Trichoderma asperellum HM-8 and its application

Abstract

The present invention relates to a Trichoderma asperellum ( Trichoderma asperellum ) HM-8, which has both antagonistic effects against Erwinia spp. and Fusarium solani f. sp. batatas, and the microbial inoculum prepared by using the Trichoderma asperellum HM-8 strain can effectively control sweet potato root rot and can also effectively control ginger basal rot.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and particularly relates to a Trichoderma asperellum HM-8 and its application. Background Art

[0002] Sweet potato root rot is a soil-borne fungal disease that seriously harms sweet potato production and occurs in both sweet potato seedbeds and fields. After sweet potatoes are infected with root rot, the rhizome part turns black and rots, resulting in fewer and smaller sweet potato tubers or no tubers being formed, leading to a decrease in the yield and quality of sweet potato roots. The main pathogen of sweet potato root rot is Fusarium solani f. sp. batatas ( Fusarium solani (Mart.) Sacc. f. sp. batatas Mc Clure ), also known as Fusarium solani f. sp. batatas. Contaminated soil and diseased residues in the soil are the main primary infection sources in the following year. The pathogen of sweet potato root rot spreads through the soil. The distribution of the pathogen is the highest in the tillage layer, and the disease is also severe. The pathogen in the soil can survive for at least 3 years, and its vertical distribution can reach 100 cm in the soil layer, but the density is the highest in the tillage layer soil.

[0003] Ginger basal rot, also known as ginger soft rot, ginger stem rot, etc., has extremely complex causes of basal rot. Reported pathogens include the bacterium Ralstonia solanacearum ( Ralstonia solanacearum ), Erwinia spp. ( Erwinia sp .), and the fungal group Pythium myriotylum and Fusarium oxysporum ( Fusarium oxysporum ). Research shows that in some ginger-growing areas in Hebei, the main pathogenic bacterium causing ginger basal rot is Erwinia spp., which mainly exists in the soil.

[0004] Currently, the measures for controlling sweet potato root rot and ginger basal rot in production mainly include agricultural control, chemical control, and biological control. The many advantages of chemical agents have made them widely used in agricultural production. However, with the gradual enhancement of human health and environmental protection awareness, the long-term and large-scale non-standard use of pesticides has gradually revealed many problems such as environmental pollution, pesticide residues, the emergence of drug resistance, and the direct toxicity to non-target organisms. Biological control, to a certain extent, makes up for the deficiencies of chemical pesticides and will play a major role in the future sustainable development of agriculture. Summary of the Invention

[0005] The object of the present invention is to provide a Trichoderma asperellum HM-8 that can effectively control sweet potato root rot and ginger basal rot.

[0006] The present invention adopts the following technical solutions:

[0007] A Trichoderma asperellum ( Trichoderma asperellum)HM-8, with the deposit number of CGMCC No. 41051, was deposited at the China General Microbiological Culture Collection Center on January 23, 2024, at the address of Beijing, China.

[0008] Furthermore, the Trichoderma asperellum HM-8 can antagonize Fusarium solani f. sp. batatas.

[0009] Furthermore, the Trichoderma asperellum HM-8 can antagonize Erwinia bacteria.

[0010] Furthermore, the Trichoderma asperellum HM-8 has the ability to antagonize both Erwinia bacteria and Fusarium solani f. sp. batatas.

[0011] A microbial inoculant, which contains the thallus, spores, metabolites, fermentation broth and / or fermentation products of the above-mentioned Trichoderma asperellum HM-8.

[0012] In the microbial inoculant, the number of spores of Trichoderma asperellum HM-8 is not less than 10 8 CFU / g.

[0013] The microbial inoculant also contains soluble starch.

[0014] An application of the above-mentioned Trichoderma asperellum HM-8 in biological control.

[0015] An application of the above-mentioned Trichoderma asperellum HM-8 in biological control of sweet potato root rot.

[0016] An application of the above-mentioned Trichoderma asperellum HM-8 in biological control of ginger basal rot.

[0017] The beneficial effects of the present invention are as follows: The Trichoderma asperellum HM-8 of the present invention has the ability to antagonize both Erwinia bacteria and Fusarium solani f. sp. batatas. The microbial inoculant prepared by using the Trichoderma asperellum HM-8 strain can effectively control sweet potato root rot and ginger basal rot, and the control effects reach 88.09% and 80.20% respectively, and the yields of sweet potato and ginger increase significantly. In comparison, its control effect and yield are significantly higher than those of chemical pesticides, and it can replace pesticides for disease control, reducing environmental pollution. Description of the Drawings

[0018] Figure 1 It is the inhibition rate test of strain HMC03 against Fusarium solani f. sp. batatas.

[0019] Figure 2 It is the inhibition rate test of strain HMC04 against Fusarium solani f. sp. batatas.

[0020] Figure 3 It is the petri dish inhibition effect of the fermentation broth of strain HMC03 against Erwinia bacteria.

[0021] Figure 4 The inhibition effect of the fermentation broth of strain HMC04 on Erwinia in a petri dish.

[0022] Figure 5 The colony morphology of Trichoderma asperellum HM-8.

[0023] Figure 6 The morphology of Trichoderma asperellum HM-8 stained with cotton blue under a microscope.

[0024] Figure 7 The phylogenetic tree of Trichoderma asperellum HM-8. Detailed implementation manners

[0025] The following combines specific embodiments to further elaborate on the present invention. It should be noted that the specific embodiments are explanations rather than limitations of the present invention.

[0026] Example 1 Screening of strains

[0027] Soil samples were collected from the rhizosphere soil of ginger plants without ginger basal rot disease in a severely ginger basal rot disease-affected plot in Liushouying Town, Funing District, Qinhuangdao City, Hebei Province. A total of 6 samples were collected, each taking 100 g, and the 6 collected soil samples were evenly mixed. 10 g of the mixed soil sample was placed into a conical flask containing 90 mL of sterile water and shaken well. The soil dilution coating method was used for treatment. After gradient dilution of the soil sample, 100 μL of the dilutions with dilution factors of 10 -4 、10 -5 、10 -6 were evenly coated on the plates containing PDA medium, and incubated in the dark at a constant temperature of 25 °C in an inverted position. After 2 days, observations were made daily. When hyphae grew out, the hyphal blocks were picked and transferred to a new PDA plate for continued cultivation. When spores were produced, single spore isolation and purification were carried out. The purified strains were numbered and stored on a slant in a 4 °C refrigerator for later use.

[0028] The formula of the above PDA medium is as follows: 200 g / L of potato, 20 g / L of agar, and 20 g / L of glucose. The preparation method is as follows: The potato is peeled and cut into pieces, boiled for 30 min, filtered through a gauze, then sucrose and agar are added, heated and stirred until the agar is completely dissolved, and then made up to 1000 mL, sterilized at 121 °C for 20 min for later use after dispensing.

[0029] The antagonistic effect of the above-numbered strains against Fusarium solani f. sp. batatas was determined by the plate confrontation method. The specific method is as follows: (1) Culture the above-purified and numbered strains on PDA medium; (2) After 4 days, use a 200 μL pipette tip to punch a 5 mm diameter fungal plug of Fusarium solani f. sp. batatas and inoculate it in the middle of the PDA medium plate; (3) Punch fungal plugs of the isolated and purified numbered strains and symmetrically inoculate them at a distance of 3 cm from the middle on the above PDA medium plate, and incubate at 28 °C for 5 - 7 days with 3 replicates for each treatment. Observe every day. When the screened strain covers the plate, measure the colony diameter of Fusarium solani f. sp. batatas, and calculate the inhibition rate of the screened strain against Fusarium solani f. sp. batatas.

[0030] Inhibition rate (%) = (9 - colony diameter of Fusarium solani f. sp. batatas) ÷ 9 × 100.

[0031] In the formula, 9 is the diameter of the plate (cm).

[0032] After detection, the inhibition rates of the strains numbered HMC03 and HMC04 against Fusarium solani f. sp. batatas were relatively high, 100% and 53.33% respectively, as Figure 1 、 Figure 2 and shown in Table 1.

[0033] Table 1 Inhibition rates of HMC03 and HMC04 against Fusarium solani f. sp. batatas

[0034] .

[0035] Antagonistic effect test of the strains numbered HMC03 and HMC04 against Erwinia sp. ( Erwinia sp .). Detect the antibacterial activity of the fermentation broth of HMC03 and HMC04. The specific steps are as follows:

[0036] 1) Prepare a bacterial suspension of the pathogen Erwinia sp. with a concentration of 10 7 CFU / mL; Pipette 1 mL of the Erwinia sp. fermentation broth onto the surface of the LB medium plate and spread the bacterial liquid evenly with a spreader.

[0037] 2) Use a 1000 μL pipette tip to punch a 5 mm diameter hole in the above medium;

[0038] 3) Add the fermentation broth of the strain HMC03 or HMC04 to be detected into the hole and fill it up without overflowing;

[0039] 4) Incubate at 37 °C for 3 days, observe the results, and measure the diameter of the inhibition zone with a vernier caliper. The experiment is repeated 3 times.

[0040] Judgment criteria: When the diameter of the inhibition zone ≥ 15 mm, it indicates that Erwinia is extremely sensitive to the fermentation broth of HMC03 or HMC04 strains; when 10 mm ≤ the diameter of the inhibition zone ≤ 15 mm, it indicates moderate sensitivity; when 6 mm ≤ the diameter of the inhibition zone ≤ 10 mm, it indicates low sensitivity, and no inhibition zone indicates insensitivity.

[0041] The test results show that the average diameter of the inhibition zone produced by the fermentation broth of HMC03 strain against Erwinia is 30.4 mm (as Figure 3 ), and the average diameter of the inhibition zone produced by the fermentation broth of HMC04 strain against Erwinia is 24.6 mm (as Figure 4 ), among which the inhibition ability of the fermentation broth of HMC03 strain against Erwinia is stronger.

[0042] Example 2 Identification of HMC03 strain

[0043] The isolated HMC03 strain was inoculated onto PDA medium. When initially cultured on the PDA plate, the colonies of the HMC03 strain were white, round, without obvious concentric rings and radial morphology. It could cover a 9-cm petri dish after being cultured on PDA medium for 72 h, with a relatively fast growth rate. After 5 days, green conidia were produced from the middle to the periphery and then covered the entire plate. Its aerial hyphae were dense, the surface was closely like a blanket, and the edge was white and flocculent. Through microscopic observation, the conidia of HMC03 were spherical, preliminarily proving that the HMC03 strain was a mold. As Figure 5 and Figure 6 shown.

[0044] The Hebei Academy of Sciences Institute of Biology was entrusted to identify the HMC03 strain. Referring to "6.5" in NY / T 1736-2009, the ITS region of this strain (as shown in SEQ ID No.1) was amplified by PCR, the amplified product was recovered and sequenced, and the sequence was compared for homology with the sequences registered in the GenBank database, and a phylogenetic tree was constructed using MEGA7.0 as Figure 7 shown. The ITS region sequence of this strain had 100% homology with Trichoderma asperellum.

[0045] Based on sequence similarity, phylogenetic analysis, and morphological identification, the HMC03 strain was identified as Trichoderma asperellum ( Trichoderma asperellum ), named HM-8. This strain was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 23, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, and its biological deposit number is CGMCC No. 41051.

[0046] Example 3 Preparation of microbial agent of strain HM-8

[0047] Inoculate the strain HM-8 into PDA medium and culture it in an incubator at 25°C for 7 days. Take 3 - 4 fungal blocks from it and add them to the seed bottle. Culture it on a shaker at 25°C and 180 rpm for 48 h to prepare the seed liquid for use.

[0048] Add the seed liquid to the seed fermenter at a volume ratio of 1:150 (the composition of the seed fermenter medium: by weight, containing 2% sucrose, 1% peptone, 0.2% NaCl, 0.01% K2HPO4, 0.01% MgSO4, 0.5% CaCO3) for culture, and then add it to the fermenter of the chlamydospore fermentation medium at a volume ratio of 1:20 (the chlamydospore 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). Culture at 28°C for 132 h until 90% of the hyphae form chlamydospores to obtain the chlamydospore fermentation broth, then perform freeze spray drying, and mix the chlamydospore dry powder of this bacterium with soluble starch to form a wettable powder. After detection, the spore count of Trichoderma asperellum HM-8 contained in this bactericide is 1.2×10 8 CFU / g. The obtained wettable powder is the HM-8 microbial bactericide used in the following examples.

[0049] Example 4 Application of HM-8 Microbial Bactericide in Controlling Ginger Rhizome Rot

[0050] The experimental site was selected in a ginger planting area in Funing District, Qinhuangdao City, Hebei Province. This plot has been continuously planted with ginger for years and has serious continuous cropping diseases (rhizome rot). Select an area of 20m×6m as an experimental area. Plant 10 ridges of ginger in each experimental area, about 500±30 plants, and the variety is Mianjiang. A total of 12 experimental areas were set up, and a protection row was established between each experimental area. Randomly select 3 experimental areas out of the 12 experimental areas for each treatment group to conduct the experiment. The experimental design is as follows:

[0051] (1) Blank control group 1: Do not apply any bactericidal powder, and directly plant ginger in the ginger ditch.

[0052] (2) Control group 2: Use 4 kg of 50% carbendazim wettable powder per mu to mix with 20 kg of soil, evenly spread it in the ginger ditch, and then plant ginger in the ginger ditch. The 50% carbendazim wettable powder was purchased from the market.

[0053] (3) Bactericidal powder treatment group: First, evenly spread the bactericidal powder of the wettable powder of the HM-8 microbial bactericide prepared in Example 3 of the present invention in the ginger ditch at different dosages, and then plant ginger in the ginger ditch. Among them: Treatment group 1: The dosage of the HM-8 microbial bactericide is 1 kg / mu, mixed with 20 kg / mu of moist soil; Treatment group 2: The dosage of the HM-8 microbial bactericide is 3 kg / mu, mixed with 20 kg / mu of moist soil.

[0054] During the growth process of ginger, other cultivation and management measures applied were all carried out in accordance with the local production technical specifications. When harvesting ginger, the incidence of ginger basal rot was counted, and the control efficiency of Trichoderma asperellum HM-8 microbial inoculum against ginger basal rot was calculated. The specific results are shown in Table 2.

[0055] Table 2 Control effect of Trichoderma asperellum HM-8 microbial inoculum against ginger basal rot

[0056] 。

[0057] Disease grading standard: Grade 0, the ginger plants are healthy without disease; Grade 1, local lesions occur on the mother ginger plants, and the offspring ginger plants are healthy without disease; Grade 2, there are lesions on the offspring ginger plants, but no withering; Grade 3, local withering of the ginger clumps (30% - 50%); Grade 4, the ginger clumps are basically withered or completely withered, and the ginger flesh turns color and rots less than 60%; Grade 5, the ginger clumps are completely withered, and the ginger flesh rots more than 60%.

[0058] The calculation formulas for disease index and relative control effect are as follows:

[0059] Disease index (%) = [∑(number of diseased plants at each level × corresponding disease grade) / (total number of plants surveyed × highest disease grade)] × 100;

[0060] Relative control effect (%) = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100.

[0061] From the results in Table 2, it can be seen that compared with the blank control group, the disease index of ginger basal rot can be greatly reduced in each treatment group. Among them, the control efficiency of treatment group 2 is as high as 88.09%, and the yield increases by 53.5% compared with the blank control; compared with control group 2, each treatment group also has a good prevention and control effect. Among them, the control effect of treatment group 2 is nearly 10% higher than that of control group 2, and the yield increases by 10.8%. This shows that the Trichoderma asperellum HM-8 microbial inoculum provided by the present invention has a significant control effect on ginger basal rot, can replace pesticides, and reduce environmental pollution.

[0062] Example 5 Application of HM-8 microbial preparation in controlling sweet potato root rot

[0063] The test plot was set in the sweet potato planting field of a grower in Lulong Town, Lulong County, Hebei Province. The test plot is a hilly mountain slope field with loam soil, medium fertility conditions, and poor water conservancy conditions. Except for the water used for planting, it basically depends on natural precipitation. The test plot has continuously planted sweet potatoes for 10 years. In the past 3 years, the root rot has occurred severely, and the yield loss is about 30%.

[0064] A total of 3 treatments were set in the test, as shown in Table 3.

[0065] Table 3 Application Test Plan of HM-8 Microbial Agent in Controlling Sweet Potato Root Rot

[0066] .

[0067] Among them, 50% carbendazim wettable powder and 50% thiram wettable powder were both purchased from the market.

[0068] Each treatment had 3 replicates. There were 5 rows of sweet potatoes planted in the test plot, with a row spacing of 0.8 m, a row length of 10 m, and a plant spacing of 0.2 m. The plots were randomly arranged, and the area of each plot was about 48 square meters. The tested sweet potato variety was Tengfei. All plots were uniformly treated for nematodes and underground pests in advance. The control agents were 35% phoxim microcapsule (for controlling underground pests) and 10% fosthiazate granules (for controlling nematodes). On May 10, 2023, the sweet potatoes in all test treatments were completed for planting. On October 11, the diagonal fixed-point 5-point investigation method was used. 10 plants were investigated for each treatment, and the sweet potato yield and diseased sweet potatoes were measured and recorded (sweet potato tubers with an individual fresh weight less than 50 g were not included in the statistics). The investigation statistical table is shown in Table 4.

[0069] Table 4 Control Effect of Each Treatment on Sweet Potato Root Rot and Sweet Potato Yield

[0070] .

[0071] The disease condition of sweet potato root rot was classified into 5 levels according to the disease symptoms of sweet potato tubers:

[0072] Level 0: The sweet potato tubers are normal without symptoms;

[0073] Level 1: Individual roots turn black (the number of diseased roots accounts for less than 10% of the total number of roots), and there are no lesions on the underground stem, which has no obvious impact on tuber formation;

[0074] Level 2: A few roots turn black (the number of diseased roots accounts for 10% - 25% of the total number of roots), and there are individual lesions on the underground stem and sweet potato tubers, which has a slight impact on tuber formation;

[0075] Level 3: Nearly half of the roots turn black (the number of diseased roots accounts for 25.1% - 50.0% of the total number of roots), and there are many lesions on the underground stem and sweet potato tubers, which has a significant impact on tuber formation, and there are woody roots;

[0076] Level 4: Most roots turn black (the number of diseased roots accounts for more than 50% of the total number of roots), the lesions on the underground stem are many and large, and no tubers are formed or even die.

[0077] The diseased tuber rate, disease index and control effect were calculated according to the following formulas:

[0078] Diseased tuber rate (%) = (number of diseased sweet potato tubers in the plot / number of harvested sweet potato tubers in the plot) × 100;

[0079] Disease index (%) = [Σ (number of sweet potato tubers at each disease level × representative value of the corresponding level) / (number of harvested sweet potato tubers in the plot × highest disease level)] × 100;

[0080] Control effect (%) = [(disease index in the blank control plot - disease index in the medicament treatment plot) / disease index in the blank control plot] × 100.

[0081] Table 4 results show that Treatments 1 and 2 both have good control effects and yield-increasing effects on sweet potato root rot. Among them, the control effect of the former is over 80.20% compared with the control, and the plot yield is also 129.45 kg higher than that of the control. Compared with Treatment 2, Treatment 1 also has a good control effect and yield-increasing effect, indicating that the Trichoderma asperellum HM-8 microbial agent of the present invention also has a significant control effect on sweet potato root rot, can replace pesticides, and reduce environmental pollution.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Trichoderma acanthosporum ( Trichoderma asperellum ) HM-8, characterized in that, Its deposit number is CGMCC No.41051.

2. A microbial agent, characterized in that: The method comprises the cells and / or spores of Trichoderma spinulosa HM-8 as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that: The spore count of Trichoderma spinulosa HM-8 is not less than 10 8 CFU / g.

4. The microbial agent according to claim 2, characterized in that: It is characterized in that Also contains soluble starch.

5. Use of the Trichoderma aspergillus HM-8 as claimed in claim 1 in biological control of sweet potato root rot.

6. Use of the Trichoderma aspergillus HM-8 as claimed in claim 1 in biological control of ginger stem base rot.

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