花生疮痂病菌毒素提取工艺及生物活性研究

Extraction Technology and Activity Analysis of Elsinoё arachidis Toxin

为明确花生疮痂病菌毒素种类、提取方法及生物活性,对花生疮痂病菌毒素进行颜色反应、紫外-可见光谱分析和薄层色谱分析,以提取液中的毒素含量为指标考察毒素的较优提取工艺,并采用离体叶片测定毒素生物活性。结果表明:花生疮痂病菌毒素提取液呈红色,遇碱变绿,遇酸恢复为红色;用紫外分光光度计进行全波长扫描,发现在460nm、525nm、565nm处出现最大吸收峰;薄层色谱分析中出现5条清晰条带,Rf值分别为0.68,0.50,0.35,0.22和0.08。初步明确花生疮痂病菌所产毒素为痂囊腔菌素(Elsinochrome)。花生疮痂病菌毒素以超声振荡法提取效果最佳,提取液中毒素含量为3.11mg·g^(-1),较优提取工艺为:提取试剂为丙酮,菌丝粉碎细度为60目,液料比为100∶1,超声温度为40~55℃,超声时间为60min,超声功率为70~100W,超声提取2次。毒素的生物活性测定结果表明,该毒素具有较强的致病性,能够产生与病原菌侵染相似症状,病斑扩展与毒素接种浓度呈正相关,表明毒素痂囊腔菌素是花生疮痂病菌的重要致病因子。

In order to identify and determine the extraction technology and bioactivity of the toxin produced by Elsinoёarachidis,investigation on coloration reaction, UV-visible spectrum, thin-layer chromatography（TLC） of the toxin were performed. The results showed that the color of the toxin produced by E. arachidis in acetone was red and turned green when dissolved in alkaline solution, then recovered red again when dissolved in acid solution. The maximum absorption peaks at 460 nm, 525 nm and 565 nm were observed under visible light scanning. The Rfvalues of five bands separated by TLC of toxin extraction were0.68, 0.5, 0.35, 0.22 and 0.08, respectively. The results showed that the toxin produced by E. arachidis was elsinochrome. The optimal extraction method was ultrasonic oscillation（3.11 mg·g^-1） and the optimal extraction technology was as follow： solvent acetone, fineness of hypha 60 meshes, ratio of liquor to material 100∶1, temperature 40-55 ℃, time 60 min, power 70-100 W and2 times. To estimate the activity of the toxin, the detached leaves were inoculated by toxin. The results showed that the toxin had pathogenicity to peanut leaves which caused same symptom as E. arachidis did and the extension of scab was positively correlated with toxin concentration. The toxin is an important pathogenic factor of E. arachidis.