CN103789234A - Bacillus amyloliquefaciens and application thereof - Google Patents
Bacillus amyloliquefaciens and application thereof Download PDFInfo
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Abstract
The invention provides bacillus amyloliquefaciens SZ-60 which is characterized in that the collection number is CGMCC No.8277. The bacterial colony formed by the strain on a beef-extract peptone (NB) medium by virtue of single-cell reproduction is irregular with oyster white color, slight upheaval at the center and wet and semitransparent surface; the microscopy is of a rod shape, and the size is (0.3-0.4)*(3.2-3.3)microns; with flagella, G- and spores, the strain can grow on the beef-extract peptone (NB) medium containing 2-5% of NaCl; the formula of the beef-extract peptone (NB) medium contains 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl, 17g of agar and 1,000ml of water, and the pH is 6.8-7.2. The bacillus amyloliquefaciens realizes a remarkable antagonistic action on the main pathogenic bacteria causing ginseng root rot, epidemic diseases, sclerotinia sclerotiorum, cylindrocarpon destructans, black spot and damping off. The invention also provides an application of the bacillus amyloliquefaciens SZ-60 in preventing plant fungal diseases, or application in preparing a microbial preparation for preventing plant fungal diseases.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens and application thereof.
Background technology
Ginseng (Panax ginseng C.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 local mainstay industry.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, have reduced ginseng crude drug's security and commodity value.Disease prevention and control problem has become one of significant problem of restriction ginseng industry Sustainable development.
Ginseng diseases approximately has 20～40 kinds, wherein, 6 class pathogenic bacterias are the main pathogenic fungi that cause ginseng Common Diseases and limit ginseng industry development below: the general sickness rate of ginseng maize ear rot being caused by fusarium solani (Fusarium solani) is in 30% left and right, 6 years strangers join the mortality ratio that root rot causes can reach 50%～80%, main harm seedling root and rhizome portion (the following stem in earth's surface), rotten ginseng root 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 (Phytophthora cactorum) causes can reach 70%, when its symptom is light for falling ill, on blade, produces irregular dark green color spot, and severe one cauline leaf is withered, butt rot, and plant is withered in flakes, and the underproduction is serious; The above ginseng of ginseng sclerotium disease main harm life in the 3 years root that Sclerotinia ginseng (Sclerotinia schinseng) causes, site of pathological change is bud bud, root and rhizome, after ginseng velamen evil, initial stage is raw a little white velvet-like 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 that forms many mouse excrement shapes of epidermis; The general sickness rate 20%-30% of black fleck disease of ginseng that ginseng alternaric bacteria (Alternaria panax) causes, reaches 100% when serious, causes early leaf fall, and plant is withered, shaky, ginseng root and the underproduction of ginseng seed; The Ginseng Rhizoctonia Solani that dry thread Pyrenomycetes (Rhizoctonia solani) 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 40% left and right, germ make seedling the stem of 3～5 centimetres of dry wet soil interfaces in ground hang contracting, rot, cut off transfusion tissue, cause seedling lodging; Destroy the ginseng rust maize ear rot that causes of post spore bacterium (Cylindrocarpon destructans) when serious sickness rate reach more than 70%, this disease betides each position of root, and scab is rust, diffuses to full root by putting to face, large, ventilative bad, the soil ulmin bed thickness of soil humidity, morbidity is heavy.
Do not reach for a long time the effect of people's anticipation far away by prevent and treat ginseng diseases with Chemical control methods such as chemical pesticides, and the micro-ecological environment of excessive use chemical pesticide meeting spoiled soil, weighting ring environment pollution, also make toxic substance accumulation 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, utilize crop rhizosphere soils screening to there is the soil microorganisms of remarkable antagonistic effect to pathogenic bacteria, and make the important means that microbial preparation is biological prevention and control research, be also the important channel of beneficial microorganism development of resources.
Biological control is take ecological principle as basis, utilizes the interaction between living species, with a kind of or class biological inhibition another kind or another kind of biology.The advantage of biological control maximum is free from environmental pollution, there is no pesticide residue, this be the abiotic method prevention and elimination of disease and pests such as agricultural chemicals cannot be than.Utilizing antagonistic microbe control pathogenic micro-organism is the important component part of biological prevention, it has avoided chemical pesticide to use the problem of a series of plant protection, environment and the energy aspect brought, avoid the harm of pesticide residue to people and animals, the more important thing is and promoted agriculture Sustainable development.The U.S. has GB03, MBI600, QsT713 conciliates the 4 bacillus subtilis biocontrol strains such as starch subtilis mutation and obtains Environmental Protection Agency's commercialization or limited commercialization production application license.Separate starch subtilis mutation FZB24 in Germany and commercially produced, for controlling the different fungal diseases of food crop.
Genus bacillus (Bacillus) is one of important microbe in biocontrol of plant disease research, and its kind to distribute extensively, is the dominant population of the micro-ecology of soil and plant.This quasi-microorganism, by secretion antibiotics and growth competition, is brought into play multiple beneficial effect aspect controlling plant diseases.Utilize at present bacillus amyloliquefaciens control ginseng fungal disease to have no report.
Summary of the invention
The object of the invention is to: the main fungal disease in producing for ginseng, especially the long-pending expanding day of ginseng soil-borne disease generating plane, Agro-chemicals control is except easily causing environmental pollution and pesticide residue, also undesirable practical situation of preventive effect, propose a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain SZ-60 and causing respectively ginseng maize ear rot, 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 6 kinds of pathogenic bacteria prevention and control of Sclerotinia ginseng (S.schinseng).
The invention provides a kind of to the above-mentioned ginseng maize ear rot that causes, 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 Bacillus amyloliquefaciensSZ-60, depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date: on September 25th, 2013, deposit number: CGMCC No.8277, its 16SrDNA sequence length is 1436bp, specifically as shown in SEQ ID No:l.Application BLAST software and DNAMAN software etc. are analyzed, and the 16SrDNA sequence of SZ-60 bacterial strain is compared by BLAST, can in GenBank, find the close bacterial strain sequence that homology is very high.With SZ-60 bacterial strain similarity the highest be Bacillus amyloliquefaciens, homology reaches 99%.Find with UPGMA method phylogenetic tree construction according to MEGA5.10Beta2 software, SZ-60 and Bacillus amyloliquefaciens belong to a hereditary branch together, and sibship is very approaching, and pro-borne has reached 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 (Bacillus amyloliquefaciens).
The bacillus amyloliquefaciens SZ-60 of the present invention bacterium colony that unicellular breeding growth forms on beef extract-peptone (NB) substratum is irregular, oyster white, slightly protuberance, surface wettability, translucent of center; It is shaft-like that microscopy is, and size is 0.3～0.4 × 3.2～3.3 μ m, tool flagellum, and G ﹣, has gemma; It can grow at the temperature of 25 ℃～40 ℃, and its optimum growth temperature is 32 ℃; Its growth pH scope is 6.5～8.0, and 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; Casein, tyrosine reaction are negative; Can not utilize D-Glucose, D-wood sugar; Starch Hydrolysis test microscopy has dextrin to generate; Gelatin liquification test is positive; Citrate trianion utilizes test medium to be acid (green); V-P (pH 7.0) measures and generates red compound; L-arabinose, N.F,USP MANNITOL tests positive; On beef extract-peptone (NB) substratum that contains 2%～5% NaCl, 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, pH 6.8～7.2.
The invention still further relates to the application of bacillus amyloliquefaciens SZ-60 in control fungal diseases of plants, or in the microbial preparation of preparation control fungal diseases of plants application, described plant optimization is ginseng, described fungi is preferably the fusarium solani (F.solani) that causes ginseng maize ear rot, cause the 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, cause the Phytophthora cactorum bacterium (Ph.cactorum) of Ginseng Blight In China and cause one or more in these 6 kinds of pathogenic bacterias of Sclerotinia ginseng (S.schinseng) of ginseng sclerotium disease.Bacillus amyloliquefaciens SZ-60 of the present invention also has somatotrophic effect to ginseng.
The microbial preparation that the invention still further relates to the spore of the full nutrient solution culture that contains bacillus amyloliquefaciens SZ-60 and bacillus amyloliquefaciens SZ-60, it can prepare by following preparation method:
(1) bacillus amyloliquefaciens SZ-60 test tube kind is inoculated in in bottled 300mL nutrient broth yeast extract paste (NBY) nutrient solution of 1000mL triangle, turns 180/min, cultivate 36 hours at 30 ℃, obtain zymocyte liquid;
(2) by zymocyte liquid fermentation culture in the fermentation culture being inoculated in seeding tank with seeding tank volume 20%, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of seeding tank, 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 while being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12～16 hours;
(3) by fermentation culture in 10% of fermented bacterium in the seeding tank NBY nutrient solution being inoculated in fermentor tank, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of fermentor tank, after inoculation fermentation bacterial classification 4 hours, every 2 hours, the fermented liquid in fermentor tank is carried out to fermented quality detection, observe bacterial content, until final cultures (comprising bacterium and the gemma) biomass in ferment tank liquid is 10
9cfu/ml is above, gemma content is while being more than or equal to 90%, immediately fermented liquid is gone out to tank, carries out packing, obtains 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 and put into 1000mL water, after fully mixing, pH is adjusted to 6.8～7.2, in 1000mL triangular flask, loading amount is 300mL nutrient solution, seal triangle bottleneck with double-deck sealed membrane, 121 ℃ of moist heat sterilizations 30 minutes, cooling rear for subsequent use.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 in proportion soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO
3)
2, fully stir, fermentation culture pH is adjusted to 7.0～7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, cooling after inoculation immediately.
In the fermenting process of seeding tank or fermentor tank, in the time there is more foam, can add defoamer, described defoamer is organosilicon, add-on is not overflowed and is as the criterion with the foam in seeding tank or fermentor tank; After fermentation cylinder for fermentation completes, in fermentor tank, add the phenylformic acid sanitas that accounts for total liquid volume 2/10000ths in tank, stir, then go out tank and carry out packing.
The fermented liquid obtaining 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, in diluent, can also add auxiliary agent organosilicon, 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 CGMCC No.8277 is to causing respectively ginseng maize ear rot, 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 6 kinds of pathogenic bacterias of Sclerotinia ginseng (S.schinseng) all have good prevention effect, ginseng is had to growth promoting function, nontoxic no pathogenicity, to person poultry safety, free from environmental pollution, simultaneously, after biocontrol fungicide dilution, be applied directly to and in soil, plant carried out to root irrigation and can bring into play its germicidal action, can significantly improve the Rational structure of microflora in Ginseng Rhizosphere environment, form a bio-diversity Soil of Ginseng Rhizomsphere micro-ecological environment, thereby control effectively, enduringly the popular of ginseng fungal disease.
Accompanying drawing explanation
Fig. 1: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to destroying the dull and stereotyped restraining effect of growth of post spore bacterium (Cylindrocarpon destructans) 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 (Fusarium solani) 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 (Alternaria panax) 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 (Sclerotinia schinseng) mycelia.
Fig. 5: bacillus amyloliquefaciens SZ-60 bacterial strain of the present invention is to the dull and stereotyped restraining effect of the growth of dry thread Pyrenomycetes (Rhizoctonia solani) mycelia.
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 (Phytophthora cactorum) mycelia.
The growth promoting function (A: blank, B: cast SZ-60 bacteria suspension) of Fig. 7: SZ-60 to ginseng.
Fig. 8: SZ-60 is to destroying the prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of the microbial ginseng rust maize ear rot of post spore.
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of Fig. 9: SZ-60 to the microbial Ginseng Blight In China of Phytophthora cactorum.
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of the ginseng sclerotium disease that Figure 10: SZ-60 causes Sclerotinia ginseng.
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of Figure 11: SZ-60 to the microbial ginseng maize ear rot of fusarium solanae.
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of the black fleck disease of ginseng that Figure 12: SZ-60 causes ginseng alternaric bacteria.
The prevention effect (A: blank, B: cast SZ-60 bacteria suspension) of the Ginseng Rhizoctonia Solani that Figure 13: SZ-60 causes dry thread Pyrenomycetes.
Embodiment
Embodiment 1
Separation and the preservation of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SZ-60 bacterial strain
This bacterial strain 30cm around the perennial Ginseng Rhizosphere in Fusong County Wan Liang town, Jilin Province culture of ginseng ground separates and obtains with interior soil.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, the 10-30min that fully vibrates, makes sample mix with sterilized water, makes sample suspension, leaves standstill 5min.Under aseptic condition, get 1mL supernatant liquor, add the 9mL0.05%SDS aqueous solution (lauryl sodium sulfate aqueous solution), 40 ℃ of insulation 20min, get 1mL, add 9mL sterilized water, make successively 10 by gradient
-3, 10
-4, 10
-5diluent.Draw respectively the each diluent of 100 μ L and join on beef extract-peptone (NB) flat board, adopt plate dilution method to be evenly coated with, every processing repeats for 3 times, and 34 ℃ of incubators are cultivated 1～2 day.Select single bacterium colony and be transferred to that NB is dull and stereotyped to be cultivated, grow after bacterium colony, with the transfering loop separation and purification of ruling, purifying bacterial strain is in 4 ℃ 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 bacterium colony that unicellular breeding growth forms on NB substratum is irregular, oyster white, slightly protuberance, surface wettability, translucent of center; It is shaft-like that microscopy is, and size is 0.3～0.4 × 3.2～3.3 μ m, tool flagellum, and G ﹣, has gemma; It can grow at the temperature of 25 ℃～40 ℃, and its optimum growth temperature is 32 ℃; Its growth pH scope is 6.5～8.0, and 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; Casein, tyrosine reaction are negative; Can not utilize D-Glucose, D-wood sugar; Starch Hydrolysis test microscopy has dextrin to generate; Gelatin liquification test is positive; Citrate trianion utilizes test medium to be acid (green); V-P (pH 7.0) measures and generates red compound; L-arabinose, N.F,USP MANNITOL tests positive; On the NB substratum that contains 2%～5% NaCl, 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 CGMCC No.8277.
Our called after SZ-60 of this bacterial strain, Classification And Nomenclature is: bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Embodiment 2
The restraining effect of bacillus amyloliquefaciens SZ-60 bacterial strain to the growth of ginseng fungal disease pathogenic bacteria
Adopt filter paper method to measure the antagonistic action of bacterial strain SZ-60 to ginseng fungal disease pathogenic bacteria: the bacterium cake of with the punch tool of diameter 8mm, the ginseng pathogenic bacteria bacterium colony having activated being made to corresponding size, aseptic inoculation is to the centre of potato dextrose agar (PDA) dull and stereotyped (diameter 90mm), and the sterilizing filter paper dick that is simultaneously 1cm by 4 diameters sticks on 4 angle points at 25mm place, anomaly plate center.Bacterial strain SZ-60 is made to bacteria suspension, and (concentration is about 10
8cfu/mL), wherein every point of 3 filter paper dicks connects 20 μ L bacteria suspensions, and 1 filter paper dick point connects 20 μ L sterilized waters, and as treatment group, each processing repeats 3 times; On 4 filter paper dicks of another 4 angle points at one flat plate, connect respectively 20 μ L sterilized waters, as a control group, all being placed in 28 ℃ of incubators cultivates 6～7 days, treat that control group pathogenic bacteria bacterium colony covers with flat board, measure treatment group pathogenic bacteria colony diameter (unit: mm), and calculate bacteriostasis rate according to following formula.Every kind of pathogenic bacteria is repeated 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 peeling potato 200g, glucose 20g, add water 1000mL, regulating pH is 7.0.On boiling water bath, heat 20min, after filtered through gauze, constant volume, to 1000mL, adds the rear packing moist heat sterilization (121 ℃, 30min) of agar 22g fusing.
The preparation method of above-mentioned bacteria suspension: the bacterial strain SZ-60 of preservation activates after 2 days by plate streaking mode, get the bacterial colony of 3～4 diameter 1cm with transfering loop, be linked into 200mL containing in the triangular pyramidal bottle of 50mL beef extract-peptone (NB) nutrient solution, 32 ℃ of shaking tables, make concentration after 180r/min cultivation 48h and are 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 fusarium solani (F.solani), destruction post spore bacterium (C.destructans), ginseng alternaric bacteria (A.panax), dry thread Pyrenomycetes (Rh.solani), Phytophthora cactorum bacterium (Ph.cactorum) and 6 kinds of pathogenic bacterias of Sclerotinia ginseng (S.schinseng) of causing respectively ginseng maize ear rot, rust rot, black spot, damping-off, epidemic disease and sclerotium disease.Particularly to causing the Phytophthora cactorum bacterium of Ginseng Blight In China and causing that the bacteriostasis rate of the destruction post spore bacterium of ginseng rust maize ear rot has reached more than 90%, to cause black spot ginseng alternaric bacteria, cause the fusarium solani of ginseng maize ear rot and cause that the bacteriostasis rate of the Sclerotinia ginseng of ginseng sclerotium disease is 71～82%, bacteriostasis rate to the dry thread Pyrenomycetes that causes Ginseng Rhizoctonia Solani has also reached 46.4%, reflects that bacterial strain SZ-60 has broad spectrum to the preventive and therapeutic effect of 6 kinds of the main pathogenic fungi of ginseng.
The restraining effect of table 1 bacterial strain SZ-60 to ginseng pathogenic fungi
Embodiment 3
The antagonistic activity experiment of bacillus amyloliquefaciens SZ-60 to ginseng pathogenic bacteria
Adopt Oxford agar diffusion method to measure the antagonistic activity of bacterial strain SZ-60 to ginseng fungal disease germ: after SZ-60 bacterial strain is made to bacteria suspension, to collect (preparation method is with example 2), under 4 ℃ of conditions, the centrifugal 20min of 12 000r/min, collect supernatant liquor through 0.22 μ m filtering with microporous membrane, 4 ℃ of gained filtrates save backup.The ginseng germ bacterium cake that is 8mm at the dull and stereotyped central authorities of PDA inoculation diameter under aseptic condition, then 4 aseptic Oxford cups are put in to 4 symmetrical angle points of the anomaly plate about 25mm in center, in every glass, drip 100 μ l filtrates, every processing repeats for 3 times, cultivates after 5 days for 28 ℃ and measures 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, the supernatant liquor that SZ-60 bacteria suspension is described is less than 5mm except the antibacterial bandwidth to dry thread Pyrenomycetes, antibacterial bandwidth to all the other 5 kinds of pathogenic bacterias is all greater than 5mm, wherein the antibacterial bandwidth of Sclerotinia ginseng is greater than to 10mm, the supernatant liquor that SZ-60 bacteria suspension is described has antagonistic activity (table 2) in various degree to 6 kinds of ginseng pathogenic bacterias.
The antagonistic activity of table 2 bacillus amyloliquefaciens SZ-60 to ginseng pathogenic fungi
Note: with reference to Vestberg method, "+", " ++ ", " +++ " represent that respectively antibacterial band radius is <5mm, 5-10mm, >10mm.
The growth-promoting functions of bacillus amyloliquefaciens SZ-60 bacterial strain to ginseng
(32 ℃, 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 ginseng woods soil: vermiculite, according to 2:1 ratio preparation 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, after 25-30min, takes out, transplant respectively in the flowerpot of sterilizing soil is housed, every basin 5 strains.With SZ-60 bacteria suspension, (bacteria containing amount is about 10 to every basin
8cfu/mL) 50 times of sterilized water diluent 30mL fill with root, every processing 3 basins, and random alignment, greenhouse moisturizing is cultivated; Aseptic beef extract-peptone for control group (NB) is cultivated immersion seedling, and processing mode is the same, No. 3 basins of every processing, and random alignment, greenhouse moisturizing is cultivated, and waters respectively the aseptic culture fluid (except not containing SZ-60, all the other compositions are identical with treatment group) of 30mL.Grow to after 30 days until ginseng-leaf, choose at random respectively the each 5 strain ginseng-leafs for the treatment of group and control group, carefully whole seedling strain is dug out, wash away root earth, measure its plant height, root length, whole strain fresh weight and root fresh weight index.Then dry to constant weight for 180 ℃, survey whole strain dry weight and root dry weight.
Indoor pot measurement result shows (shown in table 3), inoculation SZ-60 bacterial strain is in the time of sterilizing 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.
The promoter action of table 3 bacterial strain SZ-60 to Ginseng Growth
Embodiment 5
Bacillus amyloliquefaciens SZ-60 grows test surely.
(1) bacillus amyloliquefaciens SZ-60 ginseng cauline leaf determine grow
To be inoculated in NB nutrient solution with the bacillus amyloliquefaciens SZ-60 that Rifampin (300 μ g/mL) mark is crossed, 32 ℃, 180r/min, shaking table vibration 24h, makes bacteria containing amount and is about 10
8the mark bacterial strain bacteria suspension of cfu/mL.By 3 years stranger's seedling (Fusong) transplant at random in being equipped with in the flowerpot of nature soil (taking from Fusong County forest land, Jilin Province soil) and sterilizing soil (taking from Fusong County forest land soil at 121 ℃ of moist heat sterilization 2h).Height of seedling 10cm final singling, every basin 3 strains, inject to ginseng-leaf root soil the bacillus amyloliquefaciens SZ-60 bacteria suspension that 10mL mark is crossed, after 10d, gather respectively leaf and the each 1.0g of rhizome portion of ginseng-leaf, by after its surface sterilization, add respectively 2mL sterilized water and grind, get supernatant liquor and coat respectively on the NB flat board containing Rifampin (300 μ g/mL).Whether cultivate 4 days for 32 ℃, 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 explanation SZ-60 can be in ginseng body the long period exist, it has endogeny.
(2) bacillus amyloliquefaciens SZ-60 determining in soil grown
To be inoculated in NB nutrient solution with the bacillus amyloliquefaciens SZ-60 that Rifampin (300 μ g/mL) mark is crossed, 32 ℃, 180r/min, shaking table vibration 24h, makes bacteria containing amount and is about 10
8the mark bacterial strain bacteria suspension of cfu/mL.Respectively natural soil and sterilizing soil are loaded in flowerpot, every basin 1kg soil, mixes soil to injecting the bacillus amyloliquefaciens SZ-60 bacteria suspension that 100mL mark crosses in soil.Under room temperature, place, (soil first, after gradient dilution, gets 10 to the bacterium in 1 soil of separation in 7 days
-3, 10
-4, 10
-5soil diluent 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 sterilizing soil naturally afterwards
5more than cfu/g soil.This explanation 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 in bottled 300mL nutrient broth yeast extract paste (NBY) nutrient solution of 1000mL triangle, at 180r/min, cultivates 36 hours at 30 ℃, 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 fully 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 ℃ of moist heat sterilizations 30 minutes, cooling rear for subsequent use.
Embodiment 7
The preparation of bacillus amyloliquefaciens SZ-60 microbial preparation
The full nutrient solution culture that bacillus amyloliquefaciens SZ-60 microbial preparation contains bacillus amyloliquefaciens SZ-60 and the spore of bacillus amyloliquefaciens SZ-60, preparation method is as follows:
(1) by the fermentation culture in the fermentation culture being inoculated in seeding tank with seeding tank volume 20% of cultured zymocyte liquid in embodiment 6, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of seeding tank, 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 while being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12～16 hours;
(2) by fermentation culture in 10% of fermented bacterium in the seeding tank NBY nutrient solution being inoculated in fermentor tank, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of fermentor tank, after inoculation fermentation bacterial classification 4 hours, every 2 hours, the fermented liquid in fermentor tank is carried out to fermented quality detection, observe bacterial content, until final cultures (comprising bacterium and the gemma) biomass in fermented liquid is 10
9cfu/ml is above, gemma content is while being more than or equal to 90%, immediately fermented liquid is gone out to tank, carries out packing, obtains 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%, and 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, add in proportion soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO
3)
2, fully stir, fermentation culture pH is adjusted to 7.0～7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, cooling after inoculation immediately.
Embodiment 8
The field controling test of bacillus amyloliquefaciens SZ-60 bacterial strain to ginseng fungal disease
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of fusarium solani (Fusarium solani) of ginseng maize ear rot is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 10, and random district group is arranged, and every community kind has ginseng 20 strains, repeated for 4 times.Before transplanting, 100 times of sterilized water diluents of the SZ-60 microbial preparation that treatment group is prepared with 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, respectively at June 20 and June 30 filling with root twice.The contrast take 250 times of sterilized water diluents of 10% derosal as medicament, take sterilized water as blank.June 20, each investigation on July 10 occurring degree, calculate disease index, and investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of destruction post spore bacterium (Cylindrocarpon destructans) of ginseng rust maize ear rot is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted May 19.Random district group is arranged, and every community kind has ginseng 20 strains, repeats for 4 times.Before transplanting, 100 times of sterilized water diluents of the SZ-60 microbial preparation that treatment group is prepared with embodiment 7 dip in root, when ginseng starts to fall ill, ginseng are filled with to root, and above-mentioned 100 times of diluent 30mL are filled with in every strain, respectively at May 29 and June 8 filling with root twice.The contrast take 1000 times of sterilized water diluents of 50% carbendazol wettable powder as medicament, take sterilized water as blank.May 29, a disease index of each investigation on June 18, investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of ginseng alternaric bacteria (Alternaria panax) of black fleck disease of ginseng is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted July 5.Random district group is arranged, and every community kind has ginseng 20 strains, 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, respectively at spraying twice on July 15 and July 23.Reach 1500 times of sterilized water diluents take Amici as medicament contrast, take sterilized water as blank.July 15, a disease index of each investigation on August 1, investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of Phytophthora cactorum bacterium (Phytophthora cactorum) of Ginseng Blight In China is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted July 5.Random district group is arranged, and every community kind has ginseng 20 strains, 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, respectively at spraying twice on July 15 and July 23.The contrast take 1000 times of sterilized water diluents of 50% prochloraz-manganese chloride complex wettable powder as medicament, take sterilized water as blank.July 15, each investigation on August 1 occurring degree, calculate disease index, and investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of Sclerotinia ginseng (Sclerotinia schinseng) of ginseng sclerotium disease is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 10, and random district group is arranged, and every community kind has ginseng 20 strains, repeated for 4 times.Before transplanting, 100 times of sterilized water diluents of the SZ-60 microbial preparation that treatment group is prepared with 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, respectively at June 20 and June 30 filling with root twice.The contrast take 500 times of sterilized water diluents of 40% dimetachlone wettable powder as medicament, take sterilized water as blank.June 20, each investigation on July 10 occurring degree, calculate disease index, and investigation result is as table 4.
Be arranged in Jilin Agriculture University's medicinal garden ginseng experiment field in 2013 and carry out causing that the control of dry thread Pyrenomycetes (Rhizoctonia solani) of Ginseng Rhizoctonia Solani is tested, practice ground is Plain field, and soil is timbered soil.Ginseng was transplanted June 5, and random district group is arranged, and every community kind has ginseng 20 strains, 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 with embodiment 7, every strain 30mL, Ginseng Rhizoctonia Solani starts after morbidity, and July 6, July 16 was filled with root twice, the contrast take 500 times of sterilized water diluents of anilazine as medicament, take sterilized water as blank.July 6, each investigation on July 26 occurring degree, calculate disease index, and 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-processing disease index)/contrast disease index × 100
Note:
As shown in table 4, SZ-60 is to all having good preventive effect by the microbial ginseng maize ear rot of above-mentioned cause of disease, 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.
The prevention effect test of table 4 bacterial strain to above-mentioned ginseng fungal disease
Embodiment 9
Application TaKaRa MiniBEST Bacterial GenomicDNA Extraction Kit Ver.2.0 test kit (precious biotechnology (Dalian) company limited) method is extracted DNA, synthetic PCR primer sequence is 16S1F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' voluntarily, 16S1R:5 '-TACGGCTACCTTGTTACGACTT-3 '.Take SZ-60 thalline genomic dna as template, after PCR reaction amplification, detect through 1.5% agarose gel electrophoresis, obtain the specific fragment of a 1500bp left and right, measure this fragment sequence (adopting TIANGEN gel to reclaim test kit in the order-checking of life work biotechnology (Shanghai) limited-liability company), result shows, the DNA of SZ-60 bacterial strain is 1436bp, specifically as shown in SEQ ID No:1.The 16srDNA sequence application BLAST software recording and DNAMAN software and clustalx splicing software are analyzed, found that the homology of SZ-60 bacterial strain DNA sequence dna and bacillus amyloliquefaciens 16srDNA portion gene group 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 Bacillus amyloliquefaciens belong to a hereditary branch together, sibship is very approaching, and pro-borne has reached 100%.The qualification result of combining form and Physiological-biochemical Characters, can confirm that bacterial strain SZ-60 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Each embodiment is not to concrete restriction of the present invention above; as long as the scope limiting according to claim, under the enlightenment of this patent, in conjunction with the basic general knowledge of this area; control by described bacterial strain for ginseng fungal disease, all belongs to protection scope of the present invention.
Claims (10)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SZ-60, is characterized in that, its preserving number is CGMCC No.8277.
2. bacillus amyloliquefaciens SZ-60 as claimed in claim 1, is characterized in that, this bacterial strain bacterium colony that unicellular breeding growth forms on beef extract-peptone (NB) substratum is irregular, oyster white, slightly protuberance, surface wettability, translucent of center.It is shaft-like that microscopy is, and size is 0.3～0.4 × 3.2～3.3 μ m, tool flagellum, and G ﹣, has gemma.It can grow at the temperature of 25 ℃～40 ℃, and its optimum growth temperature is 32 ℃.Its growth pH scope is 6.5～8.0, and 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; Casein, tyrosine reaction are negative.Can not utilize D-Glucose, D-wood sugar.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; On beef extract-peptone (NB) substratum that contains 2%～5% NaCl, 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.
3. the application of bacillus amyloliquefaciens SZ-60 as claimed in claim 1 or 2 in control fungal diseases of plants, or in the application of preparing in the microbial preparation of preventing and treating fungal diseases of plants.
Preferably, described plant is ginseng.
4. application as claimed in claim 3, it is characterized in that, described fungi is one or more in fusarium solani (Fusarium solani), destruction post spore bacterium (Cylindrocarpon destructans), ginseng alternaric bacteria (Alternaria panax), dry thread Pyrenomycetes (Rhizoctonia solani), Phytophthora cactorum bacterium (Phytophthora cactorum) and Sclerotinia ginseng (Sclerotinia schinseng).
5. a microbial preparation, is characterized in that, the full nutrient solution culture of the bacillus amyloliquefaciens SZ-60 that contains claim 1 and the spore of bacillus amyloliquefaciens SZ-60.
6. microbial preparation as claimed in claim 5, is characterized in that, it prepares by following preparation method:
(1) bacillus amyloliquefaciens SZ-60 test tube kind as claimed in claim 1 is inoculated in in bottled 300mL nutrient broth yeast extract paste (NBY) nutrient solution of 1000mL triangle, at 180r/min, cultivates 36 hours at 30 ℃, obtain zymocyte liquid;
(2) by zymocyte liquid fermentation culture in the fermentation culture being inoculated in seeding tank with seeding tank volume 20%, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of seeding tank, 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 while being about to occur gemma, obtain fermented bacterium, the fermentation time in seeding tank is 12～16 hours;
(3) by fermentation culture in 10% of fermented bacterium in the seeding tank NBY nutrient solution being inoculated in fermentor tank, dissolved oxygen amount is 18～20%, stirring velocity 200rpm, 30～34 ℃ of the temperature of fermentor tank, after inoculation fermentation bacterial classification 4 hours, every 2 hours, the fermented liquid in fermentor tank is carried out to fermented quality detection, observe bacterial content, until final cultures (comprising bacterium and the gemma) biomass in fermentation cylinder for fermentation liquid is 10
9cfu/ml is above, gemma content is while being more than or equal to 90%, immediately fermented liquid is gone out to tank, carries out packing, obtains SZ-60 microbial preparation; Fermentation time in fermentor tank is 24～36 hours.
Preferably, the formula of described 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; 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.
More preferably, described NBY nutrient solution is like this preparation: take extractum carnis, peptone, yeast extract, malt extract, glucose and put into 1000mL water, after fully mixing, pH is adjusted to 6.8～7.2, in 1000mL triangular flask, loading amount is 300mL nutrient solution, seal triangle bottleneck with double-deck sealed membrane, 121 ℃ of moist heat sterilizations 30 minutes, cooling rear for subsequent use;
Described fermentation culture is preparation like this: in seeding tank, add required water, add in proportion soyflour, soybean cake powder, starch, yeast extract, corn steep liquor, NaCl, Ca (HCO
3)
2, fully stir, fermentation culture pH is adjusted to 7.0～7.2, sealing charge cavity, with high-temp steam sterilizing 2 hours, cooling after inoculation immediately.
7. microbial preparation as claimed in claim 6, it is characterized in that: in the fermenting process of seeding tank or fermentor tank, in the time there is more foam, add defoamer, described defoamer is organosilicon, and add-on is not overflowed and is as the criterion with the foam in seeding tank or fermentor tank; After fermentation cylinder for fermentation completes, in fermentor tank, add the phenylformic acid sanitas that accounts for total liquid volume 2/10000ths in tank, stir, then go out tank and carry out packing.
8. the application of the microbial preparation as described in any one in claim 5-7 in control ginseng fungal disease.
9. application as claimed in claim 8, it is characterized in that, described fungi is one or more in fusarium solani (Fusarium solani), destruction post spore bacterium (Cylindrocarpon destructans), ginseng alternaric bacteria (Alternaria panax), dry thread Pyrenomycetes (Rhizoctonia solani), Phytophthora cactorum bacterium (Phytophthora cactorum) and Sclerotinia ginseng (Sclerotinia schinseng).
10. application as claimed in claim 9, is characterized in that, at ginseng fungal disease their early stage, the diluent of described microbial preparation is applied in soil equably, and in described diluent, the dilution volume ratio of microbial preparation and water is 1:100.
Preferably, also added auxiliary agent organosilicon in described diluent, the volume ratio of described auxiliary agent organosilicon and diluent is 1:5000.
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| CN110423712A (en) * | 2019-08-05 | 2019-11-08 | 慕恩（广州）生物科技有限公司 | A kind of auspicious Russia's series bacillus of Pierre and its application |
| CN110423712B (en) * | 2019-08-05 | 2022-12-23 | 慕恩（广州）生物科技有限公司 | Paenibacillus porrigens and application thereof |
| CN111961701A (en) * | 2020-01-21 | 2020-11-20 | 吉林农业大学 | Fermentation liquor for producing extracellular bacteriostatic protein by bacillus amyloliquefaciens SZ-60 and fermentation method thereof |
| CN113213986A (en) * | 2020-01-21 | 2021-08-06 | 吉林农业大学 | Biological organic fertilizer containing bacillus amyloliquefaciens and preparation method and application thereof |
| CN113215011A (en) * | 2020-01-21 | 2021-08-06 | 吉林农业大学 | Fermentation culture solution for producing extracellular bacteriostatic protein by shortwave monimonas SZ-22 and fermentation method thereof |
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| KR102509861B1 (en) | 2020-11-27 | 2023-03-14 | 강원도 | Novel Bacillus amyloliquefaciens strain and composition for controlling Fusarium oxysporum or Phytophthora drechsleri of Astragalus membranaceus Bunge root comprising the same as an active ingredient |
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