CN105670958A - Low-temperature biocontrol bacterial strain and application thereof - Google Patents
Low-temperature biocontrol bacterial strain and application thereof Download PDFInfo
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Abstract
The invention discloses a low-temperature biocontrol bacterial strain and an application thereof. The low-temperature biocontrol bacterial strain is Bacillus methylotrophicus WSWGH-10, is deposited with CGMCC and is assigned the accession number CGMCC No.11541 at Oct, 21st, 2015 and at the deposition address Chaoyang District of Beijing, Beichen road, Yard 1, No.3. The biocontrol bacterial strain can grow and reproduce at low temperature, can product various antibacterial substances, such as siderophore, chitinase, lipopeptides, proteins and the like, and also has the capability of degrading cellulose. The bacterial strain has a wide antibacterial spectrum, can inhibit phytophthora sojae, sclerotinia sclerotiorum, pathogen of maize stalk rot, fusarium moniforme, ustilaginoidea virens, rice wilt pathogens, potato rhizoctonia bacteria, root rot pathogen of acanthopanax, pathogen of cucumber fusarium wilt, botrytis cinerea and such pathogenic fungus and tobacco bacterial disease pseudomonas angulate holland and such pathogenic bacteria. The bacterial strain also has the function of promoting plant growth.
Description
Technical field
The invention belongs to microbial technology field, relate to strain low temperature biocontrol bacterial strain and an application thereof.
Background technology
Corps diseases is a kind of natural disaster in agricultural production, directly affects the yield and quality of crop, and the loss caused is very huge. Day by day serious due to chemical pesticide Drug resistance and pollution problem, eco-friendly microbial pesticide becomes the important means of crop disease control. At present, microbial pesticide is based on active bacteria formulation, and in application, preventive effect instability is common problem. Can biocontrol microorganisms survive in soil environment and breed the key being to affect its actual protection effect. Owing to the common sowing time is in the soil moisture relatively low early spring or autumn, microbial pesticide bacterial strain now used is room temperature bacterium, poor growth or stop growing under cryogenic, reduce the application effect of biological pesticide, hinder the wide popularization and application of microbial pesticide, therefore the acquisition of low temperature biocontrol bacterial strain can provide the bacterium source material of high-quality for the research and development of the suitable natural disposition bio-bacterial manure of low temperature and biological pesticide, has potential research and exploitation advantages in Biological control.
Summary of the invention
It is an object of the invention to provide a strain low temperature biocontrol bacterial strain, this biocontrol microorganisms can growth and breeding under cryogenic, it is possible to produces to bite the multiple antibiotic substance such as ferrum element, chitinase, lipopeptid class and protide, also has degraded cellulose ability. This bacterial strain antimicrobial spectrum is wide, pathogenic fungi and the pathogenetic bacterias (Nicotiana tabacum L. bacterial disease angular leaf spot fungus) such as pathogen of soybean root rot, soybean sclerotinia crown rot bacterium, Causal Organism of Maize Basal Stalk bacterium, fusarium moniliforme, Ustilaginoidea virens, rice wilt pathogens, julienne potatoes core pathogenic bacteria, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, cucumber fusarium axysporum, botrytis cinerea can be suppressed, but also there is promotion plant growing function.
It is an object of the invention to be achieved through the following technical solutions:
One strain low temperature biocontrol bacterial strain, it is Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.11541, preservation date is on October 21st, 2015, and preservation address is Beichen Lu, Chaoyang District, Beijing City 1 institute 3.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 morphological character: Gram positive aerobic bacterium, shaft-like, there is spore, be sized to 0.2～0.5 × 1.0～1.5 μm; Bacteria colony white is circular, gauffer.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 physio-biochemical characteristics: utilize glucose, oxidase, catalase, M.R, gelatin liquefaction, Starch Hydrolysis, citrate utilization, nitrate reduction, indole generation and litmus milk are the positive, V.P and urease-negative; Growth temperature range 6～30 DEG C, optimum growth temperature 20 DEG C.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 molecular biology identification result: by 16SrDNA sequence and house-keeping gene gyrB sequence alignment, be 99% with Methylotrophic bacillus cereus homology.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 of the present invention can produce to bite the multiple antibiotic substance such as ferrum element, chitinase, lipopeptid class and protide, also has degraded cellulose ability.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 of the present invention can in the growth of the pathogenic fungi such as the effective suppression pathogen of soybean root rot of cryogenic conditions 10 DEG C, soybean sclerotinia crown rot bacterium, Causal Organism of Maize Basal Stalk bacterium, fusarium moniliforme, Ustilaginoidea virens, rice wilt pathogens, julienne potatoes core pathogenic bacteria, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, cucumber fusarium axysporum, botrytis cinerea and pathogenetic bacteria (Nicotiana tabacum L. bacterial disease angular leaf spot fungus), suppression ratio is more than 82%, antimicrobial spectrum is wide, and bacteriostasis rate is high.
Methylotrophic bacillus cereus (Bacillusmethylotrophicus) WSWGH-10 of the present invention has promotion plant growing function, its 50 times and 100 times of diluents can promote corn seed germination, germination percentage is 90.3% and 94.5%, more than blank (88.2%), and bud length, root length and radical are noticeably greater than blank.
Preservation information:
Classification And Nomenclature: Methylotrophic bacillus cereus (Bacillusmethylotrophicus);
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Deposit number: CGMCCNo.11541;
Preservation date: on October 21st, 2015;
Preservation address: Beichen Lu, Chaoyang District, Beijing City 1 institute 3.
Accompanying drawing explanation
Fig. 1 is thalli morphology;
Fig. 2 is colonial morphology;
Fig. 3 is bacterial strain 16srRNA systematic evolution tree;
Fig. 4 is bacterial strain gyrB phylogenetic analysis;
Fig. 5 is that strain secretes bites the detection of ferrum element ability;
Fig. 6 is the detection of strain secretes cellulase ability;
Fig. 7 is the detection of strain secretes chitinase ability;
Fig. 8 is the detection of strain secretes protease ability;
Fig. 9 is antibacterial protein Antibacterial Activity;
Figure 10 is lipopeptide antibiotic Antibacterial Activity;
Figure 11 is the bacterial strain suppression result to Tobacco Angular Leaf Spot Disease bacterium;
Figure 12 is that bacterial strain promotes corn seed germination result.
Detailed description of the invention
Below in conjunction with accompanying drawing, technical scheme is further described; but it is not limited thereto; every technical solution of the present invention modified or equivalent replaces, without deviating from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
The invention provides strain low temperature biocontrol bacterial strain and an application thereof, particular content is as follows:
1) Screening and Identification of low temperature biocontrol bacterial strain:
Low temperature biocontrol bacterial strain WSWGH-10 is that separation screening obtains from the frozen soil in winter of Hegang District, Heilongjiang Province.Screening carries out by the following method: takes after 5g frozen soil thaws and grinds, puts in the triangular flask containing 50ml sterilized water, 10 DEG C of concussion 3h, is diluted to 10 with sterilized water-7, take 10 respectively-5、10-6With 10-7The bacterium solution 0.1ml of degree of rarefication is coated on LB flat board, quiescent culture 3～7d in 10 DEG C of incubators, chooses the LB flat board that single bacterium colony line switching is new, quiescent culture 3～7d in 10 DEG C of incubators. With pathogen of soybean root rot for indicator bacteria, adopt opposite culture method screening biocontrol bacterial strain, low temperature biocontrol bacterial strain can be obtained.
Bacterial strain carries out form, cultural character and physio-biochemical characteristics identify, specifically comprise the following steps that under 400 power microscopes, after the Low-temperature culture thalline of 24 hours is carried out Gram’s staining, observe thalline size; To be coated on after bacterium solution doubling dilution on LB culture medium flat plate, observe colonial morphology and color; Bacterial strain is carried out oxidase, catalase, glucose fermentation, V.P, M.R, the utilization of Starch Hydrolysis, urease, citrate, nitrate reduction, indole generation, gelatin liquefaction and litmus milk test, result shows that this bacterial strain is Gram positive aerobic bacterium, shaft-like, there is spore, be sized to 0.2～0.5 × 1.0～1.5 μm (Fig. 1); Bacteria colony white is circular, gauffer (Fig. 2); Glucose can be utilized; Oxidase, catalase, M.R, gelatin liquefaction, Starch Hydrolysis, citrate utilization, nitrate reduction, indole generation and litmus milk are the positive, V.P and urease-negative. Growth temperature range 6～30 DEG C, optimum growth temperature 20 DEG C.
Bacterial strain is carried out 16SrDNA sequence analysis and house-keeping gene gyrB analyzes: take 1mL bacterium solution centrifugal collection thalline, genomic DNA is extracted by bacterial genomes DNA extraction kit (TIANGENBIOTECH), with genomic DNA for template, pcr amplification is carried out respectively with 16SrDNA universal primer and bacillus cereus gyrB gene universal primer, PCR primer is carried out agarose gel electrophoresis, specific DNA band is checked order, GenBank carries out homologous sequence comparison, with Mega5.0 software building phylogenetic tree (Fig. 3～4), 16SrDNA sequence and the gyrB genetic homology of result and Methylotrophic bacillus cereus are 99%, combining form, cultural character and physio-biochemical characteristics, it is accredited as Methylotrophic bacillus methylotrophicus.
2) strain secretes protease, cellulase, chitinase and bite ferrum element ability detection:
Adopt protease, cellulase, chitinase and bite ferrum element differentiate flat band method detect whether this bacterial strain can produce protease, cellulase, chitinase and bite ferrum element. Protease detects: detect on culture medium flat plate (defatted milk powder 1%, Carnis Bovis seu Bubali cream 0.3%, peptone 1%, sodium chloride 0.5%) by inoculation at protease, cultivates 3～7d for 10 DEG C, observes with or without enzymolysis transparent circle. Cellulase detect: by inoculation on Cellulose and congo red differential medium (ammonium sulfate 0.2%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.05%, cellulose powder 2%, Congo red 0.02%, agar powder 2%), cultivate 3～7d for 10 DEG C, observe with or without enzymolysis circle. Chitinase detects: by inoculation (tobacco brown spot pathogen 1% on chitin is cultivated, peptone 0.2%, yeast powder 0.2%, magnesium sulfate 0.02%, iron sulfate 0.001% dipotassium hydrogen phosphate 0.07%, potassium dihydrogen phosphate 0.03%, Congo red 0.02%, agar powder 2%) 10 DEG C cultivate 3～7d, observe with or without enzymolysis circle. Bite the detection of ferrum element: by inoculation CAS-flat board, cultivate 3～7d for 10 DEG C, observe with or without enzymolysis circle.Result show this bacterial strain can extracellular proteinase, cellulase, chitinase and bite ferrum element, illustrate that this bacterial strain not only has biological and ecological methods to prevent plant disease, pests, and erosion function and also has the ability (Fig. 5-8) of degraded cellulose and albumen.
3) antibiotic substance of low temperature biocontrol microorganisms is determined:
Antibacterial protein: by bacterial strain in LB culture medium 10 DEG C, that 170r/min cultivates 3d, 4000r/mim is centrifugal, collects supernatant, adopt ammonium sulfate precipitation method to obtain albumen, first add ammonium sulfate to 30% saturation, 4 DEG C of overnight precipitation, 10000r/mim is centrifuged 20min, collects precipitation, must precipitate I. Supernatant adds ammonium sulfate to 50%, 70% and 90% saturation, 4 DEG C of overnight precipitation, 10000r/mim is centrifuged 20min, collect precipitation II, III and IV, dissolve with PBS, desalination of dialysing, after concentration, detect bacteriostatic activity with pathogen of soybean root rot for indicator bacteria, result shows that precipitation II, III and IV have bacteriostatic activity (Fig. 9), illustrates that this bacterial strain can produce multiple antibacterial protein.
Lipopeptide antibiotic: by bacterial strain in LB culture medium 10 DEG C, that 170r/min cultivates 3d, 4000r/mim is centrifugal, collect supernatant, adopt hydrochloric acid precipitation method to obtain lipopeptide antibiotic. Concentrated hydrochloric acid is added to pH2 in supernatant, place overnight for 4 DEG C, next day, 10000r/mim was centrifuged 20min, collect precipitation, use methanol extracting, adopting paper disk method to detect bacteriostatic activity with pathogen of soybean root rot for indicator bacteria, result lipopeptide antibiotic has bacteriostatic activity (Figure 10), illustrates that this bacterial strain can also produce lipopeptide antibiotic.
4) antimicrobial spectrum of low temperature biocontrol microorganisms measures:
Adopt mycelial growth rate method to measure bacterial strain WSWHG-10 and 10 kind of plant pathogenic fungi (pathogen of soybean root rot, soybean sclerotinia crown rot bacterium, Causal Organism of Maize Basal Stalk bacterium, fusarium moniliforme, Ustilaginoidea virens, rice wilt pathogens, julienne potatoes core pathogenic bacteria, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, cucumber fusarium axysporum, botrytis cinerea) are suppressed situation. Taking 0.1ml Low-temperature culture 48h bacterium solution and coat on PDA plate, inoculate pathogen mycelia block (diameter 5mm) at plate center, coating sterilized water compares, and cultivates 7d for 10 DEG C, measures colony diameter, calculate bacteriostasis rate as follows. Bacteriostasis rate %=matched group colony diameter-matched group colony diameter/matched group colony diameter Х 100%. Result shows that 10 kinds of pathogenic fungi are had good inhibition by this bacterial strain, it is suppressed that rate reaches 82.0～90.3% (tables 1), illustrates that this bacterial strain antimicrobial spectrum is wide, and bacteriostasis is strong.
The table 1 bacterial strain antibacterial result to pathogenic fungi
Inhibition zone method is adopted to measure the suppression situation of the bacterial strain WSWHG-10 pathogen (angular leaf spot fungus) to causing Nicotiana tabacum L. bacterial disease, take 0.1ml pathogenetic bacteria and coat on LB flat board, sterilizing steel ring is put at center, steel ring adds 0.05ml biological and ecological methods to prevent plant disease, pests, and erosion bacterium solution, cultivate 7d for 10 DEG C, observing with or without inhibition zone, result has inhibition zone around steel ring, illustrates that biocontrol microorganisms WSWHG-10 can suppress this pathogenetic bacteria growth (Figure 11). These results suggest that this bacterial strain antimicrobial spectrum is wide, not only various plants pathogenic fungi is had very strong inhibitory action, bacterial disease is also had good inhibiting effect.
5) growth promotion of low temperature biocontrol microorganisms measures:
The bacterium solution sterilized water cultivating 48h being diluted 50 and 100 times, corn seed is immersed in 4d in 2 treatment fluids, takes out and be put on moisture filter paper, 3d is cultivated in 28 DEG C of moisturizings, calculates germination percentage, with clear water process for comparison. Result shows that 50 and 100 times of diluents promote germination, and germination percentage is 90.3% and 94.5%, and more than blank (88.2%), and bud length, root length and radical are noticeably greater than blank (table 2, Figure 12).
The impact on corn seed germination of table 2 bacterial strain
Claims (6)
1. a strain low temperature biocontrol bacterial strain, its Classification And Nomenclature is Methylotrophic bacillus methylotrophicus, and deposit number is CGMCCNo.11541.
2. low temperature biocontrol bacterial strain described in claim 1 suppresses the application in plant pathogenic fungi and the growth of Nicotiana tabacum L. bacterial disease angular leaf spot fungus at cryogenic conditions.
3. low temperature biocontrol bacterial strain according to claim 2 suppresses the application in plant pathogenic fungi and pathogenetic bacteria growth at cryogenic conditions, it is characterised in that described cryogenic temperature is 10 DEG C.
4. low temperature biocontrol bacterial strain according to claim 2 suppresses the application in plant pathogenic fungi and pathogenetic bacteria growth at cryogenic conditions, it is characterized in that described plant pathogenic fungi is pathogen of soybean root rot, soybean sclerotinia crown rot bacterium, Causal Organism of Maize Basal Stalk bacterium, fusarium moniliforme, Ustilaginoidea virens, rice wilt pathogens, julienne potatoes core pathogenic bacteria, Radix Et Caulis Acanthopanacis Senticosi pine root fungus, cucumber fusarium axysporum, botrytis cinerea, pathogenetic bacteria is Nicotiana tabacum L. bacterial disease angular leaf spot fungus.
5. the application in promoting plant growing of the low temperature biocontrol bacterial strain described in claim 1.
6. low temperature biocontrol bacterial strain application in degraded cellulose and albumen described in claim 1.
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