Trichoderma harzianum and microbial agent for promoting growth of forage grasses and application thereof

Abstract

The present disclosure relates to a Trichoderma harzianum and a microbial inoculant for promoting growth of forage grasses and applications thereof. The Trichoderma harzianum is named as Trichoderma harzianum Trichoderma harzianum ; the accession number of the Trichoderma harzianum is CGMCC No. 41719. The Trichoderma harzianum can effectively promote growth of forage grasses.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of microbiology, in particular, a Trichoderma harzianum and microbial inoculant for promoting the growth of pasture grasses and the use thereof. BACKGROUND

[0002] With the acceleration of urbanization and the large amount of grassland occupation and destruction caused by economic development, the growth of pasture grasses is affected. At the same time, the pasture production technology is relatively backward, and lacks modern management means and technical support, so it is difficult to meet the efficient and high-quality demand for pasture. Natural grassland mainly provides forage sources for livestock such as cattle, sheep, horses, camels, yaks and other livestock in the animal husbandry industry.

[0003] In order to further meet the needs of the animal husbandry industry, it is of great significance to seek a safe and efficient microorganism for promoting the growth of pasture grasses. SUMMARY

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

[0005] In order to achieve the above-mentioned purpose, the first aspect of the present disclosure provides a Trichoderma harzianum, the taxonomic name of which is Trichoderma harzianum Trichoderma harzianum ; the accession number of the Trichoderma harzianum is CGMCC No.41719.

[0006] Optionally, the nucleotide sequence of the rpb2 gene of the Trichoderma harzianum is shown as SEQ ID NO.1; the nucleotide sequence of the tef1a gene of the Trichoderma harzianum is shown as SEQ ID NO.2; and the nucleotide sequence of the ITS gene of the Trichoderma harzianum is shown as SEQ ID NO.3.

[0007] Optionally, the shape of the Trichoderma harzianum is round, the edge is regular, the front of the colony presents green color, has green aerial hyphae and green spores, and the back of the colony presents grayish white color.

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

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

[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 of the first aspect or the microbial inoculant of the second aspect in promoting the growth of the forage grass.

[0012] Optionally, the forage grass is selected from one or more of cocksfoot, orchardgrass, Chinese fine fescue and Canada bluegrass.

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

[0014] Optionally, the Trichoderma harzianum is used in an amount of 10 8 -10 11 CFU / m 2 .

[0015] Through the above technical solutions, the present disclosure provides a Trichoderma for promoting the growth of forage grass and a microbial inoculant, which can effectively promote the growth of forage grass, and the Trichoderma harzianum has the advantages of safety and high efficiency, and has good application prospect.

[0016] Other features and advantages of the present disclosure will be described in detail in the following specific embodiment part.

[0017] Biological material preservation

[0018] The Trichoderma harzianum of the present disclosure is a pure culture isolated by the inventors of the present disclosure from the soil of Keketuohai, Fuyun County, Altay Region, Xinjiang, and has a preservation number of CGMCC No. 41719, a preservation date of December 13, 2024, a preservation unit of China General Microbiological Culture Collection Center, an address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard, and a classification name of Trichoderma harzianum. Trichoderma harzianum . BRIEF DESCRIPTION OF DRAWINGS

[0019] The accompanying drawings are included to provide a further understanding of the present disclosure and constitute a part of the specification, and together with the specific embodiments below, serve to explain the present disclosure but do not constitute a limitation on the present disclosure. In the drawings:

[0020] Figure 1 is a colony morphology diagram of the Trichoderma of the present disclosure. DETAILED DESCRIPTION

[0021] The specific embodiments of the present disclosure are described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and do not limit the present disclosure.

[0022] The inventors of the present disclosure isolated a pure culture from the soil of Keketuohai, Fuyun County, Altay Region, Xinjiang, which was identified as belonging to the genus Trichoderma Trichoderma , named as Trichoderma harzianum Trichoderma harzianum . The strain has been preserved in the General Microbiological Center of the China Microbial Culture Collection Management Committee designated by the National Intellectual Property Bureau, with a preservation date of December 13, 2024, and a preservation number of CGMCC No. 41719.

[0023] The first aspect of the present disclosure provides a Trichoderma harzianum, which is classified and named as Trichoderma harzianum Trichoderma harzianum ; the preservation number of the Trichoderma harzianum is CGMCC No. 41719.

[0024] The Trichoderma harzianum of the present disclosure can survive and grow in a conventional bacterial culture medium. The Trichoderma harzianum of the present disclosure as described above is a Trichoderma harzianum strain capable of promoting the growth of pasture grass.

[0025] According to the present disclosure, the nucleotide sequence of the rpb2 gene of the Trichoderma harzianum is shown as SEQ ID NO. 1; the nucleotide sequence of the tef1a gene of the Trichoderma harzianum is shown as SEQ ID NO. 2; and the nucleotide sequence of the ITS gene of the Trichoderma harzianum is shown as SEQ ID NO. 3.

[0026] In an embodiment of the present disclosure, as shown in Figure 1 , the shape of the Trichoderma harzianum is round, the edge is regular, the front of the colony presents green color, has green aerial hyphae and green spores, and the back of the colony presents grayish white color. Moreover, the yield of spores is high.

[0027] The second aspect of the present disclosure provides a microbial inoculant containing the Trichoderma harzianum with a preservation number of CGMCC No. 41719 and a culture medium.

[0028] In the microbial inoculant, the viable cell count of the Trichoderma harzianum can vary within a large range. In an embodiment of the present disclosure, the viable cell count of the Trichoderma harzianum with a preservation number of CGMCC No. 41719 in the microbial inoculant is 10 8 - 10 11 CFU per gram of the microbial inoculant.

[0029] According to the present disclosure, the culture medium is potato dextrose agar medium (PDA) and potato dextrose medium (PD).

[0030] Optionally, the preparation method of the potato dextrose agar solid medium (PDA) is as follows: 20 g of peeled potato is boiled for 20 minutes to obtain juice, the juice is mixed with 2 g of glucose, 1.5 g of agar and water to make 100 mL, the pH is 6.5-7.0, 50 mL is filled into each of two triangular bottles, and the sterilized slant or plate is prepared; the preparation method of the potato dextrose liquid medium (PD) is as follows: 20 g of peeled potato is boiled for 20 minutes to obtain juice, the juice is mixed with 2 g of glucose and water to make 100 mL, the pH is 6.5-7.0, 50 mL is filled into each of two triangular bottles, and the sterilization is performed.

[0031] Alternatively, the above various culture media can be sterilized according to the conventional sterilization method and then stored, for example, sterilized at 115-125 ℃ and 1.5-2 atmospheres for 10-30 minutes.

[0032] The preparation method of the microbial agent can include the following steps: the Trichoderma harzianum with the preservation number of CGMCC No. 41719 is inoculated into the potato dextrose agar solid medium, cultured in a 25 ℃ incubator for 7 days, 3-4 pieces of mycelium are taken from the culture medium and added into a seed bottle, the seed bottle is cultured in a 25 ℃, 180 rpm shaking incubator for 48 h to prepare a seed liquid; the seed liquid is added into a seed fermentation tank at a volume ratio of 1:150, then added into a fermentation tank of chlamydospore fermentation medium (containing 2.5% of starch, 1.5% of yeast powder, 5.0% of corn syrup, 0.4% of CaCO3, 0.01% of ZnSO4 and 0.03% of MgSO4 by weight) at a volume ratio of 1:20, and cultured at 28 ℃ for d until 90% of the mycelium forms chlamydospores to obtain a chlamydospore fermentation liquid; then the chlamydospore fermentation liquid is adsorbed by diatomite, centrifuged or plate-frame filtered, and dried to form a wettable powder.

[0033] Preferably, the number of viable Trichoderma harzianum with the preservation number of CGMCC No. 41719 in each gram of the microbial agent obtained by the culture is 10 8 -10 11 CFU, more preferably 10 9 -10 10 CFU. During the culture, the number of viable bacteria can be tested by a conventional method, for example, a blood plate counting method or an OD value observation method to obtain the concentration of viable bacteria. The bacterial liquid after the culture can be directly used as the microbial agent, and preferably, the bacterial liquid is further processed into a microbial agent with a more convenient storage form by steps such as sterile filtration and freeze-drying. The culture conditions of the bacterial strain are not particularly limited and can be the conventional conditions for the culture of Trichoderma harzianum, for example, the culture is performed by using a shaking incubator, the culture temperature can be 25-30 ℃, and the culture time can be 1-2 days.

[0034] The third aspect of the present disclosure provides the use of the Trichoderma harzianum of the first aspect or the microbial inoculant of the second aspect in promoting the growth of pasture grass.

[0035] In an embodiment of the present disclosure, the pasture grass is a natural pasture grass, for example, one or more of cocksfoot, Chinese orchardgrass, Chinese meadowgrass and Canada wildrye.

[0036] The fourth aspect of the present disclosure provides a method for promoting the growth of pasture grass, the method comprising: applying the Trichoderma harzianum of the first aspect or the microbial inoculant of the second aspect to the soil at the root of the pasture grass.

[0037] In an embodiment of the present disclosure, the Trichoderma harzianum is applied in an amount of 10 8 -10 11 CFU / m 2 .

[0038] In the above embodiment, the application can be in a manner routinely used by those skilled in the art, for example, spraying, root irrigation or soil mixing.

[0039] The present disclosure is further described in detail below with reference to the following examples, but the scope of the present disclosure is not limited to the following examples.

[0040] The preparation method of the potato dextrose agar solid medium (PDA) used in the present example is as follows: 20 g of peeled potato is boiled for 20 minutes to obtain 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 two triangular bottles, each of which is 50 mL, and after sterilization, the juice is prepared into an inclined plane or a flat plate.

[0041] The formula of the fermentation tank of the chlamydospore fermentation medium is as follows: 2.5% starch, 1.5% yeast powder, 5.0% corn syrup, 0.4% CaCO3, 0.01% ZnSO4 and 0.03% MgSO4 by weight.

[0042] Example 1

[0043] The present example is used to illustrate the isolation and purification of the Trichoderma harzianum of the present disclosure.

[0044] I. Soil collection and treatment

[0045] Soil samples were collected in Keketuohai, Fuyun County, Altay Region, Xinjiang Uygur Autonomous Region.

[0046] The collection method is as follows: 10 m 2In the sample plot, five-point diagonal sampling method was used for soil collection. When sampling, the surface soil of 2 cm was removed, and the soil around the plant roots was collected. The rhizosphere soil of the host plant was collected to a depth of 20 cm, mixed, and then 200 g was placed in a sterilized self-sealing bag. The bag was taken back to the laboratory, and the records were made (including: collection location, latitude and longitude, altitude, and covered vegetation). The samples were stored at 4°C.

[0047] II. Isolation and purification of strain MCSZ-1.

[0048] 1. Soil sample treatment:

[0049] 10 g of clean soil was added to 90 mL of sterile water, and the mixture was uniformly shaken to obtain a mixed solution. 1 mL of the mixed solution was added to a test tube containing 9 mL of sterile water, and the mixture was uniformly mixed to obtain a dilution solution with a dilution of 10 -1 . The 10 -2 , 10 -3 , and 10 -3 fold dilution solutions were prepared by 10-fold dilution method. 100 μL of the 10 -3 fold dilution solution was spread on the isolation PDA culture medium plate, and the dilution solution was evenly spread with a sterile applicator. The process was repeated 3 times on the PDA culture medium. The culture was incubated at a temperature of 28°C in a mold incubator. After 10 days, the fungal colonies were observed, and the suspected Trichoderma colonies on the plate were counted and subjected to further purification.

[0050] 2. Culture and purification of the strain:

[0051] The suspected Trichoderma harzianum colonies on the plate were picked and cultured on PDA culture medium for multiple rounds of purification until the strain was completely purified, with a total culture period of more than three weeks. The purified Trichoderma harzianum strain was stored in a 4°C refrigerator on PDA slant, and 3 portions were preserved in a 30% glycerol tube in an ultra-low temperature freezer. According to the growth needs, the Trichoderma harzianum was cultured in an appropriate amount and at an appropriate time, with 10 8 CFU per strain, and was used for 40 days by root irrigation on the soil of the orchardgrass roots. The results showed that the strain MCSZ-1 had good effect on promoting the growth of pasture (see Example 2 for details), and therefore the strain MCSZ-1 was subjected to the following identification.

[0052] III. Identification of strain MCSZ-1.

[0053] 1. Morphological identification:

[0054] The same size of the fungus cake was taken from the edge of the purified colony of strain MCSZ-1 and placed in the center of the culture medium (PDA culture medium). The growth rate and growth conditions of the strain during the culture process, colony color, and mycelial morphological characteristics were observed. The results are shown in Figure 1 .

[0055] The results show that the strain MCSZ-1 obtained by the above steps of separation and purification presents a radial round colony, regular edge, green front, green aerial hypha and green spore after being cultured at 27℃ for 7 days, and the aerial hypha extends to the four directions, and the back of the colony presents a grayish white color.

[0056] 2. Molecular biology identification:

[0057] DNA extraction: scrape an appropriate amount of mycelium of the strain MCSZ-1 into a sterile centrifuge tube, and place it at 0℃ for 30 min or more, and before starting the DNA extraction, put a steel ball in each centrifuge tube, and break the cells by oscillation at a frequency of 50 Hz for 3 min by using a freeze-thaw cell disruptor, and then sequentially perform the operation steps according to the fungal genome extraction kit (Aidley fungal DNA rapid extraction kit), collect the DNA in a new centrifuge tube, and measure the concentration by using an ultramicro spectrophotometer, and the qualified DNA can be subjected to PCR amplification.

[0058] PCR amplification: the internal transcribed spacer (ITS) of ribosomal RNA encoding gene, the 4th and 5th introns and most of the last exon of translation elongation factor 1α (TEF1α), and the gene of 2 (the second subunit of RNA polymerase) are amplified, and the primer sequences are shown in Table 1: tef 1α) are amplified, and the primer sequences are shown in Table 1: rpb 2 (the second subunit of RNA polymerase) are amplified, and the primer sequences are shown in Table 1:

[0059] Table 1

[0060]

[0061] 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.

[0062] PCR reaction system: 2 μL of each of the 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.

[0063] PCR amplification procedure of RPB and TEF genes: 95℃ for 3 min, 95℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min (35 cycles), and 72℃ for 5 min.

[0064] PCR amplification procedure of ITS sequence: 95℃ for 3 min, 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min (35 cycles), and 72℃ for 5 min.

[0065] PCR product detection and sequencing: prepare 1% agarose gel, add 6 μL of DNA marker to the first or last gel well, and add 6 μL of PCR product to the other gel wells, electrophorese at 125 V for 30 min, and observe the banding condition under the gel imaging system and collect the image.

[0066] After successful amplification of the sample, it was sent to the company (Qingke Biotechnology Co., Ltd.) for sequencing, and the rpb 2sequence is shown in SEQ ID NO. 1, tef 1αsequence is shown in SEQ ID NO. 2, and ITS sequence is shown in SEQ ID NO. 3.

[0067] According to the latest standard for classification of Trichoderma species proposed by Cai and Druzhinina: it must simultaneously satisfy rpb2 ≥ 99%, tef1a ≥ 97% and ITS ≥ 76% three conditions can be determined as a Trichoderma species. Using the latest standard for identification of Trichoderma species, we compared the rpb2 gene, tef1a gene and ITS region sequence of strain MCSZ-1 in NCBI Blast (https: / / blast.ncbi.nlm.nih.gov / Blast.cg) respectively, and found that the rpb2 gene of strain MCSZ-1 had 99.02-99.43% similarity with Trichoderma harzianum, the tef 1αgene of strain MCSZ-1 had 97-99.92% similarity with Trichoderma harzianum, and the ITS sequence of strain MCSZ-1 had 99.23% similarity with Trichoderma harzianum. Therefore, we confirmed that the classification of Trichoderma species was named Trichoderma harzianum.

[0068] Through the above morphological and molecular biological identification, strain MCSZ-1 was identified as Trichoderma harzianum (T. harzianum). Trichoderma harzianum The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 13, 2024, with the address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the preservation number of CGMCC No. 41719.

[0069] Example 2

[0070] This example is used to illustrate the preparation of microbial inoculant.

[0071] The Trichoderma harzianum with the preservation number of CGMCC No. 41719 was inoculated into a potato dextrose agar solid medium (PDA) and cultured in a 25 °C incubator for 7 days, 3-4 mycelial blocks were taken from the medium and added into a seed bottle. The seed bottle was cultured in a 25 °C, 180 rpm shaker for 48 h to prepare a seed liquid. The seed liquid was added into a seed fermenter at a volume ratio of 1:150, and then added into a chlamydospore fermentation medium fermenter (containing 2.5% starch, 1.5% yeast powder, 5.0% corn syrup, 0.4% CaCO3, 0.01% ZnSO4 and 0.03% MgSO4 by weight) at a volume ratio of 1:20. The chlamydospore fermentation medium fermenter was cultured at 28 °C for 5 d until 90% of the mycelium formed chlamydospores to obtain a chlamydospore fermentation liquid. The chlamydospore fermentation liquid was adsorbed by diatomite, centrifuged or plate-and-frame filtered, and dried to form a wettable powder. The obtained wettable powder was the microbial inoculant of the present example, and the viable cell count of the Trichoderma harzianum in the microbial inoculant was 5×10 9 CFU / g.

[0072] Comparative Example 1

[0073] The Trichoderma harzianum strain with the preservation number of CGMCC No. 40016 purchased from the China General Microbiological Culture Collection Center was inoculated into a potato dextrose agar solid medium (PDA) and cultured in a 25 °C incubator for 7 days, 3-4 mycelial blocks were taken from the medium and added into a seed bottle. The seed bottle was cultured in a 25 °C, 180 rpm shaker for 48 h to prepare a seed liquid. The seed liquid was added into a seed fermenter at a volume ratio of 1:150, and then added into a chlamydospore fermentation medium fermenter at a volume ratio of 1:20. The chlamydospore fermentation medium fermenter was cultured at 28 °C for 5 d until 90% of the mycelium formed chlamydospores to obtain a chlamydospore fermentation liquid. The chlamydospore fermentation liquid was adsorbed by diatomite, centrifuged or plate-and-frame filtered, and dried to form a wettable powder. The obtained wettable powder was the microbial inoculant of the present example, and the viable cell count of the Trichoderma harzianum in the microbial inoculant was 5×10 9 CFU / g.

[0074] Comparative Examples 2-7

[0075] Six strains of Trichoderma harzianum isolated together with the Trichoderma harzianum with the preservation number CGMCC No. 41719 of the present application were inoculated into potato dextrose agar solid medium (PDA) respectively, and cultured in a 25°C incubator for 7 days, 3-4 mycelial blocks were taken from the medium and added into seed bottles. The seed bottles were cultured on a 25°C, 180 rpm shaker for 48 h to prepare seed liquid. The seed liquid was added into a seed fermenter at a volume ratio of 1:150, and then added into a chlamydospore fermentation medium fermenter at a volume ratio of 1:20, and cultured at 28°C for 5 d until 90% of the mycelium formed chlamydospores to obtain chlamydospore fermentation liquor. Then diatomite was used to adsorb, centrifuge or plate-frame filter the fermentation liquor, and dry to form wettable powder, so that the viable count of the Trichoderma harzianum was 5×10 9 CFU / g, and the obtained wettable powder was the microbial agent of the present comparative example.

[0076] Test Example 1

[0077] The present test example was used to illustrate that the Trichoderma harzianum of the present disclosure can promote the growth of pasture grass.

[0078] The wettable powders of Example 2 and Comparative Examples 1-7 were used for testing respectively.

[0079] Experimental group: conducted in a natural grassland in Haian, Qinghai, and duck grass was selected as the test object, a piece of land was specially developed for planting duck grass, the experimental group was treated with the wettable powder, and the application amount was 2 g / m 2 (that is, 10 8 CFU / m 2 ); the control group was not treated with the wettable powder; the field management and other conditions were the same. After growing for 40 days, the height of the plant above ground was measured, and the measurement was started from the base of the stem and ended at the tip of the leaf. The results are shown in Table 2.

[0080] Table 2

[0081]

[0082] As can be seen from Table 2, according to the height change of the pasture grass in Example 2 and Comparative Examples 1-7, the height and growth of the pasture grass of Example 2 can still be increased by about 39% compared with Comparative Examples 2 and 6 which have the best height and growth. It can be seen that the Trichoderma harzianum with the preservation number CGMCC No. 41719 of the present disclosure can promote the growth of pasture grass.

[0083] The preferred embodiments of the present disclosure are described in detail above with reference to the drawings, but the present disclosure is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present disclosure within the technical concept of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0084] It should be further noted that various specific technical features described in the above specific embodiments can be combined in any suitable manner, and the disclosure will not be repeated here for various possible combinations.

[0085] In addition, various different embodiments of the disclosure can also be combined with each other as long as they do not contradict the idea of the disclosure, and they should also be considered as disclosed by the disclosure.

Claims

1. A Trichoderma harzianum characterized in that, The Trichoderma harzianum is named as Trichoderma harzianum Trichoderma harzianum The preservation number of the Trichoderma harzianum is CGMCC No. 41719.

2. The Trichoderma harzianum of 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 the Trichoderma harzianum is shown as SEQ ID NO.

3.

3. The Trichoderma harzianum of claim 2, wherein, The Trichoderma harzianum has a round shape, regular edge, green front surface, green aerial hyphae and green spores, and a grayish white back surface.

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

5. The microbial inoculant of claim 4, wherein, The viable cell number of Trichoderma harzianum with the preservation number of CGMCC No. 41719 in the microbial inoculant is 10 8 -10 11 CFU per gram.

6. The microbial inoculant of claim 4, wherein, The culture medium is a potato glucose agar medium and a potato glucose medium.

7. The Trichoderma harzianum of any one of claims 1-3 or the microbial agent of any one of claims 4-6 is applied to promote the growth of a forage grass; the forage grass is a cocksfoot.

8. A method of promoting the growth of pasture grasses, characterised by, The method comprises: applying the Trichoderma harzianum of any one of claims 1-3 or the microbial agent of any one of claims 4-6 to the soil under the roots of the forage grass; the forage grass is a cocksfoot.

9. The method of claim 8, wherein, The Trichoderma harzianum is used in an amount of 10 8 -10 11 CFU / m 2 .

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