CN1952116A - Bacillusamyloliquefaciens strain and application thereof - Google Patents
Bacillusamyloliquefaciens strain and application thereof Download PDFInfo
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Abstract
The invention relates to a strain of lys-starch bacillus as well its application. The bacterium is separated from the soil on eggplant fields, anning district, Lanzhou, Gansu. The bacterium is identified to be Bacillus amyloliquefaciens. It's preserved in the Chinese typical culture preserving center (CCTCC) with a preserving name of Bacillus amyloliquefaciens I7 and preserving code of M206027. The broth of M206027 and M206027strain, non-cell culture fluid of M206027 strain's broth, and the monomer extracted from the M206027strain, M206027 broth or its non-cell culture fluid can be used in the biological control of peach anthracnose bacterium, pear nigrities artis bacterium, apple brown rot bacterium, Fusarium acuminatum, Fusarium oxysporum, Fusarium semitectum, chilli black rot bacterium, cucumber blight bacterium, tomato early blight bacterium, eggplant early blight bacterium, dry thread Pyrenomycetes, wheat foot rot bacterium, potato thread Pyrenomycetes, potato early blight bacterium, potato late blight bacterium and watermelon downy mildew bacterium. It's easy to get the material of the invention and easy to operate; besides, it can be widely used in full scale production.
Description
Technical field
The present invention relates to the bacillus amyloliquefaciens of the new screening of a strain, be preserved in Chinese typical culture collection center C CTCC, preservation name I7 (Bacillus amyloliquefaciens), preserving number M206027.This strains separation is in the soil on eggplant ground, Anning District, Lanzhou City, Gansu Province, and it has significant inhibition growth effect to the pathogenic fungi that can cause Plant diseases, and nontoxic to people and animals, to eco-friendly, can be prepared into the biological pesticide product.
Background technology
Nineteen twenty-one, it is the beginning that people utilize the antagonistic microbe controlling plant diseases that Hartely utilizes the antagonism fungi to prevent and treat cotton seedling samping off (Pythium debaryanum)
[1]Subsequently, broad research has all been carried out to this emerging field in countries in the world, attempts to filter out the control that antagonistic microbe is used for Plant diseases.After the fifties in last century, a large amount of chemical pesticides that are extensive use of cause pathogenic bacteria that chemical pesticide is produced resistance, high residue causes environmental pollution, and chemical pesticide produces to poison etc. to people and animals and quickened people and seek new, control of plant disease approach safely and effectively.Plant diseases is brought heavy losses to agriculture production every year, in the various measures of controlling plant diseases, biological control has the safer advantage of environment, ecology and human health, therefore, countries in the world are attached great importance to carrying out of biological and ecological methods to prevent plant disease, pests, and erosion research and are utilized, and it will play an increasingly important role in disease control.Wherein the biological and ecological methods to prevent plant disease, pests, and erosion microorganism have be difficult for making pathogenic bacteria produce resistance, environmentally safe, production technique is simpler and many microorganisms have the characteristics that promote plant growth simultaneously and cause people's attention and attention
[2]
Australian phytopathologist Kerr in 1973 utilizes radiation edaphic bacillus (Agrobacterium radiobacterK84) root knot of successfully having prevented and treated plant, and form the commercialization preparation rapidly, promptly apply in states such as U.S., Australia, days, greatly promoted the interest of people biocontrol of plant disease
[1]The bacteria preparation that is widely used in the plant biological and ecological methods to prevent plant disease, pests, and erosion at present has kind more than 20 approximately, is mainly the bacterial strain that Agrobacterium, Pseudomonas, Bacillus etc. belong to
[3]
Bacillus is the aerobic and amphimicrobian of a class, the G that produces gemma
+Rod-shaped bacterium, its physiological characteristic is rich and varied, and distribution and extensive is the important microbe population of soil and plant surface rhizosphere.The outstanding feature of genus bacillus is to produce the gemma that heat, ultraviolet ray, ionizing rays and some pharmaceutical chemicals is had very strong resistance, can stand various bad environment, this helps production, the formulation processing of biocontrol fungicide and survives in environment, grows surely and breeding
[3,4]It can produce the antimicrobial substance of multiple inhibition plant pathogenic fungi, the antibacterial protein and the low-molecular-weight antibacterial peptide that comprise macromolecule, it is again the non-pathogenic bacteria that nature extensively exists simultaneously, to the person poultry harmless, environmentally safe, thereby in the biological control Plant diseases, play an important role.The field applied research has confirmed that the genus bacillus biocontrol fungicide in stability, with the consistency of chemical pesticide with at the aspects such as consistence of different plant different year preventive effects, obviously is better than non-genus bacillus and fungi biocontrol fungicide
[5]Bacillus is used for mainly containing of controlling plant diseases, subtilis (B.subtilis), bacillus cereus (B.cereus), bacillus pumilus (B.pumillus), bacillus polymyxa (B.polymyxa) etc. at present.
Fungal disease is the main disease that endangers plant-growth, influences crop yield always
[6], quantity is maximum in Plant diseases, reaches more than 80%, and distributed pole is wide, is a most important class in the Plant diseases
[7], fungal diseases of plants brings serious harm for China's agriculture production always.As Hou Xianpeng point out the regional pollution-free vegetable in Gansu Province east produce in just with fungal disease most species, generation area maximum, endanger the most serious
[8]Open the clear and coherent investigation Tianshui of crossing of clever and protect the main vegetable disease in ground, identify and confirm that wherein fungal disease just reaches 37 kinds, and Micobial Disease and virus disease have only 3 kinds and 5 kinds respectively
[9]The domestic patent of utilizing bacillus amyloliquefaciens control fungal diseases of plants product that yet there are no, also rarely seen in the world United States Patent (USP) 6,960, the bacillus amyloliquefaciens bacterial strain that one strain is separated to from Taiwan lily ground soil is disclosed in 342, as biocontrol microorganisms, prevent and treat the lily gray mold that causes by oval grape spore with it
[10]In addition, biocontrol microorganisms performance biological and ecological methods to prevent plant disease, pests, and erosion effect also relates to its deciding in the different soils environment grows, and can be applicable to multiple soil type but yet there are no any bacillus amyloliquefaciens biocontrol fungicide so far.Related bacillus amyloliquefaciens I7 among the present invention, not only this bacterial strain itself has wide spectrum biological and ecological methods to prevent plant disease, pests, and erosion potentiality, and the acellular nutrient solution of I7 also has wide spectrum biological and ecological methods to prevent plant disease, pests, and erosion potentiality, and they have higher preventive effect to the pathogenic fungi that is no less than 16 strains.The researchist of same domain it is also envisioned that thus: the fermented liquid of I7 bacterial strain and the monomer that extracts from the acellular nutrient solution of I7 bacterial strain, I7 fermented liquid, I7 fermented liquid or mixture can have wide spectrum biological and ecological methods to prevent plant disease, pests, and erosion potentiality too, and the pathogenic fungi that is no less than 16 strains is had higher preventive effect.Special needs to be pointed out is that this bacillus amyloliquefaciens bacterial strain, its fermented liquid and extracting solution thereof etc. still show higher activity under northwest drought, semiarid different soils type, weather condition, intensive ultraviolet condition; can be used for preventing and treating to the serious fungal disease of agriculture production harm; reduce the consumption of chemical pesticide, the ecotope of protection the Northwest.Because pathogenic fungi involved in the present invention extensively exists, obviously, bacillus amyloliquefaciens bacterial strain of the present invention, its fermented liquid and the monomer or the mixture that from its acellular nutrient solution, extract, especially the monomer or the mixture that extract in the acellular nutrient solution are not limited only to use in above-mentioned region.
Reference
1.Baker K F.Evolving concepts of biological control of plantpathogens[J].Ann.Rev.Phytopathol.，1987，25：67-85.
2. Pei Yan etc. the separation and purification of anti-fungus polypeptide APS-1 and characteristic [J]. microorganism journal, 1999,1 (3): 114-117.
3. old middle justice etc. Plant diseases biological and ecological methods to prevent plant disease, pests, and erosion genus bacillus antibacterial mechanisms and genetic improvement research [J]. Plant Pathology, 2003,33 (2): 97-103.
4.AhimouF.，Jacques and Deleu..Surfactin and iturin A effects on Bacillussubtilis surface hydrophobicity[J].Microbiol Technol，2000，27：749-754.
5.Monica L E，Elizabeth A D J，William E B J，et al.Viability andstability of biological control agents on cotton and snap bean seeds[J].Pest.Manag.Sci.，2001，57(8)：695-706.
6. easily rely on oneself Zhou Piaohua, Zhu Yaan. plant resistance to fungal disease genetically engineered progress [J]. crop investigations, 2001 (3): 81-84.
7. Li Yan. Plant diseases ABC [J]. orchard worker's friend, 2003 (6): 34-35.
8. Hou Xian roc. the chemical prevention [J] of common fungal disease during the regional pollution-free vegetable in east, Gansu Province produces. Gansu rural science and technology, 2003 (6): 29-31.
9. it is clear to open clever. the main vegetable disease register [J] in protection ground, Tianshui. and agriculture of gansu science and technology, 2005 (1): 40-42.
10.US6，960，342 Method for inhibiting pathogenic fungi in plants usingBacillus amyloliquefaciens.
Summary of the invention
The invention provides the bacillus amyloliquefaciens bacterial strain of the new purposes of a strain, it all has the obvious suppression effect to the growth of multiple pathogenic fungi, and is adapted to the soil climate condition of the Northwest.The culture temperature of this bacterial strain in NA solid medium and NA liquid nutrient medium is 28～30 ℃, incubation time 24～48h, be 24h (inoculum size 1: 100) the best kind age of cultivating in the NA seed culture medium, be inoculated in fermention medium at 1: 20 by inoculum size again, the pH7.0 of fermented liquid, rotating speed 120rpm/min, 30 ℃ of temperature, fermentation time 72h.In the above conditions, the identification mark of this bacterial strain is: rod-shaped bacterium, G
+The property; The flagellum adnation, on each thalline 8～20 flagellums are arranged, its length is about 2～3 times of thalline; Pod membrane is arranged; Form gemma, middle life, sporangium is expanded; Can be under the temperature condition more than 45 ℃ normal growth, be high temperature microbe; Can under 12% salt concn, grow, but not belong to halophilic bacteria; The growth optimal pH is between 7～8, and the upgrowth situation in alkaline environment will be significantly better than sour environment; Be accredited as Bacillus amyloliquefaciens (bacillus amyloliquefaciens) through VITEK32 full automatic microorganism identification systems; Measured 16SrRNA sequence alignment result shows: with homology＞99% of the 16SrRNA sequence of known bacillus amyloliquefaciens; (G+C) the mol% measurement result shows: (G+C) mol% of I7 is 49.4%, generally is worth (43.5～44.9%) difference 4～5% with (G+C) mol% of bacillus amyloliquefaciens, can think of the same race in different strains.
The fermented liquid of bacillus amyloliquefaciens bacterial strain I7 of the present invention, I7, the acellular nutrient solution of I7 fermented liquid and the monomer that extracts from the acellular nutrient solution of I7 bacterial strain, I7 fermented liquid, I7 fermented liquid thereof or mixture have intensive to suppress active to the Plant diseases fungi, can be prepared into biological pesticide and use.
Embodiment
Embodiment 1
The separation and purification of bacterial strain
From the eggplant ground soil of Anning District, Lanzhou City, Gansu Province morbidity, select suitable place, take soil sample in 5～20mm place, with dilution-plate method separation of bacterial therefrom:
(1) preparation soil diluent: take by weighing soil sample 10g, put into the 100ml sterilized water, 30min vibrates on the shaking table.Leave standstill and make clarification.Aseptic technique is drawn supernatant liquor 100 μ l and is added in the 900 μ l sterilized waters, is 10 with the sterilized water dilution
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8Totally eight concentration gradients.
(2) cultivate: each weaker concn adds NA (extractum carnis 3g, peptone 5g, glucose 10g, yeast extract 1g, agar 15g, distilled water 1000ml, the pH7.0) nutrient agar of the sterilization that is chilled to 55～60 ℃, makes flat board.Be inverted 30 ℃ of cultivations in the thermostat container.
(3) picking list bacterium colony: after 3～4 days, after treating to grow bacterium colony on the flat board, according to form picking list bacterium colony in the NA liquid nutrient medium of 20ml sterilization, 30 ℃ of constant-temperature shaking culture on the shaking table.
(4) purifying: adopt plate streaking partition method purifying.Be coated with dull and stereotyped check until obtaining pure culture, called after I7.
(5) preservation: the I7 of purifying adopts the glycerine preserving process, in the bacterial classification pipe, adds/go into the glycerine of 1ml 30%, and the sterilization back adds the 1ml inoculum, make the glycerine final concentration be 15% ,-20 ℃ frozen.
This culture is white in color on the NA solid medium, and colony shape is irregular, and showing has fold, sensation thickness during with the transfering loop picking.
Embodiment 2
The identification mark of bacterial strain
1. thalli morphology is observed and staining reaction
I7 is inoculated in the suitable time poststaining microscopy of constant-temperature shaking culture in the NA liquid nutrient medium.Comprise gramstaining (Viola crystallina oxamide staining), flagella staining (silver nitrate method staining), capsule stain (wet ink method), spore staining (the Schaeffer-Fulton Albert'stain Albert method of improvement).
Coloration result: I7 is a rod-shaped bacterium, G
+The property; The flagellum adnation, on each thalline 8～20 flagellums are arranged, its length is about 2～3 times of thalline; Pod membrane is arranged; Form gemma, middle life, sporangium is expanded.
2. environmental factors is to the influence of bacterial strain I7
2.1 O
2Influence to bacterial strain I7
Adopt the deep layer agar method to measure O
2Influence to I7 growth inserts I7 in the test tube that glucose beef extract-peptone agar deep layer substratum (pH7.0) is housed, leave standstill insulation 48h in 28 ℃ of thermostat containers after beginning observe its upgrowth situation continuously, till result behind the 10d is clear.According to its growth site in test tube, judge separately to O
2Demand and tolerance.
The result shows that I7 only in the test tube surface growth, is aerobic bacteria.
2.2 temperature is to the influence of bacterial strain I7
Preparation NA agar plate, making substratum thickness after solidifying is 1.5～2 times of general substratum.I7 is coated with flat board, respectively gets two cover flat boards and be inverted under 4 ℃, 10 ℃, 30 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃ of conditions and be incubated 48h, observe its upgrowth situation.
Table 1 temperature is to the influence of I7 growth
| Temperature (℃) | 4 | 10 | 30 | 45 | 50 | 55 | 60 | 65 |
| Upgrowth situation | - | + | ++ | +++ | + | - | - | - |
"-" expression is not grown, and "+" expression growth is relatively poor, and " ++ " expression growth is general, " +++" the expression well-grown.
As seen from the above table, I7 can be under the temperature condition more than 45 ℃ normal growth, be high temperature microbe.
2.3 osmotic pressure is to the influence of bacterial strain I7
Preparation contains 0.85%, 5%, 10%, 12%, 15%, 20% and the beef extract-peptone nutrient agar flat board of 25%NaCl.I7 is coated with flat board, and each two cover is cultivated in 28 ℃ of thermostat containers.Observe and write down the growing state that contains I7 on the different concns NaCl flat board behind the 7d.
Table 2 osmotic pressure is to the influence of I7 growth
| NaCl concentration % | 0.85 | 5 | 10 | 12 | 15 | 20 | 25 |
| Upgrowth situation | +++ | ++ | + | + | - | - | - |
"-" expression is not grown, and "+" expression growth is relatively poor, and " ++ " expression growth is general, " +++" the expression well-grown.
As seen from the above table, I7 can grow under 12% salt concn, but does not belong to halophilic bacteria.
2.4 pH is to the influence of bacterial strain I7
Preparation pH 3,4,5,6,7,8,9,10,11,12 NA liquid nutrient medium.The I7 children bacterium liquid in age of aseptic technique inoculation equivalent guarantees the bacterial concentration unanimity that inserts in each culturing bottle.120rpm/min behind 30 ℃ of following shaking culture 24h, measures the OD of culture
600Value.
Table 3 pH is to the influence of bacterial strain I7 growth
| pH | pH3 | pH4 | pH5 | pH6 | pH7 | pH8 | pH9 | pH10 | pH11 | pH12 |
| OD600 | 0.008 | 0.013 | 0.018 | 0.656 | 0.855 | 0.788 | 0.756 | 0.751 | 0.479 | 0.420 |
As seen from the above table, the growth optimal pH of I7 is between 7～8, and its upgrowth situation in alkaline environment will be significantly better than sour environment.
3.I7 biochemical reactions
Carry out biochemical characteristic with full automatic microorganism identification systems VITEK 32 and supporting aerobic spore-bearing bacilli identification card (BAC card) and identify, detect 31 biochemical indicators altogether.
The biochemical identification result of table 4 I7
| Aerobic spore-bearing bacilli identification card BAC | |||||||||||||||||
| Index result | Index Result | NEG - | SUC + | TZR - | TAG - | GLU + | INO - | GAL - | ARA - | XYL - | MAN - | RAF - | SAL - | AGA - | INU - | RIB - | MLT - |
| Index result | Index Result | TRE - | PLA - | SOR + | NAG - | AMY + | KCN + | NCL - | MEN + | OLD - | NAA - | ARB - | PAS + | NAE - | ESC + | THRM - | |
Annotate: biochemical indicator abbreviation full name is with reference to VITEK aerobic spore-bearing bacilli identification card (BAC card) operation instruction.
The I7 mirror
Be decided to be Bacillus amyloliquefaciens (bacillus amyloliquefaciens), confidence level＞99%.
4.16S the rRNA oligonucleotide sequence is analyzed
Use CEQXL2000, the BECKENMAN-COUNLTERDNA sequenator carries out the 16S rRNA sequencing of I7.Sequencing primer is:
27f： 5-AGAGTTTGATCCTGGCTCAG-3
685r： 5-TCTACGCATTTCACCGCTAC-3
705f： 5-GTAGCGGTGAAATGCGTAGA-3
1492r： 5-ACGGCTACCTTGTTACGACTT-3
Measuring 2 partial sequences of this bacterial strain 16S rRNA gene, is respectively 625bp and 721bp.
The 625bp sequence:
GTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGAGGGAACTTGAGTGC
The 721bp sequence:
CGACTCTCTGGTTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCT
With this 2 partial sequence at www.ncbi.nlm.nih.govhttp: //compare with the 16S rRNA gene order of BLAST instrument and listed part bacterial strain on the www.ncbi.nlm.nih.gov website, the result show with the homology of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) all＞99%.
5.DNA the mensuration of base ratio
The instrument that uses is the SHIMADZU UV-1700 ultraviolet-visible pectrophotometer with supporting temperature control unit, and wavelength is fixed on 260nm, and I7DNA sample to be measured is diluted to OD260 between 0.3～0.6 with 0.1SSC.Do the reference bacterial strain with E.coli K12, adopt the thermal denaturation temperature method to measure:
Because the Tm value of the E.coli-K12 that measures is 75.7 (among the 0.1SSC), so calculate (G+C) mol% value of I7 by following formula:
0.1SSC:(G+C) mol%=51.2+2.08 (the unknown bacterium of Tm-Tm E.coli K12)
The Tm value of measured I7 is 74.87, and (G+C) mol% that calculates I7 is 49.4%.
(G+C) mol% of bacillus amyloliquefaciens is generally 43.5～44.9% (the outstanding Bacteria Identification handbook of uncle, the 8th editions).Since meaningless in different bacterium (G+C) the mol% difference 2%, can think same bacterium; 4～5% interior different strains of the same race;＞5% does not belong to a kind of; 10～15% belong to interior different bacterium together; 20～30% do not belong to together even not equal.(G+C) mol% of I7 is 49.4%, with the general value difference of (G+C) mol% of bacillus amyloliquefaciens not 4～5%, also can think of the same race in different strains
Embodiment 3
The preparation of the acellular nutrient solution of I7 bacterial strain
1. I7 is inoculated in the 25ml a small amount of NA liquid nutrient medium (pH7.0) 120rpm/min, 30 ℃, shaking culture 24h in 1: 100 ratio of inoculum size;
2. more above-mentioned nutrient solution is inoculated in a large amount of NA liquid nutrient mediums of 1000ml (pH7.0) 120rpm/min, 30 ℃, shaking culture 72h in 1: 20 ratio;
3. with fermented liquid through 8000～12000r/min centrifuging and taking supernatant, the concentrated solution that the supernatant liquor of collecting obtains after bacterium filter membrane (0.22 μ m) filters is coated on it on flat board, detects to there not being viable bacteria, is the acellular nutrient solution of I7.
Embodiment 4
I7 bacterial strain, the acellular nutrient solution of I7 are to the fungistatic effect of pathogenic bacteria
1.I7 bacterial strain is to the fungistatic effect of pathogenic bacteria
I7 is inoculated in the NA liquid nutrient medium in 1: 100 ratio, 120rpm/min, 30 ℃ of temperature behind the shaking culture 24h, are drawn 200 μ l bacterium liquid and are evenly coated the NA agar plate, behind the interior 30 ℃ of cultivation 48h of thermostat container, buy the I7 bacterium cake of getting 7mm with punch tool, insert PDA agar plate central authorities, the equidistant pathogenic fungi bacterium cake that inserts 7mm around 20mm is outer, contrast replaces I7 bacterium cake with the NA agar block, and every processing repeats 3 wares.28 ℃ of cultivations in the thermostat container.When treating that plate is covered with in contrast, the right-angled intersection method is measured antagonism band diameter, and every ware is surveyed 2 data, surveys 6 altogether.(D1, D2 are the 1st ware take off data, and D3, D4 are the 2nd ware take off data, and D5, D6 are the 3rd ware take off data, and D is a mean number)
Table 5 I7 is to the fungistatic effect of 16 kinds of pathogenic bacterias
1-anthracnose of peach bacterium 2-pears alternaria 3-apple brown rot bacterium 4-fusarium acuminatum
5-Fusarium oxysporum 6-Fusarium semitectum 7-capsicum black rot 8-cucumber fusarium axysporum
9-tomato early blight bacterium 10-eggplant early blight bacterium 11-dry thread Pyrenomycetes 12-root rotof flax bacterium
13-julienne potatoes pyrenomycetes 14-target bacterium 15-phytophthora infestans 16-watermelon Pseudoperonospora cubensis
2.I7 acellular nutrient solution is to the fungistatic effect of pathogenic bacteria
Employing contains the method for medium with 10 times of (1/10), 20 times (1/20) of I7 bacterial strain preparation dilution, 40 times (1/40), 100 times (1/100) and 400 times (1/400), moves into pathogenic fungi bacterium piece in the dull and stereotyped central authorities of PDA; Adopting direct titrimetric method that stoste and the diluent that respectively dilutes gradient are got 200 μ l direct titrations is on the bacterium piece of 7mm at diameter, puts 28 ℃ and cultivates down, measures pathogenic bacteria bacterium piece growth diameter behind the 72h.Drip sterilized water to impinging upon on the pathogenic fungi bacterium piece.5 repetitions are established in every processing, and test repeats 2 times.
Respectively dilute pathogenic bacteria growth diameter under the gradient behind table 6 72h
1-anthracnose of peach bacterium 2-pears alternaria 3-apple brown rot bacterium 4-fusarium acuminatum
5-Fusarium oxysporum 6-Fusarium semitectum 7-capsicum black rot 8-cucumber fusarium axysporum
9-tomato early blight bacterium 10-eggplant early blight bacterium 11-dry thread Pyrenomycetes 12-root rotof flax bacterium
13-julienne potatoes pyrenomycetes 14-target bacterium 15-phytophthora infestans 16-watermelon Pseudoperonospora cubensis
Table 7 is obtained by the data computation of table 6: inhibiting rate=100% * (CK diameter-pathogenic bacteria diameter)/CK diameter
Respectively dilute the inhibiting rate of gradient behind table 7 72h to the pathogenic bacteria growth
1-anthracnose of peach bacterium 2-pears alternaria 3-apple brown rot bacterium 4-fusarium acuminatum
5-Fusarium oxysporum 6-Fusarium semitectum 7-capsicum black rot 8-cucumber fusarium axysporum
9-tomato early blight bacterium 10-eggplant early blight bacterium 11-dry thread Pyrenomycetes 12-root rotof flax bacterium
13-julienne potatoes pyrenomycetes 14-target bacterium 15-phytophthora infestans 16-watermelon Pseudoperonospora cubensis
Claims (5)
1. a bacillus amyloliquefaciens I7 separates in the soil on eggplant ground, Anning District, Lanzhou City, Gansu Province, by China's typical culture collection center preservation, and preservation name Bacillus amyloliquefaciens I7, preserving number is M206027.
2. a bacillus amyloliquefaciens I7 according to claim 1, its feature is as follows: bacillus amyloliquefaciens I7 is at NA solid medium (beef extract 3.0g, peptone 5.0g, glucose 10g, yeast extract 1.0g, agar 15g, water 1000ml, pH7.0) and NA liquid nutrient medium (beef extract 3.0g, peptone 5.0g, glucose 10g, yeast extract 1.0g, water 1000ml, pH7.0) cultivate culture condition: 28～30 ℃ of temperature, incubation time 24～48h in, the identification mark of this bacterium is: I7 is a rod-shaped bacterium, G+; The flagellum adnation, on each thalline 8～20 flagellums are arranged, its length is about 2～3 times of thalline; Pod membrane is arranged; Form gemma, middle life, sporangium is expanded; Can be under the temperature condition more than 45 ℃ normal growth, be high temperature microbe; Can under 12% salt concn, grow, but not belong to halophilic bacteria; The growth optimal pH is between 7～8, and its upgrowth situation in alkaline environment will be significantly better than sour environment; Be accredited as Bacillus amyloliquefaciens (bacillus amyloliquefaciens) through VITEK 32 full automatic microorganism identification systems; Measured 16SrRNA sequence alignment result shows: with homology＞99% of the 16S rRNA sequence of known bacillus amyloliquefaciens; (G+C) the mol% measurement result shows: (G+C) mol% of I7 is 49.4%, generally is worth (43.5～44.9%) difference 4～5% with (G+C) mol% of bacillus amyloliquefaciens, can think of the same race in different strains.
3. according to claim 1 and 2 described bacillus amyloliquefaciens I7, it is characterized in that this bacterial strain can be used as the broad-spectrum antifungal biocontrol microorganisms and uses, in particular for the biological control of plant pathogenic fungis such as anthracnose of peach bacterium, pears alternaria, apple brown rot bacterium, fusarium acuminatum, Fusarium oxysporum, Fusarium semitectum, capsicum black rot, cucumber fusarium axysporum, tomato early blight bacterium, eggplant early blight bacterium, dry thread Pyrenomycetes, root rotof flax bacterium, julienne potatoes pyrenomycetes, target bacterium, phytophthora infestans, watermelon Pseudoperonospora cubensis.
4. according to claim 1 and 2 described bacillus amyloliquefaciens I7, the acellular nutrient solution that it is characterized in that this bacterial strain can be used as broad-spectrum antifungal biological and ecological methods to prevent plant disease, pests, and erosion agent application, in particular for the anthracnose of peach bacterium, the pears alternaria, the apple brown rot bacterium, fusarium acuminatum, Fusarium oxysporum, Fusarium semitectum, the capsicum black rot, cucumber fusarium axysporum, tomato early blight bacterium, eggplant early blight bacterium, dry thread Pyrenomycetes, the root rotof flax bacterium, the julienne potatoes pyrenomycetes, the target bacterium, phytophthora infestans, the biological control of plant pathogenic fungis such as watermelon Pseudoperonospora cubensis.
5. according to claim 1 and 2 described bacillus amyloliquefaciens I7, it is characterized in that the fermented liquid of I7 bacterial strain and from the I7 bacterial strain, the I7 fermented liquid, monomer that extracts in the acellular nutrient solution of I7 fermented liquid or mixture can be used as broad-spectrum antifungal biological and ecological methods to prevent plant disease, pests, and erosion agent and use, in particular for the anthracnose of peach bacterium, the pears alternaria, the apple brown rot bacterium, fusarium acuminatum, Fusarium oxysporum, Fusarium semitectum, the capsicum black rot, cucumber fusarium axysporum, tomato early blight bacterium, eggplant early blight bacterium, dry thread Pyrenomycetes, the root rotof flax bacterium, the julienne potatoes pyrenomycetes, the target bacterium, phytophthora infestans, the biological control of plant pathogenic fungis such as watermelon Pseudoperonospora cubensis.
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