CN106434493B - A strain of biocontrol Streptomyces and its application - Google Patents

Abstract

The invention discloses one plant of biological and ecological methods to prevent plant disease, pests, and erosion streptomycete and its applications.The biological and ecological methods to prevent plant disease, pests, and erosion streptomycete is blastmycin streptomycete, and it is CGMCC No.13270 in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center that bacterial strain number, which is JZB130180,.The bacterial strain has significant antagonism to serious 14 kinds of plant pathogenic fungis and 6 kinds of plant pathogenetic bacterias occur in agricultural production, and antimicrobial spectrum is wide.The bacterial strain generates chitinase, cellulase, protease and thermophilic iron element, the essential characteristic for having biocontrol microorganisms, the fermentation liquid of the bacterial strain has stronger control effect to graw mold of tomato and peach brown rot, and there are apparent growth-promoting functions to tomato plant, illustrate that the bacterial strain has broad application prospects in the field of biological control of plant disease.

Description

A strain of biocontrol Streptomyces and its application

technical field

The invention belongs to the technical field of microbial pesticides, and particularly relates to a biocontrol Streptomyces strain and its application.

Background technique

As people pay more and more attention to and understand the concepts of ecological balance, organic food and green health, biological control methods can effectively avoid a series of problems caused by chemical pesticides, and at the same time have profound social benefits, ecological Benefit and long-term benefits have received extensive and unprecedented attention in agricultural production. Under the background of sustainable development of agricultural science, biological control is also the only way for agricultural development. Actinomycetes are the earliest and most widely used biocontrol microorganisms and play a huge role in the biocontrol of plant diseases. Actinomycetes are still the research hotspots in the research and development of new medicines, new pesticides and live biological control agents in the world. They have a wide variety of metabolic functions and are a kind of microbial resources with a wide range of practical uses.

Peaches are one of the most popular fruits, and peach trees have become a pillar industry in many regions. Brown rot caused by Monilinia spp. is one of the most important diseases in peach production and the most important post-harvest disease of kernel fruits worldwide. The disease can cause a large amount of yield reduction in pre-harvest, post-harvest and storage and transportation periods, especially post-harvest, with losses as high as 59-90% in Europe and the United States. But peach brown rot is a common and serious disease in peach fruit growing period and storage period, which is distributed worldwide. It occurs in the north and south of China, especially in coastal areas such as Zhejiang and Shandong and the peach producing areas in the Jianghuai River Basin. Many studies have reported that fruit rot is mainly due to the invasion of pathogenic microorganisms. In order to effectively reduce the loss caused by postharvest rot of fruits, people have used physical measures such as low temperature, modified atmosphere, and decompression in combination with chemical pesticides to prevent and control the occurrence of many fungal diseases such as brown rot. However, at present, chemical agents are mainly used to prevent and control the disease. Although it can effectively and quickly achieve the purpose of prevention and control, large-scale use will cause pesticide residues, pollute the environment and have adverse effects on human health. If used alone for a long time, the bacteria will develop resistance, as above As mentioned above, biological control has become a hot spot of current research and development because it is safe to the environment, has no pollution, and has low cost, and meets the requirements of agricultural ecology and sustainable development.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to inhibit plant pathogens and promote plant growth.

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

The Streptomyces provided by the present invention, Streptomyces blastmyceticus (Streptomyces blastmyceticus), its strain number is JZB130180, and its registration number in the General Microbiology Center of the China Microorganism Culture Collection Management Committee is CGMCC No.13270. It is hereinafter referred to simply as Streptomyces blastmyceticus JZB130180 or strain JZB130180.

Streptomyces blastmyceticus JZB130180 on solid medium, the colonies are regular and round with neat edges, the cells are yellow, the surface of the colony is shriveled, the center is raised, and with the extension of the culture time, gray-white appears spore heap. Streptomyces blastmyceticus (Streptomyces blastmyceticus) JZB130180 is a Gram-positive bacteria, can liquefy gelatin, contact enzyme and oxidase positive, can coagulate and peptonize milk, hydrolyze starch, weakly utilize citrate, not Produces melanin and hydrogen sulfide. Streptomyces blastmyceticus JZB130180 has the 16S rDNA sequence of Sequence 1 in the Sequence Listing, and the atpD, recA, and rpoB gene sequences shown in Sequences 2-4 in the Sequence Listing.

In order to solve the above technical problems, the present invention provides inoculants containing metabolites of Streptomyces blastmyceticus JZB130180 or/and Streptomyces blastmyceticus JZB130180.

Specifically, the above-mentioned bacterial agent may be a pathogenic bacteria inhibitor or a disease inhibitor.

The active ingredient of the above-mentioned pathogenic bacteria inhibitor can be the metabolite of Streptomyces blastmyceticus JZB130180 or/and Streptomyces blastmyceticus JZB130180, and the active ingredient of the above-mentioned pathogenic bacteria inhibitor can also contain other biological components Or non-biological components, other active components of the above pathogenic bacteria inhibitor can be determined by those skilled in the art according to the inhibitory effect on pathogenic bacteria.

The active ingredient of the above-mentioned disease inhibitor can be the metabolite of Streptomyces blastmyceticus JZB130180 or/and Streptomyces blastmyceticus JZB130180, and the active ingredient of the above-mentioned disease inhibitor can also contain other biological components Or non-biological components, other active components of the above-mentioned disease inhibitors can be determined by those skilled in the art according to the inhibitory effect on diseases.

Any of the following applications of the metabolites of Streptomyces blastmyceticus JZB130180 or/and Streptomyces blastmyceticus JZB130180 also belong to the protection scope of the present invention:

1) Application in inhibiting pathogenic bacteria;

2) Application in the preparation of pathogenic bacteria inhibitors;

3) Application in the preparation of disease inhibitors;

4) Application in disease suppression.

In the above, the pathogenic bacteria can be all or part of the following pathogenic bacteria: Botrytis cinerea, plant brown rot bacteria, wheat sheath blight, wheat scab, wheat total rot, pepper anthracnose, grape anthracnose, plant Fusarium wilt, rice sheath blight, lily root rot, apple ringworm, pepper scab, cucumber angular leaf spot, eggplant wilt, Bacillus cereus, soil rhizobacter and Chinese cabbage black rot.

The plant Fusarium wilt can be all or part of the following pathogens: Fusarium wilt of cucumber, Fusarium wilt of cotton, Fusarium wilt of watermelon and Fusarium wilt of cabbage.

The Bacillus can be Bacillus cereus.

In the plant brown rot fungus, the fruit of the plant is a stone fruit, such as a peach. The plant brown rot fungus can specifically be Monilinia fructicola.

In the botrytis cinerea, the plant may be a Solanaceae plant, such as a tomato.

In the above, the disease may be all or part of the following diseases: botrytis cinerea, plant brown rot, wheat sheath blight, wheat scab, wheat take-all disease, pepper anthracnose, grape anthracnose, plant wilt disease, rice sheath blight, lily root rot, apple ring rot, pepper scab, cucumber angular spot, eggplant bacterial wilt and Chinese cabbage black rot.

The plant fusarium wilt may be all or part of the following four diseases: cucumber fusarium wilt, cotton fusarium wilt, watermelon fusarium wilt and cabbage fusarium wilt.

In the plant brown rot, the fruit of the plant can be a stone fruit, such as a peach.

In the botrytis cinerea, the plant may be a Solanaceae plant, such as a tomato.

In the above, in addition to the active ingredient, the inoculum also contains a carrier. The carrier may be a carrier commonly used in the field of pesticides and which 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 or/and polyglycol; the liquid carrier can be It is an organic solvent, vegetable oil, mineral oil or water; the organic solvent can be decane or/and dodecane.

The dosage form of the bacterial agent can be various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

As required, surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants), pH adjusters, etc. can also be added to the bacterial agent.

The application of the metabolites of Streptomyces blastmyceticus JZB130180 or/and Streptomyces blastmyceticus JZB130180 in promoting plant growth also belongs to the protection scope of the present invention.

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

P1) seed plants;

P2) dicotyledonous plants;

P3) Solanaceae;

P4) Tomato.

In the above application, the promoting plant growth may be increasing the length of the main root of the plant and/or increasing the number of fibrous roots of the plant and/or increasing the stem height and/or the weight of the plant.

In the above, the metabolite of Streptomyces blastmyceticus JZB130180 can be obtained from the fermentation broth of Streptomyces blastmyceticus JZB130180. The metabolite of Streptomyces blastmyceticus JZB130180 can be specifically prepared according to the following method: Cultivate Streptomyces blastmyceticus JZB130180 in a liquid medium, remove the rice in the liquid culture (fermentation broth) Streptomyces blastmyceticus JZB130180 is the metabolite of Streptomyces blastmyceticus JZB130180.

In the present application, the tomato botrytis cinerea may be Botrytis cinerea.

Streptomyces blastmyceticus (Streptomyces blastmyceticus) JZB130180 has significant antagonistic effect on 14 kinds of phytopathogenic fungi and 6 kinds of phytopathogenic bacteria which occur seriously in agricultural production, and has a broad antibacterial spectrum. The strain produces chitinase, cellulase, protease and siderophos, and has the basic characteristics of biocontrol bacteria. The fermentation broth of this strain has a strong control effect on tomato gray mold and peach brown rot, and It has obvious growth-promoting effect on tomato plants, indicating that the strain has broad application prospects in the field of biological control of plant diseases.

Preservation Instructions

Species name: Streptomyces blastmyceticus

Strain number: JZB130180

Preservation institution: General Microbiology Center of China Microorganism Culture Collection Management Committee

Abbreviation of depositary institution: CGMCC

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

Deposit date: November 14, 2016

Depository center registration number: CGMCC No.13270

Description of drawings

Figure 1 shows the separation plate.

Among them, A-E are the colonies on the plate during soil separation, and F is the actinomycetes slants preserved after purification.

Fig. 2 is the antagonism result of the plate antagonistic activity assay against Peach brown rot fungus.

Among them, CK is the control of peach brown rot.

Figure 3 shows the culture state of strain JZB130180 on three media (left image) and the mycelium morphology (right image) under a 400 magnification light microscope.

Figure 4 shows the PCR amplification results in the identification of strain JZB130180.

M: DL2000 DNA Marker, lanes 1 and 2: extracted DNA samples; lanes 3-6: gene amplification bands of 16S rDNA, atpD, recA, and rpoB in sequence.

Figure 5 is a phylogenetic tree constructed based on the 16S rDNA sequence of strain JZB130180.

Figure 6 shows the results of the antibacterial assay of strain JZB130180 against plant pathogens.

Wherein, A: wheat sheath blight, B: wheat scab, C: wheat total rot, D: pepper anthracnose, E: grape anthracnose, F: peach brown rot, G: tomato gray mold, H: Cucumber Fusarium wilt, I: Cotton Fusarium wilt, J: Watermelon Fusarium wilt, K: Cabbage Fusarium wilt, L: Rice sheath blight, M: Lily root rot, N: Apple rot, O: Capsicum scab Bacteria, P: angular leaf spot of cucumber, Q: bacterial wilt of eggplant, R: Bacillus cereus, S: Agrobacterium tumefaciens C58, T: black rot of Chinese cabbage.

Figure 7 shows the detection of extracellular enzymes related to biocontrol produced by strain JZB130180.

Among them, A: chitinase detection; B: protease detection; C: cellulase detection; D: siderophil detection

Figure 8 shows the bacteriostatic effect of the aseptic fermentation filtrate of strain JZB130180 on peach brown rot.

Figure 9 shows the growth of peach brown rot fungus in a toxic medium.

Among them, 1 to 6 indicate that the concentration of the sterile fermentation filtrate of strain JZB130180 in the toxic medium is 0 μg/mL, 12.5 μg/mL, 25.0 μg/mL, 50.0 μg/mL, 100 μg/mL, and 200 μg/mL.

Figure 10 shows the inhibitory effect of strain JZB130180 aseptic fermentation filtrate on peach brown rot.

Among them, rows 1-3 are treatment B1, treatment C1 and negative control in sequence.

Figure 11 shows the inhibitory effect of strain JZB130180 aseptic fermentation filtrate on tomato gray mold.

Among them, rows 1-4 are treatment B2, treatment C2, treatment D and negative control in sequence.

Figure 12 is the determination of the growth-promoting effect of the aseptic fermentation filtrate of strain JZB130180 on tomato plants.

Among them, AZ10, BZ10 and CZ10 are the results of seed soaking treatment for 10 days from sowing; DZ20 is the result of seed soaking treatment for 20 days after sowing; DG20 is the result of root irrigation treatment for 20 days after sowing; EZ30 is the seed soaking treatment. The results of 30 days from sowing; EG30 is the result of root irrigation treatment 30 days after sowing; FZ40 is the result of seed soaking treatment 40 days after sowing; FG40 is the result of root irrigation treatment 40 days after sowing.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. 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 obtained from the Beijing Academy of Agriculture and Forestry, so as to repeat the experiment of this application:

Fusarium wilt of cabbage - Fusarium oxysporum Schl.f.sp.conglutinans (Wollenw.) Snyder&Hansen] (Li Mingyuan et al. Cruciferous vegetable fusarium wilt and its pathogen identification. Plant Protection. 2003,29 (6):44-45);

Fusarium oxysporum f.sp.niveum (Geng Lihua et al. Establishment and verification of the identification technology system for physiological races of Fusarium oxysporum. China Vegetables. 2010, (20): 52-56)

Rhizoctonia cerealis (Ji Zhaolin et al. Effect of wheat sheath blight toxin on wheat plants. Journal of Yangzhou University (Agriculture and Life Science Edition)). 2011,3 2(3):55-59);

Gaeumannomyces graminis (Sacc.) Arx&Olivier var.triticiJ.Walker (Liu Weiguo. Effects of Chemical Treatment on Control Efficacy and Yield Factors of Wheat Take-all Disease. Hubei Agricultural Science. 2012,51(16):3483- 3487);

Tomato Botrytis cinerea - Botrytis cinerea Per.ex Fr. (Li Xinghong et al. Detection of drug resistance of Botrytis cinerea to pyrimethamine in Beijing area. Plant Protection. 2012, 38(4): 141-143 );

Peach brown rot fungus - Monilinia fructicola (wint.) Rehm (Wang Fei et al. Occurrence and control of peach brown rot. Fruit trees and flowers. 2012, 5:58);

Colletotrichum capsici (Song Genmiao et al. Synergistic effect of thiamdone and difenoconazole mixture on 4 different pathogenic bacteria. Plant Protection. 2012, 38(4): 171-174);

Grape anthracnose - Colletortrichum gloeosporioides Penz.e tSacc. (Li Lixia et al. Genetic diversity analysis of grape anthracnose S RAP. China Agricultural Science Bulletin. 2012, 2 8(1 2): 230-235);

Pseudomonas syringae pv.lachrymans (A.T.Alleyne.et.al.Identification of Pseudomonas syringae pv.lachrymans in Barbados by rep-PCR.Journal of Agricultural Science and Technology B1.2001,593-597);

Eggplant Ralstonia solanacearum (Feng Linlin et al. Identification and Character Observation of Eggplant Ralstonia Resistant Materials. Changjiang Vegetables. 2000, (10): 35-37);

Chinese cabbage black rot fungus (Xanthomonas campestris pv.campsetris) (Zhai Wenhui et al. Humidity test for identification of Chinese cabbage black rot and correlation analysis of disease resistance at seedling and adult stages. Chinese Vegetables. 2010, (10): 59-63);

Lily root rot fungus - Fusarium oxysporum Schlecht (An Zhihui et al. Identification and control methods of lily root rot pathogen. Zhongyuan Vegetables. 2010, (3): 23-24)

Agrobacterium tumefaciens C58cereon (Fan Chengming et al. Analysis of secreted protein signal peptides in Agrobacterium tumefaciens C58Cereon. Acta Microbiol. 2005, 45(4): 561-566);

Fusarium graminearum Schw. (Chen Ran et al. Research progress in biological control of wheat scab. Henan Agricultural Science. 2014, 43(12): 1-5)

Fusarium oxysporum f.sp.cucumerinum Owen (Wei Qiaojie et al. Screening and identification of antagonistic bacteria against cucumber Fusarium wilt and its biological control effect. Journal of Nanjing Agricultural University. 2013, 36(1): 40-46)

Fusarium oxysporum f.sp.vasinfectum (Zhu Weiling et al. Screening of Antagonistic Bacteria of Cotton Fusarium Wilt. 2006, 25(2): 7-9)

Rhizoctonia solani (Chen Siyu et al., Screening and identification of antagonistic bacteria against Rhizoctonia solani. Chinese Journal of Plant Protection. 2013, 40(3): 211-218)

Botryosphaeria dothidea (Liu Baoyou et al., Sensitivity and cross-resistance of Botryosphaeria dothidea to difenoconazole and flusilazole. Chinese Journal of Phytopathology. 2013, 43(5): 541-548)

The plasmids of Xanthomonas vesicatoria (Xanthomonas vesicatoria) (Xiang Ping et al., Xanthomonas vesicatoria) and X.oryzae pv.oryzicola and their relationship with streptomycin resistance and copper resistance. Plants Acta Pathology. 2003, 33(4):330-333)

Bacillus cereus (Weikun et al., Research on the degradation characteristics and enzymes of pyrene by Bacillus cereus. Journal of Environmental Science. 2016, 36(2):506-512)

Part of the culture medium involved in the following examples is as follows:

Potato dextrose agar medium (PDA): 200g of potato, washed and cut into small pieces ^of about 1cm, boiled for about 30min, filtered with four layers of gauze, the filtrate was made up to 1000ml with tap water, added 20g of glucose, 15g of agar, pH was natural, 121 Autoclave at ℃ for 30 min.

PD liquid medium: 200 g of potatoes, washed and cut into small pieces of about 1 cm ³ , boiled for about 30 minutes, filtered with four layers of gauze, the filtrate was supplemented to 1000 ml with tap water, added with 20 g of glucose, pH was natural, and autoclaved at 121 °C for 30 minutes.

Gao's No. 1 agar medium: soluble starch 20g, NaCl 0.5g, KNO ₃ 1g, FeSO ₄ ﹒ 7H ₂ O 0.01g, K ₂ HPO ₄ 0.5g, MgSO ₄ ﹒ 7H ₂ O 0.5g, agar 20g, tap water 1000ml, pH 7.2-7.4, sterilized at 121°C for 30min.

Actinomycetes isolation medium I: glucose 10g, beef extract 2g, aspartin 0.5g, K ₂ HPO ₄ 0.5g, agar 20g, tap water 1000ml, pH 6.8 or natural, sterilized at 121°C for 30min.

Actinomycetes isolation medium II: soluble starch 20g, NaCl 0.5g, KNO ₃ 1g, Fe ₂ (SO ₄ ) ₃ 0.01g, K ₂ HPO ₄ 0.5g, MgSO ₄ ﹒ 7H ₂ O 0.5g, agar 20g, tap water 1000ml, pH 7.2-7.4, sterilized at 121°C for 30min.

Actinomycetes isolation medium III: glycerol 20g, NaCl 1g, CaCO ₃ 0.1g, L-arginine 2.5g, FeSO ₄ ﹒ 7H ₂ O 0.1g, MgSO ₄ ﹒ 7H ₂ O 0.1g, agar 20g, tap water 1000ml, pH 6.8～7.0, sterilized at 121°C for 15min.

Actinomycetes isolation medium IV: sucrose 15g, NaNO ₃ 2g, yeast extract 4g, K ₂ HPO ₄ 0.5g, MgSO ₄ ﹒ 7H ₂ O 0.5g, KCl 0.5g, FeSO ₄ ﹒ 7H ₂ O 0.01g, agar 20g, tap water 1000ml, pH 7.2, sterilized at 121°C for 20min.

Actinomycetes isolation medium V: soluble starch 10g, (NH ₄ ) ₂ SO ₄ 2g, K ₂ HPO ₄ 1g, MgSO ₄ ﹒ 7H ₂ O 1g, NaCl 1g, CaCO ₃ 3g, agar 15g, tap water 1000ml, pH 7.2～7.4, sterilize at 121°C for 20min.

Actinomycetes isolation medium VI: 20g oat flour infusion, trace salt solution (FeSO ₄ ﹒ 7H ₂ O 0.1g, MnCl ₂ ﹒ 4H ₂ O 0.1g, ZnSO ₄ ﹒ 7H ₂ O 0.1g, tap water 100ml) 1ml , 15g of agar, 1000ml of tap water, pH 7.2, sterilized at 121°C for 90min.

Actinomycetes isolation medium VII: glucose 1g, soluble starch 2g, yeast extract 0.5g, NZ Amine 0.5g, CaCO ₃ 0.1g, agar 20g, tap water 1000ml, pH 7.2～7.4, sterilized at 121°C for 20min.

ISP2 medium: yeast extract 4.0 g, wort 10.0 g, glucose 4.0 g, agar 20.0 g, distilled water 1000 mL, pH 7.2-7.4, sterilized at 121° C. for 20 min.

Example 1. Isolation and identification of Streptomyces oryzae JZB130180

1.1 Strain isolation

Soil samples were collected from the high-altitude area of Leigong Mountain, Guizhou Province, China, and were subjected to dry heat treatment, dried at room temperature for 1-2 weeks, ground and sieved, and each soil sample was treated at 100 °C for 1 hour in a blast drying oven. to separate. Accurately weigh 1g of soil sample and put it into a 50ml sterile triangular flask containing 9ml sterile salt solution and small glass beads, after fully shaking on a shaker, it is a ^10-1 concentration dilution, and then use a 1ml pipette Pipette 10 ^-1 concentration diluent into 9ml sterile conical flask, shake well to obtain 10 ^-2 concentration diluent, and so on, dilute to 10 ^-3 concentration diluent. After the sample suspension is ready, use a sterile pipette to suck up 0.1ml each of the ^10-2 concentration and ^10-3 concentration dilution solution in strict accordance with the aseptic operation requirements, and put them into the pre-poured and correspondingly numbered actinomycete isolation medium respectively. Plate (the medium contains 20mg/l K ₂ Cr ₂ O ₇ as an inhibitor to inhibit fungi and bacteria in soil samples), and then gently spread the suspension on the plate with a sterile glass coating spatula , place the coated plate in a constant temperature incubator at 28°C for constant temperature cultivation until colonies grow. Observing the actinomycetes cultured in the constant temperature incubator, picking the mature colonies on the slant of Gao's No. 1 agar medium with a sterile inoculation needle in time, streaking and culturing, in order to isolate more actinomycetes, each Pour at least 25-30ml of separation medium into each petri dish to prevent the inactivation of dry-split bacteria, extend the culture time to 3-4 weeks, and pick bacteria in batches. After the same soil sample was uniformly numbered for different Actinomycete strains isolated on different media, they were temporarily stored in a refrigerator at 4°C on a slant (Figure 1).

Screening of biocontrol strains: Take peach brown rot as the target pathogen, and culture it on PDA in a constant temperature incubator at 25°C for 5-7 days. At this time, a large number of spore layers are produced on the plate. On a PDA plate with uniform thickness, first spot a few spores of peach brown rot fungus in the center, and then spot a few spores of four isolated and purified actinomycetes at a distance of 2 cm from the pathogen. Pathogens were blank controls. After labeling, place them in a 25°C constant temperature incubator for cultivation. When the control plate is covered with the entire plate, observe and record the experimental results. The experiment was repeated three times.

Test results: As a result, more than 20 antagonistic actinomycete strains with strong antibacterial effect on peach brown rot were obtained, and one strain numbered JZB130180 had the best antibacterial effect, and its antibacterial bandwidth was more than 7 mm (Figure 2). ).

1.2 Identification of strains

Morphological culture characteristics: The activated strain JZB130180 was streaked on PDA, ISP2 and Gao's No. 1 agar plates, and incubated at a constant temperature of 28 °C. The culture morphology of the strain on each medium was observed and recorded, and the bacteria were inoculated and cultured for 5-7 days by the insert method, and the morphology of the aerial hyphae was observed and photographed under an optical microscope. The results showed that the strain JZB130180 could grow on the above three media, the colony was regular and round with neat edges, the cell was yellow, the colony surface was shriveled, the center was raised, and with the extension of the culture time, gray-white spores appeared. heap. Among the above three kinds of media, the bacterium grows more vigorously on ISP2 medium, and has stronger sporulation ability, followed by PDA medium, and the growth of bacteria on Gao's No. 1 medium is poor. From the colony morphology and microscopic observation, it was preliminarily believed that the bacteria belonged to actinomycetes (Figure 3).

Physiological and biochemical characteristics: Amylase hydrolysis, hydrogen sulfide production, citrate utilization, gelatin liquefaction, contact enzymes, melanin production, nitrate reduction were performed on the methods in the reference document "Soil Microbial Research Principles and Methods" (Lin Xiangui, 2010) of strain JZB130180 , milk coagulation and peptonization and other physiological and biochemical properties determination. The results showed that the strain JZB130180 was a gram-positive bacterium, which could liquefy gelatin, was positive for contact enzymes and oxidase, could coagulate and peptonize milk, hydrolyze starch, and weakly utilize citrate without producing melanin and hydrogen sulfide.

Sequence determination and analysis: The strain JZB130180 was activated on a PDA plate, cultured at 28°C for 5 days, transferred to PD liquid medium and shaken for 24 hours, took 1 mL of bacterial liquid, centrifuged to keep the bacteria, and used the Biomed bacterial genomic DNA rapid extraction kit The genomic DNA of JZB130180 strain was extracted. The primer sequence design and PCR amplification procedure for amplifying the selected genes are detailed in Table 1. All PCRs used 25μL amplification system: 10pmoL/L upstream and downstream primers 1μL each, ultrapure dNTP Mixture (10mmol/L) 1μL, 2.5U Taq DNA polymerase 0.5μL, 10×PCR Buffer 2.5μL, genomic DNA template (10ng) 1 μL, ddH ₂ O 18 μL. PCR amplification products were detected by 1% agarose gel electrophoresis. After the target band was recovered and purified by cutting gel, it was connected to the pMD-18T vector, transformed into DH5α E. coli competent, and screened by X-gal blue and white spots, and the PCR method was positive. clones, and send the bacterial liquid of the positive clones to the company for sequencing. Primer synthesis and sequence sequencing in this study were completed by Zhongmeitaihe Biotechnology (Beijing) Co., Ltd. After the 16S rRNA gene of strain JZB130180 was amplified and sequenced, a standard strain with higher homology was selected in LPSN (http://www.bacterio.net/), with Actinoalloteichus cyanogriseus IFO 14455 ^T as the periphery, and the obtained sequence was in The NCBI website uses BLAST for alignment analysis, and uses DNAMAN 5.2.2 and MEGA 5.2.1Version 3 software to perform multiple sequence matching and alignment analysis with similar sequences in GenBank. Neighbor-joining method is used to construct phylogenetic tree. After bootstrap (1000 cycles) Verify the reliability of the phylogenetic tree. The results show that the gene amplification results of 16S rDNA, atpD, recA and rpoB of strain JZB130180 are shown in Figure 4. From the phylogenetic tree (Figure 5) constructed from the 16S rDNA sequence of strain JZB130180 (sequence 1 in the sequence listing), it can be seen that strain JZB130180 and Streptomyces blastmyceticus NRRL B-5480AY999802.1 are clustered in a branch superior. The sequences of the housekeeping genes atpD, recA and rpoB of the amplified strain JZB130180 are shown as sequences 2-4 in the sequence table, respectively. The sequences of these housekeeping genes were found by the Blast search and comparison results of the NCBI website, which were basically consistent with the results of the 16S rDNA sequence alignment analysis. Therefore, the JZB130180 strain was identified as Streptomyces blastmyceticus.

Table 1 PCR amplified genes and primer sequences

Streptomyces blastmyceticus (Streptomyces blastmyceticus) JZB130180 has been deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Management Committee (CGMCC) on November 14, 2016, and the deposit number is CGMCCNo.13270. It is hereinafter referred to as Streptomyces blastmyceticus JZB130180 or strain JZB130180.

Example 2. Determination of antibacterial spectrum of Streptomyces blastmyceticus JZB130180

Test medium:

LB agar medium: tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, agar 15.0g, distilled water 1000mL, pH 7.0～7.2;

LB liquid medium: The same as LB agar medium except that no agar is added.

KB medium: peptone 20.0 g, glycerol 10.0 mL, K ₂ HPO ₄ 1.50 g, MgSO ₄ 7H ₂ O 1.50 g, agar 15.0 g, distilled water 1000 mL, KOH adjusted to pH 7.2;

All the above media were autoclaved at 121°C for 20min.

experiment method:

(1) The antibacterial effect on phytopathogenic fungi was determined by the plate confrontation method. The specific operation steps are as follows: firstly, strain JZB130180 and various phytopathogenic fungi (Table 2) were cultured on PDA medium for 4-5d respectively, and a sterile hole puncher with a diameter of 7mm was used to make a bacterial cake, and put it on a blank PDA plate. , place the pathogenic bacteria cake in the center of the plate, the mycelial surface is close to the medium, and at 180° at a distance of 2.5cm from the pathogenic bacteria, the bacterial cake of the strain JZB1301800 is reversely attached to the medium, and cultivated in a constant temperature incubator at 25-28 °C for 5 -7d, take the plate with only pathogenic bacteria as blank control, measure the antibacterial bandwidth between the colony edge of pathogenic bacteria and strain JZB130180 and the colony diameter of each pathogenic bacteria blank control, each pathogenic bacteria is repeated three times;

(2) The antibacterial effect on phytopathogenic bacteria was measured by double-layer culture method. The specific operation steps were as follows: Spot the activated strain JZB130180 on the KB plate, cultivate at 28°C for 48h, and use 3mL of chloroform to vaporize it. Kill the bacterial strain JZB130180 and let it stand for 10-12 hours so that the chloroform vapor can be completely volatilized. Phytopathogenic bacteria (Table 2) were activated and cultured on LB liquid medium, respectively, and cultured with shaking at 180r/min at 28°C for 24h. The cultured target pathogenic bacteria were prepared into 10 ⁸ cfu/mL bacterial suspension with sterile physiological saline. Pipette 100 μl of bacterial suspension and add it to 3 mL of 1% water agar that was melted and cooled to 50 °C, quickly mixed, and immediately poured onto the plate of the strain JZB130180 that had been killed by chloroform, spread into a uniform thin layer, and each treatment was three times. Repeatedly, incubate at 28°C for 36h, observe the bacteriostatic effect and measure the diameter of the bacteriostatic zone by the cross method.

test results:

The results of the antibacterial spectrum determination (see Table 2 and Figure 6) show that the bacterium has a very obvious antibacterial effect on almost all plant pathogenic fungi, and the antibacterial bandwidth of cabbage wilt and wheat sheath blight is up to 12mm, The antibacterial effect on apple rota fungus is small, and the antibacterial bandwidth is 0.5mm. It also showed strong inhibitory effect on the selected phytopathogenic bacteria, and the diameter of the inhibition zone was between 35-50mm. This indicated that the bacteria had a broad antibacterial spectrum.

Table 2 Determination results of antibacterial spectrum of JZB130180 strain

Note: "-" indicates that the content was not tested; the data in the table are the average of three repeated values, and the same letter indicates that the difference is not significant (P < 0.05).

Example 3. Biocontrol-related activity assay of strain JZB130180

1. Test medium: CAS medium (for detection of siderophore) consists of 4 kinds of solutions (sterilized separately before mixing). Solution 1 (CAS/HDTMA solution) includes CAS solution: 60.5 mg of CAS (chrome azure) dissolved in 50 mL of water; iron solution: 10 mmol/L HCl solution containing 1 mmol/L FeCl ₃ ·6H ₂ O, pH 2.0; HDTMA Solution: 72.9 mg of hexadecyl trimethylamine bromide was dissolved in 40 mL of water; 50 mL of CAS solution was mixed with 10 mL of iron solution, 40 mL of HDTMA solution was added and stirred evenly, and the obtained blue-black liquid was sterilized. CAS/HDTMA solution; Solution 2 (Salts/Buffer solution): Dissolve 30.24 g Pipes in 750 mL Salts solution (KH ₂ PO ₄ 0.3 g, NaCl 0.5 g, NH ₄ Cl 1.0 g) at 50% (W/V) Adjust pH to 6.8 with KOH, add 15g of agar and dilute to 800mL with distilled water, autoclave and cool to 50°C; solution 3 (750mL): glucose 2g, mannitol 2g, MgSO ₄ ·7H ₂ O 493mg, CaCl ₂ 11mg, H ₃ BO ₃ 1.4 mg, ZnSO ₄ 7H ₂ O 1.2 mg, MnSO ₄ 2H ₂ O 1.17 mg, Na ₂ MoO ₄ 2H ₂ O 1 mg, CuSO ₄ 40 μg, distilled water to 750 mL, autoclaved and cooled to 50°C. Add 750 mL of solution 3 to 800 mL of solution 2, and mix with 30 mL of solution 4, that is, filter-sterilized 10% (W/V) casamino acid, and finally add 1580 mL of the mixture to 100 mL of solution 1, stirring slowly (to avoid foaming). ), lay flat.

Chitin medium (detection of chitinase): colloidal chitin 2.5g, K ₂ HPO ₄ 0.7g, KH ₂ PO ₄ 0.3g, MgSO ₄ ·7H ₂ O 0.5g, FeSO ₄ ·7H ₂ O 0.01 g, agar 15g, distilled water 1000mL, pH 7.0.

Skim milk agar medium (detecting protease): imported skim milk powder 40g, agar 10g, deionized water 1000mL, autoclaved at 115°C for 30min, mixed with LB solid medium 1:9, and spread into a plate for later use.

Congo red medium (detecting cellulase): MgSO ₄ ·7H ₂ O 0.25g, K ₂ HPO ₄ 0.5g, cellulose 1.88g, Congo red 0.2g, gelatin 2g, agar 4g, distilled water 1000mL, pH 7.0.

Pikovskaya's agar medium (detection of phosphatase): yeast extract 0.5g, glucose 10g, Ca ₃ (PO ₄ ) ₂ 5g, (NH ₄ ) ₂ SO ₄ 0.5g, KCl 0.2g, MgCl ₂ 0.1g, MnSO ₄ · H ₂ O 0.1 mg, FeSO ₄ 0.1 mg, agar 15 g, distilled water 1000 mL, pH 7.0.

Liquid seed medium and fermentation medium: corn starch 30.0g, glucose 10.0g, soybean meal powder 20.0g, peptone 6.0g, ammonium sulfate 2.50g, MgSO ₄ ﹒ 7H ₂ O 1.50 g, KH ₂ PO ₄ 0.30 g, NaCl 7.50 g, CaCO ₃ 4.0 g, high temperature amylase 0.05 g, distilled water 1000 mL, pH 7.0-7.4.

All the above media were autoclaved at 121°C for 20min.

2. Test

2.1 Detection of biocontrol-related extracellular enzymes

2.1.1 Detection of siderophil

The strain JZB130180 was first cultured on Gao's No. 1 medium at 28°C for 3-5 days, and then inoculated into CAS medium. After culturing at 28°C for 3-5 days, it was observed whether a yellow halo appeared around the colony. Three replicates were set for each treatment. The yellow halo around the colony indicated the production of siderophore due to the competition of the iron ions chelated by EDTA in the medium to the medium from blue to yellow.

2.1.2 Detection of chitinase

The strain JZB130180 was first cultured on Gao's No. 1 medium at 28°C for 3-5 days, and then inoculated into chitin medium. After culturing at 28°C for 3-5 days, the formation of a transparent circle around the colony was observed. Production of tyrosinase. Three replicates were set for each treatment.

2.1.3 Detection of protease

The strain JZB130180 was first cultured on Gao's No. 1 medium at 28°C for 3-5 days, then inoculated into skim milk agar medium, and cultured at 28°C for 3-5 days, and then the formation of a transparent circle around the colony was observed. Production of proteases. Three replicates were set for each treatment.

2.1.4 Detection of cellulase

The strain JZB130180 was first cultured on Gao's No. 1 medium at 28°C for 3-5 days, and then inoculated into Congo red medium. After culturing at 28°C for 3-5 days, the formation of red hydrolysis circles around the colonies was observed, and the appearance of red hydrolysis circles indicated that Production of cellulase. Three replicates were set for each treatment.

2.1.5 Detection of phosphatase

The strain JZB130180 was first cultured on Gao's No. 1 medium at 28°C for 3-5 days, and then inoculated on Pikovskaya's agar medium. After culturing at 28°C for 3-5 days, the formation of a transparent circle around the colony was observed. The appearance of a transparent circle indicates phosphate ester. production of enzymes. Three replicates were set for each treatment.

The results showed that strain JZB130180 was able to secrete chitinase, cellulase, siderophil and protease, but not phosphatase (Fig. 7). Among them, the activities of chitinase, cellulase and siderophore are all important reference indicators for the biocontrol function of biocontrol bacteria.

2.2 Preparation of sterile fermentation filtrate of strain JZB130180

Strain JZB130180 was cultured on ISP2 medium at a constant temperature of 28°C for 5-7 days, and then two loops of bacteria were inoculated into liquid seed medium (50mL volume/500mL conical flask), and 200r/min was shaken on a shaking table at 28°C for 20 days. ~24h, take a small amount of culture solution and observe it under a microscope, if a large amount of mycelium is observed and it is not contaminated by spherical or rod-shaped bacteria, it is the seed culture solution, and it is inoculated into the fermentation medium (100mL volume/500mL triangle with 3% inoculum volume). 200r/min shaking culture at 28°C for 5-7 d, and after the fermentation is completed, the prepared fermentation broth is obtained. The fermentation broth was centrifuged at 12,000 rpm for 10 min to take the supernatant, and the supernatant was filtered through a 0.22 μm microporous membrane for sterilization to obtain a sterile fermentation filtrate of strain JZB130180.

2.3 Antibacterial activity of strain JZB130180 against peach brown rot

Inoculate the peach brown rot fungus on the PDA plate, cultivate at 22°C for 5-7 days until the entire plate produces abundant spores, add an appropriate amount of sterile water to the spore-producing plate, scrape the spores through the homemade absorbent cotton filter layer, remove the Mycelium, dilute the spore suspension with sterile water to 10 ⁷ spores/mL for later use. Disposable petri dish (Φ=9cm) is poured into 25mL PDA medium per dish. After solidification, draw 200μL of spore suspension of peach brown rot fungus and evenly spread it on the PDA plate. Under aseptic conditions, put it on the PDA plate. The cultivated peach brown rot fungus was punched with a sterile hole punch of Φ=7mm to make a bacterial cake, and the bacterial cake was taken out, and 100 μL of the aseptic fermentation filtrate of the strain JZB130180 in step 2.2 was added to each hole, and placed at a constant temperature of 22 °C for 2 to 3 days. Observe and measure the diameter of the inhibition zone.

The results showed that the diameter of the inhibition zone of the strain JZB130180 aseptic fermentation filtrate on the plate was more than 3.0 cm (Fig. 8), indicating that the strain JZB130180 aseptic fermentation filtrate contained substances with strong antibacterial activity against the peach brown rot bacteria. .

2.4 Determination of the virulence of strain JZB130180 against Peach brown rot

2.4.1 Production of peach brown rot fungus cake

Inoculate the peach brown rot fungus on the PDA plate, cultivate at 22°C for 5-7 days until the entire plate produces abundant spores, add an appropriate amount of sterile water to the spore-producing plate, scrape the spores through the homemade absorbent cotton filter layer, remove the Mycelium, dilute the spore suspension with sterile water to 10 ⁷ spores/mL for later use. Disposable petri dish (Φ=9cm) is poured into 25mL PDA medium per dish. After solidification, draw 200μL of spore suspension of peach brown rot fungus and evenly spread it on the PDA plate. Under aseptic conditions, put it on the PDA plate. The cultivated peach brown rot fungus was punched out a certain number of peach brown rot fungus cakes with a sterile hole punch of Φ=6mm.

2.4.2 Preparation of toxic medium: When the PDA medium is melted and cooled to about 45°C, the sterile fermentation filtrate of the strain JZB130180 in step 2.2 and PDA (20 mL) are prepared according to the concentration of 1.25%, 2.5%, 5%, 10% and 20%. Proportionally mixed, the final concentration of the sterile fermentation filtrate of strain JZB130180 is 12.5 μg/mL, 25.0 μg/mL, 50.0 μg/mL, 100 μg/mL, 200 μg/mL to make uniform plates with uniform thickness and thickness to obtain a toxic medium. The PDA without the sterile fermentation filtrate of strain JZB130180 was used as the blank control. Each treatment was repeated 3 times and cooled before use.

2.4.3 Indoor toxicity assay

Place the peach brown rot fungus cake of 2.4.1 on the poisonous medium of 2.4.2 with a sterile inoculating needle, with the mycelium facing down, place a 6mm cake on the center of each plate, and incubate at 22°C Cultivated in the box.

Results record: After culturing for 5-7 days, the colony diameter was measured by the cross method, and the EC ₅₀ of the fermentation broth was obtained by data analysis and regression.

Colony diameter (mm) = actual measured colony diameter average -6

Relative inhibition rate (%)=(control colony diameter-treated colony diameter)/control colony diameter×100%

Taking the content of the sterile fermentation filtrate of strain JZB130180 in PDA as the concentration, the logarithm of the concentration as the independent variable X, and the probability of the inhibition rate as the dependent variable Y, the virulence regression equation, correlation coefficient (r), and EC ₅₀ value were obtained. See Table 3.

As can be seen from Table 3, the aseptic fermentation filtrate of strain JZB130180 has an EC ₅₀ of 40.5 μg/mL against peach brown rot pathogen, indicating that the aseptic fermentation filtrate of strain JZB130180 has a strong inhibitory effect on peach brown rot pathogen. It can be clearly seen from Fig. 9 that the growth inhibitory effect on the mycelium of peach brown rot pathogen is stronger as the concentration increases. When the concentration is 20%, the mycelium does not grow at all.

Table 3 The virulence test results of strain JZB130180 sterile fermentation filtrate to peach brown rot

2.5 Determination of the control effect of strain JZB130180 on peach brown rot and tomato gray mold

2.5.1 Preparation of spore suspension of pathogenic bacteria: Peach brown rot and tomato botrytis cinerea were incubated on PDA at a constant temperature of 23 to 25°C for 4 to 5 days, and then scraped their conidia, placed in sterile water, and placed in a shaker. Vibrate on the top to prepare a suspension of peach brown rot fungus spores (the content of peach brown rot fungus is 10 ⁸ cfu/mL) and a spore suspension of tomato gray mold fungus (the content of tomato gray mold fungus is 10 ⁸ cfu/mL).

The experiment was repeated three times, and the experimental method for each repetition was as follows: select peach fruit (variety: Kubo) and tomato fruit (variety: Jiafen 19) with neat appearance, no pests and mechanical damage. The fruit was first treated with 0.5% NaClO for 1 min, and then rinsed with sterile water for several times. A 5mm (deep) × 3mm (width) wound was punctured at the waist of the fruit with sterile inoculation needles, and 4 holes were made in each fruit. After the wound was dried, the peach fruits were randomly divided into 3 groups, with 5 fruits in each group. They are treatment A1 (negative control), treatment B1 and treatment C1, respectively. The tomato fruits were randomly divided into 4 groups with 5 fruits in each group, namely treatment A2 (negative control), treatment B2, treatment C2 and treatment D.

Each peach fruit of treatment A1 was inoculated with 50 μl of sterile water, and after complete absorption, each peach fruit was inoculated with 30 μl of the peach brown rot fungus spore suspension of step 2.5.1; each peach fruit of treatment B1 was inoculated with 50 μl of step 2.2 strain JZB130180 Aseptic fermentation filtrate, after being completely absorbed, each peach fruit was inoculated with 30 μl of the peach brown rot fungus spore suspension of step 2.5.1; each peach fruit treated with C1 was inoculated with 50 μl of step 2.2 strain JZB130180 sterile fermentation filtrate 5-fold dilution (The liquid obtained by diluting the sterile fermentation filtrate of strain JZB130180 in step 2.2 by 5 times with sterile water), after complete absorption, inoculate each peach fruit with 30 μl of the peach brown rot fungus spore suspension in step 2.5.1. Each tomato fruit of treatment A2 was inoculated with 50 μl of sterile water, and after complete absorption, each tomato fruit was inoculated with 30 μl of the spore suspension of Peach brown rot fungus in step 2.5.1; each tomato fruit of treatment B2 was inoculated with 50 μl of step 2.2 strain JZB130180 Aseptic fermentation filtrate, after being completely absorbed, each tomato fruit was inoculated with 30 μl of the peach brown rot fungus spore suspension of step 2.5.1; each tomato fruit treated with C2 was inoculated with 50 μl of step 2.2 strain JZB130180 sterile fermentation filtrate 5-fold dilution (The liquid obtained by diluting the sterile fermentation filtrate of strain JZB130180 in step 2.2 by 5 times with sterile water), after being completely absorbed, each tomato fruit was inoculated with 30 μl of the peach brown rot fungus spore suspension of step 2.5.1; Tomato fruit inoculate 50 μl of step 2.2 strain JZB130180 sterile fermentation filtrate 10 times dilution (the liquid obtained by diluting step 2.2 strain JZB130180 sterile fermentation filtrate 10 times with sterile water), after complete absorption, each tomato fruit is inoculated with 30 μl step 2.5 .1 Peach brown rot spore suspension. It was placed in a plastic packaging box, covered with a layer of plastic wrap, and cultivated at 23° C. with humidity (humidity not lower than 90%). The disease incidence and lesion diameter of each treatment were counted every 24h. According to the lesion diameter, the control effect of peach brown rot and tomato gray mold was calculated according to the following formula: control effect (%) = (negative control lesion diameter - lesion diameter of corresponding treatment)/negative control lesion diameter × 100% .

It can be seen from the incidence rate and lesion diameter of peach fruit that step 2.2 strain JZB130180 sterile fermentation filtrate (referred to as stock solution) and step 2.2 strain JZB130180 sterile fermentation filtrate 5-fold dilution (referred to as 5-fold dilution) are effective against brown rot bacteria. Both showed inhibitory effect, and the higher the concentration, the better the inhibitory effect (Table 4 and Figure 10). The control effect of the fruit treated with the stock solution reached 100% in the first 2 days. After the third day, the control effect of the stock solution on brown rot was more than 10% higher than that of the 5-fold dilution. The results indicated that the sterile fermentation filtrate of the inoculated strain JZB130180 could delay the occurrence of brown rot on peach fruit, and also had a certain inhibitory effect on the expansion of lesions, and the control effect on peach brown rot was more than 50%.

Table 4 Aseptic fermentation filtrate of strain JZB130180 aseptically fermented filtrate against peach brown rot test results

Step 2.2 strain JZB130180 sterile fermentation filtrate (referred to as stock solution) and step 2.2 strain JZB130180 sterile fermentation filtrate 5-fold dilution (referred to as 5-fold dilution) and step 2.2 strain JZB130180 sterile fermentation filtrate 10-fold dilution (referred to as 10-fold dilution) liquid) showed strong inhibitory effect on tomato Botrytis cinerea (Table 5 and Figure 11). The stock solution treatment performed the best and remained disease-free throughout the experiment, followed by the 5-fold dilution treatment, and finally the 10-fold dilution. In the first 2 days, the diameter of all treated lesions was not obvious, but the incidence rate decreased with the increase of concentration. On the 6th day, the morbidity rates of 5-fold dilution and 10-diluent were 20% and 70% significantly lower than that of CK (100%), and the control effect was still obvious with the extension of culture time. As the concentration increased, the lesion diameter was smaller and the incidence rate was lower in the treatment group compared with CK. It is indicated that the sterile fermentation filtrate of strain JZB130180 in step 2.2 can prevent the occurrence of tomato gray mold, and can effectively prevent tomato gray mold.

Table 5 Aseptic fermentation filtrate of strain JZB130180 aseptically fermented filtrate against tomato botrytis cinerea

Example 4. Determination of the growth-promoting effect of strain JZB130180 on plants

(1) Seed soaking treatment: Jiafen 19 tomato seeds were surface-sterilized with 75% alcohol for 5 minutes, rinsed with sterile water for 3 times, surface-sterilized with 10% sodium hypochlorite solution for 10 minutes, rinsed with sterile water for 5 times, and the tomato seeds with the same shape as possible were removed. It is divided into tap water seed soaking treatment (blank control CK1), 50 times seed soaking treatment, 100 times seed soaking treatment and 500 times seed soaking treatment.

50 times of fermentation diluent seed soaking treatment (50X): with step 2.2 strain JZB130180 sterile fermentation filtrate of Example 3 50 times dilution (the liquid obtained by diluting step 2.2 strain JZB130180 sterile fermentation filtrate of Example 3 50 times with tap water) The seeds were soaked for 24 hours, and the treatment solution was changed every 12 hours.

100-fold fermentation diluent seed soaking treatment (100X): use the 100-fold dilution of the sterile fermentation filtrate of the strain JZB130180 in step 2.2 of Example 3 (the liquid obtained by diluting the sterile fermentation filtrate of the strain JZB130180 of the step 2.2 of Example 3 by 100 times with tap water) The seeds were soaked for 24 hours, and the treatment solution was changed every 12 hours.

500-fold fermentation dilution treatment (500X): 500-fold dilution of sterile fermentation filtrate of step 2.2 strain JZB130180 in Example 3 (the liquid obtained by diluting step 2.2 strain JZB130180 sterile fermentation filtrate of Example 3 500 times with tap water) The seeds were soaked for 24 hours, and the treatment solution was changed every 12 hours.

Seed soaking treatment in tap water (blank control CK1): soak the seeds with an equal volume of tap water for 24 hours, and replace the treatment solution every 12 hours.

After soaking the seeds, they were sown in a nutrient pot (sterilized soil + peat = 1:1), and the seed soaking in tap water was used as a control to investigate the growth-promoting effect on the 10th, 20th, 30th and 40th days after sowing. Three replicates were set for each treatment, with 5 nutritional pots per replicate, and three tomato seeds were sown in each pot.

(2) Irrigation treatment: the seed buds of Jiafen 19 tomato germinated through pretreatment were sown in a nutrient pot, and when the seedlings grew to the two-leaf stage, they were divided into tap water root irrigation treatment (blank control CK2), 50 times root irrigation treatment, 100 times of root irrigation and 500 times of root irrigation.

50 times of fermentation dilution liquid root irrigation treatment (50X): start the first root irrigation with 20 mL of step 2.2 bacterial strain JZB130180 aseptic fermentation filtrate of 20 mL of Example 3, and then use the same volume as the first irrigation every 10d. In step 2.2 of Example 3, the 50-fold dilution of the sterile fermentation filtrate of strain JZB130180 was watered once at the roots, for a total of three times.

100 times of fermentation dilution solution root irrigation treatment (100X): start the first root irrigation with 20 mL of step 2.2 bacterial strain JZB130180 aseptic fermentation filtrate 100 times dilution of Example 3, and then use the same volume as the first irrigation every 10d. In the step 2.2 of Example 3, the 100-fold dilution of the sterile fermentation filtrate of strain JZB130180 was watered once at the roots, for a total of three times.

500 times of fermentation dilution liquid root irrigation treatment (500X): start the first root irrigation with 20 mL of step 2.2 strain JZB130180 sterile fermentation filtrate 500 times dilution of Example 3, and then use the same volume as the first irrigation every 10d. In step 2.2 of Example 3, the 500-fold dilution of the sterile fermentation filtrate of strain JZB130180 was watered once at the roots, for a total of three times.

Blank control root irrigation treatment (blank control CK2): start the first root irrigation with 20 mL of tap water, and then use the same volume of tap water as the first root irrigation every 10 days, for a total of three irrigations.

Three replicates were set for each treatment, with 5 nutritional pots per replicate, and three tomato seeds were sown in each pot.

Determination indicators and methods: seedling height and root length, root number, germination rate, fresh seedling weight, etc. The lengths of the seedlings and roots of each treatment were measured with a ruler, wherein the root was the longest root as the measurement object, and the average value of all the tested seedlings in each treatment was used as the seedling height and root length. Number of roots: count the number of all roots longer than 1 cm, and finally take the average value of all the tested seedlings in each treatment.

test results:

The test results of continuous detection for 40 days showed that compared with the blank control treated with tap water, the tomato seedlings grew vigorously after the aseptic fermentation filtrate of each concentration of strain JZB130180 was treated. Compared with the blank control, the tomato seedlings treated with root irrigation have significant differences in stem diameter, main root and stem length, number of fibrous roots, plant height, and plant fresh weight, and the above-mentioned indicators are better than those of the blank control at the later stage of growth. Tomato seedlings were treated with tap water soaking (Table 6 and Figure 12). Considering the statistical results comprehensively, 100 times fermentation dilution liquid was selected for root irrigation, and 500 times fermentation dilution liquid was the best for seed soaking effect.

Table 6 Determination of the growth-promoting effect of JZB130180 strain treatment on tomato seedlings (investigation results on the 20th day from sowing)

Note: The data in the a.b.c.d. columns of the table are the average values of the set test replicates.

<110> Beijing Academy of Agriculture and Forestry

<120> A biocontrol Streptomyces strain and its application

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gtcctccggt aagaccaccc tcaccctgca cgccgtggcc aacgcccaga aggcgggcgg 180

caccgtcgcc ttcgtcgacg cggagcacgc gctcgacccg gagtacgcga aggcgctggg 240

cgtcgacacc gacaacctca tcctctccca gccggacacc ggcgagcagg cgctggagat 300

cgtggacatg ctggtccgct ccggcgccct cgacctgatc gtcatcgact ccgtcgccgc 360

gctcgtgccg cgtgcggaga tcgagggcga gatgggcgac tcccacgtcg gcctccaggc 420

gcgactgatg agccaggcgc tccgcaagat caccagcgcg ctcaaccagt ccaagaccac 480

cgcgatcttc atcaaccagc tccgcgagaa gatcggcgtc atgttcggct cgccggagac 540

gacgaccggt ggccggggccc tgaagttcta cgcctcggtg cgcctggaca tccgccgcat 600

cgagaccctc aaggacggca cggaggccgt cggcaaccgc acccgcgtca aggtcgtcaa 660

gaacaaggtc gcgccgccct tcaagcaggc cgagttcgac atcctctacg gccagggcat 720

cagccgcgag ggcggcctga tcgacatggg cgtggagcac ggcttcgtcc gcaaggccgg 780

cgcctggtac acgtacgagg gcgaccagct cggccagggc aagga 825

<210> 4

<211> 871

<212> DNA

<213> Streptomyces blastmyceticus

<400> 4

tcaaggagtt cttcggcacc agccagctgt cccagttcat ggaccagacg aacccgctgt 60

cgggcctgac ccacaagcgc cgtctgtccg cgctgggccc cggtggtctg tcccgtgagc 120

gggccggctt cgaggtccga gacgtccacc cgtcgcacta cggccgcatg tgcccgatcg 180

aggacgcccga aggcccgaac atcggtctga tcggctcgct cgcgtcgtac ggccgcgtca 240

acgcgttcgg tttcgtcgag accccgtacc gcaaggtcgt gggcggcgtc gtcaccgacg 300

acgtggacta cctgactgcc gacgaggaag accgcttcgt catcgcgcag gcgaacgccc 360

ccctcaccga ggacatgcgt tacgccgagt cgcgcgtgct ggtccgccgc cgtggcggcg 420

agatcgacta catcgccggc gacgacgtcg actacatgga cgtctccccg cgccagatgg 480

tgtcggtcgc gaccgcgatg atccccttcc tcgagcacga cgacgccaac cgcgcgctca 540

tgggatcgaa catgatgcgc caggccgttc cgctgatcaa ggcggaggcc ccgctggtcg 600

gcaccggcat ggagtaccgc tgtgcggtcg acgccggtga cgtcatcaag gcggagaagg 660

acggtgtggt ccaggaggtc tccgcggact acgtcaccgt cgccaacgac gacggcacgt 720

acaacacgta ccgggtcgcc aagttctccc gctccaacca gggcacgtcc ttcaaccaga 780

aggtcgtcgt cgacgagggc gcccgggtcg tctccggcca ggtgctcgcc gacggtccgt 840

ccacggacga gggcgagatg gccctcggca a 871

Claims (13)

1. Streptomyces blastmyceticus ( Streptomyces blastmyceticus), its strain number is JZB130180, and its registration number in the General Microbiology Center of China Microbial Culture Collection Management Committee is CGMCC No.13270. 2. An inoculum comprising the Streptomyces oryzae according to claim 1 or/and the metabolites of Streptomyces oryzae according to claim 1. 3. A pathogenic bacteria inhibitor, characterized in that: the pathogenic bacteria inhibitor comprises the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1. 4. The pathogen inhibitor according to claim 3, wherein the pathogen inhibitor has an inhibitory effect on the following pathogens: botrytis cinerea, plant brown rot, wheat sheath blight, wheat scab , Wheat total rot, pepper anthracnose, grape anthracnose, plant fusarium wilt, rice sheath blight, lily root rot, apple ringworm, pepper scab, cucumber angular leaf spot, eggplant bacterial wilt, waxy Bacillus, soil rhizogenes and/or Chinese cabbage black rot. 5 . A disease inhibitor, characterized in that: the disease inhibitor contains the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 . 6. disease inhibitor according to claim 5, is characterized in that: described disease is botrytis cinerea, plant brown rot, wheat sheath blight, wheat scab, wheat take-all disease, pepper anthracnose, Grape anthracnose, plant fusarium wilt, rice sheath blight, lily root rot, apple ring rot, pepper scab, cucumber corner spot, eggplant bacterial wilt and/or Chinese cabbage black rot. 7. any of the following applications of the metabolite of the Streptomyces oryzae described in claim 1 or/and the Streptomyces oryzae described in claim 1: 1) Application of the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 in inhibiting pathogenic bacteria; 2) Application of the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 in the preparation of pathogen inhibitors; 3) Application of the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 in the preparation of disease inhibitors; 4) Application of the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 in disease control. 8. application according to claim 7, is characterized in that: described pathogenic bacteria is botrytis cinerea, plant brown rot, wheat sheath blight, wheat scab, wheat total rot, pepper anthracnose, grape Anthracnose, Fusarium wilt, Rhizoctonia oryzae, Lily root rot, Apple ringworm, Capsicum scab, Cucumber angular spot, Eggplant solanacearum, Bacillus cereus, Soil tumefaciens and/or Cabbage black rot fungus; The diseases are botrytis cinerea, plant brown rot, wheat sheath blight, wheat scab, wheat take-all disease, pepper anthracnose, grape anthracnose, plant fusarium wilt, rice sheath blight, lily root rot , apple ring rot, pepper scab, cucumber angular spot, eggplant bacterial wilt and/or Chinese cabbage black rot. 9. The application of the Streptomyces oryzae described in claim 1 or/and the metabolites of the Streptomyces oryzae described in claim 1 in promoting plant growth. 10. The application according to claim 9, wherein the plant is a seed plant. 11. The application according to claim 9, wherein the plant is a dicotyledonous plant. 12. The application according to claim 9, wherein the plant is a Solanaceae plant. 13. The application according to claim 9, wherein the plant is a tomato.