CN101519684A - Method for screening pseudomonas syringae pv.tabaci antagonistic bacteria - Google Patents
Method for screening pseudomonas syringae pv.tabaci antagonistic bacteria Download PDFInfo
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- CN101519684A CN101519684A CN200910094283A CN200910094283A CN101519684A CN 101519684 A CN101519684 A CN 101519684A CN 200910094283 A CN200910094283 A CN 200910094283A CN 200910094283 A CN200910094283 A CN 200910094283A CN 101519684 A CN101519684 A CN 101519684A
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Abstract
The invention discloses a method for screening pseudomonas syringae pv.tabaci antagonistic bacteria, which is characterized in that an improved asynchronism opposing culture method is adopted, bacteria to be tested are in point inoculum of 4-6 points and are cultured for 2-3 days, pseudomonas syringae pv.tabaci suspension is sprayed and dispersed, the bacteria are continuously cultured in an opposing way for 2-3 days after the water on the surface is dried in a sterile way and are observed for inhibition zones, and the bacteria with the inhibition zones are target bacteria obtained after the screening. The invention can remarkably enhance the screening efficiency, enrich the asynchronism opposing culture method and accelerate the biological control of bacterial plant diseases.
Description
Technical field
The present invention relates to the biological control technology, more particularly, the present invention relates to a kind of method of screening wildfire bacterium antagonistic bacterium.
Background technology
Wildfire is a kind of bacterial disease that is caused by the false pseudomonas bacillus of tobacco (pseudomonas syringae pv.tabaci), found in the Fo Jiniya and the North Carolina state of the U.S. early than 1917, now being distributed widely in 26 in the world and having produced the cigarette countries and regions, is one of bacterial disease important on the tobacco.China just finds this disease in Liaoning Yan Qu as far back as the 1950's, but harm is little always.Since the eighties in 20th century, this disease in Yunnan, cigarette districts such as Guizhou, Henan, Shandong, Hubei, Liaoning and Jilin all have big area to take place, and the trend that increases the weight of are year by year arranged, and have caused comparatively serious economy loss.
The prairie fire germ can also can also be propagated by tobacco seed in other various crop and the survival of weeds root system with the residual body of diseased plant at field wintering.Therefore, adopt cultivation steps such as crop rotation, field elimination invalid body to prevent the suitable difficulty of having of this disease; Chemical control to be utilizing agricultural streptomycin, but Streptomyces is in the single microbiotic of composition, and long-term continuous administration easily makes pathogen develop immunity to drugs, and has had different resistance in some cigarette districts, shows as application concentration and improves constantly.In the situation of current shortage disease-resistant variety, for effectively preventing and treating this disease, be necessary to seek beneficial bacteria, both can suppress wildfire, can delay the resistance of germ again.Therefore, screening efficient and stable antagonism bacterium is to guarantee one of condition that biological control is succeedd, and the method for screening wildfire bacterium antagonistic bacterium then is its basis.
The method of screening wildfire bacterium antagonistic bacterium mainly contains dull and stereotyped face-off culture method or metabolite activity detection method.Because remove live bacteria in the metabolite activity detection method, obtain just to carry or the smart operation steps of carrying product comparatively complicated, more loaded down with trivial details, so employing this method is few usually.Based on the screening of flat board face-off culture method, two kinds of face-offs of synchronous versus asynchronous cultural method is arranged at present.Can adopt when synchronously face-off is cultivated and in substratum, mix pathogenetic bacteria and fall flat board and put inoculation bacterium to be screened again, also can adopt sandwich flat board; Usually cultivate earlier when asynchronous face-off is cultivated and wait to screen bacterium after produce microbiotic, inoculate pathogenetic bacteria, generally can adopt sandwich flat board, also can adopt upset dull and stereotyped, also can remove bacterium to be screened, be coated with pathogenetic bacteria again.Face-off is synchronously cultivated and is suitable for treating that screening produces microbiotic than the pathogenetic bacteria antagonistic bacterium faster of growing; And produce microbiotic and synchronously or the bacterium to be screened that lags behind than the pathogenetic bacteria growth, then suitablely adopt asynchronous face-off to cultivate, and often be suitable for producing microbiotic than pathogenetic bacteria growth antagonistic bacterium faster to be screened.Therefore, it is more to adopt asynchronous face-off to cultivate.But these asynchronous face-off cultural method screening efficiencies are lower, have much room for improvement.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art part, a kind of method of improved screening wildfire bacterium antagonistic bacterium is provided.This method can improve screening efficiency, enriches asynchronous face-off cultured method, accelerates the biological control of phytobacterial disease.
Purpose of the present invention is achieved by following technical proposals.
* except as otherwise noted, the percentage ratio that is adopted among the present invention is mass percent.
The invention provides a kind of method of screening wildfire bacterium antagonistic bacterium, described method adopts following steps:
(1) cultivation of tested bacteria, wildfire bacterium
Tested bacteria, wildfire bacterium place respectively on NA or the KB flat board and cultivate, and wherein tested bacteria adopts 4～6 points to connect, and the wildfire bacterium is adopted streak inoculation, cultivate 2～3 days for 28 ± 1 ℃;
(2) preparation of wildfire bacterium suspension
Get tween 80 sterile water solution 10～15ml of 0.1% and join on the cultured wildfire bacterium flat board, scrape lawn, be transferred in the sterilization triangular flask, the tween 80 sterile water solution adjusting bacterial concentration with 0.1% is 10
4～5Cfu/ml, get final product wildfire bacterium suspension;
(3) the spraying disperse of wildfire bacterium suspension
The wildfire bacterium suspension of preparation is transferred in the small-sized spraying plant of sterilization, sterilization, on the Bechtop of having sterilized, open cultured tested bacteria flat board, under ventilation state, to dull and stereotyped top spraying wildfire bacterium suspension, by the wind-force germ with regard to even dispersion to flat board, continue to ventilate air-dry planar surface moisture content 10～15 minutes;
(4) face-off is cultivated
Build the ware lid, collect flat board, 28 ± 1 ℃ are continued to cultivate 2～3 days, observe to have or not inhibition zone, and what inhibition zone was arranged is the target bacteria that screening obtains.
Compared with prior art, the present invention has following outstanding advantage:
1. can significantly improve screening efficiency.Adopt improved asynchronous face-off cultural method, tested bacteria 4～6 point connects, and cultivates the disperse wildfire of spraying again bacterium suspension 2～3 days.The method that the inoculation pathogenetic bacteria is adopted the spraying disperse is cultivated in this face-off, can carry out in batches, can improve 20%～30% efficient when the tested bacteria more than 100 is above;
2. the present invention is except that being applicable to wildfire bacterium antagonistic bacterium screening, also applicable to the screening of other bacteria pathogeny antagonistic bacterium, and wide accommodation.
Description of drawings
Fig. 1 shows that tested bacteria has inhibition zone;
Fig. 2 shows that tested bacteria do not have inhibition zone.
Embodiment
By specific embodiment given below and Application Example, can further be well understood to the present invention.But they are not the qualifications to protection domain of the present invention.
Embodiment
(1) cultivation of tested bacteria, wildfire bacterium
Tested bacteria bacterial strain 040239,050117 is totally 2 strain bacterial strains, and wildfire bacterium pst05 bacterial strain all separates from the tobacco disease leaf.5 points of 2 strain tested bacteria bacterial strains are connected on NA or the KB flat board and (are same bacterial strain with 5 in ware in this example, the concrete upward different bacterial strain of available 5 strains of implementing), and wildfire bacterium pst05 adopts streak inoculation, cultivates 2～3 days for 28 ± 1 ℃;
(2) preparation of wildfire bacterium suspension
Get tween 80 sterile water solution 10～15ml of 0.1% and join on the cultured wildfire bacterium flat board, scrape lawn, be transferred in the sterilization triangular flask, the tween 80 sterile water solution adjusting bacterial concentration with 0.1% is 10
4～5Cfu/ml, get final product wildfire bacterium suspension;
(3) the spraying disperse of wildfire bacterium suspension
The wildfire bacterium suspension of preparation is transferred in the small-sized spraying plant of sterilization, sterilization, on the Bechtop of having sterilized, open cultured tested bacteria flat board, under ventilation state, to dull and stereotyped top spraying wildfire bacterium suspension, by the wind-force germ with regard to even dispersion to flat board, continue to ventilate air-dry planar surface moisture content 10～15 minutes;
(4) face-off is cultivated
Build the ware lid, collect flat board, 28 ± 1 ℃ are continued to cultivate 2～3 days, observe to have or not inhibition zone, and what inhibition zone was arranged is the target bacteria that screening obtains.Bacterial strain 040239 has inhibition zone (Fig. 1), bacterial strain 050117 no inhibition zone (Fig. 2).Therefore, bacterial strain 040239 is the target bacteria bacterial strain of screening acquisition.
Application Example 1
---the leaf of antagonism wildfire bacterium encloses bacteria screening
1. materials and methods
1.1 tobacco leaf sample collecting
The sample specified place is a sampling point with sampling county (city) maximum area 2 intensive growing areas in blocks, sampling point has fragmentary wildfire to take place, with full wafer ground is sample, by the sampling of five point samplings, choose 10 strains, 1 in blade is got in every strain, every strain leaf position must not be identical, merge 10 leaves as 1 sample, normal temperature is preserved, and preservation period must not be above 7 days.
1.2 the separation of sample bacteriums such as fresh tobacco leaf sample
Every leaf is by 5 point sampling clip 1cm
25～10 of big leaflet tissues, interior (being added with the grains of sand) 150r/min vibration of the 500ml triangular flask that claims 1g to place 100ml0.85% tween 80 aqua sterilisa 30min dilutes 10,100 times, coating, 28 ± 1 ℃ of constant temperature culture, picking list bacterium colony, the line purifying, number-mark, preservation.
1.3 the screening of antagonistic bacterium
Adopt screening method of the present invention to carry out.
2. result and analysis
Gather 27 parts of tobacco samples, separation of bacterial 1451 strains, screening obtains bacterial strain 42 strains of antagonism prairie fire germ, accounts for 2.89% of total mensuration bacterial strain; Antibacterial circle diameter only has 6 strains greater than 0.5mm, accounts for 0.41%.
Application Example 2
---the rhizosphere bacteria screening of antagonism wildfire bacterium
1. materials and methods
1.1 tobacco rhizosphere soil sample sample collecting
The sample specified place is a sampling point with sampling county (city) maximum area 2 intensive growing areas in blocks, and sampling point has fragmentary wildfire to take place, and is sample with full wafer ground, by the sampling of five point samplings.When gathering the rhizosphere soil sample, 5 every 2 strain the rhizosphere band soil of totally 10 strains take the side fibrous root several, and surface dust is removed in shake gently, only stays the grogs of combining closely with root, is rhizosphere soil, merges 10 strains and has the rhizosphere soil sample of side fibrous root as 1 sample.Freshness protection package is sealed up for safekeeping and is placed 4 ℃ of refrigeratores, maximum 7 days of the sample of preservation.
1.2 the separation of rhizosphere soil sample bacterium
The rhizosphere soil sample is cut into the long segment of 1cm, claim in the 500ml triangular flask that 1g places 100ml0.85% tween 80 aqua sterilisa (being added with 10 glass strains) 150r/min the 30min that vibrates, dilute 100,1000,10000 times, draw the 0.2ml diluent respectively and coat the NA flat board, choose that bacterium colony is clear, colony number is at 30～100 flat board, the careful all single bacterium colonies of picking, line purifying, be stored in the NA inclined-plane, number-mark, preservation.
1.3 the screening of antagonistic bacterium
Adopt screening method of the present invention to carry out.
2. result and analysis
Gather 27 parts of soil samples from tobacco rhizosphere, separation of bacterial 1393 strains, preliminary screening obtains bacterial strain 33 strains of antagonism prairie fire germ, and the antagonism screening rate accounts for 2.37%; Antibacterial circle diameter only has 8 strains greater than 0.5mm, accounts for 0.57%.
Claims (5)
1. method of screening wildfire bacterium antagonistic bacterium, it is characterized in that: adopt improved asynchronous face-off cultural method, tested bacteria 4～6 point connects, cultivated 2～3 days, the disperse wildfire of spraying again bacterium suspension behind the aseptic air-dry surface water, continues face-off and cultivated 2～3 days, observation has or not inhibition zone, and the target bacteria for the screening acquisition of inhibition zone is arranged.
2. method according to claim 1, it is characterized in that: the method that described tested bacteria 4～6 point connects is tested bacteria, wildfire bacterium to be placed respectively on NA or the KB flat board cultivate, wherein the wildfire bacterium is adopted streak inoculation, cultivates 2～3 days for 28 ± 1 ℃.
3. method according to claim 1, it is characterized in that: the preparation method of described wildfire bacterium suspension gets tween 80 sterile water solution 10～15ml of 0.1% to join on the cultured wildfire bacterium flat board, scrape lawn, be transferred in the sterilization triangular flask, it is 10 that the tween 80 sterile water solution with 0.1% is regulated bacterial concentration
4～5Cfu/ml, get final product wildfire bacterium suspension.
4. method according to claim 1, it is characterized in that: the method for described wildfire bacterium suspension spray disperse is that the wildfire bacterium suspension that will prepare is transferred in the small-sized spraying plant of sterilization, sterilization, on the Bechtop of having sterilized, open cultured tested bacteria flat board, under ventilation state, to dull and stereotyped top spraying wildfire bacterium suspension, by the wind-force germ with regard to even dispersion to flat board, continue to ventilate air-dry planar surface moisture content 10～15 minutes.
5. method according to claim 1 is characterized in that: described face-off cultured method is to build the ware lid, collects flat board, and 28 ± 1 ℃ are continued to cultivate 2～3 days, observes to have or not inhibition zone, and what inhibition zone was arranged is the target bacteria that screening obtains.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910094283XA CN101519684B (en) | 2009-04-01 | 2009-04-01 | Method for screening pseudomonas syringae pv.tabaci antagonistic bacteria |
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| CN200910094283XA CN101519684B (en) | 2009-04-01 | 2009-04-01 | Method for screening pseudomonas syringae pv.tabaci antagonistic bacteria |
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| CN101519684A true CN101519684A (en) | 2009-09-02 |
| CN101519684B CN101519684B (en) | 2011-11-23 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184181A (en) * | 2013-04-10 | 2013-07-03 | 西南大学 | Streptomy cesgriseochromogenes for preventing tobacco wildfire |
| CN106282299A (en) * | 2015-05-14 | 2017-01-04 | 北京中医药大学 | A kind of method of external artificial acquisition bacterial resistance bacterial strain |
| CN110923178A (en) * | 2019-09-25 | 2020-03-27 | 中国农业科学院农业资源与农业区划研究所 | Plant immunity inducing antibacterial agent and application thereof |
| CN111004743A (en) * | 2019-10-31 | 2020-04-14 | 山西大学 | Method for screening plant rhizosphere soil antagonistic bacteria |
-
2009
- 2009-04-01 CN CN200910094283XA patent/CN101519684B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184181A (en) * | 2013-04-10 | 2013-07-03 | 西南大学 | Streptomy cesgriseochromogenes for preventing tobacco wildfire |
| CN106282299A (en) * | 2015-05-14 | 2017-01-04 | 北京中医药大学 | A kind of method of external artificial acquisition bacterial resistance bacterial strain |
| CN110923178A (en) * | 2019-09-25 | 2020-03-27 | 中国农业科学院农业资源与农业区划研究所 | Plant immunity inducing antibacterial agent and application thereof |
| CN111004743A (en) * | 2019-10-31 | 2020-04-14 | 山西大学 | Method for screening plant rhizosphere soil antagonistic bacteria |
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| CN101519684B (en) | 2011-11-23 |
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