CN102337239B - Streptomyces sp. strain and application thereof for preparing formulation for inhibiting plant virus diseases - Google Patents
Streptomyces sp. strain and application thereof for preparing formulation for inhibiting plant virus diseases Download PDFInfo
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Abstract
The invention relates to a Streptomyces sp. ZX01 strain. A fermentation product obtained through fermentation culture of the Streptomyces sp. ZX01 strain can be used for preventing and controlling tobacco mosaic virus (TMV), cucumber mosaic virus (CMV) and barley yellow dwarf virus (BYDV) and various other plant virus diseases. The Streptomyces sp. ZX01 strain with the accession number of CGMCC No.4893 is preserved in China General Microbiological Culture Collection Center in May 24, 2011.
Description
Technical field
The invention belongs to microorganism and Application Areas thereof, be specifically related to a kind of streptomyces strain and for the preparation of the application of control of plant virosis preparation.
Background technology
Plant virus is a kind of cytozoicus pathogen with dip-dye activity that is comprised of nucleic acid and protein, and according to the 8th report of ICTV (ICTV), the plant virus kind of at present whole world discovery reaches 1122.The caused Plant diseases of plant virus have the title of " plant cancer ", and its hazard rating to farm crop is only second to the mycosis original, and almost every kind of farm crop all are subject to the harm of 2～3 kinds of viruses.Global Agriculture production in viroses of plant serious harm, it is reported, whole world every year, due to illness the plant loss that causes of poison harm reached 60,000,000,000 dollars unexpectedly, wherein only the financial loss in an every year of food crop just up to 20,000,000,000 dollars.
China has a lot of important viruses to spread all over national major production areas because the loss that plant virus harm causes is difficult to counting every year.Such as 70～eighties of twentieth century, the northern area of China is popular because of wheat class yellow dwart, bushy stunt, the general underproduction 20%～30%, and serious reaches 60%, even total crop failure.In recent years, cucumber mosaic virus, tobacco mosaic virus (TMV) and blister beetle mosaic virus cause many vegetables underproduction even total crop failure.Therefore, the control of crop virosis evil all is a great problem of puzzlement agriculture production all the time.
Because the special born of the same parents' endoparasitism of plant virus combines the propagation of virus self and host's metabolism together; Plant lacks complete immunity system in addition, in a single day virus is contaminated plant, just can survive indefinitely in vegetable cell, until the host is dead.Therefore, so that the control of the viroses of plant is more difficult with respect to the control of the pests such as sick (fungi, bacterium), worm, grass.In addition, the plant virus kind is many, and the route of transmission is wide and exist and change into new virus and be incorporated into the genomic risk of host, has also strengthened the difficulty of Control of Plant Virus Disease.At present, except immunological species, not yet developing has selectivity highly and the desirable preparation of or low toxicity nontoxic to the host to virus.The anti-virus formulation of present stage exploitation is greatly mainly with putting prevention first, and how undesirable drug effect is, its inhibiting rate generally only has 30%～60% under best working concentration, add pharmacy variety more single, so that virus easily produces resistance to these medicaments, the Antiphytoviral new drug of therefore seeking efficient, wide spectrum, safety is difficult point and the focus of current pesticide research.
Summary of the invention
The object of the invention is to, a streptomyces strain (Streptomyces sp.) ZX01 (hereinafter to be referred as bacterial strain ZX01) is provided, and can be used in the application of preparation anti-plant viral disease preparation by this bacterial strain of evidence ZX01.
Bacterial strain ZX01 of the present invention submits China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on May 24th, 2011, and preserving number is CGMCC No.4893.
After measured, the 16SrDNA sequence of this bacterial strain ZX01 is as follows:
tgcagtcgaa cgatgaaccc acttcggtgg gggattagtg gcgaacgggt gagtaacacg 60
tgggcaatct gcccttcact ctgggacaag ccctggaaac ggggtctaat accggatacg 120
accacttcag gcatctgatg gtggtggaaa gctccggcgg tgaaggatga gcccgcggcc 180
tatcagcttg ttggtggggt aatggcccac caaggcgacg acgggtagcc ggcctgagag 240
ggcgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaatattgca caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt 360
cgggttgtaa acctctttca gcagggaaga agcgaaagtg acggtacctg cagaagaagc 420
gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tgtccggaat 480
tattgggcgt aaagagctcg taggcggctt gtcacgtcgg atgtgaaagc ccggggctta 540
accccgggtc tgcattcgat acgggcaggc tagagtgtgg taggggagat cggaattcct 600
ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg gtggcgaagg cggatctctg 660
ggccattact gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt 720
agtccacgcc gtaaacgttg ggaactaggt gttggcgaca ttccacgtcg tcggtgccgc 780
agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840
ttgacggggg cccgcacaag cagcggagca tgtggcttaa ttcgacgcaa cgcgaagaac 900
cttaccaagg cttgacatat accggaaaca tctagagata ggtgccccct tgtggtcggt 960
atacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1020
acgagcgcaa cccttgttct gtgttgccag catgcccttc ggggtgatgg ggactcacag 1080
gagactgccg gggtcaactc ggaggaaggt ggggacgacg tcaagtcatc atgcccctta 1140
tgtcttgggc tgcacacgtg ctacaatggc cggtacaaag agctgcgaag ccgtgaggcg 1200
gagcgaatct caaaaagccg gtctcagttc ggattggggt ctgcaactcg accccatgaa 1260
gtcggagttg ctagtaatcg cagatcagca ttgctgcggt gaatacgttc ccgggccttg 1320
tacacaccgc ccgtcacgtc acgaaagtcg gtaacacccg aagccggtgg cccaacccct 1380
tgtgggaggg agct 1394。
The seed culture condition of this streptomyces strain (Streptomyces sp.) ZX01 is:
Under 28 ℃, 120rpm condition, be placed on and cultivate 36h on the substratum; Described substratum consists of: glucose 10g/L, yeast extract paste 10g/L, sodium-chlor 2.5g/L, magnesium sulfate heptahydrate 0.5g/L, three water dipotassium hydrogen phosphate 0.5g/L, pH value 7.2.
Through evidence, streptomyces strain ZX01 of the present invention can be used for preparing the anti-plant viral disease preparation after fermentation culture.
Fermentation culture conditions is as follows:
Under 28 ℃, 180rpm condition, cultivate 7d; Substratum consists of: millet 10g/L, glucose 10g/L, calcium carbonate 2g/L, sodium-chlor 2.5g/L, peptone 3g/L, pH7.2～7.4.
Through applicant's checking, the fermented liquid after the bacterial strain ZX01 fermentation contains the material that inhibition TMV, CMV and BYDV copy, breed, and can be used for preparing the anti-plant viral disease preparation.
Description of drawings
Fig. 1 is the schematic diagram to streptomycete ZX01 phylogenetic analysis.
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail.
Embodiment
The separation in the soil sample that Xinjiang of China Ka Nasi lake gathers of the application's seminar obtains a bacterial strain, and the applicant is with its called after ZX01, and the seed culture condition of this bacterial strain ZX01 is:
Under 28 ℃, 120rpm condition, cultivate 36h; Substratum consists of: glucose: 10g/L, yeast extract paste: 10g/L, sodium-chlor: 2.5g/L, magnesium sulfate heptahydrate: 0.5g/L, three water dipotassium hydrogen phosphate: 0.5g/L, pH value 7.2.
Determine tentatively that through Morphological Identification and 16SrDNA sequential analysis this bacterial strain ZX01 is streptomyces; Further research is found, the fermented liquid of this bacterial strain has stronger inhibition, passivation and therapeutic action to TMV, CMV and BYDV.
Fermentation culture conditions is as follows:
Under 28 ℃, 180rpm condition, cultivate 7d; Substratum consists of: millet 10g/L, glucose 10g/L, calcium carbonate 2g/L, sodium-chlor 2.5g/L, peptone 3g/L, pH7.2～7.4.Fermented liquid after fermentation can be directly used in preparation anti-plant viral disease preparation.
Streptomycete ZX01 of the present invention submits Chinese common micro-organisms preservation center preservation on May 24th, 2011, and preserving number is CGMCC No.4893.
1, the evaluation of bacterial strain ZX01
The sequence of the morphological specificity of bacterial strain ZX01 of the present invention, cultural characteristic, physiological and biochemical property and 16SrDNA and bacterium classification result are as follows:
Bacterial strain ZX01 is well-grown (seeing Table 1) on most of substratum, bacterium colony fine hair shape on the Gao Shi substratum, and substrate mycelium does not rupture without tabula, and the aerial hyphae growth is vigorous, spore cylindricality, smooth surface.The ZX01 bacterial strain all produces without soluble pigment on various substratum.
Table 1: the cultural characteristic of bacterial strain ZX01
The physio-biochemical characteristics of bacterial strain ZX01 and utilization of carbon source situation see Table 2.Bacterial strain ZX01 gelatine liquefication and nitrate reduction reaction are all positive.Can utilize glucose, D-ribose, sucrose, D-wood sugar, PEARLITOL 25C, D-semi-lactosi, rhamnosyl, pectinose and Mierocrystalline cellulose as sole carbon source, can not utilize D-Fructose and inositol.
Table 2: bacterial strain ZX01 part physiological and biochemical property
| Feature | H-4-2 | Feature | H-4-2 |
| Gelatine liquefication | + | Sucrose | + |
| Starch Hydrolysis | + | The D-wood sugar | + |
| Cellulose utilization | ++ | PEARLITOL 25C | + |
| Nitrate reduction | + | Inositol | - |
| D-Fructose | - | The D-semi-lactosi | + |
| Glucose | ++ | Rhamnosyl | ++ |
| D-ribose | + | Pectinose | + |
Annotate: "+" is positive reaction; "-" is negative reaction; " ++ " is the degree of strength of reaction.
The 16SrDNA sequence of streptomycete ZX01 of the present invention is as follows:
tgcagtcgaa cgatgaaccc acttcggtgg gggattagtg gcgaacgggt gagtaacacg 60
tgggcaatct gcccttcact ctgggacaag ccctggaaac ggggtctaat accggatacg 120
accacttcag gcatctgatg gtggtggaaa gctccggcgg tgaaggatga gcccgcggcc 180
tatcagcttg ttggtggggt aatggcccac caaggcgacg acgggtagcc ggcctgagag 240
ggcgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaatattgca caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt 360
cgggttgtaa acctctttca gcagggaaga agcgaaagtg acggtacctg cagaagaagc 420
gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tgtccggaat 480
tattgggcgt aaagagctcg taggcggctt gtcacgtcgg atgtgaaagc ccggggctta 540
accccgggtc tgcattcgat acgggcaggc tagagtgtgg taggggagat cggaattcct 600
ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg gtggcgaagg cggatctctg 660
ggccattact gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt 720
agtccacgcc gtaaacgttg ggaactaggt gttggcgaca ttccacgtcg tcggtgccgc 780
agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840
ttgacggggg cccgcacaag cagcggagca tgtggcttaa ttcgacgcaa cgcgaagaac 900
cttaccaagg cttgacatat accggaaaca tctagagata ggtgccccct tgtggtcggt 960
atacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1020
acgagcgcaa cccttgttct gtgttgccag catgcccttc ggggtgatgg ggactcacag 1080
gagactgccg gggtcaactc ggaggaaggt ggggacgacg tcaagtcatc atgcccctta 1140
tgtcttgggc tgcacacgtg ctacaatggc cggtacaaag agctgcgaag ccgtgaggcg 1200
gagcgaatct caaaaagccg gtctcagttc ggattggggt ctgcaactcg accccatgaa 1260
gtcggagttg ctagtaatcg cagatcagca ttgctgcggt gaatacgttc ccgggccttg 1320
tacacaccgc ccgtcacgtc acgaaagtcg gtaacacccg aagccggtgg cccaacccct 1380
tgtgggaggg agct 1394。
The 16SrDNA sequencing entrusts spun gold auspicious bio tech ltd in Nanjing to carry out, specific as follows: as to extract genomic dna with lysozyme Method from new fresh thalli, adopt universal primer to carry out the 16SrDNA amplification, the PCR product is sent to the auspicious bio tech ltd of Nanjing spun gold after testing afterwards.Effectively delivering kind of the Phylogenetic dendrogram that the 16SrDNA sequence construct goes out (Fig. 1) from bacterial strain ZX01 and streptomyces can find out: all reference bacterial strains form three large branches altogether.The 16S rDNA sequence similarity of all bacterial strains is between 98.5%～99.1% among bacterial strain ZX01 and the branch III, the closest with the Phylogenetic Relationships of Streptomyces lavendofoliae Kuchaeva, sequence similarity between them is 99.1%, and this is not less than 95% standard and is consistent with the middle 16SrDNA sequence similarity that defines that belongs at present.But this bacterial strain is not met with any known bacterial classification on systematic evolution tree, but independently becomes to prop up with higher evolution difference, illustrates that all there is larger difference in each type strain of this bacterial strain and the same III of branch in evolution.
2, bacterial strain ZX01 is active to the inhibition of TMV
(1) bacterial strain ZX01 inhibition that TMV is just infected
To cultivate the tunning of 7d at 28 ℃, 180rpm condition bottom fermentation centrifugal, supernatant liquor is for subsequent use.Choosing healthy, eugonic 5～6 leaf phase Nicotiana glutinosas is withered spot host, and the whole strain of fermented liquid is smeared, and contrasts whole strain and smears clear water, respectively at whole strain inoculation TMV virus (10 μ g/mL) behind dispenser 12h, 24h and the 48h; Measure bacterial strain ZX01 fermented liquid to the prophylactic effect of TMV virus infection Nicotiana glutinosa.The results are shown in Table 3.
Table 3: the inhibition that bacterial strain ZX01 fermented liquid just infects TMV
Annotate: viral A working concentration is 500mg/L; Fermented liquid is fermenation raw liquid.
(2) bacterial strain ZX01 is to the inhibition of TMV proliferation function
Adopt whole strain method to measure fermented liquid copies propagation in the lobus cardiacus smoke to the TMV virus particle restraining effect.Choosing healthy, eugonic 5～6 leaf phase Nicotiana glutinosas is withered spot host, with the whole blade of TMV virus frictional inoculation, behind 6h, 12h and the 24h Nicotiana glutinosa is processed in the bacterial strain fermentation liquor coating, contrasts whole strain and smears clear water.The results are shown in Table 4.
Table 4: bacterial strain ZX01 fermented liquid copies the inhibition of propagation in the lobus cardiacus smoke to TMV
Annotate: viral A working concentration is 500mg/L; Fermented liquid is fermenation raw liquid.
Adopt the leaf disk method to measure fermented liquid copies propagation in common tobacco K326 body to TMV effect.Choose well-grown common cigarette K326, behind the blade inoculation TMV 6h, break into the leaf disc that diameter is 12mm with punch tool at the inoculation tobacco leaf, leaf disc is flown in fermented liquid and the viral A liquid (mass concentration is 0.5mg/mL), virus inoculation facing up, and respectively with the leaf disc that flies at virus inoculation in the distilled water and the not positive contrast of health tobacco leaf disc and the negative control of virus inoculation, 10 leaf discs of every processing.After processing 48h the PBST damping fluid of each leaf disc with 1: 10 (w/v) is coated with, centrifugal after grinding, supernatant liquor is measured the OD value at its 405nm place with indirect elisa method, the results are shown in Table 5.
Table 5: bacterial strain ZX01 fermented liquid is to the inhibition of TMV in common cigarette propagation
| Process | Average OD 405Value | Viral level/μ g/mL | Inhibiting rate/% |
| Fermented liquid | 0.6227 | 1.4835 | 53.27 |
| Virus A | 0.6218 | 1.4809 | 53.35 |
| CK(+) | 1.2162 | 3.1744 | - |
| CK(-) | 0.1094 | - | - |
Annotate: viral A working concentration is 500mg/L; Fermented liquid is fermenation raw liquid.
3, bacterial strain ZX01 is active to the inhibition of CMV
To place the solarium to cultivate for being seeded in the seedling pan after the examination tobacco K326 seed disinfection, virus inoculation when treating that the cigarette seedling has 5～7 true leaves.Adopt artificial frictional inoculation, investigate respectively the incidence of cigarette strain behind the inoculation 20d.Can be found out by result's (table 6), the preventive effect of processing and result for the treatment of are respectively 63.91% and 52.48%.
Table 6: bacterial strain ZX01 fermented liquid is to the prevention effect of yellow stunt of wheat
Annotate: viral A working concentration is 500mg/L; Fermented liquid is fermenation raw liquid.
4, bacterial strain ZX01 is active to the inhibition of BYDV
In the wheat phase of standing up barly yellow dwarf virus GAV strain is inoculated on the wheat, in flowering stage of wheat statistics sickness rate and disease index.Can be found out by result's (table 6), the preventive effect of processing and result for the treatment of are respectively 58.36% and 46.82%.
Table 6: bacterial strain ZX01 fermented liquid is to the prevention effect of yellow stunt of wheat
Annotate: viral A working concentration is 500mg/L; Fermented liquid is fermenation raw liquid.
Result according to above example shows, bacterial strain ZX01 of the present invention is through liquid fermentation and culture, contain the material that inhibition TMV, CMV and BYDV copy, breed in its fermented liquid, can significantly alleviate the generation of virus disease, be expected to be developed as the microbial reagent of a kind of novel control plant virus.
tgcagtcgaa cgatgaaccc acttcggtgg gggattagtg gcgaacgggt gagtaacacg 60
tgggcaatct gcccttcact ctgggacaag ccctggaaac ggggtctaat accggatacg 120
accacttcag gcatctgatg gtggtggaaa gctccggcgg tgaaggatga gcccgcggcc 180
tatcagcttg ttggtggggt aatggcccac caaggcgacg acgggtagcc ggcctgagag 240
ggcgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300
gaatattgca caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt 360
cgggttgtaa acctctttca gcagggaaga agcgaaagtg acggtacctg cagaagaagc 420
gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tgtccggaat 480
tattgggcgt aaagagctcg taggcggctt gtcacgtcgg atgtgaaagc ccggggctta 540
accccgggtc tgcattcgat acgggcaggc tagagtgtgg taggggagat cggaattcct 600
ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg gtggcgaagg cggatctctg 660
ggccattact gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt 720
agtccacgcc gtaaacgttg ggaactaggt gttggcgaca ttccacgtcg tcggtgccgc 780
agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840
ttgacggggg cccgcacaag cagcggagca tgtggcttaa ttcgacgcaa cgcgaagaac 900
cttaccaagg cttgacatat accggaaaca tctagagata ggtgccccct tgtggtcggt 960
atacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1020
acgagcgcaa cccttgttct gtgttgccag catgcccttc ggggtgatgg ggactcacag 1080
gagactgccg gggtcaactc ggaggaaggt ggggacgacg tcaagtcatc atgcccctta 1140
tgtcttgggc tgcacacgtg ctacaatggc cggtacaaag agctgcgaag ccgtgaggcg 1200
gagcgaatct caaaaagccg gtctcagttc ggattggggt ctgcaactcg accccatgaa 1260
gtcggagttg ctagtaatcg cagatcagca ttgctgcggt gaatacgttc ccgggccttg 1320
tacacaccgc ccgtcacgtc acgaaagtcg gtaacacccg aagccggtgg cccaacccct 1380
tgtgggaggg agct 1394
Claims (3)
- Streptomyces strain ( Streptomyces sp.) ZX01 is for the preparation of the application of anti-plant viral disease preparation, described streptomyces strain ( Streptomyces sp.) deposit number of ZX01 is CGMCC No.4893, the described viroses of plant are: tobacco mosaic virus disease (Tobacco mosaic virus, TMV), Cucumber Mosaic Virus (Cucumber mosaic virus, CMV) and yellow stunt of wheat viral disease (Barley yellowdwarf virus, BYDV).
- 2. application as claimed in claim 1 is characterized in that, described streptomyces strain ( Streptomyces sp.) fermentation culture conditions of ZX01 is: under 28 ℃, 180rpm condition, cultivate 7d; Substratum consists of: millet: 10g/L, glucose: 10g/L, calcium carbonate: 2g/L, sodium-chlor: 2.5g/L, peptone: 3g/L, pH value 7.2 ~ 7.4.
- 3. application as claimed in claim 1 is characterized in that, described streptomyces strain ( Streptomyces sp.) the seed culture condition of ZX01 is, under 28 ℃, 120rpm condition, is placed on culture medium culturing 36h; Described substratum consists of: glucose: 10g/L, yeast extract paste: 10g/L, sodium-chlor: 2.5g/L, magnesium sulfate heptahydrate: 0.5 g/L, three water dipotassium hydrogen phosphate: 0.5g/L, pH value 7.2.
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Effective date of registration: 20141230 Address after: 712100, Shaanxi Yangling Demonstration Zone Industrial Park Yang Yang Road West of the south side Patentee after: Yangling Fu Ji Biological Technology Co. Ltd. Address before: 712100 Shaanxi Province, Xi'an city Yangling District Tai Road No. 3 demonstration Patentee before: Yangling Nongkeda Research & Development Center of Biorational Pesticide |