Streptomycete as well as related products and application thereof

Abstract

The invention discloses streptomyces as well as a related product and application thereof, and relates to the technical field of plant protection. The invention provides streptomycete FXJ4, the preservation number of which is CCTCC NO: M20241085, the streptomycete FXJ4 can effectively inhibit rotting and mildewing of tobacco products in the preparation and / or storage process, and when the streptomycete FXJ4 is applied to production of the tobacco products, the quality and safety of the tobacco products can be greatly improved, and the storage life of the tobacco products can be greatly prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of plant protection, and in particular to Streptomyces and related products and applications thereof. Background Art

[0002] In recent years, cigars have been prone to rotting and mildew during the drying and fermentation process. Tobacco leaves are easily affected by environmental humidity, absorb too much water, and are more likely to breed mold, which causes unpleasant odors in the tobacco leaves, seriously reducing the quality of cigar leaves and their products. Some molds may also produce toxins, causing health and social problems. Tobacco leaf mildew can have a great impact on fermentation and storage, and may even partially or completely lose the use value of the tobacco leaves. This has seriously affected the production of cigars, and has become a constraint on the development of the cigar industry and the increase in tobacco farmers' income.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a streptomyces and related products and applications thereof.

[0005] The present invention is achieved in that:

[0006] In a first aspect, an embodiment of the present invention provides a Streptomyces FXJ4, whose taxonomic name is Streptomyces olivochranogenes and whose deposit number is CCTCC NO: M20241085.

[0007] In a second aspect, an embodiment of the present invention provides a culture obtained by culturing the Streptomyces FXJ4 described in the above embodiment.

[0008] In a third aspect, an embodiment of the present invention provides a bacterial agent, which includes: the Streptomyces FXJ4 described in the aforementioned embodiment and / or the culture described in the aforementioned embodiment.

[0009] In a fourth aspect, an embodiment of the present invention provides the use of the Streptomyces FXJ4 described in the preceding embodiments, or the culture described in the preceding embodiments, or the bacterial agent described in the preceding embodiments in preventing and / or controlling mildew of tobacco products or in preparing products for preventing and / or controlling mildew of tobacco products.

[0010] In a fifth aspect, embodiments of the present invention provide use of the Streptomyces FXJ4 described in the preceding embodiments, or the culture described in the preceding embodiments, or the bacterial agent described in the preceding embodiments in the preparation of tobacco products.

[0011] The present invention has the following beneficial effects:

[0012] The present invention provides a Streptomyces FXJ4, which can effectively inhibit the decay and mildew of tobacco products during the preparation and / or storage process. The Streptomyces FXJ4 is applied to the production of tobacco products to greatly improve the quality, safety and storage period of tobacco products. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 The photos of the strain FXJ4 of Streptomyces olivochromogenes screened out in Example 1 of the present invention, wherein A is a sterile water control and B is a FXJ4 treatment;

[0015] Figure 2 This is the colony characteristic diagram of the biocontrol strain FXJ4;

[0016] Figure 3 This is the phylogenetic tree of the biocontrol strain FXJ4;

[0017] Figure 4 The physiological and biochemical characteristics of the biocontrol strain FXJ4; A represents the production of cellulase, B represents the production of siderophore, and C represents the production of melanin;

[0018] Figure 5 It is the control effect of biocontrol agents on cigar tobacco; among them, A is the sterile water control, B is the treatment with FXJ4 biocontrol agent, and C is 10 mg / mL sodium benzoate. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] On the one hand, an embodiment of the present invention provides a Streptomyces FXJ4, which was deposited in the China Center for Type Culture Collection on May 28, 2024, with a taxonomic name of Streptomyces olivochranogenes, a collection number of CCTCCNO: M20241085, and a collection address of Wuhan University, Wuhan, China, with a postal code of 430072.

[0021] On the other hand, an embodiment of the present invention provides a culture obtained by culturing the Streptomyces FXJ4 described in the above embodiment.

[0022] The present invention has no special restrictions on the culture conditions of Streptomyces FXJ4, and the existing culture medium and culture conditions applicable to Streptomyces olivochromatosis can be used.

[0023] In some embodiments, the culture temperature includes 20-40°C; the culture temperature can specifically be any one of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40°C or a range between any two of them.

[0024] In some embodiments, the culture medium used for the culture may include any one or more combinations of Gao's medium No. 1, potato agar medium and fermentation medium.

[0025] On the other hand, an embodiment of the present invention provides a bacterial agent, which includes: the Streptomyces FXJ4 described in the above embodiment and / or the culture described in the above embodiment.

[0026] In some embodiments, the bacterial agent includes: a bacterial suspension of Streptomyces FXJ.

[0027] In some embodiments, the bacterial agent includes: fermentation broth of Streptomyces FXJ.

[0028] On the other hand, an embodiment of the present invention provides the use of the Streptomyces FXJ4 described in the preceding embodiments, or the culture described in any preceding embodiment, or the bacterial agent described in any preceding embodiment in preventing and / or controlling mildew of tobacco products or in preparing products for preventing and / or controlling mildew of tobacco products.

[0029] In some embodiments, the prevention and / or control of tobacco product mildew comprises: spraying the Streptomyces FXJ4 described in the preceding embodiment or the culture described in any preceding embodiment or the bacterial agent described in any preceding embodiment on the tobacco product.

[0030] In some embodiments, the tobacco product comprises any one or more of shredded tobacco, cigarette tobacco leaves and cigar tobacco leaves.

[0031] The Streptomyces FXJ4 or its culture or its bacterial agent provided in the embodiment of the present invention has no special limitation on the variety of cigar tobacco leaves, and is suitable for preventing and / or controlling mildew of different varieties of cigar tobacco leaves.

[0032] In some embodiments, the mildew includes mildew caused by Penicillium citrinum.

[0033] In addition, the embodiments of the present invention also provide the use of the Streptomyces FXJ4 described in the above embodiments, or the culture described in any of the above embodiments, or the bacterial agent described in any of the above embodiments in the preparation of tobacco products.

[0034] In some embodiments, the tobacco product comprises any one of shredded tobacco, cigarette tobacco and cigar tobacco.

[0035] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Example 1, Isolation and screening of Streptomyces olivochromogenes FXJ4

[0037] PDB medium: 200g peeled potatoes, 20g glucose, 1000mL distilled water, natural pH. Cut the potatoes into pieces and boil for 20min, filter the filtrate twice through 8 layers of gauze, add glucose and distilled water to make up to 1000mL, and sterilize at 121℃ for 30min.

[0038] PDA medium: The preparation method is the same as PDB medium, add 20g agar powder, and sterilize at 121℃ for 30min.

[0039] GS medium: soluble starch 20g, K2HPO4 0.5g, KNO3 1g, FeSO4 0.01g, NaCl 0.5g, MgSO4·7H2O 0.5g, make up to 1L, pH 7.2-7.4, autoclave at 121℃ for 30min.

[0040] Fermentation medium: cornmeal 17 g, sucrose 10 g, peptone 1.9 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 2 g, FeSO4·7H2O 0.01 g, CaCO3 1 g, make up to 1 L, and autoclave at 121°C for 30 min.

[0041] Cellulase detection medium: peptone 10g, yeast powder 10g, sodium carboxymethyl cellulose (CMC-Na) 10g, sodium chloride 5g, potassium dihydrogen phosphate (KH2PO4) 1g, agar 18g, dilute to 1000mL, pH 7.0, sterilize at 121℃ for 20min.

[0042] Iron carrier detection culture medium: Solution A: 1mmol / L chrome azurol (CAS); 4mmol / L hexadecyltrimethylammonium bromide (CTAB); 0.01mmol / L FeCl3.6H2O.

[0043] Solution B: 0.01 mol / L phosphate buffer, pH 7.0.

[0044] Solution C: 2g sucrose, 3.0g acid hydrolyzed casein, 20ml 1mmol / L MgSO4, 1ml 1mmol / L CaCl2, 18-20g agar powder, dilute to 1000mL with deionized water. Sterilize at 121℃ for 20min.

[0045] Tyrosine medium (for determination of melanin production): 1 g L-tyrosine, 8.5 g NaCl, 1 g yeast extract, 20 g agar, dilute to 1000 ml with water, adjust pH to 7.2, and sterilize at 121°C for 30 min.

[0046] 2. Sample Collection

[0047] The samples were collected from cigars during the fermentation process in Chenzhou City, Hunan Province (112°14′E, 24°54′N).

[0048] 3. Isolation and purification of strains

[0049] Healthy cigar tissue was soaked in 70% (v / v) ethanol for 30s, 1% (v / v) sodium hypochlorite for 30s, and finally rinsed with sterile water for 3 times. After the tissue block was cultured on 28℃ GS medium for 1-2 days, the colonies on the culture dish were observed, and the single colonies on the culture dish were purified 3 times by plate streak separation method. After purification 3 times, they were stored in a 4℃ refrigerator according to the number for use.

[0050] 4. Screening of strains

[0051] The 32 actinomycetes obtained after separation and purification were activated and streaked on PDA agar medium (28°C, 72h). Using Penicillium citrinum as the pathogen indicator, 200 μL of Penicillium citrinum spore suspension (spore concentration 2×10 5 CFU / mL) was evenly spread on the PDA medium plate, and FXJ4 bacterial cakes were inoculated in three directions 3 cm away from the center of the plate. The treatment with PDA medium was used as the control, and each treatment was repeated 3 times. The plate was placed in a 28℃ constant temperature incubator for 2 days, and the formation of the inhibition zone was observed and the size of the inhibition zone diameter was measured.

[0052] like Figure 1 As shown, the actinomycete strain FXJ4 obtained by the above method had an inhibition zone diameter of 1.76±0.1 cm against Penicillium citrinum.

[0053] Example 2, Identification of Streptomyces olivochromogenes FXJ4

[0054] The selected antagonistic strains were inoculated on PDA medium for 24 hours of activation and morphological observation. The results showed that the colonies of the strain FXJ4 were light yellow, dry, and tightly bound to the medium. The colony characteristics of FXJ4 are shown in Figure 2 .

[0055] The strain culture method of Li Xiaofang et al. (Li Xiaofang, Tian Yehan, Peng Haiying, et al., Isolation, screening and identification of antagonistic actinomycetes for controlling Fusarium wilt of bitter melon, Chinese Journal of Applied Ecology, 2020, 31(11): 3869-3879) was used. The candidate strains were streaked and inoculated on ISP1-7 and cultured in a constant temperature incubator at 28°C for 7-14 days. The color of aerial hyphae, intrabasal hyphae, and the presence of soluble pigments were observed.

[0056] Table 1 Growth and culture characteristics of strain FXJ4

[0057] Culture medium Aerial hyphae Mycelium in the base Soluble pigments Growth status ISP 1 White White none Growing vigorously ISP 2 yellow yellow yellow Growing vigorously ISP 3 Light yellow Light yellow none Growing vigorously ISP 4 yellow Beige none Good growth ISP 5 yellow White none Good growth ISP 6 White White black Good growth ISP 7 yellow White Dark brown Good growth

[0058] The 16S rRNA gene sequence of strain FXJ4 was analyzed for homology using the NCBI BLAST database. Reference sequences were selected based on literature review and sequences in the database, and a multi-gene phylogenetic tree was constructed using MEGA 11. Figure 3 As shown, strain FXJ4 was identified as Streptomyces olivochranogenes.

[0059] Example 3 Physiological and biochemical properties of Streptomyces olivochromatus FXJ4

[0060] The utilization of different carbon and nitrogen sources by the strains was determined by referring to the method described in the "Streptomyces Identification Manual" (Actinomycetes Classification Group, Institute of Microbiology, Chinese Academy of Sciences, Streptomyces Identification Manual, Beijing: Science Press, 1975).

[0061] Table 2 Carbon source utilization of strain FXJ4

[0062] Measurement indicators FXJ 4 Xylose + Arabinose + fructose - lactose - glucose +

[0063] Table 3 Nitrogen source utilization of strain FXJ4

[0064]

[0065]

[0066] The selected antagonistic strain FXJ4 was inoculated on PDA medium for 24h activation and inoculated into:

[0067] A: Cellulase detection medium. After inoculation, place the medium in a 28℃ incubator for 7 days, stain it with 1mg / mL Congo red for 1 hour, pour out the stain, and soak it in 1mol / l NaCl for 1 hour, then observe it.

[0068] B: Iron carrier production detection medium. When the medium is heated to about 60℃, add 0.5mL solution B to 100mL solution C, and finally add 5mL solution A. Mix thoroughly and pour into a plate in a sterile environment. After inoculation, place it in a 28℃ incubator for 7 days and observe whether there is any change in the transparent circle around the colony.

[0069] C: Melanin detection medium. After inoculation, place the medium in a 28℃ incubator for 7 days, observe the changes in the medium, and use the uninoculated melanin detection medium as a blank control.

[0070] The results are as follows Figure 4 The results showed that strain FXJ4 could produce cellulase and had the ability to produce siderophore and melanin.

[0071] Example 4: Control effect of biocontrol agents on cigars

[0072] The biocontrol strain FXJ4 was inoculated on a PDA culture medium plate for activation and cultured at 28°C for 5 days, then the strain was inoculated in 100ml of Gao's No. 1 culture medium and cultured for 24 hours, and then 5ml was inoculated in a conical flask containing 100ml of fermentation medium, and cultured in a shaking table at 28°C and 180r / min for 72 hours to obtain the product.

[0073] The application method of the biocontrol agent is as follows: the sun-dried cigar tobacco leaves are cut into small pieces of 7×7 cm, and three treatments are set, each with 6 cigar tobacco leaves (QX208).

[0074] Treatment A: clean water spray;

[0075] Treatment B: biocontrol agent spray;

[0076] Treatment C: 10 mg / mL sodium benzoate;

[0077] Test method: A, B, and C treatments were implemented by spraying, with 3 mL sprayed for each treatment. After an interval of 30 minutes, 3 mL of Penicillium citrinum spore suspension (spore concentration 2×105 CFU / mL) was inoculated by spraying.

[0078] Investigation method: Investigate the diseased area of ​​each treatment 7 days after inoculation.

[0079] The test results are shown in Table 4 and Figure 5 .

[0080] Table 4 Effect of Streptomyces olivochromogenes FXJ4 on the prevention and control of cigar mildew

[0081]

[0082] The results showed that the moldy rate of cigars treated with olive-color Streptomyces FXJ4 could be reduced to 19.33%; the moldy rate of cigars could be reduced to 0% after treatment with the preservative 10 mg / mL sodium benzoate; the experimental results showed that olive-color Streptomyces FXJ4 had a good preventive effect on cigar mildew. Therefore, the promotion and application of olive-color Streptomyces FXJ4 is expected to replace chemical pesticides to prevent and control cigar mildew, protect the environment, reduce pesticide residues, improve the safety of tobacco leaves, and have broad application prospects.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Streptomyces FXJ4, characterized in that Its taxonomic name is Streptomyces olivochranogenes, and its deposit number is CCTCC NO: M20241085.

2. A culture, characterized in that The method is obtained by culturing the Streptomyces FXJ4 according to claim 1.

3. The culture according to claim 2, characterized in that The culture temperature ranges from 20 to 40°C.

4. A bacterial agent, characterized in that It includes: The Streptomyces FXJ4 according to claim 1 and / or the culture according to claim 2 or 3.

5. Use of the Streptomyces FXJ4 according to claim 1, the culture according to claim 2 or 3, or the bacterial agent according to claim 4 in preventing and / or controlling mildew of tobacco products or in preparing products for preventing and / or controlling mildew of tobacco products.

6. The use according to claim 5, characterized in that: The method for preventing and / or controlling mildew of tobacco products comprises: spraying the Streptomyces FXJ4 according to claim 1 or the culture according to claim 2 or 3 or the bacterial agent according to claim 4 on the tobacco products.

7. The use according to claim 5, characterized in that: The tobacco products include any one or more of shredded tobacco, cigarette tobacco leaves and cigar tobacco leaves.

8. The use according to any one of claims 5 to 7, characterized in that: The mildew includes mildew caused by Penicillium citrinum.

9. Use of the Streptomyces FXJ4 according to claim 1, the culture according to claim 2 or 3, or the bacterial agent according to claim 4 in the preparation of tobacco products.

10. The use according to claim 8, characterized in that: The tobacco product comprises any one of shredded tobacco, cigarette tobacco leaves and cigar tobacco leaves.

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