CN102676420B - Pseudomonas putida strain for bio-control of Tobacco mosaic virus - Google Patents

Abstract

The invention discloses a Pseudomonas putida strain for bio-control of Tobacco mosaic virus, wherein the strain code is A3; the strain is preserved in the General microbial center of China Committee for Culture Collection of Microorganisms on 7 September 2011, and the preservation number of the strain is CGMCC No.5231. The strain can restrain invasion of the Tobacco mosaic virus. The bio-control experiment shows that the Tobacco mosaic virus can be prevented and treated. Simultaneously, the invention also provides a mixture of active small molecule substances obtained from the strain, and themixture has high application value in the respect of prevention and treatment of the Tobacco mosaic virus.

Description

One strain tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion pseudomonas putida bacterial strain

Technical field

The invention belongs to biological technical field, relate to a strain tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion pseudomonas putida bacterial strain, this bacterial strain and the mixture that obtains the active small molecular material from this bacterial strain are in the application of tobacco mosaic viruses aspect preventing and treating.

Background technology

Tobacco virus venereal disease evil is one of the main disease in each cigarette district, the world, and all will cause enormous economic loss every year.The tobacco virus disease of China enters after the seventies in 20th century in existing document record in the early days of foundation, and the harm of virus disease increases the weight of year by year.The State Tobacco Monopoly Bureau that 1989-1991 carries out subsidizes scientific research project " national INVESTIGATION ON INFECTIOUS TOBACCO DISEASES IN ", find out that the virus that causes China's tobacco virus has 16 kinds, what wherein widely distributed, harm was serious has 4 kinds: comprise tobacco mosaic viruses (TMV), tobacco cucumber mosaic virus (CMV), marmor erodens (TEV), marmor upsilon (PVY), secondly also have nepovirus (TRsv), potato virus X (PVX), tobacco necrosis virus (TNv), annulus orae (TSV) etc.According to estimates, because the harm of virus disease, the whole world only just causes about more than 100,000,000 dollars financial loss by harm of tobacco mosaic virus disease every year.

The not homophyletic of tobacco mosaic viruses ties up to the interaction that usually exists gene level and non-genomic level when mix invading the cigarette strain, and homophyletic does not tie up to when successively invading the cigarette strain and can have cross protection yet.Tobacco mosaic viruses has iuntercellular to shift after infecting the cigarette strain in cigarette strain body and shifts two kinds at a distance, plastochondria mainly passes through plasmodesma when iuntercellular shifts, remote shift then by screen casing etc., also have the transport velocity of the virus of reporting relevant with the translational speed of plant nutrition main flow.

The circulation way of tobacco mosaic viruses mainly contains juice and propagates, and as juice friction propagation artificial in the farming operating process of field, mechanical friction propagation etc., also can propagate by soil, and invalid root, stem, the leaf in field all can pass poison in soil.Virus generally infects tobacco by the microtrauma mouth, can cause Ultrastructural variations such as a series of such as lesion tissues such as necrosis, deformity, dwarfing, floral leaf, variable colors, in addition can also blade cell loose, that karyon is undesired, chromatinal mass is network-like.Can cause also behind the virus infection tobacco that some Physiology and biochemistries change, infect behind the plant its coat protein as viral tobacco mosaic viruses and can produce the function of photosynthesis II and have a strong impact on; Along with the prolongation of virus infection time, the content of plant chlorophyll also can correspondingly reduce, and influences photosynthesis in addition, reduces resistibility; Report that also the Phosphoric acid esterase in the diseased plant rhizosphere soil, urease activity are lower than healthy tree rhizosphere soil behind the virus infection, and its peroxidase activity is higher than healthy tree rhizosphere soil, can in the disease resistance of kind is identified, plays directive function by these indexs.

China and even the production of global tobacco in the tobacco mosaic viruses serious harm that is called as one of " plant cancer ", cause great financial loss.It is the most serious that each opium district of China is subjected to TMV harm before the sixties in 20th century, and cucumber mosaic virus, marmor upsilon and tobacco mosaic viruses were between any two or the compound production of infecting the tobacco of causing harm of three afterwards.TMV all can take place from seedling stage to the land for growing field crops whole growth growth period in cigarette strain process of growth, mainly cause the blade gage distortion of tobacco leaf and even deformity, shrinkage and present yellowish green mottled, can make the floral organ distortion when serious, little and the shrinkage of fruit, seed can not germinate, also can make the color and luster of flue-cured tobacco, jealous, quality decline in various degree.Tobacco virus for the control of virus disease, should be strengthened the prediction of disease and pest in case generation seldom has chemical process in time to control, and it is pathogenetic dynamically in time to grasp virus, can take prophylactico-therapeutic measures targetedly.The general employing puted prevention first the strategy of integrated control combination.

Cultivate antiviral kind.This is the most basic effective ways for the treatment of TMV.China cultivates a collection of antiviral kind and is applied in the practice, and as the big gold dollar of safflower, Liaoyan No.15, anti-TMV effect such as NC89 is remarkable; The white rib of also have introducing 21, Ke Ke 86 etc., the pure and mild strain of anti-TMV that also has Fang Rongxiang etc. to cultivate by transgenic technology all shows stronger resistance.

Strengthen seedbed management, cultivate anosis strong sprout.The nutrition soil in seedbed will be through disinfecting, and the fertilizer of using can not be sneaked into the diseased plant detritus.

Carry out crop rotation, enhance field management.With non-host plant (as wheat, peanut, paddy rice etc.) crop rotation about 3 years.Note the vega hygienic condition, in time clear up the residual body of diseased plant, soil is buried in deep ploughing, in time topdresses, and pinches that hand cleans up with suds when smearing wooden fork, and utensil is in time sterilized, and can not smoke when entering vega.

Chemical control.But though use fast and simple not good especially to the TMV prevention effect medicament still at present of antiviral agent.Therefore prevent and treat to prevent that from the dispenser in seedling stage the friction of virus from propagating, and improve disease resistance that the antiviral agent of using mainly contains Ningnanmycin, cytosintetidemycin, poison and disappears etc. at present, can be used as a kind of assisting therapy.

Since over half a century, chemical pesticide occupies important status always in agriculture production, yet the characteristic of the unreasonable use of chemical pesticide and high poison thereof, high residue not only causes great threat to people and animals' safety, also havoc the eubiosis.Along with the resistance of disease and pest is strengthened year by year, the usage quantity of agricultural chemicals and frequency of utilization be corresponding increasing also, again the pollution of environment and the lethality of non-target organism is caused more serious vicious cycle simultaneously.And biological pesticide has low toxicity, breeding soon, does not injure natural enemy and controlling object is difficult for developing immunity to drugs, and is called as " public nuisance-free agricultural chemicals ".Along with the pursuit to green ecological agricultural and circulation Sustainable development, the biological pesticide of low toxicity, noresidue has caused national governments and the people's great attention, and the research and development of biological pesticide and industrialization also obtain the support energetically of country.

At present the natural product total amount of compound that obtains of people surpasses 1,000,000 kinds, wherein have kind more than 20,000 be microbial metabolites in these microorganism active products, directly use have 150-160.Chemical pesticide is that the method by combinatorial chemistry obtains, resultant velocity is fast but be difficult to obtain lead compound, and microbial pesticide separation and purification from natural product is come out, and can directly act on the prevention and control of plant diseases, pest control, also can be used as the synthetic new agricultural chemicals of lead compound.These agricultural chemicals both can keep advantages such as the low toxicity, low residue of original biologically active substance, had overcome the not high deficiency of poor stability, activity of some natural product simultaneously, made that the potentiality of biologically active substance are fully excavated.Biological pesticide is compared with chemical pesticide, the source of its effective constituent, suitability for industrialized production approach, diseases prevention mechanism and the mode of action all are not quite similar, utilize microorganism and meta-bolites thereof to prevent and treat the disease and pest that harms the crops and induce the promotion plant growth, can be by controlling worm with bacterium, controlling bacterium, carry out in modes such as bacterium weedings with bacterium.These microbial pesticides comprise fungi, bacterium, actinomycetes or its metabolite, for example Bacillus thuringiensis, muscardine, nucleopolyhedrosis virus, jingganmycin, C type Clostridium botulinum extracellular toxin etc.Chemical pesticide mainly by synthetic, comprises organophosphorus, organochlorine, amino formate, pyrethroid, its strong toxicity, and the effect specificity is strong.Compare, biological pesticide more is adapted to the application of integrated pest management.

Therefore this test will be explored from the biological control aspect, the short living effective antagonistic strain of screening preventive effect, and its antiviral-mechanism studied, by conventional and molecular biology approach, utilize these antagonistic bacteriums and antagonistic substance thereof control tobacco mosaic viruses disease that research basis and experiment material are provided in the future.

Rhizosphere, the leaf of plant enclose and other little ecosystem in, antagonistic bacterium extensively exists, and since cultivate convenient, growth cycle is short, have very big potentiality to be exploited as new antagonism bacterium source.

Domestic to the existing report of tobacco mosaic viruses diease occurrence thing control, but most screening and pot experiment stages that only limit to biocontrol microorganisms, and it is produced the domestic not further investigation as yet of all many-sides such as diseases prevention mechanism of extraction, physio-biochemical characteristics and the biocontrol microorganisms of antimicrobial substance.

Summary of the invention

The objective of the invention is to overcome the above-mentioned deficiency of existing in prior technology, and provide a strain tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion pseudomonas putida bacterial strain, be used for the biological control of tobacco mosaic viruses disease, for the control of tobacco mosaic viruses disease provides new Microbial resources.

Its technical scheme is:

Bacterial strain of the present invention separates acquisition from Jimo test farm, Qingdao, vega soil sample, identify that the A3 bacterial strain is pseudomonas putida (Pseudomonas putida).The culture condition of this bacterial strain is 28 ℃, common beef broth substratum, pH7.0.The bacterial strain code name was A3, and this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 7th, 2011, and culture presevation number is CGMCC No.5231.

The method of the invention may further comprise the steps:

1) active bacterial strain A3 is inoculated into purifying on the NA substratum, picking list bacterium colony is stored on the medium slant in the test tube, and well-grown single colony inoculation is in liquid NB substratum on the picking culture medium flat plate, and in 28 ℃, 180rpm/min cultivates 64h;

2) oxalic acid with 0.5mol/L transfers to 2.0～3.0 with fermented liquid pH value, leaves standstill 2h, egg white shape precipitation occurs, filters with sterile gauze, discards dregs;

3) extract repeatedly three times with 3 times of ethyl acetate to fermentating liquid volume, collect organic phase and water respectively, be evaporated to medicinal extract at Rotary Evaporators, the organic phase thickening temperature is controlled at 45 ℃, and the water temperature is controlled at 68 ℃;

4) with the crude extract that obtains of the acetone precipitation ethyl acetate extraction of 50 times of volumes, 4 ℃ of standing over night, 10000r/m, centrifugal 20min discards precipitation, and 40 ℃ of rotary evaporations boil off acetone and obtain medicinal extract;

5) use dissolve with methanol medicinal extract, mix with the equivalent deionized water again, cross D-101 macroporous resin Static Adsorption behind the constant volume, carry out wash-out with elutriant then, eluent system is methanol, and gradient is 3/7, its gradient composition is collected 500mL, and concentrating under reduced pressure becomes 1 medicinal extract shape material.

Beneficial effect of the present invention:

The biological and ecological methods to prevent plant disease, pests, and erosion pseudomonas putida strains A 3 of the antagonism tobacco mosaic viruses disease that the present invention obtains, can be applicable to the control to the tobacco mosaic viruses disease, adopt the withered spot host's of inoculation biology mensuration to show, its fermented liquid is 95.0% (see figure 1) to the inhibiting rate of TMV, the material that the antagonistic substance liquid-liquid extraction of Ba33 bacterial strain secretion obtains is measured through biology, the proof acetic acid ethyl ester extract has anti-TMV activity, reach 99% (seeing Table 1), after adopting GC/MS to carry out chemical analysis and structure evaluation, obtain 38 kinds of compounds, wherein ketone compounds altogether, alcohol compound is in the majority.Study on mechanism result shows that (see Fig. 2, Fig. 3), electron microscopic observation shows that the active small molecular material can destroy the integrity of TMV plastochondria, makes the virus particle fracture and arranges confused and disordered.In the mutual work of virus-host, can induce after the active small molecular material is handled the cigarette strain and produce PR albumen in the plant body, its expression amount is increased, induce plant generation systemic disease resistance.In the test of formulation development (see Fig. 4, Fig. 5, Fig. 6), 0.25% tween 20 is beneficial to strains A 3 and acts on tobacco leaf surface, inoculates TMV after spraying 1d, can better suppress the generation of tobacco virus, has actual using value.

Description of drawings

Fig. 1 is that the incarnadine bacterial strain is to the antagonistic activity design sketch of TMV;

Fig. 2 is the plastochondria aspect graph of active substance processing back TMV, a:TMV plastochondria (contrast) b: the TMV plastochondria c after mixed with the A3 fermented liquid: the TMV plastochondria after mixed with activity extract;

Fig. 3 is the influence figure of active substance processing to tobacco leaf PR gene expression amount;

Fig. 4 is that the tween of different concns mixes the design sketch to the cigarette strain with the A3 bacterial strain;

Fig. 5 is that the different vaccination time is to the inhibition figure of TMV;

Fig. 6 is that different contrasts are to the inhibition figure of TMV.

Embodiment

Below in conjunction with concrete and embodiment method of the present invention is done explanation in further detail.

The screening of control TMV antagonistic bacterium A3 bacterial strain

Obtained strains is inoculated in the liquid NB substratum with transfering loop, and 28 ℃, 180rpm cultivates 64h, and the fermented liquid of cultivation is in 10000rpm/min, centrifugal 10min, and most of thalline of leaving away is collected supernatant liquor, and the gained supernatant liquor is carried out anti-TMV Determination of biological activity.Select healthy, eugonic 6-7 leaf phase three lives-NN cigarette to inoculate.The TMV tobacco tender leaf of fully morbidity is ground to form homogenate with the sterilization mortar, with 1: 40 dilution proportion of phosphoric acid buffer (PH7.0) after the sterilization.The mixed liquid of Zuo Banye inoculation NB substratum and viral equal-volume compares, and the mixed liquid of right half leaf inoculation fermentation supernatant liquor and viral equal-volume deals with, the blade face of water sprinkling immediately after the inoculation.3-4 blade of every processing inoculation repeats 3 times, and observations as shown in Figure 1 behind the 3d.

The separation and purification of active substance

The anti-TMV active bacterial strain A3 that filters out is inoculated into purifying on the NA substratum, and picking list bacterium colony is stored on the medium slant in the test tube.Well-grown single colony inoculation is in liquid NB substratum on the picking culture medium flat plate, and in 28 ℃, 180rpm/min cultivates 64h.Oxalic acid with 0.5mol/L transfers to 2.0～3.0 with fermented liquid pH value, leaves standstill 2h, egg white shape precipitation occurs, filters with the multilayer sterile gauze, discards dregs.Extract repeatedly three times with 3 times of ethyl acetate to fermentating liquid volume, collect organic phase and water respectively, be evaporated to medicinal extract at Rotary Evaporators, the organic phase thickening temperature is controlled at 45 ℃, and the water temperature is controlled about 68 ℃.The water enriched material is transferred to 7.0 with 10% NaOH solution with the pH of water, with 7 times of freezing dehydrated alcohol precipitation water-phase extracts to the water volume, precipitation separation and ethanol supernatant liquor are concentrated into medicinal extract with gained precipitation and ethanol supernatant liquor in 45 ℃ of rotary evaporations, obtain 3 kinds of crude extracts.

The acetic acid ethyl ester extract that rotary evaporation is obtained, ethanol supernatant enriched material, ethanol sedimentation respectively with sterilized water be diluted to 1 *, 5 *, 25 *, 125 *, half leaf method inoculation 5-6 leaf phase in withered spot three lives cigarette, result's (table 1) shows, in three kinds of different extracts of originating, the biological activity of acetic acid ethyl ester extract is the strongest, thus explanation, anti-TMV active substance mainly is distributed in the organic phase ethyl acetate, therefore as seen, can come further separation and purification with acetic acid ethyl ester extract as the purification object.

Three kinds of crude extracts of table 1 are to the inhibition of TMV

Table?1?Different?antivirial?rates?of?three?crude?extract?against?TMV

The purifying of the anti-TMV active substance of crude extract

With the crude extract that obtains of the acetone precipitation ethyl acetate extraction of 50 times of volumes, 4 ℃ of standing over night, 10000r/m, centrifugal 20min discards precipitation, and 40 ℃ of rotary evaporations boil off acetone and obtain medicinal extract.Dissolve medicinal extract with small amount of methanol, mix with the equivalent deionized water again, cross D-101 macroporous resin Static Adsorption behind the constant volume, carry out wash-out with elutriant then, eluent system is methanol, gradient is methanol 0/10,1/9,2/8,3/7,4/6,5/5,6/4,7/3,8/2,9/1,10/0, every gradient composition is collected 500mL respectively, concentrating under reduced pressure becomes 11 kinds of medicinal extract shape materials, each component is inoculated withered spot three lives cigarette carry out biology mensuration, the result shows (table 2), and the anti-TMV activity of the 4th gradient elution material (namely numbering D) is the strongest.

Each elution fraction of table 2 is to the inhibition of TMV

Table?2?Inhibitory?effect?of?different?elution?components?against?TMV

The antiviral active substance constituent structure is identified

The component that antiviral activity is strong is measured composition with GC/MS, the GC/MS condition determination:

Chromatographic column: DB-5MS type fused-silica capillary column (30m * 0.25mm * 0.25 μ m).50 ℃ of initial column temperatures kept 2 minutes, were warming up to 280 ℃ with 5 ℃/min, kept 2min; 250 ℃ of vaporizer temperature; Carrier gas is high-purity He (99.999%); Press before the post to be 7.62psi, carrier gas flux is 10mL/min; Sample size 1 μ L; Splitting ratio 40: 1;

The mass spectrum condition: the EI source is ion source; 230 ℃ of ion source temperatures; Multiplier voltage 2047V; 280 ℃ of interface temperature; Solvent delay 3min; The full scan mode, sweep limit 10amu～550amu.

According to above-mentioned condition the concentrated extract of numbering D is carried out isolation identification, by the HPMSD chem workstation, in conjunction with NIST98 standard mass spectrum picture library and in conjunction with relevant document manual retrieval, carrying out material identifies, detect material in 41 altogether, by the HPMSD chem workstation, identify through carrying out material with mass-spectrometric data storehouse and the contrast of standard spectrogram, identify 38 kinds of materials altogether, wherein contain 14 kinds of ester compounds, 7 kinds of ketone compounds, 8 kinds of acid compounds, 7 kinds of alcohol compounds.

Embodiment 2

Observe A3 bacterial strain active substance under the perspective Electronic Speculum to the influence of TMV virion

After negative staining was handled the TMV that purifies, through the JEM-100CX transmission electron microscope observing, the TMV plastochondria in the visual field was typically shaft-like, the about 500nm of length, and upright and outspoken and marshalling, integrity is good; Behind A3 bacterial strain fermentation liquor and TMV plastochondria combination treatment 2min, through electron microscopic observation, in the visual field TMV plastochondria generation noticeable change almost complete rupture become little shaft-like, arrange confused and disordered; Behind A3 bacterial strain activity extract processing TMV plastochondria, the electron microscopic observation result shows that the result is the same with fermentation liquor treatment TMV plastochondria, and the TMV plastochondria also is to fragment into small segment, arranges unordered.

This shows that activity extract all has strong destruction to the TMV plastochondria in A3 bacterial strain fermentation liquor and the bacterial strain, and failure mode is identical, can infers tentatively that the A3 fermented liquid is exactly activity extract to the antagonistic substance of TMV.This active substance may be that virus is produced passivation, suppresses the polymerization of TMV coat protein, and destroys the integrity of plastochondria.

1.2A3 fermented liquid is induced NC89 tobacco PR expression of gene

Inoculate TMV behind the fermentation liquor treatment tobacco leaf 12h, different time sections is to influence such as Fig. 3 of cigarette strain PR genetic expression.It is the sample of sample time 12h, 24h, 36h, 48h, 60h, 72h shown in the figure, the NB substratum is handled the negative contrast of cigarette strain, on the result, PR gene 24 hours expression amounts behind inoculation TMV are the highest, thereafter expression amount reduced at 36 hours, and expression amount remains unchanged substantially after 48 hours.And inoculate TMV after the strain of fermentation liquor treatment cigarette, PR expression of gene amount and negative control significantly increase, and prove that A3 bacterial strain actives mass-energy induces cigarette strain PR expression of gene amount to increase.

Embodiment 3

The A3 inoculation is arrived the about 3L of NB liquid nutrient medium, 28 ℃, 180rpm, cultivate 60h-72h, obtain the A3 bacterial strain fermentation liquor, tween 20 adds in the fermented liquid as the blade face spreading agent, is mixed with tween 20 concentration respectively and is 3%, 2%, 1%, 0.5% and 0.25% medicament, each prepared at concentrations 500mL.

Medicament adopts the foliage-spray method, arrive the 4-5 leaf during phase in NC89 cigarette seedling length, on blade, spray the medicament of respective concentration equably with aseptic atomizer, each concentration is sprayed 12 strains, behind chemicals treatment 1d, 2d, 3d, 4d, inoculate TMV juice (inoculating 3 strains every day) then respectively, keep inoculation dynamics unanimity with cotton rod as far as possible.The NB liquid nutrient medium adds the tween of corresponding concentration as the negative control of each concentration; Antiviral agent-Ningnanmycin, be diluted to the suds of 3%, 2%, 1%, 0.5%, 0.25% concentration respectively as positive control.Observations shows behind the inoculation 7d, and under different concns and the processing of different vaccination time, the occurring degree of NC89 tobacco leaf has significantly different.On the whole, spray medicine and inoculation time are more short at interval, inhibition to TMV is more good, cigarette strain growing way is more good, namely spray 1d inoculation TMV behind the medicine incidence be lower than 2d behind the spray medicine) effect of inoculation TMV juice, than the inhibiting rate height of 3d, the viral inhibition of inoculation TMV juice is the poorest behind the 4d again for TMV effect of inoculation behind the 2d.Simultaneously, under the influence of the tween 20 of different concns, concentration is more low, and anti-TMV effect is also more good, the tobacco leaf growing way is also more good, i.e. the cigarette strain antiviral effect of the tween 20 of different concns processing is followed successively by from high to low: 0.25%＞0.5%＞1%＞2%＞3% (Fig. 4).The mixed solution of substratum and tween 20 inoculation TMV juice in the negative control, TMV incidence serious (Fig. 6), and tween concentration is more high, and the damage intensity on blade face is more big, and leaf margin curls more serious.The suds of respective concentration are inoculated TMV juice after spraying 1d, and along with the reduction of concentration, antiviral effect weakens gradually, and prevention effect generally is lower than the effect (Fig. 6) that sprays strains A 3.Inoculation TMV juice after Ningnanmycin sprays one day, the cigarette strain is not morbidity (Fig. 6) substantially.

Test shows, the antiviral effect of strains A 3 is higher than suds, be lower than Ningnanmycin, find also in the test that the tween 20 excessive concentration causes the blade face shrinkage easily, and be unfavorable for that strains A 3 fermented liquids infiltrate blade, 0.25% tween 20 is beneficial to strains A 3 and acts on tobacco leaf surface, better suppresses the generation of tobacco virus.

Attached: nucleotide sequence involved in the present invention

SEQ ID NO1 (A3 bacterial strain 16S rDNA complete sequence):

(16sF)GGTGGACAGATCACCGTGGTACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATTACGTGCTGGTAACTAAGGACAAGGGTTGCGCTC?GTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTTGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCAACTTAACAAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGGTGCTGCTTATTCTGTCGGTAACGTCAAAATTGCAGAGTATTAATCTACAACCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGCATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATCCAGGAGCAAGCTCCTTTCATCCGCTCGACTGCATGTGTAGCTGCCGCCATGCAG(16sR)

Claims (5)

1. strain tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion pseudomonas putida (Pseudomonas putida) bacterial strain, the bacterial strain code name is A3, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 7th, 2011, culture presevation number is CGMCC No.5231.

2. the application of the described strains A 3 of claim 1 in the control tobacco mosaic viruses.

3. the described strains A 3 of claim 1 is mixed the application of back in the control tobacco mosaic viruses with tween 20 %.

4. a method of utilizing the described tobacco mosaic viruses biological and ecological methods to prevent plant disease, pests, and erosion of claim 1 pseudomonas putida bacterial strain to prepare the mixture of active small molecular material is characterized in that, may further comprise the steps:

1) active bacterial strain A3 is inoculated into purifying on the NA substratum, picking list bacterium colony is stored on the medium slant in the test tube, and well-grown single colony inoculation is in liquid NB substratum on the picking culture medium flat plate, and in 28 ℃, 180rpm/min cultivates 64h;

2) oxalic acid with 0.5mol/L transfers to 2.0～3.0 with fermented liquid pH value, leaves standstill 2h, egg white shape precipitation occurs, filters with sterile gauze, discards dregs;

3) extract repeatedly three times with 3 times of ethyl acetate to fermentating liquid volume, collect organic phase and water respectively, be evaporated to medicinal extract at Rotary Evaporators, the organic phase thickening temperature is controlled at 45 ℃, and the water temperature is controlled at 68 ℃;

4) with the crude extract that obtains of the acetone precipitation ethyl acetate extraction of 50 times of volumes, 4 ℃ of standing over night, 10000r/m, centrifugal 20min discards precipitation, and 40 ℃ of rotary evaporations boil off acetone and obtain medicinal extract;

5) use dissolve with methanol medicinal extract, mix with the equivalent deionized water again, cross D-101 macroporous resin Static Adsorption behind the constant volume, carry out wash-out with elutriant then, eluent system is methanol, and gradient is 3/7, its gradient composition is collected 500mL, and concentrating under reduced pressure becomes 1 medicinal extract shape material.

5. the application of the mixture of the active small molecular material that obtains of the described method of claim 4 in the tobacco mosaic viruses control.

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