CN103555615B - A kind of bacillus amyloliquefaciens and uses thereof - Google Patents

A kind of bacillus amyloliquefaciens and uses thereof Download PDF

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CN103555615B
CN103555615B CN201310490095.5A CN201310490095A CN103555615B CN 103555615 B CN103555615 B CN 103555615B CN 201310490095 A CN201310490095 A CN 201310490095A CN 103555615 B CN103555615 B CN 103555615B
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bacillus amyloliquefaciens
tobacco
gray mold
bacterium colony
bacterium
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CN103555615A (en
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汪汉成
陈庆园
李文红
夏海乾
王茂胜
陆宁
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GUIZHOU PLANT PROTECTION INSTITUTE
Guizhou Institute of Tobacco Science
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GUIZHOU PLANT PROTECTION INSTITUTE
Guizhou Institute of Tobacco Science
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Abstract

The invention discloses a kind of bacillus amyloliquefaciens and uses thereof, its morphological specificity: bacillus amyloliquefaciens X-60? (<i>Bacillus? amyloliquefaciens</i>? X-60) on NA substratum, 24 are cultivated? single bacterium colony is produced after hour, bacterium colony is oyster white, circular, there is serration at edge, median rise, smooth surface, thalline is short straight rod shape, gram-positive microorganism, can gemma be produced, can extracellular proteinase, cellulase etc.The present invention has good prevention effect to tobacco gray mold, and has the prophylactic effect of long period, environmental friendliness.

Description

A kind of bacillus amyloliquefaciens and uses thereof
Technical field
The present invention relates to biological technical field, relate in particular to a kind of bacillus amyloliquefaciens, also relate to the purposes of this bacillus amyloliquefaciens in control floating seedlings tobacco in seedling stage gray mold simultaneously.
Background technology
By Botrytis cinerea ( botrytiscinereapers.:Fr) the tobacco gray mold caused is one of important disease on tobacco, and tobacco all can fall ill in whole breeding time, occurs heavier with seedling stage and squaring period.Mainly betide seedling stage on subaerial blade, scab subcircular, brown, there is unsharp light color edge; Under high humidity environment, scab is expanded rapidly, and diameter can reach more than 5 centimetres, and scab is web rot shape, it is covered with the mould layer of grey.When intensive floating seedlings, germ is fast diffusion very, and the production of cigarette seedling in serious threat, therefore to seedling stage tobacco gray mold control extremely urgent.
For a long time, in production, tobacco gray mold prevented and treated by the main chemical prevention medicament that leans on, major pharmaceutical agent has derosal, the thiophanate methyl of benzimidazoles, the procymidone (Sukeling) of dicarboximide class, iprodione, the mould prestige of second of amino formate and mixture thereof, also have the phonetic mould amine of aniline pyrimidine class etc.Because ash arrhizus bacteria has the characteristic that proliferative speed is high, heritable variation is large and fitness is high, continuous a large amount of chemical agent that uses makes this pathogenic bacteria produce resistance to some sterilant for many years, has it produces resistance report to various sterilization agent both at home and abroad.Microbiobacterial agent can compete nutrition, site, space with pathogenic bacteria, inducing plant produces disease resistance, its secondary metabolite is inhibited to pathogenic bacteria, have growth-promoting functions to plant, and green, environmental protection, safety, in the control of Plant diseases, there is important using value.Therefore, find eco-friendly, efficient, safe microbiobacterial agent, the control using it for floating seedlings tobacco in seedling stage gray mold has wide development space, is also the needs of agricultural safety Sustainable development simultaneously.
Summary of the invention
The object of the invention is to overcome the problems referred to above and the one provided has good prevention effect to tobacco gray mold, and have the prophylactic effect of long period, eco-friendly bacillus amyloliquefaciens.
Another object of the present invention is to provide the purposes of this bacillus amyloliquefaciens in control floating seedlings tobacco in seedling stage gray mold.
A kind of bacillus amyloliquefaciens, its morphological specificity: cultivate on NA substratum after 24 hours and produce single bacterium colony, bacterium colony is oyster white, circular, there is serration at edge, median rise, smooth surface, and thalline is short straight rod shape, gram-positive microorganism, can produce gemma, can extracellular proteinase, cellulase etc.
This bacterial classification is deposited in China typical culture collection center (address: Luojiashan, Wuchang, Wuhan City, Hubei Province, Wuhan University) on August 13rd, 2013, preserving number: CCTCCNO:M2013375, and name is called: bacillus amyloliquefaciens X-60 ( bacillusamyloliquefaciensx-60),
(1) separating step of bacillus amyloliquefaciens X-60 (BacillusamyloliquefaciensX-60):
" isolation by dilution method " is adopted to take 10g tobacco rhizosphere soil in clean beaker, be added on 90ml sterilized water, magnetic agitation makes it fully mix, and leaves standstill and draws 0.5ml supernatant liquor in 15 × 150mm clean tube after 20 minutes, add 4.5ml sterilized water fully mix 10 -1concentration mother liquor, then 0.5ml supernatant liquor is drawn from the test tube after dilution, then add 4.5ml sterilized water and fully mix obtained 10 -2strength solution, is diluted to 10 successively -9the soil solution of different concns is stand-by.Draw the above-mentioned different concns soil solution 100 μ l respectively, be spread evenly across on the NA flat board of culturing bacterium, 28 DEG C of dark culturing, picking list bacterium colony purifying after bacterium grows.
(2) substratum:
Beef extract-peptone solid medium (NA): extractum carnis 3g, peptone 10g, agar powder 16g, water 1000ml, pH6.8 ~ 7.2.
Beef extract-peptone liquid nutrient medium (NB): extractum carnis 3g, peptone 10g, water 1000ml, pH6.8 ~ 7.2.
(3) bacterial classification conservation and go down to posterity:
With the test tube of 18 × 180mm, contain NA substratum 9ml, beveling, inoculated bacteria, cultivate 2-3 days for 28 DEG C, put 4 DEG C of preservations, 3 months transferred speciess once.
Bacterium after purifying is streak culture on NA substratum, single bacterium colony is chosen after bacterium grows, with the toothpick of sterilizing single for bacterium bacterium colony chosen and shake training 24h to the test tube that 5mlNB substratum is housed (15 × 150mm) is inner, drawing 700 μ l bacterium liquid to 2ml bacterial strain preserves in pipe, in preservation pipe, add 700 μ l30% sterile glycerols again, be placed in-20 DEG C of medium-term and long-term preservations of refrigerator-freezer.
(4) the condition feature of bacterial classification:
The bacterium colony that this bacterium is formed, on NA flat board, 30 DEG C, cultivates 2d, colony diameter 5 ~ 15mm, bacterium colony subcircular, white, translucent, smooth moistening, slightly swell, thalline is short straight rod shape, is aerobic bacteria, and Gram-reaction is positive.Through Bacteria Identification handbook describe, in conjunction with Biolog qualification result and 16SrDNA the sequencing results, this bacterial strain is designated as bacillus amyloliquefaciens X-60 ( bacillusamyloliquefaciensx-60).
(5) cultural characters of bacterial classification
Culture temperature 20 ~ 50 DEG C, optimum temperature range 30 ~ 37 DEG C; Cultivate initial pH4.0 ~ 9.0, the suitableeest 6.0 ~ 7.0; Cultivate salt concn 0.5 ~ 5%, best 0.5%; Can cultivate carbon source and have glucose, sucrose, fructose, semi-lactosi, lactose, maltose, fiber two pool, starch, wherein glucose is best; Nitrogenous source can be cultivated and have extractum carnis, peptone, KNO 3, NH 4nO 3, (NH 4) 2sO 4and NaNO 2, wherein extractum carnis and peptone the best.
The present invention compared with prior art, has obvious beneficial effect, and as can be known from the above technical solutions: the present invention shows through isolated test, the growth of Bacillus amyloliquefaciens strain X60 to tobacco ash arrhizus bacteria has very strong restraining effect.Greenhouse floating seedlings control experiment shows: this microbial inoculum significantly can reduce the generation of the tobacco gray mold caused by pathogenic bacteria Botrytis cinerea, and has the prophylactic effect of long period.Have with the suitable prevention effect of chemical fungicide carbendazim.This microbial inoculum with wet bacterium and culture presevation liquid formulated, preservative fluid for thallus is the Tween40 nonionic surface active agent of 0.01mol/L phosphate buffered saline buffer and 0.001mol/L.This microbial inoculum can cigarette seedling on Direct spraying floating seedlings seedbed, also can fully mix rear nursery with seedling medium.The present invention is specially for the biocontrol fungicide of floating seedlings tobacco in seedling stage gray mold exploitation.Owing to being microbiobacterial agent, environmentally friendly, completely not because of the series of problems that the use of chemical pesticide brings, be conducive to No-harmful apple orchard, organic production.
Embodiment
From flue-cured tobacco ( nicotianatabacuml.) in rhizosphere soil, separation obtains.Bacillus amyloliquefaciens X60 cultivates after 24 hours and produces single bacterium colony on NA substratum, and bacterium colony is oyster white, and circular, there is serration at edge, median rise, smooth surface, thalline is short straight rod shape, gram-positive microorganism, can produce gemma, can extracellular proteinase, cellulase etc.
By the strain X 60 of above-mentioned control tobacco gray mold at NB nutrient solution 30 DEG C of 180rpm shaking culture 36-48 hour, then centrifugal 10 minutes of 6000rpm, get X60 wet thallus and preservative fluid for thallus, be that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum finished product viable bacteria concentration is 1 × 10 10-1 × 10 12cFU/ml, preservative fluid for thallus is 0.01mol/L phosphate buffered saline buffer, it is also the Tween40 nonionic surface active agent of 0.001mol/L containing concentration, NB nutrient solution compound method used is: extractum carnis 3g, peptone 10g, add deionized water to 1L, pH6.8 ~ 7.2, through 120 DEG C of sterilizing packing after 20 minutes, stand-by.
The inhibition test that bacillus amyloliquefaciens X60 grows tobacco ash arrhizus bacteria
Mixed according to 1 ︰ 1 ratio with NA substratum by the PDA substratum melted, flat board processed, after dull and stereotyped cooling, every ware central authorities' inoculation tobacco ash arrhizus bacteria bacterium cake (diameter 5mm, colony edge gets bacterium cake), cultivates under 28 DEG C of dark conditions.Adopt sterile toothpick apart from the upper inoculating strain X60 of bacterium dish about 2cm one, place " wire cross " after 2d, another line is inoculated sterilized water for contrast, cultivate under being placed in 28 DEG C of dark conditions, after 4d, observe the size of inhibition zone.X60 microbial inoculum is to the efficiency test of floating seedlings cigarette seedling gray mold
The preparation of pathogenic bacteria spore suspension: tobacco ash arrhizus bacteria is inoculated in PDA culture medium flat plate, 6d is cultivated under 25 DEG C of dark conditions, then flat board is placed in the formation of induced meristem spore under 15 DEG C of dark conditions, after 3d, obtains conidial suspension with sterile gauze elimination mycelia.Measure the concentration of spore suspension with blood counting chamber, and spore suspension concentration is adjusted to 10 5individual spore/ml, for subsequent use.
The preparation of X60 microbial inoculum: by the strain X 60 of above-mentioned control tobacco gray mold at NB nutrient solution 30 DEG C of 180rpm shaking culture 36-48 hour, then centrifugal 10 minutes of 6000rpm, get X60 wet thallus and preservative fluid for thallus, be that 1:50 is made into microbial inoculum by mass volume ratio, microbial inoculum finished product viable bacteria concentration is 1 × 10 10-1 × 10 12cFU/ml, preservative fluid for thallus is 0.01mol/L phosphate buffered saline buffer, it is also the Tween40 nonionic surface active agent of 0.001mol/L containing concentration, NB nutrient solution compound method used is: extractum carnis 3g, peptone 10g, add deionized water to 1L, pH6.8 ~ 7.2, through 120 DEG C of sterilizing packing after 20 minutes, stand-by.
Nursery in floating seedling culturing dish for tobacco (kind: the large gold dollar of safflower), for subsequent use when cigarette seedling grows to 5-6 leaf.Biocontrol microorganisms treatment group I, by above-mentioned biocontrol microorganisms liquid even spraying to cigarette seedling leaf surface, until run off; Control group spraying sterilized water II; Derosal treatment group III, 80% carbendazol wettable powder 1000 times dilution spraying, until run off; The 1500 times of liquid sprayings of the phonetic mould amine suspension agent of phonetic mould amine treatment group IV, 40%, until run off.Process after 24 hours, by above-mentioned tobacco ash arrhizus bacteria spore suspension even spraying to blade surface, often process process cigarette seedling 60 strain.Under cigarette seedling being placed in natural lighting after connecing bacterium, 15-27 DEG C, RH>80% cultivate.Observed and recorded blade incidence after 24 days, and investigate disease index, calculate preventive effect.
According to following state of an illness statistical grade, carry out state of an illness statistics according to blade onset area:
0 grade--complete stool is anosis;
1 grade--lesion area accounts for less than 5% of blade area, or stem's scab is no more than 1/3 of stem girth;
3 grades--lesion area accounts for 6% ~ 10% of blade area, or stem's scab is around stem girth 1/3 ~ 1/2;
5 grades--lesion area accounts for 11% ~ 20% of blade area, or stem's scab exceedes 1/2 of stem girth, but not all around stem girth;
7 grades--lesion area accounts for 21% ~ 40% of blade area, or stem's scab is all around stem girth;
9 grades--lesion area accounts for more than 41% of blade area, or stem's scab is all around stem girth, and plant is wilting even withered;
Indoor flat plate dual test shows, the inhibiting rate of strain X 60 pairs of tobacco ash arrhizus bacterias can reach 51.3%
Floating seedlings cigarette seedling greenhouse test result shows, X60 microbial inoculum treatment group can be good at the generation preventing tobacco gray mold, and its prevention effect reaches 82.66%
The preventive effect to floating seedlings cigarette seedling gray mold after 24 days used by table 1X60 microbial inoculum
Process Sickness rate (%) Disease index Preventive effect (%)
Contrast 86.5 84.46 --
Derosal 5.34 8.66 89.75
Phonetic mould amine 3.26 7.48 91.14
X60 microbial inoculum 8.46 14.64 82.66

Claims (2)

1. a bacillus amyloliquefaciens ( bacillusamyloliquefaciens) X-60, it is characterized in that the preserving number of described bacterial classification is CCTCCNO:M2013375.
2. bacillus amyloliquefaciens as claimed in claim 1 ( bacillusamyloliquefaciens) purposes of X-60 in control floating seedlings tobacco in seedling stage gray mold.
CN201310490095.5A 2013-10-18 2013-10-18 A kind of bacillus amyloliquefaciens and uses thereof Active CN103555615B (en)

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CN106689212B (en) * 2016-12-14 2019-08-30 贵州省烟草科学研究院 A kind of microbicide compositions containing Fluoxastrobin, preparation method and application
CN107912447A (en) * 2017-11-23 2018-04-17 贵州省烟草科学研究院 A kind of microbicide compositions containing bacillus amyloliquefaciens, preparation method and the usage
CN109797117A (en) * 2019-01-30 2019-05-24 贵州大学 It is a kind of produce multienzyme double bacteria preparations preparation and its application method
CN110622997A (en) * 2019-11-08 2019-12-31 四川龙蟒福生科技有限责任公司 Bactericidal composition, application thereof and bactericide

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CN103087951A (en) * 2013-01-14 2013-05-08 北京市农林科学院 Bacillus amyloliquefaciens and application thereof
CN103173397A (en) * 2013-04-02 2013-06-26 山东省农业科学院农产品研究所 Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof

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CN103087951A (en) * 2013-01-14 2013-05-08 北京市农林科学院 Bacillus amyloliquefaciens and application thereof
CN103173397A (en) * 2013-04-02 2013-06-26 山东省农业科学院农产品研究所 Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof

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Inventor after: Wang Hancheng

Inventor after: Li Wenhong

Inventor after: Chen Qingyuan

Inventor after: Xia Haiqian

Inventor after: Wang Maosheng

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