Trichoderma harzianum strain and application thereof

Abstract

The invention provides a Trichoderma harzianum strain, which is named Trichoderma harzianum, is preserved in China General Microbiological Culture Collection Center (CGMCC), has a preservation number of CGMCC No.41725, and has a preservation date of December 20, 2024. The Trichoderma harzianum strain is named as Trichoderma harzianum, is preserved in China General Microbiological Culture Collection Center (CGMCC), and has a preservation number of CGMCC No.41725. The sequence of the rDNA-ITS is as shown in SEQ ID NO: 1; the sequence of TEF-1alpha is as shown in SEQ ID NO: 2, and the sequence of RPB2 is as shown in SEQ ID NO: 3. The trichoderma harzianum strain disclosed by the invention can be used for inhibiting the activity of E. tucutricum, B. sorokiniana, A. alternania and B. zecoola, can be used for generating auxin and gibberellin, and can be used for promoting the growth of silage corn and preventing and treating the leaf spot disease of the corn alternaria in alpine regions of Qinghai province. The trichoderma harzianum strain disclosed by the invention has the advantages that the activity of the E. tucutricum, the B. sorokiniana, the A. alternania and the B. zecoola can be inhibited, and the growth of the silage corn can be promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a Trichoderma harzianum strain and application thereof. Background Art

[0002] Given the ecological significance of the Yellow River source region and Qinghai Province, as well as the need to reduce the use of chemical fertilizers and pesticides, finding biopesticides suitable for Qinghai's alpine regions that are low-residue, environmentally friendly, and highly effective is a promising alternative to chemical pesticides. This approach would both protect the ecological environment and enable farmers to sustainably cultivate crops. The use of bioantagonistic strains for plant disease control is a hot topic of research both domestically and internationally. Biocontrol methods are safe, effective, non-toxic, and harmless, effectively reducing environmental pollution caused by chemical pesticide use and controlling pesticide residues. Biocontrol mechanisms are based on microecological principles, encompassing a combination of nutrient competition, induced resistance, and parasitism. These methods utilize natural, environmentally friendly raw materials, posing no safety concerns for agricultural products. Furthermore, microorganisms proliferate rapidly, making them easily scalable and promising for plant disease control.

[0003] Microbial preparations primarily consist of beneficial microorganisms. Due to their narrow scope of application and limited geographical adaptability, introduced beneficial microorganisms can often prove ineffective when applied locally in Qinghai. Therefore, screening for beneficial microorganisms suitable for Qinghai is crucial for increasing crop yields and preventing and controlling diseases. Microbial preparations are based on the principles of microecology. In nature, microorganisms and plants maintain a harmonious balance, with microorganisms mutually constraining each other to maintain a stable and orderly relationship. Therefore, understanding the growth-promoting and disease-resistant properties of microorganisms and screening for those with these properties can lay a solid foundation for the subsequent preparation and development of microbial preparations. Regulating the microecology through scientific practice, adhering to the laws of nature, and adhering to the principle of "following nature, drawing from nature, and returning to nature" is a key approach to achieving sustainable agriculture.

[0004] Currently, there are no microbial agents for crop production in high-altitude cold regions. Therefore, the present invention aims to clarify the growth-promoting and disease-resistant properties of Trichoderma harzianum strain LT2-15 isolated from local soil in Qinghai, and provide basic materials for the subsequent development of microbial agents suitable for use in high-altitude cold regions in Qinghai Province. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a Trichoderma harzianum strain and its application. The Trichoderma harzianum strain can inhibit the activity of E. turcicum, B. sorokiniana, A. alternata and B. zeicola, and can produce auxin and gibberellins. The strain can be used to promote the growth of silage corn and prevent and control corn Alternaria leaf spot disease in the high-altitude and cold regions of Qinghai Province.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a Trichoderma harzianum strain, the Trichoderma harzianum strain is named Trichoderma harzianum, deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with a deposit number of CGMCC No. 41725, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is December 20, 2024; the rDNA-ITS sequence of the Trichoderma harzianum is shown in SEQ ID NO: 1; the TEF-1α sequence of the Trichoderma harzianum is shown in SEQ ID NO: 2, and the RPB2 sequence of the Trichoderma harzianum is shown in SEQ ID NO: 3.

[0007] The present invention also provides the use of the above-mentioned Trichoderma harzianum strain, which is used to inhibit the activity of E. turcicum, B. sorokiniana, A. alternata, and B. zeicola.

[0008] The present invention also provides the application of the above-mentioned Trichoderma harzianum strain, wherein the Trichoderma harzianum strain is used to promote the growth of silage corn.

[0009] The present invention also provides the use of the above-mentioned Trichoderma harzianum strain, wherein the Trichoderma harzianum strain is used for preventing and treating corn Alternaria leaf spot disease caused by Alternaria fungi.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] The Trichoderma harzianum strain of the present invention can inhibit the activities of E. turcicum, B. sorokiniana, A. alternata and B. zeicola, can produce auxin and gibberellins, and can be used for promoting the growth of silage corn and preventing and controlling corn Alternaria leaf spot disease in the high-altitude and cold regions of Qinghai Province.

[0012] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a morphological characteristic diagram of Trichoderma harzianum LT2-15 in Example 1 of the present invention.

[0014] Figure 2 This is a phylogenetic tree jointly constructed based on rDNA-ITS, TEF-1α and RPB2 gene sequences of Trichoderma harzianum LT2-15 in Example 1 of the present invention.

[0015] Figure 3 This is the determination of the antibacterial effect of Trichoderma harzianum LT2-15 on pathogenic fungi in Example 1 of the present invention.

[0016] Figure 4 This is the standard curve for determining auxin according to Example 1 of the present invention.

[0017] Figure 5 It is the gibberellin determination standard curve of Example 1 of the present invention.

[0018] Figure 6 This is the indoor growth-promoting effect of Trichoderma harzianum LT2-15 on silage corn determined in Example 1 of the present invention.

[0019] Figure 7 This is a safety test of Trichoderma harzianum LT2-15 on silage corn in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] The Trichoderma harzianum strain of this embodiment is named Trichoderma harzianum, deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 41725, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is December 20, 2024; the rDNA-ITS sequence of the Trichoderma harzianum is shown in SEQ ID NO: 1; the TEF-1α sequence of the Trichoderma harzianum is shown in SEQ ID NO: 2, and the RPB2 sequence of the Trichoderma harzianum is shown in SEQ ID NO: 3.

[0021] The above-mentioned Trichoderma harzianum strain is used to inhibit the activity of E. turcicum, B. sorokiniana, A. alternata, and B. zeicola.

[0022] The above-mentioned Trichoderma harzianum strain is used to promote the growth of silage corn.

[0023] The above-mentioned Trichoderma harzianum strain is used for preventing and controlling corn Alternaria leaf spot disease.

[0024] (1) Isolation and identification of Trichoderma harzianum strain LT2-15

[0025] Strain LT2-15 was isolated and purified from a soil sample collected from Longyang Canyon in August 2022 using the dilution plating method and mycelial tip purification method. The strain was identified using morphological characteristics and rDNA-ITS, TEF-1α, and RPB2 sequence analysis.

[0026] Primers for rDNA-ITS amplification were ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′);

[0027] TEF-1α gene amplification primers: EF-1 (5′-ATGGGTAAGGARGACAAGAC-3′) and EF-2 (5′-GGARGTACCAGTSATCATGTT-3′);

[0028] RPB2 gene amplification primers: RPB2-5F (5'-GAYGAYMGWGATCAYTTYGG-3') and RPB2-7CR (5'-CCCATRGCTTGYTTRCCCAT-3').

[0029] After culturing LT2-15 on PDA medium at 25℃ in the dark for 6 days, the colonies are army green with a slight yellowish green tint. This strain grows fast and the mycelium can cover the plate in 2-3 days. The conidia are spherical to ovoid and slightly light green ( Figure 1 ).

[0030] Strain LT2-15 was sequenced using rDNA-ITS (SEQ ID NO: 1), TEF-1α (SEQ ID NO: 2), and RPB2 (SEQ ID NO: 3). Sequencing results were compared using the NCBI BLAST website, revealing the highest similarity with Trichoderma harzianum, with a similarity of ≥99%. Sequence alignment was performed using MEGA 7.0 software, and sequence assembly was performed using SequenceMatrix 1.7.8. Protocrea farinosa was used as the outgroup, and the calculation was repeated 1000 times to construct a multi-gene joint phylogenetic tree. Figure 2 LT2-15 was found to be closely related to Trichoderma harzianum, clustering in a clade. It was more distantly related to other Trichoderma strains. Therefore, based on morphological characteristics and phylogenetic analysis, LT2-15 was identified as Trichoderma harzianum.

[0031] The strain LT2-15 was named Trichoderma harzianum and deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.41725. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is December 20, 2024.

[0032] (2) Determination of antibacterial activity

[0033] The antifungal activity against Exserohilum turcicum, Alternaria alternata, Bipolaris sorokiniana, and B. zeicola, pathogens of silage maize, a specialty crop in Qinghai Province, was determined using a stand-off culture method. Six-day-old cultured strain LT2-15 and the pathogens were used to obtain bacterial cakes using a 0.5 cm borer. The target bacteria and LT2-15 were then affixed upside down to the sides of a 9 cm Petri dish, 1 cm from the edge, with three replicates per strain.

[0034] Inhibition rate = (pathogen colony radius of the control group - pathogen colony radius of the experimental group) / pathogen colony radius of the control group × 100%

[0035] Through indoor confrontation culture, it was found that the inhibition rate of Trichoderma harzianum strain LT2-15 against four pathogenic fungi was between 61.3% and 72.3% (Table 1 and Figure 3 ), among which the inhibitory effect on Alternaria maydis was the best.

[0036] Table 1 Inhibition rate of Trichoderma harzianum LT2-15 on four pathogenic fungi (%)

[0037]

[0038] (3) Determination of antibacterial effect of strain fermentation broth

[0039] Inoculate LT2-15 onto PDA medium and incubate in a 25°C mold incubator for 5 days. Then, add 2 mL of CM liquid medium to each dish in a clean bench. Use a sterile inoculating loop to break the mycelium. Add the mycelial suspension to a flask containing 200 mL of CM medium and shake at 180 rpm for 7 days. Filter the mycelium using a sterile funnel (lined with two layers of filter paper). Extract the fermentation broth three times with ethyl acetate, and collect the organic phase. Concentrate the broth to 2-3 mL using a rotary evaporator at 55°C under reduced pressure. Dispense the broth evenly into two 2.0 mL centrifuge tubes using a pipette, dry in a 50°C oven until a paste forms. Dissolve the dried paste in 500 μL of methanol, filter through a 0.45 μm organic filter membrane, and store at 4°C until needed.

[0040] Use 9cm PDA plates to measure the antibacterial effect of fermentation broth. Place a pathogen cake (0.5cm) 1cm away from the edge of the plate, use a 0.5cm punch to punch out a cake 1cm away from the edge of the other side of the plate, add 50μL of dry paste dissolution solution here, and repeat three times. Add an equal volume of methanol as a blank control. Place in a 25℃ mold incubator for 7 days, and then measure the radius of the pathogen. Calculate the antibacterial rate of the fermentation broth according to method (2).

[0041] The crude extract of the fermentation broth was extracted and its antibacterial activity against four pathogens was determined. The results showed that the antibacterial activity was good, with inhibition rates ranging from 66.4% to 68.9%. The best inhibition rate was 68.9% against Helminthosporium macrosporum.

[0042] Table 2 Determination of antibacterial rate of Trichoderma harzianum LT2-15 fermentation broth (%)

[0043]

[0044] (IV) Determination of the ability to produce auxin

[0045] The strain was inoculated into a test tube containing 5 mL of PDA liquid culture medium, repeated three times. The culture was cultured at 25°C on a shaker at 150 rpm for 7 days. The IAA production characteristics of the strain were determined using the Salkowski colorimetric method. After centrifugation of the fermentation broth at 8000 rpm for 10 minutes, 1.5 mL of the supernatant was collected and added with 3 mL of Salkowski reagent. After mixing, the supernatant was incubated at 25°C in the dark for 30 minutes. After the reaction, the absorbance at 530 nm was measured. Sterile liquid culture medium was used as a blank. IAA solutions with concentration gradients of 0, 5, 20, 40, and 60 mg / L were prepared as standard samples and the absorbance was measured. A standard curve was constructed with absorbance as the ordinate and IAA concentration as the abscissa. The IAA content (μg / mL) in the culture medium was calculated based on the standard curve.

[0046] The auxin production capacity of Trichoderma harzianum LT2-15 was determined, and a standard curve (y = 0.0148x + 0.0127, R 2 =0.9993)( Figure 4 ). It was found that the strain can produce auxin, and the concentration of auxin produced is 1.64μg / mL.

[0047] (V) Determination of the ability of strains to produce gibberellin (GA3)

[0048] Preparation and culture of the liquid and supernatant of the strain refer to the determination of the ability to produce auxin. Take 1mL of supernatant in a 25mL centrifuge tube and add 3mL of Fe 3+ Solution (concentration) and 4mL o-phenanthroline solution and shake well. After heating in a boiling water bath for 50 minutes, cool to room temperature with cold water. Then add 3mL NaF solution and 5mL HAc-NaAc buffer with pH=5. Finally, make up to 25mL with sterile water and shake well. Use the liquid culture medium without inoculation of bacteria as a control and measure the absorbance of the solution at a wavelength of 510nm. Use the liquid culture medium without inoculation of bacteria as a control and prepare 0, 20, 40, 60, 80mg / L concentration gradient GA3 solution as a standard sample to measure the absorbance. Draw a standard curve with absorbance as the ordinate and GA3 concentration as the abscissa. Calculate the GA3 content (μg / mL) in the culture medium based on the standard curve.

[0049] The gibberellin production capacity of Trichoderma harzianum strain LT2-15 was determined, and a standard curve (y = 0.0006x-0.018, R 2 =0.994)( Figure 5 ). It was found that the strain could produce gibberellin GA3 at a concentration of 113.00 μg / mL.

[0050] (VI) Determination of the indoor growth-promoting effect of strains with high antibacterial activity

[0051] After the strain was grown on PDA medium for 7 days, 1×10 6 Place silage corn 'Tieyan 53' seeds in 12cm plastic pots filled with sterilized nutrient soil and add 100mL of the spore suspension. Place 15 seeds per pot, repeating the process with three pots per strain. Water every 7 days and measure plant height after 2 weeks.

[0052] Trichoderma harzianum LT2-15 had a good effect on promoting the growth of silage corn 'Tieyan 53' indoors. The plant height of the control group without the addition of the strain was 11.18 cm, while the plant height of the corn after adding LT2-15 was 14.67 cm (Table 3 and Figure 6 The results showed that Trichoderma harzianum LT2-15 had a good growth-promoting effect indoors.

[0053] Table 3 Determination of the growth-promoting effect of Trichoderma harzianum LT2-15

[0054] strain Corn (cm) LT2-15 14.67 CK 11.18

[0055] (7) Determination of field growth promotion effect

[0056] From April to July 2024, the field growth promotion effect was determined in the experimental field of the Department of Agriculture and Forestry of Qinghai University (No. 251, Ning Road, Ershilipu Town, Chengbei District, Xining City, Qinghai Province). The experimental plot area was 10m 2 Silage maize 'Tieyan 53' was planted using the full-film single-ridge technique. Base fertilizer application rates included 250 kg / mu of organic fertilizer, 25 kg / mu of diammonium phosphate, and 10 kg / mu of urea. No topdressing was applied.

[0057] Prepare 1×10 6 Conidia suspension (conidia / mL) was prepared. 1000 mL of this suspension was mixed with 1 kg of soil and applied evenly throughout the plot. Sterile water-mixed soil was used as a control. Three replicates were set for each treatment. Plant height and stem diameter were measured three months after planting. Thirty-five corn plants were randomly selected for each treatment; stem diameter was uniformly measured at the middle of the first node.

[0058] On July 12, 2024, the growth-promoting effect of Trichoderma harzianum LT2-15 on silage corn in the field was determined. The results showed that the plant height and stem diameter of corn silage treated with LT2-15 were 102.00 cm and 8.21 cm, respectively, compared to 96.96 cm and 7.74 cm, respectively, in the control group (Table 4). These results demonstrate that Trichoderma harzianum LT2-15 is effective in promoting growth in the field.

[0059] Table 4 Determination of the growth-promoting effect of Trichoderma harzianum LT2-15 in the field

[0060] strain Corn plant height (cm) Corn stalk diameter (cm) LT2-15 102.00 8.21 CK 96.96 7.74

[0061] (8) Determination of field control effects

[0062] In early June 2024, the field control effect was tested at the experimental field of the Department of Agriculture and Forestry of Qinghai University (No. 251, Ning Road, Ershilipu Town, Chengbei District, Xining City, Qinghai Province). The test was conducted in the afternoon without wind or rain. First, a concentration of 1×10 6 800 mL of LT2-15 conidia suspension was applied to the leaves of silage corn 'Tieyan 53' using sterile gauze. The experimental plot area was 10 m 2 Three replicates were set up, and sterile water was used as a blank control. Twenty-four hours later, the silage corn leaves were inoculated with the same volume and concentration of a conidia suspension of Alternaria maydis. One month later, the incidence and severity of the disease were investigated in the field, and the disease index was calculated.

[0063] The field efficacy of Trichoderma harzianum LT2-15 against Alternaria leaf spot disease in maize (corn) was determined. The results showed that the disease index for the blank control (Alternaria leaf spot disease) was 58.24. In the control group treated with a conidia suspension of LT2-15, the disease index was 27.09 (Table 5). These results demonstrate that Trichoderma harzianum LT2-15 is effective in field control.

[0064] Table 5 Determination of field control effect of Trichoderma harzianum LT2-15 strain

[0065] strain Corn Alternaria leaf spot disease index LT2-15 27.09 CK 58.24

[0066] (IX) Safety assessment of Trichoderma harzianum LT2-15 on crops

[0067] LT2-15 was inoculated onto a PDA plate and cultured at 25°C for 7 days. The mycelium was interrupted using a sterile inoculation loop, and 0.025% Twain 20 solution was added to prepare 20 mL of mycelial suspension. Corn was planted in plastic pots (12 cm in diameter) filled with sterilized nutrient soil, and the growth cycle was 2 weeks. The safety of the strain on corn was determined by the smear inoculation method. The mycelial suspension was smeared onto the corn leaves using a sterile cotton swab, with an inoculation volume of 2 mL / pot. Moisturized culture was carried out in the dark at 25°C for 24 hours, followed by light culture at 25°C, and the disease was observed after 7 days. Under the same conditions, corn was inoculated with the large-spotted umbilical spore as a positive control, and sterile water was inoculated as a negative control. The inoculation test was repeated 3 times.

[0068] To determine whether Trichoderma harzianum LT2-15 is pathogenic to silage corn, the strain was inoculated into silage corn 'Tieyan 53' ( Figure 7 ). It was found that the strain is not pathogenic to silage corn and can be used as material for the development of subsequent microbial preparations.

[0069] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A Trichoderma harzianum strain, characterized in that The Trichoderma harzianum strain is named Trichoderma harzianum, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 41725 and a deposit date of December 20, 2024; the rDNA-ITS sequence of Trichoderma harzianum is shown in SEQ ID NO: 1; the TEF-1α sequence of Trichoderma harzianum is shown in SEQ ID NO: 2, and the RPB2 sequence of Trichoderma harzianum is shown in SEQ ID NO:

3.

2. A use of the Trichoderma harzianum strain as claimed in claim 1, characterized in that: The Trichoderma harzianum strain is used for inhibiting the activity of E. turcicum, B. sorokiniana, A. alternata and B. zeicola.

3. A use of the Trichoderma harzianum strain as claimed in claim 1, characterized in that: The Trichoderma harzianum strain is used to promote the growth of silage corn.

4. A use of the Trichoderma harzianum strain as claimed in claim 1, characterized in that: The Trichoderma harzianum strain is used for preventing and controlling corn Alternaria leaf spot disease caused by Alternaria fungi.

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