CN102108339B - Bacillus megaterium with capability of inducing stress resistance of soybeans and application thereof - Google Patents
Bacillus megaterium with capability of inducing stress resistance of soybeans and application thereof Download PDFInfo
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- CN102108339B CN102108339B CN2010105603295A CN201010560329A CN102108339B CN 102108339 B CN102108339 B CN 102108339B CN 2010105603295 A CN2010105603295 A CN 2010105603295A CN 201010560329 A CN201010560329 A CN 201010560329A CN 102108339 B CN102108339 B CN 102108339B
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- bacillus megaterium
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Abstract
The invention relates to a culture method of bacillus megaterium Sneb69 which can induce soybeans to withstand low temperature in early spring and soybean cyst nematode after processing soybean seeds and an application thereof, and belong to the technical field of microbial pesticides. A bacterial strain Sneb69 in the invention was collected in China General Microbiological Culture Collection Center on November 2, 2010 with the collection number of CGMCC No.4296. Through liquid fermentation, culture and preparation, the soybeans can withstand the low temperature in the early spring and the soybean cyst nematode after the soybean seeds are processed by metabolites. The soybeans after being processed by the metabolites of the bacteria can be sown in the early spring, and be grown in soil with serious soybean cyst nematode. By adopting the invention, difficult problems that the effective accumulated temperature for growth of the soybeans in high-altitude regions all the year around is less, and seasons which are suitable for the growth of the soybeans are short are solved, so that the culture method has a good application and development prospects.
Description
Technical field:
The invention belongs to the microbial pesticide technical field, be specifically related to degeneration-resistant bacillus megaterium 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, can resist germ and nematode but filter out short the livings bacterium of rhizosphere that inducing soybean produces system's resistance in order to the opposing low temperature while.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 can be resisted low temperature and soybean cyst nematode at seed germination and growing period bacterial strain; 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 produce the resistance to low temperature and soybean cyst nematode.
The strain bacterial isolates that the present invention screens acquisition is bacillus megaterium (Bacillus megaterium) Sneb69, 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.4296.Bacillus megaterium is the strain that from a large amount of soil bacteria bacterial strains, filters out has strong induced activity to soybean a bacterial isolates.
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 bacillus megaterium (Bacillus megaterium) Sneb69 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 bacillus megaterium of the present invention (Bacillus megaterium) Sneb69 process routine.
Bacterial isolates bacillus megaterium of the present invention (Bacillus megaterium) Sneb69 is after cultivating through the test tube kind, again warp
Enlarge fermentation culture and prepare, its concrete grammar is following:
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, fermentation 45-50 hour, preferred 48h.
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 bacillus megaterium of the present invention (Bacillus megaterium) Sneb69 itself or its metabolite that comprises significant quantity is as the effective active composition.
Bacillus megaterium of the present invention (Bacillus megaterium) Sneb69 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 low temperature and soybean SCN infringement in early spring in seedling stage, solve northeast soybean SCN disease cause harm serious and early spring cryogenic situation.
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 bacillus megaterium (Bacillus megaterium) Sneb69
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 bacillus megaterium (Bacillus megaterium) bacterium colony was preserved separately; Expand numerous and Physiology and biochemistry and measure, cultivating proterties, to meet the preservation of bacillus megaterium (Bacillus megaterium) characteristic subsequent use.
Embodiment 2: the cultivation of bacillus megaterium (Bacillus megaterium) Sneb69
At first bacillus megaterium (Bacillus megaterium) Sneb69 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 bacillus megaterium (Bacillus megaterium) Sneb69 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, and when soybean seedling, can resist causing harm of soybean cyst nematode.
Embodiment 3: the fermentation of bacillus megaterium (Bacillus megaterium) Sneb69
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 bacillus megaterium (Bacillus megaterium) Sneb69
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 bacillus megaterium (Bacillus megaterium) Sneb69 leavened prod
With the fermented liquid dilution is 10
6Cfu/L in 1: 50 ratio treatment coatings soybean seeds, after drying, in Kangping County, Liaoning Province, in the natural occurrence soil of two places, Daqing, Heilongjiang Province, carries out the soybean seeds sowing of handling 5 revision tests and is treated to contrast with the NA liquid nutrient medium.The experimental plot is the secondary soybean field.Behind the 35d from root cyst amount, the soil in cyst amount, the root three aspects investigation of nematode amount find that the soybean SCN is had significant inhibitory effect, and all keep stable resistance against diseases in repeating in that different areas are different.(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.
Embodiment 6: it is 10 that concentration is used in the use of bacillus megaterium (Bacillus megaterium) Sneb69 leavened prod
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 ℃, 2～4 ℃, using concentration is 10
6Germinating energy and percentage of germination are all apparently higher than contrast behind the Sneb69 dressing of cfu/ml, and abnormal rate all 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％or?1％?level，respectively
Claims (8)
1. a strain has the bacterial isolates that inducing soybean produces resistance after handling soybean seeds; It is characterized in that: this bacterial strain is bacillus megaterium (Bacillus megaterium) Sneb69; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 02nd, 2010, preserving number is CGMCC No.4296.
2. the bacterium metabolite that has inducing soybean generation resistance; 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 the bacterium metabolite of resistance; 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 150rmin
-1～200rmin
-1, fermentation time 45-50h.
6. preparation method according to claim 5 is characterized in that: described fermentation time is 48h.
7. a soybean seeds treatment agent is characterized in that, the described bacterium metabolite of claim 2 that comprises significant quantity is used for inducing soybean and produces the resistance to low temperature and soybean cyst nematode as activeconstituents.
Described bacterial isolates of claim 1 or the described bacterium metabolite of claim 2 at inducing soybean in the cold tolerance of seed germination phase and the application in the anti-soybean cyst nematode.
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| CN102108339B true CN102108339B (en) | 2012-07-25 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9084428B2 (en) * | 2012-08-14 | 2015-07-21 | Marrone Bio Innovations, Inc. | Bacillus megaterium bioactive compositions and metabolites |
| CN102925387B (en) * | 2012-10-26 | 2014-01-29 | 沈阳农业大学 | Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application |
| CN105018367B (en) * | 2014-04-18 | 2019-06-18 | 浙江农林大学 | The degeneration-resistant ocean habitat bacillus of one plant of induction plant disease-resistant |
| CN104130050B (en) * | 2014-07-01 | 2018-02-09 | 山西绿图生物科技有限公司 | Multielement composite microbe fertilizer and its production method |
| CN105112340B (en) * | 2015-09-15 | 2018-08-28 | 沈阳农业大学 | One kind having the active bacterium bacterial strain of induction of resistance and its cultural method and application |
| 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 |
| CN110218665B (en) * | 2019-05-13 | 2021-12-14 | 沈阳农业大学 | Bacillus with corn leaf blight resistance inducing effect and application thereof |
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| CN101831395B (en) * | 2010-03-15 | 2012-07-04 | 沈阳农业大学 | Klebsiella pneumoniae for inducing soybean to resist atrazine and application |
| CN101818124B (en) * | 2010-03-15 | 2012-01-25 | 沈阳农业大学 | Stenotrophomonas capable of improving herbicide resistance in soybeans, method for preparing same and application thereof |
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