CN112746037A - Streptomyces castochromogenes strain CPAT-W03 and application thereof - Google Patents
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Abstract
The invention discloses a streptomyces badensis strain CPAT-W03 and application thereof, wherein the streptomyces badensis strain 2 is named in classificationStreptomyces badiusAnd the preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: year 2020, month 01, day 13, accession number: 19342 said Streptomyces auburn belongs to the kingdom of bacteria, phylum Actinomycetes, class Actinomycetes, order Streptomyces, family Streptomycetaceae, genus Streptomyces. The application is the application of the streptomyces badetanus strain 2 in biological control of the cymbidium sinense stem basal rot.
Description
Technical Field
The invention belongs to the technical field of microbial isolation culture, and particularly relates to application of a streptomyces badius bacterial strain CPAT-W03 in biological control of cymbidium sinense stem basal rot.
Background
Molan (C)Cymbidium sinense) Is terrestrial plant of orchid family, and is mainly distributed in China, India, Burma, Vietnam, Thailand, Nippon lucidus Islands, etc. The basal rot of cymbidium sinense stems is one of the main diseases of artificial cultivation and industrial production. The disease is common and is an important disease on orchid, which affects the growth and appreciation of orchid. The disease is mainly caused by the base of the stem of the cymbidium sinense. At the early stage of disease, light brown and water stain-like scabs appear on roots, and the roots become brown and rot after expansion, and stem bases die. The pathogenic bacteria of black orchid stalk base rot is Phoma (Phoma arachidicola). Because the root of the stem is damaged, the leaf apex is dry in light time, the leaf is yellow green, the growth potential is poor, and the whole plant dies in heavy time, so that serious economic loss is caused.
Due to the increasing 3R problem and the characteristics of orchid as indoor ornamental flower, chemical control is no longer suitable for the control of stalk rot of orchid, so biological control is considered as a green and ecologically feasible control method, and a biological strain which can control the stalk rot of cymbidium sinense and is harmless to the environment and human beings is necessary to be screened and separated.
Disclosure of Invention
The first purpose of the invention is to provide a streptomyces badensis strain (A)Streptomyces badius) (ii) a The second purpose is to provide the application of the streptomyces badius CPAT-W03 in the biological control of the stalk base rot of cymbidium sinense.
All percentages used in the present invention are volume percentages unless otherwise indicated.
The first purpose of the invention is realized by that the streptomyces castaneae strain CPAT-W03 is classified and named as (A)Streptomyces badius) And the preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: year 2020, month 01, day 13, accession number: 19342 said Streptomyces West belongs to the kingdom of bacteria, phylum Actinomycetes, class Actinomycetes, order Streptomyces, family Streptomycetaceae, genus Streptomyces.
Streptomyces castaneae (A) of the inventionStreptomyces badius) CPAT-W03, deposited at China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, abbreviated as: CGMCC. The address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, the institute of microbiology, Chinese academy of sciences, zip code 100080). The preservation number is CGMCC No: 19342.
the strain has a good disease inhibiting effect on cymbidium sinense stem basal rot, and can inhibit the growth of pathogenic bacteria without influencing the normal growth of cymbidium sinense.
The bacterial strain can be observed to be inhibited by the cymbidium sinense stem rot pathogenic bacteria on the 5 th day of the opposite culture method, the inhibition phenomenon is more obvious when the bacterial strain is observed on the 7 th day of the opposite culture, and the inhibition zone reaches 6 mm.
The strain is obtained by the following specific steps:
A. collecting a specimen: collecting Panzhihua cycas coralliform mycorrhiza from the cycas natural preservation area in Panzhihua city of Sichuan province;
B. and (3) isolated culture of the strain: adopting a plate marking method to separate the Panzhihua cycas coralliform mycorrhizal endophytic actinomycetes: washing the soil on the surface of coralline mycorrhiza with distilled water, sucking water, sterilizing with 75% alcohol for 1 min, sterilizing with 0.1% mercuric chloride solution for 5 min, sterilizing with 5% sodium hypochlorite solution for 5 min, and rinsing with sterile water for 3 times. Finally rinse 3 times with sterile water. And (3) sucking excess water by using sterile filter paper, fully grinding the coralline mycorrhiza by using a sterile mortar, dipping a small amount of ground tissue fluid by using a sterile inoculating ring, scribing on a Gao's first culture medium plate, and culturing for 7-10 days at 28 ℃. After the colony is formed, picking out a typical single colony by using an aseptic inoculating loop, and purifying the strain in a new Gao's No. one culture medium by using a scribing method;
C. and (3) strain preservation: inoculating pure bacterial colony of the biocontrol standby bacterial strain obtained in the experiment to a test tube inclined plane, culturing at 28 ℃ for 10 days, and storing in a refrigerator at 4 ℃. The preservation medium is a Gao's first culture medium.
The solid culture medium comprises the following components in g/L: 20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of dipotassium phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate heptahydrate, 0.1 g of potassium dichromate, 20 g of agar and 1L of distilled water, wherein the pH value is 7.4-7.6.
The specimen collection place in the step A is a cycas natural preservation area in Panzhihua city, Sichuan province.
The specimen in the step A is collected from the glehnia littoralis coralloid mycorrhiza in the cycas natural preservation area.
And step B, operating under an alcohol lamp, burning an inoculating needle and an inoculating loop on the flame of the alcohol lamp, and operating all separation steps under alcohol and the like in the sterile operating platform.
And B, sterilizing materials required by tests such as an inoculating needle, an inoculating loop, sterile water, tweezers, an empty culture medium and the like in a high-pressure steam sterilizer at 121 ℃ for 30 min.
The separation culture condition of the step B is preferable; and (3) performing static culture in the dark at the temperature of 28 ℃ for 7-10 days.
The conditions for the purification culture and the preservation in the step C are preferred; standing at 28 deg.C in dark for 10 d, and storing at 4 deg.C.
The streptomyces castanensis strain (A) is usedStreptomyces badius) The method for CPAT-W03 in biological control of stem basal rot of cymbidium sinense comprises screening of biocontrol strains and determination of biocontrol effect, and specifically comprises the following steps:
A. screening of biocontrol strains: and (4) primarily screening the antagonistic bacteria by using a cross method. The specific operation is as follows: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) Inoculating 6 mm agar blocks of pathogenic bacteria at the center of improved PDA culture medium plate for indicating bacteria, and inoculating single bacterial colony obtained by separate culture at equal distance of 2.5 cm away from pathogenic bacteriaInoculating 4 strains in a dish, setting 5 times of repeated culture at the constant temperature of 28 ℃ for 5-7 days, observing the size of a bacteriostatic zone, and selecting the strain with the optimal antagonistic effect;
B. measurement of biocontrol effect: in order to screen out strains with stronger bacteriostatic activity, the strains obtained by primary screening are re-screened by adopting a plate confronting growth method: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) For indicating bacteria, firstly, beating activated pathogenic bacteria agar blocks by using a 6 mm puncher, placing the pathogenic bacteria agar blocks on a flat plate, then inoculating antagonistic bacteria by adopting a point inoculation method, wherein the distance between the two bacteria is 2.5 cm, only inoculating the pathogenic bacteria agar blocks in a control group, setting 3 times of repetition, culturing for 5-7 days in an incubator at 28 ℃, observing the growth condition of the pathogenic bacteria, measuring an inhibition zone, calculating an inhibition rate, and finally screening out the antagonistic bacteria with the best antagonistic effect. The formula for calculating the bacteriostasis rate is as follows: bacteriostatic ratio (%) = (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter × 100%.
The improved PDA solid medium comprises the following components in g/L: 200 g of potato, 20 g of glucose, 20 g of agar, 0.5 g of dipotassium phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate and 1L of distilled water, and the pH is natural.
The second purpose of the invention is realized by that the streptomyces castanea (S.flavus) ((S.castanea))Streptomyces badius) Application of CPAT-W03 in biological control of cymbidium sinense stem basal rot.
The beneficial effects of the invention include the following aspects.
1. The strain has good effect of preventing and treating the basal stem rot of cymbidium sinense and has no influence on the normal growth of cymbidium sinense.
2. The bacterial strain can be observed to be inhibited on the 5 th day of opposite culture, and the inhibition phenomenon is more obvious on the 7 th day of opposite culture.
3. The strain is easy to obtain, short in culture time and beneficial to large-scale production, popularization and application.
Drawings
FIG. 1 shows Streptomyces castanensis strain (S.aubergiensis) in example 1Streptomyces badius) CPAT-W03 Strain cultured on Gao's No. one medium at 28 ℃ for 10 daysThe front and the back of the colony morphology;
FIG. 2 shows Streptomyces aubergiensis strains by plate-confrontation method in example 1 (Streptomyces badius) CPAT-W03 is applied to the front and the back of an antagonistic effect chart in the biological control of the cymbidium base rot;
FIG. 3 shows pathogenic bacteria of basal rot of cymbidium sinense (Moss)Phoma arachidicola) Pure culture bacterial colony front and back.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.
The streptomyces castanea strain CPAT-W03 is classified and namedStreptomyces badiusAnd the preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: year 2020, month 01, day 13, accession number: 19342 said Streptomyces auburn belongs to the kingdom of bacteria, phylum Actinomycetes, class Actinomycetes, order Streptomyces, family Streptomycetaceae, genus Streptomyces.
The application of the streptomyces badetanus strain CPAT-W03 is the application of the streptomyces badetanus strain CPAT-W03 in biological control of cymbidium sinense root and stem rot.
The streptomyces castanensis strain (A)Streptomyces badius) CPAT-W03 in the biological control of cymbidium sinense stem basal rot comprises the steps of strain activation and propagation and biocontrol effect determination, and specifically comprises the following steps:
A. strain activation and propagation: the streptomyces castanensis strain (A) is preparedStreptomyces badius) CPAT-W03 is inoculated on an activation and propagation culture medium, and the activation and propagation strain is obtained by culturing for 7-10 days at the temperature of 28 ℃;
B. and (3) biocontrol effect determination: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) And (3) inoculating the strain to an improved PDA culture medium, then inoculating the activated and expanded strain prepared in the step A to the culture medium, culturing for 5-7 days at 28 ℃, and observing the bacteriostasis effect.
The activation and multiplication culture medium is a Gao's first culture medium.
The biocontrol effect is measured by adopting a flat plate confrontation method.
The invention is further illustrated by the following specific examples:
example 1
Streptomyces castochromosis Strain (A)Streptomyces badius) CPAT-W03 was obtained, characterized and deposited.
(1) Streptomyces castochromosis Strain (A)Streptomyces badius) Obtaining and identifying CPAT-W03.
Streptomyces castaneae strain (of the invention)Streptomyces badius) CPAT-W03 is isolated from Panzhihua cycad mycorrhiza. The sample was collected from the cycas revoluta nature reserve area of Panzhihua city, Sichuan province, and stored at 4 ℃ for further use. Adopting a plate marking method to separate the Panzhihua cycas coralliform mycorrhizal endophytic actinomycetes: washing the soil on the surface of coralline mycorrhiza with distilled water, sucking water, sterilizing with 75% alcohol for 1 min, sterilizing with 0.1% mercuric chloride solution for 5 min, sterilizing with 5% sodium hypochlorite solution for 5 min, and rinsing with sterile water for 3 times. Finally rinse 3 times with sterile water. And (3) sucking excess water by using sterile filter paper, fully grinding the coralline mycorrhiza by using a sterile mortar, dipping a small amount of ground tissue fluid by using a sterile inoculating ring, scribing on a Gao's first culture medium plate, and culturing for 7-10 days at 28 ℃. After the colonies formed, a typical single colony was picked with a sterile inoculating loop and the strain was streaked in new Gao's No. one medium.
The solid culture medium comprises the following components in g/L: 20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of dipotassium phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate heptahydrate, 0.1 g of potassium dichromate, 20 g of agar and 1L of distilled water, wherein the pH value is 7.4-7.6.
For the above isolated strain CPAT-W03, the results of the experiments were recorded as follows by morphological identification and molecular biological identification:
A. morphological characteristics: bacterial colony of the strain CPAT-W03 is dark yellow, is nearly circular to irregular, has a fold protruding on the surface and a villous edge, is in a flour shape at the later stage, is dry and opaque, and has a tawny halo around the bacterial colony;
B. 16S rDNA sequence analysis: to bacteria16S rDNA sequence determination is carried out on the strain, the 16S rDNA fragment of the strain is subjected to PCR amplification by taking the total DNA of the strain CPAT-W03 as a template and an actinomycete 16S rDNA universal primer 8-27F (5'-AGAGTTTGATCCTGGCTCAG-3')/1523-1504R (5'-TTAAGGTGATCCAGCCGCA-3'), and the PCR reaction system is as follows: 2 × PowerTaq PCR MasterMix: 12.5 μ L, template DNA: 1 μ L, upstream primer (10 μmol/L): 1 μ L, downstream primer (10 μmol/L): 1 mu L, ddH2O: 9.5 μ L, total volume: 25 muL. PCR reaction procedure: pre-denaturation at 95 deg.C for 5 min, performing the following cycles of denaturation at 95 deg.C for 1 min, annealing at 55 deg.C for 1 min, extension at 72 deg.C for 2 min, 35 cycles of extension at 72 deg.C for 10 min, and storing at 4 deg.C in PCR instrument.
The PCR product is sent to Kunming Shuichi Biotechnology Limited for purification and sequencing, the sequence is spliced and then submitted to EzBioCloud (https:// www.ezbiocloud.net /) for comparison, and the comparison result shows that the strain CPAT-W03 and the streptomyces castanensis (CPAT-W03)Streptomyces badius) (AY999783) the similarity of the 16S rDNA sequence reached 100%. Identification of the strain CPAT-W03 as Streptomyces aubergiensis by sequence alignment and morphological characteristics (S. aubergiensis)Streptomyces badius). The sequence of the strain CPAT-W03 is shown in a sequence table.
(2) Streptomyces castochromosis Strain (A)Streptomyces badius) Collection of CPAT-W03.
From the above identification results, it was confirmed that the strain was Streptomyces castochromogenes (S.) (Streptomyces badius) The serial number is CPAT-W03, and the gene is preserved in China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, abbreviated as: CGMCC. The address is as follows: west road No. 1 institute 3, north academy of sciences, china, zip code 100080) in the area of yang. The accession number is 19342.
Example 2
Streptomyces castochromosis Strain (A)Streptomyces badius) Screening of CPAT-W03 strain.
(1) And (4) screening strains.
And (4) primarily screening the antagonistic bacteria by using a cross method. The specific operation is as follows: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) For indicating bacteria, in improving PDInoculating 6 mm agar blocks of pathogenic bacteria at the center of a culture medium plate A, inoculating single bacterial colonies obtained by separation culture at equal intervals at positions 2.5 cm away from the pathogenic bacteria, inoculating 4 bacterial strains in each dish, setting 5 times of repeated culture, culturing at the constant temperature of 28 ℃ for 5-7 days, observing the size of an inhibition zone, and selecting the bacterial strains with the optimal antagonistic effect.
(2) And (5) observing the result.
The cross method is adopted to screen the separated strains, and the result shows that 7 strains have antagonistic action on the stem basal rot of cymbidium sinense.
Example 3
Streptomyces castochromosis Strain (A)Streptomyces badius) CPAT-W03 strain for biocontrol effect determination.
(1) And (4) measuring the biocontrol effect.
Confrontation culture method: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) For indicating bacteria, firstly, beating activated pathogenic bacteria agar blocks by using a 6 mm puncher, placing the pathogenic bacteria agar blocks on a flat plate, then inoculating antagonistic bacteria by adopting a point inoculation method, wherein the distance between the two bacteria is 2.5 cm, only inoculating the pathogenic bacteria agar blocks in a control group, setting 3 times of repetition, culturing for 5-7 days in an incubator at 28 ℃, observing the growth condition of the pathogenic bacteria, measuring an inhibition zone, calculating an inhibition rate, and finally screening out the antagonistic bacteria with the best antagonistic effect. The formula for calculating the bacteriostasis rate is as follows: bacteriostatic ratio (%) = (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter × 100%.
(2) And (5) observing the result.
The biological control effect of the screened strains is measured by adopting a plate confronting culture method, 7 antagonistic bacteria have antagonistic activity on pathogenic bacteria of the cymbidium base rot, and the inhibition effects of different antagonistic bacteria on pathogenic bacteria hyphae have certain difference. The CPAT-W03 strain has the strongest antagonistic activity and clear boundary of a bacteriostatic circle, and has a bacteriostatic zone of 6 mm for the basal rot pathogen of cymbidium sinense and reaches a significance level. Although the culture time is prolonged, the bacteriostatic circle is always transparent, which indicates that the antagonistic bacteria antagonistic performance is stable.
SEQUENCE LISTING
<110> southwest university of forestry
<120> Streptomyces castaneae strain CPAT-W03 and application thereof
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1287
<212> DNA
<213> 16S rDNA sequence of CPAT-W03 Strain
<400> 1
ctgcccttca ctctgggaca agccctggaa acggggtcta ataccggata acactctgtc 60
ccgcatggga cggggttgaa agctccggcg gtgaaggatg agcccgcggc ctatcagctt 120
gttggtgggg taatggccta ccaaggcgac gacgggtagc cggcctgaga gggcgaccgg 180
ccacactggg actgagacac ggcccagact cctacgggag gcagcagtgg ggaatattgc 240
acaatgggcg aaagcctgat gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta 300
aacctctttc agcagggaag aagcgaaagt gacggtacct gcagaagaag cgccggctaa 360
ctacgtgcca gcagccgcgg taatacgtag ggcgcaagcg ttgtccggaa ttattgggcg 420
taaagagctc gtaggcggct tgtcacgtcg gatgtgaaag cccggggctt aaccccgggt 480
ctgcattcga tacgggctag ctagagtgtg gtaggggaga tcggaattcc tggtgtagcg 540
gtgaaatgcg cagatatcag gaggaacacc ggtggcgaag gcggatctct gggccattac 600
tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 660
cgtaaacgtt gggaactagg tgttggcgac attccacgtc gtcggtgccg cagctaacgc 720
attaagttcc ccgcctgggg agtacggccg caaggctaaa actcaaagga attgacgggg 780
gcccgcacaa gcagcggagc atgtggctta attcgacgca acgcgaagaa ccttaccaag 840
gcttgacata taccggaaag catcagagat ggtgcccccc ttgtggtcgg tatacaggtg 900
gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 960
acccttgttc tgtgttgcca gcatgccctt cggggtgatg gggactcaca ggagactgcc 1020
ggggtcaact cggaggaagg tggggacgac gtcaagtcat catgcccctt atgtcttggg 1080
ctgcacacgt gctacaatgg ccggtacaat gagctgcgat gccgcgaggc ggagcgaatc 1140
tcaaaaagcc ggtctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtt 1200
gctagtaatc gcagatcagc attgctgcgg tgaatacgtt cccgggcctt gtacacaccg 1260
cccgtcacgt cacgaaagtc ggtaaca 1287
Claims (5)
1. A streptomyces badensis strain CPAT-W03 is characterized in that the streptomyces badensis strain is classified and namedStreptomyces badiusAnd the preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: year 2020, month 01, day 13, accession number: 19342 said Streptomyces auburn strain belongs to the bacteria kingdom, Actinomycetes, Streptomyces, Streptomycetales.
2. The application of the Streptomyces aubergiensis strain CPAT-W03 as claimed in claim 1, wherein the Streptomyces aubergiensis strain CPAT-W03 is used for biological control of cymbidium sinense stem basal rot.
3. The application of the Streptomyces aubergiensis strain CPAT-W03 as claimed in claim 2, wherein the Streptomyces aubergiensis strain CPAT-W03 comprises the steps of strain activation and propagation and biocontrol effect determination in biological control of cymbidium sinense stem basal rot, and specifically comprises:
A. strain activation and propagation: inoculating the streptomyces castanea fulvidraco strain CPAT-W03 to an activation and propagation medium, and culturing at 28 ℃ for 7-10 days to obtain an activation and propagation strain;
B. and (3) biocontrol effect determination: pathogenic bacteria of basal rot of cymbidium sinense (A)Phoma arachidicola) Inoculating on modified PDA culture medium, and preparing by step AThe activated and propagated strains are also inoculated on a culture medium, cultured for 5-7 days at 28 ℃, and the bacteriostatic effect is observed.
4. The application of the Streptomyces aubergiensis strain CPAT-W03 in the biological control of the stalk rot of cymbidium sinense as claimed in claim 3, wherein the activation and propagation culture medium is a Gao's first culture medium.
5. The use of the Streptomyces aubergiensis strain CPAT-W03 in the biological control of the stem base rot of the cymbidium sinense as claimed in claim 3, wherein the biological control effect is determined by a plate counter-acting method.
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| CN113046281A (en) * | 2021-05-20 | 2021-06-29 | 福建省农业科学院植物保护研究所 | Streptomyces castochromosis JA58 for preventing and treating potato late blight and application thereof |
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