CN103789234B - A kind of bacillus amyloliquefaciens and application thereof - Google Patents

A kind of bacillus amyloliquefaciens and application thereof Download PDF

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CN103789234B
CN103789234B CN201410017081.6A CN201410017081A CN103789234B CN 103789234 B CN103789234 B CN 103789234B CN 201410017081 A CN201410017081 A CN 201410017081A CN 103789234 B CN103789234 B CN 103789234B
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ginseng
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bacillus amyloliquefaciens
bacterium
tank
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CN103789234A (en
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杨利民
孙卓
韩梅
林红梅
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Jilin Agricultural University
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Abstract

Do you the invention provides a kind of bacillus amyloliquefaciens (Bacillus? amyloliquefaciens) SZ-60, is characterized in that, does is its preserving number CGMCC? No.8277.Described bacterial strain irregular, the oyster white of bacterium colony that monoclonal growth is formed on beef extract-peptone (NB) substratum, center are slightly swelled, surface wettability, translucent; Microscopy is shaft-like, and size is 0.3 ~ 0.4 × 3.2 ~ 3.3 μm, tool flagellum, G ﹣, have gemma containing 2% ~ 5% NaCl beef extract-peptone (NB) substratum on all can grow; Does is the formula of described beef extract-peptone (NB) substratum: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 17g, water 1000ml, pH? 6.8 ~ 7.2.Bacillus amyloliquefaciens (Bacillus? amyloliquefaciens) SZ-60 has remarkable antagonistic action to causing the main pathogenic fungi of ginseng maize ear rot, epidemic disease, sclerotium disease, rust rot, black spot and damping-off.The present invention also provides the application of bacillus amyloliquefaciens SZ-60 in control fungal diseases of plants, or the application in the microbial preparation of preparation control fungal diseases of plants.

Description

A kind of bacillus amyloliquefaciens and application thereof
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens and application thereof.
Background technology
Ginseng (PanaxginsengC.A.Mey) is the negative dicotyledonous medicinal plant of Araliaceae Panax perennial root, is the rare medicinal herbs of China, has the good reputation of " kings of hundred grass ".Ginseng has establishing in large scale in countries such as China, Korea S, Korea, Russia, and wherein, Northeast China is the main producing region of ginseng, has become one of mainstay industry of locality.The long-term artificial growth of ginseng and breed breeding difficulty cause greatly that ginseng germplasm is degenerated, disease is serious, of poor quality, yield poorly.A large amount of uses of chemical pesticide, cause pesticide residue and environmental pollution, reduce security and the commodity value of ginseng crude drug.Disease prevention and control problem has become one of significant problem of restriction ginseng industry Sustainable development.
Ginseng diseases about has 20 ~ 40 kinds, wherein, 6 class pathogenic bacterias are the main pathogenic fungi causing ginseng Common Diseases and limit ginseng industry development below: the general sickness rate of ginseng maize ear rot caused by fusarium solani (Fusariumsolani) is about 30%, 6 years strangers join the mortality ratio that root rot causes can reach 50% ~ 80%, main harm seedling root and rhizome portion (below earth's surface stem), the ginseng root rotted is chocolate web rot shape, the rotten shape of later stage grain, only deposits the root skin of hollow; The Ginseng Blight In China sickness rate that Phytophthora cactorum bacterium (Phytophthoracactorum) causes can reach 70%, and its symptom is for blade producing irregular dark green color spot when falling ill light, and severe one cauline leaf is withered, butt rot, and plant is withered in flakes, and the underproduction is serious; The ginseng sclerotium disease main harm that Sclerotinia ginseng (Sclerotiniaschinseng) causes life in 3 years is above joins root, site of pathological change is bud bud, root welding technology, after ginseng velamen evil, initial stage is raw a little white fluffy mycelium on surface, after, inner corrupt, softening rapidly, cell is all cleared up totally, only leave downright bad exterior skin, the inside and outside sclerotium forming many mouse excrement shapes of epidermis; The general sickness rate 20%-30% of the black fleck disease of ginseng that ginseng alternaric bacteria (Alternariapanax) causes, reaches 100% time serious, causes early leaf fall, and plant withers, shaky, ginseng root and the underproduction of ginseng seed; The Ginseng Rhizoctonia Solani that dry thread Pyrenomycetes (Rhizoctoniasolani) causes is one of ginseng Major Diseases in seedling stage, under the condition of low temperature high humidity, developmenting spread is very rapid, general sickness rate is 8 ~ 30%, severe patient can reach about 40%, germ make the stem of seedling 3 ~ 5 centimetres of dry wet soil interfaces on ground hang contracting, rot, cut off transfusion tissue, cause seedling to lodge; When the ginseng rust maize ear rot that destruction post spore bacterium (Cylindrocarpondestructans) causes is serious, sickness rate reaches more than 70%, this disease betides each position of root, and scab is rust, diffuses to full root by point to face, large, ventilative bad, the soil ulmin thickness of soil humidity, morbidity is heavy.
The effect that ginseng diseases does not reach people's anticipation is is far away prevented and treated for a long time by Chemical control methods such as use chemical pesticides, and the micro-ecological environment of excessive use chemical pesticide meeting spoiled soil, weighting ring environment pollution, also toxic substance is made to accumulate in a large number in ginseng root, reduce safety in utilization and the commodity value of ginseng, therefore, people progressively turn to prevention emphasis in bio-control method and cultural control measure.In crop plants protection field, utilizing crop rhizosphere soils to screen soil microorganisms pathogenic bacteria to remarkable antagonistic effect, and make the important means that microbial preparation is biological prevention and control research, is also the important channel of beneficial microorganism development of resources.
Biological control is based on ecological principle, utilizes the interaction between living species, with a kind of or a class biological inhibition another kind of or another kind of biology.The maximum advantage of biological control is free from environmental pollution, does not have pesticide residue, and this is that the non-biological methods prevention and elimination of disease and pests institutes such as agricultural chemicals cannot be than.Utilizing antagonistic microbe to prevent and treat pathogenic micro-organism is the important component part of biological prevention, it avoid chemical pesticide use bring a series of plant protection, environment and energy aspect problem, avoid the harm of pesticide residue to people and animals, the more important thing is the Sustainable development facilitating agricultural.The U.S. has GB03, MBI600, QsT713 and conciliates 4 bacillus subtilis biocontrol strains acquisition Environmental Protection Agency's commercializations such as starch Bacillus subtilis var or limited merchandized handling application license.Separate starch Bacillus subtilis var FZB24 in Germany to be commercially produced, for controlling the different fungal diseases of food crop.
Genus bacillus (Bacillus) is one of the important microbe in biocontrol of plant disease research, and the many distributions of its kind are wide, are the dominant populations of soil and plant Tiny ecosystem.This quasi-microorganism, by secretion antibiotics and growth competition, plays multiple beneficial effect in controlling plant diseases.Utilize bacillus amyloliquefaciens to prevent and treat ginseng fungal disease at present and have no report.
Summary of the invention
The object of the invention is to: the main fungal venereal disease evil in producing for ginseng, especially ginseng soil-borne disease occurring area expanding day, Agro-chemicals control is except easily causing environmental pollution and pesticide residue, the practical situation that preventive effect is also undesirable, propose a kind of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) bacterial strain SZ-60 and causing ginseng maize ear rot respectively, rust rot, black spot, damping-off, the fusarium solani (F.solani) of epidemic disease and sclerotium disease, destroy post spore bacterium (C.destructans), ginseng alternaric bacteria (A.panax), dry thread Pyrenomycetes (Rh.solani), application in Phytophthora cactorum bacterium (Ph.cactorum) and Sclerotinia ginseng (S.schinseng) 6 kinds of pathogenic bacteria prevention and control.
The invention provides one and cause ginseng maize ear rot to above-mentioned, rust rot, black spot, damping-off, 6 kinds of pathogenic bacterias of epidemic disease and sclerotium disease all have the bacillus amyloliquefaciens SZ-60 of good prevention and control effect, the preservation name of this bacterial strain is called bacillus amyloliquefaciens SZ-60, Classification And Nomenclature is BacillusamyloliquefaciensSZ-60, depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on September 25th, 2013, deposit number: CGMCCNo.8277, its 16SrDNA sequence length is 1436bp, specifically as shown in SEQIDNo:l.Application BLAST software and DNAMAN software etc. are analyzed, and by the 16SrDNA sequence of SZ-60 bacterial strain by BLAST comparison, can find the close strain sequence that homology is very high in GenBank.With SZ-60 bacterial strain similarity the highest be Bacillusamyloliquefaciens, homology reaches 99%.Find with UPGMA method phylogenetic tree construction according to MEGA5.10Beta2 software, SZ-60 and Bacillusamyloliquefaciens belongs to a hereditary branch together, and sibship is very close, and pro-borne reaches 100%.The qualification result of combining form and Physiological-biochemical Characters, can confirm that bacterial strain SZ-60 of the present invention is bacillus amyloliquefaciens (Bacillusamyloliquefaciens).
Bacillus amyloliquefaciens SZ-60 of the present invention irregular, the oyster white of bacterium colony that monoclonal growth is formed on beef extract-peptone (NB) substratum, center are slightly swelled, surface wettability, translucent; Microscopy is shaft-like, and size is 0.3 ~ 0.4 × 3.2 ~ 3.3 μm, tool flagellum, and G ﹣, has gemma; It can grow at the temperature of 25 DEG C ~ 40 DEG C, and its optimum growth temperature is 32 DEG C; Its growth pH scope is 6.5 ~ 8.0, the most suitable growth pH is 7.2; Aerobic growth; Nitrate reduction reaction generates red compound; Catalase is determined as the positive; Lipase reverse should be negative; The reaction of casein, tyrosine is negative; D-Glucose, D-wood sugar can not be utilized; Starch Hydrolysis test microscopy has dextrin to generate; Gelatin liquification test is positive; Citrate trianion utilizes test medium to be acid (green); V-P (pH7.0) measures and generates red compound; L-arabinose, N.F,USP MANNITOL tests positive; Containing 2% ~ 5% NaCl beef extract-peptone (NB) substratum on all can grow.The formula of described beef extract-peptone (NB) substratum is: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 17g, water 1000mL, pH6.8 ~ 7.2.
The invention still further relates to the application of bacillus amyloliquefaciens SZ-60 in control fungal diseases of plants, or preparation control fungal diseases of plants microbial preparation in application, described plant optimization is ginseng, described fungi preferably causes the fusarium solani (F.solani) of ginseng maize ear rot, cause destruction post spore bacterium (C.destructans) of ginseng rust maize ear rot, cause the ginseng alternaric bacteria (A.panax) of black fleck disease of ginseng, cause the dry thread Pyrenomycetes (Rh.solani) of Ginseng Rhizoctonia Solani, one or more in the Phytophthora cactorum bacterium (Ph.cactorum) causing Ginseng Blight In China and these 6 kinds of pathogenic bacterias of the Sclerotinia ginseng (S.schinseng) causing ginseng sclerotium disease.Bacillus amyloliquefaciens SZ-60 of the present invention also has somatotrophic effect to ginseng.
The invention still further relates to the microbial preparation of the spore of full nutrient solution culture containing bacillus amyloliquefaciens SZ-60 and bacillus amyloliquefaciens SZ-60, it prepares by following preparation method:
(1) bacillus amyloliquefaciens SZ-60 test tube kind is inoculated in bottled 300mL nutrient broth yeast extract paste (NBY) nutrient solution of use 1000mL triangle, at 180 turns/min, cultivates 36 hours at 30 DEG C, obtain zymocyte liquid;
(2) zymocyte liquid is being inoculated in fermentation culture in the fermentation culture in seeding tank with seeding tank volume 20%, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of seeding tank 30 ~ 34 DEG C, inoculation fermentation bacterium liquid, after 4 hours, carried out fermented quality detection every 2 hours to the fermented liquid in seeding tank, until fermented liquid thalli growth is fuller when being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12 ~ 16 hours;
(3) fermentation culture in the NBY nutrient solution in fermentor tank is inoculated in by 10% of fermented bacterium in seeding tank, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of fermentor tank 30 ~ 34 DEG C, inoculation fermentation bacterial classification is after 4 hours, every 2 hours, fermented quality detection is carried out to the fermented liquid in fermentor tank, observe bacterial content, until final cultures (comprising bacterium and the gemma) biomass in ferment tank liquid is 10 9when more than cfu/ml, spore content are more than or equal to 90%, immediately fermented liquid is gone out tank, carry out packing, obtain SZ-60 microbial preparation; Fermentation time in fermentor tank is 24 ~ 36 hours.
The formula of NBY nutrient solution is: extractum carnis 3.5g, peptone 10.0g, yeast extract 5.0g, malt extract 10g, glucose 5.0g, water 1000mL, pH6.8 ~ 7.2; Preparation method is: take extractum carnis, peptone, yeast extract, malt extract, glucose puts into 1000mL water, after abundant mixing, pH is adjusted to 6.8 ~ 7.2, in 1000mL triangular flask, loading amount is 300mL nutrient solution, triangle bottleneck is sealed with double-deck sealed membrane, 121 DEG C of moist heat sterilizations 30 minutes, for subsequent use after cooling.The formula of fermentation culture is by weight percentage: soyflour 2.0%, soybean cake powder 1.5%, starch 0.5%, yeast extract paste 0.2%, corn steep liquor 0.2%, NaCl0.1%, Ca (HCO 3) 20.2%, surplus is water, pH7.0 ~ 7.2; Preparation method adds required water in seeding tank, adds soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO in proportion 3) 2, fully stir, fermentation culture pH is adjusted to 7.0 ~ 7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, inoculates after cooling immediately.
In the fermenting process of seeding tank or fermentor tank, when there is more foam, can add defoamer, described defoamer is organosilicon, and add-on is not overflowed with the foam in seeding tank or fermentor tank and is as the criterion; After fermentation cylinder for fermentation completes, in fermentor tank, add the benzoic acid preservative accounting for total liquid volume 2/10000ths in tank, stir, then go out tank and carry out packing.
The fermented liquid obtained by above-mentioned zymotechnique is a kind of microbial bactericide.Microbial preparation of the present invention can be applied in soil equably with the form of diluent, and the dilution volume ratio of microbial preparation and water is 1:100.In addition, can also add auxiliary agent organosilicon in diluent, the volume ratio of organosilicon and diluent is 1:5000.
The invention has the advantages that, preserving number is that the bacillus amyloliquefaciens SZ-60 of CGMCCNo.8277 is to causing ginseng maize ear rot respectively, rust rot, black spot, damping-off, the fusarium solani (F.solani) of epidemic disease and sclerotium disease, destroy post spore bacterium (C.destructans), ginseng alternaric bacteria (A.panax), dry thread Pyrenomycetes (Rh.solani), Phytophthora cactorum bacterium (Ph.cactorum) and Sclerotinia ginseng (S.schinseng) 6 kinds of pathogenic bacterias all have good prevention effect, to ginseng, there is growth promoting function, nontoxic no pathogenicity, to person poultry safety, free from environmental pollution, simultaneously, be applied directly to after biocontrol fungicide dilution and in soil, root irrigation carried out to plant and can play its germicidal action, significantly can improve the Rational structure of microflora in Ginseng Rhizosphere environment, form a bio-diversity Soil of Ginseng Rhizomsphere micro-ecological environment, thus effectively, enduringly control the popular of ginseng fungal disease.
Accompanying drawing explanation
Fig. 1: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth destroying post spore bacterium (Cylindrocarpondestructans) mycelia.
Fig. 2: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth of fusarium solani (Fusariumsolani) mycelia.
Fig. 3: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth of ginseng alternaric bacteria (Alternariapanax) mycelia.
Fig. 4: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth of Sclerotinia ginseng (Sclerotiniaschinseng) mycelia.
Fig. 5: the dull and stereotyped restraining effect of growth of bacillus amyloliquefaciens SZ-60 bacterial strain Rhizoctonia solani (Rhizoctoniasolani) mycelia of the present invention.
Fig. 6: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth of Phytophthora cactorum bacterium (Phytophthoracactorum) mycelia.
Fig. 7: SZ-60 to the growth promoting function of ginseng (A: blank, B: cast SZ-60 bacteria suspension).
Fig. 8: SZ-60 to the prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of destroying the microbial ginseng rust maize ear rot of post spore.
Fig. 9: SZ-60 to the prevention effect of the microbial Ginseng Blight In China of Phytophthora cactorum (A: blank, B: cast SZ-60 bacteria suspension).
Figure 10: SZ-60 to the prevention effect of the ginseng sclerotium disease that Sclerotinia ginseng causes (A: blank, B: cast SZ-60 bacteria suspension).
Figure 11: SZ-60 to the prevention effect of the microbial ginseng maize ear rot of fusarium solanae (A: blank, B: cast SZ-60 bacteria suspension).
Figure 12: SZ-60 to the prevention effect of the microbial black fleck disease of ginseng of ginseng rod method (A: blank, B: cast SZ-60 bacteria suspension).
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of the Ginseng Rhizoctonia Solani that Figure 13: SZ-60 Rhizoctonia solani causes.
Embodiment
Embodiment 1
The separation of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) SZ-60 bacterial strain and preservation
The soil of this bacterial strain around the perennial Ginseng Rhizosphere in Fusong County Wan Liang town, Jilin Province culture of ginseng ground within 30cm is separated and obtains.Gather above-mentioned pedotheque, sieve after air-dry.Take sample 10g, put into the triangular flask that granulated glass sphere and 90mL sterilized water are housed, fully vibrate 10-30min, and sample is mixed with sterilized water, and obtained sample suspension, leaves standstill 5min.Aseptically get 1mL supernatant liquor, add the 9mL0.05%SDS aqueous solution (lauryl sodium sulfate aqueous solution), 40 DEG C of insulation 20min, get 1mL, add 9mL sterilized water, make 10 successively by gradient -3, 10 -4, 10 -5diluent.Drawing each diluent of 100 μ L respectively joins on beef extract-peptone (NB) flat board, adopts plate dilution method even spread, often processes 3 times and repeats, and 34 DEG C of incubators are cultivated 1 ~ 2 day.Picking individual colonies is transferred to NB slat chain conveyor, after growing bacterium colony, carries out line separation and purification with transfering loop, and purifying bacterial strain is in 4 DEG C of preservations.The formula of beef extract-peptone (NB) substratum is: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 17g, water 1000mL, pH6.8 ~ 7.2.
This bacterial strain irregular, oyster white of bacterium colony that monoclonal growth is formed on NB substratum, center are slightly swelled, surface wettability, translucent; Microscopy is shaft-like, and size is 0.3 ~ 0.4 × 3.2 ~ 3.3 μm, tool flagellum, and G ﹣, has gemma; It can grow at the temperature of 25 DEG C ~ 40 DEG C, and its optimum growth temperature is 32 DEG C; Its growth pH scope is 6.5 ~ 8.0, the most suitable growth pH is 7.2; Aerobic growth; Nitrate reduction reaction generates red compound; Catalase is determined as the positive; Lipase reverse should be negative; The reaction of casein, tyrosine is negative; D-Glucose, D-wood sugar can not be utilized; Starch Hydrolysis test microscopy has dextrin to generate; Gelatin liquification test is positive; Citrate trianion utilizes test medium to be acid (green); V-P (pH7.0) measures and generates red compound; L-arabinose, N.F,USP MANNITOL tests positive; Containing 2% ~ 5% NaCl NB substratum on all can grow.
This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 25th, 2013, and preserving number is CGMCCNo.8277.
Our called after SZ-60 of this bacterial strain, Classification And Nomenclature is: bacillus amyloliquefaciens (Bacillusamyloliquefaciens).
Embodiment 2
Bacillus amyloliquefaciens SZ-60 bacterial strain is to the restraining effect of ginseng fungal disease growth of pathogenic bacteria
Filter paper enzyme is adopted to measure bacterial strain SZ-60 to the antagonistic action of ginseng fungal disease pathogenic bacteria: the bacterium cake with the punch tool of diameter 8mm, the ginseng pathogenic bacteria bacterium colony activated being made corresponding size, aseptic inoculation is to the centre of potato dextrose agar (PDA) dull and stereotyped (diameter 90mm), and the sterilizing filter paper dick being simultaneously 1cm by 4 diameters sticks at apart from 4 angle points at plate center 25mm place.Bacterial strain SZ-60 is made bacteria suspension, and (concentration is about 10 8cfu/mL), wherein 3 every sheet points of filter paper dick connect 20 μ L bacteria suspensions, and 1 filter paper dick point connects 20 μ L sterilized waters, and as treatment group, each process repeats 3 times; Separately on 4 filter paper dicks of 4 angle points of one flat plate, connect 20 μ L sterilized waters respectively, as a control group, all be placed in 28 DEG C of incubators to cultivate 6 ~ 7 days, treat that control group pathogenic bacteria bacterium colony covers with flat board, measurement processing group pathogenic bacteria colony diameter (unit: mm), and according to following formulae discovery bacteriostasis rate.Often kind of pathogenic bacteria repeats 3 times, results averaged.
Bacteriostasis rate (%)=[ (A-B)/(A-8) ] × 100%
Note: A is control group pathogenic bacteria colony diameter, i.e. 90mm; B is treatment group pathogenic bacteria colony diameter.
The preparation method of potato dextrose agar (PDA) substratum: take peeled potatoes 200g, glucose 20g, add water 1000mL, regulates pH to be 7.0.Boiling water bath heats 20min, and after filtered through gauze, constant volume is to 1000mL, adds agar 22g and melts rear packing moist heat sterilization (121 DEG C, 30min).
The preparation method of above-mentioned bacteria suspension: after the bacterial strain SZ-60 of preservation activates 2 days by plate streaking mode, the bacterial colony of 3 ~ 4 pieces of diameter 1cm is got with transfering loop, be linked into 200mL containing in the triangular pyramidal bottle of 50mL beef extract-peptone (NB) nutrient solution, shaking table 32 DEG C, 180r/min makes concentration after cultivating 48h and is about 10 8the Bacteria suspension of cfu/mL.The formula of NB nutrient solution is: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, water 1000mL, pH6.8 ~ 7.2.
Result is as shown in table 1, and bacterial strain SZ-60 all has obvious restraining effect to causing the fusarium solani of ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease and sclerotium disease (F.solani), destruction post spore bacterium (C.destructans), ginseng alternaric bacteria (A.panax), dry thread Pyrenomycetes (Rh.solani), Phytophthora cactorum bacterium (Ph.cactorum) and Sclerotinia ginseng (S.schinseng) 6 kinds of pathogenic bacterias respectively.Particularly to causing the Phytophthora cactorum bacterium of Ginseng Blight In China and causing the bacteriostasis rate of the destruction post spore bacterium of ginseng rust maize ear rot to reach more than 90%, to causing the ginseng alternaric bacteria of black spot, causing the fusarium solani of ginseng maize ear rot and causing the bacteriostasis rate of the Sclerotinia ginseng of ginseng sclerotium disease to be 71 ~ 82%, also 46.4% is reached to the bacteriostasis rate of the dry thread Pyrenomycetes causing Ginseng Rhizoctonia Solani, reflects that the preventive and therapeutic effect of bacterial strain SZ-60 to ginseng 6 kinds of the main pathogenic fungi has broad spectrum.
Table 1 bacterial strain SZ-60 is to the restraining effect of ginseng pathogenic fungi
Embodiment 3
Bacillus amyloliquefaciens SZ-60 tests the antagonistic activity of ginseng pathogenic bacteria
Odontothrips loti is adopted to measure bacterial strain SZ-60 to the antagonistic activity of ginseng fungal disease germ: after SZ-60 bacterial strain being made bacteria suspension, to collect (preparation method is with example 2), under 4 DEG C of conditions, the centrifugal 20min of 12000r/min, collect supernatant liquor through 0.22 μm of filtering with microporous membrane, gained filtrate 4 DEG C saves backup.Be the ginseng germ bacterium cake of 8mm at PDA dull and stereotyped central authorities inoculation diameter under aseptic condition, then 4 aseptic Oxford cups are put in 4 the symmetrical angle points being about 25mm apart from plate center, drip 100 μ l filtrates in every glass, often process 3 times and repeat, cultivate after 5 days for 28 DEG C and measure antibacterial bandwidth.
Result shows, SZ-60 is to fusarium solani (F.solani), destroy post spore bacterium (C.destructans), dry thread Pyrenomycetes (Rh.solani), ginseng alternaric bacteria (A.panax), the antibacterial bandwidth of Sclerotinia ginseng (S.schinseng) and Phytophthora cactorum bacterium (Ph.cactorum), be respectively 5.4mm, 7.8mm, 1.8mm, 5.2mm, 11.3mm, 6.7mm, illustrate that the supernatant liquor of SZ-60 bacteria suspension is less than except 5mm except the antibacterial bandwidth of Rhizoctonia solani, all 5mm is greater than to the antibacterial bandwidth of all the other 5 kinds of pathogenic bacterias, wherein 10mm is greater than to the antibacterial bandwidth of Sclerotinia ginseng, illustrate that the supernatant liquor of SZ-60 bacteria suspension has antagonistic activity (table 2) in various degree to 6 kinds of ginseng pathogenic bacterias.
Table 2 bacillus amyloliquefaciens SZ-60 is to the antagonistic activity of ginseng pathogenic fungi
Note: with reference to Vestberg method, "+", " ++ ", " +++ " represent that antibacterial band radius is <5mm, 5-10mm, >10mm respectively.
Embodiment 4
Bacillus amyloliquefaciens SZ-60 bacterial strain is to the growth-promoting functions of ginseng
(32 DEG C, 180r/m, 48h, concentration is about 10 to preparation SZ-60 bacterial strain bacteria suspension 8cfu/mL, preparation method is with embodiment 2).By new for ginseng woods soil: vermiculite, according to 2:1 proportions matrix, is loaded after sterilizing in same volume flowerpot.Application is taken at 3 years stranger's seedlings in Fusong Wan Liang county, carefully shakes off to be attached to the soil of root, random packet.Treatment group ginseng-leaf is soaked in pre-configured SZ-60 bacteria suspension, takes out after 25-30min, transplant respectively in the flowerpot that sterilized soil is housed, the strain of every basin 5.(bacteria containing amount is about 10 to every basin SZ-60 bacteria suspension 8cfu/mL) 50 times of sterilized water diluent 30mL fill with root, often process 3 basins, random alignment, and greenhouse moisturizing is cultivated; Control group aseptic beef extract-peptone (NB) cultivates immersion seedling, and processing mode is the same, often processes No. 3 basins, random alignment, and greenhouse moisturizing is cultivated, and waters the aseptic culture fluid (except not containing except SZ-60, all the other compositions are identical with treatment group) of 30mL respectively.After ginseng-leaf grows to 30 days, random selecting treatment group and each 5 strain ginseng-leafs of control group, carefully dig out whole for seedling strain, wash away root earth respectively, measures its plant height, root length, whole strain fresh weight and root fresh weight index.Then dry to constant weight for 180 DEG C, survey whole strain dry weight and root dry weight.
Indoor pot measurement result shows (shown in table 3), inoculation SZ-60 bacterial strain is when sterilized soil plantation ginseng, ginseng plant plant height, whole strain fresh weight, root fresh weight, root length, whole strain dry weight and root dry weight all have increase in various degree compared with aseptic culture fluid, prove that SZ-60 bacterial strain has significant promoter action (Fig. 7-13) to Ginseng Growth.Meanwhile, also illustrate that SZ-60 is safe to ginseng.
Table 3 bacterial strain SZ-60 is to the promoter action of Ginseng Growth
Embodiment 5
Bacillus amyloliquefaciens SZ-60 grows test surely.
(1) bacillus amyloliquefaciens SZ-60 ginseng cauline leaf determine grow
Be inoculated in NB nutrient solution, 32 DEG C, 180r/min by with Rifampin (300 μ g/mL) labeled bacillus amyloliquefaciens SZ-60, shaking table vibration 24h, makes bacteria containing amount and is about 10 8the labeled strain bacteria suspension of cfu/mL.By 3 years stranger's seedling (Fusong) transplant at random in the flowerpot that nature soil (take from Fusong County forest land, Jilin Province soil) and sterilized soil (taking from Fusong County forest land soil at 121 DEG C of moist heat sterilization 2h) are housed.Height of seedling 10cm final singling, the strain of every basin 3, the labeled bacillus amyloliquefaciens SZ-60 bacteria suspension of 10mL is injected to ginseng-leaf root soil, leaf and each 1.0g in rhizome portion of ginseng-leaf is gathered respectively after 10d, after its surface sterilization, add the grinding of 2mL sterilized water respectively, get on NB flat board that supernatant liquor coats respectively containing Rifampin (300 μ g/mL).Whether cultivate 4 days for 32 DEG C, observing flat board has bacillus amyloliquefaciens SZ-60 bacterium colony to grow.Result shows, leaf and rhizome portion all can be recovered to bacillus amyloliquefaciens SZ-60.This illustrate SZ-60 can in ginseng body the long period exist, it has endogeny.
(2) bacillus amyloliquefaciens SZ-60 determining in soil is grown
Be inoculated in NB nutrient solution, 32 DEG C, 180r/min by with Rifampin (300 μ g/mL) labeled bacillus amyloliquefaciens SZ-60, shaking table vibration 24h, makes bacteria containing amount and is about 10 8the labeled strain bacteria suspension of cfu/mL.Respectively naturally native and sterilized soil are loaded in flowerpot, every basin 1kg soil, the bacillus amyloliquefaciens SZ-60 bacteria suspension injecting 100mL labeled in soil mixes soil.Ambient temperatare is put, and (soil first, after gradient dilution, gets 10 to the bacterium in separation in 7 days 1 soil -3, 10 -4, 10 -5soil dilution liquid carry out flat board coating), calculate bacteria containing amount.Result shows, 28 days the determining the amount of growing and all can reach 10 of SZ-60 in soil and sterilized soil naturally afterwards 5more than cfu/g soil.This illustrates that SZ-60 has stronger colonization ability in soil.
Embodiment 6
The preparation of bacillus amyloliquefaciens SZ-60 zymocyte liquid
Bacillus amyloliquefaciens SZ-60 test tube kind is inoculated in bottled 300mL nutrient broth yeast extract paste (NBY) nutrient solution of use 1000mL triangle, at 180r/min, cultivates 36 hours at 30 DEG C, obtains zymocyte liquid.
The preparation method of NBY nutrient solution: take extractum carnis 3.5g, peptone 10.0g, yeast extract 5.0g, malt extract 10g, glucose 5.0g, put into 1000mL water, after abundant mixing, pH being adjusted to loading amount in 6.8 ~ 7.2,1000mL triangular flask is 300mL nutrient solution, seals triangle bottleneck with double-deck sealed membrane, 121 DEG C of moist heat sterilizations 30 minutes, for subsequent use after cooling.
Embodiment 7
The preparation of bacillus amyloliquefaciens SZ-60 microbial preparation
Bacillus amyloliquefaciens SZ-60 microbial preparation contains the full nutrient solution culture of bacillus amyloliquefaciens SZ-60 and the spore of bacillus amyloliquefaciens SZ-60, and preparation method is as follows:
(1) zymocyte liquid cultured in embodiment 6 is being inoculated in fermentation culture in the fermentation culture in seeding tank with seeding tank volume 20%, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of seeding tank 30 ~ 34 DEG C, inoculation fermentation bacterium liquid, after 4 hours, carried out fermented quality detection every 2 hours to the fermented liquid in seeding tank, until fermented liquid thalli growth is fuller when being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12 ~ 16 hours;
(2) fermentation culture in the NBY nutrient solution in fermentor tank is inoculated in by 10% of fermented bacterium in seeding tank, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of fermentor tank 30 ~ 34 DEG C, inoculation fermentation bacterial classification is after 4 hours, every 2 hours, fermented quality detection is carried out to the fermented liquid in fermentor tank, observe bacterial content, until final cultures (comprising bacterium and the gemma) biomass in fermented liquid is 10 9when more than cfu/ml, spore content are more than or equal to 90%, immediately fermented liquid is gone out tank, carry out packing, obtain SZ-60 microbial preparation; Fermentation time in fermentor tank is 24 ~ 36 hours.
The formula of fermentation culture is (by weight percentage): with soyflour 2.0%, soybean cake powder 1.5%, starch 0.5%, yeast extract 0.2%, corn steep liquor 0.2%, NaCl0.1%, Ca (HCO 3) 20.2%, surplus is water, pH7.0 ~ 7.2, and preparation method is: in seeding tank, add required water, adds soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO in proportion 3) 2, fully stir, fermentation culture pH is adjusted to 7.0 ~ 7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, inoculates after cooling immediately.
Embodiment 8
Bacillus amyloliquefaciens SZ-60 bacterial strain is to the field controling test of ginseng fungal disease
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of the fusarium solani of ginseng maize ear rot (Fusariumsolani) in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 10, and random district group arranges, and every community kind has ginseng 20 strain, repeated for 4 times.Before transplanting, 100 times of sterilized water diluents of SZ-60 microbial preparation prepared by treatment group embodiment 7 dip in root, and root rot their early stage starts to fill with root, and above-mentioned 100 times of diluent 30mL are filled with in every strain, fill with root twice respectively at June 20 and June 30.Contrasting for medicament with 250 of 10% derosal times of sterilized water diluents, take sterilized water as blank.June 20, July 10 respectively investigate occurring degree, and calculate disease index, investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of destruction post spore bacterium (Cylindrocarpondestructans) of ginseng rust maize ear rot in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted May 19.The group arrangement of random district, every community kind has ginseng 20 strain, repeats for 4 times.Before transplanting, 100 times of sterilized water diluents of SZ-60 microbial preparation prepared by treatment group embodiment 7 dip in root, fill with root when ginseng starts to fall ill to ginseng, and above-mentioned 100 times of diluent 30mL are filled with in every strain, fill with root twice respectively at May 29 and June 8.Contrasting for medicament with 1000 of 50% carbendazol wettable powder times of sterilized water diluents, take sterilized water as blank.May 29, June 18 is an investigation disease index respectively, and investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of the ginseng alternaric bacteria (Alternariapanax) of black fleck disease of ginseng in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted July 5.The group arrangement of random district, every community kind has ginseng 20 strain, repeats for 4 times.When ginseng starts to fall ill, treatment group ginseng is sprayed to 100 times of sterilized water diluents of SZ-60 microbial preparation prepared by embodiment 7, spray twice respectively at July 15 and July 23.Reaching 1500 times of sterilized water diluents with Amici to contrast for medicament, take sterilized water as blank.July 15, August 1 be an investigation disease index respectively, and investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of the Phytophthora cactorum bacterium (Phytophthoracactorum) of Ginseng Blight In China in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted July 5.The group arrangement of random district, every community kind has ginseng 20 strain, repeats for 4 times.When ginseng starts to fall ill, treatment group ginseng is sprayed to 100 times of sterilized water diluents of SZ-60 microbial preparation prepared by embodiment 7, spray twice respectively at July 15 and July 23.Contrasting for medicament with 1000 times of sterilized water diluents of 50% prochloraz-manganese chloride complex wettable powder, take sterilized water as blank.July 15, August 1 respectively investigate occurring degree, and calculate disease index, investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of the Sclerotinia ginseng of ginseng sclerotium disease (Sclerotiniaschinseng) in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 10, and random district group arranges, and every community kind has ginseng 20 strain, repeated for 4 times.Before transplanting, 100 times of sterilized water diluents of SZ-60 microbial preparation prepared by treatment group embodiment 7 dip in root, and sclerotium disease their early stage starts to fill with root, and above-mentioned 100 times of diluent 30mL are filled with in every strain, fill with root twice respectively at June 20 and June 30.Contrasting for medicament with 500 times of sterilized water diluents of 40% dimetachlone wettable powder, take sterilized water as blank.June 20, July 10 respectively investigate occurring degree, and calculate disease index, investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field to causing the controlling experiment of the dry thread Pyrenomycetes of Ginseng Rhizoctonia Solani (Rhizoctoniasolani) in 2013 to carry out, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 5, and random district group arranges, and every community kind has ginseng 20 strain, repeated for 4 times.After 7 days, 100 times of sterilized water dilution liquid irrigating roots of the SZ-60 microbial preparation that treatment group is prepared by embodiment 7, every strain 30mL, after Ginseng Rhizoctonia Solani starts morbidity, July 6, July 16 filled with root twice, contrasting for medicament with 500 of anilazine times of sterilized water diluents, take sterilized water as blank.July 6, July 26 respectively investigate occurring degree, and calculate disease index, investigation result is as table 4.
As above efficiency test method of calculation: disease index=[∑ (sick level strain number × typical value)/(total strain number × the highest sick level typical value)] × 100,
Relative control effect (%)=(contrast disease index-process disease index)/contrast disease index × 100
Note:
As shown in table 4, SZ-60 all has good preventive effect to by above-mentioned cause of disease microbial ginseng maize ear rot, ginseng rust maize ear rot, black fleck disease of ginseng, Ginseng Rhizoctonia Solani, Ginseng Blight In China and ginseng sclerotium disease, and prevention effect is equal to or slightly better with the primary medicament that contrasts of above-mentioned disease.
Table 4 bacterial strain is tested the prevention effect of above-mentioned ginseng fungal disease
Embodiment 9
Application TaKaRaMiniBESTBacterialGenomicDNAExtractionKitVer.2.0 test kit (precious biotechnology (Dalian) company limited) method extracts DNA, the PCR primer sequence of synthesizing voluntarily is 16S1F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 16S1R:5 '-TACGGCTACCTTGTTACGACTT-3 '.With SZ-60 phage gene group DNA for template, after PCR reaction amplification, detect through 1.5% agarose gel electrophoresis, obtain the specific fragment of an about 1500bp, measure this fragment sequence (adopting TIANGEN gel to reclaim test kit to check order in Sangon Biotech (Shanghai) Co., Ltd.), result shows, the DNA of SZ-60 bacterial strain is 1436bp, specifically as shown in SEQIDNo:1.The 16srDNA sequence application BLAST software recorded and DNAMAN software and clustalx are spliced software analyze, find that the homology of SZ-60 bacterial strain DNA sequence dna and bacillus amyloliquefaciens 16srDNA partial genome sequence is very high, reach 99%.Utilize MEGA5.10Beta2 software with 1000 stochastic samplings of UPGMA method phylogenetic tree construction, calculate bootstrap value (Bootstrap), find that SZ-60 and Bacillusamyloliquefaciens belongs to a hereditary branch together, sibship is very close, and pro-borne reaches 100%.The qualification result of combining form and Physiological-biochemical Characters, can confirm that bacterial strain SZ-60 is bacillus amyloliquefaciens (Bacillusamyloliquefaciens).
Each embodiment is not to concrete restriction of the present invention above; as long as according to the scope that claim limits, under the enlightenment of this patent, in conjunction with the basic general knowledge of this area; described bacterial strain is used for, in the control of ginseng fungal disease, all belong to protection scope of the present invention.

Claims (14)

1. bacillus amyloliquefaciens (Bacillusamyloliquefaciens) SZ-60, is characterized in that, its preserving number is CGMCCNo.8277.
2. bacillus amyloliquefaciens SZ-60 as claimed in claim 1, is characterized in that, this bacterial strain bacterium colony that monoclonal growth is formed on beef-protein medium is irregular, oyster white, center are slightly swelled, surface wettability, translucent; Microscopy is shaft-like, and size is 0.3 ~ 0.4 × 3.2 ~ 3.3 μm, tool flagellum, and G ﹣, has gemma; It can grow at the temperature of 25 DEG C ~ 40 DEG C, and its optimum growth temperature is 32 DEG C; Its growth pH scope is 6.5 ~ 8.0, the most suitable growth pH is 7.2; Aerobic growth; Nitrate reduction reaction generates red compound; Catalase is determined as the positive; Lipase reverse should be negative; The reaction of casein, tyrosine is negative; D-Glucose, D-wood sugar can not be utilized; Starch Hydrolysis test microscopy has dextrin to generate; Gelatin liquification test is positive; Citrate trianion utilizes test medium for green, in acid; When pH7.0, V-P measures and generates red compound; L-arabinose, N.F,USP MANNITOL tests positive; Containing 2% ~ 5% NaCl beef-protein medium on all can grow;
The formula of described beef-protein medium is: extractum carnis 3.0g, peptone 10.0g, NaCl5.0g, agar 17g, water 1000mL, pH6.8 ~ 7.2.
3. the application of bacillus amyloliquefaciens SZ-60 as claimed in claim 1 or 2 in control fungal diseases of plants, or the application in the microbial preparation of preparation control fungal diseases of plants.
4. apply as claimed in claim 3, it is characterized in that, described plant is ginseng.
5. the application as described in claim 3 or 4, it is characterized in that, described fungi is fusarium solani (Fusariumsolani), destroy in post spore bacterium (Cylindrocarpondestructans), ginseng alternaric bacteria (Alternariapanax), dry thread Pyrenomycetes (Rhizoctoniasolani), Phytophthora cactorum bacterium (Phytophthoracactorum) and Sclerotinia ginseng (Sclerotiniaschinseng) one or more.
6. a microbial preparation, is characterized in that, containing, for example the full nutrient solution culture of bacillus amyloliquefaciens SZ-60 according to claim 1 and the spore of bacillus amyloliquefaciens SZ-60 as claimed in claim 1.
7. microbial preparation as claimed in claim 6, it is characterized in that, it is prepared by following preparation method:
(1) bacillus amyloliquefaciens SZ-60 test tube kind as claimed in claim 1 is inoculated in the bottled 300mL nutrient broth yeast extract paste nutrient solution of use 1000mL triangle, at 180r/min, cultivates 36 hours at 30 DEG C, obtain zymocyte liquid;
(2) zymocyte liquid is being inoculated in fermentation culture in the fermentation culture in seeding tank with seeding tank volume 20%, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of seeding tank 30 ~ 34 DEG C, inoculation fermentation bacterium liquid, after 4 hours, carried out fermented quality detection every 2 hours to the fermented liquid in seeding tank, until fermented liquid thalli growth is fuller when being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12 ~ 16 hours;
(3) fermentation culture in the nutrient broth yeast extract paste nutrient solution in fermentor tank is inoculated in by 10% of fermented bacterium in seeding tank, dissolved oxygen amount is 18 ~ 20%, stirring velocity 200rpm, the temperature of fermentor tank 30 ~ 34 DEG C, inoculation fermentation bacterial classification is after 4 hours, every 2 hours, fermented quality detection is carried out to the fermented liquid in fermentor tank, observe bacterial content, until the final cultures biomass comprising bacterium and gemma in fermentation cylinder for fermentation liquid is 10 9when more than cfu/ml, spore content are more than or equal to 90%, immediately fermented liquid is gone out tank, carry out packing, obtain SZ-60 microbial preparation; Fermentation time in fermentor tank is 24 ~ 36 hours.
8. microbial preparation as claimed in claim 7, it is characterized in that, the formula of described nutrient broth yeast extract paste nutrient solution is: extractum carnis 3.5g, peptone 10.0g, yeast extract 5.0g, malt extract 10g, glucose 5.0g, water 1000mL, pH6.8 ~ 7.2; The formula of described fermentation culture is by weight percentage: soyflour 2.0%, soybean cake powder 1.5%, starch 0.5%, yeast extract 0.2%, corn steep liquor 0.2%, NaCl0.1%, Ca (HCO 3) 20.2%, surplus is water, pH7.0 ~ 7.2.
9. microbial preparation as claimed in claim 8, is characterized in that,
Described nutrient broth yeast extract paste nutrient solution is like this preparation: take extractum carnis, peptone, yeast extract, malt extract, glucose puts into 1000mL water, after abundant mixing, pH is adjusted to 6.8 ~ 7.2, in 1000mL triangular flask, loading amount is 300mL nutrient solution, triangle bottleneck is sealed with double-deck sealed membrane, 121 DEG C of moist heat sterilizations 30 minutes, for subsequent use after cooling;
Described fermentation culture is preparation like this: in seeding tank, add required water, add soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO in proportion 3) 2, fully stir, fermentation culture pH is adjusted to 7.0 ~ 7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, inoculates after cooling immediately.
10. microbial preparation as claimed in any one of claims 7-9, it is characterized in that: in the fermenting process of seeding tank or fermentor tank, when there is more foam, adding defoamer, described defoamer is organosilicon, and add-on is not overflowed with the foam in seeding tank or fermentor tank and is as the criterion; After fermentation cylinder for fermentation completes, in fermentor tank, add the benzoic acid preservative accounting for total liquid volume 2/10000ths in tank, stir, then go out tank and carry out packing.
The application of 11. microbial preparations according to any one of claim 6-10 in control ginseng fungal disease.
12. apply as claimed in claim 11, it is characterized in that, described fungi is fusarium solani (Fusariumsolani), destroy in post spore bacterium (Cylindrocarpondestructans), ginseng alternaric bacteria (Alternariapanax), dry thread Pyrenomycetes (Rhizoctoniasolani), Phytophthora cactorum bacterium (Phytophthoracactorum) and Sclerotinia ginseng (Sclerotiniaschinseng) one or more.
13. apply as claimed in claim 12, it is characterized in that, at ginseng fungal disease their early stage, are applied to equably in soil by the diluent of described microbial preparation, and in described diluent, the dilution volume ratio of microbial preparation and water is 1:100.
14. apply as claimed in claim 13, it is characterized in that, also added auxiliary agent organosilicon in described diluent, and the volume ratio of described auxiliary agent organosilicon and diluent is 1:5000.
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