WO2022105128A1 - 一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用 - Google Patents

一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用 Download PDF

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WO2022105128A1
WO2022105128A1 PCT/CN2021/090911 CN2021090911W WO2022105128A1 WO 2022105128 A1 WO2022105128 A1 WO 2022105128A1 CN 2021090911 W CN2021090911 W CN 2021090911W WO 2022105128 A1 WO2022105128 A1 WO 2022105128A1
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straw
parts
trichoderma harzianum
trichoderma
powder
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PCT/CN2021/090911
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French (fr)
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吴晓青
张新建
周方园
赵晓燕
张广志
范素素
王加宁
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山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

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  • the invention relates to the technical field of microorganism application, in particular to a strain of Trichoderma harzianum Ta97 in Africa and its application in the field of straw returning.
  • Crop straw is one of the main solid wastes in agricultural production, with an annual output of about 800 million tons in my country.
  • Straw contains a large amount of organic substances such as fiber, protein and fat, which has the value of resource utilization.
  • resource utilization methods of straw animal feed and composting are more commonly used.
  • composting is the main resource utilization method.
  • composting requires a special composting site, which limits the treatment of straw. Therefore, seeking A more reasonable way of resource utilization is of great significance to straw disposal.
  • the present invention provides a strain of Trichoderma harzianum Ta97 and its application in straw returning, the African Trichoderma harzianum Ta97, the Latin classification of the strain is named: Trichoderma afroharzianum, the strain On July 14, 2020, it was deposited in the China Microbial Culture Collection Management Committee as the General Microbiology Center.
  • the preservation unit is referred to as CGMCC.
  • the address is: No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the Trichoderma harzianum Ta97 has manganese peroxidase, lignin peroxidase, cellulase and hemicellulase activities at the same time.
  • the African Trichoderma harzianum Ta97 was applied to the straw returning to the field.
  • the average weight loss rate of the straw reached 80.40% within 30 days.
  • Level 4 which can fully degrade straws in the field; and obtain fertilizers that are conducive to crop absorption, provide nutrients for the growth of next crops, and achieve the purpose of saving fertilizer costs.
  • Trichoderma harzianum Ta97 An African Trichoderma harzianum Ta97, the Latin classification name of this strain is: Trichoderma afroharzianum, this strain has been deposited in the China Microbial Culture Collection Management Committee on July 14, 2020 as the General Microbiology Center, the preservation unit is referred to as CGMCC, and the address is : Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, preservation number: CGMCC No.19930.
  • tef1 gene sequence of the African Trichoderma harzianum Ta97 is:
  • the Trichoderma harzianum Ta97 has manganese peroxidase, lignin peroxidase, carboxymethyl cellulase and hemicellulase activities simultaneously; wherein, manganese peroxidase and lignin peroxide Enzymes are involved in breaking down lignin, carboxymethyl cellulase is involved in breaking down cellulose, and xylanase is involved in breaking down hemicellulose.
  • the manganese peroxidase activity was 3.52IU/mL
  • the lignin peroxidase activity was 2.43IU/mL
  • the carboxymethyl cellulose activity was 3.76IU/mL
  • the xylanase activity was 61.65IU /mL.
  • the inoculum prepared by Trichoderma harzianum Ta97 specifically Trichoderma harzianum Ta97 wettable powder, in the wettable powder, the number of effective viable bacteria is ⁇ 5 ⁇ 10 9 cfu/g.
  • Trichoderma powder preparation the seed liquid is fermented in a fermentor containing a liquid fermentation medium at a volume percentage of 12% to 22%, and the fermentation temperature is 28 to 30° C. before 12 hours and 24 to 30° C. after 12 hours. 27°C, the initial pH is 3.5 ⁇ 5.5, the stirring speed is 200 ⁇ 300r/min, the ventilation rate is 10 ⁇ 15L/min, the fermentation time is 60 ⁇ 80 hours; the number of viable bacteria in the fermentation product is 1 ⁇ 10 8 ⁇ 1 ⁇ 10 9 cfu/mL; the fermentation broth is freeze-dried by vacuum to obtain Trichoderma powder, and its viable count is ⁇ 1 ⁇ 10 10 cfu/g;
  • Trichoderma powder and auxiliary according to the following parts by weight: 30-50 parts of Trichoderma powder, 30-50 parts of diatomaceous earth, 2-4 parts of wetting agent, 3-6 parts of dispersant, 0.2-0.5 part of adhesive and 1-2 parts of zinc sulfate; mix the above components evenly.
  • the liquid fermentation medium is composed of the following components by weight: 50-100 parts of blasted corn stalk powder, 50-100 parts of blasted wheat stalk powder, and 30-50 parts of wheat bran powder , 10-30 parts of glucose, 0.5-1 part of (NH 4 ) 2 SO 4 , 0.02-0.04 part of KH 2 PO 4 , 0.03-0.05 part of MgSO 4 ⁇ 7H 2 O and 5000-8000 parts of water.
  • the present invention provides a strain of Trichoderma harzianum Ta97, which has four degrading enzymes of manganese peroxidase, lignin peroxidase, carboxymethyl cellulose and xylanase simultaneously.
  • the manganese peroxidase activity was 3.52IU/mL
  • the lignin peroxidase activity was 2.43IU/mL
  • the carboxymethyl cellulase activity was 3.76IU/mL
  • the xylanase activity was 61.65IU/mL .
  • the Trichoderma harzianum Ta97 provided by the present invention has four kinds of enzyme activities at the same time, wherein, manganese peroxidase and lignin peroxidase are involved in decomposing lignin, carboxymethyl cellulase is involved in decomposing cellulose, xylem Carbohydrase is involved in the decomposition of hemicellulose; the four enzyme activities improve the ability of the strain to degrade straw. In the laboratory degradation test of straw, the average weight loss rate of straw within 30 days reached 80.40%.
  • the straw In the field test, the straw It can be fully degraded within 30 days to reach grade 4 of decomposing; it avoids the problem that the complex bacterial strain constructed by using multiple strains in the prior art requires strict fermentation conditions during the fermentation process, and makes the straw returning to the field easier to achieve.
  • the inoculum of Trichoderma harzianum Ta97 provided by the present invention can be used directly when the straw is returned to the field, avoiding the site limitation of the existing compost, and the in-situ returning to the field can also reduce manual handling of the straw, artificial fertilization and returning to the field. It has the advantages of saving labor and cost.
  • Fig. 1 is the colony and microscopic morphological diagram of Trichoderma harzianum Ta97 cultured in PDA plate at 25°C for 7 days, in the figure, A is the front view of the colony, B is the reverse view of the colony, C is the morphological diagram of the conidiophore, D is the morphological diagram of conidia, and E is the morphological diagram of chlamydospore.
  • Fig. 2 is the enzymatic activity assay plate of Trichoderma harzianum Ta97, in the figure, A is the aniline blue plate for screening peroxidase, B is the RB brilliant blue plate for screening peroxidase, and C is the screen for cellulose Enzyme CMC plate, D is the xylan plate for screening hemicellulase.
  • the African Trichoderma harzianum Ta97 of the present invention is isolated and screened from the farmland soil of corn and wheat rotation, and the sampling depth is 20 cm.
  • A is the front view of the colony
  • B is the negative view of the colony
  • C is the morphological map of conidiophore
  • D is the morphological map of conidia
  • E is the morphological map of chlamydospore.
  • the tef1 gene sequence of Ta97 is:
  • Trichoderma harzianum Ta97 obtained in Example 1 was cultivated at a constant temperature of 25°C for 3 days on a PDA plate;
  • 1Screening strains producing peroxidase on aniline blue plate Take the cultured colonies on the PDA plate with a sterile punch, inoculate on the aniline blue plate, prepare 3 replicate plates, and invert at 25 ⁇ 1°C. , recorded on the aniline blue plate with or without decolorization and transparency, the decolorized person is recorded as +, otherwise it is recorded as -. According to the diameter of the transparent area, strains with strong peroxidase production ability were selected, and the time was recorded.
  • Aniline blue solid medium RB brilliant blue was separately filtered to make a 2% stock solution (200 times), which was added to PDA at 0.01% (V/V) under sterile conditions.
  • Trichoderma harzianum Ta97 can decolorize the dye and make it transparent in aniline blue medium, and the diameter of the transparent area is 4.72 cm on the 4th day, as shown in Figure A in Figure 2.
  • 2Screening of peroxidase-producing strains on RB bright blue plate Take the cultured colonies on the PDA plate with a sterile punch, inoculate on the PDA-RB bright blue plate, prepare 3 plates and repeat, at 25 ⁇ 1°C Invert the culture, record whether there is a yellow ring on the PDA-RB bright blue plate, the yellow ring is marked as +, otherwise it is marked as -. According to the size of the yellow circle produced, the strain with strong peroxidase production ability was selected, and the color development time was recorded.
  • PDA-RB Brilliant Blue Solid Medium RB Brilliant Blue was separately filtered to make a 2% stock solution (200 times), and mixed with PDA medium at a ratio of 625 mg/ml under sterile conditions.
  • Trichoderma harzianum Ta97 can produce a yellow ring in the PDA-RB bright blue medium, and the diameter of the yellow ring is 5.68 cm on the 4th day, as shown in Figure B in Figure 2.
  • 3CMC plate to screen cellulase-producing strains Use a sterile punch to take the cultured colonies on the PDA plate, inoculate them on the CMC plate, prepare 3 plates to repeat, and invert at 25 ⁇ 1 °C for 4 days, then Cover the plate with 0.1% Congo red staining solution, let it stand for 30 min, and then decolorize it with 1 mol/L NaCl for 1 h to observe whether each strain produces a hydrolysis transparent circle. According to the diameter of the hydrolysis transparent circle, strains with strong cellulase-producing ability were selected.
  • CMC solid medium CMC 10g, (NH 4 ) 2 SO 4 4g, KH 2 PO 4 2g, MgSO 4 ⁇ 7H 2 O 0.5g, peptone 1.0g, agar 16g, sodium deoxycholate 0.5g, water 1000ml, Autoclave at 121°C for 20min.
  • Trichoderma harzianum Ta97 can produce hydrolyzed transparent circles in CMC medium, and the diameter of the transparent circles is 3.10 cm on the 4th day, as shown in Figure C in Figure 2.
  • Xylan solid medium (NH 4 ) 2 SO 4 2g, MgSO 4 ⁇ 7H 2 O 0.5g, KH 2 PO 4 1g, NaCl 0.5g, xylan 2g, agar 20g, distilled water 1000mL, natural pH, Autoclave at 121°C for 20min.
  • Trichoderma harzianum Ta97 can produce hydrolysis transparent circle in CMC medium, and the diameter of the transparent circle is 8.51cm on the 4th day, as shown in Figure D in Figure 2.
  • Trichoderma harzianum Ta97 has an enzyme system that can degrade lignin, cellulose and hemicellulose at the same time, and all four enzymes in this enzyme system have enzymatic activity.
  • 1Lignin peroxidase activity detection activate Trichoderma harzianum Ta97 on PDA plate, pick Trichoderma harzianum Ta97 on the plate to prepare 1 ⁇ 10 7 /mL conidia suspension, press 1% ( V/V) inoculated in veratrol culture solution (culture solution includes: starch 0.6g, yeast powder 7.5g, wheat straw powder 0.5g, KH 2 PO 4 4g, MgSO 4 7H 2 O 0.244g, CaCl 2 0.066g, Veratrol 2mmol, vitamin B1 2mg, trace element solution 40mL, 0.15% Tween-80, distilled water 1000mL), cultivated for 5 days under the conditions of 25 ⁇ 1°C and 160rpm to obtain fermentation broth; Centrifuge at 12000rpm for 20min, and the supernatant is the crude enzyme solution.
  • culture solution includes: starch 0.6g, yeast powder 7.5g, wheat straw powder 0.5g, KH 2 PO 4 4g, MgSO 4 7H 2 O 0.2
  • Oxidation of veratrol to produce 1 ⁇ moL of veratrol in 1 min is 1 unit of enzyme activity. Taking the sterilized fermentation broth as a control, three replicates were set. After testing, the lignin peroxidase activity of Trichoderma harzianum Ta97 was 2.43IU/mL.
  • Manganese peroxidase activity detection activate the Ta97 strain on the PDA plate, prepare a conidia suspension of 1 ⁇ 10 7 /mL, and inoculate it into the veratrol medium at 1% (V/V). Cultivate at 25 ⁇ 1°C and 160 rpm for 5 days to obtain a fermentation broth; centrifuge the fermentation broth at 4°C and 12000 rpm for 20 min, and the supernatant is the crude enzyme liquid.
  • Carboxymethyl cellulase activity detection activate Trichoderma harzianum Ta97 on PDA plate, prepare 1 ⁇ 10 7 /mL conidia suspension, and inoculate it in CMC culture medium at 1% (V/V) (CMC solid medium minus agar), cultured at 25 ⁇ 1° C. and 160 rpm for 7 days to obtain fermentation broth. The fermentation broth was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was the crude enzyme solution.
  • V/V CMC solid medium minus agar
  • xylanase activity activate Trichoderma harzianum Ta97 on PDA plate, prepare 1 ⁇ 107/mL conidia suspension, and inoculate it in xylan culture medium at 1% (V/V) (The xylan solid medium is obtained by subtracting the agar), and cultured at 25 ⁇ 1° C. and 160 rpm for 7 days to obtain a fermentation broth. Pipette 0.5 mL of the 20-fold diluted fermentation broth, add it to a 1% xylan solution prepared with disodium hydrogen phosphate-lemon buffer (pH 4.8), and perform enzymatic hydrolysis at 50° C. for 30 min.
  • W is the content of xylose generated by enzymatic hydrolysis, mg
  • N is the dilution ratio of the fermentation broth
  • V is the volume of the reaction solution, mL.
  • Trichoderma harzianum Ta97 was 61.65IU/mL.
  • African Trichoderma harzianum Ta97 wettable powder, preparation method the process is as follows:
  • the seed liquid is fermented in a fermentor equipped with a liquid fermentation medium at a volume percentage of 18%, and the fermentation temperature is 29°C before 12 hours and 25°C after 12 hours, and the initial pH is 4.2 , the stirring speed is 200 ⁇ 300r/min, the ventilation rate is 10L/min, and the fermentation time is 70 hours; the number of viable bacteria in the fermentation product is 1 ⁇ 10 9 cfu/mL; the fermentation liquid is vacuum freeze-dried, that is, Trichoderma powder, the viable count of which is ⁇ 1 ⁇ 10 10 cfu/g;
  • the liquid fermentation medium is composed of the following components by weight: 80 parts of blasted corn stalk powder, 80 parts of blasted wheat stalk powder, 40 parts of wheat bran powder, 20 parts of glucose, 0.7 part of (NH 4 ) 2 SO 4 , KH 2 PO 4 0.03 part, MgSO 4 ⁇ 7H 2 O 0.04 part, water 7000 parts;
  • Trichoderma powder and auxiliary agent according to the following parts by weight: 40 parts of Trichoderma powder, 40 parts of diatomaceous earth, 2.6 parts of wetting agent, 4.8 parts of dispersant, 0.3 part of adhesive, 1.5 parts of zinc sulfate portion; mix the above components evenly; the number of effective viable bacteria is ⁇ 5 ⁇ 10 9 cfu/g.
  • the seed liquid is fermented in a fermenter equipped with a liquid fermentation medium at a volume percentage of 12%, and the fermentation temperature is 28°C before 12 hours and 24°C after 12 hours, and the initial pH is 3.5 , the stirring speed is 200 ⁇ 300r/min, the ventilation rate is 12L/min, and the fermentation time is 60 hours; the number of viable bacteria in the fermentation product is 1 ⁇ 10 8 cfu/mL; the fermentation liquid is vacuum freeze-dried, which is Trichoderma powder, the viable count of which is ⁇ 1 ⁇ 10 10 cfu/g;
  • the liquid fermentation medium is composed of the following components by weight: 50 parts of blasted corn stalk powder, 50 parts of blasted wheat stalk powder, 30 parts of wheat bran powder, 10 parts of glucose, 0.5 part of (NH 4 ) 2 SO 4 , KH 2 PO 4 0.02 part, MgSO 4 ⁇ 7H 2 O 0.03 part, water 5000 part;
  • Trichoderma powder and auxiliary agent according to the following parts by weight: 30 parts of Trichoderma powder, 30 parts of diatomaceous earth, 2 parts of wetting agent, 3 parts of dispersant, 0.2 part of adhesive, 1 part of zinc sulfate portion; mix the above components evenly; the number of effective viable bacteria is ⁇ 5 ⁇ 10 9 cfu/g.
  • the seed liquid is fermented in a fermenter equipped with a liquid fermentation medium at a volume percentage of 22%, and the fermentation temperature is 30°C before 12 hours and 27°C after 12 hours, and the initial pH is 5.5 , the stirring speed is 200 ⁇ 300r/min, the ventilation rate is 15L/min, and the fermentation time is 80 hours; the number of viable bacteria in the fermentation product is 5 ⁇ 10 9 cfu/mL; the fermentation liquid is vacuum freeze-dried, which is Trichoderma powder, the viable count of which is ⁇ 1 ⁇ 10 10 cfu/g;
  • the liquid fermentation medium is composed of the following components by weight: 100 parts of blasted corn stalk powder, 100 parts of blasted wheat straw powder, 50 parts of wheat bran powder, 30 parts of glucose, 1 part of (NH 4 ) 2 SO 4 , KH 2 PO 4 0.04 part, MgSO 4 ⁇ 7H 2 O 0.05 part, water 8000 parts;
  • Trichoderma powder and auxiliary agent according to the following parts by weight: 50 parts of Trichoderma powder, 50 parts of diatomaceous earth, 4 parts of wetting agent, 6 parts of dispersant, 0.5 part of adhesive, 2 parts of zinc sulfate portion; mix the above components evenly; the number of effective viable bacteria is ⁇ 5 ⁇ 10 9 cfu/g.
  • Raw materials corn stalks, the corn variety is Zhengdan 958, collected from the farmland of Caishi Town, Jinan City, Shandong Province; the inoculum is the inoculum provided in Example 4.
  • Treatment group 5 treatments, named as T1, T2, T3, T4 and T5; the treatment process was mixing corn stover with Trichoderma harzianum Ta97 inoculant; the dosage of Trichoderma harzianum Ta97 inoculant was 10 10 cfu/kg straw;
  • Control group 2 treatments, named D1 and D2; the treatment process was mixing corn stalks with commercially available straw decomposing inoculants (the main components of the commercially available straw decomposing inoculants were Bacillus subtilis, Bacillus licheniformis, yeast, mold and its metabolites); the dosage of commercial bacterial agent is 10 10 cfu/kg straw; control group: 1, only corn straw;
  • the above-mentioned treatment group, contrast control and control group are respectively degraded to corn stalk according to the following method (the degree of degradation is represented by weight loss rate), and the above-mentioned treatment group (5), contrasted with nylon mesh bags with a diameter of 20 meshes are respectively loaded into the above-mentioned treatment group.
  • the mixtures of the group (2) and the control group (1) were 100 g each, buried in a soil layer with a depth of 5-10 cm, and samples were taken on the 10th, 20th and 30th days after pre-embedding, and the weight loss rate of corn stalks in the nylon bag was determined. .
  • the results are shown in Table 1,
  • the Trichoderma harzianum Ta97 inoculum provided by the present invention has the same degradation performance on corn stalks in the same time period, which shows that the Trichoderma harzianum Ta97 of the present invention is obtained
  • the enzymatic system formed in the metabolites of T. harzianum is stable, and in the metabolites, the enzymatic activity of the enzymes is stable; it further illustrates that the metabolites of Trichoderma harzianum Ta97 of the present invention are suitable for wide application.
  • Trichoderma harzianum Ta97 inoculant on the degradation of corn stover and wheat stover in field soil in field test
  • the test site was farmland near Huangwang Road, High-tech Zone, Jining City, Shandong province, and was selected in a corn-wheat rotation farmland area with flat terrain and few human factors.
  • the soil type is yellow cinnamon soil
  • the organic matter content is about 2%
  • the total nitrogen content is about 800mg/kg
  • the total phosphorus content is about 700mg/kg
  • the total potassium content is about 18g/kg
  • the pH is about 7.
  • each cell has an area of 15 square meters, and the processing cells are randomly arranged, and the processing cells are spaced to protect rows.
  • the inoculum is the inoculum provided in Example 4.
  • Treatment group corn stalks are returned to the field after blasting + Trichoderma harzianum Ta97 inoculum, three treatment groups are set up, named T1, T2 and T3; the per mu dosage of Trichoderma harzianum Ta97 inoculum is 2.5 kg/ mu;
  • Control group corn stalks were returned to the field after blasting
  • the stalks after harvesting corn are blasted on the spot, then the blasted stalks are evenly spread on the field surface, and the aqueous solution containing bacterial agents is evenly sprayed on the surface of the straws (the weight ratio of Trichoderma harzianum Ta97 bacterial agent to water is 0.8:100). ), at the same time evenly apply an appropriate amount of urea to make the soil carbon-nitrogen ratio 30:1, press and adhere the blasted straw to the soil, water until the straw fully absorbs water, and decomposes in situ; before sowing, rotate the straw to restore Sowing and field management of the next crop of wheat are routinely carried out.
  • the difference between the control group and the treatment group was that Trichoderma harzianum Ta97 was not added.
  • the inoculum is the inoculum provided in Example 4.
  • test area and group settings are the same as those in the test method of returning corn stalks to the field, and the position of returning the corn stalks to the field is the same as that of returning the corn Degradation ability of Trichoderma harzianum Ta97 on wheat straw. Since there is generally no interval between the harvest of wheat and the sowing of the next stubble of corn in the experimental site, the use of inoculants for returning wheat straw to the field is different from that of corn straw.
  • the difference between the control group and the treatment group was that Trichoderma harzianum Ta97 was not added.
  • Trichoderma harzianum Ta97 inoculum accelerated the degradation of wheat straws returned to the field and improved the emergence rate of the next corn.
  • the enzyme in the bacterial agent has strong adaptability to the environment, strong degradation performance, and is less affected by the degradation environment, so it is suitable for wide popularization and application.

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Abstract

提供了一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用，该菌株的拉丁文分类命名为：Trichoderma afroharzianum，已于2020年07月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院，保藏编号：CGMCC No.19930。该非洲哈茨木霉Ta97可应用于秸秆还田，在实验室试验中，秸秆在30天内，秸秆平均失重率达到80.40％，在大田试验中，秸秆在30天内能够充分降解，达到腐熟4级。

Description

一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用 技术领域
本发明涉及微生物应用技术领域，具体涉及一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用。
背景技术
农作物秸秆是农业生产中主要的固体废弃物之一，我国每年产量8亿吨左右。秸秆中含有纤维、蛋白和脂肪等大量的有机物质，具有资源化利用价值。在秸秆的资源化利用方式中，以动物饲料和堆肥两种方式较为常用，其中，堆肥是主要的资源化利用方式，然而，堆肥需要有专门的堆肥场地，使得秸秆处理受限，因此，寻求一种更加合理的资源利用化方式对秸秆处理具有重要意义。
秸秆返田是一种节约成本的秸秆处理方式，然而，秸秆中的木质素通过酯键与纤维素、半纤维素结合，交织形成坚固组织以抵御逆境，导致秸秆就地还田后降解周期过长，不仅无法有效向土壤释放营养物质，未降解完全的秸秆还会阻碍下茬作物苗期发育。
现有技术中，在将秸秆就地还田时，常使用含有木质纤维素降解酶系的微生物对还田秸秆加速降解，但大部分降解菌的降解功能单一，不能满足就地还田秸秆降解的需求，因此，通常将多株菌进行配伍形成复合菌系，但是由于每株菌的生长、产酶条件不同，使复合菌系的发酵条件苛刻，另外，复合菌系的研究成本高，从而使复合菌系在秸秆就地还田中的应用难以真正实现，这也是限制秸秆就地还田的主要因素。
发明内容
针对现有技术的上述不足，本发明提供了一株非洲哈茨木霉Ta97及其在秸秆还田方面的应用，该非洲哈茨木霉Ta97，该菌株的拉丁文分类命名为：Trichoderma afroharzianum，该菌株已于2020年07月14日保藏于中国微生物菌种保藏管理委员为普通微生物中心，保藏单位简称CGMCC，地址：北京市朝阳区北辰西路1号院，保藏编号：CGMCC No.19930。该非洲哈茨木霉Ta97同时具有锰过氧化物酶、木质素过氧化物酶、纤维素酶和半纤维素酶活性。将该非洲哈茨木霉Ta97应用于秸秆还田，在实验室对秸秆的降解试验中，秸秆在30天内的平均失重率达到80.40％，在大田试验中，秸秆在30天内能够充分降解，达到腐熟4级，能够实现对秸秆就地还田进行充分降解；且获得利于作物吸收的肥料，为下茬作物生长提供营养，达到节约肥料成本的目的。
本发明的技术方案如下：
一株非洲哈茨木霉Ta97，该菌株的拉丁文分类命名为：Trichoderma afroharzianum，该菌株已于2020年07月14日保藏于中国微生物菌种保藏管理委员为普通微生物中心，保藏单位简称CGMCC，地址：北京市朝阳区北辰西路1号院，保藏编号：CGMCC No.19930。
进一步的，该非洲哈茨木霉Ta97的tef1基因序列为：
Figure PCTCN2021090911-appb-000001
Figure PCTCN2021090911-appb-000002
进一步的，该非洲哈茨木霉Ta97同时具有锰过氧化物酶、木质素过氧化物酶、羧甲基纤维素酶和半纤维素酶活性；其中，锰过氧化物酶和木质素过氧化物酶参与分解木质素，羧甲基纤维素酶参与分解纤维素，木聚糖酶参与分解半纤维素。
进一步的，该锰过氧化物酶活力为3.52IU/mL，木质素过氧化物酶活力为2.43IU/mL，羧甲基纤维素酶活力为3.76IU/mL，木聚糖酶活力为61.65IU/mL。
上述非洲哈茨木霉Ta97在秸秆还田方面的应用。
进一步的，在上述应用中，具体为非洲哈茨木霉Ta97制备的菌剂在秸秆还田方面的应用。
优选的，非洲哈茨木霉Ta97制备的菌剂，具体为非洲哈茨木霉Ta97可湿性粉剂，在该可湿性粉剂中，有效活菌数≥5×10 9cfu/g。
进一步的，非洲哈茨木霉Ta97可湿性粉剂的制备方法，过程如下：
(1)种子液的制备：将-80℃保藏的Ta97接种于PDA固体平板，25℃活化3天，在菌落边缘取菌丝再次接种于PDA平板中，25～28℃培养3天，再重复取菌丝培养一次，即为活化的菌株；将活化好的菌株在PDA平板上25～28℃、12小时光照12小时黑暗条件下培养10天，制备10 7个/mL的分生孢子悬浊液，以1:100体积比接种于PDB培养液，25℃、180rpm条件下震荡培养40～60小时，即为种子液，种子液的活菌数1×10 4～1×10 5cfu/mL；
(2)木霉菌粉制备：种子液以体积百分比12％～22％接入装有液体发酵培养基的发酵罐中发酵，发酵温度在12小时之前为28～30℃、12小时之后为24～27℃，初始pH为3.5～5.5，搅拌速度为200～300r/min，通气量为10～15L/min，发酵时间为60～80小时；发酵产物中活菌数为1×10 8～1×10 9cfu/mL；将发酵液经真空冷冻干燥，即为木霉菌粉，其活菌数≥1×10 10cfu/g；
(3)制剂：将木霉菌粉与助剂按照以下重量份数混合：木霉菌粉30～50份，硅藻土30～50份，润湿剂2～4份，分散剂3～6份，粘着剂0.2～0.5份，硫酸锌1～2份；将上述组分混匀，即可。
进一步的，在步骤(2)中，液体发酵培养基按下述重量份数的组分组成：爆破玉米秸秆粉50～100份，爆破小麦秸秆粉50～100份，麦麸粉30～50份，葡萄糖10～30份，(NH 4) 2SO 4 0.5～1份，KH 2PO 4 0.02～0.04份，MgSO 4·7H 2O 0.03～0.05份，水5000～8000份。
进一步的，在实验室条件下对秸秆的降解试验中，秸秆在30天内的平均失重率达到80.40％，在大田试验中，秸秆在30天内能够充分降解，达到腐熟4级。
相对于现有技术，本发明的有益效果在于：
1、本发明提供了一株非洲哈茨木霉Ta97，该非洲哈茨木霉Ta97同时具有锰过氧化物酶、木质素过氧化物酶、羧甲基纤维素酶和木聚糖酶四种降解酶，且锰过氧化物酶活力为3.52IU/mL，木质素过氧化物酶活力为2.43IU/mL，羧甲基纤维素酶活力为3.76IU/mL，木聚糖酶活力为61.65IU/mL。
2、本发明提供的非洲哈茨木霉Ta97同时具有四种酶活，其中，锰过氧化物酶和木 质素过氧化物酶参与分解木质素，羧甲基纤维素酶参与分解纤维素，木聚糖酶参与分解半纤维素；四种酶活提高了该菌株对秸秆的降解能力，在实验室对秸秆的降解试验中，秸秆在30天内的平均失重率达到80.40％，在大田试验中，秸秆在30天内能够充分降解，达到腐熟4级；避免了现有技术中采用多菌株构建的复合菌系在发酵过程中，对发酵条件要求苛刻的问题，使秸秆还田更易实现。
3、本发明提供的非洲哈茨木霉Ta97菌剂，可在秸秆就地还田时直接使用，避免了现有堆肥的场地限制，且就地还田还可减少人工搬运秸秆、人工施肥还田的劳动输出，具有节约劳动力和成本的优点。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为非洲哈茨木霉Ta97在PDA平板中25℃培养7天的菌落及显微形态图，图中，A为菌落正面图，B为菌落反面图，C为分生孢子梗形态图，D为分生孢子形态图，E为厚垣孢子形态图。
图2为非洲哈茨木霉Ta97的酶活测定平板图，图中，A为筛选过氧化物酶的苯胺蓝平板图，B为筛选过氧化物酶的RB亮蓝平板图，C为筛选纤维素酶CMC平板图，D为筛选半纤维素酶的木聚糖平板图。
具体实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明的实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例1菌株的分离鉴定
(1)菌株来源
本发明非洲哈茨木霉Ta97分离筛选自玉米小麦轮作的农田土壤，取样深度为20公分。
将采集的土壤样本用含有0.3％吐温-80的无菌水梯度稀释至10 -1～10 -7倍，从每个梯度稀释液中分别取100μL涂布玫瑰红钠琼脂培养基平板，在25℃恒温培养3天。从每个梯度稀释的平板中挑取单菌落，通过形态学显微镜观察和翻译延伸因子基因(translation elongation factor 1-alpha gene，tef1)比对，鉴定种属，其中编号为Ta97的菌株为分离得到的优势菌株，经鉴定为非洲哈茨木霉(Trichoderma afroharzianum)。
Ta97的菌落状态见图1，图中，A为菌落正面图，B为菌落反面图，C为分生孢子梗形态图，D为分生孢子形态图，E为厚垣孢子形态图。
Ta97的tef1基因序列为：
Figure PCTCN2021090911-appb-000003
Figure PCTCN2021090911-appb-000004
实施例2菌株筛选
(1)将实施例1获得的非洲哈茨木霉Ta97，在PDA平板上25℃恒温培养3天；
(2)筛选
采用平板筛选，过程如下：
①苯胺蓝平板筛选产过氧化物酶的菌株：用无菌打孔器取PDA平板上培养好的菌落，接种于苯胺蓝平板上，制备3个平板重复，在25±1℃条件下倒置培养，记录在苯胺蓝平板上有无脱色透明现象，脱色者记为+，反之记为-。根据透明区域直径，选出产过氧化物酶能力强的菌株，并记录时间。
苯胺蓝固体培养基：将RB亮蓝单独过滤除制成2％的母液(200倍)，无菌条件下以0.01％(V/V)加入PDA中。
经培养，非洲哈茨木霉Ta97在苯胺蓝培养基中可使染料脱色透明，第4天透明区域直径为4.72cm，见图2中A图。
②RB亮蓝平板筛选产过氧化物酶的菌株：用无菌打孔器取PDA平板上培养好的菌落，接种于PDA-RB亮蓝平板上，制备3个平板重复，在25±1℃条件下倒置培养，记录在PDA-RB亮蓝平板上有无黄色环产生，有黄色轮环者记为+，反之记为-。根据所产生黄色圈的大小，选出产过氧化物酶能力强的菌株，并记录显色时间。
PDA-RB亮蓝固体培养基：将RB亮蓝单独过滤除制成2％的母液(200倍)，无菌条件下与PDA培养基按625mg每毫升的比例混均。
经培养，非洲哈茨木霉Ta97在PDA-RB亮蓝培养基中可产生黄色环，第4天黄色环直径为5.68cm，见图2中B图。
③CMC平板筛选产纤维素酶的菌株：用无菌打孔器取PDA平板上培养好的菌落，接种于CMC平板上，制备3个平板重复，在25±1℃条件下倒置培养4天，然后用0.1％的刚果红染液覆盖平板，静置30min，再用1mol/L NaCl脱色1h，观察各菌株是否产生水解透明圈。根据水解透明圈的直径，选出产纤维素酶能力强的菌株。
CMC固体培养基：CMC 10g，(NH 4) 2SO 4 4g，KH 2PO 4 2g，MgSO 4·7H 2O 0.5g，蛋白胨1.0g，琼脂16g，去氧胆酸钠0.5g，加水1000ml，121℃高压灭菌20min。
经培养，非洲哈茨木霉Ta97在CMC培养基中可产生水解透明圈，第4天透明圈直径为3.10cm，见图2中C图。
④木聚糖平板筛选产半纤维素酶的菌株：用无菌打孔器取PDA平板上培养好的菌落，接种于木聚糖平板上，制备3个平板重复，在25±1℃条件下倒置培养4天；然后用0.1％的刚果红染液覆盖平板，静置10min，再用2mol/L NaCl脱色2～3次，观察各菌株是否产生水解透明圈。根据水解透明圈的直径，选出产半纤维素酶能力强的菌株。
木聚糖固体培养基：(NH 4) 2SO 4 2g，MgSO 4·7H 2O 0.5g，KH 2PO 4 1g，NaCl 0.5g，木聚糖2g，琼脂20g，蒸馏水1000mL，pH值自然，121℃高压灭菌20min。
经培养，非洲哈茨木霉Ta97在CMC培养基中可产生水解透明圈，第4天透明圈直径 为8.51cm，见图2中D图。
经过平板筛选，非洲哈茨木霉Ta97具有同时降解木质素、纤维素和半纤维素的酶系并该酶系中的四种酶均具有酶活性。
实施例3菌株代谢产物的酶活测定
①木质素过氧化物酶活力检测：在PDA平板上活化非洲哈茨木霉Ta97，挑取平板上非洲哈茨木霉Ta97制备1×10 7个/mL的分生孢子悬浊液，按1％(V/V)接种于藜芦醇培养液(培养液包括：淀粉0.6g，酵母粉7.5g，麦秸粉0.5g，KH 2PO 4 4g，MgSO 4·7H 2O 0.244g，CaCl 2 0.066g，藜芦醇2mmol，维生素B1 2mg，微量元素溶液40mL，0.15％吐温-80，蒸馏水1000mL)中，在25±1℃、160rpm条件下培养5天，获得发酵液；将发酵液在4℃、12000rpm离心20min，上清为粗酶液。
将待测粗酶液600μL、250mmol/L酒石酸缓冲液3.2mL和10mmol/L藜芦醇溶液0.1mL混合成反应液，于30℃水浴预热，然后加入10mmol/L H 2O 2溶液0.1mL启动反应，迅速测定310nm处的吸光度，4min后再测定1次。
1min内氧化藜芦醇产生1μmoL藜芦醛为1个酶活单位。以灭菌发酵液为对照，设置三个重复。经检测，非洲哈茨木霉Ta97的木质素过氧化物酶活力为2.43IU/mL。
②锰过氧化物酶活力检测：在PDA平板上活化Ta97菌株，制备1×10 7个/mL的分生孢子悬浊液，按1％(V/V)接种于藜芦醇培养液中，在25±1℃、160rpm条件下培养5天，获得发酵液；将发酵液在4℃、12000rpm离心20min，上清为粗酶液。
将待测粗酶液400μL、50mmol/L乳酸钠缓冲液3.4mL和1.6mmol/L硫酸锰溶液0.1mL混合成反应液，于37℃水浴预热，然后加入1.6mmol/L H 2O 2溶液0.1mL启动反应，迅速测定240nm处的吸光度，4min后再测定1次。
1min内使1μmol/L的Mn 2+转化为Mn 3+为1个酶活单位。以灭菌发酵液为对照，设置三个重复。经检测，非洲哈茨木霉Ta97的锰过氧化物酶活力为3.52IU/mL。
③羧甲基纤维素酶活力检测：在PDA平板上活化非洲哈茨木霉Ta97，制备1×10 7个/mL的分生孢子悬浊液，按1％(V/V)接种于CMC培养液(CMC固体培养基减掉琼脂即得)中，在25±1℃、160rpm条件下培养7天，得发酵液。发酵液在4℃、12000rpm离心10min，上清为粗酶液。向2mL Eppendorf管中加入含有1％(w/v)CMC的柠檬酸-Na 2HPO 4缓冲液(pH 4.5)400μL，50℃水浴锅中预热3min后，加入粗酶液100μL，保温30min后加入1mL DNS试剂终止反应，充分摇匀后沸水浴5min，冷水冷却后测定其在540nm波长下的吸光度。以灭菌的发酵液为对照。设置三个重复。经检测，非洲哈茨木霉Ta97的羧甲基纤维素酶活力为3.76IU/mL。
④木聚糖酶活力检测：在PDA平板上活化非洲哈茨木霉Ta97，制备1×10 7个/mL的分生孢子悬浊液，按1％(V/V)接种于木聚糖培养液(木聚糖固体培养基减掉琼脂即得)中，在25±1℃、160rpm条件下培养7天，得发酵液。吸取稀释20倍的发酵液0.5mL，加到磷酸氢二钠-柠檬缓冲液(pH 4.8)配制的1％木聚糖溶液中，50℃酶解30min。酶解后加入3mL DNS试剂，沸水浴中加热5min，然后用冰水迅速冷却，补加蒸馏水至25mL，测定540nm处吸光度，设置三个生物学重复，以灭菌的发酵液为对照，按照下面的公式计算：
Figure PCTCN2021090911-appb-000005
式中：W为酶解生成木糖的含量，mg；
N为发酵液的稀释倍数；
30为酶解时间，min；
V为反应液体积，mL。
经检测，非洲哈茨木霉Ta97的木聚糖酶活力为61.65IU/mL。
实施例4非洲哈茨木霉Ta97菌剂的制备1
非洲哈茨木霉Ta97可湿性粉剂，制备方法，过程如下：
(1)种子液的制备：将-80℃保藏的Ta97接种于PDA固体平板，25℃活化3天，在菌落边缘取菌丝再次接种于PDA平板中，25℃培养3天，再重复取菌丝培养一次，即为活化的菌株；将活化好的菌株在PDA平板上25℃、12小时光照12小时黑暗条件下培养10天，制备10 7个/mL的分生孢子悬浊液，以1:100体积比接种于PDB培养液，25℃、180rpm条件下震荡培养50小时，即为种子液，种子液的活菌数5×10 5cfu/mL；
(2)木霉菌粉制备：种子液以体积百分比18％接入装有液体发酵培养基的发酵罐中发酵，发酵温度在12小时之前为29℃、12小时之后为25℃，初始pH为4.2，搅拌速度为200～300r/min，通气量为10L/min，发酵时间为70小时；发酵产物中活菌数为1×10 9cfu/mL；将发酵液经真空冷冻干燥，即为木霉菌粉，其活菌数≥1×10 10cfu/g；
液体发酵培养基按下述重量份数的组分组成：爆破玉米秸秆粉80份，爆破小麦秸秆粉80份，麦麸粉40份，葡萄糖20份，(NH 4) 2SO 4 0.7份，KH 2PO 4 0.03份，MgSO 4·7H 2O 0.04份，水7000份；
(3)制剂：将木霉菌粉与助剂按照以下重量份数混合：木霉菌粉40份，硅藻土40份，润湿剂2.6份，分散剂4.8份，粘着剂0.3份，硫酸锌1.5份；将上述组分混匀，即可；有效活菌数≥5×10 9cfu/g。
实施例5非洲哈茨木霉Ta97菌剂的制备2
(1)种子液的制备：将-80℃保藏的Ta97接种于PDA固体平板，25℃活化3天，在菌落边缘取菌丝再次接种于PDA平板中，27℃培养3天，再重复取菌丝培养一次，即为活化的菌株；将活化好的菌株在PDA平板上27℃、12小时光照12小时黑暗条件下培养10天，制备10 7个/mL的分生孢子悬浊液，以1:100体积比接种于PDB培养液，27℃、180rpm条件下震荡培养40小时，即为种子液，种子液的活菌数1×10 4cfu/mL；
(2)木霉菌粉制备：种子液以体积百分比12％接入装有液体发酵培养基的发酵罐中发酵，发酵温度在12小时之前为28℃、12小时之后为24℃，初始pH为3.5，搅拌速度为200～300r/min，通气量为12L/min，发酵时间为60小时；发酵产物中活菌数为1×10 8cfu/mL；将发酵液经真空冷冻干燥，即为木霉菌粉，其活菌数≥1×10 10cfu/g；
液体发酵培养基按下述重量份数的组分组成：爆破玉米秸秆粉50份，爆破小麦秸秆粉50份，麦麸粉30份，葡萄糖10份，(NH 4) 2SO 4 0.5份，KH 2PO 4 0.02份，MgSO 4·7H 2O 0.03份，水5000份；
(3)制剂：将木霉菌粉与助剂按照以下重量份数混合：木霉菌粉30份，硅藻土30份，润湿剂2份，分散剂3份，粘着剂0.2份，硫酸锌1份；将上述组分混匀，即可；有效活菌数≥5×10 9cfu/g。
实施例6非洲哈茨木霉Ta97菌剂的制备3
(1)种子液的制备：将-80℃保藏的Ta97接种于PDA固体平板，25℃活化3天，在菌落 边缘取菌丝再次接种于PDA平板中，28℃培养3天，再重复取菌丝培养一次，即为活化的菌株；将活化好的菌株在PDA平板上28℃、12小时光照12小时黑暗条件下培养10天，制备10 7个/mL的分生孢子悬浊液，以1:100体积比接种于PDB培养液，25℃、180rpm条件下震荡培养60小时，即为种子液，种子液的活菌数1×10 5cfu/mL；
(2)木霉菌粉制备：种子液以体积百分比22％接入装有液体发酵培养基的发酵罐中发酵，发酵温度在12小时之前为30℃、12小时之后为27℃，初始pH为5.5，搅拌速度为200～300r/min，通气量为15L/min，发酵时间为80小时；发酵产物中活菌数为5×10 9cfu/mL；将发酵液经真空冷冻干燥，即为木霉菌粉，其活菌数≥1×10 10cfu/g；
液体发酵培养基按下述重量份数的组分组成：爆破玉米秸秆粉100份，爆破小麦秸秆粉100份，麦麸粉50份，葡萄糖30份，(NH 4) 2SO 4 1份，KH 2PO 4 0.04份，MgSO 4·7H 2O 0.05份，水8000份；
(3)制剂：将木霉菌粉与助剂按照以下重量份数混合：木霉菌粉50份，硅藻土50份，润湿剂4份，分散剂6份，粘着剂0.5份，硫酸锌2份；将上述组分混匀，即可；有效活菌数≥5×10 9cfu/g。
实验室试验在实验室条件下，测定非洲哈茨木霉Ta97菌剂对玉米秸秆的失重率
原材料：玉米秸秆，玉米品种为郑单958，采自山东省济南市彩石镇农田；菌剂为实施例4提供的菌剂。
试验设置：
1、原料准备
(1)将玉米秸秆爆破烘干后待用；
(2)组别设置：
处理组：5个处理，命名为T1、T2、T3、T4和T5；处理过程为玉米秸秆与非洲哈茨木霉Ta97菌剂混合；非洲哈茨木霉Ta97菌剂用量为10 10cfu/kg秸秆；
对比组：2个处理，命名为D1和D2；处理过程为玉米秸秆与市售秸秆腐熟菌剂混合(市售秸秆腐熟菌剂的主要成分为枯草芽孢杆菌、地衣芽孢杆菌、酵母菌、霉菌及其代谢产物)；市售菌剂用量为10 10cfu/kg秸秆；对照组：1个，仅为玉米秸秆；
2、试验过程
将上述的处理组、对比照和对照组分别按照下述的方法对玉米秸秆降解(降解的程度用失重率表示)，用孔径20目的尼龙网袋分别装入上述处理组(5个)、对比组(2个)、对照组(1个)的混合物各100g，埋入5～10cm深的土层中，预埋后的第10、20和30天取样，测定尼龙袋中玉米秸秆的失重率。
秸秆失重率测定为将尼龙袋中的未腐熟烂软的秸秆挑拣出来，冲洗干净，置70℃烘箱烘干至恒重，秸秆失重率＝(培养前秸秆质量-培养n天时秸秆质量)/培养前秸秆质量×100％。结果见表1，
表1施用不同菌剂各时期玉米秸秆失重率(％)
Figure PCTCN2021090911-appb-000006
结合表1可以看出，非洲哈茨木霉Ta97菌剂在第10、20和30天对秸秆的平均失重率分别即为25.75％，55.89％和80.40％，相比市售腐熟菌剂腐熟速度更快，表明该非洲哈茨木霉Ta97菌剂对玉米秸秆具有良好的降解能力，且酶活性持续性好。
另外，通过5个处理组的失重率可以看出，本发明提供的非洲哈茨木霉Ta97菌剂在相同时间内，对玉米秸秆的降解性能一致，这表明，本发明的非洲哈茨木霉Ta97获取的代谢产物中形成的酶系稳定，且在代谢产物中，酶的酶活性稳定；进一步说明了本发明的非洲哈茨木霉Ta97的代谢产物，适于广泛应用。
大田试验非洲哈茨木霉Ta97菌剂对玉米秸秆和小麦秸秆在田间土壤中降解的应用效果
试验地点及土壤特征
试验地点为山东省济宁市高新区黄王路附近农田，选在地势平坦、人为影响因素少的玉米-小麦轮作农田区域。土壤类型为黄褐土，有机质含量2％左右，全氮含量800mg/kg左右，全磷含量700mg/kg左右，全钾含量18g/kg左右，p H 7左右。
1、对玉米秸秆就地还田的试验方法
试验区域：
设置多个小区，每个小区的面积为15平方米，各处理小区间随机排列，处理小区间隔保护行。
组别设置：
菌剂为实施例4提供的菌剂。
(1)处理组：玉米秸秆爆破后就地还田+非洲哈茨木霉Ta97菌剂，处理组设置3个，命名T1、T2和T3；非洲哈茨木霉Ta97菌剂的亩用量为2.5公斤/亩；
(2)对照组：玉米秸秆爆破后就地还田；
处理组就地还田的具体过程为：
对收获玉米后的秸秆就地爆破，然后将爆破后的秸秆均匀平铺在田面上，在秸秆表面均匀喷洒含菌剂的水溶液(非洲哈茨木霉Ta97菌剂与水的重量比为0.8:100)，同时均 匀施加适量的尿素，使土壤碳氮比为30:1，将爆破后的秸秆与泥土压紧黏连，浇水至秸秆充分吸水，原地腐熟分解；播种前将秸秆旋耕还田，常规进行下茬小麦的播种和田间管理。
对照组就地还田与处理组就地还田的区别在于，不添加非洲哈茨木霉Ta97菌剂。
田间调查：在将处理组第10、20、30天调查田间秸秆腐熟效果，指标包括颜色、气味和手感，颜色分为中黄、微黄、褐黄、黑黄4个等级，气味分为霉味、氨味、酒味、腐烂味4个等级，手感分为硬、微软、软、腐烂4个等级。调查结果见表2，
表2对照和处理组玉米秸秆的腐熟效果
Figure PCTCN2021090911-appb-000007
结合表1可以看出，施用非洲哈茨木霉Ta97菌剂的小区，还田的玉米秸秆在第30天调查时从颜色、气味和手感三个指标评判已达第4级，完成腐熟，且3个处理组的处理结果一致；对照组小区在第30天时未达到完全腐熟，表明本发明提供的非洲哈茨木霉Ta97菌剂中的代谢产物能够在秸秆就地还田中对秸秆进行降解，且降解时间短，3个处理小区的降解程度一致，说明该非洲哈茨木霉Ta97菌剂在大田试验中具有良好的稳定性。
2、对小麦秸秆就地还田的试验方法
小麦秸秆来自上茬玉米秸秆腐熟时种植的小麦收获后残留田间的秸秆。菌剂为实施例4提供的菌剂。
试验区域和组别设置均与玉米秸秆就地还田的试验方法中的相同，且就地还田的位置与玉米秸秆就地还田的位置相同，用来研究连续就地还田时，该非洲哈茨木霉Ta97菌剂对小麦秸秆的降解能力。由于试验地小麦收获后至播种下茬玉米一般无间隔期，对小麦秸秆就地还田的菌剂使用方法与玉米秸秆不同。
处理组就地还田的具体过程为：
对收获小麦后的秸秆就地爆破，然后将爆破后的秸秆均匀平铺在田面上，常规翻耕播种玉米，每25cm一穴，每穴2粒。在玉米苗期早期，用常规喷洒设备均匀喷洒含1％Ta97可湿性粉剂的水溶液，同时避苗均匀施加适量的尿素，使土壤碳氮比为25:1，亩用菌量为2.6公斤/亩。浇水使土壤含水量为60％。常规中耕操作，后续田间管理常规。
对照组就地还田与处理组就地还田的区别在于，不添加非洲哈茨木霉Ta97菌剂。
田间调查：(1)腐熟效果田间调查，调查标准同上，在第10、20、30天调查田间秸秆腐熟效果，见表3；(2)玉米出苗率调查，在第10天(出苗高峰期)调查对照及各处理的出苗数，计算出苗率，见表4，出苗率％＝出苗数/播种数×100。
表3对照和处理组小麦秸秆的腐熟效果
Figure PCTCN2021090911-appb-000008
Figure PCTCN2021090911-appb-000009
表4对照和处理组第10天的玉米出苗率
  T1 T2 T3 CK
出苗率％ 90.41 90.75 89.90 85.11
结合表3可以看出，在同一位置连续就地还田时，非洲哈茨木霉Ta97菌剂仍能对小麦秸秆进行充分降解，还田的小麦秸秆在第30天调查时从颜色、气味和手感三个指标评判已达第4级，完成腐熟；
结合表4可以看出，非洲哈茨木霉Ta97菌剂的施用加速还田小麦秸秆的降解，提高了下茬玉米的出苗率。这充分说明，连续就地还田时非洲哈茨木霉Ta97菌剂对小麦秸秆的降解性能好，进一步说明该菌剂中的代谢产物对秸秆的降解性能不受季节环境和作物影响，进而可以看出，菌剂中的酶对环境的适应性强、降解性能强，受降解环境的影响小，适于广泛推广应用。
尽管通过参考优选实施例的方式对本发明进行了详细描述，但本发明并不限于此。在不脱离本发明的精神和实质的前提下，本领域普通技术人员可以对本发明的实施例进行各种等效的修改或替换，而这些修改或替换都应在本发明的涵盖范围内/任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。

Claims (10)

  1. 一株非洲哈茨木霉Ta97，其特征在于，该菌株的拉丁文分类命名为：Trichoderma afroharzianum，该菌株已于2020年07月14日保藏于中国微生物菌种保藏管理委员为普通微生物中心，保藏单位简称CGMCC，地址：北京市朝阳区北辰西路1号院，保藏编号：CGMCC No.19930。
  2. 如权利要求1所述的非洲哈茨木霉Ta97，其特征在于，该非洲哈茨木霉Ta97的tef1基因序列为：
    AGAAGGTAACCTTCAACTGATTTTCGCCTCGATTCTTCCTCTCTTCACAT
    TCAATTGTGCCCGACAATTCTGCAGAGAATTTTCGTGTCGACAATTTTTC
    ATCACCCCGCTTTCCATTACCCCTCCTTTCCAGCGACGCAAATTTTTTTT
    TCTGTCGTTTGGTTTTTAGTGGGGTTCTCTGTGCAACCCCACTAGCTCCC
    TGCTTTTTCCTGCTTCACTCTCACTTCCTCGTCATCATTCAACGTGCTCT
    GCGTCTTTGGTCATTCAGCGACGCTAACCACTTTTCCATCAATAGGAAGC
    CGCCGAACTCGGTAAGG。
  3. 如权利要求1或2所述的非洲哈茨木霉Ta97，其特征在于，该非洲哈茨木霉Ta97同时具有锰过氧化物酶、木质素过氧化物酶、羧甲基纤维素酶和半纤维素酶活性。
  4. 如权利要求3所述的非洲哈茨木霉Ta97，其特征在于，锰过氧化物酶活力为3.52IU/mL，木质素过氧化物酶活力为2.43IU/mL，羧甲基纤维素酶活力为3.76IU/mL，木聚糖酶活力为61.65IU/mL。
  5. 将权利要求1-4任一项所述的非洲哈茨木霉Ta97在秸秆还田方面的应用。
  6. 如权利要求5所述的应用，其特征在于，具体为非洲哈茨木霉Ta97制备的菌剂在秸秆还田方面的应用。
  7. 如权利要求6所述的应用，其特征在于，非洲哈茨木霉Ta97制备的菌剂，具体为非洲哈茨木霉Ta97可湿性粉剂，在该可湿性粉剂中，有效活菌数≥5×10 9cfu/g。
  8. 如权利要求7所述的应用，其特征在于，非洲哈茨木霉Ta97可湿性粉剂的制备方法，过程如下：
    (1)种子液的制备：将-80℃保藏的Ta97接种于PDA固体平板，25℃活化3天，在菌落边缘取菌丝再次接种于PDA平板中，25～28℃培养3天，再重复取菌丝培养一次，即为活化的菌株；将活化好的菌株在PDA平板上25～28℃、12小时光照12小时黑暗条件下培养10天，制备10 7个/mL的分生孢子悬浊液，以1:100体积比接种于PDB培养液，25℃、180rpm条件下震荡培养40～60小时，即为种子液，种子液的活菌数1×10 4～1×10 5cfu/mL；
    (2)木霉菌粉制备：种子液以体积百分比12％～22％接入装有液体发酵培养基的发酵罐中发酵，发酵温度在12小时之前为28～30℃、12小时之后为24～27℃，初始pH为3.5～5.5，搅拌速度为200～300r/min，通气量为10～15L/min，发酵时间为60～80小时；发酵产物中活菌数为1×10 8～1×10 9cfu/mL；将发酵液经真空冷冻干燥，即为木霉菌粉，其活菌数≥1×10 10cfu/g；
    (3)制剂：将木霉菌粉与助剂按照以下重量份数混合：木霉菌粉30～50份，硅藻土30～50份，润湿剂2～4份，分散剂3～6份，粘着剂0.2～0.5份，硫酸锌1～2份；将上述组分混匀，即可。
  9. 如权利要求8所述的应用，其特征在于，在步骤(2)中，液体发酵培养基按下述重量份 数的组分组成：爆破玉米秸秆粉50～100份，爆破小麦秸秆粉50～100份，麦麸粉30～50份，葡萄糖10～30份，(NH 4) 2SO 40.5～1份，KH 2PO 40.02～0.04份，MgSO 4·7H 2O 0.03～0.05份，水5000～8000份。
  10. 如权利要求8所述的应用，其特征在于，在实验室条件下对秸秆的降解试验中，秸秆在30天内的平均失重率达到80.40％，在大田试验中，秸秆在30天内能够充分降解，达到腐熟4级。
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