An Alternaria gossypii SWFU-MM002 and its applications

Abstract

The present invention discloses a strain of Alternaria gossypina SWFU-MM002 and its application, belonging to the technical field of microbiology. In the present invention, a strain of bacteria is screened and isolated from diseased Mikania micrantha, and it is identified as Alternaria gossypina through identification. Therefore, this strain is named Alternaria gossypina SWFU-MM002. Through experiments, it is found that this strain has the effect of inhibiting the growth of Mikania micrantha. Therefore, it can be used to prepare drugs for inhibiting the growth of Mikania micrantha, providing a new strategy for the biological control of Mikania micrantha.

Description

Technical Field

[0001] The invention relates to a cotton alternaria strain SWFU-MM002 and an application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Mikania micrantha is a malignant weed that is difficult to control. Its invasion will affect the species diversity of the invaded area. Currently, the control of Mikania micrantha mainly includes physical control by manual felling, chemical control by spraying chemical agents, and biological control using natural enemies. Among them, physical control consumes a lot of manpower and material resources, and the spraying of chemical agents will have a certain impact on the surrounding ecological environment. Biological control is currently the most effective method for ecological environmental harmony, and is used in the prevention and control of many diseases and pests.

[0003] In recent years, microbial herbicides have attracted much attention as biological control agents due to their low toxicity, easy degradation, and abundant sources. Therefore, there is an urgent need for a microorganism that can be used for the control of Mikania micrantha. Summary of the invention

[0004] In order to broaden the method of biological control of Mikania micrantha, the present invention provides a strain of Alternaria spp. SWFU-MM002, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on August 9, 2024, with a deposit number of GDMCC No. 64973 and a taxonomic name of: Alternaria gossypina The storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] The present invention also provides an application of Alternaria sinensis SWFU-MM002 in preparing a drug for inhibiting the growth of micrantha.

[0006] Preferably, the drug is a fermentation broth of Alternaria solani SWFU-MM002 or an emulsifiable concentrate containing spore powder of Alternaria solani SWFU-MM002.

[0007] Preferably, the method for preparing the fermentation broth of Alternaria spp. SWFU-MM002 is as follows: a bacterial cake of Alternaria spp. SWFU-MM002 is inoculated into a liquid PDB culture medium to obtain a fermentation broth, and the fermentation broth is centrifuged.

[0008] Preferably, the preparation method of the emulsifiable concentrate containing spore powder is as follows: solid fermentation culture obtains spore powder of Alternaria sinensis SWFU-MM002, dissolves the spore powder in an organic solvent, adds an emulsifier and a stabilizer, and stirs to obtain an emulsifiable concentrate containing spore powder, wherein the organic solvent is any organic solvent and the emulsifier is any emulsifier.

[0009] Beneficial effects of the present invention

[0010] The present invention provides a strain of Alternaria gossypii SWFU-MM002, and it is found that this strain has the effect of inhibiting the growth of Mikania micrantha. Therefore, this strain can be used to prepare drugs for inhibiting the growth of Mikania micrantha, and this strain will not affect the ecological environment, providing a new resource for the biological control of Mikania micrantha and having a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a display diagram of the colony obtained by isolation and purification in Example 1 on the culture dish.

[0012] Figure 2 It is a display diagram of the colony obtained by isolation and purification in Example 1 on the bottom of the culture dish.

[0013] Figure 3 It is a microscopic diagram of the conidia obtained by isolation and purification in Example 1.

[0014] Figure 4 It is a microscopic diagram of the catenulate spores obtained by isolation and purification in Example 1.

[0015] Figure 5 It is a phylogenetic tree of the strain SWFU-MM002 obtained by isolation and purification in Example 1.

[0016] Figure 6 It is the phenotypes of healthy plants and infected plants of Mikania micrantha 15 days after the treatment in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] The technical solutions of the present invention will be further described below in conjunction with the drawings and through specific embodiments. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0018] The equipment, materials, reagents, etc. used in the present invention can be obtained through commercial channels without special instructions.

[0019] Preparation of PDB liquid medium: 200 g of potatoes, 20 g of glucose, 1000 mL of water, autoclaved at high pressure for 30 min. Example 1

[0020] Isolation of the strain

[0021] Rinse the diseased Mikania leaves with water to remove dust from the leaf surface, cut the tissue at the junction of the diseased and healthy into small pieces [(3 ~ 5) mm × (3 ~ 5) mm], soak it in 75% ethanol solution for about 1 minute, and then rinse it with sterile water 3 times. Place it in PDA culture medium and culture it in a 27 ℃ constant temperature box under dark conditions for 4 days. After the colonies grow, pick the hyphae for purification. After the colonies grow, transfer them to a new plate for further cultivation, and save the purified strain slant for later use.

[0022] Strain identification

[0023] (1) Morphological observation and identification: Cultivate the pathogen on a plate. After 7 days of cultivation, observe the morphology of the colonies. Figure 1 As shown, from Figure 1 It can be seen that the isolated strain has the hyphae growing upward from the middle of the colony and the aerial hyphae are well developed. As time goes by, the colony becomes round, the front of the colony is gray-brown inside, the outer circle is white, and the bottom of the colony is as follows Figure 2 As shown, from Figure 2 It can be seen that the back of the colony is dark in the middle and gradually becomes lighter towards the outside, with a ring pattern. Under a 20×40 magnification microscope, the isolated conidia can be seen as follows Figure 3 As shown, from Figure 3 It can be seen that the conidia are pear-shaped, inverted round rod-shaped or oval, with 1 to 3 transverse septa and 1 to 2 longitudinal septa. Figure 4 As shown, from Figure 4 It can be seen that Alternaria cotton forms long chains of unbranched conidia, with a chain length of 3 to 11 spores.

[0024] (2) Molecular biological identification: DNA was extracted from the isolated strains using a fungal genomic DNA rapid extraction kit, and the ribosomal RNA internal transcribed spacer gene ITS and Alternaria allergen gene were used to identify the Altα Glyceraldehyde-1-phosphate dehydrogenase gene GPD Perform PCR reaction;

[0025] The ITS1 primer sequence is 5′-TCCGTAGGTGAACCTGCGC-3′,

[0026] The primer sequence for ITS4 was 5′-TCCTCCGCTTATTGATATGC-3′,

[0027] The sequence of the Alt-for primer is 5′-ATGCAGTTCACCACCATCGC-3′,

[0028] The sequence of the Alt-rev primer is 5′-ACGAGGGTGAYGTAGGCGTC-3′,

[0029] The primer sequence of GPD1 is 5′-CAACGGCTTCGGTCGCATTG-3′,

[0030] The primer sequence of GPD2 is 5′-GCCAAGCAGTTGGTTGTGC-3′.

[0031] Send the PCR reaction product to a sequencing company for sequencing. The sequencing result of Altα1 is shown in SEQ ID No.1, the sequencing result of GPD is shown in SEQ ID No.2, and the sequencing result of ITS is shown in SEQ ID No.3. Submit the sequenced sequences to NCBI to obtain accession numbers, and download the sequences of related strains of the above three genes, as well as information such as the strains for comparison and the accession numbers of related genes. In the Phylosuite software, perform sequence alignment, Gblock trimming, and Concatenation through MAFFT. On this basis, with the help of the Neighbor-joining (NJ) method in the MEGA7.0 software, construct a phylogenetic tree as shown in Figure 5 , and the result shows that the isolated strain is Alternaria gossypii ( Alternaria gossypina ), which is named Alternaria gossypii SWFU-MM002. Example 2

[0032] Preparation of the crude extract of Alternaria gossypii SWFU-MM002

[0033] (1) Strain activation

[0034] Inoculate the preserved Alternaria gossypii SWFU-MM002 on a PDA medium and culture it in a constant temperature incubator at 28 °C for 5 days.

[0035] (2) Preparation of SWFU-MM002 fungal cakes

[0036] Under aseptic conditions, punch the activated Alternaria gossypii SWFU-MM002 colonies in step (1) with a puncher with a diameter of 5 mm to obtain fungal cakes.

[0037] (3) Preparation of the fermentation broth

[0038] Under aseptic conditions, pick about 5 fungal cakes obtained in step (2) with an inoculation needle and inoculate them into 100 mL of liquid PDB seed medium. Place the medium in a constant temperature shaker and culture it for 15 days. The rotation speed of the constant temperature shaker is 200 rpm, and the temperature is 27 °C. Regularly observe the situation of the bacterial liquid. After the culture is completed, centrifuge the fermentation broth to obtain the supernatant, and the supernatant is the crude extract of Alternaria gossypii SWFU-MM002. Example 3

[0039] A fungal herbicide with the active ingredient being Alternaria gossypii SWFU-MM002 at a concentration of 1×10 8 spores / mL. The preparation method is as follows: Inoculate the activated strain into the solid medium PDA medium, with the culture temperature at 27°C. After culturing for 10 days, brush the spores on the surface of the medium with a writing brush and collect the spore powder; add the collected spore powder to soybean oil, Tween-80, and sodium carboxymethylcellulose, and stir evenly to obtain an emulsifiable concentrate containing spore powder. Among them, the concentration of Alternaria gossypii SWFU-MM002 spore powder in the obtained emulsifiable concentrate is 1×10 8 spores / mL. In the emulsifiable concentrate, the mass percentage of Tween-80 is 0.3%, the mass percentage of sodium carboxymethylcellulose is 0.3%, the mass percentage of soybean oil is 99.4%, and the total mass percentage of Tween-80, sodium carboxymethylcellulose, and soybean oil is 100% Example 4

[0040] Experiment on the inhibition of Mikania micrantha growth by Alternaria gossypii SWFU-MM002

[0041] (1) Cultivation of Mikania micrantha seedlings

[0042] Sow Mikania micrantha seeds in a nutrient soil plug tray containing substrate and vermiculite, water thoroughly, place it on a cultivation rack for seedling raising. After the seedlings grow, they can be transplanted into separate seedling pots, or cut strong branches from the original Mikania micrantha for cutting. Two to three branches can be cut into one seedling pot to ensure the survival rate of Mikania micrantha. After the branches grow into seedlings, they can be transplanted into separate seedling pots, one seedling per pot, to ensure sufficient nutrition. After transplantation, water the seedlings thoroughly and then put them back on the cultivation rack to continue growing. When the seedlings grow to 6 - 8 leaves, select seedlings with the same growth trend as the experimental objects for treatment.

[0043] (2) Spraying the microbial herbicide on Mikania micrantha

[0044] Spray the fermentation broth of Alternaria gossypii SWFU-MM002 prepared in Example 2 on the leaves of Mikania micrantha, spraying 20 mL per Mikania micrantha seedling, and spraying once every 7 days. Use clear water as a control, and observe the phenotype of Mikania micrantha after 15 days.

[0045] As Figure 6 shown Figure 6 from left to right in the figure, the first 5 pots are Mikania micrantha plants sprayed with clear water, and the last 5 pots are Mikania micrantha plants sprayed with the fermentation broth of Alternaria gossypii SWFU-MM002. It can be seen that the plant height of Mikania micrantha plants sprayed with the fermentation broth of Alternaria gossypii SWFU-MM002 is significantly lower than that of the plants sprayed with clear water. Similarly, there are significant differences in leaf area, root length, and biomass compared with the control group, as shown in Table 1. The results show that the reagent of the active substance of Alternaria gossypii SWFU-MM002 can effectively inhibit the growth of Mikania micrantha.

[0046] Table 1 Effects of Alternaria gossypii SWFU-MM002 EC on the growth of Mikania micrantha

[0047]

Claims

1. A strain of Alternaria gossypii ( Alternaria gossypina ) strain SWFU-MM002, the deposit number of the cotton Alternaria strain SWFU-MM002 is: GDMCC No.64973.

2. Use of the cotton Alternaria strain SWFU-MM002 according to claim 1 in the preparation of a herbicide for inhibiting the growth of micrantha.

3. The use according to claim 2, characterized in that: The herbicide is a fermentation liquid of the cotton Alternaria strain SWFU-MM002 or an emulsifiable concentrate containing spore powder of the cotton Alternaria strain SWFU-MM002.

4. The use according to claim 3, characterized in that: The preparation method of the fermentation liquid of the cotton Alternaria strain SWFU-MM002 is as follows: a bacterial cake of the cotton Alternaria strain SWFU-MM002 is inoculated into a liquid PDB culture medium to obtain a fermentation liquid, and the fermentation liquid is centrifuged to obtain a supernatant.

5. The use according to claim 3, characterized in that: The preparation method of the emulsifiable concentrate containing spore powder of Alternaria strain SWFU-MM002 is as follows: solid PDA fermentation culture is used to obtain spore powder of Alternaria strain SWFU-MM002, the spore powder is dissolved in an organic solvent, an emulsifier and a stabilizer are added, and the mixture is stirred evenly to obtain an emulsifiable concentrate containing the spore powder.

6. The use according to claim 5, characterized in that: The concentration of spore powder of Alternaria strain SWFU-MM002 in the emulsifiable concentrate was 1×10 8 spores / mL, the mass percentage of the surfactant in the emulsifiable concentrate is 0.3%, the mass percentage of the stabilizer is 0.3%, the mass percentage of the suspending oil is 99.4%, and the total mass percentage of the surfactant, the stabilizer and the suspending oil is 100%; the surfactant is one of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, Span or Tween-80 or they are mixed in any proportion; the stabilizer is sodium carboxymethyl cellulose; the suspending oil is one of soybean oil, rapeseed oil, cottonseed oil, palm oil, sesame oil or corn oil or they are mixed in any proportion.

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