CN107083349A - The white black streptomycete of one plant of disease prevention growth-promoting and its preparation and application of metabolite - Google Patents

Abstract

The invention discloses the preparation and application of a strain of Streptomyces albigeris for disease prevention and growth promotion and its metabolites. The strain number of the Streptomyces albonigeris is T22, and its registration number in the General Microorganism Center of China Committee for Culture Collection of Microorganisms is CGMCC No.14190. Streptomyces alboblack T22 has inhibitory effects on botrytis cinerea, peach brown rot and capsicum anthracnose in fruit and vegetable crops. The aseptic fermentation filtrate of Streptomyces alboblack T22 can effectively inhibit the mycelial growth of Botrytis cinerea, Botrytis cinerea and Fusarium wilt of cucumber. Streptomyces alboniger can form a hydrolytic circle on the enzyme production assay medium by producing cellulase and chitinase. It shows that Streptomyces alboniger T22 can inhibit plant pathogenic fungi by producing antibiotic active substances and hydrolytic enzyme substances. Appropriate dilution of the aseptic fermentation filtrate of Streptomyces albus T22 can significantly promote the elongation of tomato hypocotyls and/or radicles, and enhance the germination potential of tomato seeds.

Description

Preparation and application of a strain of Streptomyces alboniger for disease prevention and growth promotion and its metabolites

technical field

The invention belongs to the technical field of microbial pesticides, and in particular relates to the preparation and application of a Streptomyces albus and its metabolites for disease prevention and growth promotion.

Background technique

Botrytis cinera, caused by the fungus Botrytis cinera, is common and serious in many fruit and vegetable crops. The disease can harm the stems, leaves, flowers and fruits of plants, especially in humid and rainy and greenhouse environments. The prevention and control of fruit and vegetable gray mold in my country currently mainly relies on chemical agents, but the long-term use of chemical agents in large quantities has problems such as pesticide residues, pathogenic bacteria prone to drug resistance, and environmental pollution. Exploring new high-efficiency, broad-spectrum, low-toxicity, and environmental-friendly plant disease control technologies is a difficult and hot spot in agricultural production research today. Biological control is safe and non-polluting, and pathogenic bacteria are not easy to develop drug resistance. It is an effective way to prevent and control fruit and vegetable diseases. At home and abroad, there have been studies on the control effects of microorganisms such as Bacillus subtilis, Bacillus polymyxa, and Trichoderma spp. on gray mold of fruit and vegetable crops. Due to the large variation in the pathogenic bacteria of Botrytis cinerea, the disease spreads quickly after the disease occurs, and it is difficult to effectively control it. Rational development of existing microbial resources, creation of new biocontrol agents, and addition of microbial pesticides are important for green prevention and control of fruit and vegetable diseases and pollution-free agricultural production. is of great significance.

Actinomycetes (Actinomycetes) widely exist in different natural ecological environments such as soil and ocean, and have the characteristics of various types and different metabolic functions. They are a kind of microbial populations with practical application value. 70% of the biologically active substances found in microorganisms come from actinomycetes, and about 50% of them are metabolites of Streptomyces. Screening actinomycetes resources with activity against phytopathogenic fungi and developing new microbial pesticide products that can gradually replace chemical pesticides are of great significance for the green control of Botrytis cinerea in fruit and vegetable crops.

Contents of the invention

The technical problem to be solved by the invention is how to inhibit the pathogenic bacteria of the fruit and vegetable crops and promote the growth of the fruit and vegetable crops.

In order to solve the above technical problems, the invention provides a strain of Streptomyces albonigeri.

The Streptomyces alboniger provided by the present invention is Streptomyces alboniger with a strain number of T22, and its registration number in the General Microorganism Center of China Microbiological Culture Collection Management Committee is CGMCC No.14190. Hereinafter referred to as Streptomyces alboniger T22.

Streptomyces alboniger T22 has the 16S rDNA sequence shown in Sequence 1 in the Sequence Listing. The aerial hyphae of Streptomyces alboniger T22 on Gao's No. 1 medium are white, without soluble pigment or light yellow over time Pigments (Figure 1). Under the microscope, the aerial hyphae of Streptomyces alboniger T22 can be observed until they are flexible, and the spores form chains and are oval (Figure 2).

The culture of Streptomyces alboniger T22 also belongs to the protection scope of the present invention.

The culture of Streptomyces alboniger T22 is a material obtained by culturing Streptomyces alboniger T22 in a microbial liquid fermentation medium.

In order to solve the above technical problems, the present invention provides pathogenic bacteria inhibitors.

The pathogen inhibitor provided by the present invention contains Streptomyces alboniger T22 and/or a metabolite of Streptomyces alboniger T22.

The active ingredient of the above-mentioned pathogen inhibitor can be Streptomyces alboniger T22 and/or the metabolite of Streptomyces alboniger T22, the active ingredient of the above-mentioned pathogen inhibitor can also contain other biological components or non-biological components, the above-mentioned pathogenic bacteria Other active ingredients of the inhibitor can be determined by those skilled in the art according to the inhibitory effect on pathogenic bacteria.

Among the above-mentioned pathogenic bacteria inhibitors, the pathogenic bacteria inhibitors can have an inhibitory effect on at least one of the following pathogenic bacteria:

A. Botrytis cinerea;

B. Drupe brown rot fungus;

C, capsicum anthracnose bacteria;

D. Fusarium wilt;

E. Potato blight bacillus;

F, wheat sheath blight;

G, eggplant solanacearum;

H, Cucumber horn spot fungus.

In order to solve the above technical problems, the present invention provides a disease inhibitor.

The disease inhibitor provided by the invention contains Streptomyces alboniger T22 and/or a metabolite of Streptomyces alboniger T22.

The active ingredient of the above-mentioned disease inhibitor can be Streptomyces alboniger T22 and/or the metabolite of Streptomyces alboniger T22, the active ingredient of the above-mentioned disease inhibitor can also contain other biological components or abiotic components, the above-mentioned disease Other active ingredients of the inhibitor can be determined by those skilled in the art according to the inhibitory effect on the disease.

Among the above-mentioned disease inhibitors, the disease is at least one of the following:

a. Botrytis;

b. Drupe fruit brown rot;

c. Pepper anthracnose;

d. Fusarium wilt;

e. Potato early blight;

f. Wheat sheath blight;

g, eggplant bacterial wilt;

h, Cucumber horn spot.

Any of the following applications of the metabolites of Streptomyces alboniger T22 and/or Streptomyces alboniger T22 also belong to the protection scope of the present invention:

1) Application in inhibiting pathogenic bacteria;

2) Application in the preparation of pathogen inhibitors;

3) Application in disease suppression;

4) Application in the preparation of disease inhibitors.

In the above application, the pathogenic bacteria can be at least one of the following:

A. Botrytis cinerea;

B. Drupe brown rot fungus;

C, capsicum anthracnose bacteria;

D. Fusarium wilt;

E. Potato blight bacillus;

F, wheat sheath blight;

G, eggplant solanacearum;

H, Cucumber horn spot fungus.

The disease can be at least one of the following:

a. Botrytis;

b. Drupe fruit brown rot;

c. Pepper anthracnose;

d. Fusarium wilt;

e. Potato early blight;

f. Wheat sheath blight;

g, eggplant bacterial wilt;

h, Cucumber horn spot.

Any of the following applications of the metabolites of Streptomyces alboniger T22 and/or Streptomyces alboniger T22 also belong to the protection scope of the present invention:

H, application in promoting plant seed germination;

1. Application in promoting plant seed hypocotyl and/or radicle growth;

Application of J in improving the germination potential of plant seeds.

In the above application, the plant can be any of the following plants:

P1) tomato;

P2) Tomato plants;

P3) Solanaceae plants.

In the above, the metabolites of Streptomyces alboniger T22 can be obtained from the fermentation broth of Streptomyces alboniger T22. The metabolite of Streptomyces alboniger T22 may be a sterile metabolite of Streptomyces alboniger T22. The sterile metabolite (sterile fermentation filtrate) of Streptomyces alboniger (Streptomyces alboniger) T22 specifically can be prepared according to the following method: cultivate Streptomyces alboniger (Streptomyces alboniger) T22 in liquid culture medium, filter to remove the Streptomyces alboniger T22 is the sterile metabolite of Streptomyces alboniger T22.

In the present application, the Botrytis cinerea can be Botrytis cinerea, Botrytis cinerea and/or Botrytis cinerea; the brown rot of drupe can be brown rot of peach; the fusarium wilt can be Fusarium wilt of cucumber , Fusarium wilt of cabbage and/or Fusarium wilt of cotton. The gray mold can be tomato gray mold, grape gray mold and/or strawberry gray mold, and the drupe brown rot can be peach brown rot; the fusarium wilt can be cucumber wilt, cabbage wilt and/or cotton blight.

In the above, the pathogen inhibitor and disease inhibitor contain a carrier in addition to the active ingredient. The carrier can be commonly used in the field of pesticides and is biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and At least one of diatomaceous earth; the plant material can be at least one of corn flour, soybean flour and starch; the polymer compound can be polyvinyl alcohol and/or polyglycol; the liquid carrier can be Organic solvent, vegetable oil, mineral oil or water; said organic solvent may be decane and/or dodecane.

In the pathogen inhibitor and disease inhibitor, Streptomyces alboniger T22 can exist in the form of spores, hyphae or cultures containing spores and/or hyphae.

The dosage form of the pathogenic bacteria inhibitor and disease inhibitor can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the pathogen inhibitors and disease inhibitors as required.

The present invention takes the pathogenic fungus of fruit and vegetable disease as the target, isolates and screens from the soil in the special habitat of the Qinghai-Tibet Plateau to obtain a microbial strain-Streptomyces alboniger T22, which has both disease prevention and growth-promoting effects, and is effective against Botrytis cinerea including Botrytis cinerea. A variety of plant pathogenic fungi have good broad-spectrum antagonistic activity, and their sterile fermentation filtrate can effectively promote tomato seed germination, and has good in vitro control effect on tomato gray mold, so it can be applied to fruit and vegetable fungal diseases, especially Botrytis cinerea The green prevention and control of diseases provides new resources for the preparation of new microbial pesticide products.

Streptomyces alboniger T22 has inhibitory effects on botrytis cinerea, peach brown rot and capsicum anthracnose in fruit and vegetable crops (Table 3, Figure 4). Streptomyces alboniger (Streptomyces alboniger) T22 sterile fermentation filtrate can effectively inhibit the mycelial growth of Botrytis cinerea, Botrytis cinerea and Fusarium wilt of cucumber (Figure 5). At the same time, Streptomyces alboblack T22 can form a hydrolysis circle on the enzyme production activity detection medium by producing cellulase and chitinase ( FIG. 6 ). It shows that Streptomyces alboniger T22 can inhibit plant pathogenic fungi by producing antibiotic active substances and hydrolytic enzyme substances. Appropriate dilution of the aseptic fermentation filtrate of Streptomyces alboniger T22 can significantly promote the elongation of tomato hypocotyls and/or radicles, and enhance the germination potential of tomato seeds. Compared with the control, the 100-fold dilution of the aseptic fermentation filtrate of Streptomyces alboniger (Streptomyces alboniger) T22 significantly increased the length of tomato hypocotyls and radicles, and improved the germination potential of tomato seeds by 15.1%, 29.7% and 43.9% respectively; The hypocotyl, radicle length and seed germination potential of the tomato seeds treated with 200-fold dilution of the aseptic fermentation filtrate of Streptomyces alboniger T22 increased by 8.8%, 18.1% and 32.3% compared with the control (Table 4). Streptomyces alboniger (Streptomyces alboniger) T22 aseptic metabolite (sterile fermentation filtrate) spraying treatment of tomato detached leaves can significantly reduce the diameter of tomato gray mold lesions, and white and black (Streptomyces alboniger) T22 aseptic metabolite (sterile fermentation Filtrate) treatment reached 55.1% (Table 5) in vitro control effect on tomato Botrytis cinerea. It shows that the spraying treatment of the aseptic fermentation filtrate of Streptomyces alboniger T22 has a good control effect on gray mold of tomato detached leaves ( FIG. 7 ). Streptomyces alboniger T22 can be cultured artificially, the culture conditions are simple, and the spore production is good. It is suitable for industrial production and has a good development and application prospect.

Preservation instructions

Species name: Streptomyces alboniger

Strain number: T22

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Storage date: May 25, 2017

Registration number of the collection center: CGMCC No.14190

Description of drawings

Figure 1 is the colony morphology of strain T22 on Gaoshi No. 1 agar medium.

Figure 2 is the hyphae and spore morphology of strain T22 under a 40-fold optical microscope.

Figure 3 is a phylogenetic tree constructed based on the 16S rDNA sequence of strain T22, in which strain T22 and Streptomyces alboniger strains (NR043228, AB184331) clustered on the same branch, with a similarity of 99%.

Fig. 4 is the plate antagonism effect of strain T22 on pathogenic fungi, wherein a, b, and c respectively represent the inhibitory effects of strain T22 on Botrytis cinerea, peach brown rot, and capsicum anthracnose.

Figure 5 is the inhibitory effect of bacterial strain T22 aseptic fermentation filtrate on phytopathogenic fungi, a, c, and e are the colonies of cucumber fusarium wilt, tomato cinerea and strawberry cinerea cinerea on the PDA medium without adding bacterial strain T22 sterile fermentation broth respectively , b, d, f are the colonies of Fusarium wilt of cucumber, Botrytis cinerea and Botrytis cinerea of strawberry on the PDA medium added with the aseptic fermentation filtrate of strain T22, respectively.

Figure 6 shows the production of cellulase and chitinase by strain T22, a and b are the hydrolysis circles produced by strain T22 on cellulose Congo red medium and colloidal chitin Congo red medium, respectively.

Figure 7 is the in vitro control effect of bacterial strain T22 aseptic fermentation filtrate on tomato gray mold. Among them, a, b are not inoculated with liquid medium spraying + Botrytis cinerea, c, d bacterial strain T22 aseptic fermentation filtrate spraying + Botrytis cinerea Bacteria, e, f 50% fludioxonil 1000 times liquid spraying + Botrytis cinerea.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The pathogenic bacteria used in the following examples can be collected from the wild, or can be obtained from Beijing Academy of Agriculture and Forestry Sciences, to repeat the application experiment:

Botrytis cinerea, Fusarium oxysporum f.sp.cucumerinum, Monilinia fructicola, Colletotrichum capsici, Fusarium oxysporum f.sp.vasinfectum , Fusarium oxysporum f.sp.conglutinans, Rhizoctonia acereali, Fusarium moniliforme, Ralstonia solanacearum, Xanthomonas campestris pv. campsetris) (Wang Junli et al. Identification and biocontrol characteristics analysis of a Bacillus sp. QD-10. Chinese Journal of Biological Control, 2014,30(4):564-572.).

Alternaria alternata (Zheng, et al. Characterization of Alternaria species associated with potato foliar diseases in China. Plantpathology, 2015, 64:425-433.).

Strawberry gray mold (Botrytis cinerea), grape gray mold (Botrytis cinerea), cucumber angular spot fungus (Pseudomonas syringae pv. Determination of bacterial activity. Journal of North China Agricultural Science, 2014, 29(1): 195-202.).

Embodiment 1, the isolation and identification of Streptomyces alboniger (Streptomyces alboniger) T22

1.1 Strain isolation

The strain was isolated from soil samples in special habitats of Qinghai-Tibet Plateau, Qinghai Province, China. The bacterial strains were separated by the dilution plate method, and the specific operation method was as follows: Weighed 10 g of soil samples, poured them into a conical flask filled with small glass beads and 90 mL of sterile water, oscillated for 30 min, then allowed to stand for 5 min, diluted 10 times in turn, and prepared respectively 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 suspensions, draw 0.1mL of suspensions with different concentrations and add them to Gao's No. 1 medium (add K with a final concentration of 75mg/L ₂ CrO ₇ ) plates, spread evenly and culture at 28°C for observation, and pick different single colonies to streak and purify after 5-7 days. The purified strains were transferred to Gao's No. 1 slant medium for culture, and stored at 4°C for future use. The strain numbered T22 (abbreviated as strain T22) was used for the following identification.

1.2 Strain identification

1.2.1 Observation of strain morphology

Inoculate the strain T22 on the medium recommended in the Streptomyces Identification Manual, incubate at 28°C for 7-10 days to observe the growth of the strain, and record the color of the aerial hyphae, the color of the hyphae in the base, and the presence or absence and color of soluble pigments . Streak inoculation of the strain T22 on Gao's No. 1 medium, insert a sterile cover glass at an angle of 45 degrees, culture at 28°C for 7 days, take out the cover glass and observe the morphological characteristics of hyphae and spores under an optical microscope.

Strain T22 grows well on Gao's No. 1 agar, glucose asparagine agar, potato agar, yeast extract malt extract agar, oat flour agar, and the aerial hyphae are white; it can be cultured on sucrose nitrate agar and inorganic salt starch agar Basally, the aerial hyphae are white to light olive yellow, light green grayish yellow. Strain T22 has no soluble pigment on Gaoshi No. 1 agar and sucrose nitrate agar medium, but has dark soluble pigment on glucose asparagine agar, potato agar, yeast extract malt extract agar, oat flour agar and inorganic salt starch agar medium. Yellow to black (Table 1). Under the microscope, the aerial hyphae of strain T22 can be observed until they are flexible, and the spores are in chains and oval (Figure 2). Based on the above morphological characteristics, T22 was initially identified as Streptomyces sp.

Among them, Gao's No. 1 agar: soluble starch 20.0g, NaCl 0.5g, KNO ₃ 1.0g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.5g, FeSO ₄ 0.02g, agar 18.0g, distilled water 1000mL, pH 7.2-7.4, sterilize at 121°C for 30 minutes.

Sucrose nitrate agar: 50.0g sucrose, 0.2g KNO ₃ , 18.0g agar, 1000mL distilled water, pH 7.2, sterilized at 121°C for 30min.

Glucose asparagine agar: 10.0g glucose, 0.5g asparagine, 2.0g beef extract, 0.5g K ₂ HPO ₄ , 18.0g agar, 1000mL distilled water, pH 7.2, sterilized at 121°C for 30min.

Potato agar: 200mL potato infusion, 20.0g sucrose, 18.0g agar, 1000mL distilled water, pH 7.2-7.4, sterilized at 121°C for 30min.

Yeast extract malt extract agar medium (ISP2): yeast extract 4.0g, malt extract 10.0g, glucose 4.0g, trace salt solution 1.0mL, agar 18.0g, distilled water 1000mL, pH 7.2, sterilized at 121°C for 30min. Oat flour agar (ISP3): 20.0g oatmeal (add 1000mL water and boil for 20min, then filter and replenish water to 1000mL), trace salt solution 1.0mL, agar 18.0g, distilled water 1000mL, pH 7.2, sterilize at 121°C for 30min.

Inorganic salt starch agar (ISP4): soluble starch 10.0g, K ₂ HPO ₄ 2.0g, MgSO ₄ 7H ₂ O 1.0g, (NH ₄ ) ₂ SO ₄ 2.0g, CaCO ₃ 2.0g, trace salt solution 1.0mL, Agar 18.0g, distilled water 1000mL, pH 7.2, sterilized at 121°C for 30min.

Trace salt solution: FeSO ₄ ·7H ₂ O 0.1g, MnCl ₂ ·4H ₂ O 0.1g, ZnSO ₄ ·7H ₂ O 0.1g, distilled water 1000mL.

Table 1 Morphological characteristics of strain T22 on different media

culture medium aerial hyphae base hyphae Soluble pigment Gao's No. 1 Agar White light yellow to yellowish brown none Sucrose Nitrate Agar white to light green grayish yellow White none Glucose Asparagine Agar White dark grey dark grey potato agar White yellow dark green black Yeast Extract Malt Extract Agar (ISP2) White yellowish dark yellow Oatmeal Agar (ISP3) White colorless to slightly yellow dark yellow Inorganic Salt Starch Agar (ISP4) White to pale olive-yellow yellowish black

1.2.2 Detection of physiological and biochemical characteristics of strains

Physiological and biochemical characteristics mainly detect the strain's utilization of the main carbon source and the activity of producing hormones. Strain T22 can utilize D-glucose, L-arabinose, D-mannitol, inositol, maltose, and raffinose, but cannot utilize L-rhamnose, sucrose, xylose, and fructose. Strain T22 was positive for milk peptonization, starch hydrolysis, and gelatin liquefaction, and did not produce cellulose, melanin, and hydrogen sulfide (Table 2).

Table 2 Physiological and biochemical characteristics of strain T22

Carbon source utilization result Enzyme activity result D-glucose + peptonization of milk + L-arabinose + starch hydrolysis + D-Mannitol + gelatin liquefaction + Inositol + Cellulose - maltose + melanin - Raffinose + hydrogen sulfide - L-rhamnose - sucrose - Xylose - fructose -

Note: "+" is positive, "-" is negative.

1.2.3 Molecular biological identification of strains

The strain T22 was picked and inoculated on Gao's No. 1 agar medium, and cultured at 28°C for 7 days to collect the bacteria. After the genomic DNA of the strain T22 was extracted using the bacterial genome extraction kit, the bacterial universal primers 27f: 5'-AGA GTT TGA TCC TGGCTC AG-3' and 1492r: 5'-TAC GGC TAC CTT GTT ACG ACT T-3' were used to extract the strain PCR amplification of 16S rDNA sequence of T22. The PCR amplification product was recovered, and after ligation, transformation, and identification, positive clones were sent to Beijing Biomed Biotechnology Co., Ltd. for sequencing. The strain T22 has the 16S rDNA sequence of sequence 1 in the sequence list. The obtained sequences were compared in GenBank by Blast software, multiple sequence alignment by Clustal X software, and phylogenetic tree was constructed by Neighbor-Joining method in MEGA5.0 software.

BLAST analysis and comparison showed that the strains with higher homology to strain T22 belonged to the genus Streptomyces, and the 16S rDNA sequences of the strains with higher similarity were selected to construct a phylogenetic tree using MEGA5.0 software (Figure 3). The results showed that the strain belonged to the same branch as Streptomyces alboniger (NR043228, AB184331), with a similarity of 99%. Combined with morphological and cultural characteristics, the strain T22 was identified as Streptomyces alboniger.

Streptomyces alboniger T22 was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on May 25, 2017, and the preservation number is CGMCC No.14190. Hereinafter referred to as Streptomyces alboniger T22 or strain T22.

The antibacterial activity of embodiment 2, streptomyces alboniger (Streptomyces alboniger) T22

2.1 Agar block method: use a sterile pipette to absorb 5 mL of sterile water on the slant of the target pathogenic bacteria culture to be tested, scrape off the hyphae with a bamboo stick to prepare a suspension of pathogenic bacteria, and use a 1 mL sterile pipette to absorb 0.1 mL of the bacterial suspension Spread evenly on PDA (pathogenic fungi) or LB (pathogenic bacteria) plates.

The pathogenic fungi tested were Botrytis cinerea, Botrytis cinerea, Botrytis cinerea, Monilinia fructicola, Colletotrichum capsici, crosslinked Alternaria alternata, Fusarium oxysporum f.sp.cucumerinum, Fusarium oxysporum f.sp.vasinfectum, Fusarium oxysporum f.sp.conglutinans, Wheat sheath blight Rhizoctonia cereali, Fusarium moniliforme.

The pathogenic bacteria tested were Ralstonia solanacearum, Xanthomonas campestris pv. campsetris, and Pseudomonas syringaepv. lachrymans.

Cut out the Streptomyces alboniger T22 agar block with a diameter of 0.7 cm that was cultured on the Gaoshi No. 1 plate for 7 days with a puncher, and inoculate it on the plate coated with pathogenic bacteria. After the pathogenic fungi were cultured at 25°C for 4 days and the pathogenic bacteria were cultured at 28°C for 2 days, the diameter of the antagonistic circle was measured. The results showed that Streptomyces alboniger T22 exhibited good in-dish antagonism against various plant pathogens including Botrytis cinerea (Figure 4), and the diameter of the antagonism circle was 12-26 mm (Table 3).

Table 3 Streptomyces alboniger (Streptomyces alboniger) T22 is to the antagonism ring diameter of test pathogen

2.2 Growth rate method

2.2.1 Preparation of aseptic fermentation filtrate of Streptomyces alboniger T22

Pick the Streptomyces alboniger T22 grown on the plate with the inoculation loop and inoculate it into a 500mL Erlenmeyer flask containing 100mL of liquid fermentation medium, shake and culture at 180rpm at 28°C for 7 days, collect the fermentation broth, and centrifuge at 10,000rpm and 4°C for 10min Thalline was removed, and the supernatant of the fermentation broth was filtered with a 0.22 μm microporous membrane to remove the Streptomyces alboniger T22 thallus in the culture to obtain the aseptic fermentation filtrate of Streptomyces alboniger T22 (abbreviated as bacterial strain T22). bacterial fermentation filtrate). Among them, the liquid fermentation medium is Gaoshi No. 1 liquid medium: soluble starch 20g, KNO ₃ 1g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, FeSO ₄ 7H ₂ O 0.01 g Dilute to 1000 mL with distilled water, and sterilize at 121°C for 20 minutes to obtain a liquid fermentation medium.

2.2.2 Pathogenic bacteria cake preparation

Add 4 mL of sterile water to the slopes of Fusarium oxysporum f.sp. cucumerinum, Botrytis cinerea and Botrytis cinerea that have been cultured for 5 days, and separate the mycelium with a bamboo stick. Scrape off to prepare pathogenic bacteria suspension. Use a 1mL sterile pipette to draw 0.1mL of the bacterial suspension and spread it on the PDA plate. After incubating at 28°C for 5 days, the pathogenic bacteria colonies covered the PDA plate, and made a circular pathogenic bacteria cake with a diameter of 7 mm with a sterile puncher.

2.2.3 Determination of antibacterial rate of antagonistic bacteria aseptic fermentation filtrate

Mix the sterile fermentation filtrate of Streptomyces alboniger T22 and the PDA medium cooled to about 50°C in a volume ratio of 1:4, pour it onto the plate, and pick the cake of the above-mentioned pathogenic bacteria in the center of the plate with a sterilized bamboo stick. The bacterial side was facing up, and each treatment was repeated 3 times, and sterile water was used instead of sterile fermentation filtrate as a control. After culturing at 25°C for 4 days, the colony diameter was measured by the cross method, and the inhibition rate was calculated. Inhibition rate% = (control colony diameter - treated colony diameter) / control colony diameter × 100. Test pathogenic fungi hardly grow on the PDA plate containing the aseptic fermentation filtrate of bacterial strain T22, but grow normally on the sterile water control PDA plate (Fig. 5). The aseptic fermentation filtrate of strain T22 has obvious inhibitory effect on the mycelial growth of cucumber fusarium wilt (Fusarium oxysporumf.sp.cucumerinum), tomato gray mold (Botrytis cinerea) and strawberry gray mold (Botrytiscinerea), and the inhibition rate is 79.1% %, 84.6% and 85.5%.

Embodiment 3, Streptomyces alboniger (Streptomyces alboniger) T22 produces cellulase and chitinase activity

Pick the Streptomyces alboniger T22 grown on the plate with the inoculation loop in the cellulase-producing medium and chitinase-producing medium, and culture it at 28°C for 7 days, then add 2g/L Congo red staining solution to the plate 5mL, rinsed with deionized water after staining for 30min, then decolorized with 1M NaCl solution, rinsed with deionized water, and observed the formation of hydrolysis circles around the colonies.

Cellulase production medium: cellulose powder 5.0g, (NH ₄ ) ₂ SO ₄ 2.0g, KH ₂ PO ₄ 1.0g, NaCl 0.5g, MgSO ₄ ·7H ₂ O 0.5g, agar 18.0g, distilled water 1000mL.

Chitinase-producing medium: colloidal chitin 2.5g, K ₂ HPO ₄ 0.7g, K ₂ HPO ₄ 0.3g, MgSO ₄ 7H ₂ O 0.5g, FeSO ₄ 7H ₂ O 0.01g, agar 18.0g , distilled water 1000mL.

The results showed that a hydrolysis circle formed around Streptomyces alboniger T22, indicating that Streptomyces alboniger T22 could produce cellulase and chitinase (Figure 6).

Embodiment 4, the influence of white and black streptomyces (Streptomyces alboniger) T22 aseptic fermentation filtrate on seed germination

4.1 Preparation of aseptic fermentation filtrate of Streptomyces alboniger T22

Pick the Streptomyces alboniger T22 grown on the plate with the inoculation loop and inoculate it into a 500mL Erlenmeyer flask containing 100mL of liquid fermentation medium, culture it on a shaker at 180rpm at 28°C for 7 days, collect the fermentation broth, and filter it through a 0.22μm microporous filter. Streptomyces alboniger T22 cells in the culture were removed by membrane filtration of the fermentation broth to obtain a sterile fermentation filtrate of Streptomyces alboniger T22. Among them, the liquid fermentation medium is Gaoshi No. 1 liquid medium: soluble starch 20g, KNO ₃ 1g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, FeSO ₄ 7H ₂ O 0.01 g Dilute to 1000 mL with distilled water, and sterilize at 121°C for 20 minutes to obtain a liquid fermentation medium.

4.2 Preparation of Streptomyces alboniger T22 sterile fermentation filtrate dilution

The streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate of step 4.1 is diluted 10 times, 100 times, 200 times, 500 times respectively with sterile water, obtains the 10 times dilution of streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate, 100-fold dilution of Streptomyces alboniger T22 sterile fermentation filtrate, 200-fold dilution of Streptomyces alboniger T22 sterile fermentation filtrate and 500-fold dilution of Streptomyces alboniger T22 sterile fermentation filtrate.

4.3 Seed germination test

Select tomato (Jiafen No. 1) seeds with plump particles and the same size, and use the 10-fold dilution, 100-fold dilution, 200-fold dilution and 500-fold dilution of the aseptic fermentation filtrate of Streptomyces alboniger T22 in step 4.2 Soak the seeds and use an equal volume of the liquid fermentation medium (sterile) from step 4.1 as a control. Three replicates were set for each treatment, and 10 seeds were set for each replicate. Cultivate at 28°C for 24 hours, collect the seeds, rinse the seeds with sterile water, put them on a petri dish covered with two layers of sterile water-soaked filter paper, and culture them at 28°C for 5 days, count the germination rate, measure the hypocotyl, embryo root length.

The experimental results showed that compared with the control, the 100-fold dilution of the aseptic fermentation filtrate of Streptomyces alboniger T22 significantly increased the length of tomato hypocotyls and radicles, and improved the germination potential of tomato seeds, and the increases were as high as 15.1%, 29.7% and 43.9%; The tomato seeds treated with 200-fold dilution of streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate, its hypocotyl, radicle length and seed germination potential have increased by 8.8%, 18.1% and 32.3% (table 4) compared with the contrast .

Table 4 Effect of Streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate on tomato seed germination

Embodiment 5, Streptomyces alboniger (Streptomyces alboniger) T22 fermented liquid is to tomato botrytis in vitro control effect

Using the method of detached leaves, 30 top leaves of tomato plants with the same size were selected, sterilized with 2% (w/v) sodium hypochlorite for 3 minutes, rinsed with tap water and dried in the air. The test set up 3 groups of treatments, each group treated 10 leaves, including non-inoculated liquid medium spraying + Botrytis cinerea treatment, strain T22 fermentation broth spraying + Botrytis cinerea treatment, 50% fludioxonil 1000 times liquid spraying + Botrytis cinerea treatment (Figure 7).

1, bacterial strain T22 fermented liquid spraying+Botrytis cinerea processing: the streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermented filtrate of step 4.1 of embodiment 4 is used to carry out spraying treatment to tomato leaf, until covering all over leaf, after 24h, adopt extermination A bacterial cake of Botrytis cinerea with a diameter of 7 mm was cut with a fungus puncher and inoculated in the center of the treated leaves.

2, non-inoculated liquid medium spraying+Botrytis cinerea processing: replace the streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate of step 4.1 of embodiment 4 with the liquid fermentation medium (sterile) of step 4.1 of embodiment 4 , other operations are the same as step 1. as a blank control.

3, 50% fludioxonil 1000 times of liquid spraying + Botrytis cinerea spraying treatment: replace the streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate of step 4.1 of embodiment 4 with 50% fludioxonil 1000 times of liquid, other The operation is the same as step 1. as a chemical control.

After 7 days, measure the treatment and control tomato leaf lesion diameters, according to the formula: control effect %=(blank control average lesion diameter-handling average lesion diameter)/blank control average lesion diameter × 100%, calculate the effect of bacterial strain T22 fermentation broth on In vitro control efficacy of Botrytis cinerea. The results showed that Streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate (sterile metabolites) sprayed to treat the detached tomato leaves can significantly reduce the diameter of tomato gray mold lesions, and the aseptic fermentation filtrate of Streptomyces alboniger T22 (Sterile metabolite) treatment of tomato gray mold in vitro control effect reached 55.1% (Table 5).

Table 5, Streptomyces alboniger (Streptomyces alboniger) T22 aseptic fermentation filtrate is to tomato Botrytis cinerea control effect in vitro

deal with Lesion diameter cm Anti-efficacy% Non-inoculated liquid medium spray + Botrytis cinerea 2.16±0.23 - Strain T22 fermented liquid spraying + botrytis cinerea 0.97±0.21 55.1% 50% fludioxonil 1000 times liquid spray + Botrytis cinerea 0.84±0.09 61.1%

<110> Beijing Academy of Agriculture and Forestry Sciences

<120> Preparation and application of a strain of Streptomyces alboblack and its metabolites for disease prevention and growth promotion

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1310

<212>DNA

<213> Streptomyces alboniger

<400> 1

agtggcgaac gggtgagtaa cacgtggggca atctgccctt cactctggga caagccctgg 60

aaacggggtc taataccgga taacacctcc actctcctga gtggaggtta aaagctccgg 120

cggtgaagga tgagcccgcg gcctatcagc ttgttggtga ggtaatggct caccaaggcg 180

acgacgggta gccggcctga gagggcgacc ggccaacactg ggactgagac acggcccaga 240

ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac 300

gccgcgtgag ggatgacggc cttcgggttg taaacctctt tcagcaggga agaagcgaaa 360

gtgacggtac ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggtaatacgt 420

agggcgcaag cgttgtccgg aattattggg cgtaaagagc tcgtaggcgg cttgtcacgt 480

cgggtgtgaa agcccggggc ttaaccccgg gtctgcattc gatacgggct agctagagtg 540

tggtagggga gatcggaatt cctggtgtag cggtgaaatg cgcagatc aggaggaaca 600

ccggtggcga aggcggatct ctgggccatt actgacgctg aggagcgaaa gcgtggggag 660

cgaacaggat tagataccct ggtagtccac gccgtaaacg gtgggaacta ggtgttggcg 720

acattccacg tcgtcggtgc cgcagctaac gcattaagtt ccccgcctgg ggagtacggc 780

cgcaaggcta aaactcaaag gaattgacgg gggcccgcac aagcagcgga gcatgtggct 840

taattcgacg caacgcgaag aaccttacca aggcttgaca tacaccggaa agcatcagag 900

atggtgcccc ccttgtggtc ggtgtacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt 960

gagatgttgg gttaagtccc gcaacgagcg caacccttgt tctgtgttgc cagcatgccc 1020

ttcggggtga tggggactca cagaagaccg ccggggtcaa ctcggaggaa ggtggggacg 1080

acgtcaagtc atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggcaggtaca 1140

atgagctgcg ataccgtgag gtggagcgaa tctcaaaaag cctgtctcag ttcggattgg 1200

ggtctgcaac tcgacccccat gaagtcggag ttgctagtaa tcgcagatca gcattgctgc 1260

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac gtcacgaaag 1310

Claims (10)

1. Streptomyces alboniger (Streptomyces alboniger), its strain number is T22, and its registration number in the General Microbiology Center of China Committee for Microbial Culture Collection is CGMCC No.14190. 2. The culture of Streptomyces albonigeri described in claim 1 is the material obtained by culturing the Streptomyces albonigers described in claim 1 in a microbial culture medium. 3. Pathogenic bacteria inhibitor, characterized in that: said pathogenic bacteria inhibitor contains the Streptomyces albonigeris described in claim 1 and/or the metabolite of Streptomyces albonigers described in claim 1. 4. The pathogen inhibitor according to claim 3, characterized in that: the pathogen inhibitor has an inhibitory effect on at least one of the following pathogens: A. Botrytis cinerea; B. Drupe brown rot fungus; C, capsicum anthracnose bacteria; D. Fusarium wilt; E. Potato blight bacillus; F, wheat sheath blight; G, eggplant solanacearum; H, Cucumber horn spot fungus. 5. The disease inhibitor, characterized in that: the disease inhibitor contains the Streptomyces albonigeri described in claim 1 and/or the metabolite of the Streptomyces albonigers described in Claim 1. 6. The disease inhibitor according to claim 5, characterized in that: the disease is at least one of the following: a. Botrytis; b. Drupe fruit brown rot; c. Pepper anthracnose; d. Fusarium wilt; e. Potato early blight; f. Wheat sheath blight; g, eggplant bacterial wilt; h, Cucumber horn spot. 7. any of the following applications of the metabolites of the Streptomyces albonigeris described in claim 1 and/or the Streptomyces albonigers described in claim 1: 1) the application of the metabolites of the Streptomyces albo-nigeris described in claim 1 and/or the Streptomyces albo-nigeris described in claim 1 in inhibiting pathogenic bacteria; 2) the application of the metabolites of the Streptomyces albo-nigeris described in claim 1 and/or the Streptomyces albo-nigeris described in claim 1 in the preparation of pathogen inhibitors; 3) the application of the metabolites of the Streptomyces albo-nigeris described in claim 1 and/or the Streptomyces albo-nigeris described in claim 1 in suppressing disease; 4) Application of the Streptomyces albonigeris described in claim 1 and/or the metabolites of the Streptomyces albonigers described in claim 1 in the preparation of disease inhibitors. 8. The application according to claim 7, characterized in that: the pathogen is at least one of the following: A. Botrytis cinerea; B. Drupe brown rot fungus; C, capsicum anthracnose bacteria; D. Fusarium wilt; E. Potato blight bacillus; F, wheat sheath blight; G, eggplant solanacearum; H, Cucumber horn spot fungus. 9. any of the following applications of the metabolites of the Streptomyces albonigers described in claim 1 and/or the Streptomyceses albonigers described in claim 1: H, the application of the metabolite of Streptomyces albonigeri described in claim 1 and/or the Streptomyces albonigeris described in claim 1 in promoting plant seed germination; 1, the application of the metabolite of Streptomyces albonigers described in claim 1 and/or the Streptomyceses albonigers described in claim 1 in promoting plant seed hypocotyl and/or radicle growth; J, the application of the metabolites of the Streptomyces albonigeris described in claim 1 and/or the Streptomyces albonigers described in claim 1 in improving the germination potential of plant seeds. 10. The application according to claim 9, characterized in that: the plant is any one of the following plants: P1) tomato; P2) Tomato plants; P3) Solanaceae plants.