A hooked Trichoderma YN-4 and its application

Abstract

The invention discloses a hooked Trichoderma YN‑4 and its application. The fungus was deposited in the General Microbiological Center of China Microorganism Culture Collection Committee on August 8, 2022, with a deposit number of CGMCC No.40268, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Hooked Trichoderma YN‑4 can effectively prevent and treat sagittal epimedium anthracnose, and has broad application prospects in the field of biological control of sagittal epimedium diseases.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to a hooked Trichoderma YN-4 and an application thereof. Background Art

[0002] Epimedium sagittatum Epimedium sagittatum (Sieb. et Zucc.) Maxim. is a perennial herbaceous plant of the genus Epimedium in the Berberidaceae family. It is one of the sources of traditional Chinese medicine Epimedium in my country. Its leaves are used as medicine, and it has the medicinal effects of tonifying kidney yang, strengthening bones and tendons, and removing rheumatism. Modern pharmacological studies have shown that Epimedium has multiple functions such as anti-tumor, anti-inflammatory, anti-osteoporosis, beneficial to the nervous system, and cardiovascular protection. Icaritin, a derivative of the flavonol compound icariin in Epimedium, has significant anti-cancer activity. At present, Icaritin (Icaritin) soft capsules have been approved for marketing by the National Medical Products Administration (NMPA) in January 2022 for the treatment of advanced liver cancer, and the Chinese herbal medicine Epimedium is the only source of icariin. At the same time, Epimedium, as a commonly used Chinese medicine in clinical practice, is widely used in medicine, health care and other fields. It is the main raw material for a variety of drugs and health products, and is also one of the Chinese medicines with the most development potential and market potential.

[0003] In recent years, the demand for Epimedium has increased sharply and the price has continued to rise, leading to the predatory excavation of wild Epimedium, which has caused the depletion of wild resources. At present, the artificial cultivation of Epimedium sagittatum has been successfully achieved, and it has become the species with the largest cultivation area among the medicinal Epimedium plants, and has also become the main source of medicinal Epimedium at home and abroad. At present, the largest planting base of Epimedium sagittatum in Asia has been established in Zhumadian, Henan Province, and it has also become the main supplier of artificially cultivated Epimedium medicinal materials in the country. With the introduction and domestication of Epimedium sagittatum and long-term asexual reproduction, a variety of disease problems have emerged, and the more serious disease is the leaf disease of Epimedium sagittatum. The medicinal part of Epimedium is the leaves, and the occurrence of leaf diseases will seriously affect the yield and quality of Epimedium. After disease investigation and identification, it was found that the leaf diseases of sagittal epimedium include a variety of different fungal diseases, among which anthracnose is one of the main leaf diseases in the Henan production area, with an incidence rate of up to 30-40%, which has a serious impact on the yield and quality of sagittal epimedium.

[0004] At present, the main means of preventing and controlling plant fungal diseases is chemical pesticides, but the large-scale use of chemical pesticides will cause problems such as pesticide residues and environmental pollution. The medicinal part of Epimedium sagittatum is the leaves. If chemical pesticides are used in large quantities for disease prevention and control, problems such as excessive pesticide residues will occur, resulting in unqualified products. In recent years, with the rise of ecological planting, microbial control of diseases has also become an important biological control method, and the acquisition of efficient biocontrol bacteria is the basis for the prevention and control of plant microbial diseases. At present, there are no reports on biocontrol bacteria for anthracnose of Epimedium sagittatum, and the commercial biocontrol bacteria on the market have been found to be ineffective after preliminary trials by the applicant, and the control after the application of multiple pesticides has not achieved the desired effect. Summary of the invention

[0005] In order to solve the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a hooked Trichoderma YN-4 and its application, which can effectively inhibit the growth of the pathogen of Epimedium sagittatum anthracnose, and has an excellent preventive and control effect on Epimedium sagittatum anthracnose in field experiments, thereby providing a new biocontrol agent for the biological control of Epimedium sagittatum anthracnose.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: The first object of the present invention is to provide a hook-shaped Trichoderma ( Trichoderma hamatum ), named it hooked Trichoderma YN-4, and registered and preserved it at the General Microbiological Center of China Microorganism Culture Collection on August 8, 2022, with the deposit number CGMCC No.40268, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second object of the present invention is to provide the use of the above-mentioned hooked Trichoderma YN-4 in preventing and / or treating anthracnose of Epimedium sagittatum.

[0008] The third object of the present invention is to provide a biological preparation, which includes the above-mentioned hooked Trichoderma YN-4. The biological preparation can be a fermentation broth of hooked Trichoderma YN-4, or the hooked Trichoderma YN-4 bacteria can be directly made into a solid preparation such as a suspension or powder, or a preparation made with hooked Trichoderma YN-4 as the only active ingredient or the main active ingredient and then mixed with excipients.

[0009] The present invention has the following beneficial effects:

[0010] The present invention separates the endophytes from the leaves of Epimedium sagittatum and screens out an endophytic fungus that can effectively inhibit the growth of anthracnose pathogens of Epimedium sagittatum. The endophytic fungus is identified as Trichoderma spp. ( Trichoderma hamatum) named it hooked Trichoderma YN-4, and used it in field prevention experiments to achieve ideal disease control effects. The disease control effect is much higher than that of chemical pesticides carbendazim and manganese imipenem salt. It is a green and pollution-free biocontrol fungus that can reduce the use of chemical pesticides. At the same time, since the antagonistic fungus is isolated from healthy Epimedium sagittatum plants, the antagonistic fungus is very friendly to the environment and meets the national requirements for ecological agricultural planting. It has a very broad application prospect in the field of ecological prevention and control of Epimedium diseases in traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The colony morphology of hooked Trichoderma YN-4 on PDA medium, Figure 1 -A is the colony morphology, Figure 1 -B shows the morphology of spores under a microscope.

[0012] Figure 2 The plate inhibition effect of Trichoderma hooked YN-4 on the anthracnose pathogen that causes anthracnose of Epimedium sagittatum, wherein Figure 2 -A is the anthrax bacteria control, Figure 2 -B is a plate confrontation between the antagonistic fungus YN-4 and anthrax bacteria.

[0013] Figure 3 is the phylogenetic tree of strain YN-4.

[0014] Figure 4 This is a graph showing the control effect of Trichoderma hooked YN-4 on anthracnose of Epimedium sagittatum in a field trial. Figure 4 -A is a healthy Epimedium sagittatum control, Figure 4 -B is Epimedium sagittatum that became ill after being infected by anthrax bacteria. Figure 4 -C is the sagittate Epimedium after spraying the antagonistic fungus YN-4 to control anthracnose. DETAILED DESCRIPTION

[0015] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0016] Example 1: Isolation, purification and identification of strains

[0017] 1. Isolation of endogenous antagonistic bacteria

[0018] Collection of Epimedium sagittatum plant samples: In 2021, healthy Epimedium sagittatum plants were collected from the Epimedium sagittatum GAP planting base in Pingyu County, Zhumadian City, Henan Province for isolation of endophytes.

[0019] Take a fresh and healthy sagittaria plant, wash off the surface soil with clean water, soak the leaves in 75% ethanol, 0.1% mercuric chloride solution, and 75% ethanol in a clean bench, then wash with sterile water 5-7 times, place on sterile absorbent paper to dry, take the last wash water and apply it on LB medium, and culture at 37℃ to detect whether the surface disinfection is thorough. Cut the sagittaria leaves into leaf tissues with a length and width of about 0.2-0.3cm with a sterile blade, transfer the leaf tissues to PDA medium containing 0.1% chloramphenicol, and culture at 28℃. After fungal colonies grow around the leaves, pick the hyphae at the edge of the colony and inoculate them on the PDA plate. After the colony grows and produces spores, pick the spores and dilute them with sterile water and apply them on the PDA medium. After the single colony grows, pick different single colonies and culture them separately. After obtaining pure culture, save and reserve.

[0020] The PDA medium used above contained 200 g / L potato, 20 g / L glucose, 15-20 g / L agar, and natural pH.

[0021] 2. Screening of antagonistic strains by plate confrontation method

[0022] Use a hole punch to take 5mm diameter of the anthracnose pathogen of Epimedium sagittatum (Coleosporium oleraceum Colletotrichum fructicola ) were inoculated onto PDA culture medium, and then a 5mm diameter antagonistic fungus was taken with a cork puncher and inoculated into the culture medium 3cm away from the anthracnose pathogen block of Epimedium sagittatum. The culture medium was then placed in a 28℃ incubator for 3 days to observe the antagonistic effect, measure the diameter of the pathogen colony, calculate the inhibition rate, determine the antagonistic effect of the strain, and select the strain with obvious antagonistic effect for identification and preservation.

[0023] The plate inhibition rate calculation formula is: Inhibition rate (%) = (control pathogenic fungal colony diameter - pathogenic fungal colony diameter co-cultured with antibacterial) / control pathogenic fungal colony diameter × 100%

[0024] The results showed that the inhibition rate of the antagonistic fungus YN-4 on the anthracnose pathogen of Epimedium sagittatum was 96.67% after 3 days. Figure 2 shown.

[0025] 3. Identification of strains

[0026] The antagonistic bacteria screened out above were identified as follows:

[0027] 1. Morphological identification

[0028] The colonies of the selected antagonistic bacteria were white in the early stage of culture on PDA medium, and the aerial hyphae were white. In the later stage, green conidia were formed, and the conidia were spherical or short elliptical ( Figure 1 ).

[0029] 2. Molecular identification

[0030] The genomic DNA of strain YN-4 was extracted and amplified using fungal ITS universal primers (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The PCR product was sent to a sequencing company for sequencing. The sequencing results were entered into the GenBank database for comparison. The comparison results showed that the strain was classified as the same as Trichoderma hamatum The highest similarity (the phylogenetic tree is shown in Figure 3 ), which was identified as Trichoderma hookii ( Trichoderma hamatum ), named as hooked Trichoderma YN-4. The fungus was deposited in the China General Microbiological Culture Collection Center (referred to as CGMCC, address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on August 8, 2022, with the deposit number CGMCC No.40268.

[0031] Example 2: Field biocontrol effect of strains

[0032] The field biocontrol effect of hooked Trichoderma YN-4 on anthracnose of Epimedium sagittatum is verified as follows:

[0033] Five experimental plots were selected from the healthy growing Epimedium sagittatum in Pingyu County, Zhumadian City, Henan Province, including the blank control area (healthy Epimedium sagittatum), the control group 1 (sprayed with the pesticide carbendazim), the control group 2 (sprayed with the pesticide imipenem manganese salt), the experimental group 1 (inoculated with the anthracnose pathogen of Epimedium sagittatum (Coleosporium spp. Colletotrichum fructicola ) spores), experimental group 2 (experimental group inoculated with anthrax bacteria and sprayed with hooked Trichoderma YN-4, in which YN-4 spores were sprayed at a spore concentration of 1*10 6 cfu / ml, spray about 3-5 ml spore liquid evenly on each leaf, spray once), all sagittal Epimedium in the 5 experimental plots were pierced with sterilized toothpicks, and the operation was carried out according to the above experimental group design. The disease situation of sagittal Epimedium leaves was recorded every day after 3 days of inoculation. The results showed that hooked Trichoderma YN-4 had a good control effect on sagittal Epimedium anthracnose, and the field control efficiency could reach 56.33% ( Figure 4 ), which is much higher than the control agent 1 carbendazim (the prevention efficiency is 17.16%) and the control agent 2 manganese imipenem (the prevention efficiency is 33.38%).

[0034] Field prevention efficiency (%) = (incidence rate in the control area - incidence rate in the prevention and control area) / incidence rate in the control area × 100%, where incidence rate (%) = number of diseased seedling leaves / number of inspected leaves × 100%.

[0035] In summary, it can be seen that the antagonistic fungus YN-4 (strain collection number: CGMCC No. 40268) screened by the present invention has a strong antagonistic effect on the pathogenic bacteria that cause the anthracnose disease of Epimedium sagittatum, and the field control effect experiment also achieves an ideal disease control effect, and the disease control effect is much higher than that of the chemical pesticides carbendazim and manganese imipenem salt. It is a green and pollution-free biocontrol fungus, which can reduce the use of chemical pesticides. At the same time, because the antagonistic fungus is separated from a healthy Epimedium sagittatum plant, the antagonistic fungus is very friendly to the environment, meets the national requirements for ecological agricultural planting, and has a very broad application prospect in the field of ecological control of diseases of traditional Chinese medicine Epimedium.

[0036] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A hook-shaped Trichoderma ( Trichoderma hamatum ), named as hooked Trichoderma YN-4, and deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 8, 2022, with the deposit number CGMCC No.40268, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the hooked Trichoderma YN-4 described in claim 1 in treating anthracnose of Epimedium sagittatum.

3. A biological agent, characterized in that: It comprises the hooked Trichoderma YN-4 described in claim 1.

4. A biological agent, characterized in that: The invention comprises the fermentation liquid of the hooked Trichoderma YN-4 according to claim 1.

Images