CN104109645B

CN104109645B - For preventing and treating bacterial strain and the biocontrol agent of postharvest disease of fruits and vegetables

Info

Publication number: CN104109645B
Application number: CN201410251215.0A
Authority: CN
Inventors: 施俊凤; 孙常青; 王潇冉; 高伟
Original assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Current assignee: Institute Of Storage And Preservation Of Agricultural Products Shanxi Academy Of Agricultural Sciences
Priority date: 2014-06-06
Filing date: 2014-06-06
Publication date: 2016-10-12
Anticipated expiration: 2034-06-06
Also published as: CN104109645A

Abstract

The present invention relates to antagonist, a kind of bacterial strain for preventing and treating postharvest disease of fruits and vegetables, it is deposited in China typical culture collection center on March 7th, 2014, preservation registration number is CCTCC M 2014076.A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain described in claim 1, the concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds the Na of 25～100mM in described every milliliter of bacteria suspension₂SiO₃；Or, including the bacteria suspension of bacterial strain described in claim 1, the concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds the NaHCO of 25～100mM in described every milliliter of bacteria suspension₃.Bacterial strain of the present invention can suppress the generation of postharvest fruit and vegetable gray mold well, has also been considerably improved the prevention effect of postharvest disease of fruits and vegetables.

Description

For preventing and treating bacterial strain and the biocontrol agent of postharvest disease of fruits and vegetables

Technical field

The present invention relates to antagonist, a kind of bacterium for preventing and treating postharvest disease of fruits and vegetables Strain and biocontrol agent.

Background technology

The rotten loss caused that postharvest disease of fruits and vegetables causes is the hugest.At present, control The maximally effective measure of postharvest disease of fruits and vegetables is that cryopreservation combines chemical bactericide.But life-time service Chemical bactericide, the most easily makes pathogen develop immunity to drugs, and pollutes environment, to human body Health adversely affects.Therefore, hypotoxicity, efficient, environmental friendliness and residual quantity are found low The succedaneum of antibacterial or part succedaneum the most urgent.

Application Antagonistic Fungi carries out Biological control becomes study hotspot in recent years.At present, it is separated to Biocontrol microorganisms mainly have small-sized filamentous fungi, antibacterial and yeast etc..Up to now, from Citrus chachiensis Hort. 10 various fruits such as Fructus Citri tangerinae, Fructus Mali pumilae, Fructus Persicae, pears, Fructus actinidiae chinensis, fresh Fructus Jujubae filter out tens kinds short of money Antimicrobial.More biocontrol bacteria is applied mainly to have bacillus cereus (Bacillus at present Subtilis), Pseudomonas alba (Pseudomonas spp.), soil actinomyces (Agrobacterium Radiobacter) etc..Commercialization application mainly has pseudomonas syringae at present (Pseudomonas syringae), Bacillus subtillis (Bacillus subtilis), yeast (Candidoleophil) the Pichia guilliermondii bacterium (Pichiaguillier mondii) in, Ha Ci Trichoderma spp. (Trichoderma harzianum), Hyperparasite of Powdery Mildew etc..

Use antagonistic yeast preventing and treating postharvest disease of fruits and vegetables, it is possible to reduce the consumption of chemical pesticide and Residual hazard on fruit and vegetable food, to protecting health and preventing the pollution of the environment the most particularly important. Aspire^TM, Biosave110 and Biosave111 be external oneself resist through the microorganism of registration Microbial inoculum, is mainly used in controlling citrus and the postharvest decay of pip fruits.This present utilization Antagonistic Fungi is safely and effectively prevented and treated fruit postharvest diseases and is had broad prospects.

Bulbus Allii Cepae Burkholderia (Burkholderia cepacia) is that the 1950's is as one Planting phytopathogen to be recognized by people, Burkholder can this bacterium of nineteen fifty reported first To cause the bulb of Bulbus Allii Cepae to occur to rot, referred to as Pseudomonas cepacia (Pseudomonas cepacia).In agriculture field, Bulbus Allii Cepae Burkholderia have Biological control, biodegradation and Promote the several functions such as plant growing, before also show its being widely applied agriculturally Scape.In recent years, along with the development of Protocols in Molecular Biology, along with division bacteria technology is the most corresponding Be developed, Bulbus Allii Cepae Burkholderia not only as a kind, but one group of genotype is different, The complex that phenotype is close, referred to as Burkholderia are combined group (Burkholderia Cepacia complex, is called for short Bcc).Bcc can effectively prevent and treat the fungoid of various plants Disease, Abroad in Recent Years is to prevent and treat the report of the soil-borne diseases such as damping-off, reaping hook pathogenic bacteria and rotten mildew Road is more, but also has some to postharvest diseases such as pears penicilliosis, Fructus Mali pumilae gray mold and peach brown rotes And the relevant report of plants stems leaf disease evil.Fan Qing etc. find that Prunus avium brown rot is shown by Bcc Significantly inhibition, thanks to General Guan Yu's Tomb etc. and finds to significantly inhibit water in vitro Bcc bacterial strain Rhizoctonia solani Kuhn and the growth of Fusarium moniliforme Sheld.Zheng Wei etc. have isolated Bcc bacterial strain from compost CF-66, and find that pinch outs, Rhizoctonia solani Kuhn and yeast are shown the strongest by this bacterial strain Antibacterial activity.Additionally, Bcc can also utilize many kinds of substance as sole carbon source, can drop Solve having of the soil and groundwater pollutions such as phthalate, herbicide and chlorinated hydrocarbon Poisonous substance matter, is also a kind of very promising biological modeling, is a kind of very promising biocontrol microorganisms Strain.

Summary of the invention

It is an object of the invention to provide a kind of new bacterial strain for preventing and treating postharvest disease of fruits and vegetables and Biocontrol agent.

A kind of bacterial strain for preventing and treating postharvest disease of fruits and vegetables, in being deposited on March 7th, 2014 State's Type Tissue Collection, preservation center deposit number is CCTCC NO:M 2014076, address: China. Wuhan. Wuhan University.

One, the screening technique of Antagonistic Fungi of the present invention is as follows:

1, experiment material and method

1.1, experiment material

Botrytis cinerea (B.cinerea) from the tamato fruit surface isolated of natural occurrence, It is inoculated in after purified in PDA culture medium, after cultivating 7d at 26 DEG C, uses hemocytometer Number method sterilized water is made into 1 × 10⁶The spore suspension of spore/mL.

Dai County of Shanxi Province, Fanzhi County and Taiyuan City is picked up from for separating the fruit vegetable of Antagonistic Fungi Xiaodian District, or be purchased from market, Taiyuan, sample and sampling site and be shown in Table 1.

The Fructus Lycopersici esculenti of inoculation is purchased from Taiyuan Xiaodian Area.

Table 1 sample and sampling position

1.2, experimental technique

1.2.1, the separation of Antagonistic Fungi

With reference to the method for Janisiewicz, take respectively 5～10 fruits (or 2～3 dish leaves or Tomato leaf) put into and fill in 0.2M (mole) phosphate buffer, in 100r/min shaking table After rotational oscillation 10min, discard washing liquid, add phosphate buffer, at ultrasonic cleaner Middle cleaning 30s, carries out secondary washing.The liquid of secondary washing gained through 10 times, 100 times and After 1000 times of gradient dilutions, respectively take 100 μ L and coat in PDA culture medium, 26 DEG C cultivate 2～ 3d, picking list bacterium colony carries out streak culture at PDA plate again, is further purified.

1.2.2, the screening of Antagonistic Fungi

Vivo Studies on Screening method: the Antagonistic Fungi that picking Preliminary screening goes out from PDA culture medium, activated After, at 28 DEG C, cultivate 24～48h.Picking list bacterium colony is 200r/min vibration training at 28 DEG C After supporting 24h, it is made into 1 × l0¹⁰The suspension of cfu/mL is standby.By strong for Fructus Lycopersici esculenti fruit with 2% After NaClO solution surface sterilization 2min, tap water is rinsed well, and sterile wind dries up.By nothing Bacterium Inoculating needle, at the wound of fruit waist thorn 3mm × 3mm, every fruit 2 wounds of thorn, first exists The Antagonistic Fungi suspension that 50 μ L separate is inoculated in wound, inoculates the 1 × 10 of 15 μ L after 2h⁶ The pathogen spore suspension of spore/mL, using sterilized water as comparison, sterile wind will after drying up Fruit is put in plastic casing, and 26 DEG C of constant-temperature moisture-keepings are cultivated.Measure after 7d fruit sickness rate and Lesion diameter.Often process 10 fruits, repeat for 3 times.

2, result and analysis

Screen through various fruit surfaces, be separated to 89 strain antibacterials and 53 saccharomycetes altogether, Filter out the preferable 6 strain antibacterials of antagonistic effect and 5 saccharomycetes the most further, its morbidity Rate and lesion diameter are all substantially less than comparison (P < 0.05), and what wherein antagonistic effect was best is thin Bacteria strain B-1, comparison all morbidities, and after bacterial strain processes, sickness rate is 0%, can Adopt the generation of rear gray mold, as shown in table 2 completely inhibiting Fructus Lycopersici esculenti.

Sickness rate after table 2 tamato fruit wound inoculation Antagonistic Fungi and Botrytis cinerea and lesion diameter

Same column difference letter representation Duncan formula multiplexed differential range detection significant difference in P=0.05 level.

It is randomly select that this experiment is used for separating the fruit of Antagonistic Fungi, and at room temperature moisturizing is placed After a period of time, never morbidity fruit real surface separates antagonistic microbe.For carrying out biology The Antagonistic Fungi of preventing and treating, except can effectively suppress or kill pathogen, also wants to well adapt to Ecology residing for pathogen and environmental system.

In the ecosystem at the place of disease and the microorganism of fruit, when the disease of fruit occurs Time more serious, pathogen occupies catbird seat, and Antagonistic Fungi is then suppressed state.Therefore, have Effect Antagonistic Fungi just should beneficially fruit fall ill under conditions of, but fruit do not show fall ill or Morbidity is very light, may often be such that one or more Antagonistic Fungis in action, and carries out from these fruits The screening of Antagonistic Fungi, it is easier to be separated to the Antagonistic Fungi of good antimicrobial effect.

About the screening technique of Antagonistic Fungi, Many researchers is all to use in vitro and Vivo Studies on Screening phase In conjunction with method carry out.First, flat board carries out in vitro primary dcreening operation and multiple sieve, the most again On fruit, live body inoculation, has the antagonistic strain of inhibitory action with Vivo Studies on Screening to target pathogen. In vitro Screening is more convenient and quick for the determination of bacterial strain bacteriostasis.But, in vitro bar The bacterial strain having antibacterial power filtered out under part might not also can show when live test equally Fungistatic effect, so, be substantially carried out is Vivo Studies on Screening in this experiment, because screening obtains Antagonistic Fungi be the most also with the disease suppressing Fructus Lycopersici esculenti live body to produce as final purpose.

Two, the Biology identification of Antagonistic Fungi B-1

1, experimental technique:

1.1, yeast culture

Being separated at flat lining out by the bacterial strain of purification, after cultivating 20h, picking list colony inoculation arrives In 20ml fluid medium, 37 DEG C, 200rpm cultivate 15h.

1.2, the extraction of genomic DNA

Cracking with SDS-E.C. 3.4.21.64, cetyl trimethylammonium bromide (CTAB) precipitation is thin Born of the same parents' fragment and polysaccharide, then extract STb gene by the method for isopropanol precipitating.

Take single colony inoculation in the PDA culture medium of 5mL, cultivate 24h, take 1.0mL for 37 DEG C Bacterium solution is in 1.5mL centrifuge tube, and 13000r/min is centrifuged 2min, abandons supernatant；Precipitate resuspended In the NaCl of 1.0mL0.85%, room temperature 13000r/min is centrifuged 2min, abandons supernatant；Precipitation It is resuspended in 550 μ L 1 × TE.Add 17 μ L lysozyme (35mg/mL), 37 DEG C of incubation 30min； Add 3 μ L E.C. 3.4.21.64s, 37 DEG C, 30min.Add the 10%SDS of 30 μ L, 37 DEG C of incubation 30min. Mix after the NaCl of the 5M adding 100 μ L；Add the CTAB/NaCl mixed liquor of 80 μ L, mixed Even, 65 DEG C of water-bath 10min, add equal-volume chloroform/isoamyl alcohol (24:1), mixing of vibrating gently； Room temperature 13000r/min is centrifuged 10min；Supernatant is moved to a new 1.5mL centrifuge tube, adds Volume phenol/chloroform/isoamyl alcohol (25:24:1) vibrates mixing gently.Supernatant moves on to a new 1.5ml Centrifuge tube, adds 0.6 times of volume isopropanol, and rear chamber is gentle and quiet puts 1h in mixing；20 DEG C, 13000r/min Centrifugal 20min；Abandon supernatant, add 70% ethanol of 500 μ l, overturn for several times (desalinization of soil by flooding or leaching) gently； 4 DEG C, 15000r/min is centrifuged 20min；Repeat the desalinization of soil by flooding or leaching 2 times；Centrifuge tube is inverted, dry DNA Precipitation 10～15min；DNA precipitation is dissolved in 100 μ l 1 × TE buffer, and-20 DEG C of preservations are standby With.

1.3, the clone of bacterial strain 16S rDNA

Bacterial 16 S rDNA is expanded according to the method for Coenye etc..Primer is 27F:5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and 1541R:5 '-AAG GAG GTG ATC CAC CC-3’。

Pcr amplification reaction condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 40s, 55 DEG C are moved back Fire 50s, 72 DEG C extend 1min15s, totally 35 circulations；72 DEG C terminate 10min.

PCR primer uses ultraviolet gel imaging system after 1% agarose gel electrophoresis, EB dyeing Being analyzed, reclaim purpose fragment after purification, by Shanghai, Ying Jun gene company limited carries out sequence Row measure.

PCR reaction system is: Taq (5U/ μ L) 0.8 μ L；10×PCR Buffer(Mg²⁺Plus) 10μL；dNTP Mixture(2.5mM/each)8μL；Template DNA 2.5ng；Primers F 1 (10 μm oL/L) 2 μ L, primer R1 (10 μm oL/L) 2 μ L；ddH₂O supplies 100 μ L.

1.4, the RecA gene clone of bacterial strain B-1

With reference to the methods such as Mahenthiralingam E, the forward primer of clone's recA gene BCR1:5'-TgACCgCCgAgAAgAgCAA-3' and reverse primer BCR2: 5'-CTCTTCTTCgTCCATCgCCTC-3'。

Pcr amplification reaction condition is: 95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 58 DEG C are moved back Fire 45s, 72 DEG C extend 1min, totally 30 circulations；72 DEG C terminate 10min.

PCR primer is use ultraviolet gel imaging system after 1% agarose gel electrophoresis, EB dyeing System is analyzed, and reclaims purpose fragment after purification, and by Shanghai, Ying Jun gene company limited is carried out Sequencing.

RecA gene order reaction system is 100 μ L:Taq (5U/ μ L) 0.8 μ L；10×PCR Buffer(Mg²⁺Plus)10μL；dNTP Mixture(2.5mM/each)8μL；Template DNA 2.5ng；Primers F 1 (10 μm ol/L) 2 μ L, primer R1 (10 μm ol/L) 2 μ L；ddH₂O Supply 100 μ L.

1.5, Homology search and the structure of phylogenetic tree

After the gene DNA sequence of clone is checked order, after carrying out Blast tetraploid rice, With MEGA 4.0 software building sequence homology matrix table, and use adjacent method (Neighbor-Joining) phylogenetic tree construction.

1.6, physiological and biochemical index analysis

According to " Bergey ' s Manual of Determinative Bacteriology " the 9th edition to bacterium Strain B-1 carries out physiological and biochemical test.

2, result and analysis

2.1, colonial morphology

Bacterial strain B-1 is shaft-like, and corynebacterium does not moves, without spore, Gram-negative. Bacteria culture media flat board is cultivated rounded, neat in edge, creamy is opaque (such as figure Shown in 2).

2.2, physio-biochemical characteristics

The physio-biochemical characteristics of bacterial strain B-1 are determined, result such as table 3:

The physiological and biochemical property of table 3 bacterial strain B-1

2.3,16S rDNA homology analysis and the phylogenetic tree of bacterial strain B-1 builds

The genome 16S rDNA sequence that the primer amplifies is 1439bp.To be surveyed Sequence after Blast carries out tetraploid rice, use DNAMEN software carry out multisequencing Relatively.The sequence of B-1 and Burkholderia Burkholderia lata (CP000150) homology It is 99.8%, is 99.8% with Burkholderia B.arboris (AM747630) homology, with Burkholderia B.metallica (AM747632) homology is 99.7%, with B.cenocepacia And the homology of B.cepacia (U96927) is 99.5%, with B.stabilis (AF148556) And Burkholderia pyrrocinia (Burkholderia pyrrocinia) (AF097533) (U96930) homology is 99.4%, with B.diffusa (AM747629) and B.latens (AM747628) homology is 99.3%, with Vietnam's Burkholderia (B.vietnamiensis) (AF097534) homology is 99.2%, with the homology of B.ubonensis (AB030584) Property is 99.1%.With other Burkholderia pod shell Burkholderia (B.glumae) (U96931), Glanders Burkholderia (B.mallei) (AF110188), B.oklahomensis (DQ108388), Plant Burkholderia (B.plantarii) (U96933), melioidosis Burkholderia (B. Pseudomallei) (DQ108392), the homology of B.thailandensis (U91838) Property is more than 98%.It is outer group's structure with Pi Shi Rolston bacterium (Ralstonia pickettii) Build phylogenetic tree (as shown in Figure 3), knowable to phylogenetic tree, with bacterial strain B-1 relationship That relation is nearest is Burkholderia lata (CP000150), and they gather in a branch, Bootstrapping supporting rate is 46%.Just determining bacterial strain B-1 is that Burkholderia belongs to (Burkholderia), but It is owing to the 16S rDNA sequence homology of Burkholderia is higher, so can not precise Identification To planting.

2.4, bacterial strain B-1recA Genetic homology of carbapenem-resistant and phylogenetic tree build

For more precise Identification bacterial isolates B-1, by housekeeping gene recA is carried out further Checking, the sequencing fragment gained sequence length of primer BCR amplification is 804bp.In order to enter one Step identifies the kind of bacterial strain B-1, after being checked order by recA gene 16S, through Blast and Burkholderia Other genus belonged to carries out tetraploid rice, then uses DNAMEN software to carry out Multiple alignment. The bacterial strain chosen is Burkholderia group (Burkholderia cepacia Complex) bacterial strain.The sequence of B-1 and Burkholderia contaminans (AF456121) Sequence similarity is 100%, with Burkholderia lata (AF456124) homology is 98.8%, it is 98.5% with Burkholderia metallica (AF456103) homology, with Burkholderia arboris (AM748095) homology is 98.1%, with primary gram of Hall of Bulbus Allii Cepae Moral Salmonella (Burkholderia cepacia) (AF143786) homology is relatively low, is 96.3%.

Use the phylogeny facing a connection method structure bacterial strain B-1 and the recA gene of relevant kind Tree, as shown in Figure 4.The type strain LMG 23361 of B-1 with B.contaminans (steps on Record AF456121) to gather in same branch, cluster bootstrapping supporting rate is 62%, in conjunction with thin Bacterium form, physiological and biochemical property, genome and RecA sequence analysis, identify bacterial strain B-1 For: Burkholderia contaminans.Belong to antibacterial circle (Bacteria) Proteobacteria (Proteobacteria) β-deformed rod Gammaproteobacteria (β-Proteobacteria) Burkholderia mesh (Burkholderiales) Burkholderia section (Burkholderiaceae) Burkholderia belongs to (Burkholderia) compound (the Burkholderia cepacia of Burkholderia complex)。

Three, fungistatic effect research

1, variable concentrations bacterial strain B-1 bacteria suspension is to Fructus Lycopersici esculenti postharvest disease body outer suppressioning experiment

Method:

Use bacterium dish method, by bacterial strain B.contaminans after purification, picking list colony inoculation in The LB fluid medium of 100mL (yeast extract 5g, peptone 10g, NaCl 5g, Agar 15-20g, water 1000mL) in, 30 DEG C of 200r shaken cultivation 36h, use dilution plate After method measures concentration, it is respectively 1 × 10 with sterilized water compound concentration⁵、1×10⁶、1×10⁷、1×10⁸、 1×10⁹The Antagonistic Fungi suspension of cfu/mL.Equipped with PDA culture medium (Rhizoma Solani tuber osi 200g, Glucose 20g, agar 15g, add water 1000mL) a diameter of 9cm culture dish in, After PDA culture medium solidifying, it is separately added into the Antagonistic Fungi suspension 100 μ L of variable concentrations, After coating uniformly, after sterile wind dries up, access the pathogen of a diameter of 5mm then at central point Bacterium cake, comparison adds sterilized water.Constant temperature culture at 28 DEG C, measures colony diameter after 5～7d, and Calculating its suppression ratio, process 3 times every time and repeat, experiment is repeated 3 times.

Suppression ratio (%)=(dC-dT)/(dC-5) × 100

Wherein, dC: comparison colony diameter (mm)；

DT: colony diameter (mm) under the conditions of different disposal.

Result:

The B.contaminans of variable concentrations can effectively suppress Fusarium moniliforme (Fusarium Verticillioides), Alternaria tenuissima (Alternaria tenuissima) and Botrytis cinerea The mycelia normal growth of (Botrytis cinerea), and the concentration of bacterium solution is the biggest, its antibacterial work With the strongest (as shown in Figure 5).

2, the B.contaminans bacteria suspension inhibition to graw mold of tomato

Method:

Take the strong fruit of Fructus Lycopersici esculenti, use the NaClO of 2% carry out after fruit surface sterilization, in fruit Position, equator thorn a diameter of 5mm, the wound of deep 3mm, 2 wounds of every fruit.Divide at wound Do not add 1 × 10⁷、1×10⁸、1×10⁹、1×10¹⁰The bacterial strain B-1 bacteria suspension of cfu/mL, comparison Add sterilized water.1 × 10 is connect at wound after 2h⁶The Botrytis cinerea (B.cinerea) of spore/mL 20 μ L, heat insulating culture at 25 DEG C, measure lesion diameter after 7d and calculate suppression ratio.Often process 10 fruits, experiment is repeated 3 times.

Result:

After accessing the B.contaminans bacteria suspension of variable concentrations, morbidity statistics result shows, Along with the rising of concentration, the sickness rate of graw mold of tomato declines.The incidence rate of CK gray mold is 100%；1 × 10⁷During cfu/mL, its incidence rate 65.40%, 1 × 10⁸Cfu/mL is 45.36%, 1×10⁹4.42% it is only, at maximum concentration 1 × 10 during cfu/mL¹⁰During cfu/mL, can press down completely The generation of graw mold of tomato processed.

3, the B.contaminans fermentation liquid inhibition to Fructus Lycopersici esculenti Postharvest Penicillium

Method:

B.contaminans fermentation liquid preparation method is as follows: bacterial strain B.contaminans is at LB liquid In body culture medium, 30 DEG C, 20r/min shake-flask culture 48h.

Take the strong fruit of Fructus Lycopersici esculenti, use the NaClO of 2% carry out after fruit surface sterilization, red in fruit Position, road thorn diameter 5mm, the wound of deep 3mm, 2 wounds of every fruit.48h is added respectively at wound Fermentation liquid 50 μ L, comparison add 50 μ L sterilized water.1 × 10 is connect at wound after 2h⁶Spore/mL's Penicillium expansum (Penicillium expansum) 20 μ L, heat insulating culture at 26 DEG C, examine after 7d Look into incidence.Often process 20 fruits.Experiment is repeated 3 times.

Result:

After tamato fruit is respectively connected to Antagonistic Fungi and sterilized water (comparison), the morbidity of comparison fruit Rate is 100%, and the sickness rate accessing the fruit of fermentation liquor treatment is only 13.2%.

4, the inhibition that Fructus Fragariae Ananssae is rotted by B.contaminans fermentation liquid naturally

Method:

Take the strong fruit of Fructus Fragariae Ananssae, after pre-cooling 24h, be handled as follows at 0 DEG C the same day of gathering: use The fermentation liquid of the 48h of B.contaminans soaks strawberry fruit, after fully soaking 3min, aseptic Wind is done, and dries up and is placed on 4 DEG C of wet-storages, and comparison is placed directly within 4 DEG C of wet-storages.13～ Incidence is checked when 15 days.Often process 20 fruits.Experiment is repeated 3 times.

Result:

Use the strawberry fruit of Antagonistic Fungi B.contaminans fermentation liquor treatment, its rotting rate and corruption Rotten degree is all significantly lower than comparison.

5, B.contaminans combines Na₂SiO₃Fructus Lycopersici esculenti is adopted the inhibition of rear gray mold

By 1 × 10⁸The B.contaminans bacteria suspension of cfu/mL adds variable concentrations Na₂SiO₃, as shown in Figure 6 (in figure, note: 1, CK；2、1×10⁸cfu/ml B.contaminans； 3、1×10⁸cfu/ml B.contaminans+25mM Na₂SiO₃；4、1×10⁸cfu/ml B.contaminans+50mM Na₂SiO₃；5、1×10⁸cfu/ml B.contaminans+100mM Na₂SiO₃), result shows, Na₂SiO₃It is remarkably improved the life of Antagonistic Fungi B.contaminans Anti-effect, and concentration is the Na of 50mM₂SiO₃Biological and ecological methods to prevent plant disease, pests, and erosion to Antagonistic Fungi B.contaminans Effect improves most effective.Cherry tomato inoculates the pathogen of Botrytis cinerea comparison sickness rate at normal temperatures 100%.And use merely 1 × 10⁸The Antagonistic Fungi of cfu/mL, its sickness rate is 45.36%, and Add the Na of 50mM₂SiO₃After, its sickness rate then reduces to 17.97%.

6, B.contaminans combines NaHCO₃Fructus Lycopersici esculenti is adopted the inhibition of rear gray mold

With the NaHCO of variable concentrations₃With 1 × 10⁸Cfu/mL is used in combination, as shown in Figure 7 (figure In, note: 1, CK；2、1×10⁸cfu/ml B.contaminans；3、1×10⁸cfu/ml B.contaminans+25mM NaHCO₃；4、1×10⁸cfu/ml B.contaminans+50mM NaHCO₃；5、1×10⁸cfu/ml B.contaminans+100mM NaHCO₃), result table Bright NaHCO₃Can also effectively promote the biological and ecological methods to prevent plant disease, pests, and erosion effect of Antagonistic Fungi B.contaminans, and make With the NaHCO of variable concentrations₃, its inhibition is the most different, with interpolation Na₂SiO₃Effect phase With, add the NaHCO of 50mM₃Raising to antagonistic effect is the most notable.But add Add the Na of same concentrations₂SiO₃Relatively add NaHCO₃Effect is more preferable.Adding Na₂SiO₃Time Fructus Lycopersici esculenti sickness rate is 17.97%, and adds NaHCO₃Time be 21.47%.

Bacterial strain of the present invention can suppress postharvest fruit and vegetable gray mold and penicilliosis well Occur, be also considerably improved the prevention effect of postharvest disease of fruits and vegetables.

Accompanying drawing explanation

Fig. 1 is the form schematic diagram on the culture medium flat plate of bacterial strain B-1.

Fig. 2 is the microscope schematic diagram of bacterial strain B-1.

Fig. 3 is the system of the 16S rDNA sequence based on bacterial strain B-1 built by adjacent method Grow tree schematic diagram.

Fig. 4 is that the system of the recA gene order based on bacterial strain B-1 built by adjacent method is sent out Educate tree schematic diagram.

Fig. 5 is the variable concentrations B.contaminans bacteria suspension suppression cylindricality to three kinds of pathogenic fungi Figure.

Fig. 6 is Na₂SiO₃Impact on Antagonistic Fungi B.contaminans suppression graw mold of tomato.

Fig. 7 is NaHCO₃Impact on Antagonistic Fungi B.contaminans suppression graw mold of tomato.

Detailed description of the invention

Below the specific embodiment of the present invention is described in detail.

Embodiment 1

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 25mM in described every milliliter of bacteria suspension Na₂SiO₃。

Embodiment 2

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 50mM in described every milliliter of bacteria suspension Na₂SiO₃。

Embodiment 3

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 100mM in described every milliliter of bacteria suspension Na₂SiO₃。

Embodiment 4

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 25mM in described every milliliter of bacteria suspension NaHCO₃。

Embodiment 5

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 50mM in described every milliliter of bacteria suspension NaHCO₃。

Embodiment 6

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the bacteria suspension of bacterial strain B-1, The concentration of described bacteria suspension is 1 × 10⁸Cfu/mL, adds 100mM in described every milliliter of bacteria suspension NaHCO₃。

Embodiment 7

A kind of for preventing the biocontrol agent of postharvest disease of fruits and vegetables, including the fermentation liquid of bacterial strain B-1, Fermentation condition: in LB fluid medium, 30 DEG C, 20r/min shake-flask culture 48h.

Embodiment 8

The cultural method of a kind of bacterial strain for preventing and treating postharvest disease of fruits and vegetables, the composition of preparation culture medium is such as Under: Fructus Hordei Germinatus leaching powder 8～15g/L, soy peptone 8～15g/L, MnSO₄·7H₂O 0.5～ 1.5g/L, KH₂PO₄0.5～1.0g/L, in described culture medium, then carry out inoculated and cultured.

Bacterial strain 16S rDNA sequence is as follows:

1 TACCATGCAG TCGAACGGCA GCACGGGTGC TTGCACCTGG TGGCGAGTGG

51 CGAACGGGTG AGTAATACAT CGGAACATGT CCTGTAGTGG GGGATAGCCC

101 GGCGAAAGCC GGATTAATAC CGCATACGAT CTACGGATGA AAGCGGGGGA

151 CCTTCGGGCC TCGCGCTATA GGGTTGGCCG ATGGCTGATT AGCTAGTTGG

201 TGGGGTAAAG GCCTACCAAG GCGACGATCA GTAGCTGGTC TGAGAGGACG

251 ACCAGCCACA CTGGGACTGA GACACGGCCC AGACTCCTAC GGGAGGCAGC

301 AGTGGGGAAT TTTGGACAAT GGGCGAAAGC CTGATCCAGC AATGCCGCGT

351 GTGTGAAGAA GGCCTTCGGG TTGTAAAGCA CTTTTGTCCG GAAAGAAATC

401 CTTGGCTCTA ATACAGTCGG GGGATGACGG TACCGGAAGA ATAAGCACCG

451 GCTAACTACG TGCCAGCAGC CGCGGTAATA CGTAGGGTGC GAGCGTTAAT

501 CGGAATTACT GGGCGTAAAG CGTGCGCAGG CGGTTTGCTA AGACCGATGT

551 GAAATCCCCG GGCTCAACCT GGGAACTGCA TTGGTGACTG GCAGGCTAGA

601 GTATGGCAGA GGGGGGTAGA ATTCCACGTG TAGCAGTGAA ATGCGTAGAG

651 ATGTGGAGGA ATACCGATGG CGAAGGCAGC CCCCTGGGCC AATACTGACG

701 CTCATGCACG AAAGCGTGGG GAGCAAACAG GATTAGATAC CCTGGTAGTC

751 CACGCCCTAA ACGATGTCAA CTAGTTGTTG GGGATTCATT TCCTTAGTAA

801 CGTAGCTAAC GCGTGAAGTT GACCGCCTGG GGAGTACGGT CGCAAGATTA

851 AAACTCAAAG GAATTGACGG GGACCCGCAC AAGCGGTGGA TGATGTGGAT

901 TAATTCGATG CAACGCGAAA AACCTTACCT ACCCTTGACA TGGTCGGAAT

951 CCCGCTGAGA GGTGGGAGTG CTCGAAAGAG AACCGGCGCA CAGGTGCTGC

1001 ATGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG

1051 AGCGCAACCC TTGTCCTTAG TTGCTACGCA AGAGCACTCT AAGGAGACTG

1101 CCGGTGACAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC CTCATGGCCC

1151 TTATGGGTAG GGCTTCACAC GTCATACAAT GGTCGGAACA GAGGGTTGCC

1201 AACCCGCGAG GGGGAGCTAA TCCCAGAAAA CCGATCGTAG TCCGGATTGC

1251 ACTCTGCAAC TCGAGTGCAT GAAGCTGGAA TCGCTAGTAA TCGCGGATCA

1301 GCATGCCGCG GTGAATACGT TCCCGGGTCT TGTACACACC GCCCGTCACA

1351 CCATGGGAGT GGGTTTTACC AGAAGTGGCT AGTCTAACCG CAAGGAGGAC

1401 GGTCACCACG GTAGGATTCA TGACTGGGGT GAAGTCGAA

Bacterial strain recA gene order is as follows:

1 GGAATCGTCC GGTAAAACCA CGCTCACGCT GCAGGTCATT GCCGAACTGC

51 AGAAGCTGGG CGGCACCGCA GCGTTTATCG ACGCCGAGCA CGCGCTCGAC

101 GTTCAATATG CAGCGAAGCT CGGCGTGAAC GTGCCGGAGC TGCTGATCTC

151 GCAGCCGGAC ACCGGCGAGC AGGCGCTTGA AATCACCGAC GCGCTGGTGC

201 GCTCGGGCTC GATCGACATG ATCGTCATCG ACTCGGTCGC GGCGCTCGTG

251 CCGAAGGCCG AAATCGAAGG CGAGATGGGC GATTCGCTGC CGGGTCTGCA

301 GGCCCGCCTG ATGTCGCAGG CGCTGCGCAA GCTGACCGGC ACGATCAAGC

351 GCACGAACTG CCTCGTGATC TTCATCAACC AGATCCGGAT GAAGATCGGC

401 GTGATGTTCG GCAACCCGGA AACCACGACG GGCGGTAACG CACTGAAGTT

451 CTACTCGTCG GTGCGTCTCG ACATCCGCCG GATCGGCTCG ATCAAGAAGA

501 ACGACGAGGT GATCGGCAAC GAAACCCGCG TGAAGGTCGT CAAGAACAAG

551 GTGTCGCCGC CGTTCCGCGA AGCGATCTTC GACATCCTGT ATGGCGAAGG

601 AATTTCGCGC CAGGGCGAGA TCATCGATCT CGGCGTGCAG GCGAAGATCG

651 TCGACAAGGC GGGCGCCTGG TACAGCTACA ACGGCGAGAA GATCGGCCAG

701 GGCAAGGACA ACGCGCGTGA ATTCCTGCGC GAAAATCCGG AAATCGCACG

751 CGAGATCGAG AACCGCATTC GCGAATCGCT TGGCGTGGTC ACCATGCCCG

801 ATGG

Claims

1. for preventing and treating a bacterial strain for postharvest disease of fruits and vegetables, being deposited in China typical culture collection center on March 7th, 2014, preservation registration number is CCTCC M 2014076；

Bacterial strain 16S rDNA sequence is as follows:

TACCATGCAG TCGAACGGCA GCACGGGTGC TTGCACCTGG TGGCGAGTGG

CGAACGGGTG AGTAATACAT CGGAACATGT CCTGTAGTGG GGGATAGCCC

GGCGAAAGCC GGATTAATAC CGCATACGAT CTACGGATGA AAGCGGGGGA

CCTTCGGGCC TCGCGCTATA GGGTTGGCCG ATGGCTGATT AGCTAGTTGG

TGGGGTAAAG GCCTACCAAG GCGACGATCA GTAGCTGGTC TGAGAGGACG

ACCAGCCACA CTGGGACTGA GACACGGCCC AGACTCCTAC GGGAGGCAGC

AGTGGGGAAT TTTGGACAAT GGGCGAAAGC CTGATCCAGC AATGCCGCGT

GTGTGAAGAA GGCCTTCGGG TTGTAAAGCA CTTTTGTCCG GAAAGAAATC

CTTGGCTCTA ATACAGTCGG GGGATGACGG TACCGGAAGA ATAAGCACCG

GCTAACTACG TGCCAGCAGC CGCGGTAATA CGTAGGGTGC GAGCGTTAAT

CGGAATTACT GGGCGTAAAG CGTGCGCAGG CGGTTTGCTA AGACCGATGT

GAAATCCCCG GGCTCAACCT GGGAACTGCA TTGGTGACTG GCAGGCTAGA

GTATGGCAGA GGGGGGTAGA ATTCCACGTG TAGCAGTGAA ATGCGTAGAG

ATGTGGAGGA ATACCGATGG CGAAGGCAGC CCCCTGGGCC AATACTGACG

CTCATGCACG AAAGCGTGGG GAGCAAACAG GATTAGATAC CCTGGTAGTC

CACGCCCTAA ACGATGTCAA CTAGTTGTTG GGGATTCATT TCCTTAGTAA

CGTAGCTAAC GCGTGAAGTT GACCGCCTGG GGAGTACGGT CGCAAGATTA

AAACTCAAAG GAATTGACGG GGACCCGCAC AAGCGGTGGA TGATGTGGAT

TAATTCGATG CAACGCGAAA AACCTTACCT ACCCTTGACA TGGTCGGAAT

CCCGCTGAGA GGTGGGAGTG CTCGAAAGAG AACCGGCGCA CAGGTGCTGC

ATGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG TCCCGCAACG

AGCGCAACCC TTGTCCTTAG TTGCTACGCA AGAGCACTCT AAGGAGACTG

CCGGTGACAA ACCGGAGGAA GGTGGGGATG ACGTCAAGTC CTCATGGCCC

TTATGGGTAG GGCTTCACAC GTCATACAAT GGTCGGAACA GAGGGTTGCC

AACCCGCGAG GGGGAGCTAA TCCCAGAAAA CCGATCGTAG TCCGGATTGC

ACTCTGCAAC TCGAGTGCAT GAAGCTGGAA TCGCTAGTAA TCGCGGATCA

GCATGCCGCG GTGAATACGT TCCCGGGTCT TGTACACACC GCCCGTCACA

CCATGGGAGT GGGTTTTACC AGAAGTGGCT AGTCTAACCG CAAGGAGGAC

GGTCACCACG GTAGGATTCA TGACTGGGGT GAAGTCGAA

Bacterial strain recA gene order is as follows:

GGAATCGTCC GGTAAAACCA CGCTCACGCT GCAGGTCATT GCCGAACTGC

AGAAGCTGGG CGGCACCGCA GCGTTTATCG ACGCCGAGCA CGCGCTCGAC

GTTCAATATG CAGCGAAGCT CGGCGTGAAC GTGCCGGAGC TGCTGATCTC

GCAGCCGGAC ACCGGCGAGC AGGCGCTTGA AATCACCGAC GCGCTGGTGC

GCTCGGGCTC GATCGACATG ATCGTCATCG ACTCGGTCGC GGCGCTCGTG

CCGAAGGCCG AAATCGAAGG CGAGATGGGC GATTCGCTGC CGGGTCTGCA

GGCCCGCCTG ATGTCGCAGG CGCTGCGCAA GCTGACCGGC ACGATCAAGC

GCACGAACTG CCTCGTGATC TTCATCAACC AGATCCGGAT GAAGATCGGC

GTGATGTTCG GCAACCCGGA AACCACGACG GGCGGTAACG CACTGAAGTT

CTACTCGTCG GTGCGTCTCG ACATCCGCCG GATCGGCTCG ATCAAGAAGA

ACGACGAGGT GATCGGCAAC GAAACCCGCG TGAAGGTCGT CAAGAACAAG

GTGTCGCCGC CGTTCCGCGA AGCGATCTTC GACATCCTGT ATGGCGAAGG

AATTTCGCGC CAGGGCGAGA TCATCGATCT CGGCGTGCAG GCGAAGATCG

TCGACAAGGC GGGCGCCTGG TACAGCTACA ACGGCGAGAA GATCGGCCAG

GGCAAGGACA ACGCGCGTGA ATTCCTGCGC GAAAATCCGG AAATCGCACG

CGAGATCGAG AACCGCATTC GCGAATCGCT TGGCGTGGTC ACCATGCCCG

ATGG。

2. the biocontrol agent being used for preventing postharvest disease of fruits and vegetables, it is characterised in that: include that the bacteria suspension of bacterial strain described in claim 1, the concentration of described bacteria suspension are 1 × 10⁸Cfu/mL, adds the Na of 50mM in every milliliter of described bacteria suspension₂SiO₃。

3. the biocontrol agent being used for preventing postharvest disease of fruits and vegetables, it is characterised in that: include that the bacteria suspension of bacterial strain described in claim 1, the concentration of described bacteria suspension are 1 × 10⁸Cfu/mL, adds the NaHCO of 50mM in every milliliter of described bacteria suspension₃。

4. the cultural method of the bacterial strain for preventing and treating postharvest disease of fruits and vegetables described in a claim 1, it is characterised in that: the composition of preparation culture medium is as follows: Fructus Hordei Germinatus leaching powder 8～15g/L, soy peptone 8～15 g/L, MnSO₄·7H₂O 0.5～1.5 G/L, KH₂PO₄0.5～1.0 g/L, then carry out inoculated and cultured in described culture medium.