Trichoderma Guizhou GZMT12 and application thereof

Abstract

The invention discloses trichoderma Guizhou GZMT12 and application thereof, and belongs to the technical field of microorganisms. The preservation number of the trichoderma Guizhou GZMT12 screened by the invention is CGMCC (China General Microbiological Culture Collection Center) NO. 42152. According to the present invention, the bacterial strain can provide inhibition effects on seven pathogenic bacteria such as Rhodococcus solani Kuhn, Botryosphaeria dothidea, Alternania alternania, Phomopsis litsea, Colletotrichum truncatum, Phytotrichum capsici, and Phytotrichum nitianae, and further has advantages of good prevention and treatment effect, wide antibacterial spectrum, wide application range, and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to Guizhou Trichoderma GZMT12 and its application. BACKGROUND

[0002] Plants are vulnerable to plant pathogenic fungi, which are numerous and seriously affect the yield and quality of crops, resulting in huge economic losses. Globally, plant pathogenic fungi pose a serious threat to crops and cash crops. Tobacco target spot is caused by Rhizoctonia solani Kuhn, which has strong infectivity and can spread rapidly through soil, plant residues, irrigation water, and agricultural operations, easily causing widespread epidemics and posing a serious threat to tobacco production. Botryosphaeria dothidea, Alternaria alternata, and Phomopsis lithocarpus are the main pathogenic fungi causing soft rot of kiwifruit. Soft rot of kiwifruit is a fungal disease that occurs during storage and transportation of kiwifruit. The disease manifests as fruit skin collapse, tissue softening, and rotten odor. After peeling the epidermis of the lesion, a white area appears in the center, surrounded by a yellow-green transition zone, and the fruit flesh tissue shows softening and rotting symptoms. Vertical sectioning reveals that the soft rot area extends cone-shapedly towards the interior of the fruit flesh, and is extremely prone to spread. Tobacco black shank caused by Phytophthora nicotianae causes huge economic losses to tobacco crops worldwide. Phytophthora capsici is a devastating disease in pepper production, often causing complete crop failure when it occurs severely. Colletotrichum truncatum is the pathogenic fungus of pepper anthracnose, which causes severe anthracnose on peppers, greatly affecting the marketability and economic value of peppers, and is an important disease in pepper production, with an increasingly serious trend.

[0003] Trichoderma spp. belongs to the fungal kingdom, Ascomycota, Sordariomycetes, Hypocreomycetidae, Hypocreales, and Hypocreaceae, and is one of the most representative biocontrol fungi in the field of biological control. Due to its environmental friendliness, green efficiency, and other advantages, it has become a research hotspot in recent years. SUMMARY

[0004] The object of the present application is to provide a Guizhou Trichoderma GZMT12 and application thereof, so as to solve the problems in the prior art. The Guizhou Trichoderma GZMT12 is isolated, and has the advantages of good prevention and treatment effect, wide antibacterial spectrum, wide application range, and the like, and can be used for preventing and treating plant pathogenic bacteria.

[0005] To achieve the above object, the present application provides the following solutions.

[0006] In a first aspect, the present application provides a Guizhou Trichoderma GZMT12, which was preserved in the China General Microbiological Culture Collection Center on August 4, 2025, and the address is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 42152.

[0007] In a second aspect, the present application further provides a spore suspension of the Guizhou Trichoderma GZMT12.

[0008] In a third aspect, the present application further provides a preparation method of the spore suspension, characterized in that the method comprises the steps of inoculating the Guizhou Trichoderma GZMT12 into a culture medium to obtain spores, and washing and mixing the spores with sterile water to obtain the spore suspension.

[0009] In a fourth aspect, the present application further provides a microbial agent comprising the Guizhou Trichoderma GZMT12 or the spore suspension.

[0010] In a fifth aspect, the present application further provides application of the Guizhou Trichoderma GZMT12, the spore suspension or the microbial agent in prevention and treatment of plant pathogenic bacteria.

[0011] Preferably, the plant pathogenic bacteria are selected from one or more of Botryosphaeria dothidea, Phomopsis, Alternaria alternata, Colletotrichum gloeosporioides, Phytophthora capsici, Phytophthora nicotianae and Rhizoctonia solani.

[0012] In a sixth aspect, the present application further provides application of the Guizhou Trichoderma GZMT12, the spore suspension or the microbial agent in preparation of a product for preventing and treating plant pathogenic bacteria.

[0013] Preferably, the plant pathogenic bacteria are selected from one or more of Botryosphaeria dothidea, Phomopsis, Alternaria alternata, Colletotrichum gloeosporioides, Phytophthora capsici, Phytophthora nicotianae and Rhizoctonia solani.

[0014] In a seventh aspect, the present application further provides a product for preventing and treating plant pathogenic bacteria, wherein the product comprises the Guizhou Trichoderma GZMT12, the spore suspension or the microbial agent.

[0015] In an eighth aspect, the present application also provides a method for preventing and treating plant pathogenic bacteria, comprising the step of inoculating the Trichoderma guizhouense GZMT12, the spore suspension or the microbial agent into a diseased plant.

[0016] The present application discloses the following technical effects:

[0017] The present application isolates a Trichoderma guizhouense GZMT12, which is found to have a bacteriostatic rate of 75.89% on R. solani; through in vitro leaf exploration, it is found that the disease prevention effect of the strain on tobacco R. solani can reach 76.03% after treatment with the Trichoderma guizhouense GZMT12. At the same time, the broad-spectrum antibacterial property of the Trichoderma guizhouense GZMT12 is explored, and it is found that the average bacteriostatic rate of the Trichoderma guizhouense GZMT12 on Botryosphaeria dothidea, Alternaria alternata, Phomopsis lithocarpus., Colletotrichum truncatum, phytophthora capsici and Phytophthora nicotianae, which are six kinds of pathogenic bacteria, reaches 78.07%, and the Trichoderma guizhouense GZMT12 has the advantages of good prevention effect, wide bacteriostatic spectrum and wide application range.

[0018] Collection information: Trichoderma guizhouense GZMT12 was collected in the China General Microbiological Culture Collection Center on August 4, 2025, the address of the collection center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the collection number is CGMCC NO. 42152. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 It is a schematic diagram of the flat plate confrontation method; wherein the left side is a single screening colony control group, the middle is a treatment group, and the right side is a single pathogenic bacteria control group;

[0021] Figure 2 It is the inhibition effect of the biocontrol strain on tobacco target spot disease pathogen; wherein A, C and E are single R. solani growth plates, and B, D and F are respectively GZMT12, GZMT13 and GZMT14 and R. solani co-culture growth plates;

[0022] Figure 3 Figure 7 is the morphological characteristics of Trichoderma guizhouense GZMT12; wherein, A-B are the morphological characteristics of the colony front and back; C-E are the phialides and conidiophores of the colony; F is the conidium of the colony; scale = 10 μm;

[0023] Figure 4 Figure 8 is the phylogenetic tree of the multi-gene (rpb2 and tefl) sequences constructed by the maximum likelihood method;

[0024] Figure 5 Figure 9 is the control effect of Trichoderma guizhouense GZMT12 on tobacco target spot disease detached leaves; wherein, A is a blank treatment group; B is a control treatment group; C is a positive control group; D is an experimental group;

[0025] Figure 6 Figure 10 is the broad-spectrum antibacterial determination of Trichoderma guizhouense GZMT12. DETAILED DESCRIPTION

[0026] A number of exemplary embodiments of the present application are now described in detail by referring to the following figures. The detailed description is not to be considered limiting of the application, but rather a description of certain aspects, features, and embodiments of the application.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these intervening values are also specifically included within the scope of the application. These smaller ranges are not necessarily arbitrarily predetermined, or even particularly meaningful, in the context of the application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, or even upported, as useful in various embodiments of the application. All such ranges are to be understood to be encompassed within the meaning and range of values described herein.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All documents mentioned herein are incorporated herein by reference.

[0029] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations fall within the scope of the application. Additional embodiments of the application will readily occur to those skilled in the art. The disclosures of the specification and drawings are to be considered as illustrative only of the principles of the application and are to be considered not as narrowing the generality of the application.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0031] Example 1

[0032] 1. Experimental materials

[0033] 1.1 Soil sample

[0034] The soil sample was collected from the rhizosphere soil of healthy tobacco plants (mature stage) in a diseased (tobacco target spot disease) tobacco field using a five-point sampling method. The sampling site was located in Meitan County (27°41'40"N, 107°33'40"S) of Zunyi City, Guizhou Province.

[0035] 1.2 Test target pathogen

[0036] The tobacco target spot disease pathogen Rhizoctonia solani Kühn was provided by the Tobacco Institute of Guizhou University in Guiyang City, Guizhou Province.

[0037] 1.3 Test medium

[0038] PDA medium: peeled potatoes 200 g, glucose 20 g, agar powder 15-20 g, and sterile distilled water 1 L, natural pH, sterilized at 121°C for 20 min for standby.

[0039] 2. Experimental method

[0040] 2.1 Isolation, purification, and preservation of strains

[0041] The soil dilution plate coating method was used, i.e. 10 g of soil was placed in a triangular flask containing 90 mL of sterile water, incubated at 37°C with 200 rpm shaking for 30 min, and then placed at room temperature for 10-15 min. The supernatant in the shaking flask was diluted with sterile water to six gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ), 100 μL of each gradient was uniformly coated on the PDA plate with a sterile glass rod, and incubated in a 28°C constant temperature incubator in the dark. After the colonies grew, the mycelial blocks were picked from the edge of the colonies and transferred to new PDA medium for purification to obtain pure culture. Finally, the obtained pure culture was stored in a glycerol freezing tube with a final concentration of 15-20%, and stored in a 4°C refrigerator.

[0042] 2.2 Screening of biocontrol strains

[0043] The tobacco target spot pathogen Rhizoctonia solani was used as an indicator to screen biocontrol bacteria using a flat plate confrontation method. The fungus and the indicator pathogen were inoculated 2 cm apart on PDA medium Figure 1 ), and the control group was inoculated with only the indicator fungus. Each treatment was set up in triplicate. The plates were incubated in a 28°C constant temperature incubator in the dark for 5 days, and the growth of the strains was observed and photographed.

[0044] The inhibition rate was calculated using the formula: inhibition rate = [(C-T) / C] x 100%.

[0045] where C and T represent the average growth radius of the pathogen in the control and treatment groups, respectively.

[0046] 2.3 Identification of biocontrol bacteria

[0047] 2.3.1 Morphological observation

[0048] The selected strains were inoculated on PDA medium and incubated in a 28°C constant temperature incubator in the dark. The growth performance of the strains on the medium was observed during the incubation, including colony color and sporulation status. Subsequently, the morphological characteristics such as conidial phialides, conidia (n=50), and sporulation structures were observed under a Carl Zeiss Microscopy GmbH (Jena, Germany) microscope at 40x magnification (1x40).

[0049] 2.3.2 Molecular identification

[0050] The Ezup columnar fungal genomic DNA extraction kit from Shanghai Biomed was used to extract the genomic DNA of the selected strains. The ITS universal primer pair (ITS4 and ITS5), RPB2 primer pair (fRPB2-5f and fRPB2-7cr), and TEF1 primer pair (EF1-728F and TEF1LLErev) were synthesized by Beijing Qikong Biotechnology (Chongqing) Technology Co., Ltd. The 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 2x Tap Plus MaserMix, and 9.5 μL sterile water.

[0054] PCR products were purified and sequenced by Beijing Genki Biotechnology Co., Ltd. (Chongqing).

[0055] The RPB2 and TEF1-a sequences of 25 other T. harzianum 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 rapid bootstrap replicates under partitioned models and phylogenetic trees were visualized by FigTree v. 1.4.0.

[0056] Table 2 Strain gene accession number

[0057]

[0058] Note: T and ET in the strain number represent that this strain is a model strain.

[0059] 3. Determination of the in vitro leaf protection effect of biocontrol bacteria on tobacco target spot disease

[0060] Preparation method of biocontrol strain spore suspension: the selected strain was inoculated on PDA medium and cultured, the spore-producing colonies on the PDA medium were washed with sterile water, the mycelium was removed by sterile gauze filtration, and the number of spores was counted by a hemocytometer to obtain a spore suspension of 1 x 10 7 spores / mL.

[0061] Select uniform size tobacco leaves for foliar spraying. A total of 4 groups of treatments were set. Treatment group one: no bacteria were inoculated, which was set as a blank treatment group; treatment group two: R. solani was inoculated and sterile water was sprayed, which was set as a control treatment group, treatment group three: R. solani was inoculated and 10% Jingangmycin was sprayed as a positive control group, and treatment group four: R. solani was inoculated and biocontrol strain spore suspension was sprayed as an experimental group. The amount of spraying for each tobacco seedling was based on leaf wetting, and after spraying, the pathogen was inoculated, 5-6 spots of mycelial discs were inoculated on each leaf, and the leaves were wounded. Place in a plastic box, 5 leaves per treatment, 3 replicates, after 7 days of inoculation, measure the lesion size of the leaves with a vernier caliper, and calculate the disease control effect on tobacco leaves according to the following formula: Disease control effect (%) = [(diameter of lesion in control group - diameter of lesion in treatment group) / diameter of lesion in control group] x 100.

[0062] 4. Determination of the broad-spectrum inhibition of strain GUFG16

[0063] Select 6 different pathogenic bacteria (see Table 3) and use the plate confrontation method to determine the broad-spectrum inhibition of Guizhou T. harzianum GZMT12. The 6 pathogenic bacteria used were provided by the Plant Pathology Teaching and Research Office of Guizhou University in Guiyang, Guizhou Province. The inhibition rate was calculated according to the method of 2.2.

[0064] Table 3 Six pathogenic fungi used

[0065]

[0066] 5. Results and analysis

[0067] 5.1 Isolation and screening of tobacco target spot disease biocontrol strains

[0068] A total of 12 strains were obtained by dilution plating method. The tobacco target spot pathogen Rhizoctonia solani was used as an indicator, and the plate confrontation method was used for biocontrol strain screening. The results are shown in Table 4 and Table 4. Finally, three strains of Trichoderma strains were obtained, which had morphological characteristics and inhibited the tobacco target spot pathogen Rhizoctonia solani. They were named GZMT12, GZMT13, and GZMT14. Among them, the Trichoderma strain GZMT12 had the highest inhibition rate of 75.89% on the tobacco target spot pathogen Rhizoctonia solani. The Trichoderma strain GZMT12 with the best inhibition effect on the tobacco target spot pathogen Rhizoctonia solani was selected for subsequent experiments. Figure 2 Table 4 Inhibition effect of biocontrol strains on tobacco target spot pathogen

[0069]

[0070]

[0071] 5.2 Morphological identification

[0072] The strain GZMT12 with the best inhibition effect on the tobacco target spot pathogen Rhizoctonia solani was subjected to morphological identification.

[0073] After 5 days of culture on PDA medium, the strains all covered the plate, and the colonies were white, cotton-like, and slightly raised. The colonies were white, cotton-like, and slightly raised. Figure 3 On PDA medium, the colonies were slightly cotton-like, and the mycelium was tight. Green conidia formed thick and dense concentric rings. The spores near the cake were more dense and darker. The colonies did not produce pigments.

[0074] After 7 days of culture at 28°C on PDA medium, the strains all produced conidial phialides and conidia. The conidial phialides had a clear main axis, with paired or unilateral lateral branches arranged closely; the phialides were long-necked bottle-shaped, mostly 2-3 whorled, occasionally solitary, and sized 4.48-10.90 x 2.119-3.451 μm. The conidia were single-celled, spherical, and sized 2.18-2.36 x 3.05-3.80 μm, appearing yellowish green under a microscope.

[0075] 5.3 Multigene phylogenetic analysis

[0076] ​Based on the RPB2 and TEF1-a regions, the sequences were concatenated to construct a phylogenetic tree containing the isolates GZMT12, GZMT13, GZMT14 (the three strains are morphologically consistent) and 23 strains of Trichoderma and two outgroup species Trichoderma breve Figure 4 GZMT12, GZMT13 and GZMT14 are most closely related to Trichoderma guizhouense, and the three isolates are determined as Trichoderma guizhouense.

[0077] Trichoderma guizhouense GZMT12 was preserved in China General Microbiological Culture Collection Center on August 4, 2025, and the address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 42152.

[0078] 5.4 Prevention and treatment effect of Trichoderma guizhouense GZMT12 on tobacco target spot disease

[0079] The experimental results are shown in Table 4. Figure 5 After 10% of Jingangmycin and Trichoderma guizhouense GZMT12 were treated for 7 days, the disease prevention effect of 10% of Jingangmycin on tobacco target spot disease was 53.95%, and the disease prevention effect of Trichoderma guizhouense GZMT12 on tobacco target spot disease was 76.03%. It can be seen that the Trichoderma guizhouense GZMT12 screened by the application has excellent prevention and treatment effect on tobacco target spot disease.

[0080] 5.5 Antibacterial spectrum of Trichoderma guizhouense GZMT12

[0081] The experimental results are shown in Table 4. Figure 6 Trichoderma guizhouense GZMT12 has different degrees of inhibition on 6 kinds of fungal pathogens. The average inhibition rate can reach 78.07% (see Table 5), so Trichoderma guizhouense GZMT12 can be considered as a broad-spectrum bacteriostatic biocontrol fungus with development potential.

[0082] Table 5 Inhibition rate of Trichoderma guizhouense GZMT12

[0083]

[0084] The above-described embodiments are only to describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements of the technical solutions of the application made by those skilled in the art shall fall within the protection scope determined by the claims of the application.

Claims

1. A type of Trichoderma guizhouense GZMT12, characterized in that, The Guizhou Trichoderma 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.

2. A spore suspension of Trichoderma Guizhouense GZMT12 as described in claim 1.

3. A method for preparing the spore suspension as described in claim 2, characterized in that, The method includes the steps of inoculating the Guizhou Trichoderma GZMT12 of claim 1 into a culture medium to obtain spores, and rinsing and mixing with sterile water to obtain a spore suspension.

4. A fungal agent comprising the Guizhou Trichoderma GZMT12 of claim 1 or the spore suspension of claim 2.

5. The application of the Guizhou Trichoderma GZMT12 of claim 1, the spore suspension of claim 2, or the fungal agent of claim 4 in the control of plant pathogens.

6. The application according to claim 5, characterized in that, 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*.

7. The application of the Guizhou Trichoderma GZMT12 of claim 1, the spore suspension of claim 2, or the fungal agent of claim 4 in the preparation of products for controlling plant pathogens.

8. The application according to claim 7, characterized in that, 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*.

9. A product for preventing and controlling plant pathogens, characterized in that, The product contains the Guizhou Trichoderma GZMT12 as described in claim 1, the spore suspension as described in claim 2, or the fungal agent as described in claim 4.

10. A method for controlling plant pathogens, characterized in that, The method includes the step of inoculating infected plants with the Guizhou Trichoderma GZMT12 of claim 1, the spore suspension of claim 2, or the fungal agent of claim 4.

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