CN102108338B - Cold-resistance-inducing Pseudomonas aeruginosa strain and application thereof - Google Patents
Cold-resistance-inducing Pseudomonas aeruginosa strain and application thereof Download PDFInfo
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- CN102108338B CN102108338B CN201010560320A CN201010560320A CN102108338B CN 102108338 B CN102108338 B CN 102108338B CN 201010560320 A CN201010560320 A CN 201010560320A CN 201010560320 A CN201010560320 A CN 201010560320A CN 102108338 B CN102108338 B CN 102108338B
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Abstract
The invention relates to a culture method and application of a Pseudomonas aeruginosa Sneb179 strain, which can induce soybeans to resist low temperature in early spring after being used for treating soybean seeds, belonging to the technical field of microbial pesticides. The bacterial strain Sneb179 provided by the invention was preserved in the Center of Common Microorganisms in China Committee for Culture Collection of Microorganisms on November 2nd, 2010, and the preservation number is CGMCC No.4295. The Pseudomonas aeruginosa strain is fermented and cultured by using a liquid; and after the metabolite of the Pseudomonas aeruginosa strain is used for treating soybean seeds, the soybeans can resist low temperature in early spring. The soybeans treated by the metabolite of the Pseudomonas aeruginosa can be sowed in early spring, and have certain cold resistance. The invention solves the problems of less effective accumulated temperature and short seasons for soybean growth in high-latitude areas all the year around, and has wide application development prospects.
Description
Technical field:
The invention belongs to the microbial pesticide technical field, be specifically related to cold-resistant Pseudomonas aeruginosa of a kind of inducing soybean and application.
Background technology:
Northeast is China soybean main producing region, and low temperature is the main factor that reduces soybean yields.Each stage of soybean whole growing all is prone to damages to plants caused by sudden drop in temperature, thereby reduces yield and quality, and the initial stage of especially sprouting is to low-temperature sensitive.When under early spring, sowing; Because low temperature Chang Rongyi causes the seedling-growing time prolongation and causes lopsided seedling; Perhaps seed goes rotten rotten and the forfeiture germinating power, causes the soybean underproduction, strengthens the emerge correlative study of lower temperature resistance of soybean for this reason and soybean is produced has positive meaning.Adopt measures such as strong kind of dispense smoke, membrane covering, cold hardiness or the in good time early sowing of late variety can improve the cold hardiness of plant, but all limitation is arranged.Improve the cold resistant method of soybean at present and be to use some chemical substances or cold-resistance agent etc., this may affect to crop quality and soil.Because the raising of people's environmental protection consciousness, and most inductor environmentally safe, and induction of resistance has characteristics such as long, resistance expression's time of wide spectrum, time length and the relative controllability of spatial.These characteristics have determined the plant induction of resistance to have a good application prospect.Utilize bacterium to become a focus in the domestic and international biological control research as biological control factor controlling plant diseases.
The sick research of the short biological and ecological methods to prevent plant disease, pests, and erosion of PGPR has become one of focus direction, and its research mainly concentrates on pseudomonas and bacillus category, and such group energy enough suppresses particularly soil-borne disease of plurality of plant diseases.The mechanism of action mainly includes and imitates root colonization, antibiosis, rhizosphere nutrition competition, secretes the enzyme of degraded pathogenic micro-organism etc.Therefore, the PGPR preparation has major application potentiality and value at aspects such as agriculture forest and husbandry, environment protection.
This research is intended to propose to open one's minds in view of the above, produces the cold resistant bacterial isolates of system in order to opposing low temperature but filter out inducing soybean.This research will provide new approaches and novel method for the control chilling injury, agriculture prodn, especially soybean produced play absolute pushing effect, have important significance for theories and actual application value.
Summary of the invention:
But the objective of the invention is to obtain a strain inducing soybean and can resist cryogenic bacterial strain at seed germination and growing period; After bacterial strain pressed the conventional liq fermentation culture; Can use the meta-bolites of this bacterium or this bacterium; Develop the seed treatment agent of handling soybean seeds, treated soybean seeds can make soybean resist low temperature in early spring.
The strain bacterial isolates that the present invention screens acquisition is Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date: on November 02nd, 2010; The numbering that preservation is registered on the books: CGMCC No.4295.Pseudomonas aeruginosa is the strain that from a large amount of soil bacteria bacterial strains, filters out has strong induced activity to soybean a bacterial isolates.This bacterial strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179 is a yellow bacterial isolates that filters out from Chinese ShenYang, Liaoning Province city Agricultural University Of Shenyang experimental plot soil bacteria bacterial strain in July, 2008 slowly.
The present invention is through discovering, soil bacteria has a lot of strain bacteriums all to have certain activity, from these bacteriums, filter out a strain proterties relatively preferably bacterial strain Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179 carried out further research.
The invention still further relates to the bacterium metabolite that is used for the inducing soybean seed, it is to extract preparation after the liquid fermentation and culture by bacterial isolates Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) Sneb179 process routine.
Bacterial isolates Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) Sneb179 is after cultivating through the test tube kind,
Prepare through enlarging fermentation culture, its concrete grammar is following again:
1. the test tube kind is cultivated
Culture medium prescription is beef extract-peptone (NA) substratum, i.e. composition: beef extract 3g, and peptone 10g, yeast extract 1g, sucrose 10g, agar 20g adds water to 1000mL.
2. liquid fermentation and culture
Wherein the liquid fermentation medium prescription is: by weight percentage, contain beef extract 0.2%-0.4%, peptone 1%-2%, yeast extract 0.1%-0.5%, sucrose 1%-2%, remainingly be zero(ppm) water that pH is 7-9.
The test tube kind is inoculated in 250mL triangular flask (the every bottled 100mL) liquid nutrient medium, and under 25 ℃～28 ℃, shaking speed is with 150rmin
-1～200rmin
-1, the fermentation 45-50 hour.
The bacterial classification of liquid fermenting can carry out scale prodn through large fermentation tank, and its product is the finished product of fermented liquid or fermented liquid.
The invention still further relates to a kind of soybean seeds treatment agent, its Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) Sneb179 itself or its metabolite that comprises significant quantity is as the effective active composition.
Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) Sneb179 or its metabolite have the induced activity to soybean; The seed treatment that can be used for soybean; Soybean after the processing can be resisted cryogenic infringement in early spring in seedling stage; The total effective temperature that solves the annual soybeans they grow of northeast high latitude area is few, the situation that the season of suitable soybeans they grow is short.
Embodiment:
In order further to illustrate in more detail rather than, to have provided the following example in order to limit the present invention.
Embodiment 1: the acquisition of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179
Get the soil 1g in soybean field, add sterilized water 100ml, get 200 μ l; Add the 20ml NA liquid nutrient medium that is cooled to the sterilization below 35 ℃; After 25 ℃ cultivation treated that bacterium colony grows down, according to bacterium colony color and luster and rim condition, picking Pseudomonas aeruginosa (Pseudomonas aeruginosa) bacterium colony was preserved separately; Expand numerous and Physiology and biochemistry and measure, cultivating proterties, to meet the preservation of Pseudomonas aeruginosa (Pseudomonas aeruginosa) characteristic subsequent use.
Embodiment 2: the cultivation of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179
At first Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179 bacterial strain is carried out fermentation culture, then the meta-bolites after the fermentation culture is carried out the test of soybean induced activity, confirm its inducing action soybean.
The thalline of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179 is inoculated on the test tube nutrient agar, and culture medium prescription is NA substratum, i.e. composition: beef extract 3g; Peptone 10g, yeast extract 1g, sucrose 10g; Agar 20g adds water to 1000mL.Cultivate 10d for 25 ℃, obtain the test tube kind.
The test tube kind is inoculated in 250mL triangular flask (the every bottled 100mL) liquid nutrient medium, culture medium prescription is again: by weight percentage, contain beef extract 0.2%, peptone 1%, yeast extract 0.5%, sucrose 1%, remainingly be zero(ppm) water that pH is 8.Cultivate down at 25 ℃, shaking speed is with 180rmin
-1, the fermentation 48h, the fermented liquid of formation or the finished product of fermented liquid can directly or indirectly be handled soybean seeds.The soybean seeds that is processed can be planted in the soil at low temperature in early spring, resists low temperature in early spring.
Embodiment 3: the fermentation of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179
Basically with embodiment one, difference is that the bacterial classification inoculation of the liquid culture fermentor tank to 1000L is fermented, and cultivates down at 25 ℃, and pH is 8, fermentation 48h.
Embodiment 4: the fermentation of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179
Basically with embodiment one, difference is the liquid culture based formulas, and its prescription is:
By weight percentage, contain beef extract 0.3%, peptone 1%, yeast extract 0.1%, sucrose 2%, remainingly be zero(ppm) water that pH is 7.
Embodiment 5: the use of Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179 leavened prod
Using concentration is 10
3Cfu/ml, 10
6Cfu/ml, 10
9The cfu/ml fermented liquid is cooked germination test in 1: 50 ratio treatment coatings soybean seeds after drying, and 30/ware, repeat for 5 times, low temperature at night every day (establish two groups, one group is 0～2 ℃, and one group is 2～4 ℃) is handled 12h, handles 12h daytime (15～20 ℃).Handle 7d.Write down germinating energy behind the 4d, record percentage of germination and abnormal rate behind the 7d.Test-results finds that at 0～2 ℃, using concentration is 10
6Cfu/ml and 10
3Germinating energy and percentage of germination are all apparently higher than contrast behind the Sneb179 dressing of cfu/ml, and abnormal rate all is starkly lower than contrast.At 2～4 ℃, using concentration is 10
3Germinating energy and percentage of germination are apparently higher than contrast behind the Sneb179 dressing of cfu/ml, and abnormal rate is starkly lower than contrast.(like following table)
Annotate: lowercase and capitalization are illustrated respectively in remarkable on 5% and 1% level.
Note：small?letter?and?capital?letter?are?significant?at?5％or1％level，respectively
Claims (9)
1. have inducing soybean behind the strain processing soybean seeds and produce cold resistant bacterial isolates; It is characterized in that: this bacterial strain is Pseudomonas aeruginosa (Pseudomonas aeruginosa) Sneb179; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 02nd, 2010, preserving number is CGMCC No.4295.
2. have inducing soybean and produce cold resistant bacterium metabolite; It is characterized in that; Be to prepare through the fermented liquid that forms behind the liquid fermenting by the described bacterial isolates of claim 1; The liquid fermentation medium prescription is: by weight percentage, contain beef extract 0.2%-0.4%, peptone 1%-2%, yeast extract 0.1%-0.5%, sucrose 1%-2%, remainingly be zero(ppm) water that pH is 7-9.
3. the liquid fermentation process of the described bacterial isolates of claim 1; It is characterized in that: the liquid fermentation medium prescription is: by weight percentage; Contain beef extract 0.2%-0.4%, peptone 1%-2%, yeast extract 0.1%-0.5%, sucrose 1%-2%, remainingly be zero(ppm) water; PH is 7-9, and leavening temperature is 25-28 ℃, shaking speed 150rmin
-1～200rmin
-1, fermentation time 45-50h.
4. liquid fermentation process according to claim 3 is characterized in that: described fermentation time is 48h.
5. has the preparation method that inducing soybean produces cold resistant bacterium metabolite; It is characterized in that: be to prepare through the fermented liquid that forms behind the liquid fermenting by the described bacterial isolates of claim 1; The liquid fermentation medium prescription is: by weight percentage, contain beef extract 0.2%-0.4%, peptone 1%-2%, yeast extract 0.1%-0.5%, sucrose 1%-2%, remainingly be zero(ppm) water that pH is 7-9; Leavening temperature is 25-28 ℃, shaking speed 150r.min
-1～200rmin
-1, fermentation time 45-50h.
6. preparation method according to claim 5 is characterized in that: described fermentation time is 48h.
7. the substratum of the described bacterial isolates of claim 1 that is used to ferment; It is characterized in that: fermentative medium formula is: by weight percentage; Contain beef extract 0.2%-0.4%, peptone 1%-2%, yeast extract 0.1%-0.5%, sucrose 1%-2%, remainingly be zero(ppm) water, the pH of substratum is 7-9.
8. a soybean seeds treatment agent is characterized in that, the described bacterial isolates of claim 1 or the described bacterium metabolite of claim 2 that comprise significant quantity are used for inducing soybean and resist low temperature in early spring as activeconstituents.
9. described bacterial isolates of claim 1 or the application of the described bacterium metabolite of claim 2 in inducing soybean is cold-resistant.
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| CN102108338B true CN102108338B (en) | 2012-09-05 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219671A (en) * | 2015-09-15 | 2016-01-06 | 沈阳农业大学 | A kind of bacterial isolates and cultural method thereof and application |
| CN105112339B (en) * | 2015-09-15 | 2018-12-04 | 沈阳农业大学 | A kind of bacterium bacterial strain and its cultural method and application |
| CN108002924B (en) * | 2017-12-28 | 2021-11-19 | 深圳市芭田生态工程股份有限公司 | Cold-resistant microbial liquid fertilizer and preparation method thereof |
| CN111892451A (en) * | 2020-08-20 | 2020-11-06 | 黑龙江科吉生态农业开发有限公司 | Organic fertilizer for dry seedling of full-nutrition rice and preparation method thereof |
| CN117264837B (en) * | 2023-10-09 | 2024-04-02 | 东北农业大学 | Pseudomonas with growth promoting function for low-temperature stress of plants and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730649A (en) * | 2005-09-01 | 2006-02-08 | 北京未名凯拓农业生物技术有限公司 | A Pseudomonas aeruginosa strain and its culturing method and uses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730649A (en) * | 2005-09-01 | 2006-02-08 | 北京未名凯拓农业生物技术有限公司 | A Pseudomonas aeruginosa strain and its culturing method and uses |
Non-Patent Citations (4)
| Title |
|---|
| Effects of rhizobacteria on soybean cyst nematode, H eterodera glycines;Tian H L, Robert D R.;《Journal of Nematology》;20001231;第32卷(第4期);377-388 * |
| TianHL Robert D R..Effects of rhizobacteria on soybean cyst nematode |
| 周勋波 等.大豆应用抗寒剂抗寒效应的研究.《杂粮作物》.2001,(第4期),32-33. |
| 大豆应用抗寒剂抗寒效应的研究;周勋波 等;《杂粮作物》;20010430(第4期);32-33 * |
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