CN105112340A - Fungus strain with induced resistance as well as culture method and application of fungus strain - Google Patents
Fungus strain with induced resistance as well as culture method and application of fungus strain Download PDFInfo
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- CN105112340A CN105112340A CN201510607225.8A CN201510607225A CN105112340A CN 105112340 A CN105112340 A CN 105112340A CN 201510607225 A CN201510607225 A CN 201510607225A CN 105112340 A CN105112340 A CN 105112340A
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Abstract
The invention discloses a fungus strain with induced resistance as well as a culture method and application of the fungus strain. The fungus strain belongs to Bacillus thuringiensis and has a preservation number of CGMCC No.10831. A liquid culture medium of the fungus strain is prepared from the following raw materials in percentage by mass: 0.1-0.2% of a yeast extract, 0.5-0.6% of peptone, 0.3-0.4% of a beef extract, 1-2% of cane sugar and the balance of distilled water. The fungus strain B. thuringiensis Sneb1076 or a metabolite of the fungus strain B. thuringiensis Sneb1076 has induced resistance to soybeans, and can be utilized for soybean seed treatment, so that not only can the growth of soybean plants and roots be promoted, but also the inhibiting effect on encystment of soybean cyst nematodes is relatively good, and a new way is provided for controlling of the soybean cyst nematodes.
Description
Technical field
The present invention relates to bacterial strain field, be specifically related to a kind of there is induction of resistance activity bacterial isolates and cultural method and application.
Background technology
Plant nematode in the world many countries and regions occurs and causes harm, and kind is varied.In the world, the occurrence and infection of plant nematode along with the development of agroforestry and the change of tillage and cultivation system day by day serious, add up according to Esser, to nineteen ninety, the whole world has been reported and has been found that plant nematode 207 belongs to 4832 kinds of (Liu Weizhi, Duan Yuxi .2000. plant pathogeny line insect. Beijing: Chinese agriculture press, P.1-2).According to estimates, plant nematode causes the year rate of loss of world staple crops to be 12.3%, more than 1,000 hundred million dollars, and the loss of reality is considerably beyond estimation, reason be the harm that causes of many nematodes because of there is no field Visual symptoms or specialization feature and do not arouse attention (Wang Shouhua. Pomology. Beijing: Chinese agriculture Science Press, 1994.P.1-5), in addition, the interior new discovery of causing harm to nematode of world wide is also in continuous increase, and some agricultural cultivation systems upgraded make nematode problem more outstanding, moreover, nematode and other biological interaction and the direct or indirect impact that plant produces also sharply is being increased.The harm that nematode is caused is more hidden compared with other biology in this sense! About there is kind more than 3000 to the nematode of plant pest at present, mainly contain Cyst nematode, pine wood nematode, sweet potato stem nematode etc. in China.Cyst nematode is a maximum class nematode of agriculturally causing harm, after disease occurs, the general underproduction about 10%, serious in more than 75%, even have no harvest (A.G.Whitehead, 1998.PlantNematodeControl.ISBN0851991882CABInternational).At present, the control of plant nematode is still based on chemical prevention, but in recent years, the application waste of nematocide is large, contaminate environment, poor selectivity, natural disposition of going out is strong, the drawbacks such as spoiled soil biota are day by day by people are paid attention to (Zhang Keqin, He Shichuan, the effect in nematode biological and ecological methods to prevent plant disease, pests, and erosion of the .1991. bacteriums such as Zhou Wei and bacterium and progress thereof. insecticidal microorganism .3:55-66), 21 century is called environmental protection century, the application of some effective nematocides is progressively restricted, therefore hot issue is become to the biological control research nature of plant nematode.The biocontrol bacteria main groups of the prevented and treated plant nematode reported has: Bacillius, Pseudomonas, Acinebacter, Chromobacterium, Enterobacter, Serratia, Burkholderia etc., wherein with the most species in Bacillus and Pseudomonas.Past is all generally utilize BT toxalbumin to the biological control of insect to the Biocontrol Effect of bacillus thuringiensis, directly the research of killing activity is had also to have similar report to nematode, and the little of induction of resistance defence disease report is produced for process plant seed, and for utilizing bacillus thuringiensis processed soybeans Seed inducement soybean plant strain root system to produce the research of anti-Plant nematode and invention is not reported.How to utilize the metabolism induction plant of bacterial strain to produce resistance, exciting the immunity system of plant, is the key that the present invention pays close attention to.
Summary of the invention
For the problems referred to above, the present invention obtains a bacillus thuringiensis strain by screening system, utilize the meta-bolites processed soybeans seed of this bacterium or this bacterium, resistance to soybean cyst nematode Heterodera glycines can be produced by inducing soybean during Seed Germination and growth, thus provide new approach for the problem solving soybean cyst nematode evil.
For achieving the above object, the technical scheme that the present invention takes is:
Have a bacterial isolates for induction of resistance activity, described bacterial strain is Bacillus thuringiensis Sneb1076, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: China. Beijing. No. 3, No. 1, North Star West Road, Chaoyang District institute; Preservation date: on May 21st, 2015; The numbering that preservation is registered on the books: CGMCCNo.10831.B.thuringiensisSneb1076 is the strain filtered out from a large amount of soil bacteria bacterial strain has good induction of resistance bacterial isolates to soybean cyst nematode Heterodera glycines.
Above-mentioned a kind of cultural method with the bacterial isolates of induction of resistance activity, comprises the steps:
S1, to take by mass percentage: yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, after mixing, obtains liquid nutrient medium;
S2, the liquid nutrient medium of step S1 gained is sub-packed in the triangular flask of 250mL in the ratio of every bottle of 150mL, after packing completes by strain inoculation in liquid nutrient medium, pH6.8-7.0, temperature is 25-28 DEG C, shaking speed 150rmin
-1, fermentation 2-3d, must can be used for the bacterium metabolite that inducing soybean produces anti-soybean cyst nematode resistance.
Above-mentioned a kind of bacterial isolates with induction of resistance activity can be used for preparation plant nematode biological control induction of resistance preparation.Described induction of resistance preparation includes the above-mentioned bacillus thuringiensis (B.thuringiensise) of effective amount or the meta-bolites of step S2 gained.
Wherein, described plant nematode is Cyst nematode (Soybeancystnematode).
The present invention is found by research, filters out the bacterial isolates that many strains have the anti-soybean cyst nematode Heterodera glycines of inducing soybean from soil, and wherein bacillus thuringiensis (B.thuringiensise) Sneb1076 preventive effect is better, and relatively stable.
The present invention has following beneficial effect:
Bacillus thuringiensis of the present invention (B.thuringiensise) Sneb1076 or its metabolite have the induced activity to soybean, can be used for the seed treatment of soybean, not only can promote the growth of soybean plant strain and root, to the formation of soybean cyst nematode Heterodera glycines cyst, there is good restraining effect, for soybean cyst nematode Heterodera glycines control provides a new way simultaneously.
Embodiment
In order to make objects and advantages of the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
The cultivation of bacillus thuringiensis (B.thuringiensise) Sneb1076
By the inoculation of bacillus thuringiensis (B.thuringiensise) Sneb1076 on test-tube culture medium, culture medium prescription is beef-protein medium, i.e. composition: beef extract 3g, yeast extract 1g, peptone 10g, sucrose 10g, agar 17-20g, adding distil water is to 1000mL.Cultivate 3d for 28 DEG C, obtain test tube kind.
Again test tube kind is inoculated in 250mL triangular flask (every bottled 150mL) liquid nutrient medium, culture medium prescription is: by weight percentage, yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, pH is 6.8-7.0, leavening temperature is 25-28 DEG C, shaking speed 150rmin
-1, fermentation time 2-3d.The fermented liquid formed or the finished product of fermented liquid can direct or indirect processed soybeans seeds.Processed soybean seeds can alleviate the harm caused by soybean cyst nematode evil.
Embodiment 2
Bacillus thuringiensis (B.thuringiensise) Sneb1076 leavened prod control soybean cyst nematode Heterodera glycines field efficacy experiment
This test is respectively in Shenyang City, Liaoning Province Agricultural University Of Shenyang experimental plot, and field efficacy experiment is carried out in experimental plot, Kangping city, Shenyang City, Liaoning Province and experimental plot, academy of agricultural sciences, Daqing, Heilongjiang Province.By the fermented liquid stoste prepared, liquid kind 1: 70 volume ratio is adopted to carry out seed pelleting process, dry in the shade under shady and cool condition, the soybean seeds processed normally is sowed, soybean plant strain can produce the resistance to soybean cyst nematode Heterodera glycines in seedling stage, and Induced resistant effect is more stable, obtain good prevention effect.
1. test preparation
Prepare the fermented liquid of bacillus thuringiensis (B.thuringiensise) Sneb1076 by aforementioned liquids fermentation culture method, treat for subsequent use.
2. test soybean varieties
The Liao Dynasty's beans 15, Liaoning Academy of Agricultural Sciences's soybean research provided.
3. method for treating seeds
Soybean seeds is first used 5.0% chlorine bleach liquor's surface sterilization 5min, then use aseptic water washing 3-5 time.Liquid kind 1: 70 volume ratio is adopted by the fermented liquid prepared to carry out seed pelleting process, cloudy dry, for subsequent use under shady and cool condition.
4. test method
This test is respectively in Shenyang City, Liaoning Province Agricultural University Of Shenyang experimental plot, and field efficacy experiment is carried out in Kangping city, Shenyang City, Liaoning Province and experimental plot, academy of agricultural sciences, Daqing, Heilongjiang Province, and three experimental plot, place soybean cyst nematode Heterodera glycines are all very serious, main cultivation crop soybean and corn etc.With without the soybean seeds of dressing and the soybean seeds of NA liquid medium dressing for contrast, after cultivation, 45d investigates, and is grubbed out out by soybean plant strain, keeps root system complete, investigation soybean root system cyst number and root-rot disease index.
5. test-results
Use the soybean of Sneb1076 leavened prod process, soybean root system cyst digital display work is lower than contrast, and in Agricultural University Of Shenyang, the field efficacy of Kangping and grand celebration is respectively 68.1%, 79.4% and 51.5%, there is significant difference in the soybean after Cotton seeds and untreated soybean.
The field efficacy of table 1Sneb1076 leavened prod Cotton seeds soybean seeds
Embodiment 3
The test of bacillus thuringiensis (B.thuringiensise) Sneb1076 leavened prod control soybean cyst nematode Heterodera glycines pot test effect
In order to verify that it lures anti-stability, spy this has been potted plant prevention effect research.This test carries out pot test effect test at Agricultural University Of Shenyang's warmhouse booth, with without the soybean seeds of dressing and the soybean seeds of NA liquid medium dressing for contrast, test is in triplicate.
1. test preparation
Prepare the fermented liquid of bacillus thuringiensis (B.thuringiensise) Sneb1076 by aforementioned liquids fermentation culture method, treat for subsequent use.
2. test soybean varieties
The Liao Dynasty's beans 15, Liaoning Academy of Agricultural Sciences's soybean research provided.
3. method for treating seeds
Soybean seeds is first used 5.0% chlorine bleach liquor's surface sterilization 5min, then use aseptic water washing 3-5 time.Liquid kind 1: 70 volume ratio is adopted by the fermented liquid prepared to carry out seed pelleting process, aseptically dry, for subsequent use.
4. for examination nematode
Pick up from the rhizosphere cyst soil in experimental plot, Kangping city, Shenyang City, Liaoning Province, through being accredited as soybean cyst nematode Heterodera glycines (Soybeancystnematode), by eluriating the cyst of the full maturation of sieve method picking, put into the sterilizing culture dish filling sterilized water, then to sterilize 3min with 0.5%NaCLO, and aseptic water washing 3 times, the cyst that therefrom picking is complete and full, insert and fill in the sterilizing culture dish of appropriate amounts of sterilized water, put it in the thermostat container of 28 DEG C and cultivate, after 3d, the second instar larvae J2 hatched being inoculated into soybean root easily susceptible in greenhouse, to carry out expansion numerous.Adopt tray method to hatch J2 the cyst expanding numerous acquisition, be prepared into nematode suspension 200/mL.
5. test method
The fermented liquid stoste of Sneb1076 adopted liquid kind 1: 70 volume ratio to carry out seed pelleting process, be seeded into by the soybean seeds dried after dressing and educate in the seedling alms bowl of 12 × 12cm, 2000 soybean cyst nematode Heterodera glycines inoculated by every basin.With without the soybean seeds of dressing and the soybean seeds of NA liquid medium dressing for contrast, after transplanting, 45d investigates, and measures that soybean plant height, root are long, root fresh weight and root interior lines borer population.
6. test-results
Use the soybean of Sneb1076 leavened prod process, root knot number is starkly lower than contrast, and soybean plant height, root length, stem stalk weight, root fresh weight and untreated soybean exist notable difference, and potted plant preventive effect is 40.7%.
The potted plant preventive effect of table 2Snef1076 leavened prod Cotton seeds soybean seeds
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. have a bacterial isolates for induction of resistance activity, it is characterized in that, described bacterial strain is bacillus thuringiensis (Bacillusthuringiensis), and preserving number is CGMCCNo.10831.
2. a kind of cultural method with the bacterial isolates of induction of resistance activity as claimed in claim 1, is characterized in that, comprise the steps:
S1, to take by mass percentage: yeast extract 0.1%-0.2%, peptone 0.5%-0.6%, beef extract 0.3%-0.4%, sucrose 1%-2%, remainder is distilled water, after mixing, obtains liquid nutrient medium;
S2, the liquid nutrient medium of step S1 gained is sub-packed in the triangular flask of 250mL in the ratio of every bottle of 150mL, after packing completes by strain inoculation in liquid nutrient medium, pH6.8-7.0, temperature is 25-28 DEG C, shaking speed 150rmin
-1, fermentation 2-3d, obtains meta-bolites.
3. a kind of application with the bacterial isolates of induction of resistance activity as claimed in claim 1, is characterized in that, for the preparation of plant nematode biological control induction of resistance preparation.
4. a kind of application with the bacterial isolates of induction of resistance activity as claimed in claim 3, it is characterized in that, described induction of resistance preparation includes the above-mentioned bacillus thuringiensis (B.thuringiensise) of effective amount or the meta-bolites of step S2 gained.
5. a kind of application with the bacterial isolates of induction of resistance activity as claimed in claim 3, is characterized in that, described plant nematode is Cyst nematode (Soybeancystnematode).
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0303426A2 (en) * | 1987-08-12 | 1989-02-15 | Mycogen Corporation | Novel isolates of bacillus thuringiensis having activity against nematodes |
| AU656726B2 (en) * | 1990-06-11 | 1995-02-16 | Mycogen Corporation | Novel bacillus thuringiensis microbes active against nematodes, and genes encoding novel nematode-active toxins cloned from bacillus thuringiensis isolates |
| CN1849890A (en) * | 2006-04-06 | 2006-10-25 | 湖北省生物农药工程研究中心 | Agritol suspension seedling-coating agent |
| CN102108339A (en) * | 2010-11-26 | 2011-06-29 | 沈阳农业大学 | Bacillus megaterium with capability of inducing stress resistance of soybeans and application thereof |
| CN102925387A (en) * | 2012-10-26 | 2013-02-13 | 沈阳农业大学 | Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application |
-
2015
- 2015-09-15 CN CN201510607225.8A patent/CN105112340B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0303426A2 (en) * | 1987-08-12 | 1989-02-15 | Mycogen Corporation | Novel isolates of bacillus thuringiensis having activity against nematodes |
| AU656726B2 (en) * | 1990-06-11 | 1995-02-16 | Mycogen Corporation | Novel bacillus thuringiensis microbes active against nematodes, and genes encoding novel nematode-active toxins cloned from bacillus thuringiensis isolates |
| CN1849890A (en) * | 2006-04-06 | 2006-10-25 | 湖北省生物农药工程研究中心 | Agritol suspension seedling-coating agent |
| CN102108339A (en) * | 2010-11-26 | 2011-06-29 | 沈阳农业大学 | Bacillus megaterium with capability of inducing stress resistance of soybeans and application thereof |
| CN102925387A (en) * | 2012-10-26 | 2013-02-13 | 沈阳农业大学 | Bacillus simplex for inducing soybean to generate soybean cyst nematode resistance and application |
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