CN108004183A - One plant of zaomycin streptomycete for having the anti-soybean cyst nematode Heterodera glycines of inducing soybean and application - Google Patents
One plant of zaomycin streptomycete for having the anti-soybean cyst nematode Heterodera glycines of inducing soybean and application Download PDFInfo
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Abstract
The present invention relates to have cultural method and its application of the inducing soybean generation to zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 of soybean cyst nematode resistance after one plant of processed soybeans seed.Actinomycetes strain Snea182 of the present invention has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center.After bacterial strain routinely liquid fermentation and culture, the metabolite of the bacterium can be used, develops the seed treatment of processed soybeans seed, treated soya seeds can produce the resistance to soybean cyst nematode Heterodera glycines with inducing soybean.Obvious resistance can be produced to seedling stage first generation cyst nematode with inducing soybean after the zaomycin streptomycete Snea182 metabolin processed soybeans seeds of the present invention, formation to soybean cyst nematode Heterodera glycines cyst has obvious inhibitory action, it is very promising biocontrol microorganisms, new approaches is opened for prevention soybean cyst nematode.
Description
Technical field：
The invention belongs to microbial pesticide technical field, and in particular to a kind of sand for having the anti-soybean cyst nematode Heterodera glycines of inducing soybean
Adriamycin streptomycete and application.
Background technology：
Soybean cyst nematode (Soybean Cyst Nematode, SCN), also known as soybean verticillium wilt, flue seedling sprout, are
Restrict the important disease of Soybean production.The disease has wide distribution, harm weight, host range is wide, route of transmission is more, hypopus
The features such as (cyst) time-to-live is long, is a kind of soil-borne disease of extremely difficult prevention.The disease generally make the soybean underproduction 10%~
30%, up to 70%~80%, the harm caused by disease more single than any type is all big in serious plot.Soybean cyst nematode
Every year to production loss caused by world soybean up to more than 9,000,000 tons, economic loss is up to 5,500,000,000 dollars.Particularly Heilongjiang Province
As Chinese soybean main producing region, about 6.5 × 105The soybean area of hectare is endangered by soybean cyst nematode Heterodera glycines.Pass through plantation
Disease-resistant variety, chemical prevention, soybean cyst nematode can be played necessarily by changing tillage method and rational way to manage
Preventive and therapeutic effect, but have certain limitation.In recent years, biological control is increasingly taken seriously, from the generation of microorganism
Thanking to separation in product, there is anti-nematode active material to become one of most potential controlling way.
The bio-control factors of soybean cyst nematode Heterodera glycines have very much, including fungi, bacterium, virus and protozoan etc..Actinomyces make
For the important producing strains of antibiotic, bacterial strain of the screening with eelworm-killing activity is used to, is widely used in prevention elder brother in the world at present
The avermectin of worm, mite class and nematode is exactly a kind of metabolite of actinomyces.Actinomyces are plants as fungi and bacterium
One of Important Natural Enemy biology of parasitic nematode, adjusts the population quantity of nematode, and time that actinomyces produce in nature
Raw metabolite has abundant structure diversity, therefore actinomyces show huge potentiality as the bio-control factors of nematode.
Zaomycin streptomycete is easy to breed, and accommodative ability of environment is strong, nontoxic to people and animals, has very strong environmental suitability and environment friend
Good property, plays an important role in biological control, has very big value of exploiting and utilizing.Correlative study shows zaomycin chain
Mould has growth-promoting and Biocontrol Effect, and prevention of the zaomycin streptomycete to soybean cyst nematode not yet has been reported that.
For there is this in production, one plant of actinomyces being obtained by screening system, utilizes the generation of the bacterium or the bacterium
Thank to product processed soybeans seed, inducible soybean produces the resistance to soybean cyst nematode Heterodera glycines during Seed Germination and growth, from
And solves the problems, such as soybean cyst nematode.
The content of the invention：
The purpose of the present invention is one plant of bacterial strain of selection to that can induce Soybean Seedling to cyst nematode after soya seeds processing
Obvious resistance is produced, after bacterial strain routinely liquid fermentation and culture, the metabolite of the bacterium can be used, develop processed soybeans
The seed treatment of seed, treated soya seeds can produce the resistance to soybean cyst nematode Heterodera glycines with inducing soybean.
One plant of actinomycetes strain that present invention screening obtains is zaomycin streptomycete (Streptomyces
Zaomyceticus) Snea182, depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Ground
Location：The Institute of Microorganism, Academia Sinica of BeiChen West Road, Chaoyang District, BeiJing City 1；Preservation date：On October 26th, 2016；Protect
Hide the numbering registered on the books:CGMCC No.13155.Zaomycin streptomycete (Streptomyces zaomyceticus)
Snea182 is that one plant filtered out from the Soybean Field of Heilongjiang Province of China Heihe City Aihui District Xifeng mountain area has by force soybean
The actinomycetes strain of induced activity.
The present invention has found that soil actinomycete has many strain actinomyces all to have certain anti-cyst of inducing soybean by studying
The ability of nematode, filters out one plant of relatively good bacterial strain of character from these actinomyces --- zaomycin streptomycete
(Streptomyces zaomyceticus) Snea182 is further studied.
The invention further relates to the actinomycete metabolites that anti-cyst nematode resistance is produced for inducing soybean, it is by the present invention
Actinomycetes strain zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 by conventional liquid hair
Obtained after ferment culture.
The present invention actinomycetes strain zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 be
Prepared after culture dish culture, then through expanding fermented and cultured, its specific method is as follows：
1. culture dish culture
Culture medium prescription is Gause I culture medium：Soluble starch 20g, NaCl 0.5g, KNO3 1g、 K2HPO4
0.5g、MgSO4·7H2O 0.5g、FeSO40.01g, agar 15-20g, pH 7.2-7.4, distilled water 1000mL.
2. liquid fermentation and culture
Wherein liquid fermentation medium formula is：By weight percentage, soluble starch 20g, NaCl 0.5g, KNO3
1g、K2HPO4 0.5g、MgSO4·7H2O 0.5g、FeSO40.01g, pH 7.2-7.4, distilled water 1000mL.
Strain is inoculated into 250mL triangular flasks (per bottled 100mL) fluid nutrient medium, at 25 DEG C~28 DEG C, shaking table turns
Speed is with 150rmin-1~180rmin-1, fermentation 5-7d, preferably 7d.
The strain of liquid fermentation can carry out large-scale production by large fermentation tank, its product is zymotic fluid or zymotic fluid
Manufactured goods.
The invention further relates to a kind of soya seeds inorganic agent, and it includes a effective amount of zaomycin strepto- of the present invention
Bacterium (Streptomyces zaomyceticus) Snea182 is in itself or its metabolin is as effective active composition.
Zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 of the present invention or its metabolin tool
There is the induced activity to soybean, available for the seed treatment of soybean, the soybean after processing forms the cyst of soybean cyst nematode Heterodera glycines
With good inhibiting effect, solves the problem of soybean cyst nematode.
Embodiment：
It is not intended to be limiting of the invention to further elucidate in more detail, gives the following example.
Embodiment 1：The culture of zaomycin streptomycete (Streptomyces zaomyceticus) Snea182
Fermentation training is carried out to zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 bacterial strains first
Support, soybean induced activity experiment then is carried out to the metabolite after fermented and cultured, determines its inducing action to soybean.
The thalline of zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 is inoculated into culture medium
On, culture medium prescription is Gause I culture medium, i.e. component：Soluble starch 20g, NaCl 0.5g, KNO3 1g、K2HPO4
0.5g、MgSO4·7H2O 0.5g、FeSO40.01g, agar 15-20g, pH 7.2-7.4, distilled water 1000mL, 25 DEG C of cultures
2d-3d。
Strain is inoculated into 250mL triangular flasks (per bottled 100mL) fluid nutrient medium again, culture medium prescription is：With weight
Measure percentages, soluble starch 20g, NaCl 0.5g, KNO3 1g、K2HPO4 0.5g、 MgSO4·7H2O 0.5g、FeSO4
0.01g, pH 7.2-7.4, distilled water 1000mL.Fermentation temperature is 25-28 DEG C, shaking speed 150rmin-1~180r
min-1, fermentation time 5-7d.The zymotic fluid of formation or the manufactured goods of zymotic fluid can direct or indirect processed soybeans seeds.Located
The soya seeds of reason can mitigate the harm caused by soybean cyst nematode is done harm to.
Embodiment 2：The fermentation of zaomycin streptomycete (Streptomyces zaomyceticus) Snea182
Substantially with embodiment 1, it is a difference in that the fermentation tank that the strain of Liquid Culture is inoculated into 1000L ferments,
Cultivated at 25 DEG C, pH 7.2, ferment 7d.
Embodiment 3：Zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 inducing soybean systems
The verification of resistance
Bacterial strain Snea182 has been inquired into soybean cyst nematode Heterodera glycines using crack root experimental design (split-root system)
Inducible system resistance.Challenge root system and response root system in crack root experiment is spatially completely isolated each other, in response root system
Snea182 zymotic fluids do not contact directly with soybean cyst nematode Heterodera glycines, and bacterial strain places one's entire reliance upon the control action of nematode it to planting
The induction of resistance of strain.The assay method is reliable, has been widely used in Systemic induction resistance of the Biocontrol microorganism to soil-borne disease
Research.
1. prepare experiment preparation
The zymotic fluid of zaomycin streptomycete Snea182 is prepared by aforementioned liquids fermentation culture method, is treated spare.
2. preparing experiment uses nematode
Soybean cyst nematode Heterodera glycines (Heterodera glycines)：No. 3 biological strains of soybean cyst nematode Heterodera glycines are derived from Heilongjiang Province
Heihe River institute of Academy of Agricultural Sciences proving ground.Cyst is separated from the soil sample taken using improvement elutriation-crossing sieve method, in stereoscope
Fresh, full, ripe, the homogeneous cyst of lower picking.Cyst first sterilizes 3min with 0.5% liquor natrii hypochloritis, then uses sterile water
Rinse 5 times, be placed in the culture dish containing a small amount of sterile water, cultivated in 25 DEG C of insulating boxs, tray obtains 2 ages children after hatching 15 days
Worm, 2 instar larvae suspension are adjusted to 200/ml.
3. prepare experiment seed
Heihe 43：There is provided by Heihe branch of Heilongjiang Institute of Agricultural Sciences.
4. method for treating seeds
By soya seeds first with 5% liquor natrii hypochloritis surface sterilization 5min, then with aseptic water washing 5 times.
5. test method
Handled using greenhouse pot culture, take the native and husky of no soybean cyst nematode Heterodera glycines, 165 DEG C in hot air sterilization pot, 2h sterilizings,
Then with V (soil)：V (sand) is 2：1 ratio, which mixes, to be fitted into 16 × 16cm polypots.3 43 seeds of Heihe are sowed in per alms bowl,
When bean seedlings length to two panels cotyledon, 1 plant is stayed per alms bowl.Bean seedlings are taken out and cleaned, root system is divided into two parts of equalization.Take 3 together
The polypots of sample size, is placed into triangle disposition, and the polypots of bottom is put in the pan arrest of 2 16cm diameters respectively.At top
Polypots bottom open 2 1cm × 2cm, seedling is placed in the polypots at top, makes separated root system respectively by 2 cracks
In the soil for reaching two polypots of lower part, the root system grown in the polypots of bottom two is respectively to challenge root system and response
Root system, soil matrix is loaded in 3 alms bowls and fixes plant.System is placed in greenhouse (25 DEG C, 14h illumination), in the plastics of challenge root system
The following inoculation Snea182 zymotic fluid 10ml of 2cm tables soil in alms bowl, inoculation Snea182 zymotic fluids are inoculated with 10ml J2 suspension after 1 week
To response root system.Using sterile water as control, totally 2 processing, 3 repetitions.The born of the same parents in the soil in response root system alms bowl are investigated after 35d
Soybean cyst nematode Heterodera glycines sum in capsule amount and root.
The separation and counting of cyst in 5.1 soil：Each soil sample weigh 200cc (be filled with water 800mL with 1000mL beakers,
Add soil to 1000mL), cyst is collected using the graceful funnel method of elutriation-sieving-shellfish, the number of cyst is recorded under stereoscopic anatomical lens
Amount.
The dyeing and counting of 5.2 interior nematodes：Root system is cleaned up with flowing water, claims root fresh weight, then uses hypochlorous acid
Sodium-acid fuchsin staining carries out the dyeing of nematode in root, nematode population in root is observed and recorded under stereomicroscope, and roll over
Into the quantity of every gram of root fresh weight nematode.
5.3 prevention effects calculate
6 result of the tests
Measure display, Snea182 being capable of the inducing soybean generation resistances very strong to soybean cyst nematode Heterodera glycines.Bacterial strain Snea182
In challenging root system be inoculated with after, in response root system the invasion of soybean cyst nematode Heterodera glycines significantly reduce, cyst quantity in response Soil-in-Root
Substantially reduce.The invasion of response root system soybean cyst nematode Heterodera glycines reduces by 52.42%, and cyst reduces by 61.35% (table 1) in soil.
After table 1Snea182 inoculation challenge root systems cyst sum in nematode and soil is invaded in response root system
Note：Investigation result after 35d, numeral after letter for Duncan ' s multiple range tests as a result, different size writes English
Word mother stock does not represent difference extremely significantly (P<And significant difference (P 0.01)<0.05).
Embodiment 4：The field control of zaomycin streptomycete (Streptomyces zaomyceticus) Snea182 is big
Beans Cyst nematode effect test
Using zymotic fluid stoste, by 1：100 ratio treatment coatings soya seeds, after drying, by processed Soybean Species
Sub normal sowing, can induce seedling stage big in soybean cyst nematode block after Snea182 metabolin processed soybeans seeds
Beans produce obvious resistance to cyst nematode, and the formation to soybean cyst nematode Heterodera glycines cyst has obvious inhibitory action, be have very much it is latent
The biocontrol microorganisms of power, new approaches are opened for prevention soybean cyst nematode.
1. prepare experiment preparation
The zymotic fluid of zaomycin streptomycete Snea182 is prepared by aforementioned liquids fermentation culture method, is treated spare.
2. prepare experiment seed
Heihe 43：There is provided by Heihe branch of Heilongjiang Institute of Agricultural Sciences.
Close rich 50：There is provided by grand celebration branch of Heilongjiang Institute of Agricultural Sciences.
3. method for treating seeds
By soya seeds first with 5% liquor natrii hypochloritis surface sterilization 5min, then with aseptic water washing 5 times.It will prepare
Zymotic fluid using 1% grain weight carry out seed pelleting processing, it is spare after drying.
4. test method
Using field natural occurrence soil inoculation soybean.Respectively in Heilongjiang Province's Heihe City and Heilongjiang Province Daqing two places
5 repetitions are carried out to test.Soybean Field is secondary soybean field, and Heihe proving ground continuous cropping 6 years, field cyst amount is 40 born of the same parents
Capsule/100ml soil, grand celebration proving ground continuous cropping more than 6 years, field cyst amount are 55 cysts/100ml soil.Field plot
It is designed as 3 to handle, 5 repetitions, totally 15 cells, random district's groups arrangement, per 5 ridge of cell, ridge grows 5 meters, 1 meter of passageway, per ridge
Sowing 100, cell edge set protection row.Using the soya seeds that the soya seeds without coating and liquid medium are coated as
Control, the 35d after soybean planting, after soybean cyst nematode Heterodera glycines growth and development breaks through soybean rhizoplane, each cell takes 20 plants at random
Seedling, removes and plant is connected root after table native (0-5cm) digs out, keep the complete of root system, and investigation single plant soybean root system cyst raw amount,
Calculate cyst inhibiting rate.
5. result of the test
To the soybean of Snea182 bacterial strains processing, show capsule phase investigation root system cyst in soybean cyst nematode Heterodera glycines and raw situation,
Big bean seedlings after Snea182 actinomycetes strain Cotton seeds form obvious inhibitory action to the cyst of soybean cyst nematode Heterodera glycines,
As shown in Table 2, inhibiting rates of the Snea182 in big Khanh Hoa Heihe has respectively reached 59.77%, 40.06%, after Cotton seeds
Soybean has different in the result that Heihe and grand celebration two places are handled, this may be with different regions Soybean Cyst Nematode
Difference it is related.
The influence that table 2Snea182 bacterial strain Cotton seeds soya seeds form soybean root system cyst
Note：Investigation result after 35d, numeral after letter be Duncan ' s multiple range tests as a result,
Different size writes English alphabet and represents difference extremely significantly (P respectively<And significant difference (P 0.01)<0.05).
Above-described embodiment is merely preferred embodiments of the present invention, but protection scope of the present invention is not limited to
This, any one skilled in the art the invention discloses technical scope in, the change that can readily occur in or replace
Change, should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claim
Subject to enclosing.
Claims (7)
1. there is the actinomycetes strain of the anti-soybean cyst nematode Heterodera glycines of inducing soybean after one plant of processed soybeans seed, it is characterised in that：Should
Bacterial strain is zaomycin streptomycete (Streptomyces zaomyceticus), is deposited in China on October 26th, 2016
Microbiological Culture Collection administration committee common micro-organisms center, preserving number are CGMCC No.13155.
2. the liquid fermentation method of the actinomycetes strain described in claim 1, it is characterised in that：Liquid fermentation medium formula
For：By weight percentage, soluble starch 20g, NaCl 0.5g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g,
FeSO40.01g, pH 7.2-7.4, water 1000mL, fermentation temperature are 25-28 DEG C, shaking speed 150rmin-1~180r
min-1, fermentation time 7d.
3. liquid fermentation method according to claim 2, it is characterised in that：The fermentation time is 7d.
4. the preparation method of the zymotic fluid stoste of anti-soybean cyst nematode resistance is produced with inducing soybean, it is characterised in that：It is
Formed as the actinomycetes strain described in claim 1 after liquid fermentation, liquid fermentation medium formula is：With weight hundred
Divide than meter, soluble starch 20g, NaCl 0.5g, KNO31g, K2HPO40.5g, MgSO4·7H2O 0.5g, FeSO40.01g,
PH 7.2-7.4, water 1000mL, fermentation temperature are 25-28 DEG C, shaking speed 150rmin-1~180rmin-1, fermentation when
Between 7d.
5. preparation method according to claim 4, it is characterised in that：The fermentation time is 7d.
6. a kind of soya seeds inorganic agent, it is characterised in that include a effective amount of liquid fermentation side according to claim 2
Zymotic fluid stoste formed in method, the resistance to soybean cyst nematode Heterodera glycines is produced for inducing soybean.
7. the zymotic fluid stoste formed in liquid fermentation method described in claim 2 is in the anti-soybean cyst nematode Heterodera glycines of inducing soybean
Using.
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Cited By (2)
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| CN115340961A (en) * | 2022-04-26 | 2022-11-15 | 浙江师范大学 | Streptomyces saxarubiginoides for antagonizing rice bacterial streak germ and application thereof |
| CN115975846A (en) * | 2022-08-02 | 2023-04-18 | 岳阳渔美康生物科技有限公司 | Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115340961A (en) * | 2022-04-26 | 2022-11-15 | 浙江师范大学 | Streptomyces saxarubiginoides for antagonizing rice bacterial streak germ and application thereof |
| CN115340961B (en) * | 2022-04-26 | 2023-05-16 | 浙江师范大学 | Streptomyces griseus for antagonizing rice bacterial strip spot bacteria and application thereof |
| CN115975846A (en) * | 2022-08-02 | 2023-04-18 | 岳阳渔美康生物科技有限公司 | Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof |
| CN115975846B (en) * | 2022-08-02 | 2023-10-03 | 岳阳渔美康生物科技有限公司 | Streptomyces saramycin, microecological preparation and preparation method thereof |
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Application publication date: 20180508 |