CN103271092B - A kind of biofungicide Preparation Method And The Use - Google Patents
A kind of biofungicide Preparation Method And The Use Download PDFInfo
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Abstract
The invention discloses a kind of biofungicide Preparation Method And The Use, the active component of this kind of antibacterial by 3 kinds from different medicinal plants Radix Polygalaes, threeleaf akebia, Radix Salviae Miltiorrhizae endogenetic bacteria form in inoculum concentration ratio 1: 2: 6,3 kinds of antibacterials respectively produce the molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa, this antibacterial also includes 40-50 part filler, 1-10 part surfactant, 1-10 part stabilizer, filler is white carbon, surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, and stabilizer is potassium dihydrogen phosphate。The present invention is applied widely, antibacterial effect good stable, without any harmful substance, have no side effect, environmental protection, preparation method is simple, manufacturing cycle is short, with low cost, it is the ecological agriculture and the ideal product of green non-pollution food, has significant economic benefit and social benefit。
Description
Technical field
The invention belongs to technical field of pesticide, relate to a kind of biofungicide Preparation Method And The Use。
Background technology
The use of chemical pesticide can be described as a double-edged sword。On the one hand, being rationally applied in of chemical pesticide alleviates the pest and disease damage impact on crop growthing development to a certain extent really, improves crop yield, achieves certain economic worth；But then, the abuse of chemical pesticide result also in the poisonous and harmful element content overproof of crops Pesticide Residues material and degradation of pesticide, and the health of the mankind is caused huge threat by this。While modern society's science and technology high speed development and human quality promote, people are subject to polluting this present situation in various degree the crops using chemical pesticide to cause a large amount of in agricultural production, the daily life made for meeting the mankind of the Fungicidal substance substituting chemical pesticide excellent in the urgent need to a kind of green non-pollution and action effect or minimizing chemical pesticide。
As a large agricultural country, along with popularizing of ecological agriculture idea, current China structure of agricultural production is in by traditional agricultural Model to the direction transition of the green non-pollution ecological agriculture and transformation, the idea of undue weight farm output starts by the green ecological agricultural idea of safety and Health replaced gradually, actively develops eco-agriculture energetically and green non-pollution food is the encountered inexorable trend of China's agricultural development。Although most of chemical pesticides are more notable compared with the fungistatic effect of biological pesticide, but biotechnology to show tempting superiority biofungicide antibacterial effect in field of ecological agricultural excellent and do not result in subsidiary pollution。Study antibacterial excellent effect and non-harmful biofungicide is the important topic in the outer agricultural research of Present Domestic, there is far-reaching theory significance and realistic meaning。
Summary of the invention
It is an object of the invention to overcome above-mentioned technological deficiency, it is provided that a kind of biofungicide Preparation Method And The Use。Its technical scheme is:
A kind of biofungicide, the active component of this antibacterial by 3 kinds from different medicinal plants [Radix Polygalaes (RadixPolygalae), threeleaf akebia (Akebiatrifoliate (Thunb.) Koidz), Radix Salviae Miltiorrhizae (Salviamiltiorrhiza)] endogenetic bacteria in inoculum concentration ratio 1: 2: 6 form, 3 kinds of antibacterials respectively produce the molten bacillus of enzyme (Lysobacteryananisissp.nov), bacillus amyloliquefaciens (Bacillusamyloliquefacians), Paenibacillus polymyxa (Paenibacilluspolymyxa), this antibacterial also includes 40-50 part filler, 1-10 part surfactant, 1-10 part stabilizer, filler is white carbon, surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, stabilizer is potassium dihydrogen phosphate。
The preparation method of a kind of biofungicide of the present invention, comprises the following steps:
(1) the described molten bacillus of product enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa are activated respectively in LB culture medium, then carry out 37 DEG C of fermentations；
(2) as the described three kinds of ferment product OD of step (1)600When being 0.150, inoculum concentration ratio is by bacillus amyloliquefaciens: produce the molten bacillus of enzyme: Paenibacillus polymyxa is 1: 2: 6 inoculation mixed fermentation；
(3) after 48h, 40-50 part filler, 1-10 part surfactant, 1-10 part stabilizer is added in the fermentation liquid that step (2) fermentation obtains, filler is white carbon, surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, stabilizer is potassium dihydrogen phosphate, is sufficiently mixed；
(4) mixture of step (3) gained is pulverized, then sieve (aperture is 200 orders)；
(5) check the fungistatic effect of the powder obtained that sieves, if antibacterial efficiency is more than 55%, then pack on request。
The culture medium adopted in preparation process is LB fluid medium, by weight, deionized water 950 parts, tryptone 10 parts, yeast extract 5 parts, NaCl10 part, after solute fully dissolves, regulate pH to 7.4 with 5mol/LNaOH。
During composition antibacterial single culture, all show the fungistatic effect of wide spectrum, but these three antibacterial is all different to the antibacterial effect of same pathogen。It has been investigated that by bacillus amyloliquefaciens: producing the molten bacillus of enzyme: Paenibacillus polymyxa is 1: 2: 6 inoculation, after 37 DEG C of fermentation 48h, three kinds of microorganism numbers are consistent。
Compared with prior art, the invention have the benefit that
The present invention is applied widely, antibacterial effect good stable, without any harmful substance, have no side effect, environmental protection, preparation method is simple, manufacturing cycle is short, with low cost, it is the ecological agriculture and the ideal product of green non-pollution food, has significant economic benefit and social benefit。
Invention purposes
1) alleviate the pathogen impact on crop growthing development, increase crop yield；
2) pollution using chemical pesticide to cause effectively is reduced；
3) needs of green non-pollution ecological agriculture development are met。
Accompanying drawing explanation
Fig. 1 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa, the mixed vaccine antibacterial efficiency to tomato gray mould, J, S, D, H represents bacillus amyloliquefaciens respectively, produces the mixed vaccine that the molten bacillus of enzyme, Paenibacillus polymyxa and three kinds of bacterium form in inoculum concentration ratio 1: 2: 6, and error line represents corresponding standard error；
Fig. 2 is bacillus amyloliquefaciens, produce the molten bacillus of enzyme, antibacterial efficiency that Paenibacillus polymyxa, mixed vaccine are withered to stricture of vagina, J, S, D, H represents bacillus amyloliquefaciens respectively, produces the mixed vaccine that the molten bacillus of enzyme, Paenibacillus polymyxa and three kinds of bacterium form in inoculum concentration ratio 1: 2: 6, and error line represents corresponding standard error；
Fig. 3 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa, the mixed vaccine antibacterial efficiency to screw thread, J, S, D, H represents bacillus amyloliquefaciens respectively, produces the mixed vaccine that the molten bacillus of enzyme, Paenibacillus polymyxa and three kinds of bacterium form in inoculum concentration ratio 1: 2: 6, and error line represents corresponding standard error；
Fig. 4 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa, the mixed vaccine antibacterial efficiency to root-rot, J, S, D, H represents bacillus amyloliquefaciens respectively, produces the mixed vaccine that the molten bacillus of enzyme, Paenibacillus polymyxa and three kinds of bacterium form in inoculum concentration ratio 1: 2: 6, and error line represents corresponding standard error；
Fig. 5 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa and the mixed vaccine inhibition figure to tomato gray mould, wherein, S, J, D, H represent that the product molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa and three kinds of bacterium are successively in the mixed vaccine of inoculum concentration ratio 1: 2: 6 composition respectively；
Fig. 6 is bacillus amyloliquefaciens, produces the inhibition figure that the molten bacillus of enzyme, Paenibacillus polymyxa and mixed vaccine are withered to stricture of vagina, wherein, S, J, D, H represent that the product molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa and three kinds of bacterium are successively in the mixed vaccine of inoculum concentration ratio 1: 2: 6 composition respectively；
Fig. 7 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa and the mixed vaccine inhibition figure to rib-loop, wherein, S, J, D, H represent that the product molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa and three kinds of bacterium are successively in the mixed vaccine of inoculum concentration ratio 1: 2: 6 composition respectively；
Fig. 8 is bacillus amyloliquefaciens, produces the molten bacillus of enzyme, Paenibacillus polymyxa and the mixed vaccine inhibition figure to root-rot, wherein, S, J, D, H represent that the product molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa and three kinds of bacterium are successively in the mixed vaccine of inoculum concentration ratio 1: 2: 6 composition respectively。
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, technical scheme is described in more detail。In following example, method therefor is conventional method if no special instructions, and all percent concentrations are mass percent if no special instructions, and all appearance agent are deionized water。
Embodiment 1
The preparation method of a kind of biofungicide, carries out according to following steps:
(1) liquid culture produces the molten bacillus of enzyme (Lysobacteryananisissp.nov), bacillus amyloliquefaciens (Bacillusamyloliquefaciens), Paenibacillus polymyxa (Paenibacilluspolymyxa), temperature is 37 DEG C, and rotating speed is 180r/min。Culture medium is tryptone 10 parts, yeast extract 5 parts, NaCl10 part, deionized water 950 parts。After solute fully dissolves, regulate pH to 7.4 with 5mol/LNaOH；
(2) the molten bacillus of enzyme (Lysobacteryananisissp.nov), bacillus amyloliquefaciens (Bacillusamyloliquefa-ciens), Paenibacillus polymyxa (Paenibacilluspolymyxa) three strain antibacterial OD are produced600During for equal 0.150, in inoculum concentration ratio bacillus amyloliquefaciens: produce the molten bacillus of enzyme: Paenibacillus polymyxa is 1: 2: 6 inoculation, 37 DEG C of fermentations；
(3), after 48h, in mixed fermentation liquid, 40-50 part filler, 1-10 part surfactant, 1-10 part stabilizer etc. are added。Filler is white carbon, and surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, and stabilizer is potassium dihydrogen phosphate, is sufficiently mixed；
(4) said mixture is pulverized, then cross the sieve of 200 orders；
(5) effect of the powder that inspection obtains after sieving, if inspection fungistatic effect is qualified, then packs on request。
Choosing the experiment field infecting tomato gray mould, arranging 6 pieces of area bacterium wherein is 9m2Sample prescription (3 pieces be process group, 3 pieces is matched group)；
In mass ratio, with mix bacterium agent: water=1: the mass ratio of 50 adds distilled water in above-mentioned mix bacterium agent and is configured to suspension, with the distilled water of equal in quality for comparison, nebulization is adopted to make corresponding process group and matched group above-ground plant parts moisten respectively。
Effect is extremely obvious, and after first time sprinkling, 48h tomato gray mould symptom disappears, and the sterilizing rate of tomato gray mould is reached 100% by this kind of agricultural biological antibacterial。
Relative sterilizing rate (%)=(comparison microbiological contamination area one processes microbiological contamination area)/comparison microbiological contamination area × 100%
Embodiment 2
The preparation method of biofungicide is with example 1。
Choosing the experiment field infecting tomato gray mould, arranging 6 pieces of area bacterium wherein is 9m2Sample prescription, 3 pieces be process group, 3 pieces is matched group；
In mass ratio, with mix bacterium agent: water=1: the mass ratio of 100 adds distilled water in above-mentioned mix bacterium agent and is configured to suspension, with the distilled water of equal in quality for comparison, nebulization is adopted to make corresponding process group and matched group above-ground plant parts moisten respectively。
After first time sprinkling, 48h tomato gray mould symptom is seldom, and sterilizing rate is up to 93.7%；48h tomato gray mould transference cure after second time sprinkling。
Embodiment 3
The preparation method of biofungicide is with embodiment 1。
Choosing the experiment field infecting tomato gray mould, arranging 6 pieces of area bacterium wherein is 9m2Sample prescription, 3 pieces be process group, 3 pieces is matched group；
In mass ratio, with mix bacterium agent: water=1: the mass ratio of 200 adds distilled water in above-mentioned mix bacterium agent and is configured to suspension, with the distilled water of equal in quality for comparison, nebulization is adopted to make corresponding process group and matched group above-ground plant parts moisten respectively。
After first time sprinkling, 48h tomato gray mould significantly reduces, and sterilizing rate reaches 84.4%；After second time, 48h tomato gray mould symptom is seldom, is only present in pole individually upper bit；After third time is sprayed, 48h tomato gray mould symptom is wholly absent。
Embodiment 4
The preparation of biofungicide is with example 1。
Choosing the experiment field infecting tomato gray mould, arranging 6 pieces of area bacterium wherein is 9m2Sample prescription, 3 pieces be process group, 3 pieces is matched group；
In mass ratio, with mix bacterium agent: water=1: the mass ratio of 300 adds distilled water in above-mentioned mix bacterium agent and is configured to suspension, with the distilled water of equal in quality for comparison, nebulization is adopted to make corresponding process group and matched group above-ground plant parts moisten respectively。
After first time sprinkling, 48h tomato gray mould significantly reduces, and sterilizing rate reaches 78.3%；After second time and third time are sprayed, tomato gray mould symptom seldom, is only present in pole individually upper bit；48h after spraying for 4th time, tomato gray mould symptom is wholly absent。
Embodiment 5 mix bacterium agent suppresses tomato gray mould test
Prepare microbe composite bacterial liquid, produce the molten bacillus of enzyme (Lysobacteryananisissp.nov), bacillus amyloliquefaciens (Bacillusamyloliquefaciens), Paenibacillus polymyxa (Paenibacilluspolymyxa) three strain antibacterial OD600During for equal 0.150, in inoculum concentration ratio bacillus amyloliquefaciens: produce the molten bacillus of enzyme: Paenibacillus polymyxa is 1: 2: 6 inoculation, cultivates 48h for 37 DEG C；
Configuration PDA solid medium, weigh 200 parts of the fresh Rhizoma Solani tuber osi of peeling, it is cut into small pieces, it is placed in beaker, add 500 parts of distilled water heating to smashing potato block to pieces with Glass rod, by potato block with distilled water mixture by 12 layers of filtered through gauze, adding 20 parts of glucoses, 18 parts of agar powders and 700 parts of deionized waters in filtrate, heating is to fully dissolving。Sterilizing, is down flat plate, punches (every part of culture medium makes a call to 5 holes, one, culture medium central authorities, is uniformly distributed four from centre bore 1.2-1.8cm place around culture medium) with card punch after culture medium solidifying in culture medium。Then by the tomato gray mould pure culture biscuits involvng inoculation beaten with same size card punch in the hole of culture medium central authorities, mixed vaccine, bacillus amyloliquefaciens, the molten bacillus of product enzyme, Paenibacillus polymyxa being pipetted 200uL respectively and connect in four holes being beneficial to around the culture medium of tomato gray mould counterclockwise after 30 DEG C of cultivation 24h, cultivation limit, 37 DEG C of limits is observed。
By Fig. 1 bacillus amyloliquefaciens, produce the molten bacillus of enzyme, the antibacterial efficiency of tomato gray mould is found out by Paenibacillus polymyxa and mixed vaccine, mixed vaccine to the antibacterial efficiency of graw mold of tomato pathogen more than bacillus amyloliquefaciens, produce the molten bacillus of enzyme, Paenibacillus polymyxa antibacterial efficiency to graw mold of tomato pathogen respectively。
Embodiment 6 mix bacterium agent suppresses the test of banded sclerotial blight pathogen
The method of tomato gray mould cause of disease bacterium test is suppressed to suppress stricture of vagina withered according to mix bacterium agent。By Fig. 2 bacillus amyloliquefaciens, produce the molten bacillus of enzyme, Paenibacillus polymyxa and mixed vaccine to the withered antibacterial efficiency of stricture of vagina it can be seen that mixed vaccine to the antibacterial efficiency of banded sclerotial blight pathogen more than bacillus amyloliquefaciens, produce the molten bacillus of enzyme, Paenibacillus polymyxa antibacterial efficiency to banded sclerotial blight pathogen respectively。
Embodiment 7 mix bacterium agent suppresses the test of spiral disease pathogen
The method suppressing the test of tomato gray mould cause of disease bacterium according to mix bacterium agent suppresses screw thread。By Fig. 3 bacillus amyloliquefaciens, produce the molten bacillus of enzyme, Paenibacillus polymyxa and mixed vaccine to the antibacterial efficiency of screw thread it can be seen that mixed vaccine to the antibacterial efficiency of spiral disease pathogen more than bacillus amyloliquefaciens, produce the molten bacillus of enzyme, Paenibacillus polymyxa antibacterial efficiency to spiral disease pathogen respectively。
Embodiment 8 mix bacterium agent suppresses Pathogens Causing Root Rot Disease test
The method suppressing the test of tomato gray mould cause of disease bacterium according to mix bacterium agent suppresses root-rot。Fig. 4 bacillus amyloliquefaciens, produce the molten bacillus of enzyme, the antibacterial efficiency of root-rot is illustrated by Paenibacillus polymyxa and mixed vaccine, mixed vaccine to the antibacterial efficiency of root-rot more than bacillus amyloliquefaciens, the product molten bacillus of enzyme, Paenibacillus polymyxa antibacterial efficiency to Pathogens Causing Root Rot Disease respectively。
The above; it is only the present invention preferably detailed description of the invention; protection scope of the present invention is not limited to this; any those familiar with the art is in the technical scope of present disclosure, and the simple change of the technical scheme that can become apparent to or equivalence are replaced and each fallen within protection scope of the present invention。
Claims (3)
1. a biofungicide, it is characterized in that the active component of this antibacterial by 3 kinds from different medicinal plants: Radix Polygalae, threeleaf akebia, Radix Salviae Miltiorrhizae endogenetic bacteria form in inoculum concentration ratio 1: 2: 6,3 kinds of antibacterials respectively produce the molten bacillus of enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa, this antibacterial also includes 40-50 part filler, 1-10 part surfactant, 1-10 part stabilizer, filler is white carbon, surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, and stabilizer is KH2PO4；
Its preparation method, comprises the following steps:
(1) the described molten bacillus of product enzyme, bacillus amyloliquefaciens, Paenibacillus polymyxa being activated with LB fluid medium respectively, then at 37 DEG C, 180r/min, PH ferments when being 7.4；
(2) as the described three kinds of ferment product OD of step (1)600When being 0.150, inoculum concentration ratio is by bacillus amyloliquefaciens: produce the molten bacillus of enzyme: Paenibacillus polymyxa is 1: 2: 6 inoculation, at 37 DEG C, and 180r/min, mixed fermentation when PH is 7.4；
(3) after 48h, 1-10 part surfactant, 1-10 part stabilizer, 40-50 part filler it is sequentially added in the fermentation liquid that step (2) obtains, filler is white carbon, and surfactant chooses dodecyl sodium sulfate and sodium lignin sulfonate, and stabilizer is KH2PO4, it is sufficiently mixed；
(4) being pulverized by the mixture of step (3) gained, then sieve, aperture is 200 orders, and granularity is 75um；
(5) check the fungistatic effect of the powder obtained that sieves, if antibacterial efficiency is more than 60%, then pack on request。
2. biofungicide according to claim 1, it is characterized in that, the culture medium adopted in preparation process is LB fluid medium, by weight, deionized water 950 parts, tryptone 10 parts, yeast extract 5 parts, NaCl10 part, shake container, until solute dissolves, adjusts pH to 7.4 with 5mol/LNaOH。
3. biofungicide according to claim 1, it is characterised in that when inoculum concentration ratio bacillus amyloliquefaciens: produce the molten bacillus of enzyme: when Paenibacillus polymyxa is 1: 2: 6, after 37 DEG C of fermentation 48h, three kinds of number of bacteria are consistent。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106793781B (en) | 2014-05-28 | 2020-07-28 | 拜耳作物科学有限合伙公司 | Compositions and methods for controlling fungal and bacterial diseases in plants |
| CN104388351B (en) * | 2014-11-24 | 2018-02-16 | 福州科力恩生物科技有限公司 | A kind of natural preparation method without colour biological fungus-proof antisepsis microbial inoculum |
| CN105831175B (en) * | 2016-04-29 | 2018-03-20 | 鞍山禾瑞生物科技有限公司 | A kind of liquid compound bio controls bactericide and preparation method and application more |
| CN107347919A (en) * | 2017-08-14 | 2017-11-17 | 南阳市农业科学院 | A kind of composite bactericide for preventing and treating wheat scab |
| WO2019222253A1 (en) * | 2018-05-17 | 2019-11-21 | Bayer Cropscience Lp | Stabilized fungicidal composition |
| CN110669811A (en) * | 2019-10-21 | 2020-01-10 | 天津大学 | Method for improving surfactant yield |
| CN117487706B (en) * | 2023-11-02 | 2024-06-04 | 南京农业大学 | Antagonistic bacteria for preventing and controlling root rot of radix angelicae and biological organic fertilizer |
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