A strain of Penicillium foetidum CK152 and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of Penicillium vulpinum CK152 and its application. The present invention provides a strain of Penicillium vulpinum CK152, and the deposit number of the Penicillium vulpinum CK152 is CGMCCNO.41452. It has been experimentally verified that the liquid culture of the Penicillium vulpinum CK152 strain provided by the present invention has a significant inhibitory effect on rice blast fungus, and its antibacterial rate can reach 90%, and the inhibition rate of spore germination of rice blast fungus is 100%. It can be seen that the present invention provides a new strain with excellent anti-pathogenic fungi effect, which is of great significance for the prevention and treatment of pathogenic fungi.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a strain of Penicillium fowleri CK152 and an application thereof. Background Art

[0002] Rice blast, also known as rice fever, is a fungal disease of rice. The asexual generation of the pathogen is the deuteromycete Pyricularia oryzae, and the sexual generation is the ascomycete Ascomycete Pseudomonas oryzae. Rice blast is a disease caused by the rice blast fungus and can occur in all growth stages of rice. It is one of the three major rice diseases in my country and occurs in major rice producing areas in the world. It has a great impact on rice yields. Depending on the severity of the disease, it can cause a 10% to 30% reduction in yield, and even a total crop failure in severe cases. As the most devastating and widely distributed disease for rice worldwide, rice blast has significant economic importance.

[0003] The rice blast fungus mainly overwinters in diseased rice grains and diseased rice straw in the form of mycelium or conidia, becoming the primary source of infection the following year. When dry, conidia can survive for half a year to a year, and mycelium in diseased tissues can survive for more than a year; when moist, they die after 2-3 months. The infection process of rice blast fungus on rice mainly includes: (1) conidia are spread by wind and rain and adhere to the surface of rice leaves; (2) conidia germinate and form germ tubes; (3) germ tubes differentiate to form appressorium; (4) appressorium differentiates to form infection spikes; (5) infection spikes penetrate host cells, form infection hyphae inside host cells, and expand between cells. Rice blast fungus mainly infects rice leaves with conidia. When conidia contact the host epidermis, they germinate to form germ tubes, which then differentiate into appressorium. The appressorium forms infection spikes that penetrate the host epidermis and then continue to enter the host cells, causing the host plant to show symptoms about 5-7 days after infection. It can be seen that conidia are the primary link in rice blast infection. When the rice blast fungus strain cannot produce conidia, there is no subsequent infection of rice, and its pathogenicity is greatly weakened.

[0004] At present, the prevention and control methods for rice blast in the field are still mainly agricultural prevention and control and chemical prevention. Among them, the traditional chemical prevention method will produce pesticide residues and environmental pollution due to its high toxicity and difficulty in degradation in nature, thus leading to food safety and ecological safety problems. Therefore, it is urgent to find a natural strain of rice blast resistant fungi. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Penicillium fowleri CK152 and its application to solve the problems existing in the above-mentioned prior art. The Penicillium fowleri CK152 provided by the present invention can effectively inhibit the growth of pathogenic fungi, especially rice blast.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a Penicillium vulpinum CK152, and the preservation number of the Penicillium vulpinum CK152 is CGMCC NO.41452.

[0008] The present invention provides the use of the above-mentioned Penicillium fox dung CK152 in the preparation of a biological microbial agent with the efficacy of inhibiting pathogenic fungi, wherein the pathogenic fungi include rice blast pathogen, Fusarium or anthrax.

[0009] Preferably, the pathogenic fungus includes Rice blast pathogen.

[0010] The present invention provides a biological microbial agent with the efficacy of inhibiting pathogenic fungi. The biological microbial agent comprises the above-mentioned Penicillium fowleri CK152 or the fermentation liquid of the Penicillium fowleri CK152; the pathogenic fungi comprise rice blast pathogen, Fusarium or anthrax.

[0011] Further preferably, the fermentation broth of Penicillium fox feces CK152 is obtained by liquid culture of the fermentation broth of Penicillium fox feces CK152.

[0012] Further preferably, the liquid culture includes any one or a combination of several methods including shake flask culture, seed tank culture and fermentation tank culture.

[0013] Preferably, the pathogenic fungus includes Rice blast pathogen.

[0014] The present invention provides the use of the above-mentioned Penicillium fox dung CK152 or the above-mentioned biological bacterial agent in inhibiting pathogenic fungi, wherein the pathogenic fungi include rice blast pathogen, Fusarium or anthrax.

[0015] Preferably, the pathogenic fungus includes Rice blast pathogen.

[0016] The present invention provides the use of the above-mentioned Penicillium fox feces CK152 in preparing a biological bacterial agent with nitrogen fixation and phosphorus dissolving effects.

[0017] The present invention provides a biological bacterial agent with nitrogen fixation and phosphorus dissolving effects. The biological bacterial agent comprises the above-mentioned Penicillium fox feces CK152 or the fermentation liquid of the Penicillium fox feces CK152.

[0018] Further preferably, the fermentation broth of Penicillium fox feces CK152 is obtained by liquid culture of the fermentation broth of Penicillium fox feces CK152.

[0019] Further preferably, the liquid culture includes any one or a combination of several methods including shake flask culture, seed tank culture and fermentation tank culture.

[0020] The present invention provides the use of the above-mentioned Penicillium fox dung CK152 or the above-mentioned biological bacterial agent in promoting soil nitrogen fixation and phosphorus dissolution.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides a Penicillium vulpinum CK152, the deposit number of the Penicillium vulpinum CK152 is CGMCC NO.41452. It is known from experimental verification that the liquid culture of the Penicillium vulpinum CK152 strain provided by the present invention has a significant inhibitory effect on rice blast fungus, and its antibacterial rate can reach 90%, and the inhibition rate of spore germination of rice blast fungus is 100%. It can be seen that the present invention provides a new strain with excellent anti-pathogenic fungal effect, which is of great significance for the prevention and treatment of pathogenic fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is the morphology of the Penicillium fox feces CK152 strain;

[0025] Figure 2 It is the inhibitory effect of the fox feces Penicillium CK152 strain and the rice blast fungus strain on a 28℃ plate for 12 days.

[0026] Figure 3 The inhibitory effect of the liquid culture fermentation liquid of Penicillium foetidum CK152 (dosage: 300 μL) on the rice blast fungus strain at 28°C for 10 days;

[0027] Figure 4 The inhibitory effect of the liquid culture fermentation liquid of Penicillium foetidum CK152 (dosage: 600 μL) on the rice blast fungus strain at 28°C for 10 days;

[0028] Figure 5 The inhibitory effect of Penicillium fowleri CK152 on the spore germination of rice blast fungus strains;

[0029] Figure 6 This is the growth of Penicillium CK152 strain on ANF medium and PKO medium. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1 Isolation and identification of Penicillium fowleri CK152 strain

[0036] This strain was isolated from the frozen soil of Benxi. An appropriate amount of soil sample was weighed and placed in a sterile 50mL Ep tube, 25mL of sterile water was added to enrich the microorganisms in a shaker at 28°C and 110rpm for 30 minutes, the enriched sample was diluted in different proportions and spread on a PDA medium, and colonies of different morphologies were picked for purification and culture. Through plate confrontation culture, nitrogen fixation and phosphorus solubilization experiments, a fungal strain CK152 was screened out that can effectively inhibit the growth of rice blast pathogens, Fusarium or Anthracnose, and has nitrogen fixation and phosphorus solubilization effects. The ITS sequence data amplified by the strain was submitted to GenBank. The online software Blast on the NCBI website was used for homology comparison, and the phylogenetic tree software Clustalx and the Neighbor-Joining method in MEGA 5.05 software were used to construct a phylogenetic tree. Preliminary identification showed that the CK152 strain was 99.45% homologous to the genus Penicillium (Penicillium vulpinum),

[0037] The CK152 strain was preliminarily identified as a Penicillium vulpinum strain.

[0038] The nucleotide sequence of the ITS of the CK152 strain is shown in SEQ ID NO: 1, specifically:.

[0039] The main morphological characteristics of the CK152 strain: after 7 days of culture on potato glucose medium, the colony diameter is 20-23mm, the edge of the colony is white, the middle is gray-green, flat, and the aerial hyphae are sparse; the texture is granular, and the spore stalks are produced in large quantities, about 2-5mm long, the hyphae are white at first and then brown, the back is light yellow-brown, and it will secrete yellow-brown pigments. Conidial structures are produced in large quantities at the top of the spore stalks, and the broom-like branches are mostly three whorls and forked. Conidiophores are short, wavy and curved, with smooth walls, colorless, and closely arranged on the spore stalks; the phialides are columnar, with short necks, 5.0-9.3×2.0-3.0μm; the conidia are ellipsoidal or nearly spherical, smooth, and stringy, 2.5-4.0μm, such as Figure 1 shown.

[0040] According to the biological morphological characteristics and ITS sequence identification, the CK152 strain provided by the present invention is determined to be a species of Penicillium vulpinum, and is named Penicillium vulpinum CK152.

[0041] The Penicillium vulpinum CK152 strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection (CGMCC) on September 14, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41452.

[0042] Example 2 Liquid culture of Penicillium fowleri CK152 strain

[0043] Activation of strains: The preserved Penicillium fox feces CK152 strain was inoculated on PDA medium and cultured in a 28°C incubator for 4 days.

[0044] Preparation of culture medium: Prepare 30mL / bottle of PDA liquid culture medium, directly inoculate the grown Penicillium fowleri CK152 strain into a liquid shake bottle, with an inoculation amount of 3 bacterial cakes with a diameter of 5mm; culture in a shaker at 28°C and 110rpm for two to three weeks, and concentrate by natural evaporation to obtain a liquid culture. The effective viable bacteria count of Penicillium fowleri CK152 in the liquid culture is 10 5 CFU / mL.

[0045] Among them, the PDA culture medium formula: 200g potato juice, add 20g glucose, dilute to 1L, and then add 1.5wt.% agar powder; PDA liquid culture medium formula: 200g potato juice, add 20g glucose, dilute to 1L.

[0046] Example 3 Plate confrontation between Penicillium fowleri CK152 strain and Magnaporthe oryzae strain

[0047] Plate confrontation: The activated Penicillium CK152 strain was inoculated with a 5 mm diameter bacterial cake and the number of effective live bacteria of Penicillium CK152 in the bacterial solution was 10 5 CFU / mL) and the strain cake of rice blast fungus after filtration and sterilization were inoculated on PDA medium plates, and a control group was set up and placed in a 28°C incubator for 12 days. The results are as follows Figure 2 The experimental results show that under the inhibition of Penicillium fowleri, the rice blast fungus strains stopped growing and aged rapidly after a period of time, and the inhibition rate reached 80%.

[0048] Example 4 Inhibitory effect of liquid culture of Penicillium fowleri CK152 strain on rice blast fungus strains

[0049] The liquid culture prepared in Example 2 was filtered and added to the melted PDA solid culture medium, and then the culture medium was introduced into the plate. A control group and different liquid culture groups (300 μL group and 600 μL group) were set up. The rice blast fungus strain block was inoculated and placed in a 28° C. incubator for 10 days. The results are as follows: Figure 3 and Figure 4 The experimental results showed that the inhibition rate of adding 300 μL of Penicillium CK152 liquid culture to the rice blast fungus strain was 30%, and the inhibition rate of adding 600 μL of Penicillium CK152 liquid culture to the rice blast fungus strain was 90%.

[0050] Example 5 Inhibitory effect of liquid culture of Penicillium fowleri CK152 on spore germination of rice blast fungus strains

[0051] The filtered liquid culture prepared in Example 2 was mixed with the spore liquid of the rice blast fungus strain in a volume ratio of 1:1, and then dropped on a hydrophobic film and placed in a wet box to set up a control group; each treatment was repeated 3 times; the wet box was placed in a 28° C. incubator for 4 hours, then taken out and the spore germination was observed under a microscope.

[0052] Preparation of spore solution of rice blast fungus strain: Inoculate the rice blast fungus strain on OMA medium and place it in a 28℃ incubator for 8 days, then scrape off the mycelium of rice blast fungus with a sterile cotton swab and place it upside down at room temperature for 2 days. Add an appropriate amount of sterile water to the medium and then use a sterile cotton swab to cut off the newly grown mycelium. Filter the water in the medium with sterile filter paper and adjust it to a suitable concentration under a microscope to obtain the spore solution of rice blast fungus strain. The number of spores in the spore solution is 10 5 CFU / mL.

[0053] The OMA culture medium formula is: 30 g oats, 150 mL tomato juice, add water to make up to 1 L, and add 1.5 wt.% agar powder.

[0054] The results are as follows Figure 5 The experimental results show that when the liquid culture solution of Penicillium foetidum CK152 and the spore solution are mixed in equal volumes, the inhibition rate of spore germination of the rice blast fungus strain is 100%.

[0055] Example 6 Nitrogen fixation and phosphorus solubilization effect of Penicillium fowleri CK152 strain

[0056] The Penicillium fox feces CK152 strain was inoculated on Ashuby nitrogen-free medium (ANF medium) and Montgena inorganic phosphate medium (PKO medium) respectively and grown at 28°C for 5 days, with the inoculation amount being a 5 mm diameter bacterial cake.

[0057] Among them, the ANF medium formula: 0.2g KH2PO4, 0.2g NaCl, 0.2g MgSO4, 0.1g K2SO4, 5g CaCO3, 10g glucose, add water to 1L, add 1.5wt.% agar powder, pH 7.4;

[0058] PKO medium formula: 0.5g (NH4) 2SO4, 0.3g NaCl, 0.3g MgSO4·7H2O, 0.3g KCl, 0.03g MnSO4·H2O, 0.03g FeSO4·7H2O, 5.0g Ca3(PO4)2, 10.0g glucose, add water to make up to 1L, add 1.5wt.% agar powder.

[0059] The results are as follows Figure 6 The experimental results showed that the Penicillium CK152 strain had a transparent zone phenomenon on both ANF and PKO culture media, proving that the Penicillium CK152 strain had the function of nitrogen fixation and phosphorus solubilization.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Penicillium fox dung ( Penicillium vulpinum ) CK152, characterized in that, The deposit number of the Penicillium fox feces CK152 is CGMCC NO.41452.

2. The use of the fox feces Penicillium CK152 according to claim 1 in the preparation of a biological agent having the effect of inhibiting pathogenic fungi, characterized in that: The pathogenic fungus is rice blast pathogen.

3. A biological agent having the effect of inhibiting pathogenic fungi, characterized in that: The biological bacterial agent includes the fox dung mold CK152 described in claim 1; and the pathogenic fungus is the rice blast pathogen.

4. The biological agent according to claim 3, characterized in that: The pathogenic fungi include rice blast pathogen.

5. Use of the fox feces Penicillium CK152 according to claim 1 or the biological agent according to claim 3 or 4 in inhibiting pathogenic fungi, characterized in that: The pathogenic fungus is rice blast pathogen.

6. Use of the fox feces Penicillium CK152 described in claim 1 in the preparation of a biological bacterial agent with nitrogen fixation and phosphorus solubilization effects.

7. A biological agent with nitrogen fixation and phosphorus dissolution effects, characterized in that: The biological bacterial agent includes the fox feces Penicillium CK152 described in claim 1.

8. Use of the fox dung Penicillium CK152 according to claim 1 or the biological agent according to claim 7 in promoting soil nitrogen fixation and phosphorus solubilization.

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