CN102206604A - Enterobacter agglomerans strain and application thereof in weeding - Google Patents
Enterobacter agglomerans strain and application thereof in weeding Download PDFInfo
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Abstract
The invention relates to an allelopathic bacteria-enterobacter agglomerans W5 strain and application thereof as a bioherbicide, belonging to the field of microbes applied to agricultural plant protection. The allelopathic bacteria-enterobacter agglomerans W5 strain is preserved in the China general microbiological culture collection center (address: DaTun road, Chaoyang district, Beijing), with a preserving number of CGMCC No. 4565. The invention also relates to a liquid weeding bacterial agent and a solid weeding bacterial agent containing the allelopathic bacteria-enterobacter agglomerans W5 strain as effective components and applications thereof in controlling dicotyledonous weeds of a farmland or a lawn.
Description
One, technical field
The present invention relates to a kind of enterobacteria bacterial strain and the application in weeding thereof, belong to preventing and kill off of the technical field that microorganism is applied to agriculture plant protection, prevents and kill off crop weeds, particularly farmland and lawn broadleaf weed.
Two, background technology
Weeds are biological epidemics that the serious threat modern agriculture is produced.The measure that is used for weeds control at present is mainly by using chemosynthesis weedicide and cultivation transgenic plant.The former generally has problems such as the relatively poor and resistance of Environmental compatibility, and the latter is subjected to certain restriction in the use because of transformed variety limited amount and herbicidal spectrum are narrower.Therefore, research and development are efficient, environmental protection, harmless campelyco are undoubtedly current most important, the most urgent task, and the research of new bio weedicide has great social significance and economic worth.
The plant pathogenic fungi weedicide is the more campelyco of research, but these weedicides generally have usage quantity big, take effect slow, certain more high shortcoming of ecological side effect, production and use cost is arranged, majority does not enter the practical stage as yet.The commercialization microbial herbicide of having reported at present has Devine, the Collegeo of the U.S., Canadian BioMa1, and the Shandong of China is protected No. one, the Campelyo of Japan etc.Wherein Devine is the chlamydospore suspension agent of the palm mould pathogenic strains of Florida, USA, is used for not human relations rattan of controlling weeds, and preventive effect can reach more than 90%, and the lasting period can reach 2 years.Therefore, research and development novel microorganism weedicide has the very big market space, and microbial herbicide also might become the substitute of chemical herbicide, is used for the control of sustainable agriculture ecosystem weeds.
Changing sense bacterium (Allelopathic Bacteria) is can suppress plant-growth or phytotoxic harmful rhizosphere bacteria by the secretion allelochemical in the soil, is little effect plant pathogen.The more change sense bacterium of research comprises some kinds of Rhodopseudomonas (Pseadomonas), bacillus (Bacillus), enterobacter (Enterobacter), Klebsiella (Klebsiella) and Microbacterium (Microbacterium) etc. at present.Identified that the allelochemical with removing activity has plant growth regulating substance such as IAA and ethene, prussic acid, exocellular polysaccharide and plant poison etc.Efficient removing activity in view of the special biological characteristics of changing the sense bacterium (fast, the easy cultivation of growth, mutable gene operation etc.) and some change sense molecules, change the campelyco that the sense bacterium is considered to tool potentiality to be exploited, be subjected to showing great attention to of countries in the world scientist.
Three, summary of the invention
The purpose of this invention is to provide a strain enterobacteria W5 bacterial strain and as the preparation method of weeding microbial inoculum, particular content and embodiment are as follows:
W5 bacterial strain of the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and (address: the Datun Road, Chaoyang District, Beijing City), preserving number is CGMCC No.4565.The pedotheque that is used for the W5 strains separation picks up from June, 2008 and Linfen, Shanxi Province plantation year is limited to the glutinous rehmannia producing region in 1 year, and separation method adopts dilution plate bacterium colony method.The W5 bacterial strain in the dull and stereotyped last 37 ℃ of bacterium colonies after cultivating 24h of LB present rule circle, canescence, moistening, the edge is smooth.This bacterium microscopic examination is rod-short, no gemma; Gramstaining is negative; Biochemical reactions result shows, this bacterium has catalase reaction, the utilization of grape sugar, sucrose utilization, clark and Lubsreaction, nitrate reduction reaction, Citrate trianion and the V-P reaction is positive and indoles experiment, oxydase reaction and H2S produce the characteristics that experiment is negative, can meet the identification mark of enterobacter Enterobacter amnigenus (Enterobacter amnigenus) in 28 degree growths.16S rDNA sequence of this bacterium and the homology of Enterobacter amnigenus reach 100%.Combining form is learned feature, biochemical reactions result and 16S rDNA and is analyzed, and enterobacteria W5 identification of strains is Enterobacter amnigenus (Enterobacter amnigenus).Have not yet to see the report that this bacterioid has removing activity.
To common weed such as Herba Acalyphae, Herba Oxalidis Corniculatae, red the climbing in south, the humulus grass, Manyhead Clinopodium Herb, mock-strawberry, the ox chickweed, Violaceae plant, CHANGQINGTENG, zollinger gromwell, it lake Sui, Orychophragmus violaceus, alternanthera philoxeroides, green hair green bristlegrass, wheatgrass, Horseweed Herb, Eclipta prostrata, Herba Ajugae multiforae, root of Common carpesium, Stringy Stonecrop Herb, the experiment of veronica and Herba Galii Teneri etc. shows, by the liquid of W5 bacterial strain preparation and solid herbicidal microbial inoculum to the toxic effect of multiple weeds for examination, to Herba Acalyphae, Herba Oxalidis Corniculatae, south redly climb, humulus grass, Manyhead Clinopodium Herb and 6 kinds of weeds toxicity of ox chickweed are stronger.Adopt same procedure and concentration to handle monocotyledon weed, as Herba Setariae Viridis, green hair green bristlegrass, yellow hair green bristlegrass etc., all do not find toxic action, the removing activity material that showing sense bacterium W5 bacterial strain produces is higher than monocotyledons to the toxic action of dicotyledons, and there is certain plant selectivity in its removing activity.Experiment to seed germination has also confirmed this point, and is obvious to the restraining effect effect of dicotyledons mung bean seed rudiment, to the basic unrestraint effect of monocotyledons wheat.In addition, be present in the nutrient solution definition of meeting of the behavior sense bacterium of this bacterium owing to kill careless composition.
The preparation of liquid provided by the invention and solid herbicidal microbial inoculum and using method are:
(1) preparation of fermentation liquid
Improvement LB culture medium prescription: peptone 15g/L, glucose 30g/L, natural pH.
First order seed is cultivated: picking one collarium is inoculated in the 500mL triangular flask that the 50mL substratum is housed from fresh plate, and 37 ℃, 100rpm were cultivated 10 hours.
Secondary seed is cultivated: liquid amount 6L (10L jar), and inoculation 240mL (4%), 100rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 5～10 hours for 37 ℃.
The 200L fermentor cultivation: liquid amount 150L, inoculation 6L (4%), 150rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 24～36 hours for 37 ℃, the preparation fermented liquid.
Measure through colony counting method, the bacterial number in the fermented liquid should surpass 3 * 10
9CFU/ml.
(2) preparation method of liquid herbicidal microbial inoculum
Add appropriate amount of addition agent such as tensio-active agent, sanitas, antifreezing agent in the above-mentioned fermented liquid, fully bottle behind the mixing, be the liquid herbicidal microbial inoculum.。
(3) preparation method of solid herbicidal microbial inoculum
Macroporous resin carries out adorning post after the pre-treatment with reference to shop instruction.Above-mentioned fermented liquid is centrifugal or remove by filter thalline, and supernatant liquor flows through macroporous resin column with the flow velocity of 2～8mL/min, collects effusive liquid.Use 95% ethanol towards post then, flow velocity is 2～8mL/min, collects ethanol eluate.With the elutriant evaporated under reduced pressure, pass through silicagel column and Sephadex LH-20 purifying after the dissolving again, the active ingredient that obtains is the solid herbicidal microbial inoculum.
(4) using method of weeding microbial inoculum
It is foliage-spray that weeding microbial inoculum of the present invention is recommended using method.The liquid herbicidal microbial inoculum can directly be packed into and be sprayed in the atomizer, and the solid herbicidal microbial inoculum is sprayed after can adding 200～1000L water dissolution by every kilogram.
Weeding microbial inoculum of the present invention also can mix with other chemistry that are fit to or campelyco or be used, thereby reduces the consumption of chemical herbicide, reduces the pollution to environment, enlarges use range.
Microbial inoculum of the present invention can be used for preventing and kill off the broadleaf weed in farmland or the lawn.
Weedicide of the present invention is environmentally friendly, helps the popularization of green food and Organic farming, and cost is low, pollution-free, to crop safety.
Embodiment
1. 1 kinds of preparation methods that contain the liquid herbicidal microbial inoculum of W5 bacterial strain of embodiment
(1) preparation of fermentation liquid
Improvement LB culture medium prescription: peptone 15g/L, glucose 30g/L, natural pH.
First order seed is cultivated: picking one collarium is inoculated in the 500mL triangular flask that the 50mL substratum is housed from fresh plate, and 37 ℃, 100rpm were cultivated 10 hours.
Secondary seed is cultivated: liquid amount 6L (10L jar), and inoculation 240mL (4%), 100rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 5 hours for 37 ℃.
The 200L jar is cultivated: liquid amount 150L, and inoculation 6L (4%), 150rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 24 hours for 37 ℃.Be fermented liquid.Measure through colony counting method, the bacteria content in the fermented liquid surpasses 3 * 10
9CFU/ml.
(2) preparation of liquid herbicidal microbial inoculum
Fermented liquid is put into a mixing bowl, and according to 3 parts of 92 parts of fermented liquids, tensio-active agent OP-103 part, sanitas sodium salicylate, the ratio that antifreezing agent urea is 2 parts is added various assistant agents, 80rpm, and 30min, fully mixing is the liquid herbicidal microbial inoculum.
The preparation of embodiment 2. solid herbicidal microbial inoculums
Macroporous resin carries out pre-treatment with reference to shop instruction, adorns post (1000g macroporous resin, diameter are 6cm) then.The centrifugal 20min of above-mentioned fermented liquid 4000rpm removes thalline, and supernatant liquor flows through macroporous resin column with the flow velocity of 4mL/min, collects effusive liquid.Use 95% ethanol towards post then, flow velocity is 4mL/min, collects ethanol eluate.The elutriant vacuum rotary steam to doing, is passed through silicagel column and Sephadex LH-20 purifying again, and the active ingredient that obtains is the solid herbicidal microbial inoculum.
The removing activity of embodiment 3.W5 strain liquid and solid herbicidal microbial inoculum
Be respectively for the examination weeds: Herba Acalyphae, Herba Oxalidis Corniculatae, red the climbing in south, humulus grass, Manyhead Clinopodium Herb, mock-strawberry, ox chickweed, Violaceae plant, CHANGQINGTENG, zollinger gromwell, day lake Sui, Orychophragmus violaceus, alternanthera philoxeroides, green hair green bristlegrass, wheatgrass, Horseweed Herb, Eclipta prostrata, Herba Ajugae multiforae, root of Common carpesium, Stringy Stonecrop Herb, totally 22 kinds of veronica and Herba Galii Teneris.
The solid herbicidal microbial inoculum aqueous solution of liquid herbicidal microbial inoculum and different concns is sprayed on respectively on 22 kinds of grass cutting blades, observes the downright bad situation of blade.
Above-mentioned 22 kinds of grass cutting blades are behind liquid herbicidal microbial inoculum spray medicine 24h, and wilting and necrosis gradually in 6 kinds of grass cutting blade edges, is respectively Herba Acalyphae, Herba Oxalidis Corniculatae, Curcurbitaceae weeds, Manyhead Clinopodium Herb, ox chickweed and humulus grass.The solid herbicidal microbial inoculum aqueous solution is then to the toxic effect of 17 kind of plant, and working concentration is 25-50mg/mL (table 1).
Adopt same procedure and concentration to handle monocotyledon weed, as Herba Setariae Viridis, green hair green bristlegrass, yellow hair green bristlegrass etc., all do not find toxic action, show that this microbial inoculum is significantly higher than monocotyledons to the toxic action of dicotyledons, there is certain plant selectivity in its removing activity.
The removing activity of the table 1 bacterial strain W5 liquid and the solid herbicidal microbial inoculum aqueous solution
Annotate: "+" represents the lethal effect positive; "-" represents the lethal effect feminine gender
Above embodiment is only for the present invention is further illustrated, and protection scope of the present invention is not subjected to the limitation of illustrated embodiment.
Claims (5)
1. enterobacteria W5 bacterial strain, it is characterized in that: (address: No. 3, institute in BeiChen West Road, Chaoyang District, BeiJing City), preserving number is CGMCC No.4565 to be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center.Belonging to of W5 bacterial strain sense bacterium separates from the soil in 1 year of glutinous rehmannia continuous cropping, 37 ℃ of bacterium colonies after cultivating 24h on the LB flat board present rule circle, canescence, moistening, the edge is smooth; This bacterium microscopic examination is rod-short, no gemma; Gramstaining is negative; Catalase reaction, the utilization of grape sugar, sucrose utilization, clark and Lubsreaction, nitrate reduction reaction, Citrate trianion and V-P reaction are positive; Indoles experiment, oxydase reaction and H
2S produces experiment and is negative; The homology of the Enterobacter amnigenus of 16S rDNA sequential analysis and enterobacter (Enterobacter amnigenus) reaches 100%; Combining form is learned feature, biochemical reactions result and 16S rDNA and is analyzed, and enterobacteria W5 identification of strains is Enterobacter amnigenus (Enterobacter amnigenus).
2. the liquid herbicidal microbial inoculum of W5 bacterial strain according to claim 1 is characterized in that: W5 strain cell concentration 〉=3 * 10 in the weeding microbial inoculum
9CFU/ml.
3. preparation method as the liquid herbicidal microbial inoculum of W5 bacterial strain as described in the claim 2 is characterized in that: use improvement LB substratum (peptone 15g/L, glucose 30g/L, natural pH), according to the following steps preparation:
(1) first order seed is cultivated: picking one collarium is inoculated in the 500mL triangular flask that the 50mL substratum is housed from fresh plate, and 37 ℃, 100rpm were cultivated 10 hours.
(2) secondary seed is cultivated: liquid amount 6L (10L jar), and inoculation 240mL (4%), 100rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 5 hours for 37 ℃.
(3) 200L fermentor cultivation: liquid amount 150L, inoculation 6L (4%), 150rpm, air flow 1.2vvm, pressure 0.06MPa cultivated 24 hours for 37 ℃, the gained fermented liquid adds an amount of conventional assistant agent such as tensio-active agent, sanitas, antifreezing agent etc., is the liquid herbicidal microbial inoculum.
4. solid herbicidal microbial inoculum of W5 bacterial strain according to claim 1, it is characterized in that: after the described liquid herbicidal microbial inoculum of claim 2 (before not adding assistant agent) is removed thalline, the gained supernatant liquor adsorbs with the X-5 macroporous resin column, use 95% ethanol elution then, the elutriant vacuum rotary steam is to doing, pass through silicagel column and Sephadex LH-20 chromatography again and obtain to have the active ingredient of killing the grass effect, be the solid herbicidal microbial inoculum.
5. according to the application of claim 2,4 described microbial inoculums, it is characterized in that: be applied to preventing and kill off of farmland or lawn broadleaf weed as campelyco.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483207A (en) * | 2015-11-30 | 2016-04-13 | 广东省微生物研究所 | Rapid numerical identification method for enterobacteria of food-borne pathogenic bacteria |
| CN108575640A (en) * | 2018-05-03 | 2018-09-28 | 山东省花生研究所 | A kind of implantation methods preventing peanut continuous cropping obstacle |
| CN109090144A (en) * | 2018-10-17 | 2018-12-28 | 马书文 | A kind of novel environmentally-friendly biological herbicide |
| CN109182201A (en) * | 2018-09-30 | 2019-01-11 | 浙江工业大学 | Enterobacter amnigenus I type ZJB-17002 of biology and its application |
| CN115354003A (en) * | 2022-08-25 | 2022-11-18 | 安徽省农业科学院园艺研究所 | Bacterial strain for promoting high-density growth of cucurbit and reducing cadmium absorption and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1446464A (en) * | 2003-03-19 | 2003-10-08 | 浙江大学 | Method for preparing high efficiency biological weed control bacterial agent and its usage |
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| CN105483207A (en) * | 2015-11-30 | 2016-04-13 | 广东省微生物研究所 | Rapid numerical identification method for enterobacteria of food-borne pathogenic bacteria |
| CN108575640A (en) * | 2018-05-03 | 2018-09-28 | 山东省花生研究所 | A kind of implantation methods preventing peanut continuous cropping obstacle |
| CN109182201A (en) * | 2018-09-30 | 2019-01-11 | 浙江工业大学 | Enterobacter amnigenus I type ZJB-17002 of biology and its application |
| CN109182201B (en) * | 2018-09-30 | 2020-06-23 | 浙江工业大学 | Enterobacter hesheng biological I type ZJB-17002 and application thereof |
| CN109090144A (en) * | 2018-10-17 | 2018-12-28 | 马书文 | A kind of novel environmentally-friendly biological herbicide |
| CN115354003A (en) * | 2022-08-25 | 2022-11-18 | 安徽省农业科学院园艺研究所 | Bacterial strain for promoting high-density growth of cucurbit and reducing cadmium absorption and application thereof |
| CN115354003B (en) * | 2022-08-25 | 2023-12-19 | 安徽省农业科学院园艺研究所 | Strain for promoting high-density growth of cucurbits and reducing cadmium absorption and application thereof |
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