CN113025537A

CN113025537A - Fermentation method of streptomycete

Info

Publication number: CN113025537A
Application number: CN202110485908.6A
Authority: CN
Inventors: 左彤彤; 李立梅; 马琼芳; 燕红; 周勇; 张晓光; 刘晓林; 于海媛
Original assignee: Jilin Provincial Academy of Forestry Sciences
Current assignee: Jilin Provincial Academy of Forestry Sciences
Priority date: 2020-03-28
Filing date: 2020-03-28
Publication date: 2021-06-25
Anticipated expiration: 2040-03-28
Also published as: CN113025537B; CN112931534B; CN111254099B; CN112931534A; CN111254099A

Abstract

The invention relates to a fermentation method of streptomycete, which comprises the following steps: inoculating the streptomycete with the preservation number of CGMCC No.18657 to a culture medium for fermentation culture. The streptomycete and the fermentation liquor thereof fermented by the method can effectively inhibit various pathogenic bacteria such as poplar canker pathogen and the like, and have the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect, low toxicity, environmental protection and the like.

Description

Fermentation method of streptomycete

The invention is a divisional application, and the original Chinese patent application number is: 202010232732.9, filing date: 3/28/2020, with the patent names: a streptomycete.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fermentation method of streptomycete.

Background

Poplar canker is a serious biological disaster of Poplar artificial forests in China, and the damage is becoming serious day by day. At present, chemical prevention measures are mainly taken for preventing and treating the poplar canker except for improving the variety of young trees and enhancing the disease resistance of forest trees. The method has low cost and quick response, and is widely used for a long time. The disadvantages are that the long-term use of chemical pesticide can enhance the drug resistance of pathogenic bacteria, cause a great amount of pesticide residue and cause environmental pollution. The biological control has strong specificity, persistence and effectiveness on the inhibition of pathogenic bacteria, can enhance the resistance of host plants, and is the main research direction of the current forest disease control. The poplar canker is a host leading disease, has high spreading speed and great harm and is difficult to prevent and treat. The vegetative generation of the pathogenic bacteria of the bulosa buergeriana et al is reported to be the outgrowth of the small pot (dothiorella gregaria) and the sexual generation is the botryococcus dothidea (Botryosphaeria). There are reports on the biological control of poplar canker, such as Xylena et al using Bacillus subtilis and poplar bud et al using Trichoderma aureoviride and Fusarium equiseti and Streptomyces sioyaensis reported in Zhengsuan. At present, the prevention and treatment of the poplar canker is mainly chemical prevention and treatment, and environmental-friendly and efficient biological prevention and treatment are rarely reported. Poplar canker disease caused by Pythium ultimum harms 200 poplar varieties, hybrids and clones, and its variants have been found. The fossilizid is a weak parasitic bacterium, has a latent infection phenomenon, can invade a host all the year round, and is latent in the host body, and the host shows symptoms when being stressed or weak in growth, and generally forms a disease peak period in spring. Meanwhile, host selectivity exists, the quantity of fungi on the bark of the populus tomentosa is small due to the blister canker caused by the gathering of the small cave hulls, and the incidence rate on 1-year-old seedlings of the populus tomentosa is as high as 50-70%. The spawning micrococcus is distributed in different tree spaces, the upper parts of the trees are distributed less, and the distribution of the spawning micrococcus on the trunk parts is obviously increased. The biological control method for the poplar canker obtains some results only in a laboratory or a small-area forest area at present, and in the future, biocontrol bacteria with good control effect and environmental protection are continuously screened to develop and develop an environment-friendly medicament for preventing and treating the canker.

Streptomyces is the highest genus in actinomycetes phyla, is filamentous branched hypha, non-cross-membrane, gram-positive aerobic bacteria, has strong starch and protein hydrolysis capacity, can produce secondary metabolites with various biological activities, and is widely applied to various fields. In the aspect of plant diseases and insect pests, the streptomyces has good antagonistic action on pathogenic bacteria. It can produce extracellular hydrolase such as chitinase, glucanase and cellulase to crack the fungal cell wall, so that the fungal growth is hindered; the secondary metabolite of the antibiotic is mainly aminoglycoside, nucleoside, polyene, macrolide and tetracycline antibiotics, and has the inhibition effect on the cell wall synthesis and the synthesis of protein and nucleic acid of various pathogenic bacteria. The extracellular metabolite toyocamycin of Streptomyces diastatochromogenes (Streptomyces diastatochromogenes) has obvious inhibition effect on hyphal growth and sclerotium formation of Rhizoctonia solani. In addition, some streptomyces also can secrete special substances to promote the growth of plants, so that the disease resistance of the plants is indirectly improved. Schlemonghong et al found that the endogenous antagonistic streptomycete of cotton promotes photosynthesis and growth of cotton seedling in root, and the different dilution gradients of its fermentation broth can promote growth of cotton radicle and hypocotyl. In addition, one streptomycete is screened from soil by Huangbing et al, and the lethality of the metabolite furaltadone on the pine wood nematode reaches 85%. In conclusion, streptomyces has a wide development prospect in the aspect of biological control of plant diseases, and is worth continuously exploring the research and application of streptomyces.

Streptomyces spirovorans (Streptomyces spirovorticillatus) was discovered in the screening of antibiotics against sclerotinia rot of colza by scholars of Shanghai agricultural chemicals in the last 60 th century, and the tautomeric forms (TTM) were isolated from the fermentation products. In 1987, the structure was preliminarily determined in cooperation with the Isono research group in Japan. In the aspect of plant diseases, the tautomerism has good antifungal activity, especially on Sclerotinia sclerotiorum (sclerotiorum), and the antibacterial effect is small. The tautomerism causes morphological abnormality of sclerotinia sclerotiorum, hyphal swelling and abnormal branch shape observed under a microscope; the activity of the rape sclerotium Protein Phosphatase (PP) treated by the tautomerin is reduced, and the permeability of cell membranes is changed; El-Abyad et al found that S.spirovorticillitatus ultraviolet mutant has enhanced bacteriostatic activity against Pseudomonas solanacearum; in addition, the tautomerism also has a good inhibiting effect on cucumber gray mold, but reports on the application of the strain to the poplar canker are not found so far.

The symphytic caveolae causes canker, and the poplar is not the single host. The symphytic microsclera frutescens causes canker, deadwood disease, jujube fruit shrinkage disease, rotten fruit disease and the like on cedar, apple, Chinese chestnut, jujube tree and a plurality of rosaceous plants, so that the harm of the symphytic microsclera frutescens must be highly regarded, and the comprehensive treatment of the symphytic microsclera frutescens is imperative. The biological control has the advantages of high efficiency, environmental protection and the like, and is a development trend of plant disease control. At present, no biocontrol strain screened aiming at the Populus ulcer pathogen syngen caveola is reported. The problems of unsatisfactory antibacterial activity, low control efficiency and the like exist in the biological control process. Therefore, the development of durable and effective biocontrol microbial inoculum is the most critical step in the current biological control research.

Disclosure of Invention

In view of the problems existing in the prior art, the invention provides a fermentation method of streptomycete, and the fermented streptomycete is antagonistic streptomycete which can effectively inhibit various pathogenic bacteria such as poplar canker pathogen and the like. The streptomyces provided by the invention has the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect, low toxicity, environmental protection and the like.

The technical scheme for solving the technical problems is as follows:

the invention provides a streptomycete, the preservation number of the strain is CGMCC No. 18657. The strain was identified as Streptomyces spirovorterillatus and in the examples of the present invention it was named strain HS 1. Is preserved in China general microbiological culture Collection center (CGMCC) 10 months and 10 days in 2019, and is No. 3 Xilu No.1 Beijing of the Kyoho area.

The growth temperature of the streptomycete is 4-28 ℃, and 28 ℃ is the optimal growth temperature; the pH is 5-12, and the pH7 is the optimum pH for growth. Through identification, the streptomyces can utilize carbon sources such as D-xylose, D-fructose, glucose, a-lactose, maltose, guanine, glycine, L-tyrosine and L-arabinose, wherein the D-xylose, the D-fructose and the glucose are the most suitable carbon sources, and the strains grow vigorously; raffinose, mannitol, sucrose and L-rhamnose cannot be utilized. The streptomycete has catalase and can catalyze and decompose hydrogen peroxide; the ability to produce urease; producing an amylase; the nitrate reduction reaction is positive, and the nitrate can be reduced into nitrite; lipase test is negative; the gelatin cannot be liquefied; cellulose cannot be decomposed and utilized.

The bacterial strain provided by the invention has the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect and the like. Especially has obvious and stable bacteriostatic effect on the Jusheng small acupoint shell.

The invention also provides a fermentation method of streptomyces, which comprises the following steps: inoculating the streptomyces into a culture medium for fermentation culture.

The culture medium may include the following components: 7.0 percent of starch, 3.3 percent of peanut cake powder and (NH) by mass percentage₄)₂SO₄ 0.4％、CaCO₃0.4%, NaCl 0.4%, distilled water 1000ml, pH7.0.

The fermentation conditions may be: the culture temperature is 4-28 ℃, and the pH is 5-12; preferably, the culture temperature is 28 ℃ and the pH is 7.

The invention has the beneficial effects that: the streptomycete and the fermentation liquor thereof fermented by the method can effectively inhibit various pathogenic bacteria such as poplar canker pathogen and the like, and have the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect, low toxicity, environmental protection and the like.

The invention also provides a microbial inoculum, which comprises the streptomyces and/or the fermentation liquor of the streptomyces. The microbial inoculum can also comprise auxiliary materials. For example, the excipient may be a diffuser, an adsorbent, or the like. The microbial inoculum has the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect, low toxicity, environmental protection and the like.

The streptomycete and/or the microbial inoculum can be applied to the bacteriostatic activity of pathogenic bacteria.

The streptomycete has a wide antibacterial spectrum, and has antibacterial effects on the following pathogenic bacteria, such as: pathogenic bacteria of Poplar canker, Poplar canker pathogen, Dothiorella gregaria, Poplar canker pathogenic bacteria, Microcystis chrysospora chrysosporium, Microcystis botrytis dothidea, Fusarium neospora Neosporocum parum, Fusarium oxysporum, Fusarium moniliforme, Colletotrichum truncatum, Microthecium oxysporum, Microsporum oxysporum, Alternaria alternata, Pepper Colletotrichum, Microsporum trichoderma, Microsporum moniliforme, Pseudoperonospora verticillarum, Pseudoperonospora cinerea, Pythium trichothecium, Pythium trichothecium verticillium, Pseudomonas verticillicola, Pseudomonas solanacearum, Pythium trichothecoides, Pythium trichothecium trichothecoides, Pseudomonas verticillioides, Pseudomonas verticillium, Pseudomonas verticillicium nivale, Pythium verticillium, Pythium verticilli. The streptomyces has the best bacteriostatic effect on especially Sclerotinia scleotiorum, ascosphaera aurantiaca, aschersonia vinifera, Fusarium trispora, phomopsis fulvidraco and colletotrichum truncatum.

The streptomycete and/or the microbial inoculum can be applied to biological control. For example, the streptomyces and/or the microbial inoculum provided by the invention are used for preventing and treating diseases caused by the pathogenic bacteria.

For another example: the composition is used for preventing and treating poplar canker, blueberry fusarium wilt, sweet melon fusarium wilt, rice bakanae disease, small red bean anthracnose, corn northern leaf blight, tobacco brown spot, pepper anthracnose, spruce damping off, melon and fruit saprophytosis, eggplant brown spot and the like.

A method of biological control comprising the steps of: streptomyces and/or streptomyces inocula are applied to crops in need of control. The method has the advantages of good prevention and treatment effect, stable effect and the like. The crops can be poplar, blueberry, melon, rice, small red bean, corn, tobacco, pepper, spruce, eggplant, other melons and fruits and the like.

Drawings

FIG. 1 shows the inhibitory effect of HS1 living bodies on Sclerotinia sclarea.

FIG. 2 shows the morphological characteristics of colonies (A, B) and hyphae (C) and conidia (D) of strain HS 1.

Fig. 3 shows the results of the m.r./v.p. test of strain HS 1.

FIG. 4 shows electrophoresis results of HS1 genomic DNA and 16S rDNA amplification products, wherein M is DNA Marker DL 2000; total DNA of HS 1; 2, positive control; 3,4: HS1 DNA16S rDNA PCR product; 5, positive control; 6, negative control.

FIG. 5 phylogenetic analysis of strain HS1 and related strains.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

According to the invention, antagonistic streptomycete HS1 with good biological control effect on pathogenic bacteria of various important diseases of agriculture and forestry is screened out from soil under larch forest of mountain transverse protection station of Changbai mountain national level natural protection area in Jilin province, and the bacteriostatic activity of viable bacteria and fermentation liquor of the strain is respectively measured by adopting a flat plate confronting method and a cup dish method; and the classification status is researched by methods such as morphology, physiological and biochemical characteristics, 16SrRNA and the like. The results show that the bacterial strain live bacteria and the fermentation liquor thereof have inhibition effects on 13 test plant pathogenic bacteria and have wide antibacterial spectrum, wherein the inhibition effect on the poplar canker pathogenic bacteria-the synbiotics micrococcus is strongest, the live bacteria inhibition zone reaches 20.66mm, and the diameter of the antibacterial zone of the fermentation liquor reaches 40.26 mm. The bacteriostatic zone for the pathogenic bacteria of the poplar canker disease, namely the chrysosporium aurantium, is 20.30mm, the bacteriostatic diameter of the fermentation liquor is 40.02mm, the bacteriostatic zone for the pathogenic bacteria of the blueberry canker disease, namely the botrytis cinerea is 21.72mm, and the bacteriostatic diameter is 39.84 mm; according to strain morphology, physiological and biochemical characteristics and 16SrRNA sequence comparison, the strain is finally identified to be Streptomyces spirovorticillatus, the strain has good bacteriostatic action effect on pathogenic bacteria of various important diseases of agriculture and forestry, and a new biocontrol factor is provided for biological control of plant diseases.

In the examples, the experimental materials and instruments used are described below.

(1) Test phytopathogens:

pathogenic bacteria of the poplar canker gather the micrococcus punctatus gregaria, pathogenic bacteria of the poplar canker chrysosporium, separate, purify and store by Jilin province forestry scientific research institute; blueberry canker pathogenic bacteria Botryosphaeria dothidea, blueberry deadwood pathogenic bacteria Neofusicoccum parum are presented by professor Xucheng nan of Yanan university; fusarium oxysporum, Fusarium moniliforme, Colletotrichum truncatum, Exserohilum turcatum, Alternaria alternata, Colletotrichum collecticola, Fusarium trispora, Pythium aphanidermatum, Colletotrichum collecticola, Fusarium trispora, Pythium aphanidermatum, Phomopsis Phomopsis phydrogenns, Phytopsis trichoderma, Phytopsis longissima trichothecum, Phytopsis longissimum tricuspidatum, Phytopsis Phomopsis physalum, Phytopsis physals venens, which are pathogenic bacteria of blight of melon and fruit, are given by Shenyang agricultural university plant virus research room.

All of the above strains are publicly available for repeated use in the examples of the present invention for non-commercial purposes only.

(2) A test soil sample:

the soil samples to be tested are collected from twelve soil samples of a mountain natural protection area, namely a mountain transverse protective station larch forest, a protection station Maanshan picea artificial forest, a Xiguo prefecture Sichuan forest farm and the like in Jilin province.

(3) Test medium:

synthesis of Gao's IAgar medium (mouse's synthetic agar): KNO₃ 1g、K₂HPO₄ 0.5g、MgSO₄ 0.5g、NaCl 0.5g、FeSO₄0.01g, 20g of soluble starch, 20g of agar, 1000ml of water and pH of 7.2-7.4;

potato dextrose agar (Potato dextrose agar): 200g of potatoes; 20g of glucose; 20g of agar; 1000ml of distilled water;

sucrose johnson agar (Czapek's agar): NaNO₃ 3g、K₂HPO₄ 1g、MgSO₄ 0.5g、KCl 0.5g、FeSO₄0.01g, 30g of cane sugar, 20g of agar and 1000ml of distilled water;

glucose asparagus agar (Glucose agar): glucose 10g, asparagines 0.5g, K₂HPO₄0.5g, 20g of agar and 1000ml of distilled water;

kreb No.1agar (Carrot No. 1agar): k₂HPO₄ 1g、MgCO₃ 0.3g、NaCl 0.2g、KNO₃ 1g、FeSO₄ 0.01g、CaCO₃0.5g, 20g of glucose, 20g of agar and 1000ml of distilled water;

starch ammonium agar (Starch ammonium salt medium): (NH4)₂SO₄ 2g、CaCO₃ 3g、K₂HPO₄1g of soluble starch, 10g of soluble starch and MgSO₄1g, NaCl 1g, agar 20g and distilled water 1000 ml;

maltose Yeast extract agar (Yeast extract-medium extract agar): yeast extract 10g, malt extract 10g, glucose 4g, distilled water 1000ml, agar 20g, pH 7.0;

bennett agar medium (Bennett's agar): 10g of glucose, 1g of beef extract, 1g of yeast extract, 2g of hydrolyzed casein, 1000ml of distilled water and 15g of agar;

oat flour agar (Oatemeal agar): 20g of oat flour, 1ml of trace salt solution, 1000ml of distilled water, 20g of agar and pH 7.2; the formulation of the trace salt solution comprises: FeSO₄ 0.1g、MnCl₂ 0.1g、ZnSO₄0.1g and 1000ml of distilled water;

starch agar (Soluble starch agar): soluble starch 10g, NaNO₃ 1g、MgCO₃ 1g、K₂HPO₄0.3g, NaCl 0.5g, distilled water 1000ml, agar 20 g;

glucose yeast extract agar (Glucose yeast extract agar): 10g of glucose, 10g of yeast extract, 1000ml of distilled water, 20g of agar and 7.2 of pH;

glycerol aspartyl medium (glycocol-asparagine agar): 10g of glycerol, 1g of asparagine and K₂HPO₄1g, 1ml of trace salt solution, 1000ml of distilled water and 20g of agar; the formulation of the trace salt solution comprises FeSO₄ 0.1g、MnCl₂0.1g、ZnSO₄0.1g and 1000ml of distilled water;

soybean meal agar medium (Bean agar medium): 40g of soybean meal, 20g of cane sugar, 1.5g of calcium superphosphate and MgSO₄0.75 g, 1000ml of distilled water and 20g of agar;

glycerol arginine medium (Glycine arginine medium): glycerol 6g, arginine 1g, K₂HPO₄0.5g、MgSO₄0.5g, 1000ml of distilled water and 20g of agar;

soy flour peptone medium (soy bean powder peptone medium): 20g of soybean extract powder, 5g of soluble starch, 10g of sucrose, 2g of peptone, 2g of yeast extract powder, 2g of NaCl and K₂HPO₄ 0.5g、CaCO₃0.1g, 1000ml of distilled water, 20g of agar and pH 7.2.

The strain is screened and identified by selecting Gao's No. one synthetic agar.

Fermentation medium, by mass percentage, 7.0 percent of starch, 3.3 percent of peanut cake powder and (NH)₄)₂SO₄ 0.4％、CaCO₃0.4%, NaCl 0.4%, distilled water 1000ml, pH7.0.

(4) Reagent:

an Ezup column type bacterial genome DNA extraction kit, which is purchased from Shanghai biological engineering Co., Ltd; 16SrDNA forward primer (5'-AGAGTTTGATCCTGGCTCAG-3'), reverse primer (5'-GGTTACCTTGTTACGACTT-3'), Taq PCR Master Mix (2 ×, blue dye), available from Shanghai Bioengineering Co., Ltd; DNA Marker DL2000, available from Bao bioengineering (Dalian) Inc.; granular agar, purchased from BD biosciences, and all other were domestic analytical grade.

(5) The instrument comprises the following steps: model BX53 olympus optical microscope, ltd.; ABI Veriti FAST gradient PCR Instrument, ABI, USA.

The screening of the strains and the determination of the bacteriostatic activity are described below by way of specific examples.

1. Method of producing a composite material

1.1 screening of biocontrol strains and determination of antibacterial activity

Example 1.1.1 Strain screening

In 8 months in 2018, the tested soil samples are collected from a mountain natural protection area, namely, a mountain transverse mountain protection station larch forest in Changbai mountain of Jilin province, a Maanshan picea artificial forest in a Yunshan protection station in Yunshi, a Xiguo county, a Sichuan forest farm in the front of the Changsong county, and the like, and twelve soil samples are randomly collected. The acquisition method comprises the following steps: removing surface soil by using a small shovel, taking about 200g of soil at the depth of 5-10 cm, respectively filling the soil into plastic bags, and putting labels recorded with collection places and collection time for separating streptomyces. If the soil sample can not be separated immediately, the soil sample is dried in the shade and then stored in a cool place.

And (3) separating and purifying the biocontrol streptomyces by adopting a dilution separation method. Adding each weighed 10g soil sample into 100ml of sterile water, shaking for 30min, sucking 1ml of suspension, diluting with sterile water in a gradient manner from 10^-3(i.e. dilution 10)³Multiple), 10^-4(i.e. dilution 10)⁴Multiple) and 10^-5(i.e. dilution 10)⁵Multiple times) 0.1ml of the solution is respectively sucked into a dilution tube, added onto a Gao's first plate, evenly coated, and then the culture dish is placed in a thermostat at 28 ℃ upside down for culture for 3-10 days. And (3) re-purifying all the strains obtained by separation for 3-5 times by adopting a dilution separation method, transferring the strains into a Gao's first inclined plane culture medium for culture, and storing the strains in a refrigerator at 4 ℃ for later use. And transferring all the separated strains into a Gao's first culture medium for culture, and after each strain produces enough spores, manufacturing actinomycete blocks for determining the bacteriostatic activity.

Example 1.1.2 determination of the bacteriostatic Activity of the Strain

Using Sclerotium giganteum (D.gregaria) as a target bacterium, and adopting a plate confrontation culture method to carry out living body screening of antagonistic streptomyces. Preparing a 5mm fungus cake from target bacteria, inoculating the fungus cake to the center of a potato glucose agar plate (PDA plate) (the diameter of the plate is 90mm), inoculating and looping actinomycetes cultured for 72h at a position 1.75cm away from the upper part and the lower part of the fungus cake, drawing lines in parallel, culturing at a constant temperature of 28 ℃ for 72h, measuring the width of an antibacterial zone between the actinomycetes and pathogenic fungi, and repeating the treatment for 3 times. The strain of the fermentation test is determined according to the size and stability of the zone.

EXAMPLE 1.1.3 determination of bacteriostatic spectra of biocontrol strain in vivo and fermentation broth

The in vivo bacterial inhibition spectrum of the strain HS1 is determined by adopting a plate confronting culture method, the number of pathogenic bacteria of the bacterial inhibition spectrum is 13, the width of the bacterial inhibition zone between the biocontrol strain and each pathogenic fungus is measured, and each treatment is repeated for 3 times.

The fermentation medium is prepared conventionally, and the formula comprises 7.0% of starch, 3.3% of peanut cake powder and (NH) in percentage by mass₄)₂SO₄ 0.4％、CaCO₃0.4%, NaCl 0.4%, distilled water 1000ml, pH7.0. Selecting a biocontrol strain HS1 with strong bacteriostatic activity and good stability, transferring the biocontrol strain HS1 into a fermentation culture solution, loading 40ml of triangular bottled fermentation liquor with the sample size of 250ml, inoculating 5 bacterial blocks, performing constant-temperature shaking culture (28 ℃, 150r/min) for 5 days, centrifuging the fermentation liquor at 8000r/min for 15min, taking the supernatant, removing bacteria through a bacterial filter, and storing the supernatant in a refrigerator at-20 ℃ for later use.

Pathogenic bacteria blocks with the diameter of 7mm are prepared by adopting a cup-dish method, the pathogenic bacteria blocks are inverted around a PDA flat plate at equal intervals, an oxford cup is arranged in the center, the sample adding amount of fermentation liquor is 200 mu l, the fermentation liquor is cultured in a constant temperature incubator at 28 ℃ for 72h, and then the diameter of the inhibition zone is measured by adopting a cross method.

Example 1.1.4 characterization of the physiological and biochemical Properties of biocontrol strains

Example 1.1.4.1 morphological Observation of HS1 Strain

Inoculating and observing the colony morphology of the biocontrol strain HS1 on a Gao's first culture medium; observing the morphological characteristics of hyphae and spores of the strain by using an insertion method: the sterilized PDA culture medium is poured into a flat plate, generally 15ml of the culture medium is poured into each dish, after solidification, a sterile cover glass is inserted into the culture medium at an angle of 45 degrees, the depth of the cover glass inserted into the culture medium is preferably 1/2, then inoculation is carried out along the obtuse angle boundary line of the cover glass and the surface of the culture medium, the culture is carried out at 28 ℃, the cover glass is taken out at 7 days, 14 days and 28 days respectively, the morphology of hyphae, aerial hyphae and the morphological characteristics of spores in the culture medium are observed under a microscope, and photographing records are carried out.

According to the International Streptomyces program (1966) and the research method of Actinomycetes classification group (1975) of the institute of Chinese academy of sciences, actinomycetes were cultured using a medium for identification of actinomycetes (see the above "(3) test medium) for the formulation of the medium), the HS1 strain was transferred to each identification medium slant, three tubes were used per strain, two plates were simultaneously transferred, and the culture characteristics were observed after 15 days at 28 ℃. The color and characteristics of the aerial hyphae, intrabasal hyphae, soluble pigments of the strains on various identification media were recorded. Color comparison is carried out according to the Streptomyces identification handbook.

Example 1.1.4.2 identification of the tolerance Properties of the HS1 Strain

In the pH tolerance experiment, a bennett liquid culture medium is selected, and five tolerance gradients of pH2, 5, 7, 9 and 12 are set. Fresh spores of the same amount of strain HS1 were inoculated, incubated at 28 ℃ and the growth of the treated strain was recorded at 7, 14d and repeated 3 times per treatment.

The temperature tolerance experiment selects a bennett agar culture medium, and the set temperature is 4 ℃, 15 ℃, 28 ℃, 37 ℃ and 50 ℃ for five tolerance gradients. Equal amounts of strain HS1 suspension were spread evenly, and growth was recorded for the treated strain at 7, 14d and at 14d and 28d respectively for the 4 ℃ cultures. Each treatment was repeated 3 times.

The culture medium selected in the NaCl tolerance experiment and the pH tolerance experiment are set to have five tolerance gradients of 0%, 5%, 15%, 25% and 30%. Fresh spores of the same amount of strain HS1 were inoculated, incubated at 28 ℃ and the growth of the treated strain was recorded at 7, 14d and repeated 3 times per treatment.

Example 1.1.4.3 determination of carbon Source utilization by HS1 Strain

Inoculating spores of the strain HS1 into a liquid culture medium, adding equal amounts of different carbon sources respectively, observing whether the strain grows or not and recording the growth state of the strain. The basic culture medium adopts a Pogostemon culture medium: (NH)₄)₂SO₄ 2.64g、K₂HPO₄ 2.38g、MgSO₄·7H₂O 1.0g、CuSO₄·5H₂O 0.0064g、FeSO₄·7H₂O 0.0011g、MnCl₂·4H₂O 0.0079g、ZnSO₄·7H₂0.0015g of O and 1000ml of distilled water. Selection of carbon source substances: d-xylose, D-fructose, mannitol, L-rhamnose, a-lactose, glucose, sucrose, D-maltose, raffinose, guanine, glycine, L-tyrosine and L-arabinose.

Example 1.1.4.4 characterization of the enzymatic Properties of the HS1 Strain

Catalase assay: and (3) placing HS1 lawn in a logarithmic growth phase on a solid culture medium on a clean glass slide, dropwise adding 3-10% of hydrogen peroxide, and observing the result, wherein the result shows that the bacterium is positive if a large number of bubbles are generated within 0.5min, and the bacterium is negative if the bacterium does not generate bubbles.

Urease test: sterilizing 30% urea with diethyl ether, and adding sterile urea when the culture medium is cooled to 55 deg.C to make the final concentration of urea be 2%. The HS1 strain is inoculated, the color change of the culture medium is observed after the constant temperature of 28 ℃ is kept for 4 days, the culture medium is positive when the color is changed into peach color, and the culture medium is negative when the color is white. Culture medium: peptone 1g, NaCl5g, glucose 1g, KH₂PO₄2g, 0.012g of phenol red, 15g of agar and 1000ml of distilled water.

Lipase experiments: and (3) cooling the culture medium to 40-50 ℃, adding sterile Tween 20 to a final concentration of 1%, preparing a flat plate, and inoculating the HS1 strain. Culturing at the constant temperature of 28 ℃ for 7-14 days, and observing every day. Faint halos around the growing strain were positive and none were negative. Culture medium: peptone 1g, NaCl5g, CaCl₂·7H₂O0.1g, agar 9g, distilled water 1000ml, pH7.4.

Gelatin liquefaction experiment: the HS1 strain is inoculated on a gelatin culture medium, the culture is carried out at the constant temperature of 28 ℃, and the liquefaction degree is observed at 5, 10, 20 and 30 days. Before observation, the plate was placed in a refrigerator at 4 ℃ for about 30 min. The control was medium without inoculum and each group was replicated three times. Selection of the medium: 5g of peptone, 20g of glucose, 200g of gelatin and 1000ml of distilled water.

Starch hydrolysis experiments: inoculating HS1 strain to starch agar culture medium, culturing at 28 deg.C, measuring amylase activity after 10 days, dripping iodine solution on plate, and observing the color around thallus PorphyraeColor, presence or absence of transparent circles and size. Selection of the medium: soluble starch 10g, K₂HPO₄ 0.3g、MgCO₃1.0g、NaCl0.5g、KNO₃1.0g, 15g of agar, 1000ml of distilled water and pH 7.2-7.4.

Milk clotting and peptonization experiments: inoculating HS1 strain into skimmed milk, culturing at 28 deg.C, and observing whether coagulation phenomenon and peptonization phenomenon exist in 3, 6, 10, 20, and 30 days. Three replicates per group were used as controls against the non-inoculated species. And (3) solidification: a clot is generated; peptonization: the casein is hydrolyzed into a transparent or translucent liquid state. Culture medium: fresh skim milk 1000ml, CaCO₃0.02g, and 3 times of batch sterilization.

Cellulose utilization experiments: the HS1 strain was inoculated onto a filter paper strip in a test tube, half of the filter paper was soaked in a carbon source-free synthetic solution, and cultured for 30 days to see whether the strain could grow and whether the filter paper strip could be decomposed. Three replicates per group were used as controls against the non-inoculated species. Selection of the medium: MgSO (MgSO)₄0.5g、NaCl0.5g、K₂HPO₄0.5g、KNO₃1.0g, 20g of cane sugar and 1000ml of distilled water.

Nitrate reduction experiments: HS1 was inoculated into the culture medium and assayed at 7d and 14d of culture. Two drops of solution I and two drops of solution II are added into every 1ml of bacterial liquid, and the red color is positive. The control was the non-inoculated medium. Culture medium: MgSO (MgSO)₄0.5g、NaCl0.5g、K₂HPO₄0.5g、KNO₃1.0g, 20g of cane sugar and 1000ml of water. Solution I: 0.8g of aminobenzenesulfonic acid is dissolved in 100ml of 5mol/L acetic acid. Solution II: 0.1g of diphenylamine and 20ml of water, wherein the diphenylamine is dissolved in a small amount of ethanol and then added with water, and after boiling, 150ml of diluted acetic acid is added.

Example 1.1.4.5 metabolite assay of HS1 Strain

M.r. test: inoculating HS1 strain in liquid culture medium, culturing at 28 ℃ for 2-6 days, adding a drop of methyl red reagent into the culture medium, wherein red is positive and yellow is negative. Culture medium: peptone 5g, glucose 5g, K₂HPO₄5g, 0.012g of phenol red and 1000ml of distilled water. Methyl red reagent: methyl red 0.1g, 95% ethanol 300ml, distilled water 200 ml.

V.p. test: in the same M.R. test, after the HS1 strain is cultured, the culture solution and 40% NaOH solution are mixed uniformly in equal amount, a little creatine is added, and the positive reaction is that red color appears in 10min, and sometimes the culture solution needs to be placed for a longer time. The culture medium was used in the m.r. experiment.

Experiment for hydrogen sulfide formation: the strain HS1 is inoculated on Tresner culture medium, and whether melanin is produced or not is observed after 7 days of culture. The production of melanin indicates that the strain produces hydrogen sulfide, and the uninoculated medium is used as a control. Culture medium: 10g of peptone, 0.5g of ferric citrate, 15g of agar, 1000ml of distilled water and pH 7.2.

Example 1.1.5 molecular biological characterization of the Strain

DNA extraction was performed according to the instructions of the kit for extracting genomic DNA from Ezup column type bacteria. The PCR reaction system is 50 μ L: PCR mix 25. mu. L, Template 2. mu.L, Primer F1, Primer R1 2. mu. L, ddH each₂O 19μL。

And (3) PCR reaction conditions: denaturation at 94 deg.C for 4 min; denaturation at 94 deg.C for 30s, renaturation at 57 deg.C for 30s, extension at 72 deg.C for 1min, and 35 times of circulation; extending for 10min at 72 ℃, and storing at 4 ℃. The PCR product of sterile water was used as a negative control, the PCR product of a strain whose laboratory has determined the result of 16SrDNA was used as a positive control, and 2. mu.L of the sample was applied and subjected to 2% agarose gel electrophoresis. And (3) sending the PCR product to Changchun Kumai biology company for sequencing, comparing the result with an NCBI database, determining the preliminary identification of the streptomyces species, and preparing for establishing a phylogenetic tree.

Example 1.2 data analysis

SPSS 19.0 is adopted for data statistics, Duncan's new complex pole difference method is adopted for difference significance analysis, and a phylogenetic tree is established by MEGA version 6.0.

2. Results and analysis of the experiments

2.1 preliminary screening of the Strain

104 biocontrol strains with different morphological characteristics are obtained by separation and purification, and the results of a plate confrontation test show that 13 strains with the bacteriostatic bandwidth of more than 10mm and 3 strains with the bacteriostatic bandwidth of more than 19mm are provided, wherein the strain HS1 has the best bacteriostatic effect, and the bacteriostatic bandwidth reaches 20.6mm (Table 1, figure 1). As shown in FIG. 1, FIG. 1 shows the results of the experiment on the confronting of the strain HS1 and the Sclerotium accumbens in the above examples, the left graph shows the experiment on the confronting of the living body of the strain HS1 and the Sclerotium accumbens, and the right graph shows the experiment on the confronting of the fermentation liquid of the strain HS1 and the Sclerotium accumbens. From the left image of fig. 1, it can be seen that the growth of the left side of the syngeneic cavum is limited, the growth of the right side is normal, and the growth of the syngeneic cavum is obviously inhibited by the living strain HS1, which presents a clear and wide inhibition zone. The right picture shows that the clear and obvious inhibition zone with larger diameter is presented below the oxford cup containing the strain HS1 in the upper two rows, and the growth of the syngenetic small cave shell is limited. And no bacteriostatic zone is generated below the oxford cup containing the same amount of sterile water in the last row, and the growth of the incrustation bacteria is normal. The bacterial strain HS1 has obvious bacteriostatic action, is real and effective and meets the screening requirement.

TABLE 1 bacteriostatic activity of Streptomyces lividans on Hippocastella volvacea

Table 1 Inhibition of antagonistic streptomyces to Dothiorella gregaria

The data in the table are mean ± standard deviation. Data in the table area mean + -SD.

2.2 determination of biological control bacteria HS1 live bacteria and its fermentation liquor bacteriostasis spectrum

The results of the bacterial strain HS1 in vivo bacterial inhibition spectrum determination show that: the HS1 strain has a wide antibacterial spectrum and has a certain inhibition effect on all tested pathogenic bacteria strains (shown in Table 2). The HS1 viable bacteria have the best inhibition effect on Sclerotinia sclarea, chrysosporium, Staphylosporium, Fusarium triloba, Phomopsis fulva and Colletotrichum truncatum, and have obvious inhibition zones, the average width of the inhibition zones is more than 20mm, and the inhibition zones are obviously different from other pathogenic bacteria (P is less than 0.05). Has strong inhibiting effect on Helminthosporium umbiliciformis, alternaria alternata, fusarium oxysporum and colletotrichum capsici, and the average width of the antibacterial zone is more than 15 mm.

The results of the bacterial inhibition spectrum measurement of the fermentation liquor of the strain HS1 show that (as shown in Table 2): the strain HS1 fermentation liquor can inhibit the growth of all tested pathogenic bacteria, and particularly, the four pathogenic bacteria, namely, synechocystis, ascosphaera aurantiaca, botrytis cinerea and helminthosporium macrostoma, have the best bacteriostatic effect, and the bacteriostatic diameter reaches more than 39 mm; the inhibition strength of the trichomonas truncatum, phomopsis fusca, alternaria alternata and colletotrichum capsici is high, and the inhibition diameter is 30-38 mm. The fermentation liquor of the strain HS1 has high inhibition strength on the 8 pathogenic bacteria, and the control effect is superior to other pathogenic bacteria. Through analysis of the Duncan's new repolarization method, the HS1 fermentation broth has obvious difference compared with other test strains on Sclerotinia fusca, Chaetomium aurantiacum, Staphylospora viticola, Helminthosporium macranthum and Pepper Pediculus capsici. As can be seen from the screening test, although the soil contains abundant actinomycetes, the elimination rate of antagonistic bacteria is relatively high. For the indoor screening process of antagonistic strains, live bodies, fermentation liquor, stability and the like are comprehensively considered, and then the strains with real application values can be screened. The biocontrol strain HS1 and the fermentation liquor have high inhibition strength on the Sclerotium accumbens, good stability and wide antibacterial spectrum, so the strain HS1 is selected as the biocontrol strain of the poplar canker caused by the Sclerotium accumbens.

TABLE 2 bacteriostatic spectra of biocontrol strain HS1 in vivo and fermentation broth

Table 2 Antimicrobial spectra of antagonistic microorganism and fermented broth about HS1

The data in the table are mean ± standard deviation. Lower case letters after the same column of data indicate significant differences at a P <0.05level as tested by Duncan's new repolarization method. Data in the table area ± sd. differential lower cases differences in the same column index difference at P <0.05levels by Duncan's new multiple range test, respecitvity.

2.3 morphological Classification and identification of biocontrol Strain HS1

2.3.1 morphological Observation of Strain HS1

The strain HS1 radially grows on the Gao's first culture medium, aerial hyphae flourish, and when the strain is cultured for 1-2 days at 28 ℃, a circular colony is smooth and has no spore generation. White spores began to grow from the colony edge at 3d, and gradually changed to white color of the meat mussel after 4d, and the matrix mycelium appeared yellowish without the production of soluble pigment (fig. 2A). After 30d, the colony becomes prominent and large, and the color becomes gray purple pink (FIG. 2B). Under the observation of a microscope, the hyphae are slender and have a large number of branches, the hyphae are spiral or straight, have no transverse membrane and are not broken (figure 2C), small elliptic conidia are generated at different sites of the hyphae (figure 2D), and the strain HS1 is preliminarily analyzed to be streptomyces according to morphological characteristics.

2.3.2 culture characteristics of Strain HS1

The strain HS1 is inoculated on 15 test culture media, bacterial colonies appear after the strain is cultured for 24-48 hours at the constant temperature of 28 ℃, and spores begin to generate after 72-96 hours. The morphology of lawn, the color of spore pile, the color of matrix hyphae, pigment produced and growth condition on different culture media are all different, the results of observation and comparison are shown in table 3 according to the method and color plate provided by the streptomycete identification manual, and HS1 is preliminarily judged to be the lilac group streptomycete by integrating the morphological characteristics of the strains on 15 culture media and the shapes of hyphae and spores.

TABLE 3 culture characteristics of Strain HS1

Table 4 Cultural characteristic of Streptomyces strain HS1

"-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +", vigorous growth. "-", Nogrowth; "+" growth; operate growth, "+ +", and operation growth; "+ + + + +", abundantgrowth.

2.4 physiological and Biochemical characteristics of biocontrol Strain HS1

2.4.1 tolerance assay for strain HS1

The strain HS1 has good tolerance to alkali, can grow in the range of pH 5-12, and has the optimum pH for growth when the pH is 7; the tolerance to NaCl is strong, and the higher the salt concentration is, the more vigorous the strain growth is; the strain can grow at 4-28 ℃, the high temperature tolerance is weak, the low temperature tolerance is good, and 28 ℃ is the optimal growth temperature (table 4).

TABLE 4 determination of the tolerance of the strain HS1

Table 4 Tolerance to PH/temperature/NaCl of Streptomyces strain HS1

"-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +", vigorous growth. "-", Nogrowth; "+" growth; modetegrowth; "+ + + + +", abundantgrowth.

2.4.2 carbon Source utilization of Strain HS1

The strain HS1 can utilize carbon sources such as D-xylose, D-fructose, glucose, alpha-lactose, maltose, guanine, glycine, L-tyrosine and L-arabinose, wherein the D-xylose, the D-fructose and the glucose are the most suitable carbon sources, and the strain grows vigorously; raffinose, mannitol, sucrose and L-rhamnose cannot be utilized.

TABLE 5 determination of carbon Source utilization and enzymatic Properties of Strain HS1

Table 5 Utilization of carbonsources and enzyme properties of Streptomyces strain HS1

2.4.3 enzymatic Properties of Strain HS1

The results of the enzymatic properties of strain HS1 are shown in Table 5, and the results are positive in all of the catalase, urease and starch hydrolysis experiments. The strain HS1 has catalase and can catalyze and decompose hydrogen peroxide; the ability to produce urease; the activity of the produced amylase is stronger. The nitrate reduction reaction is positive, the color is rose red, and the strain HS1 can reduce the nitrate into nitrite. Lipase test is negative; the gelatin cannot be liquefied; cellulose cannot be decomposed and utilized.

2.4.4 metabolite determination of Strain HS1

This experiment was conducted mainly to determine the organic acid component of the metabolite of strain HS 1. As shown in fig. 3, the results of the m.r./v.p. test of strain HS1 are shown in the left, and the results of the v.p. test in the right. In the left panel, the in vitro medium inoculated with HS1 strain turned red, and the control tube showed the original medium color without changing color. In the right panel, tubes inoculated with HS1 strain turned red and control tubes did not change color. The results show that the M.R. test and the V.P. test of HS1 are positive, the strain HS1 is special, the test time is persistent, and false positive does not exist. The strain HS1 is shown to decompose glucose to generate pyruvic acid in the sugar metabolism process, and the pyruvic acid is further decomposed into formic acid, acetic acid, lactic acid and succinic acid or further decarboxylated acetyl methyl methanol (figure 3); the production experiment of H2S shows positive gray black, which produces melanin, indicating that strain HS1 can decompose sulfur-containing organic substances.

2.5 identification of 16S rDNA of Strain HS1

Electrophoresis results show that the DNA integrity is good, no pollution is caused, and 16S rDNA gene amplification products of the strain HS1 are shown in figure 4. The amplification effect of the target fragment is good, the size of the product is about 1.4Kb, and the size is consistent with the reported size of 16S rDNA. After sequencing, the total length of the 16S rDNA gene amplification fragment of the strain HS1 (gene accession number: MN636764) is 1398bp, and compared with the 16S rDNA sequence of the related actinomycete strain in a GenBank database, the strain has the highest homology with the Streptomyces spirovorticillicatus 16S rDNA of the known actinomycete, and the homology reaches 99 percent. The sequence with high homology of 17 strains and the strain HS1 is selected, phylogenetic analysis is carried out by using MAGA6 software (figure 5), and the result shows that the strain HS1 has the closest genetic relationship with Streptomyces spirovorticilliticus, so the strain HS1 is preliminarily determined to be Streptomyces spirogyrus.

3 conclusion and discussion

The agricultural antibiotic for plant diseases generally has lower phytotoxicity than chemical pesticides, has the characteristics of strong bactericidal action, higher prevention and treatment efficiency and high efficiency and low toxicity, and the use of the antibiotic to replace the chemical pesticides for preventing and treating the plant diseases is one of the development directions in the future. Actinomycetes are the most antibiotic-producing group, and streptomyces is one of the most antibiotic-producing genera of actinomycetes. Generally, the screening of agricultural antibiotics can directly use pathogenic bacteria of plants as a screening model. The existing primary screening method generally takes in vitro antibacterial activity determination as the main measurement. In the aspect of prevention and treatment of poplar canker, research aiming at the symphytic micrococcus is not reported at present, 104 strains of streptomycete are co-separated from a soil sample by using a dilution separation method, the symphytic micrococcus is used as a target, a multi-screening method combining living body confrontation, fermentation liquor bacteriostatic activity determination and bacteriostatic spectrum determination is adopted to obtain an antagonistic streptomycete strain HS1 with obvious bacteriostatic effect, wider bacteriostatic spectrum and stable effect on the symphytic micrococcus, and the strain HS1 is preliminarily identified as Streptomyces spirovorticilliticulus according to morphological expressions, physiological and biochemical characteristics and molecular identification of a16 SrDNA sequence in different culture media. The bacterial strain HS1 has an inhibition zone of 20.66mm for Sclerotium hybridum, the diameter of an inhibition zone of fermentation liquor is 40.26mm, the inhibition zone of chrysosporium cucumerinum is 20.30mm, the inhibition diameter is 40.02mm, the inhibition zone of Staphylospora botrytis is 21.72mm, and the inhibition diameter is 39.84 mm. The streptomycete HS1 has the advantages of wide antibacterial spectrum, obvious antibacterial effect, stable effect and the like.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A fermentation method of streptomycete is characterized by comprising the following steps: inoculating the streptomycete with the preservation number of CGMCC No.18657 to a culture medium for fermentation culture.

2. The fermentation process of Streptomyces according to claim 1, wherein the culture medium comprises the following components: 7.0 percent of starch, 3.3 percent of peanut cake powder and (NH4) by mass percentage₂SO₄0.4％、CaCO₃0.4 percent, NaCl 0.4 percent and water as solvent.

3. The fermentation process of Streptomyces according to claim 1 or 2, wherein the pH of the culture medium is 7.

4. The fermentation method of streptomycete according to any one of claims 1 to 3, wherein the temperature of the fermentation culture is 4 to 28 ℃.

5. The fermentation process of Streptomyces according to claim 4, wherein the temperature of the fermentation culture is 28 ℃.

6. The fermentation method of Streptomyces according to any of claims 1 to 3, wherein the pH of the fermentation culture is 5 to 12.

7. The fermentation process of Streptomyces according to claim 6, wherein the fermentation culture has a pH of 7.