CN100566574C - Method and control agent with the bacillus controlling plant diseases - Google Patents

Abstract

Be provided for control agent or the control control material of biological control by plant pathogenetic bacteria, fungi or virogenetic plant disease; The method of using described control agent or control material controlling plant diseases is provided; Wherein employed bacterium also is provided.In other words, plant disease controlling agent or control material contain and belong to bacillus and can suppress plant pathogenetic bacteria, fungi or the infection of virus or the bacterium of its growth; Described control agent or control material are applied to the method for plant; And wherein employed bacterium.

Description

Method and control agent with the bacillus controlling plant diseases

Technical field

The present invention relates to the method for preventing and treating and the control agent or the material of bacillary, fungoid or viral plant disease.

Background technology

In the agricultural cultivation of economic crops such as vegetables, the development that plant to pass, soil passes or gas passes or water passes plant disease is usually directed to all many-sides such as the production of high-quality crop, stable output and profit.Especially face to face when being easy to the abnormal weather condition of plant disease takes place, the commercialization farm will be subjected to devastating impact.

Think that the main pathogens that this class can be propagated the virulence factor of disease is filamentous fungi, bacterium and virus.In order to realize normal cultivation, prevent and treat plant disease due to the described pathogene, this is absolutely necessary.

In order to make agricultural product effectively resist disease, chemical technology has obtained extensive use, for example the application of insecticide (for example fungicide, bactericide or antiviral agent).In recent years, synthetic organic insecticide is extensive use of because of its effect, and the existing controlling plant diseases of imitating of described insecticide causes significantly having improved the production of agricultural product.Yet disease control greatly depends on utilization of pesticides, and this is unfavorable to global environment, society and agricultural, for example residual pesticide in the soil, the residual pesticide in the food and the drug-fast increase of pathogenic microorganism.And the excessive use of insecticide is a problem to peasant's safety.

These problems had received worldwide very big concern already, as response, were attempting various measures.For example, Montreal treaty (Montreal Protocol) banned use of soil fumigant in 2005, i.e. methyl bromide, unless when pressing for, use, because it is the reason that causes ozone layer depletion.

As a replacement scheme depending on insecticide unduly, worldwide need the integrated pest management system (IPM) of foundation and natural ecological system harmony strongly.

Utilize the abilities of microorganisms such as bacterium to come the method for controlling plant diseases, promptly relate to the pest control method that uses microorganism insecticide, expection can be used as the effective ways that solve an above difficult problem.

Relevant the application's invention, for example following prior art file record is available.United States Patent (USP) the 5th, 344, No. 647; United States Patent (USP) the 5th, 061, No. 495; Day disclosure special permission communique 8-175919 number; Day disclosure special permission communique 2003-277210 number; Day disclosure special permission communique 2002-145712 number; Day disclosure special permission communique 9-124427 number; Day disclosure special permission communique 11-302120 number; United States Patent (USP) the 5th, 935, No. 571; Day disclosure special permission communique 7-267811 number; Phae, C.G., Shoda, M., Kita, N., Nakano, M. and Ushiyama, K., Ann.Phytopath.Soc.Japan, 58,329-339,1992; MasaoYamada, Biseibutsu nouyaku-kankyou hozenkata nougyou wo mezasite (Microbial pestictdes-in pursuit of environmental conservativeagriculture), Zenkoku nouson kyouiku kyoukai, the 328th page, 2000; With (writing) such as Shunichi Iwata, Shokubutsu boueki kouza-byougai-hen (Plantprotection course-disease volume, Japan Plant Protection Association, the 281st page, 1983.

Summary of the invention

The present invention relates to be used for control agent or the material of biological control, relate to and use described control agent or material, also relate to wherein employed bacterium with the method for controlling plant diseases by plant pathogenetic bacteria, fungi or virogenetic plant disease.

The present invention includes following invention.

(1) a kind of control agent or material of plant disease, described control agent or material comprise bacillus (Bacillus) bacterium that can suppress plant pathogenetic bacteria, fungi or virus infections or propagation.

(2) the control agent or the material of (1), the bacillus that wherein can suppress plant pathogenetic bacteria, fungi or virus infections or propagation are DAIJU-SIID2550 bacterial strain or its mutant.

The DAIJU-SIID2550 bacterial strain is preserved in Independent Administrative Leged Industrial Technology Complex Inst on November 20th, 2003 and speciallys permit biological sustenance center (a kind of 1 central authorities the 6th of ripple city east 1 fourth are built in the Ibaraki, Japan, postcode 305-8566) (International Patent OrganismDepositary of the National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan), preserving number: FERM BP-10114 (changed by the FERMP-19591 bacterial strain, this bacterial strain carried out domestic preservation on November 20th, 2003).This preservation thing is by the present inventor Daiju Yuki (3-10-5, SakaeNuza, Saitama, 352-0014, Japan) carry out preservation on November 20th, 2003, and promptly be given to the application's applicant Itsuki Co., Ltd. (3-12-5, Saiwai-cho before the date of application on February 7th, 2005, Asaka, Saitama, 351-0015, Japan).

(3) the control agent or the material of (1) or (2), wherein said plant pathogenetic bacteria is Agrobacterium (Agrobacterium), rod shape Bacillus (Clavibacter), Erwinia (Erwinia), pseudomonas (Pseudomonas), the logical Bordetella (Ralstonia) in Rolls, Corynebacterium (Corynebacterium), Curtobacterium (Curtobacterium), bulkholderia cepasea genus (Burkholderia) or Xanthomonas (Xanthomonas) bacterium, and described plant disease is caused by them.

(4) the control agent or the material of (1) or (2), wherein said plant pathogenic fungi are gas fax bacterium or soil-borne fungus, and described plant disease is caused by them.

(5) the control agent or the material of (1) or (2), wherein said plant-pathogenic virus is tobacco mosaic virus (Tobacco mosaic virus, TMV), the light-duty mottle virus of capsicum (Pepper mildmottle virus, PMMoV), Tomato mosaic virus (Tomato mosaic virus, ToMV), muskmelon withered spot disease poison (Melon necrotic spot virus, MNSV), cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMMV) or Kyuri green mottle mosaic virus (Kyuri green mottle mosaic virus, KGMMV), and described plant disease cause by them.

(6) a kind of method of controlling plant diseases, described method comprise each control agent or the material host plant that is applied to plant pathogenetic bacteria, fungi or virus with (1)-(5).

(7) method of (6), wherein said plant belong to Cruciferae (Brassicaceae), Solanaceae (Salanaceae), Curcurbitaceae (Cucurbitaceae), Liliaceae (Liliaceae), pulse family (Leguminosae), composite family (Asteraceae), Chenopodiaceae (Chenopodiaceae), grass family (Gramineae), the rose family (Rosaceae), Caryophyllaceae (Caryophyllaceae), Primulaceae (Primulaceae), Rutaceae (Rutaceae), Vitaceae (Vitaceae), Actinidiaceae (Actinidiaceae), Ebenaceae (Ebenaceae), Umbelliferae (Umbelliferae), Convolvulaceae (Convolvulaceac) or Araeceae (Araceae).

(8) method of (6) or (7), wherein said control agent or material are selected from following processing by at least one and are applied to plant: give the plant seed dressing with described bacterium, plant seed is immersed in the suspension that contains described bacterium, described bacterium is irrigated plant cultivation with in the soil, described bacterium is incorporated into plant cultivation with in the soil, described bacterium is sprayed on the plant stem-leaf, and makes described bacterium contact plant damage.

(9) a kind of new Bacillus strain, i.e. DAIJU-SIID2550 (preserving number: FERM BP-10114).

According to the present invention, can carry out biological control to plant disease due to plant pathogenetic bacteria, fungi or virus infections or the propagation.

This specification comprises the specification and/or the disclosed part or all of content of accompanying drawing of Japanese patent application 2004-55059 number and 2004-216083 number, and described document is the application's a priority document.

The accompanying drawing summary

Fig. 1 shows that the BAL bacterium is to pathogenic mutation (Xanthomonas campestris pv.campestris) the inhibition of proliferation effect of xanthomonas campestris sarson.

When Fig. 2 is presented at a variety of Chinese cabbage seed germination of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson, the onset state of the pathogenic mutation of xanthomonas campestris sarson.

Fig. 3 is presented near the onset state of the pathogenic mutation of the xanthomonas campestris sarson of seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson, and these seeds were handled with the control agent that contains the BAL bacterium.

Fig. 4 shows that the experiment of embodiment 8 begins back 9 days sample appearance.

Fig. 5 shows that the experiment of embodiment 8 begins back 9 days sample appearance.

Fig. 6 shows that the experiment of embodiment 9 begins back 26 days sample appearance.

Fig. 7 A shows the influence that bactericide and insecticide are bred the BAL bacterium.

Fig. 7 B shows the influence (continuous Fig. 7 A) that bactericide and insecticide are bred the BAL bacterium.

Fig. 8 shows the effect that suppresses ulcer bacteria propagation by the BAL bacterium.

Fig. 9 shows the effect that suppresses wilting germ propagation by the BAL bacterium.

Figure 10 shows the effect that suppresses Bacteria erwinia propagation by the BAL bacterium.

Figure 11 shows that the experiment of embodiment 14 begins back 1 day sample appearance.

Figure 12 A shows the back 16 days sample appearance of rice seedling damping off experiment beginning of embodiment 15.

Figure 12 B shows the back 16 days sample appearance of paddy rice bacterium Maize Ear Rot experiment beginning of embodiment 15.

Figure 13 shows that the experiment of embodiment 16 begins back 24 hours sample appearance.Multiplication factor in the bracket shows the final dilution gfactor when sprouting test.

Figure 14 shows that the experiment of embodiment 17 begins back 24 hours sample appearance.

Figure 15 shows that the BAL bacteria culture fluid is to the effect of gray mold among the embodiment 19.

Figure 16 shows that the experiment of embodiment 20 begins back 20 days sample appearance.

Figure 17 shows that the BAL bacteria culture fluid is to the effect of black spot among the embodiment 21.

Figure 18 shows that the BAL bacteria culture fluid is to the effect of white blister among the embodiment 22.

Figure 19 shows that the experiment of embodiment 23 begins back 18 days sample appearance.

Figure 20 shows that the experiment of embodiment 24 begins back 11 days sample appearance.

Figure 21 shows that the BAL bacteria culture fluid is to the effect of TMV among the embodiment 25.

Figure 22 shows that the BAL bacteria culture fluid is to the effect of muskmelon withered spot disease among the embodiment 26.

The preferred embodiments of the invention

The present invention relates to the method for controlling plant diseases, described method comprises that the bacillus that can suppress plant pathogenetic bacteria, fungi or virus infections or propagation is applied to the plant as plant pathogenetic bacteria, fungi or virus host.

The present invention also relates to the control agent or the material of plant disease, described control agent or material comprise can suppress plant pathogenetic bacteria, fungi or the infection of virus or the bacillus of propagation.

In addition, the bacillus that the present invention relates to suppress plant pathogenetic bacteria, fungi or virus infections or propagation is used for the control agent of plant disease due to bacterium, fungi or the virus or the purposes of material in preparation.

The invention provides the method and control agent and the control material that are used for plant disease due to biological control plant pathogenetic bacteria, fungi or the virus.Up to now, developed the Several Methods (referring to the described file of above " background technology " part) of using the bacillus controlling plant diseases; Yet these methods and invention disclosed in this invention are all inequality.

The bacterial plant disease that the present invention prevented and treated is any by the plant disease due to the bacterial pathogens.The example comprises by with the plant disease due to subordinate's the bacterium: Agrobacterium, rod shape Bacillus, Erwinia, pseudomonas, the logical Bordetella in Rolls, Corynebacterium, Curtobacterium, bulkholderia cepasea genus, Xanthomonas, bacillus radicicola belong to (Rhizobacter) and Clostridium (Clostridium).Control agent of the present invention or material are to particularly useful with plant disease due to subordinate's the bacterium: Agrobacterium, rod shape Bacillus, Erwinia, pseudomonas, the logical Bordetella in Rolls, Corynebacterium, Curtobacterium, bulkholderia cepasea belong to or Xanthomonas.

Known is a kind of destructive disease that crucifer produces that influences by the black rot due to the pathogenic mutation of xanthomonas campestris sarson.As a kind of disease of danger, the control of black rot is comprising that the country of black rot did not take place the national Buddhist monk that black rot took place and the whole world of Japan receives very big concern.Up to now, in fact also do not ratify to resist the plant variety of black rot, also do not develop effective insecticide.According to the present invention, can effectively prevent and treat for example black rot of following crucifer: wild cabbage, Chinese cabbage, radish, blue and white cabbage, cauliflower, turnip, a variety of Chinese cabbage, Brassica chinensis komatsuna, tender flower stalk, colea (Brassica napus), Shirakukina, Brassica campestris L.varamplexicaulis Tanaka et Ono, rape, man who brings news of appointment's wild cabbage (Brussels sprouts), kohlrabi (kohlrabi), cabbage mustard (Chinese kale), Brassica chinensis var.rosularis, leaf mustard (Brassica juncea), rape and kale (cabbage var.acephala).

Bacterium Maize Ear Rot of paddy rice and the bacterium seedling blight of paddy rice are the seed-borne diseases from rice husk, must prevent and treat them in the wetland rice cropping, and they are important paddy bacterial diseases, and the sterilization of seed is especially essential.For this reason, chemical control technology drops into actual the use at present usually.Yet, with insecticide rice husk is carried out disinfection, relate to the problem of handling residual pesticide etc." Momigenki " (Nissan Chemical Industries Ltd.) is unique commercially available biological insecticides that are used for the disinfectant rice.According to the present invention, can effectively prevent and treat wetland paddy rice kind and pass bacterial disease.

The fungal disease that the present invention can prevent and treat can be any by the plant disease due to the fungal pathogens.The example comprises gas borne fungus diseases and silborne fungal diseases.In the present invention, the gas borne fungus diseases comprises that water passes disease.The example of gas fax bacterium includes but not limited to powdery mildew (Erysiphe (Erysiphe), Sphaerotheca (Sphaerotheca) and Leveillula (Leveillula)), Botrytis (Botrytis), Fulvia fulva, Corynespora (Corynespora), white rust belongs to (Albugo), downy mildew (false Peronospora (Pseudoperonospora), Peronospora (Peronospora), Plasmopara (Plasmopara) and Bremia (Bremia)), seasonal febrile diseases fungi (Pyricularia grisea), palace portion cochliobolus (Cochliobolus miyabeanus), Cercospora (Cercospora) and the relevant (Cercospora that belongs to, Cercosporalla (Cercosporella), pseudo-cercospora (Pseudocercospora), Paracercospora and bacterial bloom spore belong to (Mycovellosiella), Bacillus anthracis (Bacillus anthrasis) (Colletotrichum (Colletotrichum) and small cluster shell belong to (Glomerella)), rust fungi (Puccinia (Puccinia)), black spot fungi (Alternaria (Alternaria)) and Alternaria.The example of soil-borne fungus includes but not limited to club root fungi (plasmodiophora brassica bacteria (Plasmodiophorabrassicae)), Phytophthora (Phytophthora), Fusarium (Fusarium), Verticillium (Verticillium), pythium (Pythium) and Rhizoctonia (Rhizoctonia).

Gas borne fungus diseases instantiation includes but not limited to: by powdery mildew due to powdery mildew infection tomato, eggplant, radish, wild cabbage, turnip, leaf mustard, Chinese cabbage, Brassica chinensis komatsuna, carrot, cucumber, strawberry, capsicum, muskmelon, watermelon, pumpkin and the chrysanthemum; By gray mold due to Botrytis cinerea (Botrytis cinerea) infection Curcurbitaceae, Solanaceae, lettuce, beans and the strawberry; By excellent spore bacterium leaf spot due to Corynespora cassicola (Corynespora cassiicola) the infection cucumber; Infect white blister due to the multiple brassicaceous vegetable (for example Chinese cabbage, cauliflower, turnip, radish and Brassica chinensis komatsuna) by big spore white rust (Albugo macrospora); By downy mildew due to downy mildew infection cucurbit, brassicaceous vegetable, shallot, onion, horseradish, crowndaisy chrysanthemum, Japanese cryptotaenia stem and leaf (Cryptotaenia japonica), spinach and the lettuce; By rice blast due to the seasonal febrile diseases fungal infection paddy rice; By cladosporium leaf and fruit mould of tomato (tail spore leaf mold) due to the dirty false tail spore of coal (Pseudocercospora fuligena) the infection tomato; By eggplant brown spot (leaf spot) due to the Paracercospora egenula infection eggplant; By scab (frogeye leaf spot) due to capsicum tail spore (Cercospora capsici) infection pimento and the capsicum; By scab (leaf spot) due to melon tail spore (Cercosporacitrullina) infection cucumber, muskmelon, watermelon and the sponge gourd; By the hickie on Chinese cabbage due to the little tail spore of rape (Cercosporella brassicae) infection Chinese cabbage and the turnip and the turnip; By eggplant leaf mold due to gray wool eggplant bacterial bloom spore (Mycovellosiella nattrassii) the infection eggplant; By anthracnose due to anthrax bacillus infection Chinese cabbage, turnip, radish, Brassicachinensis komatsuna, beet (Beta vulgaris), a variety of Chinese cabbage, rape, bush bean, capsicum, pimento, cucurbit, tender pod Kidney bean, shallot, onion, fragrant-flowered garlic, spinach and the strawberry; By rust due to rust fungal infection shallot, onion, Chinese onion (Allium bakeri), garlic and the fragrant-flowered garlic; By chrysanthemum rust due to the rust fungal infection chrysanthemum; By black spot due to black spot fungal infection brassicaceous vegetable and the carrot (for example chain lattice spore leaf spot and chain lattice spore black rot); By alternaric bacteria black spot (sooty spot) due to the living chain lattice spores of rape (Alternaria brassicicola) the infection wild cabbage; By leaf blight due to carrot chain lattice spore (Alternaria dauci) the infection carrot; By early blight due to a kind alternaria solani sorauer (Alternaria solani) potato-infecting; By early blight of tomato due to kind alternaria solani sorauer infection tomato; By leaf blight due to Botrytis fungal infection onion and the fragrant-flowered garlic; With infect grey mold neck rot due to the onion by the rotten grape spore of green onion (Botrytis allii).The gas borne fungus diseases, for example white blister, black spot, anthracnose and downy mildew, known production to Cruciferae leaf vegetables and root vegetables has devastating impact, the control of this class serious plant disease be subjected to comprising always the national Buddhist monk that this class disease took place do not take place this class disease the country and Japan global very big concern.Up to now, in fact still do not ratify to resist the plant variety of white blister, black spot, anthracnose and downy mildew.Some insecticides with enough control efficiency (for example metalaxyl wetting powder, iprodione wettable powder, TPN wetting powder and thiophanate methyl wetting powder) are arranged.Because these insecticides directly are applied to human consumption's leaf vegetables and root vegetables, thus its standards for safe use strictness, and use these insecticides during disease control, may have a negative impact.The present invention can carry out biological control to this class disease of for example following plant: turnip, a variety of Chinese cabbage, Brassica chinensis komatsuna, tender flower stalk, radish, cabbage mustard, Brassica chinensisvar.rosularis, cauliflower, blue and white cabbage, Chinese cabbage, wild cabbage, rape and leaf mustard (referring to embodiment 21 and 22).

The instantiation of silborne fungal diseases includes but not limited to by club root due to the plasmodiophora brassica bacteria infection crop in cruciferae; The disease that phytophthora is induced, for example by paddy rice, apple, lily, strawberry, oranges and tangerines, pears, pimento, capsicum, pumpkin, taro, potato, tomato, eggplant, cucumber, watermelon, muskmelon, cucurbit, shallot, carnation (Dianthus caryophyllus), fusarium wilt due to oranges and tangerines and spinach infect, by gray blight due to tomato and the cucumber infection, by eggplant, tomato, brown rot due to watermelon and oranges and tangerines infect, by root rot due to the strawberry infection, by paddy rice, barley, wheat, oat, yellow dwarf due to corn and bent grass infect, by stem rot due to the rose infection, by paddy rice, cotton eqpidemic disease (cottonblight) due to soybean and eggplant infect, by root rot due to the oranges and tangerines infection, by onion, shallot, four seasons green onions (Alliumfistulosum caespitosum), Chinese onion, garlic, white eqpidemic disease (whife blight) due to fragrant-flowered garlic and wild green onion (Allium grayi) infect, by stem wilt due to red bean (Phaseolus angularis) and the soybean infection, by seedling leaf blight due to the eggplant infection, and by root rot due to tomato and the eggplant infection; The disease that pythium spp is induced, for example by bush bean, cotton eqpidemic disease (cottony blight) due to pumpkin and cucumber infect, by paddy rice, eggplant, tomato, spinach, pumpkin, cucumber, bush bean, pea, rice shoot damping off due to gumbo and muskmelon infect, by stalk rotten sick (cavity spot) due to the carrot infection, by rotten mould eqpidemic disease due to the bent grass infection, by soybean, cucumber, damping off due to muskmelon and spinach infect, by wheat, barley, oat, rye, brown snow mold due to perennial ryegrass and fescue grass infect, by strawberry, cucumber, root rot due to taro and tomato infect, by white rot due to the sweet potato infection, and rotten sick by seedling due to the paddy rice infection; The disease that the rhizoctonia bacterium is induced, red neck rot (red crown rot) due to for example infecting by paddy rice, by paddy rice, barley, grain (Setaria italica), dry sheath rot due to dichromatism chinese sorghum (Sorghum bicolor) and grain infect, by soybean, red bean, bush bean, pea, potato, orchardgrass, annual bluegrass, fescue grass, perennial ryegrass, leaf rot due to bent grass and Chinese sorghum infect, by bud blight due to the strawberry infection, by stem rot due to broad bean and the carnation infection, by paddy rice, tomato, eggplant, pimento, capsicum, cucumber, muskmelon, muskmelon (Cucumis melo), cucurbit (Lagenaria siceraria), wild cabbage, cauliflower, blue and white cabbage, Brassica chinensis komatsuna, onion, shallot, rice shoot damping off due to gumbo and ivyleaf cyclamen infect, by spinach, barley, seedling blight due to wheat and wild cabbage infect, the root disease (foot blight) of withering due to infecting by lettuce, by tar spot due to potato and the burdock infection, by root rot due to radish and the carrot infection, by bottom rot due to Chinese cabbage (the Brassica campestris var.pekinensis) infection, by bavin root rot due to the carrot infection, and by damping off due to the chrysanthemum infection; The disease that wheel branch spore is induced, wilt disease due to for example infecting by strawberry, edible araliae cordatae (Aralia cordata) and soybean, by a wheel branch spore wilt disease due to eggplant, capsicum (pimento), tomato, gumbo, cucumber, butterbur (Petasites japonica), muskmelon, chrysanthemum and the rose infection, by a wheel branch spore wilt disease due to wild cabbage and the lettuce infection, by verticillium sp black spot due to the radish infection, and by jaundice (icterus) due to the Chinese cabbage infection; And the sickle spore disease of inducing, sickle spore wilt disease due to for example infecting by eggplant, by cucumber, cucurbit, sponge gourd, sickle spore wilt disease due to muskmelon and watermelon infect, by stem rot due to the sweet potato infection, by wild cabbage, strawberry, radish, turnip, verticillium wilt due to Brassica chinensiskomatsuna and edible araliae cordatae infect, by root rot due to lettuce and the bush bean infection, by Chinese onion, onion, garlic, fragrant-flowered garlic, stem rot due to taro and carrot infect, by seedling blight due to the Japanese cryptotaenia stem and leaf infection, by burdock, shallot, tomato, spinach, carnation, wilt disease due to ivyleaf cyclamen and red bean infect, by damping off due to asparagus and the carnation infection, by bacterial decay disease due to the lotus infection, by brown rot due to muskmelon and the Chinese yam infection, by sickle spore hat root rot due to tomato and the shallot infection, by the rotten disease of seedling due to the paddy rice infection with by pink colour snow mold due to the wheat infection.Be difficult to control by the silborne fungal diseases due to for example Fusarium, Verticillium or the Phytophthora fungi, and these diseases has devastating impact to vegetable growing.In order to handle described disease, carried out the cultivation resistant variety, with the chemical insecticide disinfection soil, disease is predicted and other work.For example, carried out the cultivation of Solanaceae (for example tomato, eggplant, pimento and green pepper) and Curcurbitaceae (for example cucumber, watermelon and muskmelon) resistant variety or stock, with chemical insecticide (for example chloropicrin, dazomet and methyl bromide) disinfection soil or check soil pathogen, so that prevent and treat disease.The harvest cycle of brassicaceous vegetable (for example turnip, a variety of Chinese cabbage, Brassica chinensis komatsuna, Chinese cabbage, wild cabbage or radish) is shorter than Solanaceae or cucurbitaceae vegetable.Therefore, Mei Nian continuous plantation frequency is very high.For example, the continuous plantation number of times of Brassica chinensis komatsuna, a variety of Chinese cabbage or turnip is annual 5-6 time.Therefore, the infringement due to the soil-borne disease is destructive.Adopt control agent of the present invention or material in each planting season, can prevent to be difficult in the soil propagation or the propagation of the pathogene of preventing and treating and reduce development of disease (referring to embodiment 23 and 24).

The viral plant disease that the present invention prevented and treated can be any by the plant disease due to the viral pathogens.The example includes but not limited to following virogenetic plant disease: tobacco mosaic virus (TMV), the light-duty mottle virus of capsicum (PMMoV), Tomato mosaic virus (ToMV), muskmelon withered spot disease poison (MNSV), cucumber green mottle mosaic virus (CGMMV) or Kyuri green mottle mosaic virus (KGMMV).

The instantiation of viral plant disease includes but not limited to by the tobacco mosaic due to the TMV, by pimento mosaic disease due to PMMoV, ToMV or the TMV, by muskmelon withered spot disease due to the MNSV, by cucumber green mottle mosaic due to the CGMMV, refute mosaic disease and by kyuri green mottle mosaic virus disease due to the KGMMV by the muskmelon green statin due to the CGMMV.

Viral disease due to TMV, KGMMV, CGMMV and the MNSV shows that tactility metachromia, the juice more potent than disease due to other virus are propagated, seed dispersal and soil are propagated.Therefore, the control of the important viral disease of this class has been subjected to global very big concern, is cultivating the plant variety or the stock that this class disease are had resistance always, and described cultivation product drops in the practical application.In some cases, market or consumer demand have surpassed the cultivation ability, and effectively resistant variety or stock may not can use.Therefore, chemical control technology is used in particular for preventing juice propagation, seed dispersal and soil to propagate.For example, adopted Lentemin water solube powder prevention juice to propagate, propagated, perhaps propagated (referring to Iwata etc., the same) with methyl bromide prevention soil with tertiary sodium phosphate prevention seed dispersal and juice.Because these Prevention Technique are undertaken by chemical treatment, so abuse can cause result unsatisfactory, crop is undermined or human to be injured.In addition, methyl bromide (methyl bromide fumigant) is extensive use of in Japan, in case the soil of the toxicity disease of curing the disease is propagated.Banned use of this fumigant about 2005 according to the Montreal bar, because it is relevant with ozone layer depletion.At present, extensively searching its substitute.According to the present invention, can effectively prevent and treat kind of a viral disease of biography and a soil-borne disease toxicity disease (referring to embodiment 25 and 26) with biological method.

They can be used for bacillus of the present invention and there is no concrete restriction, as long as can suppress the infection or the propagation of plant pathogenetic bacteria, fungi or virus.The example comprises bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and bacillus subtilis (Bacillussubtilis).(preserving number: FERM BP-10114), this bacterial strain is preserved in Independent Administrative Leged Industrial Technology Complex Inst and speciallys permit biological sustenance center preferred especially DAIJU-SIID2550.This bacterial strain has affiliation through inferring with bacillus amyloliquefaciens, therefore is abbreviated as hereinafter " BAL bacterium ".The plant disease controlling agent or the material that preferably contain the BAL bacterium, because it can stably be colonizated in plant or the soil, effectively keep the control of plant disease activity, even and described control agent or material be exposed to when 40 ℃-100 ℃ high temperature and also can keep the disease control activity.In the present invention, can use the BAL bacterium mutant that produces through mutagenesis.Can carry out mutagenesis with any suitable mutagen.Term used herein " mutagen " should be adopted understanding by its common implication.For example, mutagen not only comprise the medicine with mutagenesis, and comprise the processing that produces the mutagenesis effect, for example application of UV.The example of suitable mutagen comprises the nucleotide base analog, for example application of ethyl methane sulfonate, UV, N-methyl-N '-nitro-N-nitrosoguanidine and bromouracil and acridine.Also can use any other effective mutagen.

Above-mentioned BAL bacterium can separate according to for example following mode.Edible mushroom is cultivated residue carry out steam sterilizing (80 ℃, 30 minutes), residue adds physiological saline and dilutes, and part by weight is 1: 9 (residue: physiological saline), reach 15 minutes with the reciprocal shaker jolting dilution of amplitude 10cm.Then, products therefrom obtains supernatant in 500rpm low-speed centrifugal 5 minutes.Perhaps, products therefrom filters with Toyo Roshi No.2, obtains filtrate.Subsequently, supernatant or filtrate are carried out serial dilution (" Saikingaku jisshu teian (Proposal regarding bacteriological laboratorytraining) with the distilled water that contains 0.05% agar, " the Alumni Association of the Institute of Infectious Disease (writing)).Preparation 1 * 10 ^1-8Times dilution carries out serial dilution, and each 100 μ l dilution is joined YPA medium (a kind of medium that contains peptone, yeast and salt in the 9cm culture dish; Referring to " Shokubutsu byougensei biseibutsu kenkyu-hou (Study ofplant pathogenic microorganisms); idenshi sousa wo fukumu kiso to ouyo (Basics and applications including genetic engineering); " SatoshiWakimoto (writing)) in, in 25 ℃ of incubators, leave standstill and cultivated 48-72 hour, then respectively to the bacterial sampling of growth.The sampling bacterium again the YPA slant medium upload be commissioned to train foster.In order to confirm the uncontaminated irrelevant bacterium of this bacterium, the bacterium of cultivating of going down to posterity is carried out serial dilution again, to obtain single bacterium colony.From the single bacterium colony of the pure bacterium that separates thus, cultivate each single bacterium colony bacterium and turnip flavivirus (turnip yellows viruse) (lupine point sickle spore (Fusarium oxysporum f.sp.conglutinans) by antagonism on the YPA flat board, a kind ofly be used to confirm) at the cause a disease simple marking of antimicrobial acivity of mutation of xanthomonas campestris sarson, and, select to have the strongest inhibiting bacterium according to the intensity of formed inhibition zone.

The bacterium of Xuan Zeing has following characteristic thus: Gram: the positive; Endospore: have; Form: shaft-like; G+C (DNA) content (mol%): 435-44.9 (this is worth than bacillus subtilis height, and bacillus subtilis is 42-43); Optimum growth temperature: 25 ℃-30 ℃; Biosafety level: 1 (it can not cause human diseases, can not cause the serious plant disease of plant or animal yet).

Use the 16S rDNA nucleotide sequence (16S rRNA gene) (referring to following details) of the about 1600bp of length to infer the taxon of this bacterium.Found that the purpose bacterium is the new bacterial strain of bacillus.This purpose bacterium is preserved in Independent Administrative Leged Industrial Technology Complex Inst on November 20th, 2003 as the DAIJU-SIID2550 bacterial strain and speciallys permit biological sustenance center (preserving number: FERM BP-10114).

Can be by reciprocating vibration cultivation, fermentation tank culture or with conventional cultivation techniques such as liquid culture such as culture tank cultivation or solid culture, cultivate and can be used for bacillus of the present invention.The medium that can be used for bacillus of the present invention there is no concrete restriction, as long as can allow bacterium effectively reach exponential phase.Adopt preferred culture medium,, in 48-96 hour, can reach maximum yield at 25 ℃-30 ℃.Adopt more preferably medium,, in 48 hours, can reach maximum yield at 25 ℃.Can use the synthetic or natural medium that comprises following composition: for example: as carbon source, carbohydrates such as glucose, sucrose, starch, dextrin, brown sugar piece, wheat bran or rice bran; As nitrogenous source, ammonium salt (for example ammonium sulfate, ammonium chloride or ammonium nitrate) and nitrate etc. are inorganic nitrogen-sourced, or organic nitrogen source, for example yeast extract, corn medicinal extract, meat medicinal extract, wheat embryo, poly-peptone, bagasse, brewer's grains, soybean meal, rice bran or fish meal; As mineral salt, the salt of phosphorous, potassium, manganese, magnesium or iron, for example potassium dihydrogen phosphate, magnesium sulfate, manganese sulphate or ferrous sulfate.When carrying out shaken cultivation, can add additives such as defoamer on demand.Because bacillus is an aerobic bacteria, so preferably under aerobic conditions cultivate preferred solid culture or liquid culture, for example air agitation cultivation or shaken cultivation.Preferably 10 ℃-50 ℃, more preferably at 15 ℃-40 ℃, preferred pH level is at 5-9, more preferably cultivate at 6-8.As described in embodiment 5, the preferred mixed culture medium that contains soybean curb residue, wheat bran and rice bran that uses is as medium, and this medium can satisfy above-mentioned requirements.Because these materials are forms of trade waste, from effectively utilizing the viewpoint of trade waste, the preferred medium that contains soybean curb residue, wheat bran and rice bran that uses is cultivated.

According to control of plant disease technology of the present invention, can use the above-mentioned culture fluid that contains bacillus, and need not to carry out any processing.In order to improve disease-controlling effect, can carry out that film separates to culture fluid, centrifugal, isolated by filtration or other technology, to obtain to have the solution of higher concentration, can preferably use the concentrate of gained.

In the present invention, can use the dehydration product of the above-mentioned culture fluid that contains bacillus.And, can will contain the above-mentioned medium centrifugal of bacillus, with the dehydration of gained supernatant, re-use its product.In addition, containing the precipitum that the medium centrifugal of bacillus obtains can wash with water and dewater, and can use its dehydration product (in the dehydration product about 50%-100% (weight) be bacterium) usually.Allow the culture fluid that contains bacillus be adsorbed onto on the porous adsorbing material (for example powdered activated carbon, diatomite or talcum powder),, also can use products therefrom (wherein to comprise 1 * 10 usually the sorbing material dehydration ⁸-10 ⁹The cfu/g bacterium).In all cases, can dewater by routine techniques such as freeze-drying or drying under reduced pressure.Dehydration product also can grind by methods such as ball millings after dehydration.Below introduce the instantiation for preparing the method for dehydration product with the BAL bacterium.To be equipped with 100ml AG medium (1% glucose (and Wako Pure Chemical Industries, Ltd.), 1% poly-peptone (NihonPharmaceutical Co., Ltd.), 0.1%KH ₂PO ₄(Wako Pure Chemical Industries, Ltd.) and 0.05%MgSO ₄7H ₂In the 300ml conical flask of O (Wako Pure Chemical Industries, Ltd.), pH7.00, autoclaving 15 minutes), add the BAL bacterium slant culture of a loopful, in 25 ℃ of shaken cultivation 24 hours (120rpm).Subsequently, (100 μ l) joins in the 100ml AG medium with the gained culture, under aerobic conditions, cultivates 48 hours at 25 ℃.With medium centrifugal, the culture precipitation is separated with culture supernatants, reclaim supernatant, sediment washes with water, obtains bacterium.Perhaps, drip this culture, allow it be adsorbed onto on the porous adsorbing material, to obtain bacterium, described sorbing material for example powdered activated carbon (Wako Pure Chemical Industries, Ltd.), diatomite (Wako Pure ChemicalIndustries, Ltd.) or talcum powder (Wako Pure Chemical Industries, Ltd.), these materials are filled in the glass tube or lucite pipe of diameter 1.8cm, long 10-15cm.The culture supernatant that obtains thus, the bacterium through washing or the bacterium that is adsorbed all dewater by freeze-drying or drying under reduced pressure, grind with ball milling again.Therefore can obtain containing the dehydration product of bacillus.

In the present invention, bacillus can culture fluid, the form of concentrate or dehydration product uses separately.Bacillus can be chosen wantonly with other any component and mix, thereby to be similar to common microorganism formulation form (but for example pulvis, wetting powder, emulsion, solution flowable (flowable agent) or inuncts) preparation.The example of other any component that plan is mixed with bacillus comprises solid excipient and adjuvant.The example of solid excipient comprises for example following organic powder: bentonite, diatomite, talcum powder compound, perlite, vermiculite, sodium carboxymethylcellulose (CMC), brewer's grains, bagasse, soybean curb residue, wheat bran, chitin, rice bran and wheat flour.The example of adjuvant comprises fixative and thickener, for example gelatin, gum Arabic, carbohydrate and gellan gum.Brewer's grains, bagasse, soybean curb residue, wheat bran and rice bran all are the trade waste forms, therefore, from effectively utilizing the viewpoint of trade waste, preferably use them in the present invention.Below introduce in detail the control agent of the present invention of trade wastes such as comprising the culture fluid that contains bacillus and soybean curb residue or the example of material.To be equipped with 100ml AG medium (1% glucose (and WakoPure Chemical Industries, Ltd.), 1% poly-peptone (Nihon Pharmaceutical Co., Ltd.), 0.1%KH ₂PO ₄(Wako Pure Chemical Industries, Ltd.) and 0.05%MgSO ₄7H ₂O (Wako Pure Chemical Industries, Ltd.), pH 7.00, autoclaving 15 minutes) in the 300ml conical flask, the BAL bacterium slant culture that adds a loopful after 25 ℃ of reciprocating vibrations are cultivated (120rpm), adjusts to 1 * 10 with the BAL bacteria concentration again ⁷Cfu/ml.With 1,300g soybean curb residue, 600g wheat bran and 100g rice bran mix (hereinafter, this mixture is called " basic growth medium ") respectively.Above-mentioned culture fluid (1ml) is joined in the basic growth medium of 100ml, in the dark leave standstill and cultivated 72-120 hour, fully stir once every day.After leaving standstill cultivation,, obtain control profit of the present invention or material with the cultured products dehydration.The content of BAL bacterium is about 4 * 10 in thus obtained control agent or the material ^{9- 10}Cfu/g.

As mentioned above, from edible mushroom cultivation residue, isolate the BAL bacterium.Therefore, the edible mushroom that contains the BAL bacterium is cultivated residue and also be can be used as control agent of the present invention or material.Edible mushroom is cultivated residue and can be dewatered on demand and grind.When bacterial concentration is low described in edible mushroom cultivation residue or its dehydration product, can be on demand to wherein adding BAL bacteria culture fluid, concentrate or dehydration product.In this case, preferably add described material, make that the final BAL bacteria concentration in the residue is 1 * 10 ^9-10More than the cfu/ml.

The BAL bacterium that breeds in trade waste also can be used as control agent of the present invention or material.The example of control agent of this class or material is included in the BAL bacterium that breeds in the following material: wheat bran, straw, wheat straw, corn stalk or with the mushroom bed of crossing (mushroom is cultivated residue, for example edible mushroom cultivation residue).From effectively utilizing the viewpoint of trade waste, preferred this embodiment of the present invention.The BAL bacteria concentration is generally 1 * 10 in thus obtained control agent or material ^7-8Cfu/g is although this concentration is different different because of the material that is mixed.CN ratio in available trade waste is 70% in straw, is 70%-90% in wheat straw, and is 80%-90% in corn stalk.

Below introduce the instantiation of above-mentioned embodiment of the present invention.Wheat bran, wheat straw (being cut into 5-10cm) and the water of equivalent are mixed and stirring, and (nitrogen content: 1.7%-2.0%), rice bran content is 5-10kg in every 100kg mixture, preferred 5-6kg to add rice bran.According to make the identical mode of compost, this mixture of indoor stacking (because indoor not raining).The fermentation heat production (contained 1 * 10 usually with the 500ml BAL bacterium liquid of cultivating on the AG medium after 6-7 days ⁷-10 ⁹The cfu/ml bacterium) evenly disperse and agitating solution then further precipitation.After, every 4-6 days, preferred per 4 or 5 days, stir 5-6 time.In case fermentation is finished,, obtain containing the control material of BAL bacterium with the tunning dehydration.The control material of making presents different fertilizer components and analytical characteristic value according to the material difference of being mixed.Generally speaking, pH is 7.0-7.5, and moisture is 35%-40%, and N content is 2.5%-3.0%, P ₂O content is 3.5%-4.0%, K ₂O content is 2.0%-2.5%, and CaO content is 1.0%, and MgO content is 1.0%-1.5%, and content of organics is 50%-60%, and C/N is than being 0.7-1.3.When using control material of the present invention, also can use the method for simply getting sun simultaneously.(when using solar energy, the face of land covers transparent mulch film, thereby by day the internal temperature at subsurface 15cm place is adjusted to 30 ℃-40 ℃, and surface temperature is adjusted to 35 ℃-40 ℃, and processing is 7-10 days under the situation of soil water content 70%-80%.) control material of the present invention passes bacterium, fungi or virus concerning host's invasion and attack or effective especially infecting for suppressing soil.Because control material of the present invention can effectively be prevented and treated the soil-borne disease poison (referring to embodiment 25) in the soil, it is the useful especially substitute of methyl bromide soil fumigant.

According to different situations, for example intend the plant disease type of control, the development of plant disease, intend the plant variety of processing or the form of bacillus, suitably select bacillus is applied to the method for plant.For example, can use by for example following different technologies: use on the ground, indoorly use, mix in the soil, irrigated soil or surface treatment (for example seed dressing or seed pelleting).Therefore, control agent of the present invention or material can be used for preventing and treating seed dispersal, soil propagation or air or water propagation (infecting by wind-force or water).(technology of control schizomycete disease can be divided into this 3 class roughly usually).More particularly, preferably be selected from following technology and use: give the plant seed dressing with multi-form bacillus by at least a, irrigate plant cultivation soil with described bacterium, it is incorporated into plant cultivation with in the soil, it is applied to plant stem-leaf, and it is partly contacted with plant damage.Can give the plant seed dressing with bacillus by for example following mode: wrap by plant seed with the suspension of bacillus bacterium in the solution that contains film forming agent (for example gellan gum, agar or sodium carboxymethylcellulose), then dehydration.Perhaps, plant seed is immersed in the similar suspension, then dehydration.

When bacillus is applied to plant, can further sneak into other conventional active component on demand, for example insecticide, nematocide, miticide, weed killer herbicide, fungicide, the thin agent of sterilization, antiviral agent, fertilizer or soil structure conditioner (for example peat).These materials also can be used alternatingly or use simultaneously, and need not to mix.In most of the cases, as described in embodiment, the activity that is used for bacillus of the present invention is not subjected to the interference of other active component of using simultaneously.

In the present invention,, for example intend the plant disease type of control, the development of plant disease, intend the plant variety of processing or the form of bacillus, suitably select bacillus is applied to the consumption of plant according to different situations.When the solution that contains the BAL bacterium was applied to the acrial part of the plant that for example infects black rot, the BAL bacteria concentration alive in the control agent was about 1 * 10 usually ^7-10Cfu/ml, preferred about 1 * 10 ^8-9Cfu/ml.The consumption of described control agent is preferably 100-2501/10a.When the dehydrated powder that will contain the BAL bacterium was incorporated in the soil that has polluted the pathogenic mutation of xanthomonas campestris sarson, the BAL bacteria concentration of living in the dehydrated powder was generally 1 * 10 ^8-10Cfu/g, preferred 1 * 10 ⁹Cfu/g.The consumption of dehydrated powder is preferably 500-2000kg/10a, more preferably 500-1000kg/10a.According to above consumption, the control agent or the material that will contain the BAL bacterium are applied to the soil that has polluted the pathogenic mutation of xanthomonas campestris sarson, and are incorporated in the soil by abundant stirring.Then, do not handle this soil, be allowed to condition at the about 30%-40% of soil water content, placed 5-7 days under 20 ℃ of-30 ℃ of conditions of soil temperature, careful attention keeps soil moisture in order to avoid drying simultaneously.If implement described processing, even then the crucifer planting seed is in such polluted soil, this plant also can not be subjected to the infection of black rot.

The check of the taxon of reference example: DAIJU-SIID2550

With the DAIJU-SIID2550 strain join CM3 agar (Oxoid, UK) in, cultivated 2 days at 30 ℃.Then, this bacterial strain is as the DNA extraction test strain.(Applied Biosystems U.S.A.) extracts genomic DNA by the PrepMan method.The genomic DNA that extracts is as template, by the 1500-1600bp district of pcr amplification 16S ribosomal RNA gene (16S rDNA) total nucleotide sequence.Then, the 16S rDNA that increases is checked order, obtain the 16S rDNA nucleotide sequence of test strain.Purified pcr product, (Applied Biosystems U.S.A.) carries out cycle sequencing to gained PCR product to use MicroSeqFull 16S rDNA bacterium sequencing kit.Use GeneAmp PCR 9600 thermo cyclers (the Applied Biosystems of system, U.S.A.), use ABI PRISM 3100DNA sequenator (Applied Biosystems, U.S.A.), use AutoAssembler 2.1 (AppliedBiosystems, U.S.A.) assembling gained nucleotide sequence fragment.According to Applied Biosystems, the scheme of P/N4308132Rev.A is carried out the basic operation from the extracting genome DNA to the cycle sequencing.

The nucleotide sequence of gained 16S rDNA (SEQ ID NO:1) is used to carry out autoploidy search, preceding 10 strains of determining to show high homology.In addition, by the neighbor-joining method, make up the molecular system tree with determined preceding 10 strains and test strain 16S rDNA, and check the taxon of relevant bacterial classification and test strain.V.1.4.1 carry out autoploidy search and constructing system tree with MicroSeq Mirobial IdentificationSystem Software.In autoploidy search, use database MicroSeq Bacterial Full Gene Library v.0001 (AppliedBiosystems, U.S.A.).

When by MicroSeq Bacterial Full Gene Library, when in the autoploidy search, not having table of discovery to reveal the bacterial strain of 100% autoploidy, carried out the search of BLAST autoploidy with dna nucleotide sequence database GenBank/DDBJ/EMBL.

The result who analyzes by MicroSeq Bacterial Full Gene Library is to find that the difference between bacillus amyloliquefaciens, bacillus subtilis withered grass subspecies (B.subtilis subsp.subtilis) and the mocha husband bacillus (B.mojavensis) is respectively 0.45%, 0.65% and 0.71%.There is not table of discovery to reveal the bacterial isolates of complete homogeneity.According to the molecular system tree, find the 16S rDNA and bacillus amyloliquefaciens cluster (cluster) and relevant of purpose bacterial strain with it.The result who carries out the search of BLAST autoploidy with GenBank/DDBJ/EMBL is, find the test strain show with bacillus Bch (Bacillus sp.Bch) (AF411118) highest homology of bacterial strain be 99.7%, although there is not table of discovery to reveal the bacterial strain of complete homogeneity.

Therefore, find that (preserving number: FERM BP-10114) bacterial strain is the new bacterial strain of bacillus to DAIJU-SIID2550.

Hereinafter introduced the relevant embodiment that carries out control of plant disease with the BAL bacterium.

Embodiment 1

The mensuration of BAL bacterium maximum growth time

To be deposited in Independent Administrative Leged Industrial Technology Complex Inst as the DAIJU-SIID2550 strain speciallys permit biological sustenance center (preserving number: the bacterial classification (being called " BAL bacterium " hereinafter) (loopful) of being correlated with of bacillus amyloliquefaciens FERM BP-10114) is suspended in the 9ml sterile water, 100 μ l gained suspensions are joined 100ml AG medium (1% glucose (Wako PureChemical Industries is housed, Ltd.), 1% poly-peptone (Nihon Pharmaceutical Co., Ltd.), 0.1%KH ₂PO ₄(Wako Pure Chemical Industries, Ltd.) and 0.05%MgSO ₄7H ₂The 300ml conical flask of O (Wako Pure Chemical Industries, Ltd.), pH 7.00, autoclaving 15 minutes) or the 100ml potato that contains 2% sucrose are soaked in the juice body medium.(potato is soaked juice and is prepared as follows: the 200g potato is cut into about 1cm ³, add about 1 liter of distilled water and also it was boiled 30-40 minute with little fire, form by the double gauze filtration again.2% sucrose is joined in the filtrate, add distilled water and reach 1 liter until volume.Products therefrom is called " PS medium ", is placed in No. 1 heating clamber.) with the reciprocal shaker of amplitude 10cm in 120rpm, 25 ℃ of shaken cultivation 144 hours.Cultivate beginning back 0 hour, 24 hours, 48 hours, 72 hours and 144 hours, take out the 1ml culture fluid as sample.Sample culturing liquid is joined in the 9ml sterile water, mixture is fully mixed, prepare 10 times of dilutions.Then, take out 1ml sample liquid and joining in the 9ml sterile water continuously.Repeat this step, preparation 10 ^7-8Times dilution (10 times of serial dilutions).Each dilution of 100 μ l is added in the YPA flat board 9cm culture dish of (comprising peptone, yeast and salt), is coated with evenly coating of rod, cultivated 72 hours, measure formed clump count then at 25 ℃ with Conradi.Obtain the BAL bacterium number (relative value) (table 1) of each time point thus.The maximum growth time is 48 hours or 72 hours after beginning.This shows, when cultivating in the presence of BAL bacterium and the pathogenic mutation of xanthomonas campestris sarson, cultivates the beginning back and can reach maximum control efficiency in 48-72 hour.

Table 1

* cell counting is represented with the relative value of each time point, and cell counting is defined as 1 (cfu/ml) when cultivating beginning.

Embodiment 2

The BAL bacterium is to the pathogenic mutation inhibition of proliferation effect of xanthomonas campestris sarson

Scrape the BAL bacterium of getting a circular rector from the YPA slant medium, be inoculated in the 300ml conical flask that the 100mlAG medium is housed.Products therefrom was with the reciprocal shaker of amplitude 10cm, in 120rpm, 25 ℃ of shaken cultivation 48 hours.The xanthomonas campestris sarson mutation suspension (100 μ l) that causes a disease is joined in this culture fluid, again shaken cultivation 48 hours under the same conditions.The xanthomonas campestris sarson used herein mutation suspension that causes a disease is prepared as follows: scrape the cultivation of getting a circular rector and soak the mutation of causing a disease of xanthomonas campestris sarson on juice agar medium (hereinafter referred is " the PSA ") slant medium the potato that contains 2% sucrose, it is suspended in the 9ml sterile water.Experiment (control group) as a comparison joined the 100 μ l xanthomonas campestris sarsons mutation suspension that causes a disease 100ml and does not add in the AG medium of BAL bacterium, 25 ℃ of same shaken cultivation 48 hours.Each culture fluid is by serial dilution technology (referring to embodiment 1) dilution, with each dilution (100 of 100 μ l, 000 times, 1,000,000 times and 10,000,000 times of dilution) joins YPA flat board (diameter: 9cm), cultivated 72 hours at 25 ℃, measure the pathogenic mutation clump count of the xanthomonas campestris sarson that is produced then.Fig. 1 shows back 72 hours bacterium colony outward appearance (the left figure: 1,000,000 times of dilution factor of cultivation beginning; Right figure: 10,000,000 times of dilution factor).In Fig. 1, upper part is represented the xanthomonas campestris sarson independent cultured products of mutation of causing a disease, and lower part is represented the xanthomonas campestris sarson cultured products of mutation with the BAL bacterium of causing a disease.Table 2 shows the pathogenic mutation clump count result of xanthomonas campestris sarson.When the mutation of causing a disease of xanthomonas campestris sarson arises from 25 ℃ of shaken cultivation in the time of 48 hours with BAL bacterium one, the propagation of the pathogenic mutation of xanthomonas campestris sarson is suppressed fully.

Table 2

The clump count of the pathogenic mutation of xanthomonas campestris sarson

Dilution gfactor	With the AG medium that contains the BAL bacterium	Use the AG medium
			100,000 times	0	744
1,000,000 times	0	136
			10,000,000 times	0	48

Embodiment 3

The preparation of the seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson

With gauze parcel a variety of Chinese cabbage seed (34g, kind: Fuyushoumi), at 500mlChemicron G (neutral calcium hypochlorite; Available chlorine: soaked 10 minutes in 150 times of dilutions 70%), so that disinfection seed.After the seed disinfection, flushing is 1 hour under flowing water, removes moisture then, spends the night 35 ℃ of dehydrations.In addition, the pathogenic mutation of xanthomonas campestris sarson was cultivated 3 days on 5 PSA flat boards in advance, scrape and get all growth bacterium and be suspended in the 100ml distilled water preparation 1 * 10 ¹²The cfu/ml xanthomonas campestris sarson mutation bacterium liquid that causes a disease.The seed (17g) of sterilizing was soaked 20 minutes in the xanthomonas campestris sarson is caused a disease mutation solution, spend the night 35 ℃ of dehydrations then, preparation artificial contamination's seed.Check its gradient of infection, found that every infectious bacteria number that infects on the seed is 10 ^5-6Cfu, and preliminary treatment seed infection percentage is 100%.When the planting seed of these infection is in disinfection soil, often observe epicotyledonary disease (Fig. 2).In Fig. 2, left figure demonstrates the result (contrast experiment) who uses the contrast seed, and right figure demonstrates the result with the pathogenic mutation artificial contamination's of xanthomonas campestris sarson seed.

Embodiment 4

Wrap by the effect of the seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson with the BAL bacterium Really

In accordance with the following methods, handle the seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson.The BAL bacterium of before cultivating on 2 YPA flat boards is suspended in 1) 50ml 0.5% gellan gum (Scott Laboratories, Inc.) in the aqueous solution or 2) 50ml 1.5% agar (WakoPure Chemical Industries, Ltd.) in the aqueous solution, gained solution evenly suspends with magnetic stirrer to BAL bacterium.The cause a disease seed of mutation of artificial contamination's xanthomonas campestris sarson that 300mg is derived from embodiment 3 is immersed in this 50ml suspension, allows its standing over night, and it is air-dry therefrom to take out the back.Experiment (control group) as a comparison, artificial contamination's xanthomonas campestris sarson caused a disease, and to be dipped in the aqueous solution of the 50ml 0.5% gellan gum aqueous solution that do not contain the BAL bacterium or 50ml 1.5% agar the back equally air-dry for the seed of mutation.Air-dry seed is placed on the YPA flat board, cultivated 72 hours, observe whether the heterogonous growth of causing a disease of xanthomonas campestris sarson is arranged near the seed at 25 ℃.The result by following formula, obtains the infringement of each group according to the observation.

Near the cause a disease seed sum of the seed amount that mutation grows seed/added of infringement=permission xanthomonas campestris sarson

According to the infringement of being calculated,, obtain control and tire by following formula.

Control is tired=100-(infringement of the infringement/control group of processed group) * 100

The results are shown in Table 3.When the seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson was used BAL bacterium dressing, the propagation that is attached to the pathogenic mutation of xanthomonas campestris sarson of seed was suppressed.This shows that the BAL bacterium can make the seed disinfection of infecting the pathogenic mutation of xanthomonas campestris sarson.

Table 3

	Control is tired
		1) with the gellan gum aqueous solution dressing that is suspended with the BAL bacterium	100
2) with the aqueous agar solution dressing that is suspended with the BAL bacterium	100

Embodiment 5

With soybean curb residue medium culture BAL bacterium

In the 100ml soybean curb residue of sterilizing 20 minutes, sneak into 1ml1 * 10 at 105 ℃ ⁷Cfu/ml BAL bacteria culture fluid was cultivated 48 hours 25 ℃ of lucifuges, obtained BAL bacterium soybean curb residue culture.The 500mg BAL bacterium soybean curb residue culture of gained is suspended in the 9ml sterile water, use Conradi to be coated with rod, to evenly join the YPA flat board in the 9cm culture dish according to 100 each dilution of μ l that the same quadrat method of embodiment 1 is carried out 10 times of serial dilution gained respectively, measure the BAL bacterium number in the BAL bacterium soybean curb residue culture powder then.Isolate a part of BAL bacterium soybean curb residue culture powder, spend the night, measure moisture 100 ℃ of dehydrations.BAL bacterium number in the BAL bacterium soybean curb residue culture powder is 4.3 * 10 ⁹Cfu/g, moisture is 87.6%.

This experimental result confirms that the BAL bacterium can be that soybean curb residue is bred as medium with trade waste.The xeothermic dehydration product of resulting in this embodiment BAL bacterium soybean curb residue culture powder has suitable bacterial concentration and moisture.Therefore, find that this product can be used as the raw material that the present invention that preparation contains the BAL bacterium prevents and treats reagent or material.

Embodiment 6

Caused by artificial contamination's xanthomonas campestris sarson with BAL bacterium soybean curb residue culture powder packets The effect of the seed of pathology kind

The BAL bacterium soybean curb residue culture powder (moisture: 87%) spend the night 35 ℃ of dehydrations, grind with ball milling, obtain BAL bacterium soybean curb residue culture powder of embodiment 5 will be derived from.BAL bacterium soybean curb residue culture powder (each 500mg) is joined 1) 50ml 0.5% sodium carboxymethylcellulose (abbreviates CMC as hereinafter, Wako Pure Chemical Industries, Ltd.) in the aqueous solution or 2) in the 50ml 0.5% gellan gum aqueous solution, then with the gained mixture with magnetic stirrer 10 minutes.Seed (300mg) with the pathogenic mutation of gauze parcel artificial contamination xanthomonas campestris sarson soaked 10 minutes in each solution, allowed the BAL bacterium firmly be adsorbed on the surface of the seed, spent the night 35 ℃ of dehydrations.Prepare seed thus with BAL bacterium soybean curb residue culture powder coating.Experiment (control group) is as a comparison soaked dehydration then with the cause a disease seed of mutation of artificial contamination's xanthomonas campestris sarson in the 0.5%CMC aqueous solution that does not add BAL bacterium soybean curb residue culture powder or the 0.5% gellan gum aqueous solution.

3) with seed and the 16 μ l 0.5%CMC aqueous solution (seed dressing) of BAL bacterium soybean curb residue culture powder (2mg) with the pathogenic mutation of 200mg artificial contamination xanthomonas campestris sarson.

The seed of handling (each 20 seed) is joined in the NSCAA medium (Randhawa, P.S. and Schaad, N.W., 1984, Phytopathology 74:268-272), cultivated 72 hours at 25 ℃.Whether observe the pathogenic mutation of xanthomonas campestris sarson grows near seed.For above experiment 1) and 2), the sample appearance after the cultivation is seen Fig. 3.In Fig. 3, one group (control group 1) that on behalf of the CMC aqueous solution, upper left handle, one group (processed group 1) that the upper right portion representative is handled with the CMC aqueous solution that contains BAL bacterium soybean curb residue culture powder, one group (control group 2) that the bottom left section representative is handled with the gellan gum aqueous solution, a group (processed group 2) that the lower right-most portion representative is handled with the gellan gum aqueous solution that contains BAL bacterium soybean curb residue culture powder.

Check near the seed amount that allows the pathogenic mutation of xanthomonas campestris sarson seed, to grow, calculate " infringement ", obtain " control is tired "." infringement " and " control is tired " is as the definition of embodiment 4.

The results are shown in Table 4.1)-3) all experiments in, confirm that all the mutation of causing a disease of xanthomonas campestris sarson is subjected to anti-system.Specifically, the pathogenic mutation of artificial contamination's xanthomonas campestris sarson also can suppress attached to the pathogenic mutation propagation of the xanthomonas campestris sarson on the seed with the seed of BAL bacterium soybean curb residue culture powder coating.As the dressing auxiliary material, find that CMC is more suitable than gellan gum.This shows that the seed of the mixture dressing of the BAL bacterium soybean curb residue culture powder and the CMC aqueous solution is also used in the pathogenic mutation of artificial contamination's xanthomonas campestris sarson, can suppress the infection that seed is subjected to the pathogenic mutation of xanthomonas campestris sarson.

Table 4

	Control is tired
		1) handles with the BAL bacterium soybean curb residue culture powder CMC aqueous solution	84.2
2) handle with the BAL bacterium soybean curb residue culture powder gellan gum aqueous solution	25.0
		3) with BAL bacterium soybean curb residue culture powder-processed (seed dressing)	60.0

Embodiment 7

(1) edible mushroom is cultivated the preparation of residue

In accordance with the following methods, the preparation edible mushroom is cultivated residue.

Cultivate edible mushroom preceding 1 month, and preparing the fresh straw that cuts off.Replenish soybean cake and rice bran in the fresh straw, make C/N, stack into the haystack of suitable width and height than for about 70%.Per 5 days water sprays and stir 4 times, decompose and make compost up to their.

In culturing room, thus obtained compost is cut into 15-18cm thickness, as the mushroom bed.When the mushroom bed tempertaure is reduced to 24 ℃, to wherein adding bacterial classification (agaricus bisporus (Agaricus bisporus) i.e. is " white mushroom "), be that 16 ℃, humidity are 96%, carbonic acid gas is 3 in temperature, cultivate edible mushroom under the condition of 000ppm.Inoculation 1 week of back in case mycelium extends out from mushroom bed surface, then covers whole mushroom bed with soybean meal, be rotated stirring.After two weeks, put into the black bog moss of 88kg on every square metre the mushroom bed.Inoculate back 60 days results edible mushrooms, will be with the mushroom bed of crossing 80 ℃ of sterilizations 30 minutes.Obtain edible mushroom thus and cultivate residue.(Agaricus brown mushroom can be used as bacterial classification, behind appropriate time after the inoculation (for example 60-90 days), and the results mushroom.)

(2) in the soil that pollutes the pathogenic mutation of xanthomonas campestris sarson, suppress the yellow list of sarson The test of the pathogenic mutation propagation of born of the same parents bacterium sarson

The pathogenic mutation of xanthomonas campestris sarson is joined in the disinfection soil, and the soil (1 * 10 of the pathogenic mutation of xanthomonas campestris sarson is polluted in preparation ¹²The mutation of causing a disease of cfu/ml xanthomonas campestris sarson).Allow the soil that pollutes the pathogenic mutation of xanthomonas campestris sarson leave standstill 1 day at 25 ℃, polluting the xanthomonas campestris sarson with respect to 1 liter causes a disease and mixes the 50g edible mushroom in the soil of mutation and cultivate residue (promptly 2,000kg residue/10a soil), allow the gained mixture leave standstill 5 days, check the pathogenic mutation bacterial population of xanthomonas campestris sarson in the soil at 25 ℃.Test as a comparison (control group),, equally the pathogenic mutation bacterial population of xanthomonas campestris sarson in the soil that does not add edible mushroom cultivation residue is checked with the pathogenic mutation contaminated soil of xanthomonas campestris sarson.

In accordance with the following methods, measure the pathogenic mutation bacterial population of xanthomonas campestris sarson in the soil.That is to say, 20g soil by 300 eye mesh screens, is joined the soil that sieved in the 80ml physiological saline (0.85%NaCl), obtain suspension, is the reciprocal shaker of 10cm with amplitude, in 120rpm with suspension jolting 30 minutes, centrifugal 5 minutes again with 500rpm.Centrifugal gained supernatant carries out 10 times of serial dilutions with the distilled water that contains 0.05% agar, 500 μ l dilutions are added drop-wise to NSCAA medium (Randhawa, P.S. and Schaad, N.W., 1984, Phytopathology 74:268-272) in, being coated with rod with Conradi is uniformly coated on it, 25 ℃ of cultivations, check the clump count that demonstrates the pathogenic peculiar outward appearance of mutation of xanthomonas campestris sarson, determine the count of bacteria of the pathogenic mutation of xanthomonas campestris sarson.

The pathogenic mutation count of bacteria of xanthomonas campestris sarson in every gram dry ground earth: when adding edible mushroom cultivation residue is 1.09 * 10 ⁷Cfu (processed group) is 7.06 * 10 when not adding edible mushroom cultivation residue ⁷Cfu (control group).

According to following formula, obtaining control, to tire be 84.6%.The result is summarized in table 5.

Control tiring=100-(the xanthomonas campestris sarson of processed group cause a disease mutation count of bacteria)/(the xanthomonas campestris sarson of control group cause a disease mutation count of bacteria) * 100

Result of the test shows, edible mushroom is cultivated residue join in the soil, can obviously suppress the propagation of the pathogenic mutation of xanthomonas campestris sarson.

Table 5

	The mutation count of bacteria (cfu/g dry ground earth) of causing a disease of xanthomonas campestris sarson	Prevent and treat tire (%)
			Processed group	1.09×10 7	84.6
Control group	7.06×10 7	-

Embodiment 8

The pathogenic mutation of artificial contamination's xanthomonas campestris sarson also is immersed in the BAL bacteria culture fluid Seed effect and edible mushroom is cultivated residue be incorporated in the soil black rot sent out Sick inhibition effect

The BAL bacterium is joined in the AG medium, shaken cultivation 7 days, preparation BAL bacteria culture fluid, its bacteria concentration is 1 * 10 ⁶Cfu/ml.Seed (200mg) with the pathogenic mutation of gauze parcel artificial contamination xanthomonas campestris sarson soaks preset time in culture fluid.Soaked 10 minutes, 20 minutes, 40 minutes and 60 minutes.After the immersion, get on except that moisture, seed is spread out, spend the night 35 ℃ of dehydrations then from seed.In order to compare, the cause a disease seed of mutation of artificial contamination's xanthomonas campestris sarson to be immersed in the AG medium that does not add the BAL bacterium and to reach the same time, dehydration then.

The seed of handling is according to the method described above joined in the soil, soil steam soil disinfector (SB-150, Marubun Seisakusyo Co.Ltd.,) sterilize 30 minutes (being called " disinfection soil " hereinafter) at 100 ℃, in disinfection soil, mix 2 again, the 000kg/10a edible mushroom is cultivated residue, preparation mixed soil (referring to embodiment 7 (1)) (being called " containing the soil that edible mushroom is cultivated residue " hereinafter).(each 150ml) joins in the resealable container with soil, adds the seed of 20 pathogenic mutation of artificial contamination's xanthomonas campestris sarsons of as above handling, then 25 ℃ of processing.

Organize in contrast, in disinfection soil, add the seed of the pathogenic mutation of artificial contamination's xanthomonas campestris sarson, prepare experimental group.

Use back 9 days sample appearance and see Fig. 4 and Fig. 5.In Fig. 4, cultivation result under upper part representative " immersion and the soil that additionally comprises edible mushroom cultivation residue in containing the AG medium of the BAL bacterium " condition, and the cultivation result under (it is 10 minutes, 20 minutes, 40 minutes and 60 minutes that the immersion duration begins from a left side) condition " is soaked and disinfection soil " in the lower part representative in containing the AG medium of BAL bacterium.In Fig. 5, Far Left is partly represented the cultivation result under " soaking and disinfection soil " condition in not containing the AG medium of BAL bacterium, mid portion is represented the cultivation result under " soaking and disinfection soil " condition in containing the AG medium of BAL bacterium, and the cultivation result (soaking the duration all is 60 minutes) under the condition " is soaked and the soil that additionally comprises edible mushroom cultivation residue " in the rightmost representative in all experiments in containing the AG medium of BAL bacterium.

Studied the disease symptom that after using the BAL bacterium, appeared at epicotyledonary black rot in 9 days.Study according to following 6 disease state standards.

0: asymptomatic

1: the infringement area accounts for below half of cotyledon

2: the infringement area accounts for a slice cotyledon

3: the infringement area accounts for 1.5 cotyledons

4: the infringement area accounts for 2 cotyledons

5: withered

According to result of study, obtain the incidence of disease according to following formula.

The incidence of disease=(1n ₁+ 2n ₂+ 3n ₃+ 4n ₄+ 5n ₅The plant sum of)/(5 * studied)

In formula, n ₁, n ₂, n ₃, n ₄And n ₅Independent separately representative is according to the quantity of the single plant of standard 1,2,3,4 and 5 classification.

According to the incidence of disease of being measured, obtain control according to following formula and tire:

Control is tired=the 100-processed group incidence of disease/control group incidence of disease * 100

More than control of each group tire and see Table 6.As shown in table 6, the cause a disease seed of mutation of artificial contamination's xanthomonas campestris sarson is immersed in the BAL bacteria culture fluid, suppressed the development of black rot.Find that when soak time overtime in the BAL bacteria culture fluid, it is higher that this suppresses effect.Wherein mix the experimental group that edible mushroom is cultivated the soil of residue for using, soak time is lacked the experimental group of (for example 10 minutes) in the BAL bacteria culture fluid, the experimental group of soaking 60 minutes in containing the AG medium of BAL bacterium with seed and joining in the disinfection soil is compared, and the control of generation is tired substantially the same.

Table 6

* have the negative experimental group of tiring of preventing and treating and show the incidence of disease higher than control group

Embodiment 9

Use of the control of BAL bacteria culture fluid on the ground to black rot

In 4 leaf phases, will in the AG medium, carry out BAL bacteria culture fluid (300ml, the bacterial concentration: 3 * 10 of shaken cultivation with sprayer ⁸Cfu/ml) be sprayed to 9 strain a variety of Chinese cabbages (kind: Fuyushoumi) on the blade face.After it's about 1 hour allowing past a variety of Chinese cabbage plant, 300ml xanthomonas campestris sarson is caused a disease the mutation solution spray to these plant with sprayer.The pathogenic mutation formulations prepared from solutions of this xanthomonas campestris sarson is as follows: the mutation of earlier the xanthomonas campestris sarson being caused a disease is cultivated on 5 PSA flat boards, scrape from flat board and to get the mutation of causing a disease of all xanthomonas campestris sarsons, be suspended in the 150ml distilled water preparation 10 ⁸The cfu/ml xanthomonas campestris sarson mutation solution for later use of causing a disease.The plant of using the BAL bacterium is moved into humid room (humidity: 80%), remain on 20 ℃.In order to compare, prepared two groups: spray the BAL bacteria culture fluid but do not use the xanthomonas campestris sarson experimental group of mutation of causing a disease, and the experimental group (i.e. 9 strain a variety of Chinese cabbages) (control group) of the mutation solution that do not spray the BAL bacteria culture fluid but the xanthomonas campestris sarson of having used same concentration is caused a disease.Fig. 6 shows the back 26 days acrial part of experiment beginning.In Fig. 6, one hurdle, the left side is presented at the cultivation result in the soil of " add the BAL bacterium but do not add the mutation of causing a disease of xanthomonas campestris sarson ", a middle hurdle is presented at the cultivation result (processed group) in the soil of " add BAL bacterium and xanthomonas campestris sarson cause a disease mutation ", and one hurdle, the right is presented at the cultivation result (control group) in the soil of " do not add the BAL bacterium but add the mutation of causing a disease of xanthomonas campestris sarson ".Evaluation experimental begins the back 33 days black rot symptoms that occur on the blade, checks the disease development.At first measure the incidence of disease (sick leaf accounts for the percentage of the blade of checking) on the blade.And then measure control by following formula and tire.

Control is tired=100-(the blade incidence of disease of the blade incidence of disease/control group of processed group) * 100

The results are shown in Table 7.Before adding the pathogenic mutation of xanthomonas campestris sarson, spray the BAL bacterium, caused suppressing the development of black rot.This shows, with the preventative acrial part that is applied to the pathogenic mutation host of xanthomonas campestris sarson of BAL bacteria culture fluid, can suppress the development and the propagation of black rot.

Table 7

	The BAL bacterium	The mutation of causing a disease of xanthomonas campestris sarson	Control is tired
				Control group	Do not add	Add	-
Processed group	Add	Add	58.6

Embodiment 10

Conventional fungicide or insecticide are to the influence of BAL bacterium

Conventional fungicide and insecticide, be Amistar 20Flowable (Syngenta, nitrile Fluoxastrobin wetting powder), Jimandaisen wetting powder (DSS Hishishou, mancozeb wettable powder), Rovral wetting powder (Yashima Kagaku, iprodione wettable powder), Rizolex wetting powder (Sumitomo Chemical Co., Ltd., the tolelofos-methyl wetting powder), Sthana wetting powder (Sumitomo Chemical Co., Ltd., the oxolinic acide wetting powder), Daconil 1000Flowable (SDS, the TPN wetting powder), Z-Bordeaux (Nihon Nohyaku Co, Ltd., the copper wetting powder), Benlate wetting powder (Sumitomo Chemical Co., Ltd., the benomyl wetting powder), Ridomyl MZ wetting powder (Syngenta, mancozeb/metalaxyl wetting powder), Bestguard water solube powder (Sumika Takeda, the nitenpyram water solube powder), DDVP emulsion (NihonNohyaku Co, Ltd., DDVP emulsion), Affirm emulsion (Syngenta, benzoic acid emamectin emulsion), Admire wetting powder (Bayer CropScience, imidacloprid wettable powder), Lannate 45 wetting powders (Sankyo Agro Co., Ltd., the Methomyl wetting powder), Pandan SG water solube powder (Sumika Takeda, the cartap water solube powder), Mospilan water solube powder (Mppon Soda Co., Ltd., pyrrole worm clear water dissolubility pulvis), Elsan emulsion (Nissan Chemical Co.Ltd., PAP emulsion), Actara particle water solube powder (Syngenta, the Thiamethoxam water solube powder), Kotetsu flowable (Nihon Nohyaku Co, Ltd, the chlorphenapyr wetting powder) and Torebon emulsion (Mitsui Chemicals, Inc., ether chrysanthemum ester emulsion), it is diluted to given concentration, the preparation chemicals, (Toyo Roshi No.2) is immersed in these chemicals with filter paper, and be dry then.Again with the suspension (2 * 10 of BAL bacterium in distilled water ⁸Cfu/ml) with 44 μ l/cm ²Consumption be sprayed onto on these filter paper, again with the filter paper drying, place it in 25 ℃ of lucifuges and spend the night.Filter paper is cut into 5-10mm ²Small pieces, on the YPA flat board, carry out lucifuge at 25 ℃ and cultivate.

After the cultivation, to putting into the 10mm of YPA flat board ²The BAL bacterium of growth was observed 3-4 days near the filter paper.

The results are shown in Table 8.In addition, the flat board of cultivating after 4 days is seen Fig. 7.The growth of BAL bacterium is not subjected to the influence of fungicide or insecticide, only is subjected to the influence of some chemicals.Yet in the presence of Jimandaisen wetting powder and Ridomyl MZ wetting powder, the growth of BAL bacterium is very slow, cultivates after 3 days, does not observe bacterium colony basically.That is to say, do not observe the growth of BAL bacterium, observe growth gradually but cultivate after 4 days.But in the presence of Sthana wetting powder and Daconil 1000 flowables, do not observe the growth of BAL bacterium fully.

These results show that plant disease controlling agent of the BAL of containing bacterium of the present invention or material can be united use with many conventional fungicides or insecticide, perhaps can be used alternatingly or use simultaneously and not mix.

Table 8

Conventional fungicide or insecticide are to the influence of BAL bacterium

*+: cultivate and observed bacterium colony in back 3 days; ±: because of poor growth, cultivate back 4 talentes and observe bacterium colony;-: do not observe bacterium colony.

Embodiment 11

The BAL bacterium is to canker fungi (rod shape bacillus Michigan, Michigan subspecies) inhibition of proliferation

Scrape from the YPA slant medium and to get a loopful BAL bacterium, join in the 300ml conical flask that the 100mlAG medium is housed, the reciprocal shaker that with amplitude is 10cm was in 120rpm, 25 ℃ of shaken cultivation 48 hours.In this culture fluid, add rod shape bacillus Michigan, 100 μ l Michigan subspecies suspension, again shaken cultivation 48 hours under similarity condition.Rod shape bacillus Michigan, Michigan used herein subspecies suspension is prepared as follows: scrape and get the Michigan rod shape bacillus Michigan subspecies of loopful cultivation on the PSA slant medium, then it is suspended in the 9ml sterile water.Experiment (control group) joins rod shape bacillus Michigan, 100 μ l Michigan subspecies suspension in the 100ml AG medium that does not wherein add the BAL bacterium as a comparison, then equally 25 ℃ of shaken cultivation 48 hours.Culture fluid dilutes (referring to embodiment 1) with the serial dilution technology, with each dilution (100 of 100 μ l, 000 times, 1,000,000 times and 10,000,000 times of dilution) join in the 9cm YPA flat board, cultivated 72 hours at 25 ℃, measure the colony counting of rod shape bacillus Michigan, the Michigan subspecies of growth.The colony counting measurement result sees Table 9.The bacterium colony outward appearance is seen Fig. 8.When rod shape bacillus Michigan, Michigan subspecies cell with the BAL bacterium 25 ℃ of shaken cultivation in the time of 48 hours, therefore the propagation of rod shape bacillus Michigan, Michigan subspecies suppressed fully.

Table 9

Dilution gfactor	The AG medium that contains bacillus	The AG medium
			10 5	0	1079
10 6	0	132
			10 7	0	55

Embodiment 12

The BAL bacterium is to wilt disease bacterium (Jialuo Er Sitong Salmonella) inhibition of proliferation

Scrape from the YPA slant medium and to get a loopful BAL bacterium, join in the 300ml conical flask that the 100mlAG medium is housed, the reciprocal shaker that with amplitude is 10cm was in 120rpm, 25 ℃ of shaken cultivation 48 hours.In this culture fluid, add 100 μ l Jialuo Er Sitong Salmonella suspensions, again shaken cultivation 120 hours under similarity condition.Jialuo Er Sitong Salmonella suspension used herein is prepared as follows: scrape and get the Jialuo Er Sitong Salmonella of loopful cultivation on the PSA slant medium, then it is suspended in the 9ml sterile water.Experiment (control group) joins 100 μ l Jialuo Er Sitong Salmonella suspensions in the 100ml AG medium that does not wherein add the BAL bacterium as a comparison, then equally 25 ℃ of shaken cultivation 120 hours.Culture fluid dilutes (referring to embodiment 1) with the serial dilution technology, with each dilution (1,000 of 100 μ l, 000 times and 10,000,000 times of dilution) join in the 9cm YPA flat board, cultivated 72 hours at 25 ℃, measure the colony counting of the Jialuo Er Sitong Salmonella of growth.The colony counting measurement result sees Table 10.The bacterium colony outward appearance is seen Fig. 9.When Jialuo Er Sitong Salmonella cell with the BAL bacterium 25 ℃ of shaken cultivation in the time of 120 hours, the propagation of Jialuo Er Sitong Salmonella is suppressed thus.

Table 10

Dilution gfactor	The AG medium that contains bacillus	The AG medium
			10 6	48	831

Embodiment 13

The BAL bacterium breeds soft rot bacterium (carrot soft rot Erwinia carrot soft rot subspecies) Suppress

Scrape from the YPA slant medium and to get a loopful BAL bacterium, join in the 300ml conical flask that the 100mlAG medium is housed, the reciprocal shaker that with amplitude is 10cm was in 120rpm, 25 ℃ of shaken cultivation 48 hours.In this culture fluid, add 100 μ l carrot soft rot Erwinia carrot soft rot subspecies suspensions, again shaken cultivation 96 hours under similarity condition.Carrot soft rot Erwinia carrot soft rot subspecies suspension used herein is prepared as follows: scrape and get a loopful and cultivate the potato that contains 2% sucrose and soak carrot soft rot Erwinia carrot soft rot subspecies on juice agar medium (abbreviating " PSA " hereinafter as) slant medium, then it is suspended in the 9ml sterile water.Experiment (control group) joins 100 μ l carrot soft rot Erwinia carrot soft rot subspecies suspensions in the 100mlAG medium that does not wherein add the BAL bacterium as a comparison, then equally 25 ℃ of shaken cultivation 96 hours.Culture fluid dilutes (referring to embodiment 1) with the serial dilution technology, with each dilution (1 of 100 μ l, 000,000 times and 10,000,000 times of dilution) joins in the 9cm YPA flat board, cultivated 72 hours, measure the colony counting of the carrot soft rot Erwinia carrot soft rot subspecies of growth at 25 ℃.The colony counting measurement result sees Table 11.The bacterium colony outward appearance is seen Figure 10.When carrot soft rot Erwinia carrot soft rot subspecies cell with the BAL bacterium 25 ℃ of shaken cultivation in the time of 96 hours, the propagation of carrot soft rot Erwinia carrot soft rot subspecies is suppressed thus fully.

Table 11

Dilution gfactor	The AG medium that contains bacillus	The AG medium
			10 6	0	1259
10 7	0	187

Embodiment 14

Inhibition to soft rot of Chinese cabbage

The petiole of Chinese cabbage is used running water, distilled water and 70% washing with alcohol successively, wipe moisture, petiole is put into big glass culture dish with Kimwipes.The soft rot bacterium is that carrot soft rot Erwinia carrot soft rot subspecies were cultivated 3 days in the YPA slant medium, is suspended in the 9ml distilled water preparation 1 * 10 then ⁷Cfu/ml bacterium liquid.Add 1ml BAL bacteria culture fluid in this bacterium liquid of 1ml, the gained mixture is fully stirred, (cotton needle) is immersed in wherein with 8 cotton pins, with pin scraping Chinese cabbage petiole.Introduce the BAL bacterium thus.Subsequently, glass culture dish is sealed with Parafilm, with the bacteriological protection drying, with culture dish overnight incubation in 30 ℃ of incubators.Experiment (control group) is as a comparison introduced the mixed liquor that contains 1ml bacterium liquid and 1rml distilled water with pin equally.Do not have the petiole (being designated as "-") of soft rot and dye the petiole (being designated as "+") that soft rot is arranged by mensuration, carry out damage appraisement.Measuring control by following formula tires.

Control is tired=100-(the processed group incidence of disease/control group incidence of disease) * 100

The results are shown in Table 12.The back 1 day bacterium colony outward appearance of experiment beginning is seen Figure 11.

Table 12

	1 group	2 groups	Loss ratio	Control is tired
					Contrast	+	+	100
Mix the BAL bacterium	-	-	0	100

Embodiment 15

Inhibition to seed dispersal plant bulkholderia cepasea and pod shell bulkholderia cepasea

The inoculum preparation that is used to prepare artificial contamination's rice paddy seed is as follows: with plant bulkholderia cepasea (original seed of protection association of Japanese plant (Japan Plant Protection Association)) at the PPG medium (in the 200g potato, 3g ten water sodium hydrogen phosphates (Wako), 0.5g dipotassium hydrogen phosphate (Wako), 5g peptone (Nihon Pharmaceutical Co., Ltd.), 3g sodium chloride (Wako), 5g glucose (Wako) and 1 liter of distilled water), 25 ℃ of shaken cultivation 3 days.With the rice paddy seed that intend to pollute earlier 250 times of Chemicron G (Nippon Soda Co. Ltd.) soaks in the solution and carried out disinfection in 30 minutes, again with the flowing water flushing after 45 minutes, drying.The culture fluid (30ml) that will contain 15g seed and above-mentioned bacterium joins in the 100ml beaker, and (Yamato MINIVAC PS-22) infects seed under reduced pressure with vavuum pump.

(the 50g soybean curb residue is suspended in the 100ml distilled water with the soybean curb residue extract with the seed (3g) that pollutes and 30ml, filtered through gauze, autoclaving reaches 20 minutes for 121 ℃) 5 days the BAL bacteria culture fluid (being called " BAL bacteria culture fluid " hereinafter) that contains of shaken cultivation joins in the 50mlFalcon pipe and soaks.The seed (1.5g) that at room temperature soaked 48 hours joined contain 100ml Kumiai-Ryujyou-Baido D (Kureha Chemical Industry Co. is in ice cream cup Ltd.) (being made diameter 10cm, high 5.5cm by polyethylene).In contrast, equally also add the pollution seed that is not immersed in the BAL bacteria culture fluid.Allow seed sprout 32 ℃ of lucifuges, turn green (greening) and at 20 ℃ of indoor sustainable managements of artificial climate, used back 16 days, to put in order the strain plant extracts, check development of disease (Sakumotsu byougenkinkenkyuu gihou no kiso-bunri/baiyo/sesshu (Basics of crop pathogenresearch techniques-separation/culture/introduction), Kanichi Ohata (writing), Japan Plant Protection Association).Withered rice shoot and brown rice shoot are considered to diseased plant.Obtaining the incidence of disease by following formula also determines to prevent and treat to tire.

Control is tired=100-(the processed group incidence of disease/control group incidence of disease) * 100

The results are shown in Table 13.The back 16 days bacterium colony outward appearance of experiment beginning is seen Figure 12 A.

Table 13

The seed of pollution pod shell bulkholderia cepasea is immersed in the effect in the BAL bacteria culture fluid

	The healthy plant number	The diseased plant number	The incidence of disease	Control is tired
					Pollute seed	1	49	98.0	-
Soak seed	30	15	33.3	66.0

Method also experimentizes to pod shell bulkholderia cepasea (original seed of Japan PlantProtection Association) as described above.For the bacterium Maize Ear Rot, withered rice shoot and the serious rice shoot (half length of health plant or shorter) of disease are considered to diseased plant.The results are shown in Table 14.The back 16 days sample appearance of experiment beginning is seen Figure 12 B.

Table 14

The seed of pollution plant bulkholderia cepasea is immersed in the effect in the BAL bacteria culture fluid

	The healthy plant number	The diseased plant number	The incidence of disease	Control is tired
					Pollute seed	4	43	91.5	-
Soak seed	33	12	26.7	70.9

Embodiment 16

Inhibition effect to the generation of rice blast fungi (Magnaporthe grisea) spore

With the BAL bacteria culture fluid of undiluted BAL bacteria culture fluid, 5 times of dilutions (being the final concentration of 2 times or 10 times when the sprouting test) and distilled water (control group) as sample.The spore of Magnaporthe grisea (allow flora grow in the oatmeal agar medium of sugaring and form with the BLB radiation) is suspended in the PS medium (containing the medium that 2% sugar and potato are soaked juice), 20 each sample of μ l are joined in the porose slide (hole glass slide), allow them fully mix, allow mixture in 25 ℃ of humid rooms, leave standstill 24 hours, whether exist at the test under microscope germ tube.Figure 13 show sample mixes back 24 hours Magnaporthe grisea spore.In 5 times of dilutions of stoste and adding culture fluid, the spore germination of Magnaporthe grisea is suppressed, and does not observe the spore of sprouting.

Embodiment 17

Inhibition effect to paddy rice brown spot fungi (palace portion cochliobolus) spore germination

To 100ml YPA medium (0.5% yeast extract (Nihon PharmaceuticalCo. is housed, Ltd.), 1% poly-peptone (Nihon Pharmaceutical Co., Ltd.) and 0.5% sodium chloride, pH7.00, autoclaving 20 minutes) in the 300ml conical flask, the BAL bacterium of cultivation in the YPA slant medium that adds a circular rector is in 25 ℃ of shaken cultivation 72 hours (120rpm).The stoste of gained culture fluid, 5 times of dilutions and 10 times of dilutions are as sample.The same solution of handling through distilled water is as control group.Add the palace portion cochliobolus spore be suspended in the distilled water (making in 7-10 days) by joining pre-incubated palace portion cochliobolus in the PSA medium and cultivating at 25 ℃, its consumption is to add 100 μ l in every 1ml sample, they are fully mixed, 50 μ l gained mixtures are joined in the porose slide, be allowed to condition in the humid room then, left standstill 24 hours, examine under a microscope the germ tube structure and whether exist at 25 ℃.24 hours palace portion cochliobolus spore is seen Figure 14 after the sample mix.In culture fluid stoste, there is not spore germination.The extension of germ tube is suppressed in 5 times and 10 times of dilutions, and the swelling distortion takes place, and does not observe germ tube and extends.

Embodiment 18

Use of the control of BAL bacteria culture fluid on the ground to the paddy rice brown spot

BAL bacteria culture fluid or distilled water (group in contrast) are applied to 6 leaf phase-7 leaf phase paddy rice (kinds: Nihonbare) of middle growth in the controlled environment chamber, allow plant have under the optical condition, to place 16 hours at 25 ℃, spraying adds palace portion cochliobolus spore suspension after 24 hours.This spore suspension is prepared as follows: in advance palace portion cochliobolus is cultivated in the PSA medium, distilled water is poured in the culture dish, with the brush flora that rubs, wash spore, filter products therefrom with double gauze, regulating then under each visual field of spore concentration to 100 power microscope has 10, adds polysorbas20, and obtaining concentration is 0.02%.Then, rice plant is covered with plastic containers, constitute 25 ℃ of humid rooms, allow plant leave standstill therein 24 hours.Used back 6 days, and be determined at infringement sum and blade area on the blade that reaches full growth when using, measure every 1cm ²Infringement mean, and measure control according to following formula and tire.

Control is tired=100-(the infringement mean of processed group)/(the infringement mean of control group) * 100

The results are shown in Table 15.It is 89.7 that the control of using the BAL bacteria culture fluid and obtaining is tired.This shows that the paddy rice brown spot can be suppressed.

Table 15

	The measured blade gross area (cm 2)	The infringement number	Every 1cm 2The infringement number	Control is tired
					Processed group (culture fluid)	264.1	102	0.39	89.7
Control group (water)	352.38	1316	3.73	-

Embodiment 19

The BAL bacteria culture fluid is to the inhibition effect of gray mold of cucumber fungi (grey grape grape spore)

Partly downcut cotyledon from the cucumber plumular axis, the cotyledon that downcuts is placed in the container that is lined with hygenic towelette, make tangent plane contact paper handkerchief.Allow Botrytis cinerea soak growth in the juice agar medium (PSA) the potato that contains 2% sucrose in advance, the spore that will be produced under the condition of using BLB is suspended in the potato that contains 2% sucrose and soaks in the juice agar medium (PG).The spore density of suspension is adjusted to 2 * 10 ⁷Spore/ml, every district are put 10 cotyledons, to each Dropwise 50 μ l suspension of its center, paper dish (Toyo Roshi is used to measure antibiotic, and is thick, diameter 8mm) are attached thereon.In addition, by the paper dish, be distilled water or iprodione wettable powder (Rovral) (dilution gfactor: 1,000) drip thereon,, be allowed to condition under the optical condition, placed 16 hours at 20 ℃ with seal of vessel with 50 μ l BAL bacteria culture fluids or control material.

The results are shown in Table 16 and Figure 15.Come ecbatic according near the brown diameter that causes by infection the paper dish.Compare by following formula and distilled water, measure control and tire.

Control is tired=100-(the average brown diameter of processed group)/(the average brown diameter of distilled water group) * 100

After using 3 days, Figure 15 is seen in the brown stain of cotyledon, and the average brown stain diameter of 10 cotyledons sees Table 16.Compare with control group, the brown stain diameter of plant of having used culture fluid is obviously littler.This shows that gray mold of cucumber is suppressed.

Table 16

	Distilled water	Iprodione wettable powder	Culture fluid
				Average brown stain diameter (mm)	19.6	11.2	3.3
Control is tired	-	42.9	83.2

Embodiment 20

Use of the control of BAL bacteria culture fluid on the ground to the cucumber brown spot

Cucumber plant (the kind of sprouting on April 28th, 2004: Matsukaze) in the greenhouse, grow.At 2 true leaf developmental stages, be distilled water or Daconil 1000 (dilution gfactor: 1,000) be applied to rice shoot with the BAL bacteria culture fluid of capacity or control material.After 5 hours, solutions employed is dry 25 ℃ of mistakes.Then spore (Laboratory ofPathology and Biotechnology of the Musashino Seed Co., the original seed of the Ltd.) suspension of Corynespora cassicola being adjusted to spore density is 10 ⁵Spore/ml, spraying adds.Suspension is prepared as follows: in advance spore is cultivated in the PSA medium, distilled water is poured in the culture dish, with the brush flora that rubs, wash spore, filter with double gauze.After using like this, cucumber plant is covered with plastic containers, constitute 25 ℃ of humid rooms, allow plant leave standstill therein 24 hours, having under the optical condition, monitoring 16 hours at 25 ℃.Used back 20 days, and be determined at the infringement sum on the blade of growing fully when using, measure the mean that damages on the every blade, and measure control by following formula and tire.

Control is tired=100-(processed group is on average damaged number)/(control group on average damages number) * 100

The results are shown in Table 17.In table 17, independently represent first blade of second blade, the second strain plant of first blade, the first strain plant of the first strain plant and second blade of the second strain plant separately about digital 1-1,1-2,2-1 and 2-2.The outward appearance of back 20 days processed group of experiment beginning is seen Figure 16.The control of using the BAL bacteria culture fluid and obtaining is tired and is used Daconil 1000 and the control that obtains is tired and is 100.This shows, uses the BAL bacteria culture fluid and uses Daconil 1000 can suppress the cucumber brown spot.

Table 17

Embodiment 21

The BAL bacteria culture fluid is to the test of the effect of black spot (alternaria brassica)

Scrape the BAL bacterium of getting a loopful from the YPA slant medium, join in the 300ml conical flask that 100ml soybean curb residue extract medium is housed, with the reciprocal shaker of amplitude 10cm in 120rpm, 25 ℃ of shaken cultivation 4 days.Use small-sized hand-operated sprayer, with this BAL bacteria culture fluid even spraying in turnip (kind: Natsumaki No.13, Kokabu; Date of application: on March 10th, 2004; Extent of growth when using: 3 trophophylls, cultivation reaches 20 days in 75mm black plastic alms bowl) on the blade, then with it in humid room, placed 24 hours at 25 ℃.The spore that before was incubated at the alternaria brassica (Laboratory of Pathologyand Biotechnology of the Musashino Seed Co., the original seed of Ltd.) on the PSA flat board is suspended in the distilled water.Use small-sized hand-operated sprayer, gained suspension even spraying on the turnip blade, in humid room, 25 ℃ of placements 24 hours, 20 ℃ of management, is developed it up to symptom.When using, regulate spore density in the suspension by the dilution suspension, thereby reach 40 spores of under microscopical each visual field of 200x, having an appointment.In order to compare, following two groups are provided: be sprayed with 1000 times of iprodione wettable powder solution one group on the blade on one group (control group) of water spray rather than BAL bacteria culture fluid and the blade as existing control agent.Study according to following standard, 3 blades of every strain plant are studied.

0: asymptomatic

1: the area that shows symptom accounts for below 25% of blade area

2: the area that shows symptom accounts for the 25%-50% of blade area

3: the area that shows symptom accounts for more than 50% of blade area

4: withered

According to result of study, obtain the incidence of disease according to following formula:

The incidence of disease=(1n ₁+ 2n ₂+ 3n ₃+ 4n ₄The plant sum of)/(4 * studied)

Wherein, n ₁, n ₂, n ₃And n ₄Independent separately representative is according to the plant quantity of above criteria classification.

According to the incidence of disease that is calculated, obtain control according to following formula and tire:

Control is tired=the 100-processed group incidence of disease/control group incidence of disease * 100

The results are shown in Table 18.Figure 17 is presented at the blade outward appearance when estimating the incidence of disease.As table 18 and shown in Figure 17, use the BAL bacteria culture fluid and cause black spot is suppressed fully.

Table 18

	The incidence of disease	Control is tired
			Water (control group)	83.3	-
The BAL bacteria culture fluid	0.0	100.0
			1000 times of iprodione solution	0.0	100.0

Embodiment 22

The BAL bacteria culture fluid is to the test of the effect of white blister fungi (big spore white rust)

Scrape the BAL bacterium of getting a loopful from the YPA slant medium, join in the 300ml conical flask that 100ml soybean curb residue extract is housed, with the reciprocal shaker of amplitude 10cm in 120rpm, 25 ℃ of shaken cultivation 4 days.Use small-sized hand-operated sprayer, with this BAL bacteria culture fluid even spraying in a variety of Chinese cabbage (kind: Fukushoumi; Date of application: on February 12nd, 2004; Extent of growth when using: 5 trophophylls, cultivation reaches 30 days in 75mm black plastic alms bowl) on the blade face, with the sample after the spraying in humid room, placed 24 hours at 25 ℃.Allow sample place again 24 hours at 25 ℃, zoosporangium that will the acervulus of the big spore white rust of spontaneous formation is gathered on Brassica chinensiskomatsuna from breeding nursery is suspended in the distilled water, use small-sized hand-operated sprayer, suspension is sprayed in the sample, allows the sample of spraying in humid room, 11 ℃ of placements 24 hours.When using in the suspension zoosporangium concentration dilution to 30 zoosporangiums of under microscopical each visual field of 200x, having an appointment.Up to the symptom development, only after darkness, use tunnel (tunnel) and heating clamber.In order to compare, provide a group (control group) of water spray rather than sprinkling BAL bacteria culture fluid.Studied the acervulus quantity on the unit are on two blades of each strain plant of using big spore white rust as mentioned above.According to result of study, obtain the control of processed group by following formula and tire.

Control is tired=100-(every 1cm in the treatment group ²The quantity/control group of acervulus in every 1cm ²The quantity of acervulus) * 100

The results are shown in Table 19.Figure 18 shows disease leaf outward appearance.Use the resulting control of the BAL bacteria culture fluid height of tiring.This shows that such using is effectively, proved its practicality.

Table 19

	Quantity/the cm of acervulus 2	Control is tired
			Water (control group)	112.0	-
The BAL bacteria culture fluid	0.515	99.5

Embodiment 23

The BAL bacteria culture fluid is to the effect of radish point sickle spore (Fusarium oxysporum f.sp.raphani) The test of fruit

(Ltd. sells, wheat bran composition: 29%N, 38%P for 40g, Chiba Seifun Co. with the Ecobran material ₂O5,2.0%K ₅O, 52.5% organic matter and 35%-40% moisture; PH 7.5; C/N ratio: 10) and the 10g rice bran fully mix, mixture is carried out autoclaving, 121 ℃ reach 1 hour, are carrying out one time autoclaving after 24 hours more subsequently under similarity condition.Join in the sterile material with sterile water (30ml) with the BAL bacterium soybean curb residue culture powder (0.6g is referring to embodiment 6) of 10 times of diatomite dilutions, they are fully mixed, placed 4 days at 30 ℃, products therefrom is called the material that contains the BAL bacterium.The radish point sickle spore of cultivating in advance on the PSA flat board in advance (Laboratory of Pathology and Biotechnology of theMusashino Seed Co, the original seed of Ltd.) is cut into 5mm ²Fritter, join in the 300ml conical flask that the 100mlPG medium is housed, with the reciprocal shaker of amplitude 10cm in 120rpm, 25 ℃ of shaken cultivation 5 days.Culture fluid is filtered by double gauze, and 3, centrifugal 10 minutes of 000rpm reclaims spore.These spores are suspended in the distilled water, centrifugal under similarity condition, reclaim sediment.This step repeats twice, is used for washing.Sediment is suspended in the distilled water, and products therefrom evenly is incorporated in the sterile soil, and preparation contains 2 * 10 ⁷The contaminated soil of spore/ml radish point sickle spore.Gained soil was placed 24 hours at 25 ℃, and the material that will contain the BAL bacterium is fully mixed to the material that concentration reaches 500kg/10a and (contains 1 * 10 with it ⁷The cfu/gBAL bacterium), mixture was placed 6 days at 25 ℃, on May 20th, 2004 add turnip (kind: Natsumaki No.13, kokabu), then 25 ℃ of management.In order to compare, also test sterile soil, do not contained the contaminated soil (control group) of BAL bacterium material and joined chemical control agent in the contaminated soil, irrigate 31/m then ²1000 times of solution of benomyl wetting powder.Using the back 18 days withered degree of research cotyledon jaundice.According to investigation result, obtain the incidence of disease by following formula.

The individual sum of the incidence of disease=diseased plant number/investigation.

According to the incidence of disease of being asked, measure control by following formula and tire.

Control tiring=incidence of disease * 100 of the incidence of disease/control group of 100-processed group

The results are shown in Table 20.Figure 19 shows the back 18 days sample appearance of experiment beginning.Use the group of the control material that contains the BAL bacteria culture fluid,, show the effectiveness of higher inhibition disease compared with the group of using the chemical control agent.

Table 20

* in the diseased plant number, "-": symptom occurs; "+" wherein: asymptomatic

Embodiment 24

The BAL bacterium is to the effect of rice shoot damping off fungi (Rhizoctonia solani Kuhn (Rhizoctonia solani)) Test

Ecobran material (40g is described in embodiment 13) is fully mixed with the 10g rice bran, carry out autoclaving, 121 ℃ reach 1 hour, are carrying out one time autoclaving after 24 hours more subsequently under similarity condition.Join in the sterile material with sterile water (30ml) with the BAL bacterium soybean curb residue culture powder (0.6g is referring to embodiment 6) of 10 times of diatomite dilutions, they are fully mixed, placed 7 days at 30 ℃, products therefrom is called the material that contains the BAL bacterium.This material is incorporated in the sterile soil, and quantity reaches 1t/10a (in the soil 5 * 10 ⁷Cfu/g BAL bacterium), be allowed to condition in the dark, placed 10 days at 25 ℃.Use card punch, to 2 days rice shoot damping off fungi (Rhizoctonia solani Kuhn AG-4 of cultivation on the PSA flat board, IIIA, rice shoot damping off fungi, Laboratory of Pathology and Biotechnology of the Musashino Seed Co., Ltd. punching original seed), it is (long: 7cm that 3 small pieces floras are placed on ice cream cup with uniform distances; Wide: 7cm; High 5cm) on the bottom surface.Filling contains the sterile soil (125ml) of described material, covers the flora sheet, adds 100mg bent grass (Yukijirusi), in the controlled environment chamber in, at 28 ℃ it is managed.In order to compare, provide following two groups: one group has been used and had not wherein both been added the BAL bacterium, also do not add the sterile soil (sterile soil group) of rice shoot damping off fungi, and another group has been used the sterile soil (control group) that does not wherein add the BAL bacterium and add rice shoot damping off fungi.For these groups, except as otherwise noted, otherwise same way as adds and manages as described above.After using the BAL bacterium 11 days, check the quantity of survival strain, the quantity of survival strain is considered to use in every experimental group the common plant quantity of BAL bacterium in the sterile soil, measures the ratio of survival strain and the ratio of withered strain by following formula.

Survival strain quantity * 100 in the ratio=survival strain quantity/sterile soil of survival strain

The ratio of the ratio of withered strain=100-survival strain

According to the ratio of withered strain, measure control by following formula again and tire.

Control is tired=100-(the withered strain ratio of the withered strain ratio/control group of processed group) * 100

The results are shown in Table 21.Figure 20 shows the back 11 days sample appearance of experiment beginning.Although control is tired low (because high pollution level is arranged), but has observed effect.

Table 21

	The ratio (%) of survival strain	The ratio (%) of withered strain	Control is tired
				Sterile soil	100	0	-
Contrast	3	97	-
				Control material (1t/10a)	30	70	27.8

Embodiment 25

In the soil that tobacco mosaic virus (TMV) infects, use the control material that contains the BAL bacterium right The inhibitory action that TMV infects

The blade (4g, weight in wet base) that TMV infects is put into mortar, grind in 10ml 1/15M phosphate buffer (pH 6.98), preparation juice is stand-by.This juice is distributed in two 50mlFalcon pipes, adds phosphate buffer and make solution amount reach 50ml.Disinfection soil (100ml), 1.25g rice bran (500kg/10a equivalent) and the 50ml juice of dehydration are joined in the 500ml beaker.As BAL bacterium processed group, will be in the soybean curb residue extract 20ml BAL bacteria culture fluid of shaken cultivation centrifugal 30 minutes of 3000rpm to separate the BAL bacterium, the BAL bacterium that is separated to is suspended in the 50ml phosphate buffer, and the gained suspension joins in the above soil, fully mixes with glass bar again.Group is mixed the 50ml phosphate buffer in contrast.Soil surface covers with vinyl (vinyl), and beaker is wrapped, and opening part is tied, and the technology of simply getting sun (solar technique) is placed on beaker in 38 ℃ of incubators.Process began back 4 days, not taking out 3g soil from each process element joins the beaker, allow beaker leave standstill, by means of carborundum powder supernatant is joined among the Nicotiana glutinosa, this plant is TMV differential plant kind and has set up 8 true leaf plant pathology experimental methods, Shoji Sato, Masao Goto and Yoji Doi (writing), Kodansha Ltd.).Nicotiana glutinosa is managed in 20 ℃ of phytotrons, and counting is used back 6 days local lesion quantity that occurs, and measures blade area, by following formula, according to the infringement number of per unit area, measures control and tires.

Control is tired=100-(the infringement number of the infringement number/control group per unit area of processed group per unit area) * 100

The results are shown in Table 22.Sample appearance when Figure 21 shows morbidity.

Table 22

Embodiment 26

Inhibition to the infection of muskmelon withered spot disease poison

With 20 muskmelon seedses (kind: Earl ' s Miyabi) put into the 9cm culture dish, be lined with filter paper in this ware and be added with 4.25ml distilled water, allow them sprout 2 days at 25 ℃.With the 50ml Kureha garden mould of packing in the 25 hole cells, the seed of sprouting is placed on this soil, cover this cell with the 50ml sterile soil then.Rice shoot is cultivated in 25 ℃ of phytotrons.Testing used inoculum is prepared as follows: the blade (viral source: the land for growing field crops of the Isahaya in Japanese Nagasaki) put into mortar, grind blade in the phosphate buffer (pH 6.98) of 10ml 1/15M that will be chilled in-30 ℃ 2g muskmelon withered spot disease poison infection.Gained grinds homogenate and filters by double gauze, carries out following test then.

With the homogenate (3ml) of grinding with mix with 8 days 3ml BAL bacteria culture fluid of soybean curb residue medium shaken cultivation, container seals with Parafilm, puts into 25 ℃ of incubators then.After 5 hours and 22 hours, it is joined (according to embodiment 25) on the muskmelon cotyledon by means of carborundum powder.Group provides and has used one group that grinds homogenate and phosphate buffer in contrast.After using, plant manages in 25 ℃ of phytotrons.After application 7 or 8 days, count out the quantity of present epicotyledonary local lesion, obtain every epicotyledonary infringement quantity, measure control by following formula and tire.Mixing that control after 5 hours tires is 3.3, and mixing that control after 22 hours tires is 80.

Control is tired=100-(every epicotyledonary infringement quantity of every epicotyledonary infringement quantity/control group of processed group) * 100

The results are shown in Table 23.Sample appearance when Figure 21 shows morbidity.

Table 23

Industrial applicability

According to the present invention, can be to carrying out biological control by plant disease due to plant pathogenetic bacteria, fungi or virus infections or the propagation.

All publications, patent and patent application that this paper quotes all are attached to herein by reference.

<110>ITSUKI Co.，Ltd.

＜120〉method of usefulness bacillus controlling plant diseases and control agent

<130>PH-2257-PCT

<150>JP 2004-55059

<151>2004-02-27

<150>JP 2004-216083

<151>2004-07-23

<160>1

<170>PatentIn version 3.1

<210>1

<211>1545

<212>DNA

<213>Bacillus sp.

<400>1

tggagagttt gatcctggct caggacgaac gctggcggcg tgcctaatac atgcaagtcg 60

agcggacaga tgggagcttg ctccctgatg ttagcggcgg acgggtgagt aacacgtggg 120

taacctgcct gtaagactgg gataactccg ggaaaccggg gctaataccg gatggttgtc 180

tgaacygcat ggttcagaca taaaaggtgg cttyggctac cacttacaga tggacccgcg 240

gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccgacctga 300

gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360

agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt 420

tttcggatcg taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac 480

cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540

taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 600

tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt 660

gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720

accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 780

gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 840

gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 900

cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960

taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga 1020

taggacgtcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080

gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc 1140

agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200

atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca 1260

gcgaaaccgc gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc 1320

aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380

cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1440

ggtgaggtaa cctttatgga gccagccgcc gaaggtggga cagatgattg gggtgaagtc 1500

gtaacaaggt agccgtatcg gaaggtgcgg ctggatcacc tcctt

Claims (8)

1. plant disease controlling agent or control material, described control agent or control material comprise bacillus (Bacillus) bacterium that can suppress plant pathogenetic bacteria, fungi or virus infections or propagation, wherein said bacillus is the DAIJU-SIID2550 bacterial strain, and the preserving number of this bacterial strain is FERM BP-10114.

The control agent of claim 1 or the control material, wherein said phytopathogen is Agrobacterium (Agrobacterium), rod shape Bacillus (C7avibacter), Erwinia (Erwinia), pseudomonas (Pseudomonas), the logical Bordetella (Ralstonia) in Rolls, Corynebacterium (Corynebacterium), Curtobacterium (Curtobacterium), bulkholderia cepasea genus (Burkholderia) or Xanthomonas (Xanthomonas), and described plant disease is caused by them.

3. the control agent of claim 1 or control material, wherein said plant pathogenic fungi is gas fax bacterium or soil-borne fungus, and described plant disease is caused by them.

The control agent of claim 1 or the control material, wherein said plant-pathogenic virus is tobacco mosaic virus (Tobacco mosaic virus), the light-duty mottle virus of capsicum (Peppermild mottle virus), Tomato mosaic virus (Tomato mosaic virus), muskmelon withered spot disease poison (Melon necrotic spot virus), cucumber green mottle mosaic virus (Cucumber greenmottle mosaic virus) or Kyuri green mottle mosaic virus (Kyuri green mottle mosaicvirus), and described plant disease is caused by them.

5. the method for a controlling plant diseases, described method comprise the host plant that the control agent of claim 1 or control material is applied to plant pathogenetic bacteria, fungi or virus.

6. the method for claim 5, wherein said plant belongs to Cruciferae (Brassicaceae), Solanaceae (Solanaceae), Curcurbitaceae (Cucurbitaceae), Liliaceae (Liliaceae), pulse family (Leguminosae), composite family (Asteraceae), Chenopodiaceae (Chenopodiaceae), grass family (Gramineae), the rose family (Rosaceae), Caryophyllaceae (Caryophyllaceae), Primulaceae (Primulaceae), Rutaceae (Rutaceae), Vitaceae (Vitaceae), Actinidiaceae (Actinidiaceae), Ebenaceae (Ebenaceae), Umbelliferae (Umbelliferae), Convolvulaceae (Convolvulaceac) or Araeceae (Araceae).

7. the method for claim 5, wherein said control agent or control material are selected from following processing and are applied to plant by at least a: with described control agent or control material to the plant seed dressing, plant seed is immersed in the suspension that contains described control agent or control material, described control agent or control material are irrigated plant cultivation with in the soil, described control agent or control material are incorporated into plant cultivation with in the soil, described control agent or control material are sprayed on the plant stem-leaf, and make described control agent or control material contact plant damage.

8. a Bacillus strain is DAIJU-SIID2550, and its preserving number is FERMBP-10114.

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