A Trichoderma PP4A strain and its application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-08
- Publication Date
- 2026-08-14
AI Technical Summary
[0005]为了解决现有技术存在的上述不足，本发明的目的是提供一种拟康宁木霉PP4A菌株及其应用，以解决现有叶枯病严重影响重楼植株生长的问题
[0013]本发明具有以下有益效果：本发明前期筛选得到引发重楼叶枯病的病原菌PP23。然后从重楼叶片中分离筛选的得到一株对其有明显拮抗活性的内生真菌。通过形态学和分子生物学鉴定，确定这株拮抗菌为拟康宁木霉Trichoderma koningiopsis，将其命名为PP4A，于2026年5月18日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2026994。盆栽实验表明，病原菌侵染3天后，接种了PP4A菌株的植株叶片，病原菌菌丝明显较少，说明病原菌生长明显受到抑制，其病斑占叶片的相对面积显著小于未接种PP4A的对照组。病原菌侵染7天后，未接种PP4A的叶片出现萎蔫脱水症状。以上结果表明，PP4A菌株能够有效防治重楼叶枯病，抑制重楼叶枯病病原菌生长且对植物无害。因此，本发明为重楼叶枯病的生防菌剂开发提供理论参考，也为保障重楼高产优产提供技术支撑。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a Trichoderma PP4A strain and its applications. Background Technology
[0002] Paris polyphylla ( Paris polyphylla Paris polyphylla, known as "Seven Leaves One Flower" in the Compendium of Materia Medica, is a perennial herbaceous medicinal plant belonging to the genus Paris of the Liliaceae family. It is mainly distributed in southwestern provinces of my country, with some distribution in Shaanxi, Hunan, and Guangxi. According to the 2020 Chinese Pharmacopoeia, the dried rhizome of Paris polyphylla is used medicinally. Its main active ingredients include steroidal saponins, sterols, and flavonoids, and it is effective in treating boils, carbuncles, sore throats, snake and insect bites, injuries from falls, and convulsions. With the increasing medicinal value of Paris polyphylla, its market demand has gradually increased. However, the rampant over-harvesting of wild Paris polyphylla has led to a sharp decline in wild resources. Combined with its long natural growth cycle, wild Paris polyphylla has become a rare, endangered, and valuable medicinal material. In recent years, artificial cultivation and propagation techniques for Paris polyphylla have been gradually promoted and emerged. However, due to its long planting cycle, difficulty in crop rotation, and unreasonable use of pesticides and fertilizers, the disease resistance of its sterile seedlings has decreased, making them susceptible to diseases. Furthermore, most pathogens can overwinter and are easily transmitted. Various diseases affecting Paris polyphylla are becoming increasingly prominent. While diseases are relatively mild in their natural habitat, they worsen and occur more frequently after large-scale cultivation. Common diseases include root rot, leaf blight, anthracnose, stem rot, gray mold, and brown spot. These diseases spread rapidly and widely, causing severe damage year after year and proving difficult to control. They significantly impact the yield and quality of Paris polyphylla, severely limiting the healthy and sustainable development of the industry.
[0003] Leaf blight is a major disease reported in the production of medicinal plants such as Paris polyphylla, Asarum heterotropoides, Panax notoginseng, Polygonum tinctorium, and Adenophora stricta, and its occurrence is widespread. Researchers conducted a disease survey of Paris polyphylla in Sichuan Province and found that the incidence of leaf blight was 15%-25%, proving that the dominant pathogen is *Pseudomonas aeruginosa*. Epicoccum sorghinu This disease primarily affects the leaves. In the early stages, brown necrotic lesions appear on the tips and margins of the leaves of Paris polyphylla. These lesions rapidly expand towards the base of the leaf. In severe cases, the lesions dehydrate and become scorched, causing the entire leaf to fall off and leading to the death of the entire plant. This severely impacts the growth of Paris polyphylla and results in a sharp decline in yield. Due to changes in the ecological environment, the influencing factors and pathogen types of Paris polyphylla leaf blight vary in different regions.
[0004] Currently, the control of Paris polyphylla leaf blight mainly employs agricultural, chemical, and biological control measures. Biocontrol microorganisms are one type of biological control, controlling diseases or increasing crop yields through competition, parasitism, and antibiosis. They can inhibit the growth and reproduction of pathogens without harming the plant. Plant endophytes can survive in roots, buds, leaves, flowers, and seeds, exhibiting symbiotic, mutualistic, and synergistic effects with the host without causing significant infection or disease, thus possessing the potential to develop into biocontrol microorganisms. As an important group among biocontrol bacteria, endophytes can trigger cellular responses by producing hydrolytic enzymes, secondary antimicrobial metabolites, plant hormones, and novel antimicrobial peptides, significantly improving the antioxidant system and inducing plant defense responses against pathogens. This makes them suitable as potentially highly effective biocontrol agents for sustainable pathogen control and high-quality plant production. However, there are currently no reports on the control of Paris polyphylla leaf blight by endophytic fungi. Therefore, fully exploring endophytic fungal resources for controlling Paris polyphylla leaf blight will help improve the quality and yield of Paris polyphylla, offering both ecological and economic benefits. Summary of the Invention
[0005] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a Trichoderma PP4A strain and its application to solve the problem that leaf blight seriously affects the growth of Paris polyphylla plants.
[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A *Trichoderma tumefaciens* (T.) Trichoderma koningiopsis The PP4A strain was deposited on May 18, 2026, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCC NO:M2026994.
[0007] This invention provides the application of the above-mentioned PP4A strain in the preparation of a formulation for controlling Paris polyphylla leaf blight.
[0008] Furthermore, Paris leaf blight is caused by pathogens. Ceratobasidium ramicola The resulting leaf blight disease in Paris polyphylla.
[0009] This invention provides a control agent for Paris polyphylla leaf blight, the control agent comprising at least one of the above-mentioned PP4A strain, its fermentation broth, and its fermentation supernatant.
[0010] This invention provides an application of the above-mentioned PP4A strain in the prevention and control of Paris polyphylla leaf blight.
[0011] Furthermore, Paris leaf blight is caused by pathogens. Ceratobasidium ramicola The resulting leaf blight disease in Paris polyphylla.
[0012] This invention provides a method for controlling Paris polyphylla leaf blight, comprising the following steps: spraying the above-mentioned PP4A strain or the above-mentioned control agent onto the surface of Paris polyphylla leaves until saturated.
[0013] This invention has the following beneficial effects: In the early stages of screening, the pathogen PP23, which causes leaf blight in Paris polyphylla, was obtained. Then, an endophytic fungus with significant antagonistic activity against it was isolated and screened from Paris polyphylla leaves. Through morphological and molecular biological identification, this antagonistic fungus was determined to be *Trichoderma tumefaciens*. Trichoderma koningiopsis The strain was named PP4A and deposited at the China Center for Type Culture Collection (CCTCC) on May 18, 2026, with accession number CCTCC NO: M2026994. Pot experiments showed that three days after pathogen infection, the leaves of plants inoculated with PP4A exhibited significantly fewer pathogen mycelia, indicating a marked inhibition of pathogen growth. The relative area of lesions on the leaves was also significantly smaller than in the uninoculated control group. Seven days after pathogen infection, leaves uninoculated with PP4A showed wilting and dehydration symptoms. These results indicate that PP4A can effectively control Paris polyphylla leaf blight, inhibiting the growth of the pathogen and being harmless to plants. Therefore, this invention provides a theoretical reference for the development of biocontrol agents for Paris polyphylla leaf blight and also provides technical support for ensuring high-yield and high-quality production of Paris polyphylla. Attached Figure Description
[0014] Figure 1 The pathogen causing Paris polyphylla leaf blight Ceratobasidium ramicola Morphology and phylogenetic tree; Figure 2 A plate image of PP4A strain grown on PDA medium for 7 days; Figure 3 Phylogenetic tree diagram of PP4A strain; Figure 4 For PP4A strain and pathogen Ceratobasidium ramicola Plate image after 7 days of cultivation; Figure 5 This image shows the biocontrol effect of strain PP4A against Paris polyphylla leaf blight. Detailed Implementation
[0015] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0016] Example 1: Isolation, purification, and molecular identification of the pathogen causing Paris polyphylla leaf blight In the early stages of this invention, leaves of Paris polyphylla with lesions were collected, and a pathogenic blight fungus, PP23, was isolated and screened. Morphological and molecular biological identification of this pathogen was then performed. Figure 1It can be seen that the hyphae of the pathogen PP23 are white and fluffy, arranged in whorls (see...). Figure 1 A). Genomic DNA was extracted from PP23 mycelia, and its ITS (the rDNA internal transcribed spacer), RPB2 (the second largest subunit of nuclear DNA-directed RNA polymerase II), and ATP6 (ATP synthase subunit 6) sequences were amplified by PCR. A phylogenetic tree was constructed by combining these sequences (see [link to phylogenetic tree]). Figure 1 B), based on morphological characteristics, the pathogen was identified as... Ceratobasidium ramicola .
[0017] Example 2: Pathogen Ceratobasidium ramicola Screening and identification of antagonistic bacteria (1) Isolation and purification of endophytic fungi from Paris polyphylla First, rinse the freshly collected Paris polyphylla leaves with clean tap water to remove surface dirt and impurities. Using a tissue separation method, cut the leaves into appropriately sized pieces suitable for subsequent disinfection. Briefly soak these pieces in a 75% ethanol solution for 30 seconds to initially remove surface impurities and microorganisms. Then, rinse with a 5% sodium hypochlorite solution for 30 seconds for further disinfection. After disinfection, rinse the diseased tissue three times repeatedly with sterile water until all residual disinfectant is completely removed. The rinsing time for each rinse should be adjusted flexibly according to the thickness of the leaf epidermis.
[0018] Ten tissue samples were placed in each petri dish to ensure a sufficient sample volume for isolation. Using a sterile inoculation needle, the tissues were gently transferred to the surface of prepared PDA medium supplemented with 0.375% lactic acid. Several samples were placed in each dish for subsequent selection based on colony morphology. Simultaneously, 20 μL of sterile water used in the final rinse was taken as a control group to confirm that the surface was thoroughly disinfected for subsequent experiments.
[0019] Invert the inoculated petri dishes and place them in a constant temperature incubator at 28℃. Observe daily for several days, periodically monitoring colony growth on the culture medium. Once colonies have grown on the medium, use a sterile inoculation needle to select single colonies with consistent morphology and good growth, and transfer them to fresh PDA medium for purification. Repeat this process 3-5 times until a pure culture is obtained. Then, preserve the strain in 50% glycerol at -80℃.
[0020] (2) Plate confrontation between antagonistic bacteria and pathogenic bacteria PP23 The antibacterial effect of endophytic bacteria was determined using the plate confrontation method. The tested pathogenic bacteria and endophytic bacterial strains were activated and cultured. Mycelial blocks of PP23 were taken using a 5mm punch and inoculated into the center of a PDA plate. Purified and isolated endophytic bacterial pellets were taken using a 5mm punch and inoculated in three directions at 2.5cm from the center. The plates were then incubated upside down at 28℃ for 5 days. Three replicates were set up. Mycelial diameter was measured, and the inhibition rate was calculated.
[0021] Inhibition rate (%) = (Diameter of pathogen control group - Diameter of pathogen treatment group) / Diameter of pathogen control group × 100% (3) Molecular identification of antagonistic bacteria Total DNA was extracted from antagonistic bacteria using the Biospin Fungal Genomic DNA Extraction Kit (Hangzhou Borui Technology Co., Ltd.) following the product instructions. The extracted DNA was dissolved in 150 μL of sterile water and stored at -20°C for later use. The internal transcribed spacer (ITS) region of the antagonistic bacteria's ribosomal DNA was amplified. The amplification system, amplification program, and PCR primers are shown in Tables 1-3. After the PCR amplification products were confirmed by agarose gel electrophoresis, they were sent to Kunming Jieteng Biotechnology Co., Ltd. for sequencing. The sequencing primers were consistent with the amplification primers. The sequencing results were analyzed using BLAST alignment on NCBI (https: / / www.ncbi.nlm.nih.gov / ). Closely related model bacteria were selected, and their sequences were downloaded. Sequence alignment was performed using MEGA-X, and a phylogenetic tree was constructed based on maximum likelihood (ML).
[0022] Table 1. PCR amplification system of antagonistic bacteria
[0023] Table 2 PCR amplification procedure for molecular identification of antagonistic bacteria
[0024] Table 3 Primer sequences for molecular identification of antagonistic bacteria
[0025] Example 2: Evaluation of the biocontrol effect of strain PP4A against Paris polyphylla leaf blight The antibacterial effect of the PP4A strain was verified using a live plant inoculation method. Three-year-old Paris polyphylla plants were selected for the experiment. The plants were placed in sterile soil mixed with the PP4A strain (experimental group), while those in ordinary sterile soil served as the control group. The plants were cultured at 80% relative humidity and 25℃ for three days. Afterward, 70% alcohol was sprayed evenly onto the leaves. Using a strictly sterilized needle, tiny, uniform holes were gently pierced into two leaves on each plant. A 5 mm diameter block of pathogen mycelium was placed over these holes, ensuring direct contact between the mycelium and the plant tissue. After inoculation, all plants were kept in a greenhouse at 25℃, 80% relative humidity, and a 12-hour photoperiod. The growth status of all plants and changes in leaf lesions were observed, and the relative area of the lesions (the percentage of lesion area to the total leaf area) was calculated to determine whether PP4A could inhibit the symptoms of Paris polyphylla leaf blight.
[0026] Experimental Results and Analysis (1) Morphological and molecular identification of PP4A strain The endophytic fungus PP4A, isolated and purified from Paris polyphylla leaves, initially appeared as white colonies with cottony aerial hyphae. After 7 days of culture, the central hyphae in the culture medium turned green as they extended. Figure 2 As shown. A BLAST search was performed on the ITS sequences obtained from sequencing strain PP4A, and strains with high gene similarity were selected to construct a phylogenetic tree. PP4A was compared with 11 model strains of the genus *Trichoderma*, and simultaneously... Escovopsis weberi Using fungi (ATCC 64542) as exogenous strains, a phylogenetic tree was constructed based on the maximum likelihood method (see [link to phylogenetic tree]). Figure ¾ ), and found that it is related to Trichoderma koningiopsis The strain sequence clustered on one branch, therefore strain PP4A was identified as *Trichoderma corniglita*. Trichoderma koningiopsis ).
[0027] (2) PP4A strain against pathogens Ceratobasidium ramicola antagonistic effect When the isolated and purified PP4A was plate-confronted with the pathogen PP23, it was found that PP4A significantly inhibited the mycelial growth of PP23 (see...). Figure 4 The average antibacterial rate was 66.43 ± 0.25%.
[0028] (3) Evaluation of the biocontrol effect of strain PP4A against Paris polyphylla leaf blight Before pathogen infection, the Paris polyphylla plants growing in soil mixed with PP4A were very healthy, indicating that PP4A had no adverse effect on the growth of Paris polyphylla. (See Table 4 and...) Figure 5 It can be seen that 3 days after infection with PP23 strain, the mycelium of PP23 strain on the leaves of plants inoculated with PP4A was significantly reduced, indicating that the growth of PP23 strain was significantly inhibited. Figure 5A and 5D), the relative area of lesions on the leaves was significantly smaller than that of the control group not inoculated with PP4A ( Figure 5 B and 5E). Seven days after infection with PP23 strain, leaves that were not inoculated with PP4A showed symptoms of wilting and dehydration. Figure 5 (C and 5F). The above results indicate that PP4A can inhibit the growth of the pathogen causing Paris polyphylla leaf blight and is harmless to plants.
[0029] Table 4. Effect of PP4A on the relative area of lesions 3 days after pathogen infection.
[0030] The ITS sequence of the PP4A strain of this invention is as follows: (SEQ ID NO.1).
[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A type of Trichoderma truncatum ( Trichoderma koningiopsis PP4A strain, characterized in that, The PP4A strain was deposited on May 18, 2026, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCC NO:M2026994.
2. The use of the PP4A strain according to claim 1 in the preparation of a formulation for controlling Paris polyphylla leaf blight.
3. The application according to claim 2, characterized in that, The aforementioned Paris polyphylla leaf blight is caused by pathogens. Ceratobasidium ramicola The resulting leaf blight disease in Paris polyphylla.
4. A control agent for Paris polyphylla leaf blight, characterized in that, The control agent includes at least one of the PP4A strain described in claim 1, its fermentation broth, and its fermentation supernatant.
5. The application of the PP4A strain according to claim 1 in the prevention and control of Paris polyphylla leaf blight.
6. The application according to claim 5, characterized in that, The aforementioned Paris polyphylla leaf blight is caused by pathogens. Ceratobasidium ramicola The resulting leaf blight disease in Paris polyphylla.
7. A method for controlling Paris polyphylla leaf blight, characterized in that, The steps include: spraying the PP4A strain of claim 1 or the control agent of claim 4 onto the surface of Paris polyphylla leaves until saturated.