CN101444216B - Application of toyocamycin in preparing plant blight control medicines - Google Patents
Application of toyocamycin in preparing plant blight control medicines Download PDFInfo
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- CN101444216B CN101444216B CN2008101897381A CN200810189738A CN101444216B CN 101444216 B CN101444216 B CN 101444216B CN 2008101897381 A CN2008101897381 A CN 2008101897381A CN 200810189738 A CN200810189738 A CN 200810189738A CN 101444216 B CN101444216 B CN 101444216B
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Abstract
The invention discloses a novel usage of a toyocamycin in medicine, namely the application of toyocamycin in preparing plant blight control medicines. A test validates the toyocamycin has better effect for restraining phytophthora fungi and can be broadly applied to the medicines for controlling plant blights, has excellent effect for controlling the plant blight and can not cause pollution to the environment when being used for a long period of time.
Description
Technical field
The invention belongs to biological pesticide technical field, relate to the new medicine use of toyokamycin, specifically is to utilize the nucleoside compound toyokamycin and produce the new purposes of microorganism aspect preparation control vegetable plague medicine.
Background technology
Vegetable plague is by Oomycete, the disease that Phytophthora (Phytophthora) fungus causes, and the Phytophthora fungus that the whole world has been reported has (the Ho of kind more than 80; 1990), the mould host range of epidemic disease is wide, and host plant reaches the (Gregory of kind more than 1000; 1983); Can cause that like camphor tree epidemic disease mould (P.cinnamomi) 900 various plants root-rot rot (Zentmyer, 1980), palm mould (P.palmivora) and P.megakarya are the important pathogen of tropical crop; Cause that cocoa leaf is withered, the stem root-rot mashed and ulcer (Appiah, 1999).Phytophthora capsici (P.capsici), Phytophthora nicotianae (P.nicotianae) are the important pathogen of tropical crops such as rubber, pepper, pineapple, sisal hemp and fruits and vegetables, flowers, forest; Cause the rot of root rot, seedling damping off and basal part of stem, stem tuber, bulb, leaf etc., all cause serious economy loss every year.To this type disease; Though inhale the control that chemical bactericide can be used for eqpidemic disease in existing metalaxyl series, white urea cyanogen series etc. are efficient; But control efficiency is not very desirable; Add long-term use chemical pesticide and not only environment has been caused very big pollution, and phytophthora also is prone to develop immunity to drugs to more used systemic fungicides.
Biological control is considered to Sustainable Control soil and passes the effective way that the phytophthora disease takes place; And can not cause environmental pollution; Has very big development potentiality; Therefore, the novel agricultural antibiotic or the biological and ecological methods to prevent plant disease, pests, and erosion active bacteria formulation of research and development broad-spectrum high efficacy are in China's agricultural production and even all have crucial meaning in the world.
The initial report of toyokamycin be by rich add streptomycete (Streptomyces toyocaensis278) produce (Intern.J.Syst.Bacteriol., 1968,18:174-176), have various active such as antitumor, antibiotic and weeding.0.018 μ g/mL toyokamycin can suppress the HeLa cell, to leukemic lymphoblastoid, zooperies such as L-1210, EC are ([P] .GB764198,1956,12.) effectively; To Candida albicans, aspergillus fumigatus, staphylococcus aureus drug-fast bacteria and enterococcus also have inhibitory action (Chinese antibiotic magazine, 2004,10:577), but do not see the report that is useful on vegetable plague control.The structure of toyokamycin is following:
Summary of the invention
A kind of new medicinal usage that the purpose of this invention is to provide toyokamycin, i.e. the application of toyokamycin on preparation vegetable plague control medicine.
Toyokamycin of the present invention has multiple preparation method, belongs to already known processes and mature technology, and multiple conventional separation means capable of using is separated acquisition or obtained through synthetic and semi-synthetic means from microorganism.
Toyokamycin of the present invention is a kind of active constituents of medicine; Preparation process according to routine; Can be active component with the toyokamycin, add conventional excipient substance, process any fit for service formulation; Like aqua, wetting powder, granula etc., be used for that plant Phytophthora (Phytophthora) fungus causes etc. the control of various plants eqpidemic disease.
Because the present invention discloses the drug effect of toyokamycin control vegetable plague first, therefore, become component or auxiliary material cooperation to make medicament separately or with other activity toyokamycin, as long as this medicine is used to prevent and treat vegetable plague, all belong to protection scope of the present invention.
Correspondingly; According to the mechanism of toyokamycin on the control vegetable plague; The microorganism that it produced also possesses the drug effect of control vegetable plague, is that active component is prepared into the control that agriculture medicine is used for vegetable plague with the microorganism that toyokamycin was produced, and also belongs to protection scope of the present invention.Microorganism that toyokamycin produces be white light gray streptomycete (Streptomycesalbogriseus) BM10, in vain sting streptomycete (Streptomyces albospinus) WZ254 bacterial strain, richly add streptomycete (Streptomyces toyocaensis) 278 bacterial strains, streptomyces rimosus Streptomyces rimosus (ATCC 14673), richly add streptomycete and do not have spinamycin mutation streptomycetes such as (Streptomyces toyocaensis var.aspiculamyceticus) or other microorganism.
In order to understand the new medicinal usage of toyokamycin of the present invention better; From the zymotic fluid of the white light gray streptomycete BM10 that phytophthora blight of pepper had strong bacteriostasis and Bai Ci streptomycete (Streptomyces albospinus) WZ254, isolate nucleoside compound-toyokamycin with the inventor below; Carried out that the antagonism phytophthora blight of pepper suppresses determination of activity and to the field control effect test of capsicum epidemic disease, explain that toyokamycin is in the new purposes of preventing and treating aspect the vegetable plague.
1, filter paper face-off cultivation is measured the inhibition activity of nucleoside compound SR-1 (toyokamycin) to phytophthora
With SR-1 (toyokamycin) sample ligand process 0.01g/ml testing sample, establish methanol solvate control group and aqua sterilisa control group, get the bacterium cake cultivating to beat on 4 days Phytophthora capsici and the Phytophthora nicotianae culture plate respectively with diameter 6mm card punch, transfer in the middle of the PDA culture plate; Placing the filter paper dick (diameter 6mm) of sterilizing in 28 ℃ of cultivations apart from colony edge 2.0cm place after 24 hours; Every ware is placed 3, in the scraps of paper, adds the SR-1 of 20 μ L water, methanol solvate and 0.01g/ml respectively, and each handles repetition 3 times; After treating that liquid on the scraps of paper volatilizes; Cover the ware lid, behind 28 ℃ of cultivation 5d, measure its antibacterial distance.Result of the test (table 1) shows that SR-1 (toyokamycin) has strong inhibitory activity to Phytophthora capsici and Phytophthora nicotianae, and its average antibacterial distance is respectively 18mm and 17mm, and contrast aqua sterilisa and methanol solvate then do not have bacteriostatic activity.
Table 1 compound S R-1 (Feng Jiahuo is plain) measures the result to the active filter paper method of the inhibition of phytophthora
2, suppress the mycelial growth rate method and measure the inhibition activity of nucleoside compound SR-1 (toyokamycin) phytophthora
Compound S R-1 (toyokamycin) is diluted to finite concentration, joins in the PDA medium that is cooled to about 50 ℃, process and contain the PDA culture medium flat plate that SR-1 concentration is 0.01mg/mL; With the PDA flat board that does not add any medicine is contrast; At Phytophthora capsici and the Phytophthora nicotianae bacterium piece of the access diameter 6mm of the dull and stereotyped central authorities of the PDA for preparing, every processing repetition 3 times was cultivated 5 days down in 28 ℃; Measure the phytophthora colony diameter with the right-angled intersection method, calculate mycelial growth inhibition rate by following formula.
Result of the test (table 2) shows that compound S R-1 (toyokamycin) has suppressed the mycelial growth of phytophthora blight of pepper and Phytophthora nicotianae bacterium fully under test concentrations 0.01mg/mL.
Table 2 compound S R-1 (toyokamycin) suppresses the mensuration result of phytophthora mycelial growth
3, toyokamycin is to the field control effect test of capsicum epidemic disease
This test is carried out in China tropical agricultural biotechnologies research institute proving ground, and test method is with reference to " pesticide field efficacy medicine test criterion ", and 4 processing are established in test: handle one, spray BM10 bacterial strain fermentation liquor supernatant 1800mL; Handle two, spray and concentrate 10 times BM10 strain fermentation supernatant 18001mL; Handle three, spray 600 times of liquid 1800mL of 72% metalaxyl-mn-zn (production of Dalian Rui Kede bio tech ltd); (4) contrast, spray clear water 1800mL.Every processing repetition 3 times, each sub-district area 6m
2, district's group is arranged at random, 45 strains of plantation capsicum seedling.One week of capsicum seedling field planting; Connecing phytophthora blight of pepper in the pepper seedling base portion (cultivated 4 days down for 28 ℃ with the V8 medium; Cut and inoculate after bacterium piece water planting waited to produce a large amount of sporangiums in 24 hours), treated to begin dispenser after the slight morbidity of part capsicum seedling, whenever sprayed once at a distance from 7 days; Spray the one all " Invest, Then Investigate " control efficiency of dispenser for the third time continuously three times.Morbidity percentage and control efficiency are calculated by following formula respectively:
Morbidity percentage and control efficiency percentage carry out variance analysis and Duncan ' s multiple ratio after the square root arcsine is changed.Streptomycete BM10 bacterial strain fermentation liquor concentrates the average capsicum epidemic disease incidence of disease, the average control efficiency statistic analysis result of four processing such as 10 * liquid, fermenation raw liquid, 600 * 72% metalaxyl and contrast and sees table 3.
The field test results of table 3 toyokamycin control capsicum epidemic disease
A, b is illustrated in the significance of difference level of P＞0.05; A, B, C is illustrated in the significance of difference level of P＞0.01
Can know from the statistic analysis result of managing the incidence of disease and control efficiency everywhere; Spray medicine for the third time after 7 days; The incidence of disease that the BM10 fermented liquid supernatant concentrates 10 times of liquid, BM10 fermenation raw liquid, 600 times of processing such as liquid of metalaxyl-mn-zn of 72% is respectively 2.3%, 3.7%, 4.44%; Control efficiency is respectively 82.61%, 78.9%, the incidence of disease of 73.68%, three kind of chemicals treatment all extremely significantly is lower than between 17.8%, three kind of chemicals treatment of the check plot incidence of disease the incidence of disease and control efficiency difference are not remarkable; Spray medicine for the third time after 14 days; The incidence of disease that the BM10 fermented liquid supernatant concentrates 10 times of liquid, BM10 fermenation raw liquid, 600 times of processing such as liquid of metalaxyl-mn-zn of 72% is respectively 3.7%, 5.2%, 9.6%; Control efficiency is respectively 88.02%, 82.5%, 68.49%; The incidence of disease that the BM10 fermented liquid supernatant concentrates 10 times of liquid and the processing of BM10 fermenation raw liquid all extremely significantly is lower than the contrast incidence of disease 30.4%; The incidence of disease that 600 times of liquid of 72% metalaxyl-mn-zn are handled does not reach significance level with contrast incidence of disease difference; The control efficiency of the concentrated 10 times of liquid of BM10 fermented liquid supernatant is significantly higher than 600 times of liquid processing of metalaxyl-mn-zn of 72%, and it is not remarkable that fermenation raw liquid is handled the control efficiency difference of handling with 600 times of liquid of metalaxyl-mn-zn of 72%; Spray medicine for the third time after 21 days; The incidence of disease that the BM10 fermented liquid supernatant concentrates 10 times of liquid, BM10 fermenation raw liquid, 600 times of processing such as liquid of metalaxyl-mn-zn of 72% is respectively 5.19%, 8.1%, 18.52%; Control efficiency is respectively 89.96%, 84.4%, 64.35%; The BM10 fermented liquid supernatant concentrates the incidence of disease that 10 times of liquid are handled and the fermenation raw liquid supernatant is handled and extremely significantly is lower than the incidence of disease 51.9% that 600 times of liquid of 72% metalaxyl-mn-zn are handled and contrasted; The incidence of disease difference that the BM10 fermented liquid supernatant concentrates 10 times of liquid and fermenation raw liquid processing is not remarkable; The control efficiency that the concentrated 10 times of liquid of BM10 fermented liquid supernatant are handled and the fermenation raw liquid supernatant is handled all utmost point is significantly higher than the control efficiency that 600 times of liquid of 72% metalaxyl-mn-zn are handled, and the control efficiency difference that concentrated 10 times of liquid processing of BM10 fermented liquid supernatant and fermenation raw liquid supernatant are handled is not remarkable.
Above test data analyzer result shows; Spray concentrated 10 times of liquid of BM10 fermented liquid supernatant and BM10 fermented liquid supernatant liquid and all capsicum epidemic disease is had good control efficiency; Control efficiency reaches more than 82%; Wherein fermented liquid supernatant concentrates the preventive effect that 10 times of liquid are significantly higher than 600 times of liquid of 72% metalaxyl-mn-zn, and the control efficiency of BM10 fermented liquid supernatant liquid is then worked as with 600 times of liquid phases of 72% metalaxyl-mn-zn.
The invention discloses the new medicine use of toyokamycin; The effect that has better inhibited Phytophthora fungus through the experimental verification toyokamycin; Can be widely used in preventing and treating in the medicine of vegetable plague; Control for vegetable plague has good effect, and long-term use can not cause very big pollution to environment.
Embodiment
Below in conjunction with instantiation the present invention is done further elaboration, but do not limit the present invention.The extraction of embodiment one nucleoside compound SR-1 (toyokamycin) separates
A, inclined-plane and plating medium: potato 200g, glucose 20g, CaCO
30.2g, MgSO
47H
2O 0.2g, agar 20.0g, water 1000mL, pH value 7.0; Soybean powder medium: analysis for soybean powder 10g, glucose 10g, CaCO
30.2g, MgSO
47H
2O 0.2g, water 1000mL, pH value 7.0.
B, bacterial strain BM10 adopt second order fermentation; At first bacterial classification BM10 is transferred on plating medium; In 28 ℃ cultivate 4～6d after, be inoculated in seed culture medium (soybean powder medium), on 28 ℃, 200r/min shaking table, cultivate 3d; Transfer in fermentation medium (with the seed medium), on 28 ℃, 200r/min shaking table, cultivate 5～6d.
C, zymotic fluid be through the centrifugal 20min of 8000r/min, and supernatant is evaporated to 1/50 of original volume in 45 ℃, and with ethyl acetate extraction three times, concentrating under reduced pressure gets ethyl acetate medicinal extract (17.3g).Ethyl acetate medicinal extract (17.3g) is through silica gel column chromatography; Obtain 8 streams part (Fr.1-8) with the chloroform-methanol gradient elution; Each component is carried out active testing through above-mentioned activity test method, confirms that component Fr.4 is an active component, and Fr.4 obtains SR-1 (3.0g) single-activity component through repeatedly being recrystallized.
The structure of embodiment dinucleotide compounds SR-1 (toyokamycin) compound is identified
Utilize spectral technique, comprise ultraviolet, infrared, nuclear magnetic resonnance and high resolution mass spectrum analysis, identified the structure of the SR-1 compound among the embodiment 1.Utilization
13C-NMR,
1The H-NMR data belong to.
The physicochemical constant of compound S R-1 (toyokamycin): white, needle-shaped crystals, potassium permanganate show light yellow,
In ESI-MS (m/z), 292 [M+H] appear
+, 314 [M+Na]
+The peak, molecular formula: C
12H
13N
5O
4, molecular weight: 291.
Nucleoside compound SR-1's (toyokamycin)
13C-NMR with
1The H-NMR data:
13C-NMR(100MHz，DMSO-d
6)δ156.9(C-4)，153.4(C-2)，150.0(C-8)，132.3(C-6)，115.2(CN)，101.2(C-9)，87.7(C-1′)，85.4(C-4′)，82.9(C-5)，74.1(C-2′)，70.1(C-3′)，61.1(C-5′)；(400MHz，DMSO-d
6)δ8.22(1H，s)，8.44(1H，s)，6.91(2H，s，4-NH
2)，6.06(1H，d，J＝5.6Hz)，5.46(1H，d，J＝6.0Hz)，4.37(1H，dd，J＝11.0，5.5Hz)，5.20(2H，m)，3.93(1H，dd，J＝7.2，3.6Hz)，4.09(1H，dd，J＝9.0，4.5Hz)，3.61(2H，m)。
Claims (2)
1. the application of toyokamycin on preparation vegetable plague control medicine.
2. application according to claim 1 is characterized in that: toyokamycin is cooperated the making medicament separately or with other active component or auxiliary material.
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| CN101961013B (en) * | 2010-03-19 | 2013-01-16 | 中国计量学院 | Application of toyocamycin in controlling tomato gray mold |
| CN101961015B (en) * | 2010-03-19 | 2013-04-24 | 中国计量学院 | Application of toyocamycin in preventing and treating tomato early blight |
| CN101940211B (en) * | 2010-03-19 | 2013-04-24 | 中国计量学院 | Application of toyocamycin in preventing and curing cucumber wilt |
| CN102027948B (en) * | 2010-11-02 | 2012-12-26 | 中国计量学院 | Application of toyocamycin in prevention and control of cucumber rhizoctonia rot |
| CN102329756B (en) * | 2011-09-28 | 2014-05-07 | 中国热带农业科学院热带生物技术研究所 | Streptomyces albospinus strain BWL15-4 for preventing and treating banana vascular wilt and application thereof |
| CN103233018B (en) * | 2013-05-08 | 2014-06-25 | 中国计量学院 | Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application |
| CN103320372B (en) * | 2013-05-08 | 2014-07-23 | 中国计量学院 | Recombinant streptomyces diastatochromogenes with reinforced toyF expression, construction method and uses thereof |
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Non-Patent Citations (2)
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| Yasuharu Sekizawa等.怎样发现新除草抗生素.《世界农药》.1984,(第04期),全文. * |
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