Actinomycete strain YC3041 and application thereof in preparation of biological agent for preventing and treating tobacco phytophthora root rot

Abstract

The invention discloses an actinomycete strain YC3041 and application of the actinomycete strain YC3041 in preparation of a biological preparation for preventing and treating tobacco phytophthora root rot. The actinomycete strain YC3041 is classified and named as Streptomyces pratensis; the strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC), and the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou; the preservation date is July 4, 2024; the preservation number of the strain is GDMCC No: 64828. Physiological and biochemical tests show that the YC3041 has the capabilities of gelatin liquefaction, milk dissolution and the like, can utilize various glycogens, and has the potassium dissolving capability. The YC3041 also has a certain inhibition effect on other pathogenic bacteria, such as YC1026 and YC1265. The invention provides a novel efficient biocontrol strain YC3041 for biological control of phytophthora root rot of tobacco, and the strain has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial and plant disease biological control, and in particular to an actinomycete strain YC3041 and application thereof in preparing a biological agent for controlling tobacco blight. Background Art

[0002] Tobacco is an important economic crop. However, during tobacco cultivation, a pathogen called Phytophthora nicotianae often poses a serious threat to tobacco growth. Phytophthora nicotianae is a soil-borne disease that attacks various parts of the tobacco plant, including the roots, stems, and leaves, causing a range of symptoms. In the early stages of the disease, the roots of the tobacco plant gradually turn brown and rot, while the leaves aboveground show yellowing and withering, slowing growth, and the plant becoming stunted. As the disease worsens, black rotten patches may appear on the stems, eventually causing the plant to fall over and die. Once infected with Phytophthora nicotianae, tobacco yields are significantly reduced, and quality also deteriorates. This results in heavy economic losses for tobacco growers and impacts the stable development of the entire tobacco industry.

[0003] Faced with the severe damage caused by tobacco blight, the main control methods currently include chemical pesticides and biological control. While chemical pesticides can quickly control the disease in the short term, this approach is a double-edged sword, with long-term use leading to numerous negative consequences. On the one hand, the extensive use of chemical pesticides can lead to environmental pollution, with residual pesticides entering the soil and water, disrupting the ecological balance and affecting the survival of other organisms. On the other hand, pathogens can easily develop resistance to chemical pesticides, resulting in a gradual decline in their effectiveness. The continued increase in pesticide use further exacerbates environmental pollution and pesticide residue problems. In this context, biological control, owing to its environmental, safety, and sustainability advantages, is gaining increasing attention.

[0004] The core of biological control is the use of antagonistic microorganisms to inhibit the growth of pathogens. Currently, the most studied biocontrol bacteria include Trichoderma, Bacillus, and Pseudomonas. These biocontrol bacteria work through various mechanisms, such as producing antimicrobial substances that directly inhibit pathogen growth, competing with pathogens for nutrients and living space, or inducing resistance responses in plants to enhance their ability to resist pathogens. However, these existing biocontrol strains still have limitations in practical application.

[0005] For example, the antibacterial effect of some biocontrol bacteria is not ideal, the inhibition rate against tobacco blight is low, and they cannot effectively control the disease; some biocontrol bacteria have poor stability under different environmental conditions, and are greatly affected by factors such as temperature, humidity, and soil type, making it difficult to maintain stable prevention and control effects in various complex environments; in addition, some biocontrol bacteria have limited adaptability and can only work in specific tobacco-growing areas or under specific planting conditions, and cannot meet the needs of widespread promotion and application.

[0006] Therefore, the search for an environmentally friendly, stable and long-acting actinomycete strain YC3041 and its application in the preparation of biological agents for controlling tobacco blight is of great practical significance for improving the level of biological control of tobacco blight. Summary of the Invention

[0007] The purpose of the present invention is to provide an environmentally friendly, stable and long-acting actinomycete strain YC3041 and its application in preparing a biological agent for preventing and treating tobacco blight.

[0008] The technical solution of the present invention is as follows: The present invention provides the following technical solution: In the first aspect, the present application provides an actinomycete strain YC3041.

[0009] In a second aspect, the present application provides an actinomycete strain YC3041 agent.

[0010] In a third aspect, the present application provides an application of an actinomycete strain YC3041 in the preparation of a biological agent for preventing and treating tobacco blight.

[0011] The first aspect of the present application provides an actinomycete strain YC3041, which is classified and named Streptomyces pratensis; it is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is July 4, 2024; the deposit number is GDMCC No: 64828.

[0012] Furthermore, the actinomycete strain YC3041 appeared off-white on different culture media, the aerial hyphae were off-white, the matrix mycelium was yellow to yellow-brown, and produced yellow soluble pigments; the surface dried after 7 days of culture on ISP2 medium; the hyphae were small and curved under an optical microscope; and the spore hyphae were loosely spiral under a scanning electron microscope, the spores were spherical or oval, with a smooth surface and straight threads.

[0013] In a second aspect, the present application provides an actinomycete strain YC3041 inoculum, the active ingredient of which is at least one of the following (a), (b), and (c): (a) Fermentation culture of actinomycete strain YC3041; (b) Supernatant of sonication lysis of actinomycete strain YC3041 cells; (c) Ultrasonic lysis pellet of actinomycete strain YC3041 cells.

[0014] A third aspect of the present application provides an application of an actinomycete strain YC3041 in the preparation of a biological agent for preventing and treating tobacco blight.

[0015] Furthermore, the biological preparation includes a culture of the YC3041 strain or a fermentation product thereof. The biological pesticide also includes an adjuvant that can promote the growth of YC3041 or enhance its antibacterial effect.

[0016] Beneficial effects: The actinomycete strain YC3041 of the present invention has a significant inhibitory effect on tobacco blight, with an inhibition rate of 68.63%. It can effectively curb the growth of tobacco blight, reduce the incidence of the disease, and reduce the yield and quality losses caused by tobacco blight.

[0017] Compared with the prior art, the present invention has the following advantages: (1) Advantages of biological control: As a biocontrol bacterium, YC3041 is more environmentally friendly and safer than chemical pesticides. It does not pollute the environment and does not cause pathogens to develop drug resistance, which is in line with the development concept of sustainable agriculture.

[0018] (2) Excellent strain characteristics: YC3041 of the present invention has stable growth characteristics on different culture media, good morphological characteristics and physiological and biochemical activities, and is easy to cultivate and apply. It has the ability to dissolve potassium, which can promote plant growth and enhance plant resistance to pathogens.

[0019] (3) Broad antimicrobial spectrum: In addition to its inhibitory effect on tobacco blight, YC3041 also has a certain inhibitory effect on other pathogens such as YC1026 and YC1265, indicating that it has a broad antimicrobial spectrum and can provide a wider application prospect for the prevention and control of diseases of tobacco and other crops.

[0020] (4) Clear identification: Through multiple identification methods such as morphological characteristics observation, 16S rRNA and rpoB gene sequence analysis, YC3041 was clearly identified as Streptomyces praecox. The identification result is reliable and provides accurate strain information for subsequent promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0022] Figure 1 This is a diagram showing the antagonistic effect of the biocontrol bacteria of the present invention on pathogenic bacteria.

[0023] Figure 2 The figures show the culture morphology and microscopic morphology of the strain YC3041 of the present invention on ISP2 medium. Figure 2 a is a colony morphology (front view) of YC3041 of the present invention on ISP2 medium; Figure 2 b is a morphological diagram (reverse side) of YC3041 of the present invention grown on ISP2 medium; Figure 2 c is the optical microscope observation result of YC3041 of the present invention; Figure 2 d is the scanning electron microscope observation result of YC3041 of the present invention.

[0024] Figure 3 This is a phylogenetic tree diagram of strain YC3041 constructed based on the 16S rRNA gene sequence of the present invention.

[0025] Figure 4 This is a phylogenetic tree diagram of the strain YC3041 constructed based on the ropB gene sequence of the present invention.

[0026] Figure 5 Graph showing the potassium-dissolving ability of YC3041 of the present invention.

[0027] Figure 6 This figure shows the inhibitory effect of YC3041 of the present invention on Fusarium (YC1026).

[0028] Figure 7 This is a control (CK) graph showing the inhibitory effect of YC3041 of the present invention on Fusarium (YC1026).

[0029] Figure 8 This is a graph showing the inhibitory effect of YC3041 of the present invention on wheat stem rot pathogen (YC1265).

[0030] Figure 9 This is a control (CK) graph of the inhibitory effect of YC3041 of the present invention on wheat stem rot pathogen (YC1265). DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0033] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0034] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can all mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can each be single or plural.

[0035] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0036] In a first aspect, an embodiment of the present application provides an actinomycete strain YC3041, which is classified and named Streptomyces pratensis; it is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is July 4, 2024; the deposit number is GDMCC No: 64828.

[0037] In some embodiments, the actinomycete strain YC3041 appears off-white on different culture media, the aerial hyphae are off-white, the matrix mycelium is yellow to yellow-brown, and yellow soluble pigment is produced; the surface is dry after culturing on ISP2 medium for 7 days; the hyphae are small and curved under an optical microscope; and the spore hyphae are loosely spiral under a scanning electron microscope, the spores are spherical or oval, with a smooth surface and straight threads.

[0038] In a second aspect of the present invention, an actinomycete strain YC3041 inoculum is provided, wherein the active ingredient is at least one of the following (a), (b), and (c): (a) Fermentation culture of actinomycete strain YC3041; (b) Supernatant of sonication lysis of actinomycete strain YC3041 cells; (c) Ultrasonic lysis pellet of actinomycete strain YC3041 cells.

[0039] A third aspect of the embodiments of the present application provides an application of an actinomycete strain YC3041 in the preparation of a biological agent for preventing and treating tobacco blight.

[0040] In some embodiments, the biological agent includes a culture of the YC3041 strain or a fermentation product thereof. The biological pesticide also includes an adjuvant that can promote the growth of YC3041 or enhance its antibacterial effect.

[0041] Example 1 The present invention discloses an actinomycete strain YC3041, which is classified as Streptomyces pratensis; the actinomycete strain YC3041 is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is July 4, 2024, and the deposit number is GDMCC No: 64828.

[0042] Actinomycete strain YC3041 appeared off-white on different culture media, with off-white aerial hyphae and yellow to yellow-brown matrix mycelium producing yellow soluble pigment. The surface dried after 7 days of culture on ISP2 medium. Under an optical microscope, the hyphae were small and curved. Under a scanning electron microscope, the spore hyphae were loosely spiral, the spores were spherical or oval, with a smooth surface and straight threads.

[0043] Example 2 The present invention provides an actinomycete strain YC3041 inoculant, wherein the active ingredient is at least one of the following (a), (b), and (c): (a) Fermentation culture of actinomycete strain YC3041; (b) Supernatant of sonication lysis of actinomycete strain YC3041 cells; (c) Ultrasonic lysis pellet of actinomycete strain YC3041 cells.

[0044] Example 3 The invention discloses an application of an actinomycete strain YC3041 in preparing a biological preparation for preventing and treating tobacco blight.

[0045] The biological preparation includes the culture of the YC3041 strain or its fermentation product. The biological pesticide also includes an adjuvant that can promote the growth of YC3041 or enhance its antibacterial effect.

[0046] Example 4 Identification of YC3041 The morphological characteristics of YC3041 on different culture media are shown in Table 2. On all cultures, the mycelia were off-white, with off-white aerial hyphae and yellow to yellow-brown matrix mycelia, producing yellow soluble pigment. After 7 days of culture on ISP2 medium, the surface was dry ( Figure 2 ); Under an optical microscope, the fungus grows well, with small and curved hyphae; under a scanning electron microscope, the spores are loosely spiral, spherical or oval, with a smooth surface and straight threads.

[0047] The 16S rRNA gene sequence of YC3041 was amplified by PCR to obtain a 1316 bp fragment, which was submitted to GenBank and obtained accession number PQ867190. The nucleotide sequence was compared with the registered sequences in GenBank using the Blast program. The homology with Streptomyces pratensis, S. anulatus, and S. cyaneofuscatus was 100%. Phylogenetic analysis showed that YC3041 and S. pratensis clustered in a single branch ( Figure 3 ). Further PCR amplification of the rpoB gene sequence yielded an 815 bp gene fragment, accession number (ropB). BLAST analysis revealed 98.16%, 91.29%, and 90.80% homology with S. pratensis, S. anulatus, and S. cyaneofuscatus, respectively. Phylogenetic analysis showed that YC3041 and S. pratensis were clustered in the same branch ( Figure 4 ), with a 100% support rate, clearly distinguishing it from other populations. Combining morphological characteristics and molecular biological identification results, strain YC3041 was identified as S. pratensis. The characteristics of strain YC3041 grown on different culture media are shown in Table 2.

[0048] Table 2

[0049] Figure 2 The figures show the culture morphology and microscopic morphology of the strain YC3041 of the present invention on ISP2 medium. Figure 2 a is a colony morphology (front view) of YC3041 of the present invention on ISP2 medium; Figure 2b is a morphological diagram (reverse side) of YC3041 of the present invention grown on ISP2 medium; Figure 2 c is the optical microscope observation result of YC3041 of the present invention; Figure 2 d is the scanning electron microscope observation result of YC3041 of the present invention. Figure 3 This is a phylogenetic tree diagram of strain YC3041 constructed based on the 16S rRNA gene sequence of the present invention. Figure 4 This is a phylogenetic tree diagram of the strain YC3041 constructed based on the ropB gene sequence of the present invention.

[0050] Physiological and biochemical assays of YC3041 The morphological characteristics and physiological and biochemical test results of strain YC3041 on different culture media are shown in Table 3. YC3041 has the ability to liquefy gelatin, molten milk, reduce nitrates, hydrolyze starch, and utilize eight glycogens, including xylose, fructose, and maltose. YC3041 can grow on potassium-depleting medium, indicating that it has potassium-depleting ability ( Figure 5 Table 3 shows the physiological and biochemical characteristics of strain YC3041.

[0051] Table 3

[0052] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction. Figure 5 Graph showing the potassium-dissolving ability of YC3041 of the present invention.

[0053] Test Example 1 Determination of Indoor Inhibition Rate of Biocontrol Strains A biocontrol actinomycete strain YC3041 with strong inhibitory activity against tobacco blight was screened by the plate standoff method, with an inhibition rate of 68.63% against the pathogen. (Table 1, Figure 1 ), the inhibitory effect of biocontrol bacteria on the growth of pathogens is shown in Table 1.

[0054] Table 1

[0055] Note: The lowercase letters after the data in the same column in the table indicate significant differences among treatments (P<0.05). Figure 1 This is a diagram showing the antagonistic effect of the biocontrol bacteria of the present invention on pathogenic bacteria.

[0056] Test Example 2 Antimicrobial spectrum of YC3041 YC3041 had a moderate inhibitory effect on the growth of both YC1026 and YC1265 on PDA medium, with an inhibition rate of 23.35% for YC1026 and 19.98% for YC1265. The antibacterial activity of YC3041 is shown in Table 4.

[0057] Table 4

[0058] Figure 6 This figure shows the inhibitory effect of YC3041 of the present invention on Fusarium (YC1026). Figure 7 This is a control (CK) graph showing the inhibitory effect of YC3041 of the present invention on Fusarium (YC1026). Figure 8 This is a graph showing the inhibitory effect of YC3041 of the present invention on wheat stem rot pathogen (YC1265). Figure 9 This is a control (CK) graph of the inhibitory effect of YC3041 of the present invention on wheat stem rot pathogen (YC1265).

[0059] The basic principles, main features and advantages of the present invention are shown and described above. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims, the description and their equivalents.

Claims

1. An actinomycete strain YC3041, characterized in that: The actinomycete strain YC3041 is classified as Streptomyces pratensis Deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Deposit date: July 4, 2024; Deposit number: GDMCC No: 64828.

2. The actinomycete strain YC3041 according to claim 1, characterized in that: The actinomycete strain YC3041 appears off-white on different culture media, with aerial hyphae being off-white and matrix mycelia being yellow to yellow-brown, producing yellow soluble pigment. The surface of the actinomycete strain is dry after being cultured on ISP2 culture medium for 7 days. The hyphae are thin and curved under an optical microscope. The spore hyphae are loosely spiral, spherical or oval, with a smooth surface and straight threads under a scanning electron microscope.

3. The actinomycete strain YC3041 agent according to claim 1, wherein the active ingredient is at least one of the following (a), (b), and (c): (a) a fermentation culture of the actinomycete strain YC3041 according to claim 1; (b) the supernatant of ultrasonic lysis of the actinomycete strain YC3041 cells according to claim 1; (c) Ultrasonic lysis precipitate of the actinomycete strain YC3041 cells according to claim 1.

4. Use of the actinomycete strain YC3041 according to claim 1 in the preparation of a biological agent for preventing and treating tobacco blight.

5. The use according to claim 4, characterized in that: The biological preparation includes the culture of YC3041 strain or its fermentation product.

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