摘要
嘧肽霉素是我校自主研发的生物农药,具有高效、低毒、低残留等优点,可以防治多种农作物病毒病害和真菌病害,已获得国家发明专利。本文以生物活性为检测手段,利用多种色谱方法获得了嘧肽霉素中两种抗真菌物质的纯品组分1和组分2,一种抗细菌物质的纯品组分3,和一种抗病毒物质的粗品组分4;通过波谱法对组分1和组分2进行了结构解析;开发了组分1和组分3的高效液相色谱分析方法;研究了组分1的抗真菌机理和组分3的抗病毒机理,结果如下:
1.组分1以烟草赤星病菌(Alternaria alternata)为指示菌分离而得。嘧肽霉素发酵液经离心得上清,草酸酸化预处理充分去除蛋白质等杂质,依次经过大孔吸附树脂Diaion HP20吸附,50%甲醇洗脱;TOSOH SP650M离子交换柱吸附,0-1mol/L NaCl梯度洗脱;Daisogel ODS-B反相柱纯化,20%甲醇作为流动相,冻干得纯品。经紫外光谱、红外光谱、质谱和核磁共振试验最终得到了组分1的分子式为C12H13N5O4,并推测了其结构式。这是首次获得嘧肽霉素抗真菌组分的纯品和结构式。
2.组分2同样以烟草赤星病菌(Alternaria alternata)为分离指示菌,经过大孔吸附树脂吸附后的活性馏分用硅胶柱层析进行进一步分离,氯仿:正丁醇:甲醇:醋酸=42:2:4:2作为流动相,并用Sephadex LH-20以20%甲醇作为流动相做最后的纯化,可得到纯品。质谱检测表明组分2的分子量是244。质谱、核磁、紫外等多种试验结果表明组分2可能是组分1合成过程的中间体,也可能是组分1由于发酵时间过度或其它原因发生降解的产物。
3.组分3以蜡样芽孢杆菌(Bacillus cereus. Frankland)作为指示菌分离得到。预处理后的发酵液先后经过大孔吸附树脂Diaion HP20和离子交换树脂SK1B去除杂质后,用732阳离子交换树脂配合0.1-0.5 mol/L氨水梯度洗脱进行浓缩分离,最后依次通过HW40和LH-20分子筛层析柱纯化,得到分子量为574的纯品。
4.组分4具有抗TMV活性,通过枯斑抑制试验分离。嘧肽霉素发酵液通过大孔吸附树脂吸附,用50%丙酮洗脱并浓缩蒸干之后用ODS-BP反相柱分离,0-100%甲醇梯度洗脱,得到组分4粗品。
5.开发了嘧肽霉素组分1的高效液相色谱定性和定量分析方法,最佳色谱条件为:色谱柱选用DIKMA DiamonsilⅡC18柱(200mm×4.6mm,φ5μm);检测波长为278nm;流动相为20%甲醇水溶液,柱温为30℃。此色谱条件下组分1保留时间约10.2min,此方法的准确度和精密度较高,通过此方法测得的嘧肽霉素发酵液中抗真菌组分含量为4.11μg/mL。组分2的性质与组分1相近,可以采用同样的分离条件进行分离。
6.明确了组分3的色谱分离条件:色谱柱选用DIKMA DiamonsilⅡC18柱(200mm×4.6mm,Φ5μm检测波长为230nm;流动相采用30mmol/L磷酸盐缓冲液,含有10mmol/L辛烷磺酸钠,pH=3。柱温为30℃。此方法可以进一步验证用于定性和定量分析。
7.抗菌谱测试表明组分1对番茄灰霉病菌(Botrytis cinerea)、西瓜炭疽病菌(Colletotrichum orbiculare)、番茄早疫病菌(Alternaria solani)黄瓜菌核病菌(Sclerotinia sclerotiorum)、烟草赤星病菌(Alternaria alternata)等16种植物病原真菌有抑菌效果。组分1的抑菌防病机理研究表明:组分1能够显著抑制赤星病菌菌丝生长和孢子萌发,并导致菌丝和孢子萌发后的芽管明显畸形,但杀菌作用很弱或基本无杀菌效果。组分1能导致菌丝体内电解质泄漏,细胞膜上麦角固醇的含量降低,菌丝内和培养液中脂质过氧化产物—丙二醛的含量升高,证明其对病菌细胞膜有一定破坏作用。与此同时,组分1还可显著抑制菌丝体内可溶性蛋白的含量。
8.在嘧肽霉素各组分针对烟草花叶病毒的防效试验中,组分4在心叶烟上对TMV的防治效果最好,枯斑抑制率达100%,组分3次之,枯斑抑制率为74.9%,而组分1和组分2没有明显的防治效果。当测试四种组分对普通烟的治疗效果时,组分4的治疗效果最好,防效达到62.8%,组分3次之,为49.8%。组分1也表现出一定的防治效果,防效为18.3%。组分2对TMV在系统寄主普通烟NC89上没有治疗作用。
抗病毒机理研究表明,嘧肽霉素组分可以在体外直接作用于病毒粒子,使其丧失侵染能力,从而保护心叶烟免受TMV的侵染。生物学方法、紫外分光光度计法以及ELISA测定结果均表明,嘧肽霉素组分3处理后烟草植株体内的病毒浓度均显著降低,这说明组分3能够降低植株体内的病毒浓度,充分说明嘧肽霉素对病毒的复制和增殖有较强的抑制作用。应用实时荧光定量PCR技术对TMV RNA在烟草中蓄积量测定结果表明组分3可以在TMV侵入寄主早期抑制其基因组RNA的复制。
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.
引文
1.陈贵斌,牛晋阳,张建勇,等.2003.反相高效液相色谱法测定抗生素AGPM发酵液中的主要成分[J].色谱,21(4):378-381.
2.陈梅,邱德文,刘峥,等.2006.植物激活蛋白对烟草花叶病毒RNA复制及外壳蛋白合成的抑制作用[J].中国生物防治,22(1):63-66.
3.陈文君,吴剑波,金启光,等.1990.农用抗生素11371各组份的分离纯化和鉴别[J].中国抗生素杂志,15(1):42-48.
4.陈伊,李舟,魏玉珍,等.2009.根际放线菌I06-03431产生的核苷类抗生素3431的研究[J].生物学杂志,26(4):17-20.
5.陈允亮,陶黎明,高菊芳.2009.磷氮霉素的研究进展及应用前景[J],现代农药,8(5):8-12.
6.崔长辉.2004.广谱抗菌素--多抗霉素(多氧霉素、宝丽安)[J].吉林蔬菜,(6):10.
7.崔佳佳,胡海峰,朱宝泉,等.2004.微生物来源的抗真菌抗生素SIPI-763-1的研究[J].中国抗生素杂志,29(10):577-578.
8.代富英,周金燕,谭红.2006.捷安肽素抗真菌作用机理研究[J].成都医学院学报,1(2):86-90.
9.刁春玲,张国平,徐广芳,等.2006.苯并噻唑衍生物亚磷酸盐对禾谷镰刀菌的作用机理初探[J].农药学学报,8(3):233-238.
10.董丹,王春梅,吴元华.2006.嘧肽霉素复配制剂06抗病毒机理初探[J].沈阳农业大学学报,37(4):593-596.
11.杜春梅,吴元华.2005.4%嘧肽霉素水剂的毒性和防效[J].黑龙江大学自然科学学报,22(3):338-341.
12.杜春梅,吴元华.2007.嘧肽霉素发酵培养基及发酵条件的优化[J].黑龙江大学自然科学学报,24(4):484-489.
13.高芬.2008.抗真菌农用抗生素KA08的研究[D].沈阳农业大学.
14.高伟,田黎,张久明,等.2010.海洋芽胞杆菌B-9987菌株对番茄灰霉病和早疫病的作用机制初探[J].植物保护,36(1):55-59.
15.高伟,田黎,周俊英,等.2009.海洋芽孢杆菌(Bacillus marinus)B-9987菌株抑制病原真菌机理[J].微生物学报,49(11):1494-1501.
16.顾觉奋,陈钢.1994.H-103大孔吸附剂分离纯化抗生素LY-92的研究[J].离子交换与吸附,10(6):504-509.
17.关洪全,李建春,吕乃群,等.1995.SY-01的抗真菌活性及其机理的探讨[J].中国微生态学杂志,7(2):19-23.
18.何学超,郭道林,兰盛斌,等.2008.粮食中麦角甾醇含量正相液相色谱法检测[J].粮食储藏,37(2):43-46.
19.贺家亮,张敏,赵胜娟.2005.纳他霉素的研究现状[J].中国食品添加剂,(3):30-32.
20.黄昌华,郭崇明,夏文胜,等.1996B-HCH菌株发酵滤液对植物病原真菌的作用[J].中国生物防治,12(3):14-16.
21.黄惠琴,郭夫江,胡永华,等.2006.海洋放线菌活性组分AM105-II的分离与结构鉴定[J].中国海洋药物,25(4):17-21.
22.姬生宝,范晋勇,元英进.2005.藤黄灰链霉菌-H103发酵液中抗真菌活性成分的分离纯化[J].微生物学通报,32(3):77-81.
23.贾红伟.1998.链霉菌1948发酵液中抗病毒活性成分的提取分离、结构测定及抗病毒作用的研究[D].中国协和医科大学.
24.江晓帆,宋颖,赵秀香等.链霉菌Streptomyces tz92发酵液的抗真菌谱及稳定性.湖北农业科学,2008.47(6):3.
25.江山,韩熹莱,郭雪柳.1995.抗植物病毒剂对烟草和甜菜病程相关蛋白的诱导作用[J].植物病理学报,25(3):243-246.
26.蒋细良,谢德龄,倪楚芳,等.1997.中生菌素对真菌作用机理的研究[J].中国生物防治,13(2):23-25.
27.金同铭,宋家祥,李香玲,等.1989.抗小麦赤霉病抗生素861-A的提取、纯化及理化性质[J].微生物学报,29(4):317-319.
28.金文藻,孙承航,姜威,等.2002.生技霉素的组分研究[J].中国抗生素杂志,27(12):705-708.
29.李春香,董占能,张召术.2001.离子交换树脂在天然产物提取分离中的应用[J].云南化工,28(5):26-28.
30.李德舜,苏忠锐,袁志刚,等.2004.山东链霉菌产抗真菌物质的理化性质的初步研究[J].山东大学学报(理学版),39(6):121-124.
31.李广敏,郭秀林,商振清,等.1998.多胺与番茄对烟草花叶病病毒抗性关系的研究[J].华北农学报,13(2):65-69.
32.李晶,安德荣,李爱荣,等.2008.荧光假单胞杆菌AbIII745-6抑菌物质分离纯化和结构鉴定[J].微生物学杂志,28(2):11-14.
33.李鹏,李绍平,龚元香,等.2004.加压溶剂提取高效液相色谱法测定天然和人工虫草中的麦角甾醇、核苷及其碱基[J].药学学报,39(11):917-920.
34.李庆华,张李桃,陶黎明,等.2005.SPRI-231菌株活性组分分离纯化及抑菌作用简报[J].农药学学报,7(4):379-382.
35.李十中,胡永平.2001.高纯度土霉素碱制备工艺研究Ⅱ.土霉素新产品中2-乙酰-2-去酰胺土霉素的去除[J].中国抗生素杂志,26(6):437-439.
36.李增利.2004Nisin抗菌作用机制及抑菌效力[J].食品科技,(10):59-62.
37.李忠琴,林海英,孟春,等.2002.从结晶母液中提取分离卡那霉素A、B的工艺优化研究[J].中国抗生素杂志,27(2):111-115.
38.梁蓉芳,周启.1987.农抗5102产生菌原生质体融合育种的研究-Ⅲ.融合子的遗传分析[J].生物工程学报,3(2):130-136.
39.梁月荣,刘祖生,陆建良,等.1999.茶树根际土壤抗酸铝真菌ALF-1 (Neurosporasp.)抗酸铝机理[J].茶叶科学,(2):119-124.
40.廖芳,严红,赵素芳,等.2010.应用实时荧光定量RT-PCR检测几种食用菌蛋白抗烟草花叶病毒的活性[J].植物病理学报,40(6):622-627.
41.刘明周,伍学纲,陶黎明,等.2002.农用抗生素94166-II、III的提取、分离、纯化及结构鉴定[J].农药学学报,4(3):83-86.
42.刘秋.抗真菌生物农药-凯地菌素的研究[D].沈阳农业大学,2002.
43.刘训理,崔云龙,张国珍,等.1995.农用抗生素S-921有效组分的分离纯化及部分理化性质研究[J].山东农业大学学报,26(2):199-204.
44.柳焕英.1996.用离子交换处理从米糠中提取植酸的研究[J].武汉水利电力大学学报,29(6):117-119.
45.龙厚茹,谢德龄.1999.中生菌素对马铃薯青枯病菌的溶菌作用[J].中国生物防治,15(2):48-49.
46.芦昕婷.2010.苯菌酮:一种新型谷类白粉病杀菌剂的作用机制研究[J].世界农药,32(6):21-26.
47.陆丹,罗芬,池玉梅,等.2011.高效液相色谱法同时测定中药材虎掌南星的核苷类成分[J].色谱,29(1):83-86.
48.吕玲玲,雷建军,宋明.2003.抗生素的研究及其在农业上的应用[J].微生物学杂志,23(1):40-42.
49.马学萍,陈建刚,刘开全,等.2008.温度对心叶烟接种病毒后产生枯斑数的影响[J].曲靖师范学院学报,27(6):40-41.
50.牛赡光,王清海,张淑静,等.2009.化学杀菌剂和生防菌对棉花黄萎病联合作用机制研究[J].山东农业大学学报(自然科学版),40(2):247-250.
51.权春善,郑维,曹治明,等.2005.洋葱伯克霍尔德菌CF-66抗菌物质的分离纯化及性质的研究[J].微生物学报,45(5):707-710.
52.邵秀玲,甘琴华,厉艳,等.2011.利用TaqMan探针实时荧光PCR方法检测香石竹细菌性萎蔫病菌[J].植物病理学报,41(1):24-30.
53.申莉莉,杨金广,王凤龙,等.2010.实时荧光RT-PCR检测By33蛋白抑制TMV复制的作用[J].中国烟草学报,16(3):86-89.
54.申莉莉,杨金广,王凤龙,等.2010.实时荧光RT-PCR检测By33蛋白抑制TMV复制的作用[J].中国烟草学报,16(3):86-89.
55.施跃峰,桑金隆,竺利红,等.2004.新微生物农药抑霉菌素的研究[J].核农学报,18(1):68-71.
56.司书毅,娄志贤,陶佩珍,等.2000.抗病毒抗生素16704A的研究Ⅰ.产生菌的分类、发酵及活性成分的提取、分离与鉴别[J].中国抗生素杂志,25(6):407-409.
57.宋风平,王树桐,胡同乐,等.2009.芒果苷对马铃薯晚疫病菌的抑菌作用机制初探[J].农药学学 报,11(2):213-218.
58.宋晓妍,陈秀兰,孙彩云,等.2005.棉花黄萎病菌拮抗木霉的筛选及其抑菌机制的研究[J].山东大学学报(理学版),40(6):98-102.
59.隋勤.2007.利迪链霉菌A02拮抗产物的分离纯化和鉴定[D].东北农业大学.
60.孙备.2000.细叶冬青中抗微生物作用的三萜类化合物及其抗真菌作用机理[J].国外医学(中医中药分册),22(2):102-103.
61.孙宁,邓丛良,么磊,等.2010.应用细菌磁颗粒实时荧光RT-PCR检测南瓜花叶病毒[J].植物病理学报,40(6):632-635.
62.孙强.2007.新型抗真菌抗生素SN06的分离纯化研究[D].中国科学院研究生院(大连化学物理研究所)
63.谭天伟,沈忠耀.1996.双水相萃取分离技术的评价和展望[J].微生物学通报,23(6):368-370.
64.汤惠英,吕伟清.1983.薄层层析定量测定方法在抗生素组份测定中的应用Ⅱ.红霉素组份薄层层析定量测定方法及应用[J],抗生素,8(3):168-177.
65.陶黎明,顾学斌,倪长春,等.2002.长川霉素的研究[J].农药,41(12):11-13.
66.田颖川,王祈楷.1982.放线菌素D对烟草花叶病毒RNA在离体烟叶中复制的影响[J].微生物学报,22(4):316-320.
67.王德茂,高玉斌,张海余,等.1986.公主岭霉素复合剂的研究[J].生物防治通报,2(2):89-91.
68.王芳,纪明山,谷祖敏,等.2009.苦参内生枯草芽孢杆菌B(36)抗菌物质对番茄叶霉病菌的作用机制[J].中国生物防治,25(3):250-254.
69.王建,李忠红,倪坤仪,等.2006.高效液相色谱-电喷雾离子阱质谱法分析6种氨基糖甙类抗生素[J].分析化学,34(7):979-982.
70.王艳红,吴元华,朱春玉,等.2006.嘧肽霉素又一抗病毒活性成分的研究[J].沈阳农业大学学报,37(1):44-47.
71.王源超,张正光,李俊,等.2003.H2O2参与棉疫病菌90kD蛋白激发子诱导的烟草过敏反应和系统获得抗性[J].植物生理与分子生物学学报,29(3):185-191.
72.魏颖颖,吴元华,张晓雯,等.2008.嘧肽霉素生物合成阻断突变株原生质体制备和再生条件的研究[J].沈阳农业大学学报,39(2):174-177.
73.吴绍熙,郭宁如,侯幼红.1998.抗真菌药物作用机理的研究进展[J].辽宁医学杂志,12(4):14-17.
74.吴元华,高芬,杜春梅,等.2008.嘧肽霉素高产菌株的选育[J].中国生物防治,24(1):58-62.
75.吴元华,朱春玉,杜春梅,等.2005.嘧肽霉素影响烟草花叶病毒RNA蓄积量的研究[J].病毒学报,21(4):311-313.
76.夏立钧,王锦华,罗国芳,等.1995.酶解液中脱氧核苷的高效液相色谱测定[J].分析化学,23(5):558-560.
77.谢小梅,许杨.2004.抗真菌中药的作用机理研究进展[J].中国中药杂志,29(3):12-14.
78.徐绍华,田颖川,覃秉益,等.1987.用电镜放射自显影术研究TMV-RNA在烟叶细胞中的复制[J].微生物学报,27(1):17-22.
79.徐铮,曹永兵,姜远英.2001.麦角甾醇生物合成途径中的抗真菌药作用靶酶[J].国外医药(抗生素分册),22(5):193-197.
80.阎国华,甘立军,周燮.1999.用H103大孔吸附树脂提取赤霉素A_3的初步研究[J].南京农业大学学报,22(1):25-28.
81.杨翠云,于翠,张舒亚,等.2006.利用实时荧光RT-PCR方法检测番茄环斑病毒和烟草环斑病毒[J].植物检疫,增刊:31-34.
82.杨伟东,郑耘,陈枝楠,等.2007.烟草环斑病毒RT-RealtimePCR检测方法[J],植物保护学报,34(2):157-160.
83.易汪雪,陈舜胜,杨翠云,等.2011.单管实时荧光RT-PCR方法同时检测大豆种子中的菜豆荚斑驳病毒和烟草环斑病毒[J].植物病理学报,41(1):85-92.
84.余凤玉,李振华,曾会才.2005.抗真菌农用抗生素的研究进展[J].热带农业科学,25(1):60-65.
85.余立,王俊秋.2005.高效液相色谱法在中国药典2005年版抗生素品种中的应用[J].中国抗生素杂志,30(12):709-711.
86.俞用川,张宽厚.1959.改进的中性红培养基的研究及其中抑菌剂的作用机制[J].微生物学报,7(3):245-250.
87.张道来,陈军辉,周明,等.2010.反相高效液相色谱法同时测定罗氏海盘车中的7种核苷化合物[J].色谱,28(8):795-799.
88.张宁波.2006.农用抗生素研究进展[J].湖北农业科学,45(6):830-833.
89.张穗,唐文华,谈文.2002.井冈霉素A处理珊西烟后对TMV枯斑数目和抗性相关酶的影响[J].中国烟草学报,8(4):39-42.
90.张纬,黄海,孙晓明,等.2009.柠檬提取物抑菌、杀灭病毒作用机制研究[J].中国微生态学杂志,21(5):430-434.
9I.章观德,刘洪月,梁意红.1986.薄盖灵芝发酵菌丝体中主要核苷及其碱基的反相高效液相色谱测定[J].药学学报,(1):35-39.
92.赵秀香,吴元华,杜春梅,等.2004.新型农药嘧肽霉素防治番茄病毒病药效[J].农药,43(12):534-536.
93.赵子翰,李俊英,杨晓雁,等.2003.SIIA-C2191-A和B,黄灰链霉菌产生的多环口山酮类新抗生素I.菌种分类,发酵,分离纯化和生物学活性(英文)[J].中国抗生素杂志,28(10):627-632.
94.郑昌亮,唐秋瑾.1986.使用高效液相色谱分析检测抗生素[J].抗生素,11(3):261-266.
95.郑裕国,虞炳钧,陈小龙,等.1996.离子交换法提取井冈霉素的研究[J].农药,35(11):9-10.
96.中国科学院微生物研究所.1973.春雷霉素[J].化学通报,2(1):2.
97.周国梁,尚琳琳,林泓,等.2011.油菜茎基溃疡病菌的实时荧光PCR检测[J].植物病理学报,41(1):I0-17.
98.周利娟,焦阳,姚正颖,等.2009.海洋链霉菌GB-2产抗真菌物质的分离纯化和稳定性研究[J].中国海洋药物.28(4):35-39.
99.朱斌.蒋受军,胡昌勤.2002.高效毛细管电泳法测定加替沙星含量[J].药物分析杂志,22(5):397-400.
100.朱昌雄,蒋细良.2004.我国农用抗生素的研发现状及其进展[J].现代化工,24(10):1-4.
101.朱昌雄,倪楚芳,谢德龄,等.1993.农抗120对水稻纹枯病菌抗生活性试验[J].生物防治通报,9(1):15-18.
102.朱昌雄,谢德龄,倪楚芳.1990.农抗120防治作物真菌病害的效果评价[J].生物防治通报,6(4):179-182.
103.朱春玉,吴元华,王春梅,等.2005.嘧肽霉素对烟草花叶病毒抑制作用研究[J].植物保护,31(4):53-55.
104.朱春玉,吴元华,赵秀香,等.2006.嘧肽霉素抗烟草花叶病毒作用机理初步研究[J].植物病理学报,36(4):314-316.
105.朱春玉,吴元华,赵秀香,等.2007.嘧肽霉素对烟草花叶病毒在不同寄主上的防效研究[J].辽宁大学学报(自然科学版),34(1):50-53.
106.朱小平,陈曦,周翼衡,等.1998.转复制酶基因抗病毒烟草的研究[J].中国烟草学报,4(1):20-23.
107.朱晓峰.2009.杀线虫菌株Snf009的产业化基础研究[D].沈阳农业大学.
108. Aoki S, Takebe I.1975. Replication of tobacco mosaic virus RNA in tobacco mesophyll protoplasts inoculated in vitro[J], Virology.65(2):343-354.
109. Auger-Buendia M A, Hamelin R, Tavitian A.1978. Influence of toyocamycin on the assembly and processing of preribosomal ribonucleoproteins in the nucleolus of mammalian cells[J]. Biochim Biophys Acta,521(1):241-250.
110. Brock M, Buckel W.2004. On the mechanism of action of the antifungal agent propionate[J]. Eur J Biochem,271(15):3227-3241.
111. Buck K W.1999. Replication of tobacco mosaic virus RNA[J]. Plil Trans R Soc Lond B,354: 613-627.
112. Chaerle L, Lenk S, Hagenbeek D, et al.2007. Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus[J]. Russ J Plant Physl,164(3):253-262.
113. Culver J N, Stubbs G, Dawson W O.1994. Structure-function Relationship Between Tobacco Mosaic Virus Coat Protein and Hypersensitivity in Nicotiana sylvestris[J]. J Mol Biol,242(2): 130-138.
114. Del Pozo O, Lam E.1998. Caspases and programmed cell death in the hypersensitive response of plants to pathogens[J]. Curr Biol,8(20):1129-1132.
115. Gao W, Tian L, Zhou J, et al.2009. Antifungal mechanism of Bacillus marinus B-9987[J]. Wei Sheng Wu Xue Bao,49(11):1494-1501.
116. Hadjiolova K V, Naydenova Z G, Hadjiolov A A.1981. Inhibition of ribosomal RNA maturation in Friend erythroleukemia cells by 5-fluorouridine and toyocamycin[J]. Biochem Pharmacol,30(13): 1861-1863.
117. Hatano K, Morishita Y, Nakai T, et al.2002. Antifungal mechanism of FK463 against Candida albicans and Aspergillus fumigatus[J]. J Antibiot (Tokyo),55(2):219-222.
118. Hayashi K, Kamio S, Oono Y, et al.2009. Toyocamycin specifically inhibits auxin signaling mediated by SCFTIR1 pathway[J], Phytochemistry,70(2):190-197.
119. Ishikawa M, Meshi T, Watanabe Y, et al.1988. Replication of chimeric tobacco mosaic viruses which carry heterologous combinations of replicase genes and 3'noncoding regions[J]. Virology, 164(1):290-293.
120. Janiak A M, Milewski S.2001. Mechanism of antifungal action of kanosamine[J]. Med Mycol, 39(5):401-408.
121. Knoester M, Linthorst H J M, Bol J F, et al.2001. Involvement of ethylene in lesion development and systemic acquired resistance in tobacco during the hypersensitive reaction to tobacco mosaic virus[J]. Physiol Mol Plant P,59(1):45-57.
122. Kocsis B, Kustos I, Kilar F, et al.2009. Antifungal unsaturated cyclic Mannich ketones and amino alcohols:study of mechanism of action[J]. Eur J Med Chem,44(5):1823-1829.
123. Kubo I, Fujita K, Lee S H.2001. Antifungal mechanism of polygodial[J]. J Agric Food Chem,49(3): 1607-1611.
124. Larsen C J, Mauchauffe M, Hamelin R, et al.1979. Effect of toyocamycin on the synthesis of the 70S RNA of a murine retrovirus[J]. Nucleic Acids Res,6(4):1547-1556.
125. Mccarty R M, Bandarian V.2008. Deciphering deazapurine biosynthesis:pathway for pyrrolopyrimidine nucleosides toyocamycin and sangivamycin[J], Chem Biol.15(8):790-798.
126. Monneron A, Burglen J, Bernhard W.1970. Action of toyocamycin on nucleolar fine structure and function[J]. J Ultrastruct Res,32(3):370-389.
127. Montalbini P.1991. Enhanced uricase activity in tobacco mosaic virus infected tobacco leaves[J]. Plant Sci,74(2):261-265.
128. Moyer S A, Holmes K S.1979. The specific inhibition of vesicular stomatitis virus replication by toyocamycin[J]. Virology,98(1):99-107.
129. Nishiyama Y.2002. Structure of fungi and the action mechanism of antifungal agents[J]. Jpn J Antibiot,55(6):902-906.
130. Ogawa T, Watanabe Y, Okada Y.1992. Cis-acting elements for in trans complementation of replication-defective mutant of tobacco mosaic virus[J]. Virology,191(1):454-458.
131. Osbourn J K, Watts J W, Beachy R N, et al.1989. Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein[J]. Virology,172(1):370-373.
132. Pritchard D W, Ross A F.1975. The relationship of ethylene to formation of tobacco mosaic virus lesions in hypersensitive responding tobacco leaves with and without induced resistance[J]. Virology,64(2):295-307.
133. Rezaian M A, Francki R I B.1974. Replication of tobacco ringspot virus:II. Differences in nucleotide sequences between the viral RNA components[J]. Virology,59(1):275-280.
134. Saito T, Yamanaka K, Watanabe Y, et al.1989. Mutational analysis of the coat protein gene of tobacco mosaic virus in relation to hypersensitive response in tobacco plants with the N'gene[J]. Virology,173(1):11-20.
135. Shin R, Lee G, Park C, et al.2001. Isolation of pepper mRNAs differentially expressed during the hypersensitive response to tobacco mosaic virus and characterization of a proteinase inhibitor gene[J]. Plant Sci,161(4):727-737.
136. Smart T E, Dunigan D D, Zaitlin M.1987. In vitro translation products of mrnas derived from TMV-infected tobacco exhibiting a hypersensitive response[J]. Virology,158(2):461-464.
137. Stange C, Matus J T, Domnguez C, et al.2008. The N-homologue LRR domain adopts a folding which explains the TMV-Cg-induced HR-like response in sensitive tobacco plants[J]. J Mol Graphics Modell,26(5):850-860.
138. Sverak L, Bonar R A, Langlois A J, et al.1970. Inhibition by toyocamycin of RNA synthesis in mammalian cells and in normal and avian tumor virus-infected chick embryo cells[J]. Biochim Biophys Acta,224(2):441-450.
139. Venkov P V, Stateva L I, Hadjiolov A A.1977. Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic-sensitive adenosine-utilizing Saccharomyces cerevisiae mutant[J]. Biochim Biophys Acta,474(2):245-253.
140. Wang Y C, Hu D W, Zhang Z G, et al.2003. Purification and immunocytolocalization of a novel Phytophthora boehmeriae protein inducing the hypersensitive response and systemic acquired resistance in tobacco and Chinese cabbage[J]. Physiol Mol Plant P,63(4):223-232.
141. Yan R, Hou J, Ding D, et al.2008. In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi[J]. J Basic Microbiol,48(4):293-301.