CN101720772A - Macrolide composition for preventing and controlling fungal disease of crop and preparation process thereof - Google Patents
Macrolide composition for preventing and controlling fungal disease of crop and preparation process thereof Download PDFInfo
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- CN101720772A CN101720772A CN200810233435A CN200810233435A CN101720772A CN 101720772 A CN101720772 A CN 101720772A CN 200810233435 A CN200810233435 A CN 200810233435A CN 200810233435 A CN200810233435 A CN 200810233435A CN 101720772 A CN101720772 A CN 101720772A
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Abstract
The invention provides an agricultural fungicide WYLZ-1349 taking azalomycin F3, azalomycin F4 and a nyphimycin composition as effective constituents, as well as preparation method and application thereof. The preparation method of the agricultural fungicide WYLZ-1349 comprises the following steps of: firstly culturing Streptomyces malaysiensis ECO 00002 with the preservation number of CGMCCNo. 1680; then separately extracting a fermentation culture; breaking the wall of mycelium with acetone or methanol and mixing with a fermentation supernate; and absorbing by macroporous absorption resin for separation; eluting by the acetone or the methanol; concentrating or further purifying an eluent so as to obtain the agricultural fungicide WYLZ-1349 or the azalomycin F3, the azalomycin F4 and nyphimycin; and then adding a carrier or an auxiliary agent so as to obtain a finished product. Test results show that the agricultural fungicide WYLZ-1349 has good effect especially in preventing and controlling rice blast, tobacco brown spot, tomato gray mold and pepper anthracnose and has small using amount, low using cost for peasants, wide usable range, and the like compared with the traditional medicament.
Description
Technical field
The invention belongs to pesticide production technology field, particularly relate to a kind of farm antibiotics preparation technology and purposes of preventing and treating crop fungal disease.
Background technology
Current, human more and more stricter to environment requirement, and the development difficulty of chemical pesticide is also increasing, success rate is more and more lower.Screening through decades has almost spreaded all over the imaginabale chemical constitution of people.For this reason, world industry developed country and the new exploitation point of each big numerous and confused searching of agricultural chemicals company, also in order to seek new lead compound, the research and development of agricultural chemicals have entered the epoch of back to nature simultaneously, promptly seek developable agricultural chemicals or lead compound from natural products.Because natural products passes through the evolution of long-term and bio-molecular interaction and selects at aspects such as molecular size and character, thereby has good quasi-medicated property; In addition, as the part of the ecosystem, natural products possesses a kind of special advantage: the adaptability of environment and compatibility.How from natural products, to seek novel pesticide compound or lead compound, become one of theme of current pesticide developing.
Studies show that in a large number, chemical constitution by biosynthetic microorganism secondary metabolite is extremely complicated and diversified, they not only act on target organisms with the mechanism of action of uniqueness, and has an inherent biodegradability, the clear superiority of low-residual, the side effect to non-target organism when effectively preventing and treating crop pest is less.Therefore can from the microorganism secondary metabolite, develop bactericide efficiently, to overcome resistance and the pollution problem that causes by many old kind chemosynthesis bactericide.Successfully develop at present both at home and abroad the disinfectant use in agriculture of various microbial sources, blasticidin-S S for example, kasugarnycin, polyoxin, valida, midolthromycin, pyrrolnitrin and strobilurins etc. are used to prevent and treat fungal diseases of plants.In addition, the antimycotic material of microbial source can also be as the lead compound of synthetic high activity analog, for example respectively with pyrrolnitrin and strobilurins as lead compound, synthesize active higher fenpiclonil and fluorine fludioxonil, and Fluoxastrobin and methyl kresoxim-methyl, and all successfully listings.Recently, constantly there is new Antibiotique composition from various microorganism secondary metabolites, to be separated to,, can be used for preventing and treating some crop pest as gopalamicin and thiobutacin etc.In addition, the native compound quantity of sustainable growth shows that the multifarious great potential of occurring in nature has also been excavated far away.
Actinomycetes are various antibiotic microorganisms of getting bumper crops most, it produces the unique and diversified antibiotic of structure as mechanisms of action such as aminoglycoside, anthracene nucleus, glycopeptide, beta-lactam, macrolide, nucleosides, peptide, polyenoid and polyethers, and the antibiotic of its generation accounts for more than 75% of natural antibiotics.Especially it should be noted that, the antibiotics that streptomycete produces aspect quantity and structure diversity all above actinomycetic other genus, in the antibiotic that particularly uses on agricultural, about 60% separates from streptomycete, shows that streptomycete has stronger antibiotics synthesis capability.The present invention formulates under this background.
Summary of the invention
1. goal of the invention: the invention provides a kind of active ingredient is the antifungal agricultural antibiotics of azalomycin F3, azalomycin F4 and nifimycin composition, called after WYLZ-1349, and a kind of fermentation from the microorganism of streptomyces is provided, separates, extract azalomycin F3, azalomycin F4 and nifimycin composition, add suitable stabilizing agent and auxiliary agent again and be prepared into the method for antifungal agricultural antibiotics product, and utilize this product to prevent and treat the application of crop fungal disease, thereby develop the novel pesticide of a kind of good stability, efficient, safety and low toxicity.
The inventor separates various microorganisms and screens the microorganism secondary metabolite in order to seek the more more effective antifungal agricultural antibiotics than in the past from soil, then to these secondary metabolites make with extra care respectively, evaluation and Application and Development.In this process, the inventor finds: belonging to the secondary metabolite that Malaysian streptomycete (Streptomyces malaysiensis) ECO 00002 of streptomyces produced has good control efficiency to the various crop disease, by making with extra care to active ingredient, identify, and compare with the spectral data of the related compound of bibliographical information, find the planar structure of 3 main active in the active ingredient and the azalomycin F3 (Azalomycin F3) of bibliographical information, azalomycin F4 (Azalomycin F4) and the identical (Chem.Pharm.Bull. of nifimycin (Niphimycin), 1967,15:1657-1661,1726-1732; 1982,30:1653-1657,1658-1668,1669-1673,4006-4014.J.Antibiot., 1970,23:107-112; 1981,34:1107-1118; 1982,35:1480-1494; 1984,37:103-109,1167-1169,1170-1186; 1985,38:1363-1369; 1986,39:713-716; 1990,43:639-647; 1993,46:1912-1915; 1994,47:688-696; 1995,48:293-299,896-898,1173-1175,1350-1352; 1996,49:765-769; 1997,50,194-200,484-489,965-969.J.BasicMicrobiol., 1998,38:415-419.J.Am.Chem.Soc., 1982,104:4129-4141), be 36 yuan of ring macrocyclic lactone compounds.
2. technical scheme: the present invention is achieved through the following technical solutions.
Refining and the authentication method of the preparation technology of WYLZ-1349, active component is characterized in that carrying out successively as follows:
(1) seed culture process:
Adopt Malaysian streptomycete (Streptomyces malaysiensis) ECO 00002, preservation is registered on the books and is numbered CGMCC No.1680, in seeding tank, add following ingredients, by weight the percentage composition meter: yeast extract 0.4～0.8, glucose 0.4～1.0, malt extract 1.0～2.0, B B-complex 0.001～0.002, trace salt 0.005～0.01, and 0.05～1.0% (V/V) silicone defoaming agent, all the other content are water, and 121 ℃ of autoclavings are standby after 40 minutes.
Condition of culture: cultivated in seeding tank 36～48 hours, cultivation temperature is 26～32 ℃, and pH 6.5～7.0, throughput 1: (0.5～0.8) (V/V), tank pressure 0.03～0.05Mpa, 100～150 rev/mins of stir speed (S.S.)s.
(2) antibiotic fermentation process:
In fermentation tank, add following ingredients, by weight the percentage composition meter: mannitol 2.0～3.0, soybean meal 2.0～4.0, dipotassium hydrogen phosphate 0.01% and 0.05～1.0% (V/V) silicone defoaming agent, all the other content are water.After above-mentioned material was put into fermentation tank, it was standby to adjust the pH value before the sterilization and be 7.5,121 ℃ of autoclavings 40 minutes.Step (1) cultured seed liquid is inserted fermentation tank, and last jar of inoculum concentration is 10%～20% (V/V).
Condition of culture: incubation time 72～96 hours, cultivation temperature are 26～32 ℃, and pH 6.5～7.0, throughput 1: (0.5～1.0) (V/V), tank pressure 0.03～0.05Mpa, stir speed (S.S.) 100-200 rev/min.
(3) tunning post processing, carry out successively according to the following steps:
A. zymotic fluid is filtered, filter supernatant and mycelium; B. mycelium is put into extractor, adds organic solvents such as acetone or methyl alcohol and stir broken walls after 6～10 hours, filter mycelial extract; C. mycelial extract is pumped in the concentration tank, in 700mmHg vacuum, vapour pressure 0.02MPa, concentrating under reduced pressure reclaims acetone under 20～30 ℃ of conditions of temperature; After treating that acetone has reclaimed, with mycelial extraction clear liquid with steps A. the supernatant of gained adopts macroporous absorbent resin absorption, and with organic solvent wash-outs such as acetone or methyl alcohol, the eluent that contains active component obtains the crude extract of WYLZ-1349 after concentrated; D. in crude extract, get finished product WYLZ-1349 behind the various agricultural chemicals universal supports of adding proper proportion or the auxiliary agent.
(4) the refining and evaluation of active component among the WYLZ-1349: get step C. obtains in the step (3) part crude extract process silica gel and Sephadex LH-20 gel filtration chromatography, can obtain half highly finished product.Use the high performance liquid chromatography preparation to obtain the pure product of 3 main active respectively then, purity can reach 98-99%.Measure respectively these 3 pure product of main active one dimension (
1H,
13And physicochemical constants such as fusing point, optically-active and dissolubility C NMR) and two dimension (HMQC, COSY, HMBC, ROESY and HMQC-TOCSY) nuclear magnetic resonance spectrum, mass spectrum (MS), infrared spectrum (IR) and ultraviolet spectra (UV).By the various spectral datas of labor, and compare with the spectral data of bibliographical information azalomycin F3, azalomycin F4 and nifimycin, determine that these 3 main active are azalomycin F3, azalomycin F4 and nifimycin, its molecular formula is respectively C
56H
95N
3O
17, C
57H
97N
3O
17And C
59H
103N
3O
18, molecular weight is respectively 1082.36,1096.39 and 1142.46, infers it and has following planar structure respectively:
R=H, the azalomycin F3 nifimycin
R=CH
3, azalomycin F4
Azalomycin F3, azalomycin F4 and nifimycin belong to the antifungal agricultural antibiotics of 36 yuan of ring macrocyclic lactone class formations with guanidine radicals and malonic acid base, and its physicochemical property sees Table 1～table 3 respectively, and the nuclear magnetic resonance spectroscopy data see Table 4～table 6 respectively.
The physicochemical property of table 1 azalomycin F3
| Outward appearance | The unformed powder of white |
| Fusing point (℃) | ??133-135 |
| HRESI(+)-MS(m/z) | ??1082.6734[M+H] +，1104.6562[M+Na] + |
| HRESI(-)-MS(m/z) | ??1080.6584[M-H] -，2162.3253[2M-H] - |
| Molecular formula, molecular weight | ??C 56H 95N 3O 17，1082.36 |
| UVλ max(MeOH)，nm | ??241，271 |
| IRν max(KBr)cm -1 | ??3437，3129，3005，2356，1634，1400 |
| [α] D 25(c?1.0，MeOH) | ??+40° |
| Outward appearance | The unformed powder of white |
| Dissolubility | Be soluble in methyl alcohol and ethanol.Be slightly soluble in water.Be insoluble in chloroform and ethyl acetate. |
The physicochemical property of table 2 azalomycin F4
| Outward appearance | The unformed powder of white |
| Fusing point (℃) | 127-129 |
| HRESI (+)-MS (m/z) | 1096.6892[M+H] +, 1118.6721[M+Na] + |
| HRESI (-)-MS (m/z) | 1094.6749[M-H] -, 2190.3559[2M-H] - |
| Molecular formula, molecular weight | C 57H 97N 3O 17, 1096.39 |
| UV λ Max(MeOH), nm | 241,271 |
| IR ν Max(KBr) cm -1 | 3438,3131,3006,2356,1635,1403 |
| [α] D 25(c 1.0, MeOH) | + 45 ° |
| Dissolubility | Be soluble in methyl alcohol and ethanol.Be slightly soluble in water.Be insoluble in chloroform and ethyl acetate. |
The physicochemical property of table 3 nifimycin
| Outward appearance | The unformed powder of white |
| Fusing point (℃) | ??136-138 |
| HRESI(+)-MS(m/z) | ??1142.7319[M+H] +，1164.7131[M+Na] + |
| HRESI(-)-MS(m/z) | ??1140.7160[M-H] -，2282.4404[2M-H] - |
| Molecular formula, molecular weight | ??C 59H 103N 3O 18，1142.46 |
| UVλ max(MeOH)，nm | ??233 |
| IRν max(KBr)cm -1 | ??3379，2932，2357，1719，1647，1595，1456，1378，1305，1259 |
| [α] D 25(c?1.0，MeOH) | ??+34° |
| Outward appearance | The unformed powder of white |
| Dissolubility | Be soluble in methyl alcohol and ethanol.Be slightly soluble in water.Be insoluble in chloroform and ethyl acetate. |
Table 4 azalomycin F3
1H,
13C nuclear magnetic resonance spectroscopy data (solvent: methyl alcohol-d
4)
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??1 | ??170.2(s) | ??- | ??29 | ??74.3(d) | ??3.91(1H，m) |
| ??2 | ??126.8(s) | ??- | ??30 | ??140.2(s) | ??- |
| ??3 | ??140.4(d) | ??7.08(1H，d，11.0) | ??31 | ??125.2(d) | ??5.98(1H，d，10.3) |
| ??4 | ??127.7(d) | ??6.43(1H，dd，14.7，11.7) | ??32 | ??128.7(d) | ??6.20(1H，dd，14.7，11.0) |
| ??5 | ??146.2(d) | ??6.04(1H，dd，15.4，8.8) | ??33 | ??136.3(d) | ??5.39(1H，m) |
| ??6 | ??44.7(d) | ??2.45(1H，m) | ??34 | ??41.1(d) | ??2.54(1H，m) |
| ??7 | ??75.9(d) | ??3.76(1H，m) | ??35 | ??80.8(d) | ??4.78(1H，dd，7.7，3.3) |
| ??8 | ??39.3(t) | ??1.77，1.48(2H，m) | ??36 | ??35.2(d) | ??1.82(1H，m) |
| ??9 | ??75.0(d) | ??3.88(1H，m) | ??37 | ??34.6(t) | ??1.33，1.16(2H，m) |
| ??10 | ??44.6(d) | ??1.53(1H，m) | ??38 | ??28.0(t) | ??1.42(2H，m) |
| ??11 | ??72.4(d) | ??3.90(1H，m) | ??39 | ??33.7(t) | ??1.98(2H，m) |
| ??12 | ??29.9(t) | ??1.65(2H，m) | ??40 | ??132.6(d) | ??5.45(1H，m) |
| ??13 | ??30.7(t) | ??2.06，1.32(2H，m) | ??41 | ??130.3(d) | ??5.44(1H，m) |
| ??14 | ??40.9(d) | ??1.59(1H，m) | ??42 | ??30.7(t) | ??2.06，1.42(2H，m) |
| ??15 | ??72.4(d) | ??3.85(1H，m) | ??43 | ??41.8(t) | ??1.91，1.68(2H，m) |
| ??16 | ??46.6(t) | ??2.85，1.48(2H，m) | ??44 | ??42.2(t) | ??3.15，1.64(2H，m) |
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??17 | ??99.9(s) | ??- | ??45 | ??13.0(q) | ??1.91(3H，s) |
| ??18 | ??77.2(d) | ??3.92(1H，m) | ??46 | ??17.2(q) | ??1.11(3H，d，6.6) |
| ??19 | ??65.6(d) | ??3.86(1H，m) | ??47 | ??10.6(q) | ??0.87(3H，d，7.3) |
| ??20 | ??33.7(t) | ??2.84，1.91(2H，m) | ??48 | ??14.4(q) | ??0.91(3H，d，6.6) |
| ??21 | ??65.5(d) | ??3.84(1H，m) | ??49 | ??13.4(q) | ??1.63(3H，s) |
| ??22 | ??42.2(t) | ??1.78，1.65(2H，m) | ??50 | ??17.7(q) | ??1.01(3H，d，6.6) |
| ??23 | ??69.8(d) | ??5.24(1H，m) | ??51 | ??14.9(q) | ??0.95(3H，d，6.6) |
| ??24 | ??41.9(t) | ??1.81，1.68(2H，m) | ??52 | ??158.3(s) | ??- |
| ??25 | ??70.8(d) | ??3.87(1H，m) | ??53 | ??28.6(q) | ??2.84(3H，s) |
| ??26 | ??41.3(t) | ??1.91，1.29(2H，m) | ??1′ | ??171.7(s) | ??- |
| ??27 | ??66.3(d) | ??4.07(1H，m) | ??2′ | ??46.2(t) | ??3.22(2H，s) |
| ??28 | ??44.2(t) | ??1.69，1.48(2H，m) | ??3′ | ??174.2(s) | ??- |
Table 5 azalomycin F4
1H,
13C nuclear magnetic resonance spectroscopy data (solvent: methyl alcohol-d
4)
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??1 | ??170.2(s) | ??- | ??30 | ??140.3(s) | ??- |
| ??2 | ??126.7(s) | ??- | ??31 | ??125.1(d) | ??5.97(1H，d，10.3) |
| ??3 | ??140.3(d) | ??7.08(1H，d，11.0) | ??32 | ??128.6(d) | ??6.20(1H，dd，14.3，10.6) |
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??4 | ??127.6(d) | ??6.42(1H，dd，14.7，11.7) | ??33 | ??136.2(d) | ??5.39(1H，m) |
| ??5 | ??146.3(d) | ??6.04(1H，dd，14.7，8.8) | ??34 | ??41.1(d) | ??2.54(1H，dd，14.3，7.0) |
| ??6 | ??44.7(d) | ??2.44(1H，m) | ??35 | ??80.7(d) | ??4.77(1H，dd，7.0，2.6) |
| ??7 | ??75.8(d) | ??3.76(1H，m) | ??36 | ??35.1(d) | ??1.82(1H，m) |
| ??8 | ??39.3(t) | ??1.77，1.47(2H，m) | ??37 | ??34.6(t) | ??1.33，1.16(2H，m) |
| ??9 | ??74.9(d) | ??3.88(1H，m) | ??38 | ??28.0(t) | ??1.42(2H，m) |
| ??10 | ??44.5(d) | ??1.53(1H，m) | ??39 | ??33.7(t) | ??1.98(2H，m) |
| ??11 | ??72.3(d) | ??3.90(1H，m) | ??40 | ??132.5(d) | ??5.44(1H，m) |
| ??12 | ??29.9(t) | ??1.65(2H，m) | ??41 | ??130.4(d) | ??5.43(1H，m) |
| ??13 | ??30.7(t) | ??2.06，1.33(2H，m) | ??42 | ??30.7(t) | ??2.06，1.44(2H，m) |
| ??14 | ??40.8(d) | ??1.59(1H，m) | ??43 | ??41.8(t) | ??1.91，1.67(2H，m) |
| ??15 | ??72.4(d) | ??3.85(1H，m) | ??44 | ??42.2(t) | ??3.17，1.65(2H，m) |
| ??16 | ??46.6(t) | ??2.85，1.49(2H，m) | ??45 | ??13.0(q) | ??1.91(3H，s) |
| ??17 | ??99.8(s) | ??- | ??46 | ??17.2(q) | ??1.10(3H，d，5.9) |
| ??18 | ??77.1(d) | ??3.92(1H，m) | ??47 | ??10.6(q) | ??0.86(3H，d，6.6) |
| ??19 | ??65.5(d) | ??3.86(1H，m) | ??48 | ??14.4(q) | ??0.91(3H，d，5.9) |
| ??20 | ??33.7(t) | ??2.85，1.91(2H，m) | ??49 | ??13.4(q) | ??1.63(3H，s) |
| ??21 | ??65.5(d) | ??3.84(1H，m) | ??50 | ??17.7(q) | ??1.01(3H，d，5.9) |
| ??22 | ??42.2(t) | ??1.77，1.65(2H，m) | ??51 | ??14.9(q) | ??0.95(3H，d，5.9) |
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??23 | ??69.8(d) | ??5.22(1H，m) | ??52 | ??157.4(s) | ??- |
| ??24 | ??41.8(t) | ??1.82，1.69(2H，m) | ??53 | ??28.6(q) | ??2.85(3H，s) |
| ??25 | ??70.8(d) | ??3.87(1H，m) | ??54 | ??28.6(q) | ??2.85(3H，s) |
| ??26 | ??41.2(t) | ??1.91，1.29(2H，m) | ??1′ | ??171.7(s) | ??- |
| ??27 | ??66.1(d) | ??4.06(1H，m) | ??2′ | ??46.2(t) | ??3.23(2H，s) |
| ??28 | ??44.3(t) | ??1.69，1.49(2H，m) | ??3′ | ??174.0(s) | ??- |
| ??29 | ??74.2(d) | ??3.91(1H，m) |
Table 6 nifimycin
1H,
13C nuclear magnetic resonance spectroscopy data (solvent: methyl alcohol-d
4)
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??1 | ??176.7(s) | ??- | ??31 | ??132.0(d) | ??6.18(1H，dd，14.7，10.3) |
| ??2 | ??48.0(d) | ??2.44(1H，m) | ??32 | ??132.0(d) | ??6.06(1H，dd，14.7，10.3) |
| ??3 | ??65.9(d) | ??4.09(1H，m) | ??33 | ??137.0(d) | ??5.53(1H，dd，14.7，8.1) |
| ??4 | ??133.0(d) | ??5.44(1H，dd，15.4，8.8) | ??34 | ??40.7(d) | ??2.54(1H，m) |
| ??5 | ??136.6(d) | ??5.70(1H，dd，15.4，8.8) | ??35 | ??79.9(d) | ??4.75(1H，dd，7.4，4.4) |
| ??6 | ??43.4(d) | ??2.32(1H，m) | ??36 | ??32.6(d) | ??1.91(1H，m) |
| ??7 | ??75.7(d) | ??3.75(1H，m) | ??37 | ??42.5(t) | ??1.32，0.95(2H，m) |
| ??8 | ??39.3(t) | ??1.70，1.50(2H，m) | ??38 | ??40.7(d) | ??1.60(1H，m) |
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??9 | ??72.4(d) | ??3.75(1H，m) | ??39 | ??33.5(t) | ??1.62(2H，m) |
| ??10 | ??44.5(d) | ??1.53(1H，m) | ??40 | ??27.8(t) | ??1.38(2H，m) |
| ??11 | ??72.4(d) | ??3.90(1H，m) | ??41 | ??33.9(t) | ??1.98(2H，m) |
| ??12 | ??30.7(t) | ??1.65(2H，m) | ??42 | ??132.4(d) | ??5.47(1H，m) |
| ??13 | ??37.6(t) | ??1.32，1.07(2H，m) | ??43 | ??130.0(d) | ??5.44(1H，m) |
| ??14 | ??30.7(d) | ??1.55(1H，m) | ??44 | ??30.4(t) | ??2.08(2H，m) |
| ??15 | ??75.3(d) | ??4.07(1H，m) | ??45 | ??30.0(t) | ??1.65(2H，m) |
| ??16 | ??44.5(t) | ??1.53(2H，m) | ??46 | ??41.8(t) | ??3.16(2H，m) |
| ??17 | ??99.9(s) | ??- | ??47 | ??14.9(q) | ??1.00(3H，d，6.6) |
| ??18 | ??77.5(d) | ??3.36(1H，dd，8.8) | ??48 | ??16.9(q) | ??1.08(3H，d，6.6) |
| ??19 | ??69.8(d) | ??3.90(1H，m) | ??49 | ??10.7(q) | ??0.89(3H，d，5.9) |
| ??20 | ??41.3(t) | ??1.91(2H，m) | ??50 | ??11.4(q) | ??0.85(3H，d，6.6) |
| ??21 | ??76.0(d) | ??4.07(1H，m) | ??51 | ??20.6(q) | ??0.87(3H，d，5.9) |
| ??22 | ??42.0(t) | ??1.83，1.77(2H，m) | ??52 | ??18.0(q) | ??1.02(3H，d，6.6) |
| ??23 | ??71.2(d) | ??5.20(1H，m) | ??53 | ??15.2(q) | ??0.93(3H，d，6.6) |
| ??24 | ??42.1(t) | ??1.83，1.77(2H，m) | ??54 | ??15.1(q) | ??0.91(3H，d，5.9) |
| ??25 | ??65.8(d) | ??3.86(1H，m) | ??55 | ??158.3(s) | ??- |
| ??26 | ??43.2(t) | ??1.65，1.58(2H，m) | ??56 | ??28.5(q) | ??2.84(3H，s) |
| ??27 | ??69.5(d) | ??4.13(1H，m) | ??1′ | ??171.6(s) | ??- |
| ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) | ??No | ??δ C(mult.)??(125MHz) | ??δ H(int.，mult.，J HH?Hz)??(500MHz) |
| ??28 | ??45.4(d) | ??1.53(1H，m) | ??2′ | ??46.2(t) | ??3.23(2H，s) |
| ??29 | ??76.1(d) | ??4.07(1H，m) | ??3′ | ??174.3(s) | ??- |
| ??30 | ??135.3(d) | ??5.67(1H，dd，15.4，8.8) |
The preparation technology of described WYLZ-1349 is characterized in that: the inoculum concentration that is inserted fermentation tank by seeding tank is 10%～20% (V/V); Bacterial strain is 36～48 hours at the incubation time of seeding tank; All add 0.05～1.0% (V/V) silicone defoaming agent in seeding tank and the fermentation tank.
Among the described WYLZ-1349 active component refining and authentication method, it is characterized in that: the molecular formula of 3 main active azalomycin F3s, azalomycin F4 and nifimycins is respectively C among the WYLZ-1349
56H
95N
3O
17, C
57H
97N
3O
17And C
59H
103N
3O
18, molecular weight is respectively 1082.36,1096.39 and 1142.46, has following planar structure respectively:
The preparation technology of described WYLZ-1349 is characterized in that: azalomycin F3, azalomycin F4 and nifimycin composition are used to prevent and treat crop fungal disease.
3. advantage and effect
The fermentative medium formula that the present invention adopts is simple, raw materials enjoy stable sources, and cost is low, is fit to large-scale industrial production.When guaranteeing that raw material is stable, can control the quality that every fermentation parameter is controlled finished product by strictness, thus the complete quality system of formation science.This product is chafe, eye etc. not, to the people, animal nonhazardous; Crops there is not poisoning, resistance of rainwater washing against; To light, thermally-stabilised.This product control crop fungal disease effect is obvious.
Embodiment:
Embodiment 1
A kind of bacterial strain that produces antifungal agricultural antibiotics, its formal name used at school is Malaysian streptomycete (Streptomycesmalaysiensis) ECO 00002, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 17th, 2006, and preservation is registered on the books and is numbered CGMCC No.1680.This strain cell wall contains L, the L-diaminopimelic acid, and full cell sugar hydrolysate contains galactose and arabinose, cell wall I type.This bacterial strain soaks well-grown on juice and the nutrient agar Gause I, glycerine-asparagine, yeast extract-malt extract, oats flakes, potato; Morphological feature on the Gause I agar medium is: bacteria colony white, and circle, surperficial lint shape, there is more mycelial growth at the edge; Substrate mycelium and aerial hyphae are thread, and irregular branch does not generally rupture; Form long spore chain on the aerial hyphae, straight shape, waveform or spirality, spore circle, oval to shaft-like.This bacterial strain is the energy liquefy gelatin on peptone-gelatin culture medium, solidify milk, utilization to nitrate reduction and urea is positive, the energy hydrolyzed starch, do not grow on the cellulose, do not produce hydrogen sulphide and melanin, can utilize glucose, rhamnose, arabinose, sucrose, fructose, galactose, maltose, mannose, raffinose, mannitol, sorbierite, glycerine, sodium oxalate, sodium acetate and citric acid.
The preparation technology of WYLZ-1349, refining and authentication method, carry out as follows successively:
(1) seed culture process:
Adopt Malaysian streptomycete (Streptomyces malaysiensis) ECO 00002, in seeding tank, add following ingredients, percentage composition meter by weight: yeast extract 0.4, glucose 0.4, malt extract 1.0, B B-complex 0.001, trace salt 0.005, and 0.05% (V/V) silicone defoaming agent, all the other content are water, 121 ℃ of autoclavings are standby after 40 minutes.
Condition of culture: cultivated 48 hours in seeding tank, cultivation temperature is 28 ± 1 ℃, and pH 6.5～7.0, throughput 1: 0.6 (V/V), tank pressure 0.03Mpa, 120 rev/mins of stir speed (S.S.)s.
(2) antibiotic fermentation process:
In fermentation tank, add following ingredients, by weight the percentage composition meter: mannitol 2.0, soybean meal 2.0, dipotassium hydrogen phosphate 0.01% and 0.05% (V/V) silicone defoaming agent, all the other content are water.After above-mentioned material was put into fermentation tank, it was standby to adjust the pH value before the sterilization and be 7.5,121 ℃ of autoclavings 40 minutes.The seed liquor that step (1) forms is inoculated into fermentation tank, and inoculum concentration is 15% (V/V).Tank pressure is controlled at 0.03Mpa during fermentation, throughput 1: 0.5 (V/V), and cultivation temperature is 28 ± 1 ℃, 180 rev/mins of stir speed (S.S.)s, incubation time 72 hours.
(3) tunning post processing, carry out successively according to the following steps:
A. zymotic fluid is filtered, filter supernatant and mycelium; B. mycelium is put into extractor, added 75% acetone-water (V/V) solution stirring 6 hours, filter mycelial extract; C. mycelial extract is pumped in the concentration tank, in 700mmHg vacuum, vapour pressure 0.02MPa, concentrating under reduced pressure reclaims acetone under 20～30 ℃ of conditions of temperature; After treating that acetone has reclaimed, with mycelial extraction clear liquid with steps A. the supernatant of gained adopts macroporous absorbent resin absorption, and 40% acetone-water (V/V) wash-out, the eluent that contains active component obtain the crude extract I of WYLZ-1349 after concentrated; D. get 50 parts of crude extract I and draw back powder, 2 parts of size LS and 56 parts of diatomite and mix, pulverize, promptly get the wetting powder of 100 parts of desired contents with 4 parts.
(4) among the WYLZ-1349 active component refining with identify: get step C. obtains in the step (3) 200 gram crude extract I through silica gel column chromatographies, 85% chloroform-methanol (V/V) wash-out is collected active component, obtains 60 and restrain yellow powder II after concentrating; Get 20 gram II through Sephadex LH-20 gel filtration chromatography, methanol-eluted fractions is collected active component, obtains 7 gram buff powder III after concentrating; Get 200 milligrams of III, the preparation of utilization Waters 1525 high performance liquid chromatography, chromatographic column: HederaODS-P (250 * 20mm, 5 μ m), flowing phase: 75% methanol-water (V/V), flow velocity: 10.0 ml/min, detect wavelength: 233 nanometers, collect retention time respectively and be the unimodal of 22.47 minutes, 26.20 minutes and 33.84 minutes, after concentrating, obtain 32 milligrams, 29 milligrams and 43 milligrams of purity respectively and be 99% IV, V and the pure product of VI.The one dimension of mensuration IV, V and the pure product of VI (
1H,
13And physicochemical constants such as fusing point, optically-active and dissolubility C NMR) and two dimension (HMQC, COSY, HMBC, ROESY and HMQC-TOCSY) nuclear magnetic resonance spectrum, mass spectrum (MS), infrared spectrum (IR) and ultraviolet spectra (UV).By the various spectral datas of labor, and compare with the spectral data of bibliographical information azalomycin F3, azalomycin F4 and nifimycin, determine that IV, V and VI are respectively azalomycin F3, azalomycin F4 and nifimycin, its molecular formula is respectively C
56H
95N
3O
17, C
57H
97N
3O
17And C
59H
103N
3O
18, molecular weight is respectively 1082.36,1096.39 and 1142.46, has following planar structure respectively:
R=H, the azalomycin F3 nifimycin
R=CH
3, azalomycin F4
Embodiment 2
To be diluted to 50～200 mg/litre by embodiment 1 prepared WYLZ-1349 finished product, use tricyclazole (75% wetting powder) respectively, dimethachlon (40% wetting powder), iprodione (50% wetting powder) and tpn (75% wetting powder) be medicament in contrast, clear water is as blank, test WYLZ-1349 finished product is to rice blast, Alternaria alternate, the control efficiency of graw mold of tomato and pepper anthracnose, result of the test shows that WYLZ-1349 can effectively prevent and treat rice blast, Alternaria alternate, graw mold of tomato and pepper anthracnose, dosage are starkly lower than the contrast medicament.Result of the test sees Table 7～table 10.
Table 7WYLZ-1349 control rice blast result of the test
| Handle medicament | Working concentration (mg/litre) | Average preventive effect (%) |
| ??WYLZ-1349 | ??100 | ??73.3 |
| ??WYLZ-1349 | ??200 | ??81.9 |
| ??WYLZ-1349 | ??300 | ??95.1 |
| Tricyclazole | ??400 | ??97.6 |
| The clear water contrast | ??- | ??- |
Table 8WYLZ-1349 control Alternaria alternate result of the test
| Handle medicament | Working concentration (mg/litre) | Average preventive effect (%) |
| ??WYLZ-1349 | ??50 | ??71.0 |
| ??WYLZ-1349 | ??100 | ??80.7 |
| ??WYLZ-1349 | ??200 | ??91.6 |
| Dimethachlon | ??400 | ??86.8 |
| The clear water contrast | ??- | ??- |
Table 9WYLZ-1349 control graw mold of tomato result of the test
| Handle medicament | Working concentration (mg/litre) | Average preventive effect (%) |
| ??WYLZ-1349 | ??50 | ??72.8 |
| ??WYLZ-1349 | ??100 | ??83.3 |
| ??WYLZ-1349 | ??200 | ??92.7 |
| Iprodione | ??400 | ??87.9 |
| The clear water contrast | ??- | ??- |
Table 10WYLZ-1349 control pepper anthracnose result of the test
| Handle medicament | Working concentration (mg/litre) | Average preventive effect (%) |
| ??WYLZ-1349 | ??50 | ??71.9 |
| ??WYLZ-1349 | ??100 | ??81.6 |
| ??WYLZ-1349 | ??200 | ??93.5 |
| Tpn | ??400 | ??91.4 |
| The clear water contrast | ??- | ??- |
Claims (3)
1. disinfectant use in agriculture is characterized in that it is is the bactericide WYLZ-1349 of active ingredient with azalomycin F3, azalomycin F4 and nifimycin composition, and the molecular formula of azalomycin F3, azalomycin F4 and nifimycin is respectively C
56H
95N
3O
17, C
57H
97N
3O
17And C
59H
103N
3O
18, molecular weight is respectively 1082.36,1096.39 and 1142.46, has following planar structure respectively:
R=H, the azalomycin F3 nifimycin
R=CH
3, azalomycin F4
Producing the used bacterial strain of WYLZ-1349 is Malaysian streptomycete (Streptomyces malaysiensis) ECO00002, and deposit number is CGMCC No.1680.
2. the preparation method of the described disinfectant use in agriculture of claim 1, it is characterized in that cultivating earlier Malaysian streptomycete (Streptomycesmalaysiensis) ECO 00002, separation and Extraction fermentation culture medium again, with acetone or methyl alcohol with the mycelium broken wall after, merge with fermented supernatant fluid, through the macroporous absorbent resin adsorbing separation, and with acetone or methanol-eluted fractions, eluent concentrate or be further purified new phosphorus azomycin, add again behind carrier or the auxiliary agent finished product.
3. the application of the described disinfectant use in agriculture of claim 1 is characterized in that described bactericide can prevent and treat following crop fungal disease: rice blast, Alternaria alternate, graw mold of tomato and pepper anthracnose.
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| CN103044439A (en) * | 2012-12-21 | 2013-04-17 | 江西农业大学 | De-malonate monoacyl azalomycin F, preparation method thereof and application thereof to preparation of MRSA infection therapeutic drug |
| CN103232964A (en) * | 2013-05-24 | 2013-08-07 | 武汉大学 | High-yield azalomycin F compound strain streptomycete TKPJ3039 and application thereof |
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| CN103232964A (en) * | 2013-05-24 | 2013-08-07 | 武汉大学 | High-yield azalomycin F compound strain streptomycete TKPJ3039 and application thereof |
| CN103232964B (en) * | 2013-05-24 | 2014-12-17 | 武汉大学 | High-yield azalomycin F compound strain streptomycete TKPJ3039 and application thereof |
| CN106719770A (en) * | 2016-11-23 | 2017-05-31 | 云南省农业科学院农业环境资源研究所 | A kind of purposes of the method and azalomycin of the method for suppressing rape knee bacteria growing and preventing and treating crop in cruciferae clubroot |
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| CN107354103B (en) * | 2017-04-21 | 2020-05-26 | 浙江师范大学 | Streptomyces lunalinharesii ZJNU968 strain and application thereof |
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| CN114164135A (en) * | 2021-10-15 | 2022-03-11 | 中国热带农业科学院热带生物技术研究所 | Preparation method and application of banana vascular wilt resistant compound |
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