CN110881480B - Trichoderma harzianum biological agent for preventing and treating wheat scab - Google Patents

Abstract

The invention provides a trichoderma harzianum biological preparation for preventing and treating wheat scab, which is prepared from the following components: 40 to 70 percent of trichoderma harzianum, 3 to 8 percent of cyanesoxim-methyl and 25 to 55 percent of auxiliary material. The Trichoderma harzianum is Trichoderma harzianum BACC 0957.

Description

Trichoderma harzianum biological agent for preventing and treating wheat scab

Technical Field

The invention relates to a trichoderma harzianum biological agent for preventing and treating wheat scab, and belongs to the field of biological agents.

Background

Trichoderma (Trichoderma) is an important group of biocontrol fungi for plant diseases, belonging to Deuteromycotina (Deuteromycetina), Hyphomycetes (Hyphomycetes), Hyphomycetales (Hyphomycetales), and Hyphomycetes (Hyphomycetaceae), and widely distributed in soil, rotten wood and other substrates.

Wheat scab is an important disease in wheat production in China and even in the world. Seedlings can be damaged from the seedling stage to the ear stage, and seedling withering, stem base rot, stem rot and ear rot are mainly caused, wherein the most serious damage is ear rot, and the quality and the yield of wheat are seriously influenced. The wheat scab has great influence on the quality of wheat, and the diseased grains basically lose the seed use and industrial value. The wheat with diseased grains has low flour yield, high ash content, low whiteness and brightness, and low processing precision of the flour. Research shows that the content of pentosan in the diseased wheat flour is increased, the content of crude protein, crude starch, wet gluten and dry gluten is reduced, the quality of gluten is also reduced, the fluidity is strong, the gas-holding performance is poor, the dough is easy to flow and collapse and deform, the dough is difficult to process, the baking quality is poor, and the rheological property of the dough is reduced. Besides, the infected seeds can also produce various toxins such as Deoxynivalenol (DON), Zearalenone (ZEA) and the like, and after people and animals eat the infected seeds, toxic reactions with different degrees, such as skin allergy, gastrointestinal dysfunction, appetite reduction, diarrhea, vomiting, abdominal pain and other symptoms can be caused, the growth is delayed, the immune function of the body is reduced, and the infected seeds also have the effects of carcinogenesis, teratogenesis and mutagenesis, so that the pregnant women can be aborted or even die.

Chemical control is the main way of wheat scab control at present. But biological control is the only way to solve the 3R problem caused by chemical control. With the enhancement of environmental protection consciousness and the attention on green food, the biological control of plant diseases is increasingly becoming a development trend. The biological control of wheat scab has the advantages of no environmental pollution, safety to people and other organisms, lasting control effect, no residue in products, strong killing specificity to germs, easy coordination with other plant protection measures, energy conservation and the like. The promotion of biological control is a main means for controlling wheat scab.

The role of Trichoderma harzianum in biological control has been studied at home and abroad. It has competitive action, heavy parasitic action, antibiotic action, and plant growth regulating action. According to previous researches, the bacterial wilt resistance of cowpea, powdery mildew prevention and control, field tomato gray mold prevention and control and the like are provided, and the bacterial wilt resistance of cowpea is also used for the research of fruit fresh keeping and the like. But the control of wheat scab is not yet involved. Therefore, the search for a good biological control method is the core of wheat scab control.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a trichoderma harzianum biological agent for preventing and treating wheat scab.

As one aspect of the present invention, the present invention provides a trichoderma harzianum biological agent, which is prepared from the following components: 40 to 70 percent of trichoderma harzianum, 3 to 8 percent of cyanesoxim-methyl and 25 to 55 percent of auxiliary material.

Preferably, the preparation is prepared from the following components: 45 to 65 percent of trichoderma harzianum, 3 to 8 percent of cyanesoxim-methyl and 30 to 50 percent of auxiliary materials.

Further preferably, the preparation is prepared from the following components: 50% of trichoderma harzianum, 5% of cyanesoxim-methyl and 45% of auxiliary materials.

In specific embodiments, the trichoderma harzianum can be trichoderma harzianum spore powder, trichoderma harzianum spore powder extract, and a culture medium after trichoderma harzianum cultivation, including a liquid culture medium and a solid culture medium.

In a specific embodiment, the auxiliary materials comprise a carrier, a wetting agent, a dispersing agent, an emulsifying agent, an adsorbent, a regulator and an ultraviolet inhibitor. Specifically, the carrier is diatomite, the wetting agent is nekal BX or sodium dodecyl sulfate K12, the dispersing agent is selected from one or more of nonylphenol polyoxyethylene ether NP-10, nonylphenol polyoxyethylene ether NP-40, sodium methyl dinaphthalene sulfonate NNO, sodium lignosulfonate M11, sodium carboxymethylcellulose CMC-Na and fatty alcohol sulfate, the emulsifier is Tween 20, the adsorbent is activated carbon, and the regulator is potassium dihydrogen phosphate.

In a preferred embodiment, the formulation is made from the following components: 40-70% of trichoderma harzianum spore powder, 3-8% of cyanoenamine, 7-20% of diatomite, 2-15% of nekal BX, 2-8% of sodium methyl dinaphthalenesulfonate NNO, 203-7% of tween, 8-18% of activated carbon, 3-6% of monopotassium phosphate and 0.1-0.3% of ultraviolet inhibitor.

In a most preferred embodiment, the formulation is made from: 50% of trichoderma harzianum spore powder, 5% of cyantraniliprole, 15% of diatomite, 5% of nekal BX, 5% of sodium methyl dinaphthalenesulfonate NNO, 205% of tween, 11.9% of activated carbon, 3% of potassium dihydrogen phosphate and 0.1% of ultraviolet inhibitor.

As another aspect of the present invention, the present invention provides a method for preparing the Trichoderma harzianum biological agent, comprising the steps of:

step 1, mixing spore powder, a carrier, a wetting agent, a dispersing agent, an adsorbent, an emulsifier, a regulator and an ultraviolet inhibitor for later use;

and 2, adding the mixture prepared in the step 1 into a proper amount of water to prepare an aqueous solution, adding the phenamacril, and uniformly mixing to obtain the composition.

As another aspect of the invention, the invention provides application of the Trichoderma harzianum biological agent in preventing and treating wheat scab.

The preferred strain BACC0957 of trichoderma harzianum is preserved in CGMCC (China general microbiological culture Collection center) of China Committee for culture Collection of microorganisms in Beijing in China, and the preservation number is as follows: CGMCC NO: 15981, date of deposit: 6 and 21 months in 2018. The strain is screened from more than 200 plant pathogenic fungi, and the inhibition rate of the strain on fusarium graminearum is 62.97%, which is obviously higher than that of other screened strains.

The trichoderma harzianum biological preparation prepared by mixing trichoderma harzianum and phenamacril has a remarkable effect on preventing and treating wheat scab, the disease ear rate prevention effect reaches 62.67%, and the disease index prevention effect reaches 73.91%.

Detailed Description

Example 1 isolation and screening of Trichoderma harzianum BACC0957

The method for detecting and separating the fungi in the soil is adopted to separate soil samples of various samples, and the separation method refers to a method for detecting and separating the fungi in the soil, page P132 (7) of plant disease research method, which is compiled in the formula. The separation culture medium is as follows: martin's (Martin) medium: 10g of glucose, 5g of peptone and KH₂PO₄ 1g，MgSO₄ 7H₂0.5g of O, 20g of agar, 30.0mg of streptomycin, 1/30000 ml of aqueous solution of rose bengal and 900ml of water. Dissolving the components in water, heating, adding Bengal red at boiling, packaging, sterilizing, and adding streptomycin before pouring into plate. Used for separating fungi from soil.

The method comprises the following steps of measuring the inhibition rate of more than 200 plant pathogenic fungi separated from soil to main pathogen Fusarium graminearum of wheat scab, and measuring the bacteriostasis capacity of Trichoderma harzianum to Fusarium graminearum by adopting a colony confrontation method, wherein the specific method comprises the following steps:

firstly, preparing a PDA flat plate, respectively culturing strains to be detected and fusarium graminearum, after the plate is basically full of the strains, punching holes (equidistant from the edge of the plate) on the positions where the two culture media grow consistently by using a sterilized 4mm puncher.

Secondly, picking the perforated culture medium blocks on the PDA empty plate by using sterilized tweezers respectively, inoculating two kinds of fungi in a reverse mode, enabling the distances to be basically consistent, and measuring the size of the inhibition zone once every 12 hours (a cross method). The bacteriostatic rate was calculated according to the following formula.

Inhibition rate ═ [ (control colony diameter-4) - (treated colony diameter-4) ]/(control colony diameter-4) × 100% note: "4" is the diameter of the colony taken by the punch.

The inhibition of Fusarium graminearum by the different strains is shown in Table 1 (only strains with an inhibition of more than 20% are shown in the table, the inhibition of the remaining strains is less than 20%).

TABLE 1 inhibition of Fusarium graminearum by different strains

Selecting strains (strain)	Inhibition of Fusarium graminearum%
		Trichoderma harzianum (BACC0957)	62.97
Trichoderma 47	55.81
		Trichoderma 84	44.76
Trichoderma 22	31.00
		Trichoderma reesei No. 19	36.91
Trichoderma 76	24.70

As can be seen from Table 1, the highest inhibition rate of the strain BACC0957 on Fusarium graminearum is 62.97%. The PDA plate can show that Trichoderma harzianum BACC0957 shows obvious inhibition effect on fusarium graminearum which is pathogen of gibberellic disease.

The strain is judged to be trichoderma harzianum by a fungus morphology identification method, and the culture characters and morphological characteristics of the strain are as follows:

culturing on potato-glucose agar (PDA) culture medium at 25 deg.C for 3 days, wherein the colony grows over the culture dish, and has round shape, white color, radial shape, rare hyphae, and thicker hyphae at colony edge; culturing for 5 days to generate white granular spore-forming clusters, and generating green spore-forming clusters at the edges of the colonies; after 7 days of culture, radial and green granular spore-forming clusters are formed on the surface of the colony, more spore-forming clusters are formed on the edge, the colony is cushion-shaped, and the back is white. The hyphae are colorless, branched and smooth, have a diaphragm and are 2.5-5.5 mu m wide. Spore cells are produced singly, in pairs or in rounds on conidiophores. The spore-forming cells are flask-shaped, have narrow base parts and wide middle parts, are thinned upwards and are colorless, and the size of the spore-forming cells is 6.5-9.0 multiplied by 2.5-4.0 mu m. Conidiophores are single-grown, nearly spherical to oval, single-grown, green and smooth, and a plurality of spores are gathered into a head shape with the diameter of 2.5-3.5 multiplied by 2.5-3.0 mu m. Has no obvious odor. Chlamydospores are not seen.

Example 2 cultivation of spore powder of Trichoderma harzianum BACC0957

Inoculating the spores on the inclined plane into a shake flask for culture and shake flask culture, wherein the culture solution is 200g of boiled juice of potatoes, adding 2% of glucose, supplementing water to 1L, culturing at 25 ℃ and 180rpm for 7 days. And stopping culturing when the culture solution in the shake flask is sticky and green to dark green.

Then inoculating the seed liquid to a solid culture medium for fermentation for 7d, wherein the fermentation conditions are as follows: the temperature is 25 ℃, and the humidity is 95 percent; the solid state fermentation culture medium comprises: 4.6% of bran, 13.9% of rice hull, 13.9% of corn flour, 4.6% of wood chip and 63.0% of water. Inoculating 20%, culturing in dark at 24 deg.C for 2-3 days, culturing at 25 deg.C for 5-7 days, culturing at 55 deg.C for full spore filament, and sieving with double-layer sieve to obtain spore powder for preparing biological preparation.

The spore powders of Trichoderma harzianum described in examples 3-7 below were all spore powders of Trichoderma harzianum BACC0957 obtained by culturing in example 2.

Example 3

A Trichoderma harzianum biological agent, which is prepared from the following components:

50% of trichoderma harzianum spore powder, 5% of cyantraniliprole, 15% of diatomite, 5% of nekal BX, 5% of different types of dispersants, 205% of tween, 11.9% of activated carbon, 3% of potassium dihydrogen phosphate and 0.1% of ultraviolet inhibitor.

The dispersant types are: nonylphenol polyoxyethylene ether NP-10 (liquid), nonylphenol polyoxyethylene ether NP-40 (crystal), sodium methyl dinaphthalenesulfonate NNO (solid powder), sodium lignosulfonate M11 (solid powder), sodium carboxymethylcellulose CMC-Na and fatty alcohol sulfate (liquid).

Different types of dispersants were investigated as follows:

step a, weighing a proper amount of sample (accurate to 0.0002g), placing the sample in a 250mL beaker containing 50mL of standard hard water (30 +/-1 ℃), and shaking the sample by hand to make circular motion for 2min at about 120 times per minute.

And b, placing the suspension in a water bath at the same temperature for 13min, then completely washing the suspension into a 100mL measuring cylinder by using standard hard water at the temperature of 30 +/-1 ℃, diluting to a scale, covering a plug, and turning the measuring cylinder upside down for 30 times within 1min by taking the bottom of the measuring cylinder as an axis.

And c, opening the plug, vertically placing the plug into a constant-temperature water bath without vibration, and standing for 30 min.

Step d, remove 9/10 (i.e., 90mL) suspension of the contents with a pipette within 10s-15s without shaking or stirring up the sediment in the graduated cylinder. The 10mL suspension remaining at the bottom of the cylinder was washed with a certain amount of distilled water into a 250mL beaker, heated, the water was evaporated to near dryness, oven-dried at 50 ℃ to constant weight, taken out of the beaker and weighed.

Step e, repeat 5 times each, finally calculate the average.

The results are shown in Table 2.

TABLE 2 screening of dispersants in wettable powders

The results show that NNO dissolution rate is highest, so NNO was chosen as the dispersant.

Example 4

A Trichoderma harzianum biological agent, which is prepared from the following components:

50% of trichoderma harzianum spore powder, 5% of cyantraniliprole, 15% of diatomite, different types of wetting agents, 5% of NNO, 205% of tween, 11.9% of activated carbon, 3% of monopotassium phosphate and 0.1% of ultraviolet inhibitor.

The dispersant types are: nekal BX, sodium dodecyl sulfate K12.

Different types of wetting agents were investigated as follows:

step a, adding different wetting agents according to the formula, and selecting a plurality of samples with different concentrations.

And step b, carrying out a wetting experiment on the preparations.

And c, recording the complete wetting time.

The results are shown in Table 3:

TABLE 3 screening of wetting agents in wettable powders

Sample (I)	Mean value of complete wetting time
		BX 3% finished product sample	30min
BX 5% finished product sample	23min
		BX 8% finished product sample	17min
K128% finished product sample	38min
		K1210% sample of finished product	28min
K1215% finished product sample	21min

The results in table 3 show that the wetting agent selected in example 2 has better wetting effect than K12 in terms of the dosage of nekal BX and sodium dodecyl sulfate K12 and the time for complete wetting, i.e., BX is selected as the wetting agent. The wetting times of 5% and 8% of BX addition were not very different, and finally the wetting agent was determined to be BX 5%.

Example 5

A Trichoderma harzianum biological agent, which is prepared from the following components:

45% of trichoderma harzianum spore powder, 3% of cyantraniliprole, 17% of diatomite, 5% of BX, 7% of NNO, 203% of tween, 16.9% of activated carbon, 3% of monopotassium phosphate and 0.1% of ultraviolet inhibitor.

Example 6

A Trichoderma harzianum biological agent, which is prepared from the following components:

60% of trichoderma harzianum spore powder, 6% of cyantraniliprole, 7% of diatomite, 8% of BX, 2% of NNO, 205% of tween, 8.9% of activated carbon, 3% of monopotassium phosphate and 0.1% of ultraviolet inhibitor.

Example 7

A Trichoderma harzianum biological agent, which is prepared from the following components:

55% of trichoderma harzianum spore powder, 15% of diatomite, 5% of nekal BX, 5% of NNO, 205% of tween, 11.9% of activated carbon, 3% of monopotassium phosphate and 0.1% of ultraviolet inhibitor.

Example 8

The biological preparations prepared in the examples 3 and 7 are respectively used for preventing and controlling wheat scab in fields (the biological preparation of the example 3 takes NNO as a dispersant).

The field control experiment is set as follows:

the pesticide is used for preventing and treating the disease in the full-bloom stage (40% -50% of blossoming), and 3 square meters of wheat field is selected for each treatment and the dosage is equal. Each group was dosed as follows:

1. CK, not using medicine, only spraying equal amount of water;

2. the biological preparation prepared in example 7;

3. the biological preparation prepared in example 3.

The results are shown in Table 4.

Table 4 field experimental results

Numbering	1(CK)	2	3
				Ear rate of disease	75.00％	44.00％	28.00％
Index of disease condition	28.75％	15.50％	7.50％
				Preventing effect of ear disease rate		41.33±6.25％	62.67±6.29％
Disease index prevention effect		46.08±8.74％	73.91±9.28％

The results in table 4 show that, in the control of field wheat scab, the trichoderma harzianum and cyanomycobactam biological agent prepared in example 3 can significantly reduce the ear rate and disease index, and the control effect is obviously better than that of trichoderma harzianum used alone.

Claims (8)

1. A Trichoderma harzianum biological agent, which is prepared from the following components: 40% -70% of trichoderma harzianum, 3% -8% of cyanamide and 25% -55% of auxiliary materials; the trichoderma harzianum is spore powder of trichoderma harzianum BACC 0957; the Trichoderma harzianum BACC0957 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC NO: 15981.

2. the biological agent according to claim 1, wherein said agent is prepared from: 45-65% of trichoderma harzianum, 3-8% of cyantraniliprole and 30-50% of auxiliary materials.

3. The biological agent according to claim 2, wherein said agent is prepared from: 50% of trichoderma harzianum, 5% of cyanesoxim-methyl and 45% of auxiliary materials.

4. The biological agent according to any one of claims 1 to 3, wherein the adjuvant comprises a carrier, a wetting agent, a dispersing agent, an emulsifying agent, an adsorbent, a regulator, and an ultraviolet inhibitor.

5. The biological preparation as claimed in claim 4, wherein the carrier is diatomaceous earth, the wetting agent is nekal BX or sodium dodecyl sulfate K12, the dispersing agent is selected from one or more of nonylphenol polyoxyethylene ether NP-10, nonylphenol polyoxyethylene ether NP-40, sodium methyl dinaphthalenesulfonate NNO, sodium lignosulfonate M11, sodium carboxymethylcellulose CMC-Na and fatty alcohol sulfate, the emulsifier is Tween 20, the adsorbent is activated carbon, and the regulator is potassium dihydrogen phosphate.

6. The biological agent according to claim 5, wherein said agent is prepared from: 40-70% of trichoderma harzianum spore powder, 3-8% of cyantraniliprole, 7-20% of kieselguhr, 2-15% of nekal BX, 2-8% of sodium methyl dinaphthalene sulfonate NNO, 203-7% of tween, 8-18% of activated carbon, 3-6% of potassium dihydrogen phosphate and 0.1-0.3% of ultraviolet inhibitor.

7. A method of preparing a biological agent as claimed in claim 4, wherein the method comprises the steps of:

step 1, mixing spore powder, a carrier, a wetting agent, a dispersing agent, an adsorbent, an emulsifier, a regulator and an ultraviolet inhibitor for later use;

and 2, adding the mixture prepared in the step 1 into a proper amount of water to prepare an aqueous solution, adding the phenamacril, and uniformly mixing to obtain the composition.

8. The use of a Trichoderma harzianum biological agent as claimed in any one of claims 1 to 6 for the control of wheat scab.