Streptomyces sp.Zm3-3, microbial agent and application of microbial agent

Abstract

The invention discloses Streptomyces sp.Zm3-3, a microbial agent and application of the microbial agent, and belongs to the technical field of microorganisms. According to the invention, a strain of Streptomyces sp.Zm3-3 is separated and screened from corn leaves, and the strain can antagonize the growth of hyphae of Zea maydis and curvularia lunata. Indoor potting and field control effect determination results show that the control effect of the Zm3-3 fungicide on corn southern leaf blight and corn curvularia leaf spot exceeds 70%, and the control effect on corn rust exceeds 60%. The Zm3-3 microbial inoculum and the fermentation filtrate thereof can digest hyphae of Zea maydis and curvularia lunata and inhibit germination of conidia of pathogenic bacteria. The biocontrol microbial inoculum prepared from the strain can replace or reduce the use of chemical pesticides, accords with the new idea of green plant protection development, is beneficial to guarantee of food and ecological safety, and has good economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a Streptomyces sp. Zm3-3, a microbial agent and applications thereof. Background Art

[0002] Corn is my country's largest crop and a vital feed and raw material. In 2024, the national corn planting area reached 673 million mu (approximately 1.67 billion hectares), with a production of 589.83 billion jin (approximately 1.6 billion kg). In addition, 13.64 million tons of corn were imported. my country has a large population and limited land, resulting in a severe shortage of arable land reserves, and food supply and demand remain in a long-term "tight balance." In 2024, corn diseases affected an estimated 260 to 290 million mu (approximately 1.6 to 2.7 hectares) of land nationwide, posing a serious threat to food production.

[0003] Leaf spot is an important disease of corn, including southern rust, large leaf spot, small leaf spot, gray leaf spot, Curvularia leaf spot, brown leaf spot, etc. Among them, small leaf spot Curvularia leaf spot and southern rust occur year-round in the summer-sown corn areas of Huanghuaihai in my country, causing serious damage and difficult to prevent and control. Corn small leaf spot is caused by Helicoverpa maydis and can occur from seedling to adult stage. The disease is mild in the seedling stage and gradually worsens after the corn tasting. In 2022, the occurrence area in Henan Province alone reached 8 million mu. Corn Curvularia leaf spot is a disease that broke out in the summer-sown corn areas of Huanghuaihai after the 1990s. In the early stage of infection, small chlorotic spots appear on the leaves, which gradually expand into round or oval chlorotic transparent plates with withered white to yellow-brown in the middle. After the corn tasting, it quickly spreads and causes the leaves to dry up. Southern corn rust broke out in the Huanghuai and Haihe River basins in 2015, 2017, 2021, and 2023, and has become a major disease of summer corn in these regions. It was added to my country's list of Class I crop diseases in 2023. Due to changing climate conditions, the narrow resistance spectrum of popular corn varieties, and weak overall resistance to multiple leaf spot diseases, corn leaf spot has been prevalent year after year, seriously impacting grain yield and quality.

[0004] With growing awareness of environmental protection and a growing pursuit of a green lifestyle, biological control has garnered increasing attention due to its environmentally friendly and residue-free properties. Furthermore, biological control has the potential to offset the adverse effects of chemical fungicides on non-target organisms (Soo et al., 2020), making it a crucial measure for crop disease control in recent years. While there has been extensive research and reports on antagonistic microorganisms against Fusarium diseases such as corn stalk rot and ear rot, biocontrol efforts targeting corn leaf spot, particularly those simultaneously antagonizing multiple leaf spot diseases, are relatively limited, primarily focusing on Bacillus species. Xie Lanfen et al. identified a strain of Bacillus amyloliquefaciens B9601-Y2 that secretes iturin and polyketide antibiotics, antagonizing the growth of both northern and southern leaf spot pathogens, demonstrating its potential as a biocontrol agent against corn leaf spot. Chen Yali et al. found that a composite microbial agent containing three effective biocontrol bacteria, Bacillus subtilis, Bacillus amyloliquefaciens and Paenibacillus gelatinosa, can effectively prevent and control corn leaf blight and corn rust. In addition, Trichoderma harzianum powder has a good preventive effect on corn leaf blight, and kasugamycin water-dispersible granules have a good preventive effect on corn rust.

[0005] In order to further improve the comprehensive prevention and control effect of major corn leaf spot diseases, it is crucial to develop green prevention and control technology for corn leaf spot diseases and screen microorganisms with good prevention and control effects on corn leaf spot diseases. Summary of the Invention

[0006] The purpose of the present invention is to provide a Streptomyces sp. Zm3-3, a microbial agent and applications thereof, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a Streptomyces sp. Zm3-3, which was deposited in the China Center for Type Culture Collection on January 14, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: M2025121.

[0009] The second technical solution of the present invention is a microbial agent, including the Streptomyces sp. Zm3-3.

[0010] The third technical solution of the present invention is the use of the Streptomyces sp. Zm3-3 or the microbial agent in preventing and controlling plant leaf spot diseases.

[0011] The fourth technical solution of the present invention is the use of the Streptomyces sp. Zm3-3 or the microbial agent in the preparation of products for preventing and treating plant leaf spot diseases.

[0012] The fifth technical solution of the present invention is a product for preventing and treating plant leaf spot disease, comprising the Streptomycess p. Zm3-3 or the microbial agent.

[0013] The sixth technical solution of the present invention is a method for preventing and controlling plant leaf spot disease, which comprises spraying a product containing the Streptomyces p. Zm3-3 or the microbial agent during the seedling and trumpeting stages of corn.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] 1. Currently, no endophytic Streptomyces rocheri strains have been shown to simultaneously control corn leaf spot caused by Helicoverpa maydis, Curvularia lunata, and corn rust caused by Puccinia maydis. The Streptomyces rocheri strain Zm3-3 in the present invention is isolated from corn leaves, which facilitates its colonization and activity on corn leaves.

[0016] 2. Streptomyces rocheri Zm3-3 of the present invention significantly antagonized the growth of Helminthosporium maydis and Curvularia lunata. It demonstrated excellent control efficacy against corn leaf spot, with efficacy rates of 72.45% in indoor potted plants and 70.73% in the field. It also demonstrated excellent control efficacy against corn Curvularia leaf spot, with efficacy rates of 84.63% in indoor potted plants and 74.73% in the field. It also demonstrated excellent control efficacy against corn rust, with efficacy rates of 64.28% in indoor potted plants and 60.95% in the field.

[0017] 3. The genome of Streptomyces louchelii Zm3-3 of the present invention contains abundant glycosyl hydrolase genes and secondary metabolite gene clusters. The bacterial agent and metabolites can decompose the hyphae of Helicoverpa maydis and Curvularia lunata and inhibit spore germination.

[0018] 4. The biocontrol agent prepared using the Bacillus cereus of the present invention can replace or reduce the use of chemical pesticides, improve food and ecological environmental safety, meet the strategic requirements of green plant protection, and have good economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is the inhibitory effect of Zm3-3 strain on fungal growth.

[0021] Figure 2Shown are the colony morphology and physiological and biochemical characteristics of strain Zm3-3.

[0022] Figure 3 This is a phylogenetic tree constructed based on the 16S rDNA sequences of strain Zm3-3 and other Streptomyces.

[0023] Figure 4 This is the control effect of Zm3-3 fungicide on corn leaf spot in indoor potted plants.

[0024] Figure 5 The control effect of Zm3-3 fungicide on corn Curvularia leaf spot in indoor potted plants.

[0025] Figure 6 This is the control effect of Zm3-3 fungicide on corn rust in indoor potted plants.

[0026] Figure 7 This is the effect of Zm3-3 inoculant and its fermentation filtrate on the spore germination and hyphae of Helminthosporium maydis.

[0027] Figure 8 The effect of Zm3-3 inoculant and its fermentation filtrate on the spore germination and mycelium of Curvularia lunata.

[0028] Figure 9 This is the circle map of the genome of strain Zm3-3.

[0029] Figure 10 This is a phylogenetic tree constructed based on the average amino acid identity (AAI) and average nucleotide identity (ANI) of the Zm3-3 strain and other Streptomyces.

[0030] Figure 11 This is the GO functional annotation classification result of the genes of Zm3-3 strain.

[0031] Figure 12 The KEGG pathway enrichment analysis results of Zm3-3 strain genes.

[0032] Figure 13 This is the identification result of the carbohydrate active enzyme (CAZy) gene of the Zm3-3 strain. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0039] The present invention provides a strain of Streptomyces sp. Zm3-3, which was deposited in the China Center for Type Culture Collection on January 14, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2025121.

[0040] The embodiment of the present invention also provides a microbial agent, including the Streptomyces sp. Zm3-3.

[0041] The embodiments of the present invention also provide the use of the Streptomyces sp. Zm3-3 or the microbial agent in preventing and treating plant leaf spot diseases.

[0042] In some specific embodiments, the plant leaf spot diseases include southern blight, Curvularia leaf spot, and rust; and the plant includes corn.

[0043] In some specific embodiments, the leaf spot disease includes the leaf spot disease caused by Helicoverpa maydis; the Curvularia leaf spot disease includes the Curvularia leaf spot disease caused by Curvularia lunata; and the rust disease includes the rust disease caused by Puccinia maydis uredospores.

[0044] The embodiments of the present invention also provide the use of the Streptomyces sp. Zm3-3 or the microbial agent in preparing a product for preventing and treating plant leaf spot disease.

[0045] In some specific embodiments, the plant leaf spot diseases include southern blight, Curvularia leaf spot, and rust; and the plant includes corn.

[0046] In some specific embodiments, the leaf spot disease includes the leaf spot disease caused by Helicoverpa maydis; the Curvularia leaf spot disease includes the Curvularia leaf spot disease caused by Curvularia lunata; and the rust disease includes the rust disease caused by Puccinia maydis uredospores.

[0047] An embodiment of the present invention further provides a product for preventing and treating plant leaf spot disease, comprising the Streptomyces p. Zm3-3 or the microbial agent.

[0048] In some specific embodiments, the plant leaf spot diseases include small spot disease, Curvularia leaf spot and rust disease; the plant includes corn; the small spot disease includes small spot disease caused by Helicoverpa maydis; the Curvularia leaf spot disease includes Curvularia leaf spot disease caused by Curvularia lunata; the rust disease includes rust disease caused by uredia spores of Puccinia maydis.

[0049] The present invention also provides a method for preparing a biocontrol agent, comprising the following steps: inoculating Streptomyces rocherii Zm3-3 into Gao's No. 1 liquid culture medium, shaking at 25°C and 150 rpm for 7 days, and obtaining a fermented bacterial liquid, which is the biocontrol agent. The Gao's No. 1 liquid culture medium comprises: 20 g / L soluble starch, 1 g / L potassium nitrate (KNO3), 0.5 g / L dipotassium hydrogen phosphate (K2HPO4), 0.5 g / L sodium chloride (NaCl), 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O, and a pH of 7.0.

[0050] An embodiment of the present invention also provides a method for preventing and controlling plant leaf spot disease, which comprises spraying a product containing the Streptomyces p. Zm3-3 or the microbial agent during the seedling and trumpeting stages of corn.

[0051] The present invention isolated and screened an endophytic strain of Streptomyces rochei Zm3-3 from corn leaves. This strain is capable of antagonizing the mycelial growth of Helicoverpa maydis and Curvularia lunata. Zm3-3 was prepared using Gao's No. 1 culture medium. Indoor potted plant and field tests showed that Zm3-3 demonstrated over 70% efficacy against corn leaf spot and Curvularia leaf spot, and over 60% efficacy against corn rust. Zm3-3 and its fermentation filtrate were able to disintegrate the mycelia of Helicoverpa maydis and Curvularia lunata and inhibit the germination of conidia of the pathogens. Genomic analysis of the Zm3-3 strain further confirmed its membership in the Streptomyces rochei strain, with 141 glycoside hydrolase genes and 18 secondary metabolite gene clusters identified in its genome. The biocontrol agent prepared using the strain of the present invention can replace or reduce the use of chemical pesticides, conforms to the new method of developing green plant protection, is beneficial to ensuring food and ecological safety, and has good economic and social benefits.

[0052] Example 1

[0053] Obtaining the antagonistic microorganism Zm3-3 strain

[0054] The following helminth spores of maize, Curvularia lunata, helminth spores of wheat root decay, Rhizoctonia graminearum and Fusarium graminearum used were all isolated, identified and preserved by the Innovation Team of Grain Crop Pest Monitoring and Control at Henan Agricultural University.

[0055] Corn seeds (Zhengdan 958) were surface disinfected and germinated, then planted in sterile soil and cultured at 25°C with 16 h light / 8 h dark for 10 days. Corn leaves were surface disinfected, cut into small pieces approximately 3 mm long, and placed on LB medium. The culture was incubated at 25°C in the dark, and 200 bacterial colonies were screened.

[0056] The pathogens of corn leaf spot (Bipolaris sorokiniana) and Curvularia lunata were activated on PDA plates. A 5 mm diameter bacterial cake was then taken from the edge of the colony and placed in the center of the PDA medium. Symmetrical streaks of bacteria were made 2.5 cm from the bacterial cake. A control group was streaked with LB at the same location. Each treatment was replicated three times. Incubation was performed at 25°C in the dark until the control colonies exceeded half of the plate. The colony diameters of the treated and control groups were measured, and the inhibition rate was calculated.

[0057] Inhibition rate = (colony diameter of control group - colony diameter of treated group) / colony diameter of control group × 100%.

[0058] like Figure 1As shown in FIG, a microorganism with significant antagonistic activity against the growth of Helminthosporium maydis and Curvularia lunata was screened and named Zm3-3 ( Figure 1 In addition, Zm3-3 had a certain inhibitory effect on the growth of Bipolarissorokiniana and Rhizoctonia Cerealis, but had no antagonistic effect on the growth of Fusarium graminearum.

[0059] Example 2

[0060] Identification of the antagonistic microorganism strain Zm3-3

[0061] Compared with LB medium, Zm3-3 grows better on Gao's medium No. 1. Figure 2 As shown, strain Zm3-3 was grown on Gao's medium No. 1 at 25°C in the dark for 3 days. The bacteria were tightly attached to the medium and difficult to pick up. The single colony was round, about 2 mm in diameter, with a grayish-white surface, irregular edges, and microvilli. Physiological and biochemical tests showed that strain Zm3-3 could produce melanin, β-1,3-glucanase, amylase, and cellulase, while H2S production, gelatin liquefaction, methyl red test, and nitrate reduction test were all negative ( Figure 2 ).

[0062] Compared with LB medium, Zm3-3 grows better on Gao's medium No. 1. Figure 2 As shown, strain Zm3-3 was grown on Gao's medium No. 1 at 25°C in the dark for 3 days. The bacteria were tightly attached to the medium and difficult to pick up. The single colony was round, about 2 mm in diameter, with a grayish-white surface, irregular edges, and microvilli. Physiological and biochemical tests showed that strain Zm3-3 could produce melanin, β-1,3-glucanase, amylase, and cellulase, while H2S production, gelatin liquefaction, methyl red test, and nitrate reduction test were all negative ( Figure 2 ).

[0063] Using Zm3-3 genomic DNA as a template, PCR amplification with primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) yielded a 1454-bp 16S rDNA sequence. A Blastn search on NCBI revealed a 99% sequence similarity to the 16S rDNA of Streptomyces rocherii. A phylogenetic tree was subsequently constructed, showing ( Figure 3), strain Zm3-3 was in the same branch as Streptomyces rochei. Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence comparison, strain Zm3-3 was finally identified as Streptomyces rochei.

[0064] This example provides a Streptomyces sp. Zm3-3 strain, which was deposited with the China Center for Type Culture Collection on January 14, 2025, at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M2025121.

[0065] Example 3

[0066] Preparation of Streptomyces rocherii Zm3-3 bacterial agent

[0067] The strain Zm3-3 was activated on Gao's No. 1 solid culture medium. After 3 days, a single colony was picked with a toothpick and inoculated into Gao's No. 1 liquid culture medium. The culture was shaken at 25°C and 150 rpm for 4 days. The resulting fermentation liquid was the biocontrol agent of the strain Zm3-3.

[0068] Gao's solid culture medium / liquid formula No. 1 is: 20g soluble starch, 1g potassium nitrate (KNO3), 0.5g potassium dihydrogen phosphate (K2HPO4), 0.5g sodium chloride (NaCl), 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 15g agar powder, and distilled water is added to 1L; if agar powder is not added, it is culture medium.

[0069] Example 4

[0070] Effect of Zm3-3 fungicide on corn leaf spot in potted plants

[0071] Preparation of spore solution of Zea mays: Use a 5mm puncher to punch 3-5 fresh fungus cakes on the edge of the activated Zea mays colony, put them into 300g sterile sorghum culture medium, and culture them in the dark at 25℃ for 5-7d. During the culture period, pay attention to shake and mix every day to avoid caking of sorghum culture medium and uneven mycelial expansion. Culture until the sorghum grains are covered with mycelium. After washing with sterile water, spread the sorghum grains flat on a sterile tray and cover with moist gauze to keep them moist. Culture them in the dark at 25℃ until a large number of conidia are produced. Rinse the spores on the sorghum grains with sterile water before inoculation, filter and make a concentration of 1×10 5 Spore suspension with a concentration of 100 μg / mL.

[0072] Indoor inoculation of corn leaf blight: Zhengdan 958 corn seeds were washed three times with distilled water and incubated using the moisturizing germination method until the seeds germinated. The germinated corn seeds were sown into sterile culture medium and incubated in a greenhouse at 25°C with 16 hours of light and 8 hours of darkness for 10 days. Watering was done every three days. When the corn reached the three-leaf stage, the leaves were sprayed with the biocontrol agent Zm3-3. The control group was sprayed with an equal volume of Gao's No. 1 liquid culture medium. Two days later, each corn plant was sprayed with 3-5 mL of a prepared spore suspension of Helminthosporium maydis, ensuring moisture retention. Incubation continued until clear necrotic lesions formed on the leaves of the control group. The occurrence of corn leaf blight was photographed and investigated. The data were recorded and the disease index and biocontrol efficacy were calculated. The experiment was repeated five times, with at least five replicates per test.

[0073] like Figure 4 The results in Table 1 show that the corn leaves in the control group had numerous necrotic lesions, with an average disease index of 36.00. However, the corn leaves sprayed with the Zm3-3 biocontrol agent showed almost no necrotic lesions, with only a few small white spots, and the average disease index dropped to 9.92. The Zm3-3 biocontrol agent achieved a 72.45% efficacy against southern corn leaf blight. Compared to the control group, the corn treated with the Zm3-3 biocontrol agent also showed significant increases in plant height and fresh weight. This demonstrates that strain Zm3-3 has a highly effective control effect against southern corn leaf blight.

[0074] Table 1 The control effect of Zm3-3 fungicide on corn leaf spot in indoor potted plants

[0075]

[0076] Example 5

[0077] Control Effect of Zm3-3 Microbial Agent on Curvularia Leaf Spot of Corn

[0078] Preparation of spore solution of C. lunata: Use a 5mm puncher to punch 3-5 fresh fungus cakes at the edge of the activated C. lunata colony and inoculate them into 300g sterile sorghum culture medium. Culture in the dark at 25℃ for 5-7 days. During the culture period, pay attention to shaking and mixing every day to avoid caking of sorghum culture medium and uneven mycelial expansion. Culture until all sorghum grains are covered with mycelia. After rinsing with sterile water, spread the sorghum grains on a clean stainless steel tray and cover with moist gauze to keep them moist. Culture at 25℃ in the dark and keep them moist until a large number of conidia are produced. Rinse the spores on the sorghum grains with sterile water before inoculation, filter and prepare a concentration of 1×10 5 Spore suspension with a concentration of 100 μg / mL.

[0079] Indoor inoculation of corn Curvularia leaf spot: Zhengdan 958 corn seeds were washed three times with distilled water, germinated using the moist germination method, and then sown into sterile culture medium. The seeds were incubated in a greenhouse at 25°C with 16 hours of light and 8 hours of darkness for 10 days, watered every three days. When the corn reached the three-leaf stage, the leaves were sprayed with the biocontrol agent Zm3-3. The control group was sprayed with an equal amount of Gao's No. 1 liquid culture medium. Two days later, each corn plant was inoculated with 3-5 mL of a prepared spore suspension of Curvularia lunata. Incubation continued until distinct necrotic lesions formed on the control corn leaves. The occurrence of corn leaf spot was photographed and investigated. The data were recorded and the disease index and biocontrol efficacy were calculated. The experiment was repeated five times, with at least five replicates per experiment.

[0080] like Figure 5 The results in Table 2 show that the control group had numerous white, circular lesions on corn leaves, with a disease index of 21.67. However, corn leaves sprayed with the Zm3-3 biocontrol agent in advance had very few white, circular lesions, with a disease index of only 3.33. The Zm3-3 biocontrol agent demonstrated an 84.63% efficacy against Curvularia leaf spot in maize. Compared to the control group, corn plants sprayed with Zm3-3 exhibited significantly increased plant height and fresh weight. This demonstrates that strain Zm3-3 has a significant control effect against Curvularia leaf spot in maize.

[0081] Table 2 The control effect of Zm3-3 fungicide on corn leaf spot in indoor potted plants

[0082]

[0083] Example 6

[0084] The control effect of Zm3-3 microbial agent on corn rust

[0085] Zhengdan 958 corn seeds were washed three times with distilled water, germinated using the moist germination method, and then sown into sterile culture medium. The seeds were incubated in a greenhouse at 25°C under 16 h light / 8 h darkness for 10 days, watered every three days. At the three-leaf stage, the leaves were sprayed with the biocontrol agent Zm3-3. A control group was sprayed with an equal volume of Gaoshi No. 1 liquid culture medium. Two days later, corn rust was inoculated: fresh uredus spores of Puccinia maydis (isolated and identified by the Innovation Team for Monitoring and Control of Grain Crop Diseases and Insect Pests at Henan Agricultural University and propagated using the Zhengdan 958 variety) were gently scraped from the inoculated corn leaves with a toothpick. The collected uredus spores were collected by rotating the toothpick into a 2 mL centrifuge tube. The collected uredus spores were mixed with 0.01% Tween and 20-40 μL of the spore suspension was pipetted onto the leaf surface with the tip gently touching it (being careful not to scratch it). The spore suspension was then slowly dispensed from the leaf base to the tip, moving the pipette tip forward. A fine mist of water was sprayed from above using a watering can, evenly applied to the inoculated leaf surfaces. After moisturizing, the corn plants were placed in a sealed moisturizing box (humidity above 80%, with mist sprayed on all sides, leaving as much water as possible at the bottom) and kept moisturized at 25°C in the dark for 24 hours. The corn seedlings were then cultured in a greenhouse at 25°C with a 16-hour light / 8-hour dark cycle. When new uredia formed on the leaves of the control corn, photographs were taken and the occurrence of corn rust was investigated. The data were recorded and the disease index and control efficacy were calculated. The experiment was repeated five times, with at least five replicates per test.

[0086] like Figure 6 The results in Table 3 show that numerous yellow, circular lesions formed on corn leaves in the control group, some of which had already developed clear uredia. The average disease index was 38.89. Leaves inoculated with the rust fungus after spraying with the Zm3-3 biocontrol agent showed only a few small, chlorotic spots and uredia, and the disease index dropped to 13.89. The Zm3-3 biocontrol agent demonstrated a 64.28% efficacy against corn rust. Compared to the control group, the fresh weight of corn plants sprayed with Zm3-3 increased. This demonstrates that strain Zm3-3 has a strong control effect against corn rust.

[0087] Table 3 The control effect of Zm3-3 fungicide on corn rust in indoor potted plants

[0088]

[0089] Example 7

[0090] Field control effect of Zm3-3 microbial agent on corn leaf spot, Curvularia leaf spot and rust

[0091] The test was conducted in Xiangfu District, Kaifeng City, Henan Province, using a wheat-corn year-round planting model. Base fertilizer and topdressing were applied according to routine procedures. No fungicides were used during the entire growing period except for the test.

[0092] Corn variety: Zhengdan 958, density is 5,000 plants / mu.

[0093] A biocontrol agent was prepared by mixing Zm3-3 fermentation broth with sterile water at a ratio of 1:5. This agent was sprayed on windless evenings during the corn season. A control group was sprayed with Gao's No. 1 culture medium at the same concentration. Five days later, on windless evenings, spores of Helminthosporium maydis, Curvularia lunata, and Uredia pyralis were sprayed. Twenty corn plants were inoculated with each pathogen in the control and treatment groups. Disease incidence was assessed at the end of August according to the disease classification criteria (Table 4), and the disease index and control efficacy were calculated.

[0094] Table 4 Classification of disease levels of different corn leaf spot diseases

[0095]

[0096] The results showed that compared with the control group, the spraying treatment with Zm3-3 strain significantly reduced the disease index of corn leaf spot, corn Curvularia leaf spot and corn rust, with the control effects being 70.73, 74.73 and 60.95, respectively (Table 5).

[0097] Table 5 Field control effect of Zm3-3 fungicide on different corn leaf spot diseases

[0098]

[0099] Example 8

[0100] Prepare the Zm3-3 bacterial agent with reference to Example 3, and obtain the fermentation filtrate of Zm3-3 after filtering with a bacterial filter. Prepare the spore suspension of Zea maydis with reference to Example 4, and prepare the spore suspension of Curvularia lunata with reference to Example 5. Mix the Zea maydis spore suspension or Curvularia lunata spore suspension with the Zm3-3 bacterial agent or fermentation filtrate in a 1:1 ratio, and mix the fungal spore suspension with Gao's No. 1 culture medium in a 1:1 ratio as a control. Place the culture in a 25°C incubator with shaded light, and after the conidia in the control group have fully germinated, observe the germination of Zea maydis spores and Curvularia lunata spores under a microscope. The experiment was repeated three times.

[0101] Three pieces of cake were collected from the edge of an activated Helminthosporium maydis or Curvularia lunata colony and placed in 5 mL of Zm3-3 inoculum or fermentation filtrate. A control group consisted of three fresh Helminthosporium maydis or Curvularia lunata cakes placed in 5 mL of liquid Gao's No. 1 medium. The mixture was incubated in a darkened incubator at 25°C for 12 hours, and the mycelial morphology was observed under a microscope. This experiment was repeated three times.

[0102] like Figure 7As shown, spores of the control group of Helminthosporium maydis germinated and had intact hyphae. However, spores treated with the Zm3-3 inoculant failed to germinate, and many spore tips were disintegrated. The hyphae treated with the Zm3-3 inoculant were fragmented and darkened. Spores of the Helminthosporium maydis treated with the Zm3-3 fermentation filtrate formed bulges at the tips but were unable to produce further hyphae. The hyphae treated with the Zm3-3 fermentation filtrate also disintegrated into fragments and darkened.

[0103] like Figure 8 As shown, spores of C. lunata in the control group germinated and had intact hyphae. However, spores treated with the Zm3-3 inoculant failed to germinate. The hyphae treated with the Zm3-3 inoculant were fragmented and darkened. Some C. lunata spores treated with the Zm3-3 fermentation filtrate germinated, but the germ tube length was significantly shortened. The hyphae treated with the Zm3-3 fermentation filtrate also fragmented and darkened.

[0104] Example 9

[0105] Genome sequencing and functional gene analysis of Zm3-3 strain

[0106] Zm3-3 bacteria were collected and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for whole-genome sequencing. De novo sequencing was performed using the Illumina HiSeq × 10 platform. DNA samples that passed quality control were sequenced using inserts ranging from 0 to 400 bp. PE150 (pair-end sequencing) was performed, resulting in the assembly of multiple genome scaffolds. Prodigal software was used to predict the genome coding sequence, CGView software was used to construct the genome circle map, and the antiSMASH online tool was used to rapidly identify, annotate, and analyze gene clusters involved in secondary metabolite synthesis.

[0107] The genome of strain Zm3-3 was precisely assembled and the genome circle map was drawn ( Figure 9 ), with a genome size of 8,376,602 bp, 77 scaffolds, an average GC content of 72.45%, and 7,388 annotated coding genes. A phylogenetic tree was constructed based on average amino acid identity (AAI) and average nucleotide identity (ANI). Strain Zm3-3 had the highest identity with Streptomyces rocheri, with 99.14 and 99.15 ( Figure 10 ).

[0108] GO functional annotation analysis showed that a total of 3407 genes in the Zm3-3 strain genome were successfully classified, mainly distributed in three functional categories: cellular component (CC, 1307 genes), molecular function (MF, 2809 genes) and biological process (BP, 1544 genes) ( Figure 11 KEGG pathway analysis showed that a total of 4933 genes in the Zm3-3 strain genome were annotated to multiple metabolic and regulatory pathways, including metabolic pathways such as secondary metabolite biosynthesis, xenobiotic degradation and metabolism, glycan biosynthesis and metabolism, as well as pathways related to cellular processes and environmental information processing ( Figure 12 ).

[0109] Metabolic system analysis revealed that the Zm3-3 strain genome contained abundant carbohydrate active enzymes and secondary metabolite synthesis gene clusters. Figure 13 As shown in the figure, 141 glycoside hydrolases were identified in the Zm3-3 genome, which may be related to the function of Zm3-3 in digesting pathogenic fungi. In addition, 18 metabolic gene clusters (similarity ≥ 50%) were identified in the Zm3-3 genome, including 4 terpenes, 4 NRPSs, 2 T2PKSs, 2 T1PKSs, 2 lanthipeptides, 1 ectoine, 1 indole, 1 siderophore, and 1 RiPP (Table 6).

[0110] Table 6 Prediction of secondary metabolite synthesis gene clusters in the genome of strain Zm3-3

[0111]

[0112] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A Streptomyces sp. Zm3-3, characterized in that: This strain was deposited in the China Center for Type Culture Collection on January 14, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 2025121.

2. A microbial agent, characterized in that: The invention comprises the Streptomyces sp. Zm3-3 according to claim 1.

3. Use of Streptomyces sp. Zm3-3 according to claim 1 or the microbial agent according to claim 2 in preventing and controlling plant leaf spot disease.

4. The use according to claim 3, characterized in that The plant leaf spot diseases include leaf spot, Curvularia leaf spot and rust; and the plant includes corn.

5. The use according to claim 4, characterized in that The leaf spot disease includes the leaf spot disease caused by Helicoverpa maydis; the Curvularia leaf spot disease includes the Curvularia leaf spot disease caused by Curvularia lunata; and the rust disease includes the rust disease caused by the uredospores of Puccinia maydis.

6. Use of the Streptomyces sp. Zm3-3 according to claim 1 or the microbial agent according to claim 2 in the preparation of a product for preventing and treating plant leaf spot disease.

7. The use according to claim 6, characterized in that The plant leaf spot diseases include leaf spot, Curvularia leaf spot and rust; and the plant includes corn.

8. The use according to claim 6, characterized in that The leaf spot disease includes the leaf spot disease caused by Helicoverpa maydis; the Curvularia leaf spot disease includes the Curvularia leaf spot disease caused by Curvularia lunata; and the rust disease includes the rust disease caused by the uredospores of Puccinia maydis.

9. A product for preventing and treating plant leaf spot disease, characterized in that: It includes the Streptomycess p.Zm3-3 according to claim 1 or the microbial agent according to claim 2.

10. A method for preventing and controlling plant leaf spot disease, characterized in that: A product comprising the Streptomycess p. Zm3-3 according to claim 1 or the microbial agent according to claim 2 is sprayed on corn at the seedling stage and the bell-opening stage.

Images