CN100560716C - Grass green streptomycete - Google Patents
Grass green streptomycete Download PDFInfo
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- CN100560716C CN100560716C CNB2008100316051A CN200810031605A CN100560716C CN 100560716 C CN100560716 C CN 100560716C CN B2008100316051 A CNB2008100316051 A CN B2008100316051A CN 200810031605 A CN200810031605 A CN 200810031605A CN 100560716 C CN100560716 C CN 100560716C
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Abstract
The invention discloses a streptomycete bacterial strain, this bacterium is grass green streptomycete (Streptomycesherbaricolor strain) HNS2-2, carried out preservation on June 16th, 2008 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC NO.2540.Its 16SrDNA is included by GenBank, and login sequence number is EU035296.Separation screening obtains in the actinomycetes of this bacterial strain from the shenlongjia pedotheque; its ferment filtrate has the effect of resisting tobacco mosaic virus (TMV); in the potted plant test, TMV there is the obvious suppression effect, the host is had provide protection and therapeutic action; disease index is respectively 7.21,8.49,10.91; prevention effect is respectively 61.88%, 54.06% and 42.37%, can be applicable in the control of tobacco mosaic virus disease of each kind of plant.
Description
Technical field:
The present invention relates to microbial technology field, refer to a kind of microorganism grass green streptomycete of Antiphytoviral especially.
Background technology:
The viroses of plant are main diseases of farm crop, have 1000 various plants virus diseases to be familiar with by common people at present, because it is of a great variety, generation is general, harm is serious, become the important object that the investigator pays close attention to and studies already.Come the controlling plant virus disease although developed multiple control strategy at present, but, virus relies on the host fully because duplicating required material, energy and place in vegetable cell, and plant does not have complete immunity system, up to now, almost there is not chemical agent effectively the virus on the disease plant to be removed fully, chemical agent has brought some negative impacts, its use to be subjected to increasing restriction for ecotope, people and animals' life quality, non-target organism existence etc. simultaneously; In addition, adopt the harm of organizing traditional methods such as detoxification, antiviral breeding also can't fundamentally alleviate virus disease; The antiviral transgenic plant that grew up in nearly 20 years; by the coat protein gene of virus, rdrp gene, proteinase gene, floating preteins gene etc. are imported in the plant materials; to plant generation provide protection in various degree; aspect agriculture production, have a extensive future; but there is certain environmental risk in the antiviral transgenic plant of using the viral genome source.Therefore, development and utilization prevents and treats the focus that the viroses of plant are undoubtedly 21 century from the actives of plant and other Biological resources.
Summary of the invention:
Technical problem to be solved by this invention is: at above-mentioned the deficiencies in the prior art, provide a kind of and can produce plant virus TMV actives, have the microorganism grass green streptomycete that industrial fermentation production forms novel, efficient, nontoxic (low toxicity) agricultural chemicals potentiality.
In order to solve the problems of the technologies described above; the technical solution adopted in the present invention is: the application of a kind of ferment filtrate of grass green streptomycete in antibacterial; this grass green streptomycete (Streptomycesherbaricolor strain) is HNS2-2, separates obtaining from the pedotheque that gather China shenlongjia national natural reserves.Carried out preservation on June 16th, 2008 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC NO.2540, be characterized in: the bio-active substance of the ferment filtrate of this bacterial strain pH4-9,40 ℃-80 ℃, and 2-8 hour condition of 40w fluorescent lamp illumination under the chemical property performance stable, the ferment filtrate of this bacterial strain has the effect of the gram-positive bacteria activity of inhibition.
This grass green streptomycete bacterial strain HNS2-2 has following character:
1. morphological specificity:
Go up in 28 ℃ of these bacterial strains of cultivation after 15 days with the inserted sheet method at No. 1 nutrient agar of Gao Shi (GAU), under opticmicroscope, observe form and the spore shape of mycelia, referring to Fig. 1, Fig. 2, aerial hyphae physically well develops, the straight shape of spore chain, cylindric, substrate mycelium is woven into net, and branch, no diaphragm, non-cracking phenomenon are arranged.Observe under electron microscope, referring to Fig. 3, Fig. 4, spore chain is straight, smooth surface when spore is cylindric, ripe.
From Fig. 1-Fig. 4 as can be known, bacterial strain meets in No. 1 nutrient agar cultivation of Gao Shi 15 days morphological feature and the actinomycetic morphological specificity of streptomyces.
2. the feature on various substratum:
Bacterial strain of the present invention aerial hyphae prosperity on most of substratum, substrate mycelium are enriched, aerial hyphae whitewash, pale purple to light grey, and substrate mycelium is pale yellow to brown.Outstanding, the fine hair shape of bacterium colony on No. 1 nutrient agar of Gao Shi, the pale purple grey of gas silk, base silk is yellow, produce grass green to blackish green soluble pigment, and on inorganic salt Starch Agar substratum the little red and white of gas silk, base silk almond yellow, the shallow colour of camel's hair of soluble pigment; Bacterium colony surface warty produces the brown soluble pigment on the Chai Nasi nutrient agar.Specifically see the following form 1.
The cultural characteristic of table 1 bacterial strain of the present invention
3. physiological and biochemical property and utilization of carbon source situation:
The physiological and biochemical property of bacterial strain of the present invention and utilization of carbon source situation see the following form 2,
The physio-biochemical characteristics of table 2 bacterial strain of the present invention and utilization of carbon source situation
Annotate: "-" expression negative reaction, the strong and weak degree of "+" " ++ " " +++" expression reaction.
By last table 2 as can be known, its physio-biochemical characteristics meet the feature of streptomyces
[119,120]The HNS2-2 bacterial strain can make gelatine liquefication, starch hydrolysis, milk solidifies and peptonize, and can produce hydrogen sulfide, and can strong starch-splitting, but can not decomposition of cellulose, can not utilize the tyrosine chromogenesis, and the nitrate that can not reduce is nitrite.Utilize D-semi-lactosi, glucose, fructose, sucrose, wood sugar, do not utilize N.F,USP MANNITOL, inositol, maltose, rhamnosyl.
Further the employing molecular biology method carries out the evaluation on the molecule, analyzes through the 16SrDNA gene sequencing, and the 16SrDNA partial sequence that gets bacterial strain of the present invention is as follows:
gagtttgatc?ctggctcagg?acgaacgctg?gcggcgtgct?taacacatgc?aagtcgaacg
atgaagccct?tcggggtgga?ttagtggcga?acgggtgagt?aacacgtggg?caatctgccc
ttcactctgg?gacaagccct?ggaaacgggg?tctaataccg?gatacgactg?cgggaggcat
ctcctgtggt?ggaaagctcc?ggcggtgaag?gatgagcccg?cggcctatca?gcttgttggt
ggggtaatgg?cccaccaagg?cgacgacggg?tagccggcct?gagagggcga?ccggccacac
tgggactgag?acacggccca?gactcctacg?ggaggcagca?gtggggaata?ttgcacaatg
ggcgaaagcc?tgatgcagcg?acgccgcgtg?agggatgacg?gccttcgggt?tgtaaacctc
tttcagcagg?gaagaagcga?aagtgacggt?acctgcagaa?gaagcgccgg?ctaactacgt
gccagcagcc?gcggtaatac?gtagggcgca?agcgttgtcc?ggaattattg?ggcgtaaaga
gctcgtaggc?ggcttgtcac?gtcggatgtg?aaagcccgag?gcttaacctc?gggtctgcat
tcgatacggg?ctagctagag?tgtggtaggg?gagatcggaa?ttcctggtgt?agcggtgaaa
tgcgcagata?tcaggaggaa?caccggtggc?gaaggcggat?ctctaggcca?ttactgacgc
tgaggagcga?aagcgtgggg?agcgaacagg?attagatacc?ctggtagtcc?acgccgtaaa
cgttgggaac?taggtgttgg?cgacattcca?cgtcgtcggt?gccgcagcta?acgcattaag
ttccccgcct?ggggagtacg?gccgcaaggc?taaaactcaa?aggaattgac?gggggcccgc
acaagcggcg?gagcatgtgg?cttaattcga?cgcaacgcga?agaaccttac?caaggcttga
catataccgg?aaagcattag?agatagtgcc?ccccttgtgg?tcggtataca?ggtggtgcat
ggctgtcgtc?agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgca
Correlated series among the 16S rDNA sequence of HNS2-2 bacterial strain and the GenBank is carried out the BLAST similarity analysis, adopt MEGA software with close sequence construct phylogenetic tree, the results are shown in Figure 5 with 14 high strain bacterial strains of homology.
The BLAST comparison result of correlated series shows among the 16SrDNA sequence of HNS2-2 bacterial strain and the GenBank, HNS2-2 belongs to streptomyces (Streptomyces), produce the sequence homology of look streptomycete S.racemochromogenes strain NRRL B-5430 (DQ026656.1) and voluminous look streptomycete S.polychromogenes strain NRRL B-3032 up to 99% with spike, with the sequence homology of lilac grey streptomycete S.lavendulae DNA strain IFO 14028 (D85114.1) be 98%, with the sequence homology of the type strain S.herbaricolor strain NRRL B-3299T of grass green streptomycete be 100%, grow on the tree at phyletic evolution and be in same branch.
By streptomycete of the present invention being carried out the mensuration of morphological specificity, cultural characteristic, physio-biochemical characteristics and 16SrDNA sequence, show that the characteristic feature that bacterial strain HNS2-2 has a type strain S.herbaricolor strain NRRL B-3299T of grass green streptomycete promptly produces grass green diffustivity pigment, the straight shape of fibrillae of spores in substratum; Therefore both sequence homologies are 100%, it are included into grass green streptomycete, and in the GenBank registration, login sequence number is EU035296.
Description of drawings:
Fig. 1 is the mycelia form photo of grass green streptomycete bacterial strain HNS2-2 of the present invention on No. 1 nutrient agar of Gao Shi.
Fig. 2 is the spore shape photo of grass green streptomycete bacterial strain HNS2-2 of the present invention on No. 1 nutrient agar of Gao Shi.
Fig. 3 is the spore electromicroscopic photograph one of grass green streptomycete bacterial strain HNS2-2 of the present invention on No. 1 nutrient agar of Gao Shi.
Fig. 4 is the spore electromicroscopic photograph two of grass green streptomycete bacterial strain HNS2-2 of the present invention on No. 1 nutrient agar of Gao Shi.
Fig. 5 is that grass green streptomycete HNS2-2 of the present invention is according to the bacterial strain of 16SrDNA sequence construct and the phylogenetic tree of relevant bacterial strain.
Fig. 6 is that the ferment filtrate of grass green streptomycete bacterial strain HNS2-2 of the present invention is to the preventive effect figure of TMV on the systemic infection host.Wherein: a: contrast b: handle c: handle d through therapeutic action: handle through restraining effect through provide protection.
Fig. 7 is the influence of the antibacterial bio-active substance stability of acid base pair.
Fig. 8 is the influence of temperature to antibacterial bio-active substance stability.
Embodiment:
Below specify the separation screening process and the application example of bacterial strain of the present invention.
Bacterial strain of the present invention adopts dilution-plate method to separate the actinomycetes with anti-plant pathogenic microorganisms from the shenlongjia pedotheque and obtains, and separating screening method is as follows:
1. strains separation
Adopt dilution-plate method, the earth sample 10g that fetches earth adds sterilized water 90mL and mixes the preparation sample suspension, carries out 10 times of gradient dilutions subsequently;
Get the proper concn diluent and (be generally 10
3, 10
4, or 10
5Sample diluting liquid) coating is inoculated in (No. 1 synthetic agar of Gao Shi: Zulkovsky starch 20g, KNO in No. 1 substratum of Gao Shi
31g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, NaCL0.5g, FeSO
40.01g, agar 20g, distilled water 1000mL, pH7.2～7.4.) put 28 ℃ to cultivate 4d.The single bacterium colony slant preservation of picking is divided into from preservation and counts the strain actinomycetes.
2. the primary dcreening operation of actinomycetes strain and multiple sieve
1) preparation of soil actinomycete ferment filtrate
Respectively with the single colony inoculation of actinomycetes in No. 1 liquid nutrient medium of Gao Shi, put 28 ℃ of shaking tables, 220rpm cultivates 4d, fermented liquid has special aroma than thickness, and microscopy do not have living contaminants, then acidifying is filtered, the filtrate refrigerator is preserved standby.
2) inoculation method
Getting TMV infects the disease leaf and adds an amount of 0.01mol/L pH7.2 phosphoric acid buffer (7mL phosphoric acid buffer/g sick leaf), put in the sterilization mortar and grind to form homogenate, 4 layers of filtered through gauze, spray 400 order quartz sands, adopt conventional juice frictional inoculation, inoculation back 5min-10min washes blade gently with clear water, and test is carried out in 25 ℃ of-30 ℃ of no worm greenhouses.
3) try cause of disease with tobacco mosaic virus (TMV) TMV as confession, the common tobacco K326 of systemic infection host, withered spot host thorn apple are as test plant, each strain fermentation filtrate is as disease-resistant bio-active substance, employing frictional inoculation method inoculation system infects the common tobacco K326 of host, withered spot host thorn apple carries out primary dcreening operation, from the primary dcreening operation result, carry out multiple sieve again, obtain bacterial strain HNS2-2 of the present invention.
Bacterial strain of the present invention is to the preventive effect of TMV on withered spot host thorn apple
The withered spot host thorn apple of getting growing way unanimity, 4-5 sheet leaf is divided into 3 groups respectively; carry out handling by following vitro inhibition effect, provide protection and 3 kinds of inoculations of therapeutic action: behind 5 times of dilutions of bacterial strain HNS2-2 ferment filtrate (20%) and TMV equal-volume the mixings 1h inoculation, the preceding 24h of inoculation and inoculate after 24h spray 20% ferment filtrate respectively; adopt ordinary method mechanical friction inoculation; every processing 5 strains (3 true leaves of every strain); repeat 2 times; other is provided with contrast; inoculation back 5d investigation scab number is averaged and is calculated withered spot inhibiting rate.
The result shows: 20% ferment filtrate be seeded on the thorn apple after TMV prepares liquid equal-volume mixing 1h, average withered spot number is 4.38.Not adopting the average withered spot number of thorn apple blade of the ferment filtrate of bacterial strain of the present invention is 56.55.Thereby withered spot inhibiting rate is 92.62%.Ferment filtrate is respectively 9.16,18.65 in the average withered spot number that the forward and backward 24h of inoculation uses, and withered spot inhibiting rate is respectively 83.78%, 67.26%.And relatively carry out the withered spot host thorn apple potted plant behind the different treatment 5d, the withered spot number amount has notable difference, but the influence of plant growing way is not had obvious difference.Test-results illustrates in the ferment filtrate of this bacterial strain and contains the meta-bolites that plant virus TMV is had biological action that virus is had stronger vitro inhibition effect, simultaneously the host is had certain protection effect and therapeutic action.
Bacterial strain of the present invention is to the preventive effect of TMV on the systemic infection host
Get the consistent common cigarette K326 of growing way; every processing 30 strains; repeat 3 times; carrying out 3 kinds of inoculations of following vitro inhibition effect, provide protection and therapeutic action handles: behind 5 times of dilutions of bacterial strain HNS2-2 ferment filtrate (20%) and TMV equal-volume the mixings 1h inoculation, the preceding 24h of inoculation and inoculate after 24h spray the ferment filtrate of 5 times of dilutions (20%) respectively; adopt ordinary method mechanical friction inoculation; morbidity " Invest, Then Investigate " disease index, the calculating prevention effect of averaging.
The result shows: on common tobacco K326; the HNS2-2 ferment filtrate has the obvious suppression effect, the host is had provide protection and therapeutic action TMV; disease index is respectively 7.21,8.49,10.91, and prevention effect is respectively 61.88%, 54.06% and 42.37%.Common tobacco K326 behind provide protection, therapeutic action, 3 kinds of processing of restraining effect 30d, as can be seen from Figure 6, all there is the morbidity phenomenon on common cigarette K326, but the state of an illness is lower than contrast, control group part plant complete stool blade floral leaf, leaf malformation also forms linear leaf, and diseased plant is downgraded; And lobus cardiacus veinclearing, slight floral leaf or 1/3-1/2 blade floral leaf only appear in treatment group, do not have plant to downgrade and the leaf malformation phenomenon.
As from the foregoing, the ferment filtrate of bacterial strain of the present invention infects TMV and has prophylactic effect, restraining effect and therapeutic action, has Antiphytoviral TMV activity.
Further the bio-active substance to bacterial strain of the present invention carries out the chemical property stability test, promptly adopt pH1-12 (result is referring to Fig. 7) respectively, 40 ℃, 60 ℃, 80 ℃, 100 ℃, ferment filtrate is handled in 120 ℃ (result is referring to Fig. 8) and the illumination of 40w fluorescent lamp and sunlight illumination (the results are shown in Table 3), with the streptococcus aureus is the indication bacterial classification, (step of cup-plate method is: will melt the nutrient agar that is cooled to 45 ℃ to adopt cup-plate method to measure the ferment filtrate bacteriostatic activity, by 0.2% (V/V) inoculum size, insert for examination bacterial liquid culture, pour into after mixing in the sterilized culture dish, promptly in each culture dish, place the Oxford cup after waiting to solidify, the Oxford cup is equidistantly placed, if sterilized water is done blank, in each Oxford cup, insert the fermented liquid that 200 μ L had handled through film filter then, each is handled and repeats 3 times, averages.Place 36 ℃ constant incubator, measure antibacterial circle diameter behind the cultivation 24h.)。
Table 3 light is to the influence of antibacterial bio-active substance stability
The bio-active substance that the result shows bacterial strain of the present invention respectively under 2-8 hour condition of pH4-9,40 ℃-80 ℃, the illumination of 40w fluorescent lamp performance stable.
In adopting the strain fermentation filtrate bacteriostatic activity test of the present invention of cup-plate method mensuration, show that also its ferment filtrate has the obvious suppression effect to streptococcus aureus, Bacillus subtillis, and form bigger inhibition zone; And it is little to the inhibition circle of intestinal bacteria and Salmonella.Its this bacterial strain of presentation of results activeconstituents is effective to the gram-positive microorganism restraining effect.Sickle-like bacteria, mould are had the obvious suppression effect, and its inhibiting power is gibberella＞sickle-like bacteria＞mould.
Sequence table
＜110〉Agricultural University Of Hunan
＜120〉grass green streptomycete
<130>PHW08443
<140>200810031605.1
<141>2008-06-27
<160>1
<170>PatentIn?version?3.1
<210>1
<211>1074
<212>DNA
＜213〉streptomyces (Streptomyces)
<400>1
gagtttgatc?ctggctcagg?acgaacgctg?gcggcgtgct?taacacatgc?aagtcgaacg 60
atgaagccct?tcggggtgga?ttagtggcga?acgggtgagt?aacacgtggg?caatctgccc 120
ttcactctgg?gacaagccct?ggaaacgggg?tctaataccg?gatacgactg?cgggaggcat 180
ctcctgtggt?ggaaagctcc?ggcggtgaag?gatgagcccg?cggcctatca?gcttgttggt 240
ggggtaatgg?cccaccaagg?cgacgacggg?tagccggcct?gagagggcga?ccggccacac 300
tgggactgag?acacggccca?gactcctacg?ggaggcagca?gtggggaata?ttgcacaatg 360
ggcgaaagcc?tgatgcagcg?acgccgcgtg?agggatgacg?gccttcgggt?tgtaaacctc 420
tttcagcagg?gaagaagcga?aagtgacggt?acctgcagaa?gaagcgccgg?ctaactacgt 480
gccagcagcc?gcggtaatac?gtagggcgca?agcgttgtcc?ggaattattg?ggcgtaaaga 540
gctcgtaggc?ggcttgtcac?gtcggatgtg?aaagcccgag?gcttaacctc?gggtctgcat 600
tcgatacggg?ctagctagag?tgtggtaggg?gagatcggaa?ttcctggtgt?agcggtgaaa 660
tgcgcagata?tcaggaggaa?caccggtggc?gaaggcggat?ctctaggcca?ttactgacgc 720
tgaggagcga?aagcgtgggg?agcgaacagg?attagatacc?ctggtagtcc?acgccgtaaa 780
cgttgggaac?taggtgttgg?cgacattcca?cgtcgtcggt?gccgcagcta?acgcattaag 840
ttccccgcct?ggggagtacg?gccgcaaggc?taaaactcaa?aggaattgac?gggggcccgc 900
acaagcggcg?gagcatgtgg?cttaattcga?cgcaacgcga?agaaccttac?caaggcttga 960
catataccgg?aaagcattag?agatagtgcc?ccccttgtgg?tcggtataca?ggtggtgcat 1020
ggctgtcgtc?agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag?cgca 1074
Claims (1)
1, the application of a kind of ferment filtrate of grass green streptomycete in antibacterial, this grass green streptomycete (Streptomyces herbaricolor strain) is HNS2-2, carried out preservation on June 16th, 2008 at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC NO.2540, it is characterized in that: the bio-active substance of the ferment filtrate of this bacterial strain is at pH4-9,40 ℃-80 ℃, and the chemical property performance is stable under 2-8 hour condition of 40w fluorescent lamp illumination, and the ferment filtrate of this bacterial strain has the effect of the gram-positive bacteria activity of inhibition.
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| CN101921815B (en) * | 2010-09-01 | 2011-10-05 | 湖南农业大学 | Method for producing flavonoid compound by streptomyces |
| CN111066516B (en) * | 2019-12-19 | 2022-04-22 | 扬州大学 | Seedling leaf rapid inoculation method for barley yellow mosaic virus |
| CN117050919B (en) * | 2023-10-10 | 2024-01-02 | 南昌大学 | Application of rhodococcus strain ND011 and tobacco planting method |
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Non-Patent Citations (2)
| Title |
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| 链霉菌菌株HNS222 的分类鉴定及其代谢产物抗烟草花叶病毒活性. 陈力力等.中国生物防治，第24卷第1期. 2008 |
| 链霉菌菌株HNS222 的分类鉴定及其代谢产物抗烟草花叶病毒活性. 陈力力等.中国生物防治，第24卷第1期. 2008 * |
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