A strain of Lecanococcus scaber for controlling pepper gray mold

Abstract

The invention discloses a strain of Lecanococcus scaber for preventing and treating pepper gray mold. Lecanicillium psalliotae )Lps‑ivf2024 strain. In the experiment of controlling pepper gray mold, the fungus showed a good control effect on Botrytis cinerea in greenhouse potted plants. When treated with the fermentation liquid of the Lps‑ivf2024 strain, the pepper gray mold disease index of the treatment group dropped to 61.48, compared with the disease index of the control group of 95.56, and the control efficiency was 35.66%. The blade spore wax scale fungus can be used to control pepper gray mold caused by Botrytis cinerea. This strain plays a certain role in promoting the development of new biological pesticides to solve the harm of gray mold in pepper production.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to a ceroplastid strain for preventing and controlling pepper gray mold. Background Art

[0002] Botrytis cinerea is caused by a fungus called Botrytis cinerea ( Botrytis gray ) is a low-temperature airborne disease caused by infection with Botrytis cinerea, which can infect flowers, fruits, leaves, and stems. Botrytis cinerea ) belongs to the phylum Ascomycota, class Hammerglossum, orders Mollicutes, family Sclerotinia, genus Sclerotinia ( Botryotinia ), is recognized as one of the world's major pathogenic fungi. Under suitable environmental conditions, one of the early symptoms of Botrytis cinerea infection is the formation of water-soaked spots on the infected parts of the plant, which gradually evolve into brown spots and local necrosis, and then cause leaf blight, blossom blight and post-harvest fruit rot.

[0003] Gray mold of pepper mainly harms pepper seedlings. In the early stage of the disease, the tip of the cotyledon turns yellow, and then spreads to the stem; gradually brown spots are formed. Under low temperature and high humidity conditions, gray mold will be produced. Gray mold of pepper is one of the main diseases of pepper cultivation in my country. It has the characteristics of early onset, fast spread, and large area. It can cause pepper production to reduce by 30%-50%, and even a total crop failure in severe cases.

[0004] Gray mold is a common necrotrophic disease. The pathogen usually exists in the soil or on diseased plant residues in the form of sclerotia, hyphae, spores, etc. The early infection stage may inhibit host cell death, thereby achieving successful colonization. Once the pathogen enters the host tissue through direct penetration or wounds, it will secrete lytic enzymes (such as cutinases, lipases, and pectin lyases) to destroy the plant cell wall for further infection, and then produce phytotoxins, organic acids, and reactive oxygen species (ROS), triggering oxidative bursts and programmed cell death (PCD), leading to host cell death.

[0005] At present, chemical pesticides are mainly used to control gray mold in production. However, due to the diversity of hosts, short life cycle, genetic variability, and easy resistance to a variety of chemical agents of gray mold pathogens, as well as the serious pesticide residue problems caused by chemical control, it is urgent to implement green biological control. Biological control is one of the important means to control gray mold disease. It can reduce agricultural production costs, reduce the selectivity of gray mold pathogens for pesticides, and reduce the amount of pesticide residues in the environment. It has the characteristics of being green, environmentally friendly, safe, low in drug resistance, renewable and sustainable, and plays an increasingly important role in modern green agriculture and sustainable agricultural production. Summary of the invention

[0006] The invention aims to provide a strain of Lecanopsis serrata for preventing and controlling pepper gray mold.

[0007] A strain of Lecanopsis scaber Lecanicillium psalliotae ) strain, with the deposit number of CGMCC NO.41214, named Lps-ivf2024.

[0008] The cecropinia scaber Lecanicillium psalliotae The ITS sequence (fungal internal transcribed spacer) of the fungus is shown in the sequence listing as SEQ ID NO: 1.

[0009] The cecropinia scaber Lecanicillium psalliotae ) in the prevention and control of pepper gray mold.

[0010] A pesticide for preventing and treating pepper gray mold, comprising the ceroplast fungus ( Lecanicillium psalms ).

[0011] A fermentation product, comprising the Lecanicillium psalliotae ) obtained by fermentation.

[0012] A microbial fungicide for preventing and treating pepper gray mold, the fungicide containing the scalycococcus spores fungus ( Lecanicillium psalliotae ) and / or their fermentation products.

[0013] Preferably, the microbial fungicide is used by spraying.

[0014] The classification name of the scaly spore lecanthus fungus Lps-ivf2024 of the present invention is: Lecanicillium psalms ; Deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on May 7, 2024, and with proof of survival. Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, the culture deposit number is CGMCC NO. 41214.

[0015] The beneficial effects of the present invention are as follows: the present invention discovered a strain of Lecanops scalycoides, which, in an experiment on preventing and controlling pepper gray mold, showed a greenhouse potted control effect on Botrytis cinerea. When the strain was treated with the fermentation liquid of the Lps-ivf2024 strain, the pepper gray mold disease index of the treatment group was reduced to 61.48, compared with the disease index of the control group of 95.56, and the control efficiency was 35.66%. The Lecanops scalycoides can be used for preventing and controlling pepper gray mold caused by Botrytis cinerea. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For Lecanococcus scaber Lecanicillium psalliotae ) Phylogenetic tree of strain Lps-ivf2024 based on ITS sequence.

[0017] Figure 2 For Lecanococcus scaber Lecanicillium psalliotae ) Colony morphology of strain Lps-ivf2024; A is the morphology of sporangium and spores, and B is the colony morphology of Lecanopsis spp.

[0018] Figure 3 For Lecanococcus scaber Lecanicillium psalliotae ) strain Lps-ivf2024 and pepper gray mold pathogen strain; A is pepper gray mold pathogen strain (CK), B on the left is the blade spore wax scale ( Lecanicillium psalms ) strain, and the one on the right is the pepper gray mold pathogen strain. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] Example 1 Strain screening and identification

[0021] (1) Strain isolation and purification

[0022] This example is isolated from the eggs of loofah root-knot nematodes in Shandong Province, and the strain is purified by single hypha separation method, and the specific method is as follows:

[0023] First, wash the roots of the diseased loofah plants with sterile water, pick the egg masses from the roots, disinfect them with 1% sodium hypochlorite for 2 min, and rinse them repeatedly with sterile water until there is no irritating odor. Use a sterile blade to crush the egg masses, transfer them to a PDA plate, and culture them at 28°C. After colonies grow on the plate, use a sterile inoculation needle to pick the hyphae at the edge of the colony and transfer them to a new PDA plate. After purification by single hyphae separation method, transfer them to a PDA slant and culture them at 28°C.

[0024] (2) Strain identification

[0025] The strain was transferred to a PDA plate and cultured in a dark incubator at 28°C, and the colony color, hyphae morphology and other characteristics were recorded and observed. Using the slide method, a sterilized cover glass was inserted obliquely into the middle of the PDA plate and cultured at 28°C. When a small amount of hyphae grew onto the cover glass, the conidia, conidiophores and other characteristics of the strain were observed and recorded under a microscope and measured.

[0026] The genomic DNA of the strain was extracted, separated and purified using the CTAB (Hexadecyl trimethyl ammonium Bromide) method, and then the universal primers ITS1 and ITS4 in the internally transcribed spacer of the fungus were selected for amplification and sequencing. The sequencing results were compared in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the sequences with high credibility were downloaded to construct a phylogenetic tree for strain identification.

[0027] The primer sequence:

[0028] F: 5'-TCCGTAGGTGAACCTGCGG-3' (ITS1);

[0029] R: 5'-TCCTCCGCTTATTGATATGC-3' (ITS4);

[0030] The amplified ITS sequence is shown in the sequence listing SEQ ID NO: 1; the strain phylogenetic tree ( Figure 1 ).

[0031] (3) Screening process

[0032] The identified fungi were compared with the pepper gray mold pathogen ( Botrytis cinerea ) were cultured in confrontation and the strain Lps-ivf2024 of Lecanus scaber was found ( Figure 2 ) has an inhibitory effect on it. The pathogen and the strain to be tested were cultured in bacterial cakes, with a distance of 6 cm between the bacterial cakes and cultured at 25°C. The pathogen was inoculated alone as a control group. When the pathogen was observed to be full of the plate, it was found that the Lps-ivf2024 strain could form an obvious inhibition zone ( Figure 3 ).

[0033] Lecanus scaber Lecanicillium psalliotae) has a round, convex colony with white cotton-like appearance. The back of the colony is pink or lavender red. The pigment produced by the strain often diffuses into the agar and has no pungent smell. The phialode cells are attached to the creeping conidiophores formed by the hyphae of the strain, which are solitary or in whorls of 3-4, sometimes up to 6. The conidiophores are slender, with a swollen base and gradually tapering outward. Conidia are usually solitary or clustered vertically to the top. Large conidia are usually sickle-shaped with sharp ends, and can also form oval or elliptical microspores. The sporangium is 25.46 μm (13.67-32.05 μm) in length; the sickle-shaped macroconidia are 5.65 μm (4.15-7.53 μm) × 2.23 μm (1.37-3.74 μm); the oval or elliptical microspores are 2.86 μm (2.7-3.7 μm) × 1.17 μm (1-1.5 µm).

[0034] Example 2 Potted control test of pepper gray mold with fermentation liquid of Lecanopsis spp. Lps-ivf2024

[0035] Rinse repeatedly with distilled water Botrytis cinerea Plate, double-layer cotton gauze filter to collect spores, dilute the spore solution to 1×10 6 After the spores / mL were 2% sucrose and 0.3% potassium dihydrogen phosphate were added and mixed thoroughly to prepare a suspension of Botrytis cinerea spores for inoculating pepper plants. After the Lps-ivf2024 strain was fermented in PDB medium at 200 r and 28°C for 14 days, the mycelium in the fermentation broth was removed by centrifugation at 10000 r and 4°C for 10 min. After centrifugation, the supernatant was filtered with a 0.22 μm sterile filter membrane to obtain the fermentation filtrate.

[0036] The pepper gray mold inoculation method is foliar spraying. Botrytis cinerea The spore liquid was evenly sprayed on the pepper leaves and dried, and then the fermentation filtrate of Lps-ivf2024 strain was evenly sprayed on the pepper leaves and dried. Three different treatments were set up in the experiment: 1) Botrytis cinerea After foliar spraying, the inoculation was followed by treatment with Lps-ivf2024f fermentation filtrate; 2) only inoculation Botrytis cinerea (disease control); 3) water treatment (negative control). Ten seedlings were treated for each treatment, and the results were repeated three times. Each treatment was kept moisturized for 1 day under dark conditions at 23°C and 90% relative humidity, and then transferred to normal light conditions for 4 days to investigate the disease index. During this period, water was applied appropriately to maintain high soil humidity.

[0037] Grading of severity of pepper gray mold at seedling stage: 0: no symptoms; 1: 3 lesions on a single leaf; 3: 4-6 lesions on a single leaf; 5: 7-10 lesions on a single leaf; 7: 11-20 lesions on a single leaf, some of which are densely packed; 9: 1 / 4 of the area of ​​​​the single leaf is densely packed. After the positive control is investigated, the disease index and control effect are calculated according to the following formula.

[0038] Disease index = ∑[(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100

[0039] Prevention and treatment effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0040] Test results:

[0041] The experiment showed that the biocontrol effect of Lps-ivf2024 fermentation liquid on pepper gray mold was clarified through potted plant experiments. The results showed: Botrytis cinerea The disease index of the group treated with Lps-ivf2024 fermentation liquid after foliar spraying was 61.48. Botrytis cinerea The susceptible control group had a more severe disease, with more water-soaked spots and mold on the leaves, and stems breaking. The disease index was 95.56. The peppers in the control group treated with only water grew healthily, and the plants showed no symptoms. Botrytis cinerea The average protection rate of the plants treated with the fermentation liquid of Lps-ivf2024 strain was 35.66%, and the disease symptoms of the plants were relatively mild.

[0042] Table 1 Survey on disease index and control effect of different treatments

[0043]

[0044] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Lecanus scaber Lecanicillium psalliotae ) strain, whose deposit number is CGMCC NO.41214, named Lps-ivf2024; the scaly spore lecanthus ( Lecanicillium psalliotae ) Prevent and control pepper gray mold.

2. A pesticide for preventing and treating pepper gray mold, characterized in that: Containing the lecanthus spores fungus according to claim 1 ( Lecanicillium psalliotae ).

3. A microbial fungicide for preventing and controlling pepper gray mold, characterized in that: The fungicide contains the Lecanococcus scaber as claimed in claim 1 ( Lecanicillium psalliotae ).

4. The microbial fungicide for preventing and controlling pepper gray mold according to claim 3, characterized in that: The microbial bactericide is used by spraying.

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