CN1519310A - Strain of streptomycete for preventing sheath blight of rice - Google Patents
Strain of streptomycete for preventing sheath blight of rice Download PDFInfo
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Abstract
A streptomyces globisporus H50 (CCTCC No.M203034) for preventing and eliminating the hypochnus and blast of rice is obtained from soil by screening with PAD and YPTG plate method. Its advantage is high effect (more than 65% for its bacterium inhibiting rate).
Description
Technical field:
The present invention relates to a kind of bacterial strain of streptomyces, particularly relate to a kind of triage techniques that utilizes streptomycete, by the dull and stereotyped point of application connection of PAD and YPTG primary dcreening operation, obtain the streptomycete bacterial strain that rice sheath blight disease pathogenic bacteria and rice blast pathogenic bacteria are had strong antagonistic action.
Background technology:
Rice sheath blight disease (Thanatephorus cucumeris), rice blast (Pyricularia) are the two big diseases in China rice district.Sichuan Dao Qu high temperature, high humidity, since 1987, a situation arises just reaches the degree of laying particular stress on every year for rice sheath blight disease, rice blast.Because the rice varieties of promoting lacks high anti-banded sclerotial blight gene at present, and utilize disease-resistant variety not obtain satisfied protection effect as yet, in addition, promote and be subjected to the influence of measure such as rice field management along with the big area of short-stalked variety and hybrid rice, therefore the generation area of rice sheath blight disease enlarges year by year, and the rate of loss that causes China's paddy rice annual production thus is more than 10%.Current, mainly be to prevent and treat this disease in actual production with jingganmycin, though its effect is better,, caused sheath blight fungus to develop immunity to drugs because of its unicity, life-time service.And at Sichuan Dao Qu, rice blast is the strongest with the hazardness of ZB15 physiological strain, but at present the gene of cloning this blast resisting from rice plant also has one section very long distance, so, the genetically engineered of changeing anti-rice blast rice is limited, and general chemical pesticide all can cause great harm to human health and environment.As everyone knows, the streptomycete mushroom is the important mushroom that produces the small molecules antibiotic substance, but utilizes microorganism to carry out in the control of diseases and pests of agronomic crop, and relevant report is not also seen in the research and the utilization of styreptomyces globispotus strain.
Summary of the invention:
Purpose of the present invention is intended to overcome the deficiencies in the prior art, provide a kind of Sichuan rice sheath blight disease pathogenic bacteria AG-1 and rice blast pathogenic bacteria ZB15 of utilizing as indicator strain, disengaging latch fungal strain from soil is to obtain bacterial strain that rice sheath blight disease, rice blast are had strong antagonistic action.
Concrete technical scheme of the present invention is as follows:
The invention belongs to the bacterial classification of streptomyces, this bacterial strain is a styreptomyces globispotus strain (Streptomycesglobisporus H50) (being designated hereinafter simply as the H50 bacterial strain), from the ecological geographic soil in China Sichuan Province, separate, filter out, in on May 2nd, 2003 in China's typical culture collection center preservation, deposit number is CCTCC NO:M 203034.
H50 bacterial strain of the present invention has following character:
A, morphological specificity
On Gause I agar and the glucose asparagine agar 28 ℃ cultivate 7～70 days after, observe the form of mycelia and spore shape, surface tissue respectively with opticmicroscope and electron microscope.The result shows that this bacterial strain substrate mycelium does not have tabula, does not rupture; The aerial hyphae branch of this bacterium is more, and fibrillae of spores is upright or gentle bent; Spore sphere or oval, smooth surface.
B, cultural characteristic
The growth result of bacterial strain on eight kinds of substratum
A, Gao Shi synthesize an agar
Aerial hyphae is a yellow-white, and substrate mycelium is chromogenesis not, is colourless, does not produce soluble pigment.
B, glucose asparagine agar
Aerial hyphae is a light gray, and substrate mycelium is light yellow, does not produce soluble pigment.
C, sucrose nitrate agar
Aerial hyphae is a yellowish green, and substrate mycelium is light yellow, does not produce soluble pigment.
D, starch ammonium salt agar
Aerial hyphae is a straw grass look, and substrate mycelium is a lark, does not produce soluble pigment.
E, inorganic salt Starch Agar
Aerial hyphae is a beige, and substrate mycelium is light yellow, does not produce soluble pigment.
F, yeastex malt extract agar
Aerial hyphae is a beige, and substrate mycelium is a beige, does not produce soluble pigment.
G, oatmeal agar
Aerial hyphae is the ecru green, and substrate mycelium is simple brown, does not produce soluble pigment.
H, potato are soaked juice agar
Aerial hyphae is a light tan, and substrate mycelium is light brown, does not produce soluble pigment.
C, cell cell wall chemical composition analysis
The strain whole-cell hydrolyzed solution contains LL-diaminopimelic acid (LL-DAP), glycine; Atypism sugar (sugared type C); It is the II type that bacterial strain contains phosphatidylethanolamine (PE).
D, physiological and biochemical property
In gelatine liquefication, iced milkization, starch hydrolysis, nitrate reduction reaction, be positive, and milk is solidified, can not on Mierocrystalline cellulose, grow, do not produce H2S, can not utilize tyrosine.But can utilize D-glucose, L-arabinose, D-fructose, D-N.F,USP MANNITOL, L-rhamnosyl, D-wood sugar and semi-lactosi, can not utilize inositol and raffinose.
E, strain identification result
Decide the genus principle according to what morphological specificity combined with chemical classification, above-mentioned experimental result shows: " H50 bacterial strain " substrate mycelium does not have tabula, do not rupture, no sporangiocyst, the aerial hyphae branch is more, and fibrillae of spores is straight or gentle bent, the cell walls chemical composition is the I type, sugar type C, phosphate lipid II type (containing PE), its essential characteristic meet streptomyces (Streptomyces).Surely plant principle according to cultural characteristic and Physiology and biochemistry experiment, its feature conforms to the styreptomyces globispotus strain feature, names to be styreptomyces globispotus strain (Streptomyces globisporus).
The pathogenic bacteria of the present invention's energy antagonism rice sheath blight disease and rice blast, especially Sichuan Dao Qu Rhizoctonia solani is merged mycelia group AG-1 and endangers serious ZB15 rice blast physiological strain and have the intensive antagonistic action, its prevention effect is good, bacteriostasis rate reaches more than 65%, has solved two big disease and pests of hazard rice effectively.
Description of drawings:
Fig. 1 is the form (comprising A and B) of H50 bacterial strain
Fig. 2 is the antagonistic action (comprise C, D and E) of H50 bacterial strain to rice sheath blight disease, rice blast pathogen
Fig. 3 is the teratogenic effect (comprise F and G) of H50 bacterial strain to pathogenic bacteria
Wherein, A is the spore surface feature (* 10000) of bacterial strain, B is the mycelium morphology (* 750) of bacterial strain, C is bacterial strain is stepped sheath blight fungus to water a antagonism, D is bacterial strain is stepped the seasonal febrile diseases bacterium to paddy rice a antagonism, E is the CK sheath blight fungus, and F hangs for the H50 mycelia and contracts fracture (2780 *), and G is that the H50 protoplasma overflows (2500 *)
Embodiment:
The screening method of embodiment 1:H50 bacterial strain
To place 250 milliliters of Erlenmeyer flasks that sterilized water is housed in right amount by each soil picking of experimental field gathering from Wenjiang, Sichuan, rotating speed with 120 rev/mins under 25 ℃ was cultivated 12 hours, draw 1ml bacterium liquid then respectively, coat on the PDA flat board (1000 milliliters of potato 200 grams, sucrose 10～20 grams, agar 17～20 grams, water) with E-test, place 26～28 ℃ of constant incubators to cultivate.After treating 48 hours, the single bacterium colony that the picking cultural characteristic is different, to be measured behind purifying repeatedly.Earlier with the primary election of some connection, then having the bacterial strain of inhibition zone to adopt dull and stereotyped face-off culture method to rice sheath blight disease and rice blast pathogen.
Embodiment 2: the research of antagonism bacterium antibiosis
A, antagonism bacterium are measured the bacteriostasis rate of sheath blight fungus and rice blast pathogen: place the ferment product through millipore filtration to soak 5 minutes mycelia piece of the same age, the point of application connection places PDA and the dull and stereotyped last 28 ℃ of cultivations of YPTG then, bacteriostasis rate is calculated in the clear water contrast.
By the mensuration to the banded sclerotial blight mycelia piece growing state of the supernatant liquor effect that contains antagonistic substance of collecting after PAD liquid nutrient medium shaking culture, the bacteriostasis rate that draws the H50 bacterial strain is 67.3%; On the YPTG substratum, the bacteriostasis rate of H50 bacterial strain reaches 62.5%.
B, antagonism bacterium are measured the teratogenesis of banded sclerotial blight mycelia and rice blast: the antagonism bacterium is used the inoculation of face-off method, and the sheath blight fungus filament and the normal mycelium at picking face-off edge are observed under the ordinary optical mirror, and detect through electron-microscope scanning.
As can be seen, use the face-off cultural method, show antibacterial band, antibacterial width reaches 9.0 millimeters and 8.7 millimeters respectively.Simultaneously, the protoplasma that contrasts normal mycelia is even, and mycelia is rounded, stalwartness, and the mycelia joint is longer.H50 banded sclerotial blight mycelia internode shortens, and vegetative point expands, the protoplastis aggegation, and the mycelia ultimate swelling, mycelia has phenomenon of rupture, and the mycelium twisted phenomena is serious, and ghost is more.
The influence that C, antagonism bacterium sprout the banded sclerotial blight sclerotium: aseptic slide glass is immersed in the aseptic PAD substratum that melts, make it the surface and enclose one deck medium agar, taking-up is placed in the culture dish, and 2 in every ware adds 5 ml sterile waters and preserves moisture in the ware.From the flat board screening size of cultivating sheath blight fungus in advance, the sclerotium that formation time is consistent, placing concentration is 10
8Handled 10 seconds in the zymocyte liquid of individual/milliliter, be placed on then on the agar of slide glass, every 3, grain is handled 8 (24) apart from being 1.5 centimetres at every turn, cultivates the sprouting situation of microscopy sclerotium after 24 hours in 30 ℃ of incubators.
The influence that D, antagonistic strain form sclerotium: will lead to the age diameter is that 5 millimeters banded sclerotial blight mycelia piece places and collects on liquid (ferment filtrate is overanxious repeatedly through millipore filtration) and the PDA blended substratum, place 30 ℃ of incubators to cultivate, detect the time that sclerotium forms.
The result shows: the sclerotium after bacterium liquid is handled is sprouted the comparison of mycelia index according to all having reduced.H50 has reduced by 66.7% than the sclerotium contrast of handling with clear water.Sclerotium is being formed in the influence, and the clear water contrast forms and needs 60 hours, and H50 needs 84 hours, obvious difference.
E, bacterium liquid seed soaking sub-district biological and ecological methods to prevent plant disease, pests, and erosion test-results: each antagonistic bacterium 28 ℃ of shaking culture in the YSP substratum were diluted to concentration and are about 10 after 48 hours
8The bacterium liquid of individual/milliliter was soaked seed 48 hours down at 28 ℃, and presprouting of seeds 2 days is sowed at seedling Tanaka, membrane covering.The spore suspension of yellow green trichoderma of antagonistic effect and clear water are arranged in contrast with jinggangmeisu, to Rhizoctonia solani Kuhn, wooden mould spore concentration is 10
9Individual/milliliter, the concentration of jingganmycin is 40 mcg/ml.After treating 25 days, transfer is in cement pit, and individual plant is inserted and carried, and strain, line-spacing are 26.6 centimetres of 23.3 cm x, and the sub-district random alignment is isolated with guard rows between the sub-district, and the experimental plot is managed routinely.The Sheng of tillering phase inoculation sheath blight fungus.Randomly drawed plant height, tiller number and the disease index of 50 strains investigation rice plant apart from 20 days harvesting times.
Handle with the tunning of H50 bacterial strain, can reach 60.75%, be higher than the processing preventive effect 53.14% of jingganmycin the preventive effect of banded sclerotial blight; Preventive effect to rice blast reaches 74.8%, is higher than the preventive effect 68.3% of Kitazine.Show that the H50 bacterial strain has big application potential on control paddy rice two big diseases.
Embodiment 3: the evaluation of bacterial strain
A, morphological specificity: cultivate thalli morphology observation and the photograph of carrying out routine after 2～4 days with electron microscope and opticmicroscope for 30 ℃ on protein culture medium, the ox meat extract agar.
B, cultural characteristic: Gao Shi synthetic agar, glucose asparagine agar, sucrose nitrate agar, starch ammonium salt agar, inorganic salt Starch Agar yeastex malt agar, oatmeal agar, a potato are soaked on the juice nutrient agar 30 ℃ and cultivate and observe its colony shape and thalline color after 2～4 days.
C, chemical classification: the amino acid and the sugared type analysis of full cell hydrolyzed solution carried out in the analysis of cell walls chemical composition by the improved quick thin plate chromatography of H asegawa (TCL).
D, Physiology and biochemistry experiment: with reference to the content of " Bergeys Manual of SustematicBacteriology " VO1.II bacterial strain is carried out Physiology and biochemistry and identify.The phosphate lipid type is pressed the Lechevalier experimental technique.
Claims (2)
1, the streptomycete bacterial strain of a kind of anti-rice sheath blight disease and rice blast is characterized in that: styreptomyces globispotus strain (Streptomyces globisporus) H50 CCTCC NO:M 203034.
2, the streptomycete bacterial strain of a kind of anti-rice sheath blight disease according to claim 1 and rice blast, it is characterized in that: described H50 bacterial strain has following character:
A, morphological specificity
On Gause I agar and the glucose asparagine agar 28 ℃ cultivate 7～70 days after, observe the form of mycelia and spore shape, surface tissue respectively with opticmicroscope and electron microscope.The result shows that this bacterial strain substrate mycelium does not have tabula, does not rupture; The aerial hyphae branch of this bacterium is more, and fibrillae of spores is upright or gentle bent; Spore sphere or oval, smooth surface.
B, cultural characteristic
The growth result of bacterial strain on eight kinds of substratum
A, Gao Shi synthesize an agar
Aerial hyphae is a yellow-white, and substrate mycelium is chromogenesis not, is colourless, does not produce soluble pigment.
B, glucose asparagine agar
Aerial hyphae is a light gray, and substrate mycelium is light yellow, does not produce soluble pigment.
C, sucrose nitrate agar
Aerial hyphae is a yellowish green, and substrate mycelium is light yellow, does not produce soluble pigment.
D, starch ammonium salt agar
Aerial hyphae is a straw grass look, and substrate mycelium is a lark, does not produce soluble pigment.
E, inorganic salt Starch Agar
Aerial hyphae is a beige, and substrate mycelium is light yellow, does not produce soluble pigment.
F, yeastex malt extract agar
Aerial hyphae is a beige, and substrate mycelium is a beige, does not produce soluble pigment.
G, oatmeal agar
Aerial hyphae is the ecru green, and substrate mycelium is simple brown, does not produce soluble pigment.
H, potato are soaked juice agar
Aerial hyphae is a light tan, and substrate mycelium is light brown, does not produce soluble pigment.
C, cell cell wall chemical composition analysis
The strain whole-cell hydrolyzed solution contains LL-diaminopimelic acid (LL-DAP), glycine; Atypism sugar (sugared type C); It is the II type that bacterial strain contains phosphatidylethanolamine (PE).
D, physiological and biochemical property
In gelatine liquefication, iced milkization, starch hydrolysis, nitrate reduction reaction, be positive, and milk is solidified, can not on Mierocrystalline cellulose, grow, do not produce H2S, can not utilize tyrosine.But can utilize D-glucose, L-arabinose, D-fructose, D-N.F,USP MANNITOL, L-rhamnosyl, D-wood sugar and semi-lactosi, can not utilize inositol and raffinose.
E, strain identification result
Decide the genus principle according to what morphological specificity combined with chemical classification, above-mentioned experimental result shows: " H50 bacterial strain " substrate mycelium does not have tabula, do not rupture, no sporangiocyst, the aerial hyphae branch is more, and fibrillae of spores is straight or gentle bent, the cell walls chemical composition is the I type, sugar type C, phosphate lipid II type (containing PE), its essential characteristic meet streptomyces (Streptomyces).Surely plant principle according to cultural characteristic and Physiology and biochemistry experiment, its feature conforms to the styreptomyces globispotus strain feature, names to be styreptomyces globispotus strain (Streptomyces globisporus).
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Cited By (7)
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| CN101525587B (en) * | 2009-03-25 | 2011-06-01 | 中国农业大学 | Streptomyces strain and application thereof |
| CN101422170B (en) * | 2008-10-28 | 2011-12-28 | 云南省烟草科学研究所 | Streptomyces globisporus microbial agent for controlling Alternaria alternate and preparation method thereof |
| CN101781626B (en) * | 2007-08-07 | 2012-08-22 | 吉林省农业科学院 | Streptomyces avermitilis with antagonism to pyriculariagrisea and preparation method thereof |
| CN102732460A (en) * | 2012-06-13 | 2012-10-17 | 浙江师范大学 | Streptomyces hygroscopicus Sh-43 strain, its fermentation solution production method and application |
| CN109526985A (en) * | 2018-11-19 | 2019-03-29 | 黑龙江省农垦科学院 | Application of the soil class bacillus albumen EsxA as rice blast biocontrol agent |
| CN110607249A (en) * | 2018-12-27 | 2019-12-24 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
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|---|---|---|---|---|
| CN101781626B (en) * | 2007-08-07 | 2012-08-22 | 吉林省农业科学院 | Streptomyces avermitilis with antagonism to pyriculariagrisea and preparation method thereof |
| CN101422170B (en) * | 2008-10-28 | 2011-12-28 | 云南省烟草科学研究所 | Streptomyces globisporus microbial agent for controlling Alternaria alternate and preparation method thereof |
| CN101525587B (en) * | 2009-03-25 | 2011-06-01 | 中国农业大学 | Streptomyces strain and application thereof |
| CN102732460A (en) * | 2012-06-13 | 2012-10-17 | 浙江师范大学 | Streptomyces hygroscopicus Sh-43 strain, its fermentation solution production method and application |
| CN102732460B (en) * | 2012-06-13 | 2013-06-26 | 浙江师范大学 | Streptomyces hygroscopicus Sh-43 strain, its fermentation solution production method and application |
| CN109526985A (en) * | 2018-11-19 | 2019-03-29 | 黑龙江省农垦科学院 | Application of the soil class bacillus albumen EsxA as rice blast biocontrol agent |
| CN109526985B (en) * | 2018-11-19 | 2021-03-12 | 黑龙江省农垦科学院 | Application of geobacillus protein EsxA as biological rice blast preventing and treating agent |
| CN110607249A (en) * | 2018-12-27 | 2019-12-24 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
| CN110607249B (en) * | 2018-12-27 | 2022-05-17 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
| CN115216430A (en) * | 2022-08-01 | 2022-10-21 | 湖南科技大学 | Streptomyces biocontrol strain and application thereof |
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| CN1233818C (en) | 2005-12-28 |
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