A strain of Streptomyces GS2 and its application

Abstract

This invention relates to a Streptomyces sp. GS2 strain and its applications, belonging to the field of agricultural microbiology technology. To screen for Streptomyces strains that can serve as biocontrol agents and produce beneficial compounds, this invention provides a Streptomyces sp. GS2 strain. Plate confrontation experiments, pot experiments, and germination experiments demonstrated that this strain has a strong inhibitory effect on various plant pathogenic microorganisms and exhibits good control effects against plant diseases caused by various pathogenic microorganisms. Furthermore, it was found that this strain can promote plant growth. This strain has high application value and promising prospects as a biological agent. In addition, extraction of the fermentation products of Streptomyces sp. GS2 revealed that this strain can produce lycorine-like compounds, making it a novel strain for the production and preparation of lycorine-like compounds.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and relates to a strain of Streptomyces GS2 and its applications, specifically to a strain of Streptomyces GS2 and its applications in promoting plant growth, preventing and controlling plant diseases, and preparing lydimycin-like compounds. Background Technology

[0002] Plant diseases caused by plant pathogens have a serious impact on agricultural production. Traditional plant disease control relies mainly on chemical pesticides, but the extensive use and overuse of chemical pesticides have caused environmental pollution, and the impact of pesticide residues in crop products on human health is increasingly attracting widespread attention. Biocontrol using microorganisms is cost-effective and environmentally friendly, making it an important component of integrated agricultural pest management and a key research focus both domestically and internationally. Screening and constructing high-performance microbial strains and developing biocontrol agents have become important directions for plant disease control.

[0003] Streptomyces are a valuable microbial resource, producing a rich variety of secondary metabolites with antifungal activity. They also secrete hydrolytic enzymes that disrupt fungal cell walls, generating plant-growth-promoting substances such as indoleacetic acid, playing a crucial role in plant disease control. Some actinomycete resources, such as *Streptomyces griseoviridis* K61, *Streptomyces lydicus* WYEC108, and *Streptomyces saraceticus* KH400, have been successfully developed into biocontrol agents for plant disease control. Therefore, *Streptomyces* hold significant research importance in the development of biocontrol agents. Summary of the Invention

[0004] In order to screen for Streptomyces that can be used as biocontrol agents and produce beneficial compounds, this invention provides a Streptomyces sp. GS2 strain. This strain can promote plant growth, prevent and control plant diseases caused by plant pathogens, and can ferment to produce lydimycin-like compounds, which has broad application prospects.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0006] The first objective of this invention is to provide a Streptomyces sp. GS2 strain deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28479, deposited on September 19, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] A second objective of this invention is to provide the application of the aforementioned Streptomyces GS2 in promoting plant growth.

[0008] In one embodiment of the present invention, the concentration of Streptomyces GS2 is 10. 5 cfu / mL ~10 8 It can promote plant growth within the range of cfu / mL.

[0009] In one embodiment of the present invention, the plant is any one of soybean, rapeseed, cucumber, rice, chili pepper, pumpkin, potato, wheat, and corn.

[0010] The third objective of this invention is to provide the application of the aforementioned Streptomyces GS2 in the prevention and control of plant diseases, which are plant diseases caused by any one of the following plant pathogens: Sclerotinia sp., Rhizoctonia sp., Colletotrichum sp., Botrytis sp., Alternaria sp., Phytophthorasp., and Pythium sp.

[0011] In one embodiment of the present invention, the plant disease is a plant disease caused by any one of the following plant pathogens: Phytophthorainfestans, Phytophthora capsici, Phytophthora sojae, Pythium ultimum, Pythium aphanidermatum, Sclerotinia sclerotiorum (causal agent of rapeseed sclerotinia), Alternaria alternata, Rhizoctonia solani (causal agent of rice sheath blight), Colletotrichum orbiculare (causal agent of cucumber anthracnose), and Botrytis cinerea (causal agent of tomato gray mold).

[0012] The fourth object of the present invention is to provide a microbial agent containing the above-mentioned Streptomyces GS2.

[0013] A fifth objective of this invention is to provide the application of the above-mentioned microbial agent in promoting plant growth.

[0014] In one embodiment of the present invention, the plant is any one of soybean, rapeseed, cucumber, rice, chili pepper, pumpkin, potato, wheat, and corn.

[0015] The sixth objective of this invention is to provide the application of the above-mentioned microbial agent in the prevention and control of plant diseases, wherein the plant diseases are caused by any one of the following plant pathogens: Sclerotinia sp., Rhizoctonia sp., Colletotrichum sp., Botrytis sp., Alternaria sp., Phytophthorasp., and Pythium sp.

[0016] In one embodiment of the present invention, the plant disease is a plant disease caused by any one of the following plant pathogens: Phytophthorainfestans, Phytophthora capsici, Phytophthora sojae, Pythium ultimum, Pythium aphanidermatum, Sclerotinia sclerotiorum (causal agent of rapeseed sclerotinia), Alternaria alternata, Rhizoctonia solani (causal agent of rice sheath blight), Colletotrichum orbiculare (causal agent of cucumber anthracnose), and Botrytis cinerea (causal agent of tomato gray mold).

[0017] In one embodiment of the present invention, the microbial agent is applied by any one or a combination of two or more of the following methods: mixing with soil, drenching roots, spraying, soaking seeds, and coating.

[0018] In one embodiment of the present invention, the microbial agent further contains agriculturally acceptable adjuvants.

[0019] A seventh object of the present invention is to provide the application of the above-mentioned Streptomyces GS2 in the preparation of lidecynoid compounds, characterized in that the structural formula of the lidecynoid compounds is shown in Formula I:

[0020]

[0021] In Formula I, R is CH3 or H. When R = CH3, the lidimycin compound is lidimycin; when R = H, the lidimycin compound is desmethyllidimycin.

[0022] In one embodiment of the present invention, the specific method for preparing lidocycin-like compounds is as follows:

[0023] 1) Inoculate Streptomyces GS2 into seed culture medium, and obtain seed liquid by shaking flask culture. Then inoculate the seed liquid into fermentation culture medium and obtain fermentation broth by shaking flask culture.

[0024] 2) Centrifuge the fermentation broth obtained in step 1) to collect the cells, add an extractant for ultrasonic extraction, and collect the extractant to obtain the crude extract.

[0025] 3) The crude extract was separated and purified to obtain lidocimic acid and demethyllidocimic acid.

[0026] In one embodiment of the present invention, the seed culture medium and the fermentation culture medium in step 1) are both ISP3 liquid culture medium.

[0027] In one embodiment of the present invention, the specific method of step 1) is to inoculate the activated Streptomyces GS2 into ISP3 liquid culture medium and culture it at 28°C with shaking for 2-4 days to obtain seed liquid; then inoculate the seed liquid into ISP3 liquid culture medium at a volume percentage of 2%-10% and culture it at 28°C with shaking for 5-10 days to obtain fermentation broth.

[0028] In one embodiment of the present invention, the extractant in step 2) is methanol, and the ultrasonic time is 30-50 min.

[0029] In one embodiment of the present invention, the specific method of separation and purification in step 3) is to mix the crude extract obtained in step 2) with silica gel powder and then dry-load it onto a column, followed by dichloromethane / methanol gradient elution, gel chromatography, dichloromethane / methanol elution, and then semi-preparative HPLC separation and purification to obtain lidocimil and demethyllidocimil.

[0030] The beneficial effects of this invention are:

[0031] The *Streptomyces GS2* provided by this invention exhibits strong inhibitory effects against various plant pathogenic microorganisms, particularly showing near 100% inhibition rates against *Phytophthora indicum*, *Phytophthora capsici*, and *Sclerotinia sclerotinia*, and demonstrates excellent control over plant diseases caused by these pathogens. Furthermore, *Streptomyces GS2* can promote plant growth. Compared to traditional chemical control methods, *Streptomyces GS2* can safely, efficiently, and pollution-free control plant diseases, reducing environmental pollution, improving the quality and safety of agricultural products, and protecting human health. As a biological agent, it has high application value and promising prospects for development.

[0032] In addition, Streptomyces GS2 can produce lidimycin-like compounds, including encapsulated lidimycin and demethyl lidimycin, through fermentation. Streptomyces GS2 can be used as a new production strain for lidimycin-like compounds. Attached Figure Description

[0033] Figure 1 The graph shows the results of the antagonistic activity assay of Streptomyces GS2 against plant pathogens.

[0034] Figure 2 The graph shows the results of the growth-promoting effect of Streptomyces GS2 on wheat and corn; among them, Figure 2 In the figure, 'a' represents the results of the growth-promoting effect of Streptomyces GS2 on wheat. Figure 2 Figure b in the figure shows the results of the growth-promoting effect of Streptomyces GS2 on maize.

[0035] Figure 3 The HR-ESI-MS spectrum of lidimycin;

[0036] Figure 4 For lidimycin 1 H-NMR spectrum;

[0037] Figure 5 For lidimycin 13 C-NMR spectrum;

[0038] Figure 6 The HR-ESI-MS spectrum of demethyllidocycin;

[0039] Figure 7 For demethyllidimycin 1 1H-NMR spectrum;

[0040] Figure 8 For demethyllidimycin 13 C-NMR spectrum. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0042] The plant pathogens used in this invention, namely Phytophthora infestans, Phytophthora capsici, Phytophthora sojae, Pythium ultimum, Pythium aphanidermatum, Sclerotinias clerotiorum (causal agent of sclerotinia rot in rapeseed), Alternaria alternata, Rhizoctonia solani (causal agent of rice sheath blight), Colletotrichum orbiculare (causal agent of anthracnose in cucumber), and Botrytiscinerea (causal agent of gray mold in tomato), are preserved at Northeast Agricultural University.

[0043] The preparation method of the V8 liquid culture medium used in this invention is as follows:

[0044] Add 3.4g of calcium carbonate powder to 340mL of V8 mixed fruit and vegetable juice, centrifuge at 4℃ and 4000rpm for 5min, measure 300mL of the juice without solid suspension and add it to 2700mL of deionized water to prepare 3L of V8 liquid culture medium, and sterilize at 121℃ for 30min. (The V8 mixed fruit and vegetable juice was purchased from the United States and is a 340mL can of "Kampo V8 Vegetable Juice 100%" standard product).

[0045] The preparation method of the PDA culture medium used in this invention is as follows:

[0046] Peel 200g of potatoes, cut them into small pieces by hand, boil them in boiling water for 30 minutes, filter them with cheesecloth, and keep the juice; add 20g of agar and 20g of glucose to the filtered juice, and make up to 1000mL; sterilize at 121℃ for 30 minutes.

[0047] The method for preparing the carrot culture medium used in this invention is as follows:

[0048] Cut 200g of carrots into approximately 2×2cm cubes by hand, boil in boiling water for 30 minutes, filter through cheesecloth, and retain the juice; add 20g of agar to the filtered juice and bring the volume to 1000mL; sterilize at 121℃ for 30 minutes.

[0049] Example 1: Inhibitory effect of Streptomyces GS2 on plant pathogens

[0050] Strain GS2 was isolated from forest soil in Mianyang City, Sichuan Province. 16S rRNA sequencing was performed, and the resulting DNA sequence was compared using BLAST (https: / / blast.ncbi.nlm.gov / Blast.cgi) with bacterial 16S rRNA in the NCBI public database. Homology detection and comparison confirmed that this strain is *Streptomyces*. Strain GS2 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28479, deposited on September 19, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0051] The antagonistic activity of Streptomyces GS2 against plant pathogens was determined using the plate confrontation culture method. The plant pathogens included *Phytophthora infestans*, *Phytophthora capsici*, *Phytophthora sojae*, *Pythium ultimum*, *Pythium apharidae*, *Sclerotinia sclerotiorum* (causal agent of rapeseed sclerotinia rot), *Alternaria alternata*, *Rhizoctonia solani* (causal agent of rice sheath blight), *Colletotrichum orbiculare* (causal agent of cucumber anthracnose), and *Botrytis cinerea* (causal agent of tomato gray mold). *Phytophthora sojae* was cultured on carrot solid medium, while the other pathogens were cultured on PDA solid medium. Streptomyces GS2 was inoculated onto the left side of the corresponding solid culture medium plate and incubated at 28°C for 4 days until single colonies of Streptomyces GS2 reached a suitable size. The pathogen was then punched into a mycelial cake using a punch (d = 5 mm) and attached to the right side of the solid culture medium plate with a toothpick, so that Streptomyces GS2 and the pathogen were opposite each other. A plate without Streptomyces GS2 served as a control. The entire plate was then incubated at 28°C until the pathogen in the control plate had completely covered the plate. The inhibition rate of Streptomyces GS2 was then calculated. The formula for calculating the inhibition rate is as follows:

[0052]

[0053] The results are as follows Figure 1As shown in the experimental results, Streptomyces GS2 exhibited significant inhibitory effects on all tested plant pathogens. The inhibition rates against Phytophthora infestans, Phytophthora capsici, Phytophthora sojae, Pythium ultimum, Pythium apharidae, Sclerotinia sclerotiorum (causal agent of rapeseed sclerotinia rot), Alternaria alternata, Rhizoctonia solani (causal agent of rice sheath blight), Colletotrichum orbiculare (causal agent of cucumber anthracnose), and Botrytis cinerea (causal agent of tomato gray mold) were 100%, 91%, 93.1%, 51.1%, 51.4%, 91.7%, 79.4%, 71%, 74.8%, and 65.9%, respectively.

[0054] Example 2: Application of Streptomyces GS2 in the control of Phytophthora in soybean

[0055] (1) Preparation of Streptomyces GS2 spore suspension and Phytophthora soybean spore suspension

[0056] Preparation of Streptomyces GS2 spore suspension: Streptomyces GS2 plates were streaked to cover ISP3 plates. After growth, all spores were scraped off and added to a 250mL Erlenmeyer flask containing 10mL of sterile water and glass beads. The flask was shaken at 250rpm for 30min to disperse and mix the spores. The mixed spore suspension was then diluted sequentially to 10... -7 Choose 10 -5 10 -6 and 10 -7 For each of the three concentrations, take 100 μL of the solution and spread it on a plate. Incubate until all single colonies have grown, and count the number of colonies to obtain the number of spores in the plate. Each concentration is the corresponding concentration of spore suspension. Dilute the spore suspension appropriately according to the experimental requirements.

[0057] Preparation of Phytophthora soybeanis spore suspension: Phytophthora soybeanis stored at 4℃ was transferred to carrot solid medium and cultured at 28℃ for 3 days. After a second transfer and culture under the same conditions for 5-6 days, holes were punched in the plates contaminated with Phytophthora soybeanis. The punched mycelial discs were placed into carrot liquid medium and allowed to stand for 24 hours. The liquid medium was then discarded, and sterile water was added for washing three times. Finally, the plates were soaked in sterile water and cultured for 12 hours to release zoospores. The filtrate was filtered and the concentration of the released zoospores was adjusted to 1×10⁻⁶. 5 CFU / mL.

[0058] (2) Surface disinfection of soybean seeds

[0059] The steps for disinfecting soybean seeds are as follows: Select seeds of uniform size and plumpness, immerse them in 70% alcohol (by volume) for 1 minute, and then pour out the alcohol; rinse three times with sterile water; immerse in 5% sterile NaClO solution for 3 minutes; finally, rinse several times with sterile water to ensure that there is no residual disinfectant on the surface.

[0060] (3) Soybean seedling pot biocontrol experiment

[0061] The soybean seedling disease control experiment consisted of four treatment groups: pathogen treatment, treatment with both Streptomyces GS2 and the pathogen, Streptomyces GS2 treatment, and sterile water treatment. The specific experimental method for the soybean Phytophthora infestation control experiment at the seedling stage is as follows: A Streptomyces GS2 spore suspension was prepared (spore concentration of 10...). 7 To prepare a solution (CFU / mL), 1 mL of spore suspension was evenly mixed with 100 g of soil using a soil mixing method. Soybean seeds were then planted in the prepared soil, four seedlings per pot. Once the seedlings reached stage V3, a concentration of 10 CFU / mL was obtained. 5 Soybeans were infected with CFU / mL soybean Phytophthora spores via root drenching. Disease incidence was assessed 10 days later, and the incidence rate and control efficacy were calculated using the following formula.

[0062]

[0063]

[0064] The results showed that after inoculation with spore suspension of Phytophthora soybeanis pathogen, the control group treated only with Phytophthora soybeanis pathogen began to show symptoms one week later, with obvious irreversible wilting of leaves, some leaves falling off, and light brown, irregularly shaped leaf spots. The disease incidence rate was 100%, and the disease index was 41.9±3.5. In contrast, the control group treated with Phytophthora soybeanis pathogen inoculated with Streptomyces GS2 spore suspension in the soil showed good growth, with almost no disease symptoms, and the plants were significantly stronger than those treated without active fungal spore suspension. The disease incidence rate was 13.9%, the disease index was 14.2±1.5, and the control effect reached 76.5%. Compared with the control group, the soybean plant height of the experimental group inoculated only with Streptomyces GS2 without pathogen inoculation was increased by 14.6%.

[0065] The above results indicate that Streptomyces GS2 can not only effectively prevent infection by Phytophthora in soybean, but also promote the growth of soybean.

[0066] Example 3: Application of Streptomyces GS2 in the control of Phytophthora capsici

[0067] (1) Preparation of Streptomyces GS2 spore suspension and Phytophthora capsici spore suspension

[0068] Preparation of Streptomyces GS2 spore suspension: Same as in Example 2.

[0069] Preparation of Phytophthora capsici spore suspension: The activation steps for Phytophthora capsici are the same as those for Phytophthora sacchariformis. After successful transfer culture, the Phytophthora capsici spores are punched, and the punched mycelial cakes are placed in 10% V8 liquid medium. After culturing at 28℃ for 3 days, the mycelial cakes are poured off and soaked in sterile water. The water is changed every 12 hours for one week. After this process, the mycelial cakes are placed in environments of 4℃ and 28℃ for 30 minutes each, filtered, and the filtrate is used to adjust the spore concentration to 1×10⁻⁶. 5 CFU / mL.

[0070] (2) Surface disinfection of chili seeds:

[0071] The steps for disinfecting the surface of chili seeds are as follows: Select seeds of uniform size and plumpness, place them in 70% alcohol (by volume) and soak for 1 minute, then pour out the alcohol; rinse 3 times with sterile water; soak in 5% sterile NaClO solution for 3 minutes; finally rinse several times with sterile water to ensure that there is no residual disinfectant on the surface.

[0072] (3) Potted biocontrol experiment on chili seedlings

[0073] The pepper seedling disease control experiment consisted of four treatment groups: pathogen treatment, treatment with both Streptomyces GS2 and pathogen, Streptomyces GS2 treatment, and sterile water treatment. The specific experimental method for the pepper seedling Phytophthora disease control experiment is as follows: Pepper seedlings need to be raised in advance, cultivated at room temperature, and then transplanted into soil, with 4 seedlings per pot. They were cultivated until the seedlings reached the 7-8 leaf stage, and the root inoculation concentration was 10. 5 After culturing capsicum spores at CFU / mL for 24 hours, a concentration of 10 CFU / mL was then inoculated onto the roots. 8 Streptomyces GS2 spore suspension at CFU / mL. After 7 days, the disease incidence was investigated according to the grading standard for Phytophthora capsulatum, and the disease index and control effect were calculated using the following formula.

[0074] Grading standards for Phytophthora blight of pepper: Grade 0: No disease; Grade 1: Slight blackening of the root and stem, leaves do not wilt or wilt reversibly; Grade 2: Blackening of the root and stem for 1-2 cm, leaves wilt irreversibly, and occasional shedding of lower leaves; Grade 3: Blackening of the root and stem for more than 2 cm, leaves wilt significantly or leaf drop is obvious; Grade 4: Blackening and shrinkage of the roots, all leaves except the growing point fall off or the whole plant wilts; Grade 5: Plant dies.

[0075]

[0076]

[0077] The results showed that 24 hours after inoculation with *Phytophthora capsici*, followed by inoculation with live spore suspension and bagging for 3 days, disease symptoms appeared approximately 5-7 days later. The group treated only with *Phytophthora capsici* showed significant disease, with extensive browning and severe leaf drop, wilting of the entire plant, and slow overall growth. The disease incidence rate was as high as 100%, with a disease index of 46.1±1.9. The disease-inoculated group treated with live spore suspension showed slight yellowing of leaves and minor leaf drop of the lower leaves, but the plants were generally healthy. The disease incidence rate was 20.2%, the disease index was 23.4±2.1, and the control effect reached 61.3%. Compared with the control group, the experimental treatment group inoculated only with *Streptomyces GS2* without pathogen inoculation showed a 10.9% increase in plant height.

[0078] The above results indicate that Streptomyces GS2 can not only effectively prevent infection by Phytophthora capsici, but also promote the growth of peppers.

[0079] Example 4: Application of Streptomyces GS2 in controlling cucumber diseases caused by Pythium spp.

[0080] The preserved *Pythium spp.* strain was activated and grown on 10% V8 medium and cultured in the dark at 25°C for 1-2 days. A 10×15mm mycelial block was cut with a scalpel and placed in 15mL of sterile tap water, mycelial side facing up. The water was changed every 30 minutes. This process was repeated three times. Then, sterile water was added, just enough to cover the mycelial surface, and the mixture was incubated at 25°C for 20-24 hours to induce zoospore production. The concentration was then adjusted to 10% with sterile water. 5 10 spores / mL. Fill 10cm diameter plastic pots with sterilized sand and potting soil, add water until saturated, flatten the surface, and evenly poke one sowing hole in each pot. Disinfect cucumber seeds with 10% sodium hypochlorite for 5 minutes, rinse 4-5 times with sterile water, and then air dry. Place one seed in each hole, cover with a layer of sterilized substrate soil, and place in a greenhouse for cultivation. Treat each seedling with Streptomyces GS2 spores at a rate of 10 spores / mL. 6 Three days later, inject zoospores of *Pythium spp.* into the cucumber roots, with an inoculation amount of approximately 10 spores per pit. 5 Each spore. After 11 days, the disease index of the plant and the control effect are calculated using the following formula:

[0081]

[0082]

[0083] The representative values ​​for each level are as follows: 0 = The plant remains green and healthy; 1 = The leaf sheath changes color and the lower leaves turn yellow; 2 = The plant survives, but the leaves are completely yellowed or die; 3 = The entire plant dies.

[0084] The results showed that the disease index of cucumbers treated with Streptomyces GS2 was lower than that of the control group inoculated with Pythium spp. The average disease index of the treatment group was 26.7, and the relative control efficacy was 59.3%.

[0085] Example 5: Application of Streptomyces GS2 in the control of Sclerotinia sclerotinia rot in rapeseed caused by S. sclerotiorum

[0086] Select healthy rapeseed plants with 2-3 true leaves, choosing those with uniform plant size and leaves. Add a suspension of Streptomyces GS2 spores (10... 7 Spray the rapeseed leaves evenly with spores / mL and allow them to air dry indoors overnight. Then, place a 5mm diameter mycelium cake of rapeseed sclerotinia pathogen of the same age in the center of each leaf, with the mycelial side facing down. After inoculation, incubate the rapeseed in an environment at 24℃ and relative humidity greater than 80% for 5 days. The disease incidence in the control group is then graded according to the following criteria:

[0087] Grade 0: No symptoms appear; Grade 1: Lesion area on leaves ≤ 5%; Grade 3: Lesion area on leaves ≤ 10% (6% <); Grade 5: Lesion area on leaves ≤ 20% (10% <); Grade 7: Lesion area on leaves ≤ 40% (20% <); Grade 9: Lesion area on leaves > 40%.

[0088] The disease index is calculated based on the area of ​​diseased leaves, and then the control effect is statistically analyzed.

[0089] The formulas for calculating the disease index and control efficacy in detached leaves are as follows:

[0090]

[0091]

[0092] The results showed that Streptomyces GS2 had a control efficacy of 68.2% against Sclerotinia sclerotinia rot caused by Sclerotinia sclerotiorum.

[0093] Example 6: Application of Streptomyces GS2 in the control of gray mold disease in tomatoes caused by B. cinerea

[0094] Tomato fruits of similar size and maturity were selected and the biocontrol effect of Streptomyces GS2 against tomato gray mold was determined using the wound inoculation method. The specific steps were as follows: Tomato fruits were soaked in 75% ethanol for 5 minutes, then in 3% sodium hypochlorite solution for 5 minutes, rinsed three times with sterile water, and then dried with sterile filter paper. 5mm diameter holes were gently punched into the tomato surface using a 5mm diameter punch, taking care to prevent the skin from cracking. Streptomyces GS2 spore suspension (10... 6 Inoculate the fruit wound with a bacterial sample (number of bacteria per mL). After the liquid is completely absorbed by the fruit, use an inoculation needle to pick up a 5 mm diameter pathogenic fungal block and inoculate it into the fruit wound. Finally, place the tomato fruit on a sterile porcelain dish, and place two pieces of water-soaked absorbent cotton in the blank space for moisture retention. Seal with plastic wrap and incubate in a 25℃ constant temperature incubator for 7 days. Measure the lesion area and calculate the control efficacy.

[0095] Efficacy % = (Control lesion area - Treated lesion area) × 100 / Control lesion area

[0096] The results showed that Streptomyces GS2 had good in vitro control efficacy against tomato gray mold caused by Botrytis cinerea, with a control effect of up to 76.2%.

[0097] Example 7: Application of Streptomyces GS2 in the control of rice sheath blight caused by R. solani

[0098] Rice seeds were soaked for 24 hours and then placed in a 25℃ incubator to promote germination. Once the seeds showed signs of sprouting, they were sown in plastic pots filled with sterilized soil and watered regularly. After 20 days, they were transplanted into plastic buckets, with 3 seedlings per bucket and 3 seedlings per hole. Rice plants with uniform growth at the late tillering stage were selected, and rice sheath blight pathogenic fungal cakes (5mm in diameter) were inoculated at equal positions using the short toothpick embedding method. Subsequently, a suspension of Streptomyces GS2 spores (10...) was sprayed. 6 Apply spray (each leaf spot / mL) until the leaves drip. Spray once every 7 days, for a total of 2 applications. Investigate and record the length of lesions after 21 days. Record the severity of the disease according to a scale of 0 to 9. 0: Healthy plant, no disease; 1: Lesion area less than 1 / 4 of the leaf sheath area; 3: Lesion area occupies 1 / 4 to 1 / 2 of the leaf sheath area (excluding 1 / 2); 5: Lesion area occupies 1 / 2 to 3 / 4 of the leaf sheath area (excluding 3 / 4); 7: Lesion area is greater than or equal to 3 / 4 of the leaf sheath area; 9: Lesions reach the top of the plant, and all leaves are severely infected.

[0099]

[0100]

[0101] The results showed that Streptomyces GS2 had no pathogenic effect on rice, but its control effect on rice sheath blight was 58.7%.

[0102] Example 8: Application of Streptomyces GS2 in the control of Alternaria leaf spot disease in rapeseed

[0103] Rapeseed leaves are of suitable size, regular shape, easy to cultivate, and susceptible to infection by *Alternaria alternata*. Therefore, rapeseed leaves of similar size, shape, and growth status were selected as test leaves. After harvesting, the leaves were soaked in sterile water for 30 minutes and then air-dried for later use. A suspension of *Streptomyces GS2* spores was sprayed onto both sides of the rapeseed leaves, forming tiny water droplets that did not drip. The leaves were then air-dried and laid flat in a petri dish containing filter paper, with an appropriate amount of sterile water added to maintain moisture. *Alternaria alternata* fungal cakes were collected using a 9mm diameter punch and inoculated into the center of the leaf, with the mycelial surface in contact with the leaf. The leaves were cultured in a plant light incubator at 28℃, with alternating light and dark periods of 12 hours each. After 5 days, the disease incidence on the leaves was observed. The diameter of lesions on the leaves was measured using the cross-sectional method and photographed for recording.

[0104]

[0105] The results showed that Streptomyces GS2 had a good control effect on rapeseed leaf spot disease, with a control efficacy of 57.9%.

[0106] Example 9: Application of Streptomyces GS2 in promoting plant growth

[0107] Streptomyces GS2 was streaked across the entire surface of ISP3 solid medium. After growth, all spores were scraped off and added to a 250mL Erlenmeyer flask containing 10mL of sterile water and glass beads. The flask was shaken at 250rpm for 30min to disperse and mix the spores. The mixed spore suspension was then diluted sequentially to 10... -7 Choose 10 -5 10 -6 and 10 -7 For each of the three concentrations, 100 μL of the solution was spread onto plates and incubated until all single colonies had grown. The number of colonies was counted to determine the number of spores in the plate. Each concentration represents a spore suspension. The spore suspensions were then diluted to prepare spore concentrations of 10-1. 5 10 6 10 7 10 8 and 10 9 CFU / mL spore suspension.

[0108] Select wheat and corn seeds of normal color and size, soak them in sterile water for 6 hours, wash them with a 70% ethanol solution for 1 minute, then wash them with a 3% NaClO solution for 5 minutes, followed by washing them twice with a 2% Na2S2O3 solution, and finally washing the seed surface three times with sterile water. Soak the sterilized seeds in pre-prepared solutions of different concentrations (10... 5 -10 9 The seeds were soaked in GS2 spore suspension (CFU / mL) for 30 min, with sterile water as the control group. The soaked seeds were evenly placed on plates lined with double-layered filter paper, and 5 mL of sterile water was added to each plate as the moisture required for seed germination. The wheat germination plates were incubated in the dark at 18℃ for 7 days, and the root and shoot lengths of the wheat were measured. The corn germination plates were incubated in the dark at 30℃ for 7 days, and the root and shoot lengths of the corn were measured.

[0109] Experimental results are as follows Figure 2 As shown in Table 1, the experimental results indicate that even at low concentrations, Streptomyces GS2 significantly increased the shoot and root lengths of wheat and corn, with the growth-promoting effect increasing with increasing spore concentration. However, when the spore concentration reached 10... 9 At CFU / mL, Streptomyces GS2 has an inhibitory effect on the growth of wheat and corn. Therefore, a concentration of 10 CFU / mL is considered ineffective for Streptomyces GS2. 5 CFU / mL ~10 8 It can promote plant growth within the range of CFU / mL.

[0110] Table 1. Growth-promoting effects of Streptomyces GS2 on wheat and corn.

[0111]

[0112] Example 5: Application of Streptomyces GS2 in the preparation of lidimycin compounds

[0113] (1) Fermentation culture of Streptomyces GS2 to prepare fermentation broth:

[0114] Fermentation strain: The fermentation strain is Streptomyces GS2.

[0115] Slant culture: Sterilize ISP2 medium (4.0g yeast extract, 10.0g malt extract, 4.0g glucose, 20.0g agar, 1000mL distilled water, pH 7.0-7.2) at 121℃ for 20min, and then incubate at 28℃ for 6-8 days after inoculation.

[0116] Seed culture: Seed culture medium composition: glucose 4.0g, malt extract powder 10.0g, yeast powder 4.0g, CaCO3 0.2g, distilled water 1000mL, pH 7.0-7.2. Dispense 250mL into each 1000mL Erlenmeyer flask and sterilize at 121℃ for 20min. Then, wash the spores of Streptomyces GS2 from the slant culture with 10mL of sterile water to prepare a spore suspension with a concentration of 10%. 7 -10 8 Add 2 mL of spore suspension to each bottle, place on a shaker, and incubate at 28°C and 250 rpm for 48 hours.

[0117] Fermentation culture: The fermentation medium ISP3 consisted of 20 g / L oat flour, 0.001 g / L FeSO4·7H2O, 0.001 g / L MnCl2·4H2O, and 0.001 g / L ZnSO4·7H2O, prepared with distilled water, pH 7.0-7.2, and sterilized at 121℃ for 20 min. A 10% inoculum was added to a 5 L fermenter and cultured at 28℃ with a stirring rate of 100 rpm and an aeration rate of 120 m³ / h. 3 / h, culture for 5-10 days.

[0118] (2) Obtaining crude extract:

[0119] The fermentation broth obtained in step (1) was centrifuged to obtain cell bodies and supernatant. The active ingredients in the cell bodies were extracted by ultrasonication with methanol for 30-50 min. After recovering the methanol solvent, the extract was concentrated to obtain crude extract.

[0120] (3) Preparation of the compound:

[0121] The crude extract obtained in step (2) was mixed with silica gel powder (100-200 mesh) and then dry-loaded onto a column. Gradient elution with dichloromethane / methanol was performed, with the volume ratios of dichloromethane to methanol being 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50, respectively. This yielded six elution fractions: Fraction A to Fraction F. Fraction E was further analyzed by Sephadex LH20 gel chromatography using a dichloromethane:methanol (v / v) ratio of 1:1 as the eluent. The eluent was divided into three portions (each 1 / 3 was a portion, for a total of three portions), yielding three fractions: Fraction E1 to Fraction E3. Fraction E1 was further analyzed by semi-preparative HPLC (using an Agilent SB-C18 column, 250 × 9.4 mm). The sample was purified by separation and purification using methanol:water (0.1% triethylamine) as the mobile phase at a flow rate of 1.5 mL / min to obtain lidocymidine (retention time t).R =16.3min) and demethylidemycin (retention time t R =18.2min).

[0122] (4) Structural identification of compounds lidocimine and demethyllidocimine:

[0123] The HRESIMS spectrum of lidimycin is as follows Figure 3 As shown, the NMR spectrum is as follows Figure 4-5 As shown, the HRESIMS spectrum of desmethylidemamycin is as follows: Figure 6 As shown, the NMR spectrum is as follows Figure 7-8 As shown. The structures of lidecyn and desmethyllidecyn were determined by 1D and 2D NMR, MS and other spectroscopic analyses as follows:

[0124]

[0125] Lidimycin: R=CH3

[0126] Demethyllidimycin: R=H

[0127] Lidimycin is a white powder with the molecular formula C. 47 H 74 N4O 10 High-resolution mass spectrometry (HRESIMS): m / z [M+H] + 855.5483 (calcd for C) 47 H 74 N4O 10 ,855.5478).

[0128] Desmethyllidimycin is a white powder with the molecular formula C. 46 H 71 N4O 10 High-resolution mass spectrometry (HRESIMS): m / z [M+H] + 841.5322(calcd for C 46 H 73 N4O 10 ,841.5321).

[0129] Lidimycin and desmethyllidimycin 1 H and 13 The C NMR (MeOD) data are as follows.

[0130] Lidimycin: 1H NMR(400MHz,MeOD)δH 5.58(1H,m,H-23),5.51(1H,m,H-27),5.45(1H,m,H-22),5.50(1H,m,H-26),5.42(1H,m,H-19),4.85(1H,m,H-11),3.64(1H,m,H-21),4.03(1H,m,H-29),4.02(1H,m,H-35),3.80(1H,t,J=6.6Hz,H-25),3.65(1H,m,H-33),3.80(1H,m,H-9),3.58(1H,m,H-17),3.51(2H,m,H2-40),3.48(1H,m,Ha-38),3.43(1H,m,H-8),3.35(1H,m,Hb-38),3.07(1H,m,H-5),2.62(1H,m,H-13),2.49(1H,m,H-10),2.32(1H,m,Ha-20),1.66(1H,m,H-28),1.83(1H,m,Ha-32),1.13(1H,m,Hb-32),2.21(1H,m,H-24),2.20(1H,m,Hb-20),2.07(1H,m,Ha-36),2.07(2H,m,H2-37),1.90(1H,m,Hb-36),1.84(2H,m,H2-14),1.54(2H,m,H2-15),1.72(3H,s,H3-42),1.70(1H,m,Hb-7),1.37(1H,m,Ha-7),2.30(2H,m,H2-34),1.62(3H,s,H3-46),1.58(3H,s,H3-44),2.20(1H,m,H-16),1.39(3H,s,H3-41),1.37(1H,m,Ha-6),1.72(1H,m,Hb-6),1.54(2H,m,H2-15),1.84(2H,m,H2-14),0.93(3H,d,J=6.8Hz,H3-45),0.76(3H,d,J=6.7Hz,H3-43); 13C NMR(100MHz,MeOD)δC 203.86(C-3),192.62(C-39),180.97(C-1),155.79(C-47),141.28(C-18),140.44(C- 12),139.68(C-30),134.95(C-23),134.29(C-22),134.34(C-26),129.80(C-27),123. 97(C-31),123.01(C-19),120.19(C-11),102.99(C-2),83.72(C-17),77.62(C-9),78 .32(C-25),75.59(C-21),73.69(C-29),71.15(C-8),70.21(C-33),57.61(C-35),54.4 3(C-4),50.69(C-40),48.24(C-38),44.99(C-13),43.99(C-24),43.71(C-10),42.3( C-28),41.65(C-34),37.83(C-32),37.96(C-15),37.38(C-16),36.74(C-20),33.55(C -5),32.22(C-36),29.60(C-7),29.97(C-14),29.60(C-7),24.14(C-37),23.48(C-6) ,23.37(C-42),17.87(C-41),17.13(C-43),16.50(C-45),11.99(C-46),11.96(C-44).

[0131] Demethyllidimycin: 1H NMR(400MHz,MeOD)δH 5.63(1H,m,H-31),5.59(1H,m,H-23),5.51(1H,m,H-27),5.57(1H,m,H-30),5.50(1H,m,H-22),5.50(1H,m,H-26),5.33(1H,t,J=6.8Hz,H-19),4.85(1H,m,H-11),4.06(1H,m,H-29),4.03(1H,m,H-21),4.03(1H,m,H-35),3.84(1H,t,J=6.7Hz,H-25),3.65(1H,m,H-33),3.64(1H,m,H-9),3.57(1H,m,H-17),3.50(2H,m,H2-40),3.48(1H,m,Ha-38),3.43(1H,m,H-8),3.35(1H,m,Hb-38),3.06(1H,m,H-5),2.63(1H,m,H-13),2.50(1H,m,H-10),2.34(1H,m,Ha-20),2.19(1H,m,Hb-20),1.65(1H,m,H-32),2.23(1H,m,H-24),2.21(1H,m,H-28),2.07(1H,m,Ha-36),2.06(2H,m,H2-37),1.90(1H,m,Hb-36),1.54(2H,m,H2-15),1.85(2H,m,H2-14),1.73(3H,s,H3-42),1.55(2H,m,H2-7),2.28(1H,m,Ha-34),2.21(1H,m,Hb-34),1.58(3H,s,H3-44),1.84(1H,m,H-16),1.40(3H,s,H3-41),1.73(2H,m,H2-6),0.93(3H,d,J=6.8Hz,H3-45),0.76(3H,d,J=6.7Hz,H3-43); 13C NMR(100MHz,MeOD)δC 203.88(C-3),192.44(C-39),181.02(C-1),155.75(C-47),141.22(C-12),139.65 (C-18),136.91(C-30),134.90(C-23),134.71(C-26),134.27(C-22),129.50(C-27 ),128.02(C-31),123.99(C-19),120.19(C-11),102.99(C-2),83.71(C-17),77.53 (C-25),75.59(C-9),73.66(C-21),73.14(C-29),71.13(C-8),69.86(C-33),57.54 (C-35),54.44(C-4),50.69(C-40),48.21(C-38),44.98(C-13),77.53(C-25),43. 76(C-10),42.15(C-28),41.66(C-34),41.46(C-32),37.90(C-15),37.90(C-16),3 6.71(C-20),33.54(C-5),32.14(C-36),29.92(C-14),29.57(C-7),24.11(C-37),2 3.38(C-42),23.38(C-6),17.86(C-41),17.11(C-43),16.35(C-45),11.96(C-44).

[0132] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A strain of Streptomyces ( Streptomyces sp.) GS2, characterized in that, The accession number is CGMCC NO. 28479.

2. The application of Streptomyces GS2 as described in claim 1 in promoting plant growth, characterized in that, The plant in question is either wheat or corn.

3. The application of Streptomyces GS2 as described in claim 1 in the prevention and control of plant diseases, characterized in that, The plant disease is caused by Phytophthora capsici ( Phytophthora capsici ), soybean phytotoxicum ( Phytophthora sojae ), Pythium spp. ( Pythium aphanidermatum ), the pathogen of rapeseed sclerotinia stem rot ( Sclerotinia sclerotiorum ) 、 Alternating Alternaria ( Alternaria alternata ), rice sheath blight pathogen ( Rhizoctonia solani ), tomato gray mold pathogen ( Botrytis cinerea Plant diseases caused by any one of the plant pathogens in the list.

4. A microbial agent containing Streptomyces GS2 as described in claim 1.

5. The application of the microbial agent according to claim 4 in promoting plant growth, characterized in that, The plant in question is either wheat or corn.

6. The application of the microbial agent according to claim 4 in the prevention and control of plant diseases, characterized in that, The plant disease is caused by Phytophthora capsici ( Phytophthora capsici ), soybean phytotoxicum ( Phytophthora sojae ), Pythium spp. ( Pythium aphanidermatum ), the pathogen of rapeseed sclerotinia stem rot ( Sclerotinia sclerotiorum ) 、 Alternating Alternaria ( Alternaria alternata ), rice sheath blight pathogen ( Rhizoctonia solani ), tomato gray mold pathogen ( Botrytis cinerea Plant diseases caused by any one of the plant pathogens in the list.

7. The application according to any one of claims 5 or 6, characterized in that, The microbial agent can be applied by any one or a combination of two or more of the following methods: mixing with soil, drenching roots, spraying, soaking seeds, and coating.

8. The use of Streptomyces GS2 as described in claim 1 in the preparation of lidocycin compounds, characterized in that, The structural formula of the lidimycin-like compounds is shown in Formula I: Equation I; In Formula I, R is CH3 or H. When R = CH3, the lidimycin compound is lidimycin; when R = H, the lidimycin compound is demethyllidimycin.

9. The application according to claim 8, characterized in that, The specific methods for preparing lidimycin-like compounds are as follows: 1) Inoculate Streptomyces GS2 into seed culture medium, and obtain seed liquid by shaking flask culture. Then inoculate the seed liquid into fermentation culture medium and obtain fermentation broth by shaking flask culture. 2) Centrifuge the fermentation broth obtained in step 1) to collect the cells, add an extractant for ultrasonic extraction, and collect the extractant to obtain the crude extract; 3) The crude extract was separated and purified to obtain lidocimic acid and demethyllidocimic acid.

10. The application according to claim 9, characterized in that, Step 2) The extractant is methanol; Step 3) The specific method of separation and purification is to mix the crude extract obtained in Step 2) with silica gel powder and then dry-load it onto a column, and sequentially perform dichloromethane / methanol gradient elution, gel chromatography, dichloromethane / methanol elution, and then separate and purify it by semi-preparative HPLC to obtain lidocimil and demethyllidocimil.

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