Application of trichoderma harzianum in biological control of wild apple tree canker

Abstract

The invention discloses an application of trichoderma harzianum in biological prevention and treatment of wild apple tree canker, the classification name of the trichoderma harzianum is Bacillus velezensis Rifai, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO. 42231, and the preservation time is September 29, 2025. Experimental results show that the trichoderma harzianum has remarkable antagonistic activity on parasitic cyst, apple cyst, nutshell cyst, cyst hormaechei, prunus cyst and pear cyst, is environment-friendly and pollution-free, and also provides a safe and reliable new way for preventing and treating the valsa mali of the wild apples; in addition, the research also finds that the fermentation product of the trichoderma harzianum has a certain biocontrol effect on the in-vitro branches infected by the physosporium piricola, so that the trichoderma harzianum and the fermentation product of the trichoderma harzianum provided by the invention can be used for preventing and treating the wild apple tree canker, and have good development and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to the application of a Trichoderma harzianum strain in the biological control of wild apple tree rot. Background Technology

[0002] Xinjiang wild apple (Malus sieversii), a dominant species in the wild fruit forests of the Tianshan Mountains in Xinjiang, is a Tertiary relict plant of the genus Malus in the Rosaceae family and is listed as a national second-class protected wild plant. This species is widely recognized as the original ancestor of modern cultivated apple (Malus domestica). Its germplasm resources possess rich genetic diversity and unique stress resistance traits, including drought tolerance, cold resistance, and resistance to various diseases and pests. It is an irreplaceable natural gene pool of wild fruit tree germplasm resources in my country and has significant value in global apple germplasm resource innovation and biodiversity conservation.

[0003] In recent years, the ecosystem stability of wild apple forests in Xinjiang has been severely threatened by multiple factors, including climate change, habitat fragmentation, and increased pests and diseases. Among these threats, branch and trunk canker, caused by fungi of the genus *Cytospora* spp., is particularly prominent. This disease is characterized by rapid onset, severe damage, and difficulty in control, leading to branch and trunk canker, bark necrosis, and even the death of the entire plant. It has become a major contributing factor to the degradation of wild fruit forests in the Tianshan Mountains and the sharp decline in wild apple populations. However, current control measures for this disease still rely primarily on chemical agents, lacking efficient and ecologically safe new control methods.

[0004] Trichoderma is a group of biocontrol fungi widely distributed in rhizosphere soil. They antagonize plant pathogenic fungi through various mechanisms, including nutrient space competition, hyperparasitism, secretion of antibiotic metabolites, and induction of systemic resistance in plants. They have shown good application potential in the control of various agricultural and forestry crop diseases, possessing advantages such as strong environmental compatibility, long-lasting efficacy, and safety for non-target organisms. However, highly efficient Trichoderma strains targeting the specific pathological system of *Trichoderma spp.* (Xinjiang wild apple) have not yet been systematically explored, and their biocontrol potential and application feasibility require further evaluation.

[0005] Therefore, developing a Trichoderma strain with significant inhibitory activity against the pathogen causing wild apple rot in Xinjiang is of great significance for promoting the green control of this disease. Summary of the Invention

[0006] In view of the technical problems in the background art, the purpose of this invention is to provide the application of a Trichoderma harzianum strain in the biological control of wild apple tree rot disease.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a strain of Trichoderma harzianum, which is classified and named as follows: Trichoderma harzianum Rifai, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 42231, on September 29, 2025. A second aspect of the present invention is to provide the fermentation product of the aforementioned Trichoderma harzianum, wherein the fermentation product of Trichoderma harzianum is a sterile fermentation broth of Trichoderma harzianum, which is obtained by inoculating Trichoderma harzianum with preservation number CGMCC No. 42231 and culturing it in PDB liquid medium for fermentation.

[0008] Preferably, the specific steps for preparing the fermentation product of Trichoderma harzianum include: activating Trichoderma harzianum and inoculating it into PDB liquid culture medium, placing it in a constant temperature shaker at 25-28℃ and shaking at 150-180 r / min for 3-5 days to obtain the fermentation culture broth of the strain, centrifuging, filtering and sterilizing to obtain the fermentation product of Trichoderma harzianum.

[0009] A third aspect of the present invention is to provide the application of the above-mentioned Trichoderma harzianum and its fermentation product as an antibacterial agent in the biological control of wild apple tree rot disease.

[0010] A fourth aspect of the present invention is to provide the application of the above-mentioned *Trichoderma harzianum* in the biological control of plant fungal diseases, wherein the fungal pathogen is *Trichoderma parasiticum* (…). Cytospora parasitica ), Apple shell cystis ( Cytospora mali ), drupecarpa ( Cytospora leucostoma ), Hochnis spp. ( Cytospora hoffmannii ), Prunus spp. ( Cytospora pruinopsis ) and pear shell cystis ( Cytospora populina ).

[0011] The present invention has the following beneficial effects: (1) This invention provides a Trichoderma harzianum strain XJYGL-M07, which can inhibit the growth of various pathogens such as parasitic cystidia, malocystidia, drupecystidia, holocystidia, Prunus spp., and pear cystidia. In other words, the Trichoderma harzianum provided by this invention has a significant antagonistic effect on the above six cystidia pathogens and is environmentally friendly and pollution-free. Therefore, it can be used in the biological control of wild apple tree rot disease, which provides a safe and reliable new approach for the control of wild apple rot disease. Therefore, this strain has good application prospects in the development of microbial pesticides.

[0012] (2) The present invention also provides the fermentation product of Trichoderma harzianum XJYGL-M07 strain. The experimental results show that the fermentation product of Trichoderma harzianum XJYGL-M07 strain can effectively inhibit the infection and spread of pathogenic fungi, specifically Pyrocystis pyriformis. Therefore, it can be concluded that the fermentation product of XJYGL-M07 strain, i.e. its sterile fermentation broth, can be used for the prevention and treatment of wild apple rot.

[0013] (3) The *Trichoderma harzianum* provided by this invention is isolated from the rhizosphere soil of wild apple trees. It has good environmental adaptability and control effect, is simple to cultivate and easy to preserve, and has good prospects for development and application. This invention utilizes *Trichoderma harzianum* to construct a biological control system, which not only helps to reduce the intensity of chemical pesticide use and alleviate environmental pressure, but also provides technical support for the in-situ protection of wild apple germplasm resources and the sustainable management of wild fruit forest ecosystems. It has important practical significance and prospects for promotion and application in biodiversity conservation and ecological restoration. Preservation Information Preservation period: September 29, 2025; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Accession number: CGMCC NO. 42231; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing; Postal code: 100101; Category Naming: Trichoderma harzianum Rifai. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying 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.

[0015] Figure 1 The colony morphology of Trichoderma harzianum strain XJYGL-M07 on PDA medium is as follows: (A) Front colony morphology after 3 days of growth on PDA medium; (B) Back colony morphology after 3 days of growth on PDA medium; (C) Front colony morphology after 7 days of growth on PDA medium; (D) Back colony morphology after 7 days of growth on PDA medium. Figure 2 Microscopic morphology of strain XJYGL-M07: (A) aerial hyphae; (B) hyphal mass; (C) chlamydospores; (D) chlamydospores and conidia. Scale bars in the figures are all 20 μm. Figure 3 The maximum likelihood phylogenetic tree for strain XJYGL-M07 was constructed based on the ITS, RPB2, and TEF1-a gene sequences (where Trichoderma harzianum strain XJYGL-M07 is shown in bold, with nodes marked with bootstrapping support of more than 50%, and T indicating the type strain). Figure 4 The antagonistic effect of Trichoderma harzianum strain XJYGL-M07 on parasitic cystidia, malocystidia, drupecystidia, holocystidia, Prunus spp. and pear cystidia. Figure 5 The image shows the biocontrol effect of aseptic fermentation broth of Trichoderma harzianum strain XJYGL-M07 on detached branches inoculated with Cytospora populina. From left to right: mechanical damage control group, Cytospora populina inoculated control group, therapeutic treatment group, and protective treatment group. Detailed Implementation

[0016] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0017] Example 1 I. Isolation and Identification of Trichoderma harzianum strain XJYGL-M07 1. Separation and screening 1.1 Source of wild apple rhizosphere soil samples From June 2023 to June 2024, soil samples from the roots of healthy and diseased wild apples were collected from a wild apple resource nursery in Xinjiang.

[0018] 1.2 Main Reagents Potato dextrose agar (PDA): 200g potatoes (peeled), 20g glucose, 15g agar powder 20g, 1000mL distilled water.

[0019] 1.3 Isolation, Screening, Purification and Preservation of Trichoderma Using the dilution plate method, 5g of each soil sample was placed in a 45mL Erlenmeyer flask containing sterile water. The flask was shaken for 30 minutes at 25℃ and 180 rpm. After standing, the supernatant was aspirated and serially diluted to 10⁻⁶. -1 10 -2 10 -3100 μL of each dilution was plated on PDA plates, with each concentration replicated three times. The plates were incubated in the dark at 25°C. After colonies grew, mycelial fragments were picked from the colony edge and transferred to fresh PDA medium for purification. Colonies with morphology similar to *Trichoderma* were selected and preserved. The purified strain was labeled as strain XJYGL-M07 and stored in cryovials containing 15% glycerol. Store at 80℃. 2. Observation of the morphological characteristics of Trichoderma Strains XJYGL-M07 were cultured in the dark at 25°C for 3 days. Mycelial fragments from the colony edges were picked and transferred to the center of a new PDA plate, and cultured in the dark at 25°C for three consecutive days. The growth of colonies on the PDA medium was recorded daily (hyphae density, colony color, and sporulation, etc.). After 2 days of culture on PDA medium, sterilized coverslips were inserted at a 45° angle at the colony edge. Once the mycelia had grown onto the coverslip, their microscopic morphological characteristics were observed using a Nikon upright fluorescence microscope. The results are shown below. Figure 1 and Figure 2 .

[0020] Figure 1 The results showed that strain XJYGL-M07, on PDA medium, produced pale yellow pigment in colonies, with no obvious odor, and the hyphae exhibited a whorled radial pattern. After 3 days of culture, white conidial clusters appeared at the mycelial cake, mainly distributed near the cake with fewer at the edges. After 7 days, conidia covered the entire culture dish, with spores near the cake showing a green color, and the green spore clusters were distributed in a whorled pattern. On PDA medium, the colony growth rate was relatively fast, reaching 3 days at 25℃. It can grow to fill a 90mm diameter petri dish in 4 days.

[0021] Depend on Figure 2 Microscopic morphological results revealed that the conidiophores have a distinct main axis, with paired or unilateral lateral branches growing on the main axis, arranged relatively sparsely; the conidia are green, elliptical, and 2.7 mm in size. 4.2)×(2.3 3.9) μm, aspect ratio is 1 1.49 μm (n = 50); chlamydospores, thick-walled, nearly spherical, terminal or intercalary, measuring (5.0 μm). 9.2)×(4.8 8.7) μm, aspect ratio 1 1.49μm (n=50).

[0022] 3. Construction of a molecular phylogenetic tree of Trichoderma 3.1 Trichoderma DNA Extraction The strain XJYGL-M07 was cultured on PDA medium for 7 days (25℃), and then DNA was extracted using the CTAB method.

[0023] 3.2 PCR amplification The ITS universal primer pair (ITS4 and ITS5) and the RPB2 primer pair (fRPB2) synthesized by Sangon Biotech (Shanghai) Co., Ltd. 5f and fRPB2 7cr) and TEF1 primer pair (EF1) PCR was performed on the gene sequence of strain XJYGL-M07 using primers 728F and TEF1LLErev. The specific primer information is shown in Table 1.

[0024] Table 1. Primer Information

[0025] Note: In the primers above, Y represents T or C; R represents G or A; W represents A or T; and S represents C or G. PCR reaction system: 2 μL each of upstream and downstream primers, 25 μL of TaqPCRStarMix (with Loading Dye), 1 μL of bacterial DNA template, 20 μL of ddH2O, and a total system of 50 μL.

[0026] PCR amplification program for RPB and TEF genes: 95℃ for 3 min, 95℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min (35 cycles), 72℃ for 5 min. PCR amplification program for the ITS sequence: 95℃ for 3 min, 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min (35 cycles), 72℃ for 5 min.

[0027] PCR product detection and sequencing: Prepare a 1% agarose gel, add 6 μL of DNA marker to the first or last well, and add 6 μL of PCR product to the other wells. Electrophoresis is performed at 125V for 30 min, and the bands are observed and images are acquired using a gel imaging system.

[0028] After successful sample amplification, sequencing was performed, and the rpb2 sequence of strain XJYGL-M07 was shown in SEQ ID NO.1, the TEF1-a sequence in SEQ ID NO.2, and the ITS sequence in SEQ ID NO.3.

[0029] 3.3 System Evolution Analysis According to the latest standards for Trichoderma species classification proposed by Cai and Druzhinina, three conditions must be met simultaneously for a Trichoderma species to be identified: rpb2 ≥ 99%, TEF1-a ≥ 97%, and ITS ≥ 76%. Using these latest standards, the rpb2, TEF1-a, and ITS sequences of strain XJYGL-M07 were compared with those in NCBIBlast (https: / / blast.ncbi.nlm.nih.gov / Blast.cg). The results showed that the rpb2 gene of strain XJYGL-M07 shared 99.38% similarity with *Trichoderma harzianum*, exceeding 99% GEF1-a. The TEF1-a gene of strain XJYGL-M07 showed over 97% similarity to *Trichoderma harzianum*, and the ITS sequence of strain XJYGL-M07 showed over 98% similarity to *Trichoderma harzianum*. A maximum likelihood phylogenetic tree of strain XJYGL-M07 was constructed based on the ITS, RPB2, and TEF1-a gene sequences, as shown below. Figure 3 As shown in Table 2, and based on the morphological, molecular biological identification, and multigene phylogenetic tree results above, strain XJYGL-M07 was identified as *Trichoderma harzianum*, and classified as... Trichoderma harzianum Rifai was then preserved.

[0030] II. Prevention and Control Trials 1. Antagonistic effects of Trichoderma harzianum XJYGL-M07 against different pathogens The plate confrontation method was used to determine the resistance of strain XJYGL-M07 to the pathogen (see Table 2, parasitic cystis) Cytospora parasitica ) 、 Apple cystis ( Cytospora mali ) 、 Drupecarpa spores ( Cytospora leucostoma ) 、 Hochnis spp. ( Cytospora hoffmannii ) 、 Prunus spp. ( Cytospora pruinopsis ) 、 pear shell cysts ( Cytospora populinaThe antagonistic effect of strain XJYGL-M07 on the pathogen was investigated using the following steps: Activated strain XJYGL-M07 and pathogenic bacterial pellets (5 mm in diameter) were inoculated on opposite sides of a PDA plate, spaced 6 cm apart. A PDA plate inoculated only with the pathogen served as a control. Each treatment was repeated in triplicate. The plates were incubated at 25°C, and the colony radii opposite each other were recorded after 6 days. The antibacterial effect of strain XJYGL-M07 was evaluated by calculating its inhibition rate against the pathogen. The inhibition rate was calculated using the formula: Inhibition rate = [(CT) / C] × 100%, where C and T represent the average growth radius of the pathogen in the control and treatment groups, respectively. Results are shown below. Figure 4 And Table 3.

[0031] Table 2. Pathogens

[0032] Table 3. Antagonistic effects of strain XJYGL-M07 against various pathogens

[0033] Note: N / A indicates that the pathogen was covered by Trichoderma on day 6 of the confrontation culture, making it impossible to measure its effective radius.

[0034] Depend on Figure 4 As shown in Table 3, the confrontation plate test revealed that after 6 days of culture, strain XJYGL-M07 exhibited strong antibacterial effects against six pathogens, with inhibition rates all exceeding 80%. Specifically, the inhibition rates were 81.08% ± 0.17 against *Carya caryopsis*, 94.71% ± 0.17 against *Carya caryopsis drupe*, and 90.92% ± 0.32 against *Carya caryopsis parasiticus*, *Carya caryopsis holodermidis*, and *Carya caryopsis przewalskii*, with strain XJYGL-M07 completely covering the pathogen radius, making it impossible to measure their effective radius. After 6 days of co-culture, significant pigment changes were observed at the junction of the hyphae of the six pathogens and strain XJYGL-M07. Among them, *Carya caryopsis parasiticus*... Cytospora parasitica ) and pear shell cystis ( Cytospora populina There was obvious sporulation at the junction. The above results indicate that strain XJYGL-M07 showed significant antagonistic effects against all six pathogens, therefore strain XJYGL-M07 can be used for the biological control of wild apple tree canker.

[0035] 2. Biocontrol effect of aseptic fermentation broth of strain XJYGL-M07 on detached branches of wild apple. Through antagonistic experiments, the novel pathogen of wild apple rot in Xinjiang was selected as *Pyripoda spp.* (Pyripoda spp.). Cytospora populina This study aimed to evaluate the therapeutic and protective effects of strain XJYGL-M07 on detached branches of wild apple.

[0036] Three to five mycelial cakes of the activated XJYGL-M07 strain were punched using a 5mm punch and placed in a 300mL Erlenmeyer flask containing 150mL PDB medium. The flasks were then incubated for 5 days at 25℃ and 180rpm on a shaker to obtain the fermentation broth of strain XJYGL-M07. The fermentation broth was then centrifuged at 10000rpm for 5 minutes. The supernatant was collected and initially filtered through a 0.45μm microporous membrane for sterilization, followed by a second sterilization using a 0.22μm microporous membrane to obtain sterile fermentation broth of strain XJYGL-M07. This sterile broth was stored at 4℃ for later use.

[0037] Select two-year-old wild apple branches of uniform thickness, cut them into sections of the same length, wash them with clean water, and let them dry. Place them in a clean bench, soak them in 2% sodium hypochlorite for 10 minutes, soak them in 75% alcohol for 1 minute, rinse them three times with sterile water, let them dry on sterile filter paper, seal the top and bottom ends of the branches with paraffin, and then use a scrubbing wheel to make pinhole-like wounds in the center of the treated branches, making the wounds small and deep.

[0038] Therapeutic treatment: *Pyrus pyrifolia* mycelium was first inoculated onto the treated branch wounds, then wrapped and fixed with plastic wrap. After 48 hours of constant temperature and humidity incubation at 25°C, the mycelium was removed. XJYGL-M07 sterile fermentation broth (200 μL each time, dried and then reapplied, repeated 5 times) was applied to the wound. An equal volume of PDB medium was applied as a control. The wounds were then wrapped with plastic wrap and incubated at constant temperature and humidity for another 5 times. Each treatment was repeated 5 times.

[0039] Protective measures: First, apply 200 μL of XJYGL-M07 sterile fermentation broth to the wound (reapply after drying, repeat 5 times), wrap with plastic wrap, and incubate at 25°C for 48 hours. Then, inoculate the wound with 5 mm pear shell cysteine ​​cake, seal the wound with plastic wrap, and use an equal amount of PDB medium as a control. Continue incubation at constant temperature and humidity. Each treatment was repeated 5 times.

[0040] Fifteen days after inoculation with the pathogen, the long and short diameters of the lesions were measured, and the lesion area and control effect were calculated. Lesion area = 1 / 4 × π × major axis × minor axis Control efficacy (%) = [(Lesion area in control group - lesion area in treatment group) / lesion area in control group] × 100% In this experiment, the control effect of sterile fermentation broth of strain XJYGL-M07 was determined, and the disease control potential of strain XJYGL-M07 against detached branches of wild apple was evaluated. The results are as follows: Figure 5 As shown.

[0041] Figure 5The results showed that 15 days after inoculation with *Pyrophyllum demersum* sp. cake, obvious lesions appeared on the branches of wild apple trees in the control group. The diseased bark of the branches in each treatment group was scraped off to expose the xylem. The long and short diameters of the lesions in each treatment group were measured and counted, and the lesion area was calculated. The lesion area in the control group was 1136.11 mm². 2 Compared to the treatment group treated with sterile fermentation broth of strain XJYGL-M07, the lesion area (508.60 mm²) was significantly smaller. 2 The lesion area in the protective treatment group was significantly reduced (119.85 mm²). 2 The significant reduction in [something] indicates that the aseptic fermentation broth of strain XJYGL-M07 can effectively inhibit the infection and spread of the pathogen, specifically *Trichoderma harzianum*. This demonstrates that the aseptic fermentation broth of strain XJYGL-M07 has significant preventive, protective, and therapeutic effects against wild apple rot. The above experimental results show that the aseptic fermentation broth of *Trichoderma harzianum* XJYGL-M07 provided by this invention has good biocontrol effects on detached branches of wild apple, with a therapeutic efficacy of 55.23% and a protective efficacy of 89.45%.

[0042] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. Use of a strain of Trichoderma harzianum or a fermentation product of Trichoderma harzianum for biological control of Armillaria mellea, characterized in that, The Trichoderma harzianum is named as Trichoderma harzianum Rifai, preserved in China General Microbiological Culture Collection Center, with a preservation number of CGMCC NO. 42231, and a preservation time of September 29, 2025; the fermentation of the Trichoderma harzianum is a sterile fermentation liquid of the Trichoderma harzianum, prepared by inoculating and culturing the Trichoderma harzianum in a PDB liquid medium after activation.

2. Use according to claim 1, characterized in that, The fermentation product of Trichoderma harzianum is obtained by activating Trichoderma harzianum, inoculating it into PDB liquid medium, and culturing it in a constant temperature shaker at 25-28℃ and 150-180 r / min for 3-5 days to obtain the fermentation culture broth of the strain. After centrifugation and filtration to remove bacteria, the fermentation product of Trichoderma harzianum is obtained.

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