Streptomyces LZZY-S3 and application thereof

Abstract

The invention provides streptomyces LZZY-S3 and application thereof, and belongs to the field of microorganisms. The streptomyces LZZY-S3 provided by the invention enriches strains for biological control, and realizes the dual functions of plant pathogenic bacterium resistance and plant growth promotion. The streptomyces LZZY-S3 has a broad-spectrum fungus inhibition function and can be applied to plant pathogenic bacterium resistance; pathogenic bacteria include: European tea leaf disease pathogen, Botryosphaeria spp., Gibberella zeae pathogen, fusarium oxysporum pathogen, wheat root rot pathogen, rice seedling rot pathogen, pumpkin gummy stem blight pathogen, wheat stem rot pathogen, Trichothecium roseum, grape white rot pathogen, and Colletotrichum graminum pathogen. The pathogenic bacteria include: European tea leaf disease pathogen, Botryosphaeria spp. Wherein the inhibition rate of tea cake disease germs reaches 91.2%. The streptomyces LZZY-S3 can also be applied to plant growth promotion, preparation of nitrogen-fixing bacterial agents, preparation and production of ACC deaminase bacterial agents and the like.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Streptomyces with broad-spectrum antibacterial activity and application thereof. Background Art

[0002] Plant pathogenic fungi are the main cause of crop diseases. Their infection can cause a significant reduction in yield, and in severe cases even lead to crop failure, which seriously threatens global food security. Current disease control mainly relies on chemical and physical methods. However, chemical control is prone to produce pesticide residues and drug resistance problems, causing ecological risks and hidden dangers to the quality and safety of agricultural products. Physical control has disadvantages such as high cost and low efficiency. In recent years, with the advancement of ecological civilization construction, biological control has gradually become the mainstream. Compared with chemical and physical control, biological control has the advantages of being environmentally friendly, low cost and sustainable. Screening microorganisms with application potential and developing fungicides have become an important direction for crop disease control, laying the foundation for the realization of green agriculture.

[0003] Streptomyces has attracted much attention in the field of biological control research due to its powerful ability to synthesize secondary metabolites. At present, the secondary metabolites synthesized by Streptomyces account for about 80% of the natural metabolites of actinomycetes, of which there are more than 100,000 antibiotic compounds, accounting for 70%-80% of agricultural natural biological active substances. In addition, studies have shown that Streptomyces can also promote plant seed germination and plant growth and development through nitrogen fixation, reducing ethylene content and effectively alleviating abiotic stress. However, there are relatively few existing Streptomyces strains with broad-spectrum antifungal activity and growth-promoting functions. In order to enrich the strain library for biological control, it is of great significance to explore some new strains with more comprehensive functions and better effects. Summary of the invention

[0004] The invention provides a novel strain of Streptomyces LZZY-S3, which enriches the strains used for biological control and achieves the dual functions of inhibiting plant pathogens and promoting plant growth.

[0005] A Streptomyces strain, named Streptomyces sp.LZZY-S3, was deposited on April 12, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration, located in Beijing, China, with the deposit number: CGMCC No.30319.

[0006] Application of strain LZZY-S3 in plant growth promotion.

[0007] Furthermore, the plant includes wheat.

[0008] Furthermore, the concentration of the strain LZZY-S3 is 10 5CFU / mL.

[0009] Furthermore, the culture medium used by the strain LZZY-S3 is ISP3; the ISP3 culture medium includes, by mass percentage, 2.0% oatmeal powder, 0.1% trace element solution and 2.0% agar; the trace element solution includes, by mass percentage, 0.1% FeSO4, 0.1% MnCl2 and 0.1% ZnSO4; the pH of the culture medium is 7.0-7.2.

[0010] Application of strain LZZY-S3 in the preparation of nitrogen-fixing bacterial agent.

[0011] Application of strain LZZY-S3 in the preparation of bacterial agent for producing ACC deaminase.

[0012] Application of strain LZZY-S3 in plant resistance to pathogens.

[0013] Further, the pathogens include: tea cake disease pathogen (Exobasidium vexans), grape seat disease pathogen (Botryosphaeria spp.), wheat fusarium wilt pathogen (Gibberella), cucumber wilt pathogen (Fusarium oxysporum), wheat root rot pathogen (Pythium), rice seedling rot pathogen (Fusarium graminearum), pumpkin vine blight pathogen (Didymella), wheat stem base rot pathogen (Fusariumpseudograminearum), apple rotten heart pathogen (Trichothecium roseum), grape white rot pathogen (Coniella diplodiella), apple anthracnose pathogen (Colletotrichum graminicola) or wheat sheath blight pathogen (Rhizoctonia cerealis).

[0014] Beneficial Effects

[0015] The present invention provides a novel strain of Streptomyces LZZY-S3, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 12, 2024, with the deposit number: CGMCC No.30319.

[0016] The streptomyces LZZY-S3 provided by the invention has good broad-spectrum inhibitory activity against a variety of plant pathogenic fungi, including tea cake disease pathogen (Exobasidium vexans), grape seat disease pathogen (Botryosphaeria spp.), wheat head blight pathogen (Gibberella), cucumber wilt pathogen (Fusarium oxysporum), wheat root rot pathogen (Pythium), rice seedling rot pathogen (Fusarium graminearum), pumpkin vine blight pathogen (Didymella), wheat stem base rot (Fusariumpseudograminearum), apple rotten heart disease pathogen (Trichothecium roseum), grape white rot pathogen (Coniella diplodiella), apple anthracnose pathogen (Colletotrichum graminicola) or wheat sheath blight pathogen (Rhizoctonia cerealis), wherein the inhibition rate of tea cake disease pathogen reaches 91.2%.

[0017] The Streptomyces LZZY-S3 provided by the invention can promote the germination of wheat seeds.

[0018] The Streptomyces LZZY-S3 provided by the invention can produce ACC deaminase and nitrogenase, and has the potential to promote plant nitrogen fixation and plant growth.

[0019] The Streptomyces LZZY-S3 provided by the present invention has the advantages of high antibacterial activity, environmental friendliness, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the scanning electron micrograph of strain LZZY-S3;

[0021] Figure 2 This is a neighbor-joining phylogenetic tree constructed based on 16S rRNA gene sequences;

[0022] Figure 3 The phospholipid composition analysis of strain LZZY-S3, where a is the result of the ninhydrin colorimetric method; b is the result of the molybdenum blue colorimetric method; c is the result of the molybdenum phosphate colorimetric method; Chromatographic system: one-dimensional (chloroform: methanol: water = 65:25:4, v / v); two-dimensional (chloroform: acetic acid: methanol: water = 80:18:12:5, v / v). DPG: diphosphatidylglycerol; PE: phosphatidylethanolamine; PME: phosphatidylmethylethanolamine; PL1-2: unknown lipid;

[0023] Figure 4 This is the result of the quinone group composition analysis of strain LZZY-S3;

[0024] Figure 5 This is the result diagram of the effect of different concentrations of bacterial solution on wheat seed germination;

[0025] Figure 6 This is the result of nitrogenase and ACC deaminase activity test of strain LZZY-S3;

[0026] Figure 7 This is a graph showing the results of the antagonistic activity test of strain LZZY-S32 against plant pathogens. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific examples, but it should not be construed as limiting the scope of the present invention. The materials, reagents, methods and instruments used in the following examples, unless otherwise specified, are conventional materials, reagents, methods and instruments in the art and can be obtained through commercial channels.

[0028] Example 1: Strain identification.

[0029] The strain LZZY-S32 was registered and deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on April 12, 2024, with the deposit number: CGMCC No.30319.

[0030] (1) Morphological and cultural characteristics.

[0031] The well-growing strain LZZY-S3 was inoculated into ISP 2 medium by plate streaking method and cultured at 28°C for 2 weeks. The samples were prepared by insert method and the morphology of aerial and intrabasal hyphae, spores and sporangium structures were observed under optical microscope (Nikon ECLIPSE E200). The morphology of spores / spore chains was further observed by scanning electron microscope.

[0032] The culture characteristics study used internationally accepted culture media, namely ISP2 medium (whose formula is as follows in mass percentage: yeast extract powder 0.4%, malt extract powder 1%, glucose 0.4%, agar 2.0%, pH 7.0-7.2), ISP3 medium (whose formula is as follows in mass percentage: oatmeal 2.0%, trace element solution 0.1%, agar 2.0%, pH 7.0-7.2), ISP4 medium (whose formula is as follows in mass percentage: soluble starch 1.0%, (NH4)2SO4 0.1%, NaCl 0.1%, K2HPO4 0.1%, CaCO3 0.2%, trace element solution 0.1%, agar 2.0%, pH 7.0-7.2), ISP5 medium (whose formula is as follows in mass percentage: glucose 1.0%, K2HPO4 0.1%, asparagine 0.1%, trace element solution 0.1%, glycerol 1.0%, agar 2.0%, pH 7.0-7.2) and ISP6 medium (whose formula is as follows: peptone 2.0%, sodium thiosulfate 0.008%, yeast powder 0.1%, ferric citrate amine 0.05%, K2HPO4 0.1%, agar 2.0%, pH 7.0-7.2), wherein the components of the trace solution are as follows: FeSO4 0.1%, MnCl2 0.1%, ZnSO4 0.1% by mass percentage. The strain LZZY-S3 was inoculated into the above culture medium by plate streaking method, and after culturing at 28°C for 1 week, the growth of aerial hyphae and base hyphae of LZZY-S3 and the production of soluble pigments were statistically observed.

[0033] The morphology of spores / spore chains was observed by scanning electron microscopy. Figure 1 , strain LZZY-S3 exhibited well-developed branched stroma hyphae and produced aerial hyphae that differentiated into linear or spiral spore chains. These chains consisted of cylindrical spores (0.55-0.81 μm × 0.75-1.22 μm) with a rough surface.

[0034] The growth of strain LZZY-S3 in different media was vigorous on ISP3 medium, moderate on ISP4 and ISP6 medium, and weak on ISP2 and ISP5 medium. The color of aerial hyphae ranged from white to light gray, while the color of matrix hyphae ranged from light yellow to bright yellow. No soluble pigments were detected on all tested media, and the specific characteristics are shown in Table 1.

[0035] Table 1 Culture characteristics of strain LZZY-S3

[0036] Culture medium Growth status Aerial hyphae Mycelium in the base Soluble pigments ISP2 Weaker none Bright Yellow none ISP3 good Light Gray Light yellow none ISP4 medium white Light yellow none ISP5 Weaker none Light yellow none ISP6 medium white Dark yellow none

[0037] (2) Physiological and biochemical characteristics.

[0038] The physiological and biochemical characteristics of strain LZZY-S3 were tested, including carbon and nitrogen source utilization, H2S production, catalase activity, gelatin liquefaction, starch hydrolysis, milk coagulation, cellulose hydrolysis, urease synthesis, nitrate reduction, pH, temperature and NaCl tolerance range. The detection method is based on "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng, Huang Ying. Rapid Identification and Systematic Classification of Actinomycetes [J]. Science Press, 2011.).

[0039] The results showed that strain LZZY-S3 could grow in the pH range of 6.0-8.0, with an optimum pH of 7.0. Strain LZZY-S3 could grow in the range of 20℃-40℃, with an optimum growth temperature of 28℃. In addition, strain LZZY-S3 showed tolerance to NaCl concentrations of 0%-5%, with an optimum growth concentration of 0%-1%. Strain LZZY-S3 was positive for starch degradation, nitrate reduction, gelatin liquefaction, and urease production, but negative for milk coagulation, H2S production, catalase activity, and cellulose degradation. The strain was able to use L-asparagine, L-threonine, glycine, L-serine, L-alanine, L-glutamic acid, L-glutamine, L-arginine, and L-aspartic acid as the sole nitrogen source, but could not utilize creatine, L-proline, or L-tyrosine. In terms of carbon metabolism, strain LZZY-S3 was able to utilize D-fructose, D-galactose, D-glucose, D-mannitol, D-maltose, D-mannose, D-raffinose, L-rhamnose, and D-sucrose as sole carbon sources. However, it was unable to utilize L-arabinose, D-sorbitol, D-ribose, lactose, D-xylose, or meso-inositol.

[0040] (3) Molecular biological characteristics.

[0041] The genome of strain LZZY-S3 was used as a template, and 5′-AGAGTTTGATCCTGGCTCAG-3′ was used as an upstream primer; 5′-AAGGAGGTGATCCAGCCGCA-3′ was used as a downstream primer to amplify its 16S rRNA gene by PCR. The target fragment was recovered using GeneStar's DNA Rapid Gel Recovery Kit. The gel recovery product was connected to the PMD-19T vector, transformed, and the plasmid was extracted and sequenced.

[0042] The 16S rRNA gene sequence of strain LZZY-S3 is shown in SEQ ID No. 1. EzBioCloud analysis showed that strain LZZY-S3 belongs to the genus Streptomyces. Phylogenetic analysis based on 16S rRNA sequences and using the neighbor-joining method further confirmed the classification of LZZY-S3 in the genus Streptomyces. The results are shown in Figure 2 .

[0043] (4) Chemical classification characteristics.

[0044] The chemical classification characteristics of strain LZZY-S3 were analyzed, including the composition of cell wall amino acids, phospholipids, fatty acids and quinone groups. The analysis method was based on "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng, Huang Ying. Rapid Identification and Systematic Classification of Actinomycetes [J]. Science Press, 2011.).

[0045] The results showed that the cell wall amino acid of strain LZZY-S3 was LL-diaminopimelicacid. Its phospholipids mainly included diphosphoglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylmethylethanolamine (PME) and two unknown types of phospholipids (PL) ( Figure 3 ). The fatty acids of the strain mainly include anteiso-C 15:0 (17.15%), iso-C 16:0 (16.15%), anteiso-C 17:0 (13.82%) and iso-C 15:0 (10.61%). In addition, it also contains a small amount of iso-C 16:0 (9.72%), iso-C17:0(9.35%), C18:1ω9c(2.82%), C15:0(1.87%), anteiso-C17:1w9c(1.56%), iso-C14:0(1.52%) and C17:0cyclo(1.19%), and the fatty acid type belongs to type IIc. The quinone composition of strain LZZY-S3 is MK-9(H8)(44.74%) and MK-9(H6)(55.26%), as shown in Figure 4 The above chemical classification characteristics are consistent with the typical characteristics of Streptomyces.

[0046] Example 2: Effect of Streptomyces LZZY-S3 on promoting seed germination.

[0047] Preparation of test strains: strain LZZY-S3 was inoculated on ISP3 medium by plate streaking method. After culturing at 28°C for 7 days, 5 mL of sterile water was added to the culture dish and the bacteria were scraped with a sterile cotton swab to obtain a spore suspension. After counting under a microscope, the spore suspension was diluted to 1×10 4 , 1×10 5 , 1×10 6 CFU / mL is reserved.

[0048] Seed treatment: Select wheat seeds of uniform size and fullness, rinse with 70% alcohol for 30 seconds and then rinse with sterile water 5 times. Soak in 5% sodium hypochlorite solution for 15 minutes and then rinse with sterile water 5 times, and use sterile filter paper to absorb the surface moisture. Put the disinfected seeds into sterile culture dishes, put 50 wheat seeds in each dish, and then add 10mL of bacterial solution of different concentrations respectively. Add the same volume of sterile water to the control group, and set 3 replicates for each treatment. Soak the seeds completely in the bacterial solution, and rinse with sterile water 5 times after soaking for 5 hours. Take two sterile filter papers, place them in a sterile culture dish, soak the filter paper with sterile water, spread the seeds on the filter paper, and cover them with 2 layers of sterile gauze. Place the culture dish in an incubator with a relative humidity of 70% to 80% and culture at a constant temperature of 25°C. The germination standard is that the length of the embryo axis extending out of the seed coat reaches 1 / 2 of the seed length. The number of seeds germinating is observed and recorded regularly every day until the number of seeds germinating does not change, and the germination potential and germination rate of the seeds are calculated. The calculation formula is as follows:

[0049]

[0050] The results are as follows Figure 5 As shown, the concentration is 10 4 and 10 6 CFU / mL spore suspension had no significant effect on seed germination rate and germination potential. 5 CFU / mL, the germination potential and germination rate of wheat seeds were significantly improved compared with the control group, by 15.8% and 14.9% respectively.

[0051] Example 3: Nitrogenase and 1-aminocarbonyl-1-cyclopropanecarboxylic acid (ACC) deaminase activities of Streptomyces LZZY-S3.

[0052] In order to study the nitrogenase and 1-aminocyclopropane-1-carboxylate deaminase activities of strain LZZY-S3, the strain was inoculated into Ashby nitrogen-free solid medium (including, by mass percentage, 1% mannitol, 0.02% KH2PO4, 0.02% MgSO4, 0.02% NaCl, 0.02% CaSO4, 0.01% CaCO3, 2% agar, pH 7.2) and ADF solid medium (LEAGENE) by the plate streak method and cultured at 28°C for 7 days.

[0053] The results are as follows Figure 6As shown, strain LZZY-S3 showed good growth on both Ashby nitrogen-free solid medium and ADF medium. This indicates that the strain has both nitrogenase and ACC deaminase activities. Studies have shown that soil microorganisms use nitrogenase to convert nitrogen in the atmosphere into ammonia or ammonium ions that are easily absorbed by plants, thereby promoting plant nitrogen fixation and supporting plant growth. In addition, 1-aminocarbonyl-1-cyclopropanecarboxylic acid (ACC) is a precursor of ethylene, and the accumulation of ethylene inhibits plant growth. Soil microorganisms produce ACC deaminase (ACCD), which degrades ACC and thus reduces ethylene levels, alleviates its inhibitory effect on plant growth, and promotes plant growth. These results indicate that strain LZZY-S3 has the potential to promote plant growth.

[0054] Example 4: Inhibitory effect of Streptomyces LZZY-S3 on plant pathogens.

[0055] The plate confrontation culture method was used to determine the antagonistic effect of strain LZZY-S3 on plant pathogens. The plant pathogens include: tea cake disease pathogen (Exobasidium vexans), grape cavity disease pathogen (Botryosphaeria spp.), wheat fusarium rust pathogen (Gibberella), cucumber wilt pathogen (Fusarium oxysporum), wheat root rot pathogen (Pythium), rice rot seedling disease pathogen (Fusarium graminearum), pumpkin vine blight pathogen (Didymella), wheat stem base rot pathogen (Fusariumpseudograminearum), apple rot heart disease pathogen (Trichothecium roseum), grape white rot pathogen (Coniella diplodiella), apple anthracnose pathogen (Colletotrichum graminicola), wheat sheath blight pathogen (Rhizoctonia cerealis), all pathogens were cultured on PDA solid medium at a constant temperature of 28℃ for use. The strain LZZY-S3 was inoculated on the left side of the PDA solid culture medium and cultured at 28°C for 3 days. The pathogen was punched out with a hole puncher (d = 5mm) to make a cake, and inoculated on the right side of the plate containing the strain LZZY-S3, so that the strain LZZY-S3 was opposite to the above pathogen. At the same time, the PDA culture medium inoculated with only the cake was used as a control, and was placed at a constant temperature of 28°C to culture until the pathogenic microorganisms in the control plate covered the entire plate, and the antibacterial rate of the strain LZZY-S3 was calculated. The calculation formula for the antibacterial rate is as follows:

[0056]

[0057] The results showed that strain LZZY-S3 was effective against Exobasidium vexans, Botryosphaeria spp., Gibberella, Fusarium oxysporum, Pythium, Fusarium graminearum, Didymella, Fusariumpseudograminearum, Trichothecium roseum, Coniella diplodiella, Colletotrichum graminicola, Rhizoctonia wilt, and Exobasidium vexans. cerealis) were 91.20%, 55.70%, 58.95%, 63.70%, 54.20%, 64.60%, 73.30%, 56.10%, 89.40%, 56.55%, 69.75% and 60.25% respectively. The highest inhibition rate was against Exobasidium vexans ( Figure 7 ). These findings indicate that strain LZZY-S3 has good application value and prospects as a biocontrol strain.

Claims

1. A Streptomyces strain, characterized in that The strain was named LZZY-S3 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 12, 2024, with the deposit number: CGMCC No.30319.

2. Use of the strain LZZY-S3 according to claim 1 in promoting plant growth.

3. The application according to claim 2, characterized in that: The plants include wheat.

4. The application according to claim 2, characterized in that: The concentration of the strain LZZY-S3 was 10 5 CFU / mL.

5. The application according to claim 2, characterized in that: The culture medium used for the strain LZZY-S3 is ISP3; The ISP3 culture medium comprises, by mass percentage, 2.0% oatmeal powder, 0.1% trace element solution and 2.0% agar; the trace element solution comprises, by mass percentage, 0.1% FeSO4, 0.1% MnCl2 and 0.1% ZnSO4; the pH of the culture medium is 7.0-7.

2.

6. Use of the strain LZZY-S3 according to claim 1 in the preparation of a nitrogen-fixing bacterial agent.

7. Use of the strain LZZY-S3 according to claim 1 in the preparation of a bacterial agent for producing ACC deaminase.

8. Use of the strain LZZY-S3 according to claim 1 in plant resistance to pathogenic bacteria.

9. The use according to claim 8, characterized in that: The pathogens include: tea cake disease pathogen (Exobasidiumvexans), grape seat disease pathogen (Botryosphaeria spp.), wheat fusarium wilt pathogen (Gibberella), cucumber wilt pathogen (Fusarium oxysporum), wheat root rot pathogen (Pythium), rice seedling rot pathogen (Fusariumgraminearum), pumpkin vine blight pathogen (Didymella), wheat stem base rot pathogen (Fusariumpseudograminearum), apple rotten heart pathogen (Trichothecium roseum), grape white rot pathogen (Coniella diplodiella), apple anthracnose pathogen (Colletotrichum graminicola) or wheat sheath blight pathogen (Rhizoctonia cerealis).

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