Streptomyces murinus FJSM-07 and application thereof

Abstract

The invention discloses streptomyces murinus FJSM-07 and an application of the streptomyces murinus FJSM-07. Relates to the technical field of microbial biocontrol. The invention provides a fermentation product and application thereof. The streptomyces FJSM-07 provided by the invention is separated from sea mud, and at present, related researches of the strain are few; a fermentation liquor crude extract and a strain of the streptomyces FJSM-07 have bacteriostatic activity on various plant disease pathogenic fungi, and have the characteristics of low toxicity, low residue, no pollution and difficult generation of drug resistance; the streptomyces FJSM-07 has a good inhibition effect on pathogenic bacteria of corn stalk rot, pathogenic bacteria of leaf spot disease and the like. The streptomyces FJSM-07 is easy to culture, and the fermentation liquor crude extract is easy to extract and prepare. Therefore, the streptomyces FJSM-07 can be easily developed into a biocontrol bacterium preparation, and has a good market application prospect in prevention and treatment of plant fungal diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microbial biocontrol, and more specifically to Streptomyces japonicus FJSM-07 and application thereof. Background Art

[0002] Corn is an important food crop and feed crop, and is also the crop with the highest total output in the world, with its planting area second only to rice and wheat. However, in the process of corn cultivation, pests and diseases have always been a major problem faced by the corn industry.

[0003] Current status and problems of corn pest and disease control technology:

[0004] 1. There are many kinds of pests and diseases, the main ones include corn leaf spot (large and small spot, brown spot, rust, white spot, etc.), stem rot, ear rot, grassland leaf moth, corn borer, aphids and red and yellow spiders, etc. These main pests and diseases, especially corn stem base rot and leaf spot, have complex pathogens and multiple pathogens. The compound infection poses a great threat to the growth and yield of corn. If they are not effectively controlled in time, the corn yield will drop sharply or even fail.

[0005] 2. The prevention and control technology is not advanced enough. Most growers still use traditional methods of fertilization, spraying and weeding. Although these methods are simple and convenient, they are not efficient and environmentally friendly. It is necessary to introduce and promote more advanced prevention and control technologies, such as biological control, plant protection agents and other green prevention and control technologies, to reduce the pollution of chemical pesticides to the environment and the harm to the human body.

[0006] 3. Lack of scientific knowledge. Farmers generally lack a deep understanding of pests and diseases, and lack scientific knowledge of prevention and control. Farmers do not fully understand and master some new pest and disease prevention and control technologies. Therefore, pest and disease infection is still an important reason for the reduction of corn quality and yield, and is one of the main limiting factors restricting the healthy development of my country's corn industry.

[0007] At present, the prevention and control of corn diseases mainly rely on chemical control and disease-resistant varieties, but due to the diversity and differences of corn varieties, it is difficult to achieve targeted variety disease resistance; and chemical control mainly uses triazole fungicides. The long-term application of such preparations can easily lead to the development of pathogen resistance, as well as a series of problems such as pesticide residues, environmental pollution and reduction of soil microbial populations. Biological control has attracted much attention as one of the development directions of plant disease control, but the available biological control strain resources are still relatively small.

[0008] Therefore, it is an urgent problem for technicians in this field to screen out more efficient biocontrol strains that have antagonistic effects on the pathogens of corn stalk rot and leaf spot. Summary of the invention

[0009] In view of this, the present invention provides a Streptomyces japonicus FJSM-07 and applications thereof.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] Preservation information: Streptomyces murinus FJSM-07 is deposited in the General Microbiology Center of China Culture Collection Administration, the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC: NO.25867, and the preservation date is September 30, 2022.

[0012] A Streptomyces murinus FJSM-07, with a deposit number of CGMCC: NO.25867.

[0013] The present invention also provides a fermentation product of the Streptomyces muridarum FJSM-07.

[0014] The present invention also provides an antibacterial agent containing the above-mentioned Streptomyces muridarum FJSM-07 or the fermentation product of claim 2.

[0015] The present invention also provides a pesticide containing the Streptomyces japonicus FJSM-07 or the fermentation product thereof.

[0016] The present invention also provides the use of the above-mentioned antibacterial agent in controlling or preventing plant diseases.

[0017] The invention also provides the use of the pesticide in controlling or preventing plant diseases.

[0018] Preferred: The plant is corn.

[0019] Preferably, the plant disease is a plant pathogenic fungal disease.

[0020] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a Streptomyces muridarum FJSM-07 and its application, and the technical effects achieved are:

[0021] (1) The Streptomyces FJSM-07 provided by the present invention was isolated from sea mud collected from the mangrove tidal flat wetland in Yunxiao County, Zhangzhou City, Fujian Province. There are few reports on the isolation of antagonistic Streptomyces from sea mud and their application in corn disease prevention and control, and there are currently very few related studies on this type of strain;

[0022] (2) The crude extract and strain of the fermentation broth of Streptomyces FJSM-07 have antibacterial activity against a variety of plant pathogenic fungi. Compared with the currently used chemical fungicides, they are low in toxicity, low in residue, non-polluting, and not prone to drug resistance.

[0023] (3) Streptomyces FJSM-07 has a good inhibitory effect on the pathogens of corn stalk rot and leaf spot.

[0024] (4) Streptomyces FJSM-07 is easy to culture, and its fermentation broth is easy to extract and prepare. Therefore, Streptomyces FJSM-07 can be easily developed into a biocontrol agent, which has a good market application prospect for the prevention and control of plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The accompanying drawing is a diagram showing the culture characteristics of the strain FJSM-07 provided by the present invention on PDA medium.

[0027] Figure 2 The attached drawing is a diagram showing the morphological characteristics of conidia of strain FJSM-07 provided by the present invention.

[0028] Figure 3 The accompanying drawing is a phylogenetic tree diagram constructed from the strain FJSM-07 provided by the present invention.

[0029] Figure 4 The accompanying drawings are antibacterial effect diagrams provided by the present invention, wherein A: pathogenic bacterial colony characteristics of corn leaf spot pathogens such as Echinococcus nigricans, Echinococcus kaoliang, Helminthosporium maydis, Alternaria alternata, and Helminthosporium macrospotted; B: antibacterial effect of Streptomyces muridarum FJSM-07 on 5 pathogens. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiment of the invention discloses Streptomyces japonicus FJSM-07 and application thereof.

[0032] Example 1

[0033] Isolation and Culture of Biocontrol Actinomycetes

[0034] 1. Biological samples and culture media: The sample sea mud was collected from the mangrove tidal flat wetland in Yunxiao County, Zhangzhou City, Fujian Province. The collected samples were brought back to the laboratory for isolation and culture of actinomycetes. A total of 6 culture media were selected for strain analysis and identification. The components are shown in Table 1. Nalidixic acid 15 μg / mL, nystatin 50 μg / mL and cycloheximide 50 μg / mL were added to the 6 culture media.

[0035] Table 1 Composition of culture medium

[0036]

[0037] 2. Sample processing: The collected sea mud samples were isolated and cultured using the following processing methods. Processing method: Take 1g of sea mud sample, put it into 4mL of sterile water, heat shock at 55℃ for 10min, shake vigorously, dilute the sample 1:4, take 20μL of the dilution and evenly spread it on 6 isolation culture medium plates, culture at 28℃ for 7d-12d, and wait for actinomycetes to grow.

[0038] 3. Purification and culture of actinomycetes: single colony hyphae grown on the isolation culture medium plate were selected for transfer purification. According to the culture and morphological characteristics of the strains, duplicate strains were removed, and finally 19 marine actinomycete strains were isolated. For the purified strains, hyphae were selected for transfer and inoculation into Gao's No. 1 (formula: soluble starch 20g, KNO31 g, K2HPO40.5 g, MgSO4·7H2O 0.5g, FeSO4·7H2O0.01g, NaCl 0.5g, agar 20g, distilled water 1000mL, pH 7.2-7.4) (slant) culture medium, cultured in a 28℃ constant temperature incubator for 5d-7d, and then stored in a 4℃ refrigerator for later use.

[0039] Example 2

[0040] Screening of Antagonistic Actinomycetes to Biocontrol

[0041] 1. Preparation of indicator bacteria plate: The pathogens of corn stalk rot and leaf spot were isolated from the rhizomes and leaves of diseased corn and belonged to Fusarium verticilliordes and Bipolaris maydis. The preserved pathogens of corn stalk rot and leaf spot were transferred to PDA medium (preparation method: weigh 200g of fresh peeled potatoes, chop them, add 1000mL of water and boil for half an hour, filter with gauze, take the filtrate to 1000mL, add 20g of glucose and 20g of agar, heat until fully dissolved, and then dispense into conical flasks or glass test tubes, 121℃, autoclave for 28min) and cultured in a microbial incubator at 28℃. After the colony produces spores, add 10mL of sterile water to the culture dish to collect the spores. After filtering with sterile gauze, adjust the concentration of the spore suspension to 10 with sterile water. 6 -10 9 spores / mL, and 20 μL of spore suspension of different concentrations was evenly spread on the PDA plate for use (the experiment was repeated 3 times).

[0042] 2. Screening of strains with inhibitory activity: The 19 strains of marine actinomycetes (labeled as 1#-19#) isolated in Example 1 were inoculated on PDA culture medium plates, respectively, and cultured at 28°C for 7 days. A 5mm diameter bacterial cake was taken from the bacterial growth site with a hole puncher and placed in the center of a plate inoculated with indicator bacteria, and then cultured in a microbiological incubator at 28°C. After 48 hours, the presence and size of transparent inhibition zones around the bacterial cakes were observed, and 6 strains with inhibition zones larger than 15 mm were rescreened, namely 3#, 7#, 8#, 11#, 15#, and 17# (Table 2).

[0043] Through the above method, a strain with strong inhibitory activity against corn stalk rot and leaf spot pathogens was screened out from the 19 isolated marine actinomycetes, numbered 07# (Table 2), and named Streptomyces FJSM-07.

[0044] Table 2 Inhibitory effects of biocontrol strains on maize stalk rot and leaf spot pathogens

[0045]

[0046] Example 3

[0047] Cultivation and morphological, physiological and biochemical characteristics of Streptomyces FJSM-07

[0048] 1. Culture and morphological characteristics of strain FJSM-07: Six internationally used culture media were used, namely inorganic salt starch agar (ISP-4, formula: soluble starch 10g, K2HPO41g, MgSO4·7H2O1g, NaCl 1g, (NH4)2SO42g, CaCO32g, FeSO4·7H2O 0.001g, MnCl2·7H2O0.001g, ZnSO4·7H2O 0.001g, agar 20g, distilled water 1000mL, pH 7.0-7.4), glucose asparagine agar (ISP-5, formula: L-aspartic acid 1g, glucose 10g, K2HPO41g, trace element solution 1mL, agar 20g, distilled water 1000mL, pH 7.2, trace element solution: FeSO4·7H2O0.1g, MnCl2·4H2O 0.1g, ZnSO4·7H2O 0.1g), oatmeal agar (ISP-3, formula: oatmeal 20g, agar 20g, trace element solution 1mL, pH 7.2, trace element solution: FeSO4·7H2O 0.1g, MnCl2·4H2O 0.1g, ZnSO4·7H2O 0.1g), yeast extract maltose agar (ISP-2, formula: yeast extract 4g, malt extract 10g, glucose 4g, agar 20g, distilled water 1000mL, pH 7.0), nutrient agar (NA, formula: peptone 10g, beef extract 3g, sodium chloride 5g, agar 20g, distilled water 1000mL, pH 7.3) and glycerol asparagine agar (formula: L-asparagine 1g, K2HPO41 g, trace salt solution 1mL, glycerol 10g, agar 20g, distilled water 1000mL, pH 7.0-7.4), inoculate strain FJSM-07 onto the above culture medium, and then place it in an inverted incubator at 28℃ for culture. After 7d, observe the growth of aerial hyphae, intrabasal hyphae and the production of soluble pigments of FJSM-07.

[0049] The results showed that strain FJSM-07 could grow on all six culture media. Except for the absence of aerial mycelium and less matrix mycelium on nutrient agar, the other media produced aerial mycelium and matrix mycelium. No soluble pigment was produced on the six culture media (Table 3). Strain FJSM-07 grew well on PDA medium ( Figure 1 ), the spores are loose, regular short spirals, sometimes only in the initial spiral, and the spore surface is covered with fine hairs up to 2 μm long ( Figure 2 ).

[0050] Table 3 Culture and morphological characteristics of strains

[0051]

[0052] 2. Physiological and biochemical characteristics of strain FJSM-07: Referring to the methods described in "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng, Huang Ying. Rapid Identification and Systematic Classification of Actinomycetes [M]. Beijing: Science Press, 2011) and "Streptomyces Identification Manual" (Actinomycetes Classification Group, Institute of Microbiology, Chinese Academy of Sciences. Streptomyces Identification Manual [M]. Beijing: Science Press, 1975), the physiological and biochemical characteristics of strain FJSM-07, such as carbon source utilization, melanin production, H2S production, gelatin liquefaction, starch hydrolysis, milk coagulation peptoneization and other indicators were measured.

[0053] The results showed that in the test of physiological and biochemical characteristics, strain FJSM-07 could utilize D-glucose, D-fructose, L-arabinose, D-xylose, sucrose, galactose, maltose and glycerol as carbon sources; it could not utilize L-rhamnose, sucrose, raffinose, D-mannitol, mannose and lactose; it could hydrolyze starch, coagulate and peptone milk and reduce nitrate, but could not liquefy gelatin and did not produce melanin and tyrosinase (Table 4).

[0054] Table 4 Physiological and biochemical characteristics of strains

[0055]

[0056] In summary, through culture and detection and analysis of morphological characteristics, physiological and biochemical characteristics, Streptomyces FJSM-07 was preliminarily identified as Streptomyces murinus.

[0057] Example 4

[0058] 16S rDNA Sequence Analysis of Streptomyces FJSM-07

[0059] The strain FJSM-07 was cultured in Gao's No. 1 liquid medium (soluble starch 20g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, distilled water 1L, pH 7.4-7.6) for 4-5 days, and the bacterial solution was collected after centrifugation in a sterile centrifuge tube. 0.1g-0.2g of bacterial cells were weighed in a sterile mortar, and a small amount of PVP was added and ground into powder with liquid nitrogen. The total bacterial DNA was extracted using an actinomycete genome extraction kit. The universal primer 27F:

[0060] 5′-AGAGTTTGATCCTGGCTCAG-3′, 1541R: 5′-AAGGAGGTGATCCAGCCGCA-3′ to amplify actinomycete 16S rDNA.

[0061] The PCR product was loaded into an agarose gel containing GelRed dye and the target band was recovered after electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The measured sequences were compared and analyzed using NCBI Blast, and the 16S rDNA sequences of strains with high homology in GenBank were selected and multiple alignments were performed using Clustal X software to analyze the sequence similarity between strain FJSM-07 and the reference strains, and the phylogenetic tree was constructed using the Neighborjoining method in Mega4.1 software ( Figure 3 ).

[0062] The results showed that the 16S rDNA sequence of Streptomyces FJSM-07 was subjected to Blast comparison analysis on the website of the National Center for Biotechnology Information (NCBI) of the United States. It was found that the measured sequence had high homology with multiple species of Streptomyces in Genbank, among which it had the highest homology with Streptomyces murinus (GeneBank accession numbers: NR_112445, NR_041072 and OQ561298), and the sequence similarity reached 100%.

[0063] Based on the preliminary identification results of strain FJSM-07 based on culture, morphological characteristics, physiological and biochemical characteristics, Streptomyces FJSM-07 was finally identified as Streptomyces murinus and then deposited through patent procedures.

[0064] Example 5

[0065] Preparation of crude extract from fermentation broth of Streptomyces FJSM-07

[0066] 1. Activation culture of strains: The isolated and preserved Streptomyces FJSM-07 was transferred to Gao's medium No. 1 plate and cultured in a 28°C incubator for 7 days.

[0067] 2. Fermentation culture of the strain: Take the Streptomyces FJSM-07 activated on Gao's medium No. 1, inoculate it into Gao's liquid medium No. 1, and ferment and culture it at 28°C with shaking at 200 rpm / min for 6 days.

[0068] 3. Preparation of crude extract of fermentation broth: Take the fermentation broth of shaking culture, filter it through sterile filter paper, and obtain the supernatant and mycelium respectively. Take the supernatant, extract it with ethyl acetate three times, combine the extracts, and obtain the ethyl acetate extract of the supernatant. After soaking the mycelium in 80% acetone aqueous solution overnight, use ultrasonic crushing to extract three times, combine the extracts, and concentrate the extracts under reduced pressure until there is no acetone. The obtained aqueous phase is extracted three times with an equal volume of ethyl acetate to obtain the ethyl acetate extract of the mycelium. Combine the supernatant and the ethyl acetate extract of the mycelium, and concentrate them under reduced pressure to dryness to obtain the ethyl acetate extract of the strain. Weigh 1g of the extract, add 1mL of dimethyl sulfoxide to dissolve it, and then add sterile water to make up to 10mL, which is the crude extract of Streptomyces FJSM-07 fermentation broth.

[0069] Example 6

[0070] Determination of the antibacterial spectrum of Streptomyces FJSM-07

[0071] 1. Test strains and culture medium: The plant pathogens tested include the pathogens of corn stalk rot (Fusarium verticilliordes, Fusarium solani, Fusarium oxysporum, Colletotrichum gloeosporioides), the pathogen of corn leaf spot Epicoccum nigrum, the pathogen of corn leaf spot Micrococcus sorghum, the pathogen of corn leaf spot Bipolaris maydis, the pathogen of corn leaf spot Helminthodes panicularis, and the pathogen of corn leaf spot Exserohilum turcicum. The culture medium used for culturing plant pathogens is PDA culture medium (its preparation method is shown in Example 2).

[0072] 2. Activation of test strains: Heat and dissolve the PDA culture medium and pour it into a sterile culture dish with a diameter of 9 cm to prepare a PDA culture medium plate. Use a sterile inoculation needle to pick up the plant pathogenic bacteria blocks stored in the slant culture medium in a 4°C refrigerator, inoculate them on the PDA culture medium plate, and place it in a 28°C constant temperature incubator for culture and activation.

[0073] 3. Antibacterial effect of strain FJSM-07 on five kinds of corn leaf spot pathogens: The plate confrontation method was used to select the corn leaf spot pathogens Echinococcus nigrofaciens, Echinococcus kaoliang, Helicoverpa zeae, Alternaria alternata, and Helicoverpa maxima for the antibacterial spectrum experiment of corn leaf spot pathogens. After 7 days of confrontation culture, the results showed that strain FJSM-07 had excellent antibacterial effect ( Figure 4 ,Table 5).

[0074] Table 5 Inhibitory bandwidth (7d) of strain FJSM-07 against five maize leaf spot pathogens

[0075]

[0076]

[0077] 4. Determination of the antibacterial activity of the crude extract of strain FJSM-07: Four plant pathogens, including Fusarium oxysporum, Fusarium solani, Fusarium oxysporum, and Colletotrichum gloeosporioides, were used as target bacteria, and the antibacterial spectrum of Streptomyces FJSM-07 was screened by the punching method. A 5mm diameter target bacterial cake was taken with a puncher and placed 2.5cm away from the center of the culture dish. Then a hole was punched in the center of the PDA plate mixed with the target bacteria. 200μL of sterile fermentation broth crude extract was added to the hole, and the plates were placed in a HGZ-150 type light incubator at (28±1)℃ and a photoperiod of 12L:12D. The size of the inhibition zone was determined after 6 days, and the experiment was repeated 3 times. The test results showed that strain FJSM-07 had a strong inhibitory effect on Fusarium oxysporum and Fusarium gloeosporum, and also had a strong inhibitory effect on Fusarium solani and Colletotrichum gloeosporioides (Table 6).

[0078] Table 6 Inhibitory effect of crude extract of Streptomyces FJSM-07 fermentation broth on plant pathogens

[0079]

[0080] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0081] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Streptomyces murinus FJSM-07, characterized in that: The deposit number is: CGMCC: NO.25867.

2. The fermentation product of Streptomyces muridarum FJSM-07 according to claim 1.

3. An antibacterial agent containing the Streptomyces muridarum FJSM-07 according to claim 1 or the fermentation product according to claim 2.

4. A pesticide containing the Streptomyces muridarum FJSM-07 according to claim 1 or the fermentation product according to claim 2.

5. Use of the antibacterial agent according to claim 3 in controlling or preventing plant diseases.

6. Use of the pesticide according to claim 4 in controlling or preventing plant diseases.

7. The use according to claim 5 or 6, characterized in that The plant is corn.

8. The use according to claim 7, characterized in that The plant diseases are plant pathogenic fungal diseases.

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