Trichoderma harzianum for promoting growth of pasture, microbial agent and application of trichoderma harzianum and microbial agent

Abstract

The invention relates to trichoderma harzianum for promoting growth of pasture, a microbial agent and application of the trichoderma harzianum and the microbial agent. The trichoderma harzianum is classified and named as trichoderma harzianum, and the trichoderma harzianum is named as trichoderma harzianum. The preservation number of the trichoderma harzianum is CGMCC (China General Microbiological Culture Collection Center) No.41719. The trichoderma harzianum can effectively promote the growth of forage grass.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular to a Trichoderma harzianum and a microbial agent for promoting forage growth and applications thereof. Background Art

[0002] The acceleration of urbanization and economic development has led to the occupation and destruction of a large number of grasslands, affecting the growth of forage grass. At the same time, forage grass production technology is relatively backward, lacking modern management methods and technical support, making it difficult to meet the demand for efficient and high-quality forage grass. Natural grasslands mainly provide forage grass for cattle, sheep, horses, camels, yaks and other livestock in the animal husbandry industry.

[0003] In order to further meet the needs of animal husbandry, it is of great significance to seek a safe and efficient microorganism that promotes grass growth. Summary of the invention

[0004] The purpose of the present disclosure is to provide a Trichoderma harzianum and a microbial agent for promoting the growth of forage.

[0005] In order to achieve the above-mentioned object, the present disclosure provides a first aspect of Trichoderma harzianum, the classification of which is named as Trichoderma harzianum Trichoderma harzianum ; The deposit number of the Trichoderma harzianum is CGMCC No.41719.

[0006] Optionally, the Trichoderma harzianum rpb2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; tef1α The nucleotide sequence of the gene is shown in SEQ ID NO.2; the nucleotide sequence of the ITS gene of Trichoderma harzianum is shown in SEQ ID NO.3.

[0007] Optionally, the Trichoderma harzianum is round in shape with regular edges, the front of the colony is green with green aerial hyphae and green spores, and the back of the colony is off-white.

[0008] A second aspect of the present disclosure provides a microbial agent, which contains Trichoderma harzianum with a deposit number of CGMCC No. 41719 and a culture medium.

[0009] Optionally, per gram of the microbial agent, the number of viable bacteria of Trichoderma harzianum with a deposit number of CGMCC No.41719 is 10 8 -10 11 CFU.

[0010] Optionally, the culture medium is potato dextrose agar medium and potato dextrose medium.

[0011] The third aspect of the present disclosure provides the use of the Trichoderma harzianum described in the first aspect or the microbial agent described in the second aspect in promoting forage growth.

[0012] Optionally, the forage grass is selected from one or more of Dactylis grass, Festuca australis, Festuca sinensis and Agropyron ovata.

[0013] A fourth aspect of the present disclosure provides a method for promoting forage growth, the method comprising: applying the Trichoderma harzianum described in the first aspect or the microbial agent described in the second aspect to the soil at the root of the forage.

[0014] Optionally, the dosage of the Trichoderma harzianum is 10 8 -10 11 CFU / m 2 .

[0015] Through the above technical scheme, the present disclosure provides a Trichoderma and a microbial agent for promoting the growth of forage grass. The fungus and the agent can effectively promote the growth of forage grass, and the Trichoderma harzianum has the advantages of safety and high efficiency, and has good application prospects.

[0016] Other features and advantages of the present disclosure will be described in detail in the following detailed description.

[0017] Biomaterial Deposit The Trichoderma harzianum disclosed in the present invention is a pure culture isolated by the inventor of the present invention from the soil of Keketuohai in Fuyun County, Altay Prefecture, Xinjiang. Its preservation number is CGMCC No.41719, and the preservation date is December 13, 2024. The preservation unit is the General Microbiological Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Trichoderma harzianum Trichoderma harzianum . BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 is a colony morphology diagram of the Trichoderma disclosed herein. DETAILED DESCRIPTION

[0019] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0020] The inventors of the present disclosure isolated a pure culture from the soil of Keketuohai in Fuyun County, Altay, Xinjiang, and identified that the fungus belongs to the genus Trichoderma ( Trichoderma), named Trichoderma harzianum Trichoderma harzianum The strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, a depository designated by the State Intellectual Property Office, on December 13, 2024, with a deposit number of CGMCC No.41719.

[0021] The present disclosure provides a first aspect of Trichoderma harzianum, the classification of which is named Trichoderma harzianum Trichoderma harzianum ; The deposit number of the Trichoderma harzianum is CGMCC No.41719.

[0022] The Trichoderma harzianum disclosed in the present invention can survive and grow in a conventional bacterial culture medium. The Trichoderma harzianum disclosed in the present invention as described above is a Trichoderma harzianum strain that can promote the growth of forage.

[0023] According to the present disclosure, the Trichoderma harzianum rpb2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; tef1α The nucleotide sequence of the gene is shown in SEQ ID NO.2; the nucleotide sequence of the ITS gene of Trichoderma harzianum is shown in SEQ ID NO.3.

[0024] In one embodiment of the present disclosure, Figure 1 As shown, the shape of the Trichoderma harzianum is round with regular edges, the front of the colony is green, has green aerial hyphae and green spores, and the back of the colony is grayish white. The spore yield is relatively high.

[0025] A second aspect of the present disclosure provides a microbial agent, which contains Trichoderma harzianum with a deposit number of CGMCC No. 41719 and a culture medium.

[0026] The number of viable bacteria of Trichoderma harzianum in the microbial agent can vary within a wide range. In one embodiment of the present disclosure, the number of viable bacteria of Trichoderma harzianum with a deposit number of CGMCC No. 41719 per gram of the microbial agent is 10 8 -10 11 CFU.

[0027] According to the present disclosure, the culture medium is potato dextrose agar medium (PDA) and potato dextrose medium (PD).

[0028] Optionally, the preparation method of the potato dextrose agar solid culture medium (PDA) is as follows: take 20 g of peeled potatoes, boil them for 20 minutes to extract juice, mix the juice with 2 g of glucose, 1.5 g of agar and water to make 100 mL, the pH is 6.5-7.0, put it into 2 Erlenmeyer flasks, each with 50 mL, and make it into a slant or a flat plate after sterilization; the preparation method of the potato dextrose liquid culture medium (PD) is as follows: take 20 g of peeled potatoes, boil them for 20 minutes to extract juice, mix the juice with 2 g of glucose and water to make 100 mL, the pH is 6.5-7.0, put it into 2 Erlenmeyer flasks, each with 50 ml, and sterilize.

[0029] Alternatively, the above-mentioned various culture media can be sterilized according to conventional sterilization methods and used for later use, for example, sterilized at 115-125° C. and 1.5-2 standard atmospheric pressures for 10-30 minutes.

[0030] Among them, the preparation method of the microbial agent may include: inoculating Trichoderma harzianum with a deposit number of CGMCC No. 41719 into a potato glucose agar solid culture medium, culturing in an incubator at 25°C for 7 days, taking 3-4 bacterial blocks therefrom and adding them to a seed bottle. Cultivate in a shaker at 25°C and 180rpm for 48 hours to prepare a seed solution for standby use. Add the seed solution to a seed fermentation tank at a volume ratio of 1:150 for cultivation, and then add it to a fermentation tank of a thick-walled spore fermentation medium (by weight, containing 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, 0.03% MgSO4) at a volume ratio of 1:20, and cultivate at 28°C for d until 90% of the mycelium forms thick-walled spores to obtain a thick-walled spore fermentation liquid; then use diatomaceous earth to adsorb the fermentation liquid, centrifuge or plate and frame filter, and dry it to form a wettable powder.

[0031] Preferably, the viable count of Trichoderma harzianum with a deposit number of CGMCC No. 41719 per gram of the microbial agent obtained by culturing can be 10 8 -10 11 CFU, preferably 10 9 -10 10 CFU. During the culture process, the number of viable bacteria can be tested by conventional methods, such as the blood cell plate counting method or the OD value observation method to obtain the concentration of viable bacteria. The cultured bacterial liquid can be used directly as a microbial agent. Preferably, the bacterial liquid is further processed into a microbial agent in a dosage form that is more convenient to store through steps including sterile filtration and freeze drying. Among them, the culture conditions of the strain are not particularly limited, and can be the conditions commonly used in the culture process of Trichoderma harzianum, such as shaking culture on a shaker, the culture temperature can be 25-30°C, and the culture time can be 1-2 days.

[0032] The third aspect of the present disclosure provides the use of the Trichoderma harzianum described in the first aspect or the microbial agent described in the second aspect in promoting forage growth.

[0033] In one embodiment of the present disclosure, the forage grass is natural forage grass, for example, one or more of Dactylis grass, Festuca australis, Festuca sinensis and Agropyron ovata.

[0034] A fourth aspect of the present disclosure provides a method for promoting forage growth, the method comprising: applying the Trichoderma harzianum described in the first aspect or the microbial agent described in the second aspect to the soil at the root of the forage.

[0035] In one embodiment of the present disclosure, the dosage of Trichoderma harzianum is 10 8 -10 11 CFU / m 2 .

[0036] In the above embodiment, application can be carried out in a manner conventionally adopted by those skilled in the art, such as spraying, root irrigation or soil mixing.

[0037] The present invention is further described in detail below in conjunction with examples, but the scope of the present invention is not limited to the following examples.

[0038] The preparation method of the potato dextrose agar solid medium (PDA) used in this example is as follows: 20 g of peeled potatoes are boiled for 20 minutes to extract the juice, 2 g of glucose and 1.5 g of agar are added to the juice, water is added to 100 mL, the pH is 6.5-7.0, and the juice is filled into 2 Erlenmeyer flasks, each with 50 mL, and sterilized to form a slant or a plate.

[0039] The formula of the fermentation tank of the chlamydospore fermentation medium is: by weight, it contains 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, and 0.03% MgSO4.

[0040] Example 1 This example is used to illustrate the separation and purification of Trichoderma harzianum disclosed in the present invention.

[0041] 1. Soil collection and treatment.

[0042] Soil samples were collected in Keketuohai, Fuyun County, Altay Prefecture, Xinjiang Uygur Autonomous Region.

[0043] The collection method is as follows: 10m away from the vegetation community at each sampling point 2In the sample plot, the five-point diagonal sampling method was used for soil collection. When sampling, after removing 2 cm of the topsoil, the soil around the plant roots was collected, and the rhizosphere soil of the host plant at a depth of 20 cm was taken. After mixing, 200 g was taken and placed in a sterilized self-sealing bag, brought back to the laboratory, and records were made (including: collection location, longitude and latitude, altitude, and covering vegetation), and stored at 4°C.

[0044] 2. Isolation and purification of strain MCSZ-1.

[0045] 1. Soil sample processing: Take 10g of sieved soil and add it to 90mL of sterile water. Shake evenly to obtain a mixture. Take 1mL of the mixture and add it to a test tube containing 9mL of sterile water. Mix evenly to obtain a dilution of 10. -1 The dilution was prepared by 10-fold dilution method to obtain 10 -2 , 10 -3 Dilutions of different dilutions. -3 100 μL of the soil sample dilution was applied on the separation PDA medium plate, and then the dilution was evenly applied with a sterile applicator, and repeated 3 times on the PDA medium. Cultured in a fungus incubator at 28°C, observed the fungal colonies after 10 days, counted the suspected Trichoderma colonies on the plate, and carried out the next step of purification.

[0046] 2. Cultivation and purification of strains: Pick suspected Trichoderma harzianum colonies on the plate and place them on PDA medium for multiple rounds of purification culture until the strain is completely purified. The total culture period is more than three weeks. The purified Trichoderma harzianum strain is temporarily stored on the PDA slant in a 4°C refrigerator. At the same time, three copies are collected in 30% glycerol tubes and stored in an ultra-low temperature refrigerator. According to the growth needs, culture in appropriate amounts and at appropriate times, with 10 per strain of Trichoderma harzianum. 8 The amount of CFU was measured and the soil at the root of Dactylis was irrigated for 40 days. The results showed that strain MCSZ-1 had a good effect in promoting forage growth (see Example 2 for details). Therefore, strain MCSZ-1 was identified as follows.

[0047] 3. Identification of strain MCSZ-1.

[0048] 1. Morphological identification: Take a bacterial cake of the same diameter from the edge of the purified colony of strain MCSZ-1 and place it in the center of the culture medium (PDA culture medium) to observe the growth rate of the strain, growth status during the culture process, colony color, hyphae morphology and other characteristics. Figure 1 shown.

[0049] The results showed that after culturing at 27°C for 7 days, the strain MCSZ-1 isolated and purified in the above steps had radially circular colonies with regular edges. The front of the colonies was green and had green aerial hyphae and green spores, and the aerial hyphae extended in all directions. The back of the colonies was grayish white.

[0050] 2. Molecular biology identification: DNA extraction: scrape an appropriate amount of mycelium of strain MCSZ-1 and put it into a sterile centrifuge tube, place it at 0℃ for more than 30 minutes, and before starting DNA extraction, put a steel bead in each centrifuge tube, oscillate it at a frequency of 50Hz for 3 minutes using a freeze-thaw cell disruptor. After taking it out, follow the operating steps of the Fungal Genome Extraction Kit (Adlai Fungal DNA Rapid Extraction Kit) in sequence. Collect the DNA in a new centrifuge tube and determine the concentration using an ultra-micro spectrophotometer. Qualified DNA can be amplified by PCR.

[0051] PCR amplification: internal transcribed spacer (ITS) of ribosomal RNA encoding genes, translation elongation factor 1α ( tef 1α) introns 4 and 5 and most of the last exon, and rpb 2 (the second subunit of RNA polymerase) gene was amplified, and the primer sequences are shown in Table 1: Table 1

[0052] In the above primers, Y is T or C; R is G or A; W is A or T; and S is C or G.

[0053] PCR reaction system: 2 μL of upstream and downstream primers, 25 μL of Taq PCR StarMix (with Loading Dye), 1 μL of strain DNA template, 20 μL of ddH2O, and a total system of 50 μL.

[0054] PCR amplification program for RPB and TEF genes: 95°C for 3 min, 95°C for 30 s, 52°C for 30 s, 72°C for 1 min (35 cycles), and 72°C for 5 min.

[0055] PCR amplification program for ITS sequence: 95°C for 3 min, 95°C for 30 s, 50°C for 30 s, 72°C for 1 min (35 cycles), and 72°C for 5 min.

[0056] PCR product detection and sequencing: prepare a 1% agarose gel, add 6 μL of DNA marker to the first or last gel well, add 6 μL of PCR product to other gel wells, run electrophoresis at 125 V for 30 min, observe the bands under a gel imaging system, and collect images.

[0057] After the sample was successfully amplified, it was sent to the company (Qingke Biotechnology Company) for sequencing, and the strain MCSZ-1 was measured. rpb 2 The sequence is shown in SEQ ID NO.1, tef The 1α sequence is shown in SEQ ID NO.2, and the ITS sequence is shown in SEQ ID NO.3.

[0058] According to the latest standards for the classification of Trichoderma species proposed by Cai and Druzhinina: rpb2 ≥99%, tef1α ≥97% and ITS≥76% can be identified as a Trichoderma species. Using the latest standards for Trichoderma species identification, we identified strain MCSZ-1 rpb2 Gene, tef1α The gene and ITS region sequences were compared in NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cg) and it was found that strain MCSZ-1 rpb2 The gene similarity with Trichoderma harzianum is 99.02-99.43%, and the strain MCSZ-1 tef The 1α gene has a similarity of 97-99.92% with that of Trichoderma harzianum, and the ITS sequence of strain MCSZ-1 has a similarity of 99.23% with that of Trichoderma harzianum. Therefore, we confirmed the classification of the Trichoderma species and named it Trichoderma harzianum.

[0059] Through the above morphological and molecular biological identification, strain MCSZ-1 was identified as Trichoderma ( Trichoderma harzianum ), the strain was deposited on December 13, 2024 in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, with the address at: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No.41719.

[0060] Example 2 This example is used to illustrate the preparation of microbial inoculants.

[0061] The Trichoderma harzianum with the deposit number of CGMCC No.41719 was inoculated into a potato dextrose agar solid medium (PDA), cultured in an incubator at 25°C for 7 days, and 3-4 bacterial blocks were taken from the PDA and added to a seed bottle. The culture was shaken at 25°C and 180rpm for 48 hours to prepare a seed solution for standby use. The seed solution was added to a seed fermentation tank at a volume ratio of 1:150 for culture, and then added to a fermentation tank of a thick-walled spore fermentation medium (containing 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, and 0.03% MgSO4 by weight) at a volume ratio of 1:20, and cultured at 28°C for 5 days until 90% of the hyphae formed thick-walled spores to obtain a thick-walled spore fermentation liquid; then the fermentation liquid was adsorbed on diatomaceous earth, centrifuged or plate-frame filtered, and dried to form a wettable powder. The obtained wettable powder is the microbial agent of this embodiment, and the number of viable bacteria of Trichoderma harzianum in the microbial agent is 5×10 9 CFU / g.

[0062] Comparative Example 1 The strain of Trichoderma harzianum purchased from the China Center for Type Culture Collection with the collection number of CGMCC No.40016 was inoculated into a potato dextrose agar solid medium (PDA), cultured in an incubator at 25°C for 7 days, and 3-4 bacterial blocks were taken from it and added to a seed bottle. The seed solution was prepared by shaking and cultured on a shaker at 25°C and 180rpm for 48 hours. The seed solution was added to a seed fermentation tank at a volume ratio of 1:150 for culture, and then added to a fermentation tank of thick-walled spore fermentation medium at a volume ratio of 1:20, and cultured at 28°C for 5 days until 90% of the hyphae formed thick-walled spores to obtain thick-walled spore fermentation liquid; then the fermentation liquid was adsorbed, centrifuged or plate-frame filtered, and dried using diatomaceous earth to form a wettable powder. The obtained wettable powder is the microbial agent of this comparative example, and the number of viable bacteria of Trichoderma harzianum in the microbial agent is 5×10 9 CFU / g.

[0063] Comparative Examples 2-7 The six strains of Trichoderma harzianum isolated together with the Trichoderma harzianum with the deposit number of CGMCC No.41719 of the present application were inoculated into potato dextrose agar solid medium (PDA) respectively, and cultured in an incubator at 25°C for 7 days, and 3-4 bacterial blocks were taken from them and added to the seed bottle. Shake and culture on a shaker at 25°C and 180rpm for 48 hours to prepare the seed liquid. The seed liquid was added to the seed fermentation tank at a volume ratio of 1:150 for culture, and then added to the fermentation tank of thick-walled spore fermentation medium at a volume ratio of 1:20, and cultured at 28°C for 5 days until 90% of the hyphae formed thick-walled spores to obtain thick-walled spore fermentation liquid; then the fermentation liquid was adsorbed on diatomaceous earth, centrifuged or plate-and-frame filtered, and dried to form a wettable powder, so that the number of viable bacteria of Trichoderma harzianum was 5×109 CFU / g, and the obtained wettable powder is the bacterial agent of this comparative example.

[0064] Test Example 1 This test example is used to illustrate that the Trichoderma harzianum disclosed in the present invention can promote the growth of forage.

[0065] The wettable powders of Example 2 and Comparative Examples 1-7 were respectively used for the tests.

[0066] Experimental group: The experiment was conducted in an outdoor natural grassland in Haiyan, Qinghai. Duckgrass was selected as the test object. A piece of open space was specially opened to plant Duckgrass. The experimental group was treated with wettable powder at an application rate of 2g / m 2 (i.e. 10 8 CFU / m 2 ); the control group was not treated with wettable powder; field management and other conditions were the same. After 40 days of growth, the height of the plants above ground was measured, starting from the base of the stem and ending at the tip of the leaf. The results are listed in Table 2.

[0067] Table 2

[0068] As shown in Table 2, according to the changes in the height of the grass in Example 2 and Comparative Examples 1-7, the height of the grass in Example 2 can still be increased by about 39% compared with the best growing Comparative Examples 2 and 6. It can be seen that the Trichoderma harzianum with the deposit number of CGMCC No. 41719 disclosed in the present invention can promote the growth of grass.

[0069] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0070] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0071] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A Trichoderma harzianum, characterized in that Trichoderma harzianum Trichoderma harzianum ; The deposit number of the Trichoderma harzianum is CGMCC No.41719.

2. The Trichoderma harzianum according to claim 1, wherein The Trichoderma harzianum rpb2 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The Trichoderma harzianum TEF1α The nucleotide sequence of the gene is shown in SEQ ID NO.2; The nucleotide sequence of the ITS gene of Trichoderma harzianum is shown in SEQ ID NO.

3.

3. The Trichoderma harzianum according to claim 2, wherein The shape of the Trichoderma harzianum is round with regular edges, the front of the colony is green, has green aerial hyphae and green spores, and the back of the colony is grayish white.

4. A microbial agent, characterized in that: The microbial agent contains Trichoderma harzianum with a preservation number of CGMCC No.41719 and a culture medium.

5. The microbial agent according to claim 4, wherein In each gram of the microbial agent, the number of viable bacteria of Trichoderma harzianum with a preservation number of CGMCC No. 41719 is 10 8 -10 11 CFU.

6. The microbial agent according to claim 4, wherein The culture medium is potato dextrose agar medium and potato dextrose medium.

7. Use of the Trichoderma harzianum described in any one of claims 1 to 3 or the microbial agent described in any one of claims 4 to 6 in promoting forage growth.

8. The use according to claim 7, wherein: The forage grass is selected from one or more of Dactylis grass, Festuca australis, Festuca sinensis and Agropyron ovata.

9. A method for promoting forage growth, characterized in that: The method comprises: applying the Trichoderma harzianum described in any one of claims 1 to 3 or the microbial agent described in any one of claims 4 to 6 to the soil at the root of the forage grass.

10. The method according to claim 9, wherein: The dosage of the Trichoderma harzianum is 10 8 -10 11 CFU / m 2 .

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