 into slant culture medium, and culturing at 25-28 deg.C for 5-7 days;
(2) liquid culture in eggplant bottles: inoculating the trichoderma pseudokoningii after slant culture into a liquid culture medium, and culturing for 3-5 days in an incubator at the temperature of 25-28 ℃;
(3) and (3) medium-sized fermentation tank amplification liquid culture: flushing down the trichoderma pseudokoningii in the step (2) by using sterile water, inoculating the trichoderma pseudokoningii into a medium-sized fermentation tank, and keeping the dissolved oxygen concentration at 18-22% in the fermentation process, the temperature at 28-30 ℃, the stirring speed at 200-250r/min and the ventilation volume at 10-15L/min;
(4) solid culture: inoculating the liquid strain obtained in the step (3) into a solid culture medium according to the inoculation amount of 10% of the volume ratio, transferring the solid culture medium into a culture room for culture after inoculation, wherein the temperature of the culture room is controlled to be 28-31 ℃, the temperature of the culture medium is controlled to be 25-29 ℃, the relative humidity of air is controlled to be 95-98%, the thickness of the culture medium is 5-7cm, and the culture time is 5-7 days;
(5) and (3) drying: after the solid fermentation is finished, naturally drying the organic fertilizer until the water content is controlled to be 7-10%, and obtaining the Trichoderma pseudokoningii TG-102 solid culture;
(6) performing solid fermentation on a mixture of trichoderma pseudokoningii, pre-decomposed cassava residues and livestock and poultry manure, adjusting the water content, adding 20 kg of trichoderma pseudokoningii TG-102 solid culture into each ton of solid organic materials, performing turning after fermenting for two days, performing turning every day, and finishing the fermentation after 5-7 days; drying at 60 deg.C or below to make its water content less than 30%, and packaging.
6. The fermentation process of claim 5, wherein the slant medium formulation in step (1) is: 200g of potato, 15g of glucose, 20g of agar and 1000 ml of distilled water.
7. The fermentation process of claim 5, wherein the liquid medium formulation in step (2) is: 2.2 percent of wheat bran, 1.0 percent of glucose, 0.45 percent of magnesium sulfate, 0.35 percent of monopotassium phosphate, 0.3 percent of calcium chloride and the balance of water, wherein the PH value is 6.5-6.8.
8. The fermentation method according to claim 5 or 7, wherein the liquid medium is sterilized at 115-125 ℃ for 25-35 min in step (2).
9. The fermentation process of claim 5, wherein the solid medium formulation in step (4) is: the weight ratio of wheat bran to wheat straw powder is 8: 2, sterilizing at 121 ℃ for 30min with water content of 70 percent and pH of 6.5.
10. The application of the microbial organic fertilizer as claimed in claim 4 in preventing and treating aspergillus flavus on corn.
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