Streptomyces chrysosporium HC307 and application of metabolite of streptomyces chrysosporium HC307 in prevention and treatment of plant diseases

Abstract

The invention discloses application of streptomyces chrysosporium HC307 and metabolites thereof in prevention and treatment of plant diseases, and belongs to the technical field of microbial prevention and treatment. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 23, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.32047. The novel streptomyces chrysosporium HC307 is screened from special habitat soil, carbon and nitrogen sources can be widely utilized, the growth pH range is 5-9, the optimum temperature is 28 DEG C, and the salt tolerance degree is not higher than 6%; in addition, the strain can generate various enzymes, can secrete secondary metabolites to inhibit growth of actinomucor elegans, and has a good application prospect in the fields of enzyme preparation industry and agricultural biological control.

Description

Technical Field

[0001] The invention relates to the technical field of microbial control, and in particular to application of a strain of Streptomyces chrysosporium HC307 and its metabolites in preventing and controlling plant diseases. Background Art

[0002] Codonopsis pilosula is a perennial herbaceous vine of the genus Codonopsis in the Campanulaceae family. It is rich in bioactive substances such as polyacetylenes, polyenes, flavonoids, lignans, alkaloids, coumarins, terpenes, steroids, organic acids and polysaccharides. It has been recorded in the "New Compendium of Materia Medica" that it can strengthen the spleen and lungs, nourish blood and produce body fluid. Modern pharmacological studies have shown that it can enhance the body's immunity, protect the gastrointestinal mucosa and resist ulcers, promote hematopoiesis, regulate blood sugar, delay aging and other pharmacological effects. In addition, Codonopsis pilosula, as a plant with both medicinal and edible properties, can also be made into health wine. Fresh Chinese herbal medicines have higher effective ingredients and pharmacological activities than dried Chinese herbal medicines. However, fresh Codonopsis pilosula may be infected with pathogenic fungi such as Mucor elegans in a short period of time after being picked, thus causing Mucor rot, causing epidermal rot and juice leakage, affecting the pharmacological properties of Codonopsis pilosula, and even producing and accumulating fungal toxins, losing its application value. At present, the research on Mucor elegans mainly focuses on its advantages as fermented bean curd, but its harm to Codonopsis pilosula has not been taken seriously and solved. Therefore, how to inhibit Actinobacillus elegans to reduce its damage to Codonopsis pilosula has become an urgent problem to be solved.

[0003] As the main source of natural products, Streptomyces can produce a variety of biologically active substances, including a variety of antibiotics, alkaloids, organic acids, peptides, etc. It is a microbial resource with great application value and economic value, and has attracted widespread attention. More than a thousand species of Streptomyces have been discovered and identified. Based on this, it is very difficult to isolate and identify a new species of Streptomyces. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Streptomyces chrysosporium HC307 and its metabolites for use in preventing and controlling plant diseases, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a strain of Streptomyces chrysosporium ( Streptomyces flavusporus ) HC307, the strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 23, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32047.

[0006] The second technical solution of the present invention is that the secondary metabolite of Streptomyces chrysosporium HC307 is prepared by: (1) The seed liquid of Streptomyces chrysosporium HC307 was transferred to TSB medium at a volume ratio of 5%, and cultured at 28°C and 180 rpm / min for 5 days to obtain a fermentation liquid; (2) After fermentation is completed, the bacterial cells and the bacterial liquid are separated by centrifugation, and the secondary metabolites are extracted separately to obtain the secondary metabolites.

[0007] The third technical solution of the present invention is a microbial preparation, comprising the Streptomyces chrysosporium HC307 or the secondary metabolite.

[0008] A fourth technical solution of the present invention is the use of the Streptomyces chrysosporium HC307, the secondary metabolite or the microbial preparation in the preparation of a product that inhibits Actinobacillus elegans.

[0009] A fifth technical solution of the present invention is a method for preventing and treating Mucor rot, which utilizes the Streptomyces chrysosporium HC307, the secondary metabolites or the microbial preparation to inhibit the growth of Actinomucor elegantis.

[0010] Based on the above technical solution, the present invention has the following technical effects: The invention screens out a novel Streptomyces chrysosporium HC307 from soil in a special habitat, which can widely utilize carbon and nitrogen sources, has a growth pH range of 5-9, an optimum temperature of 28°C, and a salt tolerance of no more than 6%. In addition, the strain produces a variety of enzymes and can secrete secondary metabolites to inhibit the growth of Actinomucor elegantis. As a microbial preparation, the strain has no pollution to the environment and no residue, can prevent and control the problem of Mucor rot after the harvesting of Codonopsis pilosula, protect plants from pathogenic fungi, and better protect its pharmacological value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0012] Figure 1 Streptomyces chrysosporium ( Streptomyces flavusporus ) Morphological characteristics of HC307 cultured in different culture media for 14 days; A is Gao's medium I; B is Czapek's medium; C is ISP1; D is IPS2; E is ISP3; F is ISP4; G is Bennett's medium; and H is PDA.

[0013] Figure 2 Streptomyces chrysosporium ( Streptomyces flavusporus ) Morphological image of HC307; in the figure, A is the mycelium image of Streptomyces chrysosporium under an optical microscope, and B is the mycelium image of Streptomyces chrysosporium under a scanning electron microscope.

[0014] Figure 3 A 16S rRNA-based Streptomyces chrysosporium strain ( Streptomyces flavusporus ) Phylogenetic tree of HC307.

[0015] Figure 4 Streptomyces chrysosporium ( Streptomyces flavusporus ) The results of the secondary metabolites of HC307 inhibiting the activity of Actinomucor elegans at a concentration of 200 μg / mL; in the figure, A is Actinomucor elegans-CK; B is Actinomucor elegans-treated group. DETAILED DESCRIPTION

[0016] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0019] 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0022] The present invention provides a strain of Streptomyces chrysosporium ( Streptomyces flavusporus ) HC307, the strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 23, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32047.

[0023] The present invention also provides a secondary metabolite of Streptomyces chrysosporium HC307, and the preparation method thereof comprises: (1) The seed liquid of Streptomyces chrysosporium HC307 was transferred to TSB medium at a volume ratio of 5%, and cultured at 28°C and 180 rpm / min for 5 days to obtain a fermentation liquid; (2) After fermentation is completed, the bacterial cells and the bacterial liquid are separated by centrifugation, and the secondary metabolites are extracted separately to obtain the secondary metabolites.

[0024] In some specific embodiments, the centrifugation condition is: centrifugation at 4000 rpm / min for 10 min.

[0025] In some specific embodiments, the extraction condition is ultrasonic extraction three times with an equal volume of ethyl acetate.

[0026] In some specific embodiments, the extraction further includes a step of concentration under reduced pressure.

[0027] The embodiment of the present invention further provides a microbial preparation, comprising the Streptomyces chrysosporium HC307 or the secondary metabolite.

[0028] The embodiment of the present invention also provides the use of the Streptomyces chrysosporium HC307, the secondary metabolite or the microbial preparation in the preparation of a product for inhibiting Actinobacillus elegans.

[0029] The embodiment of the present invention also provides a method for preventing and controlling Mucor rot, which utilizes the Streptomyces chrysosporium HC307, the secondary metabolites or the microbial preparation to inhibit the growth of Actinomucor elegantis.

[0030] The present invention screened and obtained a novel Streptomyces strain, Streptomyces chrysosporium HC307, which can produce amylase, esterase, protease and gelatinase and has potential application ability in the field of enzyme engineering.

[0031] The Streptomyces chrysosporium HC307 provided by the present invention can widely utilize carbon and nitrogen sources and has good environmental adaptability.

[0032] The Streptomyces chrysosporium HC307 provided by the invention can grow in the range of 0%-6% sodium chloride and has a certain salt tolerance.

[0033] The present invention also provides a study on the enzymatic properties of the Streptomyces chrysosporium, and it is found that the Streptomyces chrysosporium can produce amylase, esterase, protease and gelatinase, and can be applied to the field of enzyme engineering.

[0034] The present invention cultivates the Streptomyces chrysosporium under conditions rich in carbon source, nitrogen source or sodium chloride, with a pH of 5-9 and an optimum temperature of 28°C.

[0035] Preferred carbon sources include: D-trehalose, D-cellobiose, lactose, mannitol, arabinose, sucrose, maltose, glucose, inositol, and β-cyclodextrin.

[0036] Preferred nitrogen sources include: glycine, tyrosine, histidine, diammonium phosphate, proline, ammonium nitrate, and methionine.

[0037] The preferred sodium chloride concentration range is 0%-6%. Example 1

[0038] Isolation, Purification and Identification of Streptomyces chrysosporium HC307 1.1 Sources (1) Samples were collected from Kangga, Naidong District, Shannan City, Tibet Autonomous Region (N29°29'38", E91°68'91", altitude: 3650m).

[0039] (2) Pathogenic fungus: Actinomyces elegans, provided by Gansu Agricultural University.

[0040] (3) Test culture medium: Table 1 Test culture medium , (4) Aseptic operating conditions: All utensils and equipment must be sterilized in an autoclave (121°C, 30 min), and inoculation and other operations must be performed in a clean bench.

[0041] 1.2 Isolation and identification of target strains The target strain was isolated from the soil of Kangga Arakawa. The actinomycetes in the soil were isolated by gradient dilution method and inverted in a 28℃ constant temperature incubator for 3-7 days. From the third day after incubation, the morphology and color of the culture medium colonies were observed, and a single colony was picked with an inoculation loop. The single colony was inoculated on Gao's No. 1 medium by the three-zone streak method, and inverted for 3-7 days to obtain pure culture actinomycetes. This operation was repeated on the unpurified medium until a single colony HC307 was isolated. The strain was preserved by glycerol preservation method. The single colony was inoculated into TSB medium and cultured at 180rpm / min for 3-7 days. An equal volume of 50% glycerol was mixed with the bacterial solution into a preservation tube and stored in a -20℃ refrigerator.

[0042] 1.3 Identification of target strains (1) Morphological identification The strain HC307 was transferred to Gao's ager, Czapek's medium, ISP1, ISP2, ISP3, ISP4, Bennett's ager and PDA medium. Each medium was repeated 3 times and cultured in a 28℃ biochemical incubator for 14 days. The surface morphology of the strain on each medium was observed and recorded, including the presence of water absorption spots and mature spores, the color of spore piles, and the color and characteristics of aerial hyphae, intrabasal hyphae, and soluble pigments (see Table 2). The sterilized cover glass was inserted at a 45-degree angle into Gao's ager medium using the insert method, so that the hyphae extended and grew onto the glass slide, and then the hyphae morphology was observed under an optical microscope. After natural drying for 2-3 days, it was sent to the center of the instrument for gold spraying for 45-60 seconds, and the hyphae morphology was observed using a scanning electron microscope.

[0043] Table 2 Colony characteristics of strain HC307 cultured on different media for 14 days , The strain HC307 of the present invention grows vigorously on Gao's I, ISP1, ISP3, ISP4, and Czapek's medium, and can produce pink pigment on Gao's I medium, grows weakly on Bennett's medium and ISP2, and basically does not grow on PDA medium (see Figure 1 ). The strain HC307 of the present invention grows tightly on Gao's medium No. 1, and the surface is dry and powdery. The strain produces yellow aerial hyphae and brown-yellow base hyphae. Due to its full utilization of carbon and nitrogen sources, HC307 can produce pink pigment. The hyphae of HC307 were observed by optical microscope and scanning electron microscope. The hyphae grew radially from the center to the surroundings. The hyphae were mostly straight with a little bend. The aerial hyphae were well developed and the hyphae had branches (see Figure 2 ).

[0044] (2) Identification of physiological and biochemical characteristics The effects of different pH values ​​(3, 4, 5, 6, 7, 8, 9 and 10, 11, 12) on the growth of Streptomyces HC307 were determined in Gao's liquid medium No. 1. The effects of different temperatures (25℃, 28℃, 30℃, 33℃, 37℃, 40℃) and different NaCl concentrations (w / v, 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% and 9%) on the growth of Streptomyces HC307 were determined on Gao's solid medium No. 1. The growth of the colonies on the culture medium was observed after 7-14 days of cultivation to ensure a single factor variable. In order to identify the biochemical characteristics of HC307, according to the Manual of Identification of Common Bacterial Systems (2001), the enzymatic properties of strain HC307 were evaluated by gelatin liquefaction, starch hydrolysis, protease hydrolysis, Tween decomposition, and the biochemical characteristics of strain HC307 were evaluated by the utilization of carbon and nitrogen sources. The specific results are shown in Table 3.

[0045] Table 3 Some physiological and biochemical characteristics of strain HC307 , Note: + represents positive, - represents negative.

[0046] Strain HC307 can grow at a NaCl concentration of 0%-6%, and has the best growth at 0%. As the NaCl concentration increases, the growth of strain HC307 becomes weaker and weaker, and at a NaCl concentration of 7%, strain HC307 does not grow. HC307 can grow at 25℃-40℃, and grows vigorously at 28℃. As the temperature rises and the culture time increases, strain HC307 can produce pigments that make the surrounding of the colony turn pink, and the color gradually deepens. Strain HC307 can grow in the pH range of 5-9, and has a good growth state in the pH range of 5-7. Strain HC307 can liquefy gelatin, hydrolyze starch, decompose Tween-20 and Tween-80, and decompose urea, but cannot produce hydrogen sulfide, indole, and tryptophan.

[0047] (3) Molecular biological identification Extraction of strain DNA: After the strain is activated and cultured, 1-2 mL of bacterial liquid is taken, and about 0.5 g of bacterial cells are obtained by centrifugation. A certain concentration of lysozyme is added, and then the DNA is extracted using the bacterial genomic DNA extraction kit of Beijing Tiangen Biochemical Technology Co., Ltd. After extraction, it is stored at -20°C. The PCR amplification system of 16S rRNA is shown in Table 4. Universal primers 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCCTTGTTACGACTT-3') are used, and genomic DNA is used as a template. Amplification is performed according to the cycle program in Table 5.

[0048] Table 4 PCR reaction system , Table 5 PCR cycle process , The PCR products were cut after 1% agarose gel electrophoresis and purified by gel recovery using the agarose gel DNA recovery kit of Beijing Tiangen Biochemical Technology Co., Ltd. The recovered products were sent to Shanghai Shenggong Biological Co., Ltd. for sequencing after electrophoresis detection. Molecular phylogenetic analysis: After the sequences of the two-way test were spliced, BLAST comparison was performed in the NCBI database, and the strain sequences with higher homology in GenBank were selected. Multiple sequence alignment analysis was performed using MEGA 11, and the Neighbor-Joining method was used to construct a phylogenetic tree. Bootstrap was set to 1000 times to test its credibility.

[0049] The PCR product of strain HC307 was purified, recovered and sequenced to obtain a gene sequence of 1411 bp. A phylogenetic tree was constructed using the neighbor-joining method (see Figure 3 ). The obtained DNA sequence was compared by Blast on NCBI. After comparison, it was found that the strain had the highest similarity with Streptomyces chlororosans, which was 97.5%. At the same time, in the phylogenetic tree, strain HC307 belongs to the genus Streptomyces, but forms an independent branch. Combined with the identification of the strain morphology, it can be determined that strain HC307 is Streptomyces.

[0050] In addition, based on the morphological, molecular biological, physiological and biochemical characteristics, the strain HC307 was named Streptomyces chrysosporium ( Streptomyces flavusporus ) HC307, deposited on September 23, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No: 32047. Example 2

[0051] Determination of the antibacterial activity of Streptomyces chrysosporium against Actinobacillus elegans (1) Preparation of crude extracts of secondary metabolites of Streptomyces chrysosporium strains: Use an inoculation needle to pick up a single colony of Streptomyces chrysosporium HC307 and put it into TSB medium, culture it at 28°C, 180 rpm / min for 3 days to obtain seed liquid, then transfer it to TSB medium at a volume ratio of 5% inoculum, culture it at 28°C, 180 rpm / min for 5 days to obtain fermentation liquid.

[0052] The fermentation liquid of Streptomyces chrysosporus cultured for 5 days was centrifuged at 4000 rpm / min for 10 minutes, the bacterial cells and liquid were separated, and the extracts were repeatedly ultrasonically extracted 3 times with equal volumes of ethyl acetate, and the extracts were concentrated under reduced pressure to obtain its metabolites. The Streptomyces chrysosporus produced a large amount of secondary metabolites, and the concentration of the secondary metabolites was 233.7-429.3 mg / L after culturing on TSB medium for 5 days.

[0053] (2) Antibacterial activity study: The crude extract was dissolved in 5% DMSO and added to PDA agar plates, diluted to a final concentration of 200 µg / mL. 5% DMSO was added to PDA medium as a control. A 6 mm fungus block was placed in the center of the plate and incubated at 30°C for 5-7 days. The inhibitory rate of the crude extract against Actinomucor elegans was calculated according to the following formula.

[0054] ; Among them, D 对照 A represents the diameter of fungi after adding 5% DMSO; D experiment represents the diameter of fungi after adding 200 μg / mL crude extract.

[0055] The inhibitory rate of crude extract of strain HC307 against Actinobacillus elegans was as follows Figure 4 As shown, the inhibitory rate of the crude extract of secondary metabolites of strain HC307 against Actinobacillus elegans was 50.7%.

[0056] The present invention provides a new species of Streptomyces - Streptomyces chrysosporium ( Streptomyces flavusporus ) HC307, which can produce a variety of enzyme active substances, and its metabolites have a certain inhibitory effect on Actinocorus elegantis, which is of great significance for the prevention and control of Mucor rot during the storage of Codonopsis pilosula.

[0057] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Streptomyces chrysosporium ( Streptomyces flavusporus ) HC307, characterized in that, The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 23, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32047.

2. The secondary metabolite of Streptomyces chrysosporium HC307 according to claim 1, characterized in that: The preparation method thereof comprises: (1) The seed liquid of Streptomyces chrysosporium HC307 was transferred to TSB medium at a volume ratio of 5%, and cultured at 28°C and 180 rpm / min for 5 days to obtain a fermentation liquid; (2) After fermentation is completed, the bacterial cells and the bacterial liquid are separated by centrifugation, and the secondary metabolites are extracted separately to obtain the secondary metabolites.

3. The secondary metabolite according to claim 2, characterized in that The centrifugal conditions are: 4000 rpm / min for 10 min.

4. The secondary metabolite according to claim 2, characterized in that The extraction conditions are ultrasonic extraction with equal volume of ethyl acetate for 3 times.

5. The secondary metabolite according to claim 2, characterized in that The method further comprises the step of reducing pressure and concentrating after the extraction.

6. A microbial preparation, characterized in that: The method comprises the Streptomyces chrysosporium HC307 according to claim 1 or the secondary metabolite according to any one of claims 2 to 5.

7. Use of the Streptomyces chrysosporium HC307 according to claim 1, the secondary metabolite according to any one of claims 2 to 5, or the microbial preparation according to claim 6 in the preparation of a product for inhibiting Actinobacillus elegans.

8. A method for preventing and treating Mucor rot, characterized in that: The growth of Actinomucor elegans is inhibited by using the Streptomyces chrysosporium HC307 described in claim 1, the secondary metabolite described in any one of claims 2 to 5, or the microbial preparation described in claim 6.

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