CN101423812B - Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof - Google Patents
Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a Bacillus amyloliquefaciens YN-1 the preserving number of which is CGMCC NO.2328, a biological antimicrobial agent and a method for preparing the same. In terms of weight portion, the biological antimicrobial agent comprises 82 to 95 portions of bacillus fermentation broth, 2 to 8 portions of surfactant, and 1 to 4 portions of preservative; and the method for preparing the biological antimicrobial agent comprises the steps of the preparations of test tube strains, seed liquid and fermentation medium, liquid fermentation production, the preparation of agents and the like. The Bacillus amyloliquefaciens has broad antimicrobial spectrum and good antimicrobial effect; a microbial agent prepared from the Bacillus amyloliquefaciens can effectively control fungal diseases such as apple brown spot, Rhizoctonia cerealis and the like, and the control effect is significant and stable and the control efficiency on the diseases such as apple target spot, brown spot and the like can be as high as 81.4 percent. The method for producing the biological antimicrobial agent is simple and reasonable, and screens out a low-cost industrial culture medium, thus the industrial production has low cost, and the method is easy to popularize and apply.
Description
Technical field
The present invention relates to the biological control field, relate in particular to a kind of new bacillus amyloliquefaciens and microbial preparation and the method for using this genus bacillus preparation.
Technical background
As everyone knows, because the technical characterictic of microbial pesticide and developing direction and the human following mode of production, food safety, nutrient health, the eubiosis and species diversity all have good blending, the microbiological industry production technology system based on modern fermentation engineering and biotechnology that adds is gradually improved, and the research and development of microbial pesticide have caused that the researchist pays close attention to widely and payes attention to.
Utilize grow nonparasitically upon another plant on the plant or rhizosphere soil in antagonistic bacterium or its meta-bolites regulation and control harmful microbe balance can reach the sick purpose of producing of protecting of control, also be the important of biocontrol of plant disease and valid approach, screening efficient and activity stabilized antagonistic strain then is the prerequisite that guarantees that biological control is succeedd.Genus bacillus (Bacillus spp.) is the bacterioid that nature extensively exists, some bacterial strains wherein can produce tens kinds of antimicrobial substances such as bacitracin, big ring grease, ring grease and bacteriophage-like particle, have a wide range of applications in biocontrol of plant disease.Simultaneously, genus bacillus is a gram positive bacterium, can form gemma, and cell walls does not contain intracellular toxin, and is most nontoxic to people and animals except that indivedual kinds, therefore has very strong environmental compatibility and environment friendly.It is an emerging industry that microbial pesticide is compared with chemical pesticide market, the development microbial pesticide has enormous and latent market in China, it is considerable that the achievement in research of novel microorganism agricultural chemicals is transferred the possession of market potential, and will advance traditional agricultural chemicals industry restructuring and skill upgrading, obtain huge scale and benefit, be the Sustainable development of modern agriculture production and ecotope, for the adjustment of related industries structures such as agricultural provides the important techniques guarantee.
Summary of the invention
The technical problem to be solved in the present invention provides that a kind of new antimicrobial spectrum is wide, the genus bacillus bacterial classification (Bacillus amyloliquefaciens) of good antimicrobial effect, and a kind of microbial preparation of efficient, nontoxic, safe controlling plant diseases and preparation method cheaply thereof also are provided.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YN-1, its deposit number is CGMCC NO.2328.
More than one state the microbial preparation of bacillus amyloliquefaciens preparation, and in weight part, this microbial preparation contains 82～95 parts of bacillus amyloliquefaciens YN-1CGMCC NO.2328 fermented liquids, 2～8 parts in tensio-active agent, 1～4 part of sanitas.
Described microbial preparation also contains 2～6 parts of antifreezing agents.
Described bacillus amyloliquefaciens fermented liquid is to make by the following method:
Described bacillus amyloliquefaciens YN-1 is inoculated in nutrient solution, fermentation culture at least 36 hours, with filtering fermentation liquor, the sterilization, again it is concentrated into original volume 40～60% the back; Contain glucose 8～12g/L, Semen Maydis powder 10～16g/L, soybean cake powder 10～16g/L in the described nutrient solution.
Above-mentioned biotechnological formulation can be by routine method such as filling root, seed soaking, spraying use.Can use separately, also can mix use with other sterilant, plant modifying agent that this biocontrol strain is had no side effect.Generally use, before method is sowing, adopt the seed dressing of fermentation preparation and seed in crop sowing time or the disease phase of starting; Or start the phase in disease, apply crop rhizosphere or spray application by irritating root.
A kind of preparation method of microbial preparation may further comprise the steps:
(1) the test tube strains preparation is inoculated into bacillus amyloliquefaciens YN-1CGMCC NO.2328 on the test tube NYDA slant medium, cultivates down for 26～30 ℃ and obtains test tube strains in 24～48 hours;
(2) the seed liquor preparation inserts above-mentioned test tube strains in the NYD nutrient solution, under 26～30 ℃, cultivate and made primary seed solution in 12～16 hours, with 1: 100～150 volume ratio primary seed solution is inserted in the NYD nutrient solution then, under 26～30 ℃, carry out fermentation culture 12～14 hours, and obtained secondary seed solution;
(3) fermention medium preparation is got glucose, Semen Maydis powder, soybean cake powder and is added water and be mixed with the substratum that contains glucose 8～12g/L, Semen Maydis powder 10～16g/L, soybean cake powder 10～16g/L;
(4) liquid fermenting production to fermentation equipment and medium sterilization after, secondary seed solution is inoculated in nutrient solution by 1: 20～30 volume ratio, after 26～30 ℃ of bottom fermentations are cultivated at least 36 hours, with filtering fermentation liquor, sterilization is concentrated into it 40～60% of original volume again and promptly gets the YN-1 bacterial strain fermentation liquor;
(5) formulation preparation is got 82～95 parts of step gained YN-1 bacterial strain fermentation liquors, 2～8 parts in tensio-active agent, 1～4 part of sanitas, abundant mixing.
In above-mentioned steps (5), also can further sneak into 2～6 parts of antifreezing agents.
Above-mentioned antifreezing agent is at least a in ethylene glycol, urea, calcium chloride, the sodium-chlor.
Above-mentioned tensio-active agent is 0204, at least a among the SilwetL-77, Mon 0818, OP-10.
Foregoing preservatives is a sodium salicylate.
The present invention has actively useful effect:
1. the B.amyloliquefaciens YN-1 antimicrobial spectrum that is filtered out is wide, good antimicrobial effect, it is to ring rot of apple bacterium (Physalospora piricola Nose.), apple rot pathogen (Valsa mali Miyabe etYamada), marssonina coronaria (Marssonina mali (P.Henn.) Tto.), botrytis cinerea pers (Botrytiscinerea) and capsicum wilt (Fusarium oxysporum f.sp.capsicum) have stronger restraining effect, indoor YN-1 bacterial strain reaches 90% (referring to embodiment 3) to the fungistatic effect of brown patch germ, illustrate that this bacterium and meta-bolites thereof can be used for preparing wide spectrum, the high-performance bio preparation has very wide suitability for industrialized production prospect.
2. the main controlling object of microbial preparation of the present invention is apple marssonina leaf spot, canker of apple fruit etc.In addition, with the biotechnological formulation of this bacterial strain preparation fungal diseases such as the ring spot bacterium of medicinal plants such as farm crop such as cucumber, capsicum, watermelon, cotton, wheat and glutinous rehmannia, Chinese yam, rotten pathogenic bacteria, brown patch germ, ash arrhizus bacteria, wilt, anthrax also there is the better prevention effect; The efficiency test that carries out in the sub-district, field on ground such as Wangwu Shan Mountain forest farm, Jiyuan City, Wen County shows: this biological prevention and control agent effect is remarkable, good stability, can the effectively preventing apple marssonina leaf spot, fungal disease such as wheat hypochnus, can be to the prevention effect of diseases such as apple zonate spot, brown spot up to 81.4% (seeing embodiment 10).
3. the preparation method of biotechnological formulation of the present invention is simple, reasonable, has filtered out industrial substratum with low cost, greatly reduces industrial production cost, the application popularization of favourable this biotechnological formulation.
Bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) YN-1, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 9th, 2008, it abbreviates CGMCC as, and deposit number is CGMCC NO.2328.
Embodiment
Embodiment 1 bacterial screening
Indicator: with ring rot of apple bacterium (Physalosporapiricola Nose.), Valsa mali (Valsamali Miyabe et Yamada), marssonina coronaria (Marssonina mali (P.Henn.) Tto.), withered germ of water-melon (Fusarium oxysporum (Schl.) f.sp.niveum (Smith) Snyder), cucumber fusarium axysporum (Fusarium oxysporum (Schl.) f.sp.Cucumerinum Owen.), capsicum blight (Fusariumoxysporum f.sp.capsicum), cotton wilt (Fusarium oxysporium Schl.f.sp.vasinfectum), wheat hypochnus (Rhizotonia cerealisvan der Hoeven apud.Boerema﹠amp; Verhoeven) as indicator.
Substratum: NYDA substratum (extractum carnis 8g, peptone 8g, glucose 10g, agar 15g, moisturizing is to 1000ml), NYD substratum (extractum carnis 8g, peptone 8g, glucose 10g, moisturizing is to 1000ml) and PDA substratum (peeling potato 200g, glucose 10g, agar powder 15g, moisturizing is to 1000ml), dual-purpose substratum (peeling potato 200g, extractum carnis 5g, peptone 5g, glucose 10g, agar powder 15g, moisturizing is to 1000ml).
Screening method: romaine lettuce ground, the Huiji District hair village, Zhengzhou City, adopt five point samplings, scalp top layer 10cm soil layer, behind the abundant mixing of gained soil sample, get 500g and dry standby.Strain separating: air-dry soil sample is crossed 40 mesh sieves, and each soil sample is got 15g, adds to fill in the triangular flask of 250ml sterilized water and some granulated glass spherees, puts 120r/min concussion 20min on the shaking table, and getting supernatant liquor after leaving standstill is mother liquor.Adopt the gradient dilution method with mother liquor weaker concn to 10 successively
-3, 10
-4, 10
-5Stand-by; Dull and stereotyped coating: after drawing 100 μ l bacterium liquid and being coated with to the NYDA flat board, cultivate 24h in 28 ℃ of incubators; Choose single bacterium colony line: select the single bacterium colony with different colony characteristicses and rule on the NYDA flat board, succeeding transfer culture is four times in 28 ℃ of incubators, noticeable change person inclined-plane is not taken place growth characteristics preserve standby.Adopt following general paper dish method then, contain toxic medium method and carry out the screening of antagonism bacterium.
Paper dish method: (1) will cultivate the inclined-plane antagonism bacterial classification of 2 days (28 ℃ of temperature) and make bacteria suspension with sterilized water, and will be stand-by; Filter paper breaks into the double-layer circular scraps of paper of Φ=6mm with punch tool, and it is stand-by to sterilize.The PDA plate culture medium is cultivated pathogenic bacteria (90mm culture dish), and (diameter 6mm) breaks into the bacterium cake along colony edge with punch tool, and be stand-by; (2) dual-purpose substratum is poured into culture dish (120mm), connect 3 kinds of pathogenic bacteria bacterium cakes in every ware, two bacterium cakes of every kind of bacterium insert the paper dish that had soaked bacteria suspension, a kind of antagonism bacterium of every ware between the bacterium cake.Compare with not connecing antimicrobial pathogenic bacteria short of money simultaneously, treat the contrast cover with after, the range estimation fungistatic effect; (3) dual-purpose substratum is poured into culture dish (120mm), connect a kind of pathogenic bacteria bacterium cake in every ware, 3 bacterium cakes of every ware insert the paper dish that had soaked bacteria suspension between the bacterium cake, and every ware connects the antagonism bacterium of a kind of primary dcreening operation.Compare with not connecing antimicrobial pathogenic bacteria short of money simultaneously, treat that contrast is covered with after, measure antibacterial circle diameter.
Contain toxic medium method: the reasonable antagonism bacterial classification that will filter out in paper dish method inserts the NYD nutrient solution, and shaking table is cultivated (28 ℃, 120r/min) 72h, and (121 ℃, 20min) back is stand-by in sterilization.In culture dish (90mm), add earlier 1mL stoste, add 19mL again and be cooled to PDA substratum about 45 ℃, mixing, after treating culture medium solidifying, insert similar pathogenic bacteria bacterium cake, repeat 6 times, do contrast with the flat board that does not contain stoste simultaneously, treat to measure colony diameter after contrast is covered with, calculate bacteriostasis rate.
Bacteriostasis rate calculation formula: bacteriostasis rate (%)=(contrast colony diameter-processing colony diameter) * 100/ (contrast colony diameter-6)
Present embodiment obtains the bacterial strain of 22 preliminary screening altogether, wherein there are 13 bacterial strains that the pathogenic bacteria of participating in the experiment is had in various degree restraining effect, to these effects preferably antagonistic strain be that indicator is further cooked antagonistic effect with marssonina coronaria etc., filter out the YN-1 bacterial strain that it is had fine prevention effect.
Table 1 separated portions bacterial strain is to the bacteriostatic action of marssonina coronaria
And the YN-1 bacterial strain further made bacteriostatic test with ring rot of apple bacterium, Valsa mali, marssonina coronaria, withered germ of water-melon, cucumber fusarium axysporum, capsicum wilt, cotton-wilt fusarium, rhizoctonia cerealis etc. as indicator, the result is as follows:
Table 2YN-1 antagonism bacterium is to the bacteriostatic action of fungal diseases of plants pathogenic bacteria
Above result shows, not only marssonina coronaria had significant antagonistic action by separating the bacterial strain YN-1 that obtains in the vegetable soil, and other fungal diseases of plants pathogenic bacteria also had antagonistic action preferably, show that the YN-1 bacterial strain has wide spectrum, bacteriostatic action efficiently.
Embodiment 2 identification of strains
To the strain separated YN-1 of institute among the embodiment 1, observe by biological characteristics, carry out further identification of morphology, method is as follows:
(1) dyeing: dyeing process routinely dyes.
(2) form and cultural characteristic: on NYDA and NYD substratum, cultivate 48h for 28 ℃, observe its colony characteristics and color etc., with observation by light microscope thalline feature.
(3) physiological and biochemical property: carry out with reference to " common bacteria system identification handbook " method.
(4) The stability of strain: place-70 ℃ of refrigerators to preserve this bacterial strain, take out, go down to posterity to cultivate and measure its bacteriostasis rate at 3,6,12,24 months different times.
(5) 16S rDNA and analysis: bacterial strain adopt the LB liquid nutrient medium 28 ℃ of shaking culture to logarithmic phase, centrifugal with 9000r/min, collect thalline, extract bacterial genomes DNA according to a conventional method, the universal PC R primer of synthetic be used to increase bacterial 16 S rDNA and ITS sequence increases, and the purpose fragment utilizes the method for molecular biology routine to clone and check order.By BLAST (http://www.ncbi.nlm.nih.gov/) sequencing result is analyzed, carried out homology relatively, determine the classification position of this bacterial strain with the sequence in the GenBank database.
Above experimental result record is as follows:
1. morphological specificity: this bacterium is in 28 ℃ of cultivations, and at microscopically, thalline is shaped as direct rod shape, and the thalline mean size is 21.5 * 2.82 μ m, and gramstaining is positive.Have pod membrane and gemma, gemma is shaped as ellipse, at the center, expands slightly.The flagellum end is given birth to, single flagellum.Has mobility.
2. cultural characteristic: this bacterium is cultivated 24h in 28 ℃ on the NYDA plate culture medium, produce circular bacterium colony, and the edge is irregular, the bacterium colony quality is moistening, white, the inoculating needle picking colony is used in tarnish, the thalline adhesion, have viscosity, the substratum color does not change, no special odor, continue to cultivate after 48 hours, it is dry that bacterium colony becomes.
3. physiological and biochemical property
The physiological and biochemical property of table 3 bacterial strain YN-1
Annotate: "+" expression positive reaction, "-" expression negative reaction.
4. The stability of strain places-70 ℃ of refrigerators to preserve this bacterial strain, goes down to posterity still to have the activity of bacterial classification for 6 times, and the growth temperature range of this bacterium is very wide, and between 10～55 ℃, optimum temperature is 28 ℃.Its growth potential of hydrogen is between 5～8, and optimum pH is 7.
5.16S rDNA and ITS sequential analysis are bacillus (Bacillus) according to 16S rDNA sequential analysis YN-1 bacterial strain, show also that from every morphological specificity, cultural characteristic and the physiological and biochemical property of bacterial strain this bacterial strain belongs to bacillus, identical with the feature of control group B.amyloliquefaciens bacterial classification.And further the ITS sequential analysis shows that also YN-1 bacterial strain ITS sequence and the B.amyloliquefaciensITS sequence of having reported have 99% homology.So with the YN-1 identification of strains is B.amyloliquefaciens.16S 1513bp
AGAGTTTGATCATGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGA
TGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGAC
TGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAA
AGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTC
ACCAAGGCGACGATGCGTAGCCGACCTGAGGGGGTGATCGGCCACACTGGGACTGAGACACGGCCC
AGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCC
GCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAAT
AGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAA
TACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCT
GATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAG
GAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGC
GACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT
GGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTA
ACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGC
CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT
CCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCG
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAG
CATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAA
TCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACC
GCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGA
AGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACC
GCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCC
GAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAACCGT
ITS?264bp
GTGCCAAGGCATCCACCGTGCGCCCTTTCCAACTTAACCGTTAAAAAGAATCACTACGTGATATCTTG
CATTACTAATTGAATGTGAATTACTTCTGTTATCTAGTTTTCAAAGAACAATATCTCGAAGAATCGAAT
GATCCTTCAAAACTAAACAAGACAGGGAACGTTCTGTTTATAAGACCCAAGGTCTTATATTCCGTTAA
AATCCTTAGAAAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACT
264bp
AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGATTTTAACGGAA
TATAAGACCTTGGGTCTTATAAACAGAACGTTCCCTGTCTTGTTTAGTTTTGAAGGATCATTCGATTCT
TCGAGATATTGTTCTTTGAAAACTAGATAACAGAAGTAATTCACATTCAATTAGTAATGCAAGATATCA
CGTAGTGATTCTTTTTAACGGTTAAGTTAGAAAGGGCGCACGGTGGATGCCTTGGCAC
321bp
AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGATTTTAACGGAA
TATAAGACCTTGGGTCTTATAAACAGAACGTTCCCTGTCTTGTTTAGTTTTGAAGGATCATTCCTTCAA
AACATAGGAACACCTGCTAAATGCGCCACTTCCTGTGGCAACGCATGTGTTAGCATGTCCTATGCGTT
GTTCTTTGAAAACTAGATAACAGAAGTAATTCACATTCAATTGTAATGCAAGATATCACGTAGTGATTC
TTTTTAACGGTTAAGTTAGAAAGGGCGCACGGTGGATGCCTTGGCAC
321bp
GTGCCAAGGCATCCACCGTGCGCCCTTTCTAACTTAACCGTTAAAAAGAATCACTACGTGATATCTTG
CATTACAATTGAATGTGAATTACTTCTGTTATCTAGTTTTCAAAGAACAACGCATAGGACATGCTAACA
CATGCGTTGCCACAGGAAGTGGCGCATTTAGCAGGTGTTCCTATGTTTTGAAGGAATGATCCTTCAAA
ACTAAACAAGACAGGGAACGTTCTGTTTATAAAACCCAAGGTCTTATATTCCGTTAAAATCCTTAGAA
AGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACT
264bp
AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGATTTTAACGGAA
TATAAGACCTTGGGTCTTATAAACAGAACGTTCCCTGTCTTGTTTAGTTTTGAAGGATCATTCGATTCT
TCGAGATGTTGTTCTTTGAAAACTAGATAACAGAAGTAATTCACATTCAATTAGTAATGCAAGATATCA
CGTAGTGATTCTTTTTAACGGTTAAGTTAGAAAGGGCGCACGGTGGATGCCTTGGCAC
322bp
AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGATTTTAACGGAA
TATAAGACCTTGGGTTTTATAAACAGAACGTTCCCTGTCTTGTTTAGTTTTGAAGGATCATTCCTTCAA
AACATAGGAACACCTGCTAAATGCGCCACTTCCTGTGGCAACGCATGTGTTAGCATGTCCTATGCGTT
GTTCTTTGAAAACTAGATAACAGAAGTAATTCACATTCAATTAGTAATGCAAGATATCACGTAGTGATT
CTTTTTAACGGTTAAGTTAGAAAGGGCGCACGGTGGATGCCTTGGCAC
438bp
AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTCTAAGGATTTTAACGGAA
TATAAGACCTCGGGTCTTATAAACAGAACGTTCCCTGTCTTGTTTAGTTTTGAAGGAACTTTAATTCTT
TCATATGGTAGATGGGCCTGTAGCTCAGCTGGTTAGAGCGCACGCCTGATAAGCGTGAGGTCGGTGG
TTCGAGTCCACTCAGGCCCACCATCTTTACCCAATACGGGGCCTTAGCTCAGCTGGGAGAGCGCCTG
CTTTGCACGCAGGAGGTCAGCGGTTCGATCCCGCTAGGCTCCACCAACGTGTTCTTTGAAAACTAGA
TAACAGAAGTAATTCACATTCAATTAGTAATGCAAGGTATCACGTAGTGATTCTTTTTAACGGTTAAGT
TAGAAAGGGCGCACGGTGGATGCCTTGGCAC
Embodiment 3YN-1 bacterial strain was measured the bacteriostatic activity of marssonina coronaria, ring rot of apple bacterium in the different fermentations stage
With marssonina coronaria, ring rot of apple bacterium is indicator.
Bacteriostatic activity is measured with containing toxic medium method.Cultivate marssonina coronaria, ring rot of apple bacterium flat board stand-by.Beginning in 6 hours after the access seed liquor, until different time sections sampling in 84 hours, after the sample sterilization of taking out, get to pour in the 90mm culture dish in the 1mL adding 19mLPDA substratum and make flat board, insert marssonina coronaria, ring rot of apple bacterium bacterium cake then respectively, repeat 6 times, do contrast with the PDA marssonina coronaria that does not contain fermented liquid, ring rot of apple bacterium flat board, treat to measure colony diameter after contrast is covered with, calculate bacteriostasis rate, average, antibacterial result is as shown in the table:
Table 4YN-1 bacterial strain fermentation liquor is to ring rot of apple bacterium and marssonina coronaria inhibiting rate (%)
The contained bacteriostatic activity material of the used biocontrol strain YN-1 of present embodiment fermented liquid as can be seen from Table 4, play the obvious suppression effect since 12 hours to indicator, along with prolonging bacteriostasis rate, fermentation time increases gradually, peak up to 48 hours bacteriostasis rates, ring rot of apple bacterium bacteriostasis rate is reached 92.4%, the marssonina coronaria bacteriostasis rate reaches 90.3%, after this still keeps than high bacteriostatic activity.This shows the antagonistic substance that this bacterial strain produces, after fermentation, produce gradually up to 48 hours and peak.Laboratory test results shows that active substance that YN-1 produces has higher bacteriostatic action to ring rot of apple bacterium and marssonina coronaria.
The preparation method of 4 one kinds of microbial preparations of embodiment, step is as follows:
1. the test tube strains preparation is inoculated into above-mentioned bacterial strains B.amyloliquefaciens YN-1CGMCC NO.2328 on the test tube NYDA slant medium, and 28 ℃ of following activation culture obtained test tube strains in 24 hours;
2. the seed liquor preparation inserts above-mentioned test tube strains in the NYD nutrient solution, shaking table cultivate 16 hours (28 ℃, 130 rev/mins) make primary seed solution, then primary seed solution was inoculated in 1: 100 and carries out (28 ℃ of fermentation culture in the 100mLNYD nutrient solution, 130 rev/mins) 12 hours, obtain secondary seed solution;
3. the fermention medium preparation is a raw material with glucose, Semen Maydis powder, soybean cake powder, adds water and is mixed with the nutrient solution that contains glucose 10g/L, Semen Maydis powder 13g/L, soybean cake powder 13g/L;
4. liquid fermenting production
Behind fermentation equipment and medium sterilization, secondary seed solution is inoculated in nutrient solution by 1: 25 volume ratio, 28 ℃ of following cultivation and fermentation 48 hours, with filtering fermentation liquor, sterilization promptly got the YN-1 bacterial strain fermentation liquor with its vacuum concentration to 50% of original volume again;
Sterilization: the sterilization of pipeline, air filter, fermentor tank, substratum is undertaken by the production technique standard.
Fermentation condition: 28 ℃ of temperature, carry out temperature regulation with the tap water method of cooling; Tank pressure 0.06MPa; Air flow 6slpm; Stirring velocity 120rpm; The pH value is measured in sampling in per 2 hours 1 time, violet staining microscopy thalli morphology, and observation has or not assorted bacterium etc.Carry out the viable bacteria detection with the culture medium flat plate method.Fermented 48 hours, and stopped when fungistatic effect is basicly stable cultivating.
5. formulation preparation is with above-mentioned YN-1 bacterial strain fermentation liquor 90kg and 0204 2kg, OP-103kg, sodium salicylate 0.5kg, sodium-chlor 1.5kg, the abundant mixing of calcium chloride 3kg.
To contain toxic medium method above-mentioned biotechnological formulation is carried out indoor determination of activity, and carry out field test in the apple orchard, its quality guaranteed period test shows that this biotechnological formulation at room temperature can be preserved 2 years.
The preparation method of 5 one kinds of microbial preparations of embodiment, substantially the same manner as Example 4, difference is: be raw material with glucose, Semen Maydis powder, soybean cake powder 1., add water and be mixed with the nutrient solution that contains glucose 8g/L, Semen Maydis powder 15g/L, soybean cake powder 15g/L; 2. to after fermentation equipment and the nutrient solution sterilization, secondary seed solution is inoculated in above-mentioned nutrient solution by 1: 22 volume ratio, 28 ± 1 ℃ of following cultivation and fermentation 36 hours, with filtering fermentation liquor, sterilization promptly gets the YN-1 bacterial strain fermentation liquor with its vacuum concentration to 40% of original volume again; 3. formulation preparation YN-1 bacterial strain fermentation liquor 85kg, SilwetL-772kg, sodium salicylate 2kg, urea 6kg, fully mixing.
The preparation method of 6 one kinds of microbial preparations of embodiment, substantially the same manner as Example 4, difference is: 1. to contain glucose 11g/L, Semen Maydis powder 10g/L, soybean cake powder 16g/L in the nutrient solution; 2. to after fermentation equipment and the nutrient solution sterilization, the second order fermentation seed liquor is inoculated in above-mentioned nutrient solution by 1: 30 volume ratio,, filters the nutrient solution after the fermentation 28 ± 1 ℃ of following cultivation and fermentation 48 hours, and sterilization, again its vacuum concentration to 60% of original volume is promptly got the YN-1 bacterial strain fermentation liquor; 3. formulation preparation adds OP-10 7kg, sodium salicylate 0.9kg, ethylene glycol 2kg, fully mixing in the 82kgYN-1 bacterial strain fermentation liquor.
The preparation method of 7 one kinds of microbial preparations of embodiment, substantially the same manner as Example 4, difference is: 1. to contain glucose 9g/L, Semen Maydis powder 16g/L, soybean cake powder 11g/L in the nutrient solution; 2. to behind fermentation equipment and the medium sterilization, the second order fermentation seed liquor is inoculated in above-mentioned nutrient solution by 1: 30 volume ratio,, filters the nutrient solution after the fermentation 28 ± 2 ℃ of following cultivation and fermentation 48 hours, and sterilization, again its vacuum concentration to 55% of original volume is promptly got the YN-1 bacterial strain fermentation liquor; 3. formulation preparation adds Mon 0818 6kg, sodium-chlor 1kg, ethylene glycol 2kg, calcium chloride 4kg, fully mixing in the 95kgYN-1 bacterial strain fermentation liquor.
The preparation method of 8 one kinds of microbial preparations of embodiment, substantially the same manner as Example 4, difference is: 1. to contain glucose 12g/L, Semen Maydis powder 12g/L, soybean cake powder 13g/L in the nutrient solution; 2. formulation preparation, with YN-1 bacterial strain fermentation liquor 94kg and OP-10 8kg, sodium salicylate 1kg, sodium-chlor 1kg, urea 2kg, the abundant mixing of calcium chloride 2kg.
The preparation method of 9 one kinds of microbial preparations of embodiment, substantially the same manner as Example 4, difference is: be raw material with glucose, Semen Maydis powder, soybean cake powder 1., add water and be mixed with the nutrient solution that contains glucose 12g/L, Semen Maydis powder 15g/L, soybean cake powder 15g/L; 2. to behind fermentation equipment and the medium sterilization, secondary seed solution is inoculated in above-mentioned nutrient solution by 1: 28 volume ratio, 28 ± 1 ℃ of following cultivation and fermentation 36 hours, with filtering fermentation liquor, sterilization is concentrated into it 40% of original volume again and promptly gets the YN-1 bacterial strain fermentation liquor; 3. formulation preparation YN-1 bacterial strain fermentation liquor 88kg, SilwetL-77 4kg, sodium salicylate 0.3kg, sodium-chlor 3kg, fully mixing.
Embodiment 10 microbial bactericides are prevented and treated the field test of apple marssonina leaf spot
(1) experimental field situation and time
Situation experimental field: test being located at Wangwu Shan Mountain forest farm, Jiyuan City, and brown spot takes place more serious.5 mu of orchard areas, Cultivar Fuji, the age of tree 10 years, density 65 strain/mus.
Test period: on August 26,8 to 2006 years July in 2006.
(2) test medicine: YN-1 strain fermentation liquid formulation and active bacteria formulation compare with the 80% new big wettable powder (U.S. believes company admittedly) of giving birth in addition.
(3) test is handled: after the YN-1 bacterial strain fermentation liquor is filtered, sterilizes, concentrates, be processed into aqua, its essentially consist: active substance (effective ingredient), solvent (water or other organism), auxiliary agent (tensio-active agent, antifreezing agent, sanitas etc.).With microbial preparation YN-1 described in embodiment 4 and the embodiment 5 is that mother liquor is an example, establishes 25 times, 50 times, 100 times diluents respectively; The viable bacteria that contains of viable bacteria is calculated with viable count/ml; 80% is new big living 800 times; The clear water contrast compares; Use with foliar spray, 9 processing, every processing 5 strains, totally 50 strains are established in test; Three medications, be 15 days interval.
(4) control time and method: time: carry out for the first time the investigation of disease radix before the medication, investigation for the second time is after the last medication the 10th day; Method: 3 of every processing investigation, every tree is from East, West, South, North, 5 orientation samplings of interior thorax, and 50 leaves of every orientation investigation are investigated the disease number of sheets, sick level, and calculate disease index, preventive effect.
The brown spot grade scale:
0 grade: anosis;
1 grade: lesion area accounts for the leaf total area less than 10%;
3 grades: lesion area 11%～25%;
5 grades: lesion area 26%～40%;
7 grades: lesion area 41%～65%;
9 grades: lesion area is greater than 65%.
Calculation formula:
(5) test-results
The fermented liquid of antagonism bacterium YN-1 adds different auxiliary agents or viable bacteria is used on apple, and apple marssonina leaf spot is had the better prevention effect.The prevention effect of the survey showed that (table 5) embodiment 4 is respectively 67.2%, 81.4%, 73.2%; ((preventive effect that contains viable count/ml) is respectively 79.2% and 53.2% to contain viable count/ml), 100 times for 50 times of viable bacterias.With execute water contrast and compare disease index tangible reduction is all arranged; The new big prevention effect 76.3% of giving birth to of control group chemical pesticide, peasant household is 68.1% from the prevention effect of pipe.
Table 5YN-1 is to the field control effect questionnaire of apple marssonina leaf spot
Claims (9)
- A bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) YN-1, its deposit number is CGMCC NO.2328.
- 2. microbial preparation with the preparation of the described bacillus amyloliquefaciens of claim 1, it is characterized in that, in weight part, this microbial preparation contains 82～95 parts of bacillus amyloliquefaciens YN-1 CGMCC NO.2328 fermented liquids, 2～8 parts in tensio-active agent, 1～4 part of sanitas, described bacillus amyloliquefaciens fermented liquid is to make by the following method: above-mentioned bacillus amyloliquefaciens YN-1 is inoculated in nutrient solution, fermentation culture at least 36 hours, with filtering fermentation liquor, the sterilization, again it is concentrated into original volume 40～60% the back; Contain glucose 8～12g/L, Semen Maydis powder 10～16g/L, soybean cake powder 10～16g/L in the described nutrient solution.
- 3. microbial preparation according to claim 2 is characterized in that, described microbial preparation also contains 2～6 parts of antifreezing agents.
- 4. microbial preparation according to claim 3 is characterized in that, described antifreezing agent is an ethylene glycol.
- 5. microbial preparation according to claim 4 is characterized in that, described tensio-active agent is 0204, at least a among the SilwetL-77, Mon 0818, OP-10.
- 6. microbial preparation according to claim 5 is characterized in that, described sanitas is a sodium salicylate.
- 7. the preparation method of a microbial preparation as claimed in claim 2 is characterized in that, it may further comprise the steps:(1) the test tube strains preparation is inoculated into bacillus amyloliquefaciens YN-1CGMCC NO.2328 on the test tube NYDA slant medium, cultivate down for 26～30 ℃ and obtained test tube strains in 24～48 hours, described NYDA slant medium is to be raw material with extractum carnis 8g, peptone 8g, glucose 10g, agar 15g, makes to 1000ml through moisturizing;(2) the seed liquor preparation inserts above-mentioned test tube strains in the NYD nutrient solution, under 26～30 ℃, cultivate and made primary seed solution in 12～16 hours, with 1: 100～150 volume ratio primary seed solution is inserted in the NYD nutrient solution then, under 26～30 ℃, carried out fermentation culture 12～14 hours, obtain secondary seed solution, described NYD nutrient solution is to be raw material with extractum carnis 8g, peptone 8g, glucose 10g, makes to 1000ml through moisturizing;(3) fermention medium preparation is got glucose, Semen Maydis powder, soybean cake powder and is added water and be mixed with the substratum that contains glucose 8～12g/L, Semen Maydis powder 10～16g/L, soybean cake powder 10～16g/L;(4) liquid fermenting production to fermentation equipment and medium sterilization after, secondary seed solution is inoculated in nutrient solution by 1: 20～30 volume ratio, after 26～30 ℃ of bottom fermentations are cultivated at least 36 hours, with filtering fermentation liquor, sterilization is concentrated into it 40～60% of original volume again and promptly gets the YN-1 bacterial strain fermentation liquor;(5) formulation preparation is got 82～95 parts of step gained YN-1 bacterial strain fermentation liquors, 2～8 parts in tensio-active agent, 1～4 part of sanitas, abundant mixing.
- 8. the preparation method of microbial preparation according to claim 7 is characterized in that, in described step (5), further sneaks into 2～6 parts of antifreezing agents, and described antifreezing agent is an ethylene glycol.
- 9. according to the preparation method of claim 7 or 8 described microbial preparations, it is characterized in that described tensio-active agent is 0204, at least a among the SilwetL-77, Mon 0818, OP-10; Described sanitas is a sodium salicylate.
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