CN104498395B - D amino acid suppresses banana bacterial soft rot bacterium biomembrane and its culture medium - Google Patents

D amino acid suppresses banana bacterial soft rot bacterium biomembrane and its culture medium Download PDF

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CN104498395B
CN104498395B CN201410715110.6A CN201410715110A CN104498395B CN 104498395 B CN104498395 B CN 104498395B CN 201410715110 A CN201410715110 A CN 201410715110A CN 104498395 B CN104498395 B CN 104498395B
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amino acid
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soft rot
methionine
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张景欣
林壁润
黄宁
沈会芳
蒲小明
潘群英
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Plant Protection Research Institute Guangdong Academy of Agricultural Sciences
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Abstract

The invention belongs to crop disease control field, application and its culture medium of a kind of D amino acid in banana bacterial soft rot bacterium bacterial biof iotalm is suppressed are disclosed.The D amino acid of present invention screening significantly can effectively suppress the growth of the bacterial biof iotalm of banana bacterial soft rot bacterium.Present invention screening obtains 4 kinds of D amino acid that can significantly inhibit the bacterial biofilm growth of bacterial soft rot bacterium, D tyrosine, D valines, D methionine, D tryptophans；And obtain its minimum inhibitory concentration.Present invention also offers the mixture formula of above-mentioned 4 kinds of D amino acid, the concentration of various D amino acid is greatly reduced.Present invention also offers a kind of D amino acid medias for above-mentioned application.New method and thinking can be provided based on present disclosure to develop the new technology of preventing and treating of bacterial soft rot bacterium；Used with reference to D amino acid, improve the using effect of the prevention and controls such as chemical pesticide, biological pesticide.

Description

D- amino acid suppresses banana bacterial soft rot bacterium biomembrane and its culture medium
Technical field
The invention belongs to crop disease control field, more particularly to a kind of D- amino acid is suppressing the bacillary soft corruption of banana Application and its culture medium in germ bacterial biof iotalm.
Background technology
Banana is mainly grown in tropical and subtropical region, at least 107 country's production bananas；2013, banana was The fourth-largest food plant in the output value whole world, is only second to rice, wheat and corn, and nearly 10 years whole world banana planting area is in becoming for increasing Gesture.However, the crop has been subject to the serious harm of banana bacterial soft rot；From the Dickeya that this seminar reports first Sp. generation (Lin et al, 2010) of the banana bacterial soft rot for causing in China's Mainland rises, the expansion of bacterial soft rot Exhibition rate of propagation is rapid, and the hair of the disease is found in the banana producing region such as Banana In Guangdong Province of China major production areas and Hainan, Guangxi, Fujian It is raw.The disease Field diseases 20%~30%, seriously up to more than 100%, and cause whole strain dead, serious harm banana Industry.Comprehensive biochemical character, conserved genetic sequences etc., the pathogen be accredited as Dickeya zeae (Zhang et al, 2010)。
Bacterium is attached to biological or non-biological material surface and can form many bacteriums of film sample to adapt to living environment and inhaling and is combined Body, i.e. bacterial biof iotalm (Bacterial biofilm, BBF) (Stoodley et al, 2002).The bacterium of biofilm states Than the ability that the bacterium under floating state has stronger drug resistance, virulence and confrontation host immune defenses, can be antibacterial in host Survived in composition, bacterial biof iotalm has turned into the important virulence factor (Morris and Monier, 2003) of pathogenetic bacteria, and Play a role (Danhom and Fuqua, 2007) in the dynamic process of plant and pathogen interaction.Bacterial biof iotalm is copper Green pseudomonad infects the key factor of various hosts, and scanning electron microscopic observation finds that it forms bacterium in host plant is organized Biomembrane (Tan et al, 1999)；Causing the xyllela fastidiosa of grape Pi Ershi diseases can form bacterial biof iotalm simultaneously in xylem Occlusion catheter, plant dehydration and nutrient transport is obstructed and show symptom (Guilhabert and Kirkpatrick, 2005)；Meanwhile, plant protection equally faces the increased problem of the resistance to the action of a drug of bacterial biof iotalm.Due to the extracellular thing of bacterial biof iotalm The barrier action of matter, bacterial density and by reducing membrane passage etc., reduce antibacterial medicines and enter intracellular and increase Strong intracellular drug efflux so that bacterium has the resistance to the action of a drug that 1,100 times are higher by than state of swimming in biofilm states.Therefore, pin To plant pathogenetic bacteria, the research related to the inhibiting factor screening of bacterial biof iotalm can be provided for the exploitation of plant protection technology New approaches, it is also significant for prevention and control bacterial plant disease.
The content of the invention
In order to overcome the shortcomings and deficiencies of the prior art described above, primary and foremost purpose of the invention is to provide D- amino acid in suppression Application in banana bacterial soft rot bacterium bacterial biof iotalm processed.The D- amino acid that the present invention is provided is to banana bacterial soft rot The bacterial biof iotalm of bacterium has significant inhibitory action.
Another object of the present invention is to provide a kind of D- amino acid medias for above-mentioned application.
The purpose of the present invention is realized by following proposal：
Application of the D- amino acid in banana bacterial soft rot bacterium (Dickeya zeae) bacterial biof iotalm is suppressed, it is described D- amino acid be at least one in D-Tyrosine, D-trp, D-Val and D- methionine.
In order to the present invention is further better achieved, when the D-Tyrosine is used alone, its effective inhibition concentration is 420 μMol/L；
In order to the present invention is further better achieved, when the D-trp is used alone, its effective inhibition concentration is 5mMol/L；
In order to the present invention is further better achieved, when the D-Val is used alone, its effective inhibition concentration is 11mMol/L；
In order to the present invention is further better achieved, when the D- methionine is used alone, its effective inhibition concentration is 15mMol/L。
Of the invention to be preferably used in mixed way two kinds, three kinds or four kinds above-mentioned D- amino acid, mix and match uses above-mentioned D- amino Acid, each other with synergistic effect, is effectively reduced the consumption of various D- amino acid.
It is highly preferred that consumption when four kinds of D- amino acid collocation are used in the application can be 120 μM of D- junket ammonia of ol/L Acid, the D- methionine of the D-Val of 5mMol/L, 7mMol/L, the tryptophan of 0.5mMol/L；
Or the consumption in the application when collocation of four kinds of D- amino acid is used can be 160 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 5mMol/L, the tryptophan of 0.5mMol/L；
Or the consumption in the application when collocation of four kinds of D- amino acid is used can be 120 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 5mMol/L, the tryptophan of 3mMol/L；
Or the consumption in the application when collocation of four kinds of D- amino acid is used can be 320 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 3mMol/L, the tryptophan of 3mMol/L.
It is above-mentioned that D- amino acid is applied in suppression banana bacterial soft rot bacterium bacterial biof iotalm, bacterium can be significantly inhibited Biofilm development, for the new technology of preventing and treating for developing bacterial soft rot bacterium provides new method and thinking
Suppressing banana bacterial soft rot bacterium bacterium for above-mentioned D- amino acid medias present invention also offers one kind It is the D- amino acid medias of application in biomembrane, is to be added with D- amino acid in fluid nutrient medium commonly used in the art to obtain Culture medium, such as NA fluid nutrient mediums.Described D- amino acid is D-Tyrosine, D-trp, D-Val and D- egg ammonia At least one in acid.
In order to the present invention is further better achieved, above-mentioned four kinds of D- ammonia is preferably added in described D- amino acid medias Base acid.
It is highly preferred that the content of four kinds of D- amino acid is 120 μM of D- junket ammonia of ol/L in described D- amino acid medias Acid, the D- methionine of the D-Val of 5mMol/L, 7mMol/L, the tryptophan of 0.5mMol/L；
Or in described D- amino acid medias the content of four kinds of D- amino acid be 160 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 5mMol/L, the tryptophan of 0.5mMol/L；
Or in described D- amino acid medias the content of four kinds of D- amino acid be 120 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 5mMol/L, the tryptophan of 3mMol/L；
Or in described D- amino acid medias the content of four kinds of D- amino acid be 320 μM of D-Tyrosines of ol/L, The D-Val of 3mMol/L, the D- methionine of 3mMol/L, the tryptophan of 3mMol/L.
The D- amino acid medias that the present invention is provided can significantly inhibit the life of banana bacterial soft rot bacterium bacterial biof iotalm It is long, for the new technology of preventing and treating for developing banana bacterial soft rot bacterium provides new method and thinking；Used with reference to D- amino acid, carried The using effect of the prevention and controls such as chemical pesticide high, biological pesticide, develops new efficient, low toxicity, environment-protection pesticide.
The present invention has the following advantages and beneficial effect relative to prior art：
The D- amino acid of present invention screening significantly can effectively suppress the life of the bacterial biof iotalm of banana bacterial soft rot bacterium It is long.The present invention obtains the D- amino acid of the bacterial biofilm growth that 4 kinds can significantly inhibit bacterial soft rot bacterium, D- by screening Tyrosine, D-Val, D- methionine, D-trp；And screen the their own minimum inhibitory concentration of acquisition.Meanwhile, this hair It is bright to suppress the notable of banana bacterial soft rot bacterium also by violet staining and the clear and definite D- amino acid of laser confocal microscope Effect and cytologic characteristic.Present invention also offers the mixture formula of above-mentioned 4 kinds of D- amino acid, various D- amino are greatly reduced The concentration of acid, and can equally significantly inhibit the growth of bacterial biof iotalm.It is used for above-mentioned application present invention also offers one kind D- amino acid medias.New side can be provided based on present disclosure to develop the new technology of preventing and treating of bacterial soft rot bacterium Method and thinking；Used with reference to D- amino acid, improve the using effect of the prevention and controls such as chemical pesticide, biological pesticide.
Brief description of the drawings
Fig. 1 is the bacterial biof iotalm biomass relative quantification of Dickeya zeae MS1, wherein, D.zeae biofilms, D.zeae planktonic represent the relative quantification of bacterial biof iotalm and planktonic bacteria respectively.
Fig. 2 is the morphological feature laser co-focusing micrograph of Dickeya zeae MS1 bacterial biof iotalms.
Fig. 3 is Dickeya zeae MS1 bacterial biof iotalm violet staining analysis charts after D- amino acid treatments.
Fig. 4 is the laser co-focusing analysis chart that D-Val suppresses Dickeya zeae MS1 bacterial biofilm growths.
Fig. 5 is that 4 kinds of D- amino acid mixtures formulas suppress the analysis chart that the bacterial biof iotalm of Dickeya zeae MS1 is formed.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited In this.
Banana bacterial soft rot bacterium (Dickeya zeae MS1) is in document " Identification of Dickeya zeae as a Causal Agent of Bacterial Soft Rot in Banana in China.Plant Disease,2014,98(4):Disclosed in 436-442. ", carried by Inst. of Plant Protection, Guangdong Prov. Academy of Agricultural Sciences laboratory For；
D- amino acid is bought in Sigma-Aldrich companies.
Embodiment 1：The culture of bacterium and the culture of bacterial biof iotalm
Take bacterium bacterial strain Dickeya zeae MS1 (the Identification of Dickeya that glycerine freezes (- 80 DEG C) zeae as a Causal Agent of Bacterial Soft Rot in Banana in China.Plant Disease,2014,98(4):NA solid mediums (beef extract 3g, yeast extract 1g, peptone 436-442.) are inoculated into respectively 5g, glucose 10g, agar 18g, water are settled to 1000mL, and pH is 7.2~7.4) on flat board, line culture；Picking single bacterium falls within Liquid NA culture mediums (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, water are settled to 1000mL, and pH is 7.2~ 7.4) culture in, 180rpm, 32 DEG C of cultures.
Culture Dickeya zeae MS1 to 108CFU/mL；NA fluid nutrient mediums dilute bacterium solution to 1:1000.Take 2mL dilute The bacterium solution released is added in the microwell plate of polystyrene 24, per 3 repetitions of sample, 32 DEG C of quiescent cultures, under making after culture different time One step is detected.After culture 6h, 12h, 24h, 36h, 48h, 60h, 72h, 96h, 144h, 216h, planktonic bacteria is first extracted in 96 holes In plate, 600nm (OD are measured in ELIASA600) absorbance (Multiskan Spectrum, Thermo Lab Systems)；Next step mixes remaining planktonic bacteria and bacterial biof iotalm, and 600nm (OD are measured again600) absorbance. Calculate the bacterium OD values after mixing and subtract planktonic bacteria OD values, the as relative quantification of bacterial biof iotalm biomass.
Culture Dickeya zeae MS1 to 108CFU/mL；NA culture mediums dilute bacterium solution to 1:1000.Add 5mL dilutions Bacterium solution in the orifice plate of polystyrene 6, be soaked into bacteria culture media using the optics slide of polystyrene, trained jointly with bacterium Support 6h, 24h.Removal planktonic bacteria is cleaned using PBS, the bacterial cell of adhering substrate slide uses 5% glutaraldehyde 1h is fixed, then is cleaned with PBS；PI is dyeed for DNA, and Alexa-Fluor Concanavalin A (ConA) are used In combining exocellular polysaccharide, dyestuff respectively in connection with for analyzing Dickeya zeae MS1 bacterial biof iotalms in different time sections shape Into feature.Excited using following argon laser, receive wavelength parameter：PI 630-633nm are excited and received with 650-800nm, ConA 346nm are excited and received with 442nm, confocal laser scanning microscope.
Result is shown in Fig. 1 and Fig. 2.As seen from the figure, 0~12h, flcating germ increment is more than bacterial biof iotalm；Reached to 24h dynamic State is balanced, and the speed of growth is consistent；After 48h, flcating germ becomes feeble and die and bacterial biof iotalm is constant；Show that Dickeya zeae MS1 are in vitro 24h can form the bacterial biof iotalm of stabilization after culture.Confocal laser scanning microscope result is similar to, and what is formed in 6h is thin Bacterium biomembrane is leaner, and during to 24h, bacterium has formed complete, intensive bacterial biof iotalm.
Embodiment 2：Different D- amino acid suppress the effect analysis of banana bacterial soft rot bacterium
Prepare D-Val, D- methionine, D-trp, D-Thr, D-Arg, D-PROLINE, D-Leu, D- Serine, D-His, D-Asp, D-Glu, D-Gln, D-phenylalanine, D-alanine etc. The storing liquid of 100mMol/L；The storing liquid of the 10mMol/L of D-Tyrosine is prepared, wherein D-Tyrosine storing liquid contains 0.1Mol/L HCl.Matched according to various concentrations, prepare the NA fluid nutrient mediums of series D- amino acid concentrations；Be respectively configured 200,220, 240th, 260,280,300,320,340,360,380,400,420,440,460,480 and 500 μM of ol/L (write a Chinese character in simplified form μM) concentration (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, water are settled to 1000mL, pH to the NA fluid nutrient mediums of D-Tyrosine For 7.2~7.4)；It is respectively configured 1,3,5,6,7,8,9,10,11,12,13,14,15,17.5 and 20mMol/L's (writing a Chinese character in simplified form mM) (beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, water are settled to 1000mL, pH to the NA culture mediums of other D- amino acid For 7.2~7.4).
Take glycerine and freeze the bacterium bacterial strain Dickeya zeae MS1 of (- 80 DEG C) and be inoculated into NA solid medium (beef extracts 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water are settled to 1000mL, and pH is 7.2~7.4) on flat board, draws Line culture；Picking single bacterium is cultivated in falling within NA fluid nutrient mediums, 180rpm, 32 DEG C of cultures.
Culture Dickeya zeae MS1 to 108CFU/mL；Bacterium is diluted using the NA culture mediums of various concentrations D- amino acid Liquid is to 1:1000, set without D- amino acid bacterium solution control (CK), and aseptic NA fluid nutrient mediums control (NA).Take 2mL dilutions Bacterium solution is added in the microwell plate of polystyrene 24,32 DEG C of quiescent cultures.After culture 24h, planktonic bacteria is first extracted in 96 orifice plates, often 3 repetitions of sample, 600nm (OD are measured in ELIASA600) absorbance (Multiskan Spectrum, Thermo Lab Systems)；Next step mixes remaining planktonic bacteria and bacterial biof iotalm, and 600nm (OD are measured again600) absorbance. Calculate the bacterium OD values after mixing and subtract planktonic bacteria OD values, as the relative quantification of bacterial biof iotalm biomass, the results are shown in Table 1 ~2.
Squirter bacterium can form obvious bacterial biof iotalm after cultivating 24h in 24 orifice plates in gas-liquid interface, its Relative quantification value (OD600) it is 0.52 to 0.83.From table 1 and the result of table 2, after D- amino acid treatments, different D- amino Acid shows completely different effect for squirter bacterium；The performance of D-Val, D- methionine, D-trp and D-Tyrosine Different degrees of inhibitory action；Remaining 9 kinds of D- amino acid, the bright ammonia of D-Thr, D-Ser, D-PROLINE, D-Arg, D- Acid, D-phenylalanine, D-His, D-Gln, D-alanine etc. are then for squirter bacterium bacterial biof iotalm without suppression Make and use.
Wherein, the ability of D-Tyrosine suppression bacterial biofilm growth is most strong, fragrant in 420 μM of concentration and higher concentration The significant suppressed trend of bacterial biof iotalm performance of any of several broadleaf plants Bacteria erwinia, when being acted on to 480 μM of concentration, bacterial biof iotalm is in 24 holes Do not grown substantially in plate.The inhibitory action of D-trp is taken second place, under 5mM and higher concentration effect, bacterial biofilm growth Appearance is decreased obviously trend.D-Val and D- methionine for bacterial biof iotalm a certain degree of inhibitory action of also performance, Under 11mM and the D-Val of higher concentration, or the D- methionine effect of 15mM and higher concentration, squirter bacterium The growth of bacterial biof iotalm is similarly subjected to significantly inhibit.Compare down, bacterium of remaining 9 kinds of D- amino acid for squirter bacterium The basic unrestraint effect of biomembrane or weaker inhibitory action, when their activity reaches 20mM, bacterial biof iotalm still may be used Normal growth.
Embodiment 3：D- amino acid suppresses the violet staining analysis of bacterial biof iotalm effect
Take glycerine and freeze the bacterium bacterial strain Dickeya zeae MS1 of (- 80 DEG C) and be inoculated into NA solid medium (beef extracts 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water are settled to 1000mL, and pH is 7.2~7.4) on flat board, draws Line culture；Picking single bacterium is cultivated in falling within NA fluid nutrient mediums, 180rpm, 32 DEG C of cultures.
Culture Dickeya zeae MS1 to 108CFU/mL；It is respectively configured the D-Thr and D-alanine of 20mM concentration NA fluid nutrient mediums, the 200 μM and 420 μM D-Tyrosine NA fluid nutrient mediums of concentration, the D-trp NA of 3mM and 5mM concentration Culture medium, the D-Val NA culture mediums of 5mM and 11mM concentration, the D- methionine NA culture mediums of 7mM and 15mM concentration；With difference The NA culture mediums of concentration D- amino acid dilute bacterium solution to 1:1000, set without the control of D- amino acid bacterium solution, and sterile liquid NA trainings Support base control.Take in the bacterium solution addition microwell plate of polystyrene 96 of 0.2mL dilutions, 32 DEG C of quiescent cultures make next after culture 24h Step detection.
Culture is discarded, is used Bouin's liquid (picric acid saturated aqueous solution 75mL, glacial acetic acid 5mL, formalin 25mL) After room temperature fixes 15min, washed away with PBS, add the μ L of 0.5% crystal violet 50, dye 15min, microwell plate is cleaned with distilled water, treated Ethanol acetone mixture (70 is added after drying:30) 200 μ L, 3min is stored at room temperature after fully mixing again, measures 590nm (OD590) Absorbance.
Fig. 3 is shown in bacterial biof iotalm violet staining interpretation of result after D- amino acid treatments.In figure, CK is left for bacterium solution is compareed, CK is right for NA culture mediums are compareed；D-Thr, D-Ala, D-Val, D-Tyr, D-Met, D-Trp represent D-Thr, the ammonia of D- third respectively Acid, D-Val, D-Tyrosine, D- methionine, D-trp, upper values represent D- amino acid concentrations.From result, CK The bacterium solution control on a left side, 20mMD- threonines, D-alanine can form complete bacterial biof iotalm；420 μM of D- junket ammonia of concentration It is soft that the D- methionine of acid, the D-trp of 5mM concentration, the D-Val of 11mM concentration and 15mM concentration can significantly inhibit banana The formation of maize ear rot bacterium bacterium living beings.
Embodiment 4：D- amino acid suppresses the laser co-focusing analysis of bacterial biof iotalm effect
Take glycerine and freeze the bacterium bacterial strain Dickeya zeae MS1 of (- 80 DEG C) and be inoculated into NA solid medium (oxen respectively Meat extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water are settled to 1000mL, and pH is 7.2~7.4) flat board On, line culture；Picking single bacterium is cultivated in falling within NA fluid nutrient mediums, 180rpm, 32 DEG C of cultures.
Culture Dickeya zeae MS1 to 108CFU/mL, is respectively configured the D- figured silk fabrics ammonia of 5mM, 7mM, 11mM, 15mM concentration The NA culture mediums of acid, dilution bacterium solution to 1:1000, set without the control of D- amino acid bacterium solution, and the control of sterile liquid NA culture mediums. Add the bacterium solution of 5mL various concentrations D-Val configuration in the orifice plate of polystyrene 6, soaked using the optics slide of polystyrene In entering bacteria culture media, with bacterium co-incubation 24h.Removal planktonic bacteria is cleaned using PBS, adhering substrate slide Bacterial cell is fixed 1h using 5% glutaraldehyde, then is cleaned with PBS；PI is used for DNA dyeing (red fluorescence), Alexa-Fluor Concanavalin A (ConA) (green fluorescence) are used to combine exocellular polysaccharide, dyestuff respectively in connection with being used for Analysis D- amino acid suppresses the effect of squirter bacterium bacterial biof iotalm.Excited using following argon laser, receive wavelength ginseng Number：PI 630-633nm are excited and received with 650-800nm, and ConA 346nm are excited and received with 442nm, laser confocal microscope Observation.
Various concentrations D-Val suppresses the laser co-focusing analysis of banana bacterial soft rot bacterium bacterial biofilm growth Result is shown in Fig. 4.In figure, each concentration for the treatment of is indicated in the left of figure, and CK is the bacterium solution control for being not added with D- amino acid.Can by Fig. 4 See, CK can form complete dense bacterium living beings membrane structure, exocellular polysaccharide density is larger；The treatment of 5mM and 7mM D-Vals Bacterial biofilm growth is normal, and biofilm structure is complete；11mM D-Vals are the inhibition concentration of bacterial biof iotalm, and laser is common Focus analysis show that the bacterial biof iotalm after concentration treatment is imperfect, many places exist substantially it is empty every, but exocellular polysaccharide is still Largely it is present in bacterial biof iotalm, is the important skeleton for constituting bacterial biof iotalm；After the treatment of 15mM D-Vals, bacterium life Thing film is further suppressed, and biomembrane is imperfect, and the bacterium of large area is exposed in bacterial biof iotalm periphery, bacterial biof iotalm Integrated degree is further reduced.
Embodiment 5：D- amino acid mixtures formula suppresses the effect analysis of banana bacterial soft rot bacterium
Take glycerine and freeze the bacterium bacterial strain Dickeya zeae MS1 of (- 80 DEG C) and be inoculated into NA solid medium (beef extracts 3g, yeast extract 1g, peptone 5g, glucose 10g, agar 18g, water are settled to 1000mL, and pH is 7.2~7.4) on flat board, draws Line culture；Picking single bacterium is cultivated in falling within NA fluid nutrient mediums, 180rpm, 32 DEG C of cultures.
Culture Dickeya zeae MS1 to 108CFU/mL；With 4 kinds of D- amino acid (D-Tyrosine, D-Val, D- eggs Propylhomoserin, D-trp) growth inhibiting concentration component design is not caused to bacterial biof iotalm, by a large amount of screenings, this research choosing Fixed following 4 kinds of different D- amino acid is formulated, a.120 μM D-Tyrosine, 5mM D-Vals, 7mM D- methionine, 0.5mM D-trp；B.160 μM D-Tyrosine, 3mM D-Vals, 5mM D- methionine, 0.5mM D-trps；c.120μM D- Tyrosine, 3mM D-Vals, 5mM D- methionine, 3mM D-trps；D.320 μM D-Tyrosine, 3mM D-Vals, 3mM D- methionine, 3mM D-trps.With above-mentioned mixture formula, the NA training amino acid for configuring various concentrations D- amino acid is supported Base, dilution bacterium solution to 1:1000, set without the control of D- amino acid bacterium solution.The bacterium solution for taking 2mL dilutions adds the micropore of polystyrene 24 In plate, 32 DEG C of quiescent cultures.After culture 24h, directly observation, photograph.
Same culture Dickeya zeae MS1 to 108CFU/mL, and use above D- amino acid recipe configuration differences are dense The D- amino acid medias of degree, are set without the control of D- amino acid bacterium solution.The bacterium solution for taking 0.2mL dilutions adds the micropore of polystyrene 96 In plate, 32 DEG C of quiescent cultures make next step detection after culture 24h.
Culture is discarded, is used Bouin's liquid (picric acid saturated aqueous solution 75mL, glacial acetic acid 5mL, formalin 25mL) After room temperature fixes 15min, washed away with PBS, add the μ L of 0.5% crystal violet 50, dye 15min, microwell plate is cleaned with distilled water, treated Ethanol acetone mixture (70 is added after drying:30) 200 μ L, 3min is stored at room temperature after fully mixing again, measures 590nm (OD590) Absorbance.
The result that the bacterial biof iotalm of 4 kinds of D- amino acid mixture influence banana bacterial soft rot bacterium is formed is shown in Fig. 5.Figure In, it is after the acid treatment of D- amino and control treatment, to cultivate the direct observation result before the bacterial biof iotalm dyeing of 24h above figure； It is that the violet staining of bacterial biof iotalm is analyzed after the acid treatment of D- amino and control treatment below figure；1 is formula a, and 2 is formula B, 3 is formula c, and 4 is formula d, and CK is control treatment.It can be seen that at 4 kinds of differences D- amino acid mixture formulas of the invention The growth of the bacterial biof iotalm of reason is substantially suppressed, even bacterial biof iotalm forms and leaner is difficult to observe.By contrast, it is right Bacterial biof iotalm according to treatment is dense and more complete.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (3)

  1. Application of the 1.D- amino acid in banana bacterial soft rot bacterium (Dickeya zeae) bacterial biof iotalm is suppressed, its feature It is：Described D- amino acid is at least one in D-Tyrosine, D-trp, D-Val and D- methionine.
  2. 2. D- amino acid according to claim 1 is suppressing the life of banana bacterial soft rot bacterium (Dickeya zeae) bacterium Application in thing film, it is characterised in that：Effective inhibition concentration of the D-Tyrosine is 420 μM of ol/L；The D-trp Effective inhibition concentration is 5mMol/L；Effective inhibition concentration of the D-Val is 11mMol/L；The D- methionine it is effective Inhibition concentration is 15mMol/L.
  3. 3. D- amino acid according to claim 1 is suppressing the life of banana bacterial soft rot bacterium (Dickeya zeae) bacterium Application in thing film, it is characterised in that：Consumption when D- amino acid collocation is used be 120 μM of D-Tyrosines of ol/L, The D-Val of 5mMol/L, the D- methionine of 7mMol/L, the tryptophan of 0.5mMol/L；
    Or for 160 μM of D-Tyrosines of ol/L, the D-Val of 3mMol/L, the D- methionine of 5mMol/L, 0.5mMol/L color Propylhomoserin；
    Or for 120 μM of D-Tyrosines of ol/L, the D-Val of 3mMol/L, the D- methionine of 5mMol/L, 3mMol/L color ammonia Acid；
    Or for 320 μM of D-Tyrosines of ol/L, the D-Val of 3mMol/L, the D- methionine of 3mMol/L, 3mMol/L color ammonia Acid.
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D-Amino Acids Trigger Biofilm Disassembly;lana Kolodkin-Gal,Diego Romero,Shugeng Cao,Richard Losick;《Science》;20100430;第328卷;627-629 *

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