Penicillium oxalicum and application thereof

Abstract

The invention relates to penicillium oxalicum and application of the penicillium oxalicum. The penicillium oxalicum is penicillium oxalicum BYZHCG-1. The invention further relates to a preparation method of the penicillium oxalicum. The endophytic biocontrol bacterium penicillium oxalicum BYZHCG-1 disclosed by the invention has very strong bacteriostatic activity on aquaculture source fusarium solani, can provide reference for green prevention and treatment of fungal diseases in aquaculture, effectively reduces the use of antibiotics and chemical drugs, and improves the quality safety of drugs and aquatic products.

Description

Technical Field

[0001] The invention belongs to the field of biological strains, and particularly relates to Penicillium oxalicum and applications thereof. Background Art

[0002] In recent years, studies have isolated Fusarium solani from diseased areas of aquatic animals such as California bass, Litopenaeus vannamei shrimp, and sea turtles. This fungus can cause visceral swelling and bleeding, fin ulceration in California bass, black gill disease in Litopenaeus vannamei shrimp, carapace necrosis, and low hatchability in sea turtles, severely impacting both freshwater and marine aquaculture species. Malachite green, a highly effective treatment for fungal diseases, has been banned, and other drugs pose safety and quality risks to aquatic products. Therefore, it is urgent to identify biocontrol agents for Fusarium solani from aquaculture sources to provide a reference for green control of this fungus. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a Penicillium oxalicum and an application thereof.

[0004] The invention provides a Penicillium oxalicum, which is Penicillium oxalicum BYZHCG-1, and its preservation number is CGMCC No.41918.

[0005] The present invention provides an application of the oxalicum Penicillium in preparing a Fusarium solani inhibitor.

[0006] The present invention provides an application of the oxalicum Penicillium in preparing an aquatic-sourced Fusarium solani inhibitor.

[0007] The present invention provides an application of the Penicillium oxalicum in preparing medicines for treating and / or preventing aquatic animal diseases caused by Fusarium solani.

[0008] The present invention provides an application of the Penicillium oxalicum in preparing a medicine for treating and / or preventing leukoplakia on turtle back.

[0009] The invention provides a medicine for treating and / or preventing leukoplakia on turtle back, and the medicine contains the Penicillium oxalicum.

[0010] The medicine contains the fermentation liquid of the Penicillium oxalicum.

[0011] The number of viable bacteria in the fermentation broth of Penicillium oxalicum is 1×10 7 -1×10 8 cfu / mL.

[0012] The oxalic acid Penicillium is Penicillium oxalic acid BYZHCG-1, which was deposited on April 17, 2025 at the General Microbiology Center of the China Culture Collection Administration, located at: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, abbreviated as CGMCC, and the deposit number is CGMCC No. 41918.

[0013] Beneficial effects

[0014] The MIC (minimum inhibitory concentration) of most existing antifungal drugs is much greater than the recommended dosage in the drug instructions, and these drugs will seriously affect the food intake and vitality of aquatic animals, and even cause death. In particular, fungal diseases are prone to occur in low temperature environments, and aquatic animals eat less or even do not eat at low temperatures, and their immunity will be significantly reduced. If irritating drugs are used, it is very easy to cause the death of aquatic animals. The endophytic biocontrol fungus Penicillium oxalicum BYZHCG-1 in the present invention has strong antibacterial activity against Fusarium solani from aquaculture, and the inhibition rate in the plate confrontation test is 86.9%; its fermentation liquid can effectively treat white spot disease on the carapace of Chinese grass turtles. The present invention can provide a reference for the green prevention and control of fungal diseases caused by Fusarium solani in aquaculture, effectively reduce the use of antibiotics and chemical drugs, and improve the quality and safety of drugs and aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The growth morphology of Penicillium oxalicum BYZHCG-1, including (a) front view; (b) back view; (c) microscope view;

[0016] Figure 2 This is a graph showing the antagonism of fungi to Fusarium solani;

[0017] Figure 3 Images of Penicillium oxalicum BYZHCG-1 and Fusarium solani confronting each other; (a) Microscopic image of Fusarium solani hyphae during the confrontation between the two bacteria; (b) and (c) Microscopic images of Penicillium oxalicum BYZHCG-1 during the confrontation between the two bacteria; (d) Microscopic image of the boundary between the two bacteria; (e) Microscopic image of Fusarium solani hyphae at the late stage of the confrontation between the two bacteria;

[0018] Figure 4 This is a graph showing the inhibition of the mycelial growth of Fusarium solani by the fermentation liquid of Penicillium oxalicum BYZHCG-1; CK is the blank control;

[0019] Figure 5 This is a graph showing the inhibition of spore growth of Fusarium solani by the fermentation liquid of Penicillium oxalicum BYZHCG-1; CK is the blank control;

[0020] Figure 6 Comparison of the white spots on the carapace of Chinese soft-shelled turtle before and after using the fermentation liquid of Penicillium oxalicum BYZHCG-1. DETAILED DESCRIPTION

[0021] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0022] Example 1

[0023] 1. Strain Isolation

[0024] White spots appeared on the carapaces of young Chinese soft-shelled turtles (Pelagic turtles) from a turtle hatchery in Baoying County. However, some hatchlings from the same batch had normal carapace color. Hatchlings with normal carapace color and their hatching medium were selected for study. The carapaces of the hatchlings were disinfected with 75% alcohol, and a small amount of the carapace was aseptically removed and placed on a 9-cm diameter potato dextrose agar (PDA) plate. After incubation at 26°C for 3 days, the dominant strain was selected for purification, and a dominant fungal strain (subsequently named BYZHCG-1) was isolated and cultured. One gram of vermiculite (from the hatchery) was mixed with 20 mL of sterile water and shaken at 26°C at 180 rpm for 1 hour. The mixture was centrifuged (12,000 rpm, 4°C for 5 minutes), and 100 μL of the supernatant was spread on a PDA plate. The PDA plates were incubated at 26°C for 3 days, and two dominant fungal strains (subsequently named BYZHCG-2 and BYZHCG-3) were isolated. Potato dextrose agar (PDA) medium: 200 g potatoes, 20 g glucose, 15 g agar powder, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH.

[0025] 2. Biocontrol bacteria screening methods

[0026] Using Fusarium solani as the target, a plate standoff method was used to screen for biocontrol agents against Fusarium solani from aquaculture sources. The Fusarium solani isolate was obtained from the laboratory of the Baoying County Aquatic Animal Disease Prevention and Control Center and was isolated from a Chinese soft-shell turtle with white spots on its carapace. Particles of Fusarium solani hyphae were inoculated onto polystyrene-derived dilution (PDA) plates and incubated at 26°C for 7 days. A 6 mm diameter F. solani cake was then inoculated onto the center of a new PDA plate using a sterile hole punch (6 mm diameter). Two 6 mm diameter cakes of the same test fungi, BYZHCG-1, BYZHCG-2, and BYZHCG-3, were then inoculated 2 cm from each side of the F. solani cake. Three replicates were set for each test fungus, with a PDA plate inoculated only with F. solani as the control. The plates were incubated upside down at 26°C for 7 days. Growth was recorded, and the inhibition rate was calculated. The mycelium growth inhibition rate (%) = (the diameter of Fusarium solani in the control group - the diameter of Fusarium solani in the treatment group) / the diameter of Fusarium solani in the control group × 100%.

[0027] Results: The isolated fungal strains were subjected to growth confrontation tests with Fusarium solani. The three test fungi all had different degrees of inhibition on Fusarium solani ( Figure 2 ), BYZHCG-1 had an inhibition rate of 86.9%, BYZHCG-2 had an inhibition rate of 54.8%, and BYZHCG-3 had an inhibition rate of 57.1%. BYZHCG-1 had the highest inhibition rate against Fusarium solani and was obtained from the carapace of a healthy Chinese soft-shell turtle.

[0028] Optical microscope was used to observe the confrontation between BYZHCG-1 and Fusarium solani plates ( Figure 3 When BYZHCG-1 confronts Fusarium solani, the hyphae of both fungi will become thicker ( Figure 3 a and 3b). BYZHCG-1 grows faster, and the hyphae produce many branches, especially at the junction of the two fungi, where a large number of spores are produced ( Figure 3 c and 3d), quickly occupying the nutrients and living space, invading the colony of Fusarium solani, entangling its hyphae and inhibiting its growth. As time goes by, the hyphae of Fusarium solani stop growing, gradually turn red, gradually wither and eventually die ( Figure 3 e).

[0029] 3. Identification

[0030] A small amount of BYZHCG-1 hyphae was extracted using the Ezup Column Fungal Genomic DNA Extraction Kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed using the universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', as shown in SEQ ID NO:1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', as shown in SEQ ID NO:2). The PCR reaction system consisted of 25 μL of the following: 12.5 μL of premix, 9.5 μL of ddH2O, 1 μL each of primers ITS1 and ITS4, and 1 μL of template DNA (lysate supernatant). PCR reaction conditions included 35 cycles of pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 15 s, and extension at 72°C for 10 s, followed by extension at 72°C for 10 min. A portion of the amplified products was detected by 1% agarose gel electrophoresis, and the remaining PCR amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0031] Based on morphological and molecular biological identification, the fungus BYZHCG-1 was identified as Penicillium oxalicum. Its growth morphological characteristics are as follows: Figure 1 As shown, it has the characteristics of rapid reproduction and a short growth cycle. The colonies are initially white, gradually turning turquoise, fuzzy, and wrinkled on the surface. The back of the colonies is yellow-brown. Microscopic observation shows that the hyphae are septate, with prominent conidiophores and broom-like branches, and the spores are oval.

[0032] Gene sequence

[0033] , as shown in SEQ ID NO:3.

[0034] IV. Inhibitory test of Penicillium oxalicum BYZHCG-1 fermentation broth on Fusarium solani

[0035] Preparation of the fermentation broth of Penicillium oxalicum BYZHCG-1: Add 0.85% physiological saline to a PDA plate covered with mycelium of Penicillium oxalicum BYZHCG-1, use an inoculating spatula to scrape the Penicillium oxalicum BYZHCG-1 on the surface of the PDA plate, place it in a 50mL centrifuge tube, add 10mL of 0.85% physiological saline, shake and centrifuge (5000r / min, 10min), discard the supernatant, and add 20mL of 0.85% physiological saline to make a spore solution, and detect the concentration of the spore solution by a hemocytometer. Take an appropriate amount of the above-mentioned Penicillium oxalicum BYZHCG-1 spore solution in 200mL of potato dextrose liquid medium (PDB), culture it in a shaker at 26°C and 180rpm for 7 days, so that the number of viable bacteria in the bacterial solution reaches 1×10 7 -1×10 8cfu / mL, centrifuge (12000r / min, 4℃, 10min), collect the supernatant, and filter through a 0.22μm sterile filter membrane to obtain sterile fermentation broth.

[0036] Potato dextrose broth (PDB): 200 g potatoes, 20 g glucose, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH.

[0037] (1) Effect of Penicillium oxalicum BYZHCG-1 fermentation broth on the mycelial growth of Fusarium solani

[0038] When the sterilized PDA medium is cooled to 50°C, it is mixed with the above-mentioned sterile fermentation liquid of Penicillium oxalicum BYZHCG-1 at a ratio of 1:1 to prepare a plate containing the fermentation liquid. A 6mm diameter Fusarium solani cake is inoculated in the center of the medium containing the fermentation liquid. The plate is inverted and incubated in a 26°C incubator for 7 days. Three replicates are set up. The growth of Fusarium solani on the PDA plate without fermentation liquid is used as the control group to observe the mycelial growth ( Figure 4 The inhibition rate of mycelial growth of the fermentation broth (%) = (diameter of Fusarium solani in the control group - diameter of Fusarium solani in the treatment group) / diameter of Fusarium solani in the control group × 100%. The inhibition rate of mycelial growth of Fusarium solani by the fermentation broth of Penicillium oxalicum BYZHCG-1 was 52.9%.

[0039] (2) Effect of Penicillium oxalicum BYZHCG-1 fermentation broth on the growth of Fusarium solani spores

[0040] The preparation process of Fusarium solani spore solution is the same as that of Penicillium oxalicum BYZHCG-1, and then add appropriate amount of 0.85% normal saline to adjust the concentration of spore solution to 1×10 7 cfu / mL. The sterile fermentation liquid of Penicillium oxalicum BYZHCG-1 was mixed with the spore liquid of Fusarium solani at a ratio of 1:1. After mixing, 150 μL of the mixture was evenly spread on a PDA plate. Three replicates were set up. A mixture of 0.85% saline and spore liquid at a ratio of 1:1 was used as the control group. The plate was inverted and cultured in a 26°C incubator for 7 days to observe the growth of Fusarium solani spores ( Figure 5 The inhibition rate of spore growth by the fermentation broth (%) = (spore germination area of Fusarium solani in the control group - spore germination area of Fusarium solani in the treatment group) / spore germination area of Fusarium solani in the control group × 100%. The inhibition rate of the fermentation broth of Penicillium oxalicum BYZHCG-1 on spore germination of Fusarium solani was 75.9%.

[0041] 5. Effect of Penicillium oxalicum BYZHCG-1 fermentation liquid on the treatment of white spots on the carapace of Chinese grass turtles

[0042] Thirty young Chinese soft-shelled turtles with similar white spots on their carapaces were selected from a turtle hatchery in Baoying County as experimental subjects and divided equally into three groups: A1, A2, and A3. Group A1 was treated with a commonly used antifungal chemical drug in aquaculture. The drug solution was prepared according to the MIC (minimum inhibitory concentration), and 10 young turtles were immersed in the drug solution for 1 hour every day for 15 consecutive days. Group A2 was treated with the fermentation liquid of Penicillium oxalicum BYZHCG-1, and 10 young turtles were immersed in the fermentation liquid every day (the number of viable bacteria in the fermentation liquid reached 1×10 7 -1×10 8 cfu / mL) for 1 hour for 15 consecutive days; Group A3 served as the control group and did not receive any treatment. The turtles were fed normally during the experiment. After 15 days, although the antifungal chemical drugs used in Group A1 had a therapeutic effect on the white spots on the carapace of young turtles, they were more irritating to the young turtles, and their activity ability decreased significantly, and they stopped eating, and 6 of them died; the untreated group A3 had a gradually larger area of white spots on the carapace of young turtles, a significantly decreased activity ability, a decreased feeding ability, and 3 deaths; the group A2, which used the fermentation liquid of Penicillium oxalicum BYZHCG-1, had a significantly smaller area of white spots on the carapace of young turtles, a lighter color, normal activity and feeding ability, and no deaths. The treatment effect was significantly better than that of Group A1 and Group A3 ( Figure 6 ).

Claims

1. A Penicillium oxalicum, characterized in that The oxalicum Penicillium is Penicillium oxalicum BYZHCG-1, and its preservation number is CGMCC No.41918.

2. Use of the Penicillium oxalicum according to claim 1 in the preparation of a Fusarium solani inhibitor.

3. Use of the Penicillium oxalicum according to claim 1 in the preparation of an inhibitor of Fusarium solani from aquatic sources.

4. Use of the Penicillium oxalicum according to claim 1 in the preparation of a medicament for treating and / or preventing aquatic animal diseases caused by Fusarium solani.

5. Use of the Penicillium oxalicum according to claim 1 in preparing a medicament for treating and / or preventing leukoplakia on turtle back.

6. A drug for treating and / or preventing leukoplakia on tortoise shell, characterized in that: The medicine contains the Penicillium oxalicum according to claim 1.

7. The drug according to claim 6, characterized in that The medicine contains the fermentation broth of the Penicillium oxalicum according to claim 1.

8. The medicine according to claim 7, characterized in that The number of viable bacteria in the fermentation broth of Penicillium oxalicum is 1×10 7 -1×10 8 cfu / mL.

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