CN103087944A - Bacilluscereus and application thereof - Google Patents
Bacilluscereus and application thereof Download PDFInfo
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- CN103087944A CN103087944A CN2012105218189A CN201210521818A CN103087944A CN 103087944 A CN103087944 A CN 103087944A CN 2012105218189 A CN2012105218189 A CN 2012105218189A CN 201210521818 A CN201210521818 A CN 201210521818A CN 103087944 A CN103087944 A CN 103087944A
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- bacilluscereus
- bacillus cereus
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Abstract
The invention discloses a bacilluscereus (Bacilluscereus1205) and an application thereof. The morphological characteristics are as follows: the 1205 bacterial strain is cultivated on an ordinary agar plate culture medium at 37 DEG C for 36 hours, the bacterial colony is nearly round, grey white, non-transparent and as large as 3-7mm, the edge is irregular and expanded, the surface is rough, granulated and is like ground glass or wax, no pigment is secreted; the bacilluscereus is a gram positive bacteria, rod-shaped, 1.0-1.3*3.0-5.0 micrometer, chaining and moveable; the spore is ellipse, terminal or secondary terminal and unclearly expanded; and the bacilluscereus can endure the cultivation of 7% NaCl. The bacilluscereus has the characteristics of strong stress resistance, simple nutritional requirement, stabilization, easy colonization, stability to light and ultraviolet, low cost, long storage period, is easy to apply and realize industrialization, and has a certain growth promoting effect on tobaccos.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of bacillus cereus bacterial strain, also relate to simultaneously the purposes of its bacillus cereus bacterial strain aspect the inhibition black shank.
Background technology
Tobacco is important cash crop, is also one of the most serious crop of disease hazard, and from being seeded into results, whole process all can be subject to infecting of pathogen, and causes infectious disease.Black shank (
Phytophthora parasitica var nicotianae(Breda de Hean) Tuker) be one of serious disease of tobacco disserve to produce, fragmentary generation, comprise that south China, Central China, East China and the plantation cigarette district, northwest in Taiwan Province all extensively occurs except cigarette district, northeast in China, financial loss is comparatively serious.
Produce at present upper general adopt disease-resistant variety, cultural control, chemical prevention and bio-control method, speed is fast and quantity is large but because the zoospore of this germ forms, infect short incubation period after the cigarette strain, rapid onset, can occur repeatedly to infect again a season of growth, should disease sometimes also mix generation with other disease simultaneously, thereby cause the forfeiture of some anti-balck shank varietal resistances; The employing of chemical pesticide in chemical prevention, effective in a short time.But prolonged and repeated using can cause cigarette strain poisoning, cigarette strain pesticide residue and germ to the resistance of agricultural chemicals.Biological control is to carry out at present in the world the first-selected prophylactico-therapeutic measures of control of plant disease, this from occurring in nature separate, screening beneficial microorganism and be used for antagonism, the inhibiting method of preventing and treating of pathogenic micro-organism, have efficient, nontoxic, noresidue and the characteristics such as low-cost.
Report from the research of existing biocontrol of plant disease, the microbe species of employing has the microorganisms such as bacterium, fungi, actinomycetes.But biological and ecological methods to prevent plant disease, pests, and erosion bacterial classification ubiquity and is surely grown difficultly, easily is subjected to the impact of ambient conditions, as comparatively responsive to light, ultraviolet, drying.
Summary of the invention
The object of the invention is to overcome the problems referred to above and provide a kind of have strong stress resistance, nutritional requirement simply, stablize, be easy to surely to grow, to light and UV stable, the characteristics such as easily industrialization, cost is lower, the shelf lives is long, be easy to use, and tobacco is had the bacillus cereus bacterial strain of certain growth promoting function.
Another object of the present invention is to provide the purposes of this bacillus cereus bacterial strain aspect the inhibition Phytophthora nicotianae.
A kind of bacillus cereus 1205(
Bacillus cereus1205), its morphological specificity: 1205 bacterial strains 37 ℃, are cultivated 36 h on the plain agar plate culture medium, the bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, the surface irregularity particulate state is not secreted pigment like ground-glass appearance or wax-like; Be gram positive bacterium, shaft-like, 1.0～1.3 * 3.0～5.0 μ m, chaining, mobility, gemma is oval, and end is given birth to or inferior end is given birth to, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, positive, the semi-solid puncture test positive that catalase is positive, glucose fermentation is positive, Starch Hydrolysis is positive, nitrate reduction is positive, gelatine liquefication is positive, maltose ferments, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment feminine gender, sucrose fermentation feminine gender, lactose fermentation is negative, the N.F,USP MANNITOL hydrolysis is negative.
This bacterial classification is deposited in Chinese Typical Representative culture collection center (address: Wuhan, China Wuhan University) on September 26th, 2012, and preserving number: CCTCC NO:M2012382, name is called: bacillus cereus 1205(
Bacillus cereus1205).
Phytophthora nicotianae there is inhibiting bacillus cereus 1205(
Bacillus cereus1205) separating step:
(1) isolation and purification of Antagonistic Fungi: take the 10g that picks up from Dafang County, Bijie City, Guizhou Province and plant the crude opium sample in the sterilized water of 90mL with granulated glass sphere, 160rpm vibration 20min on shaking table, standing 5min draws Soil Slurry with sterilized water gradient dilution step by step.Draw 0.1mL diluent coating beef extract-peptone dull and stereotyped, be inverted for 37 ℃ and cultivate 36h, after flat board grows bacterium colony, picking list bacterium colony purifying, numbering is preserved, and is to be measured.
(2) screening of antagonistic bacterium: adopt dull and stereotyped face-off method, good oat medium is dull and stereotyped, beat with the 5mm punch tool phytophthora silk piece of getting on 10% V8 juice substratum and be placed on dull and stereotyped central authorities, inoculate bacterial strain to be measured apart from 2.5cm place is symmetrical around it, take the flat board that do not connect thalline as contrast.Triplicate was cultivated 7 days in 28 ℃ of incubators, had observed a strain bacterium and had produced obvious inhibition zone on every side, and Phytophthora nicotianae is had obvious antagonistic effect, chose this bacterial isolates, and it is numbered 1205.
(3) evaluation of Antagonistic Fungi: 1205 bacterial strain points are connected on the plain agar flat board, 37 ℃, cultivate 36 h, the bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, the surface irregularity particulate state is not secreted pigment like ground-glass appearance or wax-like; Be gram positive bacterium, shaft-like, 1.0～1.3 * 3.0～5.0 μ m, chaining, mobility, gemma is oval, and end is given birth to or inferior end is given birth to, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, positive, the semi-solid puncture test positive that catalase is positive, glucose fermentation is positive, Starch Hydrolysis is positive, nitrate reduction is positive, gelatine liquefication is positive, maltose ferments, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment feminine gender, sucrose fermentation feminine gender, lactose fermentation is negative, the N.F,USP MANNITOL hydrolysis is negative.With reference to " uncle's outstanding Bacteria Identification handbook (the 8th edition), " common bacteria system identification handbook and " bacillus " are initially identified as bacillus.
In the beef extract-peptone liquid nutrient medium, 28 ℃ of 160rpm cultivate 12h, centrifugal collection thalline with 1205 inoculation.Adopt Shanghai to give birth to work Ezup pillar genome DNA extraction test kit (bacterium) and extract genomic dna.Take the DNA that extracts as template, use forward primer
16SF(5'-GAGAGTTTGATCCTGGCTCAG-3')
With reverse primer 16SR (5'-AAGGAGGTGATCCAGCCGCA-3'), the 16S rDNA gene of bacterial strain is carried out pcr amplification.The PCR condition is: 94 ℃ of 3min; 94 ℃ of 45s, 54 ℃ of 30s, 72 ℃ of 1min, 25 circulations; 72 ℃ are extended 5min eventually, and 4 ℃ stop.The PCR product send by Shanghai living work biotechnology limited-liability company and checks order, and length is 1468bp.Sequencing result in GenBank with
Bacillus cereusSimilarity is 97%.Comprehensive colonial morphology, Physiology and biochemistry and Molecular Identification, with this bacterial strain be designated as bacillus cereus (
Bacillus cereus1205).
Bacillus cereus bacterial strain 1205 suppresses the application of Phytophthora nicotianae aspect:
1205 bacterial strains that (1) will activate are chosen an articulating and are entered in NYBD nutrient solution (extractum carnis 8g, yeast extract paste 5g, glucose 10g, water 1L, pH nature, 121 ℃, sterilization 45min), and 28 ℃ of cultivation 48h of 160rpm are as the fermented liquid of antagonistic effect mensuration.
Adopt dull and stereotyped face-off method, (rolled oats 30g adds 10L distilled water to good oat medium, and 100 ℃ of water-baths are 1h approximately, 8 layers of filtered through gauze remove slag, adding distil water is supplied 1L again, agar 18g, pH nature, 121 ℃, sterilization 30min) flat board is beaten with the 5mm punch tool and is got phytophthora silk piece and be placed on dull and stereotyped central authorities, around it apart from three of 2.5cm place symmetry placements soaked bacillus cereus (
Bacillus cereus1205) filter paper of fermented liquid (diameter is 5mm), take not with fermented liquid as contrast, triplicate was cultivated in 28 ℃ of incubators 7 days, the antibacterial circle diameter of 1205 pairs of tobacco black shank bacteriums has reached 20.5mm.
The present invention compared with prior art has obvious beneficial effect, as can be known from the above technical solutions: bacillus cereus of the present invention (
Bacillus cereus1205) from the horse's mouth, to the wide accommodation of the natural environmental conditions such as temperature, pH, strong to the tolerance of poor environment, can keep the eubiosis, can adapt to the disease ecosystem, easily cultivate and preserve, suppress Phytophthora nicotianae effective, very large application prospect is agriculturally arranged.This technological line is reasonable, can be large-scale industrial production reliable pilot scale data and design considerations are provided, realize liquid fermenting suitability for industrialized production bacillus cereus (
Bacillus cereus1205), lay the foundation for developing the popularization of associated biomolecule preparation and land for growing field crops.
Embodiment
Phytophthora nicotianae there is inhibiting bacillus cereus 1205(
Bacillus cereus1205) separating step:
(1) strains tested: black shank pathogenic bacteria---Phytophthora nicotianae (
Phytophthora nicotianae), Microbiological Lab of Guizhou University separates, screens, identifies, preserves.
(2) isolation and purification of Antagonistic Fungi: take the 10g that picks up from Dafang County, Bijie City, Guizhou Province and plant the crude opium sample in the sterilized water of 90mL with granulated glass sphere, 160rpm vibration 20min on shaking table, standing 5min draws Soil Slurry with sterilized water gradient dilution step by step.Draw 0.1mL diluent coating beef extract-peptone (extractum carnis 5.0 g, peptone 10.0g, NaCl 5.0g, agar 20g, water 1L, pH7.2,121 ℃, sterilization 45min) flat board, be inverted for 37 ℃ and cultivate 36h, after flat board grows bacterium colony, picking list bacterium colony purifying, numbering is preserved, and is to be measured.
(3) screening of antagonistic bacterium: adopt dull and stereotyped face-off method, (rolled oats 30g adds 1L distilled water to good oat medium, and 100 ℃ of water-baths are 1h approximately, 8 layers of filtered through gauze remove slag, adding distil water is supplied 1L again, agar 18g, pH nature, 121 ℃, sterilization 30min) flat board is beaten with the 5mm punch tool and is got the 10%V8 substratum (10mL V8 adds deionized water 90 mL, CaCO
30.02g, agar 2g, pH nature, 121 ℃, sterilization 30min) on phytophthora silk piece be placed on dull and stereotyped central authorities, inoculate bacterial strain to be measured apart from 2.5cm place is symmetrical around it, take the flat board that do not connect thalline as contrast.Triplicate was cultivated 7 days in 28 ℃ of incubators, and having observed a strain bacterium has obvious antagonistic effect to Phytophthora nicotianae, and this strain bacterium is numbered 1205.
1205 bacterial strains of activation are chosen an articulating enter in NYBD nutrient solution (extractum carnis 8g, yeast extract paste 5g, glucose 10g, water 1L, pH nature, 121 ℃, sterilization 45min), 160rpm cultivates 48h for 28 ℃, as the fermented liquid of antagonistic effect mensuration.
Adopt dull and stereotyped face-off method, good oat medium is dull and stereotyped, beat with the 5mm punch tool and get phytophthora silk piece and be placed on dull and stereotyped central authorities, around it apart from three filter papers (diameter is 5mm) that soaked 1205 fermented liquids of the symmetrical placement in 2.5cm place, take not with fermented liquid as the contrast, triplicate was cultivated 7 days in 28 ℃ of incubators, and the antibacterial circle diameter of 1205 pairs of Phytophthora nicotianaes has reached 20.5mm.Simultaneously, picking colony edge and antibacterial mycelia film-making with the contact site, microscopic examination be to the Phytophthora nicotianae myceliolysis, fracture, the protoplasma phenomenon of condensing.
The evaluation of Antagonistic Fungi: 1205 on the plain agar plate culture medium, and 37 ℃, cultivate 36 h, the bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, the surface irregularity particulate state is not secreted pigment like ground-glass appearance or wax-like; Be gram positive bacterium, shaft-like, 1.0～1.3 * 3.0～5.0 μ m, chaining, mobility, gemma is oval, and end is given birth to or inferior end is given birth to, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, positive, the semi-solid puncture test positive that catalase is positive, glucose fermentation is positive, Starch Hydrolysis is positive, nitrate reduction is positive, gelatine liquefication is positive, maltose ferments, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment feminine gender, sucrose fermentation feminine gender, lactose fermentation is negative, the N.F,USP MANNITOL hydrolysis is negative.With reference to " uncle's outstanding Bacteria Identification handbook (the 8th edition), " common bacteria system identification handbook and " bacillus " are initially identified as bacillus.
In beef extract-peptone liquid nutrient medium (extractum carnis 5.0 g, peptone 10.0g, NaCl 5.0g, water 1L, pH7.2), 28 ℃ of 160rpm cultivate 12h, centrifugal collection thalline with 1205 inoculation.Adopt Shanghai to give birth to work Ezup pillar genome DNA extraction test kit (bacterium) and extract genomic dna.Take the DNA that extracts as template, use forward primer
16SF(5'-GAGAGTTTGATCCTGGCTCAG-3')
With reverse primer 16SR (5'-AAGGAGGTGATCCAGCCGCA-3'), the 16S rDNA gene of bacterial strain is carried out pcr amplification.The PCR condition is: 94 ℃ of 3min; 94 ℃ of 45s, 54 ℃ of 30s, 72 ℃ of 1min, 25 circulations; 72 ℃ are extended 5min eventually, and 4 ℃ stop.The PCR product send by Shanghai living work biotechnology limited-liability company and checks order, and length is 1468bp.Sequencing result in GenBank with
Bacillus cereusSimilarity is 97%.Therefore, comprehensive colonial morphology, Physiology and biochemistry and Molecular Identification, with this bacterial strain be designated as bacillus cereus (
Bacillus cereus1205).
By bacteriostatic test is illustrated bacillus cereus of the present invention (
Bacillus cereusThe purposes of inhibition Phytophthora nicotianae 1205):
(1) preparation of fermented liquid:
To activate bacillus cereus (
Bacillus cereus1205) bacterial strain is chosen an articulating and is entered in NYBD nutrient solution (extractum carnis 8g, yeast extract paste 5g, glucose 10g, water 1L, pH7.2,121 ℃, sterilization 45min), and 28 ℃ of 160rpm cultivate 12h, as seed liquor.By 3% inoculum size, 1205 seed liquor accesses are equipped with in the 250ml culturing bottle of 50ml NYBD fermentation culture, 28 ℃, 160rpm, shake-flask culture 48h gets the Antagonistic Fungi fermented liquid.
(2) with aseptic round filter paper (diameter 5mm), draw fermented liquid slightly dry before spirit lamp, be placed on gently dull and stereotyped (the rolled oats 30g of oat of inoculation Phytophthora nicotianae, add 1L distilled water, 100 ℃ of water-baths are 1h approximately, and 8 layers of filtered through gauze remove slag, then adding distil water is supplied 1L, agar 18g, the pH nature, 121 ℃, sterilization 30min) on, be inverted in 28 ℃ of incubators and cultivated 7 days, the antibacterial circle diameter of 1205 pairs of Phytophthora nicotianaes reaches 20.5mm.
Claims (2)
1. bacillus cereus 1205(
Bacillus cereus1205) preserving number that, it is characterized in that described bacterial classification is CCTCC NO:M2012382.
2. bacillus cereus 1205 as claimed in claim 1 is in the purposes that suppresses aspect Phytophthora nicotianae.
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Cited By (5)
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| CN104230563A (en) * | 2014-09-11 | 2014-12-24 | 红云红河烟草(集团)有限责任公司 | Microbial organic fertilizer containing Bacillus cereus |
| CN111500653A (en) * | 2020-05-26 | 2020-08-07 | 樟树市狮王生物科技有限公司 | Production process of polyglutamic acid |
| CN112340856A (en) * | 2020-11-03 | 2021-02-09 | 苏州汉风科技发展有限公司 | Biological environment-friendly wastewater treatment method |
| CN115093983A (en) * | 2022-04-21 | 2022-09-23 | 云南农业大学 | Growth-promoting disease-resistant biological agent |
| CN115322930A (en) * | 2022-08-19 | 2022-11-11 | 遵义大兴复肥有限责任公司 | Soil probiotics for promoting tobacco seedling growth |
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| CN115322930B (en) * | 2022-08-19 | 2023-06-09 | 遵义大兴复肥有限责任公司 | Soil probiotics for promoting tobacco seedling growth |
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Inventor after: Li Zhu Inventor after: Zhang Chuanping Inventor after: Ge Yongyi Inventor after: Huang Huagang Inventor after: Shen Yan Inventor after: Hu Wei Inventor after: Gao Dongdong Inventor after: Zhang Longlin Inventor before: Zhang Chuanping Inventor before: Li Zhu Inventor before: Ge Yongyi Inventor before: Huang Huagang Inventor before: Shen Yan Inventor before: Hu Wei Inventor before: Gao Dongdong Inventor before: Zhang Longlin |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG CHUANPING LI ZHU GE YONGYI HUANG HUAGANG SHEN YAN HU WEI GAO DONGDONG ZHANG LONGLIN TO: LI ZHU ZHANG CHUANPING GE YONGYI HUANG HUAGANG SHEN YAN HU WEI GAO DONGDONG ZHANG LONGLIN |
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