CN119214172B - A biocontrol marine-derived Penicillium and its application - Google Patents

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 tropical crop pathogens such as rubber wilt pathogens, coconut gray spot pathogens, wheat fusarium pathogens, cucumber phytophthora root rot pathogens, avocado phytophthora root rot pathogens, rubber anthracnose pathogens, mango anthracnose pathogens, sisal zebra pattern pathogens, rubber red root pathogens, rubber brown root pathogens, coffee anthracnose pathogens, stylo anthracnose pathogens, banana anthracnose pathogens, etc. The invention provides a new direction for the control of tropical crop pathogens, and has a good development and application prospect.

Description

Biocontrol marine source penicillium and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to biocontrol marine penicillium and application thereof.

Background

The world tropical crop industry is mainly concentrated in southeast asia and south asia, amazon river basin in south america, congo river basin in africa, and the fjord coastal region in guinea. Among them, southeast Asia is the world's tropical cash crop's place of maximum yield, such as Thailand is the world's largest natural rubber producing country and export country, where the highest yield was 40% of the world's total rubber production, indonesia is the world's largest oil palm and coconut producing country, and Philippines is the world's largest coconut export country. In recent years, tropical agriculture in the world has been rapidly developed, and tropical agriculture has become an important economic source and the pillar industry in the major tropical countries of the world. Worldwide 95% of tropical crop production is in developing countries, 80% of consumption is mainly concentrated in developed countries. It can be said that there is a great demand for tropical crop products in the world, which will strongly drive the world's tropical crop industry to develop rapidly. The world major tropical crops comprise natural rubber, cash crops such as cassava, sugarcane, oil palm and the like, tropical fruits such as bananas, pineapples, mangoes, coconuts and the like, beverage crops such as coffee, cocoa and the like, and tropical medicinal plants represented by betel nuts.

Mango, for example, is widely planted in more than 70 countries worldwide. Mango anthracnose caused by colletotrichum gloeosporioides (Colletotrichum gloeosporioides) is one of the fungal diseases that lead to gradual degradation of fruit quality. The pathogen can infect plants during growth and development, and can attack harvested mangoes. Currently, several chemicals such as benomyl, carbendazim, mancozeb, and prochloraz are available to control anthracnose. However, prolonged use can lead to environmental pollution and chemical residues in the fruit, and in addition, colletotrichum gloeosporioides can be resistant to many fungicidal components. Therefore, there is a need to explore effective and safe bactericides as alternatives to traditional bactericides.

The biological pesticide is used as a novel green pesticide, can regulate the growth of plants and induce the resistance of the plants to some pathogenic bacteria besides killing insects and bacteria, is easy to degrade, and has small influence on human and animal safety and environment. Compared with other biopesticides, the microbial biopesticide has the advantages of simple fermentation process, low production cost, short production period, multiple development and utilization paths, difficult resistance generation, easy genetic improvement and the like, and becomes a research hotspot in the field of biopesticides.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a biocontrol marine penicillium and application thereof, so as to solve the problems in the prior art.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The application of biocontrol marine penicillium in preventing and controlling tropical crop pathogenic bacteria, wherein the tropical crop pathogenic bacteria are rubber fusarium wilt pathogenic bacteria, coconut gray spot pathogenic bacteria, wheat scab pathogenic bacteria, cucumber phytophthora root rot pathogenic bacteria, pyriform pear phytophthora root rot pathogenic bacteria, rubber anthracnose pathogenic bacteria, mango anthracnose pathogenic bacteria, sisal hemp zebra stripe pathogenic bacteria, rubber red root disease pathogenic bacteria, rubber brown root disease pathogenic bacteria, coffee anthracnose pathogenic bacteria, cylindrical flower anthracnose pathogenic bacteria and banana anthracnose pathogenic bacteria;

The biocontrol marine source Penicillium is named Penicillium sp.DSF059 and is preserved in China Center for Type Culture Collection (CCTCC) with a preservation date of 2023, 11 and 23 days and a preservation number of CCTCC NO: M20232310.

Preferably, the rubber fusarium wilt pathogen is fusarium oxysporum (Fusarium oxysporum), the coconut gray spot pathogen is trichoderma pseudodisc (Pestalotiopsis palmarum), the wheat scab pathogen is fusarium graminearum (Fusarium graminearum), the cucumber phytophthora root rot pathogen is phytophthora cucumber (Phytophthora melonis), the pyritic root rot pathogen is phytophthora camphorata (Phytophthoracinnamomi), the rubber anthracnose pathogen is colletotrichum gloeosporum (Colletotrichum gloeosporioides strainRC) the mango anthracnose pathogen is colletotrichum gloeosporioides (Colletotrichum gloeosporioides strain 171-1), the sisal zebra stripe pathogen is phytophthora nicotianae (Phytophthora nicothianae), the rubber red root pathogen is rubber ganoderma lucidum (Ganoderma pseudoferreum), the rubber brown root pathogen is harmful wood layer pore fungus (Phellinus noxius), the coffee anthracnose pathogen is cercospora (Colletotrichum coffeanum), the cylindrical flower anthracnose pathogen is colletotrichum gloeosporum (Colletotrichum gloeosporioides strainCH) and the banana anthracnose pathogen is banana anthracnose (Colletotrichum musae).

Preferably, the biocontrol marine source penicillium can inhibit germination of pathogenic bacterial spores by producing ferriphile, glucanase, caseinase, cellulase, chitinase and chitinase, and hydrolyze pathogenic bacterial cell walls to promote tropical crop growth.

Preferably, the tropical crop is one of rubber, coconut, wheat, cucumber, avocado, mango, sisal, coffee, colza, banana.

Preferably, the control method for the mango anthracnose pathogenic bacteria comprises the step of treating in-vitro leaves or harvested mangoes by using a biocontrol marine source penicillium culture solution.

Preferably, the preparation method of the biocontrol marine-source penicillium culture solution comprises the following steps:

(1) The spore suspension is prepared by inoculating biocontrol marine source penicillium strain into PDA solid culture medium, culturing at 28deg.C for 5d to activate the strain, eluting spores with sterile Tween 80 solution with volume fraction of 0.05%, and preparing spore suspension;

(2) Fermenting, namely inoculating spore suspension into a PDB culture medium according to the inoculum size of 3% by volume, and fermenting and culturing for 7d at 28 ℃ and 180r/min to obtain fermentation liquor;

(3) Filtering, namely filtering and sterilizing the fermentation liquor obtained in the step (2), and collecting filtrate, namely the marine-origin-biocontrol penicillium culture solution.

Preferably, the PDA solid culture medium in the step (1) is composed of 200g of potatoes, 20g of glucose, 15-30g of agar, 400mL of aged seawater and the balance of deionized water per liter.

Preferably, the spore suspension of step (1) has a concentration of 1.0X10 ⁸ cfu/mL.

Preferably, the PDB medium in the step (2) is composed of 200g of potato, 20g of glucose, 400mL of aged seawater and the balance of deionized water per liter.

Preferably, the PDA solid medium of step (1) can also be replaced with the following medium for the cultivation of penicillium, the reagent components and medium information involved are as follows:

Charles mother liquor is 150g of nitric acid, 25g of potassium chloride, 25g of magnesium sulfate heptahydrate, 0.5g of ferrous sulfate heptahydrate and deionized water to 500mL.

The trace element mother liquor contains 0.25g of anhydrous copper sulfate, 0.5g of zinc sulfate heptahydrate and deionized water to 500mL.

Martin culture medium, glucose 10g, peptone 5g, potassium dihydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g,0.1% Bengalum red solution 3.3mL, agar 15-20g, aged seawater 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

PDA culture medium, potato 200g, glucose 20g, agar 15-20g, aged sea water 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

MEA culture medium comprises malt extract 20g, peptone 1g, glucose 20g, microelement mother liquor 10mL, agar 15-20g, aged seawater 400mL, distilled water to 1L, and pH is natural. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

CYA culture medium comprises 1g of dipotassium hydrogen phosphate, 5g of yeast extract, 30g of sucrose, 10mL of Charles mother liquor, 15-20g of agar, 400mL of aged seawater, and distilled water with constant volume to 1L and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

OA culture medium, namely 30g of oat, 10mL of trace element mother liquor, 15-20g of agar, 400mL of aged seawater, distilled water to a volume of 1L and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

Yes culture medium comprises yeast extract 20g, sucrose 150g, magnesium sulfate heptahydrate 0.5g, trace element mother liquor 10mL, agar 15-20g, aged seawater 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

V8 culture medium, V8 juice filtrate 100mL, calcium carbonate 1g, agar 15-20g, distilled water to 1L, pH is natural. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

PDB culture medium, namely 200g of potato, 20g of glucose, 400mL of aged seawater, distilled water to 1L and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

CYB culture medium, namely 1g of dipotassium hydrogen phosphate, 5g of yeast extract, 30g of sucrose, 10mL of Charles mother liquor, 400mL of aged seawater, and distilled water with constant volume to 1L and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

G25NB medium, charles mother liquor 10mL, dipotassium hydrogen phosphate 1G, yeast extract 5G, sucrose 30G, glycerol 250G, aged seawater 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

MEB culture medium, malt extract 20g, peptone 1g, glucose 20g, trace element mother liquor 10mL, aged seawater 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

OB culture medium 30g, trace element mother liquor 10mL, aged seawater 400mL, distilled water to 1L, and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

Martin liquid culture medium comprises glucose 10g, peptone 5g, potassium dihydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g,0.1% Bengalum red solution 3.3mL, aged seawater 400mL, distilled water to constant volume to 1L, and pH is natural. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

CB culture medium, namely 30g of sucrose, 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 05g of magnesium sulfate heptahydrate, 0.01g of ferrous sulfate heptahydrate, 400mL of aged seawater, distilled water to 1L of constant volume and natural pH. Sterilizing at 121deg.C for 20min. After cooling to 65 ℃ or below, 100mg/mL streptomycin sulfate, 100mg/mL chloramphenicol, and 100mg/mL ampicillin were added to each 1mL of the solution, which was sterilized by ultrafiltration with a 0.22um filter membrane, on an ultra clean bench.

Cellulase detection Medium ：(NH₄)₂SO₄2.0g,MgSO₄·7H₂O 0.5g,K₂HPO₄1.0g,NaCl 0.5g, sodium carboxymethylcellulose 2.0g, agar 22.0g, ddH ₂ O600 mL, seawater 400mL,121 ℃ sterilization 20min, natural pH.

Amylase detection medium comprises starch 20.0g,KCl 0.5g,NaNO ₃2.0g,K₃PO₄1.0g,MgSO₄·7H₂ O0.5 g, agar 18.0g, ddH ₂ O600 mL, seawater 400mL, sterilizing at 121deg.C for 20min, and natural pH.

Chitosan detection medium comprises chitosan 10.0g,(NH₄)₂SO₄2.5g,MgSO₄·7H₂O 0.25g,K₂HPO₄0.02g,NaCl 4.0g,CaCO₃6.0g, agar 20.0g, pH6.0, ddH ₂ O600 mL, seawater 400mL, sterilizing at 121deg.C for 20min, and natural pH.

Chitinase detection medium comprises soluble starch 20g, colloid chitin 100g with 2%, K ₂HPO₄1g,KNO₃1g,MgSO₄0.5g,NaCl 0.5g,FeSO₄ 0.01.01 g, agar 20g, ddH ₂ O600 mL, seawater 400mL, sterilizing at 121deg.C for 20min, and natural pH.

Protease detection culture (MM 6051) of Casein detection, peking Lei Corp.

Glucanase assay beta-1, 3-glucan culture (MM 6220) from Beijing Cool Lei technology Co.

Siderophilic assay improved CAS solid medium (PM 0821) from beiku labs science and technology limited.

Compared with the prior art, the invention has the beneficial effects that:

(1) The marine environment is different from the land environment, and as the largest ecological system in the world, various microorganisms and active substances are contained in the marine environment. The biological resources in the biological pesticide precursor are greatly different from terrestrial organisms in terms of growth metabolism due to different living environment factors, so that the biological pesticide precursor becomes a huge resource library to be developed, and particularly in recent years, the development and utilization of the marine microbial resources are promoted along with the development of the marine biotechnology and the application of the modern biotechnology, so that the novel biological pesticide precursor provides rich candidate resources. The invention discloses a method for preventing and controlling pathogenic bacteria of tropical crops by using deep sea fungi, which not only can inhibit the growth of the pathogenic bacteria, but also can condition the growth of plants and induce the resistance of the plants to some pathogenic bacteria, and is easy to degrade, and has little influence on the safety of people and livestock and the environment;

(2) The invention discloses a biocontrol marine penicillium which has control effects on pathogenic bacteria of rubber wilt, pathogenic bacteria of coconut gray, pathogenic bacteria of wheat scab, pathogenic bacteria of cucumber phytophthora root rot, pathogenic bacteria of pear phytophthora root rot, pathogenic bacteria of rubber anthracnose, pathogenic bacteria of mango anthracnose, pathogenic bacteria of sisal hemp zebra stripes, pathogenic bacteria of rubber red roots, pathogenic bacteria of rubber brown roots, pathogenic bacteria of coffee anthracnose, pathogenic bacteria of column flower anthracnose, pathogenic bacteria of banana anthracnose and other tropical crops.

Drawings

FIG. 1 shows the colony morphology of Penicillium sp.DSF059, wherein A shows the colony morphology (front MEA, CYA, OA and YES, back MEA, CYA, OA and YES in order from left to right), B-C shows the conidiophore and conidium under an optical microscope, D-E shows the conidiophore under an electron microscope, F shows the conidiophore under an electron microscope;

FIG. 2 is a polygenic joint phylogenetic tree of Penicillium profundum sp.DSF059;

FIG. 3 shows the biological characteristics of Penicillium sp.DSF059, penicillium abyssal, A temperature, B pH, C light, D spore death temperature;

FIG. 4 is an enzyme activity profile of Penicillium sp.DSF059 of deep sea origin;

FIG. 5 shows the results of a plate-stand test of Penicillium sp.DSF059 from deep sea on a variety of plant pathogens; oxysporum, fusarium oxysporum, banana vascular wilt; N. dimidiatum. Nannochloris, P. palmarum. Cocois pseudodisc polyzoon, F. Graminearum, wheat scab Fusarium graminearum, L. theobromae. Cokokumi, P.melonis, cucumber phytophthora, P.cinneaminom, phytophthora pyriformis, C. gloeosporioides strainRC 178. Rubber anthracnose, C. gloeosporioides strain-1. Mango anthracnose, P. nicothianae. Sisal, nicotiana tabacum, G. pseudoferreum. Rubber red root, P. noxius. Rubber brown pest wood layer pore fungus, C.Coffenum, coffee anthracnose, C.34008. Azoma anthracnose, C. musae. Banana anthracnose;

FIG. 6 is a schematic diagram showing the bacteriostatic activity of Penicillium profundum sp.DSF059 on mango anthracnose;

FIG. 7 is a bar graph showing the antibacterial activity of Penicillium profundum sp.DSF059 on different media of mango anthracnose;

FIG. 8 shows the control effect of Penicillium profundum sp.DSF059 PDB fermentation broth on mango in vitro leaves, wherein A is sterile water (front side), B is Penicillium sp.DSF059 PDB fermentation broth (front side), C is pyraclostrobin 2000-fold liquid (front side), D is sterile water (back side), E is Penicillium sp.DSF059 PDB fermentation broth (back side), and F is pyraclostrobin 2000-fold liquid (back side);

FIG. 9 shows the control effect of Penicillium profundum sp.DSF059 PDB fermentation broth on harvested mango fruits.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the specific embodiments.

The marine source Penicillium biocontrol is named Penicillium sp.DSF059 and is preserved in China Center for Type Culture Collection (CCTCC), the preservation unit address is No. 299 of Wuchang district of Wuhan, hubei province, the preservation date is 2023, 11 months and 23 days, and the preservation number is CCTCC NO: M20232310.

EXAMPLE 1 isolation and purification of strains

1.1 Soil samples were collected from marine sediments of depth 2595m under water, 112 ° 30'00 "E, 17 ° 55' 00" N, south china sea, sampled with pushcore, and surface and deep sediments mixed together for treatment. The strain is separated by a Martin culture medium, inoculated with a soil sample by a seal method and purified by a PDA culture medium.

1.2 The specific operation method comprises the steps of placing a deep sea sediment sample into an ultra-clean workbench for air drying, grinding the deep sea sediment sample into powder after the deep sea sediment sample is dried, then pressing an autoclaved foam plug on the sediment powder, then stamping the sediment powder on the surface of a separation culture medium in a clockwise direction, and continuously pressing for 3 times to generate a continuous dilution effect. After inoculation, the plates were incubated at 28℃for 1-3 weeks in dark. Each medium was observed daily for strain growth, and when colonies grew, small colonies were picked and inoculated onto Potato Dextrose Agar (PDA) medium for purification culture. And (3) when the strain grows to a certain size, performing monospore separation culture to obtain a purified strain. Purified strains were stored to-80 ℃ using 20% glycerol and numbered according to strain isolation.

Example 2 morphological and molecular biological identification of Penicillium sp.DSF059 of marine origin

2.1 Bacterial colony morphology identification, namely, a three-point inoculation method is adopted, the purified bacterial strain DSF059 is respectively inoculated into MEA, CYA, OA and YES culture mediums for culture, and after 7 days of dark culture at 28 ℃, the growth condition of the bacterial strain, the colors of aerial hyphae and hyphae in the basal medium, the existence of soluble pigments, the colors and the like are observed and recorded.

The colony morphological characteristics of Penicillium sp.DSF059 as shown in FIG. 1, DSF059 was cultured for 7d at 28℃in MEA medium, the colony diameter was 28-31mm, the colony was thinner, the texture was velvet-like and powdery, the conidium structure was produced in large amounts in gray, the mycelium was light gray, no exudate, no soluble pigment, and the reverse yellow. Culturing in CYA culture medium at 28deg.C for 7d, wherein the colony has a diameter of 35-43mm, moderate thickness, concave middle, small amount of radioactive grooves, fine texture, sparse conidium structure, white color, no exudates, yellow-green soluble pigment, and yellow-green reverse surface. Culturing in OA culture medium at 28deg.C for 7d, wherein the colony has a diameter of 36-42mm and moderate thickness, has trace radioactive grooves, has a velvet-like texture and powder-like texture, has a large amount of conidium structure, is gray, has white mycelium, has no exudates, has yellow-green soluble pigment, and has yellow-green back surface. Culturing in YES culture medium at 28deg.C for 7d, wherein the colony has diameter of 38-40mm, is thicker, has a large number of radial grooves, has umbilical protrusion in the center, has a velvet shape and flocculent shape, has a large number of conidium structures, is grey green, has white mycelium, has no exudates, has no soluble pigment, and has golden yellow reverse surface.

2.2 Microscopic morphological characterization after 7d incubation of the strain at 28℃by slide wet chamber method, conidiophores and conidia were observed with Nikon scientific grade microscope (Ni-U, NIKON, tokyo, japan) and thermal field emission scanning electron microscope (SEM; JSM-7610FPlus, JEOL, tokyo, japan).

The slide wet chamber method is operated by ① pouring the PDA film, aseptically inverting the PDA film in a thickness of 2-3mm in a petri dish. ② The culture chamber is manufactured by manufacturing a culture chamber in another new culture dish, paving a layer of round filter paper which is slightly smaller than the plate on the bottom layer of the culture chamber, placing a U-shaped glass rod on the round filter paper, placing a clean glass slide on the glass rod, covering the culture dish cover, wrapping with newspaper, and taking out for sterilization. ③ Inoculating, namely cutting the PDA thin layer into agar blocks with the side length of about 1.0cm by using a scalpel, and transferring the agar blocks to two ends of a glass slide in the small chamber. A small amount of spores or mycelia were picked up with a sterile inoculating loop and inoculated onto the edge of the agar block, and a cover slip was covered on the agar block with sterile forceps. ④ Culturing, namely transferring 2-3 mL of sterilized 20% glycerol to filter paper of a plate (used for maintaining humidity in the plate) by using a pipetting gun, covering a culture medium and a plate cover, sealing by using a sealing film, and continuously culturing in a 28 ℃ incubator for 5-10d.

Before observing the strain morphology with a scanning electron microscope, a coverslip in which the penicillium morphology has been observed under an optical microscope is treated. The coverslips were first soaked in 2.5% glutaraldehyde and left to stand in a refrigerator at 4 ℃ overnight. After washing the coverslips 3-5 times with phosphate buffer (pH 7.4), the coverslips were dehydrated with 30%, 50%, 70%, 80%, 90% and 100% ethanol gradients, each gradient being treated for 20min. After that, the cover glass was wrapped in tin foil and dried for 10min with a vacuum freeze dryer. The dried material was fixed to aluminum pins with conductive carbon tape, sputtered with gold, and observed for strain and spore morphology with a thermal field emission scanning electron microscope.

The microcosmic morphological characteristics of Penicillium sp.DSF059 are that conidium stems occur in a matrix, the stems are 70-140 multiplied by 1.7-2.8 mu m, the walls are smooth, broom branches are single-round, the number of bottle stems is 4-8 per round, 5.5-8.2 multiplied by 1.4-2.4 mu m, the conidium is nearly round, and the walls are smooth. No sclerotium was observed.

2.3 Molecular biological identification, namely, hyphae on an MEA flat plate are picked, DNA is extracted by using a genome DNA extraction kit, and gene interval sequences (The nuclear ribosomal internal transcribed spacer region, ITS), beta-tubulin genes (BenA), calmodulin genes (CaM) and RNA polymerase II second large subunit (RPB 2) gene fragments of housekeeping genes 5.8S rDNA and 28S rDNA are amplified by using the hyphae as templates.

ITS PCR amplification primer sequences were ITS1:5'-tccgtaggtgaacctgcgg-3' (SEQ ID NO. 5) and lTS4:5'-tcctccgcttattgatatgc-3' (SEQ ID NO. 6), benA PCR amplification primer sequences were Bt2a:5'-ggtaaccaaatcggtgctgctttc-3' (SEQ ID NO. 7) and Bt2b:5'-accctcagtgtagtgacccttggc-3' (SEQ ID NO. 8), caMPCR amplification primer sequences were CF1:5'-GCCGACTCTTTGACYGARGAR-3' (SEQ ID NO. 9) and CF 4:5'-TTTYTGCATCATRAGYTGGAC-3' (SEQ ID NO. 10), and RPB2 PCR amplification primer sequences were 5F:5'-GAYGAYMGWGATCAYTTYGG-3' (SEQ ID NO. 11) and 7CR 5'-CCCATRGCTTGYTTRCCCAT-3' (SEQ ID NO. 12).

The PCR reaction system was 25uL 2X TAQ MASTER Mix,19uL ddH ₂ O,2uL template DNA, 2uL primers each, and total volume 50 uL.

PCR amplification conditions for ITS, ben A and CaM genes were 94℃for 5min, 94℃for 45s,55℃for 45s,72℃for 60s,35 cycles, and 72℃for 7min.

The RPB2 gene PCR amplification conditions were 94℃for 5min, 94℃for 45s,48℃for 45s,72℃for 60s,5 cycles, 94℃for 45s,50℃for 45s,72℃for 60s,5 cycles, 94℃for 45s,52℃for 45s,72℃for 60s,30 cycles, and 72℃for 7min.

The ITS gene sequence of the marine Penicillium sp.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.

Sequencing results were subjected to Blast comparison analysis on NCBI and a polygenic joint phylogenetic tree was constructed (FIG. 2), and by combining morphological analysis, the strain was identified as a potential new species of Penicillium, and was deposited in China center for type culture Collection with a accession number of CCTCC NO: M20232310.

EXAMPLE 3 biological Properties of Penicillium sp.DSF059 of marine origin

3.1 Effect of temperature on Penicillium sp.DSF 059 hyphae growth and conidium production Strain DSF059 was inoculated onto PDA plates by three-point inoculation, placed in constant temperature incubators at 4 ℃, 15 ℃, 22 ℃, 28 ℃, 30 ℃, 33 ℃ and 37 ℃ respectively, after 7d of dark inversion culture, colony diameters were measured by the crisscross method, and 3 cakes were taken equidistant from the center to the edge of the colonies by a puncher, placed in 2mL centrifuge tubes containing 1mL of 0.05% Tween 80 solution, shaken on a vortex shaker for 1min, diluted 10-fold or 100-fold, and the spore amounts were counted by a hemocytometer.

3.2 Effect of pH on the growth of Penicillium sp.DSF 059 hyphae and conidium production plates were prepared after adjusting the pH of PDA medium to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 using 3mol/L HCl and NaOH, respectively, and colony diameters and spore amounts were determined after inoculating the strains DSF059 in the center of the plates in a three-point inoculation method, and culturing in the dark at 28℃for 7 d.

3.3 Effects of photoperiod on the growth of hyphae and conidium production of Penicillium sp.DSF 059 Strain DSF059 was inoculated onto PDA plates in a three-point inoculation method and colony diameter and spore amount were determined after inversion culture in 0, 8, 12, 16 and 24h light incubators, respectively, for 7 d.

3.4 Lethal temperature of Penicillium sp.DSF 059 Strain DSF059 was inoculated into triangular flasks containing PDA culture, spores were eluted with 0.05% Tween 80 solution after 5d of dark culture at 28℃and counted by using a hemocytometer, and the test concentration of spore suspension was adjusted to 2X 10 ³ spores/mL, spore suspension was packed into 2mL centrifuge tubes, 1mL each tube was treated in thermostatic waterbaths at 45℃50℃55℃60℃and 65℃for 10min, 100. Mu.L each treatment was applied to PDA plates, and the number of plate colonies was counted after 48h, and the plate colony-free treatment temperature was the lethal temperature.

Conclusion that Penicillium sp.DSF 059 can grow at 15-33 ℃, the optimum growth temperature is 28-30 ℃ (figure 3A), penicillium sp.DSF 059 can grow at pH5-pH12, the pH is 11 (figure 3B), the Penicillium sp.DSF 059 can grow under dark and light conditions, the colony and spore yield are maximum (figure 3C) under continuous illumination for 12-16h, and the DSF059 spores can resist a certain high temperature, and the spore death temperature is 65 ℃ (figure 3D).

Example 4 enzymatic Activity Properties of Marine Penicillium sp.DSF059

4.1, Adopting a single colony inoculation method, picking a single colony of Penicillium sp.DSF059, inoculating to the centers of a cellulase detection medium, an amylase detection medium, a chitosan detection medium, a chitinase detection medium, a protease detection medium, a glucanase detection medium and a ferrite detection medium, repeating for three times, and culturing for 7 days at a 28 ℃ in an inversion mode.

4.2 Biological characteristics of Penicillium sp.DSF059 are shown in FIG. 4. Penicillium sp.DSF059 can produce ferrite, dextranase, casein enzyme, cellulase, chitinase and chitinase, thereby inhibiting the germination of pathogenic fungi spores and hydrolyzing the cell walls of the pathogenic fungi to achieve the purpose of biocontrol.

Example 5 Marine Penicillium sp.DSF059 inhibits growth of mango anthracnose hyphae

5.1 Using plate counter method to detect the influence of Penicillium sp.DSF059 on the growth of mango anthracnose pathogen hypha. Mango anthracnose pathogenic bacteria and Penicillium sp.DSF059 are respectively inoculated on two PDA culture mediums, and fungus cakes (phi=5 mm) are respectively picked after the two PDA culture mediums are cultured for 5 days under the dark condition of 28 ℃. Firstly inoculating mango anthracnose pathogenic bacteria cakes in the middle of a PDA culture medium, inoculating Penicillium sp.DSF059 strains at intervals of 2cm up and down and left and right, simultaneously independently inoculating mango anthracnose pathogenic bacteria serving as a blank control, repeating for 3 times, culturing at a constant temperature of 25 ℃ for 7 days, and then observing a bacteriostatic effect.

Hypha growth inhibition (%) = (control colony diameter-treated colony diameter)/(control colony diameter) ×100.

5.2 Results are shown in FIG. 5 (upper right, C.gloosporioides strain 171-1), and compared with the control group, mango anthracnose pathogenic bacteria in the treatment group are obviously inhibited by Penicillium sp.DSF059, and the growth inhibition rate of the mango anthracnose pathogenic bacteria hyphae is 65.33 +/-0.58%.

Example 6 Marine Penicillium sp.DSF059 control Effect on mango anthracnose

6.1 Determination of bacteriostatic Activity of different cultures Penicillium sp.DSF059 was inoculated into triangular flasks containing PDA culture, spores were eluted with 0.05% Tween 80 solution after 5d of dark culture at 28℃and counted using a hemocytometer, and the test concentration of the spore suspension was adjusted to 1X 10 ⁸ spores/mL. Inoculating spore suspension of Penicillium sp.DSF059 strain into CB, CYB, G25NB, MEB, OB, martin and PDB culture medium according to 3% (v/v), culturing at 28deg.C and 180r/min for 7d, collecting fermentation liquor, sterilizing with 0.22 μm filter membrane, collecting filtrate, mixing fermentation filtrate with PDA culture medium 1:4 uniformly to prepare medicated plate, inoculating mango anthracnose pathogenic bacteria cake (diameter 5 mm) at the center of plate without fermentation filtrate as control, placing plate upside down at 25deg.C for 7d, measuring colony diameter by crisscross method, and calculating inhibition rate;

inhibition (%) = (control colony diameter-treated colony diameter)/(control colony diameter) ×100.

6.2 In vitro leaf blade control effect determination, inoculating spore suspension of Penicillium sp.DSF059 strain into PDB at 3% (v/v), culturing at 28deg.C and 180r/min for 7d, collecting fermentation broth, sterilizing with 0.22 μm filter membrane, and collecting filtrate. Picking fresh-tipped leaves of mango without diseases, carrying out surface disinfection by 75% alcohol, needled by sterile plum blossom to hurt leaf tissues, spraying the surface of the leaf by the sterile fermentation liquid of Penicillium sp.DSF059, airing, inoculating a mango anthracnose fungus cake with the diameter of 0.5cm at the wound, spraying the corresponding fermentation liquid for the 2 nd time after inoculation of the fungus, airing, and placing in a moisturizing box with the humidity of 90%. Each leaf was inoculated at 3 points and each treatment was inoculated at 3 leaves with a 2000-fold solution of 25% pyraclostrobin suspension as test control and sterile water as blank control. 7d after inoculation, measuring the diameter of the lesion by adopting a crisscross method;

control effect (%) = (sterile water control plaque spread area-treated plaque spread area)/sterile water control plaque spread area×100.

6.3, After the mango is picked, the mango control effect is measured, namely, in-vitro mango inoculation is adopted, and the screened Tainong No. 1 mango is sterilized by using 2.0% (W/V) sodium hypochlorite solution and then repeatedly washed by using sterile water. The method comprises the steps of randomly dividing disinfected and cleaned mangoes into 4 groups (4 mangoes per serving), standing for 24 hours in a 150mm disinfection culture dish after airing, soaking the first group in the disinfection deionized water for 5 minutes, airing to serve as a control group, soaking the second group in the PDB fermentation liquor for 5 minutes, airing to serve as an antagonistic bacteria control group, soaking the third group and the fourth group in spore suspension of colletotrichum gloeosporioides with the concentration of 10 ⁷ CFU/mL, airing the third group serving as an pathogenic bacteria control group, soaking the fourth group in the antagonistic bacteria fermentation liquor for 5 minutes, airing to serve as an anti-effect treatment group, finally, placing the mangoes in the 150mm disinfection culture dish (4 per dish), culturing under the conditions of constant temperature and constant humidity (25 ℃ and 60% relative humidity), and observing experimental results after 5 days;

control effect (%) = (area of pathogen-attached control plaque-area of treated plaque)/area of pathogen-attached control plaque x 100%.

6.4 Results of antibacterial activity measurement on different culture solutions show that the antibacterial activity of fermentation filtrate obtained by fermenting the strain Penicillium sp.DSF059 in a PDB culture medium is strongest to mango anthracnose pathogenic bacteria, and the antibacterial activity is 48.62% (see figure 6 and figure 7 for details). After being treated by Penicillium sp.DSF059 PDB fermentation liquid, the anthracnose of the in-vitro leaves and the picked fruits of mango is obviously inhibited, the control effect on the in-vitro leaves is 94.46% (figure 8), and the control effect on the picked fruits is 73.67% (figure 9);

The analysis shows that the Penicillium sp.DSF059 has remarkable inhibition effect on mango anthracnose, can be used for developing birth control products, is applied to biological control of mango anthracnose, and discovers that PDB fermentation liquor of Penicillium sp.DSF059 has no adverse effect on mango.

EXAMPLE 7 inhibition of Penicillium sp.DSF059 of marine origin against other pathogenic fungi

7.1 Detecting the inhibitory Effect of Penicillium sp.DSF059 on other Tropical crop pathogenic fungi by plate counter method

Firstly, respectively inoculating banana wilt, dragon fruit canker, coconut gray spot, wheat scab, rubber coke rot, phytophthora root rot of cucumber, phytophthora root rot of pear, anthracnose of rubber tree, red root disease of rubber tree, brown root disease of rubber tree, anthracnose of coffee, anthracnose of colpitis and pathogenic bacteria of banana anthracnose and Penicillium sp.DSF059 on two PDA culture mediums, inoculating pathogenic bacteria of sisal hemp zebra disease on a V8 culture medium, culturing for 5 days under the dark condition of 28 ℃, and respectively taking bacterial cakes (phi=5 mm). Firstly inoculating a pathogenic bacteria cake to be detected in the middle of a PDA culture medium (sisal zebra disease pathogenic bacteria are inoculated in a V8 culture medium), inoculating Penicillium sp.DSF059 strains at intervals of 2cm from top to bottom to left to right, simultaneously singly inoculating the pathogenic bacteria as a blank control, repeating for 3 times, and culturing at a constant temperature of 25 ℃ for 7 days to observe the antibacterial effect.

7.2 Experimental results

Referring to FIG. 5, penicillium sp.DSF059 of marine origin has significant inhibitory effects on Fusarium oxysporum (F.oxysporum), fusarium pseudodish multiple of coconut gray, fusarium graminearum (F.graminearum), fusarium cucumerinum (P.melonis), fusarium linked immunosorbent (P.cinamomum), fusarium linked immunosorbent (P.cinamomi), rubber tree anthracnose gum spore RC178 (C.gloosporides sstran RC 178), sisal zebra-point tobacco pest (P.nicothianae), rubber tree red root rubber ganoderma lucidum (G.psudoferreum), rubber tree brown root harmful wood layer pore bacteria (P.nonxius), coffee anthracnose coffee thorium (C.coenum), rubber tree anthracnose gum CH008 (C.glooosporisoxin) and banana anthracnose C008, but has no inhibition effect on new darkness aschersonia (N.dimidiatum) and rubber scorch rot cocoa bisporus (L.theobromae). Wherein Penicillium sp.DSF059 has the best inhibiting effect on rubber ganoderma lucidum strain of rubber tree red root disease, which reaches 87 percent, and has better inhibiting effect on rubber tree anthracnose gum spore anthracnose RC178, coffee anthracnose coffee thorn dish spore bacteria, rubber tree brown root disease harmful wood layer pore bacteria, banana anthracnose bacteria, colpitis anthracnose gum spore anthracnose CH008, coconut gray spot pseudodisc multiple spore bacteria and sisal hemp zebra stripe disease tobacco phytophthora, and the inhibiting rate is more than 60 percent.

Example 8 control Effect of different types of mould on pathogenic bacteria of tropical crops

8.1 Experimental materials Streptomyces griseus (Streptomyces) from Bohua Biotechnology Co., ltd., trichoderma viride (Trichoderma viride) from Hubei Xingzhou Kogyo Co., ltd., jin Huiqing mold (Penicillium aurantiogriseum) from Wuhan Huaerna Biotechnology Co., ltd., penicillium bei-ril (Penicillium bilaiae) from Guangzhou Eumicroorganism Biotechnology Co., ltd., penicillium quinquefolium (Penicillium herquei) from Shanghai Xuan Ke Biotechnology Co., ltd., samson (Paecilomyces lilacinus (Thorn.)) from Ji Xinwang arrive Biotechnology Co., ltd., penicillium griseum (Penicillium griseofulvum) from Shanghai Limited.

8.2 Experimental methods

The inhibition effect on the pathogenic fungi of the tropical crops is detected by referring to the 7.1 flat plate counter method.

8.3 Experimental results

As shown in table 1:

TABLE 1 inhibition of pathogenic fungi of tropical crops by different types of mould

From table 1, it can be known that the inhibition effects of different types of mold on the pathogenic fungi of tropical crops are different, and the best effect is that the marine Penicillium sp.DSF059 disclosed by the application is the paecilomyces lilacinus, and the types of different Penicillium are different, so that the control effect is greatly influenced. Compared with penicillium which is commonly used for inhibiting pathogenic fungi of tropical crops in the prior art, the marine penicillium has better control effect, and the marine resources are rich. Therefore, the marine penicillium can be considered to be prepared into the bacteriostatic agent for preventing and treating the pathogenic fungi of the tropical crops, and has higher economic value and popularization prospect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (1)

1. An application of marine-derived Penicillium in controlling coffee anthrax pathogens, characterized in that the marine-derived Penicillium is deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20232310; The coffee anthracnose pathogen is 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.