CN101822272A - Streptomyces griseoflavus and application thereof in biological prevention and control of plant diseases - Google Patents

Abstract

The invention discloses application of streptomyces griseoflavus in biological prevention and control of plant diseases, particularly streptomyces griseoflavus NMG6-3-9 CGMCC No. 3441 in biological prevention and control of plant diseases. The provided strains have good antagonism effect on pathogenic bacteria of alfalfa root rot, can be used for the biological prevention and control of alfalfa root rot and other plant diseases, and have wide application potential in the field of biological prevention and control of plant diseases. The invention lays a foundation for the biological prevention and control of alfalfa root rot and further provides a scientific basis for the development and the application of a biological prevention actinomycete preparation.

Description

Ash yellow streptomycete and the application in biocontrol of plant disease thereof

Technical field

The invention belongs to microbial technology field, be specifically related to the application of grey yellow streptomycete in biocontrol of plant disease.

Background technology

Since the fifties in last century, a large amount of chemical pesticides that are extensive use of cause pathogen that chemical pesticide is produced resistance, high residue causes environmental pollution, and chemical pesticide produces to poison etc. to people and animals and quickened people and seek new, control of plant disease approach safely and effectively.In the various measures of controlling plant diseases, biological control has the safer advantage of environment, ecology and human health, therefore, and the carrying out and utilize of countries in the world attention biological and ecological methods to prevent plant disease, pests, and erosion microbe research very.The biological and ecological methods to prevent plant disease, pests, and erosion microorganism have be difficult for making pathogen produce drug resistance, environmentally safe, production technology is simpler and many microorganisms have the characteristics that promote plant growth simultaneously and cause people's attention and attention, it will play an increasingly important role in disease control.Utilize the effects such as systemic disease resistance of antagonistic microbe to antibiosis, nutrition and competition for space, hyperparasitism and the inducing plant generation thereof of pathogen, coming controlling plant diseases is a most important component of biocontrol of plant disease.In the biological control of plant disease, actinomycetes are one of biocontrol microorganisms of tool competitive ability in numerous antagonistic microbes, and actinomycetes are widely used in the Strategies of Agricultural Bio-control owing to producing the various metabolite with antibiotic activity of chemical constitution.

Find actinomycetes so far from Cohn (1872), reported that 69 belong to 1687 kinds.In known actinomycetes, discovery tool antagonisms more than half, what wherein be applied to biological control mainly is some kinds of streptomyces Streptomyces, and next has nocardia Nocardia, actinoplanes Actinoplanes, micromonospora Micromonospora etc.Actinomycetes produce and account for more than 70% in the new active substance of finding from microorganism over nearly 10 years, and actinomycetic screening of biological and ecological methods to prevent plant disease, pests, and erosion and application have become the research emphasis in plant pathology field.

Actinomycetes generally pass through two kinds of approach to target bacterium performance biological and ecological methods to prevent plant disease, pests, and erosion effect: by antibiosis, competition effect, predation and hyperparasitism, cause the pathogenic of pathogen and infect the efficient reduction outside pin main body; Actinomycetes also can be brought out it and produce resistance or produce antibacterial substance in the host plant tissue, influence growth of pathogenic bacteria, breeding, cause its death at last.

Utilize actinomycetes controlling plant diseases, particularly soil-borne disease to have many successful examples.As far back as nineteen fifty, Cooper V E just finds that actinomycetes have significant antagonism to sugarcane root rot Fusarium arrkenomnes.With the living cells preparation that actinomycetic spore and mycelia are made, have nontoxic, noresidue, do not injure non-target microorganism,, advantages such as diseases prevention lasting period long good with environment compatibility.The actinomycetes live body preparation Mycostop of extensive use in the world, the spore of the grayish green streptomycete S.griseovidis that from mud coal, is separated to by the Kemira (1989) of Finland and the living cells preparation that mycelia is made exactly, be mainly used to prevent and treat sickle spore bacterium Fusarium sp., some common soil-borne pathogens such as phytophthora Phytophthora sp. and rhizoctonia Rhizoctonia sp., pass through seed treatment, after measures such as soil sprinkling or filling root are handled, Mycostop can grow surely at plant root, growth and breeding, stop the intrusion of pathogen, and can produce the growth that hormone promotes host plant, thereby improve the output of crop.The Chinese Academy of Sciences used from the Radix Medicaginis soil of Jingyang County, China Shaanxi and to separate the area maximum that " 5406 " antibiotic bacteria that the streptomyces microflavus Streptomycesmicroflavus that obtains makes is promoted nineteen fifty-three.It is imposed on the field, the effect that has diseases prevention, keeps a full stand of seedings and increase production by seed dressing.

In recent years, the antagonism actinomycetes screening of soil-borne diseases such as capsicum epidemic disease, cotton seedling blight, cotton wilt, cucumber fusarium axysporum, take-all, banana blight and soybean phytophthora root rot and biological and ecological methods to prevent plant disease, pests, and erosion application etc. are studied both at home and abroad.Mark A.Roberts (2001) separates, filters out 700 strains and has the actinomycetes that suppress and kill various plants pathogen ability, and is especially remarkable to fungus-caused root disease effect.Yi Long etc. separate actinomycetes 7 strains that capsicum epidemic disease had strong antagonism from the capsicum rhizosphere soil, wherein the sick effect of the greenhouse pot culture control of bacterial strain CQ21-3 reaches 73.2%; The 58 strain actinomycetes that Zong Zhaofeng etc. separate from soil have filtered out 2 strains has the chitinase of better prevention effect to produce bacterium to cotton seedling blight, verticillium wilt; Pan Rongguang etc. filter out from 208 strain actinomycetes has 12 strains of antagonism bacterial strain to cucumber fusarium axysporum, and wherein the antimicrobial spectrum of bacterial strain PM-1 is wide, and target bacterium mycelia is had obvious teratogenesis; The actinomycetes that separation screening 12 strains have antagonism to take-all from the soil sample of Xinjiang protection ground vegetables and cotton field collection such as Qiao Hongping, 3 strain fungistatic effects reach more than 80%, and the disease prevention growth-promoting effect is obvious; (2003) such as Cao's ideals analyze discovery to living actinomycetes in the banana, and rose light gray streptomycete has tangible antagonistic activity to Fusarium oxysporum.

In addition, the antibiotic that actinomycetes produce is widely used in industry, agricultural and medical domain, is also playing crucial effects aspect the crop pest control greatly, and is obtaining huge social benefit, economic benefit and ecological benefits.Since its have pollution-free, noresidue, renewable, be difficult for making pest to develop immunity to drugs, cost is low, be easy to characteristics such as industrialization has become one of the main body of public nuisance-free agricultural chemicals and developing direction of following agricultural chemicals.In view of actinomycetes and metabolite thereof prevent and treat disease, promote crop growth, improve the important function of aspects such as crop yield, just more and more by people's development and application.

Clover Medicago sativa L. is important high-quality legume forage, is described as " king of herbage ", has ecological functions such as improving the soil, reduce water and soil loss concurrently, has important function in the western ecological construction of China.In recent years, along with the variation of crop allocation, weather conditions and ecotope, and kind unification in the ALFALFA PRODUCTION and continuous cropping for many years, be the generation and the popular condition of having created of disease.The clover root rot generally betides each clover cultivation area, and being failed by the clover meadow of its initiation causes its period of use to shorten, and this disease has become one of key constraints of ALFALFA PRODUCTION.How this disease is carried out scientific and effective control, make establishment of alfalfa grassland bring into play production, economic and social benefit to greatest extent, become the problem that more and more presses for solution.Rely on seed selection resistant variety and chemical agent to be controlled to the control of clover root rot is main at present.But lack real effectively disease-resistant variety in producing at present, or the disease resistance of disease-resistant variety is lost very easily.The excessive application of chemical bactericide easily makes the pathogen pesticide resistance, and the grass product residue of pesticide exceed standard, and ecological environmental pollution increases the weight of, and causes its application to be limited to.Therefore the research that meets the biopesticide of environmental protection, health requirements receives publicity day by day with application.

Be exploitation antagonism actinomycetes bactericide, need set up the effective screening model of a cover carries out reliable results to a large amount of actinomycetes strains resistant activity screening.In the prior art, only adopt single vitro Screening models such as bacteriostatic test plate method (as flat board face-off method, inhibition zone method, Oxford agar diffusion method, cylinder-plate method, dig piece method, agar block method or filter paper method), spore germination method that active bacterial strain is screened mostly, its The selection result is unreliable, make the follow-up study result can not real service in agricultural production.Therefore, need to seek more effectively antagonism actinomycetes screening technique.

Summary of the invention

An object of the present invention is to provide the purposes of grey yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9CGMCC No.3441.

The purposes of grey yellow streptomycete provided by the present invention (Streptomyces griseoflavus) NMG6-3-9CGMCC No.3441 is the application of grey yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9CGMCC No.3441 in the biological control plant disease.

In the above-mentioned application, described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, banana blight, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, capsicum epidemic disease, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

In the above-mentioned application; described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusarium avenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusariumsemitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoria medicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomycesgraminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotinia sclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichotheciumroseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Erwiniacarotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

Another object of the present invention provides a kind of microbial inoculum that is used for the biological control plant disease.

The microbial inoculum that is used for the biological control plant disease provided by the present invention, its active component are the zymotic fluid that grey yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9 CGMCC No.3441 or the grey yellow streptomycete that ferments (Streptomyces griseoflavus) NMG6-3-9 CGMCC No.3441 obtain.

In the above-mentioned microbial inoculum, described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

In the above-mentioned microbial inoculum; described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusarium avenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusariumsemitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoria medicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomycesgraminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotinia sclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichothecium roseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Erwiniacarotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

Ash yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9 CGMCC No.3441 is used for the microbial inoculum of biological control plant disease in preparation application also belongs to protection scope of the present invention.

In the above-mentioned application, described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

In the above-mentioned application; described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusarium avenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusariumsemitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoriamedicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomycesgraminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotinia sclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichothecium roseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Erwiniacarotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

In above-mentioned application or the above-mentioned microbial inoculum, described oat sickle spore bacterium Fusarium avenaceum is oat sickle spore bacterium Fusarium avenaceum ACCC 30065, described Rhizoctonia solani Kuhn Rhizoctonia solani is Rhizoctonia solani Kuhn Rhizoctonia solani ACCC 30332, described Fusarium graminearum Peronosporaaestivalis is Fusarium graminearum Peronospora aestivalis ACCC 30068, described fusarium semitectum Fusarium semitectum is fusarium semitectum Fusarium semitectum ACCC 31945, described banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense is banana Fusarium oxysporum Fusarium oxysporumf.sp.cubense ACCC 36369, the rotten mould Pythium aphanidermatum of described melon and fruit is the rotten mould Pythium aphanidermatum ACCC 36125 of melon and fruit, described melon shell two spore Ascochyta citrullina are melon shell two spore Ascochyta citrullina ACCC 36440, a described big beautiful spore Verticilliumdahlia that takes turns is a big beautiful spore Verticillium dahlia ACCC 36109 that takes turns, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomyces graminis ACCC 30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunata ACCC36580 of standing grain, described phytophthora blight of pepper Phytophthora capsici is phytophthora blight of pepper Phytophthora capsiciACCC 36279, described Botrytis cinerea Botrytis cinerea is Botrytis cinerea Botrytis cinerea ACCC30091, described sclerotinite Sclerotinia sclerotiorum is sclerotinite Sclerotinia sclerotiorumACCC 30096, described pink poly-end spore Trichothecium roseum is pink poly-end spore Trichotheciumroseum ACCC 36459, described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora ACCC01443, the withered Lei Er Salmonella of described green grass or young crops Ralstonia solanacearum is blue or green withered Lei Er Salmonella Ralstoniasolanacearum ACCC 01470, and described staphylococcus aureus Staphylococcus aureaus is staphylococcus aureus Staphylococcus aureaus ACCC 01336.

In the bacteriostatic test plate, bacterial strain of the present invention is 32.68mm to the antibacterial circle diameter of target bacterium clover eggplant fusarium (Fusarium solani); Spore germination suppresses in the experiment, and bacterial strain of the present invention is 100% to the inhibiting rate of target bacterium clover eggplant fusarium (F.solani); In the potted plant protection effect test experience of live body, bacterial strain effect group of the present invention, the plant disease index is 16.35, and the incidence of disease is 39.68%, and preventive effect is 81.50%, and effect significantly is better than control group (jinggangmycin, fermentation medium, clear water).In addition, bacterial strain of the present invention has the antibiotic property of wide spectrum, following bacterium all had good antagonism: clover eggplant sickle spore pine root fungus F. solani, clover point sickle spore pine root fungus F. oxysporum, oat sickle spore bacterium F. avenaceum, the clover mould Stemphyllium botryosum of handle that crawls, Rhizoctonia solani Kuhn Rhizoctonia solani, Fusarium graminearum F. graminearum, fusarium semitectum F. semitectum, banana Fusarium oxysporum F. oxysporum f.sp.cubense, the rotten mould Pythium aphanidermatum of melon and fruit, clover septoria musiva Septoria medicaginis, melon shell two spore Ascochyta citrullina, a big beautiful spore Verticillium dahlia that takes turns, gaeumannomyce Gaeumannomyces graminis, colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides, standing grain is bent spore mould Curvularia lunata, the prominent navel spore Exserohilumleonard turcicum of big spot, phytophthora blight of pepper Phytophthora capsici, Botrytis cinerea Botrytis cinerea, sclerotinite Sclerotinia sclerotiorum, pink poly-end spore Trichotheciumroseum; Pseudomonas syringae Pseudomonas syringae; carrot soft rot Erwinia Erwiniacarotovora var.carotovora; clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; blue or green withered Lei Er Salmonella Ralstonia solanacearum; oat acidophil watermelon subspecies Acidovoraxavenae subsp.citrulli, staphylococcus aureus Staphylococcus aureaus.

Therefore, bacterial strain NMG6-3-9 of the present invention has extraordinary antagonism to clover root-rot disease pathogen bacterium, can be used in biological control clover root rot and other plurality of plant diseases, have broad application prospects in biological control disease field, also staphylococcus aureus is had good inhibition effect simultaneously, illustrate that this bacterial strain antimicrobial spectrum is wide.The present invention lays the foundation for the biological control of clover root rot, and then provides scientific basis for the anti-actinomyces preparations of this diease occurrence and application.

Description of drawings

Fig. 1 is bacterial strain NMG6-3-9 aerial hyphae form (under the light microscope 200 *).

Fig. 2 is bacterial strain NMG6-3-9 spore shape (under the ESEM 10000 *).

Fig. 3 is bacterial strain NMG6-3-9 fibrillae of spores form (under the ESEM 10000 *).

Fig. 4 is the phylogenetic tree according to the bacterial strain NMG6-3-9 of 16S rDNA gene order structure.

Embodiment

Employed experimental technique is conventional method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

The separation of embodiment 1, bacterial strain and evaluation

One, separates

No. 1 medium of Gao Shi: agar 15g, soluble starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄3H ₂O0.5g, MgSO ₄7H ₂O 0.5g, FeSO ₄7H ₂O 0.01g, distilled water 1000mL, pH 7.2-7.4.

PDA agar medium: potato (peeling) 200g, sucrose (or glucose) 20g, distilled water 1000mL, pH value nature.

Clover eggplant fusarium (Fusarium solani); (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (06): 105-107.)

(1) separation obtains the actinomycetes pure culture from soil

Adopt dilution plate method isolated strains from pick up from the domestic Mu Us Shadi soil specimen in Ordos City, the Inner Mongol.

Gather soil, the soil sample of gathering through natural air drying, is sieved, weigh.Adopt agar plate dilution method to obtain main actinomycetes monoid in the soil.Concrete grammar: pretreated soil specimen is diluted to 10 with sterile water ^-1, 10 ^-2, 10 ^-3, 10-4,10-5,10-6 suspension doubly, put 200r/min shaking table vibration 5min, get the 0.2mL supernatant, with the triangle glass rod make on No. 1 agar plate of Gao Shi, smear even.Place 28 ℃ of constant incubators to cultivate 7-10d.Observe the colony growth situation, dominant bacterium colony purifying in test tube in the picking plate, with single bacterium colony of purifying be transferred to cultivate on the Gause I inclined-plane after, numbering is preserved, and obtains 294 strain pure culture actinomycetes altogether.

(2) dull and stereotyped bacteriostatic method primary dcreening operation active bacterial strain

The cultural method of clover eggplant fusarium (Fusarium solani): (F. solani) is inoculated in the PDA medium with the clover eggplant fusarium, cultivates 5-6d in 28 ℃ of constant incubators, obtains clover eggplant fusarium bacterium cake.

The 294 strain actinomycetes that are separated to are inoculated in respectively on No. 1 agar plate of Gao Shi in 28 ℃ of following 5d of cultivation, treat that long good back breaks into lawn with card punch the bacterium cake of diameter 5mm, and actinomycetes bacterium cake placed the dull and stereotyped center of PDA, make the one side that has mycelia be attached to media surface, evenly instead paste onesize 4 clover eggplant fusariums (F. solani) bacterium cake at distance center 2.5cm behind the 3d, face-off is connected to the actinomycetes periphery, each bacterial strain repeats 5 times, cultivate down for 25 ℃, observe whether producing antibacterial (bacteriolyze) circle, the diameter of routine observation, measurement antibacterial (bacteriolyze) circle.Adopt the right-angled intersection method to measure the diameter of antibacterial (bacteriolyze) circle.Choice according to diameter decision bacterial strain.Actinomycetic antagonism intensity is embodied in the diameter of antibacterial (bacteriolyze) circle of its generation.

Actinomycetic antagonism intensity statistics standard: R is the antagonism loop diameter, divides following 4 grade separation: 10mm＜R≤20mm (antagonism is extremely strong), 5mm＜R≤10mm (better resistance short of money), 0mm＜R≤5mm (antagonism is weak) and R=0 (no antagonism).Under above standard, choose extremely strong, the stronger actinomycetes of antagonism and carry out follow-up screening.

Antagonism is the resistance of actinomycetes to the clover pine root fungus.

The result shows, screens altogether from 294 strain bacterium in this step and obtains the actinomycetes strain that 8 strains meet the antagonism requirement, and wherein 3 strains are that antagonism is extremely strong, and 5 strains are better resistance short of money.

Above-mentioned bacteriostatic test plate method primary dcreening operation active bacterial strain adopts conventional flat board face-off method, Oxford agar diffusion method, cylinder-plate method, dig piece method, agar block method or filter paper method etc. all can.

(3) spore germination method is sieved active bacterial strain again

1. the preparation of clover pine root fungus spore suspension

The PDA medium: potato 200g, glucose (or sucrose) 10～20g, agar 17～20g, distilled water 1000mL, pH 7.0.

(Fusarium solani) is inoculated in the PDA medium with the clover eggplant fusarium, in 28 ℃ of constant incubators, cultivate 5-6d, treat to use sterile water flush away aerial hyphae after mycelia is covered with medium, the irradiation of 400nto fluorescent lamp is cultivated down and is produced spore, after treating that 2-3d produces spore, wash spore with sterile water, remove mycelia and add 0.02%Tween-20 through biofilter aseptic filtration and make spore suspension and preserve down standbyly at 4 ℃, spore suspension miospore concentration is 10 ⁶Cfu/mL.

2. the actinomycetic preparation of antagonism

(1) preparation of actinomycetes strain

The 8 strain bacterium of bacterial strain uses therefor for obtaining in the experiment two in this experiment.

No. 1 synthetic medium of slant medium: Gao Shi.Agar 15g, soluble starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄3H ₂O 0.5g, MgSO ₄7H ₂O 0.5g, FeSO ₄7H ₂O 0.01g, distilled water 1000mL, pH7.3.

Seed culture medium: yeast extract 4g, glucose 10g, peptone 3g, beef extract 3g, CaCO ₃4g, distilled water 1000mL, pH 7.2-7.4.

Fermentation medium: glucose 5g, glycerine 5g, soluble starch 5g, peptone 5g, yeast extract 5g, analysis for soybean powder 5g, KH ₂PO ₄0.5g, CaCO ₃0.5g, NaCl 0.5g, MgSO ₄0.5g, distilled water 1000mL, pH 7.0-7.2.Soluble starch is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is HX169; Peptone is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is SH0010; Yeast extract is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is SH0348; Analysis for soybean powder is available from the supermarket.

1) slant culture: respectively with inoculation on No. 1 inclined-plane of Gao Shi, cultivate 5-6d at 28 ℃;

2) seed culture: be inoculated in the 100mL seed culture medium from the good actinomycetes inclined-plane of growing, 220r/min, 28 ℃ of shaken cultivation 48h obtain seed culture fluid;

3) fermented and cultured: the inoculum concentration by 10% (v/v) is inoculated in seed culture fluid in the 100mL fermentation medium, and 220r/min, 28 ℃ of shaken cultivation 120h obtain containing the zymotic fluid of actinomycetes strain.

3. the actinomycetic screening of antagonism

With the zymotic fluid that makes in the said method 2, mix with the spore suspension equal-volume that makes in the said method 1, get an above-mentioned mixed liquor and drip on the cover glass of handling with collodion on the surface 5 repetitions of every processing.Behind the cultivation 6h that preserves moisture under 25 ℃, check the sprouting of blank spore, after the germination rate of blank spore reaches 85%, check the clover pine root fungus spore germination rate (is sprouting greater than half person of spore length with spore germ tube length) of all processing.Blank group: obtain with the spore suspension equal-volume mixed preparing that makes in sterile water and the said method 1.Test repeats 5 times.

Calculate the spore germination inhibiting rate as follows:

In the 8 strain antagonism actinomycetes that obtain in the experiment two and the processing of clover pine root fungus, clover pine root fungus spore germination inhibiting rate is followed successively by 33.85%, 49.46%, 62.72%, 68.98%, 76.45%, 81.36%, 84.60%, 100%; Choose the spore germination inhibiting rate and carry out following experiment four, totally 3 strains greater than 80% bacterial strain.

(4) greenhouse pot culture live test

This experiment is further screened the 3 strain bacterium that screening in the step 3 obtains.

(1) actinomycetes suspension preparation: the 4 strain actinomycetes strains that step 3 is obtained are shaken cultivation 7d in fermentation medium respectively, and making concentration is 10 ⁸The spore suspension of cfu/mL.

(2) preparation of target bacterium spore suspension: indicator strain (F.solani) is cultivated 5-6d with medium oatmeal in 28 ℃ of constant incubators, after treating that mycelia is covered with medium, alternately cultivate (dark-illumination) 7d, produce a large amount of conidiums, wash spore with sterile water, remove mycelia through biofilter aseptic filtration, it is 10 that adding 0.02%Tween-20 makes concentration ⁶The spore suspension of cfu/mL.

Medium oatmeal: oats flakes 30g, agar 17-20g, distilled water 1000mL (oats flakes 30g adding distil water 1000mL heats 1h on boiling water bath, add water after the filtered through gauze and supply 1000mL, adds agar fusing back packing, 121 ℃ of sterilization 20min).

(3) greenhouse pot culture efficiency test

Clover (No. 1, middle lucerne) seed is provided by national germplasm herbage storehouse in mid-term, and catalog number is 00068.

With clover (No. 1, middle lucerne) seed disinfection, the bromogeramine surface sterilization 3min with 0.1%, distilled water flushing 3 times, vernalization in 25-28 ℃ of incubator, bunch plating when treating that seed shows money or valuables one carries unintentionally.The soil of growing seedlings is then mixed thoroughly with actinospore suspension earlier through 121 ℃ of following autoclaving 2h, and actinomycetic inoculum concentration is 10 in the soil ⁸Cfu/g.Emerge and hinder root with target bacterium spore suspension behind the 30d and connect bacterium, inoculum concentration is 10 ⁶Cfu/g is contrast 1 (CK1) with the clear water, cultivates the used fermentation medium of actinomycetes and is contrast 2 (CK2), and every processing repeats 10 times.Check clover seedling incidence behind the inoculation target bacterium 45d, calculate disease index and control rate.By the preventive effect analysis, from the high activity bacterial strain that sifts out again, filter out the resistant activity bacterial strain of indoor control rate 〉=75%.

State of an illness grade scale:

0 grade-healthy tree, apparent asymptomatic;

There is scab 1 grade-rhizome or main root part, but not in flakes;

There are scab and in flakes in 2 grades-main root, lateral root and rhizome portion, but are no more than 1/3;

3 grades-1/3～/ 2 rhizomes and root are infected variable color, and lateral root is infected variable color, and lateral root obviously reduces;

4 grades-rhizome and root variable color, partial decomposition, the obvious browning of vascular bundle, plant strain growth are suppressed and are short and small withered and yellow;

5 grades-butt rot, vascular bundle blackening and plant are wilted dead.

The computing formula of the incidence of disease, disease index and control efficiency:

(the contrast disease index is meant the water contrast)

When the control efficiency that calculates (%) 〉=75%, can think that this bacterial strain fermentation liquor has resisting alfalfa root rot activity preferably for test agent, this bacterial strain is the purpose bacterial strain.

As a result, obtain 1 strain to clover root rot control efficiency greater than 75% antagonism actinomycetes strain, with this bacterial strain called after NMG6-3-9.

Two, identify

(1) morphological feature is observed

Bacterial strain to be identified is made inserted sheet cultivate, carry out morphological feature and observe.Viewed aerial hyphae form is carried out optical microscopy camera, fibrillae of spores is carried out SEM take a picture.

Bacterial strain NMG6-3-9 bacterium colony gray suede powdery, cultivate observation through inserted sheet: the mycelia of NMG6-3-9 is longer, aerial hyphae multi-branched, spirality (Fig. 1).Spore circle, ellipse or long column shape, fibrillae of spores is straight, softening bent or being pine opens spirality, its stereoscan photograph such as Fig. 2, shown in Figure 3.

(2) cultural characteristic is observed

With bacterial strain to be identified, be seeded in respectively Gause I, Cha Shi, glucose-asparagine, Ke Shi-number, potato is soaked on juice, the glucose yeast cream agar slant and the potato ball medium on, place 28 ℃ of cultivations, observe its cultural characteristic and change color.Get stable ripe color characteristic as its cultural characteristic, as identifying the foundation of planting.Observe color, the color of matrix filament and the color of soluble pigment of record aerial hyphae.The result charges to the evaluation table.In addition, still should observe the upgrowth situation of bacterial strain, as the quality of growing, aerial hyphae presents how appearance-velvet-like, powdery or cotton-shaped etc. as with reference to feature.

Bacterial strain NMG6-3-9 is well-grown on most of medium, cultivation properties and characteristics such as the following table 1 of this bacterium on different medium; This bacterium be except that can producing on czapek agar medium the soluble pigment in addition, other several for examination medium on chromogenesises not.

The cultural characteristic of table 1 bacterial strain NMG6-3-9

Annotate: be as the criterion with last observed result.

(3) physio-biochemical characteristics are measured

The gelatin liquefaction ability is measured: inoculation in cylindricality gelatin culture medium surface, is cultivated down for 28 ℃, respectively 5,10,20, and 30d, each observes the degree that once liquefies.Should be before observing with the freezing 20～30min of bacterial classification pipe.Not liquefying as gelatin is still solid state, is liquefaction if present liquid.

Milk solidifies and peptonize mensuration: with inoculation to be determined in skim milk, 28 ℃ of cultivations, respectively the 3rd, 6,10,20, and 30d each observe once.

The starch hydrolysis is measured: after the medium fusing, be cooled to 50 ℃ and fall dull and stereotyped, after solidifying with the bacterial classification dibbling on flat board, thermophilic is cultivated 2～4d, drip road Ge Shi iodine liquid after forming lawn on flat board, be advisable to be paved with periphery of bacterial colonies, it is blue that flat board is, and periphery of bacterial colonies has or not transparent circle to occur illustrating whether starch is hydrolyzed, and the size of transparent circle can illustrate the size of hydrolyzed starch ability.

Cellulose hydrolysis test: preparation is fit to the actinomycetes growth and the synthetic basic culture solution of carbonaceous sources not, behind the packing test tube, make cellulose (carbon source) with filter paper of Xinhua, it is cut into wide 1cm, long 6cm filter paper bar, add in the test tube, half is immersed in the liquid, and half is exposed at outside the liquid, to identify inoculation outside liquid on one section filter paper bar, and put 28 ℃ and observe after cultivating 30d.If this bacterium can be resolved into the filter paper bar loose fibres, or makes it to fracture, it is positive to be fragmented into matter shape person, illustrates that this bacterial strain produces cellulase and makes it hydrolysis.Otherwise the person is negative for the filter paper no change.

Hydrogen sulphide produces test: inoculation to be measured to the organic inclined-plane that contains ironic citrate, is put 28 ℃ and cultivated (looking the lawn growth and maturity is advisable) observed result about 10d.If occur the pitchy precipitation in the medium, the expression experiment is positive.Otherwise nondiscolouring person is negative.

Utilization of carbon source test: with 1～2 of the spore suspension of bacterial strain to be identified, be seeded on the no carbon source medium, be coated with evenly with aseptic spreading rod, the ditching of ruling then is the boundary with the ditch, and plating medium is divided into some sub-districts, and every sub-district adds a kind of carbon source respectively.Carbon source commonly used has 9 kinds: arabinose, galactose, rhamnose, wood sugar, fructose, sucrose, sorbose, mannitol and inositol etc.Every ware need be established the sugar-free check plot.After putting 28 ℃ of cultivations 7,14d, observe the record growth respectively and utilize situation.

Result of study sees Table 2: bacterial strain NMG6-3-9 energy liquefy gelatin, and milk is solidified, it is peptonized, a little less than the starch hydrolysis, do not utilize cellulose, nitrate reduction does not produce tyrosinase, H ₂S.Except that glucose and inositol are utilized relatively poor, can both well utilize arabinose, fructose, sucrose, rhamnose, wood sugar, mannitol and galactose.

The physiological and biochemical property of table 2 bacterial strain NMG6-3-9

Annotate: "-" expression unreacted, "+", " ++ ", " +++" be the power of expression reaction respectively.

(4) evaluation of cell wall chemical constituent

Paper chromatography cell wall amino acid analysis: according to G ⁺The kind of the 3rd amino acids in the bacteria cell wall peptide glycan molecule is divided into nine monoids (seeing Table 3) with actinomycetes.

The main type of table 3 actinomycetes cell wall

Annotate: LL-DAP: racemic diaminourea acidic group pimelic acid; Meso-DAP: meso diaminopimelic acid; DAB:1,4-dihydroxy butyric acid.

Key step is:

(1) thalline preparation: scrape and get the centrifuge tube that cultured bacterial strain is put into 1.5mL;

(2) thalline hydrolysis: the HCl 100 μ L at the thalline adding 6mol/L that is used for full cell amino acid analysis, put into 120 ℃ of autoclaves, 15min;

(3) point sample: get hydrolyzate point sample on No. 1 filter paper of Xinhua that 4 μ L are used for amino acid analysis, get standard amino acid sample diaminopimelic acid DAP (containing LL-DAP, Meso-DAP and DD-DAP), lysine, ornithine, aspartic acid, glycine, glutamic acid, each 1 μ L of alanine interval point sample on filter paper successively more respectively.

(4) exhibition layer: the exhibition layer system that is used for the analysis of full cell amino acid hydrolyticsolution is a methyl alcohol: water: HCl (6N): pyridine=16: 5.2: 0.8: 2, and exhibition layer 2 times;

(5) colour developing: amino acid analysis is with 0.4% ninhydrine acetone soln, 100～110 ℃ of heating 2～3min, observation.

Paper chromatography cell wall sugar is analyzed: chemical constituent and full cell hydrolyzate sugar type according to the actinomycetes cell wall are divided into four sugared types (seeing Table 4) with actinomycetes.

Key step is

(1) the thalline preparation is scraped and is got the centrifuge tube that cultured bacterial strain is put into 1.5mL;

(2) thalline hydrolysis: add the HCl 100 μ L of 0.25mol/L in the thalline, put into 120 ℃ of autoclaves, 15min;

(3) point sample: get hydrolyzate point sample on No. 1 filter paper of Xinhua that 4 μ L are used for amino acid analysis, get the standard sugar sample more respectively: wood sugar, arabinose, galactose, glucose, rhamnose, mannose, each 1 μ L of ribose be interval point sample on filter paper successively.

(4) exhibition layer: the exhibition layer system that is used for the analysis of full cell amino acid hydrolyticsolution is a n-butanol: water: pyridine: methyl alcohol=10: 6: 6: 1, and exhibition layer 2 times;

(5) colour developing: developer: phthalic acid: aniline: water saturated n-butanol (3.25: 2: 100), 120 ℃ of heating 3min.

Bacterial strain NMG6-3-9 is carried out paper chromatography cell wall amino acid analysis, result such as table 5:

The full cell amino acid analysis of table 5 bacterial strain NMG6-3-9 result

Annotate: "-" expression unreacted, "+" expression reaction.

Bacterial strain NMG6-3-9 is carried out the analysis of paper layer chromatography cell wall sugar, result such as table 6:

The full cell wall sugar of table 6 bacterial strain NMG6-3-9

Annotate: "-" expression unreacted, "+" expression reaction.

Consolidated statement 5, table 6 result be as can be known: containing amino acid whose kind in the bacterial strain NMG6-3-9 cell wall is: LL-DAP (LL-diaminopimelic acid), glycine (GLY) and glutamic acid (GLU), in the contrast literary composition table 3 as can be known this strain cell wall belong to the I type; Except that containing glucose, do not contain other sugared kind in the table 4 in the cell wall, atypism sugar, this bacterial strain sugar type is the C type, meets the chemical classification characteristic of streptomycete Streptomyces.

(5) 16S rDNA sequence analysis

The preparation of DNA lamina membranacea:

DNA extraction:

Biological and ecological methods to prevent plant disease, pests, and erosion actinomyces strain NMG6-3-9 behind the purifying is inoculated in the Gause I culture dish, and 28 ℃ are cultured to the mid-term of growing, standby.

The key step of extracting is as follows:

(1) scrapes the thalline that takes a morsel in the ware, add 60 μ L2 * CATB buffer solution, be ground to slurries in the ice bath mortar, move into (every bacterium is cooked 3 pipes) in the 1.5mL centrifuge tube.

(2) add 500 μ L lysozyme treatment fluids (sucrose 0.3M, Tris-HCl 1M, pH 8.0 for lysozyme 2mg/mL, RNase solution 50 μ g/mL) and place 37 ℃ of insulation 2h.

(3) add 250 μ L20%SDS solution, concussion mixes.

(4) 55 ℃ of insulation 60min.

(5) add isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1) fully shakes up, 4 ℃ of Tris-HCl 1M, pH 8.0 times centrifugal (8000rpm, 5min).

(6) get supernatant and move in another centrifuge tube, add 10%NaAC liquid, 2.5 times of freezing precipitation with alcohols of volume.

(7) 4 ℃ centrifugal, and (10,000rpm 5min), abandons supernatant.

(8) repeating step (5)～(7) is 1 time.

(9) wash 2～3 times with 70% ethanol, dry, add TE buffer solution 60 μ L, fully get 3 μ L after the dissolving and detect also-20 ℃ preservation down.

Pcr amplification 16S rDNA:

(1) PCR instrument: model: ALD-1244, rev, C.B

(2) amplimer: 16S rDNA universal primer (50 μ M) forward Pf is 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (corresponding to E.coli8～27 bit bases), oppositely Pr is 5 '-TACGGCTACCTTGTTACGACTT-3 ' (corresponding to E.coli1492～1514 bit bases), and is synthetic by Institute of Micro-biology of Chinese Academy of Sciences gene engineering center.

(3) amplification system:

Table 716S rDNA pcr amplification system

Annotate: 25 μ L systems

Pcr amplification parameter and program:

Add each component successively by amplification system (table 7), instantaneous centrifugal mixing, instantaneous centrifugal mixing behind the adding Taq enzyme.95 ℃ of pre-sex change 5min enter circulation: 94 ℃ of sex change 1min, and 50 ℃ of annealing 1min, 72 ℃ are extended 2min.After 35 circulations, 72 ℃ are extended 10min.Amplified production-20 ℃ storage.

The electrophoretic examinations of pcr amplification product:

Deposition condition is 0.8% Ago-Gel (containing EB 0.5 μ g/mL), 1 * TAE electrophoretic buffer, and 80V voltage electrophoresis 40min, PCR product applied sample amount are 4 μ L, with point sample behind the 2 μ L sample-loading buffer mixings.

Observed result under the 254nm ultraviolet is with the DNA Marker[DL2000 of TaKaRa company] be nucleic acid standard molecular weight object of reference, determine expanding fragment length.The amplified production band should be on position, the reference material 1500bp left and right sides.

16S rDNA PCR product complete sequence determination:

The purifying of PCR product and sequencing are undertaken by Shanghai Ying Jun Bioisystech Co., Ltd.During order-checking with amplimer as forward and reverse primer, by Shanghai Ying Jun Bioisystech Co., Ltd with calculating power traction thing design software, primer and synthetic in the middle of designing according to the sequence search of measured sample.

The structure of systematic evolution tree:

The procaryotic 16S rDNA sequence of having measured in the 16S rDNA sequence surveyed and the GenBank gene pool is compared, access 16S rDNA sequence with the higher bacterial strain of its sequence homology.Adopt CLUSTAL X 1.8 softwares that the homologous sequence of being measured is carried out many couplings and arrange, carry out the structure of systematic evolution tree by Treeview software.The bacterial strain higher with bacterial strain NMG6-3-9 sequence homology sees Table 8.Further adopt the Protocols in Molecular Biology means that bacterial strain NMG6-3-9 is carried out Molecular Identification, analyze through 16S rDNA gene sequencing, measure 1433 effective bases altogether, shown in sequence table 1, the systematic evolution tree of structure as shown in Figure 4.

The bacterial strain higher with bacterial strain NMG6-3-9 sequence homology sees Table 8.As can be seen from Table 8, reach 99% bacterial strain that is streptomyces, can learn that bacterial strain NMG6-3-9 belongs to streptomyces with the strains tested similitude.Find that by the 16SrDNA sequence analysis bacterial strain NMG6-3-9 and grey yellow streptomycete S.griseoflavus affiliation are closer, autoploidy has reached 99.90%, and in the independent branch of systematic evolution tree that coexists.

Table 8 constructing system is grown the used Streptomyces 16S rDNA GenBanK number of landing of tree

To sum up, by morphological feature, cultural characteristic, physio-biochemical characteristics and cell wall constituent analysis, and binding molecule Biology identification result, at last bacterial strain NMG6-3-9 is decided to be streptomyces ash yellow streptomycete (Streptomycesgriseoflavus).

This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 12nd, 2009 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3441.The classification called after ash yellow streptomycete (Streptomyces griseoflavus) of this bacterial strain, strain name is NMG6-3-9.

Four, the cultivation of bacterial strain NMG6-3-9

No. 1 synthetic medium of slant medium: Gao Shi.Agar 15g, soluble starch 20g, NaCl 0.5g, KNO ₃1g, K ₂HPO ₄3H ₂O 0.5g, MgSO ₄7H ₂O 0.5g, FeSO ₄7H ₂O 0.01g, distilled water 1000mL, pH 7.3.

Seed culture medium: yeast extract 4g, glucose 10g, peptone 3g, beef extract 3g, CaCO ₃4g, distilled water 1000mL, pH 7.2-7.4.

Fermentation medium: glucose 5g, glycerine 5g, soluble starch 5g, peptone 5g, yeast extract 5g, analysis for soybean powder 5g, KH ₂PO ₄0.5g, CaCO ₃0.5g, NaCl 0.5g, MgSO ₄0.5g, distilled water 1000mL, pH 7.0-7.2.

Soluble starch is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is HX169; Peptone is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is SH0010; Yeast extract is available from the favour commerce and trade Co., Ltd of Inner Mongol letter, and catalog number is SH0348; Analysis for soybean powder is available from the supermarket.

1) slant culture: bacterial strain NMG6-3-9 is inoculated on No. 1 inclined-plane of Gao Shi, cultivates 5-6d at 28 ℃;

2) seed culture: be inoculated in the 100mL seed culture medium from the good actinomycetes inclined-plane of growing, 220r/min, 28 ℃ of shaken cultivation 48h obtain seed culture fluid;

3) fermented and cultured: the inoculum concentration by 10% (v/v) is inoculated in seed culture fluid in the 100mL fermentation medium, and 220r/min, 28 ℃ of shaken cultivation 120h obtain zymotic fluid.

The application of embodiment 2, bacterial strain NMG6-3-9

One, the antimicrobial spectrum of bacterial strain NMG6-3-9 is measured:

PDA agar medium: potato (peeling) 200g, sucrose (or glucose) 20g, distilled water 1000mL, pH nature.

Actinomycetes strain NMG6-3-9 is inoculated on No. 1 agar plate of Gao Shi in 28 ℃ cultivates 5d down, obtain lawn; Lawn is broken into the bacterium cake of diameter 5mm with card punch, and the bacterium cake placed the dull and stereotyped central authorities of PDA, make the one side that has mycelia be attached to media surface, cultivate 3d down for 30 ℃, onesize target bacterium bacterium cake face-off is connected to around the bacterial strain NMG6-3-9 then, cultivate down for 28 ℃, cover with culture dish to the target bacterium till.Adopt the right-angled intersection method to measure the diameter of antibacterial (bacteriolyze) circle.

Above-mentioned bacteriostatic test plate adopts conventional flat board face-off method, Oxford agar diffusion method, cylinder-plate method, dig piece method, agar block method or filter paper method etc. all can.

Every kind of target bacterium is repeated results averaged 3 times.

The result shows, bacterial strain NMG6-3-9 has in various degree inhibitory action (table 9) to 20 kinds of dissimilar plant pathogenic fungis, and this bacterial strain also has certain inhibition effect (table 10) to 6 kinds of bacteriums.The antimicrobial spectrum of proof bacterial strain NMG6-3-9 is wider, and the variety classes pathogen is had selectivity.

Table 9 bacterial strain NMG6-3-9 is to the fungistatic effect of 20 plant species disease funguses

The target bacterium	Antibacterial circle diameter (mm)	The target bacterium	Antibacterial circle diameter (mm)
				Clover eggplant fusarium Fusarium solani	??32.68	Melon shell two spore Ascochyta citrullina	??21.38
Clover point sickle spore bacterium F.oxysporum	??30.54	A big beautiful spore Verticillium dahlia that takes turns	??20.88
				Oat sickle spore bacterium F. avenaceum	??29.24	Gaeumannomyce Gaeumannomyces graminis	??20.36
The clover mould Stemphylium botryosum of handle that crawls	??28.60	Colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides	??19.97
				Rhizoctonia solani Kuhn Rhizoctonia solani	??27.96	Standing grain is bent spore mould Curvularia lunata	??19.24
Fusarium graminearum Peronospora aestivalis	??27.45	The prominent navel spore Exserohilumleonard turcicum of big spot	??17.65
				Fusarium semitectum F.semitectum	??25.48	Phytophthora blight of pepper Phytophthora capsici	??17.08
Banana Fusarium oxysporum F. oxysporum f.sp.cubense	??24.69	Botrytis cinerea Botrytis cinerea	??16.57
				The rotten mould Pythium aphanidermatum of melon and fruit	??23.75	Sclerotinite Sclerotinia sclerotiorum	??15.43
Clover septoria musiva Septoria medicaginis	??22.95	Pink poly-end spore Trichothecium roseum	??11.05

Table 10 bacterial strain NMG6-3-9 is to the fungistatic effect of 6 kinds of bacteriums

The target bacterium	Antibacterial circle diameter (mm)	The target bacterium	Antibacterial circle diameter (mm)
				Pseudomonas syringae Pseudomonas syringae	??23.12	Blue or green withered Lei Er Salmonella Ralstonia solanacearum	??18.56
Carrot soft rot Erwinia	??21.96	Oat acidophil watermelon subspecies	??16.70
				??Erwinia?carotovora?var.??carotovora		??Acidovorax?avenae?subsp.??citrulli
Clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae	??20.18	Staphylococcus aureus Staphylococcus aureaus	??12.85

Following bacterial strain in the table 9 and 10 is all available from Chinese agriculture microorganism fungus kind preservation administrative center (being called for short ACCC): oat sickle spore bacterium Fusarium avenaceum ACCC 30065, Rhizoctonia solani Kuhn Rhizoctonia solaniACCC 30332, Fusarium graminearum Peronospora aestivalis ACCC 30068, fusarium semitectum Fusarium semitectum ACCC 31945, banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense ACCC 36369, the rotten mould Pythium aphanidermatum ACCC 36125 of melon and fruit, melon shell two spore Ascochyta citrullina ACCC 36440, a big beautiful spore Verticillium dahliaACCC 36109 that takes turns, gaeumannomyce Gaeumannomyces graminis ACCC 30310, standing grain is bent spore mould Curvularia lunata ACCC 36580, phytophthora blight of pepper Phytophthora capsici ACCC36279, Botrytis cinerea Botrytis cinerea ACCC 30091, sclerotinite Sclerotiniasclerotiorum ACCC 30096, pink poly-end spore Trichothecium roseum ACCC 36459, carrot soft rot Erwinia Erwinia carotovora var.carotovora ACCC 01443, blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC 01470, staphylococcus aureus Staphylococcusaureaus ACCC 01336.

Clover septoria musiva Septoria medicaginis (Hou Tianjue. northern China meadow disease survey and main disease control [J]. Chinese meadow, 1993,3:56-60.) (grassland research institute, China Agriculture academy of sciences)

Colletotrichum gloeosporioides Penz Colletotrichum gloeosporioides (Liu Zhengping, Hu Jun, the Gao Xiang, etc. matrimony vine anthrax bacteria biological characteristic research [J]. Beijing Agricultural College's journal, 2005,20 (3): 36-39.) (grassland research institute, China Agriculture academy of sciences)

The prominent navel spore Exserohilumleonard turcicum of big spot (Zhou Hongyou, Liu Zhengping, Hu Jun, etc. the biological characteristic research [J] of Sudan grass leaf blight bacterium. North China agronomy newspaper, 2009,24 (1): 174-177.) (grassland research institute, China Agriculture academy of sciences)

Pseudomonas syringae Pseudomonas syringae (Hou Tianjue. northern China meadow disease survey and main disease control [J]. Chinese meadow, 1993,3:56-60.) (grassland research institute, China Agriculture academy of sciences)

Clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae (day rank of nobility. China's clover disease generation present situation and Preventing Countermeasures [J]. Inner Mongol grass cultivation, 1994,3:4-8.) (grassland research institute, China Agriculture academy of sciences)

Oat acidophil watermelon subspecies Acidovorax avenae subsp.Citrulli (Hu Jun, HUANG Junxia, Liu Shuanping, Deng. different Hami melon kinds are to the research [J] of bacillary fruit blotch disease resistance and development trend. North China agronomy newspaper, 2006,21 (6): 107-110.) (grassland research institute, China Agriculture academy of sciences)

Clover eggplant fusarium Fusarium solani (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (6): 105-107.) (grassland research institute, China Agriculture academy of sciences)

Clover point sickle spore bacterium Fusarium oxysporum (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (6): 105-107.) (grassland research institute, China Agriculture academy of sciences)

Clover crawl the mould Stemphylium botryosum of handle (Hou Tianjue. northern China meadow disease survey and main disease control [J]. Chinese meadow, 1993,3:56-60.) (grassland research institute, China Agriculture academy of sciences)

Two, the sprouting to target bacterium spore suppresses

1. the preparation of clover pine root fungus spore suspension

The PDA medium: potato 200g, glucose 20g, agar 15g, distilled water 1000mL, pH 7.0.

(Fusarium solani) is inoculated in the PDA medium with the clover eggplant fusarium, in 28 ℃ of constant incubators, cultivate 5-6d, treat to use sterile water flush away aerial hyphae after mycelia is covered with medium, the irradiation of 400nto fluorescent lamp is cultivated down and is produced spore, after treating that 2-3d produces spore, wash spore with sterile water, remove mycelia and add 0.02%Tween-20 through biofilter aseptic filtration and make spore suspension and preserve down standbyly at 4 ℃, concentration is 10 ⁶Cfu/ml.

2. the actinomycetic preparation of antagonism

(1) preparation of bacterial strain fermentation liquor

Bacterial strain NMG6-3-9 is inserted fermentation medium, and 28 ℃ of following shaken cultivation 4-5d with zymotic fluid centrifuging and taking supernatant, with supernatant liquid filtering, collect filtrate, and the filtrate note is made bacterial strain NMG6-3-9 ferment filtrate, and the spore concentration in the bacterial strain NMG6-3-9 ferment filtrate is 10 ⁸Cfu/mL.

3. the actinomycetic screening of antagonism

The spore suspension equal-volume that makes in ferment filtrate and the above-mentioned experiment 1 is mixed, get an above-mentioned mixed liquor and drip on the cover glass of handling with collodion on the surface 5 repetitions of every processing.Under 25 ℃, preserve moisture and cultivate the sprouting of checking clover eggplant fusarium (Fusarium solani) spore in the blank behind the 6h, after the germination rate of clover eggplant fusarium in the blank (Fusarium solani) spore reaches 85%, check the spore germination rate (is sprouting greater than half person of spore length with spore germ tube length) of all processing clover eggplant fusariums (Fusarium solani).Test repeats 5 times.

Calculate the spore germination inhibiting rate as follows:

The blank group of methods: method is consistent with experimental group, just zymotic fluid is replaced with sterile water.

3 repetitions are established in experiment.

The result: control group, 85% spore germination is arranged, form elongated and uniform mycelia; Experimental group: bacterial strain NMG6-3-9 reaches 100% to the spore germination inhibiting rate, and spore germination speed is reduced, and makes the conidium cell and sprout the germ tube deformity that, and then can reduce the ability that it infects the host.

Three, bacterial strain NMG6-3-9 is to the biological control of plant disease

Adopt and irritate the root inoculation method indoor seedling stage NMG6-3-9 is prevented and treated clover root rot test.The clover root rot is caused by clover eggplant fusarium Fusarium solani.

Clover eggplant fusarium Fusarium solani (Cao Lixia, Zhao Cunhu, Bai Quanjiang, etc. middle of Inner Mongolia area clover root-rot disease pathogen research (English) [J]. North China agronomy newspaper, 2008,23 (06): 105-107.) (grassland research institute, China Agriculture academy of sciences).

Clover (No. 1, middle lucerne) seed is available from national germplasm herbage storehouse in mid-term, and catalog number is 00068.Jinggangmycin is available from Agricultural Materials shop; Czapek ' s culture fluid (sucrose 30g, NaNO ₃2g, KCl 0.5g, K ₂HPO ₄3H ₂O 1g, MgSO ₄7H ₂O 0.5g, FeSO ₄7H ₂O 0.01g, distilled water 1000mL, the pH nature.

The preparation of target bacterium spore suspension: pathogen is used 28 ℃ of Czapek ' s culture fluids, and 120r/min shake-flask culture 96h obtains the spore suspension of pathogen, calculates spore suspension concentration with blood counting chamber, and spore suspension miospore concentration is 10 ⁶Cfu/mL.

The preparation of bacterial strain NMG6-3-9 ferment filtrate: bacterial strain NMG6-3-9 is inserted fermentation medium, 28 ℃ of following shaken cultivation 4-5d, with zymotic fluid centrifuging and taking supernatant, with supernatant liquid filtering, collect filtrate, the filtrate note is made bacterial strain NMG6-3-9 ferment filtrate, and the spore concentration in the bacterial strain NMG6-3-9 ferment filtrate is 10 ⁸Cfu/mL.

For trying soil sample: soil sterilization back is stand-by naturally.Soil after the sterilization is sub-packed in (high 15cm, diameter 8cm) in the different seedling-raising cup, and every cup native 200g that goes into to sterilize is placed in the higher enamel tray in edge.Sowing is a few days ago fully watered along the basin edge, and it is absorbed water naturally.

Seedling management: alfalfa seed is with 0.1% bromogeramine sterilization 3min, and is clean with flushing with clean water, puts into 28-30 ℃ of incubator, sows when treating the long 0.5cm of its show money or valuables one carries unintentionally back or bud.Interior in the controlled environment chamber growing seedlings, day temperature remain on 24-26 ℃, and at 15-18 ℃ of night, soil moisture remains on 60%-80%.

Inoculation method: after treating clover growth of seedling 30d (will sow the same day note do the 0th day), with the ferment filtrate (10 of bacterial strain NMG6-3-9 ⁸Cfu/mL) irritate in the seedling root every strain 10mL; Behind the 3d equally with pathogen suspension (10 ⁶Cfu/mL) irritate root inoculation, every strain 10mL.With the plant of 5% jinggangmeisu aqua (concentration is 50 μ g/mL) root irrigation be the agricultural chemicals contrast, with the plant of fermentation medium and clear water root irrigation as blank.Per 20 seedling-raising cup are a processing, every glass 4 strain, and every processing repeats for 3 times.The periodic investigation incidence, statistics disease index and relative control efficiency behind the 45d.The disease classification is carried out with reference to following standard:

State of an illness grade scale:

0 grade-healthy tree, apparent asymptomatic;

There is scab 1 grade-rhizome or main root part, but not in flakes;

There are scab and in flakes in 2 grades-main root, lateral root and rhizome portion, but are no more than 1/3;

Variable color is infected by 3 grades-1/3～1/2 root and rhizome portions, and lateral root obviously reduces;

The variable color of 4 grades-root and rhizome portion, partial decomposition, the obvious browning of vascular bundle, plant strain growth are suppressed and are short and small withered and yellow;

5 grades-butt rot, vascular bundle blackening and plant are wilted dead.

Contrast all refers to clear water in the aforementioned calculation formula.

At different levels to represent a level value be 0,1,2,3,4,5, and the highest grade value represented is 5.

Result of the test (table 11) shows, can reduce the incidence of disease and the disease index of clover root rot for the zymotic fluid of examination antagonism bacterium NMG6-3-9, disease index is 16.35, and relative control effect reaches 81.50%, is higher than the processing preventive effect 60.78% of CK3 (5% jinggangmycin aqua).In addition from table the result as can be seen CK2 (fermentation medium) though disease index low than clear water contrast, but difference is very little, though its morbidity to plant is influential but is not the principal element of diseases prevention, and the principal element of diseases prevention is the metabolite of antagonism bacterium NMG6-3-9.In pot experiment, the antagonism bacterium can effectively alleviate the state of an illness, demonstrates comparatively desirable application potential.

Table 11 antagonism bacterium NMG6-3-9 is to the potted plant protection effect of clover root rot

Handle	The incidence of disease (%)	Disease index	Control efficiency (%)
				CK1 (clear water)	??93.56	??88.39	??-
CK2 (fermentation medium)	??76.76	??85.70	??3.03％
				CK3 (5% jinggangmeisu aqua)	??53.34	??34.67	??60.78％
??NMG6-3-9	??39.68	??16.35	??81.50％

Sequence table

＜110〉grassland research institute, China Agriculture academy of sciences

 

＜120〉application of grey yellow streptomycete in the biological control plant disease

 

<160>1

 

<210>1

 

<211>1433

 

<212>DNA

 

＜213〉grey yellow streptomycete (Streptomyces griseoflavus)

 

<400>1

tgctgcgggt?gcttacacat?gcaagtcgaa?cgatgaacca?cttcggtggg?gattagtggc?????60

gaacgggtga?gtaacacgtg?ggcaatctgc?cctgcactct?gggacaagcc?ctggaaacgg????120

ggtctaatac?cggatactga?tccgcttggg?catccaggcg?gttcgaaagc?tccggcggtg????180

caggatgagc?ccgcggccta?tcagcttgtt?ggtgaggtag?tggctcacca?aggcgacgac????240

gggtagccgg?cctgagaggg?cgaccggcca?cactgggact?gagacacggc?ccagactcct????300

acgggaggca?gcagtgggga?atattgcaca?atgggcgaaa?gcctgatgca?gcgacgccgc????360

gtgagggatg?acggccttcg?ggttgtaaac?ctctttcagc?agggaagaag?cgaaagtgac????420

ggtacctgca?gaagaagcgc?cggctaacta?cgtgccagca?gccgcggtaa?tacgtagggc????480

gcaagcgttg?tccgggaatt?attgggcgta?aagagctcgt?aggcggcttg?tcacgtcggt????540

tgtgaaagcc?cggggcttaa?ccccgggtct?gcagtcgata?cgggcaggct?agagttcggt????600

aggggagatc?ggaattcctg?gtgtagcggt?gaaatgcgca?gatatcagga?ggaacaccgg????660

tggcgaaggc?ggatctctgg?gccgatactg?acgctgagga?gcgaaagcgt?ggggagcgaa????720

caggattaga?taccctggta?gtccacgccg?taaacggtgg?gcactaggtg?tgggcaacat????780

tccacgttgt?ccgtgccgca?gctaacgcat?taagtgcccc?gcctggggag?tacggccgca????840

aggctaaaac?tcaaaggaat?tgacgggggc?ccgcacaagc?ggcggagcat?gtggcttaat????900

tcgacgcaac?gcgaagaacc?ttaccaaggc?ttgacataca?ccggaaagca?ttagagatag????960

tgcccccctt?gtggtcggtg?tacaggtggt?gcatggctgt?cgtcagctcg?tgtcgtgaga???1020

tgttgggtta?agtcccgcaa?cgagcgcaac?ccttgtcccg?tgttgccagc?aagcccttcg???1080

gggtgttggg?gactcacggg?agaccgccgg?ggtcaactcg?gaggaaggtg?gggacgacgt???1140

caagtcatca?tgccccttat?gtcttgggct?gcacacgtgc?tacaatggcc?ggtacaatga???1200

gctgcgatac?cgcgaggtgg?agcgaatctc?aaaaagccgg?tctcagttcg?gattggggtc???1260

tgcaactcga?ccccatgaag?tcggagtcgc?tagtaatcgc?agatcagcat?tgctgcggtg???1320

aatacgttcc?cgggccttgt?acacaccgcc?cgtcacgtca?cgaaagtcgg?taacacccga???1380

agccggtggc?ccaacccctt?gtgggaggga?gctgtcgaag?gtgacgcgat?ttc??????????1433

Claims (10)

1. the application of grey yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9 CGMCC No.3441 in the biological control plant disease.

2. application according to claim 1 is characterized in that: described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, banana blight, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, capsicum epidemic disease, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

3. application according to claim 2; it is characterized in that: described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusariumavenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusarium semitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoriamedicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomyces graminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichumgloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotiniasclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichothecium roseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Erwinia carotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

4. microbial inoculum that is used for the biological control plant disease, its active component is the zymotic fluid that grey yellow streptomycete (Streptomycesgriseoflavus) the NMG6-3-9 CGMCC No.3441 or grey yellow streptomycete (Streptomycesgriseoflavus) the NMG6-3-9 CGMCC No.3441 that ferments obtain.

5. microbial inoculum according to claim 4 is characterized in that: described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

6. according to claim 4 or 5 described microbial inoculums; it is characterized in that: described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusariumavenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusarium semitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoriamedicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomyces graminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichumgloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotiniasclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichothecium roseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Erwinia carotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

7. grey yellow streptomycete (Streptomyces griseoflavus) NMG6-3-9 CGMCC No.3441 is used for the application of the microbial inoculum of biological control plant disease in preparation.

8. application according to claim 7 is characterized in that: described plant disease is the clover root rot, the clover mould leaf spot of handle of crawling, clover Rhizoctonia solani Kuhn fusarium wilt, dogstail sickle spore fusarium wilt, the prairie milk vetch root rot, the rotten mould root rot of clover, clover septoria musiva leaf spot, the climing rot of watermelon, eggplant verticillium wilt, the dogstail full rot, take-all, matrimony vine anthracnose, dogstail is bent the mould leaf blight of spore, the Sudan grass leaf blight, the tomato gray mould bacterium, stem rot of cucumber, maize kernel rot, melon and fruit is adopted the back rot, the bacillary cane blight of clover, vegetable bacterial soft rot, the clover bacterial leaf spot, solanaceous crops bacterial wilt or melon bacterial fruit blotch.

9. according to claim 7 or 8 described application; it is characterized in that: described clover root rot is by clover eggplant fusarium Fusarium solani; clover point sickle spore bacterium Fusarium oxysporum or oat sickle spore bacterium Fusariumavenaceum cause; the described clover mould leaf spot of handle of crawling is caused by the clover mould Stemphyllium botryosum of handle that crawls; described clover Rhizoctonia solani Kuhn fusarium wilt is caused by Rhizoctonia solani Kuhn Rhizoctonia solani; described dogstail sickle spore fusarium wilt is caused by Fusarium graminearum Peronospora aestivalis; described prairie milk vetch root rot is caused by fusarium semitectum Fusarium semitectum; described banana blight is caused by banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense; the rotten mould root rot of described clover is caused by the rotten mould Pythium aphanidermatum of melon and fruit; described clover septoria musiva leaf spot is caused by clover septoria musiva Septoriamedicaginis; the climing rot of described watermelon is caused by melon shell two spore Ascochyta citrullina; described eggplant verticillium wilt is caused by a big beautiful spore Verticillium dahlia that takes turns; described dogstail full rot is caused by gaeumannomyce Gaeumannomyces graminis; described take-all is caused by gaeumannomyce Gaeumannomyces graminis; described matrimony vine anthracnose is caused by colletotrichum gloeosporioides Penz Colletotrichumgloeosporioides; the curved mould leaf blight of spore of described dogstail is caused by the curved mould Curvularia lunata of spore of standing grain; described Sudan grass leaf blight is caused by the prominent navel spore Exserohilumleonard turcicum of big spot; described capsicum epidemic disease is caused by phytophthora blight of pepper Phytophthora capsici; described tomato gray mould bacterium is caused by Botrytis cinerea Botrytis cinerea; described stem rot of cucumber is caused by sclerotinite Sclerotiniasclerotiorum; described maize kernel rot is caused by pink poly-end spore Trichothecium roseum; described melon and fruit is adopted the back rot and is caused by pink poly-end spore Trichothecium roseum; the bacillary cane blight of described clover is caused by clover pseudomonas syringae Pseudomonas syringae; described vegetable bacterial soft rot is caused by carrot soft rot Erwinia Frwinia carotovora var.carotovora; described clover bacterial leaf spot is caused by clover Xanthomonas campestris Xanthomonas campestris pv.alfalfae; described solanaceous crops bacterial wilt is caused that by the withered Lei Er Salmonella of green grass or young crops Ralstonia solanacearum described melon bacterial fruit blotch is caused by oat acidophil watermelon subspecies Acidovorax avenae subsp.citrulli.

10. according to arbitrary described application or microbial inoculum among the claim 1-9, it is characterized in that: described oat sickle spore bacterium Fusarium avenaceum is oat sickle spore bacterium Fusarium avenaceum ACCC 30065, described Rhizoctonia solani Kuhn Rhizoctonia solani is Rhizoctonia solani Kuhn Rhizoctonia solani ACCC 30332, described Fusarium graminearum Peronospora aestivalis is Fusarium graminearum Peronospora aestivalisACCC 30068, described fusarium semitectum Fusarium semitectum is fusarium semitectum Fusariumsemitectum ACCC 31945, described banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense is banana Fusarium oxysporum Fusarium oxysporum f.sp.cubense ACCC 36369, the rotten mould Pythium aphanidermatum of described melon and fruit is the rotten mould Pythium aphanidermatum ACCC 36125 of melon and fruit, described melon shell two spore Ascochyta citrullina are melon shell two spore Ascochyta citrullina ACCC36440, a described big beautiful spore Verticillium dahlia that takes turns is a big beautiful spore Verticillium dahliaACCC 36109 that takes turns, described gaeumannomyce Gaeumannomyces graminis is gaeumannomyce Gaeumannomycesgraminis ACCC 30310, the curved spore mould Curvularia lunata of described standing grain is the curved spore mould Curvularia lunata ACCC 36580 of standing grain, described phytophthora blight of pepper Phytophthora capsici is phytophthora blight of pepper Phytophthora capsici ACCC 36279, described Botrytis cinerea Botrytis cinerea is Botrytis cinerea Botrytis cinerea ACCC 30091, described sclerotinite Sclerotinia sclerotiorum is sclerotinite Sclerotinia sclerotiorum ACCC 30096, described pink poly-end spore Trichothecium roseum is pink poly-end spore Trichothecium roseum ACCC 36459, described carrot soft rot Erwinia Erwinia carotovora var.carotovora is carrot soft rot Erwinia Erwinia carotovora var.carotovora ACCC 01443, the withered Lei Er Salmonella of described green grass or young crops Ralstoniasolanacearum is blue or green withered Lei Er Salmonella Ralstonia solanacearum ACCC 01470, and described staphylococcus aureus Staphylococcus aureaus is staphylococcus aureus Staphylococcus aureausACCC 01336.

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