A polyoxin-producing strain and application thereof

Abstract

The present application relates to a kind of polyantimycin production strain and its application, the polyantimycin production strain is Streptomyces Streptomyces Sp.MKL-0403, the polyantimycin production strain not only has polyantimycin production activity, and its fermentation product has higher bacteriostatic activity and more extensive bacteriostatic range.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agriculture, and particularly relates to a polyoxin production strain and application thereof. BACKGROUND

[0002] Polyoxin is also called polyoxin, which is a pesticide antibiotic with good systemic conductive effect. The main prevention and control objects include wheat powdery mildew, tobacco brown spot, cucumber downy mildew and the like.

[0003] Under the background of actively practicing the goal of reducing the amount and increasing the efficiency in the current pesticide industry, screening and cultivating an excellent strain with high polyoxin yield, strong antibacterial activity and wide antibacterial range has become a key path to achieve the goal, and plays an important supporting role in promoting the application of pesticide reduction. SUMMARY

[0004] In order to solve the above technical problems, the application provides a polyoxin production strain, which can not only produce polyoxin, but also has high antibacterial activity and a wider antibacterial range.

[0005] According to one aspect of the application, a polyoxin production strain is provided, and the polyoxin production strain is Streptomyces sp. Streptomyces MKL-0403, which is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 35576.

[0006] According to one aspect of the application, a polyoxin production strain is provided, and the polyoxin production strain is Streptomyces sp. Streptomyces MKL-0403, which satisfies any one or a combination of the following conditions:

[0007] (1) contains a molecular marker, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1;

[0008] (2) contains a molecular marker, and the nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1;

[0009] (3) the genomic DNA is amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and a specific band can be obtained by agarose gel electrophoresis analysis of the amplification product;

[0010] (4) using primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 to amplify the genomic DNA, and sequencing the amplified product to obtain a nucleotide sequence shown in SEQ ID NO: 1; or

[0011] The obtained nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0012] According to an aspect of the present application, a molecular marker is provided, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1; or

[0013] The nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0014] According to an aspect of the present application, the use of the above-mentioned molecular marker in identifying the above-mentioned polyoxin-producing strain is provided.

[0015] According to an aspect of the present application, a primer pair is provided, and the nucleotide sequence of the forward primer is shown in SEQ ID NO: 2;

[0016] The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0017] According to an aspect of the present application, the use of the above-mentioned primer pair in identifying the above-mentioned polyoxin-producing strain is provided.

[0018] According to an aspect of the present application, a method for identifying the above-mentioned polyoxin-producing strain is provided, and the method comprises the following steps:

[0019] (1) sequencing the polyoxin-producing strain;

[0020] (2) obtaining a sequence containing a specific molecular marker, and the specific molecular marker satisfies any one or a combination of the following conditions:

[0021] 1) the nucleotide sequence is shown in SEQ ID NO: 1; and / or

[0022] 2) the nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0023] Optionally, the sequencing is selected from:

[0024] (1) genome sequencing; and / or

[0025] (2) PCR product sequencing.

[0026] Optionally, the PCR product is obtained by amplification using primers shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0027] According to an aspect of the present application, there is provided a use of the above-mentioned polyoxin production strain in the preparation of a pesticide product.

[0028] According to an aspect of the present application, there is provided a pesticide product containing a fermentation product of the above-mentioned polyoxin production strain, wherein the effective component of the fermentation product contains polyoxin.

[0029] Optionally, the pesticide product is selected from any one of a fermentation stock solution, a fermentation dilute solution, a fermentation concentrate solution, a technical material, a mother material, a preparation.

[0030] Optionally, the polyoxin content of the fermentation stock solution, the fermentation dilute solution or the fermentation concentrate solution is 10-300000 μg / mL.

[0031] The polyoxin content of the technical material or the mother material is 5-99%.

[0032] The polyoxin content of the preparation is 0.01-30%.

[0033] Optionally, the dosage form of the preparation is selected from any one of a powder, a granule, an oil, a seed treatment suspension.

[0034] Optionally, the dosage form of the preparation is selected from a sustained release agent.

[0035] Optionally, the dosage form of the preparation is selected from a suspension.

[0036] Optionally, the dosage form of the preparation is selected from a suspension seed coating agent.

[0037] Optionally, the dosage form of the preparation is selected from any one of a seed treatment dispersible powder, a seed treatment soluble powder, a seed treatment liquid, a seed treatment emulsion, a seed treatment microcapsule suspension.

[0038] Optionally, the dosage form of the formulation is selected from any one of the following: large granules, fine granules, microparticles, microcapsules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsions, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, liposomes, microcapsule suspensions, oil suspensions, and suspension emulsions.

[0039] According to one aspect of this application, a method for preparing the above-mentioned pesticide product is provided, the method comprising the following steps:

[0040] Using Streptomyces Streptomyces The pesticide product was obtained by fermentation of sp. MKL-0403.

[0041] Optionally, the preparation method includes the following steps:

[0042] (1) Utilizing Streptomyces Streptomyces The pesticide product is obtained by fermentation with sp. MKL-0403 to obtain the fermentation stock solution; or

[0043] The preparation method further includes the following steps:

[0044] (2) Further processing of the fermentation liquid to obtain any one of the following: fermentation dilution, fermentation concentrate, technical material, mother drug, or formulation, which is the pesticide product.

[0045] According to one aspect of this application, the use of the above-described strain, the above-described pesticide product, or the pesticide product obtained according to the above-described method in the prevention and control of plant diseases is provided.

[0046] According to one aspect of this application, the use of the above-described strain, the above-described pesticide product, or the pesticide product obtained according to the above-described method in the preparation of plant disease control agents is provided.

[0047] The positive and beneficial effects achieved by this application are as follows: the Streptomyces provided in this application Streptomyces sp. MKL-0403 has high polyoxin production activity, as well as higher antibacterial activity and antibacterial range.

[0048] Preservation instructions:

[0049] Streptomyces Streptomyces sp. MKL-0403 was deposited at the China General Microbiological Culture Collection Center on August 11, 2025, and classified as Streptomyces. Streptomyces sp., accession number CGMCCNo. 35576.

[0050] Golden-colored Streptomyces MKL-4 Streptomyces aureochromogenes MKL-4 was deposited at the China Center for Type Culture Collection on December 5, 2022, and classified as *Streptomyces aureomarginata*. Streptomyces aureochromogenes, The accession number is CCTCC NO: M 20221883. Attached Figure Description

[0051] Figure 1 The inhibitory effect of single colonies 1-5 in Example 1 on cucumber powdery mildew;

[0052] Figure 2 Streptomyces Streptomyces Phylogenetic tree of 16S rDNA of sp. MKL-0403;

[0053] Figure 3 Streptomyces Streptomyces Liquid chromatography-mass spectra of fermentation broth for sp. MKL-0403;

[0054] Figure 4 Streptomyces Streptomyces Mass spectrum of component 1 of sp. MKL-0403;

[0055] Figure 5 Streptomyces Streptomyces H-NMR spectrum of component 1 of sp. MKL-0403;

[0056] Figure 6 Streptomyces Streptomyces sp. MKL-0403 component 1 13 C-NMR spectrum;

[0057] Figure 7 The diagram shows the effect of controlling powdery mildew in cucumbers;

[0058] Figure 8 The image shows the inhibitory effect of *Fusarium wilt* on pumpkin vine.

[0059] Figure 9 The agarose gel electrophoresis results are for the amplification products of SEQ ID NO: 2 and SEQ ID NO: 3. Detailed Implementation

[0060] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings:

[0061] The term "active ingredient" refers to the component in pesticide products that has toxic and killing activity against diseases, insects, and weeds.

[0062] The term "technical material" means a product obtained in the course of production consisting of the active ingredient and the relevant impurities, to which a small amount of additives can be added, if necessary.

[0063] The term "technical material" means a product obtained in the course of production consisting of the active ingredient and the relevant impurities, to which a small amount of additives can be added, if necessary.

[0064] The term "formulation" means a product which is stable in its state, processed from a pesticide technical material (mother drug) and suitable adjuvants, or processed from biological fermentation, plant extraction, etc.

[0065] The term "plant disease" means that during the growth and development of plants, due to the influence of other organisms and adverse non-biological factors, the growth and development of plants is significantly hindered, pathological changes occur in the interior and exterior of plants, and the growth and development of plants are hindered, even death, resulting in reduced yield and quality. The "plant disease" of the present application refers specifically to the disease caused by the infection of other organisms on plants, which include but are not limited to fungi (such as cucumber powdery mildew fungus, pumpkin vine wilt disease fungus), or bacteria (such as cucumber bacterial angular spot disease fungus) and the like.

[0066] The reagents and solvents used in the following examples can be purchased by commercial channels, or prepared by conventional methods known to those skilled in the art. The experimental methods not specified in the following examples are carried out according to conventional methods and conditions.

[0067] Preparation of PDA medium: 200 g of potatoes were washed, peeled, chopped, and boiled in 800 ml of distilled water for half an hour, then filtered with gauze, and 20 g of glucose and 20 g of agar were added, and distilled water was added to make up to 1000 ml, and sterilized at 121°C for 30 minutes.

[0068] Preparation of LB medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 20 g of agar were dissolved and made up to 1000 ml with distilled water, and the pH was adjusted to 7.0, and sterilized at 121°C for 20 min.

[0069] Seed medium preparation: corn flour 15 g / L, low-temperature soybean cake powder 20 g / L, beer yeast powder 4 g / L, fructose glucose syrup 10 mL / L, NaCl 1 g / L, CaCO3 3 g / L, KH2PO4 1 g / L, the rest is water, pH 6.5-6.8, sterilized at 121°C for 30 min.

[0070] Fermentation medium preparation: corn flour 100 g / L, low-temperature soybean meal 25 g / L, beer yeast powder 4 g / L, fish meal 5 g / L, NaCl 3 g / L, CaCO3 3 g / L, KH2PO4 0.5 g / L, α-amylase 0.015% of the mass of corn flour, and the rest is water, pH 6.5-6.8, sterilized at 121°C for 30 min.

[0071] The liquid phase detection method (HPLC) and the biological titer detection method refer to the enterprise standard Q / SMS 083-2024 "10% polyoxin soluble granules" of Farmland Crop Science (Shaanxi) Co., Ltd. (formerly Shaanxi Maikelu Biological Technology Co., Ltd.).

[0072] Genomic DNA extraction was performed using the bacterial genomic DNA extraction kit of Tiangen Biosciences Co., Ltd., and the extraction method referred to its instruction manual.

[0073] Specific amplification was performed using the rapid PCR polymerase PrimeSTAR Max DNA Polymerase of Takara Bio Inc., and the amplification system was as follows:

[0074]

[0075] The amplification program was as follows:

[0076]

[0077] The streptomyces strain used in the present application:

[0078]

[0079]

[0080] Example 1 Streptomyces Streptomyces Isolation and identification of sp. MKL-0403 strain

[0081] 1. Strain isolation

[0082] 9 soil samples collected from the mangrove nature reserve in Longhai District, Zhangzhou City, Fujian Province, China were taken, 10 g of each was dissolved in 90 mL sterile water, and gradient diluted with sterile water to 100 times and 1000 times, and an appropriate amount of 1000 times dilution was spread on PDA medium plates, and cultured at 28°C for 3 days. 13 single colonies that grew out were further purified and cultured, and the purified single colonies were inoculated into triangular flasks containing fermentation medium, and fermented at a temperature of 28°C and a rotation speed of 180 rpm for 3 days to obtain fermentation broth. The fermentation broth was filtered through a 0.22 μm bacterial filter to remove the bacterial cells, and was ready for use.

[0083] Forty-two cucumber leaves with consistent powdery mildew disease symptom were collected (half of each leaf was cut off for the experiment to ensure the leaves could be laid flat in the humidity chamber). These were divided into 14 groups, each sprayed with 6 mL of the fermentation broth from one of the 13 single colonies and sterile water (negative control). The groups were incubated at 28°C for 2 days, and the disease development was observed (the control effects of strains numbered 1-5 are shown in [reference needed]). Figure 1 The strain with the best control effect on powdery mildew (strain number 3) was selected for further research.

[0084] 2. Strain identification

[0085] The above-mentioned strain No. 3 was identified using the 16S rDNA identification method. The specific identification steps are as follows:

[0086] (1) Genomic DNA extraction: After activating and culturing strain No. 3 on PDA medium plates, genomic DNA was extracted.

[0087] (2) 16S rDNA amplification: Using genomic DNA as a template, 16S rDNA fragments were amplified from genomic DNA using universal 16S rDNA primers (27F sequence as shown in SEQ ID NO: 4 and 1492R sequence as shown in SEQ ID NO: 5). The amplification products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for analysis.

[0088] (3) 16S rDNA sequence analysis: The 16S rDNA sequence obtained from sequencing was compared with the 16S rDNA sequence in GenBank using BLAST. The results showed that it was consistent with... Streptomyces The 16S rDNA sequence of sp. strain SEP3 showed the highest similarity (>99%). A phylogenetic tree was constructed using Mega 5.0 software, and this strain was related to... Streptomyces sp. strainSEP3 clusters on a single branch (see...) Figure 2 ).

[0089] Based on the above identification results, strain No. 3 was identified as Streptomyces. Streptomyces sp., named Streptomyces Streptomyces sp. MKL-0403. It is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35576.

[0090] Example 2 Streptomyces Streptomyces sp. MKL-0403 Active Substance Identification

[0091] 1. Liquid chromatography detection

[0092] Streptomyces Streptomyces The fermentation broth of sp. MKL-0403 was analyzed by liquid chromatography, and the active substances were identified using polyoxin B standard. The chromatogram is shown below. Figure 3 As shown. The results indicate that Streptomyces Streptomyces In the fermentation broth of sp. MKL-0403, the component with a peak elution time of 8.892 (component 1) was polyoxin B.

[0093] A golden-colored Streptomyces 980# was used to prepare a fermentation broth according to the method in Example 1. The active substances in the broth were identified using polyoxin B standard. The results showed that component 2 with a peak time of 8.802 was polyoxin B.

[0094] Take golden-colored Streptomyces MKL-4 Streptomyces aureochromogenes MKL-4, the fermentation broth was prepared according to the method of Example 1, and the active substances in it were identified by using polyoxin B standard. The results showed that component 3 with a peak time of 8.849 was polyoxin B.

[0095] 2. Identification of the structure of the active ingredient

[0096] Separation, concentration, and drying were performed on Streptomyces using gel column chromatography. Streptomyces sp. MKL-0403, golden-colored Streptomyces 980#, golden-colored Streptomyces MKL-4 Streptomyces aureochromogenes Components 1-3 were obtained from the fermentation broth of MKL-4. After mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, it was finally determined that components 1-3 were all polyoxin B.

[0097] The mass spectrum of component 1 is as follows Figure 4 As shown, the H-NMR spectrum is as follows Figure 5 As shown, 13 C-NMR spectra as follows Figure 6 As shown.

[0098] The above results indicate that Streptomyces Streptomyces sp. MKL-0403, golden-colored Streptomyces 980#, golden-colored Streptomyces MKL-4 Streptomyces aureochromogenes MKL-4 can produce multiple antibiotics.

[0099] Example 3 Streptomyces Streptomyces Sp. MKL-0403 Fermentation Level Determination

[0100] Streptomyces Streptomyces sp. MKL-0403, golden-colored Streptomyces MKL-4 Streptomyces aureochromogenesMKL-4, Streptomyces aureofaciens 980# were inoculated into seed culture medium respectively for 1 day, then transferred into 5L fermenter for fermentation for 7 days, and the samples were taken for bioactivity detection. The results showed that the bioactivity of Streptomyces sp. MKL-0403 was 35010 U / mL; the bioactivity of Streptomyces aureofaciens MKL-4 was 31254 U / mL; and the bioactivity of Streptomyces aureofaciens 980# was 3701 U / mL. Streptomyces sp. MKL-0403 was 35010 U / mL; the bioactivity of Streptomyces aureofaciens MKL-4 Streptomyces aureochromogenes MKL-4 was 31254 U / mL, and the bioactivity of Streptomyces aureofaciens 980# was 3701 U / mL.

[0101] From the above results, it can be seen that the bioactivity of Streptomyces sp. MKL-0403 was significantly higher than that of Streptomyces aureofaciens MKL-4 Streptomyces MKL-4 and Streptomyces aureofaciens 980#. Streptomyces aureochromogenes

[0102] Example 4 Bioassay of Streptomyces sp. MKL-0403 Streptomyces Example 4 Bioassay of Streptomyces sp. MKL-0403

[0103] 1. Field control effect of different strains on cucumber powdery mildew

[0104] (1) Preparation of the pesticide

[0105] 5mL of the fermentation broth of Streptomyces sp. MKL-0403, 5mL of the fermentation broth of Streptomyces aureofaciens MKL-4 Streptomyces MKL-4 prepared in Example 3 were taken respectively, and diluted 500 times for standby. Streptomyces aureochromogenes MKL-4 prepared in Example 3 were taken respectively, and diluted 500 times for standby.

[0106] (2) Method of applying the pesticide

[0107] A field in the early stage of cucumber powdery mildew was selected, and divided into 12 plots, each plot was 6.5m 2 , and 4 treatments were set, each treatment had 3 plots. Three groups of treatments were sprayed with the above fermentation broth diluents respectively, and the other group was sprayed with water as a blank control group, and the spraying amount of each plot was 1L.

[0108] (3) Investigation of the control effect

[0109] After half a month of applying the pesticide, the disease conditions of each group were as shown in the table: A was the fermentation broth treatment group of Streptomyces sp. MKL-0403, only a few white spots were observed on a small number of leaves; B was the fermentation broth treatment group of Streptomyces aureofaciens MKL-4 Figure 7 Streptomyces Streptomyces aureochromogenes ​​​A few spots of white powder on the leaves of MKL-4 fermentation liquid treatment group could be observed; C was Streptomyces aureofaciens 980# fermentation liquid treatment group, and more obvious spots of white powder on the leaves of most leaves could be observed; D was the control group, and most of the leaves could observe the white powder connected into a piece.

[0110] Randomly take 4 points per cell, investigate 2 strains per point, and investigate all leaves per strain. Record according to the following grading method.

[0111] Leaf injury grading method (in leaf units):

[0112] 0, no lesion;

[0113] 1, lesion accounts for less than 5% of the entire leaf area;

[0114] 3, lesion accounts for 6%~10% of the entire leaf area;

[0115] 5, lesion accounts for 11%~20% of the entire leaf area;

[0116] 7, lesion accounts for 21%~40% of the entire leaf area;

[0117] 9, lesion accounts for more than 40% of the entire leaf area.

[0118] Disease index =∑{(number of leaves at each level x relative level value) / (total number of leaves surveyed x 9)}x100

[0119] Control effect = (disease index of blank control area - disease index of treatment area) / disease index of blank control area x 100%

[0120] The control effect calculation results are as follows:

[0121]

[0122] 2, Inhibition effect of different strains on P. cucurbitacearum

[0123] Take 1 mL of Streptomyces MKL-0403 fermentation liquid prepared in Example 3, 1 mL of Streptomyces aureofaciens MKL-4 fermentation liquid prepared in Example 3, and 1 mL of Streptomyces aureofaciens 980# fermentation liquid prepared in Example 3, respectively, and add them to 100 mL of melted PDA medium, pour into culture dishes to prepare toxicity plates, and reserve. Streptomyces Streptomyces aureochromogenes MKL-0403 fermentation liquid prepared in Example 3, 1 mL of Streptomyces aureofaciens MKL-4 fermentation liquid prepared in Example 3, and 1 mL of Streptomyces aureofaciens 980# fermentation liquid prepared in Example 3, respectively, and add them to 100 mL of melted PDA medium, pour into culture dishes to prepare toxicity plates, and reserve.

[0124] Use a puncher with a diameter of 6 mm to punch P. cucurbitacearum (Podosphaera xanthii) with a colony diameter of about 6 cm after activation. Stagonosporopsis cucurbitacearum ​The colony edge was punched to obtain a bacterial cake, and was inoculated on the above-mentioned toxic PDA plates, with a blank PDA plate without adding any agent as a control. The above-mentioned process was repeated for three times, and was placed at 28°C for culture. After 7 days, the inhibition rates of the fermentation products were investigated and calculated.

[0125] The calculation formula was as follows: inhibition rate of colony growth = (diameter of control colony - diameter of treated colony) / (diameter of control colony - diameter of bacterial cake) x 100%.

[0126] The results are shown in Figure 8 (A: Streptomyces sp. MKL-0403, B: Streptomyces aureofaciens MKL-4 Streptomyces , C: Streptomyces aureofaciens 980#, and D: blank control). Streptomyces aureochromogenes

[0127] The specific statistical data are shown in the following table:

[0128]

[0129] The above-mentioned results show that Streptomyces aureofaciens 980# has very weak inhibitory effect on P. cucumerina, with an inhibition rate of only 10.0%. Streptomyces aureofaciens MKL-4 Streptomyces aureochromogenes has an inhibition rate of only 18.9%. However, Streptomyces sp. MKL-0403 has very good inhibitory effect on P. cucumerina, with an inhibition rate of 83.3%. Streptomyces

[0130] From the above-mentioned efficacy experiments, it can be seen that Streptomyces sp. MKL-0403 has better control effect on cucumber powdery mildew than Streptomyces aureofaciens MKL-4 Streptomyces and Streptomyces aureofaciens 980#. Streptomyces aureochromogenes Applicants further expanded the inhibition detection and unexpectedly found that Streptomyces sp. MKL-0403 can effectively inhibit P. cucumerina, while Streptomyces aureofaciens MKL-4 Streptomyces and Streptomyces aureofaciens 980# have very weak inhibitory ability on P. cucumerina. Streptomyces aureochromogenes Therefore, Streptomyces sp. MKL-0403 has stronger inhibitory ability and wider inhibitory spectrum. Streptomyces

[0131] Example 5: SCAR molecular marker analysis of Streptomyces sp. MKL-0403 Streptomyces

[0132] Streptomyces sp. MKL-0403, Streptomyces lavendulae var. hainanensis Streptomyces , Streptomyces sp. MKL-0403, Streptomyces lavendulae var. hainanensis Streptomyces ​​​​lavendulae var. hainanensis) UV-11, Streptomyces sp. 8130, Streptomyces aureofaciens MKL-4 Streptomyces aureochromogenes MKL-4, Streptomyces microflavus MKL-2 Streptomyces microaureaus MKL-2), Streptomyces offalvus Streptomyces caniferus NEAU6, Streptomyces aureofaciens 980#, Streptomyces The genomic DNA of the 8 strains of Streptomyces was used as a template, and 20 random primers (TGCCGAGCTG, AGTCAGCCAC, AATCGGGCTG, AGGGGTCTTG, GTTTCGCTCC, CATCCCCCTG, CTGCTGGGAC, GTGAGGCGTC, CCGCATCTAC, GTGTGCCCCA, GGACCCAACC, TTGGCACGGG, AGCGCCATTG, GGTGCGGGAA, GTGACATGCC, TGCGCCCTTC, GGACTGCAGA, CTCACCGTCC, GGTGATCAGG, CTGAGACGGA) were used for random amplification. After agarose gel electrophoresis analysis of the amplification products, the Streptomyces Streptomyces MKL-0403 was cut off, recovered, and sequenced to obtain the sequence of the specific band.

[0133] Primers were designed according to the obtained specific band sequence (the forward primer is shown as SEQ ID NO: 2; the reverse primer is shown as SEQ ID NO: 3), and the genomic DNA of the above 8 strains of Streptomyces was used as a template for amplification. The amplification products were subjected to agarose gel electrophoresis analysis, and the results are shown in Figure 9 MKL-0403 (lane 1 in the figure) could amplify a specific band, and the other 7 strains could not amplify a band, verifying that the primers (SEQ ID NO: 2 and SEQ ID NO: 3) could specifically identify Streptomyces sp. MKL-0403. Streptomyces Streptomyces The specific band obtained by specifically amplifying Streptomyces sp. MKL-0403 using SEQ ID NO: 2 and SEQ ID NO: 3 was recovered and sequenced, and the sequence is shown as SEQ ID NO: 1.

[0134] The specific band obtained by specifically amplifying Streptomyces sp. MKL-0403 using SEQ ID NO: 2 and SEQ ID NO: 3 was recovered and sequenced, and the sequence is shown as SEQ ID NO: 1. Streptomyces

[0135] ​​The specific band sequence shown in SEQ ID NO: 1 was subjected to Blast comparison in GenBank, and no identical or similar sequence was retrieved in all publicly disclosed strains of Streptomyces in NCBI (No significant similarity found), which further indicates that the specific sequence shown in SEQ ID NO: 1 can be used to identify Streptomyces Streptomyces sp. MKL-0403.

[0136] The above results show that the primers of SEQ ID NO: 2 and SEQ ID NO: 3 are specific primers for identifying Streptomyces Streptomyces sp. MKL-0403 strain, and the sequence of the PCR amplification product thereof, i.e., the sequence shown in SEQ ID NO: 1, is a SCAR molecular marker of Streptomyces Streptomyces sp. MKL-0403. The primers of SEQ ID NO: 2 and SEQ ID NO: 3, and the specific band sequence (SEQ ID NO: 1) can quickly and effectively identify Streptomyces sp. MKL-0403.

[0137] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A polyoxin-producing strain, characterized in that, The polyoxin-producing strain is Streptomyces. Streptomyces sp. MKL-0403, the polyoxin-producing strain, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35576.

2. The polyoxin-producing strain according to claim 1, characterized in that, Satisfy any one or a combination of the following: (1) Contains a molecular marker, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) Genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3. The amplification products were analyzed by agarose gel electrophoresis to obtain specific bands. (3) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3. The amplified products were sequenced and analyzed, and the nucleotide sequence obtained was shown in SEQ ID NO:

1.

3. A molecular marker, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

1.

4. Use of the molecular marker of claim 3 in identifying the polyoxin-producing strain of any one of claims 1-2.

5. Primer pairs, characterized in that, The nucleotide sequence of the forward primer is shown in SEQ ID NO: 2; The nucleotide sequence of the reverse primer is shown in SEQ ID NO:

3.

6. Use of the primer pair according to claim 5 in identifying the polyoxin-producing strain according to any one of claims 1-2.

7. A method for identifying the polyoxin-producing strain according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Sequencing of the polyoxin-producing strain; (2) Obtain a sequence containing a specific molecular marker, wherein the specific molecular marker satisfies the following: 1) The nucleotide sequence is shown in SEQ ID NO:

1.

8. The method according to claim 7, characterized in that, The sequencing data were selected from: (1) Genome sequencing; and / or (2) Sequencing of PCR products.

9. The method according to claim 8, characterized in that, The PCR product was obtained by amplification using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3.

10. Use of the polyoxin-producing strain according to any one of claims 1-2 in the preparation of pesticide products.

11. A pesticide product, characterized in that, It contains the fermentation stock solution, fermentation dilution solution, and fermentation concentrate of the polyoxin-producing strain as described in any one of claims 1-2.

12. The pesticide product according to claim 11, characterized in that, The polyoxin content of the fermentation stock solution, fermentation dilution solution, or fermentation concentrate solution is 10-300000 μg / mL.

13. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of powder, granule, oil, or seed treatment suspension.

14. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from slow-release formulations.

15. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from suspension concentrates.

16. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from suspension seed coating agents.

17. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, and seed treatment microcapsule suspension.

18. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of the following: large granules, fine granules, microparticles, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsion granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, greases, microcapsule suspensions, oil suspensions, and suspension emulsions.

19. A method for preparing the pesticide product according to any one of claims 11-18, characterized in that, The preparation method includes the following steps: Using Streptomyces Streptomyces The pesticide product was obtained by fermentation of sp. MKL-0403.

20. The preparation method according to claim 19, characterized in that, The preparation method includes the following steps: (1) Utilizing Streptomyces Streptomyces The pesticide product is obtained by fermentation with sp. MKL-0403 to obtain the fermentation stock solution; or The preparation method further includes the following steps: (2) Further processing of the fermentation liquid to obtain either fermentation dilution or fermentation concentrate is the pesticide product.

21. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18, or the pesticide product obtained by the method according to any one of claims 19-20 in the prevention and control of plant diseases.

22. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18, or the pesticide product obtained by the method according to any one of claims 19-20 in the preparation of plant disease control agents.

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