A strain of dark green Trichoderma Ta102, multifunctional organic fertilizer and its application

Abstract

The invention relates to the field of microbial application technology, and specifically to a dark green Trichoderma Ta102, a multifunctional organic fertilizer and an application thereof. The dark green Trichoderma (Trichoderma atroviride) Ta102 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit address of No. 1 Beichen West Road, Chaoyang District, Beijing, a deposit date of July 13, 2022, and a deposit number of CGMCC No. 40242. The dark green Trichoderma Ta102 has four lignocellulose degrading enzymes, namely, high-activity laccase and manganese peroxidase, hemicellulase and cellulase. The dark green Trichoderma Ta102 is used to prepare a multifunctional organic fertilizer, and the decomposition time is shortened to 9 to 12 days, and the fermentation conditions are required to be relatively low, which is helpful to simplify the organic fertilizer preparation process and reduce the cost. The prepared multifunctional organic fertilizer has the functions of inhibiting soil-borne diseases such as damping-off, wilt and root rot and promoting growth.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a dark green Trichoderma Ta102, a multifunctional organic fertilizer and applications thereof. Background Art

[0002] Organic fertilizers are usually made from plant and / or animal residues, which are processed to make them safe for use. They are used to improve soil fertility and provide nutrients for plant growth. Organic fertilizers contain a high amount of organic matter, which can increase and renew soil organic matter, promote microbial reproduction, improve soil physical and chemical properties and biological activity, and maintain and improve soil fertility.

[0003] At present, the main raw materials used in organic fertilizer production are straw (according to statistical research, mainly crop straw) and livestock and poultry manure (according to statistical research, mainly cow dung and sheep dung), both of which contain a large amount of lignocellulose. In the composition of lignocellulose, lignin is an aromatic amorphous polymer composed of three alcohol monomers. As a binder between the cross-links of cellulose and hemicellulose, the degradation of lignin is the rate-limiting factor in the degradation of lignocellulose. The natural degradation of lignin includes two stages: depolymerization and mineralization. The depolymerization stage mainly catalyzes the breaking of carbon-carbon bonds, carbon-hydrogen bonds and ether bonds between lignin structural units. The mineralization stage mainly deconstructs the aromatic compounds produced in the depolymerization stage into linear forms (Zhao Yiquan et al., 2020). Some microorganisms have enzymes such as laccase and peroxidase that can degrade lignin. Among them, laccase is the only enzyme known to participate in the depolymerization process of lignin and can further utilize aromatic compounds to participate in the mineralization process (Li Chunfeng, 2010). High laccase activity plays an important role in the rapid degradation of lignocellulose.

[0004] Common organic fertilizer fermentation agents are made by compounding live bacteria with degradation ability and adding carriers, which can, to a certain extent, increase the accumulated temperature of material decomposition and accelerate the softening and decomposition of organic fertilizer. However, due to the limitations of bacterial composition and environmental differences, especially the low decomposition efficiency of lignin, the fermentation efficiency of organic fertilizer is not ideal and the use effect is unstable. Therefore, finding strains that produce highly active laccase is of great significance to improve the decomposition efficiency of organic fertilizer. Summary of the invention

[0005] In view of the technical problem that the currently selected lignin-degrading bacteria have poor degradation ability and lead to low decomposition efficiency of organic fertilizer, the present invention provides a dark green Trichoderma Ta102, a multifunctional organic fertilizer and applications thereof. The dark green Trichoderma Ta102 is separated and screened from the fermentation product of corn straw-cow dung compost at the peak period of laccase activity, and can efficiently and stably produce high-activity laccase, thereby greatly improving the decomposition efficiency of organic fertilizer.

[0006] In the first aspect, the present invention provides a dark green Trichoderma Ta102, wherein the dark green Trichoderma (Trichoderma atroviride) Ta102 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, the storage address is No. 1 Beichen West Road, Chaoyang District, Beijing, the storage date is July 13, 2022, and the storage number is CGMCC No.40242.

[0007] In a second aspect, the present invention provides an application of the above-mentioned dark green Trichoderma Ta102 in producing highly active laccase, manganese peroxidase, hemicellulase and cellulase.

[0008] The test showed that the enzyme activity of highly active laccase in Trichoderma viridiflora Ta102 was 396.50IU / mL, the activity of manganese peroxidase was 2.96IU / mL, the activity of hemicellulase was 60.41IU / mL, and the activity of cellulase was 3.25IU / mL. Manganese peroxidase participates in the depolymerization of lignin, hemicellulase participates in the decomposition of hemicellulose and cellulase participates in the decomposition of cellulose. Highly active laccase participates in both the depolymerization process of lignin and the mineralization process. Trichoderma viridiflora Ta102 with this composite degradation enzyme system can efficiently prepare organic fertilizer, and the decomposition time only takes 9 to 12 days.

[0009] In a third aspect, the present invention provides an application of the above-mentioned Trichoderma aureum Ta102 in producing indoleacetic acid. Experiments show that the production level of indoleacetic acid of Trichoderma aureum Ta102 is 153.42 μg / L.

[0010] In a fourth aspect, the present invention provides an application of the dark green Trichoderma Ta102 for inhibiting Rhizoctonia solani, Fusarium oxysporum or Fusarium moniliforme.

[0011] The inhibition rates of Trichoderma aureum Ta102 against Rhizoctonia solani were 100.00%±0.00%, against Fusarium oxysporum was 100.00%±0.00%, and against Fusarium moniliforme was 75.85%±4.62%.

[0012] In a fifth aspect, the present invention provides an application of preparing organic fertilizer by fermenting the above-mentioned dark green Trichoderma Ta102.

[0013] In a sixth aspect, the present invention provides a multifunctional organic fertilizer, wherein the raw materials include 0.1 to 0.5 parts of dark green Trichoderma Ta102 wettable powder, 100 to 150 parts of cow dung, and 30 to 40 parts of crushed corn stalks, wherein the effective viable bacteria count of the dark green Trichoderma Ta102 wettable powder is ≥ 8×10 9 cfu / g;

[0014] The preparation method is as follows: the water-to-material ratio is controlled to be 1:(0.7±0.05), the initial pH value is 6±0.5, the pile is piled and fermented at room temperature for 9 to 12 days, the pile is turned over every 3 days, and after the fermentation is completed, the pile is dried and crushed.

[0015] Furthermore, the dark green Trichoderma Ta102 wettable powder is prepared according to the following preparation method:

[0016] (1) Preparation of seed solution: Dark green Trichoderma Ta102 stored at -80°C was inoculated on a PDA plate, activated at 26-30°C for 3 days, hyphae were taken from the edge of the colony and inoculated on a PDA plate, cultured at 26-30°C for 3 days, and the hyphae culture was repeated once to obtain an activated strain; the activated strain was cultured on a PDA plate at 26-30°C, 12 h light-12 h dark conditions for 7-12 days to prepare 10 7 The conidia suspension of 1 × 10 / mL was inoculated into PDB culture medium at a volume ratio of 1:100 and cultured at 26°C and 180 rpm for 48 to 72 hours to obtain seed solution with a viable count of 1 × 10 5 ~9×10 5 cfu / mL;

[0017] (2) Preparation of Trichoderma viride Ta102 powder: The seed liquid was added to a fermentation tank filled with liquid fermentation medium at a volume percentage of 15% to 25%, and the fermentation temperature was 27 to 33°C before 12 hours and 25 to 30°C after 12 hours. The initial pH was 4.0 to 5.0, the stirring speed was 200 to 300 rpm, the ventilation volume was 10 to 15 L / min, and the fermentation time was 72 to 96 hours. The number of viable bacteria in the fermentation product was 1×10 9 ~8×10 9 cfu / mL; the fermentation broth was vacuum freeze-dried to obtain dark green Trichoderma Ta102 powder, the number of viable bacteria of which was ≥2×10 10 cfu / g;

[0018] (3) Preparation of dark green Trichoderma Ta102 wettable powder: Mix dark green Trichoderma Ta102 powder and adjuvants according to the following weight parts: 50-70 parts of dark green Trichoderma Ta102 powder, 40-60 parts of diatomaceous earth, 3-5 parts of wetting agent, 4-6 parts of dispersant, 0.3-0.6 parts of adhesive, and 1.5-2.5 parts of zinc sulfate, and mix well to obtain dark green Trichoderma Ta102 wettable powder.

[0019] Furthermore, in step (2), the liquid fermentation medium comprises the following components in parts by weight: 70-200 parts of exploded corn stalk powder, 40-80 parts of exploded vegetable stalk powder, 45-65 parts of wheat bran powder, 15-40 parts of glucose, 1-1.5 parts of (NH4)2SO4, 0.03-0.05 parts of KH2PO4, 0.04-0.06 parts of MgSO4·7H2O, and 6000-8000 parts of water.

[0020] In a seventh aspect, the present invention provides an application of the multifunctional organic fertilizer to inhibit strawberry Rhizoctonia root rot, strawberry Fusarium root rot, tomato damping-off and / or tomato Fusarium wilt.

[0021] In an eighth aspect, the present invention provides an application of the multifunctional organic fertilizer to promote the growth of strawberries or tomatoes.

[0022] The beneficial effects of the present invention are:

[0023] 1. The dark green Trichoderma Ta102 provided by the present invention has a highly active laccase and a composite degradation enzyme system, which can shorten the preparation and decomposition time of organic fertilizer to 9 to 12 days.

[0024] 2. The dark green Trichoderma Ta102 provided by the present invention can inhibit Rhizoctonia solani, Fusarium oxysporum or Fusarium moniliforme, and can also produce 153.42 μg / L indoleacetic acid. The organic fertilizer prepared by fermentation of the dark green Trichoderma Ta102 has the function of inhibiting soil-borne diseases such as damping-off and root rot and promoting growth.

[0025] 3. The process for preparing multifunctional organic fertilizer using dark green Trichoderma Ta102 provided by the present invention has lower requirements on fermentation conditions compared with the composite bacterial system constructed by multiple strains, which helps to simplify the organic fertilizer preparation process and reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a colony status diagram of Ta102 in Example 1 of the present invention.

[0028] Figure 2 This is a diagram of the plate screening results in Example 2 of the present invention.

[0029] In the figure, A is a front view of the colony, B is a back view of the colony, C is a view of the guaiacol plate screening result, D is a view of the aniline blue plate screening result, E is a view of the CMC plate screening result, and F is a view of the xylan plate screening result. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] Example 1 Isolation and identification of strains

[0032] (1) Strain source: Fermentation product of corn straw-cow dung compost at the peak period of laccase activity, collected from a composting site in Jinan, Shandong in May 2020.

[0033] (2) Strain Isolation: The fermentation samples were diluted to 10% with sterile water containing 0.3% Tween-80. -1 ~10 -7 100 μL of each gradient dilution was taken to coat a rose bengal sodium agar plate and cultured at 28°C for 3 days. Single colonies were picked from each gradient dilution plate for identification.

[0034] (3) The species was identified by morphological microscopic observation and internally transcribed spacer (ITS) comparison. Among them, the strain numbered Ta102 was the dominant strain isolated.

[0035] The colony status of Ta102 is shown in Figure 1 , the ITS gene sequence of Ta102 is:

[0036] .

[0037] After identification, the strain numbered Ta102 is Trichoderma atroviride, and it is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration. The storage address is No. 1 Beichen West Road, Chaoyang District, Beijing. The storage date is July 13, 2022, and the storage number is CGMCC No.40242.

[0038] Example 2 Qualitative analysis and identification of the degradative enzyme activity of Trichoderma aureum Ta102

[0039] (1) The dominant strain Trichoderma aureum Ta102 isolated in Example 1 was cultured on a PDA plate at 26° C. for 3 days;

[0040] (2) Qualitative detection of lignocellulose degrading enzymes using a plate, the process is as follows:

[0041] ① Screening of laccase-producing strains using guaiacol plates

[0042] a. Preparation of guaiacol plates: Guaiacol was sterilized by filtration alone to prepare a 1% stock solution (25 times), and aseptically added to a PDA medium containing 0.1 g / L CuSO4 at a concentration of 0.01% by volume, and the plates were cooled and poured.

[0043] b. Use a sterile cork puncher to take the colonies cultured on the PDA plate and inoculate them on the guaiacol plate. Prepare three replicate plates and invert them at 27±1℃. Record whether brick-red pigment is produced on the guaiacol plate. Brick-red pigments are marked as +, and negative pigments are marked as -. Select strains with strong laccase production ability based on the diameter of the pigment-producing area and record the time.

[0044] After cultivation, dark green Trichoderma Ta102 can produce obvious brick red pigment in guaiacol plate medium. On the 4th day, the diameter of the pigment produced was 9 cm (filling the plate). Figure 2 Figure C.

[0045] ② Screening of strains producing manganese peroxidase using aniline blue agar plates

[0046] a. Preparation of aniline blue plates: RB brilliant blue was sterilized by filtration alone to prepare a 2% stock solution (200 times), and aseptically added to a PDA medium containing 0.1 g / L CuSO4 at a concentration of 0.01% by volume, and the plates were cooled and poured.

[0047] b. Use a sterile cork puncher to take the colonies cultured on the PDA plate and inoculate them on the aniline blue plate. Prepare 3 replicate plates and invert them at 27±1℃. Record whether there is decolorization and transparency on the aniline blue plate. Decolorization is recorded as +, and vice versa as -. Select the strain with strong manganese peroxidase production ability according to the diameter of the transparent area and record the time.

[0048] After cultivation, dark green Trichoderma Ta102 can decolorize the dye in aniline blue plate culture medium. The diameter of the transparent area on the 4th day is 4.04cm. Figure 2 Figure D.

[0049] ③CMC plate screening of cellulase-producing strains:

[0050] a. CMC plate culture medium: CMC 10g, (NH4)2SO4 4g, KH2PO4 2g, MgSO4·7H2O 0.5g, peptone 1.0g, agar 16g, sodium deoxycholate 0.5g, distilled water 1000ml, autoclave at 121℃ for 20min.

[0051] b. Use a sterile punch to take the colonies cultured on the PDA plate, inoculate them on the CMC plate, prepare 3 replicate plates, invert and culture at 27±1℃ for 4 days, then cover the plate with 0.1% Congo red dye, let it stand for 30 minutes, and then decolorize it with 1mol / L NaCl for 1 hour to observe whether each strain produces a hydrolysis transparent zone. According to the diameter of the hydrolysis transparent zone, select the strain with strong cellulase production ability.

[0052] After cultivation, dark green Trichoderma Ta102 can produce hydrolysis transparent zone in CMC medium. The diameter of the transparent zone on the 4th day is 3.09cm. Figure 2 Figure E.

[0053] ④ Screening of hemicellulase-producing strains on xylan plates

[0054] a. Xylan plate medium: (NH4)2SO4 2g, MgSO4·7H2O 0.5g, KH2PO4 1g, NaCl 0.5g, xylan 2g, agar 20g, distilled water 1000mL, natural pH, autoclave at 121℃ for 20min.

[0055] b. Use a sterile hole puncher to take the colonies cultured on the PDA plate, inoculate them on the xylan plate, prepare 3 replicate plates, and invert and culture them at 25±1℃ for 4 days; then cover the plate with 0.1% Congo red dye, let it stand for 10 minutes, and then decolorize it with 2mol / L NaCl for 2-3 times to observe whether each strain produces a hydrolysis transparent zone. According to the diameter of the hydrolysis transparent zone, select the strain with strong hemicellulase production ability.

[0056] After cultivation, dark green Trichoderma Ta102 can produce hydrolysis transparent zone in xylan medium. The diameter of the transparent zone is 6.24cm on the 4th day. Figure 2 Figure F.

[0057] Plate screening showed that Trichoderma aureum Ta102 had four enzymes: laccase, manganese peroxidase, cellulase and hemicellulase.

[0058] Example 3 Quantitative detection of the degradative enzyme activity of Trichoderma aureum Ta102

[0059] ① Laccase activity detection: Activate Ta102 strain on PDA plate and prepare 1×10 7 The conidia suspension of 1000 g / mL was cultured at 27±1°C and 160 rpm for 5 days to obtain a fermentation liquid; the fermentation liquid was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant was a crude enzyme liquid.

[0060] Take 0.7mL crude enzyme solution and add 0.7mL ABTS buffer (3mmol / L ABTS, 0.1mol / L acetic acid-sodium acetate buffer pH=4.5) to start the reaction, and measure the change of absorbance within 10min at a wavelength of 420nm. Define the amount of enzyme required to convert 1μmolABTS per minute as one activity unit. Set three replicates. After testing, the laccase activity of dark green Trichoderma Ta102 was 396.50IU / mL,

[0061] ② Manganese peroxidase activity detection: Activate Ta102 strain on PDA plate and prepare 1×10 7 The conidia suspension of 1000 g / mL was inoculated into resveratrol culture medium at a volume ratio of 1%, and cultured at 26±1°C and 160 rpm for 5 days to obtain fermentation liquid; the fermentation liquid was centrifuged at 4°C and 12000 rpm for 20 minutes, and the supernatant was the crude enzyme liquid.

[0062] Mix 400 μL of the crude enzyme solution to be tested, 3.4 mL of 50 mmol / L sodium lactate buffer and 0.1 mL of 1.6 mmol / L manganese sulfate solution into a reaction solution, preheat in a 37°C water bath, then add 0.1 mL of 1.6 mmol / L H2O2 solution to start the reaction, quickly measure the absorbance at 240 nm, and measure it again after 4 minutes.

[0063] Within 1 minute, 1 μmol / L of Mn 2+ Converted to Mn 3+ 1 unit of enzyme activity. The sterilized fermentation broth was used as the control and three replicates were set. The manganese peroxidase activity of Trichoderma aureum Ta102 was 2.96 IU / mL.

[0064] ③ Cellulase activity detection: Activate Ta102 strain on PDA plate and prepare 1×10 7 The conidia suspension of 1% / mL was inoculated into CMC culture medium (CMC solid culture medium minus agar) at a volume ratio of 1%, and cultured at 26±1℃, 160rpm for 7 days to obtain fermentation liquid. The fermentation liquid was centrifuged at 4℃, 12000rpm for 10min, and the supernatant was the crude enzyme liquid. 400μL of citric acid-Na2HPO4 buffer (pH=4.5) containing 1% (w / v) CMC was added to a 2mL Eppendorf tube, preheated in a 50℃ water bath for 3min, and then 100μL of the crude enzyme liquid was added. After keeping warm for 30min, 1mL DNS reagent was added to terminate the reaction, and the mixture was fully shaken and boiled in a water bath for 5min. After cooling with cold water, the absorbance at a wavelength of 540nm was measured. The sterilized fermentation liquid was used as a control. Three replicates were set. After testing, the cellulase activity of dark green Trichoderma Ta102 was 3.25IU / mL.

[0065] ④ Hemicellulase activity detection: Activate dark green Trichoderma Ta102 on PDA plate and prepare 1×10 7 The conidia suspension of 100 / mL was inoculated into xylan culture medium (xylan solid culture medium minus agar) at a volume ratio of 1%, and cultured at 26±1℃ and 160rpm for 7 days to obtain fermentation liquid. 0.5mL of the fermentation liquid diluted 20 times was taken and added to a 1% xylan solution prepared with disodium hydrogen phosphate-lemon buffer (pH=4.8), and enzymolyzed at 50℃ for 30min. After enzymolysis, 3mL of DNS reagent was added, heated in a boiling water bath for 5min, then quickly cooled with ice water, and distilled water was added to 25mL. The absorbance at 540nm was measured, and three biological replicates were set up. The sterilized fermentation liquid was used as the control, and the calculation was performed according to the following formula:

[0066]

[0067] Where: W is the content of xylose generated by enzymatic hydrolysis, mg;

[0068] N is the dilution factor of the fermentation broth;

[0069] 30 is the enzymatic hydrolysis time, min;

[0070] V is the volume of the reaction solution, mL.

[0071] After testing, the hemicellulase activity of dark green Trichoderma Ta102 was 60.41 IU / mL.

[0072] ⑤ Determination of indoleacetic acid content: Activate Ta102 strain on PDA plate and prepare 1×10 7 The conidia suspension of 100 μg / mL was cultured at 27±1℃ and 160rpm for 5 days to obtain the fermentation broth; the fermentation broth was centrifuged at 4℃ and 12000rpm for 20min, and the supernatant was taken. The indoleacetic acid content in the fermentation supernatant was detected by GC-MS. The indoleacetic acid production of dark green Trichoderma Ta102 was 153.42μg / L.

[0073] Example 4 Antagonistic effect of Trichoderma solani Ta102 on Rhizoctonia solani, Fusarium oxysporum or Fusarium moniliforme

[0074] Dark green Trichoderma Ta102 was inoculated into PDA plates with Rhizoctonia solani, Fusarium oxysporum or Fusarium moniliforme respectively, activated twice at 25℃, and after the second activation and growth for 3 days, a bacterial cake was punched from the edge of the colony with a 5mm puncher. Dark green Trichoderma Ta102 and Rhizoctonia solani were placed at both ends of the center line of the same 9cm PDA plate, and the plate number was recorded as A; Dark green Trichoderma Ta102 and Fusarium oxysporum were placed at both ends of the center line of the same 9cm PDA plate, and the plate number was recorded as B; Dark green Trichoderma Ta102 and Fusarium moniliforme were placed at both ends of the center line of the same 9cm PDA plate, and the plate number was recorded as C; Plates numbered A, B and C were all cultured at 25℃ for 6 to 8 days, and the distance T (unit: cm) from the front end of Ta102 growth to the pathogen cake was measured. The plates cultured with the three pathogens alone were used as controls, and the distance C (unit: cm) from the front end of the pathogen growth to the pathogen cake was measured as a control. Each plate was set up with 5 biological replicates, and the inhibition rate was calculated as follows:

[0075]

[0076] The results show that the inhibition rate of Trichoderma viride Ta102 against Rhizoctonia solani is 100.00%±0.00%, the inhibition rate against Fusarium oxysporum is 100.00%±0.00%, and the inhibition rate against Fusarium moniliforme is 75.85%±4.62%. The results show that Trichoderma viride Ta102 has a significant antagonistic effect on the above three pathogens.

[0077] Example 6 Preparation of dark green Trichoderma Ta102 bacterial agent and multifunctional organic fertilizer

[0078] (1) Preparation of seed solution: Dark green Trichoderma Ta102 stored at -80°C was inoculated on a PDA plate, activated at 26°C for 3 days, hyphae were taken from the edge of the colony and inoculated on a PDA plate, cultured at 26°C for 3 days, and the hyphae culture was repeated once to obtain an activated strain; the activated strain was cultured on a PDA plate at 26°C, 12 h light-12 h dark conditions for 7 days to prepare 10 7 The conidia suspension of 100 / mL was inoculated into PDB culture medium at a volume ratio of 1:100 and cultured at 26°C and 180 rpm for 48 hours to obtain seed solution with a viable count of 1×10 5 cfu / mL;

[0079] (2) Preparation of dark green Trichoderma Ta102 powder:

[0080] a. Prepare a liquid fermentation medium comprising the following components in parts by weight: 70 parts of blasted corn stalk powder, 40 parts of blasted vegetable stalk powder, 45 parts of wheat bran powder, 15 parts of glucose, 1 part of (NH4)2SO4, 0.03 parts of KH2PO4, 0.04 parts of MgSO4·7H2O, and 6000 parts of water.

[0081] b. The seed liquid was added to the fermentation tank filled with liquid fermentation medium at a volume percentage of 15%. The fermentation temperature was 27°C before 12 hours and 25°C after 12 hours. The initial pH was 4.0, the stirring speed was 200 rpm, the ventilation volume was 10 L / min, and the fermentation time was 72 hours. The number of viable bacteria in the fermentation product was 1×10 9 cfu / mL; the fermentation broth was vacuum freeze-dried to obtain dark green Trichoderma Ta102 powder, the number of viable bacteria of which was ≥2×10 10 cfu / g;

[0082] (3) Preparation of dark green Trichoderma Ta102 wettable powder: Dark green Trichoderma Ta102 powder and adjuvants were mixed according to the following weight parts: 50 parts of dark green Trichoderma Ta102 powder, 40 parts of diatomaceous earth, 3 parts of Tween-80, 4 parts of sodium methylene dinaphthalene sulfonate, 0.5 parts of starch paste, and 1.5 parts of zinc sulfate. The dark green Trichoderma Ta102 wettable powder was prepared, and the effective viable count was ≥ 8×10 9 cfu / g.

[0083] (4) Preparation of multifunctional organic fertilizer: The raw materials were fully mixed according to the following weight proportions: 0.1 part of dark green Trichoderma Ta102 wettable powder, 100 parts of cow dung, and 30 parts of crushed corn stalks, with a water-to-material ratio of 1:0.7 and an initial pH of 6. The fertilizer was piled and fermented at room temperature for 12 days, with the pile turned every 3 days. After fermentation, the fertilizer was dried and crushed.

[0084] Example 7 Preparation of dark green Trichoderma Ta102 bacterial agent and multifunctional organic fertilizer

[0085] (1) Preparation of seed solution: Dark green Trichoderma Ta102 stored at -80°C was inoculated on a PDA plate, activated at 30°C for 3 days, hyphae were taken from the edge of the colony and inoculated on a PDA plate, cultured at 230°C for 3 days, and the hyphae culture was repeated once to obtain an activated strain; the activated strain was cultured on a PDA plate at 30°C, 12 h light-12 h dark conditions for 12 days to prepare 1×10 7 The conidia suspension of 100 / mL was inoculated into PDB culture medium at a volume ratio of 1:100 and cultured at 26°C and 180 rpm for 72 hours to obtain seed solution. The number of viable bacteria in the seed solution was 9×10 5 cfu / mL;

[0086] (2) Preparation of dark green Trichoderma Ta102 powder:

[0087] a. Prepare a liquid fermentation medium comprising the following components in parts by weight: 200 parts of blasted corn stalk powder, 80 parts of blasted vegetable stalk powder, 65 parts of wheat bran powder, 40 parts of glucose, 1.5 parts of (NH4)2SO4, 0.05 parts of KH2PO4, 0.06 parts of MgSO4·7H2O, and 8000 parts of water.

[0088] b. The seed liquid was added to the fermentation tank filled with liquid fermentation medium at a volume percentage of 25%. The fermentation temperature was 33°C before 12 hours and 30°C after 12 hours. The initial pH was 5.0, the stirring speed was 300 rpm, the ventilation volume was 15 L / min, and the fermentation time was 96 hours. The number of viable bacteria in the fermentation product was 8×10 9 cfu / mL; the fermentation broth was vacuum freeze-dried to obtain dark green Trichoderma Ta102 powder, the number of viable bacteria of which was ≥2×10 10 cfu / g;

[0089] (3) Preparation of dark green Trichoderma Ta102 wettable powder: Dark green Trichoderma Ta102 powder and adjuvants were mixed according to the following weight parts: dark green Trichoderma Ta102 powder 70 parts, diatomaceous earth 60 parts, Tween-80 5 parts, sodium methylene dinaphthalene sulfonate 6 parts, starch paste 0.6 parts, zinc sulfate 2.5 parts, and mixed to obtain dark green Trichoderma Ta102 wettable powder, the effective viable count of which was ≥ 8 × 10 9 cfu / g.

[0090] (4) Preparation of multifunctional organic fertilizer: The raw materials were fully mixed according to the following weight proportions: 0.5 parts of dark green Trichoderma Ta102 wettable powder, 150 parts of cow dung, and 40 parts of crushed corn stalks. The water-to-material ratio was controlled to be 1:0.75, and the initial pH was 6±0.5. The fertilizer was piled and fermented at room temperature for 9 days, with the pile turned every 3 days. After fermentation, the fertilizer was dried and crushed.

[0091] Example 8 Preparation of dark green Trichoderma Ta102 bacterial agent and multifunctional organic fertilizer

[0092] (1) Preparation of seed solution: Dark green Trichoderma Ta102 stored at -80°C was inoculated on a PDA plate, activated at 28°C for 3 days, hyphae were taken from the edge of the colony and inoculated on a PDA plate, cultured at 28°C for 3 days, and the hyphae culture was repeated once to obtain an activated strain; the activated strain was cultured on a PDA plate at 28°C, 12 h light-12 h dark conditions for 9 days to prepare 10 7 The conidia suspension of 5 × 10 / mL was inoculated into PDB culture medium at a volume ratio of 1:100 and cultured at 26°C and 180 rpm for 48 to 72 hours to obtain seed solution with a viable count of 5 × 105 cfu / mL;

[0093] (2) Preparation of dark green Trichoderma Ta102 powder:

[0094] a. Prepare a liquid fermentation medium comprising the following components in parts by weight: 100 parts of blasted corn stalk powder, 60 parts of blasted vegetable stalk powder, 50 parts of wheat bran powder, 25 parts of glucose, 1.2 parts of (NH4)2SO4, 0.04 parts of KH2PO4, 0.05 parts of MgSO4·7H2O, and 7000 parts of water.

[0095] b. The seed liquid was added to the fermentation tank filled with liquid fermentation medium at a volume percentage of 20%. The fermentation temperature was 30°C before 12 hours and 27°C after 12 hours. The initial pH was 5.0, the stirring speed was 250 rpm, the ventilation volume was 12 L / min, and the fermentation time was 85 hours. The number of viable bacteria in the fermentation product was 5×10 9 cfu / mL; the fermentation broth was vacuum freeze-dried to obtain dark green Trichoderma Ta102 powder, the number of viable bacteria of which was ≥2×10 10 cfu / g;

[0096] (3) Preparation of dark green Trichoderma Ta102 wettable powder: Dark green Trichoderma Ta102 powder and adjuvants were mixed according to the following weight parts: 60 parts of dark green Trichoderma Ta102 powder, 50 parts of diatomaceous earth, 4 parts of Tween-80, 5 parts of sodium methylene dinaphthalene sulfonate, 0.5 parts of starch paste, and 2 parts of zinc sulfate. The dark green Trichoderma Ta102 wettable powder was prepared, and the effective viable count was ≥ 8×10 9 cfu / g.

[0097] (4) Preparation of multifunctional organic fertilizer: The raw materials were fully mixed according to the following weight proportions: 0.3 parts of dark green Trichoderma Ta102 wettable powder, 120 parts of cow dung, and 35 parts of crushed corn stalks. The water-to-material ratio was controlled to be 1:0.7, and the initial pH was 6±0.5. The fertilizer was piled and fermented at room temperature for 10 days, and the pile was turned every 3 days. After the fermentation was completed, the fertilizer was dried and crushed.

[0098] Example 9 Field test of multifunctional organic fertilizer applied to strawberry planting

[0099] (1) Experimental location: Strawberry greenhouse in Licheng District, Jinan City, Shandong Province. The strawberry variety used for the experiment was Hongyan.

[0100] (2) Test content:

[0101] ① Set up multiple cells, each with an area of ​​10m 2 , protection rows are spaced between cells.

[0102] ② The plots were randomly divided into four groups, namely, treatment group, control group 1, control group 2 and blank control group, with five plots in each group. The specific treatments were as follows:

[0103] Treatment group: The organic fertilizer applied was the multifunctional organic fertilizer prepared in Example 6;

[0104] Control group 1: The organic fertilizer used was the organic fertilizer prepared by the same method as in Example 6, but the dark green Trichoderma Ta102 agent was not added during the preparation process;

[0105] Control group 2: The organic fertilizer used was the organic fertilizer prepared by the same method as in Example 6, but during the preparation process, the dark green Trichoderma Ta102 agent was replaced with a commercial fermentation agent (active ingredients: Bacillus subtilis + Saccharomyces cerevisiae + Trichoderma longifolia, total number of viable bacteria> 2×10 9 cfu / g).

[0106] Blank control group: no organic fertilizer was applied.

[0107] The fertilizer application methods in strawberry planting for the treatment group and control groups 1 and 2 were unified and conventional, i.e., 400 kg / mu was applied before transplanting, 20 kg / mu was applied before flowering, and 40 kg / mu was applied during the fruiting period.

[0108] ③Results statistics and analysis:

[0109] a. At the strawberry seedling stage, 10 plants were randomly selected from each treatment to determine the seedling index and yield per mu.

[0110] in,

[0111]

[0112] b. During the strawberry harvest period, the strawberry disease situation was investigated and the control effect of each treatment was statistically calculated. The formula is as follows:

[0113]

[0114]

[0115] Among them, the disease index I is the disease index of the blank control group, and the disease index II refers to the disease index of the treatment group or control group 1 or control group 2.

[0116] Among them, the disease classification of strawberry Rhizoctonia root rot (caused by Rhizoctonia solani) refers to the "Forecasting and Prevention of Major Crop Diseases and Pests", and the disease classification of strawberry Fusarium root rot (caused by Fusarium oxysporum) refers to the "NY / T1464.16-2007 Pesticide Field Efficacy Test Guidelines".

[0117] The results are shown in Table 1:

[0118] Table 1 Effect of multifunctional organic fertilizer on strawberry planting

[0119]

[0120] Note: Inconsistent letters indicate significant differences (P<0.05).

[0121] The multifunctional organic fertilizer (treatment group) prepared by using the dark green Trichoderma Ta102 of the present invention is significantly higher than that of each control group in terms of seedling index, yield and the prevention and control effect on strawberry Rhizoctonia root rot and Fusarium root rot, indicating that the multifunctional organic fertilizer has excellent strawberry Rhizoctonia root rot, strawberry Fusarium root rot inhibitory function and strawberry growth promotion function.

[0122] Example 10 Field test of multifunctional organic fertilizer applied to tomato planting

[0123] (1) Experimental location: Tomato greenhouse in Licheng District, Jinan City, Shandong Province. The tomato variety used for the experiment was pink-fruited tomato.

[0124] (2) Test content:

[0125] ① Set up multiple cells, each with an area of ​​10m 2 , protection rows were placed between the plots, and the treatment plots were randomly arranged.

[0126] ② The plots were divided into four groups, namely, treatment group, control group 1, control group 2 and blank control group, with five plots in each group. The specific treatments were as follows:

[0127] Treatment group: The organic fertilizer applied was the multifunctional organic fertilizer prepared in Example 7;

[0128] Control group 1: The organic fertilizer used was the organic fertilizer prepared by the same method as in Example 7, but the dark green Trichoderma Ta102 agent was not added during the preparation process;

[0129] Control group 2: The organic fertilizer used was prepared in the same manner as in Example 7, but during the preparation process, the dark green Trichoderma Ta102 agent was replaced with a commercial fermentation agent (active ingredients: Bacillus subtilis + Saccharomyces cerevisiae + Trichoderma longifolia, total number of viable bacteria> 2×10 9 cfu / g)

[0130] Blank control group: no organic fertilizer was applied.

[0131] The fertilizer application methods in tomato planting for the treatment group and control groups 1 and 2 were unified and conventional, i.e., 500 kg / mu was applied before planting.

[0132] ③Results statistics and analysis:

[0133] a. At the tomato seedling stage, 10 plants were randomly selected from each treatment to determine the seedling index and yield per mu.

[0134] in,

[0135] b. During the tomato harvest period, the tomato disease situation was investigated and the control effect of each treatment was calculated and statistically analyzed. The formula is as follows:

[0136]

[0137]

[0138] Among them, the disease index I is the disease index of the blank control group, and the disease index II refers to the disease index of the treatment group or control group 1 or control group 2.

[0139] Among them, the disease classification of tomato damping-off disease (caused by Rhizoctonia solani) refers to the "Forecasting and Prevention of Major Crop Diseases and Pests", and the disease classification of tomato wilt disease (caused by Fusarium oxysporum and Fusarium moniliforme) refers to the "NY / T1464.16-2007 Guidelines for Field Efficacy Tests of Pesticides".

[0140] The results are shown in Table 2:

[0141] Table 2 Effect of multifunctional organic fertilizer on tomato planting

[0142]

[0143] Note: Inconsistent letters indicate significant differences (P<0.05).

[0144] The multifunctional organic fertilizer (treatment group) prepared using the dark green Trichoderma Ta102 of the present invention is significantly higher than that of each control group in terms of seedling index and the control and prevention effects on tomato damping-off disease and tomato wilt disease, and is significantly higher than that of the blank control group and control group 1 in terms of yield, and is improved but not significantly higher than that of control group 2 using the commercial decomposition and fermentation group.

[0145] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A dark green Trichoderma Ta102, characterized in that Dark green Trichoderma Trichoderma atroviride ) Ta102 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is July 13, 2022, and the deposit number is CGMCC No.40242.

2. A use of dark green Trichoderma Ta102 as claimed in claim 1, characterized in that: Highly active laccase, manganese peroxidase, hemicellulase and cellulase were produced using Trichoderma virens Ta102.

3. A use of dark green Trichoderma Ta102 as claimed in claim 1, characterized in that: Production of indoleacetic acid using Trichoderma aureum Ta102.

4. A use of dark green Trichoderma Ta102 as claimed in claim 1, characterized in that: Dark green Trichoderma Ta102 was used to inhibit Rhizoctonia solani, Fusarium oxysporum or Fusarium moniliforme.

5. A use of dark green Trichoderma Ta102 as claimed in claim 1, characterized in that: Organic fertilizer was prepared by fermentation with dark green Trichoderma Ta102.

6. A multifunctional organic fertilizer, characterized in that: In terms of weight, the raw materials include 0.1-0.5 parts of dark green Trichoderma Ta102 wettable powder, 100-150 parts of cow dung, and 30-40 parts of crushed corn stalks, wherein the effective viable bacteria count of dark green Trichoderma Ta102 wettable powder is ≥8×10 9 cfu / g; the deposit number of Trichoderma aureum Ta102 is CGMCC No.40242; The preparation method is to control the water-to-material ratio to 1: (0.7±0.05), the initial pH value to 6±0.5, pile fermentation at room temperature for 9 to 12 days, turn the pile every 3 days, and dry and crush after fermentation is completed.

7. The multifunctional organic fertilizer according to claim 6, wherein Dark green Trichoderma Ta102 wettable powder was prepared according to the following preparation method: (1) Preparation of seed solution: Inoculate dark green Trichoderma Ta102 stored at -80°C on a PDA plate, activate at 26-30°C for 3 days, take mycelium from the edge of the colony and inoculate it on a PDA plate, culture at 26-30°C for 3 days, repeat the culture once to obtain an activated strain; culture the activated strain on a PDA plate at 26-30°C, 12 h light-12 h dark conditions for 7-12 days to prepare 10 7 The conidia suspension of 100 / mL was inoculated into PDB culture medium at a volume ratio of 1:100 and cultured at 26°C and 180 rpm for 48-72 hours to obtain seed solution with a viable count of 1×10 5 ~9×10 5 cfu / mL; (2) Preparation of Trichoderma Ta102 powder: The seed liquid was added to a fermentation tank filled with liquid fermentation medium at a volume percentage of 15% to 25%. The fermentation temperature was 27 to 33°C before 12 hours and 25 to 30°C after 12 hours. The initial pH was 4.0 to 5.

0. The stirring speed was 200 to 300 rpm, the ventilation volume was 10 to 15 L / min, and the fermentation time was 72 to 96 hours. The number of viable bacteria in the fermentation product was 1 × 10 9 ~8×10 9 cfu / mL; the fermentation broth was vacuum freeze-dried to obtain dark green Trichoderma Ta102 powder, the number of viable bacteria of which was ≥2×10 10 cfu / g; (3) Preparation of dark green Trichoderma Ta102 wettable powder: Mix dark green Trichoderma Ta102 powder and adjuvants in the following weight proportions: 50-70 parts of dark green Trichoderma Ta102 powder, 40-60 parts of diatomaceous earth, 3-5 parts of wetting agent, 4-6 parts of dispersant, 0.3-0.6 parts of adhesive, and 1.5-2.5 parts of zinc sulfate, and mix well to obtain dark green Trichoderma Ta102 wettable powder.

8. The multifunctional organic fertilizer according to claim 7, wherein In step (2), the liquid fermentation medium comprises the following components in parts by weight: 70-200 parts of exploded corn stalk powder, 40-80 parts of exploded vegetable stalk powder, 45-65 parts of wheat bran powder, 15-40 parts of glucose, 1-1.5 parts of (NH4)2SO4, 0.03-0.05 parts of KH2PO4, 0.04-0.06 parts of MgSO4·7H2O, and 6000-8000 parts of water.

9. An application of the multifunctional organic fertilizer as claimed in claim 6, characterized in that: Apply multifunctional organic fertilizers to inhibit strawberry Rhizoctonia root rot, strawberry Fusarium root rot, tomato damping-off and / or tomato Fusarium wilt.

10. An application of the multifunctional organic fertilizer as claimed in claim 6, characterized in that: Apply multifunctional organic fertilizer to promote the growth of strawberries or tomatoes.

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