Trichoderma koningii, strain fermentation liquor of trichoderma koningii and application of trichoderma koningii in prevention and treatment of pomegranate fusarium wilt
By using biocontrol agents prepared from Trichoderma koningii and its fermentation broth, the problems of chemical resistance and high agricultural control costs in the treatment of pomegranate wilt have been solved, achieving efficient and safe biological control.
Patent Information
- Application Number
- CN202610135724.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-30
- Publication Date
- 2026-03-31
AI Technical Summary
In the existing technologies, chemical control of pomegranate wilt disease has problems of drug resistance and environmental pollution, while agricultural control measures such as crop rotation are costly and have limited effectiveness, and there is a lack of effective endophytic strains for the control of pomegranate wilt disease.
Using Trichoderma Corning and its fermentation liquid, biological control was carried out on pomegranate plants through root irrigation. By utilizing the antagonistic effect of Trichoderma Corning on sweet potato scab, a biocontrol agent was prepared to inhibit the disease.
It significantly inhibits the incidence of pomegranate wilt disease, with an antibacterial rate of up to 88.3%, and has high biological safety, reducing the occurrence of disease and plant loss.
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Figure CN121759314A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural microbial technology, and relates to a strain of Trichoderma koningii, its fermentation broth, and its application in the prevention and control of pomegranate wilt disease. Background Technology
[0002] Pomegranate( Punica granatum Pomegranate is a fruit tree with significant economic and ornamental value. Its fruit is rich in vitamin C, antioxidants, and other nutrients, and is widely used in the food and pharmaceutical industries. However, pomegranate wilt disease is seriously threatening the growth and yield of pomegranates. This disease is caused by *Pycium chinense* (a type of fungus). Ceratocystis fimbriata This disease, caused by wilt, typically manifests as yellowing, drying, and shedding of leaves, withered branches, and smaller, prematurely dropped, or rotten fruits. It severely impacts plant growth and fruit yield, leading to economic losses. The disease spreads rapidly; if effective measures are not taken promptly, it can cause widespread damage and even death of pomegranate plants within a short period, posing a serious threat to the sustainable development of the pomegranate industry.
[0003] Traditional methods for controlling pomegranate wilt mainly include chemical pesticide control and agricultural control. While chemical pesticide control can control the disease to some extent, long-term use can lead to increased pathogen resistance, environmental pollution, and pesticide residues in agricultural products. Agricultural control measures, such as crop rotation or soil disinfection, are relatively environmentally friendly, but are complex to implement, costly, and have limited effectiveness.
[0004] Endophytic bacteria are microorganisms that live inside plant tissues and do not cause obvious disease symptoms. They have complex interactions with the host plant. Studies have shown that some endophytic bacteria can produce secondary metabolites that inhibit pathogens, thereby enhancing the plant's disease resistance. Currently, there are few biocontrol bacteria that can be used to control pomegranate wilt, and their effectiveness in actual field applications is poor. Therefore, there is an urgent need to discover a safe and efficient endophytic bacterial strain for the control of pomegranate wilt. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a strain of Trichoderma Corning, its fermentation broth, and its application in the prevention and control of pomegranate wilt disease.
[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides a strain of Trichoderma Corningensis, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC NO: 67115.
[0007] A second aspect of the present invention provides a bacterial fermentation broth containing the aforementioned Trichoderma cornigans or antibacterial active substances secreted by Trichoderma cornigans.
[0008] Furthermore, the fermentation broth of the strain is prepared by the following steps: The *Trichoderma koningense* strain was inoculated into a liquid culture medium and cultured at 23°C to 27°C for 5 to 7 days to obtain the fermentation broth of the strain.
[0009] Furthermore, the liquid culture medium is a PDA liquid culture medium.
[0010] A third aspect of the present invention provides a biocontrol agent containing the fermentation broth of *Trichoderma koningii* or its strains as described above, and agriculturally acceptable adjuvants.
[0011] Furthermore, the biocontrol agent is a liquid preparation or a powder.
[0012] Furthermore, the number of viable spores of *Trichoderma koningense* per milliliter of the biocontrol agent is ≥1×10⁻⁶. 7 indivual.
[0013] The fourth aspect of the present invention provides the application of the above-described Trichoderma harzianum, its fermentation broth, or biocontrol agent in the prevention and control of pomegranate wilt disease.
[0014] Furthermore, the pomegranate wilt disease is caused by *Phytophthora stolonifera*.
[0015] Furthermore, the application method is as follows: when the plant shows symptoms of pomegranate wilt disease, the root drench is performed on the diseased plant and the healthy plants around the diseased plant using the aforementioned Trichoderma koreanum, strain fermentation liquid or biocontrol agent.
[0016] Furthermore, in the early stages of pomegranate wilt disease, the diluted Trichoderma koreanum, its fermentation liquid, or biocontrol agent can be directly applied to the root zone of the diseased pomegranate plants and the surrounding healthy plants to ensure that the bacterial solution fully penetrates the main root layer.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a strain of *Trichoderma koningii*, its fermentation broth, and its application in controlling pomegranate wilt disease. A strain of *Trichoderma koningii* was isolated and screened from microbial resources on pomegranate branches, with the preservation number GDMCC NO: 67115. *Trichoderma koningii* exhibits strong antagonistic activity against *Trichoderma koningii*, the main pathogen causing pomegranate wilt disease, with an inhibition rate as high as 88.3%, demonstrating powerful biocontrol capabilities. Pot experiments showed that the fermentation broth and biocontrol agent prepared based on *Trichoderma koningii* can effectively reduce the incidence of pomegranate wilt disease.
[0018] Instructions for the Preservation of Biological Materials Trichoderma koningii XCXY-N10, referred to in this invention as *Trichoderma Corning* strain N10, is classified and named as follows: Trichoderma koningii Its Latin name is: Trichoderma koningii, It was deposited on October 16, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67115. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. 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 description of the embodiments or the prior art 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 Endophytic bacterial plate confrontation experiment Figure 1 Photo A in the image represents the control group. Figure 1 B in the figure represents the plate confrontation results between strain P1 and strain N10 of Trichoderma koningii.
[0021] Figure 2 The culture characteristics and microscopic morphology of strain N10 are shown. Figure 2 In this context, A represents the plate culture trait. Figure 2 B in the figure represents the characteristic microscopic morphology of conidia.
[0022] Figure 3 This is the phylogenetic tree of strain N10, with strain N10 shown in the red box.
[0023] Figure 4 To observe the experimental results of microbial morphological changes using electron microscopy, Figure 4 In the figure, A represents the hyphal morphology of the control group P1 strain; Figure 4 In the diagram, B represents the hyphae after the confrontation. Figure 4 C in the text represents the conidia of the control group P1 strain; Figure 4 D in the diagram represents the conidia after the confrontation.
[0024] Figure 5 The results of the pot experiment after treatment with Corning Trichoderma strain N10 are shown. Figure 5 In the diagram, A represents the front view of the control group plants; Figure 5 B in the diagram is a top view of the control group plants; Figure 5 C in the diagram represents the front view of the plants in the treatment group; Figure 5 D in the diagram is a top view of the plants in the treatment group. Detailed Implementation
[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0026] The pathogen of pomegranate wilt in this invention is *Pseudomonas stolonifera* (sweet potato fusarium). Ceratocystis fimbriata The P1 strain is referred to as the P1 strain in this invention. For specific information about the strain, please refer to: Zheng Xiaohui, Xu Biao, He Ping, et al. "Pathogens of Fusarium Wilt in Sichuan Pomegranate" [J]. Acta Mycologica Sinica, 2012, 31(4): 523-530.
[0027] Pomegranates are widely cultivated due to their high nutritional and economic value. However, Fusarium wilt caused by *Fusarium wiltum* severely damages their growth and yield, leading to yellowing leaves, branch dieback, fruit drop, and even plant death, thus hindering the sustainable development of the industry. Traditional chemical control methods easily lead to drug resistance and residual pollution, while agricultural measures such as crop rotation are costly and have limited effectiveness. Endophytic bacteria, as symbiotic microorganisms within the host plant, can produce antibacterial active substances, enhancing plant disease resistance. However, currently, biocontrol bacteria suitable for pomegranate Fusarium wilt are scarce and have poor efficacy, necessitating the exploration of safe and efficient endophytic bacteria resources to achieve green control.
[0028] This invention provides a strain of *Trichoderma koningii*, its fermentation broth, and its application in the control of pomegranate wilt disease. First, this invention isolates and screens a strain that significantly antagonizes *Trichoderma koningii* from microbial resources on pomegranate branches, naming it strain N10. Identification confirms that strain N10 belongs to *Trichoderma koningii*. The N10 strain and its fermentation broth provided by this invention exhibit excellent antibacterial activity, significantly inhibiting the growth of *Trichoderma koningii*, the cause of pomegranate wilt disease, with an inhibition rate as high as 88.3%. It possesses advantages such as strong antibacterial ability, good control effect, and high biosafety, making it widely applicable in the biological control of plant diseases caused by *Trichoderma koningii*.
[0029] Example 1: Corning Trichoderma ( Trichoderma koningiopsis Obtaining strain N10 The isolation and purification steps of Trichoderma Corning strain N10 are as follows.
[0030] 1. Organ separation This invention collects samples from pomegranate orchards in Yaozhangou, Huili City, Liangshan Yi Autonomous Prefecture, Sichuan Province (altitude: 1723.8 m, latitude and longitude: 26.446020°N, 102.231912°E), including branches of pomegranate plants infected with wilt disease, branches of healthy plants, and healthy branches of infected plants.
[0031] Samples were isolated and inoculated onto PDA medium and incubated in a 25°C incubator. Colony morphology was observed daily, and bacterial contamination and rapidly spreading saprophytic fungi were promptly removed.
[0032] 2. Separation and purification The fungal strain was isolated by single-spore isolation and then transferred to the center of a PDA medium plate. It was cultured at a constant temperature of 25℃ for 5 days to obtain a pure strain.
[0033] 3. Screening of antagonistic strains Strain activation: Activate strain P1 on PDA plates.
[0034] Confrontation culture: Plate confrontation culture was used. The P1 strain and the isolated endophytic fungi were inoculated into the same PDA medium with the two mycelial cakes 5 cm apart. The medium was placed at 25℃ and cultured for 5 days. The plate confrontation was observed, and the inhibition rate of the endophytic fungi on the P1 strain was calculated according to the following formula.
[0035] Control group treatment: N10 Trichoderma Corning was inoculated into the left side of PDA medium with 5 mm holes, and 5 mm holes were punched in the blank PDA medium and inoculated into the right side. The medium was incubated at 25 degrees Celsius for 5 days, and the colony growth status and colony diameter were continuously observed.
[0036] The inhibition rate is calculated using the following formula: Inhibition rate = [(Rck-Rn) / Rck]×100%, where Rck is the radius of the pathogen during normal growth (control group) and Rn is the radius of the pathogen when it is inhibited (experimental group).
[0037] Table 1. Statistical table of inhibition rates of antagonistic strains against strain P1. like Figure 1 As shown in Table 1, a and h To identify statistically significant differences, this invention isolated and screened a strain N10 that exhibited significant antagonistic activity against *Saccharomyces cerevisiae* strains. The N10 strain showed an inhibition rate of up to 88.3% against *Saccharomyces cerevisiae* strain P1.
[0038] 3. Strain identification 3.1 Morphological identification The selected N10 strain was cultured on PDA plates, and the color, texture and other characteristics of the colonies were observed. The morphological characteristics of the hyphae, conidia and conidiophores of the N10 strain were also observed under a microscope.
[0039] like Figure 2 As shown, the N10 strain colonies initially consist of white mycelium, which gradually turns pale green or dark green with white edges. The mycelium is cottony or fluffy, and the conidia are symmetrical at both ends or slightly pointed at one end, and are elliptical or nearly spherical.
[0040] 3.2 Molecular biological identification DNA was extracted from strain N10 using a DNA extraction kit (purchased from Sangon Biotech Co., Ltd.) following the instructions.
[0041] The gene sequence of strain N10 was amplified by PCR using the universal primers ITS1 and ITS4 for fungal identification, and the amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.
[0042] The sequencing results are shown in SEQ ID NO.1.
[0043] The sequencing results were entered into the NCBI database for BLAST homology comparison. Highly homologous rDNA ITS sequences were selected, analyzed using MEGA 6.0 software, and a phylogenetic tree was constructed. Figure 3 ), N10 strain and Corning Trichoderma ON921655.1 ( Trichoderma koningiopsis The N10 strain is most closely related to *Trichoderma koningense*, with 99% homology. Based on morphological identification and 16S rDNA sequence analysis, it can be determined that the N10 strain is *Trichoderma koningense*. Trichoderma koningiopsis ).
[0044] Example 2: Electron microscopic observation of hyphal inhibition between Trichoderma Corning strain N10 and Mycorrhizal hygroscopicis spp. 1. Cultivating a confrontation The experimental group was treated as follows: strain P1 and the isolated Trichoderma koningii strain N10 were inoculated into the same PDA medium, with the two mycelial cakes 5 cm apart, and cultured at 25°C for 7 days.
[0045] The control group was treated as follows: the same plate confrontation culture was used, but the P1 strain was inoculated into PDA medium, and the other culture conditions (25℃, 7d) were the same as those of the experimental group.
[0046] 2. Mycelial sample delivery The P1 strain, which had been cultured in confrontation with Corning Trichoderma N10 strain, and the P1 strain of the control group, were each placed in glutaraldehyde to fix them, and then packaged in a foam box with ice packs and sent to the company.
[0047] 3. Electron microscopy observation like Figure 4 As shown, Figure 4 In the diagram, A represents untreated, normal hyphae, which are long, spreading filaments with a uniform texture, smooth surface, and good extensibility; while... Figure 4 B in the image shows that the hyphae after confrontation not only shrink significantly, but also exhibit local structural distortion and breakage, with some areas showing collapse and deformation, forming a stark contrast to the extended shape of normal hyphae. Figure 4 C in the text represents normal conidia before confrontation treatment, which are regular long rods, plump, smooth, structurally intact, and evenly distributed; after confrontation treatment with the strain... Figure 4 The D-value shows that the conidia are obviously shrunken and deformed, with a shriveled overall structure, rough and disordered surface texture, and the original regular shape is destroyed. This clearly demonstrates that the Corning Trichoderma N10 strain has a significant inhibitory effect on the morphology and structure of Sweet Potato Long-beaked Scale.
[0048] Example 3: Pot experiment of *Trichoderma harzianum* strain P1 and *Trichoderma koningii* strain N10 1. Activation of *Trichoderma harzianum* strain P1 and *Trichoderma koningii* strain N10 Pick a small amount of bacterial sludge from the preserved bacterial culture slant and inoculate it onto PDA medium. Incubate at 25°C for 3 days, until the colonies cover the entire plate.
[0049] 2. Preparation of spore suspension Single colonies of activated *Trichoderma tumefaciens* strain P1 and *Trichoderma koningii* strain N10 were picked using a sterile inoculation loop and inoculated into Erlenmeyer flasks containing 100 mL of PDA liquid medium. The flasks were then incubated in a shaker at 180 rpm and 25°C for 3 days until the fermentation broth reached the logarithmic growth phase, yielding the fermentation broths of strains P1 and N10.
[0050] 3. Preparation of biocontrol agents Take a small amount of Trichoderma koningii N10 strain fermentation broth and dilute it with sterile physiological saline to a concentration of 1×10⁻⁶. 7 A biocontrol agent containing 1 live spore / mL.
[0051] 4. Experimental treatment Two treatment groups were set up, with each group being repeated 3 times. Treatment 1 (control): After root injury, only 80 mL of P1 strain fermentation liquid was applied and evenly sprayed around the root system; Treatment 2 (experimental group): After root injury, 80 mL of biocontrol agent was applied first, and after standing for 24 hours, 80 mL of P1 strain fermentation liquid was applied to ensure that the strain fermentation liquid fully contacted the root system.
[0052] Figure 5 In the control group, plants A and B were inoculated with fermentation broth of strain P1 after root injury. They showed extensive yellowing of branches and leaves, severe decay of roots and above-ground parts, exhibiting typical pathogen infection phenotypes. Figure 5 Plants in groups C and D, treated with biocontrol agents applied before root injury, exhibited vibrant green leaves and healthy growth, without significant wilting or decline, demonstrating the protective effect of the biocontrol agent prepared from the Corning Trichoderma N10 strain. The significant difference in phenotypes between the two groups directly reflects the inhibitory effect of the Corning Trichoderma N10 strain on diseases caused by *Trichoderma longifolia*.
[0053] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0054] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A strain of *Trichoderma cornuta*, characterized in that, The *Trichoderma koningense* strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67115.
2. A fermentation broth for a bacterial strain, characterized in that, The fermentation broth of the strain contains the *Trichoderma koningense* as described in claim 1 or antibacterial active substances secreted by *Trichoderma koningense*.
3. The fermentation broth of the strain according to claim 2, characterized in that, The fermentation broth of the strain was prepared by the following steps: The *Trichoderma koningense* strain was inoculated into a liquid culture medium and cultured at 23°C to 27°C for 5 to 7 days to obtain the fermentation broth of the strain.
4. A biocontrol agent, characterized in that, The biocontrol agent contains the fermentation broth of *Trichoderma koningii* as described in claim 1 or the strain as described in claim 2, as well as agriculturally acceptable adjuvants.
5. The biocontrol agent according to claim 4, characterized in that, The biocontrol agent is a liquid preparation or a powder.
6. The biocontrol agent according to claim 5, characterized in that, The number of viable spores of *Trichoderma koningense* per milliliter of the aforementioned biocontrol agent is ≥1×10⁻⁶. 7 indivual.
7. The application of the *Trichoderma harzianum* of claim 1, the fermentation broth of the strain of claim 2, or the biocontrol agent of claim 4 in the control of pomegranate wilt disease.
8. The application according to claim 7, characterized in that, The pomegranate wilt disease is caused by *Pseudomonas aeruginosa*.
9. The application according to claim 7, characterized in that, When plants exhibit symptoms of pomegranate wilt, the roots of the diseased plants and the surrounding healthy plants should be drenched with the aforementioned Trichoderma harzianum, its fermentation liquid, or biocontrol agent.