Application of trichoderma nigrum in straw returning to field

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of Trichoderma nigricans T32781 in straw returning to the field. The strain T32781 disclosed by the invention can be massively propagated in farmland soil, and straw decomposition can be accelerated by adopting the strain T32781 disclosed by the invention, so that the morbidity of wheat stem rot (soil-borne germs) after straw returning to the field is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Trichoderma nigricans T32781 in returning straw to fields. Background Art

[0002] Returning straw to the field is currently the most important way to utilize straw resources. It can effectively increase soil carbon and fertilize the soil, but there are also a series of environmental problems in the production area. Returning straw to the field can provide a favorable living place for soil-borne pathogens and pests, thereby increasing the risk of pests and diseases. Generally speaking, the incidence of soil-borne diseases such as wheat stem base rot has increased after returning straw to the field, affecting the normal growth of crops. When the amount of straw returned to the field is too high, a large amount of straw cannot be decomposed in time, which can provide a carrier for the reproduction of soil-borne pathogens, while creating a suitable growth environment such as water and temperature, aggravating the occurrence of diseases. Therefore, screening microorganisms with cellulose and lignin degradation properties to accelerate straw decomposition, reduce the reproduction carriers of soil-borne pathogens, and reduce the occurrence of diseases is an urgent problem that needs to be solved in this industry.

[0003] Cadmium (Cd) is a heavy metal element with high biotoxicity. Cadmium in the soil is easily absorbed by plants and then transferred to the human body through the food chain, affecting human health. Therefore, how to limit the entry of cadmium in the soil into the food chain and reduce the accumulation of cadmium in the edible parts of crops is also one of the urgent problems to be solved. Screening low-cadmium accumulation crop varieties and applying physical and chemical regulation techniques to reduce the absorption and accumulation of cadmium in crops are one of the ways to achieve efficient and sustainable production.

[0004] Trichoderma is an important type of biocontrol fungus. With its low cost, high efficiency and environmental protection characteristics, it has become a highly respected biological control agent and is used to control plant diseases, promote plant growth, improve soil fertility and soil bioremediation.

[0005] In the field of biological control of various plant fungal diseases, the application of Trichoderma is particularly common. In the biological control of plant diseases, Trichoderma relies on competition, parasitism, antibiotics, and induced resistance to achieve control of plant diseases. Competition refers to the competition for space, nutrition, oxygen, etc. between two or more microorganisms existing in the same microhabitat. Among them, spatial competition is the competition of biocontrol bacteria for the surface space or internal space of the plant, especially the site where pathogens invade the plant. The pre-colonization of biocontrol bacteria on the plant surface prevents the colonization of pathogens; nutritional competition is the competition between biocontrol bacteria and pathogens for limited available materials such as plant secretions, plant residues, and root shedding, including carbohydrates, amino acids, inorganic salts, vitamins, and secondary metabolites necessary for the growth and development of microorganisms. Competition is one of the important biocontrol mechanisms of Trichoderma. Trichoderma grows and reproduces rapidly, and can decompose cellulose, lignin, chitin, etc., occupying the surface space or internal space of plants, blocking the living space and infection and colonization sites of harmful pathogens, thereby effectively reducing the infection of plant pathogens. In addition, iron, as a cofactor of many enzymes, is of indispensable importance for the normal growth and development of plants and microorganisms. However, under aerobic conditions, iron tends to form insoluble iron oxide, which makes it impossible to be effectively utilized. As a low molecular weight chelating agent, iron carriers show a very high affinity for iron. They can bind to insoluble iron (Fe 3+ ) and converts it into soluble Fe 2+ , which is convenient for plants and microorganisms to absorb and utilize. In the competition between Trichoderma and pathogens, siderophores play a vital role. Trichoderma that can secrete siderophores can not only improve the availability of iron to plants, but also the siderophores of Trichoderma effectively consume the available iron sources in the soil, thereby effectively inhibiting the growth of pathogens. Antibiotic effect refers to the phenomenon that the metabolites produced by one microorganism have a direct inhibitory effect on another microorganism, so that the growth of the other microbial population is inhibited, while the microorganism itself is not affected. Antibiotic effect is one of the important biocontrol mechanisms of Trichoderma. Trichoderma inhibits the growth and reproduction of pathogenic fungi by producing metabolites with antibacterial activity, thereby reducing the occurrence of diseases.

[0006] The invention of CN116376714A, "A Trichoderma for Alleviating Tobacco Cadmium Toxicity and Its Application", discloses the Trichoderma nigricans strain T32781 (CGMCC NO.40314) and its application --- for alleviating tobacco cadmium toxicity. After inoculation with T32781, plants can reduce the absorption of cadmium from the soil, thereby alleviating the toxic effects of heavy metal cadmium pollution in the soil on plant growth. However, no other uses are disclosed.

[0007] Most existing Trichoderma fungi only have the function of cellulose, for example, the invention of CN109112072A "Application of Trichoderma fungi in promoting plant growth": the Trichoderma fungi are Trichoderma asperellum, strain number: HTTA-Z0002, deposit number CGMCC No.15677. The HTTA-Z0002 strain can degrade phosphorus, potassium and cellulose, produce plant hormones and iron carriers, and promote plant growth in many aspects, and can induce plant resistance to stress and salt. It is a strain with multifunctional effects on plant growth and can reduce the use of chemical fertilizers. The patent does not inform that HTTA-Z0002 has the function of degrading lignin. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a new use of Trichoderma nigricans strain T32781, namely, application in returning straw to fields.

[0009] In order to solve the above technical problems, the present invention provides a new use of Trichoderma nigricans strain T32781 (with a deposit number of CGMCC NO.40314): application in returning straw to fields.

[0010] As an improvement on the use of the strain T32781 of the present invention: reducing the incidence of wheat stem base rot after returning straw to the field.

[0011] As a further improvement on the use of the strain T32781 of the present invention: it has cellulose degradation activity and lignin degradation activity, so it can be used for straw decomposition; that is, it accelerates straw decomposition, thereby reducing the incidence of wheat stem base rot (soil-borne pathogens) after straw is returned to the field.

[0012] As a further improvement of the use of the strain T32781 of the present invention: inhibiting plant pathogenic fungi and preventing and controlling plant diseases.

[0013] As a further improvement of the use of the strain T32781 of the present invention: plant pathogenic fungi include Botrytis cinerea, Alternaria angustiovoidea, Botrytis cinerea, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani, and Sclerotinia sclerotiorum.

[0014] As a further improvement of the use of the strain T32781 of the present invention: reducing the cadmium content in cereal grains.

[0015] As a further improvement of the use of the strain T32781 of the present invention: promoting the growth of crops.

[0016] The strain T32781 of the present invention has the following properties that conventional Trichoderma does not have:

[0017] 1. Degrade cellulose;

[0018] 2. Produce iron carriers;

[0019] 3. Used to prevent and control soil-borne wheat stem rot;

[0020] 4. It has antibacterial activity against plant pathogenic fungi such as Alternaria sp., Botrytis aclada, Botrytis cinerea, Fusarium graminearum, Plectosphaerella cucumerina, Sclerotinia sclerotiorum, Rhizoctonia solani, and Pythium sp., which belong to the oomycete.

[0021] 6. The bacterial strain of the present invention can be reproduced in large quantities in farmland soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 This is a diagram showing the hydrolysis transparent circle formed by Trichoderma nigricans strain T32781 on sodium carboxymethyl cellulose medium.

[0024] Figure 2 This is the filter paper enzyme activity curve of T32781.

[0025] Figure 3 This is the laccase activity graph of T32781 cultured on Trichoderma guaiac-PDA.

[0026] Figure 4 The figure shows the formation of hydroxamate siderophore by T32781 in CAS medium.

[0027] Figure 5 This is a diagram for determining the repopulation activity of T32781 in plate confrontation culture.

[0028] Figure 6 This is a diagram for determining the antibacterial activity of T32781 using the membrane filter method.

[0029] Figure 7 The effect of T32781 inoculation on rhizosphere microbial communities in the field and potted plants;

[0030] A is the relative abundance (%) of the top 10 genera with the largest number of rhizospheric fungi;

[0031] B shows the down-regulation and up-regulation of rhizosphere microorganisms by field inoculation T32781 at the genus level. After t-test, the identified fungi with Log2(FC)|>1 and P value <0.05 were marked with colors, with down-regulation marked in blue and up-regulation marked in red;

[0032] C shows the down-regulation and up-regulation of rhizosphere microorganisms by potted plants inoculated with T32781 at the genus level. After t-test, the identified fungi with Log2(FC)|>1 and P value <0.05 were marked with colors, with down-regulation marked in blue and up-regulation marked in red.

[0033] Figure 8 The effect of T32781 inoculation on agronomic traits and Cd concentrations of barley plants in the field and pots is shown;

[0034] A shows the effects of pot inoculation with T32781 on agronomic traits and Cd concentrations of barley at heading stage;

[0035] B shows the effects of pot inoculation with T32781 on agronomic traits and Cd concentrations of barley at maturity;

[0036] C is the effect of inoculation of T32781 on agronomic traits and Cd concentration of barley at heading stage;

[0037] D shows the effects of inoculation of T32781 in the field on agronomic traits and Cd concentrations of barley at maturity. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] In the present invention, the preparation method of the spore suspension is:

[0040] The strain T32781 was activated and cultured on a PDA medium at 26-28°C for 7 days, the spores were washed with sterile water and the concentration was adjusted with sterile water to prepare a spore suspension of corresponding concentration.

[0041] Spore content 2×10 8 The method for preparing the fermentation agent of Trichoderma T32781 of 100 g / g is as follows: rice husk: rice bran = 1:9 (w:w) is used as a culture medium, and Trichoderma T32781 is cultured at 28° C. for 7 days.

[0042] For Alternaria angustiovoidea, CPCC 810078 of China Pharmaceutical Microorganism Culture Collection can be used; for Botrytis aclada, CCTCC HF 2008152 of China Type Culture Collection can be used; for Plectosphaerella cucumerina, CGMCC 3.17050 of China General Microorganism Culture Collection can be used; for Botrytis cinerea, Fusarium graminearum, Rhizoctonia solani and Sclerotinia sclerotiorum, ACCC 36027, ACCC 39294, ACCC 36246 and ACCC 39269 of China Agricultural Microorganism Culture Collection can be used respectively.

[0043] The Pythium aristosporum can be selected from ACCC 39248 of China Agricultural Microbiological Culture Collection Center.

[0044] Example 1. Determination of cellulose degradation activity

[0045] 1. Screening of cellulose degradation activity

[0046] Carboxymethyl cellulose sodium (CMC-Na) culture medium: 10 g of sodium carboxymethyl cellulose, 5 g of glucose, 2.0 g of ammonium sulfate, 1.0 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 0.01 g of ferrous sulfate heptahydrate, 15 g of agar, 1000 ml of distilled water, sterilize at 121°C for 15 min.

[0047] Take a 5mm Trichoderma T32781 cake cultured in PDA medium at 28℃ for 2 days, inoculate it in a CMC-Na medium plate, culture it in the dark at 28℃ for 48h, record the colony diameter, stain it with 1 mg / mL Congo red dye for 2h, discard the dye, and then wash it with 1 MNaOH solution. Observe and record the diameter of the hydrolysis transparent zone. Each treatment is repeated 3 times. The activity of the strain in degrading cellulose is qualitatively evaluated based on the ratio of the hydrolysis transparent zone diameter to the colony diameter. The hydrolysis transparent zone diameter of Trichoderma T32781 on CMC-Na medium is 64.8mm, the colony diameter is 64.0mm, and the ratio of the hydrolysis transparent zone diameter to the colony diameter is about 1.0, indicating that Trichoderma T32781 has cellulose degradation activity ( Figure 1 ).

[0048] 2. Cellulase activity assay

[0049] Filter paper culture medium ( / L): qualitative filter paper 10g, (NH 4 ) 2 SO 4 2.0g, K 2 HPO 4 1.0 g, MgSO 4 7H 2 O 0.5 g, KCl 0.5 g, FeSO 4 7H 2 O 0.01g, natural pH, distilled water to volume. Conventional sterilization.

[0050] The cellulase activity was determined by filter paper enzyme (FPase) activity. The strain T32781 was activated and cultured on PDA medium at 28°C for 7 days, and the spores were washed and resuspended with sterile water to prepare 1×10 5 / mL spore suspension. Inoculate 2ml of spore suspension into a 500mL Erlenmeyer flask containing 200mL filter paper culture medium, culture at 28℃ and 200rpm with shaking, take 1mL of fermentation liquid on the 6th, 8th, 10th, 13th and 15th day of culture, centrifuge at 14,000rpm and 4℃ for 10min, the supernatant is the crude enzyme solution, which is used for cellulase activity determination.

[0051] The measurement steps are as follows:

[0052] (1) Preparation of 3,5-dinitrosalicylic acid (DNS) reagent

[0053] Solution A: Dissolve 6.9 g of crystalline phenol in 15.2 mL of 10% NaOH solution, dilute to 69 mL with distilled water, and add 6.9 g of sodium bisulfite to the solution.

[0054] Solution B: Dissolve 255 g of sodium potassium tartrate in 300 mL of 10% NaOH solution, and then add 880 mL of 1% 3,5-dinitrosalicylic acid solution.

[0055] Mix all the A and B solutions to obtain a yellow reagent, which should be stored in a brown bottle for 7-10 days before use.

[0056] (2) Preparation of glucose standard curve: Take 12 25mL volumetric flasks and add 0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL, 1.6mL, 1.8mL, and 2.0mL of 1.0mg / mL glucose standard solution, add distilled water to 2.0mL, add 1.5mL of DNS reagent, mix well and immediately boil in water bath for 5min, take out and immediately rinse with running water, cool to room temperature, dilute to the scale with distilled water, shake well, measure the absorbance at 520nm, and draw a standard curve with mg of glucose as the ordinate and the light absorption value as the abscissa.

[0057]

[0058] (3) Determination of FPase activity: In a 25 mL volumetric flask, add 0.5 mL of appropriately diluted crude enzyme solution, 1 mL of acetic acid-sodium acetate buffer solution (0.2 M, pH 4.8), and then add 50 mg of filter paper disc. Incubate in a 50°C water bath for 60 min. Immediately after taking out, add 1.5 mL of DNS reagent. Incubate in a boiling water bath for 5 min. Immediately after taking out, cool to room temperature with running water. Add distilled water to make up to 25 mL. Shake well. Measure the absorbance A520 with a spectrophotometer. s . Replace the enzyme solution to be tested with the inactivated enzyme solution at 100℃ for 10 minutes, and operate according to the above method as a blank control to measure the absorbance A520 ck .

[0059] ΔA520=A520 s -A520 ck

[0060] At 50°C and pH 4.8, 1 mL of enzyme solution hydrolyzes filter paper for 1 min to produce 1 μg of glucose, which is one unit of enzyme activity, expressed as U / mL.

[0061]

[0062] U: enzyme activity (U / mL); P: glucose content (mg) obtained from the standard curve obtained in step (2); K: enzyme solution dilution factor; T: constant temperature water bath time; 0.5: volume of crude enzyme solution added to the reaction solution.

[0063] The results of the filter paper enzyme activity assay showed that the filter paper enzyme activities of T32781 cultured on filter paper medium for 6, 8, 10, 13 and 15 days were 4.1990U / mL, 4.5053U / mL, 16.2733U / mL, 16.2733U / mL, 15.1177U / mL and 15.2715U / mL, respectively. Figure 2 ).

[0064] Example 2: Determination of lignin degradation activity

[0065] 1. Screening of lignin degradation activity

[0066] Guaiacol-PDA medium ( / L): 0.04% filter-sterilized guaiacol was added to PDA medium.

[0067] Laccases were used to determine the lignin degradation activity: a 5 mm cake of Trichoderma T32781 cultured in PDA medium at 28°C for 2 days was inoculated on a guaiacol-PDA medium plate and cultured in the dark at 28°C for 5 days. The generation of red-brown color change circles around the colonies and the size of the color change circles were observed and recorded. Each treatment was repeated 3 times. The laccase activity of the strain was qualitatively evaluated based on the ratio of the red-brown color change circle diameter to the colony diameter ( Figure 3 ).

[0068] 2. Laccase activity assay

[0069] Laccase induction medium ( / L): 28g PDB powder (Hangzhou Microbiological Reagent Co., Ltd.), add 800mL water to dissolve, adjust pH to 4.8 with 0.2mol / L acetic acid solution, add water to 1000mL. Dispense into 50mL Erlenmeyer flasks, 30mL per bottle, add 0.45g rice husk powder per bottle. Conventional sterilization.

[0070] The strain T32781 was activated and cultured on PDA medium at 26°C for 7 days, and the spores were washed and resuspended with sterile water to prepare the required spore suspension. 1×10 7 1 mL of spore suspension with spores / mL was cultured at 26°C and 150 rpm with shaking. On the 8th day of fermentation, 1 mL of fermentation broth was taken and centrifuged at 14,000 rpm and 4°C for 10 min. The supernatant was the crude enzyme solution, which was used for laccase activity determination.

[0071] The laccase activity was determined using the 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS) method. The assay steps are as follows:

[0072] (1) 100 mM malonic acid-sodium malonate buffer (pH 4.5), 0.6 mM ABTS solution, and crude enzyme solution in a 30°C water bath for 5 min;

[0073] (2) In a 4-mL cuvette (optical path 1 cm), add 1.6 mL of 100 mM malonic acid-sodium malonate buffer (pH 4.5) and 0.2 mL of sample solution (crude enzyme solution) as a test tube, mix well, and equilibrate at 30°C until the absorbance value of the spectrophotometer stabilizes.

[0074] A blank tube was prepared by adding 1.8 mL of 100 mM malonic acid-sodium malonate buffer (pH 4.5) without adding sample solution (crude enzyme solution).

[0075] (3) Add 0.2 mL of 0.6 mM ABTS solution to the test tube and the blank tube respectively, mix well, and record the absorbance value every 30 seconds for 10 minutes. Calculate the absorbance change per minute (A 420 / min).

[0076]

[0077] Laccase activity is defined as the oxidation of 1 nmol of substrate ABTS (ε 420 =36000(mol / L) -1 cm -1 )The amount of enzyme required is one enzyme activity unit (U / mL).

[0078]

[0079] A 420 / min: absorbance change; d: cuvette light path (1cm); ε 420 :Molar extinction coefficient (=36000(mol / L) -1 cm -1 );V 1 : total reaction volume (2 mL); V 2 : Sample solution (crude enzyme solution) volume (0.2 mL).

[0080] Laccase decomposes the substrate ABTS to produce blue-green ABTS + Free radicals, ABTS + The molar absorption coefficient of free radicals at 420 nm (ε 420 ) is 36000 (mol / L) -1 cm -1 .

[0081] The results of laccase activity assay showed that the laccase activity of T32781 cultured in laccase induction medium for 8 days was 1.444 U / mL.

[0082] Example 3: Determination of siderophore activity

[0083] CAS dye solution: add 100mL ferric chloride solution to 500mL CAS solution and shake well to mix, slowly add the mixed solution to 400mL HDTMA solution along the wall of the beaker, gently shake to mix the solution, and you can get a blue CAS dye solution. CAS solution: Chrome azurol S (CAS) 0.7g, deionized water 500mL. Ferric chloride solution: ferric chloride 0.270g, concentrated hydrochloric acid (12M) 0.83mL, deionized water 1000mL. Hexadecyltrimethyl ammonium Bromide (HDTMA) solution: HDTMA 1.5g, deionized water 1000mL.

[0084] CAS medium: Take 100 mL of 20% sucrose solution, 30 mL of 10% (mass%) acid hydrolyzed casein, 1 mL of 1 mM calcium chloride, 200 mL of 1 mM magnesium sulfate heptahydrate solution, and 18 g of agar, add deionized water to 900 mL, and sterilize at 121°C for 15 min. When the CAS medium is cooled to about 60°C, slowly add 50 mL each of the separately sterilized 0.1 M phosphate buffer and CAS dye solution, avoid generating bubbles, mix well, and pour the melted medium into a 90 mm diameter plate to obtain a blue CAS medium plate.

[0085] LNM culture medium: 2 mL of 1 mM magnesium sulfate heptahydrate, 0.1 mL of 1 mM calcium chloride, 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolyzed casein, and 0.1 M phosphate buffer to make up to 100 mL.

[0086] Take a 5mm Trichoderma T32781 cake cultured in PDA medium at 25℃ for 2 days, inoculate it in a CAS medium plate, and culture it at 25℃ for 5 days to observe whether it produces a color change circle to determine whether it produces siderophores. The color of the CAS medium plate changes from blue to orange or yellow, indicating the production of hydroxamic acid siderophores, and from blue to purple-red, indicating the production of catechol siderophores; measure the colony diameter and the color change circle diameter, calculate the ratio of the color change circle diameter to the colony diameter, and qualitatively evaluate the siderophore production activity of the strain according to the ratio of the color change circle diameter to the colony diameter. Repeat each treatment 3 times. Trichoderma T32781 changes the CAS medium from blue to purple-red, indicating that it can produce catechol siderophores; the colony diameter of Trichoderma T32781 on the CAS medium plate is 60mm, the color change circle diameter is 67mm, and the ratio of the color change circle diameter to the colony diameter is 1.1( Figure 4 ).

[0087] Take a 5mm Trichoderma T32781 cake cultured in PDA medium at 25℃ for 2 days, inoculate it in LNM medium, and culture it at 25℃ and 120r / min for 6 days. Take samples on the 4th, 5th and 6th days, centrifuge at 10000r / min for 10min, aspirate the supernatant, add an equal volume of CAS dye, mix evenly, keep it in the dark for more than 1h, and use a spectrophotometer to measure the absorbance (As) at 680nm. Add an equal volume of liquid culture medium to which uninoculated PDA medium cakes are added and CAS dye is mixed, and its absorbance is used as the reference value (Am). Each treatment is repeated 3 times. Siderophore production is expressed as the relative content of siderophore activity units (SU), SU (%) = [(Am-As) / Am] × 100%. The content of Trichoderma T32781 on the 4th, 5th and 6th days was 51.59%, 44.80% and 46.07%, respectively.

[0088] Example 4: Determination of parasitism ability by plate confrontation culture

[0089] The antifungal activity of Trichoderma sp. T32781 against pathogenic fungi and pathogenic oomycetes was determined by plate confrontation culture, with Alternaria angustiovoidea, Botrytis aclada, Botrytis cinerea, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani and Sclerotinia sclerotiorum as target pathogenic fungi. 5 mm Trichoderma T32781 cake and target pathogen cake cultured on PDA medium at 25°C for 5 days were taken, and inoculated on the edges of both sides of the PDA medium plate respectively, and cultured at 25°C in light and dark (light:dark=12h:12h, light intensity 15000Lux). The pathogen cake inoculated alone was used as the control. Each treatment was repeated 3 times. After 10 days, the contact between the pathogen and Trichoderma on the front of the culture plate was observed. The grading standard is as follows: 45 Trichoderma hyphae completely covered the pathogen; 30 Trichoderma hyphae moderately covered the pathogen; 15 Trichoderma hyphae weakly covered the pathogen; 0 Trichoderma and pathogen hyphae did not cover each other. The confrontation culture experiment found that the hyphae coverage of Trichoderma T32781 on Botrytis cinerea was 15, and the hyphae coverage on Alternaria angustiovoidea, Botrytis aclada, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani and Sclerotinia sclerotiorum was 45, indicating that Trichoderma T32781 has good parasitic ability against pathogenic fungi ( Figure 5 ).

[0090] Example 5: Determination of antibacterial activity by cellophane method

[0091] The antibacterial activity of the metabolites produced by Trichoderma T32781 against Botrytis aclada, Botrytis cinerea, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani, Sclerotiniasclerotiorum and Ustilaginoidea virens were determined by the cellophane method.

[0092] Cut the cellophane into 9 cm pieces and soak them in sterile water, sterilize them at 121°C for 15 min, and spread a double layer of cellophane on a 9 cm diameter PDA culture medium plate.

[0093] A 5 mm cake of Trichoderma T32781 cultured in PDA medium at 25℃ for 2 days was pre-inoculated on a double layer of glass paper on a PDA medium plate, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 2 days. The glass paper and the Trichoderma growing on it were removed with sterile tweezers (the metabolites produced by Trichoderma can penetrate into the culture medium through the glass paper, but Trichoderma itself cannot penetrate into the culture medium). Then a 5 mm cake of the target pathogenic fungus was inoculated into the center of the PDA medium plate; the target pathogenic fungus not pre-inoculated with Trichoderma was used as the control, and cultured at 25℃ with light and dark (light: dark = 12h: 12h, light intensity 15000Lux) for 5 days, and each treatment was repeated 3 times. The colony diameter of the pathogen was determined by the cross method, and the inhibition rate of Trichoderma against the pathogenic fungus was calculated.

[0094] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0095] The cellophane method showed that the growth inhibition rates of Trichoderma T32781 against Botrytis aclada, Botrytis cinerea, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Ustilaginoidea virens were 80.0%, 33.3%, 83.3%, 30.0%, 100%, 100%, 79.8% and 100%, respectively, indicating that Trichoderma T32781 has good antibacterial activity against pathogenic fungi ( Figure 6 ).

[0096] Example 6: Potted plant experiment to determine the effect on cucumber growth

[0097] The effect of Trichoderma T32781 on cucumber growth was determined using potted vegetable soil. Each tissue culture bottle contained: 100 g of vegetable soil, 50 g of substrate (peat: vermiculite = 3:1, w:w), and a large amount of water-soluble compound fertilizer (N:P 2 O 5 :K 2 O+TE, 20:20:20) 400 mg, water 60 mL. Each tissue culture bottle was inoculated with 1×10 7 1mL of Trichoderma spore suspension with spores / mL was stirred evenly with a triangular stick, and no Trichoderma inoculation was used as a control. The cucumber seeds were disinfected with 75% alcohol and 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 3 to 4 times, placed in a clean bench to air dry, and the cucumber seeds were placed in a 1 / 5MS medium (a medium in which the content of all components in the MS medium was reduced to 1 / 5 of the original content) plate, and germinated in the dark at 28°C until 70% of the cucumber seeds germinated. The cucumber seeds with consistent germination were sown in the above-mentioned tissue culture bottle garden soil, with 1 cucumber seed per bottle. The plant height, stem thickness and dry weight of the cucumber seedlings were measured on the 14th day after sowing, and each treatment was repeated 3 times. Calculate the growth potential, growth potential = (stem thickness / plant height) × dry weight. The results showed that the growth vigor of cucumber seedlings treated with CK was about 0.12 (stem diameter was about 2.4 mm, plant height was about 8.4 cm, and dry weight was about 0.42 g), and the growth vigor of cucumber seedlings treated with Trichoderma T32781 was about 0.183 (stem diameter was 3.63 mm, plant height was 8.53 cm, and dry weight was 0.429 g), which was about 53% higher than the control, significantly promoting the growth of cucumber plants.

[0098] Example 7: Effects on the microbial community structure of barley rhizosphere soil and cadmium content in barley grains in cadmium-contaminated soil

[0099] Pot experiments and field experiments were conducted to determine the effects of Trichoderma T32781 on the structure of barley rhizosphere microbial community in cadmium-contaminated soil and the cadmium concentration in barley grains.

[0100] Pot experiments with artificial cadmium-contaminated soil:

[0101] Uncontaminated silt loam (total Cd content 0.12 mg kg -1 , the effective Cd content is 0.03 mg kg -1 ) The topsoil was air-dried and passed through a 2 mm sieve. A large plastic bucket (63 cm long, 36 cm wide, 25 cm high) was filled with 35 kg of soil (the amount of soil for each potted plant). CdCl was added to the uncontaminated silt loam soil. 2 To a concentration of 10 mg kg -1 CdCl 2 , the soil must be artificially polluted.

[0102] The experiment included three treatments: 1) CK, barley variety “Hua 30” was planted in uncontaminated soil; 2) Cd, 10 mg kg - 1 CdCl 2 Barley cultivated in artificially contaminated soil; 3) Cd+T, 10 mg kg -1 CdCl 2 Artificially contaminated soil and inoculated with T32781 (5×10 6 Each treatment was carried out with 60 barley plants, divided into 3 replicates. Samples were taken at the heading stage and the maturity stage to detect the structure of the rhizosphere soil microbial community.

[0103] Field trials in cadmium-contaminated rice fields:

[0104] The effect of inoculant T32781 on improving barley tolerance to Cd and reducing Cd content in grains in Cd-contaminated soil was studied under open field conditions in Hangzhou, China. -1 , the effective Cd content is 1.00 mg kg -1 A completely randomized experimental design was used, with two treatments set up in four replicate plots (each replicate plot had an area of ​​4 square meters):

[0105] Mock: no application of Trichoderma as a control;

[0106] T: Apply 2×10 per mu of land 8 T32781 microbial agent 1.5kg,

[0107] There were 10 rows per 2 square meters plot, with 100 barley seeds per row. The field experiment followed the local traditional farming method. Samples were taken at the heading and maturity stages to test the structure of the rhizosphere soil microbial community.

[0108] Calculate the response:

[0109] Response (%) = (characteristic +T -Characteristics -T ) / Characteristics -T ×100%

[0110] Among them, the characteristics +T is the value of T32781 in cadmium-contaminated soil (potted plants are treated with "Cd+T" and fields are treated with "T"). -T are the values ​​of cadmium-contaminated soil without using T32781 (potted plants are treated with “Cd” and fields are treated with “Mock”).

[0111] The results are as follows:

[0112] The ITS high-throughput sequencing technology was used to determine the effect of Trichoderma T32781 on the microbial community structure of the rhizosphere soil during the heading period of barley. After the application of T32781, Trichoderma showed a significant advantage in the rhizosphere soil, accounting for 42% in the field test and 94% in the pot test ( Figure 7 A). Nineteen microbial genera showed significant changes in the field experiment, of which 15 were down-regulated (including Fusarium, Cladosporium, etc.) and 4 were up-regulated (Trichoderma, Pyrenochaetopsis, Microbotryomycetes gen_Incertae_sedis and Rozellomycota_gen_Microbotryomycetes gen_Incertae sedis) ( Figure 7 B). In the pot experiment, 18 microbial genera were significantly down-regulated (Fusarium, Neocosmospora, Bisifusarium, Paraphoma, Fusicolla, Neopyrenochaeta) and 2 were significantly up-regulated (Trichoderma and Meyerozyma) ( Figure 7C). Fusarium, Cladosporium, Neocosmospora, Bisifusarium, Paraphoma, Fusicolla, Neopyrenochaeta, etc. are typical pathogenic fungi. Therefore, the application of T32781 significantly changed the composition of rhizosphere fungal community and reduced the abundance of pathogenic fungi in soil.

[0113] Pot experiments showed that in artificial Cd-contaminated soil, the number of effective panicles and grain yield of the plant inoculated with T32781 ("Cd+T" treatment) increased by 85% and 74.12%, respectively, and the Cd concentration in the grains and the effective Cd concentration in the soil decreased by 21.31% and 45.05%, respectively. Figure 8 A, B).

[0114] Field experiments showed that in Cd-contaminated soil, inoculation of T32781 ("T" treatment) significantly increased spike length, pedicel length and grain traits, and grain yield increased by 35.89% compared with the control; it also significantly reduced the Cd concentration in barley grains and the concentration of available Cd in soil, by 10.47% and 8.58%, respectively ( Figure 8 C, D).

[0115] These results showed that under potted and field conditions, inoculation with T32781 significantly changed the composition of rhizosphere fungal communities and reduced the abundance of pathogenic fungi in the soil; significantly increased the number of effective ears and grain yield of barley, and reduced the Cd concentration in grains and the effective Cd concentration in soil.

[0116] Example 8: Field test to determine the effect of normal farmland soil conditions on cadmium accumulation in crop seeds

[0117] The field experiment was conducted to determine the effect of Trichoderma T32781 on cadmium accumulation in crop grains under normal farmland soil conditions. Zhongjin brand rice seedling medium (Hangzhou Jinhai Agricultural Technology Co., Ltd.) was used for seedling transplanting, and Trichoderma T32781 fermentation agent (spore content 2×10 8 The cadmium content and thousand-grain weight of rice were measured after harvest. The results showed that the Cd content and thousand-grain weight of rice in the control treatment were 0.14 mg / kg and 22.31 g, respectively, while the Cd content and thousand-grain weight of rice in the treatment of Trichoderma T32781 were 0.10 mg / kg and 22.56 g, respectively; the Cd content of rice in the treatment of Trichoderma T32781 was 28.57% lower than that in the control, and reached the national limit standard for cadmium in rice.

[0118] Example 9, application of Trichoderma T32781 in returning straw to fields:

[0119] The amount of straw returned to the field is generally 5-6t·hm -2 .

[0120] Before wheat sowing, evenly spread Trichoderma T32781 inoculant at a dosage of 2×10 8 1.5kg / mu, shallowly applied into the soil in combination with land preparation; the straw decomposition rate of the control group (without spreading Trichoderma T32781) was 66.2%, and the straw decomposition rate of the group treated with Trichoderma T32781 was 74.8%, an increase of 8.6%; the disease index of wheat stem base rot in the control group was 10.3, and the disease index of wheat stem base rot in the group treated with Trichoderma T32781 was 2.0, and the prevention and control effect was 80.58%.

[0121] Straw decomposition rate (Rd, %) = (W1-W2) / W1×100%. In the formula, W1 is the initial straw dry mass (g); W2 is the dry mass of the straw after decomposition (g).

[0122] Wheat stem rot is a fungal disease caused by multiple pathogens such as Fusarium graminearum and F. pseudograminearum. Due to factors such as the increase in pathogens in the soil caused by returning straw to the fields, wheat stem rot is common in my country and is showing a trend of increasing severity and spread.

[0123] Wheat stem rot investigation method: 180 days after sowing, the wheat is graded according to the length of browning at the first internode: Grade 0 internode has no browning, Grade 1 internode has browning length accounting for 0-25%, Grade 2 internode has browning length greater than 25-50%, Grade 3 internode has browning length greater than 50-75% and Grade 4 internode has browning length greater than 75-100%. Calculate the disease index and control effect.

[0124] Disease index: Disease index = (∑(number of diseased plants at each level × grading value) / (total number of surveyed plants × highest disease level) × 100

[0125] Control effect (%) = (disease index of control area - disease index of treated area) / disease index of control area × 100%.

[0126] Hyperparasitism, antibiotics, competition, etc. are the main principles of Trichoderma to achieve plant disease control in plant disease biological control. The above-mentioned Examples 1, 2 and 3 can prove that Trichoderma T32781 of the present invention has spatial competition and nutritional competition; Example 4 can prove that Trichoderma T32781 of the present invention has hyperparasitism; Example 5 can prove that Trichoderma T32781 of the present invention has antibiotics; Example 7 can prove that Trichoderma T32781 of the present invention reduces the abundance of pathogenic fungi including Fusarium in the soil, thereby reducing the occurrence of soil-borne diseases. In addition, since cellulose and lignin are the main components of straw, Examples 1 and 2 can prove that Trichoderma T32781 of the present invention has the degradation effect of cellulose and lignin. Therefore, Trichoderma T32781 of the present invention can accelerate the decomposition of straw, reduce the breeding carriers of soil-borne pathogens, and reduce the occurrence of diseases.

[0127] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Use of Trichoderma nigricans strain T32781, whose deposit number is CGMCC NO.40314; Features: Applied to returning straw to fields.

2. The use of Trichoderma nigricans strain T32781 according to claim 1, Features: Reduce the incidence of soil-borne diseases after returning straw to the fields.

3. The use of Trichoderma nigricans strain T32781 according to claim 2, Features: The soil-borne disease is wheat stem base rot.

4. The use of the Trichoderma nigricans strain T32781 according to any one of claims 1 to 3, Features: It has cellulose degradation activity and lignin degradation activity, and can be used for straw decomposition.

5. Use of the Trichoderma nigricans strain T32781 according to any one of claims 1 to 4, Features: Inhibit plant pathogenic fungi and prevent and control plant diseases.

6. Use of the Trichoderma nigricans strain T32781 according to claim 5, Features: Plant pathogenic fungi include Botrytis cinerea, Alternaria angustiovoidea, Botrytis aclada, Fusarium graminearum, Plectosphaerella cucumerina, Pythium aristosporum, Rhizoctonia solani, and Sclerotinia sclerotiorum.

7. Use of the Trichoderma nigricans strain T32781 according to any one of claims 1 to 6, Features: Reduce the cadmium content in cereal grains.

8. Use of the Trichoderma nigricans strain T32781 according to any one of claims 1 to 6, Features: Promote crop growth.

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