Trichoderma reesei F8287 and application thereof

Abstract

The invention discloses a Trichoderma reesei F8287 strain and an application of the Trichoderma reesei F8287 strain. The Trichoderma reesei F8287 strain comprises a Trichoderma reesei strain and a Belongs to the technical field of biocontrol. According to the invention, the Trichoderma reesei F8287 with efficient biocontrol potential is successfully separated and provided, and the discovery of the Trichoderma reesei F8287 effectively overcomes the defects of research and development of the Trichoderma reesei strain in the prior art. Experiments prove that the strain and the prepared microbial preparation show remarkable and stable prevention and treatment effects on botrytis cinerea and muskmelon gray mold caused by botrytis cinerea. Therefore, the strain provides a safe, efficient and brand-new microbial resource for green prevention and control of gray mold, meets the urgent demand of the field for novel biocontrol strains, and has an important application prospect in the aspect of developing biological prevention and control preparations for replacing chemical pesticides.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biocontrol technology, and more particularly to a Trichoderma parareesei F8287 and application thereof. BACKGROUND

[0002] Botrytis blight is a worldwide plant disease that is widespread and harmful. Its pathogen is Botrytis cinerea Pers., a necrotrophic fungus. The pathogen is known for its wide host range, which can infect hundreds of fruits, vegetables, flowers, and field crops, causing significant yield and economic losses in the field and post-harvest storage and transportation. More notably, the pathogen exhibits excellent survival ability and persistence in the environment, and can survive in plant residues or soil in the form of sclerotia or conidia, which facilitates its year-round cyclic infection and makes control work extremely difficult.

[0003] Currently, the prevention and control of Botrytis blight in agricultural production still relies heavily on chemical pesticides. However, this single chemical control strategy faces multiple serious challenges. First, the excessive and unreasonable use of chemical agents has led to increasingly prominent environmental residue and food safety problems, posing potential threats to ecosystems and human health. Second, and more problematic, the pathogen has high genetic and physiological flexibility and plasticity, enabling it to rapidly evolve resistance to commonly used fungicides through various mechanisms, leading to decreased or even complete loss of efficacy. In addition, the pathogen exhibits excellent adaptability during infection, effectively responding to and evading the plant's basic defense response by secreting effector proteins and activating detoxification mechanisms, further weakening the control effect of chemical agents. Therefore, seeking efficient, safe, and sustainable alternative or auxiliary control strategies has become an urgent and important task in the field of agricultural plant pathology.

[0004] Under this background, biological control, as an environmentally friendly and sustainable approach to plant disease management, has attracted extensive attention. Among them, Trichoderma spp. fungi are recognized as one of the most promising biocontrol agents. Trichoderma is a group of saprophytic filamentous fungi widely existing in natural environments such as soil and phyllosphere. They can establish a symbiotic relationship with plant roots and play multiple beneficial roles in the rhizosphere ecosystem. Its biocontrol mechanisms are diverse and efficient, mainly including: ① Through rapid growth and colonization, it competitively excludes pathogenic fungi in space and nutrition, and occupies ecological niche; ② It can secrete a variety of secondary metabolites with antibacterial activity to directly inhibit or kill pathogenic fungi; ③ It can produce cell wall-degrading enzymes (such as chitinase and glucanase) to directly lyse pathogenic fungal cell walls; ④ At the same time, Trichoderma can also act as an "immune activator" for plants, systematically inducing plants to produce disease resistance, thereby enhancing their own defense ability against pathogen invasion. Importantly, the action of Trichoderma is usually targeted, causing minimal damage to beneficial microbial community structure in soil, and helping to maintain a healthy soil microecological balance.

[0005] Currently, research and development of Trichoderma at home and abroad mainly focuses on several commercialized model species, such as T. harzianum, T. atroviride, T. virens, T. reesei and T. longibrachiatum. The research on these species has been relatively in-depth, and their biocontrol potential has been fully verified. In contrast, the resource exploration and functional research on other species in the genus Trichoderma are still insufficient, especially for T. parareesei. The relevant research reports are extremely scarce, and its control potential against Botrytis cinerea, an important pathogen, is still an unknown field in agricultural disease biological control.

[0006] Therefore, how to develop new strains of Trichoderma with control effect on Botrytis cinerea is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0007] Therefore, the present application provides a Trichoderma parareesei F8287 and its application. The strain shows significant and stable control effect on tomato gray mold and melon gray mold caused by Botrytis cinerea.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The first object of the present application is to provide a Trichoderma parareesei F8287, which is deposited in the China General Microbiological Culture Collection Center on March 28, 2024, and has a deposit number of CGMCC No. 41132 and a deposit address of No. 3, Beichen West Road, Chaoyang District, Beijing, and is taxonomically named as Trichoderma parareesei.

[0010] The further object of the present application is to provide an application of the Trichoderma parareesei F8287 in the preparation of a microbial preparation.

[0011] The further object of the present application is to provide a microbial preparation, which comprises the Trichoderma parareesei F8287, a fermentation product and at least one of spores.

[0012] As a preferred technical solution, the fermentation product is prepared by inoculating the Trichoderma parareesei F8287 into a fermentation medium for fermentation.

[0013] The further object of the present application is to provide an application of the Trichoderma parareesei F8287 or the microbial preparation in the prevention and treatment of gray mold.

[0014] The further object of the present application is to provide an application of the Trichoderma parareesei F8287 or the microbial preparation in the preparation of a medicine for preventing and / or treating gray mold.

[0015] The further object of the present application is to provide a medicine for preventing and / or treating gray mold, which comprises the Trichoderma parareesei F8287 and / or the microbial preparation.

[0016] As a preferred technical solution, the medicine for preventing and / or treating gray mold further comprises other active ingredients and / or pharmaceutical excipients.

[0017] The further object of the present application is to provide a method for preventing and treating gray mold of crops, which comprises applying the Trichoderma parareesei F8287 and / or the microbial preparation and / or the medicine to the rhizosphere of the crops.

[0018] Preferably, the crops are melons and / or tomatoes.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] The application successfully isolates and provides a new strain of Trichoderma parareesei F8287 with high biocontrol potential. The discovery of the strain effectively makes up for the deficiency in the research and development of the Trichoderma parareesei strain in the prior art. Experimental verification shows that the strain has significant and stable control effects on tomato botrytis and melon botrytis caused by Botrytis cinerea. Therefore, the strain provides a safe, efficient and new microbial resource for the green control of botrytis, solves the urgent need for new biocontrol strains in the field, and has important application prospects in the development of biological control agents to replace chemical pesticides. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of the provided drawings.

[0022] Figure 1 Figs. 1 and 2 are the colony and microscopic characteristics of Trichoderma parareesei F8287 on a plate and under a microscope, respectively.

[0023] Figure 2 Fig. 3 is a phylogenetic tree of Trichoderma parareesei F8287 constructed based on ITS sequences.

[0024] Figure 3 Fig. 4 is a plate antagonistic evaluation of Trichoderma parareesei F8287 on Botrytis cinerea.

[0025] Figure 4 Fig. 5 is fermentation and spore production evaluation of Trichoderma parareesei F8287 on a solid culture medium.

[0026] Figure 5 Fig. 6 is an in vitro plant protection evaluation of Trichoderma parareesei F8287 preparation on tomato botrytis.

[0027] Figure 6The application relates to a preparation of Trichoderma parareesei F8287 for evaluating the plant in-vitro prevention effect on Botrytis cinerea of muskmelon. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be clearly and completely described in combination with the drawings in the embodiments of the application. Apparently, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the application.

[0029] The reagents involved in the embodiments of the application are all purchased from a market channel, and the method not mentioned is a conventional experimental method, which will not be described one by one here.

[0030] Embodiment 1

[0031] Separation and identification of Trichoderma parareesei F8287 and preservation

[0032] (1) Separation and identification of Trichoderma parareesei F8287

[0033] The biocontrol Trichoderma is separated and screened from soil in a facility muskmelon greenhouse in Yanliang district, Xi'an city, and the specific separation and screening process is as follows:

[0034] The specific separation method is as follows: the soil sample in the facility greenhouse is placed in a ventilated place for natural air drying, and then ground into powder with a sterile mortar, 1 g of the soil sample is added into 9 mL of sterile water, and then gradient step-by-step dilution is carried out, 100 muL of the diluted sample with dilution of 1000 and 10000 times is coated on PDA culture medium added with 100 U / mL of akazimycin sulfate, and then 28 DEG C culture is carried out for 5-7 days, and the colonies are picked and purified to obtain.

[0035] Through the above method, a suspected Trichoderma strain F8287 is obtained. In the early stage of culture on PDA culture medium, the mycelium of F8287 is white, spreads outward like a spider web, after 48 hours, the colony shows rich aerial mycelium, the center of the colony begins to turn green, a large amount of spores are generated, the conidial phialides are densely arranged and are white or green, the conidia are oval to subspherical (Figure 1), which are similar to the colony morphology and microscopic characteristics of Trichoderma, and the strain is preliminarily determined as a Trichoderma strain.

[0036] In order to further determine the classification status of the strain, ITS sequencing and construction of a phylogenetic tree are carried out, and the specific method is as follows:

[0037] The strain was cultured on PDA culture for 7 days, and the mycelium was scraped with a sterile toothpick and placed in a sterile mortar, ground in liquid nitrogen to extract DNA, and the DNA was extracted using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit. After extraction, the DNA was used as a template for PCR amplification using ITS1 and ITS4 primers. TProfessional Standard 96 Gradient was used during amplification.

[0038] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO. 1;

[0039] ITS4: 5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO. 2.

[0040] The PCR amplification reaction system is shown in Table 1:

[0041] Table 1 PCR amplification reaction system

[0042] The PCR amplification reaction program is shown in Table 2:

[0043] Table 2 PCR amplification reaction program

[0044] 5 microliters of the PCR product obtained after amplification were electrophoresed in a 1% agarose gel and stained with GelRed. The PCR product was then purified and sequenced by BGI Biotechnology Co., Ltd. The raw sequences obtained were aligned using MEGA 5.05, manually edited, and then searched for the best match in the GenBank database using BLAST. Sequences with a similarity of more than 97% belong to the same genus. All sequences were aligned using MEGA 5.05 by Clustal W prepared alignment, and all positions containing gaps and missing data were deleted. Finally, the maximum likelihood phylogenetic tree was constructed for each family using MEGA software 5.05.

[0045] Results analysis: It was found through research that the ITS gene sequence of the strain had a similarity of 99.35% with the effective strain Trichoderma parareesei ATCC MYA-4777 (NR_120297.1), and clustered with it on the phylogenetic tree (Fig. 1) Figure 2 ), therefore, the strain was identified as Trichoderma parareesei.

[0046] (2) Preservation of Trichoderma parareesei F8287

[0047] The applicant preserved Trichoderma parareesei F8287 in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No. 41132, on March 28, 2024, and the taxonomic name Trichoderma parareesei.

[0048] Example 2

[0049] Plate inhibition of Trichoderma parareesei F8287 on Botrytis cinerea

[0050] Botrytis cinerea, the pathogen isolated from the Botrytis cinerea of melon, was inoculated on PDA medium and cultured for 5 days for standby, and Trichoderma parareesei F8287 was inoculated on PDA medium and cultured for 5 days for standby; a sterile pipette was used to punch a hole on the colony of the Botrytis cinerea pathogen, and a sterile toothpick was used to pick up the mycelium and inoculate it at the center of a new PDA culture dish, which was then cultured in a 28°C incubator, serving as the control group.

[0051] A sterile pipette was used to punch a hole on the colony of the Botrytis cinerea pathogen, and a sterile toothpick was used to pick up the mycelium and inoculate it at the center of a new PDA culture dish, and then a sterile pipette was used to punch a hole on the colony of Trichoderma parareesei F8287, and a sterile toothpick was used to pick up the mycelium and inoculate it at a position 3 cm from the center of the 9 cm culture dish, and the PDA plate had been inoculated with the Botrytis cinerea pathogen at the center, which was then cultured in a 28°C incubator, serving as the treatment group.

[0052] When the mycelium of the control group was about to cover the plate, the colony diameter was measured by cross intersection, and the inhibition rate of Trichoderma parareesei F8287 on the Botrytis cinerea pathogen was calculated, and the formula for calculating the mycelium inhibition rate was as follows:

[0053] Pathogen inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter) x 100%

[0054] Through the above method, the inhibition rate of Trichoderma parareesei F8287 on Botrytis cinerea is 82%, which indicates that Trichoderma parareesei F8287 has strong antagonistic characteristics to Botrytis cinerea in vitro Figure 3 ].

[0055] Example 3

[0056] Preparation of Trichoderma parareesei F8287 microbial preparation

[0057] (1) Trichoderma parareesei F8287 was inoculated on PDA medium for 5 days for standby, then the cultured Trichoderma parareesei F8287 was inoculated on sterile YPD medium (composition of the culture medium (g / L): yeast extract 10, peptone 20, glucose 20, deionized water 1L, pH natural) in a 250 mL shake flask by agar punching and sterile toothpick, and then cultured at 230 rpm and 28°C for 48 h to prepare seed liquid.

[0058] (2) Prepare bran solid fermentation medium, the composition of the medium is: bran 30g, glucose 2g, water 50mL, after stirring uniformly, pour into a 50mm glass culture dish, the filling amount is two-thirds of the depth of the culture dish, then cover the lid, sterilize at 121°C for 20 minutes for standby;

[0059] (3) The seed liquid prepared in step (1) is inoculated into the sterilized bran solid fermentation medium at an amount of 5%, then cover the lid, and place it in a constant temperature incubator for Trichoderma parareesei F8287 shallow plate fermentation at 28°C, and the fermentation period is 7-12d.

[0060] Trichoderma parareesei F8287 produces a large amount of green spores on the solid medium during the initial growth stage, and as the fermentation time prolongs, Trichoderma parareesei F8287 gradually produces green spores on the solid medium Figure 4 ).

[0061] After about 6 days of fermentation, the cover of the solid fermentation medium is opened, the solid medium is stirred once, and the culture is continuously cultured until about 12 days, and then the culture is naturally air-dried and crushed to obtain the Trichoderma parareesei F8287 microbial preparation, which contains Trichoderma parareesei F8287 spores.

[0062] Example 4

[0063] Evaluation of the Trichoderma parareesei F8287 microbial preparation on the biological control of gray mold

[0064] (1) Healthy tomato / melon leaves are collected, the surface of the leaves is washed with tap water, and then naturally air-dried, and then wounds are created at key positions of the leaves using needles, blades, etc.

[0065] (2) Control group: The mycelium / spore complex of the gray mold pathogen is inoculated at the wound, the petiole position is wrapped with wet cotton, the whole leaf is placed in a culture dish with water, and is placed in a 28°C incubator for culture;

[0066] (3) Treatment group: The Trichoderma parareesei F8287 microbial preparation prepared in Example 3 is diluted, under the guidance of a hemocytometer, the spore concentration is adjusted to a concentration of 1×10 8 cfu / mL, and then the spore suspension is sprayed onto the leaves, and after being placed flat and air-dried, the mycelium / spore complex of the gray mold pathogen is inoculated at the wound, the petiole position is wrapped with wet cotton, the whole leaf is placed in a culture dish with water, and is placed in a 28°C incubator for culture;

[0067] (4) After 5-7 days of culture, the wound position at the center of the leaf is observed, the diameter of the diseased tissue is recorded, the control efficiency is calculated, and the experimental results are shown in Table 1 and Table 2. Figure 5 and Figure 6

[0068] The calculation formula of the control efficiency is as follows:

[0069] Control efficiency (%) = (diameter of the diseased tissue of the control group - diameter of the diseased tissue of the treatment group) / diameter of the diseased tissue of the control group) × 100%.

[0070] Result analysis: Through the above method, it is shown that the Trichoderma parareesei F8287 microbial preparation prepared in Example 3 exhibits strong antagonistic properties against tomato gray mold on tomato leaves in vitro (Table 1 and Table 2), and the inhibition rate is 60%. Figure 5

[0071] ​​The Trichoderma parareesei F8287 microbial preparation prepared in Example 3 showed a higher control effect on the muskmelon gray mold disease on the muskmelon leaves in vitro (Table 2) Figure 6 ), with an inhibition rate of 68%.

[0072] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0073] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Trichoderma reesei F8287, characterized in that, Trichoderma parareesei F8287, characterized in that, the trichoderma reesei Trichoderma parareesei F8287 was preserved in China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 41132, the preservation date of 2024.03.28, the preservation address of No. 3, Xili, Beichen, Chaoyang District, Beijing, and the taxonomic name of Trichoderma reesei Trichoderma parareesei.

2. The H. resinaecola of claim 1 Trichoderma parareesei Use of F8287 in the preparation of a microbial preparation.

3. A microbial preparation, characterized in that, The microbial preparation comprises the Trichoderma reesei of claim 1 Trichoderma parareesei F8287, at least one of a fermentation product and a spore.

4. The microbial preparation according to claim 3, characterized in that, The fermentation product is prepared by inoculating Trichoderma reesei Trichoderma parareesei F8287 is fermented in a fermentation medium.

5. The H. reesei of claim 1 Trichoderma parareesei Use of the microbial inoculant of F8287 or any of claims 3-4 in the control of gray mold.

6. The H. reesei of claim 1 Trichoderma parareesei Use of the microbial inoculant of F8287 or any one of claims 3-4 in the manufacture of a medicament for the prevention and / or treatment of gray mold.

7. A medicine for preventing and / or treating gray mold, characterized by, The Trichoderma reesei of claim 1 Trichoderma parareesei The microbial preparation of F8287 and / or any one of claims 3-4.

8. The medicine for preventing and / or treating gray mold according to claim 7, characterized by, Further active ingredients and / or pharmaceutically acceptable adjuvants are also included.

9. A method of controlling gray mold of agricultural crops, characterized by, The Trichoderma reesei of claim 1 Trichoderma parareesei F8287 and / or the microbial preparation of any one of claims 3-4 and / or the pharmaceutical of any one of claims 7-8 is applied to the rhizosphere of a crop.

10. The method for controlling gray mold of crops according to claim 9, characterized in that, The crop is melon and / or tomato.

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