Structure Determination And Disease Control Mechanisms Of Cytosinpeptidemycin

Cytosinpeptidemycin is a high efficient, low-toxic and low-residue bio-pesticide used to control multiple crop fungi and virus diseases. Cytosinpeptidemycin was independently developed by Shenyang Agricultural University, and it was registered as a national patent. Under the guidance of its biological activities, two pure antifungal compounds 1 and 2, one pure anti-bacterial compound 3 and one impure antiviral compound 4 were obtained by different separation techniques such as column chromatography; The molecular formula of the compound 1 and compound 2 was elucidated by spectra analysis; The HPLC qualitative or quantitative analysis method was developed, and the anti-fungal and antiviral mechanism of compound 1 and 3 was investigated respectively. The results were:1. Compound 1 was purified under the guidance of anti- Alternaria alternata activity. The crude fermentation broth was purified to eliminate protein and other mess, absorbed by Diaion HP20 and eluted by 50% methanol. Then the bio-active fractions were further purified through TOSOH SP650M ion exchange chromatography with 0-1 mol/L NaCl as eluent. Finally, the pure compound 1 was obtained by Daisogel ODS-B reverse phase chromatography in 20% methanol and lyophillzed. The molecular formula was elucidated as C12H13N5O4 by interpretation of UV, IR, MS and NMR data. It was the first time to obtain the pure antifungal compound and elucidate its molecular structure.2. The anti- Alternaria alternata compound 2 was obtained through absorption of SP825L and eluted by 50% methanol, followed by silica gel and Sephadex LH-20 chromatography with Chloroform:1-butanol:methanol:acetic acid=90:5:3:2 (by vol.) and 70% methanol as mobile phase respectively. MS analysis showed the compound 2 molecular weight is 244. Results from UV and NMR analysis indicated compound 2 may be an intermediate during the compound 1 synthesis or a degradation substance in the compound 1 fermentation process due to the over time fermentation or other reason.3. Compound 3 was obtained based on the antibacterial activity on Bacillus cereus. Frankland. Some impurities were eliminated by macros absorption resin Diaion HP20 and ion exchange resin SK1B, then bio-active substance was enriched by 732 strong cation exchange resin and gradient eluted by 0.1-0.5 mol/L ammonia water. The molecular weight of pure compound 3 is 574 after the purification by HW40 and LH-20.4. Compound 4 has the activity of anti-virus and was isolated by bioassay. The crude compound 4 was isolated through a simple procedure of absorption and reverse phase chromatography. 5. The study developed the qualitative and quantitative analysis method of compound 1. The HPLC method mainly uses DIKMA DiamonsilⅡC18 column and UV detector by using methanol/water (20:80, by vol) as mobile phase with 1 mL/min flow rate and UV detection at 278 nm. The column temperature was 30℃, and the retention time was 10.2 min. The liner correlation of this method was 0.9998. The average recovery rate and coefficient variation was 99.4 and 0.25%, respectively.6. The conditions for HPLC separating compound 3 are studied:DIKMA DiamonsilⅡC18 column (200mm×4.6mm,Φ5μm) with UV detection at 230nm,30mmol/L phosphate buffer contain 10mmol/L sulfonic acid sodium salt as moble phase with column temperature at 30℃. It can be used as a quantitative and qualitative analysis method.7. Antimicrobial spectrum test showed that compound 1 was against 20 phytopathogenic fungi such as Botrytis cinerea, Colletotrichum orbiculare, Alternaria solani, Sclerotinia sclerotiorum and Alternaria alternate. Compound 1 has a obvious inhibition effect on mycelial growth and spore germination. It also can cause the hypha and the tube to grow in abnormal shapes. The test of antibiosis showed that compound 1 has inhibition action on Alternaria alternata, but has little or even no fungicide function. Studies of compound 1 active mechanism were conducted, and the result showed that compound 1 can cause the protoplasm leaked out from hypha. At the same time, the ergosterol content in mycelia of Alternaria alternata decreased remarkably and MDA content in mycelia and cultural filtrate increased significantly. These results revealed the action site of compound 3 is on the cell membrane, and it changed the content of protein in mycelia.8. The TMV control effect tests were conducted on Nicotiana glutinosa in green house, the results showed that compound 4 had a significant control effect on TMV at almost100% on Nicotiana glutinosa, compound 3 had a control effect at 74.9%. Compound 1 and 2 didn’t have a clear control effect. When examining the Nicotiana tobacum curative effects, compound 4 showed a result of 62.8%, compound 3 was 49.8%, compound 1 was 18.3, and compound 2 didn’t have any curative effect.Research on anti-viral mechanism showed that Cytosinpeptidemycin can directly act on TMV particles and depress its ability of infection. Bioassay, ultraviolet spectrophotometry and ELISA test showed compound 3 can significantly reduce the concentration of TMV virions. In addition, the Real-time RT-PCR revealed that compound 3 can inhibit TMV genome replication in early stage.