CN102876613A

CN102876613A - Method taken diffusible signal factor (DSF) as substrate and used for screening degrading bacteria

Info

Publication number: CN102876613A
Application number: CN 201210388524
Authority: CN
Inventors: 胡白石; 田艳丽; 赵玉强; 刘凤权
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2012-10-15
Filing date: 2012-10-15
Publication date: 2013-01-16

Abstract

The invention relates to a method for screening degrading bacteria for degrading a diffusible signal factor (DSF) which is taken as a substrate, and belongs to the technical field of biology. The method for screening the degrading bacteria is self-designed according to the principle of a BIOLOG bacterium identification system; the DSF is taken as a carbon source to be determined; and if bacteria are subjected to oxidation-reduction reaction by utilizing the carbon source, electrons are transferred, and colorless tetrazolium violet becomes purple, a positive strain which can degrade the DSF is screened. A Screening result that bacteria in different habitats degrade the DSF by the method is shown in a drawing.

Description

A kind of screening method of the degradation bacteria take DSF as substrate

(1) technical field

The present invention relates to screening degraded diffusivity signaling molecule (DSF) for the screening method of the degradation bacteria of substrate, belong to biological technical field.

(2) background technology

Xanthomonas campestris (Xanthomonas spp.) is one of relevant with the plant the most widely bacterial disease original that distributes.Studies show that, the member of this genus can infect 124 kinds of monocotyledonss and 268 kinds of dicotyledonss (Leyns et al., 1984).Wherein have most several kinds of economic implications, (Xanthomonas campestris) is the absolute predominance kind such as xanthomonas campestris, it has 141 pathogenic mutation at least, can infect many plants, comprise important farm crop, such as (Hayward, 1993) such as wild cabbage, Cauliflower, cabbage, tomato, pepper, cotton, soybean and English walnuts; The yellow unit cell of rice (Xanthomonas oryzae) comprises 2 pathogenic mutation, infects paddy rice and causes heavy losses; The carpetweed Xanthomonas campestris pv.citri is the most serious pathogenetic bacterias of harm oranges and tangerines.For phytobacterial disease, produce at present and there is no highly effective measure of control.Although seed treatment can stop disease by seed dispersal effectively, for the chronic illness district, Controlling effect is unsatisfactory; Still there is not at present very effectively chemical agent; Therefore, in case Micobial Disease big area generation and popular.Be difficult to take out effective prevention and control measure in short period of time.

The evidence that day by day increases shows: bacterium is not an isolated organism, but utilizes some little chemical signal molecules to carry out positive information interchange.Quorum sensing (Quorum Sensing, QS) be exactly one of iuntercellular Information exchange mechanism, bacterium is monitored the accumulation that it is secreted into the signaling molecule in the environment by QS mechanism, replys thereby make, calculate its Population (Whitehead et al., 2001; Fuqua and Greenberg, 2002).Similar to other bacteriums, yellow unit cell has also been evolved out and has been carried out the QS system that heredity is regulated on the population level.Early stage transposon mutagenesis analysis has disclosed the gene cluster of an xanthomonas campestris, and is defined as rpfABCDEFG, and this gene cluster is regulated virulence factor, comprises the generation (Tanget al., 1991) of EPS and extracellular enzyme.After this, the rpfF mutant strain is united cultivation near wild type strain, the generation of finding rpfF mutant protein enzyme returns to the wild-type level, this result shows, the yellow unit cell wild type strain of bird rape can produce a kind of diffusible signal factor (Diffusible singal factor, DSF) (Barber et al., 1997).Determined that at present DSF is a kind of short chain fatty acid, its structure is cis-11-methyl-2-dodecenoic acid (Wang et al., 2004).DSF has represented new QS signaling molecule family in the bacterium.In recent years, comprised the little Pseudomonas of xylem from multiple Xanthomonas campestris detecting the DSF activity, and clear and definite its to pathogenic remarkable regulating and controlling effect (Newman et al., 2004).

Recent studies show that, the QS signaling molecule DSF that yellow unit cell produces can be degraded by various bacteria, and these bacteriums comprise Bacillus, Paenibacillus, Microbacterium, the genus such as Staphylococcus and Pseudomonas.Based on the specific function of bacterium QS system, the focus of scientist's research concentrates in the anti-bacteria quorum sensing system.By the generation, the degraded QS signaling molecule that suppress the QS signaling molecule, the number of ways such as cascade reaction of blocking-up QS system weaken the pathogenic of bacterium.

The main reference document

Barber?C.E.，Tang?J.L.，Feng?J.X.，et?al.1997.a?novel?regulatory?system?required?for?pathogenicity?ofXanthomonas?campestris?is?mediated?by?a?small?diffusible?signal?molecule.Mol?Microbiol24：556-566.

Fuqua?C&Greenberg?EP(2002)Listening?in?on?bacteria：acylhomoserine?lactone?signaling.Nat?RevMol?Biol3：685-695

Leyns?et?al.1984.The?host?range?of?the?genus?Xanthomonas.Bot?Rev50：308-355Newman?et?al.2008.virulence?of?planthogenic?bacteria?attenuated?by?degration?of?fatty?acidcell-to-cell?signaling?factors.Mol?Plant?Microbe?Interact?21：326-334.

(3) summary of the invention

Technical problem

The objective of the invention is to invent a kind of utilize tetrazolium violet for cue mark can be fast, sensitive, the screening method of the degradation bacteria take DSF as substrate accurately.

Technical scheme

The foundation of " carbon source---tetrazolium violet-96 porocyte culture plate " screening method

This screening method is according to BIOLOG Bacteria Identification system principle designed, designed.Tetrazolium violet (tetrazolium violet) is a kind of oxidation-reduction indicator, the electronics in can the competitive oxidation reduction reaction, and behind the electron gain, tetrazolium violet is by the colourless purple that becomes, and the electronics of acquisition is more, and purple is darker.This research with DSF (diffusible signal factor) as carbon source to be measured, if bacterium can utilize this carbon source just redox reaction can occur, transfer transport is arranged, by tetrazolium violet by the colourless purple that becomes, and then can the degrade positive strain of DSF of screening.

Test materials

Tetrazolium violet (tetrazolium violet) is available from SIGMA company (69H1223), the DSF standard substance are available from Cayman company (10008123), 96 porocyte culture plates are available from BOYANG company, and bacterial strain G has reported the DSF that can degrade from Karyn L. Newman.

Test method

The preparation of inoculation liquid

1) with bacterial strain G on the NA substratum 28 ℃ cultivate 24h after, get aseptic cotton carrier and in inoculation liquid (the 0.4%NaCl aqueous solution), dip in wet.

2) cotton swab is rolled on the bacterium colony surface, the sticking thalline of getting notes not taking out of substratum.

3) on inoculation liquid pipe liquid level, rotate cotton swab along inwall, thalline is attached on the inwall, simultaneously thalline is evenly broken up.

4) inclination inoculation liquid pipe is scattered in thalline in the inoculation liquid with cotton swab.

5) adjust OD ₆₀₀=0.6.

The preparation of screening liquid

DSF (diffusible signal factor) is a kind of short chain fatty acid, solubleness in water is 0.5mg/mL (25 ℃), therefore, DSF is mixed with successively concentration is 0.025,0.05,0.1,0.2,0.3,0.4, the aqueous solution of 0.5mg/mL, get respectively 100 μ L and add in the 96 porocyte culture plates, and with sterilized water as negative control.Every hole adds the inoculation liquid of tetrazolium violet (0.1mg/mL, H2O) and the 150 μ L of 50 μ L, observes colour-change after placing 28 ℃ to cultivate 12-16h.

(4) Figure of description

Fig. 1. the sensitivity detected result of " carbon source---tetrazolium violet-96 porocyte culture plate " screening method.

Fig. 2. the specific detection result of " carbon source---tetrazolium violet-96 porocyte culture plate " screening method.

1-10: the bacterial strain of the DSF that can not degrade that has reported, 11:G bacterium, 12: sterilized water.

Fig. 3. utilize " carbon source---tetrazolium violet-96 porocyte culture plate " screening method to the detected result of actual sample.

1-94: the bacterial strain to be measured of separation; The 95:G bacterium, 96: sterilized water.

(5) embodiment

Screening to different habitats bacterium

Sampling spot

1) from the rice field soil sampling of generation bacterial blight of rice and the rice plant of morbidity;

2) from the soil soil sampling of service station's oil material contamination;

3) from the pond water water sampling;

4) near the soil sampling cloaca;

5) outside restaurant's smoke exhaust ventilator comb over against greasy dirt ground soil sampling.

6) with bacterial strain to be measured on the NA substratum 28 ℃ cultivate 24h after, get aseptic cotton carrier and in inoculation liquid (the 0.4%NaCl aqueous solution), dip in wet.

7) cotton swab is rolled on the bacterium colony surface, the sticking thalline of getting notes not taking out of substratum.

8) on inoculation liquid pipe liquid level, rotate cotton swab along inwall, thalline is attached on the inwall, simultaneously thalline is evenly broken up.9) inclination inoculation liquid pipe is scattered in thalline in the inoculation liquid with cotton swab.

10) adjust OD ₆₀₀=0.6.

The preparation of screening liquid

DSF (diffusible signal factor) is a kind of short chain fatty acid, solubleness in water is 0.5mg/mL (25 ℃), therefore, DSF is mixed with the aqueous solution that concentration is 0.5mg/mL, get respectively 100 μ L and add in the 96 porocyte culture plates, and with sterilized water as negative control.Every hole adds tetrazolium violet (0.1mg/mL, the H of 50 μ L ₂O) and the inoculation liquid of 150 μ L, observe colour-change after placing 28 ℃ to cultivate 12-16h.

The selection result

So far, obtain more than the 5000 strain bacteriums from different habitats, filter out altogether the positive strain that 57 strains have degraded DSF, identify through the 16SrRNA order-checking to belong to respectively Bacillus, Paenibacillus, Microbacterium, Staphylococcus, Pseudomonas genus.

Claims

1. be used for screening the method that diffusible signal factor is the DSF degradation bacteria, what its screening method mainly utilized is the colour-change of tetrazolium violet, if bacterium can utilize DSF just redox reaction can occur for carbon source, transfer transport is arranged, by tetrazolium violet by the colourless purple that becomes, and then can the degrade positive strain of DSF of screening.

2. the screening method of claim 1 the steps include:

(1) with bacterial strain to be measured on the NA substratum 28 ℃ cultivate 24h after, get aseptic cotton carrier inoculation liquid be dip in the 0.4%NaCl aqueous solution wet; (2) cotton swab is rolled on the bacterium colony surface, glue and get thalline, shift in inoculation liquid, adjust OD ₆₀₀=0.6; (3) get the DSF aqueous solution of 100 μ L 0.5mg/mL, add in the 96 porocyte culture plates, every hole adds the 0.1mg/mL tetrazolium violet aqueous solution of 50 μ L, adds the inoculation liquid of 150 μ L, observes colour-change after placing 28 ℃ to cultivate 12-16h.