CN104450560A - Nematode-killing sphingosine bacillus strain and application thereof - Google Patents
Nematode-killing sphingosine bacillus strain and application thereof Download PDFInfo
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- CN104450560A CN104450560A CN201410629500.1A CN201410629500A CN104450560A CN 104450560 A CN104450560 A CN 104450560A CN 201410629500 A CN201410629500 A CN 201410629500A CN 104450560 A CN104450560 A CN 104450560A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
Abstract
The invention relates to a nematode-killing sphingosine bacillus strain and an application thereof, and belongs to the technical field of microbial pesticides. The preservation number of the nematode-killing sphingosine bacillus strain is CGMCC No.9689. The invention discloses the application of the nematode-killing sphingosine bacillus strain in preparation of a bactericide for preventing and controlling tobacco bacterial wilt and root rot of panax notoginseng. The nematode-killing sphingosine bacillus strain disclosed by the invention has the beneficial effects that (1) a biocontrol inoculant prepared from the M-SX103 strain has a prevention and control effect above 67% on the tobacco bacterial wilt and the root rot of panax notoginseng and is applicable to seed treatment, transplanting time and adult-plant stage; the biocontrol inoculant having industrial application prospects provide new microbial resources for prevention and control of plant bacterial wilt and root rot of panax notoginseng; (2) a biocontrol inoculant prepared from the nematode-killing sphingosine bacillus strain has the characteristics of being low in cost, high in prevention effect, free of residues, convenient to use and safe to human and livestock and is a high-efficiency biopesticide for preventing and controlling plant bacterial wilt diseases in organic agricultural planting.
Description
Technical field:
The present invention relates to strain nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain and an application thereof, microorganism belonging to genus technical field of pesticide.
Background technology:
Plant-bacterial-wilt (Bacterial Wilt) is that the interior destructiveness soil occurred of a kind of world wide caused by green grass or young crops withered Raul Salmonella (Ralstonia solanacearum) passes bacterial disease, be called as " cancer " of plant, plant underproduction 30-100% can be made, 50 200 various plants belonged to can be infected, wherein endanger main host plants heavier, distributed more widely and have (Hayward, 1991 such as tobacco, tomato, eggplant, capsicum; 1994).Tobacco (Nicotiana tabacum) is the important cash crop of China, one of mainstay industry of Ye Shi Yunnan Province development landlord economy.Tobacco bacterial wilt is the important soil-borne disease in tobacco planting district, tropical and subtropical region, and harm is serious, and the financial loss caused is huge, has become the serious hindrance (Chen Ruitai etc., 1997) of restriction tobacco planting industry development at present.Pseudo-ginseng (Panax notoginseng) is araliaceae ginseng plant, is the traditional famous and precious famous-region drug of China.Pseudo-ginseng main product, in Wenshan Prefecture of Yunnan Province, is the Biological resources shown unique characteristics in Yunnan, one of mainstay industry of Ye Shi Yunnan Province development local economy (Wei et al., 1996).By the destructive disease that the microbial notoginseng root rot of bacterial wilt (Root-rot disease) is in notoginseng planting, its loss caused accounts for the 70-85% of the whole disease loss of pseudo-ginseng, become the serious hindrance (Dong not million etc., 1998) of restriction pseudo-ginseng industry Sustainable development.Plant-bacterial-wilt and notoginseng root rot are soil-borne disease.So far, to the control of these two kinds of diseases mainly to use chemical pesticide.Due to chemical prevention easily cause pesticide residue, contaminate environment and pathogenic bacteria produce the problems such as resistance, exploitation biological pesticide has become one of the leading measure of disease control of environmental protection, safety, holding effect.
Nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 is the new species delivered in 2012, and M-SX103 bacterial strain is Endophytic Bacteria of Tobacco, has good colonization ability at plant root.This microorganism has antagonistic activity for tobacco black shank bacterium (Phytophthora nicotianae), and Panagrellus redivivus (Panagrellus redivivus) is demonstrated to the nematode lethality rate (Liu et al., 2012) of 100%.This microorganism is to the biological and ecological methods to prevent plant disease, pests, and erosion characteristic of the former bacterium of plant-bacterial-wilt and notoginseng root rot and prevention effect so far there are no open report.
Reference:
Hayward AC.1991.Biology and epidemiology of bacterial wilt caused by Ralstonia solanacearum.Annu Rev Phytopathol 29:65-87.
Hayward AC.1991.The hosts of Pseudomonas solanacearum.In:Bacterial Wilt:The Disease and its Causative Agent,Pseudomonas solanacearum.(Eds.Hayward AC and Hartman GL.)CAB International,Wallingford,UK.pp.9-24.
Liu J,Yang LL,Xu CK,Xi JQ,Yang FX,Zhou F,Zhou Y,Mo MH,Li WJ.2012.Sphingobacterium nematocida sp.nov.,a nematicidal endophytic bacterium isolated from tobacco.International Journal of Systematic and Evolutionary Microbiology 62(8):1809-1813.
Wei JX,Du YC.1996.Modern Science Research and Application of Panax notoginseng.Yunnan Science and Technology Press,Kunming,China.
.1997. 16, the whole nation main product cigarettes such as Chen Ruitai economize (district) tobacco infectious disease report of survey. Chinese Tobacco Science, (4): 1-7.
Dong is million Yunnan notoginsengs such as .1988. such as grade not. Yunnan Science Press, Kunming, pp.103-114.
Miao does to wait clearly the research of .2006. notoginseng root rot cause of disease. Scientia Agricultura Sinica, 39 (7): 1371-1378.
Summary of the invention:
The object of the present invention is to provide strain nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain and an application thereof.
Nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX10 bacterial strain of the present invention, the biocontrol strain with commercial application prospect filtered out from tobacco endophyte, for the control of Zhi Wu Celadon rot and notoginseng root rot provides new Microbial resources.
The present invention is specifically achieved in that
Nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain, be separated in the healthy leaves of Yunnan Flue-cured Tobacco kind NC297, this bacterial strain is stored in China Microbial Culture Preservation Commission's common micro-organisms center on September 18th, 2014, No. 3, No. 1, Bei Chen West Road, Chaoyang District, Beijing City, address institute, Institute of Microorganism, Academia Sinica, preserving number: CGMCC No.9689.
Nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain, at beef-protein medium (extractum carnis 3g, peptone 10g, NaCl 5g, agar 20g, moisturizing to 1,000mL, pH7.2) on, bacterium colony is in yellow, circle, edge protuberance, cell is shaft-like, diameter 0.4-0.8 μm, long 1.0-1.5 μm, spore does not move about, Gram-negative.This bacterial strain grows in the temperature range of 10-40 DEG C, optimum growth temperature 37 DEG C; Grow in the scope of pH 6.0-9.0, the most suitable growth pH is 7.0; Grow in NaCl (w/v) scope that concentration is 0-3%, the most suitable growth concentration is the NaCl of 0-1%.Utilize primer 5 '-GGT TAC CTT GTT ACG ACT T-3 ', 5 '-AGA GTT TGA TCM TGG CTC AG-3 ' expands the 16S rRNA gene of this bacterial strain through PCR, the sequence obtained is submitted to GeneBank public database, its sequence number is JF731241, and this sequence is the principal character foundation of this bacterial strain of qualification.The main fatty acid of M-SX103 bacterial strain is different formula 15 carbon saturated fatty acid (57.7%), different formula 17 carbon saturated fatty acid (7.7%) and summed feature 3 (9.6%).It is the accidental quality foundation differentiating this bacterium.
The application of nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain of the present invention in preparation control tobacco bacterial wilt and notoginseng root rot microbial inoculum.
Beneficial effect of the present invention is:
1, the biocontrol fungicide prepared with M-SX103 bacterial strain is blue or green sick to control tobacco, and the prevention effect of notoginseng root rot, more than 67%, is applicable to seed treatment, transplanting time and Adult plant.There is the biocontrol strain of commercial application prospect, for the control of Zhi Wu Celadon rot and notoginseng root rot provides new Microbial resources.
2, with biocontrol fungicide prepared by nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain have that cost is low, preventive effect is high, noresidue, easy to use, to the feature of person poultry safety, it is the high-performance bio agricultural chemicals preventing and treating plant-bacterial-wilt evil in Organic farming plantation.
Embodiment:
Embodiment nematicide ammonia used sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain, be separated in the healthy leaves of Yunnan Flue-cured Tobacco kind NC297, this bacterial strain is stored in China Microbial Culture Preservation Commission's common micro-organisms center, preserving number on September 18th, 2014: CGMCC No.9689.
The cultivation of embodiment 1:M-SX103 bacterial strain and microbial inoculum preparation
The cultivation of M-SX103 strain tube inclined-plane seed: by inoculation on the inclined-plane of beef extract-peptone solid medium, obtains M-SX103 bacterial strain inclined-plane seed under 15-37 DEG C of condition after cultivating 2-3 days, suitable culture condition cultivates 2 days at 32 DEG C.
The liquid culture of M-SX103 strain liquid seed: get a ring M-SX103 inoculation in the beef extract-peptone liquid nutrient medium (beef extract-peptone solid medium does not add agar hereinafter referred to as NB) in triangular flask from inclined-plane seed, at 37 DEG C, rotating speed 200rpm, cultivate 2-3 days, obtained seed liquor.
M-SX103 strain fermentation tank mass propgation: the ratio access of described seed liquor in 3-5% (V/V) is cultivated containing in the fermentor tank of NB substratum, at 20-1, culture condition in 000 liter of fermentor tank controls: temperature 32-37 DEG C, stirring velocity 150-250rpm, fermentation time 48-72 hour.
The preparation of M-SX103 wetting properties microbial inoculum: the microbial cells obtained through fermentor cultivation and metabolism thereof make M-SX103 wettable powder biocontrol fungicide according to a conventional method without as former medicine and agricultural chemicals conventional absorbtion carrier and auxiliary agent.Microbial inoculum need ensure that the viable count content of M-SX103 is 1 × 10
10individual/gram more than.
Embodiment 2:M-SX103 microbial inoculum is to the efficiency test of tobacco bacterial wilt
The cultivation of tobacco bacterial wilt pathogenic bacteria (Ralstonia solanacearum): at TTC substratum (peptone 10.0g, casein hydrolyzate 1.0g, 2,3,5-chlorinated triphenyl base four nitrogen 0.05g, glucose 10.0g, agar 20.0g, distilled water 1,000mL, pH 6.8-7.2) solid plate inoculates tobacco bacterial wilt pathogenic bacteria, picking list colony inoculation is cultivated after 3 days in NB liquid nutrient medium at 32 DEG C, at 37 DEG C, under 200rpm, shaking table cultivates 2 days, and tobacco ralstonia solanacearum is made into 2.0 × 10
8the bacteria suspension of cfu/mL concentration is for subsequent use.
Reagent agent: a. viable count content is 2.5 × 10
10individual/gram M-SX103 wetting properties microbial inoculum; B.25% blue or green withered clean wettable powder (development of crop institute tobacco room, academy of agricultural sciences of Guangdong Province).
Efficiency test method: during flue-cured tobacco routine floating seedlings, rear for the dilution of the M-SX103 wetting properties microbial inoculum 400mL water of 60g and 1kg are educated matrix and mixes; After positive control is treated to 25% blue or green withered clean wettable powder 1g 400mL water dilution and 1kg educate matrix and mix; Blank is that 400ml water and 1kg educate matrix and mix.When cigarette seedling grows to 5-6 sheet leaf, diameter be 20cm, each flowerpot that 500g soil (mass ratio is: sandy soil: vegetable mould=1:1) is housed transplants a strain cigarette seedling, at cigarette root inoculation tobacco bacterial wilt pathogenic bacteria 6ml, often process three repetitions, often repeat 5 basins.Rich water quality management carries out according to a conventional method, within 90 days, adds up the sickness rate of the tobacco bacterial wilt of each process afterwards, calculates sickness rate as follows, disease index and protection effect at room temperature temperature indoor growing:
Disease index=[∑ (the corresponding progression of symptom typical values at different levels)] × 100/ highest symptom typical value × investigate total strain number) × 100
Protection effect (%)=(blank disease refer to-chemicals treatment disease refer to/contrast that disease refers to) × 100
Severity Scaling: 0 grade is asymptomatic; L level is l-2 sheet leaf withering; 2 grades is the wilting of 1/3-1/2 blade or the blackening of cane base portion; 3 grades be 2/3-3/4 blade wilt, downright bad and cane blackening is downright bad; 4 grades is that whole strain is dead.
Test-results shows: the disease index of blank is 77.4; The disease index of 25% blue or green withered clean wetting properties microbial inoculum process is 25.4, and preventive effect is 67.1%; The disease index of M-SX103 wetting properties microbial inoculum process is 23.7%, and preventive effect is 69.4%, suitable with the prevention effect of the blue or green withered clean wettable powder of chemical pesticide 25%.
The field test of embodiment 3:M-SX103 microbial inoculum control notoginseng root rot
This test is experimental field carried out at Wenshan Prefecture of the Yunnan Province graduate booth of pseudo-ginseng, and this is continuous cropping pseudo-ginseng 20 years experimental field, and the sickness rate of notoginseng root rot is more than 80%.When pseudo-ginseng nursery, be 2.5 × 10 by 100g thalline content
10m-SX103 wetting properties microbial inoculum 1L water-solubleization of individual/g, adds the mixing of 1kg Panax notoginseng seeds, makes microbial inoculum stick on seed, then breeding method management routinely.Being watered 200kg with 10kg microbial inoculum and evenly filling with root of every mu of pseudo-ginseng seedling during transplanting, transplants and uses once by the microbial inoculum of same dose and mode afterwards for 1 year again.Contrast medicament is the vinclozolin wettable powder (BASF Aktiengesellschaft) of 50%, and consumption is every mu of 2kg, and application process is with M-SX103 pulvis.Blank does not use any medicament, often processes three repetitions, often repeats 100 young plants.Rich water quality management after pseudo-ginseng transplanting carries out routinely, transplants the sickness rate adding up each process for 2 years afterwards, calculates sickness rate and preventive effect as follows:
Sickness rate (%)=morbidity strain number/investigation strain number × 100
The sickness rate of the sickness rate-chemicals treatment of prevention effect (%)=blank
Test-results shows: the sickness rate of blank is 100%; The sickness rate of vinclozolin process is 30.6%, and preventive effect is 69.4%; The sickness rate of M-SX103 wettable powder process is 28.2%, and preventive effect is 71.8%, suitable with the vinclozolin wettable powder preventive effect of 50%.
Claims (2)
1. nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain, the preserving number of this bacterial strain is: CGMCC No.9689.
2. the application of nematicide ammonia sheath alcohol bacillus (Sphingobacterium nematocida) M-SX103 bacterial strain according to claim 1 in preparation control tobacco bacterial wilt and notoginseng root rot microbial inoculum.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108467839A (en) * | 2018-03-12 | 2018-08-31 | 山西大学 | Radix Astragali growth-promoting endophyte and its growth promotion method and application |
| CN108753659A (en) * | 2018-06-21 | 2018-11-06 | 云南大学 | One plant of carbendazim degrading bacterium and its application |
| CN108841764A (en) * | 2018-07-23 | 2018-11-20 | 云南大学 | One plant of biocontrol bacteria and its application |
| CN110964675A (en) * | 2020-01-02 | 2020-04-07 | 山东农业大学 | Application of Sphingobacterium montanense in prevention and treatment of parasitic nematode disease |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108004165B (en) * | 2017-12-11 | 2021-02-02 | 云南中烟工业有限责任公司 | Application of bacterium and fermentation product thereof in preventing feed liquid for cigarettes from being rotten and deteriorated |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108467839A (en) * | 2018-03-12 | 2018-08-31 | 山西大学 | Radix Astragali growth-promoting endophyte and its growth promotion method and application |
| CN108467839B (en) * | 2018-03-12 | 2021-09-28 | 山西大学 | Astragalus root growth-promoting endophyte and growth promoting method and application thereof |
| CN108753659A (en) * | 2018-06-21 | 2018-11-06 | 云南大学 | One plant of carbendazim degrading bacterium and its application |
| CN108841764A (en) * | 2018-07-23 | 2018-11-20 | 云南大学 | One plant of biocontrol bacteria and its application |
| CN108841764B (en) * | 2018-07-23 | 2021-07-09 | 云南大学 | Biocontrol bacterium and application thereof |
| CN110964675A (en) * | 2020-01-02 | 2020-04-07 | 山东农业大学 | Application of Sphingobacterium montanense in prevention and treatment of parasitic nematode disease |
| CN110964675B (en) * | 2020-01-02 | 2021-10-08 | 山东农业大学 | Application of Sphingobacterium montanense in prevention and treatment of parasitic nematode disease |
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