CN112877261A - 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 preservation number of the streptomyces biocontrol strain is CGMCC No. 18658. When in culture, the biocontrol streptomyces is inoculated to a fermentation medium for fermentation culture. The biocontrol streptomyces and/or fermentation liquor 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: 11/27/2019, with the patent names: a biocontrol streptomyces.
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 bilberry, belongs to the plant of Vaccinium (Vaccinium Spp.) of Ericaceae, and has sour, sweet and delicious fruit, and can be processed into fruit juice, fruit wine and beverage besides fresh food. The blueberry fruit has ultrahigh nutritional value, is rich in a large amount of anthocyanin, various antioxidants and flavonoids besides fructose and vitamins, is more and more favored by people due to unique and rare nutritional and health-care values, and has continuously increased global demand on blueberries.
In recent years, the planting area of blueberries is increased year by year, the blueberry diseases are serious day by day, the economic loss is heavy, and blueberry canker is an important disease of blueberries caused by Botryosphaeria dothidea. The disease is widely distributed in the world, mainly harms 1-year-old branches and mostly occurs from wounds and pruning mouths; red brown disease spots are generated at the early stage of disease occurrence, and as the disease spots expand, the branch tips begin to dry up, so that the whole young branch gradually dies; in the later stage of the disease, small densely buried black spots (conidiophores) can be seen on the disease spots; the damaged tissue is dissected, the vascular bundle tissue turns light brown, and white mycelium can be seen. If the blueberry canker is easy to occur at low temperature or in large temperature difference between day and night, the blueberry canker greatly influences the yield of blueberries and even can cause plant death.
In recent years, reports about blueberry diseases are frequently found in China, for example, Jiangxing reports the disease conditions of blueberry canker; yanglin and the like report the damage trend of common plant diseases and insect pests in blueberry cultivation and the cultivation technology of blueberry branches, and the occurrence of blueberry diseases is greatly restrained. At present, the following measures are taken for preventing and treating the disease: chemical prevention and control, breeding disease-resistant variety, cultivating disease-free strong seedling, optimizing cultivation field and cultivation mode, etc. Among a plurality of disease prevention means, chemical prevention and breeding of disease-resistant varieties are most easily accepted by the public, but the two prevention and treatment methods have obvious defects, on one hand, breeding of the disease-resistant varieties takes longer time, and problems of resistance degeneration and the like exist, and the expected disease prevention effect cannot be achieved; on the other hand, the problems of environmental pollution, ecological balance damage, high toxicity, easy residue and the like are easily caused by the large amount of chemical pesticide input. Therefore, research on sustainable control technology mainly based on ecological system regulation and control utilizes antagonistic microorganisms with inhibiting effect on pathogenic bacteria to prevent and treat the disease, and bacteria are treated by bacteria, so that the aims of environmental protection and high-efficiency comprehensive prevention and treatment are achieved.
Actinomycetes are the earliest discovered type of microorganism with biological control action. Streptomyces occupies an important position 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 prevention and control.
Disclosure of Invention
In view of the problems in the prior art, the invention provides the biocontrol streptomyces and the fermentation method thereof, and the biocontrol streptomyces 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 technical scheme for solving the technical problems is as follows:
the invention provides a biocontrol streptomyces, and the preservation number of a strain is CGMCC No. 18658. The strain is preserved in China general microbiological culture Collection center in 2019, 10 months and 10 days.
The inventor collects soil in different areas of Jilin province to separate and obtain a plurality of actinomycetes, and finally and unexpectedly obtains 1 antagonistic strain capable of effectively inhibiting blueberry canker pathogen by adopting multiple screening, wherein the antagonistic strain is marked as Streptomyces sp.strain CX3(Streptomyces sp. strain CX3), which is CX3 for short. The streptomyces biocontrol 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 invention has the beneficial effects that: the antagonistic strain CX3 obtained by the method has wide development prospect, wide antibacterial spectrum and great biological control potential, the active antibacterial component in the fermentation liquor can be used as a biological pesticide after separation and purification and other processes, and the like, and can be developed and applied to the control of various plant diseases, and meanwhile, compared with a chemical pesticide, the fermentation liquor of the strain CX3 has multiple advantages of low toxicity, no residue, environmental friendliness and the like, and is more in line with the environmental protection concept of sustainable control.
Specifically, the biocontrol streptomyces grows vigorously on the Gao's I culture medium, aerial mycelia are abundant, and mycelia grow radially in all aspects; when the culture is carried out for 1 to 3 days at the temperature of 28 ℃, the bacterial colony is smooth and has no spore generation, after 7 days, the spore generation begins, and the spore is observed under a microscope to be mostly in a chain shape; the spore silk is not autolyzed and does not absorb water; at 15 days of culture, the color of the sporophyte is close to pink, and the spores are mostly straight and sometimes have hook-shaped tops.
The biocontrol streptomyces can utilize 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; the urease test is negative; lipase test positive, translucent halo is generated around the strain; the gelatin can be liquefied; the inability to utilize decomposed cellulose; the milk can be coagulated, but the peptonization phenomenon does not occur; the starch hydrolysis test is positive, and amylase can be produced.
The M.R. test of the biocontrol streptomyces is negative; h2The test of S is positive; v.p. experiment CX3 showed a positive red color; positive pink color was observed in the nitrate reduction reaction.
The bacteriostatic spectrum of the biocontrol streptomyces comprises the following strains: blueberry canker pathogen, poplar canker disease, corn northern leaf blight, alternaria alternata, spruce rhizoctonia solani, melon fusarium wilt, red bean colletotrichum, pythium aphanidermatum, rice bakanae pathogen, pepper colletotrichum, eggplant phomopsis fulvidraco and poplar canker disease.
The fermentation of the biocontrol streptomyces is used during fermentation cultureThe formula of the culture medium comprises the following components: every 1000mL of water, 70g of soluble starch, 33g of peanut cake powder and (NH)4)2SO4 4 g、NaCl 4g、CaCO3 4 g，pH7.0。
When the biocontrol streptomyces is cultured in a fermentation way, the fermentation culture temperature is 15-28 ℃. Preferably, the fermentation culture temperature is 28 ℃. The temperature is more beneficial to the fermentation culture of the biocontrol streptomyces.
The invention provides a biocontrol product, which comprises the biocontrol streptomyces and/or the biocontrol streptomyces fermentation liquor.
The beneficial effect who adopts above-mentioned scheme is: the biocontrol product obtained by the invention has wide development prospect, wide antibacterial spectrum and great biological control potential, can be developed and applied to the control aspect of various plant diseases as a biological pesticide, has multiple advantages of low toxicity, no residue, environmental friendliness and the like, and is more in line with the environmental protection concept of sustainable control.
The invention provides a fermentation method of the biocontrol streptomyces, which comprises the following steps: the preservation number of the streptomyces biocontrol strain is CGMCC No. 18658.
The invention has the beneficial effects that: the biocontrol streptomyces and/or fermentation liquor 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 can comprise the following steps: inoculating the biocontrol streptomyces with the preservation number of CGMCC No.18658 to a fermentation medium, and performing fermentation culture.
In the fermentation process, the culture can be carried out by adopting a fermentation tank fermentation mode or a shaking flask fermentation mode.
The fermentation temperature can be 15-28 deg.C, preferably, the fermentation culture temperature is 28 deg.C. The fermentation time may be 5-7 days.
The method can also comprise a step of centrifuging fermentation liquor obtained by fermentation, and strains of the streptomyces biocontrol strain and the fermentation liquor of the strains without the streptomyces biocontrol strain are separated and obtained by the method. The conditions for centrifugation may be suitably selected depending on the fermentation conditions, and in the present embodiment, the rotation speed of centrifugation is 8000r/min and the centrifugation time is 15min, for example.
The fermentation culture medium in the fermentation process can be prepared according to the following proportion that every 1000mL of water contains 70g of soluble starch, 33g of peanut cake powder and (NH)4)2SO4 4g、NaCl 4g、CaCO3 4g，pH7.0。
The beneficial effect who adopts above-mentioned scheme is: the proper temperature, culture time and culture medium are favorable for the fermentation culture of the biocontrol streptomyces.
The invention also provides a biocontrol product, which comprises the biocontrol streptomyces prepared by the fermentation method and/or the biocontrol streptomyces fermentation liquor.
The invention provides application of the biocontrol streptomyces in pathogenic bacteria prevention and treatment. Preferably, the invention provides application of the streptomyces biocontrol in preventing and treating phytopathogens.
Further, the plant pathogenic bacteria comprise one or more of blueberry canker pathogen, poplar canker disease, corn northern leaf blight, alternaria alternata, spruce rhizoctonia solani, melon wilt pathogen, red bean anthracnose pathogen, pythium aphanidermatum, rice bakanae pathogen, pepper anthracnose pathogen, eggplant phomopsis fulvidraco and poplar canker disease. Preferably, the prevention and treatment effect on blueberry canker germs is more obvious, and the blueberry canker germs can be used for biological prevention and treatment of blueberry canker germs.
The invention provides application of the biocontrol product in pathogenic bacteria control. Preferably, the invention provides the application of the biocontrol product in the control of phytopathogens.
Further, the plant pathogenic bacteria comprise one or more of blueberry canker pathogen, poplar canker disease, corn northern leaf blight, alternaria alternata, spruce rhizoctonia solani, melon wilt pathogen, red bean anthracnose pathogen, pythium aphanidermatum, rice bakanae pathogen, pepper anthracnose pathogen, eggplant phomopsis fulvidraco and poplar canker disease. Preferably, the prevention and treatment effect on blueberry canker germs is more obvious, and the blueberry canker germs can be used for biological prevention and treatment of blueberry canker germs.
The invention provides a method for biological control by using the streptomyces biocontrol, which comprises the following steps: and (3) the streptomyces biocontrol is fermented and cultured and then acts on plants needing biological control, or fermentation liquor separated after the streptomyces biocontrol is fermented and cultured 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.
The invention provides a method for biological control by using the biocontrol product, which comprises the following steps: and (3) the biocontrol product is fermented and cultured and then acts on the plant to be biologically controlled, or fermentation liquor separated from the biocontrol product after the biocontrol product is fermented and cultured acts on the plant 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.
Drawings
FIG. 1 shows the inhibition effect of CX3 live bacteria on blueberry canker pathogen.
FIG. 2 shows the optical microscopic morphology of strain CX 3.
Fig. 3 shows the results of the m.r. test.
FIG. 4 shows the results of the nitrate reduction test.
FIG. 5 shows the V.P test results.
FIG. 6 is H2S yields the test results.
FIG. 7 shows the phylogenetic analysis results of strain CX3 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.
In the present invention, the test phytopathogens:
blueberry ulcer bacteria Botryosphaeria dothidea, blueberry branch blight bacteria Neofusicoccum parum, poplar canker pathogenic bacteria Valsa sordida, poplar ulcer bacteria Botryosphaeria dothidea, Yunnan Rhizoctonia solani, muskmelon Fusarium oxysporum, rice bakanae bacteria F.moniliforme, red bean anthracnose bacteria Colletotrichum truncatum, corn northern leaf blight bacteria Exserohiluttricum, tobacco Alternaria alternata, pepper anthracnose bacteria Colletotrichum, eggplant Phomopsis xanthophyllus xanthans
The above test phytopathogens are all publicly available and are used only to replicate the experimental procedures and results of the invention, and are not used commercially.
The soil samples to be tested are collected from a Pinus sylvestris artificial forest at a horizontal mountain protection station in Jilin province Jichangbai mountain protection area, a Picea kuwanensis artificial forest at a western protection station in Changbai mountain protection area pool, and forest soil under forest lands in Sichuan forest lands before Fusong county, and are more than ten parts.
Test medium:
gauss No. one synthetic agar medium (mouse' ssynthetic 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 and 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;
iloxan agar (emerson 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 agar): glucose 10g, asparagines 0.5g, K2HPO40.5g, 20g of agar and 1000mL of distilled water;
bouillon Wasser agar (Broth agar): 5g of peptone, 10g of glucose, 10g of beef extract, 5g of NaCl and 20g of agar; 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): (NH)4)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): 10g of yeast extract, 10g of malt extract, 4g of glucose, 1000mL of distilled water, 20g of agar and pH 7.0;
bennett agar medium (Bennett's agar): 10g of glucose, 1g of beef extract, 1g of yeast extract, 2g of hydrolyzed casein and 15g of agar; 1000mL of distilled water
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;
formulation of trace salt solution: FeSO4 0.1g、MnCl2 0.1g、ZnSO40.1g and 100mL of distilled water;
starch agar (Soluble starch agar): soluble starch 10g, NaNO3 1g、MgCO3 1g、K2HPO40.3g, NaCl 0.5g, 1000mL of distilled water and 20g of agar;
glucose yeast extract agar (Glucose yeast extract agar): 10g of glucose, 10g of yeast extract, 1000mL of distilled water, 20g of agar and pH 7.2;
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;
formulation of trace salt solution: FeSO4 0.1g、MnCl2 0.1g、ZnSO40.1g and 100mL of distilled water;
soybean meal agar medium (Bean agar medium): 40g of soybean meal, 20g of cane sugar, 1.5g of calcium superphosphate and MgSO40.75g, 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 a Gao's No. I synthetic agar culture medium.
Fermentation medium comprising 70g of soluble starch, 33g of peanut cake powder and (NH)4)2SO4 4g、NaCl 4g、CaCO34g, 1000mL of distilled water, pH 7.0.
Reagents used in examples: ezup column type bacterial genome DNA extraction kit, biological engineering (Shanghai) corporation; taq PCR Master Mix, BBI Life sciences and DNA Marker DL2000, all of which are domestic analytical purifiers.
The instrument comprises the following steps: model BX53 olympus optical microscope, ltd.
Example 1
1.1.1 Strain selection
Selecting a Gauss-I synthetic culture medium, separating and screening strains by adopting a multi-concentration gradient dilution coating method, observing and selecting the strains with different colony morphologies every day, transferring the strains to a Gauss-I inclined plane for culture, re-purifying for 3-5 times by adopting a dilution separation method, numbering and storing in a refrigerator at 4 ℃ for later use.
1.1.2 determination of Actinomycetes bacteriostatic Activity
Transferring all the strains obtained by separation into a Gao's first culture medium for culture, taking blueberry ulcer germs as target bacteria after each strain generates enough spores, measuring the bacteriostatic activity of the actinomycetes obtained by 1.1.1 separation by a plate confrontation culture method, preparing a 7mm blueberry ulcer germ cake, inoculating the cake in the center of a PDA (personal digital assistant) plate (diameter is 90mm), inoculating rings at a position 1.5cm away from the upper part and the lower part of the cake, picking and culturing actinomycetes for 72 hours, drawing lines in parallel, taking the plate without inoculating the actinomycetes as a control group, culturing at a constant temperature of 28 ℃ until the control group plate is full, measuring the width of a bacteriostatic zone between the actinomycetes and pathogenic fungi, and repeating for 3 times every treatment. According to the size of the antibacterial zone, the bacterial strain with the strongest antagonistic activity and the most vigorous growth is taken as the selected bacterial strain to carry out the determination of the antibacterial spectrum.
1.1.3 preparation of fermentation broth and determination of its antibacterial spectrum
Fermentation medium: 70g of soluble starch, 33g of peanut cake powder and (NH)4)2SO4 4g、NaCl 4g、CaCO34g, 1000mL of distilled water, pH 7.0. Preparing 200mL of fermentation culture solution, filling the fermentation culture solution into a 500mL triangular flask, selecting a bacterial strain CX3 with strong bacteriostatic activity according to a result of 1.1.2, transferring the bacterial strain CX3 into the fermentation culture solution, carrying out constant-temperature shaking culture at 28 ℃ and 150r/min for 5-7 days, centrifuging the obtained fermentation solution for 15min at 8000r/min, and taking supernatant fluid and storing the supernatant fluid in a refrigerator at-20 ℃ for later use.
Preparing 7mm pathogenic bacteria cake by cup and dish method, symmetrically inverting the bacterial disease around PDA culture medium, placing oxford cup in the center of the plate, inoculating 200 μ L fermentation liquid in the oxford cup, culturing at constant temperature of 28 deg.C until the control group bacteria grows over the culture dish, and measuring the diameter of the inhibition zone by cross method.
1.1.4 morphological Observation of CX3 Strain
Inoculating the strain to Gauss No.1agar plate, obliquely inserting sterilized cover glass into culture medium at 45 deg.C, culturing at 28 deg.C for 5-7d, taking out the cover glass, and directly observing its form under optical microscope.
According to the international Streptomyces program and the research methods of actinomycetes classification groups of the institute of microbiology of Chinese academy of sciences (the actinomycetes classification groups of the institute of microbiology of Chinese academy of sciences, the manual of Streptomyces appraisal, Beijing: the scientific press, 1975.), the culture medium for identifying actinomycetes is used for culture, and the growth conditions and the colors of aerial hyphae, intrabasal hyphae and soluble pigments are observed.
1.1.5 identification of physiological and biochemical characteristics of CX3 Strain
In the PH tolerance experiment, a bennett liquid culture medium is selected, and five tolerance gradients of pH 2, 5, 7, 9 and 12 are set. Fresh spores of strain CX3 were inoculated, incubated at 28 ℃ and the growth of each treated strain was recorded on days 7 and 14, and 3 replicates per experiment were used.
The temperature tolerance experiment selects a culture medium and a pH tolerance experiment, and sets five tolerance gradients of 4 ℃, 15 ℃, 28 ℃, 37 ℃ and 50 ℃. Fresh spores of strain CX3 were inoculated separately, growth was recorded on days 7 and 14 for each of the treated strains, and incubation at 4 ℃ was recorded at 14d and 28d, respectively. Each set of experiments was repeated in 3 groups.
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% (mass volume percentage, the same below). Fresh spores of strain CX3 were inoculated, incubated at 28 ℃ and the growth of each treated strain was recorded on days 7 and 14, and 3 replicates per experiment were used.
1.1.6 measurement of carbon Source utilization by CX3 Strain
Inoculating fresh spores of strain CX3 into liquid culture medium, adding equal amount of different carbon sources, observing whether the strain grows and recording the growth state of the strain. The basic culture medium adopts a Pogostemon culture medium: (NH)4)2SO4 2.64g、K2HPO42.38g、MgSO4·7H2O 1.0g、CuSO4·5H2O 0.0064g、FeSO4·7H2O 0.0011g、MnCl2·4H2O 0.0079g、ZnS04·7H20.0015g of O and 1000mL of distilled water. Selection of carbon source: d-xylose, D-fructose, mannitol, L-rhamnose, a-lactose, glucose, sucrose, D-maltose, raffinose, guanine, glycine, L-tyrosine and L-arabinose.
1.1.7 characterization of enzymatic Properties of CX3 Strain
Catalase assay: and (3) selecting CX3 hyphae in logarithmic growth phase on the solid culture medium, dropping 3-10% hydrogen peroxide on a clean glass slide, and observing the result, wherein the hyphae with a large amount of bubbles generated within 5min are positive, and the hyphae without bubbles are negative.
Urease test: sterilizing 30% urea with diethyl ether, and adding sterile urea when the culture medium is cooled to 55 deg.C to make its final concentration 2%. CX3 strain was inoculated, and the color of the medium was observed after 4 days of cultivation, and the medium was positive when it became pink and negative when it became white. Culture medium: peptone 1g, sodium chloride 5g, glucose 1g, KH2PO42g, 0.012g of phenol red, 15g of agar and 1000mL of distilled water.
Lipase experiments: the substrate is Tween-20 (sterilized at 121 ℃ for later use). The medium was cooled to 40-50 ℃, sterile tween was added to a final concentration of 1%, and plates were prepared and inoculated with CX3 strain. Culturing for 7-14 days, and observing every day. Faint halos around the growing strain were positive and none were negative. Culture medium: peptone 1g, NaCl 5g, CaCl2·7H20.1g of O, 9g of agar, 1000mL of distilled water, and pH 7.4.
Gelatin liquefaction experiment: the bacteria were streaked on a medium and cultured at 28 ℃ and the degree of liquefaction was observed for 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. Culture medium: 5g of peptone, 20g of glucose, 200g of gelatin and 1000mL of distilled water.
Starch hydrolysis experiments: the strain is inoculated on a starch agar culture medium. After 10 days, amylase activity was measured, and iodine was dropped on the plate to observe the color around the lawn, the presence or absence of a transparent circle, and the size of the circle. Selection of the medium: soluble starch 10g, K2HPO4 0.3g、MgCO3 1.0g、NaCl 0.5g、KNO31.0g, agar 15g, distilled water 1000mL, pH 7.2-7.4.
Milk clotting and peptonization experiments: inoculating CX3 strain into skimmed milk, culturing at 28 deg.C, and observing whether coagulation and peptonization occur for 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: 1000mL of fresh skim milk and CaCO30.02g, and 3 times of batch sterilization.
Cellulose utilization experiments: inoculating the strain onto the filter paper strip in the test tube, soaking half of the filter paper in the carbon source-free synthetic solution, and culturing for 30 days to see whether the strain can grow and decompose the filter paper strip. Three replicates per group were used as controls against the non-inoculated species. Selection of the medium: MgSO (MgSO)4 0.5g、NaCl 0.5g、K2HPO4 0.5g、KNO31g, 20g of cane sugar and 1000mL of distilled water.
1.1.8 CX3 Strain metabolite assay
M.r. test: inoculating the strain into liquid culture medium, culturing at 28 deg.C 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: methyl Red 0.1g, glucose 5g, K2HPO45g, 0.012g of phenol red and 1000mL of distilled water.
V.p. test: the culture solution and 40% sodium hydroxide are mixed in equal amount, a little creatine is added, and the positive reaction is that red color appears within 10 min. The culture medium was used in the m.r. experiment.
Experiment for hydrogen sulfide formation: the strain is inoculated on a Tresner culture medium, and whether melanin is produced or not is observed after the strain is cultured for 7 days. The production of melanin indicates that the strain produces hydrogen sulfide, and the uninoculated culture 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.
Nitrate reduction experiments: the strain is inoculated into the culture medium and cultured for 7 days and 14 days for measurement. Solution 1, 2 was added in two drops per 1mL of the bacterial suspension. The red color was positive, and the uninoculated medium was used as a control. Culture medium: MgSO (MgSO)4 0.5g、NaCl 0.5g、K2HPO4 0.5g、KNO31.0g, 20g of cane sugar and 2000mL of distilled water. Solution 1: aminobenzenesulfonic acid 0.5g, dissolved in acetic acid (80% acetic acid, diluted 2 times with water). Solution II: 0.1g of diphenylamine and 20mL of distilled water, wherein the diphenylamine is dissolved in a small amount of ethanol, water is added, and 150mL of diluted acetic acid is added after boiling.
1.1.9 CX3 Strain identification
Inoculating the strain to Gauss No.1agar plate, obliquely inserting sterilized cover glass into the culture medium at 45 deg.C, culturing at 28 deg.C for 5-7d, taking out the cover glass, and directly observing under optical microscope.
DNA extraction was performed according to the instructions of the Ezup column type bacterial genome DNA extraction kit. The PCR reaction system is 50 microliter: PCR mix 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, ddH2O 19μL。
And (3) PCR reaction conditions: denaturation at 94 deg.C for 4 min; denaturation at 94 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10min, storage at 4 ℃, taking PCR product of sterile water as negative control, loading 2 mu L of sample, and carrying out 2% agarose gel electrophoresis. All operations are operated according to the operation flow of the kit, and the recovered product is sent to Jilin province, Kuumei, biotechnology, Inc. for sequencing analysis.
1.2 data analysis
SPSS 17.0 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.
Example 2 results and analysis
2.1 preliminary screening of the Strain
After purification, 84 purified strains with consistent shape, size and color are obtained, and the results of plate confrontation tests show that 12 strains (shown in table 1) with the bacteriostatic bandwidth of more than 10mm and 8 strains with the bacteriostatic bandwidth of more than 15mm are obtained, wherein the strain CX3 has a good bacteriostatic effect (shown in figure 1), and the bacteriostatic bandwidth reaches 16.16mm, so that the strain CX3 (obtained by screening samples collected from Picea javanica Linn of West protection station of Changbai mountain protection district) is selected as the strain for the next test.
TABLE 1 bacteriostatic activity of Streptomyces lividans against bacterial canker of blueberry
The data in the table are mean ± standard deviation.
2.2 determination of antagonistic bacteria CX3 live bacteria and its fermentation liquor bacterial inhibition spectrum
Antagonistic bacteria CX3 has certain inhibiting effect on all the strains to be tested. CX3 has the strongest inhibition effect on blueberry canker pathogenic bacteria, an obvious inhibition zone can be seen, the width of the inhibition zone reaches 16.16mm, and the difference effect with other tested pathogenic bacteria is obvious; the inhibitor has strong inhibiting effect on small red bean anthracnose bacteria, corn northern leaf blight bacteria and pepper anthracnose bacteria, the inhibiting bandwidth is respectively 13.42 mm, 13.88 mm and 12.84mm, the inhibiting effect on tobacco alternaria alternata bacteria, melon fusarium wilt bacteria and the like is relatively weak, but the inhibiting bandwidth is also more than 9.30mm (Table 2).
The bacteriostatic activity of the antagonistic strain CX3 fermentation liquor is determined by a cup and dish method, the fermentation liquor still keeps better bacteriostatic activity, the bacteriostatic effect of the fermentation liquor of the strain on blueberry canker germs is strongest, reaches 31.73mm, and has obvious difference effect with other test strains; the antagonistic bacterium has strong antagonistic effect on eggplant phomopsis fulvidraco and pepper colletotrichum, the average inhibitory diameter reaches 30.37mm, the inhibitory effect on corn northern leaf blight, red bean colletotrichum and poplar skin rot germ is good, the inhibitory diameters are respectively 28.82 mm, 25.74 mm and 26.94mm, the fermentation liquor of the antagonistic bacterium has obvious inhibitory effect on all tested pathogenic bacteria (table 2), and the minimum inhibitory diameter also reaches 22.60 mm.
TABLE 2 bacteriostasis spectra of antagonistic bacteria CX3 living body and fermentation broth
The 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 Duncan's new repolarization method. Data in the table area mean + -SD, the letters in the sample column index differences at the P <0.01level by Duncan's new complex range method.
2.3 morphological Observation of Strain CX3
The strain CX3 grows vigorously on the Gao's first culture medium, the aerial hyphae are abundant, and the hyphae grow radially in all aspects; when the culture is carried out for 1 to 3 days at the temperature of 28 ℃, the bacterial colony is smooth and has no spore generation, after 7 days, the spore generation begins, and the microscopic observation shows that the spore is mostly in a chain shape as shown in figure 2; the spore silk is not autolyzed and does not absorb water; when cultured for 15 days, the color of the spore pile is close to pink, the spores are mostly straight, and sometimes the top ends are in a hook shape, according to the characteristics, the streptomycete identification manual (edited by actinomycete classification group of institute of microbiology of Chinese academy of sciences. streptomycete identification manual. Beijing: scientific publishing house, 1975) is referred, and the strain CX3 is preliminarily analyzed to be the pink spore group of streptomycete.
2.3.1 culture Properties of Strain CX3
After the strain CX3 was inoculated on 10 test media, colonies began to appear after culturing at 28 ℃ for 36-48h, and spores began to produce 7 days later. The morphology of the lawn, the color of the spore mass, the color of the substrate hyphae, the pigment produced, and the growth varied from medium to medium, and as a result, strain CX3 did not produce soluble pigments on most media, as shown in table 3. By consulting streptomycete search manual (compiled by actinomycetes classification group of institute of microbiology of Chinese academy of sciences. streptomycete identification manual. Beijing: scientific Press, 1975.), the aerial hypha of strain CX3 on Gao's I culture medium is pink, and the matrix hypha is white, and can be further determined to be a Pink spore group of Streptomyces.
TABLE 3 cultural characteristics of Strain CX3
"-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +", vigorous growth.
2.4 physiological and biochemical characteristics of antagonistic bacteria CX3
2.4.1 determination of tolerance to the antagonist bacteria CX3
The strain CX3 showed good stress tolerance, and the results are shown in Table 4. The strain is favored by high-salt environment, grows on a culture medium containing 5% NaCl and grows vigorously under the condition of high salt; the temperature tolerance is good, the growth can be carried out at 15-28 ℃, and 28 ℃ is the optimal growth temperature; the compound has good tolerance to alkali, can grow in the pH range of 5-12, and has the optimum pH value of 7.
TABLE 4 determination of the tolerance of the Strain CX3
"-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +", vigorous growth.
2.4.2 utilization of carbon Source that antagonizes bacteria CX3
The results of the utilization of various carbon sources by strain CX3 are shown in Table 5, and the strain can utilize 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 strain can better utilize monosaccharide and disaccharide, cannot 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; the urease test is negative; lipase test positive, translucent halo is generated around the strain; the gelatin can be liquefied; the inability to utilize decomposed cellulose; the milk can be coagulated, but the peptonization phenomenon does not occur; the starch hydrolysis test is positive, and amylase can be produced.
2.4.4 determination of metabolite of antagonistic bacterium CX3
The results of the metabolite assay of strain CX3 are shown in table 5; m.r. test negative; h2The test of S is positive; v.p. experiment CX3 showed a positive red color; the control in the nitrate reduction reaction is transparent and colorless, and the strain CX3 shows positive pink. By consulting the manual of streptomyces classification and identification, strain CX3 was further determined to be a Fowler subgroup of Streptomyces, Pink roselle cluster.
TABLE 5 determination of the Strain CX3 by physiological and biochemical experiments
"-", no growth; growing "+"; "+ + +" indicates good growth; "+ + + +", 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 adding bacteria liquid and with adding equal amount of sterile water), and the right test tube is an experimental sample (CX3 strain culture liquid), and it can be seen from fig. 3 that both test tubes are negative yellow, which indicates that the strain can decompose glucose to produce pyruvic acid, the pyruvic acid is further metabolized and decomposed into lactic acid, formic acid and acetic acid, and the final pH of the mixed solution is reduced to below 4.5, and then the m.r. experiment is negative.
The results of the nitrate reduction experiments are shown in FIG. 4. In FIG. 4, the test tube on the left is the test sample (CX3 strain culture solution), the test tube on the right is the negative control sample (the negative control sample is a sample without adding bacteria solution and with adding equal amount of sterile water), and it can be seen from FIG. 4 that the test tube on the test sample is pink, and the negative control tube is colorless, which indicates that the mixed solution contains nitrite and is positive for nitrate reduction.
V.P the results of the test 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 adding bacteria liquid and with adding equal amount of sterile water), the right test tube is an experimental sample (CX3 strain culture liquid), it can be seen from FIG. 5 that the experimental sample test tube is red, and the negative control test tube is yellow, which shows that the pyruvic acid generated by glucose decomposition by the strain is further decarboxylated to form acetyl methyl methanol, the substance is oxidized to diacetyl under alkaline condition, and then combined with guanidino contained in arginine and the like in the culture medium peptone to form a red compound, namely, the V.P. test is positive.
H2The results of the S generation experiment are shown in fig. 6. In FIG. 7, the left plate is a negative control sample (the negative control sample is a sample without added bacteria solution and with an equal amount of sterile water added), the right plate is an experimental sample (CX3 strain medium), and it can be seen from FIG. 6 that the negative control plate is yellow and the experimental plate is tan, indicating that H is present2S production, H2S and ferric citrate are combined to generate FeS, and the culture medium presents positive tan.
2.5 grouping biological identification of antagonistic bacteria CX3
2.5.1 identification of 16S rDNA of Strain CX3
The 16S rDNA gene amplified fragment of the strain CX3 has a total length of 1385bp (gene accession number: MN636760) after sequencing. The sequence with higher homology between 9 strains and the strain CX3 is selected, a phylogenetic tree is established, the result of phylogenetic analysis of the strain CX3 and related strains is shown in figure 7, and the strains which are relatively close to the strain CX3 belong to the genus Streptomyces, so that the strain CX3 is preliminarily determined to be a Freund subgroup of the subgroup Streptomyces and Pink spore.
The invention separates 84 actinomycetes from the under-forest soil of each forest region of Jilin province, screens blueberry canker pathogenic bacteria as target pathogenic bacteria, finally selects 1 actinomycetes of the under-forest soil of Maanshan picea spruce in West region of Jilin province, and determines that the strain CX3 is a Freund subgroup of streptomyces and pinkish spore group by morphological observation, physiological and biochemical identification, molecular biological analysis and identification methods such as 16SrDNA analysis and the like. The streptomyces identification handbook is examined to find that no corresponding species exists, but the analysis finds that the physicochemical properties of the two strains are similar to those of the antagonistic strain CX3, namely, the streptomyces polycarbophilus and the streptomyces mediterraneans have slight differences in culture characteristics; the physicochemical properties are similar, and the starch can be hydrolyzed, but the strain CX3 can not decompose and utilize cellulose. Streptomyces mediterranean belongs to a variety of roses of the Pink roseospora group, and can produce rifamycin (Rifosycin) as a broad-spectrum antibiotic. Streptomyces polycarbophil is capable of producing antibiotic wan-135 and inhibiting positive bacteria, including Mycobacterium.
The CX3 strain has antifungal activity, the live bacteria and the fermentation liquid thereof have stronger inhibiting effect on blueberry canker bacteria, and the analysis is probably because the strain generates antifungal substances in the physiological metabolic process. The strain CX3 can well utilize monosaccharide and disaccharide but cannot utilize polysaccharide, and the analysis also has important relation with the physiological metabolic pathway. In conclusion, the gene is tentatively named as Streptomyces CX 3.
A series of experiments prove 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.
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.
Sequence listing
Scientific research institute for forestry in <110> Jilin province
<120> fermentation method of biocontrol streptomyces
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggttaccttg ttacgactt 19
Claims (10)
1. A fermentation method of biocontrol streptomyces is characterized by comprising the following steps: the preservation number of the streptomyces biocontrol strain is CGMCC No. 18658.
2. The fermentation method of the streptomyces biocontrol as claimed in claim 1, characterized in that the method comprises the following steps of inoculating the streptomyces biocontrol with the preservation number of CGMCC No.18658 into a fermentation medium, and fermenting and culturing.
3. The fermentation method of the biocontrol streptomyces as claimed in claim 1, characterized in that fermentation is performed by using a fermenter or a shake flask.
4. The fermentation method of Streptomyces biocontrol strain according to any of claims 1-3, wherein the fermentation temperature is 15-28 ℃.
5. The fermentation method of the biocontrol streptomyces as claimed in claim 4, wherein the fermentation temperature is 28 ℃.
6. The fermentation method of Streptomyces biocontrol according to any one of claims 1-3, wherein the fermentation time is 5-7 days.
7. The fermentation method of Streptomyces biocontrol strain according to any of claims 1-3, further comprising the step of centrifuging the fermentation broth obtained from the fermentation.
8. The fermentation method of the biocontrol streptomyces as claimed in claim 7, wherein the rotation speed of the centrifugation is 8000r/min, and the centrifugation time is 15 min.
9. The fermentation method of the streptomyces biocontrol as claimed in any one of claims 1-3, wherein the fermentation medium used in the fermentation culture of the streptomyces biocontrol comprises the following components: every 1000mL of water, 70g of soluble starch, 33g of peanut cake powder and (NH)4)2SO44 g、NaCl 4g、CaCO3 4 g，pH7.0。
10. A biocontrol product comprising streptomyces biocontrol and/or streptomyces biocontrol fermentation broth produced by the fermentation process of any of claims 1-9.
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