Streptomyces strain LZZY-S3 and application thereof

Abstract

The application provides a streptomyces LZZY-S3 and application thereof, and belongs to the field of microorganisms.The streptomyces LZZY-S3 provided by the application enriches strains for biological control, and realizes the dual functions of plant resistance to pathogenic bacteria and plant promotion.The streptomyces LZZY-S3 has the function of inhibiting fungi in a wide range, and can be applied to plant resistance to pathogenic bacteria, and the pathogenic bacteria include Exobasidium vexans, Botryosphaeria spp., Gibberella, Fusarium oxysporum, Pythium, Fusarium graminearum, Didymella, Fusarium pseudograminearum, Trichothecium roseum, Coniella diplodiella, Colletotrichum graminicola or Rhizoctonia cerealis, wherein the inhibition rate of Exobasidium vexans reaches 91.2%.The streptomyces LZZY-S3 can also be applied to plant promotion, preparation of nitrogen-fixing bacterial agents and preparation of ACC deaminase bacterial agents and the like.

Description

Technical Field

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

[0002] Plant pathogenic fungi are the primary cause of crop diseases. Their infection can lead to significant yield reductions, or even complete crop failure in severe cases, posing a serious threat to global food security. Current disease control methods primarily rely on chemical and physical methods. However, chemical control methods are prone to pesticide residues and resistance, posing ecological risks and potential safety hazards to agricultural products. Physical control methods, on the other hand, suffer from high costs and low efficiency. In recent years, with the advancement of ecological civilization, biological control has gradually become the mainstream. Compared with chemical and physical control methods, biological control offers advantages such as environmental friendliness, low cost, and sustainability. Screening for promising microorganisms and developing biopesticides have become important areas of crop disease control, laying the foundation for the realization of green agriculture.

[0003] The genus Streptomyces has attracted much attention in the field of biocontrol research due to its powerful ability to synthesize secondary metabolites. Currently, 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 bioactive 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 biocontrol, it is of great significance to discover some new strains with more comprehensive functions and better effects. Summary of the Invention

[0004] The present invention provides a novel 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 the China Culture Collection Administration, 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; and 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 and production of ACC deaminase bacterial agent.

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

[0013] Furthermore, 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 the China Culture Collection Administration on April 12, 2024, with the deposit number: CGMCC No.30319.

[0016] The Streptomyces LZZY-S3 provided by the present invention has good broad-spectrum inhibitory activity against a variety of plant pathogenic fungi, including the pathogens of tea cake disease (Exobasidium vexans), grape seat disease (Botryosphaeria spp.), wheat head blight (Gibberella), cucumber wilt (Fusarium oxysporum), wheat root rot (Pythium), rice seedling rot (Fusarium graminearum), pumpkin vine blight (Didymella), wheat stem rot (Fusariumpseudograminearum), apple core rot (Trichothecium roseum), grape white rot (Coniella diplodiella), apple anthracnose (Colletotrichum graminicola) or wheat sheath blight (Rhizoctonia cerealis), with the inhibition rate against the tea cake disease pathogen reaching 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 present 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 and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a 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 Figure 2 shows the phospholipid composition analysis of strain LZZY-S3, where a is the result of ninhydrin colorimetry; b is the result of molybdenum blue colorimetry; and c is the result of molybdenum phosphate colorimetry. 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 lipids.

[0023] Figure 4 This is the result of 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 the result of the antagonistic activity test of strain LZZY-S32 against plant pathogens. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific examples, but this 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 are all commercially available.

[0028] Example 1: Strain identification.

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

[0030] (1) Morphological and cultural characteristics.

[0031] Well-growing strain LZZY-S3 was inoculated onto ISP 2 medium using the plate streak method and cultured at 28°C for two weeks. Samples were prepared using the insert method and observed under an optical microscope (Nikon ECLIPSE E200) to determine the morphology of aerial and intrabasal hyphae, spores, and sporangia. Spore and spore chain morphology was further observed using a scanning electron microscope.

[0032] The culture characteristics study used internationally accepted culture media, namely ISP2 medium (its 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 (its 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 (its 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 (its 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 (formulated by weight as follows: peptone 2.0%, sodium thiosulfate 0.008%, yeast extract 0.1%, ferric citrate 0.05%, K2HPO4 0.1%, agar 2.0%, pH 7.0-7.2), wherein the trace solution comprises FeSO4 0.1%, MnCl2 0.1%, and ZnSO4 0.1% by weight. The strain LZZY-S3 was inoculated onto each of the above media using the streak plate method. After culturing at 28°C for one week, the growth of aerial and basal mycelia of LZZY-S3, as well as the production of soluble pigments, was 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 on different media showed vigorous growth on ISP3, moderate growth on ISP4 and ISP6, and weak growth on ISP2 and ISP5. The color of the aerial hyphae ranged from white to light gray, while the color of the matrix hyphae ranged from pale yellow to bright yellow. No soluble pigment was detected on any of the media tested. Specific characteristics are shown in Table 1.

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

[0036] culture medium Growth status Aerial hyphae Mycelium within 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, and pH, temperature, and NaCl tolerance. The testing methods were based on the "Rapid Identification and Systematic Classification of Actinomycetes" (Ruan Jisheng, Huang Ying. Rapid Identification and Systematic Classification of Actinomycetes [J]. Science Press, 2011).

[0039] Results showed that strain LZZY-S3 could grow in the pH range of 6.0-8.0, with an optimum pH of 7.0. It could grow in the temperature range of 20°C-40°C, with an optimum growth temperature of 28°C. Furthermore, strain LZZY-S3 exhibited tolerance to NaCl concentrations between 0% and 5%, with an optimum growth concentration between 0% and 1%. Strain LZZY-S3 tested positive for starch degradation, nitrate reduction, gelatin liquefaction, and urease production, but was negative for milk coagulation, H₂S production, catalase activity, and cellulose degradation. The strain was able to utilize L-asparagine, L-threonine, glycine, L-serine, L-alanine, L-glutamic acid, L-glutamine, L-arginine, and L-aspartic acid as its sole nitrogen source, but was unable to 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 16S rRNA gene of strain LZZY-S3 was amplified by PCR using the genome of strain LZZY-S3 as a template, with primers 5′-AGAGTTTGATCCTGGCTCAG-3′ as the upstream primer and 5′-AAGGAGGTGATCCAGCCGCA-3′ as the downstream primer. The target fragment was recovered using the GeneStar DNA Rapid Gel Extraction Kit. The gel-extracted product was ligated with 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 the 16S rRNA sequence and using the neighbor-joining method further confirmed the classification of LZZY-S3 within the genus Streptomyces. Figure 2 .

[0043] (4) Chemical taxonomic characteristics.

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

[0045] The results showed that the cell wall amino acids of strain LZZY-S3 were LL-diaminopimelic acid. 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 included 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 contained 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 type of fatty acid belonged to type IIc. The quinone group of strain LZZY-S3 consisted of MK-9(H8) (44.74%) and MK-9(H6) (55.26%), as shown in Figure 4 . The above chemical taxonomic characteristics were consistent with the typical characteristics of Streptomyces.

[0046] Example 2: Promoting effect of Streptomyces LZZY-S3 on seed germination.

[0047] Preparation of test strain: The strain LZZY-S3 was inoculated on ISP3 medium by plate streaking method, and cultured at 28°C for 7 days. Then 5 mL of sterile water was added to the culture dish, and the bacterial body was scraped with a sterile cotton swab to obtain a spore suspension. After microscopic counting, the spore suspension was diluted to 1×10 4 , 1×10 5 , 1×10 6 CFU / mL for standby.

[0048] Seed treatment: Select uniformly sized, plump wheat seeds, rinse them with 70% alcohol for 30 seconds, then rinse them five times with sterile water. Soak them in 5% sodium hypochlorite solution for 15 minutes, then rinse them five times with sterile water. Use sterile filter paper to absorb the surface moisture. Place the sterilized seeds in a sterile Petri dish, with 50 wheat seeds per dish. Add 10 mL of bacterial solution of varying concentrations. Add an equal volume of sterile water to the control group. Each treatment has three replicates. Soak the seeds completely in the bacterial solution for 5 hours, then rinse them five times with sterile water. Take two pieces of sterile filter paper, place them in a sterile Petri dish, soak the filter paper with sterile water, lay the seeds flat on the filter paper, and cover with two layers of sterile gauze. Place the Petri dish in an incubator with a relative humidity of 70% to 80% and incubate at 25°C. The germination standard is when the length of the embryonic axis extending from the seed coat reaches 1 / 2 of the seed length. Observe and record the number of seeds germinating at regular intervals every day until the number of seeds germinating remains unchanged. Calculate the germination potential and germination rate of the seeds. 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, increasing by 15.8% and 14.9% respectively.

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

[0052] To investigate the nitrogenase and 1-aminocyclopropane-1-carboxylate deaminase activities of strain LZZY-S3, the strain was inoculated onto Ashby nitrogen-free solid medium (containing, 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 streak plate 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 atmospheric nitrogen 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, thereby reducing ethylene levels, alleviating its inhibitory effect on plant growth, and promoting 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 antagonistic activity of strain LZZY-S3 against plant pathogens was determined using a plate confrontation culture method. The plant pathogens included: Exobasidium vexans, Botryosphaeria spp., Gibberella fusarium, Fusarium oxysporum, Pythium root rot, Fusarium graminearum, Didymella spp., Fusarium umpseudograminearum, Trichothecium roseum, Coniella diplodiella, Colletotrichum graminicola, and Rhizoctonia cerealis. All pathogens were cultured on PDA solid medium at 28°C. Strain LZZY-S3 was inoculated onto the left side of a PDA solid culture medium and cultured at 28°C for 3 days. A bacterial cake was punched out of the pathogen using a hole punch (d = 5 mm) and inoculated onto the right side of the plate containing strain LZZY-S3, aligning the strain LZZY-S3 with the pathogen. A PDA culture medium inoculated with only the bacterial cake was used as a control and cultured at 28°C until the pathogen in the control plate completely covered the entire plate. The inhibition rate of strain LZZY-S3 was calculated. The inhibition rate was calculated as follows:

[0056]

[0057] The results showed that the strain LZZY-S3 was effective against Exobasidium vexans, Botryosphaeria spp., Gibberella spp., Fusarium oxysporum, Pythium spp., Fusarium graminearum, Didymella spp., Fusarium spp., Trichothecium roseum, Coniella diplodiella, Colletotrichum graminicola, Rhizoctonia spp., and Pythium spp. 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, and the highest inhibition rate was found against the pathogen of tea cake disease (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 ( Streptomyces sp. ), characterized in that, The strain was named LZZY-S3 and was deposited in the General Microbiology Center of China 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 plant resistance to pathogenic bacteria, wherein the pathogenic bacteria include: Tea cake disease pathogen ( Exobasidium vexans )‌、Botrytis cinerea ( Botryosphaeria spp. )‌、Wheat fusarium 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 rot ( Fusarium pseudograminearum )‌、Apple core disease pathogen ( Trichothecium roseum )‌, grape white rot pathogen ( Coniella diplodiella )‌、Apple Anthracnose pathogen ( Colletotrichum graminicola ) or wheat sheath blight pathogen ( Rhizoctonia cerealis ).

Images