CN103589673A - Bacillus subtilis A053 and use of bacillus subtilis A053 in preparation of fermentation liquor for preventing and controlling plant pathogenic fungi - Google Patents
Bacillus subtilis A053 and use of bacillus subtilis A053 in preparation of fermentation liquor for preventing and controlling plant pathogenic fungi Download PDFInfo
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- CN103589673A CN103589673A CN201310636419.1A CN201310636419A CN103589673A CN 103589673 A CN103589673 A CN 103589673A CN 201310636419 A CN201310636419 A CN 201310636419A CN 103589673 A CN103589673 A CN 103589673A
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Abstract
The invention relates to bacillus subtilis A053 and a use of the bacillus subtilis A053 in preparation of fermentation liquor for preventing and controlling plant pathogenic fungi, and relates to bacillus subtilis. The bacillus subtilis A053 has a preservation number of CCTCC NO: M2010269. The bacillus subtilis A053 can be used for preparation of the fermentation liquor for preventing and controlling plant pathogenic fungi. The fermentation liquor can be directly used for preventing and controlling plant pathogenic fungi such as rhizoctonia solani, sclerotinia sclerotiorum, paecilomyces variotii, fusaria, trichoderma viride, alternaria longipes and gloeosporium musarum. The bacillus subtilis A053 can be prepared by simple processes and has a wide fungus inhibition spectrum and good fusarium biological-prevention effects.
Description
Technical field
The present invention relates to subtilis, particularly relate to a kind of subtilis A053 and the application in preparation control phytopathogenic fungi fermented liquid thereof.
Background technology
The Plant diseases that plant pathogenic fungi causes occupies first of plant 3 class diseases (fungal disease, Micobial Disease and virus disease), and it frequently causes disaster, and causes great rural economy loss, is one of outstanding problem hindering Chinese agriculture Economic development.China is Yi Ge large agricultural country, Ye Shiyige ocean big country, and from Marine Microorganisms, finding the active substance with specific function, to be applied to land agricultural be a newer research direction, has very strong novelty and huge application potential.Because the agricultural antibiotic of trial-production can directly be tested on plant materials, do not need through animal indirect test, so research process will shorten greatly compared with medical research.Also obtained now some valuable active substances, what have is own in being applied to the control test of plant pest, obtains good effect.
The interest of finding novel active due to polar region environment singularity and people grows with each passing day, and in recent years, polar microorganism resource has more and more caused people's attention.The arctic, due to its exclusive geography and climate characteristic, is potential, a untapped Microbial resources storehouse.It is not only the existence procreation ground of unique ecosystem microorganism, is also the potential provenance of the synthetic bacterial strain of novel bioactive material (as enzyme, microbiotic, polysaccharide and lipid etc.) and lead compound.The microorganism surviving in this special ecological diversity environment, may have the unique heredity and the metabolism diversity that are different from Lu Sheng microorganism.Therefore, arctic Microbial resources may be the potential sources of novel cpd or novel active meta-bolites.
Research shows, many marine microorganisms can produce antifungus active substance, comprise streptomyces, Aeromonas, Rhodopseudomonas, Bacillus, Flavobacterium, micrococcus sp, arthroderma, Dendrochium etc. and many uncertain bacterium (Shen Yinchu. the new development of agricultural antibiotic research and development. external medical microbiotic fascicle .1998,19 (2): 155-160).Although known, have several bacteriums to have anti-mycotic activity, genus bacillus remains best selection (Handelsman J, Stabb EV.Biocontrol of soil borne plant pathogens.Plant Cell1996; 8:1855-1869), because nontoxic to people and animals, free from environmental pollution, can produce multiple antibiotic and enzyme, have broad spectrum antibiotic activity and extremely strong anti-adversity ability (Huanghai Sea the moon, Qiu Juanping. the progress of subtilis controlling plant diseases [J]. Zhejiang Agriculture science, 2005, (3): 213-215), therefore it becomes the focus of control of plant disease research in recent years.Especially from the genus bacillus under the extreme condition of the arctic, its growth conditions, metabolic type and product are different from land or other marine sources, thereby likely develop the biologically active substance of novel structure.
Summary of the invention
One of object of the present invention is to provide a bacillus subtilis (Bacillus subtilis) A053.
Two of object of the present invention is to provide application and the method thereof of a kind of subtilis (Bacillus subtilis) A053 in preparation control phytopathogenic fungi fermented liquid.
The separated seawater sample gathering during Chinese Arctic Research Expedition for the third time from China of described subtilis (Bacillus subtilis) A053 (N78 ° 57 ' 09.2 ", E12 ° of 00 ' 02.3 "); This bacterium has been preserved in Chinese Typical Representative culture collection center on October 18th, 2010, the address at Chinese Typical Representative culture collection center is China, Wuhan, and Wuhan University, postcode is 430072, preservation center deposit number is CCTCC NO:M2010269.
Described subtilis (Bacillus subtilis) A053 is by polymerase chain reaction (PCR) its 16S rDNA sequence that increases, compare with corresponding sequence in the GenBank of American National biotechnology information center (NCBI) database, found that, itself and subtilis (Bacillus subtilis) have 99% sequence similarity.
Described subtilis (Bacillus subtilis) A053 can be used for preparation control phytopathogenic fungi fermented liquid.Its preparation method is as follows:
First subtilis (Bacillus subtilis) A053 is carried out to activation culture in the test tube that contains 10ml LB substratum, then the inoculation of activation is carried out to fermentation culture in 5L fermentor tank (substratum 3L), obtain preventing and treating phytopathogenic fungi fermented liquid.
Described LB substratum composed as follows: glucose 20g/L, KH
2pO
46g/L, K
2hPO
414g/L, MgSO
40.2g/L, (NH4)
2sO
44g/L, 2ml trace element, 0.1MPa sterilizing 30min, described trace element comprises FeSO
44H
2o, CaCl
22H
2o, MnSO
44H
2o, ZnCl
2each 1mM, puts into aseptic reagent bottle standby after filtration sterilization.
The time of described activation culture is 8～12h; The condition of described fermentation culture can be: temperature is 30～37 ℃, and pH is 6.0～8.0, and the time is 36～72h.
The using method of described control phytopathogenic fungi fermented liquid is as follows:
Control phytopathogenic fungi fermented liquid is sprayed directly on plant.
In the potted plant experiment of cotton-wilt fusarium (fusarium infection causes) control, A053 fermented liquid has good biological control effect.
Described phytopathogenic fungi comprises: dry thread Pyrenomycetes, sclerotinite, paecilomyces varioti, sickle-like bacteria, viride, long handle chain lattice spore, banana anthrax-bacilus etc.In the pot experiment of cotton-wilt fusarium (sickle-like bacteria) control, subtilis (Bacillus subtilis) A053 fermented liquid has good biological control effect, process after 7 days, the morbidity of control group is in rising trend, and sickness rate is elevated to 69.23% from 36.67%; And the incidence trend of experimental group is curbed, sickness rate drops to 37.93% from 45.95%.
Compared with prior art, the present invention has the following advantages: the preparation technology of subtilis (Bacillus subtilis) A053 fermented liquid is simple, and antifungal spectrum is wide, good to sickle-like bacteria biocontrol effect.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of subtilis (Bacillus subtilis) A053.
Fig. 2 is the fermented supernatant fluid of subtilis (Bacillus subtilis) the A053 fungistatic effect figure to different phytopathogenic fungis.In Fig. 2: A, dry thread Pyrenomycetes, B, sclerotinite, C, paecilomyces varioti, D, sickle-like bacteria, E, viride, F, long handle chain lattice spore, G, banana anthrax-bacilus
Fig. 3 is the pot experiment biocontrol effect of the fermented liquid control cotton-wilt fusarium of subtilis (Bacillus subtilis) A053.In Fig. 3, experimental group: subtilis (Bacillus subtilis) A053 fermentation liquor treatment is crossed cotton plants; Control group: fermention medium was processed cotton plants (as negative control).
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1: the separation screening of subtilis (Bacillus subtilis) A053
The present invention relates to subtilis (Bacillus subtilis) A053 derive from the seawater sample that the applicant gathers during Chinese Arctic Research Expedition for the third time from China (N78 ° 57 ' 09.2 "; in E12 ° of 00 ' 02.3 "), institute is separated obtains, this bacterium grows on LB substratum, cultivate after 24h for 30 ℃, it is light yellow that thalline is, bacterium colony is larger, sees Fig. 1.
Embodiment 2: subtilis (Bacillus subtilis) A053 strain identification
Utilize 16S rDNA universal primer 27F and 1495R (27F:AGAGTTTGATCCTGGCTCAT; 1495R:ACGGCTACCTTGTTACGACTT) subtilis (Bacillus subtilis) A05316S rDNA sequence is carried out to PCR(polymerase chain reaction) amplification.The arctic total DNA of source subtilis A053 of take is pcr amplification template, in the enterprising performing PCR reaction of Eppendorf pcr amplification instrument.Reaction conditions is: 94 ℃ of sex change 1min; 55 ℃ of annealing 1min; 72 ℃ extend 1.5min, 30 circulations.Amplification gene send the order-checking of order-checking company, obtain after this bacterial strain 16S rDNA sequence, this sequence is analyzed by American National biotechnology information center (NCBI) blast search engine (http://ncbi.nlm.nih.gov/blast) and GeneBank amplifying nucleic acid data.Found that, the sequence similarity of the bacterial strains such as subtilis (Bacillus subtilis) A05316S rDNA sequence and Bacillus subtilis (CP002905.1), Bacillus subtilis (EU047884.1), Bacillus subtilis (HQ327126.1), Bacillus subtilis (AB325584.1) is 99%, therefore, by its called after subtilis (Bacillus subtilis) A053.
Embodiment 3: the mensuration of phytopathogenic fungi antimicrobial spectrum
Adopt filter paper method to measure subtilis (Bacillus subtilis) A053 active to the inhibition of 7 kind of plant pathomycetes.With diameter, be that the aseptic punch tool of 6mm is made bacterium piece by tested pathomycete, dull and stereotyped central in solid potato culture medium (PDA) with toothpick picking one bacterium piece, under the condition of 28 ℃, cultivate 1～2 day, wait for after tested pathomycete mycelium diffusion growth, in the surrounding of each bacterium piece, place the double-deck filter paper of the diameter 6mm of some sterilizings, filter paper is apart from Jun Kuai center 0.5～1cm, on each filter paper, add again the aseptic fermented supernatant fluid of 50 μ L, continue to cultivate 2～3 days, by the comparison with contrast culture, observe mycelial growth whether can be suppressed, when if the mycelium of tested pathomycete is suppressed, mycelium can form selenodont or line style edge.As shown in Figure 2, subtilis (Bacillus subtilis) A053 fermented supernatant fluid has and suppresses active 7 kinds of tested phytopathogenic fungis such as dry thread Pyrenomycetes, sclerotinite, paecilomyces varioti, sickle-like bacteria, viride, long handle chain lattice spore, banana anthrax-bacilus result.
Embodiment 4: the biological control pot experiment of cotton-wilt fusarium (fusarium infection causes)
When cotton morbidity is more obvious, every alms bowl waters subtilis (Bacillus subtilis) A053 zymocyte liquid 50mL, and totally 6 alms bowls, are made as experimental group; Separately establish control group 6 alms bowls, water clear water.Before processing, the sickness rate of experimental group reaches 45.95%, and contrast sickness rate is 36.67%, and the occurring degree of experimental group is compared with control group weight.Process latter 7 days, the morbidity of control group is in rising trend, and sickness rate reaches 69.23%; And the incidence trend of experimental group is curbed, sickness rate is 37.93%, sees Fig. 3.Experimental result shows, waters root can to a certain degree contain increasing the weight of of blight after cotton morbidity with subtilis (Bacillus subtilis) A053 zymocyte liquid, thereby be conducive to the recovery in cotton later stage, grows.
Claims (8)
1. subtilis (Bacillus subtilis) A053, is preserved in Chinese Typical Representative culture collection center on October 18th, 2010, and preservation center deposit number is CCTCC NO:M2010269.
2. the application of subtilis (Bacillus subtilis) A053 in preparation control phytopathogenic fungi fermented liquid as claimed in claim 1.
3. application as claimed in claim 2, is characterized in that the preparation method of described control phytopathogenic fungi fermented liquid is as follows:
First subtilis (Bacillus subtilis) A053 is carried out to activation culture in the test tube that contains 10ml LB substratum, then the inoculation of activation is carried out to fermentation culture in 5L fermentor tank, obtain preventing and treating phytopathogenic fungi fermented liquid.
4. application as claimed in claim 3, is characterized in that the composed as follows of described LB substratum: glucose 20g/L, KH
2pO
46g/L, K
2hPO
414g/L, MgSO
40.2g/L, (NH4)
2sO
44g/L, 2ml trace element, 0.1MPa sterilizing 30min, described trace element comprises FeSO
44H
2o, CaCl
22H
2o, MnSO
44H
2o, ZnCl
2each 1mM, puts into aseptic reagent bottle standby after filtration sterilization.
5. application as claimed in claim 3, the time that it is characterized in that described activation culture is 8～12h.
6. application as claimed in claim 3, is characterized in that the condition of described fermentation culture is: temperature is 30～37 ℃, and pH is 6.0～8.0, and the time is 36～72h.
7. application as claimed in claim 3, is characterized in that the using method of described control phytopathogenic fungi fermented liquid is as follows:
Control phytopathogenic fungi fermented liquid is sprayed directly on plant.
8. application as claimed in claim 3, is characterized in that phytopathogenic fungi comprises: dry thread Pyrenomycetes, sclerotinite, paecilomyces varioti, sickle-like bacteria, viride, long handle chain lattice spore, banana anthrax-bacilus.
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| CN105132338A (en) * | 2015-10-15 | 2015-12-09 | 国家海洋局第三海洋研究所 | Bacillus DY26-020 and application thereof in preparation of fermented supernatant fluid for preventing and controlling plant pathogenic fungi |
| CN105132339A (en) * | 2015-10-15 | 2015-12-09 | 国家海洋局第三海洋研究所 | Broad-spectrum fungus-resistant bacillus and application thereof to preventing wheat scab |
| CN105132341A (en) * | 2015-10-15 | 2015-12-09 | 国家海洋局第三海洋研究所 | Broad-spectrum plant-pathogenic-fungus-resistant bacillus and application thereof |
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| CN105132341A (en) * | 2015-10-15 | 2015-12-09 | 国家海洋局第三海洋研究所 | Broad-spectrum plant-pathogenic-fungus-resistant bacillus and application thereof |
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| CN105132339B (en) * | 2015-10-15 | 2018-10-09 | 国家海洋局第三海洋研究所 | One plant of broad-spectrum antifungal bacillus and its application in preventing wheat scab |
| CN105238715B (en) * | 2015-10-15 | 2018-10-09 | 国家海洋局第三海洋研究所 | Bacillus DY26-004 and its prevention phytopathogenic fungi application |
| CN105475360A (en) * | 2015-12-28 | 2016-04-13 | 国家海洋局第三海洋研究所 | Composition containing bacillus subtilis and dimethachlon and application thereof |
| CN105475360B (en) * | 2015-12-28 | 2019-01-11 | 国家海洋局第三海洋研究所 | Composition and application containing bacillus subtilis and dimethachlon |
| CN105746579A (en) * | 2016-02-02 | 2016-07-13 | 安建慧 | Application of bacillus subtilis HMB19198 preparation in control of rhizoctonia silani |
| CN105861396A (en) * | 2016-06-13 | 2016-08-17 | 国家海洋局第三海洋研究所 | Marine-derived bacillus antimicrobial protein MD and preparation method thereof |
| CN105861396B (en) * | 2016-06-13 | 2023-10-13 | 自然资源部第三海洋研究所 | Marine-source bacillus antibacterial protein MD and preparation method thereof |
| CN106212505A (en) * | 2016-07-27 | 2016-12-14 | 吉林农业科技学院 | Bacillus pumilus purposes in preventing and treating Ganoderma Trichoderma atroviride disease |
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