Trichoderma guizhouense gzm t12 and application thereof
By isolating and identifying the Guizhou Trichoderma GZMT12 strain, preparing its spore suspension and inoculant, the problem of controlling various plant pathogens was solved, and a highly efficient plant disease control effect was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUIZHOU UNIV
- Filing Date
- 2025-10-13
- Publication Date
- 2026-06-16
AI Technical Summary
Plant pathogens pose a serious threat to crops and cash crops. Existing technologies are insufficient to effectively control many plant pathogens, leading to losses in yield and quality.
The Guizhou Trichoderma strain GZMT12 was isolated and identified, and its spore suspension and inoculant were prepared for the control of plant pathogens such as *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.
Trichoderma Guizhouense GZMT12 exhibits highly effective control against a variety of plant pathogens, with an inhibition rate of 75.89%-78.07%, and is widely used in products for the prevention and control of plant pathogens.
Smart Images
Figure CN121006284B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a Trichoderma guiyuanensis GZMT12 and its applications. Background Technology
[0002] Plant production is highly susceptible to damage from plant pathogens. These pathogens are diverse and can severely impact crop yield and quality, leading to significant economic losses. Globally, pathogens pose a serious threat to agricultural and cash crops. Tobacco target spot is caused by *Rhizoctonia solani* Kühn, a highly contagious bacterium that can spread rapidly through soil, diseased plant debris, irrigation water, and agricultural operations, easily causing large-scale epidemics and posing a serious threat to tobacco production. *Botryosphaeria dothidea*, *Alternaria alternata*, and *Phomopsis lithocarpus* are the main pathogens causing soft rot in kiwifruit. Kiwifruit soft rot is a fungal disease that occurs during the storage and distribution of kiwifruit. On infected fruit, it manifests as collapsed peel, softened tissue, and a rotten odor. After peeling away the epidermis at the lesion site, a milky-white area is revealed in the center of the lesion, surrounded by a yellowish-green transition zone. The flesh tissue shows signs of softening and rotting. Longitudinal sectioning reveals that the soft rot area extends cone-shaped into the flesh, making it highly susceptible to spread. Tobacco black shank caused by *Phytophthora nicotianae* causes significant economic losses to tobacco crops globally. *Phytophthora capsici* is a devastating disease in chili pepper production, often causing total crop failure in severe cases, and it occurs in various regions. *Colletotrichum truncatum* is the pathogen causing anthracnose in chili peppers. The resulting anthracnose is severe in chili peppers, greatly affecting their marketability and economic value. It is an important disease in chili pepper production, and its occurrence and damage are showing an increasingly serious trend.
[0003] Trichoderma spp. belongs to the phylum Fungi, subphylum Deuteromycetes, class Hyphomycetes, order Trichodermophycetes, family Trichodermophyceae. It is one of the most representative biocontrol fungi in the field of biological control. Due to its advantages such as environmental friendliness, green and efficient production, it has become a research hotspot in recent years. Summary of the Invention
[0004] The purpose of this invention is to provide a *Trichoderma guiyuan* strain, GZMT12, and its applications to solve the problems existing in the prior art. This invention isolates a *Trichoderma guiyuan* strain, GZMT12, which has advantages such as good control effect, broad antibacterial spectrum, and wide application range, and can be used to control plant pathogens.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] In the first aspect, the present invention provides a *Trichoderma guizhouense* GZMT12, which was deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.42152.
[0007] Secondly, the present invention also provides a spore suspension of the aforementioned *Trichoderma guiyuan* GZMT12.
[0008] Thirdly, the present invention also provides a method for preparing the spore suspension, characterized in that it includes the steps of inoculating the Guizhou Trichoderma GZMT12 into a culture medium to obtain spores and rinsing and mixing with sterile water to obtain a spore suspension.
[0009] Fourthly, the present invention also provides a fungal agent comprising the aforementioned *Trichoderma guiyuan* GZMT12 or the aforementioned spore suspension.
[0010] Fifthly, the present invention also provides the application of the Guizhou Trichoderma GZMT12, the spore suspension, or the fungal agent in the control of plant pathogens.
[0011] Preferably, the plant pathogen is selected from one or more of the following: *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.
[0012] In a sixth aspect, the present invention also provides the application of the Guizhou Trichoderma GZMT12, the spore suspension, or the fungal agent in the preparation of products for the prevention and control of plant pathogens.
[0013] Preferably, the plant pathogen is selected from one or more of the following: *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.
[0014] In a seventh aspect, the present invention also provides a product for preventing and controlling plant pathogens, the product comprising the aforementioned Trichoderma Guizhouense GZMT12, the aforementioned spore suspension, or the aforementioned fungal agent.
[0015] Eighthly, the present invention also provides a method for preventing and controlling plant pathogens, comprising the step of inoculating the aforementioned Trichoderma Guizhouense GZMT12, the aforementioned spore suspension, or the aforementioned fungal agent into infected plants.
[0016] The present invention discloses the following technical effects:
[0017] This invention isolated a strain of *Trichoderma guiyuan* GZMT12, which showed an inhibition rate of 75.89% against *R. solani*. An investigation of the strain's disease control effect on *R. solani* in tobacco using detached leaves revealed a control efficacy of 76.03% after treatment with *Trichoderma guiyuan* GZMT12. Simultaneously, the broad-spectrum antibacterial activity of *Trichoderma guiyuan* GZMT12 was investigated, showing an average inhibition rate of 78.07% against six pathogens: *Botryosphaeria dothidea*, *Alternaria alternata*, *Phomopsis lithocarpus*, *Colletotrichum truncatum*, *phytophthora capsici*, and *Phytophthora nicotianae*. This demonstrates advantages such as good control efficacy, broad antibacterial spectrum, and wide applicability.
[0018] Preservation Information: Trichoderma guizhouense GZMT12 was deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.42152. Attached Figure Description
[0019] 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.
[0020] Figure 1 This is a schematic diagram of the plate confrontation method; where the left side is the control group for screening colonies alone, the middle side is the treatment group, and the right side is the control group for pathogens alone.
[0021] Figure 2 The inhibitory effect of biocontrol strains on the pathogen of tobacco target spot disease is shown; among them, A, C, and E are the growth plates of R. solani alone, and B, D, and F are the growth plates of GZMT12, GZMT13, GZMT14 and R. solani co-cultured, respectively.
[0022] Figure 3 Morphological characteristics of Trichoderma guizhouense GZMT12; where AB are the morphological images of the colony on the front and back; CE are the conidiophores and phialides of the colony; F is the conidia of the colony; scale bar = 10 μm;
[0023] Figure 4 A phylogenetic tree was constructed using the maximum likelihood method based on the sequences of multiple genes (rpb2 and tef1).
[0024] Figure 5 The study aimed to assess the control efficacy of Trichoderma Guizhouense GZMT12 against target spot disease on detached tobacco leaves. Group A represented the blank treatment group; Group B the control group; Group C the positive control group; and Group D the experimental group.
[0025] Figure 6 Determination of the broad-spectrum antibacterial activity of Trichoderma guizhouense GZMT12. Detailed Implementation
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] Example 1
[0032] 1. Experimental Materials
[0033] 1.1 Soil Sample
[0034] Soil samples were collected using a five-point sampling method from the rhizosphere soil of healthy tobacco plants (mature stage) in the affected tobacco-growing area (tobacco target spot disease). The sampling location was in Meitan County, Zunyi City, Guizhou Province (27°41′40″N, 107°33′40″S).
[0035] 1.2 Target pathogens
[0036] The pathogen of tobacco target spot disease, Rhizoctonia solani Kühn, was provided by the Tobacco College of Guizhou University, Guiyang City, Guizhou Province.
[0037] 1.3 Test Culture Medium
[0038] PDA medium: 200 g peeled potatoes, 20 g glucose, 15-20 g agar powder and 1 L sterile distilled water, natural pH, sterilized at 121℃ for 20 min before use.
[0039] 2. Experimental Methods
[0040] 2.1 Isolation, purification and preservation of strains
[0041] The soil dilution plate method was used, where 10 g of soil was placed in an Erlenmeyer flask containing 90 mL of sterile water and incubated at 37°C and 200 rpm for 30 min on a shaker, followed by standing at room temperature for 10-15 min. The supernatant from the shake flasks was then diluted with sterile water to six different gradients (10T) in a clean bench. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take 100 μL of each dilution and spread it evenly on a PDA plate using a sterile glass rod. Incubate in the dark at 28°C. After colonies grow, pick hyphal fragments from the colony edge and transfer them to a fresh PDA medium for purification to obtain pure cultures. Finally, store the obtained pure cultures in cryovials with a final concentration of 15-20% glycerol at 4°C.
[0042] 2.2 Screening of biocontrol strains
[0043] Using *Rhizoctonia solani*, the tobacco target spot pathogen, as an indicator bacterium, the plate confrontation method was employed for screening biocontrol bacteria. The isolated fungal and indicator pathogen mycelial discs were inoculated separately at 2 cm margins on PDA medium. Figure 1 The control group was inoculated with only indicator bacteria pellets, and each treatment was repeated in triplicate. The bacteria were incubated in the dark at 28°C for 5 days, and the growth of the strains was observed and recorded by photograph.
[0044] The formula for calculating the antibacterial rate is: Antibacterial rate = [(CT) / C] × 100%.
[0045] Where C and T represent the average growth radius of the tested pathogens in the control group and treatment group, respectively.
[0046] 2.3 Identification of biocontrol bacteria
[0047] 2.3.1 Morphological observation
[0048] The selected strains were inoculated into PDA medium and cultured in the dark at 28°C. During this period, the growth performance of the strains on the medium was observed, including colony color and sporulation status. Subsequently, the morphological characteristics of conidiophores, conidia (n=50), and sporulation structures were observed under a Zeiss microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at 40x magnification (1×40).
[0049] 2.3.2 Molecular identification
[0050] Genomic DNA was extracted from the screened strains using the Ezup column-based fungal genomic DNA extraction kit from Shanghai Sangon Biotech. ITS universal primer pairs (ITS4 and ITS5), RPB2 primer pairs (fRPB2-5f and fRPB2-7cr), and TEF1 primer pairs (EF1-728F and TEF1LLErev), synthesized by Beijing Qingke Biotechnology (Chongqing) Co., Ltd., were used for amplification. Specific primer information is shown in Table 1.
[0051] Table 1. Amplification primer sequence information
[0052]
[0053] Polymerase chain reaction (PCR) was performed in a 25 μL reaction system containing 1 μL DNA template, 1 μL forward primer, 1 μL reverse primer, 12.5 μL 2× Tap Plus MaserMix, and 9.5 μL sterile water.
[0054] The PCR products were purified and sequenced by Beijing Qingke Biotechnology Co., Ltd. (Chongqing).
[0055] RPB2 and TEF1-α sequences of 25 other Trichoderma strains were downloaded from GenBank (see Table 2), and phylogenetic trees were constructed using the maximum likelihood (ML) method. ML analysis was performed on the IQ-TREE web server (http: / / iqtree.cibiv.univie.ac.at) with 1000 fast guide replicates under a partitioning model. The phylogenetic trees were visualized using FigTree v.1.4.0.
[0056] Table 2. Gene accession numbers of strains
[0057]
[0058] Note: The presence of T and ET in the strain number indicates that this strain is a type strain.
[0059] 3. Determination of the efficacy of biocontrol bacteria against tobacco target spot disease on detached leaves
[0060] Preparation method of biocontrol strain spore suspension: The screened strain was inoculated onto PDA medium and cultured. The spore-producing colonies on the PDA medium were washed with sterile water. The mycelia were removed by filtration through sterile gauze. The mycelia were counted using a hemocytometer to obtain 1×10⁻⁶ spores. 7 Spore suspension at spores / mL.
[0061] Tobacco leaves of uniform size were selected for foliar spraying. Four treatment groups were established. Treatment group 1: no inoculation (blank); Treatment group 2: inoculated with *R. solani* and sprayed with sterile water (control); Treatment group 3: inoculated with *R. solani* and sprayed with 10% jinggangmycin (positive control); Treatment group 4: inoculated with *R. solani* and sprayed with a suspension of biocontrol spores (experimental). The amount of spray applied to each tobacco seedling was sufficient to moisten the leaves. After spraying, the leaves were inoculated with the pathogen, with 5-6 mycelial cakes inoculated on each leaf, followed by puncture treatment. The leaves were placed in plastic containers for cultivation, with 5 tobacco leaves per treatment and 3 replicates. Seven days after inoculation, the size of the leaf lesions was measured using calipers. The disease control effect on tobacco leaves was calculated using the following formula: Disease control effect (%) = [(control group lesion diameter - treatment group lesion diameter) / control group lesion diameter] × 100.
[0062] 4. Determination of the broad-spectrum antibacterial activity of strain GUFG16
[0063] Six different pathogens (see Table 3) were selected, and the broad-spectrum antibacterial activity of *Trichoderma guiyuan* GZMT12 was determined using the plate confrontation method. All six pathogens were provided by the Department of Plant Pathology, Guizhou University, Guiyang City, Guizhou Province. The inhibition rate was calculated according to the method in section 2.2.
[0064] Table 3. Six pathogens used
[0065]
[0066] 5. Results and Analysis
[0067] 5.1 Isolation and Screening of Biocontrol Strains for Tobacco Target Spot Disease
[0068] Twelve fungal strains were obtained using the dilution plating method. Using *Rhizoctonia solani*, the tobacco target spot causal agent, as an indicator, the biocontrol strains were screened using the plate confrontation method. Results are as follows: Figure 2 As shown in Table 4, three Trichoderma strains with morphological characteristics and inhibitory effects against Rhizoctonia solani, the causal agent of tobacco target spot disease, were finally obtained and named GZMT12, GZMT13, and GZMT14. Among them, Trichoderma strain GZMT12 had the highest inhibition rate against Rhizoctonia solani, reaching 75.89%. Trichoderma strain GZMT12, which had the best inhibitory effect on Rhizoctonia solani, was selected for subsequent experiments.
[0069] Table 4. Antimicrobial effect of biocontrol strains on tobacco target spot pathogen.
[0070]
[0071] 5.2 Morphological identification
[0072] The strain GZMT12, which showed the best inhibitory effect against Rhizoctonia solani, the pathogen of tobacco target spot disease, was morphologically identified.
[0073] After 5 days of culture on PDA medium, all strains of the bacteria had completely filled the plates. Figure 3 On PDA medium, the colonies appear slightly cottony with tightly packed hyphae. Green conidia form thick, dense concentric rings. The conidia are more concentrated and darker in color near the mycelial cake. No pigment is produced in the colonies.
[0074] All strains produced conidiophores and conidia after culturing in PDA medium at 28°C for 7 days. The conidiophores had a distinct main axis with paired or unilateral lateral branches arranged relatively closely. The flask-shaped phialides were long-necked, mostly whorled in groups of 2-3, occasionally solitary, and measured 4.48-10.90 × 2.119-3.451 μm. The conidia were unicellular, spherical, measuring 2.18-2.36 × 3.05-3.80 μm, and appeared pale yellowish-green under a microscope.
[0075] 5.3 Multigene phylogenetic analysis
[0076] Based on the RPB2 and TEF1-α regions, sequences were tandemly constructed to create a phylogenetic tree containing isolates GZMT12, GZMT13, and GZMT14 (three morphologically identical strains) and 23 Trichoderma strains belonging to two outgroups, Trichoderma breve. Figure 4 GZMT12, GZMT13, and GZMT14 are most closely related to Trichoderma guizhouense, and these three isolates were identified as Trichoderma guizhouense.
[0077] Trichoderma guizhouense GZMT12 was deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.42152.
[0078] 5.4 Control effect of Trichoderma Guizhouense GZMT12 on tobacco target spot disease
[0079] The experimental results are shown in Figure 5 After treatment with 10% jinggangmycin and *Trichoderma guiyuan* GZMT12 for 7 days, the control efficacy of 10% jinggangmycin against tobacco target spot disease was 53.95%, while the control efficacy of *Trichoderma guiyuan* GZMT12 against tobacco target spot disease was 76.03%. Therefore, the *Trichoderma guiyuan* GZMT12 obtained in this invention has excellent control efficacy against tobacco target spot disease.
[0080] 5.5 Antibacterial spectrum of Trichoderma guizhouense GZMT12
[0081] The experimental results are shown in Figure 6 Trichoderma guiyuan GZMT12 showed varying degrees of inhibitory effects against six fungal pathogens. The average inhibition rate reached 78.07% (see Table 5). Therefore, Trichoderma guiyuan GZMT12 can be considered a broad-spectrum antibacterial biocontrol bacterium with development potential.
[0082] Table 5. Broad-spectrum antibacterial inhibition rate of Trichoderma Guizhouense GZMT12
[0083]
[0084] 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. Trichoderma guizhouense ( Trichoderma guizhouense The application of GZMT12, a spore suspension of Trichoderma guiyuanense GZMT12, or an inoculant containing GZMT12 or a spore suspension of GZMT12 in the control of plant pathogens, characterized in that... The Guizhou Trichoderma GZMT12 was deposited at the China General Microbiological Culture Collection Center on August 4, 2025, at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 42152. The plant pathogens are selected from one or more of the following: *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.
2. The application according to claim 1, characterized in that, The method for preparing the spore suspension of Trichoderma Guizhouense GZMT12 includes the steps of inoculating Trichoderma Guizhouense GZMT12 into a culture medium to obtain spores, and rinsing and mixing with sterile water to obtain a spore suspension.
3. The application of the Guizhou Trichoderma GZMT12 as described in claim 1, the spore suspension of the Guizhou Trichoderma GZMT12, or the inoculant containing the Guizhou Trichoderma GZMT12 or the spore suspension of the Guizhou Trichoderma GZMT12 in the preparation of products for controlling plant pathogens; The plant pathogens are selected from one or more of the following: *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.
4. A product for preventing and controlling plant pathogens, characterized in that, The product contains the Guizhou Trichoderma GZMT12 as described in claim 1, a spore suspension of the Guizhou Trichoderma GZMT12, or a fungal agent containing the Guizhou Trichoderma GZMT12 or a spore suspension of the Guizhou Trichoderma GZMT12.
5. A method for controlling plant pathogens, characterized in that, The steps include inoculating the infected plant with the Guizhou Trichoderma GZMT12 as described in claim 1, the Guizhou Trichoderma GZMT12 spore suspension, or the inoculum containing the Guizhou Trichoderma GZMT12 or the Guizhou Trichoderma GZMT12 spore suspension; The plant pathogens are selected from one or more of the following: *Botrytis cinerea*, *Pseudomonas stolonifer*, *Alternaria alternata*, *Anthracis chinensis*, *Phytophthora capsici*, *Phytophthora nicotine*, and *Rhizoctonia solani*.