CN101695306B - Compound bactericide of active crude extract of baumannii bacteria liquid and tebuconazole and application thereof - Google Patents
Compound bactericide of active crude extract of baumannii bacteria liquid and tebuconazole and application thereof Download PDFInfo
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- CN101695306B CN101695306B CN2009100358491A CN200910035849A CN101695306B CN 101695306 B CN101695306 B CN 101695306B CN 2009100358491 A CN2009100358491 A CN 2009100358491A CN 200910035849 A CN200910035849 A CN 200910035849A CN 101695306 B CN101695306 B CN 101695306B
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Abstract
The invention relates to a compound bactericide of an active crude extract of baumannii bacteria liquid and tebuconazole. A finished medicament of the bactericide comprises the following components: an effective active component, an assistant and a solvent, wherein the effective active component is 1-50 wt% of the finished medicament; and the effective active component comprises 25-75 wt% of the active crude extract of the baumannii bacteria liquid and the tebuconazole. The invention has the advantages that: by compounding the active crude extract of the baumannii bacteria liquid and the tebuconazole, the evident synergy is achieved, the bactericidal activity is greatly improved, and the germ drug resistance is reduced, in comparison with the current use of single bactericide; by compounding plant disease biological control bacteria and chemical pesticide, the dose of the chemical pesticide is greatly reduced; and particularly, the active crude extract of the baumannii bacteria liquid can be degraded easily in the environment, thereby not only reducing the use cost of the chemical pesticide, but also lessening the residual of the chemical pesticide and environmental pollution caused by the residual.
Description
One, technical field:
The invention belongs to a kind of biological metabolite-chemical synergic pear tree ring spot bactericide agricultural chemicals, the composite bactericide that to relate to a kind of effective active composition be Bao Man rhzomorph and Tebuconazole.
Background technology:
Black rot of pear (Botryosphaeria berengeriana de.Not.f.sp.Piricola (Nose) Koganecawa et.Sokwwwa) also claims scurf, knurl skin disease, the mashed disease of water etc., is one of important disease of China pear tree.In recent years, along with the change of main breed, of fine quality but area under cultivation susceptible variety constantly enlarges, and the generation and the harm of this disease are on the rise, and production has caused very big loss to pears, has had a strong impact on quality and the output of the operatic circle.China causes the pear fruit underproduction about 25% because of black rot of pear harm every year, the decayed fruit rate is up to more than 80% when serious, and pear fruit, blade and the limb of should disease not only infecting field growing, also can cause and adopt the back fruit rot, become one of field and important disease of fruit storage phase.Current pear tree ring spot is still based on chemical control, but along with being on the rise of problems such as " pesticide hazards ", " pesticide resistance ", and the applying of biological control technology, and people also deeply expand to biological control the research field of disease control.
Endophyte of plant (Endophyte) is meant finishes the part or all of of its history of life in plant corpus, grow in the iuntercellular of plant tissue, be distributed in leaf sheath, seed, flower, stem, blade and the root, but the microorganism that does not cause any illness.Endophyte of plant is the very abundant biological group of a species diversity, and it is wide to distribute, and kind is many, has extremely abundant bio-diversity.Endophyte of plant comprises endogenetic bacteria, interior living actinomycetes and endogenetic fungus.From the later stage seventies 20th century, endophyte is at some important commodity trees, as being found and having obtained extensive studies and application in succession in plant bark, the branches and leaves such as dragon spruce, Chinese larch, Japanese yew, eucalyptus, robur.In plants such as multiple shrub, herbaceous plant and raise crop, also found endophyte in succession.As from crops such as cotton, tomato, beet, eggplant, melon, potato, being separated to Pseudomonas, Serratia, Alcaligenes, Shigella, Citrobacter, Herbaspedicae, Clavibacter, Aumobacterium, Hydrogenophaga, more than 50 endogenetic bacterias that belong to such as Flavimonas.It is various wherein to separate the antibacterial substance structure type that obtains from endophyte of plant, mainly contain compounds such as terpene, alkaloids, aromatics, peptide class, having stronger antibacterial, bactericidal activity, is the new resources of clinical antibacterials and biopesticide research and development.
Bao Man rhzomorph (Acinetobacter baumannii) is the secondary metabolites that Nanjing University separates from medicinal plant camphor tree endogenetic bacteria, this Bao Man bacterial strain is LCH0606, it is a kind of plant biological control strain, and openly (preserving number is: CGMCC No.1752) by the CN1973631 patent documentation.The Bauman bacteria solution secondary metabolites that adopts the centrifugal back of this bacterial strain to obtain has the better prevention effect to diseases such as wheat scabs.Antibiotic kind and active substance quantitative analysis at the Bauman bacteria solution generation, obtain 0.5g/L Bao Man bacterium active coarse extractum through centrifugal, macroporous resin adsorption after concentrating with the 13L Bauman bacteria solution, silica gel column chromatography, the sephadex Sephadex-LH20 antibacterial substance in the purifying Bauman bacteria solution repeatedly again, and through analyses such as nuclear-magnetism, mass spectrums, the antibiotic main component that draws the Bao Man rhzomorph is a lipopeptid class material, its active component is Iturin A1, iturin A2, iturin A3 and Iturin A6, three's content ratio is 1: 1: 1, and the reactive compound amount is 3mg/L.LCH0606,37 ℃ of bacterium colonies after cultivating 24h on the PSA flat board present rule circle, canescence, surperficial indentation, opaque, dry easy picking, inviscid, the edge is smooth.To this bacterium microscopic dyeing observed result is rod-short, no gemma, atrichia, and about 1.0～1.5 μ m of diameter remove from office blue Albert'stain Albert and are negative.Neutral and alkalescence and＜relatively stable under 100 ℃ of conditions, antibacterial activity can keep more than 80%; The antibacterial activity of Bao Man rhzomorph easily is damaged under acid condition.Add Ca
2+Can significantly improve antibacterial activity.Be easy to become solution in water.Pathogen shows as spore and does not sprout after treatment, and mycelia is short and small, cell rupture and inclusion flows out.So LCH0606 is that a strain has the agricultural biocontrol microorganisms than the great development potentiality.
Tebuconazole (tebuconazole) chemical name: (1-(4-chlorphenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-yl methyl) penta-3-alcohol) is a kind of ethoxy triazole derivative.Physicochemical character: clear crystal; 102.4 ℃ of fusing points; Vapour pressure 0.013mPa (20 ℃); Dissolubility (20 ℃), water 32mg/L (the pH value is 7); Carrene＞200g/L, hexane＜0.1g/L, isopropyl alcohol, toluene 50～100g/L.At 4～9,22 ℃ of hydrolysis DT of pH value
50＞1 year.Rat acute per os LD
504000mg/kg.Toxicity category belongs to the low toxicity bactericide.This change platform thing is succeeded in developing in 1986 at first by Bayer A.G.At one of wide-spectrum bactericide of China exploitation, inhale therapeutic action in having in recent years, and partially absorbing and pass different to the top by plant growing power, suppress germ wheat sterol demethylation, promote plant growth, make it well developed root system, the leaf look dark green, robust plant, thus improve output.All can prevent and treat effectively Erysiphe, Puccinia, Rhynchosporium spp, nuclear cavity Pseudomonas and the microbial disease of Septoria, but lack the prevention protective effect, increase agriculture use cost.
Produce at present and go up the main chemical control black rot of pear that adopts, serious environment pollution so not only, also easily cause simultaneously the pathogen pesticide resistance to produce, especially the appearance of resistance or pesticide resistance pathogen makes people constantly rise to the various demands that can effectively suppress or kill the medicine of all kinds of pathogenic bacteria.Biopesticide is not only good to person poultry safety, environment compatibility, and damage by disease and insect is difficult for developing immunity to drugs, and has become one of focus of current main research.But biopesticide also comes with some shortcomings, as: field control effect instability, drug action slowly, problem such as the pesticide-germicide spectrum is narrow.Thereby improve preventive effect and expanding prevention object, slow down disease drug resistance, reduce the best approach of polluting, reducing residue of pesticide with biological combining with chemical pesticide, present, do not appear in the newspapers as yet at the composite black rot of pear of preventing and treating of Bao Man rhzomorph and Tebuconazole.
Two, summary of the invention:
The object of the invention: the objective of the invention is to use problems such as disease drug resistance and cost height, provide a kind of by Bao Man rhzomorph and the composite composite bactericide of Tebuconazole at the agent of Tebuconazole list.
The present invention realizes like this: a kind of Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide, comprise effective active component, auxiliary agent and solvent in the finished product medicament, it is characterized in that: the percentage by weight that the effective active composition accounts for the finished product medicament is 1～50%, described effective active composition is made up of Bauman bacteria solution active coarse extractum and Tebuconazole, in the effective active composition, the percentage by weight of Bauman bacteria solution active coarse extractum is: 25-75%.
In the effective active composition of the present invention, the percentage by weight of Bauman bacteria solution active coarse extractum is: 50-70%.
Auxiliary agent and the shared percentage by weight of solvent are respectively 15-40% and 10～60% in the finished product medicament of the present invention.
In the finished product medicament of the present invention, described auxiliary agent is conventional emulsifier and promoter, and described solvent is a water.
In the finished product medicament of the present invention, described auxiliary agent comprises emulsifier and promoter, also comprises thickener simultaneously, or defoamer, or antifreezing agent, or their combination.
The formulation of finished product medicament of the present invention is an aqueous suspension agent.
A kind of application of above-mentioned composite bactericide is characterized in that: be used to prevent and treat black rot of pear on the pear tree with being sprayed at behind the finished medicines dilution agent.
In the concrete application of composite bactericide: with 100～3500 times of finished medicines dilution agents, the content of dilution back active drug composition is at least 100mg/L.
In the concrete application of composite bactericide: the optimum content of dilution back active drug composition is 160～300mg/L
The invention has the advantages that: with Bauman bacteria solution active coarse extractum and Tebuconazole carry out composite after, compare with existing single agent use, has notable synergistic, improved bactericidal activity greatly, reduced the germ pesticide resistance, especially Bauman bacteria solution active coarse extractum more easily degraded in environment has not only reduced the chemical pesticide use cost, and has reduced the residual of chemical pesticide and to the pollution of environment.
Embodiment:
Embodiment 1:
(1) for examination germ and crop:
With the black rot of pear bacterium serves as for examination bacterium (plant protection research institute in Jiangsu Province Agriculture Science Institute provides), is " abundance of water " for examination pears kind.
(2) reagent agent:
Bauman bacteria solution is provided by Nanjing University, and 99% Bauman bacteria solution active coarse extractum is to obtain (self-control, every liter of Bauman bacteria solution can obtain 0.53g) by Bauman bacteria solution after concentrating through centrifugal, macroporous resin adsorption.99% Bauman bacteria solution active coarse extractum is soluble in water, and it is standby to make 1000 μ g/ml mother liquors; 95% Tebuconazole (Nanjing expects that Feng Huagong Co., Ltd provides) is dissolved in the ethanol, and it is standby to make 1000 μ g/ml mother liquors.
(3) the plate mycelial growth suppresses method
1. preliminary experiment
Single agent toxicity test test adopts the inhibition growth rate method to measure circle.Get 99% Bauman bacteria solution active coarse extractum of 1.01g and 95% Tebuconazole of 1.05g and be mixed with 10 with spilling essence
-2, 10
-3, 10
-4, 10
-5, 10
-6Deng 5 gradient concentrations, draw in the PSA medium of the molten 100ml of 1ml medicament at each concentration test tube.
2. composite test
On the basis of preliminary experiment, select each medicament to 5 concentration of germ mycelial growth inhibition rate in 10～90% scopes.Be single agent A (99% Bauman bacteria solution active coarse extractum) and single agent B (95% Tebuconazole), by the proportioning of carrying out in 2: 1,1: 2,3: 1,1: 3,1: 1,2: 3,3: 2.The reagent agent mother liquor for preparing being joined in the PSA medium about 50 ℃ according to the concentration ratio of setting, fully pour in the culture dish of 5 sterilizations behind the mixing, make the pastille PSA flat board of series concentration, is contrast to add ethanol PSA flat board.Be that the card punch of 5mm is beaten on the same circumference of pre-incubated ring spot colony edge and got the bacterium cake with diameter then, mycelia faces down and is inoculated into pastille culture medium flat plate central authorities, place and carry out dark culturing under 25 ± 1 ℃, colony edge to be contrasted is measured colony diameter that each is handled with the right-angled intersection method during near the ware wall, and obtain the mean value that repeats for 5 times, draw each average bacterium colony of handling and increase diameter (average colony diameter one 5mm), and calculate the mycelial growth inhibition rate (%) of each chemicals treatment germ.
Relative bacteriostasis rate=(contrast bacterium cake diameter one is handled bacterium cake diameter)/contrast bacterium cake diameter * 100
Concentration logarithm with setting in the test is abscissa (x), and the inhibiting rate value is ordinate (y), obtains each medicament to toxicity regression curvilinear equation y=a+bx, correlation coefficient r and concentration (EC in effective inhibition for the examination germ
50Value).
The synergy computational methods are with reference to agricultural chemicals indoor bioassay test rule: (NY/T 1156.6-2006) measured in the synergy of fungicide compounding.Result of the test sees Table 1.
Each medicament of table 1 is to the toxicity test of black rot of pear bacterium
Annotate: when SR＞1.5 expressions have synergistic effect; When SR＜0.5 expression has antagonism; When 0.5≤SR≤1.5 are summation action.
The compound synergic effect of bactericide uses synergy index SR to weigh usually, because the coefficient of synergism of mixture is between 0.5≤SR≤1.5, composite dose has addition, greater than 1.5, composite dose has synergistic effect, the proportioning that is A, B component is 1: 2,1: 3,2: 3 and 3: 1 o'clock, and composite dose has addition, when the proportioning of A, B component is that 2: 1,3: 2 and 1: 1 have synergistic effect for composite dose.
Embodiment 2:
The preparation of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent
The proportioning of A, B component is that 2: 1,3: 2 and 1: 1 o'clock its synergy index are respectively 1.53,1.76 and 1.63, and wherein the content with Tebuconazole in 2: 1 is minimum, so we select 2: 1 bases as present embodiment of best proportioning.
Auxiliary agent: emulsifier adopts peaceful breast 34
#Promoter is adopted the desugar calcium lignosulfonate; Thickener adopts xanthans and aluminium-magnesium silicate; Antifreezing agent adopts ethylene glycol; Defoamer adopts organic silicone.
Solvent: running water.
Each component is got the raw materials ready according to following percentage by weight:
The effective active composition:
Bauman bacteria solution active coarse extractum 26.7%, Tebuconazole 12.3% is equivalent to 40% of total amount;
Auxiliary agent:
Peaceful breast 34
#5.0%, desugar calcium lignosulfonate 10.0%, xanthans 0.5%, aluminium-magnesium silicate 3.6%, organic silicone 0.01%, ethylene glycol 5.0% is equivalent to 23.11% of total amount;
Solvent:
Surplus is a solvent, and solvent adopts running water, is equivalent to 36.89% of total amount.
Preparation process:
Adopt wet processing.With Bauman bacteria solution active coarse extractum, Tebuconazole, desugar calcium lignosulfonate and peaceful breast 34
#It is good to press the proportioning weighing, joins together with 70% of solvent (running water) total amount and carries out barreling in the sand milling still, after ground, add load weighted xanthans, aluminium-magnesium silicate, organic silicone and ethylene glycol, and residual solvent, after stirring, obtain white homogeneous phase suspending agent.
Physicochemical property
Measure the compound medicine dispersiveness with the graduated cylinder bulk method, under standard hard water condition, (the hard water compound method is with reference to agricultural chemicals emulsion-stabilizing assay method GB/T 1603-2001), 500～3000 times of liquid of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent dilution are observed suspensibilities all greater than 80%, and present embodiment can satisfy in the dilution ratio below 500～3000 times and uses.
Cold storage stability
Freeze-10 ± 2 ℃ of storage nothings down.
Heat storage stability
40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent is packed in the 50ml bottle, and sealing is preserved 14d in 54 ± 2 ℃ of insulating boxs, takes out cooling.In 24h, detect effective component content behind the leakage detection and meet standard-required.
Be categorized as low toxicity level in the agricultural chemicals hazard rating standard of present embodiment according to world health organisation recommendations.Rat acute per os LD
50Be respectively 4800mg/kg.To rabbit skin, eyes nonirritant.In test dose, there are not teratogenesis, carcinogenic, mutagenesis.
Embodiment 3:
Field control effectiveness test:
(1) medicament:
Tested agricultural chemicals:
Handle 1.: with 1500 times of dilutions (active drug composition 266mg/L) of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent (embodiment 2 provides);
Handle 2.: with 2000 times of dilutions (active drug composition 200mg/L) of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent (embodiment 2 provides);
Handle 3.: with 2500 times of dilutions (active drug composition 160mg/L) of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent (embodiment 2 provides);
The contrast agricultural chemicals:
Handle 4.: 3000 times of dilutions of 99% Bauman bacteria solution active coarse extractum (self-control) (active drug composition 330mg/L);
Handle 5.: 2000 times of liquid of 25% Tebuconazole SC (Zhangjiagang seven continent pesticide chemical Co., Ltds provide) (active drug composition 125mg/L);
Handle 6.: 1000 times of liquid of 80% carbendazol wettable powder (Zhenjiang Co., Ltd of insecticide factory provides) (active drug composition 800mg/L);
Blank
Handle 7.: clear water.
(2) test method:
Field control effectiveness test carries out in Jiangsu Province Agriculture Science Institute gardening research institute experimental field.Experimental cultivar is the abundance of water pears, life in 9 years, seeding row spacing 4 * 5m.The different medicaments of experimental evidence are established 7 processing altogether, and wherein three is tested agricultural chemicals group, and three are contrast agricultural chemicals group, and one is the blank group.Every processing repeats for 4 times, and every the repetition is a sub-district, and 2 trees in every sub-district amount to 28 sub-districts, and district's group is arranged at random.Every sub-district water consumption is 4kg, and spraying equipment is the sharp agricultural HD-400 type backpack high pressure manual sprayer of Singapore.Spray medicine first and thank the operatic circle and spend back 10d to carry out, later on according to rain day distribution situation every 20d spray 1 time, spray altogether 5 times.Efficacy survey carried out respectively at picking time and storage period.Picking time, every processing random searching 200 fruits write down total fruit number, sick fruit number.Gather the same day, worm is rejected in every processing and mechanical injury is really adorned basket, and every processing is no less than 100 fruits, puts storage 15d under the natural room temperature, the investigation drug effect.Calculate the preventive effect method with reference to national pesticide field efficacy medicine test criterion.Adopt the new multipole difference of Deng Kenshi Duncan method that test data is analyzed.(referring to table 2)
Table 2 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent is to black rot of pear control efficiency picking time
As shown in Table 2: the preventive effect of 15d pear fruit ring spot is respectively 89.3%, 85.1%, 82.3% behind the high, medium and low dosage medicine of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent; 3000 times of dilution preventive effects of contrast medicament 99% Bauman bacteria solution active coarse extractum are that 83.6%, 25% a Tebuconazole SC2000 times dilution preventive effect is 80.1%; 1000 times of dilution preventive effects of 80% carbendazol wettable powder are 72.2%.By variance analysis as can be known: there were significant differences for preventive effect between the high, medium and low dosage of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent, and high, middle dosage is significantly higher than contrast medicament (25% Tebuconazole SC2000 times dilution and 1000 times of dilutions of 80% carbendazol wettable powder).
Table 3 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent is to black rot of pear storage period control efficiency
As shown in Table 3: the preventive effect of pear fruit ring spot is respectively 87.2%, 82.2%, 75.9% during the high, medium and low dosage storage of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent 15d; 3000 times of dilution preventive effects of contrast medicament 99% Bauman bacteria solution active coarse extractum are that 2000 times of dilution preventive effects of 75.9%, 25% Tebuconazole SC are 73.1%; 1000 times of dilution preventive effects of 80% carbendazol wettable powder are 65.9%.By variance analysis table as can be known: there were significant differences for preventive effect between the high, medium and low dosage of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent, and high, middle dosage is significantly higher than contrast medicament (2000 times of liquid of 25% Tebuconazole SC and 1000 times of liquid of 80% carbendazol wettable powder).
Evaluating drug effect: composite dose of 1500～2500 times of liquid of 40% Bauman bacteria solution active coarse extractum Tebuconazole aqueous suspension agent all have the better prevention effect to black rot of pear, and field efficacy can reach more than 82%, and indoor preventive effect can reach more than 72%, and to pear tree growth safety.
Claims (9)
1. a Bauman bacteria solution (Acinetobacter baumannii) active coarse extractum Tebuconazole composite bactericide, comprise effective active component, auxiliary agent and solvent in the finished product medicament, it is characterized in that: the percentage by weight that the effective active composition accounts for the finished product medicament is 1～50%, described effective active composition is made up of Bauman bacteria solution active coarse extractum and Tebuconazole, in the effective active composition, the percentage by weight of Bauman bacteria solution active coarse extractum is: 25-75%;
Described Bauman bacteria solution active coarse extractum is the CE of Bao Man bacterial strain LCH0606, and it is to be obtained after concentrating through centrifugal, macroporous resin adsorption by Bauman bacteria solution, and containing the reactive compound amount is 3mg/L.
2. Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide according to claim 1 is characterized in that: in the effective active composition, the percentage by weight of Bauman bacteria solution active coarse extractum is: 50-70%.
3. Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide according to claim 1 and 2 is characterized in that: the shared percentage by weight of auxiliary agent and solvent is respectively 15-40% and 10～60% in the finished product medicament.
4. Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide according to claim 3 is characterized in that: described auxiliary agent is conventional emulsifier and promoter, and described solvent is a water.
5. Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide according to claim 3, it is characterized in that: described auxiliary agent comprises emulsifier and promoter, also comprises thickener simultaneously, or defoamer, or antifreezing agent, or their combination.
6. Bauman bacteria solution active coarse extractum Tebuconazole composite bactericide according to claim 1 is characterized in that: the formulation of described finished product medicament is an aqueous suspension agent.
7. the application as composite bactericide as described in claim 1 or 4 or 5 or 6 is characterized in that: be used to prevent and treat black rot of pear on the pear tree with being sprayed at behind the finished medicines dilution agent.
8. the application of composite bactericide according to claim 7 is characterized in that: with 100～3500 times of finished medicines dilution agents, the content of dilution back active drug composition is at least 100mg/L.
9. the application of composite bactericide according to claim 8 is characterized in that: the optimum content of dilution back active drug composition is 160～300mg/L.
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| CN105256061A (en) * | 2015-11-24 | 2016-01-20 | 江苏省农业科学院 | Primer pair and detection method for physalospora piricola virus detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5879682A (en) * | 1995-11-24 | 1999-03-09 | Peya Biotech Inc | Aframonum seeds for improving penile activity |
| CN1973631A (en) * | 2006-07-12 | 2007-06-06 | 南京大学 | Biocontrol fungus for preventing and controlling plant mycosis and its prepn process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5879682A (en) * | 1995-11-24 | 1999-03-09 | Peya Biotech Inc | Aframonum seeds for improving penile activity |
| CN1973631A (en) * | 2006-07-12 | 2007-06-06 | 南京大学 | Biocontrol fungus for preventing and controlling plant mycosis and its prepn process |
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