A purple Streptomyces strain and its application

Abstract

The present invention discloses a purple Streptomyces strain and its application, belonging to the field of plant growth regulators and pest repellents. The strain is classified and named as purple Streptomyces ( Streptomyces violaceus ), deposited in the General Microbiology Center of China Microbiological Culture Collection Committee on October 19, 2021, with the deposit number: CGMCC 23629, and the address of the deposit unit: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. In vitro resistance tests have shown that the strain has a broad-spectrum fungicidal effect. Potted plant experiments have shown that the fermentation liquid of the strain has a good preventive effect on cucumber gray mold, cucumber powdery mildew and cucumber downy mildew, and the strain has the effect of promoting the growth of cucumber seedlings. Therefore, the strain has the potential to be developed as a microbial fungicide and microbial fertilizer.

Description

Technical Field

[0001] The invention relates to a purple streptomyces strain and application thereof, and belongs to the field of plant growth regulators and pest repellents. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Streptomyces is a family of actinomycetes, widely distributed in nature. Compared with other microorganisms, Streptomyces can produce abundant and diverse active substances, including antibiotics, immunosuppressants and enzyme inhibitors, many of which have good control effects on bacterial and fungal diseases. At the same time, Streptomyces can also promote the growth of crops by degrading soil nutrients and producing growth-promoting substances such as auxins. Therefore, Streptomyces has the potential to be developed into microbial fungicides and microbial fertilizers, and is also a hot topic in the current microbial research field.

[0004] In a potted experiment, the Streptomyces strain F32 had a 30-day prevention effect on watermelon wilt of 66.7%, and had a broad antibacterial spectrum; Streptomyces A034 had a good control effect on pepper root-knot nematode disease. The application of this strain could significantly reduce the number of nematode eggs in the soil and promote the growth of pepper buds and roots; Streptomyces leucospora XS1-5 had a significant antagonistic effect on soybean blight bacteria, and after coating soybean seeds, it could significantly promote soybean growth, increase leaf chlorophyll content, root vitality, and increase leaf and root protective enzyme activity.

[0005] At present, the use of Streptomyces to control plant diseases and insect pests is mainly focused on the control of soil-borne diseases and insect pests, such as watermelon wilt, tomato wilt, root-knot nematode disease, etc. There are fewer examples of Streptomyces controlling foliar diseases. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention screens a purple Streptomyces strain, which is different from other Streptomyces with disease resistance and growth promotion effects. The strain has a good inhibitory effect on rice sheath blight, wheat fusarium, tomato early blight, strawberry gray mold, rice seedling pathogen, and rice blast, showing a broad-spectrum prevention effect; it can also have a wide range of preventive effects on cucumber foliar diseases such as cucumber powdery mildew, cucumber gray mold, and cucumber downy mildew, and has a growth-promoting effect on cucumber seedlings, and has the potential to be developed into a microbial fungicide and microbial fertilizer. In the process of plant disease and insect pest control, Streptomyces SDNY-40 can be compounded with other streptomyces agents for controlling soil-borne diseases, thereby achieving the purpose of comprehensive control of crops.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, a strain of Streptomyces violaceus SDNY-40 is provided, which is classified as Streptomyces violaceus ( Streptomyces violaceus ), deposited in the General Microbiology Center of China Microorganism Culture Collection on October 19, 2021, with the deposit number: CGMCC 23629, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The colony characteristics and bacterial morphology of the purple Streptomyces are as follows:

[0010] The colony characteristics and bacterial morphology of the purple Streptomyces SDNY-40 are as follows: the colony diameter is 1-2 mm, round, gray or slightly yellow, and white after long-term cultivation. The colony has white hyphae neatly around it, a bulge in the middle, a smooth surface, a thick texture, and is not easy to pick up.

[0011] The physiological and biochemical characteristics of the purple Streptomyces include:

[0012] Physiological and biochemical characteristics of Streptomyces violaceus SDNY-40

[0013]

[0014] Qualitative and quantitative determination of the function of Streptomyces violaceus:

[0015] Functional characterization: Streptomyces violaceus SDNY-40 is a disease-resistant and growth-promoting strain that can promote the growth of roots and leaves of cucumber seedlings, and has a wide range of preventive effects on cucumber foliar diseases such as cucumber powdery mildew, cucumber gray mold and cucumber downy mildew. It has the potential to be developed into a microbial fungicide and microbial fertilizer.

[0016] Quantitative determination: According to the corresponding national standards, the potting method was used to evaluate the preventive effect of Streptomyces violaceus SDNY-40 on cucumber fungal diseases.

[0017] In a second aspect of the present invention, a microbial mixture is provided, characterized in that it contains at least the strain of the present invention. The microbial mixture may contain other biocontrol bacteria or plant growth promoting strains and microorganisms that are symbiotic with the strain of the present invention.

[0018] In the third aspect of the present invention, a product comprising the strain of the present invention or the microbial mixture of the present invention is provided. The product may be a bacterial agent, a bacterial fertilizer or a composition.

[0019] Preferably, the product includes a carrier; the carrier may be a solid carrier or a liquid carrier. The solid carrier or the liquid carrier are conventional carrier materials, wherein the solid carrier may be selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, corn flour, soybean flour, polyvinyl alcohol and / or polyglycol; the liquid carrier may be vegetable oil, mineral oil or water.

[0020] Preferably, the product is in powder form, solution form, suspension form, tablet form, granule form or other forms.

[0021] Further preferably, the powder form may be a freeze-dried powder form; and the tablet form may be a tablet form containing the freeze-dried powder and optional nutrients (such as vitamins, mineral salts, etc.).

[0022] The active ingredient of the product is Streptomyces violaceus, which can be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate.

[0023] In a fourth aspect of the present invention, a fermentation broth, fermentation product or metabolite of Streptomyces violaceus is provided.

[0024] In a fifth aspect of the present invention, a method for fermenting the purple Streptomyces is provided, which comprises inoculating the purple Streptomyces into a fermentation medium for fermentation culture.

[0025] In the sixth aspect of the present invention, there is provided a strain of the present invention, a microbial mixture of the present invention, or a product of the present invention comprising the above strain or the above microbial mixture, or a fermentation liquid or fermentation product or metabolite of the present invention for use in preventing and controlling diseases or promoting plant growth.

[0026] Preferably, the diseases are cucumber powdery mildew, cucumber gray mold and cucumber downy mildew.

[0027] Preferably, the plant growth comprises promoting the growth of cucumber seedlings.

[0028] In the seventh aspect of the present invention, a method for preventing and controlling cucumber powdery mildew, cucumber gray mold and / or cucumber downy mildew is provided, the method comprising applying the above-mentioned Streptomyces violaceus, the above-mentioned microbial mixture, the above-mentioned product, the above-mentioned fermentation liquid or fermentation product or metabolite to the growth environment of cucumbers.

[0029] Preferably, the growth environment includes but is not limited to rhizosphere soil and leaves of plants.

[0030] In an eighth aspect of the present invention, a method for promoting the growth of cucumber seedlings is provided, the method comprising the step of contacting the rhizosphere surrounding environment of cucumbers to be grown with the strain of the present invention, Streptomyces violaceus.

[0031] Preferably, the rhizosphere surrounding environment is cucumber rhizosphere soil.

[0032] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0033] The Streptomyces SDNY-40 screened by the present invention is a purple Streptomyces strain that is different from other Streptomyces strains and has disease resistance and growth-promoting effects. There are no reports on purple Streptomyces strains with disease resistance and growth-promoting effects in patents and literature reports. Most Streptomyces strains mainly have preventive effects on soil-borne pests and diseases such as cucumber root-knot nematode disease and tomato bacterial wilt, and there are few Streptomyces strains with good preventive effects on foliar diseases. This strain is a purple Streptomyces strain with disease resistance and growth-promoting effects that is different from existing strains; in vitro resistance tests have shown that this strain has a broad-spectrum fungicidal effect; potted plant tests have shown that this strain has a good preventive effect on a variety of cucumber foliar diseases such as cucumber powdery mildew, cucumber gray mold and cucumber downy mildew; this strain has a growth-promoting effect on cucumber seedlings. Therefore, this strain has the potential to be developed into a microbial fungicide and microbial fertilizer. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0037] Example 1

[0038] Screening of Streptomyces violaceus of the present invention

[0039] (1) Screening process

[0040] Take the root of the cucumber seedling, gently brush off the outer soil, cut the root with sterile scissors and put it in a triangular bottle filled with sterile water. Oscillate at 180r / min at room temperature for 30 minutes to make the root completely dissolved in sterile water. Then, use the spread plate method to spread the root soil mixture according to 8 concentration gradients (10-2 ~10 -9 g / ml) on LB plates, set up one replicate for each, and culture in a 30°C incubator for 3-5 days. Select the appropriate concentration plate, pick the typical single colonies of different strains according to their morphology, size, and color, and after purification in LB medium, transfer the screened strains to the slant and store at 4°C for later use.

[0041] (2) Colony characteristics and bacterial morphology

[0042] The colony characteristics and morphology of the purple Streptomyces SDNY-40 are as follows: the colony diameter is 1-2 mm, round, gray or slightly yellow, white after long-term cultivation, with white hyphae neatly around, raised in the middle, smooth surface, thick texture, and not easy to pick up.

[0043] (3) Physiological and biochemical characteristics

[0044] Physiological and biochemical characteristics of Streptomyces violaceus SDNY-40

[0045]

[0046] (4) 16S rDNA sequence analysis

[0047] Combined with the colony morphology, physiological and biochemical characteristics and 16SrDNA sequence analysis, the strain of the present invention was identified as purple Streptomyces strain ( Streptomyces violaceus ).

[0048] The following sequences are from Streptomyces violaceus strains ( Streptomyces violaceus ) as shown in SEQ ID NO: 1:

[0049]

[0050] Example 2

[0051] 1. Culture medium

[0052] Bacteria activation medium (Japanese medium): 5g soluble starch, 5g glucose, 1g peptone, 1g beef extract, 1g yeast extract, 15g agar, 1000ml distilled water, pH 7.2.

[0053] Bacterial fermentation medium (C4 medium): 10g soluble starch, 20g glucose, 25g peanut cake powder, 1g beef extract, 4g yeast powder, 2g sodium chloride, and 0.05g sodium dihydrogen phosphate.

[0054] Fermentation conditions: The activated colony was inoculated into a 250 ml conical flask containing 100 ml C4 medium, at 28°C, 220 r / min, and fermented for 4 days.

[0055] 2. In vitro resistance test: The in vitro resistance test adopts the toxic medium method, with the following specific steps: (1) Cool the PDA medium that has been sterilized to about 50°C; (2) Add 1 ml of sample fermentation liquid to the sterilized culture dish; (3) Add 10 ml of PDA medium to each culture dish, cool to room temperature, and sterilize with ultraviolet light for at least 30 minutes; (4) Place rice blast pathogen, wheat fusarium head blight pathogen, tomato early blight pathogen, strawberry gray mold pathogen, rice seedling pathogen, and rice blast pathogen in the center of the culture dish; (5) Invert the inoculated culture dish and culture it in an incubator at 28°C, with the culture dish inoculated with only pathogen cake as the control and 50% carbendazim wettable powder as the positive control. Each test is repeated 3 times. When the mycelium of the pathogen in the control group grows all over the plate, measure the diameter of the pathogen in the test group and calculate the inhibition rate.

[0056] 3. Potted plant test

[0057] (1) Prevention and control of cucumber gray mold: Select cucumber seedlings at the 2-cotyledon stage with consistent growth, spray 10 ml of fermentation liquid evenly on the leaves of the cucumber seedlings, wait 24 hours, inoculate the leaves with pre-cultured cucumber gray mold cakes, treat the control group with an equal amount of clean water, and use a pyrimethanil suspension as the control agent. Place the control group in a dark moisturizing box for disease treatment. When the leaves around the pathogenic bacteria on the control leaves begin to rot and rot, begin to count the incidence and calculate the prevention effect.

[0058] (2) Prevention and control of cucumber powdery mildew: Select cucumber seedlings with two true leaves of the same growth, spray 10 ml of fermentation liquid evenly on the leaves of the cucumber seedlings. After 24 hours, inoculate with a powdery mildew spore suspension with a spore concentration of about 100,000 / ml. The control group is treated with an equal amount of clean water and a control agent of pyraclostrobin suspension. Place the cucumber seedlings under a constant temperature light at 24°C, and calculate the disease index and control effect at 7 and 14 days.

[0059] (3) Prevention and control of cucumber downy mildew: Select cucumber seedlings with two true leaves of the same growth, spray 10 ml of fermentation liquid evenly on the leaves of the cucumber seedlings. After 24 hours, inoculate with a spore suspension with a spore concentration of about 100,000 / ml. The control group is treated with an equal amount of clean water and a control agent of 25 mg / L of cyazofamid. Move the seedlings to an artificial climate chamber and maintain a humidity of more than 90% and a temperature of 20°C. After 5 days, investigate and calculate the disease index and control effect.

[0060] 4. Growth promotion test

[0061] Growth-promoting effect on cucumber seedlings: Before sowing cucumbers, Streptomyces SDNY-40 was pre-inoculated in the soil. The control treatment was not inoculated with actinomycetes. When the seedlings grew to two leaves and one heart, the seedlings with consistent growth were selected, the roots were carefully cleaned, the surface moisture was absorbed, the stem diameter was measured with a vernier caliper, and then the above-ground fresh weight, underground fresh weight and whole-plant fresh weight were measured with an analytical balance.

[0062] 5. Test results

[0063] (1) Results of in vitro resistance test

[0064] The antagonistic effect of Streptomyces SDNY-40 on six pathogens, including rice sheath blight, wheat fusarium, tomato early blight, strawberry gray mold, rice seedling pathogen, and rice blast, was determined by in vitro resistance test. The results are shown in Table 1:

[0065] Table 1 Results of in vitro resistance test of Streptomyces SDNY-40

[0066]

[0067] The results showed that the fungus had an inhibition rate of more than 90% against wheat ergot, tomato early blight, strawberry gray mold, rice seedling pathogen, and rice blast, showing a broad-spectrum anti-fungal disease capability.

[0068] (2) Results of the cucumber powdery mildew potted test

[0069] Table 2 The control effect of Streptomyces SDNY-40 on cucumber powdery mildew

[0070]

[0071] The results of potted plant tests showed that after treatment with the fermentation liquid of Streptomyces SDNY-40, as well as its 20-fold and 40-fold fermentation dilutions, the disease index of cucumber powdery mildew was 2.7, 3.8 and 6.7 at 7 days after application, and the prevention efficacy was 95.3%, 93.4% and 88.3%, respectively; the disease index 14 days after application was 10.1, 13.7, 16.5, and the prevention efficacy was 85.4%, 80.3% and 76.2%, showing excellent performance in preventing cucumber powdery mildew.

[0072] (3) Results of the potted test on cucumber gray mold

[0073] Table 3 The control effect of Streptomyces SDNY-40 on cucumber gray mold

[0074]

[0075] The results of potted plant tests showed that Streptomyces SDNY-40 had a strong preventive effect on cucumber gray mold and was superior to the commonly used chemical agent pyrimethanil. Three days after inoculation, a 5-fold dilution of the fermentation broth of Streptomyces SDNY-40 still had 90% inhibitory activity against cucumber gray mold, which was significantly superior to the chemical agent pyrimethanil suspension.

[0076] (4) Results of the potted test on cucumber downy mildew

[0077] Table 4 The control effect of Streptomyces SDNY-40 on cucumber downy mildew

[0078]

[0079] The results of potted plant tests showed that the fermentation liquid of Streptomyces SDNY-40 had significant effect on the prevention of cucumber downy mildew. The prevention and control effect of the 12.5-fold fermentation dilution liquid was above 90%, and the prevention and control effect of the 60-fold fermentation dilution liquid was still above 60%, showing a good preventive effect on cucumber downy mildew.

[0080] (5) Cucumber growth promotion experiment

[0081] Table 5 Effects of Streptomyces SDNY-40 on cucumber seedlings

[0082]

[0083] From the above data, it can be seen that after the cucumber seedlings were inoculated with Streptomyces SDNY-40, the fresh quality of the cucumber seedlings was significantly improved. Among them, the stem diameter increased by 16%, the above-ground quality increased by 37%, the underground root quality increased by 78%, and the whole plant quality increased by 37%.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Streptomyces violaceus SDNY-40, which is classified as Streptomyces violaceus ( Streptomyces violaceus ), deposited in the General Microbiology Center of China Microorganism Culture Collection on October 19, 2021, with the deposit number: CGMCC 23629, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial mixture, characterized in that: The method comprises the Streptomyces violaceus SDNY-40 according to claim 1.

3. A product comprising the Streptomyces violaceus SDNY-40 according to claim 1 or the microbial mixture according to claim 2.

4. The product according to claim 3, characterized in that: The product is in the form of powder, suspension, tablet or granules.

5. A fermentation broth comprising the Streptomyces violaceus SDNY-40 according to claim 1.

6. A fermentation method of Streptomyces violaceus SDNY-40 according to claim 1, characterized in that: The purple Streptomyces SDNY-40 is inoculated into a fermentation medium for fermentation culture.

7. Use of the Streptomyces violaceus SDNY-40 according to claim 1, the microbial mixture according to claim 2, the product according to claim 3, or the fermentation liquid according to claim 5 in preventing and controlling diseases or promoting plant growth, characterized in that: The diseases are cucumber powdery mildew, cucumber gray mold and cucumber downy mildew; and the plant growth is to promote the growth of cucumber seedlings.

8. A method for preventing and controlling cucumber powdery mildew, cucumber gray mold and / or cucumber downy mildew, characterized in that: The method comprises applying the Streptomyces violaceus SDNY-40 of claim 1, the microbial mixture of claim 2, the product of claim 3 or the fermentation liquid of claim 5 to the growth environment of the cucumber.

9. A method for promoting the growth of cucumber seedlings, characterized in that: The method comprises the steps of contacting the root zone surrounding environment of cucumbers to be grown with the purple Streptomyces SDNY-40 according to claim 1, the microbial mixture according to claim 2, the product according to claim 3 or the fermentation liquid according to claim 5.

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