CN108165506B - Streptomyces aureoflavus and application thereof - Google Patents
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Abstract
The invention provides an actinomycete, which is streptomyces aureoflavus XJC-SDSIM1 with the preservation number of CCTCC NO: m2017491. The streptomyces aureoflavus XJC-SDSIM1 has the growth pH range of 5-10, the growth temperature of 22-32 ℃, can not grow on a culture medium with the NaCl content of more than 5 percent, has broad-spectrum bacteriostatic activity, has good antagonistic action on pepper anthracnose pathogen, cucumber fusarium wilt pathogen, mango anthracnose pathogen, rice blast pathogen, wheat gibberellic disease, apple ring rot pathogen, strawberry anthracnose pathogen and the like, has wide development space, and has good development and application prospects.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to streptomyces aureoflavus and application thereof.
Background
One important direction in the research of biopesticides is to find new substances from microbial metabolites for controlling harmful organisms. With the demands of environmental protection, green food and agricultural sustainable development, agricultural antibiotics are increasingly gaining attention as low-toxicity and low-residue biopesticides. Currently, nearly 70% of the approximately 22500 bioactive substances found in microorganisms are produced by actinomycetes. Actinomycetes are the main source of antibiotics, and 60% of the known antibiotics are produced by actinomycetes, wherein streptomyces, such as streptomycin, aureomycin, tetracycline, paromomycin, mitomycin and other important antibiotics are mainly concentrated.
With the intensive research on the metabolic characteristics of soil actinomycetes and the further improvement of the separation and culture methods, more and more soil actinomycetes are separated. Streptomyces has been considered a major source for the production of various antibiotics. Genetic diversity analysis has shown that about ten thousand antibiotics can be produced by Streptomyces, and only a small fraction of those currently found.
Although actinomycetes are initially used in medicine, they are widely used in agriculture because of their low ecological environmental impact and low pollution. Many researches prove that the actinomycete has obvious effect on biological prevention and control of plant diseases, insect pests and weeds and has good application prospect.
Therefore, it is important to find new strains having antibacterial activity.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an actinomycete, which is streptomyces aureoflavus, has broad-spectrum antibacterial activity, has good antagonism on various germs, and has wide development space and application prospect.
The first aspect of the invention provides an actinomycete, which is streptomyces aureoflavus XJC-SDSIM1 with the preservation number of CCTCC NO: m2017491.
A second aspect of the present invention provides the use of an actinomycete according to the first aspect of the invention for antagonising Colletotrichum capsici and/or Fusarium oxysporum and/or Pyricularia oryzae and/or Fusarium graminearum and/or Phyllostachys malorum and/or Francisella fragilis.
A third aspect of the present invention provides the use of the actinomycetes according to the first aspect of the present invention for the control of anthracnose of capsicum, and/or fusarium wilt of cucumber, and/or rice blast, and/or wheat scab, and/or apple ring spot, and/or strawberry anthracnose.
A fourth aspect of the present invention provides a fermentation broth or filtrate of the fermentation broth of the actinomycete according to the first aspect of the present invention.
A fifth aspect of the present invention provides the use of the fermentation broth or filtrate of the fermentation broth according to the fourth aspect of the present invention for antagonizing Colletotrichum capsici and/or Fusarium oxysporum and/or Pyricularia oryzae and/or Fusarium graminearum and/or Phyllostachys malorum and/or Phyllostachys niponicus and/or Fragaria anachoriformis.
A sixth aspect of the invention provides the use of the fermentation broth or filtrate of the fermentation broth according to the fourth aspect of the invention for the control of anthracnose of pepper, and/or cucumber fusarium wilt, and/or rice blast, and/or wheat scab, and/or apple ring rot, and/or strawberry anthracnose.
The seventh aspect of the present invention provides a microbial inoculum comprising the actinomycetes according to the first aspect of the present invention.
The streptomyces aureoflavus XJC-SDSIM1(streptomyces sanglieri XJC-SDSIM1) has the growth pH range of 5-10, the growth temperature of 22-32 ℃, can not grow on a culture medium with the NaCl content of more than 5 percent, has broad-spectrum antibacterial activity, has good antagonistic action on pepper colletotrichum gloeosporioides, cucumber fusarium wilt, mango colletotrichum gloeosporioides, rice blast, wheat gibberella, apple ring rot, strawberry colletotrichum gloeosporioides and the like, has wide development space and has good development and application prospects.
Drawings
FIG. 1 shows the morphology of spore hyphae (A) and spores (B) of strain XJC-SDSIM 1.
FIG. 2 is a phylogenetic tree of strain XJC-SDSIM1 and related strains constructed based on the 16S rDNA sequence.
Detailed Description
The invention will be better understood by reference to the following examples.
The invention provides an actinomycete, which is streptomyces aureoflavus XJC-SDSIM1(streptomyces sanglieri XJC-SDSIM1) and has a preservation number of CCTCC NO: m2017491, the preservation date is 2017, 9 and 11 days, the preservation unit is China center for type culture Collection, and the address is Wuhan university in Wuhan, China. The streptomyces aureoflavus XJC-SDSIM1 is separated from the rhizosphere soil of tea trees collected from Puer city of Yunnan province.
1 materials of the experiment
1.1 test soil
Collecting rhizosphere soil of tea trees in Puer city of Yunnan province, placing in a sterile sealing bag, mixing, sealing, and storing in an ice box. Collecting, removing impurities such as root system and stone, and storing in refrigerator at 4 deg.C for use.
1.2 test Medium
The test medium of the present experiment included a separation medium, a medium for observing culture characteristics, a physiological and biochemical medium, a fermentation medium, and the like (xuli, 2007; huangxiaolong, 2009).
TABLE 1 isolation Medium for Actinomycetes and its formulation
TABLE 2 culture characteristic observation culture medium and its formulation
TABLE 3 culture media for physiological and biochemical characteristics
TABLE 4 fermentation media and their formulations
1.3 reagents and instrumentation used in this experiment
(1) Primary reagent
TABLE 5 major Biochemical reagents and sources
(2) Apparatus and device
TABLE 6 Instrument and apparatus
1.4 test pathogens
The No. 4 physiological race of banana Fusarium oxysporum (FOC 4) is used for screening the anti-Fusarium oxysporum and measuring the bacteriostatic activity. Banana Chang ban-Sha (Curvularia fallax), Banana Grey rot (Curvularia lunata), Banana Tremella (Btoyophora dothidea), Colletotrichum capsici (Colletotrichum acridum), Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), Fragaria ananasi (Colletotrichum fragaria), Botrytis cinerea (Botrytis cinerea), cucumber Fusarium wilt (Fusarium oxysporum (Schl.) F.sp. cumerin), mango anthracnose (Colletotrichum acridum), Pyricularia oryzae (Pyricularia oryzae Cav), Gibberella cerealis (Fusarium graminearum), Botrytis canea (Fusarium graminearum), apple ring rot (apple ring rot) for antimicrobial spectrometry.
2 method of experiment
2.1 isolation and identification of rhizosphere soil Actinomycetes
2.1.1 isolation and purification of rhizosphere soil Actinomycetes
Naturally air drying the soil sample, fully grinding and sieving, weighing 1g of the soil sample, dissolving in 10mL of sterile water, heating at 55 ℃ for 20min, and placing in a shaking table at 180r/min for culturing for 20min to prepare suspension. Taking supernatant, diluting by 10 times dilution method to obtain 10-1、10-2And 10-3The soil suspension is respectively sucked with 0.1mL of suspension and coated on a separation culture medium, inverted culture is carried out for 2-4 weeks at 28 ℃, 3 times of gradient are arranged, different single colonies are selected and repeatedly purified on a YE culture medium by a scribing method.
2.1.2 screening of antagonistic bacteria
Plate confrontation culture (grandson waves, 2010): using a puncher with the diameter of 5mm to take the fungus cakes inoculated with 5d and consistent growth force of FOC4 pathogenic bacteria edges, inoculating the fungus cakes to the center of each PDA plate, inoculating the bacteria to be tested at a position 2.5cm away from the center of the colony of the pathogenic bacteria, inoculating 4 test strains to each dish, taking the pathogenic bacteria inoculated with FOC4 only as a control, placing the inoculated pathogenic bacteria in an incubator at 28 ℃ for inverted culture for 7d, and observing the result.
2.1.3 determination of in-dish antibiogram
The bacterial strain is subjected to bacteriostasis spectrum determination by adopting a confrontation culture method (plum blossom King, 1997): a5 mm puncher is used for taking a bacterial cake of 12 purified plant pathogenic bacteria, the bacterial cake is inoculated to the center of a PDA (personal digital assistant) flat plate, a small amount of bacteria to be detected are respectively inoculated to four points 2.5cm away from the bacterial cake of the pathogenic bacteria, a culture dish only inoculated with the pathogenic bacteria is used as a blank control group, and each treatment is repeated for 3 times. After culturing in an incubator for 4-7 days, measuring the colony growth diameter of pathogenic bacteria to be tested by adopting a cross measurement method, and counting the bacteriostasis rate (Xiexing, 2011; summer dragon fungus, 2013) according to the following formula:
colony diameter (mm) — average value of colony diameters measured-5.0
2.1.4 culture characteristic Observation of antagonistic Strain
Observations of the culture characteristics were made by reference to the standard medium used in the international Streptomyces program for the culture characteristics of Actinomycetes (Cui BS, 2008). Inoculating antagonistic actinomycetes on ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7 culture media, culturing at 28 ℃ for 7-21d, and observing and recording culture characteristics of the strains on each culture media, including characteristics of colony morphology, aerial hypha production, spore color and intrastromal hypha color.
2.1.5 scanning Electron microscopy
Soaking the cover glass with potassium dichromate with the concentration of 0.05g/L, soaking and eluting with alcohol, washing with ultrapure water, blow-drying, and sterilizing at 121 ℃ for 20 minutes. The sterilized coverslip was inserted at 45 ℃ onto the culture medium of Goodpasture No. I inoculated with actinomycete strain, and cultured at 28 ℃ for 7-10 days. And (4) conveying the cover glass sample to a detection center for observation of a scanning electron microscope, placing the cover glass sample with the bacteria in a vacuum coating machine for spraying gold coating, and observing the fine structures on the surfaces of hyphae and spores of the strains by using the scanning electron microscope.
2.1.6 measurement of physiological and biochemical Properties
The physiological and biochemical identification of the strain was carried out by referring to the methods of Shirking (1966) and Xuelihua et al (2007) mainly in the following respects.
(1) Determination of enzymatic Properties
Firstly, urease experiments:
inoculating the strain on a urease culture medium, culturing for 4d at 28 ℃, and observing whether the culture medium is discolored. The test strains were tested for their ability to produce urease, and the medium was positive for pink and negative for no white.
② esterase (Tween 20, Tween 80) experiment:
streaked onto esterase medium for 1-2 weeks with daily observation. Positive if there is a faint halo around its growth, and negative if there is no halo.
③ hydrolyzing starch:
nutrient agar is used as a basic culture medium, and 1.0% of soluble starch is added. Inoculating the test strain on a flat plate, adopting a point inoculation method (the inoculation diameter is not more than 5mm), and when the strain grows well, dripping iodine solution around the bacterial colony for detection. If a transparent circle is generated around the strain, the generation of amylase is indicated, and the size of the circle indicates the strength of the amylase activity; if no amylase is produced, the color is blue.
Liquefaction of gelatin:
the strain was inoculated on the surface of gelatin medium without puncturing the medium, cultured at 28 ℃ and observed for the degree of liquefaction of the medium at 5d, 10d, 20d and 30d, respectively. Before observation, the test tube needs to be cooled for 20-30min or washed by tap water for 30min, and the liquefaction degree of the culture medium can be observed.
Cellulose decomposition:
immersing one end of the filter paper strip in a liquid culture medium, inoculating the strain to be detected on the filter paper sheet above the liquid level after sterilization, and observing whether the filter paper strip is decomposed or not after one month.
Sixthly, nitrate reduction:
the bacterial strain to be tested is inoculated in a nitrate reduction culture medium and cultured for 7 and 14 days at the temperature of 28 ℃, and the culture medium without inoculation is used as a control. A small amount of culture medium cultured for 7d and 14d was added to the test tube, and one drop of solution A and solution B was added, as was the case with the control. When the solution becomes pink, rose red, orange or brown, etc., the nitrate is positive in reduction; if no red color appears, 1 or 2 drops of diphenylamine reagent is dripped, and if the reagent is blue, the reduction effect is negative; if not blue, it is still treated as positive.
(2) Experiment for Using Single carbon Source
In the identification of actinomycetes, one of the important research indexes is the utilization of carbon sources, and different actinomycetes have different carbon source utilization capacities for sugars, alcohols, organic acids, fatty acids, and the like. Carbon source selected for the test: d-fructose, xylose, rhamnose, arabinose, raffinose, melezitose, anhydrous lactose, D-galactose, alpha-lactose, D-trehalose, D-mannose, D-ribose, inositol, sorbitol, mannitol, salicin and soluble starch are added into a basic culture medium of Pugodi according to the concentration of a carbon source of 1%. And inoculating the strain to be detected, culturing at constant temperature of 28 ℃ for 7-14d, taking the strain inoculated by a basic culture medium without any carbon source as a blank control, and observing the growth condition of the strain. If the strain can grow, the strain can utilize the carbon source; if the strain can not grow, the strain can not utilize the carbon source.
(3) Experiment for utilization of Single Nitrogen Source
Nitrogen sources selected for the test: histidine, methionine, serine, oxamic acid, glycine, hydroxyproline, phenylalanine, glutamic acid, cysteine, arginine, valine, ammonium molybdate tetrahydrate, ammonium acetate, ammonium nitrate, ammonium sulfate were added to the basal medium at a concentration of 0.5%. Inoculating strains, culturing at constant temperature of 28 deg.C for 7-14d, taking strains inoculated by basal medium without any nitrogen source as blank control, and observing growth condition of the strains. If the strain can grow, the strain can utilize the nitrogen source; if the strain can not grow, the strain can not utilize the nitrogen source.
(4) Measurement of other physiological and biochemical parameters
Temperature tolerance experiment:
inoculating the strain to be tested in the same culture medium, culturing at 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C and 36 deg.C for 7-14d under the condition of identical culture conditions, observing and recording the growth condition of bacterial colony, thereby determining the optimum temperature for strain growth.
② pH tolerance experiment:
the strains to be tested were inoculated into liquid media with pH values of 4, 5, 6, 7, 8, 9, and 10, respectively, and the other culture conditions were kept consistent, and the culture was carried out at 28 ℃ with observation every other week for four weeks. The growth of the strain was observed and recorded each time to determine the optimum pH for growth of the strain.
Salt tolerance test:
respectively inoculating the strains to be detected on NaCl (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) culture media with different concentrations, wherein other nutrient components of the culture media are the same, culturing at 28 ℃, taking 7 days as an observation period, observing for 4 weeks, and recording whether the strains can grow so as to determine the upper and lower limit concentrations of NaCl which can be tolerated by the strains.
Hydrogen sulfide production experiment:
inoculating a strain to be detected on a hydrogen sulfide culture medium containing ammonium ions in a certain proportion, and culturing for 2 weeks at 28 ℃, wherein if the culture medium is black, hydrogen sulfide is generated; and if no black color exists, no hydrogen sulfide is generated. 2.1.7 molecular biological identification of antagonistic strains
(1) Extraction of actinomycete genomic DNA
Total DNA was extracted using biotek's kit for rapid extraction of bacterial genomic DNA (DP1301, Beijing Baitach Biotech Co., Ltd., China).
(2) Sequencing and analysis of 16S rDNA
PCR amplification of 16S rDNA:
PCR amplification was performed using actinomycete genomic DNA as a template and universal primers 27F and 1492R. The primer sequences are as follows: the upstream primer 27F (5 'AGAG TTTG ATCC TGGC TCAG 3'), and the downstream primer 1492R (5 'TACG GCTA CCTT GTTACGAC TT 3'). The specific reaction system is shown in Table 7, and the reaction procedures (Zhoujun nu, 2014; Heufaphenanthrene, 2015; Na Yua, 2014) are shown in Table 8.
PCR reaction system of table 716S rDNA gene
TABLE 816 PCR amplification reaction conditions for S rDNA genes
Secondly, electrophoresis detection of PCR products:
after the PCR reaction is finished, 5 mu L of PCR amplification product is taken to carry out electrophoresis detection on the PCR product of the strain on 1% agarose gel, and whether the connection is successful is determined according to the length of the target fragment.
Sequencing and constructing phylogenetic tree:
the PCR product of the strain was sent to Huada Gene Co for sequencing. The determined gene sequences were aligned using BLAST software and compared for homology to the known 16S rDNA in GenBank and ezbiocoud databases. Sequences with higher homology are found out for multiple matching array analysis, and MEGA5.1 software is adopted to carry out clustering analysis and phylogenetic tree construction by a Neighbor-Joining method (Na Yua, 2014; Jianghua, 2015).
2.2 evaluation of bacteriostatic Activity of antagonistic Strain
2.2.1 determination of plate-confronted broad-spectrum antibacterial activity
Performing broad-spectrum determination on the strains by adopting a plate confrontation method: and (3) taking a purified bacterial cake of 12 plant pathogenic bacteria by using a 5mm puncher, inoculating the bacterial cake to the center of a PDA (personal digital assistant) flat plate, respectively inoculating a small amount of bacteria to be detected at four points 2.5cm away from the bacterial cake of the pathogenic bacteria, taking a culture dish only inoculated with the pathogenic bacteria as a blank control group, and repeating the treatment for 3 times. After culturing in an incubator for 4-7 days, measuring the colony growth diameter of pathogenic bacteria to be tested by adopting a cross method, and counting the bacteriostasis rate (Xiexing, 2011; Xiongdan, 2013) according to the following formula:
colony diameter (mm) — average value of colony diameters measured-5.0
3 results and analysis
3.1 screening and morphological characterization of Actinomycetes
Carrying out plate coating separation and streaking on the purified strains, removing the weight according to the colony morphology and the color of the strains on a purification culture medium, and carrying out primary screening by a plate opposition culture method and secondary screening by an Oxford cup method to obtain 1 actinomycete generating the largest inhibition zone; numbered XJC-SDSIM 1. The forms of spore filament (A) and spore (B) of strain XJC-SDSIM1 are shown in figure 1 and table 9, intrabasal hyphae of strain XJC-SDSIM1 are developed and not broken, and aerial hyphae are branched; spore silks are wavy and spore surfaces are smooth.
TABLE 9 culture characteristics of Strain XJC-SDSIM1 on 6 media
3.2 physiological and Biochemical characteristics of the Strain
TABLE 10 part of the physiological and biochemical characteristics of Strain XJC-SDSIM1
Positive result; negative result.
3.3 phylogenetic characteristics of the strains
Extracting total DNA of the strain XJC-SDSIM1, obtaining a 16S rDNA sequence of about 1.5kb through PCR amplification, sequencing to obtain a sequence thereof, submitting sequence information to EzTaxon for gene sequence similarity search to obtain sequence information of 16 strain model bacteria which have highest homology with the strain XJC-SDSIM1 and are named totally, performing phylogenetic analysis, and constructing a phylogenetic tree (figure 2). As can be seen from the figure, XJC-SDSIM1 is polymerized with Streptomyces to form a branch, and the homology of the 16S rDNA sequence is between 98.97 percent and 99.78 percent; among them, the homology with Streptomyces sanglieri is the highest and is 99.78%. Combining morphological characteristics, physiological and biochemical characteristics and the analysis result of 16s DNA molecular sequence, the strain XJC-SDSIM1 is preliminarily identified as Streptomyces aureoflavus (Streptomyces sanglieri).
3.4 evaluation of antibacterial Activity of Strain XJC-SDSIM1
3.4.1 antagonistic action of the plates on pathogenic bacteria
TABLE 11 inhibitory Effect of Strain XJC-SDSIM1 on 12 pathogenic fungi
The data in the table are mean ± standard deviation. The different small letters in the same column indicate significant differences at P < 0.05 as tested by Duncan's New Complex Pole Difference method.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (7)
1. An actinomycete, which is streptomyces aureoflavus (Streptomyces aureoflavus) ((Streptomyces aureoflavus))Streptomyces Sanglieri) XJC-SDSIM1, with the preservation number of CCTCC NO: m2017491.
2. Use of the actinomycetes according to claim 1 for antagonizing colletotrichum capsici, and/or fusarium oxysporum, and/or pyricularia oryzae, and/or fusarium graminearum, and/or verticillium malorum, and/or anthracnose of strawberry.
3. Use of the actinomycete according to claim 1 for controlling pepper anthracnose and/or cucumber fusarium wilt and/or rice blast and/or wheat scab and/or apple ring rot and/or strawberry anthracnose.
4. A fermentation broth of the actinomycete of claim 1.
5. Use of the fermentation broth according to claim 4 for antagonism of Colletotrichum capsici, and/or Fusarium oxysporum, and/or Pyricularia oryzae, and/or Fusarium graminearum, and/or Phyllostachys malorum, and/or Fragaria ananasi.
6. Use of the fermentation broth according to claim 4 for the control of pepper anthracnose, and/or cucumber fusarium wilt, and/or rice blast, and/or wheat scab, and/or apple ring rot, and/or strawberry anthracnose.
7. A microbial preparation comprising the actinomycetes according to claim 1.
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