CN117965371A - Streptomyces fusiformis and application thereof - Google Patents
Streptomyces fusiformis and application thereof Download PDFInfo
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Abstract
The invention relates to streptomyces fusiformis and application thereof. The strain is named as streptomyces fusiformis JN4-2 (Streptomyces netropsis JN 4-2), and the preservation number is CGMCC No.28157. The strain and the microbial inoculum thereof can be used for biological control of pathogenic bacteria such as Korean pine rhizoctonia rot, red bean anthracnose, poly-generation small hole shell bacteria, apple shell single color septoria canker, corn big spot bacteria, tobacco target spot bacteria, rice bakanae disease bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, larch tip blight bacteria, blueberry canker bacteria, poplar bark rot bacteria, apple rot bacteria, poplar canker bacteria, eggplant brown spot bacteria, melon fruit rot bacteria, tobacco brown spot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotinia rot bacteria, pear boehmeria, and the like, and have the advantages of good biological control effect, wide 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 fusiformis and application thereof.
Background
Korean pine (Pinus koraiensis) belongs to Pinaceae (Pinaceae) Pinus (Pinus), is native to North region of European Asia continent, and is the main colonisation tree species and dominant tree species in northeast forest area of China. Korean pine is a light-loving plant, has strong cold resistance and good corrosion resistance, is suitable for being planted in wet slightly acidic soil or neutral soil, and has the effect of promoting the growth of Korean pine when the atmospheric humidity is above 0.7.
The Korean pine damping off is caused by fungi (mainly rhizoctonia and fusarium), and is common and seriously damaged in seedling infectious diseases, after seedlings are damaged, the death rate is extremely high, the incidence rate of 1-2 year old seedlings is extremely high, and the serious incidence rate reaches more than 60 percent, even the absolute production rate is high. Chemical control and soil disinfectant treatment are still mainly adopted in production to prevent the occurrence and hazard of the diseases. The excessive use of chemical pesticides causes the pathogenic bacteria to generate drug resistance and simultaneously causes irreversible damage to the ecological environment, and the frequent use of soil disinfectants causes the soil environment degradation of the nursery garden and the damage of microbial communities, so that the problems of poor nutrition condition and weak growth potential of the nursery stock, low survival rate, poor adaptability and the like in the follow-up forestation and ecological restoration are further caused. In recent 40 years, agricultural production in China depends on chemical throwing, and the use amount of pesticides is greatly increased. According to the statistics of the national environmental protection agency, about 25 ten thousand tons of pesticide raw materials are produced in China in 2007, the pesticide raw materials are processed into more than 80 ten thousand tons of preparations, the preparations are applied to the environment in various ways, 10% -20% of the preparations are attached to plant bodies, and 80% -90% of the preparations are scattered in soil, water and atmosphere (Yang Yonghua, yao Jian, hua Xiaomei. The effect of pesticide pollution on the functional diversity of soil microbial communities [ J ]. J. J.Microbiol., 2000, (02): 23-25+47.). In view of the above problems, biological control is becoming more and more popular.
The disease is reported for the first time in the last century, wu You and the like in 60 th China, and research on the occurrence and control of the disease is frequent, the occurrence of seedling blight disease of pine seedlings in northeast China is affected by soil temperature, rhizoctonia solani occurs more at 18-22 ℃, pythium is 17-22 ℃ and fusarium is 23-28 ℃. The epidemic of seedling blight in northeast is mainly affected by the water content of soil and rainfall, and the disease is benefited when the rainfall is large and the water content of soil is high. Furthermore, the longer the onset is delayed, the higher the overall incidence (Wu Yousan, elegance, gu Sifang, etc.. Study II. Pine seedling damping off. Disease epidemics [ J ]. Protect plant school, 1963, (04): 399-408.). Ying et al (1979) found through experiments that the pathogenic bacteria causing seedling blight of pine were mainly rhizoctonia, fusarium and saprophytic fungi, and that rhizoctonia was the strongest in the pathogenicity aspect (4: ying. Seedling blight of pine and its prevention and control J. Jilin forestry science and technology, 1979, (02): 117-120.). Zhang Lijun et al (2012) found that Rhizoctonia solani overwintered in residues and soil of various hosts with hyphae and spores, and spread among lines by hyphae. Poor environmental conditions such as overcast and rainy at low temperature and insufficient illumination can promote the infection of germs, especially the influence of low temperature on diseases is the greatest (Zhang Lijun, wangping, ni Tianbo, li Chuncheng, gu Junying. The occurrence rule of Korean pine damping off and control technology [ J ]. Agriculture and technology, 2012,32 (09): 116).
The damage caused by plant diseases to agriculture and forestry production is more serious, economic loss is immeasurable, and along with the gradual aggravation of the defects of chemical agent control, biological control is in general attention of the whole society. The method has the advantages of high efficiency, no toxicity, environmental protection and the like, and becomes a main stream means for preventing and controlling plant diseases. Among the 16500 antibiotics reported in the world, actinomycetes account for more than half of the total number of antibiotics. Actinomycetes are the earliest discovered class of microorganisms with biological control effects. Streptomyces among actinomycetes is the genus that produces the most bacteriostatic substances. The new agricultural antibiotic doramectin is reported by Gao Yu hong et al (2009) at northeast agricultural university, and the abamectin antibiotics produced by the new species of fermentation of recombinant Streptomyces avermitilis (S.avermitilis) are one of the most excellent antiparasitic drugs in the current abamectin family as an internal and external compatible insecticide, and have good killing effects on nematodes and arthropods (Jiang Wei. Mutant biosynthesis and fermentation condition optimization [ D ] at northeast agricultural university, 2009. Gao Yuhong, guo Zhaocheng, liu Fangyue, and the like. Research progress of doramectin and application [ J ].2009,40 (4): 141-144). Boukaew et al (2017) report that low concentration metabolites of S.philinthi RM-1-138, S.philinthi RL-1-178 and S.mycarofaciens SS-2-243 have an inhibition rate of 100％([16]Sawai Boukaew,Poonsuk Prasertsan,Claire Troulet,Marc Bardin.Biological control of tomato gray mold caused by Botrytis cinerea by using Streptomyces spp.[J].BioControl,2017,62(6).). on germination of spores of Botrytis cinerea in biological control, and that a proper strain needs to be selected for achieving a better control effect. Therefore, it is important to provide a strain that can be effectively used for biological control.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides streptomyces fusiformis 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, the name of the strain is streptomyces fusiformis JN4-2 (Streptomyces netropsis JN 4-2), the preservation number is CGMCC No.28157, the strain is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month 11 days 2023, and the preservation address is the institute of microorganisms at the 3 rd national academy of sciences of China of the national institute of sciences of the sciences of China, the Chaoyang area North Star of Beijing, china.
According to the invention, a plurality of actinomycetes are obtained by separating soil collected from different areas of Jilin province, and multiple screening and prevention and control effect measurement are adopted, so that 1 antagonistic strain with the best effect for inhibiting the damping-off of Korean pine is finally obtained, and is marked as JN4-2, breakthrough of actinomycetes in the field of preventing and controlling the damping-off of Korean pine is realized, and a new strain and a new product are provided for biological prevention and control of the damping-off of Korean pine.
The invention provides a microbial inoculum comprising the streptomyces fusiformis and/or fermentation products of the streptomyces fusiformis. 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 fusiformis 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 fusiformis, which comprises the following steps: and (3) inoculating the streptomyces fusiformis into a culture medium for fermentation culture. The formulation of the fermentation medium may include: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate and 7.0 pH value per 1000mL of distilled water. 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 fusiformis into a fermentation culture medium for fermentation culture. The formulation of the fermentation medium may include: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate and 7.0 pH value per 1000mL of distilled water. 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 seed solution of the streptomyces fusiformis into a fermentation culture medium, culturing for 6d at a constant temperature of 28 ℃ and 150r/min, centrifuging, and taking the supernatant.
The formulation of the fermentation medium may include: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate and 7.0 pH value per 1000mL of distilled water.
The preparation method of the seed liquid can comprise the following steps: culturing the streptomyces fusiformis on a flat plate in a streaking way, taking spores, scraping the spores into sterile water to prepare spore suspension, and obtaining seed liquid.
The invention provides application of the streptomyces fusiformis in prevention and control of plant pathogenic bacteria. The strain provided by the invention can be used for preparing biocontrol agents and is used for preventing and controlling one or more of red pine damping-off bacteria, red bean anthracnose bacteria, poly-generation small-hole-shell bacteria, apple shell single-color septoria canker bacteria, corn big spot bacteria, tobacco target spot bacteria, rice bakanae bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, larch tip bacteria, blueberry canker bacteria, poplar skin rot bacteria, apple canker bacteria, poplar canker bacteria, eggplant brown spot bacteria, melon and fruit pythium bacteria, tobacco brown spot bacteria, blueberry branch blight bacteria, apple fruit rot bacteria, cucumber sclerotium bacteria and pear sclerotium 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 Korean pine rhizoctonia rot, red bean anthracnose, coniopsis pinicola, apocynum venetum, corn maculosa, tobacco target maculosa, rice bakanae disease, melon vine cutting bacteria, pepper anthracnose bacteria, larix Gmelini, blueberry canker bacteria, poplar bark rot bacteria, apple rot bacteria, poplar bark rot bacteria, eggplant brown rot bacteria, pythium aphanidermatum, tobacco brown rot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotium bacteria and pear fruit rot bacteria. 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 in prevention and control of plant pathogenic bacteria. The supernatant may be prepared by the following method: inoculating the seed solution of the streptomyces into a fermentation culture medium, culturing for 6d at a constant temperature of 28 ℃ and 150r/min, centrifuging, and taking the supernatant.
The formulation of the fermentation medium may include: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate and 7.0 pH value per 1000mL of distilled water.
The preparation method of the seed liquid comprises the following steps: culturing Streptomyces in a streak culture manner on a plate, taking spores, and scraping the spores into sterile water to prepare spore suspension, namely seed liquid.
The supernatant 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 experimental results of example 1 of the present invention, wherein A is the inhibitory effect of JN4-2 live bacteria on Rhizoctonia solani and B is Rhizoctonia solani.
FIG. 2 shows the culture properties of JN4-2 in Gaoshi agar medium No. 1.
FIG. 3 shows the morphology under a JN4-2 optical microscope.
FIG. 4 is a phylogenetic analysis of strain JN4-2 and related strains.
FIG. 5 shows the results of detection of the bacteria inhibition zone of JN4-2 live bacteria against Chaetomium agglutinans, wherein A is the bacteria inhibition zone of JN4-2 live bacteria against Chaetomium agglutinans, and B is the control (Chaetomium agglutinans).
FIG. 6 shows the detection result of JN4-2 live bacteria on apple fruit rot bacteria, wherein A is JN4-2 live bacteria on apple fruit rot bacteria, and B is control (apple fruit rot bacteria).
FIG. 7 shows the detection result of the bacterial inhibition zone of JN4-2 live bacteria on tobacco target spot bacteria, wherein A is the bacterial inhibition zone of JN4-2 live bacteria on tobacco target spot bacteria, and B is the control (tobacco target spot bacteria).
FIG. 8 shows the detection result of the bacterial inhibition zone of JN4-2 viable bacteria on Pythium aphanidermatum, wherein A is the bacterial inhibition zone of JN4-2 viable bacteria on Pythium aphanidermatum, and B is the control melon (Pythium muricatum control).
FIG. 9 shows the results of detection of the damping-off zone of Korean pine damping-off by the JN4-2 fermentation broth, wherein A is the damping-off zone of Korean pine damping-off by the JN4-2 fermentation broth, and B is the control (Korean pine damping-off).
FIG. 10 shows the results of detection of the zone of inhibition of the bacterial strain of the red bean anthracnose by the JN4-2 fermentation broth, wherein A is the zone of inhibition of the red bean anthracnose by the JN4-2 fermentation broth, and B is the zone of inhibition of the red bean anthracnose by the control (red bean anthracnose).
FIG. 11 shows the results of detection of the antibacterial zone of the JN4-2 fermentation broth against the melon vine cutting bacteria, wherein A is the JN4-2 fermentation broth against the melon vine cutting bacteria, and B is the control (melon vine cutting bacteria).
FIG. 12 shows the results of detection of the zone of inhibition of Laurencia largehead by JN4-2 fermentation broth, wherein, A is JN4-2 fermentation liquor and B is control (Larix Gmelini).
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.
According to the invention, 87 actinomycetes are separated from the soil under the forest in each forest area of Jilin province, and Korean pine damping-off bacteria are used as target pathogenic bacteria for screening, so that bacterial strain JN4-2 with the best inhibition effect on Korean pine damping-off bacteria is screened. The strain JN4-2 was identified as Streptomyces fusiformis by morphological observation and molecular biological analysis identification methods such as 16SrDNA analysis. Therefore, the strain is named as streptomyces fusiformis JN4-2 (Streptomyces netropsis JN 4-2), and is preserved in China general microbiological culture Collection center (CGMCC) at 8-11 of 2023, wherein the preservation address is CGMCC No.28157 of China academy of sciences of China, north Star Xiya 1, kogyo, china.
In order to better understand the biological control effect and classification status of antagonistic actinomycetes capable of effectively inhibiting the damping-off bacteria of Korean pine, the invention respectively determines the antibacterial activity and antibacterial spectrum of the JN4-2 live bacteria and the fermentation liquor by a plate counter method and a cup and saucer method. The research result shows that the strain has inhibition effect on 21 plant pathogenic bacteria to be tested and has wide antibacterial spectrum. Particularly, the strain has extremely strong inhibition effect on pathogenic bacteria of the damping-off of Korean pine, the antibacterial bandwidth of live bacteria reaches 22.54mm, and the diameter of a antibacterial circle of fermentation liquor reaches 34.60mm. The invention reports the streptomyces fusiformis for the first time, and the application of the streptomyces fusiformis to biological control research of the rhizoctonia solani, and has good application and development prospects.
Test plant pathogenic bacteria: korean pine damping-off bacteria Fusarium oxysporum, poplar skin rot bacteria Valsa sordida, poly-generation cave shell bacteria Dothiorella gregari, apple shell single color septoria canker bacteria Botryosphaeria stevensii, poplar canker bacteria Botryosphaeria dothidea, larix Gmelini Neofusicoccum laricinum, apple fruit rot bacteria Monilinia polystroma and apple rot bacteria VALSA MALI are separated and stored by Jilin province forestry science institute; the plant virus researches of the Shenyang agricultural university are presented with red bean anthracnose germ Colletotrichum sp, corn big spot germ Exserohilumturcicum, tobacco target spot germ Rhizoctoniasolani, rice bakanae germ F.moniliforme, melon vine cutting germ Fusarium oxysporum, pepper anthracnose germ C.gloeosporioides, eggplant brown spot germ Phomopsis vexans, pythium aphanidermatum germ Pythiumaphanidermatum, tobacco brown spot germ ALTERNARIAALTERNATA and cucumber sclerotium germ Sclerotiniascleroriorum; the blueberry canker germ Botryosphaeria dothidea and the blueberry cladosporium cucumerinum germ Neofusicoccum parvum are given by the professor Xu Chengnan of the university of Yan student's academy of life; the fruit rot of pear Phytophthora cactorum 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 taken from 26 parts of soil samples of the places such as Hunchun and Dunhua of Jilin province, longnan forest lands and the like.
Test medium:
gaoshi agar medium (gause's No.1agar)：KNO31g、K2HPO40.5g、MgSO40.5g、NaCl 0.5g、FeSO40.01g、 soluble starch 20g, agar 20g, water per 1000mL, pH 7.2-7.4.
Potato dextrose agar medium (potato dextrose agar, PDA): 200g of potato, 20g of glucose, 20g of agar and every 1000mL of distilled water.
Fermentation medium: 40g of wheat bran, 20g of corn flour, 60g of sucrose, 4g of potassium nitrate, 0.4g of calcium carbonate, 0.2g of dipotassium hydrogen phosphate and 7.0 pH value per 1000mL of distilled water.
Test agent:
Ezup column type bacterial genome DNA extraction kit is purchased from the biological engineering (Shanghai) limited company (abbreviated as the biological limited company); 16S rDNA Bacterial Identification PCR Kit, agarose Gel DNA Purification Kit Ver 2.0 and DNA MARKER DL2000 from Takara Bio-engineering (Dalian) Inc.; the others are all domestic analytical pure.
The universal primers 27F, 1429R were purchased from vinca biosciences, inc.
Instrument: BX53 olympus light microscope was purchased from olympus corporation.
In the embodiment, if data processing is involved, SPSS23.0 is adopted for data statistics; phylogenetic tree was established using MEGA version X.
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.
The following is presented by way of specific examples.
EXAMPLE 1 screening of Streptomyces biocontrol strains
26 Parts of soil samples of the Jilin provinces Hunchun and Dunhua, the Longshan, jilin province, jiangnan forest land of Jilin City, jiangshi, and the like are collected in 6-7 months of 2022. 100ml of sterile water and 10g of soil sample are fully mixed and then are kept stand, diluted to 10 -3、10-4、10-5 different concentrations, the mixture is coated on a Gao's first agar culture medium, bacterial strains are separated and screened, after the culture medium grows out single bacterial colonies, the bacterial strains are transferred to the Gao's first agar culture medium for purification culture, the purification culture is repeated for 3 times, and the screened bacterial strains are transferred to an inclined plane for storage in a refrigerator at 4 ℃ for standby.
After separation, purification and obtaining 87 purified strains with different forms, sizes and colors by a dilution culture method. The antibacterial activity of the strain is detected by adopting a flat plate counter test and taking Korean pine damping off as a target strain.
A test method for a plate counter test comprises the following steps: culturing the obtained strain until the strain grows vigorously, taking the Korean pine damping-off bacteria as a target strain, and measuring the inhibition effect of the separated strain on the Korean pine damping-off bacteria. The specific method comprises the following steps: making 7mm fungus cake on Korean pine damping-off fungus plate with puncher, placing pathogenic fungus cake on one side of the upper and lower 2cm distance from PDA plate (diameter 90 mm), picking cultured strain to be tested with inoculating loop on one side, streaking, and culturing at 28deg.C with plate without inoculating actinomycetes as control group. When the colony of the control group grows to be full of the flat plate, the average distance between the pathogenic bacteria cake and the strain to be detected is measured by a vernier caliper, and each group is treated for 3 times. And selecting the strain with vigorous growth and the best antibacterial effect as a next research object.
The results of the plate-facing test show that 14 strains in total show obvious antibacterial effects (table 1) among 87 strains, wherein the antibacterial effect of JN4-2 extracted from a forest farm in the south of the Yangtze river on Rhizoctonia solani is best, and the antibacterial zone of the JN4-2 can reach 22.54mm (figure 1). Thus, JN4-2 was chosen as the subject of further investigation.
TABLE 1 bacteriostatic Activity of Streptomyces live bacteria against Rhizoctonia solani
Data in the table are mean ± standard deviation.
EXAMPLE 2 morphological observations of JN4-2 Strain
Bacterial strain JN4-2 is inoculated on a Gao's agar medium, a sterilizing cover glass is obliquely inserted in the medium at 45 degrees, the bacterial strain is cultured for about 5 days at 28 ℃, and the bacterial strain is taken out and placed under a BX53 type Olinbas optical microscope for observation. According to the study method of actinomycetes classification group by microorganisms of the national academy of sciences of China, the growth condition and the appearance characteristic of the actinomycetes are observed by using culture medium for identifying actinomycetes.
Morphology observations of aerial hyphae and sporostrips: the strain JN4-2 grows radially on a Gaoshi agar medium, and when the aerial hypha is not Chang Maocheng and is cultured for 1-3 days at the constant temperature of 28 ℃, the colony is round and smooth and has no sporulation. White spores grow from the edge of the colony at the 5d, and gradually change to be white in the mussel along with the increase of the growing days, and the matrix mycelium is yellow in color and does not generate soluble pigment. The incubation was continued for 30d and the colonies were buttermilk (FIG. 2). The hyphae were found to have a large number of branches, elongated, straight shapes, no breaks, no transverse membranes, and a large number of spores dispersed around the hyphae under the microscope (fig. 3).
EXAMPLE 3 identification of JN4-2 Strain
Molecular biology identification of JN4-2 using 16SrDNA sequencing, comprising the steps of: the Ezup column type bacterial genome DNA extraction kit (Bio Inc.) was selected to extract the genome DNA of JN 4-2.
PCR amplification was performed using 16S rDNA Bacterial Identification PCR Kit. The PCR reaction system (50. Mu.L) comprises: PCR Mixture 25. Mu. L, template. Mu.L, universal primers 27F, 1429R 2. Mu. L, ddH 2 O19. Mu.L each. 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 deg.C for 10min, preserving at 4 deg.C, taking the PCR product of sterile water as negative control, taking the PCR product of the strain with the result of measuring 16SrRNA in laboratory as positive control, loading 2 μl, and performing electrophoresis on 2% agarose gel (DNA MARKER is DL 2000). The PCR product was recovered using Agarose Gel DNA Purification Kit Ver 2.0. The recovered product was sent to Jilin provincial treasury Mei Biotechnology Co.Ltd for sequencing analysis. The obtained strain 16s sequences were spliced, the sequences were subjected to homology comparison analysis with the BLAST program of NCBI and sequences of known similar pattern species downloaded in GenBank, and sequence alignment was performed by the adjoining method (NJ) of MEGA X software and a phylogenetic tree was drawn.
After PCR amplification, purification and sequencing of the strain JN4-2, the length of the 16S rDNA sequence of the strain JN4-2 is measured to be 1409bp. The sequence of 12 strains with higher homology with the strain JN4-2 was selected, and a phylogenetic tree was established, as shown in FIG. 4, with the highest homology with Streptomyces fusiformis Streptomyces netropsis, and a similarity value of 100% compared with the 16S rDNA sequence of the GenBank database. As a result, it was revealed that strains JN4-2 and Streptomycesnetropsis (GenBank: EU 119185) were collected in one branch, and that strain JN4-2 (OR 758449) was identified as Streptomyces fusiformis.
Therefore, it was named as Streptomyces fusiformis JN4-2 (StreptomycesnetropsisJN 4-2) and was preserved in China general microbiological culture Collection center (CGMCC) at 8/11/2023 with a preservation address of CGMCC No.28157, which is China academy of sciences of China at North Star West Lu No. 1, chapter No. 3 of Beijing, china.
EXAMPLE 4 determination of antibacterial Activity of antagonistic Strain JN4-2
The antibacterial activity of antagonistic strain JN4-2 on the tested strain is detected by adopting a plate counter test. The test method of the plate counter test is described in example 1.
The test strains were respectively: korean pine damping-off bacteria, red bean anthracnose bacteria, polyporus umbellatus, apple hull single color septoria, corn macula, tobacco target spot bacteria, rice bakanae disease bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, leaf spot bacteria, blueberry ulcer bacteria, poplar rot bacteria, apple rot bacteria, poplar ulcer bacteria, eggplant brown spot bacteria, melon and fruit rot bacteria, tobacco brown spot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotium bacteria, pear boulder fruit rot bacteria.
The experimental result shows that the antagonistic bacteria JN4-2 has an inhibitory effect on all tested strains. The JN4-2 has the strongest inhibition effect on the Korean pine damping-off bacteria, and obvious antibacterial bands with the width of 22.54mm are visible, so that the difference effect between the antibacterial bands and other tested pathogenic bacteria is obvious (Table 2); the composition also has strong inhibition effect on the chaetomium cupreum (figure 5), the blueberry canker and the apple fruit rot (figure 6), the inhibition bandwidths are respectively 17.19mm, 16.23mm and 16.33mm, the inhibition effect on the tobacco target spot bacteria (figure 7), the corn big spot bacteria, the melon fruit rot bacteria (figure 8) and the like is relatively weak, but the inhibition band width still reaches more than 10.13 mm.
TABLE 2 antibacterial spectrum of antagonistic JN4-2 live bacteria
Data in the table are mean ± standard deviation.
Example 5 preparation of fermentation broth of Strain JN4-2 and determination of its bacteriostasis Spectrum
The strain JN4-2 is selected to be cultured by adopting a fermentation medium because the bacterial inhibition activity of the strain JN4-2 is strongest. Culturing strain JN4-2 on a plate of a Gaoshan agar culture medium in a streaking way, and scraping spores into sterile water to prepare spore suspension serving as seed liquid after the strain grows vigorously; absorbing 2mL of seed liquid, adding into 50mL of fermentation medium, culturing at 28deg.C under constant temperature of 150r/min for 6d, centrifuging at 6000rpm/min at 4deg.C for 10min, collecting supernatant, placing into 50mL centrifuge tube, sealing, and storing in a refrigerator at-20deg.C.
The bacteriostasis spectrum of the strain JN4-2 fermentation broth is detected by adopting a cup and disc method, and the method comprises the following steps of: 7mm pathogenic bacteria cakes are prepared, the bacteria cakes are symmetrically poured around a PDA culture medium, an oxford cup is placed in the center of a flat plate, fermentation liquor (namely, supernatant prepared by the method) is inoculated in the oxford cup, the inoculation amount is 200 mu L, distilled water is taken as a reference, the culture is carried out at a constant temperature of 28 ℃, and the diameter of a bacteria inhibition zone of a treatment group is measured by a cross method after the bacteria of the reference group grow up a culture dish.
The pathogenic bacteria tested were: korean pine damping-off bacteria, red bean anthracnose bacteria, polyporus umbellatus, apple hull single color septoria, corn macula, tobacco target spot bacteria, rice bakanae disease bacteria, melon vine cutting bacteria, pepper anthracnose bacteria, leaf spot bacteria, blueberry ulcer bacteria, poplar rot bacteria, apple rot bacteria, poplar ulcer bacteria, eggplant brown spot bacteria, melon and fruit rot bacteria, tobacco brown spot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotium bacteria, pear boulder fruit rot bacteria.
The results show that: the antibacterial activity of antagonistic strain JN4-2 fermentation liquor is measured by adopting a cup and disc method, the fermentation liquor still maintains good antibacterial activity, the fermentation liquor of the strain has the strongest antibacterial effect on Korean pine damping-off bacteria (figure 9) and has obvious difference effect with other tested strains, and the antibacterial effect is 34.60 mm; the antagonistic bacteria have extremely strong antagonism to red bean anthracnose bacteria (figure 10) and apple rot bacteria, the antibacterial diameters reach 31.92mm and 29.87mm respectively, the antagonistic bacteria also have relatively good inhibition to eggplant brown streak bacteria, melon vine cutting bacteria (figure 11) and Larix Gmelini bacteria (figure 12), the antibacterial diameters are 27.60mm, 27.07mm and 28.46mm respectively, and the antagonistic bacteria fermentation liquor has obvious inhibition to all tested pathogenic bacteria (table 3), wherein the antibacterial diameter of tobacco target spot bacteria is minimum, but still reaches 18.25mm.
TABLE 3 antibacterial spectrum of antagonistic JN4-2 fermentation broths
Data in the table are mean ± standard deviation.
In conclusion, the JN4-2 strain provided by the invention has fungicidal activity, and the live bacteria and the fermentation liquor of the strain have strong inhibition effects on Korean pine damping-off bacteria, and the analysis is probably due to the fact that the strain generates antifungal substances in the physiological metabolic process.
The antagonistic strain JN4-2 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 JN4-2 has multiple advantages of low toxicity, no residue, environmental friendliness and the like, and accords with the sustainable control environment protection concept better.
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. Streptomyces fusiformis is characterized by being named as Streptomyces fusiformis JN4-2 (Streptomyces netropsis JN 4-2) and having a preservation number of CGMCC No.28157.
2. A microbial agent comprising the fermentation product of the streptomyces fusiformis of claim 1 or the streptomyces fusiformis of claim 1.
3. Use of the Streptomyces fusiformis according to claim 1 and/or the microbial inoculum according to claim 2 for controlling phytopathogenic fungi.
4. The use according to claim 3, characterized in that the plant pathogenic bacteria are selected from one or more of the group consisting of rhizoctonia solani, anthracnose of red bean, chaetomium globosum, aphalocrocis apple, pholiosis corn, pholiota, bakanae disease, melon vine cutting, anthracnose of capsicum, larch tip of larch, blueberry ulcer, poplar skin rot, apple rot, poplar ulcer, eggplant brown spot, aphanidermatum of melon, brown spot of tobacco, blueberry cladosporium, apple fruit rot, cucumber sclerotium, pear boulder rot.
5. Use of the streptomyces fusiformis according to claim 1 and/or the fungicide 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 the group consisting of rhizoctonia solani, anthracnose of red bean, chaetomium globosum, aphalocrocis apple, pholiosis monocarum, phoma zeylanicum, phoma tabacum, bakanae disease, melon vine cutting, anthracnose of capsicum, larch tip of larch, blueberry ulcer, poplar skin rot, apple rot, poplar ulcer, eggplant brown spot, aphalocrocis melon, fusarium graminearum, blueberry cladosporium, apple canker, cucumber sclerotium, pear brucella.
7. The use of Streptomyces fusiformis according to claim 1 for the preparation of a biocontrol agent.
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 Korean pine rhizoctonia rot, red bean anthracnose, coniopsis pinicola, apocynum venetum, corn maculosa, tobacco target maculosa, rice bakanae disease, melon vine cutting bacteria, pepper anthracnose bacteria, larix Gmelini, blueberry canker bacteria, poplar bark rot bacteria, apple rot bacteria, poplar bark rot bacteria, eggplant brown rot bacteria, pythium aphanidermatum, tobacco brown rot bacteria, blueberry branch rot bacteria, apple fruit rot bacteria, cucumber sclerotium bacteria and pear fruit rot bacteria.
9. Use of the streptomyces fusiformis according to claim 1 and/or the microbial inoculum according to claim 2 for controlling damping-off of Korean pine.
10. A biological control method, comprising the steps of: biological control is carried out using the Streptomyces fusiformis according to claim 1 and/or the microbial inoculum according to claim 2.
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