CN108624537B - The microbacterium of one plant of anti-soybean cyst nematode of inducing soybean and its application - Google Patents
The microbacterium of one plant of anti-soybean cyst nematode of inducing soybean and its application Download PDFInfo
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- CN108624537B CN108624537B CN201810534442.2A CN201810534442A CN108624537B CN 108624537 B CN108624537 B CN 108624537B CN 201810534442 A CN201810534442 A CN 201810534442A CN 108624537 B CN108624537 B CN 108624537B
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Abstract
The present invention relates to the microbacterium of one plant of anti-soybean cyst nematode of inducing soybean and its applications, technical points are the microbacterium of the anti-soybean cyst nematode of inducing soybean, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: on November 03rd, 2015, deposit number: CGMCC No. 11566.The microbacterium can get the fermentation liquid that inducing soybean generates anti-cyst nematode by conventional liq fermented and cultured, inducing soybean is generated to cyst nematode resistance, biological control microbial inoculum can be made in the present invention, for killing cyst nematode second instar larvae, and resistance can be generated with inducing soybean, while the soybean Jing Guo seed treatment can generate apparent disease resistance, significant effect to soybean cyst nematode Heterodera glycines, soil environment will not be destroyed, is had a good application prospect in prevention and treatment soybean cyst nematode Heterodera glycines harm.
Description
Technical field
The invention belongs to microbial pesticide technical fields, and in particular to one plant of anti-soybean cyst nematode of inducing soybean it is micro-
Bacillus and its application.
Background technique
Soybean cyst nematode be by soybean cyst nematode Heterodera glycines (Heterodera glycines) caused by disease, be a kind of
Global soybean diseases all cause to seriously threaten to the yield of countries in the world Soybean-planting, it is annual caused by loss be more than
1000000000 dollars.Disease distribution is wide, and harm weight, the cyst time-to-live is longer, is a kind of soil-borne disease of extremely difficult prevention and treatment.China soybean
Production is mainly distributed on two producing regions in northeast and Huang-Huai-Hai, less serious case's underproduction 20%-30% after soybean is caused harm by cyst nematode, weight
Person is up to 70%-80%, or even No kernels or seeds are gathered, as in a year of scarcity.
The prevention and treatment of soybean cyst nematode Heterodera glycines mainly has cultivation disease-resistant variety, improves agricultural measures, chemical prevention, biological control.
Cultivate the disease-resistant variety period it is longer, improvement planting patterns can with mitigation but also can not thoroughly control soybean cyst nematode Heterodera glycines, change
Although learning prevention and treatment effectively, because its is seriously polluted, destroy the ecological balance, can not implemented for long periods, microbial control is main
Disease resistance is generated to the poisoning of nematode and induction plant using microorganism, the influence to plant and environment is smaller, is following big
The developing direction of beans cyst nematode prevention and treatment.
Microbacterium maritypicum It is a kind of bacterium circle, Firmicutes, actinobacteria guiding principle, actinobacteria
One of subclass, Actinomycetal, Micrococcineae, microbot Cordycepps gram-positive bacteria.Microbacterium is the dominant microflora of soil
One of, it is distributed widely in varying environment, Microbacteriumarabinogalactanolyticum,
Microbacterium keratanolyticum, Microbacterium luteolum,
Microbacteriumschleiferi, Microbacterium terrae, Microbacterium
Trichothecenolyticum is present in soil, Microbacterium barkeri, Microbacterium
Laevaniformans is present in sewage more, and Microbacterium arborescens is present in lake water,
Microbacteriumlacticum and Microbacteriumliquefaciens can have found in milk and cheese,Microbacterium maritypicumIt is then to be found in seawater and ooze earliest.Microbacterium is generally used for environmental contaminants
It decomposes, drug modified, biological desulphurization etc., the report acted on about microbacterium control of plant disease is less.Microbacterium Sp. there is inhibiting effect to rice blinds blight；Microbacterium oxydansTo banana root-rot line
Worm second instar larvae has high virulence；Microbacterium maritypicumTo black leg of beet, snake eye disease, root rot
All there is preferable control efficiency with brown spot；But it is not yet it was reported that brightMicrobacterium maritypicumIn soybean
Effect in cyst nematode prevention and treatment.
Summary of the invention
To make up for the shortcomings of the above existing technologies, the purpose of the present invention is to propose to the anti-soybean cyst lines of one plant of inducing soybean
The microbacterium of parasitosis and its application, to the effect of soybean cyst nematode Heterodera glycines second instar larvae tagged and induce soybean anti-cyst nematode
On all show advantage outstanding, have good commercial exploitation prospects.
The purpose of the present invention is what is be achieved through the following technical solutions:
The microbacterium of one plant of anti-soybean cyst nematode of inducing soybean and its application, technical points are as follows: inducing soybean is anti-
The microbacterium of soybean cyst nematode isMicrobacterium maritypicum, depositary institution: Chinese microorganism strain is protected
Administration committee's common micro-organisms center is hidden, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date:
On November 03rd, 2015, deposit number: CGMCC No. 11566, classification naming:Microbacterium maritypicum。
The microbacteriumMicrobacterium maritypicumAfter culture dish culture, fermented by conventional liq
Cultivating can get inducing soybean and generates the fermentation liquid of anti-cyst nematode, specific method the following steps are included:
(1) culture dish culture:
Culture medium prescription be LB culture medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 5 g of agar powder,
Distilled water 1000 mL, pH 7.0 or so；
(2) liquid fermentation and culture:
LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, distilled water 1000 mL, pH
7.0 or so, 28-30 DEG C of culture 24-48 h；
The strain that step (1) obtains is seeded in fluid nutrient medium, 28-30 DEG C, after revolving speed 175-200 rpm, 48 h
It can get and obtain the fermentation liquid that inducing soybean generates anti-cyst nematode, bacterium amount is adjusted to 108-109CFU；
The fermentation liquid or fermented product of formation can direct or indirect processed soybeans seed, processed soya seeds can be with
Mitigate and endangered as caused by soybean cyst nematode evil, inducing soybean is generated to cyst nematode resistance.
Further, the fermentation liquid is used as seed treatment, method particularly includes: soya seeds are disappeared with 0.5% sodium hypochlorite
5 min of poison, aseptic water washing 5-10 times；It is that 1:70 mass ratio carries out seed pelleting processing (i.e. 70 grams of kinds by fermentation liquid and seed
1 gram of fermentation liquid is added in son), shady place air-dries, and Soybean Seedling can be induced to generate to cyst nematode resistance.
Beneficial effects of the present invention:
Have the present invention provides one kind and kill the active microbacterium of line, biological control microbial inoculum can be made, for killing born of the same parents
Capsule nematode second instar larvae, and resistance can be generated with inducing soybean, while the soybean Jing Guo seed treatment can be to soybean cyst
Nematode generates apparent disease resistance, and significant effect will not destroy soil environment, has in prevention and treatment soybean cyst nematode Heterodera glycines harm good
Good application prospect.
Specific embodiment
It is not intended to be limiting of the invention to further elucidate in more detail, gives the following example.
1 microbacterium of embodimentMicrobacterium maritypicum The culture of (bacterial strain Sneb159)
MicrobacteriumMicrobacterium maritypicum The depositary institution of (bacterial strain Sneb159): China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation day
Phase: on November 03rd, 2015, deposit number: CGMCC No. 11566.
MicrobacteriumMicrobacterium maritypicum Sneb159 carry out fermented and cultured, then to fermented and cultured after
Metabolite carry out the test of soybean induced activity, determine its inducing action to soybean.
(1) culture dish culture:
Culture medium prescription be LB culture medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 5 g of agar powder,
Distilled water 1000 mL, pH 7.0 or so；
(2) liquid fermentation and culture:
By above-mentioned strain, picking single colonie is inoculated into the liquid training in 250 mL triangular flasks (per bottled 100 mL culture medium)
It supporting in base, culture medium prescription are as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride add distilled water to 1000 mL,
PH 7.0 or so.Fermentation temperature is 28-30 DEG C, and shaking speed is 175 rpm-200 rpm, and fermentation time is 48 h, bacterium amount tune
To 108-109CFU obtains fermentation liquid.The fermentation liquid or fermented product of formation can direct or indirect processed soybeans seed, located
The soya seeds of reason can mitigate the harm as caused by soybean cyst nematode evil.
2 microbacterium of embodimentMicrobacterium maritypicum Sneb159 fermented product kills cyst nematode activity
Test
(1) test preparation is prepared
Tunning is obtained according to 1 method is implemented, by 10,000 rpm of tunning, 10 min is centrifuged, takes supernatant, lead to
0.22 μm of sterilised membrane filter is crossed, thallus is removed, filtered fluid is collected and obtains processing post-fermentation liquid, it is spare.
(2) for trying soybean cyst nematode Heterodera glycines
Soybean cyst nematode Heterodera glycines pick up from Agricultural University Of Shenyang, the proving ground nematode postgraduate Hou Shan, the north.Using improvement elutriation-
Crossing sieve method separates cyst from the soil sample of acquisition, and fresh, full, mature cyst is selected under Stereo microscope.Cyst is first
3 min are sterilized with 0.5% liquor natrii hypochloritis, then with aseptic water washing 5 times or more, until thoroughly removing sodium hypochlorite, is placed on and incubates
Change and concentration is added in pond as 3 mM solution of zinc sulfate, 25 DEG C of insulating box cultures, daily culture solution of replacing collects second instar larvae.
(3) cyst nematode activity test is killed in interior
500 μ L processing fermentation liquid is added in 1.5 mL centrifuge tubes, 30-40 J2 nematode suspension (10 of each addition
μ L), it is control with clear water, in triplicate, independent experiment in triplicate, observes nematode in 24 h and 48 h respectively for each processing
Death condition calculates the death rate, judges death condition by hair needle stimulus method.Test result is as shown in table 1:
Insecticidal activity of 1 microbacterium of table to soybean cyst nematode Heterodera glycines
| Processing | The 24 h death rates | The 48 h death rates |
| Sneb159 | 89.2% | 94.1% |
| CK | 9.4% | 13.4% |
As shown in Table 1, in present invention eelworm-killing activity test indoors, Sneb159 treated fermentation liquid in 24 h and
The 48 h death rates respectively reach 89.2% and 94.1%, and the death rate is respectively 9.4% and 13.4% in the control group of sterile water process,
Sneb159 is significant difference compared with the control group to the death rate of soybean cyst nematode Heterodera glycines 24 h and 48 h.
3 microbacterium of embodimentMicrobacterium maritypicum Sneb159 prevents and treats basin in soybean cyst nematode Heterodera glycines room
Plant effect test
(1) test preparation
The fermentation liquid for preparing Sneb159 as described in Example 1 is spare.
(2) soybean varieties are used in test
Distant beans 15, the purchase of seed sale market.
(3) method for treating seeds
Soya seeds 0.5% hypochlorite disinfectant, 5 min, aseptic water washing 5-10 times.By the fermentation liquid prepared and kind
Son is 1:70 mass ratio (i.e. 1 gram of fermentation liquid is added in 70 grams of seeds) to seed pelleting processing, and shady place air-dries, spare.
(4) test method
It is handled using potting, sand is 165 DEG C in hot air sterilization pot, and sterilize 2 h, is fitted into 12x12 cm polypots.It will
Soya seeds after Sneb159 Cotton seeds are seeded into the polypots for filling sterile sand, one seed of every basin, two panels to be grown
Nematode is inoculated with after true leaf, every basin is inoculated with 1500 soybean cyst nematode Heterodera glycines.As a control group with the seed of sterile water coating.After inoculation
It investigates within 30th day, gently digs out whole, bean seedlings, keep root system integrality, investigate cyst number on root system, eluriate crossing sieve method meter
Nematode population in root is investigated in cyst number in number soil, acid fuchsin dyeing.
(5) test result
It is significantly reduced using the cyst quantity on the soybean plant strain after Sneb159 seed treatment, root middle line borer population is significantly low
In control, the results showed that Sneb159 has good control efficiency to soybean cyst nematode Heterodera glycines.
Control efficiency of the 2 Sneb159 seed treatment of table to soybean cyst nematode Heterodera glycines
| Processing | Cyst sum | Root middle line borer population |
| Sneb159 | 94.75±16.40b | 12.75±1.6b |
| CK | 175±14.18a | 22.5±3.21a |
Note: data are average ± standard error in table, are indicated with letters different after column dataP0.05 level difference of < is aobvious
It writes (Duncan duncan's new multiple range method).
Claims (3)
1. the microbacterium of one plant of anti-soybean cyst nematode of inducing soybean, it is characterized in that: the depositary institution of the microbot bacteria strain:
China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, preservation date: on November 03rd, 2015, deposit number: CGMCC No.11566, classification naming: Microbacterium
maritypicum。
2. the application of microbacterium according to claim 1, it is characterized in that: the microbot bacteria strain Microbacterium
Maritypicum obtains fermentation liquid after culture dish culture, through conventional liq fermented and cultured, and the fermentation liquid can induce soybean
Seedling generates anti-cyst nematode activity, specific method the following steps are included:
(1) culture dish culture
Culture medium prescription is LB culture medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar powder 5g, distilled water
1000mL, pH7.0 or so；
(2) liquid fermentation and culture
LB liquid medium: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH7.0 or so,
28-30 DEG C of culture 24-48h；
The strain that step (1) obtains is seeded in fluid nutrient medium, 28-30 DEG C, revolving speed 175-200rpm, culture 24-48h is obtained
Soybean seedling must be can induce and generate the anti-active fermentation liquid of cyst nematode, bacterium amount is adjusted to 108-109CFU；
The direct or indirect processed soybeans seed of the fermentation liquid or fermented product of formation mitigates processed soya seeds due to big
It is endangered caused by beans cyst nematode disease, inducing soybean seedling generates resistance to cyst nematode.
3. it applies according to claim 2, it is characterized in that: the fermentation liquid is used as seed treatment, method particularly includes: soybean
Seed 0.5% hypochlorite disinfectant 5min, aseptic water washing 5-10 times；Fermentation liquid and seed are carried out according to 1:70 mass ratio
Seed pelleting processing, shady place air-dry, for inducing Soybean Seedling to generate resistance to cyst nematode.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058289C (en) * | 1995-12-29 | 2000-11-08 | 中国农业科学院生物防治研究所 | Strain for controlling cytocyst nematode disease of soybean and its preparation |
| WO2015069708A1 (en) * | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| CN105050402A (en) * | 2013-03-27 | 2015-11-11 | 诺维信生物农业公司 | Compositions and methods for enhancing plant growth |
| CN107427012A (en) * | 2014-09-09 | 2017-12-01 | 伊诺库克技术股份有限公司 | Microbial composite and method |
-
2018
- 2018-05-29 CN CN201810534442.2A patent/CN108624537B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058289C (en) * | 1995-12-29 | 2000-11-08 | 中国农业科学院生物防治研究所 | Strain for controlling cytocyst nematode disease of soybean and its preparation |
| CN105050402A (en) * | 2013-03-27 | 2015-11-11 | 诺维信生物农业公司 | Compositions and methods for enhancing plant growth |
| WO2015069708A1 (en) * | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| CN107427012A (en) * | 2014-09-09 | 2017-12-01 | 伊诺库克技术股份有限公司 | Microbial composite and method |
Non-Patent Citations (2)
| Title |
|---|
| Bacteria Associated with Cysts of the Soybean Cyst Nematode;Sarah M. Nour et al.;《AEM》;20030131;第69卷(第1期);第607-615页 * |
| 高效杀香蕉根腐线虫微生物资源的开发及活性物质的鉴定;张凤娟等;《中国植物病理学会2015年学术年会》;20150731;第423页 * |
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