CN103087944B - Bacilluscereus and application thereof - Google Patents
Bacilluscereus and application thereof Download PDFInfo
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- CN103087944B CN103087944B CN201210521818.9A CN201210521818A CN103087944B CN 103087944 B CN103087944 B CN 103087944B CN 201210521818 A CN201210521818 A CN 201210521818A CN 103087944 B CN103087944 B CN 103087944B
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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 the purposes of its bacillus cereus bacterial strain aspect inhibition black shank simultaneously.
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, China except cigarette district, northeast is fragmentary occur, comprise that Nei south China, Taiwan Province, Central China, East China and plantation cigarette district, northwest all extensively occur, financial loss is comparatively serious.
Produce at present upper general adopt disease-resistant variety, cultural control, chemical prevention and bio-control method, but because the zoospore of this germ forms, speed is fast and quantity is large, infect short incubation period after cigarette strain, rapid onset, can occur repeatedly to infect again a season of growth, this disease is also mixed generation with other disease sometimes simultaneously, thereby causes 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 the resistance of germ to agricultural chemicals.Biological control is the first-selected prophylactico-therapeutic measures of carrying out at present in the world control of plant disease, this from occurring in nature separate, screening beneficial microorganism for the antagonism of pathogenic micro-organism, the inhibiting method of preventing and treating, there is the features such as efficient, nontoxic, noresidue and 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, is easily subject to the impact of ambient conditions, as to light, ultraviolet, dry comparatively responsive.
Summary of the invention
The one that the object of the invention is to overcome the problems referred to above and provide have strong stress resistance, nutritional requirement simply, stablize, be easy to surely to grow, to light and UV stable, the features such as easily industrialization, cost is lower, the shelf lives is long, be easy to use, and tobacco is had to 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 inhibition Phytophthora nicotianae.
A kind of bacillus cereus 1205(
bacillus cereus1205), its morphological specificity: 1205 bacterial strains, on plain agar plate culture medium, 37 DEG C, are cultivated 36 h, bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, surface irregularity particulate state, like ground-glass appearance or wax-like, is not secreted pigment; For gram positive bacterium, shaft-like, 1.0～1.3 × 3.0～5.0 μ m, chaining, mobility, gemma ellipse, end is raw or inferior end is raw, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, the catalase positive, the glucose fermentation positive, the Starch Hydrolysis positive, the nitrate reduction positive, the gelatine liquefication positive, the maltose fermentation positive, the semi-solid puncture test positive, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment is negative, sucrose fermentation is negative, lactose fermentation is negative, N.F,USP MANNITOL hydrolysis is negative.
This bacterial classification has been deposited in Chinese Typical Representative culture collection center (address: Wuhan, China Wuhan University), preserving number on September 26th, 2012: CCTCC NO:M2012382, name is called: bacillus cereus 1205(
bacillus cereus1205).
Phytophthora nicotianae is had to 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 crude opium sample in 90mL in the sterilized water with granulated glass sphere, on shaking table, 160rpm vibration 20min, leaves standstill 5min, draws sterilized water gradient dilution step by step for Soil Slurry.Draw 0.1mL diluent coating beef extract-peptone flat board, be inverted for 37 DEG C and cultivate 36h, after flat board grows bacterium colony, picking list bacterium colony purifying, numbering is preserved, to be measured.
(2) screening of antagonistic bacterium: adopt dull and stereotyped face-off method, good oat medium flat board, beat with 5mm punch tool the phytophthora silk piece of getting on 10% V8 juice substratum and be placed on dull and stereotyped central authorities, at it around, distance 2.5cm place symmetry is inoculated bacterial strain to be measured, taking the flat board that do not connect thalline as contrast.In triplicate, in 28 DEG C of incubators, cultivate 7 days, observed a strain bacterium and produced obvious inhibition zone around, Phytophthora nicotianae is had to obvious antagonistic effect, choose this bacterial isolates, it is numbered to 1205.
(3) qualification of Antagonistic Fungi: 1205 bacterial strain points are connected on plain agar flat board, 37 DEG C, cultivate 36 h, bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, surface irregularity particulate state, like ground-glass appearance or wax-like, is not secreted pigment; For gram positive bacterium, shaft-like, 1.0～1.3 × 3.0～5.0 μ m, chaining, mobility, gemma ellipse, end is raw or inferior end is raw, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, the catalase positive, the glucose fermentation positive, the Starch Hydrolysis positive, the nitrate reduction positive, the gelatine liquefication positive, the maltose fermentation positive, the semi-solid puncture test positive, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment is negative, sucrose fermentation is negative, lactose fermentation is negative, N.F,USP MANNITOL hydrolysis is negative.With reference to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition), " common bacteria system identification handbook " and " bacillus ", be initially identified as bacillus.
By 1205 inoculation, in beef extract-peptone liquid nutrient medium, 28 DEG C of 160rpm cultivate 12h, centrifugal collection thalline.Adopt the raw work Ezup pillar genome DNA extraction test kit in Shanghai (bacterium) to extract genomic dna.Taking 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 to pcr amplification.PCR condition is: 94 DEG C of 3min; 94 DEG C of 45s, 54 DEG C of 30s, 72 DEG C of 1min, 25 circulations; 72 DEG C are extended 5min eventually, and 4 DEG C stop.PCR product send by Shanghai Sheng Gong 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, by this bacterial strain be designated as bacillus cereus (
bacillus cereus1205).
Bacillus cereus bacterial strain 1205 suppresses the application of Phytophthora nicotianae aspect:
(1) 1205 bacterial strains of activation are chosen to an articulating and entered in NYBD nutrient solution (extractum carnis 8g, yeast extract paste 5g, glucose 10g, water 1L, pH nature, 121 DEG C, sterilizing 45min), 28 DEG C of cultivation 48h of 160rpm, the fermented liquid of measuring as antagonistic effect.
Adopt dull and stereotyped face-off method, (rolled oats 30g, adds 10L distilled water to good oat medium, 100 DEG C of about 1h of water-bath, 8 layers of filtered through gauze remove slag, adding distil water is supplied 1L again, agar 18g, pH nature, 121 DEG C, sterilizing 30min) flat board, beat and get phytophthora silk piece and be placed on dull and stereotyped central authorities with 5mm punch tool, at it around three of distance 2.5cm place symmetry placements soaked bacillus cereus (
bacillus cereus1205) filter paper of fermented liquid (diameter is 5mm), taking not with fermented liquid as contrast, in triplicate, in 28 DEG C of incubators, cultivate 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 condition such as temperature, pH, strong to the tolerance of poor environment, can maintain the eubiosis, can adapt to the disease ecosystem, easily cultivate and preserve, suppress Phytophthora nicotianae effective, agriculturally have very large application prospect.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), promote and lay the foundation for exploitation associated biomolecule preparation and land for growing field crops.
Embodiment
Phytophthora nicotianae is had to 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 crude opium sample in 90mL in the sterilized water with granulated glass sphere, on shaking table, 160rpm vibration 20min, leaves standstill 5min, draws sterilized water gradient dilution step by step for Soil Slurry.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 DEG C, sterilizing 45min) flat board, be inverted for 37 DEG C and cultivate 36h, after flat board grows bacterium colony, picking list bacterium colony purifying, numbering is preserved, 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, 100 DEG C of about 1h of water-bath, 8 layers of filtered through gauze remove slag, adding distil water is supplied 1L again, agar 18g, pH nature, 121 DEG C, sterilizing 30min) flat board, beat and get 10%V8 substratum (10mL V8 adds deionized water 90 mL, CaCO with 5mm punch tool
30.02g, agar 2g, pH nature, 121 DEG C, sterilizing 30min) on phytophthora silk piece be placed on dull and stereotyped central authorities, at it around, distance 2.5cm place symmetry is inoculated bacterial strain to be measured, taking the flat board that do not connect thalline as contrast.In triplicate, in 28 DEG C of incubators, cultivate 7 days, having observed a strain bacterium has obvious antagonistic effect to Phytophthora nicotianae, and this strain bacterium is numbered to 1205.
1205 bacterial strains of activation are chosen to an articulating and enter in NYBD nutrient solution (extractum carnis 8g, yeast extract paste 5g, glucose 10g, water 1L, pH nature, 121 DEG C, sterilizing 45min), 160rpm, cultivates 48h for 28 DEG C, the fermented liquid of measuring as antagonistic effect.
Adopt dull and stereotyped face-off method, good oat medium flat board, beat and get phytophthora silk piece and be placed on dull and stereotyped central authorities with 5mm punch tool, around it, the filter papers (diameter is 5mm) of 1205 fermented liquids were soaked in three of the symmetrical placements in distance 2.5cm place, taking not with fermented liquid as contrast, in triplicate, in 28 DEG C of incubators, cultivate 7 days, the antibacterial circle diameter of 1205 pairs of Phytophthora nicotianaes has reached 20.5mm.Meanwhile, picking colony edge and the antibacterial mycelia film-making with contact site, microscopic examination is to Phytophthora nicotianae myceliolysis, fracture, the protoplasma phenomenon of condensing.
The qualification of Antagonistic Fungi: 1205 on plain agar plate culture medium, 37 DEG C, cultivate 36 h, bacterium colony subcircular, canescence, opaque, bacterium colony is large, 3-7mm, the irregular expansion shape that is in edge, surface irregularity particulate state, like ground-glass appearance or wax-like, is not secreted pigment; For gram positive bacterium, shaft-like, 1.0～1.3 × 3.0～5.0 μ m, chaining, mobility, gemma ellipse, end is raw or inferior end is raw, and gemma expands not obvious; Can tolerate the cultivation of 7%NaCl, the catalase positive, the glucose fermentation positive, the Starch Hydrolysis positive, the nitrate reduction positive, the gelatine liquefication positive, the maltose fermentation positive, the semi-solid puncture test positive, the casein hydrolysis positive, VP negative, Citrate trianion utilize negative, indoles experiment is negative, sucrose fermentation is negative, lactose fermentation is negative, N.F,USP MANNITOL hydrolysis is negative.With reference to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition), " common bacteria system identification handbook " and " bacillus ", be initially identified as bacillus.
By 1205 inoculation, in beef extract-peptone liquid nutrient medium (extractum carnis 5.0 g, peptone 10.0g, NaCl 5.0g, water 1L, pH7.2), 28 DEG C of 160rpm cultivate 12h, centrifugal collection thalline.Adopt the raw work Ezup pillar genome DNA extraction test kit in Shanghai (bacterium) to extract genomic dna.Taking 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 to pcr amplification.PCR condition is: 94 DEG C of 3min; 94 DEG C of 45s, 54 DEG C of 30s, 72 DEG C of 1min, 25 circulations; 72 DEG C are extended 5min eventually, and 4 DEG C stop.PCR product send by Shanghai Sheng Gong 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, by this bacterial strain be designated as bacillus cereus (
bacillus cereus1205).
By bacteriostatic test is illustrated bacillus cereus of the present invention (
bacillus cereus1205) purposes of inhibition Phytophthora nicotianae:
(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 DEG C, sterilizing 45min), and 28 DEG C 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 DEG C, 160rpm, shake-flask culture 48h, obtains Antagonistic Fungi fermented liquid.
(2) with aseptic round filter paper (diameter 5mm), draw fermented liquid slightly dry before spirit lamp, be placed on gently oat flat board (the rolled oats 30g of inoculation Phytophthora nicotianae, add 1L distilled water, 100 DEG C of about 1h of water-bath, 8 layers of filtered through gauze remove slag, then adding distil water is supplied 1L, agar 18g, pH nature, 121 DEG C, sterilizing 30min) on, be inverted in 28 DEG C of incubators and cultivate 7 days, the antibacterial circle diameter of 1205 pairs of Phytophthora nicotianaes reaches 20.5mm.
Claims (1)
- A bacillus cereus 1205 ( bacillus cereus1205), the preserving number of described bacterial classification be bacillus cereus 1205 described in CCTCC NO:M2012382 for suppressing Phytophthora nicotianae, it is characterized in that:To activate bacillus cereus ( bacillus cereus1205) bacterial strain is chosen an articulating and is entered in NYBD nutrient solution, and 28 DEG C 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 DEG C, 160rpm, shake-flask culture 48h, obtains Antagonistic Fungi fermented liquid;With aseptic round filter paper, diameter 5mm, draws fermented liquid slightly dry before spirit lamp, is placed on gently on the oat flat board of inoculation Phytophthora nicotianae, is inverted in 28 DEG C of incubators and cultivates 7 days, and the antibacterial circle diameter of 1205 pairs of Phytophthora nicotianaes reaches 20.5mm.
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| CN104230563B (en) * | 2014-09-11 | 2018-03-06 | 红云红河烟草(集团)有限责任公司 | A kind of microbial organic fertilizer containing bacillus cereus |
| CN111500653B (en) * | 2020-05-26 | 2021-01-12 | 江西和天然科技有限公司 | 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 |
| 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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Granted publication date: 20140716 Termination date: 20181207 |