A biocontrol marine-derived Penicillium and its application

Abstract

The invention discloses a biocontrol marine-source Penicillium and an application thereof, and relates to the field of microbial technology. The biocontrol marine-source Penicillium is named Penicillium sp.DSF059, and is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20232310. The invention discloses that the biocontrol marine-source Penicillium has a control effect on pathogens of rubber wilt, coconut gray spot, wheat fusarium, cucumber phytophthora root rot, avocado phytophthora root rot, rubber anthracnose, mango anthracnose, sisal zebra pattern, rubber red root, rubber brown root, coffee anthracnose, stylo anthracnose, banana anthracnose, and other tropical crop pathogens. The invention provides a new direction for the control of tropical crop pathogens, and has a good development and application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a biocontrol marine-derived Penicillium and an application thereof. Background Art

[0002] The world's tropical crop industry is mainly concentrated in Southeast Asia and South Asia, the Amazon River Basin in South America, the Congo River Basin in Africa and the Gulf of Guinea. Among them, Southeast Asia is the world's largest producer of tropical cash crops. For example, Thailand is the world's largest producer and exporter of natural rubber, and its output once accounted for 40% of the world's total rubber production at its peak; Indonesia is the world's largest producer of oil palm and coconut; the Philippines is the world's largest exporter of coconut. In recent years, the world's tropical agriculture has developed rapidly, and tropical agriculture has become an important economic source and pillar industry for major tropical countries in the world. 95% of the world's tropical crops are produced in developing countries, and 80% of consumption is mainly concentrated in developed countries. It can be said that the world has a huge demand for tropical crop products, which will strongly promote the rapid development of the world's tropical crop industry. The world's major tropical crops include cash crops such as natural rubber, cassava, sugarcane and oil palm, tropical fruits such as bananas, pineapples, mangoes and coconuts, beverage crops such as coffee and cocoa, and tropical medicinal plants represented by betel nuts.

[0003] Take mango as an example. Mango is widely grown in more than 70 countries around the world. Colletotrichum gloeosporioides ) is one of the fungal diseases that causes a gradual decline in fruit quality. The pathogen can infect plants during growth and development, and can also attack harvested mangoes. Currently, some chemicals such as benomyl, carbendazim, mancozeb, and prochloraz are available for the control of anthracnose. However, long-term use can lead to environmental pollution and chemical residues in fruits. In addition, C. gloeosporioides may develop resistance to many fungicidal ingredients. Therefore, it is necessary to explore effective and safe fungicides as an alternative strategy to traditional fungicides.

[0004] As a new type of green pesticide, biopesticides can not only kill insects and bacteria, but also regulate plant growth and induce plant resistance to some pathogens. They are easily degraded and have little impact on human and animal safety and the environment. Compared with other biopesticides, microbial-derived biopesticides have many advantages, such as simple fermentation process, low production cost, short production cycle, multiple development and utilization channels, low resistance, and easy genetic improvement, making them a research hotspot in the field of biopesticides. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention proposes a biocontrol marine-derived Penicillium and its application to solve the problems raised by the above-mentioned background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A biocontrol marine-derived Penicillium for controlling tropical crop pathogens, wherein the tropical crop pathogens are rubber wilt pathogens, coconut gray spot pathogens, wheat fusarium scab pathogens, cucumber phytophthora root rot pathogens, avocado phytophthora root rot pathogens, rubber anthracnose pathogens, mango anthracnose pathogens, sisal zebra stripe pathogens, rubber red root disease pathogens, rubber brown root disease pathogens, coffee anthracnose pathogens, stylo anthracnose pathogens, and banana anthracnose pathogens;

[0008] The biocontrol marine-derived Penicillium is named Penicillium sp. DSF059, and is deposited in the China Center for Type Culture Collection (CCTCC). The deposit date is November 23, 2023; the deposit number is CCTCC NO: M 20232310.

[0009] Preferably, the rubber wilt pathogen is Fusarium oxysporum ( Fusarium oxysporum ), the pathogen of coconut gray spot disease is Polychaeta pseudodiscus ( Pestalotiopsis palmarum ), the pathogen of wheat fusarium wilt is Fusarium graminearum ( Fusarium gramineae ), the pathogen of cucumber Phytophthora root rot is Phytophthora cucumeris ( Phytophthora melon ), the pathogen of avocado Phytophthora root rot is Phytophthora camphora ( Phytophthora cinnamomi ), the pathogen of rubber anthracnose is Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides strain RC178), the pathogen of mango anthracnose is Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides strai n 171-1), the pathogen of sisal zebra stripe disease is Phytophthora nicotianae ( Phytophthora nicothianae ), the pathogen of rubber red root disease is Ganoderma lucidum ( Ganoderma pseudo-iron ), the pathogen of rubber brown root disease is harmful wood phyllotoxin ( Noxious weevil ), the pathogen of coffee anthracnose is Colletotrichum coffeae ( Colletotrichum coffeanum ), the pathogen of stylosus anthracnose is Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides strain CH008), banana anthracnose pathogen is Colletotrichum banana ( Colletotrichum musae ).

[0010] Preferably, the marine-derived biocontrol Penicillium can inhibit the germination of pathogen spores by producing siderophore, glucanase, tyrosinase, cellulase, chitosanase and chitinase, and hydrolyze the cell wall of pathogens to promote the growth of tropical crops.

[0011] Preferably, the tropical crop is one of rubber, coconut, wheat, cucumber, avocado, mango, sisal, coffee, stylo and banana.

[0012] Preferably, the method for controlling mango anthracnose pathogens is: treating detached leaves or harvested mangoes with a culture solution of the biocontrol marine-derived Penicillium.

[0013] Preferably, the method for preparing the biocontrol marine-derived Penicillium culture solution comprises the following steps:

[0014] (1) Preparation of spore suspension: The marine-derived Penicillium biocontrol strain was inoculated into PDA solid culture medium and cultured at 28°C for 5 days to activate the strain. A sterile 0.05% Tween 80 solution was added to elute the spores to prepare a spore suspension.

[0015] (2) Fermentation treatment: Take the spore suspension and inoculate it into PDB medium at a volume ratio of 3%, and ferment it at 28°C and 180 r / min for 7 days to obtain a fermentation liquid;

[0016] (3) Filtration: The fermentation liquid obtained in step (2) is filtered and sterilized, and the filtrate is collected to obtain the biocontrol marine-derived Penicillium culture liquid.

[0017] Preferably, the PDA solid culture medium in step (1) is composed of the following ingredients per liter: 200 g potato, 20 g glucose, 15-30 g agar, 400 mL aged sea water, and the balance deionized water.

[0018] Preferably, the concentration of the spore suspension in step (1) is 1.0×10 8 cfu / mL.

[0019] Preferably, the PDB culture medium in step (2) is composed of the following ingredients per liter: 200 g potato, 20 g glucose, 400 mL aged sea water, and the balance deionized water.

[0020] Preferably, the PDA solid culture medium in step (1) can also be replaced by the following culture medium for the cultivation of Penicillium. The reagent components and culture medium information involved are as follows:

[0021] Cha's mother solution: 150 g nitric acid, 25 g potassium chloride, 25 g magnesium sulfate heptahydrate, 0.5 g ferrous sulfate heptahydrate, and dilute to 500 mL with deionized water.

[0022] Trace element mother solution: anhydrous copper sulfate: 0.25 g, zinc sulfate heptahydrate: 0.5 g, and deionized water to make up to 500 mL.

[0023] Martin medium: 10g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 3.3mL 0.1% Bengal red solution, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane in a clean bench.

[0024] PDA medium: 200g potato, 20g glucose, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0025] MEA medium: 20g malt extract, 1g peptone, 20g glucose, 10mL trace element mother solution, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0026] CYA medium: 1g potassium hydrogen phosphate, 5g yeast extract, 30g sucrose, 10mL Cha's mother liquor, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane in a clean bench.

[0027] OA culture medium: 30g oats, 10mL trace element stock solution, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. Sterilize at 121℃ for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane in a clean bench.

[0028] YES medium: 20g yeast extract, 150g sucrose, 0.5g magnesium sulfate heptahydrate, 10mL trace element mother solution, 15-20g agar, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0029] V8 medium: 100 mL of V8 juice filtrate, 1 g of calcium carbonate, 15-20 g of agar, distilled water to 1 L, pH natural. Sterilize at 121°C for 20 min. Cool to below 65°C, and add 1 mL each of 100 mg / mL streptomycin sulfate, 100 mg / mL chloramphenicol, and 100 mg / mL ampicillin sterilized by ultrafiltration with a 0.22 um filter membrane in a clean bench.

[0030] PDB medium: 200g potato, 20g glucose, 400mL Chenhai water, distilled water to 1L, pH natural. Sterilize at 121℃ for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0031] CYB medium: 1g potassium hydrogen phosphate, 5g yeast extract, 30g sucrose, 10mL Cha's mother liquor, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane in a clean bench.

[0032] G25NB medium: 10 mL of Cha's mother liquor, 1 g of potassium dihydrogen phosphate, 5 g of yeast extract, 30 g of sucrose, 250 g of glycerol, 400 mL of Chenhai water, distilled water to 1 L, pH natural. Sterilize at 121°C for 20 min. Cool to below 65°C, and add 1 mL each of 100 mg / mL streptomycin sulfate, 100 mg / mL chloramphenicol, and 100 mg / mL ampicillin sterilized by ultrafiltration with a 0.22 um filter membrane in a clean bench.

[0033] MEB medium: 20g malt extract, 1g peptone, 20g glucose, 10mL trace element mother solution, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0034] OB medium: 30g oats, 10mL trace element mother solution, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0035] Martin liquid medium: 10g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 3.3mL 0.1% Bengal rose solution, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with 0.22um filter membrane in a clean bench.

[0036] CB culture medium: 30g sucrose, 3g sodium nitrate, 1g potassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 0.01g ferrous sulfate heptahydrate, 400mL Chenhai water, distilled water to 1L, pH natural. 121℃, sterilize for 20min. Cool to below 65℃, add 1mL each of 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane in a clean bench.

[0037] Cellulase detection medium: (NH4)2SO42.0g, MgSO4·7H2O 0.5g, K2HPO41.0g, NaCl 0.5g, sodium carboxymethyl cellulose 2.0g, agar 22.0g, ddH2O 600mL, seawater 400mL, sterilized at 121℃ for 20min, natural pH.

[0038] Amylase detection medium: starch 20.0 g, KCl 0.5 g, NaNO3 2.0 g, K3PO4 1.0 g, MgSO4·7H2O 0.5 g, agar 18.0 g, ddH2O 600 mL, seawater 400 mL, sterilized at 121 ° C for 20 min, natural pH.

[0039] Chitosan detection medium: chitosan 10.0 g, (NH4)2SO4 2.5 g, MgSO4·7H2O 0.25 g, K2HPO4 0.02 g, NaCl 4.0 g, CaCO3 6.0 g, agar 20.0 g, pH 6.0, ddH2O 600 mL, seawater 400 mL, sterilized at 121 °C for 20 min, natural pH.

[0040] Chitinase detection medium: soluble starch 20g, 2% colloidal chitin 100g, K2HPO41g, KNO31g, MgSO40.5g, NaCl 0.5g, FeSO40.01g, agar 20g, ddH2O 600mL, seawater 400mL, sterilization at 121℃ for 20min, natural pH.

[0041] Protease detection: Casein assay culture (MM6051) from Beijing Coolbo Technology Co., Ltd.

[0042] Glucanase assay: β-1,3-Glucan culture (MM6220) from Beijing Coolaibo Technology Co., Ltd.

[0043] Siderophore detection: modified CAS solid medium (PM0821) from Beijing Coolbo Technology Co., Ltd.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The marine environment is different from the terrestrial environment. As the world's largest ecosystem, the marine environment contains a variety of microorganisms and active substances. Due to different living environment factors, the biological resources therein are very different from terrestrial organisms in terms of growth and metabolism, and have become a huge resource library waiting to be developed. In particular, with the development of marine biotechnology in recent years and the application of modern biotechnology, the development and utilization of marine microbial resources have been promoted, which provides a rich candidate resource for the development of new biopesticide precursors. The present invention discloses the use of deep-sea fungi to control tropical crop pathogens, which can not only inhibit the growth of pathogens, but also regulate the growth of plants and induce plant resistance to some pathogens. Moreover, it is easily degraded and has little impact on human and animal safety and the environment.

[0046] (2) The present invention discloses a marine-derived biocontrol Penicillium which has control effects on pathogens of tropical crops such as rubber wilt, coconut gray spot, wheat fusarium, cucumber phytophthora root rot, avocado phytophthora root rot, rubber anthracnose, mango anthracnose, sisal zebra stripe, rubber red root, rubber brown root, coffee anthracnose, stylo anthracnose, and banana anthracnose. The present invention provides a new direction for the control of tropical crop pathogens and has a good development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Penicillium sp. . Colony morphology of DSF059; A is the colony morphology (from left to right, MEA, CYA, OA and YES on the front, MEA, CYA, OA and YES on the back); BC: conidiophores and conidia under light microscope; DE: conidiophores under electron microscope; F: conidia under electron microscope;

[0048] Figure 2 Penicillium sp. . Multi-gene joint phylogenetic tree of DSF059;

[0049] Figure 3 Penicillium sp. . Biological characteristics of DSF059; A: temperature; B: pH; C: light; D: spore lethal temperature;

[0050] Figure 4 Penicillium sp. . Enzyme activity characteristics of DSF059;

[0051] Figure 5 The results of the plate confrontation test of deep-sea Penicillium sp. DSF059 against various plant pathogens; F.oxysporum : Banana wilt Fusarium oxysporum; N. halved : New dark-colored columnar spores of pitaya canker; P. palmarum: Coconut gray spot disease pseudodiscus multichaete; F. graminearum : Wheat head blight Fusarium graminearum; L. Theobroma cassava : Rubber scorch rot, Diplodia cocoides; P. melonis : Cucumber Phytophthora root rot Phytophthora cucumeris; P. cinnamon : Avocado Phytophthora root rot Phytophthora camphora; C. gloeosporioides strain RC178: Rubber anthracnose Colletotrichum gloeosporioides; C. gloeosporioides strain 171-1: Mango Anthracnose Colletotrichum gloeosporioides; P. nicothianae : Sisal zebra stripe disease, Phytophthora tobacco; G. pseudoferreum : Rubber red root disease Ganoderma lucidum; P. noxious : Rubber brown root disease harmful wood phylloporin; C.Coffeanum :Coffee Anthracnose Colletotrichum coffeae; C. gloeosporioides strain CH008: Colletotrichum gloeosporioides; C. musae : Banana Anthracnose Colletotrichum banana;

[0052] Figure 6 Penicillium sp. . Schematic diagram of the antibacterial activity of DSF059 in different culture media against mango anthracnose pathogens;

[0053] Figure 7 Penicillium sp. . The bar graph of the antibacterial activity of DSF059 on mango anthracnose pathogens in different culture media;

[0054] Figure 8 Penicillium sp. . The control effect of DSF059 PDB fermentation liquid on mango detached leaves; A: sterile water (front); B: Penicillium sp . DSF059 PDB fermentation broth (front); C: 2000-fold dilution of pyraclostrobin (front); D: sterile water (back); E: Penicillium sp . DSF059 PDB fermentation broth (back); F: 2000-fold dilution of pyraclostrobin (back);

[0055] Figure 9 Penicillium sp. . The protective effect of DSF059 PDB fermentation liquid on postharvest mango fruit. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0057] A biocontrol marine origin Penicillium, named Penicillium sp. DSF059, is deposited in the China Center for Type Culture Collection (CCTCC). The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is November 23, 2023; the deposit number is CCTCC NO: M 20232310.

[0058] Example 1 Isolation and purification of strains

[0059] 1.1 Soil samples were collected from marine sediments at 112°30′00″E, 17°55′00″N, 2595m underwater in the South China Sea. Pushcore was used for sampling, and surface sediments and deep sediments were mixed together for processing. The strains were isolated using Martin medium, inoculated with soil samples using the stamp method, and purified using PDA medium.

[0060] 1.2 Specific operation method: Place the deep-sea sediment sample in the clean bench for air drying. Grind it into powder after it is completely dry. Then press the autoclaved foam plug on the sediment powder, and then press it in a clockwise direction on the surface of the separation medium. Press it three times in a row to produce a continuous dilution effect. After inoculation, place the plate in a dark culture at 28°C for 1-3 weeks. Observe the growth of the strain on each culture medium every day. When the colony grows, pick a small piece of colony and transfer it to potato dextrose agar (PDA) medium for purification and culture. When the strain grows to a certain size, single spore separation and culture are carried out to obtain purified strains. The purified strains are stored at -80°C using 20% ​​glycerol and numbered according to the strain isolation situation.

[0061] Example 2 Morphological and molecular biological identification of marine-derived Penicillium sp. DSF059

[0062] 2.1 Identification of colony morphology: The purified strain DSF059 was inoculated into MEA, CYA, OA and YES culture media respectively using the three-point inoculation method. After culturing in the dark at 28°C for 7 days, the growth status of the strain, the color of aerial hyphae, the color of intrabasal hyphae, the presence of soluble pigments and their color were observed and recorded.

[0063] Penicillium sp . Colony morphology of DSF059: Figure 1As shown, DSF059 was cultured in MEA medium at 28℃ for 7 days, and the colony diameter was 28-31mm, which was relatively thin; the texture was velvety and powdery; the conidial structure was produced in large quantities, gray; the mycelium was light gray; there was no exudate; there was no soluble pigment; the reverse side was yellow. After being cultured in CYA medium at 28℃ for 7 days, the colony diameter was 35-43mm, the thickness was moderate, the middle was concave, and there were a few radioactive grooves; the texture was velvety; the conidial structure was sparse, white; there was no exudate; there was yellow-green soluble pigment; the reverse side was yellow-green. After being cultured in OA medium at 28℃ for 7 days, the colony diameter was 36-42mm, the thickness was moderate, there were trace radioactive grooves; the texture was velvety and powdery; the conidial structure was produced in large quantities, gray; the mycelium was white; there was no exudate; there was yellow-green soluble pigment; the reverse side was yellow-green. After culturing in YES medium at 28℃ for 7 days, the colony diameter is 38-40mm, relatively thick, with a large number of radial grooves and an umbilical protrusion in the center; the texture is velvety and flocculent; the conidial structure is numerous and gray-green; the mycelium is white; there is no exudate; there is no soluble pigment; the reverse side is golden yellow.

[0064] 2.2 Microscopic morphological identification: After the strain was cultured at 28°C for 7 days using the slide wet chamber method, conidiophores and conidia were observed using a Nikon scientific microscope (Ni-U, NIKON, Tokyo, Japan) and a thermal field emission scanning electron microscope (SEM; JSM-7610FPlus, JEOL, Tokyo, Japan).

[0065] The operating steps of the slide wet chamber method are as follows: ① Pour PDA thin layer: Under sterile conditions, invert a PDA thin layer with a thickness of 2-3mm in the culture dish. ② Make a culture chamber: Make a culture chamber in another new culture dish. The bottom of the chamber is covered with a layer of round filter paper slightly smaller than the flat dish. Put a U-shaped glass rod on the round filter paper, and then put a clean slide on the glass rod. Cover the culture dish with a lid, wrap it with newspaper and sterilize it. ③ Inoculation: Use a scalpel to cut the PDA thin layer into agar blocks with a side length of about 1.0cm, and transfer them to both ends of the slide in the above chamber. Use a sterile inoculation loop to pick a small amount of spores or hyphae and inoculate them on the edge of the agar block, and use sterile tweezers to cover the cover glass on the agar block. ④ Cultivation: Use a pipette to transfer 2-3 mL of sterilized 20% glycerol onto the filter paper of the plate (to maintain the humidity in the plate), cover with culture medium and plate lid, seal with sealing film, and place in a 28°C incubator for continuous cultivation for 5-10 days.

[0066] Before observing the morphology of the strain with a scanning electron microscope, the coverslips on which the morphology of Penicillium has been observed under an optical microscope should be processed. First, soak the coverslips in 2.5% glutaraldehyde and place them in a refrigerator at 4°C for overnight fixation. After rinsing the coverslips 3-5 times with phosphate buffer (pH7.4), dehydrate them with a gradient of 30%, 50%, 70%, 80%, 90% and 100% ethanol, with each gradient treatment lasting 20 minutes. Afterwards, wrap the coverslips in tin foil and dry them in a vacuum freeze dryer for 10 minutes. Fix the dried material to an aluminum pin with conductive carbon tape, sputter gold, and observe the morphology of the strains and spores with a thermal field emission scanning electron microscope.

[0067] Penicillium sp . The microscopic morphological characteristics of DSF059 are: conidiophores are generated in the matrix, with conidiophore stems of 70-140×1.7-2.8μm and smooth walls; broom branches are arranged in a single whorl; phialides are 4-8 in each whorl, 5.5–8.2×1.4–2.4μm, and bottle-shaped; conidia are nearly round, 1.2-1.5×1.3-1.6μm, and smooth walls. No sclerotia were observed.

[0068] 2.3 Molecular biological identification: Pick the hyphae on the MEA plate, use the genomic DNA extraction kit to extract DNA, and use it as a template to amplify the gene interval sequence of the housekeeping gene 5.8S rDNA and 28S rDNA (The nuclearribosomal internal transcribed spacer region, ITS), β-tubulin gene (BenA), calmodulin gene (CaM), and RNA polymerase II second largest subunit (RPB2) gene fragment.

[0069] The primer sequences for ITS PCR amplification were ITS1: 5′-tccgtaggtgaacctgcgg-3′ (SEQ ID NO.5) and lTS4: 5′-tcctccgcttattgatatgc-3′ (SEQ ID NO.6); the primer sequences for BenA PCR amplification were Bt2a: 5′-ggtaaccaaatcggtgctgctttc-3′ (SEQ ID NO.7) and Bt2b: ​​5′-accctcagtgtagtgacccttggc-3′ (SEQ ID NO.8); the primer sequences for CaM PCR amplification were CF1: 5′-gccgactctttgacygargar-3′ (SEQ ID NO.9) and CF4: 5′-tttytgcatcatragytggac-3′ (SEQ ID NO.10); the primer sequence for RPB2 PCR amplification was 5F: 5′-gaygaymgwgatcayttygg-3′ (SEQ ID NO.11) and 7CR: 5′-cccatrgcttgyttrcccat-3′ (SEQ ID NO.12).

[0070] PCR reaction system: 25uL 2×Taq Master Mix, 19uL ddH2O, 2uL template DNA, 2uL each primer, total volume 50uL.

[0071] PCR amplification conditions for ITS, Ben A and CaM genes were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 60 s, 35 cycles; post-extension at 72°C for 7 min.

[0072] PCR amplification conditions for RPB2 gene were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 48°C for 45 s, and extension at 72°C for 60 s, for 5 cycles; denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 60 s, for 5 cycles; denaturation at 94°C for 45 s, annealing at 52°C for 45 s, and extension at 72°C for 60 s, for 30 cycles; post-extension at 72°C for 7 min.

[0073] The marine source Penicillium sp . The ITS gene sequence of DSF059 is the nucleotide sequence shown in SEQ ID NO.1, the BenA gene sequence is the nucleotide sequence shown in SEQ ID NO.2, the CaM gene sequence is the nucleotide sequence shown in SEQ ID NO.3, and the RPB2 gene sequence is the nucleotide sequence shown in SEQ ID NO.4.

[0074] The sequencing results were analyzed by Blast analysis on NCBI and a multi-gene joint phylogenetic tree was constructed ( Figure 2 ), combined with morphological analysis, the strain was identified as a potential new species of Penicillium, and the strain was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232310.

[0075] Example 3 Penicillium sp. . Biological properties of DSF059

[0076] 3.1 Effect of temperature on mycelial growth and conidia production of Penicillium sp. DSF059: The strain DSF059 was inoculated onto PDA plates by the three-point inoculation method and placed in a constant temperature incubator at 4℃, 15℃, 22℃, 28℃, 30℃, 33℃ and 37℃ for dark inversion culture for 7 days. The colony diameter was measured by the cross method, and three bacterial cakes were taken from the center to the edge of the colony at equal distances with a cork puncher and placed in a 2mL centrifuge tube containing 1mL of 0.05% Tween 80 solution. The plates were oscillated on a vortex oscillator for 1 min, diluted 10 times or 100 times, and the spores were counted using a hemocytometer.

[0077] 3.2 Effect of pH on mycelial growth and conidia production of Penicillium sp. DSF059: The pH of the PDA medium was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 using 3 mol / L HCl and NaOH, and plates were prepared respectively. The strain DSF059 was inoculated in the center of the plate by the three-point inoculation method, and the colony diameter and spore amount were measured after inverted culture at 28°C in the dark for 7 days.

[0078] 3.3 Effect of photoperiod on mycelial growth and conidia production of Penicillium sp. DSF059: The strain DSF059 was inoculated onto PDA plates by the three-point inoculation method and placed in an incubator with 0, 8, 12, 16 and 24 h of light for 7 days and then inverted and cultured. The colony diameter and spore amount were measured.

[0079] 3.4 Lethal temperature of Penicillium sp. DSF059: Strain DSF059 was inoculated into a triangular flask containing PDA culture and cultured in the dark at 28°C for 5 days. Spores were then eluted with 0.05% Tween 80 solution, the spore count was counted using a hemocytometer, and the test concentration of the spore suspension was adjusted to 2×10 3spores / mL. The spore suspension was dispensed into 2 mL centrifuge tubes, 1 mL in each tube. The centrifuge tubes were placed in a constant temperature water bath at 45°C, 50°C, 55°C, 60°C and 65°C for 10 min respectively. 100 μL of each treatment was spread on a PDA plate. After 48 h, the number of colonies on the plate was counted. The treatment temperature at which there was no colony on the plate was the lethal temperature.

[0080] Conclusion: Penicillium sp. DSF059 can grow between 15℃ and 33℃, and the optimal growth temperature is 28-30℃ ( Figure 3 A); Penicillium sp. DSF059 can grow between pH5-pH12 and has very good acid-base adaptability, with the optimal growth pH being 11 ( Figure 3 B); It can grow in both dark and light conditions, and the colony and spore production are the largest when the continuous light is 12-16h ( Figure 3 C); DSF059 spores can withstand a certain high temperature, and the spore lethal temperature is 65℃ ( Figure 3 D).

[0081] Example 4 Enzyme activity characteristics of marine Penicillium sp. DSF059

[0082] 4.1 Using the single colony inoculation method, pick a single colony of Penicillium sp. DSF059 and inoculate it into the center of cellulase detection medium, amylase detection medium, chitosan detection medium, chitinase detection medium, protease detection medium, glucanase detection medium and siderophore detection medium. Repeat three times and incubate at 28℃ for 7 days.

[0083] 4.2 Penicillium sp . The biological characteristics of DSF059 are as follows Figure 4 Penicilliumsp . DSF059 can produce siderophore, glucanase, tyrosinase, cellulase, chitosanase and chitinase, thereby inhibiting the germination of pathogenic fungal spores and hydrolyzing the cell walls of pathogenic fungi to achieve the purpose of biocontrol.

[0084] Example 5 Penicillium sp. DSF059 from marine origin inhibits the growth of mango anthracnose mycelium

[0085] 5.1 The plate confrontation method was used to detect the effect of Penicillium sp. DSF059 on the mycelial growth of mango anthracnose pathogen. Mango anthracnose pathogen and Penicillium sp. DSF059 were first inoculated on two PDA culture media, respectively, and cultured at 28℃ in the dark for 5 days, and then the bacterial cakes (φ=5mm) were taken. First, the mango anthracnose pathogen cake was inoculated in the middle of the PDA culture medium, and Penicillium sp. DSF059 was inoculated with 2cm intervals above, below, left and right. . The strain DSF059 was inoculated with the mango anthracnose pathogen alone as a blank control, repeated three times, and the antibacterial effect was observed after constant temperature cultivation at 25°C for 7 days.

[0086] Mycelium growth inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter) × 100.

[0087] 5.2 Results Figure 5 (Upper right, C.gloeosporioides strain 171-1), compared with the control group, the mango anthracnose pathogen in the treatment group was affected by Penicillium sp. . DSF059 significantly inhibited the mycelial growth of mango anthracnose pathogen, with the inhibition rate of mycelial growth being 65.33±0.58%.

[0088] Example 6 Penicillium sp. . Effect of DSF059 on the control of mango anthracnose

[0089] 6.1 Determination of antibacterial activity of different culture media: Penicillium sp . DSF059 was inoculated into a triangular flask containing PDA culture and cultured in the dark at 28°C for 5 days. The spores were then eluted with 0.05% Tween 80 solution, the spore count was counted using a hemocytometer, and the experimental concentration of the spore suspension was adjusted to 1×10 8 Spores / mL. The strain Penicillium sp . The spore suspension of DSF059 was inoculated into CB, CYB, G25NB, MEB, OB, Martin and PDB culture media at 3% (v / v). After culturing at 28°C and 180 r / min for 7 days, the fermentation liquid was sterilized by filtering through a 0.22 μm filter membrane and the filtrate was collected. The fermentation filtrate and PDA culture medium were mixed in a ratio of 1:4 to prepare drug-containing plates. The PDA plates without fermentation filtrate were used as controls. The mango anthracnose pathogen cake (5 mm in diameter) was inoculated in the center of the plates. After the plates were inverted and cultured at 25°C for 7 days, the colony diameter was measured by the cross method to calculate the inhibition rate.

[0090] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter) × 100.

[0091] 6.2 Determination of control effect on detached leaves: Penicillium sp . The spore suspension of DSF059 was inoculated into PDB at 3% (v / v). After culturing at 28°C and 180 r / min for 7 days, the fermentation liquid was sterilized by filtering through a 0.22 μm filter membrane and the filtrate was collected. Disease-free mango leaves were picked, the surface was disinfected with 75% alcohol, the leaf tissue was pierced with a sterile plum needle, and then the above-mentioned Penicillium sp. . Spray the surface of the leaves with DSF059 sterile fermentation liquid, and inoculate the wound with a 0.5 cm diameter mango anthracnose fungus cake after drying; spray the corresponding fermentation liquid for the second time after inoculation, and place it in a moisturizing box at 28°C and 90% humidity after drying. Inoculate 3 points on each leaf, and 3 leaves for each treatment. Use 2000 times diluted 25% pyraclostrobin suspension as the test control, and sterile water as the blank control. The diameter of the lesion was measured by the cross method 7 days after inoculation;

[0092] Control effect (%) = (extension area of ​​lesions in sterile water control group - extension area of ​​lesions in treatment group) / extension area of ​​lesions in sterile water control group × 100.

[0093] 6.3 Determination of the control effect of post-harvest mangoes: In vitro mango inoculation was used, and the selected Tainong No. 1 mangoes were disinfected with 2.0% (W / V) sodium hypochlorite solution and then repeatedly rinsed with sterile water. The disinfected and washed mangoes were randomly divided into 4 groups (4 mangoes in each group), dried and placed in a 150mm sterilized culture dish for 24 hours; then, the first group was soaked in sterile deionized water for 5 minutes and then dried as the control group, the second group was soaked in the above-mentioned PDB fermentation liquid for 5 minutes and then dried as the antagonistic bacteria control group, and the third and fourth groups of mangoes were placed in a concentration of 10 7 The mangoes were immersed in the spore suspension of CFU / mL Colletotrichum gloeosporioides for 5 minutes and dried. The third group was used as the pathogen control group. The fourth group was dried and then immersed in the antagonistic bacteria fermentation liquid for 5 minutes and then dried as the prevention effect treatment group. Finally, the mangoes were placed in a 150mm sterilized culture dish (4 per dish) and cultured under constant temperature and humidity (25℃, relative humidity 60%). The experimental results were observed after 5 days.

[0094] Control effect (%) = (lesion area of ​​the control group inoculated with pathogens - lesion area of ​​treatment) / lesion area of ​​the control group inoculated with pathogens × 100%.

[0095] 6.4 The results of the antibacterial activity test of different culture broths showed that the strain Penicillium sp .The fermentation filtrate obtained by fermenting DSF059 in PDB medium had the strongest antibacterial activity against mango anthracnose pathogens, which was 48.62% (see Figure 6 and Figure 7 ). Penicillium sp. . After treatment with DSF059 PDB fermentation liquid, anthracnose of mango detached leaves and harvested fruits was significantly inhibited, and the control effect on detached leaves was 94.46% ( Figure 8 ), the control effect on post-harvest fruits was 73.67% ( Figure 9 );

[0096] The analysis showed that deep-sea Penicillium sp. . DSF059 has a significant inhibitory effect on mango anthracnose and can be used to develop biocontrol products for the biological control of mango anthracnose. In addition, it was found that Penicillium sp . The PDB fermentation broth of DSF059 had no adverse effects on mango.

[0097] Example 7 Penicillium sp. . Inhibitory effect of DSF059 on other pathogenic fungi

[0098] 7.1 Detection of Penicillium sp by plate standoff method . Inhibitory effect of DSF059 on pathogenic fungi of other tropical crops

[0099] First, the pathogens of banana wilt, dragon fruit canker, coconut gray spot, wheat fusarium, rubber scorch rot, cucumber phytophthora root rot, avocado phytophthora root rot, rubber tree anthracnose, rubber tree red root disease, rubber tree brown root disease, coffee anthracnose, stylo anthracnose and banana anthracnose and Penicillium sp. . DSF059 was inoculated on two PDA mediums, and the pathogen of zebra stripe disease of sisal hemp was inoculated on V8 medium. After culturing for 5 days at 28℃ in the dark, bacterial cakes (φ=5mm) were obtained. First, the pathogen cake to be tested was inoculated in the middle of the PDA medium (the pathogen of zebra stripe disease of sisal hemp was inoculated in V8 medium), and Penicillium sp. . The strain DSF059 was inoculated with pathogens alone as a blank control, repeated three times, and the antibacterial effect was observed after constant temperature cultivation at 25°C for 7 days.

[0100] 7.2 Experimental Results

[0101] See also Figure 5 , Penicillium sp .DSF059 is effective against banana wilt fungus Fusarium oxysporum ( F.oxysporum )、Coconut gray spot disease Pseudomonas aeruginosa( P. palmarum graminearum ( F. graminearum )、Cucumber Phytophthora root rot Cucumber Phytophthora ( P. melonis )、Avocado Phytophthora root rot, Camphor Phytophthora ( P.cinnamomi )、Hevea brasiliensis anthracnose Colletotrichum gloeosporioides RC178( C.gloeosporioides strain RC178), sisal zebra pattern disease of tobacco Phytophthora nicotianae ( P.nicothianae )、rubber tree red root disease rubber ganoderma lucidum ( G. pseudoferreum )、The harmful wood-forming fungus of rubber tree brown root disease ( P.noxius )、Coffee Anthracnose Colletotrichum coffeae ( C.Coffeanum )、Stylosus anthracnose Colletotrichum gloeosporioides CH008( C.gloeosporioides strain CH008) and banana anthracnose Colletotrichum spp. C.musae ) all had significant inhibitory effects, but had no significant inhibitory effects on Pitaya canker ( N. halved ) and Lasiosporium cocophyllum ( L.theobromae ) had no inhibitory effect. Among them, Penicillium sp . DSF059 has the best inhibitory effect on the rubber tree red root disease Ganoderma lucidum, reaching 87%. It also has a good inhibitory effect on the rubber tree anthracnose Colletotrichum gloeosporioides RC178, coffee anthracnose Colletotrichum coffeae, rubber tree brown root disease Poria, banana anthracnose Colletotrichum banana anthracnose, stylosanthes anthracnose Colletotrichum gloeosporioides CH008, coconut gray spot disease Polychaeta pseudodiscus and sisal zebra stripe disease Tobacco Phytophthora, with an inhibition rate of more than 60%.

[0102] Example 8 Control Effects of Different Types of Fungi on Tropical Crop Pathogens

[0103] 8.1 Experimental Materials: Streptomyces griseus ( Streptomyces ): purchased from Shanghai Bohu Biotechnology Co., Ltd.; Trichoderma viride ( Trichoderma green ): purchased from Hubei Xinghengye Technology Co., Ltd.; Penicillium aureum ( Penicillium aurantiogriseum ): purchased from Wuhan Huaerna Biotechnology Co., Ltd.; Penicillium bilairum ( Penicillium bilaiae ): purchased from Guangzhou Zhenwei Microbiology Technology Co., Ltd.; Penicillium quinquefolium ( Penicillium hercules ): purchased from Shanghai Xuanke Biotechnology Co., Ltd.; Paecilomyces lilacinus ( Paecilomyces lilacinus (Thorn.) Samson): purchased from Jinan Xinwangda Biotechnology Co., Ltd.; Penicillium griseofulvum ( Penicillium gray-yellow ): purchased from Shanghai Yushao Biotechnology Co., Ltd.

[0104] 8.2 Experimental methods

[0105] Refer to the above 7.1 plate confrontation method to detect the inhibitory effect on tropical crop pathogenic fungi.

[0106] 8.3 Experimental Results

[0107] As shown in Table 1:

[0108] Table 1 Inhibition rate of different types of molds on tropical crop pathogenic fungi

[0109]

[0110] It can be seen from Table 1 that different types of molds have different inhibitory effects on tropical crop pathogenic fungi, and the best effect is the marine source Penicillium sp. . DSF059, followed by Paecilomyces lilacinus. Different types of Penicillium greatly affect the control effect. It can be seen that the present application has found a marine Penicillium with better control effect than the Penicillium commonly used in the prior art to inhibit tropical crop pathogenic fungi, and the marine resources are abundant. Therefore, it can be considered that the marine Penicillium of the present application can be prepared into an antibacterial agent for preventing and controlling tropical crop pathogenic fungi, which has high economic value and promotion prospects.

[0111] 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. An application of marine-derived Penicillium to control coffee anthrax pathogens, characterized in that: The marine-derived Penicillium biocontrol strain is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20232310; The coffee anthracnose pathogen is Colletotrichum coffeae Colletotrichum coffeanum ; The marine-derived biocontrol Penicillium can inhibit the germination of pathogenic bacteria spores by producing siderophore, glucanase, caseinase, cellulase, chitosanase and chitinase, and hydrolyze the cell walls of pathogenic bacteria to promote the growth of tropical crops. The tropical crop is coffee.

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