CN105132341A - Broad-spectrum plant-pathogenic-fungus-resistant bacillus and application thereof - Google Patents
Broad-spectrum plant-pathogenic-fungus-resistant bacillus and application thereof Download PDFInfo
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Abstract
The invention discloses broad-spectrum plant-pathogenic-fungus-resistant bacillus and application thereof and belongs to bacillus. The preservation number of the bacillus (Bacillus sp.) DY26-014 is CCTCC NO:M2015408. The bacillus (Bacillus sp.)DY26-014 can be used for preparing plant pathogenic fungus fermentative supernatant liquid. Plant pathogenic fungi prevented by the bacillus DY26-014 comprise fusarium spp, gloeosporium musarum, paecilomyces variotii, sclerotinia sclerotiorum, alternaria longipes, trichoderma viride, rhizoctonia, mutualism alternaria, botrytis cinerea and colletotrichum dematium. The broad-spectrum plant-pathogenic-fungus-resistant bacillus has a good effect in a potted experiment for preventing sclerotinia rot of colza, and the preventing effect can be 61.9%. Fermentative supernatant liquid of the bacillus (Bacillus sp.)DY26-014 has the advantages that the preparation technology is simple, the fungus suppression spectrum is broad, and the action effect is remarkable.
Description
Technical field
The present invention relates to genus bacillus, especially relate to the genus bacillus of the anti-phytopathogenic fungi of a strain wide spectrum and the application on preparation control phytopathogenic fungi thereof.
Background technology
Ocean has and is equivalent to the land area of 2.5 times nearly, the biology that is richly stored with, chemical resource.The particular surroundingss such as marine organisms are oozed because it survives in height, high salt, low temperature, high pressure, hypoxemia, (higher primary school is peaceful can to produce the various active substance with special physiological function in metabolism and growth process, Chen Jinyan, Huang Lili, Deng. the screening of sclerotinia rot of colza Endophytic antagonistic bacteria and prophylaxis effect research [J]. Pesticide Science journal, 2010 (2): 7-10).In these Living marine resources, rich and varied, novel unique marine microorganism is the desirable resource finding novel material, New function, new gene, new mechanism, the new secondary metabolite with physiological significance is constantly found in recent years from marine microorganism, this kind of material has the unexistent new unique texture of a land microorganism (Yvonne usually, Zhang Changsheng, Tian Xinpeng, Deng. Chinese Sea Microbial diversity Journal of Sex Research [J]. Bulletin of Chinese Academy of Sciences, 2010 (6): 651-658).
The sclerotinia rot of colza caused by sclerotinite (Sclerotiniasclerotiorum) is the important disease in Rape-seed production, causes the rape underproduction 10% ~ 70%, and oleaginousness reduces by 1% ~ 5%, has a strong impact on the yield and quality of rape.Spray chemical agent and be still the method that at present control sclerotinia rot of colza is direct, the easiest, be also most effectively preventing method simultaneously.But long-term single use chemical agent of the same race, make pathogenic bacteria develop immunity to drugs to chemical agent, preventive effect declines year by year.In recent years, the emphasis of research has been turned to biological control (PanlilioAL by people gradually, CulverDH, GaynesRP, etal.Methicillin-resistantStaphylococcusaureusinUShospit als, 1975 – 1991 [J] .InfectionControl, 1992,13 (10): 582-586).Biological pesticide, to the nonstaining property of air, water source, soil and farm crop, becomes the important directions of agricultural sustainable development.
The Bacillus strain reported has preventive and therapeutic effect to the pathogenic fungi of different plant and bacterium, as the rice sheath blight disease in food crop, rice blast, wheat hypochnus, beans root rot and take-all etc., (the Cheng Liang such as the leaf muld of tomato in vegetable disease, cucumber fusarium axysporum, cucumber downy mildew, grey mould of egg plant and capsicum epidemic disease, go on a spring outing flat, Xiao Aiping. the progress [J] of antagonistic bacterium. Agricultural University Of Jiangxi's journal, 2004,25 (5): 732-737).Genus bacillus has that growth is fast, simple, the easily separated cultivation of nutrition, strong stress resistance feature (Zhu Yue beautiful, Liu Jiao, Du Chunmei. genus bacillus biological control research of plant disease progress [J]. Agriculture of Anhui science, 2013,40 (34): 16635-16638) demand of modern society to agriculture production and integrated pest management, is more met.
Summary of the invention
An object of the present invention is genus bacillus (Bacillussp.) DY26-014 providing the anti-phytopathogenic fungi of a strain wide spectrum.
Two of object of the present invention is to provide the application of genus bacillus (Bacillussp.) DY26-014 of the anti-phytopathogenic fungi of described wide spectrum in preparation control phytopathogenic fungi fermented supernatant fluid.
Described genus bacillus (Bacillussp.) DY26-014 is separated the 26th science from Chinese ocean and examines or check sedimentary clay sample (the S003 website gathered; W63 ° 45.3496 ', S3 ° 39.8693 '); This bacterium is preserved in China typical culture collection center on June 27th, 2015, and the address of China typical culture collection center is China, and Wuhan, Wuhan University, postcode is 430072, and preservation center deposit number is CCTCCNO:M2015408.
Described genus bacillus (Bacillussp.) DY26-014 to be increased its 16SrDNA sequence by polymerase chain reaction (PCR), compare with sequence corresponding in American National Biotechnology Information center (NCBI) GenBank database, find that itself and bacillus (Bacillussp.) have the sequence similarity of 99%; The GenBank accession number of 16SrDNA sequence is KT270354.
The 16SrDNA partial sequence of described genus bacillus (Bacillussp.) DY26-014 is as follows:
Described genus bacillus (Bacillussp.) DY26-014 can prevent and treat the application in phytopathogenic fungi fermented supernatant fluid in preparation.
Described phytopathogenic fungi includes but not limited to sickle-like bacteria, musae, paecilomyces varioti, sclerotinite, long handle rod method, viride, dry thread Pyrenomycetes, alternate rod method, botrytis cinerea and colletotrichum gloeosporioides Penz.It has good biological control effect to Sclerotinia sclerotiorum (sclerotinite), and in pot experiment, preventive effect can reach 61.9%.
The using method of described control phytopathogenic fungi fermented supernatant fluid can be: control phytopathogenic fungi fermented supernatant fluid is sprayed directly on the control that namely plant can be used for pathomycete.
The preparation method of described control phytopathogenic fungi fermented supernatant fluid is as follows:
Be seeded to by genus bacillus (Bacillussp.) DY26-014 in fermention medium after shaking table cultivation, centrifugal removing thalline, must prevent and treat phytopathogenic fungi fermented supernatant fluid.
The inoculum size of described genus bacillus (Bacillussp.) DY26-014 can be 1% ~ 10% of fermention medium by mass percentage; The condition that described shaking table is cultivated can be: fermention medium liquid amount is 500mL/1L triangular flask, the temperature of cultivation 25 ~ 30 DEG C, the time 48 ~ 72h of cultivation, and shaking speed is 180 ~ 220r/min.
The composition of described fermention medium can be: sodium-chlor 10g/L, Tryptones 10g/L, yeast extract 5g/L, the 121 DEG C high pressure bacterium 20min that go out; The condition of described centrifugal removing thalline can under 10000r/min centrifugal 15 ~ 20min.
Compared with prior art, the present invention has the following advantages: the preparation technology of genus bacillus (Bacillussp.) DY26-014 fermented supernatant fluid is simple, and antifungal spectrum is wide, good to sclerotinite biocontrol effect.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of genus bacillus (Bacillussp.) DY26-014.
Fig. 2 is that genus bacillus (Bacillussp.) DY26-014 fermented supernatant fluid is to the fungistatic effect figure of different phytopathogenic fungi.In fig. 2: A: botrytis cinerea; B: paecilomyces varioti; C: sclerotinite; D: viride; E: dry thread Pyrenomycetes; F: long handle rod method; G: colletotrichum gloeosporioides Penz; H: alternate rod method; I: sickle-like bacteria; J: musae.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1: the separation screening of genus bacillus (Bacillussp.) DY26-014
The present invention relates to genus bacillus (Bacillussp.) DY26-014 derive from from China's the 26th ocean science to examine or check the sedimentary clay sample gathered be separated and obtain.This bacterium grows on LB substratum, and after 28 DEG C of cultivation 24h, thalline is light yellow, and bacterium colony is comparatively large, and early growth period bacterium colony is drops, sees Fig. 1.
Embodiment 2: genus bacillus (Bacillussp.) DY26-014 strain identification
Design and synthesize the general forward primer 27F of 16srDNA gene and general reverse primer 1492R, their nucleotide sequence is respectively 27F:AGAGTTTGATCCTGGCTCAT; 1492R:ACGGCTACCTTGTTACGACTT.Collected by centrifugation cultivates the DY26-014 thalline of 24h, and extracts STb gene with genome DNA extraction test kit.Be that the 16SrDNA sequence of pcr amplification template to DY26-014 increases with STb gene.Reaction conditions is: 94 DEG C of sex change 1min; 55 DEG C of annealing 1min; 72 DEG C extend 1.5min, 30 circulations.Amplification gene send order-checking company to check order, and after obtaining this bacterial strain 16SrDNA sequence, this sequence is passed through, be analyzed with American National Biotechnology Information center (NCBI) and GeneBank amplifying nucleic acid data (
http:// ncbi.nlm.nih.gov/blast).Found that, the sequence similarity of the bacterial strains such as genus bacillus (Bacillussp.) DY26-01416SrDNA sequence and Bacillusamyloliquefaciens (KF933607.1), Bacillussubtilis (EU871706.1), Bacillusamyloliquefaciens (CP011686.1), Bacillussp. (KP128090.1) is 99%, i.e. DY26-014 and genus bacillus homology, therefore, by its called after genus bacillus (Bacillussp.) DY26-014.
Embodiment 3: the mensuration of phytopathogenic fungi antimicrobial spectrum
Filter paper enzyme is adopted to measure genus bacillus (Bacillussp.) DY26-014 to the inhibit activities of 10 kind of plant pathomycetes.Be that tested pathomycete is made bacterium block by the aseptic punch tool of 6mm with diameter, the dull and stereotyped central authorities of solid potato culture medium (PDA) are placed in toothpick picking one bacterium block, cultivate 1 ~ 2 day under the condition of 28 DEG C, after waiting for tested pathomycete mycelium diffusion growth, the double-layer filter paper sheet of the diameter 6mm of some sterilizings is placed in the surrounding of bacterium block, filter paper distance Jun Kuai center 0.5 ~ 1cm, the more aseptic fermented supernatant fluid adding 50 μ L on each filter paper.Continue cultivation 2 ~ 3 days, by with contrast the comparison of cultivating, whether observe mycelial growth can be suppressed, if when the mycelium of tested pathomycete is suppressed, mycelium can form selenodont or line style edge.Result as shown in Figure 2, genus bacillus (Bacillussp.) DY26-014 fermented supernatant fluid to sickle-like bacteria, musae, paecilomyces varioti, sclerotinite, long handle rod method, viride, dry thread Pyrenomycetes, alternate rod method, botrytis cinerea and colletotrichum gloeosporioides Penz totally 10 strain subject plant pathomycetes there is inhibit activities.
Embodiment 4: the biological control pot experiment of sclerotinia rot of colza (sclerotinite infects and causes)
Sclerotinia sclerotiorum cultivates the bacterium dish making diameter 5mm after 5 days on potato sucrose nutrient agar, waits until for subsequent use.Rape stem flowering period, blade and cane are inoculated Sclerotinia sclerotiorum bacterium dish, and postvaccinal basin alms bowl is placed in 25 DEG C, under the environment of relative humidity 80%.Rape is contained and is spent the later stage, and genus bacillus (Bacillussp.) DY26-014 fermented supernatant fluid utilizes miniaturised nebuliser (500ml) to carry out even spraying, and aperture <7mm, medicament does not stay raffinate.3 days " Invest, Then Investigate " blades investigation lesion diameter (d), calculate lesion area, lesion area=π × (d/2)
2, stem stalk measures scab length, is calculated as follows preventive effect:
Calculation result shows, genus bacillus (Bacillussp.) DY26-014 reaches 61.9% to sclerotinia rot of colza preventive effect.
Claims (10)
1. genus bacillus (Bacillussp.) DY26-014, be preserved in China typical culture collection center on 06 27th, 2015, preservation center deposit number is CCTCCNO:M2015408.
2. genus bacillus (Bacillussp.) DY26-014 as claimed in claim 1, is characterized in that 16SrDNA partial sequence is as follows:
1TGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGG
51TAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCG
101GATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTAC
151CACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT
201CACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG
251GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTT
301CCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGT
351TTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAAT
401AGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTG
451CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
501GCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG
551CTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGG
601AGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAC
651ACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAA
701AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAAC
751GATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAAC
801GCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAG
851GAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAG
901CAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGA
951TAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTC
1001AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT
1051GATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGA
1101CAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGAC
1151CTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGC
1201GAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGC
1251AACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCC
1301GCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAG
1351AGTTTGTAACACCCGAAGTCGGTGAGGTA。
3. genus bacillus (Bacillussp.) DY26-014 applies in preparation control phytopathogenic fungi fermented supernatant fluid as claimed in claim 1.
4. apply as claimed in claim 3, it is characterized in that described phytopathogenic fungi includes but not limited to sickle-like bacteria, musae, paecilomyces varioti, sclerotinite, long handle rod method, viride, dry thread Pyrenomycetes, alternate rod method, botrytis cinerea and colletotrichum gloeosporioides Penz.
5. apply as claimed in claim 3, it is characterized in that the using method of described control phytopathogenic fungi fermented supernatant fluid is: control phytopathogenic fungi fermented supernatant fluid is sprayed directly on the control that namely plant can be used for pathomycete.
6. apply as claimed in claim 3, it is characterized in that the preparation method of described control phytopathogenic fungi fermented supernatant fluid is as follows:
Be seeded to by genus bacillus (Bacillussp.) DY26-014 in fermention medium after shaking table cultivation, centrifugal removing thalline, must prevent and treat phytopathogenic fungi fermented supernatant fluid.
7. apply as claimed in claim 6, it is characterized in that the inoculum size of described genus bacillus (Bacillussp.) DY26-014 is 1% ~ 10% of fermention medium by mass percentage.
8. apply as claimed in claim 6, it is characterized in that the condition that described shaking table is cultivated is: fermention medium liquid amount is 500mL/1L triangular flask, the temperature of cultivation 25 ~ 30 DEG C, the time 48 ~ 72h of cultivation, and shaking speed is 180 ~ 220r/min.
9. apply as claimed in claim 6, it is characterized in that consisting of of described fermention medium: sodium-chlor 10g/L, Tryptones 10g/L, yeast extract 5g/L, the 121 DEG C high pressure bacterium 20min that go out.
10. apply as claimed in claim 6, it is characterized in that the condition of described centrifugal removing thalline is centrifugal 15 ~ 20min under 10000r/min.
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Cited By (5)
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| 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 |
| CN105567605A (en) * | 2016-02-19 | 2016-05-11 | 黑龙江省科学院大庆分院 | Lysinibacillus sp. and application thereof, degumming auxiliary containing lysinibacillus sp. and preparation method of degumming auxiliary |
| CN110791445A (en) * | 2019-10-29 | 2020-02-14 | 西北农林科技大学 | Bacillus amyloliquefaciens and biological pesticide fertilizer |
| CN112048457A (en) * | 2020-10-12 | 2020-12-08 | 福建农林大学 | Bacillus 31309 and application thereof in preventing and treating sclerotinia rot of colza |
| CN112175873A (en) * | 2020-10-12 | 2021-01-05 | 福建农林大学 | Bacillus W608 and application thereof in preventing and treating sclerotinia rot of colza |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105567605A (en) * | 2016-02-19 | 2016-05-11 | 黑龙江省科学院大庆分院 | Lysinibacillus sp. and application thereof, degumming auxiliary containing lysinibacillus sp. and preparation method of degumming auxiliary |
| CN110791445A (en) * | 2019-10-29 | 2020-02-14 | 西北农林科技大学 | Bacillus amyloliquefaciens and biological pesticide fertilizer |
| CN110791445B (en) * | 2019-10-29 | 2022-09-02 | 西北农林科技大学 | Bacillus amyloliquefaciens and biological pesticide fertilizer |
| CN112048457A (en) * | 2020-10-12 | 2020-12-08 | 福建农林大学 | Bacillus 31309 and application thereof in preventing and treating sclerotinia rot of colza |
| CN112175873A (en) * | 2020-10-12 | 2021-01-05 | 福建农林大学 | Bacillus W608 and application thereof in preventing and treating sclerotinia rot of colza |
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| CN105132341B (en) | 2018-10-09 |
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