CN112877261B - Fermentation method of biocontrol streptomyces - Google Patents
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Abstract
The invention relates to a fermentation method of biocontrol streptomyces, which comprises the following steps: the biocontrol streptomyces is fermented, and the preservation number of the biocontrol streptomyces strain is CGMCC No.18658. During the culture, the biocontrol streptomyces is inoculated to a fermentation culture medium for fermentation culture. The biocontrol streptomyces and/or fermentation broth prepared by the method has the advantages of good antibacterial effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like.
Description
The invention is a divisional application, and the original Chinese patent application number is: 201911183884.8, filing date: the patent name at the time of application is 2019, 11, 27: streptomyces biocontrol.
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a fermentation method of biocontrol streptomyces.
Background
Blueberry, also known as cowberry, belongs to the genus Vaccinium (Vaccinium) of the family Ericaceae (Ericaceae), and has sweet and sour fruits, and can be processed into fruit juice, fruit wine, and beverage in addition to fresh food. The fruit of the blueberry fruit has ultrahigh nutritional value, is rich in a large amount of anthocyanin, various antioxidants and flavonoids besides fructose and vitamins, has unique and rare nutritional health care value which is more and more favored by people, and the demand of blueberries is continuously increased worldwide.
In recent years, the planting area of blueberries is increased year by year, the diseases of the blueberries are serious, the economic loss is serious, and the blueberry canker is an important disease of the blueberries caused by the Botrytis cinerea (Botryosphaeria dothidea). The disease is widely distributed worldwide, mainly endangers 1-year old branches and mostly occurs from wounds and pruning wound; reddish brown disease spots are generated in the early stage of disease, and as the disease spots are expanded, branch tips start to dry up, and the whole young branches gradually die; in the later stage of the disease, densely buried small black spots (conidiophore discs) are visible on the disease spots; the damaged tissue was dissected and the vascular bundle tissue turned light brown, and white mycelia were seen. If the blueberry canker is easy to occur when the temperature difference between the low temperature and day and night is large, the blueberry yield is greatly affected by the disease, and even the plant death can be caused.
In recent years, domestic reports about the occurrence of blueberry diseases are frequent, such as Jiang Ping reports on the incidence condition of blueberry canker; yang Yanlin reports the hazard trend of common diseases and insect pests in blueberry cultivation and the cultivation technology of blueberry branches, and greatly suppresses the occurrence of blueberry diseases. At present, the prevention and treatment of the disease take the following measures: chemical control, breeding disease-resistant varieties, cultivating disease-free strong seedlings, optimizing cultivation sites and cultivation modes, and the like. Among a plurality of disease prevention means, chemical prevention and control and breeding of disease-resistant varieties are most accepted by masses, but the two prevention and control methods have obvious disadvantages, on one hand, the breeding of the disease-resistant varieties takes longer time, has the problems of resistance degradation and the like, and cannot achieve the expected disease prevention effect; on the other hand, a large amount of chemical pesticides are put into the environment to cause environmental pollution, destroy ecological balance, have high toxicity, and are easy to remain. Therefore, sustainable control technology mainly based on ecological system regulation is studied, antagonistic microorganisms with inhibition effect on pathogenic bacteria are utilized to control the disease, bacteria are used for controlling the disease, and the purposes of green, environment-friendly and high-efficiency comprehensive control are achieved.
Actinomycetes are the earliest discovered class of microorganisms with biological control effects. Streptomyces play an important role in actinomycetes. However, the existing actinomycetes have the problems of unsatisfactory control effect, unstable field control effect and the like in the field of blueberry canker.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides the biocontrol streptomycete and the fermentation method thereof, which have the advantages of good antibacterial effect, stable field control effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like.
The technical scheme for solving the technical problems is as follows:
the invention provides a biocontrol streptomycete, the preservation number of which is CGMCC No.18658. The strain is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms in 10 and 10 days of 2019.
The inventor collects soil in different areas of Jilin province to obtain a plurality of actinomycetes, and finally unexpectedly obtains 1 antagonistic strain capable of effectively inhibiting blueberry canker by adopting multiple screening, and marks the antagonistic strain as Streptomyces CX3 (Streptomyces sp. Strain CX 3), which is abbreviated as CX3 in the embodiment of the invention. The biocontrol streptomyces provided by the invention provides a new biocontrol factor for biological control of important diseases in blueberry production, and realizes breakthrough of actinomycetes in the field of blueberry canker.
The beneficial effects of the invention are as follows: the antagonistic strain CX3 obtained by the invention has wide development prospect, wide bacteriostasis spectrum and extremely high biological control potential, active bacteriostasis components in fermentation liquor can be used as biological pesticides to be developed and applied to the control of various plant diseases after separation and purification and other processes, and meanwhile, compared with chemical pesticides, the fermentation liquor of the strain CX3 has multiple advantages of low toxicity, no residue, environmental friendliness and the like, and accords with the sustainable control environment protection concept better.
Specifically, the biocontrol streptomyces grows luxuriantly on a culture medium of Gao's first, aerial hyphae are plentiful, and the hyphae grow radially in all aspects; culturing at 28deg.C for 1-3 days, wherein the colony is smooth and free of sporulation, and spores are generated after 7 days, and observed under microscope, and the spores are chain-shaped; the spore filaments are not autolyzed, and the phenomenon of water absorption is avoided; the color of the spore pile is close to pink when the spores are cultured for 15 days, and most of the spores are straight, and sometimes the top ends of the spores are hooked.
The biocontrol streptomycete can use D-xylose, D-fructose, mannitol, L-rhamnose, glucose, sucrose, maltose, guanine, glycine, L-tyrosine and L-arabinose; a-lactose and raffinose cannot be utilized.
The catalase experiment of the biocontrol streptomyces generates a large amount of bubbles; urease experiment is negative; positive lipase test, semitransparent halos are generated around the strain; gelatin can be liquefied; the use of cellulolytic enzymes is not possible; the milk can be coagulated, but peptone phenomenon does not occur; starch hydrolysis experiments were positive and amylase could be produced.
The M.R. test of the biocontrol streptomyces is negative; h 2 S is positive in experiment; v.p. experiment CX3 presents a positive red color; positive pink color was seen in the nitrate reduction reaction.
The bacteriostasis spectrum of the biocontrol streptomyces comprises the following strains: blueberry canker, poplar rot, corn leaf spot, tobacco brown spot, spruce fusarium, melon fusarium, red bean anthracnose, melon and fruit pythium, rice bakanae, pepper anthracnose, eggplant brown spot and poplar canker.
The formula of the fermentation medium used in the fermentation culture of the biocontrol streptomyces comprisesThe components are as follows: every 1000mL of water, 70g of soluble starch, 33g of peanut cake powder and (NH) 4 ) 2 SO 4 4 g、NaCl 4g、CaCO 3 4 g，pH7.0。
When the biocontrol streptomyces is fermented and cultured, the fermentation and culture temperature is 15-28 ℃. Preferably, the fermentation temperature is 28 ℃. The adoption of the temperature is more beneficial to the fermentation culture of the biocontrol streptomycete.
The invention provides a biocontrol product, which comprises the biocontrol streptomyces and/or the biocontrol streptomyces fermentation liquid.
The beneficial effects of adopting above-mentioned scheme are: the biological control product obtained by the invention has wide development prospect, wide antibacterial spectrum and extremely high biological control potential, can be used as biological pesticide to be developed and applied to the control aspect of various plant diseases, has multiple advantages of low toxicity, no residue, environmental friendliness and the like, and is more in line with the sustainable control environment-friendly concept.
The invention provides a fermentation method of the biocontrol streptomyces, which comprises the following steps: the biocontrol streptomyces is fermented, and the preservation number of the biocontrol streptomyces strain is CGMCC No.18658.
The beneficial effects of the invention are as follows: the biocontrol streptomyces and/or fermentation broth prepared by the method has the advantages of good antibacterial effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like.
Specifically, the method comprises the following steps: the biocontrol streptomyces with the preservation number of CGMCC No.18658 is inoculated into a fermentation culture medium for fermentation culture.
In the fermentation process, the culture can be performed by adopting a fermentation tank fermentation or shake flask fermentation mode.
The fermentation temperature may be 15-28deg.C, preferably 28 deg.C. The fermentation time may be 5-7 days.
The method may further comprise a step of centrifuging a fermentation broth obtained by the fermentation, and separating a fermentation broth obtained by the method from a strain containing Streptomyces biocontrol and a strain not containing Streptomyces biocontrol. The conditions for centrifugation may be appropriately selected depending on the fermentation conditions, and in this embodiment, the rotational speed of centrifugation is 8000r/min and the centrifugation time is 15min, for example.
The fermentation medium in the fermentation process can be prepared according to the following proportion, wherein each 1000mL of water contains 70g of soluble starch, 33g of peanut cake powder and (NH) 4 ) 2 SO 4 4g、NaCl 4g、CaCO 3 4g，pH7.0。
The beneficial effects of adopting above-mentioned scheme are: the proper temperature, culture time and culture medium are beneficial to the fermentation culture of the biocontrol streptomyces.
The invention also provides a biocontrol product, which comprises the biocontrol streptomyces and/or the biocontrol streptomyces fermentation broth prepared by the fermentation method.
The invention provides application of the biocontrol streptomyces in pathogen control. Preferably, the invention provides application of the biocontrol streptomyces in controlling plant pathogenic bacteria.
Further, the plant pathogenic bacteria comprise one or more of blueberry canker, poplar canker, corn leaf spot, tobacco brown spot, spruce rhizoctonia, melon fusarium wilt, red bean anthracnose, melon and fruit pythium, rice bakanae disease, pepper anthracnose, eggplant brown spot and poplar canker. Preferably, the prevention and treatment effect on the blueberry canker is more remarkable, and the method can be used for biological prevention and treatment of the blueberry canker.
The invention provides application of the biocontrol product in pathogen control. Preferably, the invention provides application of the biocontrol product in controlling plant pathogenic bacteria.
Further, the plant pathogenic bacteria comprise one or more of blueberry canker, poplar canker, corn leaf spot, tobacco brown spot, spruce rhizoctonia, melon fusarium wilt, red bean anthracnose, melon and fruit pythium, rice bakanae disease, pepper anthracnose, eggplant brown spot and poplar canker. Preferably, the prevention and treatment effect on the blueberry canker is more remarkable, and the method can be used for biological prevention and treatment of the blueberry canker.
The invention provides a method for biological control by using the biocontrol streptomyces, which comprises the following steps: the fermentation culture of the biocontrol streptomyces is then applied to plants to be biologically controlled, or the fermentation broth separated from the fermentation culture of the biocontrol streptomyces is applied to the plants to be biologically controlled.
The method has the advantages of good antibacterial effect, stable field control effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like.
The invention provides a method for biological control by using the biological control product, which comprises the following steps: the biocontrol product is fermented and cultured and then acts on plants needing biological control, or the fermentation liquor separated from the biocontrol product is fermented and cultured and then acts on the plants needing biological control.
The method has the advantages of good antibacterial effect, stable field control effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like.
Drawings
FIG. 1 shows the inhibitory effect of CX3 viable bacteria on blueberry canker.
FIG. 2 shows the morphology of strain CX3 under an optical microscope.
Fig. 3 is an m.r. test result.
FIG. 4 shows the results of the nitrate reduction test.
Fig. 5 shows V.P test results.
FIG. 6 is H 2 S produces test results.
FIG. 7 shows the results of phylogenetic analysis of strain CX3 and related strains.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
In the present invention, the plant pathogenic bacteria to be tested:
blueberry canker Botryosphaeria dothidea, blueberry cladosporium cucumerinum Neofusicoccum parum, poplar bark rot germ Valsa sordida, poplar canker Botryosphaeria dothidea, spruce rhizoctonia cerealis Rhizoctonia solani, melon fusarium wilt Fusarium oxysporium, rice bakanae germ F.moniliforme, red bean colletotrichum truncatum, corn big spot germ Exserohilummturcum, tobacco brown spot germ Alternaria alternata, pepper brown spot germ Colletotrichum capsici, eggplant brown spot germ Phomopsis vexans
The above-mentioned test plant pathogens are all publicly available and are used only for repeated experimental procedures and results of the present invention, not for commercial use.
The soil sample to be tested is taken from the artificial forest of larch in the protection station of the cross mountain in the protection area of Jilin Ji-Changbai mountain, the artificial forest of horse saddle mountain spruce in the pond of the protection area of Changbai mountain, and the soil under the forest farm in front of Fu Song county is ten in total.
Test medium:
high's synthetic agar medium (Gause' sssynthetic agar): KNO (KNO) 3 1g、K 2 HPO 4 0.5g、MgSO 4 0.5g、NaCl 0.5g、FeSO 4 0.01g of soluble starch 20g, agar 20g, 1000mL of water and pH 7.2-7.4;
potato dextrose agar (Potato dextrose agar): 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water;
sucrose agar (Czapek's agar): naNO 3 3g、K 2 HPO 4 1g、MgSO 4 0.5g、KCl 0.5g、FeSO 4 0.01g, 30g of sucrose, 20g of agar and 1000mL of distilled water;
emmerson agar (Emenoson agar): 10g of glucose, 10g of yeast extract, 4g of beef extract, 4g of peptone, 2.5g of NaCl, 20g of agar and 1000mL of distilled water;
glucose asparagus agar (Glucose asparagine): glucose 10g, asparagines 0.5g, K 2 HPO 4 0.5g, 20g of agar and 1000mL of distilled water;
watt gravy agar (Broth agar): 5g of peptone, 10g of glucose, 10g of beef extract, 5g of NaCl and 20g of agar; distilled water 1000mL
Kjeldahl agar No.1agar (Carrot No.1 agar): k (K) 2 HPO 4 1g、MgCO 3 0.3g、NaCl 0.2g、KNO 3 1g、FeSO 4 0.01g、CaCO 3 0.5g, glucose 20g,20g of agar and 1000mL of distilled water;
starch ammonium agar (Starch ammonium salt medium): (NH) 4 ) 2 SO 4 2g、CaCO 3 3g、K 2 HPO 4 1g, soluble starch 10g, mgSO 4 1g, 1g of NaCl, 20g of agar and 1000mL of distilled water;
malt Yeast extract agar (Yeast extract-medium extract agar): yeast extract 10g, malt extract 10g, glucose 4g, distilled water 1000mL, agar 20g and pH 7.0;
bennett agar medium (Bennett's agar): 10g of glucose, 1g of beef extract, 1g of yeast extract, 2g of casein hydrolysate and 15g of agar; distilled water 1000mL
Oat flour agar (oat agar): oat flour 20g, trace salt solution 1mL, distilled water 1000mL, agar 20g, pH 7.2;
the formula of the trace salt solution comprises: feSO 4 0.1g、MnCl 2 0.1g、ZnSO 4 0.1g, 100mL of distilled water;
starch agar (Soluble starch agar): soluble starch 10g, naNO 3 1g、MgCO 3 1g、K 2 HPO 4 0.3g, 0.5g of NaCl, 1000mL of distilled water and 20g of agar;
glucose yeast extract agar (Glucose yeast extract agar): glucose 10g, yeast extract 10g, distilled water 1000mL, agar 20g and pH 7.2;
glycerol asparagine medium (Glycerol-asparagine agar): glycerol 10g, asparagine 1g, K 2 HPO 4 1g, 1mL of trace salt solution, 1000mL of distilled water and 20g of agar;
the formula of the trace salt solution comprises: feSO 4 0.1g、MnCl 2 0.1g、ZnSO 4 0.1g, 100mL of distilled water;
soybean meal agar medium (Bean agar medium): 40g of soybean powder, 20g of sucrose, 1.5g of superphosphate and MgSO 4 0.75g, 1000mL of distilled water and 20g of agar;
glycerol arginine medium (Glycerine arginine medium): glycerol 6g, arginine 1g, K 2 HPO 4 0.5g、MgSO 4 0.5g, 1000mL of distilled water and agar20g of fat;
soybean meal peptone medium (Soybean powder peptone medium): 20g of soybean soaked powder, 5g of soluble starch, 10g of sucrose, 2g of peptone, 2g of yeast soaked powder, 2g of NaCl and K 2 HPO 4 0.5g、CaCO 3 0.1g, 1000mL of distilled water, 20g of agar and pH7.2.
The strain is screened and identified by using a Gaoshan first synthetic agar culture medium.
Fermentation medium, soluble starch 70g, peanut cake powder 33g, (NH) 4 ) 2 SO 4 4g、NaCl 4g、CaCO 3 4g, 1000mL of distilled water, pH7.0.
Reagents used in the examples: ezup column type bacterial genome DNA extraction kit, division of biological engineering (Shanghai); taq PCR Master Mix BBI Life sciences and DNA Marker DL2000, all of which are commercially available.
Instrument: BX 53-type olympus optical microscope, olympus corporation.
Example 1
1.1.1 Strain screening
Selecting Gao's first synthetic culture medium, separating and screening bacterial strain by adopting a multi-concentration gradient dilution coating method, observing and selecting bacterial strains with different colony morphologies every day, timely transferring the bacterial strains to a Gao's first inclined plane for culture, purifying again for 3-5 times by adopting a dilution separation method, numbering and storing in a refrigerator at 4 ℃ for standby.
1.1.2 determination of the bacteriostatic Activity of actinomycetes
Transferring all the separated strains into a Gao's first culture medium for culture, taking blueberry canker bacteria as target bacteria after each strain generates enough spores, measuring the antibacterial activity of actinomycetes separated by 1.1.1 by a plate counter culture method, preparing 7mm blueberry canker bacteria cakes, inoculating the 7mm blueberry canker bacteria cakes in the center of a PDA plate (with the diameter of 90 mm), inoculating a loop at the position 1.5cm above and below the bacteria cakes, picking actinomycetes for 72h for culture, drawing lines in parallel, taking a plate without actinomycetes as a control group, culturing at constant temperature of 28 ℃ until the control group plate is full, and measuring the width of an antibacterial zone between actinomycetes and pathogenic fungi, wherein each treatment is repeated for 3 times. According to the size of the antibacterial zone, the strain with the strongest antagonistic activity and the strongest growth vigor is used as the selected strain to carry out the determination of the antibacterial spectrum.
1.1.3 preparation of fermentation broth and determination of bacteriostasis Spectrum thereof
Fermentation medium: 70g of soluble starch, 33g of peanut cake powder and (NH) 4 ) 2 SO 4 4g、NaCl 4g、CaCO 3 4g, 1000mL of distilled water, pH7.0. 200mL of fermentation culture solution is prepared and filled into a 500mL triangular flask, a strain CX3 with strong antibacterial activity is selected according to the result of 1.1.2, and is transferred into the fermentation culture solution, the fermentation culture solution is subjected to constant-temperature shaking culture at 28 ℃ and 150r/min for 5-7 days, the obtained fermentation solution is centrifuged at 8000r/min for 15min, and the supernatant is taken and stored in a refrigerator at-20 ℃ for standby.
Preparing 7mm pathogenic bacteria cakes by adopting a cup and disc method, symmetrically pouring the bacterial diseases around a PDA culture medium, placing an oxford cup in the center of a flat plate, inoculating fermentation liquor into the oxford cup, taking non-inoculated fermentation liquor as a reference, culturing at a constant temperature of 28 ℃ until the bacteria of the reference group grow up on a culture dish, and measuring the diameter of a bacteria inhibition zone of the treatment group by using a cross method.
1.1.4 Morphological observations of CX3 Strain
The strain is inoculated on a Gaoshi No.1agar plate, a sterilizing cover glass is obliquely inserted into a culture medium at 45 degrees, after the culture is carried out for 5 to 7 days at 28 ℃, the cover glass is taken out, and the shape of the cover glass is directly observed under an optical microscope.
According to the study method of the International Streptomyces program and the actinomycetes Classification group of microorganisms of the national academy of sciences (actinomycetes Classification group of microorganisms of the national academy of sciences of China. Streptomyces appraisal handbook, beijing: scientific Press, 1975.), culture media for actinomycetes identification were used to observe the growth conditions and the colors of aerial hyphae, endohyphae and soluble pigments.
1.1.5 Identification of physiological and Biochemical Properties of CX3 Strain
The PH tolerance experiment is carried out by selecting a Bennett liquid culture medium, and setting five tolerance gradients of pH 2,5,7,9 and 12. Fresh spores of the strain CX3 were inoculated respectively, cultured at constant temperature of 28℃and the growth of each treated strain was recorded for 7 and 14 days, and 3 groups of experiments were repeated.
The temperature tolerance experiment adopts five tolerance gradients of culture medium and pH tolerance experiment, wherein the temperature is set to be 4 ℃,15 ℃,28 ℃,37 ℃ and 50 ℃. Fresh spores of strain CX3 were inoculated, respectively, and growth of each treated strain was recorded on days 7 and 14, and incubation at 4℃was recorded at 14d and 28d, respectively. Each set of experiments was repeated in 3 sets.
The culture medium used for the NaCl tolerance test is the same as the pH tolerance test, and five tolerance gradients of 0%,5%,15%,25% and 30% (mass volume percentage, the same as the following) are set. Fresh spores of the strain CX3 were inoculated respectively, cultured at constant temperature of 28℃and the growth of each treated strain was recorded for 7 and 14 days, and 3 groups of experiments were repeated.
1.1.6 Determination of CX3 Strain carbon source utilization
Fresh spores of the strain CX3 are inoculated into a liquid culture medium, equal amounts of different carbon sources are respectively added, whether the strain grows or not is observed, and the growth state of the strain is recorded. The basal medium adopts a Prgotwo culture medium: (NH) 4 ) 2 SO 4 2.64g、K 2 HPO 4 2.38g、MgSO4·7H 2 O 1.0g、CuSO 4 ·5H 2 O 0.0064g、FeSO 4 ·7H 2 O 0.0011g、MnCl 2 ·4H 2 O 0.0079g、ZnS0 4 ·7H 2 0.0015g of O and 1000mL of distilled water. Selection of a carbon source: d-xylose, D-fructose, mannitol, L-rhamnose, a-lactose, glucose, sucrose, D-maltose, raffinose, guanine, glycine, L-tyrosine, L-arabinose.
1.1.7 Identification of enzymatic Properties of CX3 Strain
Catalase assay: the CX3 hypha in the logarithmic growth phase on the solid culture medium is picked up and is dripped on a clean glass slide, 3-10% hydrogen peroxide is dripped, the observation result shows that a large amount of bubbles are generated within 5min, and the result is positive, and the result shows that no bubbles are generated is negative.
Urease experiment: sterilizing 30% urea with diethyl ether, and adding sterile urea to final concentration of 2% when the culture medium is cooled to 55deg.C. The CX3 strain was inoculated, the color of the medium was observed after 4 days of cultivation, the medium became pink, and the medium was positive, and the white was negative. Culture medium: peptone 1g, sodium chloride 5g, glucose 1g, KH 2 PO 4 2g, phenol red 0.012g, agar 15g,distilled water 1000mL.
Lipase experiment: the substrate was Tween-20 (sterilized at 121 ℃ C.). The medium was cooled to 40-50 ℃, sterile tween was added to a final concentration of 1%, plates were prepared, and CX3 strain was inoculated. Culturing for 7-14 days, and observing every day. Those with a faint halo around the growing strain were positive, and those without were negative. Culture medium: peptone 1g, naCl 5g and CaCl 2 ·7H 2 O0.1 g, agar 9g, distilled water 1000mL, pH7.4.
Gelatin liquefaction experiments: bacteria were streaked on the medium at 28℃and the degree of liquefaction was observed at 5, 10, 20, 30 days. Before observation, the plate is placed in a refrigerator at 4 ℃ for about 30 min. The culture medium without strain is used as a control, and each group is repeated three times. Culture medium: peptone 5g, glucose 20g, gelatin 200g, distilled water 1000mL.
Starch hydrolysis experiments: the strain was inoculated onto starch agar medium. After 10 days, amylase activity was measured, iodine was dropped on a plate, and the color around the lawn, the presence or absence of a transparent ring, and the size were observed. Selection of the culture medium: 10g of soluble starch, K 2 HPO 4 0.3g、MgCO 3 1.0g、NaCl 0.5g、KNO 3 1.0g, 15g of agar, 1000mL of distilled water, pH7.2-7.4.
Milk coagulation and peptonization experiments: CX3 strain is inoculated into skimmed milk, cultured at constant temperature of 28 ℃, and observed for 3, 6, 10, 20 and 30 days to see whether coagulation phenomenon and peptonization phenomenon exist. With the unvaccinated strain as a control, each group was replicated three times. And (3) solidification: clot formation; peptone: casein is hydrolyzed to a transparent or translucent liquid state. Culture medium: 1000mL of defatted fresh milk, caCO 3 0.02g, and the mixture was sterilized 3 times.
Cellulose utilization experiment: the strain was inoculated onto a filter paper in a test tube, half of the filter paper was immersed in a solution synthesized without a carbon source, and cultured for 30 days to see whether the strain can grow and whether the filter paper can be decomposed. With the unvaccinated strain as a control, each group was replicated three times. Selection of the culture medium: mgSO (MgSO) 4 0.5g、NaCl 0.5g、K 2 HPO 4 0.5g、KNO 3 1g, 20g of sucrose and 1000mL of distilled water.
1.1.8 CX3 Strain metabolite experiment
M.r. test: inoculating strain into liquid culture medium, culturing at 28deg.C for 2-6 days, adding one drop of methyl red reagent into the culture solution, and reacting in positive red and negative yellow. Culture medium: methyl red 0.1g, glucose 5g, K 2 HPO 4 5g, phenol red 0.012g and distilled water 1000mL.
V.p. test: and (3) testing culture conditions by using M.R. and mixing the culture solution with 40% sodium hydroxide in equal quantity, adding a little creatine, and taking red color as positive reaction within 10 min. Culture medium was used in M.R. experiments.
Hydrogen sulfide formation experiments: the strain was inoculated on a Tresner medium, and after culturing for 7 days, it was observed whether melanin was produced. Melanin production indicates that the strain produced hydrogen sulfide, with unvaccinated medium as a control. Culture medium: 10g of peptone, 0.5g of ferric citrate, 15g of agar and 1000mL of distilled water, pH7.2.
Nitrate reduction experiments: the strain was inoculated into the culture solution and cultured for 7 days and 14 days. 1,2 drops of each solution were added to 1mL of the bacterial solution. The red color was positive, and the uninoculated medium was used as a control. Culture medium: mgSO (MgSO) 4 0.5g、NaCl 0.5g、K 2 HPO 4 0.5g、KNO 3 1.0g, 20g of sucrose and 2000mL of distilled water. Solution 1: aminobenzenesulfonic acid 0.5g was dissolved in acetic acid (80% acetic acid, diluted with 2-fold water). Solution II: 0.1g of diphenylamine and 20mL of distilled water, wherein the diphenylamine is dissolved in a small amount of ethanol, then water is added, and 150mL of diluted acetic acid is added after boiling.
1.1.9 CX3 strain identification
The strain is inoculated on a Gaoshi No.1agar plate, a sterilizing cover glass is obliquely inserted into a culture medium at 45 degrees, and after the culture is carried out for 5-7d at 28 ℃, the cover glass is taken out and directly observed under an optical microscope.
DNA extraction was performed according to the instructions of the Ezup column bacterial genomic DNA extraction kit. The PCR reaction system was 50. Mu.l: PCR Mixture 25. Mu. L, template 2. Mu.L, forward primer (SEQ NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') 2. Mu.L, reverse primer (SEQ NO.2: 5'-GGTTACCTTGTTACGACTT-3') 2. Mu. L, ddH 2 O 19μL。
PCR reaction conditions: denaturation at 94℃for 4min; denaturation at 94℃for 30s, renaturation at 58℃for 30s, extension at 72℃for 1min,35 cycles; extending at 72 ℃ for 10min, preserving at 4 ℃, taking a PCR product of sterile water as a negative control, loading 2 mu L of the sample, and performing 2% agarose gel electrophoresis. All operations were performed according to the operational procedure of the kit, and the recovered product was sent to the Jilin Cookimei biotechnology Co., ltd for sequencing analysis.
1.2 data analysis
SPSS 17.0 is adopted for data statistics, duncan's new complex polar error method is adopted for difference significance analysis, and phylogenetic tree is established by adopting MEGA 6.0.
Example 2 results and analysis
2.1 Strain Primary screening
After purification, 84 purified strains with consistent morphology, size and color are obtained, and the plate-facing test results show that 12 strains with the antibacterial band width being larger than 10mm (shown in table 1) and 8 strains with the antibacterial band width being larger than 15mm are obtained, wherein the antibacterial effect of the strain CX3 is good (figure 1), the antibacterial band width reaches 16.16mm, and therefore the strain CX3 (obtained by screening samples collected by a man forest of Mashan mountain spruce in a protection station of a pond of a Changbai mountain) is selected as the strain of the next test.
TABLE 1 antibacterial Activity of Streptomyces living organisms against blueberry canker bacteria
Data in the table are mean ± standard deviation.
2.2 determination of the antibacterial spectrum of the active CX3 bacteria and the fermentation liquor thereof
The antagonistic bacteria CX3 has a certain inhibition effect on all the tested strains. CX3 has the strongest inhibition effect on blueberry canker, and obvious antibacterial zone is visible, the width of the antibacterial zone reaches 16.16mm, and the difference effect between the antibacterial zone and other tested pathogenic bacteria is obvious; the composition also has strong inhibition effects on red bean anthracnose, corn macula and pepper anthracnose, the inhibition bandwidths are 13.42, 13.88 and 12.84mm respectively, the inhibition effects on tobacco red star, melon fusarium wilt and the like are relatively weak, but the inhibition bandwidth is also more than 9.30mm (table 2).
The antibacterial activity of antagonistic strain CX3 fermentation liquor is measured by adopting a cup and disc method, the fermentation liquor still maintains good antibacterial activity, the antibacterial effect of the fermentation liquor of the strain on blueberry canker is strongest and reaches 31.73mm, and the difference effect with other tested strains is obvious; the antibacterial agent has extremely strong antagonism to brown spot bacteria and pepper anthracnose bacteria, the average antibacterial diameter reaches 30.37mm, the antibacterial agent also has better inhibition to corn big spot bacteria, red bean anthracnose bacteria and poplar skin rot bacteria, the antibacterial diameters are 28.82, 25.74 and 26.94mm respectively, the antagonistic fermentation liquor has obvious inhibition to all tested pathogenic bacteria (Table 2), and the minimum antibacterial diameter also reaches 22.60mm.
TABLE 2 antibacterial spectra of antagonistic CX3 living organisms and fermentation broths
Data in the table are mean ± standard deviation. The letters after the same column of data indicate significant differences at P <0.01 levels as tested by the Duncan's new complex polar error method. Data in the table are mean + -SD.the letters in the same column indicate significant differences at the P <0.01level by Duncan's new complex range method.
2.3 morphological observations of Strain CX3
The strain CX3 grows luxuriantly on the culture medium of Gaoshan No. one, aerial hyphae are luxuriant, and mycelia grow radially in all aspects; culturing at 28deg.C for 1-3 days, wherein the colony is smooth and free of sporulation, and spores are generated after 7 days, and the spores are chain-shaped when observed under a microscope as shown in figure 2; the spore filaments are not autolyzed, and the phenomenon of water absorption is avoided; the spore pile color is nearly pink when cultured for 15 days, the spores are mostly straight, and sometimes the top is hooked, and the Streptomyces identification manual (the Streptomyces identification manual by the national institute of sciences of China, the actinomycetes classification group, beijing: scientific Press, 1975) is referred to according to the characteristics, and the preliminary analysis strain CX3 is a pink spore group of Streptomyces.
2.3.1 Strain CX3 culture trait
After inoculation of the strain CX3 on 10 test media, colonies began to appear after 36-48h of incubation at 28℃and spores began to develop after 7 days. The form of the lawn, the color of the spore stack, the color of the matrix mycelium, the pigment produced, and the growth were all different on different media, and as shown in Table 3, the strain CX3 did not produce soluble pigment on most media. The strain CX3 was found to be a pink spore group of Streptomyces by reference to the Streptomyces search handbook (Proc. Natl. Acad. Sci. Biol.Acad. Sci. Actinomycetes Classification, streptomyces identification handbook, beijing: scientific Press, 1975.) and the aerial hyphae on the culture medium of Gao's first, and the matrix hyphae exhibited litchi flesh white color.
TABLE 3 culture characteristics of Strain CX3
"-", does not grow; "+" growth; "++" grows well; "+". Plus ""; vigorous growth.
2.4 physiological and Biochemical characteristics of antagonistic bacteria CX3
2.4.1 determination of tolerance of antagonistic bacteria CX3
The strain CX3 exhibited excellent stress tolerance, and the results are shown in Table 4. The strain is loved to a high-salt environment, grows on a culture medium containing 5% NaCl, and grows vigorously under the high-salt condition; the temperature tolerance is good, the growth can be carried out at 15-28 ℃, and the 28 ℃ is the optimal growth temperature; the resistance to alkali is good, the growth can be carried out in the range of pH5-12, and the pH7 is the optimum pH for the growth.
TABLE 4 determination of tolerance of Strain CX3
"-", does not grow; "+" growth; "++" grows well; "+". Plus ""; vigorous growth.
2.4.2 carbon Source utilization of antagonistic bacterium CX3
The results of the use of various carbon sources by the strain CX3 are shown in Table 5, and D-xylose, D-fructose, mannitol, L-rhamnose, glucose, sucrose, maltose, guanine, glycine, L-tyrosine and L-arabinose can be used by the strain; a-lactose and raffinose cannot be utilized. The strain can better utilize monosaccharide and disaccharide, and can not utilize polysaccharide, and the most suitable carbon source can be xylose, fructose and maltose.
2.4.3 enzymatic Properties of antagonistic bacteria CX3
The results of the enzymatic properties of strain CX3 are shown in Table 5, and the catalase test of this strain produced a large amount of bubbles; urease experiment is negative; positive lipase test, semitransparent halos are generated around the strain; gelatin can be liquefied; the use of cellulolytic enzymes is not possible; the milk can be coagulated, but peptone phenomenon does not occur; starch hydrolysis experiments were positive and amylase could be produced.
2.4.4 metabolite assay of antagonistic bacteria CX3
The results of the metabolite measurement of strain CX3 are shown in Table 5; the test is negative; h 2 S is positive in experiment; v.p. experiment CX3 presents a positive red color; the control in the nitrate reduction reaction was clear and colorless, and strain CX3 exhibited a positive pink color. The strain CX3 was further judged to be a French subgroup of the Streptomyces, pink spore group by referring to the Streptomyces classification and identification manual.
TABLE 5 physiological biochemical experimental determination of strain CX3
"-", does not grow; "+" growth; "++" grows well; "+". Plus ""; vigorous growth.
The m.r. test results are shown in fig. 3. In fig. 3, the left test tube is a negative control sample (the negative control sample is a sample without bacterial solution and with equal amount of sterile water added), the right test tube is an experimental sample (CX 3 strain culture solution), and both test tubes are negative yellow, which shows that the strain can decompose glucose to generate pyruvic acid, the pyruvic acid is further decomposed into lactic acid, formic acid and acetic acid by metabolism, the final pH of the mixed solution is reduced to below 4.5, and the m.r. experiment is finally negative.
The results of the nitrate reduction experiment are shown in fig. 4. In fig. 4, the left test tube is an experimental sample (CX 3 strain culture solution), the right test tube is a negative control sample (the negative control sample is a sample without bacterial solution and with equal amount of sterile water added), and as can be seen in fig. 4, the experimental sample test tube is pink, the negative control test tube is colorless, which indicates that nitrite exists in the mixed solution and is positive for reduction of nitrate.
The V.P test results are shown in FIG. 5. In fig. 5, the left test tube is a negative control sample (the negative control sample is a sample without bacterial solution and with equal amount of sterile water added), the right test tube is an experimental sample (CX 3 strain culture solution), and the experimental sample test tube is red, and the negative control test tube is yellow, which indicates that pyruvic acid generated by decomposing glucose by the strain is further decarboxylated to form acetomethyl methanol, and the substance is oxidized to diacetyl under alkaline condition, and then is combined with guanidine groups contained in arginine and the like in the culture medium peptone to form a red compound, namely, the v.p. test is positive.
H 2 The experimental results of S production are shown in fig. 6. In FIG. 7, the left plate is a negative control sample (negative control sample is a sample without bacteria solution and with equal amount of sterile water added), the right plate is an experimental sample (CX 3 strain medium), and it can be seen from FIG. 6 that the negative control plate is yellow, the experimental plate is tan, and H is indicated 2 S generation, H 2 S combines with ferric citrate to produce FeS, and the culture medium has positive tan.
2.5 group biological identification of antagonistic bacteria CX3
2.5.1 16S rDNA identification of Strain CX3
The full length of the amplified 16S rDNA gene fragment of strain CX3 was 1385bp (Gene accession number: MN 636760) by sequencing. The sequences with higher homology of 9 strains and the strain CX3 are selected, a phylogenetic tree is established, the phylogenetic analysis results of the strain CX3 and related strains are shown in figure 7, and the strains with closer relativity to the strain CX3 are seen to belong to Streptomyces from the figure, so that the strain CX3 is primarily determined to be Streptomyces and the French subgroup of the Pink spore subgroup.
84 actinomycetes are separated from the woodland soil of each woodland of Jilin province, blueberry canker is used as target pathogenic bacteria for screening, 1 actinomycetes of the woodland soil of Maanshan spruce in pond west region of Jilin province are finally selected, and the strain CX3 is determined to be a French subgroup of Streptomyces and Pink spore group by morphological observation, physiological and biochemical identification, 16SrDNA analysis and other molecular biological analysis and identification methods. The Streptomyces identification manual was consulted to find that no corresponding species exists, but in the analysis, two strains were found to have physicochemical properties similar to those of the antagonistic strain CX3, namely, a multi-carbon chain-phaga (S.polycarbophilus) and a Streptomyces mediterraneaus (S.medians) with only slight differences in culture traits; the physicochemical properties are similar, and starch can be hydrolyzed, but the strain CX3 can not decompose and utilize cellulose. Wherein Streptomyces mediterraneans belongs to a variant of the rose subgroup which is also the Pink spore group, and which can produce rifamycin (Rifomycin) is a broad-spectrum antibiotic. The multi-carbon chain-phagocytosis mould can produce antibiotic ten thousand-135, and inhibit positive bacteria including bifidobacterium.
The CX3 strain has fungicidal activity, and the living bacteria and the fermentation liquor of the strain have strong inhibition effect on the blueberry canker, and the analysis is probably due to the fact that the strain generates antifungal substances in the physiological metabolic process. The strain CX3 can well utilize mono-and disaccharides but not polysaccharides, and analysis has important relation to its physiological metabolic pathways. In summary, it is tentatively named streptomyces CX3.
The invention proves that the strain has the advantages of good antibacterial effect, stable field control effect, wide antibacterial spectrum, low toxicity, no residue, environmental friendliness, wide development prospect and the like through a series of experiments.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> Jilin province forestry science institute
<120> a fermentation method of Streptomyces biocontrol
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<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial Sequence)
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Claims (10)
1. Streptomyces biocontrolStreptomyces spA method of fermentation, comprising the steps of: the biocontrol streptomyces is fermented, and the preservation number of the biocontrol streptomyces strain is CGMCC No.18658.
2. The fermentation method of Streptomyces biocontrol strain according to claim 1, comprising the steps of inoculating Streptomyces biocontrol strain with the preservation number of CGMCC No.18658 to a fermentation medium for fermentation culture.
3. The fermentation method of Streptomyces biocontrol according to claim 1, wherein fermentation is performed in a fermenter or in a shake flask.
4. A fermentation process of Streptomyces biocontrol according to any one of claims 1 to 3 wherein the fermentation temperature is 15 to 28 ℃.
5. The method for fermenting Streptomyces biocontrol according to claim 4, wherein the fermentation temperature is 28 ℃.
6. A fermentation process of Streptomyces biocontrol according to any one of claims 1 to 3, wherein the fermentation time is 5 to 7 days.
7. A fermentation method of Streptomyces biocontrol according to any one of claims 1 to 3, further comprising the step of centrifuging the fermentation broth obtained by the fermentation.
8. The fermentation method of biocontrol streptomyces according to claim 7, wherein the centrifugal speed is 8000r/min and the centrifugal time is 15min.
9. A fermentation process of a Streptomyces biocontrol strain according to any one of claims 1 to 3, wherein the fermentation medium used in the fermentation culture of Streptomyces biocontrol strain comprises the following ingredients: every 1000mL of water, 70g of soluble starch, 33g of peanut cake powder and (NH) 4 ) 2 SO 4 4 g、NaCl 4g、CaCO 3 4 g，pH7.0。
10. A biocontrol product comprising the biocontrol streptomyces and/or biocontrol streptomyces fermentation broth prepared by the fermentation process of any one of claims 1-9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113061551A (en) * | 2019-11-27 | 2021-07-02 | 吉林省林业科学研究院 | Application of biocontrol streptomyces in preventing and treating plant disease pathogenic bacteria |
| CN112931534A (en) * | 2020-03-28 | 2021-06-11 | 吉林省林业科学研究院 | Application of streptomycete in biological control |
| CN113025537A (en) * | 2020-03-28 | 2021-06-25 | 吉林省林业科学研究院 | Fermentation method of streptomycete |
Non-Patent Citations (3)
| Title |
|---|
| Antagonistic activity and mechanism of an isolated Streptomyces corchorusii stain AUH‑1 against phytopathogenic fungi;Yong Yang等;《World Journal of Microbiology and Biotechnology》;20190906;第35卷(第9期);第1-9页 * |
| Streptomyces emeiensis sp. nov., a novel streptomycete from soil in China;Wei Sun等;《International Journal of Systematic and Evolutionary Microbiology》;20070731(第57期);第1635-1639页 * |
| 蓝莓溃疡病生防链霉菌CX3的抑菌效果及其鉴定;丁芮涵等;《农药》;20200410;第59卷(第4期);第308-312页 * |
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