CN105420164A - Antarctic-derived Bacillus sp.N311 and application of Bacillus sp.N311 for preventing and treating phytopathogenic fungi - Google Patents
Antarctic-derived Bacillus sp.N311 and application of Bacillus sp.N311 for preventing and treating phytopathogenic fungi Download PDFInfo
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention relates to Antarctic-derived Bacillus sp.N311 and an application of Bacillus sp.N311 for preventing and treating phytopathogenic fungi, which relate to Bacillus sp. A preservation number of the Bacillus sp.N311 is CCTCC NO: M 2015607. The Bacillus sp.N311 can be used for preparing a fermentation supernatant for preventing and treating phytopathogenic fungi. The phytopathogenic fungi comprise fusarium, gloeosporium musarum, Paecilomyces variotii, sclerotinia sclerotiorum, Alternaria longipes, Trichoderma viride, Rhizoctonia solani, alternaria alternate, botrytis cinerea and Colletotrichum gloeosporioides. The Bacillus sp.N311 has good biological prevention and treatment effects in Rape Sclerotinia Rot pot culture prevention and treatment tests, and prevention and treatment effects can reach more than 50%. The Bacillus sp.N311 fermentation supernatant has wide fungus-inhibiting spectrum and good biocontrol effect, has advantages of no toxicity, difficult generation of drug resistance, environment safety and biosafety, and has wide application prospect.
Description
Technical field
The present invention relates to genus bacillus, especially relate to a kind of genus bacillus N311 and the application in preparation control phytopathogenic fungi fermented supernatant fluid thereof.
Background technology
Plant diseases is the important factor of serious threat agriculture production, according to Food and Argriculture OrganizationFAO (FAO) statistics, and 10% ~ 15% of the underproduction caused because suffering Plant diseases every year loss average out to ultimate production.At present, due to a large amount of uses of chemical pesticide, not only bring the problem that phytopathogen develops immunity to drugs, and potential hazard be also result in ecotope and human health.Develop wide spectrum, biological pesticide that is efficient, low toxicity has become the common objective (CalvoJ, CalventeV, etal.2007) of researchist and Pesticide use person.
In recent years, from terrestrial soil microorganism, find that the situation of obvious decline has appearred in the active substance of novel structure.Ocean accounts for more than 70% of earth surface, and be richly stored with Biological resources.Find in marine organisms so far to comprise the various new active substances such as alkaloids, terpene, Macrocyclic polyester class, peptide class and polyose.Research shows, many marine biomaterial have antitumor, antiviral, antimycotic, anti-AIDS, antifatigue and strengthen effects such as immunity (Miao Huinan, Fang Xudong, burnt bright magnificent .1999).Due to geography and the climate characteristic of South Pole uniqueness, define the physical environment of a drying, bitter cold, severe radiation, accumulate and educated rich and varied, that function is special Microbial resources, mainly comprise the microorganism (Zeng Yinxin living in seawater, oceanic sediment and grow nonparasitically upon another plant altogether with marine animal and plant, Chen Bo, 1999).These novel unique South Pole marine microorganisms are the ideal source (the good .2005 of Zhu Jiangang, Yan Qide, Ling Xiao) finding novel bioactive product.
Bacillus (Bacillusspp.), owing to can produce the inverse brood cell of heat-resistant, is the dominant population of soil and plant microbial ecological.Bacillus produces antimicrobial substance by assimilation, suppresses the growth of harmful pathogenic micro-organism or direct pathogenic microbe killing (Yan Wanrong, Zhao Tingchang wait .2013).The bacterial strain of bacillus of many natural separation is successfully applied to biocontrol of plant disease, as the commodity bacterium B.subtilisRB14 of Tokyo institute research and development and the commodity bacterium B.amyloliquefaciensFZB42 of Taensa company of U.S. research and development, be mainly used in blight and the root rot of preventing and treating farm crop.Agricultural University Of Nanjing of China develops B.subtilisG1 and B.subtilsB3, and the former is mainly used in control pepper virus disease and tobacco virus, and latter is for preventing and treating wheat hypochnus (Deng Jianliang, Liu Hongyan wait .2010).
Summary of the invention
An object of the present invention is to provide a bacillus (Bacillussp.) N311.
Two of object of the present invention is to provide the application of a kind of genus bacillus (Bacillussp.) N311 in preparation control phytopathogenic fungi fermented supernatant fluid.
Described genus bacillus (Bacillussp.) N311 is separated from China's seawater sample (59 ° of 00'22.95 " W, 62 ° of 13'03.316 " S) that the 31st time scientific investigation in the Antarctic gathers; This bacterium is preserved in China typical culture collection center on October 15th, 2015, and the address of China typical culture collection center is China, and Wuhan, Wuhan University, postcode 430072, preservation center deposit number is CCTCCNO:M2015607.
Described genus bacillus (Bacillussp.) N311 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 KT962248.
The 16SrDNA partial sequence of described genus bacillus (Bacillussp.) N311 is as follows:
Described genus bacillus (Bacillussp.) N311 can apply in preparation control phytopathogenic fungi fermented supernatant fluid.
The preparation method of described control phytopathogenic fungi fermented supernatant fluid is as follows: first genus bacillus (Bacillussp.) N311 is carried out activation culture in the test tube containing 5mLLB substratum, then fermentation culture is carried out by the inoculation of activation to 500mLLB substratum, fermentation culture terminates rear centrifugal removing thalline, obtains control phytopathogenic fungi fermented supernatant fluid.
Consisting of of described LB substratum: sodium-chlor 10g/L, Tryptones 10g/L, yeast extract 5g/L, the 121 DEG C high pressure bacterium 20min that go out.
Described centrifugal removing thalline is centrifugal 15 ~ 20min under 10,000r/min.
The time of described activation culture is 12 ~ 24h.
The temperature of described fermentation culture is 28 ~ 30 DEG C, and the time of fermentation culture is 48 ~ 60h, and the shaking speed of fermentation culture is 180 ~ 220r/min.
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.
The using method of described control phytopathogenic fungi fermented supernatant fluid is as follows: directly or after dilution be sprayed on plant by control phytopathogenic fungi fermented supernatant fluid.
Compared with prior art, the present invention has the following advantages:
The preparation technology of fermented supernatant fluid is simple, and antifungal spectrum is wide, and biocontrol effect is good.In the potted plant controlling experiment of sclerotinia rot of colza (sclerotinite), demonstrate good biological control effect, preventive effect reaches more than 50%.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of genus bacillus (Bacillussp.) N311.
Fig. 2 is the fungistatic effect figure of fermented supernatant fluid to different phytopathogenic fungi of genus bacillus (Bacillussp.) N311.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.) N311
The present invention relates to genus bacillus (Bacillussp.) N311 to derive from the applicant and screen from China's seawater sample that the 31st time scientific investigation in the Antarctic gathers and obtain.This bacterium grows on LB substratum, and after 28 DEG C of cultivation 24h, thalline is faint yellow, bacterium colony surface irregularity, drying, and edge is irregular, sees Fig. 1.
Embodiment 2: genus bacillus (Bacillussp.) N311 strain identification
Utilize 16SrDNA universal primer 27F and 1492R (27F:AGAGTTTGATCCTGGCTCAT; 1492R:ACGGCTACCTTGTTACGACTT) source, South Pole bacterial strain N31116SrDNA sequence is increased.With N311 STb gene for pcr amplification template, in the enterprising performing PCR reaction of PCR amplification instrument.Reaction conditions is: 94 DEG C of sex change 1min; 55 DEG C of annealing 1min; 72 DEG C extend 1.5min, 30 circulations.Extension increasing sequence, after the order-checking of order-checking company, obtains this bacterial strain 16SrDNA sequence.By this sequence by comparing (http://ncbi.nlm.nih.gov/blast) with American National Biotechnology Information center GenBank amplifying nucleic acid data.Result shows, the sequence similarity of the bacterial strains such as the 16SrDNA sequence of N311 and Bacillusmethylotrophicus (HQ662596.1), Bacillusamyloliquefaciens (JF460737.1) and Bacillusamyloliquefaciens (JQ062537.1) is 99%, i.e. genus bacillus (Bacillussp.) N311 and genus bacillus homology, therefore, by its called after genus bacillus (Bacillussp.) N311.
Embodiment 3: the mensuration of phytopathogenic fungi antimicrobial spectrum
Paper disk method is adopted to measure genus bacillus (Bacillussp.) N311 to the inhibit activities of 10 kind of plant pathomycetes.With aseptic punch tool, tested pathomycete is made bacterium block, be inoculated in the dull and stereotyped central authorities of solid potato culture medium with toothpick picking one bacterium block, cultivate under the condition of 28 DEG C.After tested pathomycete mycelium diffusion growth, the double-layer filter paper sheet (diameter 6mm) of sterilizing is positioned over bacterium block surrounding, filter paper distance mycelia edge is about 1cm.Each filter paper adds the aseptic fermented supernatant fluid of 50 μ L, continuing to cultivate 24h, by comparing with control group, observing mycelial growth whether can be suppressed, if the mycelium of tested pathomycete is suppressed, mycelium can form selenodont or line style edge.Result as shown in Figure 2, genus bacillus (Bacillussp.) N311 fermented supernatant fluid has inhibit activities to 10 kinds of subject plant pathomycetes, is respectively: sickle-like bacteria, musae, paecilomyces varioti, sclerotinite, long handle rod method, viride, dry thread Pyrenomycetes, alternate rod method, botrytis cinerea and colletotrichum gloeosporioides Penz.
Embodiment 4: the biological control pot experiment of sclerotinia rot of colza (sclerotinite)
Sclerotinia sclerotiorum cultivates the bacterium dish making diameter 5mm after 5 days on potato sucrose substratum, 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.) N311 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)
2if stem stalk then measures scab length, is calculated as follows preventive effect:
Claims (10)
1. genus bacillus (Bacillussp.) N311, be preserved in China typical culture collection center on October 15th, 2015, preservation center deposit number is CCTCCNO:M2015607.
2. genus bacillus (Bacillussp.) N311 as claimed in claim 1, is characterized in that its 16SrDNA partial sequence is as follows:
3. genus bacillus (Bacillussp.) N311 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 the preparation method of described control phytopathogenic fungi fermented supernatant fluid is as follows: first genus bacillus (Bacillussp.) N311 is carried out activation culture in the test tube containing 5mLLB substratum, then fermentation culture is carried out by the inoculation of activation to 500mLLB substratum, fermentation culture terminates rear centrifugal removing thalline, obtains control phytopathogenic fungi fermented supernatant fluid.
5. apply as claimed in claim 4, it is characterized in that consisting of of described LB substratum: sodium-chlor 10g/L, Tryptones 10g/L, yeast extract 5g/L, the 121 DEG C high pressure bacterium 20min that go out.
6. apply as claimed in claim 4, it is characterized in that described centrifugal removing thalline is centrifugal 15 ~ 20min under 10,000r/min.
7. apply as claimed in claim 4, it is characterized in that the time of described activation culture is 12 ~ 24h.
8. apply as claimed in claim 4, it is characterized in that the temperature of described fermentation culture is 28 ~ 30 DEG C, the time of fermentation culture is 48 ~ 60h, and the shaking speed of fermentation culture is 180 ~ 220r/min.
9. apply as claimed in claim 4, 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.
10. apply as claimed in claim 4, it is characterized in that the using method of described control phytopathogenic fungi fermented supernatant fluid is as follows: control phytopathogenic fungi fermented supernatant fluid directly or after dilution is sprayed on plant.
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Cited By (5)
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| CN106635885A (en) * | 2016-11-17 | 2017-05-10 | 黑龙江省农业科学院耕作栽培研究所 | Symbiotic bacterium of entomopathogenic nematode and application of symbiotic bacterium |
| CN106635885B (en) * | 2016-11-17 | 2019-09-20 | 黑龙江省农业科学院耕作栽培研究所 | A kind of entomopathogenic nematode symbiotic bacteria and its application |
| CN106978351A (en) * | 2017-02-24 | 2017-07-25 | 河南省农业科学院园艺研究所 | Have pale purple purple spore bacterium and its application of High pathogenicity to rape endophytic bacterial |
| 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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