CN117965370A - Streptomyces hygroscopicus and application thereof - Google Patents
Streptomyces hygroscopicus and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/28—Streptomyces
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
- C12R2001/55—Streptomyces hygroscopicus
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- Microbiology (AREA)
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- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention relates to streptomyces hygroscopicus and application thereof. The strain is named as streptomyces hygroscopicus JN3-4 (Streptomyces hygroscopicusJN 3-4), the preservation number is CGMCC No.27649, and the strain and the microbial inoculum thereof can be used for biological control of pathogenic bacteria such as conidiophore parviflora, red bean anthracnose pathogen, korean pine damping-off pathogen, corn big spot pathogen, tobacco target spot pathogen, rice bakanae pathogen, melon vine cutting pathogen, pepper anthracnose pathogen, pear sclerotium pathogen, cucumber sclerotium pathogen, blueberry canker pathogen, larix Gmelinii and the like, and have the advantages of good biological control effect, broad antibacterial spectrum, wide application range and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to streptomyces hygroscopicus and application thereof.
Background
Larch belongs to larch genus of Pinaceae, has good light preference, cold resistance, strong adaptability, quick growth and high economic value of wood, and is a good tree species for artificially constructing fast-growing and high-yield wood forests. Larix Gmelini Neofusicoccum laricinum can cause Larix Gmelini. In recent years, the larch tip blight is a main disease of the larch artificial forest, the Jilin province in 1973 reports the occurrence of the disease for the first time, and as the disease incidence rule of the disease is not mastered at the moment, no effective prevention and quarantine method exists, the disease rapidly spreads, the disease is expanded to the places such as Shandong, hebei, shanxi and the like at present, the occurrence area has a tendency to increase year by year, and the hazard is also more serious. The larch tip blight mainly damages the current new tips of 1-35-year old larch artificial forests, and the disease of 6-15-year old young forests is heavier and common; onset is 7 months, and peak period is 8 and 9 months. Generally, the disease is developed from the top of the main tree, and gradually spreads downwards from the top of the tree crown. The diseased young shoots fade gradually, the top of the young shoots is bent and sagged from yellow green to brown gray to form a hook shape, the young shoots gradually drop down to leave, dry, shrink and thin the stem, a cluster of leaves remains at the top end of the tip head, and the tips wither to be purple gray. In the continuous disease, most of the tips are dead, the crowns are broom-shaped, and the growth of the harmful serious trees is stopped, so that the young trees or the whole plants die. After 15-20 days of onset, the top withered leaf clumps develop densely small black spots on the back of the leaf, namely conidium organs of pathogenic bacteria and a small amount of immature ascus shells.
In recent years, domestic reports about the occurrence and damage of fallen leaf tip disease are frequent, and at present, the prevention and treatment of the fallen leaf tip disease adopt the following measures: chemical control, breeding disease-resistant varieties, cultivating disease-free strong seedlings, optimizing cultivation sites and cultivation modes and the like (to Xiaxia, wang Zhiming, wang Xuan remote and the like. Larix Gmelin and Larix GmbH control research [ J ]. Jilin forestry science and technology, 2016,45 (02): 30-34+40.DOI: 10.16115/j.cnki.issn.1005-7129.2016.02.010.). 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, the use of a large amount of chemical pesticides is easy to cause environmental pollution, destroy ecological balance and other problems. 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 microorganisms with biological control effect, and are widely used for controlling plant diseases in recent years. Streptomyces plays an important role in actinomycetes, and studies such as antagonism (Nath R P,Gandhi K B,Manjunatha L,et al.In vitro evaluation of actinobacteria for biocontrol of dry root rot and Fusarium wilt diseases in chickpea(Cicer arietnum L.)[J].Journal of Food Legumes,2021,34(4):245-247.),Samac of isolated actinomycetes such as Pandey on the root rot of chickpea revealed that most of the strains of Streptomyces that produce antibiotics have an inhibitory effect on the growth of 3 different strains of Phytophthora medicago and that the active secondary metabolite of actinomycetes has an inhibitory effect on tomato bacterial wilt in the study in 2020, the university of northeast (Ling Ling. Streptomyces sp.NEAU-HV9 has an inhibitory effect on tomato bacterial wilt and an active metabolite study [ D ]. Northeast university of agriculture, 2021.DOI: 10.27010/d.cnki.gddnu.2020.000800); the monomeric compound produced by Streptomyces Foc TR, isolated from soil samples of banana garden at the university of Huaqiao, 2023, has a strong inhibitory effect on banana vascular wilt (Niexia. Identification of active ingredients of Streptomyces H3-2 against banana vascular wilt and its antibacterial mechanism research [ D ]. University of Huaqiao, 2022.DOI:10.27155/D. Cnki. Ghqiu. 2022.000789.).
The bacteriostasis spectrum and the bacteriostasis activity of different strains are different, and proper strains need to be selected in biological control so as to achieve better control effect. Thus, providing a suitable strain is critical for effective biocontrol.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a streptomyces hygroscopicus and application thereof. The strain is discovered for the first time, and further verification shows that the strain has the advantages of good biocontrol effect, wide antibacterial spectrum, wide application range and the like.
The technical scheme for solving the technical problems is as follows:
The invention provides a streptomyces hygroscopicus, the name of which is streptomyces hygroscopicus JN3-4 (Streptomyces hygroscopicusJN 3-4), the preservation number is CGMCC No.27649, the strain is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms for 6 months and 16 days in 2023, and the preservation address is the institute of microorganisms of China academy of sciences No. 3 of West way 1 of North Star in the Korean region of Beijing, china.
According to the invention, a plurality of actinomycetes are obtained by separating soil collected from different areas of Jilin province, and through multiple screening, 1 antagonistic strain capable of effectively inhibiting Laurencia lardii is finally obtained, and is marked as JN 3-4.
The invention provides a microbial inoculum, which comprises the streptomyces hygroscopicus and/or a fermentation product of the streptomyces hygroscopicus.
The formulation of the microbial inoculum is not particularly limited, and for example, the microbial inoculum can be a liquid preparation, a solid preparation or other types of preparations. Besides the fermentation product of streptomyces or streptomyces provided by the invention, components commonly used in the field for preparing biocontrol agents can be added so as to be beneficial to the application of the biocontrol agents.
The streptomyces hygroscopicus and the microbial inoculum thereof provided by the invention have the advantages of good biocontrol effect, wide antibacterial spectrum, wide application range and the like.
The invention provides a fermentation method of the streptomyces hygroscopicus, which comprises the following steps: and (3) inoculating the streptomyces hygroscopicus into a culture medium for fermentation culture.
The formulation of the fermentation medium may include: peanut cake powder 2.5%, starch 5.0%, yeast powder 0.08%, glucose 0.02%, (NH 4)2SO40.08％、CaCO3 0.32.32%, naCl 0.2%, pH 7.0-7.2.
Fermentation culture conditions may include: culturing at 28℃for 6d.
The invention provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating the streptomyces hygroscopicus into a fermentation culture medium for fermentation culture.
The formulation of the fermentation medium may include: peanut cake powder 2.5%, starch 5.0%, yeast powder 0.08%, glucose 0.02%, (NH 4)2SO40.08％、CaCO3 0.32.32%, naCl 0.2%, pH 7.0-7.2.
Fermentation culture conditions may include: culturing at 28℃for 6d.
The invention provides a supernatant for biological control, and the preparation method of the supernatant can comprise the following steps: inoculating the streptomyces hygroscopicus into a fermentation culture medium for fermentation culture, centrifuging and filtering.
The formulation of the fermentation medium may include: peanut cake powder 2.5%, starch 5.0%, yeast powder 0.08%, glucose 0.02%, (NH 4)2SO40.08％、CaCO3 0.32.32%, naCl 0.2%, pH 7.0-7.2.
Fermentation culture conditions may include: culturing at 28℃for 6d.
The invention provides application of the streptomyces hygroscopicus in prevention and control of plant pathogenic bacteria. The strain provided by the invention can be used for preparing a biocontrol agent and is used for preventing and controlling one or more of chaetomium globosum, red bean anthracnose, korean pine damping-off bacteria, corn maculopathy bacteria, tobacco target spot bacteria, rice bakanae bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, pear boulder fruit rot bacteria, cucumber sclerotium bacteria, blueberry canker bacteria and larch tip bacteria; is used for preventing and treating diseases caused by the pathogenic bacteria.
The invention provides application of the microbial inoculum in prevention and control of plant pathogenic bacteria. The plant pathogenic bacteria are selected from one or more of Confucius comatus, bacillus natto anthracnose, rhizoctonia solani, rhizoctonia cerealis, and Rhizoctonia solani. The microbial inoculum provided by the invention can be used for preventing and treating diseases caused by the pathogenic bacteria.
The invention provides application of supernatant of fermentation liquor in prevention and control of plant pathogenic bacteria. The supernatant of the fermentation broth may be prepared by the following method:
the supernatant of the fermentation liquor provided by the invention can be used for preventing and treating diseases caused by the plant pathogenic bacteria.
The invention has the advantages of good control effect, broad antibacterial spectrum, wide application range and the like when in application.
The invention provides a biological control method, which comprises the following steps: biological control is carried out by adopting the streptomycete and/or the microbial inoculum. Biological control can also be performed using the supernatant.
The method provided by the invention has the advantages of good control effect, wide antibacterial spectrum, wide application range and the like.
Drawings
FIG. 1 shows the inhibitory effect of JN3-4 live bacteria on Larix Gmelini.
FIG. 2 shows the results of observation of strain JN3-4, wherein A is the culture trait on JN3-4 Gaoshi No. 1 agar medium, and B is the morphology under JN3-4 electron microscope.
FIG. 3 is a phylogenetic analysis of strain JN3-4 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.
The invention takes Laurencia largehead as a target, and 1 biocontrol strain JN3-4 with better resistance is obtained through preliminary screening. The identification by morphology and 16S rDNA sequence analysis shows that the strain belongs to streptomyces hygroscopicus Streptomyces hygroscopicus. In order to clearly antagonize the biocontrol effect of the strain JN3-4, the antibacterial activity of the live bacteria and the fermentation broth of the strain were measured by a plate counter method and a cup and disc method, respectively. The result shows that the strain has inhibition effect on 12 plant pathogens to be tested, has a wide antibacterial spectrum, has inhibition effect on Larix Gmelini, convolvulus arvensis and Pyricularia pyrifolia, and the like, wherein the inhibition effect on Larix Gmelini is strongest, the antibacterial band is 19.50mm, and the diameter of the antibacterial ring of the fermentation liquor is 31.44mm. The strain has remarkable inhibition effect on various tested pathogenic bacteria and has good application and development prospects.
Test plant pathogenic bacteria:
The meristematic chaetomium Dothiorella gregari, the Korean pine damping-off bacteria Fusarium oxysporum, the Larix Gmelini Neofusicoccum laricinum and the blueberry canker bacteria Botryosphaeriadothidea are separated and stored in the laboratory; the corn big spot germ Exserohilum turcicum, the tobacco target spot germ Rhizoctonia solani, the rice bakanae germ Fusariummoniliforme, the red bean anthracnose germ Colletotrichum truncatum, the melon vine cutting germ Fusarium oxysporum, the pepper anthracnose germ Colletortrichum capsici and the cucumber sclerotium germ Sclerotinia scleroriorum are presented by plant virus research laboratories of Shenyang agricultural university; the fruit rot pathogen Potebniamycespyri is provided by the Dalian customs technical center Li Xin researchers; the public may have access to embodiments that repeat the description of the present invention for non-commercial purposes only.
The soil sample to be tested is 18 parts of soil samples taken from regions such as Dunhua and Jiaohe of Jilin province, 6 months to 7 months in 2022, jiangnan forest land of Jilin City, jiangshi, jiangnan, etc.
Test medium:
Gao's agar medium No. one (gause's No.1 agar), formulated in the following proportions: KNO 3 1g、K2HPO40.5g、MgSO4 0.5g、NaCl 0.5g、FeSO4 0.01.01 g, soluble starch 20g, agar 20g, water 1000mL, pH 7.2-7.4.
Potato dextrose agar medium (potato dextrose agar, PDA) was prepared in the following proportions: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water.
Fermentation medium: 2.5% of peanut cake powder, 5.0% of starch, 0.08% of yeast powder, 0.02% of glucose, (NH 4)2SO40.08％、CaCO3 0.32.32% of NaCl, 0.2% of pH 7.0-7.2, and the mass percentages are all.
Test agent: resin type TM genome DNA extraction kit is purchased from Shanghai Saighur Gene technologies Co., ltd; 16S rDNA Bacterial Identification PCR Kit, agarose Gel DNA Purification Kit Ver 2.0 and DNA MARKER DL2000, all available from Takara Bio Inc.
Instrument: BX53 olympus light microscope was purchased from olympus corporation; hitachi S-3400N scanning electron microscope was purchased from Hitachi, inc.
In the invention, unless specified, the experimental methods used are all conventional experimental methods in the field; the materials, reagents and equipment used are all conventional materials, reagents and equipment in the field and can be obtained commercially or prepared by conventional methods.
In the examples, if data processing is involved, statistical analysis is performed using SPSS23.0 software and the Duncan new complex pole difference method is used for difference significance test.
The following is presented by way of specific examples.
Example 1
The strain screening is carried out by adopting a progressive dilution coating separation method: baking 10g of soil sample at 120 ℃ for 1h, adding into 100mL of sterile water, shaking for 30min, sucking 1mL of suspension, carrying out gradient dilution by using sterile water, respectively sucking 0.1mL of suspension from 10 -3、10-4 and 10 -5 dilution pipes, adding the suspension onto a flat plate of Gao's first agar culture medium, uniformly coating, inverting a culture dish into a 28 ℃ incubator, culturing for 3-10d, observing and selecting strains with different colony forms every day, timely transferring the strains onto an inclined plane of the Gao's first agar culture medium for culturing, purifying for 3-5 times by adopting a dilution separation method, and storing the serial number in a refrigerator at 4 ℃ for standby.
After purification, a purified strain 87 strain with consistent morphology, size and color is obtained.
Inoculating activated Larix Gmelini to PDA plate, and culturing at 28deg.C for 7 d. Transferring all the separated strains into a flat plate of a Gaoshan agar medium for culture, taking the Larix Gmelini as a target bacteria after each strain generates enough spores, and measuring the antibacterial activity of the separated actinomycetes by a flat plate counter culture method, wherein the measuring method comprises the following steps: 7mm fungus cakes are made on a Korean pine damping-off fungus flat plate by using a puncher, the pathogenic fungus cakes are placed on one side of the upper and lower 2cm away from the center of a PDA flat plate (diameter 90 mm), the cultured strain to be detected is selected by using an inoculating loop on one side to line, after the strain to be detected is cultivated for 72 hours at the constant temperature of 28 ℃, the width of a bacteriostatic zone between actinomycetes and pathogenic fungi is measured, and each treatment is repeated for 3 times. According to the size of the antibacterial zone, the strain with the strongest antagonistic activity is used as a next research object.
The results of the plate-facing test showed that 11 strains with a width of inhibition zone greater than 13mm and 3 strains with a width of inhibition zone greater than 17mm were the best, and that the inhibition zone width of the strain JN3-4 was 19.50mm (FIG. 1), so that JN3-4 (isolated from the soil sample collected from the Jiannan forest farm) was selected as the strain for the next test (Table 1).
TABLE 1 antibacterial Activity of Streptomyces living organisms against Larix Gmelini
Data in the table are mean ± standard deviation.
Example 2
Observation of JN3-4 strain: the JN3-4 strain is inoculated on a flat plate of a Gaoshan agar medium, a sterilizing cover glass is obliquely inserted in the medium at 45 degrees, after the culture is carried out for 5-7d at 28 ℃, the cover glass is taken out, and the BX53 Olympic optical microscope and the Hitachi S-3400N scanning electron microscope are adopted for observation.
As shown in FIG. 2, strain JN3-4 grew luxuriantly on Gaoshan agar medium number one; when the culture is carried out for 1-3d at 28 ℃, bacterial colonies are smooth and have no sporulation, and single bacterial colonies are generated at most when the culture is observed under a microscope; and the spore silk is autolyzed and has the water absorption phenomenon; obvious spiral spore chains are visible under an electron microscope, and the characteristic of the streptomycete is achieved.
EXAMPLE 3 16S rDNA identification of Strain JN3-4
DNA of strain JN3-4 was extracted with reference to the instructions of the resin type TM genomic DNA extraction kit. The 16S rDNA of strain JN3-4 was PCR amplified using 16S rDNA Bacterial Identification PCR Kit, and the upstream and downstream primers used for amplifying 16S rDNA were purchased from Takara Bio (Dalian) engineering Co.
The PCR reaction system (50. Mu.L) comprises: PCR Mixture 25. Mu. L, template. Mu.L, 1. Mu. L, ddH 2 O21. Mu.L each for the upstream and downstream primers.
PCR reaction conditions: denaturation at 94℃for 5min; denaturation at 94℃for 1min, renaturation at 56℃for 1min, extension at 72℃for 2min,35 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.
The PCR product of sterile water was used as a negative control, the PCR product of the known 16S rDNA strain was used as a positive control, the sample loading amount was 2. Mu.L, and 2% agarose gel electrophoresis was performed using DNA MARKER as DNA MARKER DL. The product was recovered using Agarose Gel DNA Purification Kit Ver 2.0.0 and sent to Takara engineering Co., ltd for two-way sequencing. After the sequences were analyzed by software such as bioedit7.0.1 and manually corrected, the sequences thus obtained were subjected to homology comparison analysis with sequences of known similar-pattern strains downloaded in GenBank using the BLAST program of NCBI, and sequence alignment was performed by the adjoining method (NJ) of MEGA 6.0 software and a phylogenetic tree was drawn.
The full length of the amplified fragment of the 16S rDNA gene of the strain JN3-4 is 1217bp after sequencing. 9 sequences with higher homology with the JN3-4 strain are selected, a phylogenetic tree is established, and as shown in figure 3, the strain JN3-4 is primarily determined to be streptomyces hygroscopicus (Streptomyces hygroscopicus).
The strain is preserved in China general microbiological culture collection center (CGMCC) at 6 months and 16 days of 2023, and has the preservation name of Streptomyces hygroscopicus JN3-4 (Streptomyces hygroscopicus JN 3-4), the preservation number of CGMCC No.27649 and the preservation address of China academy of China microbiological study, the West Song No.1 and 3 of the Korean area North Star of Beijing, china.
Example 4 determination of Streptomyces JN3-4 live bacteria inhibition spectrum
Bacterial inhibition spectra of streptomycete JN3-4 live bacteria are measured by taking the bacterial strains of the group consisting of chaetomium album, red bean anthracnose, korean pine damping-off bacteria, corn big spot bacteria, tobacco target spot bacteria, rice bakanae disease bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, pear boulder fruit rot bacteria, cucumber sclerotinia bacteria, blueberry canker bacteria and Larix Gmelinii as test strains and adopting a flat plate opposite culture method. The plate counter culture method is described in example 1.
The results show that: antagonistic bacteria JN3-4 inhibited all of the tested strains. The JN3-4 has the strongest inhibition effect on the Laurencia lardii, and obvious antibacterial bands with widths reaching 19.50mm are visible, so that the difference effect between the antibacterial bands and other tested pathogenic bacteria is obvious; the composition also has strong inhibition effects on the Consumer fungus, the fruit rot of pear and the bakanae disease of rice, the inhibition bandwidths are 19.31mm, 17.06mm and 15.05mm respectively, the inhibition effects on the sclerotinia sclerotiorum, the red bean anthracis and the like are relatively weak, but the inhibition bandwidth is also more than 13.24mm (Table 2).
TABLE 2 antibacterial spectrum of antagonistic JN3-4 living organism
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.
Example 5 preparation of fermentation broth and determination of its bacteriostasis Spectrum
200ML of fermentation medium is prepared, and is filled into a triangular flask with the volume of 500mL, and is sterilized for later use. Transferring strain JN3-4 into fermentation medium, shaking culture at constant temperature of 28deg.C 150r/min for 6d, centrifuging at 8000r/min for 15min, filtering supernatant with 0.22 micrometer bacterial filter, removing thallus, and storing in refrigerator at-20deg.C.
The method comprises the steps of taking the chaetomium album, the red bean anthracnose pathogen, the Korean pine damping-off pathogen, the corn big spot pathogen, the tobacco target spot pathogen, the rice bakanae pathogen, the melon vine cutting pathogen, the pepper anthracnose pathogen, the pear boulder fruit rot pathogen, the cucumber sclerotinia, the blueberry canker pathogen and the larch tip pathogen as test strains, activating all the test plant pathogenic bacteria by adopting a cup dish method to prepare spore suspension liquid under the condition of 10 multiplied by 15 times of microscope, absorbing 300 mu L of the spore suspension liquid, adding 70mL of PDA culture medium to prepare a mixed bacteria plate, placing an oxford cup in the center of the culture dish, adding 100 mu L of fermentation liquor (namely supernatant prepared by the method), and measuring the diameter of a bacteriostasis ring by a cross method after culturing at the constant temperature of 28 ℃ for 72 hours.
The antibacterial activity of antagonistic strain JN3-4 fermentation liquor is measured by adopting a cup and disc method, and the result shows that the fermentation liquor still maintains good antibacterial activity, and the fermentation liquor of the strain has the strongest antibacterial effect on the Laurencia largehead, which reaches 31.44mm, and has obvious difference effect with other tested strains; the antagonistic bacteria have extremely strong antagonistic action on sclerotinia sclerotiorum and corn big spot bacteria, the antibacterial diameter is more than 24.69mm, the antagonistic bacteria also have relatively good inhibitory action on pepper anthracnose bacteria, poly-chaetomium globosum and rice bakanae bacteria, the antibacterial diameters are 24.34mm, 23.36mm and 22.68mm respectively, the antagonistic bacteria fermentation liquor has obvious inhibitory action on all tested pathogenic bacteria (Table 3), and the minimum antibacterial diameter is also up to 10.77mm.
TABLE 3 antibacterial spectrum of antagonistic JN3-4 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.
In conclusion, the JN3-4 strain provided by the invention has fungicidal activity, and the living bacteria and the fermentation liquor of the strain have good inhibition effects on the Laurencia largehead, probably because the strain generates antifungal substances in the physiological metabolic process.
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.
Claims (10)
1. The Streptomyces hygroscopicus is characterized by being named as Streptomyces hygroscopicus JN3-4 (Streptomyces hygroscopicusJN-4) and having a preservation number of CGMCC No.27649.
2. A microbial agent comprising the fermentation product of streptomyces hygroscopicus according to claim 1 and/or streptomyces hygroscopicus according to claim 1.
3. Use of the streptomyces hygroscopicus according to claim 1 and/or the microbial inoculum according to claim 2 for controlling phytopathogenic fungi.
4. The use according to claim 3, wherein the plant pathogenic bacteria are selected from one or more of chaetomium globosum, colletotrichum glomeratum, rhizoctonia solani, alternaria corn, phoma tabacum, bakanae disease, melon vine cutting, pepper anthracnose, sclerotinia pyriformis, cucumber sclerotium bacteria, blueberry canker bacteria, and larch tip bacteria.
5. Use of the streptomyces hygroscopicus according to claim 1 and/or the microbial inoculum according to claim 2 for controlling symptoms caused by phytopathogenic bacteria.
6. The use according to claim 5, wherein the plant pathogenic bacteria are selected from one or more of chaetomium globosum, colletotrichum glomeratum, rhizoctonia solani, alternaria corn, phoma tabacum, bakanae disease, melon vine cutting, pepper anthracnose, sclerotinia pyriformis, cucumber sclerotium bacteria, blueberry canker bacteria, and larch tip bacteria.
7. The use of the streptomyces hygroscopicus of claim 1 in the preparation of biocontrol agents.
8. The use according to claim 7, wherein the biocontrol agent is used for controlling diseases caused by phytopathogenic bacteria; the plant pathogenic bacteria are selected from one or more of Confucius comatus, bacillus natto anthracnose, rhizoctonia solani, rhizoctonia cerealis, and Rhizoctonia solani.
9. Use of the streptomyces hygroscopicus of claim 1 and/or the fungicide of claim 2 for controlling larch tip blight.
10. A biological control method, comprising the steps of: biological control is carried out using the Streptomyces hygroscopicus according to claim 1 and/or the microbial inoculum according to claim 2.
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| CN101979609A (en) * | 2010-09-27 | 2011-02-23 | 山东农业大学 | Method for extracting antifungal active substances of Streptomyces hygroscopicus |
| CN102732460A (en) * | 2012-06-13 | 2012-10-17 | 浙江师范大学 | Streptomyces hygroscopicus Sh-43 strain, its fermentation solution production method and application |
| CN108676750A (en) * | 2018-05-29 | 2018-10-19 | 重庆大学 | A kind of streptomyces hygroscopicus producing salicylic acid and rapamycin and its application in prevention plant Oomycete and fungal disease |
| CN113957003A (en) * | 2021-08-30 | 2022-01-21 | 中国热带农业科学院海口实验站 | Antifungal streptomyces hygroscopicus and application thereof |
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| CN101979609A (en) * | 2010-09-27 | 2011-02-23 | 山东农业大学 | Method for extracting antifungal active substances of Streptomyces hygroscopicus |
| CN102732460A (en) * | 2012-06-13 | 2012-10-17 | 浙江师范大学 | Streptomyces hygroscopicus Sh-43 strain, its fermentation solution production method and application |
| CN108676750A (en) * | 2018-05-29 | 2018-10-19 | 重庆大学 | A kind of streptomyces hygroscopicus producing salicylic acid and rapamycin and its application in prevention plant Oomycete and fungal disease |
| CN113957003A (en) * | 2021-08-30 | 2022-01-21 | 中国热带农业科学院海口实验站 | Antifungal streptomyces hygroscopicus and application thereof |
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