Trichoderma viride strain rm9 for antagonizing sclerotinia libertiana and its use

Abstract

This invention provides a *Trichoderma viride* RM9 strain antagonizing jasmine white mold disease and its application. The *Trichoderma viride* RM9 strain of this invention is protected under the accession number CCTCC M 20221442. *Trichoderma viride* RM9 of this invention is isolated from the rhizosphere of jasmine plants, exhibits strong rhizosphere colonization ability, and demonstrates good control efficacy against jasmine white mold disease, achieving a control effect of 83.34%. It also promotes the growth of jasmine seedlings. Furthermore, its cultivation conditions are relatively simple, and its development into a solid inoculant is beneficial for improving jasmine yield and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and specifically relates to the Trichoderma viride strain RM9, which antagonizes jasmine white rot disease, and its application. Background Technology

[0002] Trichoderma spp., belonging to the class Hyphomycetes, order Trichoderma, family Trichodermacetes, is an important biocontrol fungus widely used for the biological control of various plant diseases. As an internationally recognized biocontrol agent, Trichoderma has become a hot topic in recent years. Reports indicate it has good control effects against soil-borne diseases caused by Rhizoctonia solani, Sclerotium truncatum, Fusarium, and Phytophthora, while also promoting the growth of seedlings of fruits, vegetables, trees, and flowers.

[0003] Jasmine white mold is a soil-borne disease of jasmine caused by *Sclerotium rolfsii*. It occurs near the base of the stem close to the ground and is widespread in jasmine-growing areas, causing large-scale death of jasmine plants and a sharp reduction in jasmine flower yield, resulting in severe economic losses. The pathogen has a wide host range, affecting not only jasmine but also peanuts, rapeseed, sunflowers, and other grain and oil crops; peppers, konjac, apples, and other fruits and vegetables; as well as medicinal plants such as *Codonopsis pilosula*, *Sarcandra glabra*, and *Aster tataricus*. Currently, the control of jasmine white mold mainly relies on chemical agents. The overuse of these agents pollutes the environment and harms human health. Therefore, finding highly effective *Trichoderma* strains for controlling jasmine white mold is crucial. Summary of the Invention

[0004] In view of the above requirements, the purpose of this invention is to provide a Trichoderma virens strain RM9 with antagonistic effect against jasmine white mold disease and its application, which has a strong inhibitory effect on jasmine white mold disease and a good growth-promoting effect on jasmine plants.

[0005] To achieve the objectives of this invention, the inventors isolated and purified a fungal strain RM9 from the rhizosphere of collected jasmine plants. This strain exhibits antagonistic activity against the pathogen causing jasmine white rot. Based on the morphological characteristics and rDNA-ITS sequencing results (SEQ ID NO: 1), strain RM9 was identified as *Trichoderma virens*. Strain RM9 was deposited on September 16, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20221442, located at Wuhan University, Wuhan, China.

[0006] In a first aspect, the present invention provides Trichoderma virens strain RM9, accession number CCTCC M20221442.

[0007] Another object of the present invention is to provide the application of Trichoderma viride strain RM9, which antagonizes jasmine white rot, in the prevention and control of jasmine white rot or in the promotion of jasmine plant growth.

[0008] Secondly, the present invention provides an agent for preventing and treating jasmine white rot, comprising a solid agent of Trichoderma viride strain RM9, which antagonizes the jasmine white rot pathogen Sclerotium rolfsii.

[0009] The Trichoderma RM9 solid microbial agent is a solid microbial agent prepared by the Trichoderma RM9 strain through a process of strain activation, seed culture, and solid fermentation.

[0010] It is prepared using the following steps:

[0011] Step S1: Activate the *Trichoderma viride* strain RM9 to obtain the strain;

[0012] Step S2: Obtain seeds by liquid fermentation of the activated inoculum;

[0013] Step S3: The seeds are subjected to solid-state fermentation to prepare a solid microbial agent;

[0014] In the solid inoculant, the number of *Trichoderma viride* strain RM9 per gram of solid culture medium is 3.28 × 10⁻⁶ spores. 8 indivual.

[0015] Another object of the present invention is to provide the use of the said formulation in the prevention and treatment of jasmine white mold disease or in promoting the growth of jasmine plants.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: First, the *Trichoderma viride* RM9 strain of this invention, being isolated from the rhizosphere of jasmine, exhibits strong antagonistic ability against the pathogen causing jasmine white rot; Second, the solid inoculant prepared with *Trichoderma viride* RM9 in this invention achieves a control effect of 83.34% against jasmine white rot; Third, the solid inoculant prepared with *Trichoderma viride* RM9 in this invention has a significant effect on promoting the growth of jasmine, with a growth promotion rate of 37.14% to 161.04% on various indicators such as crown width, root length, number of leaves, number of flowers, fresh weight of stems and leaves, fresh weight of roots, total fresh weight, dry weight of roots, dry weight of stems and leaves, and total dry weight.

[0017] Preservation information for Trichoderma virens strain RM9:

[0018] Classification and nomenclature of the fungal species: Trichoderma virens

[0019] Strain preservation number: CCTCC M 20221442

[0020] Depository: China Center for Type Culture Collection, Wuhan University, Wuhan, China

[0021] Deposit date: September 16, 2022 Attached Figure Description

[0022] Figure 1 The images show colonies of Trichoderma viride strain RM9 and the pathogen of white rot of jasmine JR29-1 in confrontation culture in Example 1 (A: Day 5 of confrontation culture, nutrient competition of Trichoderma viride strain RM9 on JR29-1, B: control).

[0023] Figure 2 The images show the colony morphology and spore morphology of the Trichoderma viride strain RM9 screened in Example 1 (scale bar = 10 μm).

[0024] Figure 3 The effect of the Trichoderma viride RM9 solid inoculant prepared in Example 2 on the prevention of white rot of jasmine;

[0025] Figure 4 The effect of the Trichoderma viride RM9 solid inoculant prepared in Example 2 on the growth promotion of jasmine plants. Detailed Implementation

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] The following strains are all preserved in the applicant's laboratory and can be distributed externally.

[0028] Example 1: Isolation, screening and identification of Trichoderma viride RM9

[0029] 1. Isolation of Trichoderma viride strains

[0030] Healthy jasmine plants were collected from 16 jasmine plantations, each more than 3 kilometers apart and with over 10 years of continuous cultivation. Rhizosphere tissue was taken and surface-sterilized. The tissue was cut into 3×1 mm samples using sterilized small scissors. The samples were immersed in 75% alcohol for 10 seconds, then in 10% sodium hypochlorite solution for 2 minutes, and rinsed three times with sterile ultrapure water. The sterilized samples were placed on potato dextrose agar (PDA) medium and incubated in the dark at 28°C. During incubation, the samples were observed regularly, and Trichoderma colonies with significant differences in appearance and color were purified. A total of 16 Trichoderma strains were isolated and preserved for later use.

[0031] PDA medium formula: 200g potato, 20g glucose, 20g agar, 1000ml distilled water.

[0032] 2. Screening of biocontrol Trichoderma fungi

[0033] 2.1 Confrontation Culture: Using a sterile punch (5 mm diameter), holes were made at the edges of colonies of *Sclerotium rolfsii*, the pathogen of jasmine white rot, and different *Trichoderma* species. Mycelial discs were inoculated onto both sides of a fresh 90 mm PDA plate, with a distance of 5.5 cm between discs. Plates inoculated only with the pathogen served as a control. Each treatment was repeated three times, and the plates were incubated in the dark at 28°C. When the control colonies had fully colonized the plate, the colony diameter was measured using the cross-hatching method. The inhibition rate was calculated, using the pathogen-only inoculation as a control.

[0034] Calculation formula: Inhibition rate (%) = (Control colony radius - Treatment colony radius) / Control colony radius × 100%

[0035] In confrontation cultures, 16 Trichoderma strains showed different inhibitory effects on the pathogen of jasmine white rot (Table 1), with RM9 exhibiting the highest inhibition rate. During the confrontation culture, it was observed that the colony growth rate of Trichoderma RM9 was generally faster than that of the pathogen, rapidly occupying growth space and inhibiting the growth of the jasmine white rot pathogen JR29-1. Figure 1 When Trichoderma hyphae come into contact with pathogen hyphae, Trichoderma gradually surrounds the pathogen colony until the pathogen colony completely shrinks and is covered by the Trichoderma colony. The confrontation culture results show that Trichoderma strain RM9 can inhibit the growth of the pathogen through competition and parasitism.

[0036] Table 1. Inhibition rates of 16 Trichoderma strains against JR29-1, the pathogen of jasmine white rot.

[0037]

[0038] 3. Identification of biocontrol Trichoderma

[0039] Strain RM9 grows rapidly on PDA plates, covering a 9 cm diameter PDA dish within 3 days. It produces dense aerial hyphae, and the colonies initially appear white or light gray, gradually turning green and forming concentric rings. Under a 60x microscope, the conidia of *Trichoderma* are observed to be round or oval. Figure 3 It is transparent or pale green.

[0040] The ITS sequence was compared with relevant homologous sequences in the GenBank database (http: / / www.ncbi.nlm.nih.gov / blast / ), showing 100% similarity to *Trichoderma virens*. Based on morphological and cultural characteristics, strain RM9 was identified as *Trichoderma virens* and named *Trichoderma virens* RM9. The rDNA-ITS sequence of strain RM9 is shown in SEQ ID NO:1. Strain RM9 was deposited on September 16, 2022, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20221442, located at Wuhan University, Wuhan, China.

[0041] Example 2 Solid bacterial agent and its preparation method

[0042] The solid microbial agent containing *Trichoderma viride* RM9 provided in this embodiment was prepared by the following method: (1) Strain activation: *Trichoderma viride* RM9 was inoculated onto PDA solid culture medium and cultured in the dark at 28°C for 5 days; (2) Seed culture: The activated strain was inoculated into 100ml PDB liquid culture medium with the following formula: 2g glucose, 20g potato, 100ml distilled water, and pH value at natural; fermented in an Erlenmeyer flask at 28°C and shaken at 120rpm for 4 days, and the spore solution was diluted with sterile ultrapure water to a concentration of 1×10⁻⁶. 8 (3) Solid-state fermentation: 1000g of wheat bran was added to 900ml of PDB liquid medium and placed in a culture bag for high-temperature sterilization for 1h. After inoculation at 5%, solid-state fermentation was carried out for 7 days under dark conditions at 28℃. At this time, the seeds grew fully in the solid medium and produced a large number of spores. The results were monitored by the gradient dilution method. The results showed that the number of Trichoderma viride spores per gram of solid medium was 3.28×10⁻⁶. 8 indivual.

[0043] Example 3: Disease prevention and growth promotion effect of solid bacterial agent on jasmine plants

[0044] 1. The effect of solid inoculants on the prevention of jasmine white rot.

[0045] Cooked and sterilized oat grains were used to culture the pathogen of white mold on jasmine. Trichoderma was picked up with a sterilized toothpick and placed on 20g of wheat culture medium, then cultured in the dark at 28℃ until the mycelium was fully colonized. Solid inoculum agents were prepared as in Example 2. Trichoderma harzianum RM11 and Trichoderma viride JM2, which have relatively high inhibition rates, were selected, and solid inoculum agents were prepared using the same method as in Example 2, serving as controls A and B. The experiment was conducted in pots. Healthy jasmine plants propagated by cuttings (1 year old) were prepared in pots. Infected oat grains were buried 2cm deep in the soil around the base of the stem, 15 grains per pot, for a total of 70 pots. Two days later, 5g of Trichoderma harzianum RM9 solid inoculum was buried in the soil using the same method. Control groups A and B were buried in the soil using 5g of Trichoderma harzianum RM11 and Trichoderma harzianum JM2 solid inoculum, respectively, using the same method. Control group C had 5g of cooked and sterilized wheat bran and 15 infected oat grains buried in each jasmine plant. Control group D had 5g of cooked and sterilized wheat bran and 15 sterilized oat grains buried in each jasmine plant. Each treatment had 10 replicates. After inoculation, the plants were incubated in an artificial incubator at 28℃, 90% relative humidity, and a photoperiod of 14L:10D. Disease index and incidence rate were investigated every 3 days for 14 consecutive times. The disease index and incidence rate of the plants were calculated, and the control effect was statistically analyzed. Disease grading was carried out according to the disease grading standards and calculation method of Du Chanjuan et al. (2014). The disease grading standards were as follows: Grade 0: no yellowing of diseased leaves and branches; Grade 1: less than 25% of the whole plant wilted or died; Grade 3: 25-50% of the whole plant wilted or died; Grade 5: 50% wilted but not dead; Grade 7: the whole plant died. Incidence rate % = number of diseased plants × 100 / number of plants investigated; Disease index = Σ(each level of diseased plants × corresponding disease grade value) / total number of plants investigated × highest disease grade; Control effect % = (disease index of control group - disease index of treatment group) × 100 / disease index of control group.

[0046] In an experiment on the control effect of solid fungicides on jasmine white mold, it was found that control groups A and B developed varying degrees of diseased leaves or branches after 14 days, and some plants died after 60 days. Control group C showed leaf wilting after 14 days and all plants died after 42 days. Jasmine plants treated with Trichoderma RM9 solid fungicide were healthy, with virtually no yellowing or withered leaves or branches, showing the best control effect (as shown in Table 2). After 60 days, most jasmine plants treated with Trichoderma RM9 solid fungicide showed no yellowing or withered leaves or branches, indicating that this fungicide has a good control effect on jasmine white mold. Figure 4 ).

[0047] Table 2 Comparison of the control effects of Trichoderma viride RM9 solid inoculum on jasmine white rot disease.

[0048] deal with Incidence rate (%) Disease index Prevention and control effect CK_A 80 29.4 42.83 CK_B 90 31.5 38.75 CK_C 100 51.43 / CK_D 0 / / RM9 solid microbial agent 40 8.57 83.34

[0049] 2. The effect of solid inoculants on the growth promotion of jasmine seedlings

[0050] Two treatments were set up in the experiment: 1) inoculation with 5g of Trichoderma viride RM9 solid inoculant from Example 2; 2) inoculation with 5g of cooked and sterilized wheat bran as a blank control. Each treatment was replicated 10 times. After inoculation, the plants were placed in an artificial climate chamber at 28℃ and 14L:10D. 60 days after treatment, the jasmine plants were carefully dug up intact, and the plant height, crown width, root length, and number of flowers were measured. The fresh weight (fresh weight of stems and leaves, fresh weight of roots, and total fresh weight) was also measured. The plants were then dried in an oven at 45℃ for 72 hours, and their dry weight (dry weight of stems and leaves, dry weight of roots, and total dry weight) was measured. The growth-promoting effect of Trichoderma viride RM9 solid inoculant on strawberry plants was statistically evaluated, and the growth-promoting rate of each index was calculated. Growth-promoting rate (%) = (biomass of treatment area - biomass of blank control group) / biomass of blank control group × 100.

[0051] The results of the investigation on the growth-promoting effect of RM9 solid inoculant on jasmine seedlings 60 days after inoculation showed that, compared with the control CK, the treatment with solid inoculant significantly increased the plant height, crown width, root length, number of leaves, number of flowers, fresh weight of stems and leaves, total fresh weight, root dry weight, dry weight of stems and leaves, and total dry weight of jasmine seedlings, with a growth promotion rate ranging from 37.14% to 161.04%. However, the increase in plant height was not significant, and the growth promotion rate was only 10.00% (Table 3).

[0052] Table 3. Growth-promoting effect of Trichoderma viride RM9 solid inoculant on jasmine seedlings.

[0053] deal with RM9 solid microbial agent Comparison fertility rate Plant height 18.23cm 14.3cm 10.00％ Crown 27.97cm 17.05cm 64.05％ Root length 21.70cm 9.30cm 133.33％ Number of leaves 21.30 pieces 9.00 pieces 136.67％ Number of flowers 7.00 5.00 40.00％ Fresh weight of stems and leaves 10.26g 4.95g 107.27％ Fresh root weight 14.40g 10.50g 37.14％ Total Fresh Weight 24.66g 15.45g 59.61％ Dry weight of stems and leaves 4.02g 1.54g 161.04％ Root weight 2.41g 1.74g 38.51％ Total weight 6.43g 3.28g 96.04％

[0054]

Claims

1. A strain of Trichoderma virens (G. Harz) Buisman, Sheld. ex. Fries, antagonistic against the white thread disease of jasmine, characterized in that it is the strain RM9. Trichoderma virens ) characterized in that it is the strain RM9. The strain was deposited in China Center for Type Culture Collection on September 16, 2022, and the deposit number is CCTCC NO: M 20221442.

2. The use of the Trichoderma virens strain RM9 antagonizing jasmine Cephalosporium anguillotum in preventing and treating jasmine Cephalosporium anguillotum or in promoting the growth of jasmine plants.

3. An agent for controlling white thread blight of jasmine, characterized by comprising: the compound of claim 1 or 2. The application further provides a solid bacterial agent of the Trichoderma virens strain RM9 antagonizing jasmine Cephalosporium anguillotum.

4. The preparation for controlling jasmine white-vein leaf blight according to claim 3, characterized by: The solid bacterial agent is prepared from the Trichoderma virens strain RM9 through a process of strain activation, seed culture and solid fermentation.

5. The preparation for controlling jasmine white-armed fungus according to claim 4, characterized in that: The application further provides a preparation method of the solid bacterial agent. Step S1: activating the Trichoderma virens strain RM9 to obtain a strain; Step S2: obtaining a seed through liquid fermentation of the activated strain; Step S3: preparing a solid bacterial agent through solid culture fermentation of the seed.

6. The preparation for controlling jasmine white-vein leaf blight according to claim 5, wherein the number of Trichoderma viride spores of the strain RM9 is 3.28 x 10 8 4 spores / g of the solid medium in the solid inoculum.​ 7. The use of the preparation of any one of claims 3-6 in preventing and treating jasmine Cephalosporium anguillotum or in promoting the growth of jasmine plants.

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