A method for enhancing the ability of trichoderma to control plant diseases

Abstract

This invention discloses a method for enhancing the ability of Trichoderma to control plant diseases, belonging to the field of biocontrol technology. This invention is the first to identify the rice black spore divirus NoPV1-COTG-19 carried by *Trichoderma guiyuan* COTG-19, and confirms that this virus can enhance the inhibitory ability of *Trichoderma* against plant pathogens. Plate confrontation experiments show that the virus-carrying COTG-19 strain has a significantly higher inhibition rate against *Pseudomonas aeruginosa* than the virus-free COTG-19-FV strain. Pot and field trials show that the NoPV1-COTG-19 strain has excellent control effects against *Tetracentron sinense* canker, and the NoPV1-COTG-19 virus can be horizontally transmitted to other *Trichoderma* strains, significantly enhancing the sporulation capacity of infected *Trichoderma* strains. This invention provides a novel technical approach to improving the biocontrol efficacy of *Trichoderma*, and has broad application prospects in the field of green control of plant diseases.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to a method for enhancing the ability of Trichoderma to control plant diseases. Background Technology

[0002] Berry tea, also known as vine tea, is scientifically named Ampelopsis thaliana (…). Ampelopsis grossedentata *Amaryllis*, a tendril-like vine belonging to the genus *Amaryllis* in the family Vitaceae, is rich in flavonoids and polyphenols. Its leaf infusions and extracts possess various effects, including anti-inflammatory, antibacterial, antioxidant, hypoglycemic, and hypotensive properties, and are widely used in health products, pharmaceuticals, and cosmetics. As a distinctive industry in the Wuling Mountains region of western Hunan, *Amaryllis* plays a vital role in promoting rural revitalization, ecological prosperity, and improving national health. However, with the gradual expansion of *Amaryllis* cultivation and the changing growing environment, *Amaryllis* canker disease is becoming increasingly prevalent and its harm is growing. Therefore, finding effective and green methods to control *Amaryllis* canker disease and selecting biocontrol bacteria with good efficacy and strong resistance to environmental stress is one of the effective ways to achieve this goal.

[0003] Current research both domestically and internationally focuses on describing the effects of viruses on Trichoderma, with few reports on the mechanisms by which viruses enhance the virulence of Trichoderma, thereby improving the effectiveness of prevention. Summary of the Invention

[0004] The purpose of this invention is to provide a method to enhance the ability of Trichoderma to control plant diseases, thereby solving the problems existing in the prior art. By using fungal viruses to mediate the enhancement of Trichoderma's antagonistic ability against the causal agent of tea canker, a green and efficient control method for tea canker is achieved, providing a theoretical basis for the green and healthy development of the tea industry and the research and development of biological agents.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for enhancing the ability of Trichoderma to control plant diseases, comprising the following steps: Inducing the target Trichoderma strain to carry rice black spore divirus ( Nigrospora oryzae partitivirus NoPV1-COTG-19.

[0006] Preferably, the amino acid sequence of the rice black spore divirus NoPV1-COTG-19 is shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4.

[0007] Preferably, the target Trichoderma strain is Trichoderma guilderii (Guizhou Trichoderma). Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

[0008] Preferably, the plant disease is berry tea canker, and its pathogen is *Pseudomonas gracilis* (a type of fungus). P. winemaker ).

[0009] Preferably, the method for making the target Trichoderma strain carry rice black spore divirus NoPV1-COTG-19 is as follows: a donor Trichoderma strain carrying the virus is subjected to confrontation culture with a target Trichoderma strain not carrying the virus, and the target Trichoderma strain acquires rice black spore divirus NoPV1-COTG-19 through horizontal transmission.

[0010] The present invention also provides the application of a Trichoderma strain carrying rice black spore divirus NoPV1-COTG-19 in the preparation of a biocontrol agent for the prevention and control of plant diseases.

[0011] Preferably, the Trichoderma strain is Trichoderma guiyuanense (Guizhou Trichoderma). Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

[0012] Preferably, the plant disease is berry tea canker, and its pathogen is *Pseudomonas gracilis* (a type of fungus). P. winemaker ).

[0013] The present invention also provides a biological control agent for plant diseases, comprising a suspension of Trichoderma spores carrying rice black spore virus NoPV1-COTG-19.

[0014] Preferably, the Trichoderma strain is Trichoderma guiyuanense (Guizhou Trichoderma). Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

[0015] The present invention discloses the following beneficial effects: (1) This invention is the first to identify the RNA virus NoPV1-COTG-19 carried by *Trichoderma guiyuanensis* COTG-19, and confirms that this virus can significantly enhance the inhibitory ability of *Trichoderma guiyuanensis* against plant pathogens. Plate confrontation experiments showed that the virus-carrying COTG-19 strain was effective against *Trichoderma guiyuanensis* (…). P. viticola The inhibition rate of the virus was significantly higher than that of the COTG-19-FV strain after virus elimination, indicating that the virus is a key factor in enhancing the virulence of Trichoderma.

[0016] (2) The results of the pot experiment showed that the COTG-19 strain carrying the virus had excellent control effect on mulberry tea canker.

[0017] (3) This invention demonstrates through plate confrontation culture experiments that the virus NoPV1-COTG-19 can infect commercial Trichoderma viride strains from Trichoderma guiyang COTG-19 and significantly enhance the virulence of the infected Trichoderma viride strains. This finding indicates that the virus is not limited to a single Trichoderma strain, but can also be used to enhance the biocontrol capabilities of other Trichoderma strains, and has broad applicability and promotional value.

[0018] (4) Fungal viruses are generally considered to be harmful or neutral to the host. This invention is the first to discover that rice black spore divirus NoPV1-COTG-19 can enhance the virulence of Trichoderma. By inducing the target Trichoderma strain to carry this virus, its ability to control plant diseases can be significantly improved. The operation is simple and the effect is significant, providing a new technical path for improving the biocontrol efficacy of Trichoderma.

[0019] (5) This invention utilizes a virus-Trichoderma symbiotic system to control plant diseases. It does not rely on chemical pesticides, has no pesticide residue risk, is environmentally friendly, meets the requirements of green agriculture and sustainable development, and has good prospects for industrial application. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Phylogenetic tree of RNA virus NoPV1-COTG-19 in Trichoderma guiyang COTG-19; Figure 2 Verification results of virus-mediated enhancement of Trichoderma COTG-19 virulence; (a) viral dsRNA extraction; (b) colony morphology of COTG-19 carrying NoPV1-COTG-19 and virus-eliminated strain COTG-19-FV after 5 days of PDA culture; (c) COTG-19 carrying NoPV1-COTG-19 and virus-eliminated strain COTG-19-FV on the virus-mediated enhancement of Trichoderma COTG-19 virulence. P. viticola The plate antibacterial rate; Figure 3 Prevention of COTG-19 carrying NoPV1-COTG-19 and the virus-eliminated strain COTG-19-FV P. winemakerEffects on day 7; (a) Blank control group treated with sterile water; (b) Experimental group treated with COTG-19-FV spore suspension; (c) Experimental group treated with COTG-19 spore suspension carrying NoPV1-COTG-19; (d) Enlarged view of leaf in Figure a; (e) Enlarged view of leaf in Figure b; (f) Enlarged view of leaf in Figure c; Figure 4 Field control of COTG-19 carrying NoPV1-COTG-19 and the virus-eliminating strain COTG-19-FV. P. winemaker Effects; (a) Experimental field for the control of scabies in tea; (b) Leaves of the blank control group treated with sterile water; (c) Leaves of the experimental group treated with COTG-19-FV spore suspension; (d) Leaves of the experimental group treated with COTG-19 spore suspension carrying virus NoPV1-COTG-19; Figure 5 The results of the horizontal transmission of the virus NoPV1-COTG-19 are as follows: (a) commercial green Trichoderma used to control tea canker; (b) Trichoderma Guizhoui carrying the virus NoPV1-COTG-19; (c) conidia of commercial green Trichoderma before horizontal transmission; (d) conidia of commercial green Trichoderma after horizontal transmission. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The Guizhou Trichoderma involved in the embodiments of the present invention ( Trichoderma guizhouense COTG-19, with accession number CGMCC No. 41486, was provided by Associate Professor Liu Hong, a co-author of this invention. This strain was deposited at the China General Microbiological Culture Collection Center on August 9, 2024, and is disclosed in the invention patent "CN118909797B A strain of *Trichoderma guiyuan* and its application in the prevention and control of anthracnose in *Camellia oleifera*".

[0028] Example 1: Identification of the virus in Trichoderma guiyuan COTG-19 (1) The Guizhou Trichoderma COTG-19 strain was cultured in PDB liquid medium for 5-7 days, and viral nucleic acid was extracted by cellulose adsorption method (Morris et al., 1979) and sent to BGI for whole genome sequencing.

[0029] (2) The viral whole genome sequence was compared with the NCBI database using Blast homology, and a phylogenetic tree was constructed using MEGA X software; the taxonomic position of the virus was clarified. Figure 1 As shown). It was identified as rice black spore divirus (…). Black spore rice partitivirus NoPV1-COTG-19 is a dichotomous linear double-stranded RNA (dsRNA) composed of two gene fragments of similar size and independent functions. The longer fragment, dsRNA1, is the coding strand for RNA-dependent RNA polymerase (RdRp), and the shorter fragment, dsRNA2, is the coding strand for the outer shell protein (CP). The two nucleotide fragments are 1872 bp and 1653 bp in size, respectively. The sequences have been registered in the NCBI database with accession numbers PZ111274 and PZ111275, respectively.

[0030] The Guizhou Trichoderma (Trichoderma guiyuanensis) was determined using a plate-based culture method with two opposing plates. Trichoderma guizhouenseThe transfer ability of dsRNA virus in COTG-19 was investigated. The recipient strain was a COTG-19 strain without dsRNA virus (COTG-19-FV, the preparation method of which is detailed in Example 2). A mycelial cake was collected from the edge of the activated fresh test strain using a 5 mm diameter sterile punch. The COTG-19-FV strain and the pathogen *Pseudomonas aeruginosa* were then collected separately. Phomopsis viticola One mycelial block of the pathogen *Pseudomonas aeruginosa* was inoculated onto a PDA plate, and this was repeated three times. Similarly, one mycelial block of the non-virus-free COTG-19 strain and one mycelial block of the pathogen *Pseudomonas aeruginosa* were inoculated onto a PDA plate, and this was repeated three times. All samples were cultured in a constant temperature incubator at 25℃ using a cross-hatching method. The position of mycelial growth was recorded every 12 hours, and the mycelial growth rate was calculated. After 5 days of culture, the colony morphology of the strains was observed and photographed.

[0031] The results are as follows Figure 2 As shown in Figure c, the COTG-19 strain containing dsRNA virus is effective against... P. viticola The inhibition rate was significantly higher than that of the COTG-19-FV strain that did not contain dsRNA virus.

[0032] Example 2: Comparison of the effects of COTG-19 strains containing and without the virus on the pathogen of tea scab (Pseudomonas aeruginosa). Phomopsis viticola The inhibitory effect of ) 1. Obtain the virus-free COTG-19 strain (named COTG-19-FV) using protoplast regeneration technology. The specific method is as follows: (1) Mycelial culture: Collect COTG-19 strain mycelia on PDA plates, grind the mycelia into fragments, inoculate them into Erlenmeyer flasks containing 100 mL PDB liquid medium, and culture at 20℃ and 150 rpm for 36 h until uniform mycelial balls are formed.

[0033] (2) Enzyme solution preparation: Dissolve Lysing enzyme to a final concentration of 0.01 g / mL and snail enzyme to a final concentration of 0.001 g / mL in 0.8 M MgSO4 (pre-cooled). Filter sterilize using a 0.22 µm filter membrane and pre-cool on ice.

[0034] (3) Mycelial treatment: Collect the mycelia and wash them 2-3 times with 0.8 M MgSO4. Blot dry with sterile filter paper and cut the mycelial clumps into a paste.

[0035] (4) Lysis: Add 1 g (wet weight) of pasty mycelium to 20 mL of pre-cooled enzyme solution and lyse by slow shaking at 30℃ and 60-80 rpm for 2-3 h. During this period, examine under a microscope every 30 min to observe the formation of protoplasts.

[0036] (5) Termination and purification: Dilute the enzyme solution with 100 μL of STC solution.

[0037] Filter the solution through three layers of lens paper or a sterile 20µm nylon membrane to remove unlysolved hyphal fragments and collect the filtrate. Centrifuge the filtrate at 4°C, 5000 rpm for 10 min. Carefully discard the supernatant and gently resuspend the protoplast precipitate in STC solution.

[0038] (6) Regeneration: The purified protoplasts were serially diluted with STC solution and spread on RM regeneration medium (a hypertonic solid medium used for cell wall regeneration, cell division and callus formation in protoplast culture, with a formula of 1 L: 200 g cooked and filtered potato juice, 10 g glucose, 5 g maltose, 5 g yeast powder, 109.32 g mannitol, 20 g agar, pH 5.8-6.5, sterilized at 121℃ for 30 min). After culturing for 36-48 h, single colonies were picked to obtain the protoplast regenerated strain. The specific primers (NoPV1-RdRp-F: GAAACAGGACGGAAAAGGTAG, SEQ ID NO.5; NoPV1-RdRp-R: AGATTGATCGCCAAAAGAGAC, SEQ ID NO.6) were used to verify whether the protoplast regenerated strain carried the virus and to obtain the virus-free strain COTG-19-FV.

[0039] 2. Validation of antibacterial function Trichoderma guiyuan COTG-19 and COTG-19-FV P. viticola Inhibitory effect: Mycelial blocks of *Trichoderma guilentii* COTG-19 carrying the virus and the virus-eliminating strain COTG-19-FV were collected using a punch and then compared with the pathogen of *Tetracentron sinense* var. *guilentii* (for tea canker disease). Phomopsis viticola The bacterial blocks were cultured in PDA medium at a constant temperature of 25°C. After 5 days of culture, the colony radius was determined by the cross method, and the inhibition rate was calculated according to the following formula (1).

[0040] Antibacterial rate (%) = (XY) / X×100 (1) In formula (1), X is the radius of the control colony and Y is the radius of the treated colony.

[0041] The results are as follows Figure 2 As shown in Figure bc, the Guizhou Trichoderma COTG-19 virus-carrying bacteria... P. viticola The inhibition rate reached over 90%, while the detoxified strain COTG-19-FV showed an inhibition rate of over 90%. P. viticola The inhibition rate is around 62%.

[0042] Example 3: The preventive and therapeutic effects of COTG-19 and COTG-19-FV on mulberry tea ulcer disease 1. Potted Plant Experiment: Potted *Tea japonica* plants with good growth and similar size were selected and divided into 3 groups of 3 plants each. The experimental groups were sprayed with COTG-19 and COTG-19-FV spore suspensions, respectively, with an equal volume of sterile water as the control group. After 48 hours, equal volumes of pathogens were inoculated into each group. Phomopsis viticola Spore suspension. Disease incidence was observed and recorded by taking photos at 3, 5, 7, and 10 days after inoculation.

[0043] The results are as follows Figure 3 As shown, the observation records revealed significant differences on day 7. Plants treated with COTG-19 spore suspension did not develop the disease, while plants treated with COTG-19-FV spore suspension showed mild lesions. The control group treated with sterile water showed obvious severe lesions.

[0044] 2. Field Trial: The experimental site was selected at the Berry Tea Plantation in Jiyun Village, Fenghuang County, Hunan Province. Berry tea plants were selected in 3 rows and divided into 3 groups, with 9 plants in each group. The treatment method was the same as that in the pot experiment described above.

[0045] like Figure 4 As shown in the field comparative experiment, the control efficacy of virus-mediated Trichoderma Guizhouis COTG-19 against Berry Tea Ulcer Disease was significantly improved by treatment with COTG-19 spore suspension.

[0046] Example 4: Horizontal transmission of the virus NoPV1-COTG-19 The Guizhou Trichoderma (Trichoderma guiyuanensis) was determined using a plate-based culture method with two opposing plates. Trichoderma guizhouense The transfer ability of dsRNA virus in COTG-19 was investigated, with the recipient strain being a commercially available Trichoderma viride strain that tested negative for the virus. Trichoderma virens Using a 5 mm diameter sterile punch, mycelial discs were collected from the edge of the activated fresh test strain. One mycelial block was taken from each of the COTG-19 and commercial *Trichoderma viride* colonies and inoculated onto PDA plates, repeating three times. The plates were incubated at 25°C, with colony growth observed daily. After 5 days of incubation, mycelial discs from two different locations of the commercial *Trichoderma viride* strain were picked and placed in PDB liquid medium for shaking. Total RNA was extracted from the commercial *Trichoderma viride* strain, and virus-specific primers were used to verify whether it carried the virus. The specific primer sequences used were NoPV1-RdRp-F and NoPV1-RdRp-R, as described above.

[0047] like Figure 5 As shown, the results indicate that the NoPV1-TG19 virus can be horizontally transmitted to other *Trichoderma viride* species through plate confrontation culture, and can significantly enhance the sporulation capacity of the infected *Trichoderma viride* species. Figure 5c, which is a virus-free commercial Trichoderma conidia; Figure 5 The conidia of commercial Trichoderma (d) after infection with the NoPV1-COTG19 virus (these are conidia of commercial Trichoderma) were shown to have significantly enhanced virulence in plate tests.

[0048] The fungal virus NoPV1-COTG-19 provided by this invention has the following amino acid sequence (SEQ ID NO.1): .

[0049] The fungal virus NoPV1-COTG-19 provided by this invention has the following amino acid sequence (SEQ ID NO.2): MADHQSQITSTVAPSDSASAAGSKKSKPGKAERAARRAATGSQAGAPASSSKASAFASSVAAPKPQPGKFPVVFQTGAGEPSRDEFFGLDPDVLSSTLGGFAPRFKDHMRYSEFLSYSEYDDDDFKKQLTSAGLLRLAQQLVHAHVNMGLPQGDFAPVASTEVRVPGSMSAAISQFGELSVPALGTRFLLKDYASSVKSIIWAAHCASNEDNVEAVISRSWLPVSKTDGHTKQIIASRLNAILKIAEIQYDASTLEKCVLSGNRPDSWDQLKVLLGDTEKRQNRFDFLFQSHKDAPEFVTSFTGNEGLAVLKELGLDWCMPSASHVDWTFNPKETFSSVADEWARKSATYAQFFELASSQTNRTAASGSQAQFALIDTRDSVTVVKARLALSAPEFSLAACFPASVCLSEPCLRNVVVTTPLSVSQRATKFVQLDWR。

[0050] The nucleotide sequence of virus NoPV1-COTG-19 (SEQ ID NO.3) is as follows:

[0051] The nucleotide sequence (SEQ ID NO.4) of virus NoPV1-COTG-19 is as follows:

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for enhancing the ability of Trichoderma to control plant diseases, characterized in that, Includes the following steps: Inducing the target Trichoderma strain to carry rice black spore divirus ( Nigrospora oryzae partitivirus NoPV1-COTG-19.

2. The method according to claim 1, characterized in that, The amino acid sequence of the rice black spore divirus NoPV1-COTG-19 is shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. The method according to claim 1, characterized in that, The target Trichoderma strain is Trichoderma guiyuan ( Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

4. The method according to claim 1, characterized in that, The plant disease is berry tea canker, and its pathogen is *Pseudomonas aeruginosa* (a type of fungus). Phomopsis viticola ).

5. The method according to claim 1, characterized in that, The method for making the target Trichoderma strain carry rice black spore divirus NoPV1-COTG-19 is as follows: a donor Trichoderma strain carrying the virus is subjected to confrontation culture with a target Trichoderma strain not carrying the virus, and the target Trichoderma strain acquires rice black spore divirus NoPV1-COTG-19 through horizontal transmission.

6. Application of a Trichoderma strain carrying rice black spore divirus NoPV1-COTG-19 in the preparation of biocontrol agents for plant diseases.

7. The application according to claim 6, characterized in that, The Trichoderma strain is Trichoderma guiyuan ( Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

8. The application according to claim 6, characterized in that, The plant disease is berry tea canker, and its pathogen is *Pseudomonas aeruginosa* (a type of fungus). P. viticola ).

9. A biological control agent for plant diseases, characterized in that, Contains a suspension of Trichoderma spores carrying rice black spore divirus NoPV1-COTG-19.

10. The biological control agent according to claim 9, characterized in that, The Trichoderma strain is Trichoderma guiyuan ( Trichoderma guizhouense ) or green Trichoderma ( Trichoderma virens ).

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