A β-glucosidase from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K _m and V _max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly inhibited by Fe²⁺ and activated about 35% by Ca²⁺. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and ¹³C nuclear magnetic resonance spectroscopy.     

柑桔内生拮抗细菌的发酵液对油菜菌核病的田间防治效果

从柑桔健康组织中分离得到两株内生拮抗细菌"重桔1号"和"元石45",制成细菌悬浮液, 25℃恒温水浴振荡培养72h, 喷雾于接有油菜菌核病菌的油菜成株叶片上,以喷灭菌水为对照. 结果表明,喷有"重桔1号"和"元石45"两株细菌的发酵液的叶片菌核病菌病斑比对照叶片病斑扩展明显减小,与对照相比其病斑面积平均少扩大值分别为9.75cm2和10.62cm2 , 两株细菌的发酵液对油菜菌核病的绝对防治效果分别为19.5%和47.5%.     

向日葵品系对菌核病的抗性鉴定

在2002～2003年连续两年对经初步抗性筛选所获得的儿个向日葵品系进行了菌核病的抗性鉴定．结果表明：在所有的参鉴材料中没有发现免疫类型，但各品系间抗性有一定的差异．经两年的抗性筛选后发现AS02、AS03、AS04、AS07和AS08等5个品系对根腐型和盘腐型菌核病的抗性显著高于生产上中抗杂交种龙葵杂1号等对照品种．     

基于热红外成像技术的油菜菌核病早期检测研究

将热红外成像技术和低空遥感技术相结合,基于冠层和叶片两个尺度对菌核病侵染油菜的过程进行检测研究。从冠层尺度分析,首先获取整株样本的温度值(平均温度与最大温差),并采集其生理指数(气孔导度、CO₂浓度、蒸腾速率及光合速率)。然后,将染病样本与健康样本的温度值进行判别分析,并对其进行单因素方差分析。从结果可知,平均温度和最大温差值都可以对染病样本与健康样本进行区分,且最大温差相较平均温度结果较明显。同时单因素方差分析也显示,最大温差三次检测中均存在显著性差异。对获取的生理指数进行分析,发现染病样本与健康样本之间可以通过生理指数进行明显区分。另外,将生理指数与叶片温度进行相关性分析,结果表明二氧化碳浓度与叶片温度之间的三次检测均存在显著性差异。基于叶片尺度,首先从单一叶片来看健康区域和染病区域的温度差异,可以明显区分出染病区域和健康区域的温度差异。然后,提取健康区域与染病区域的的温度值(最大温度、最小温度、平均温度以及最大温差)对进行对比分析,并对其进行单因素方差分析。结果表明,以上四个温度指标均可以区分叶片的染病区域和健康区域。但根据单因素方差分析结果可知,与冠层尺度相同,最大温差三次检测中均存在显著性差异,可以实现对油菜菌核病的早期识别。     

核盘菌交配型基因mat1-1敲除载体的构建及转化

利用根癌农杆菌介导真菌遗传转化方法对核盘菌的交配型基因mat1-1进行基因同源重组的敲除对比实验,获得了缺失mat1-1基因的转化菌株.先利用PCR方法获得mat1-1基因的左右两侧片段,将测序正确的两个侧翼片段分别重组到农杆菌转化载体PBI-G3C中,并将新霉素抗性标记引入农杆菌转化载体PBI-G3C中,构建成农杆菌介导转化核盘菌的打靶载体ΔPBI-G3CN-mat1-1,再将ΔPBI-G3CN-mat1-1质粒转化至根癌农杆菌EHA105中.利用核盘菌菌丝进行转化,将得到的转化菌株,利用PCR方法进行验证,证实有敲除菌株存在.通过对敲除菌株进行生理表型的测定发现,缺失mat1-1基因的敲除菌株其生长速度与野生核盘菌菌株相比无明显差异,但敲除菌株不能产生菌核与子囊盘.     

周口地区油菜核盘菌分离鉴定及侵染分析

从河南省周口市采集感染菌核病的油菜,对菌核进行分离和鉴定.采用单孢分离法分离得到1个单菌系,对该单菌系进行了菌落和显微形态分析,初步鉴定该菌系为Sclerotinia sclerotiorum.利用真菌通用引物ITS1和ITS4对所分离的单菌系内转录间隔区(inter transcribed spacer,ITS)进行PCR扩增,得到540bp大小的片段,经过克隆测序和在GenBank中进行BLASTn分析,发现与部分S.sclerotiorum的ITS序列同源性达到100%,进一步鉴定该分离菌株为S.sclerotiorum.进化树分析显示,该菌系与来自意大利、日本及我国武汉地区的菌系聚为一支,说明周口菌系来源广泛.对该菌系侵染油菜叶片的细胞学和组织化学分析表明,该菌系对耐病和感病油菜品种的细胞反应相似,都没有H2O2积累,差别主要表现在耐病油菜上菌丝长度较短.     

肉豆蔻中赭曲霉毒素的测定及赭曲霉毒素产毒菌的分离鉴定

建立了同时测定肉豆蔻中3种赭曲霉毒素的超高效液相色谱-串联质谱法(UPLC-MS/MS).样品经70％甲醇超声提取,HLB柱净化,采用Agilent Proshell 120 EC C18色谱柱,以乙腈(含0.1％甲酸)-水(含0.1％甲酸)为流动相进行梯度洗脱,于电喷雾离子源正负离子模式下多反应监测模式检测.结果表明...     

Production, purification, and biochemical characterization of two β-glucosidases from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum prudces ß-glucosidases in liquid culture with a variety of carbon sources, including cellulose (filter paper), xylan, barley straw, oat meal, and xylose. Analysis by native polyacrylamide gel electrophoresis (PAGE) followed by an activity staining with the specific chromogenic substrate, 5-bromo 4-chloro 3-indolyl ß-1,4 glucoside (X-glu) showed that two extracellular β-glucosidases, designated as ß-glul1 and \-glu2, were in the filter paper culture filtrate. Only one enzyme designated as ß-glus was revealed by the same method in the xylose culture filtrate. ß-glu1 and ß-glu2 were purified to homogeneity. The purification procedure consist of a common step of anion-exchange chromatography on DEAE-Sepharose CL6B, both high-performance liquid chromatography (HPLC) anion-exchange and gel filtration columns for ß-glu1 and only HPLC gel filtration for ß-glu2. ß-glu1 has a molecular mass of 196 kDa and 96.5 kDa, as estimated by gel filtration and sodium dodecyl sulfate (SDS)-PAGE, respectively, suggesting that the native enzyme may consist of two identical subunits. The same analysis showed that ß-glu2 is a monomeric protein with an apparent molecular mass of about 76.5 kDa. ß-glu1 and ß-glu2 hydrolyses PNPG1c and cellobiose, with apparent K _m values respectively for PNPGlc and cellobiose of 0.1 and 1.9 mM for ß-glu1 and 2.8 and 8 mM for ß-glu2. Both enzymes exhibit the same temperature and pH optima for PNPG1c hydrolysis (60°C and pH 5.0). ß-glu1 was stable over a pH range of 3–8 and kept 50% of its activity after 30 min of heating at 60°C without substrate. It was further characterized by studying the effect of some cations and various reagents on its activity.     

Transmissible hypovirulent element in isolate Ep-1PN ofSclerotinia sclerotiorum

The cause of virulence decline of hypovirulent isolate Ep-1PN ofSclerotinia sclerotiorum (Lib.) de Bary has been studied through transmitting test and analysing its offsprings derived from single ascospore. When the colony of a normal strain Ep lPNA 183 intermingled with the colony of Ep-lPN, its margin became abnormal, just like EplPN. Cultures isolated from toptip of the colony of Ep-1PNA 183 showed the hypovirulence and its associated properties of Ep-1PN. Moreover, a series of cultures which have characteristics between EplPN and normal strain could be isolated from stipes of Ep-1PN. More than 200 offsprings derived from single ascospore of Ep-1PN were normal strains. According to those results, there exists a transmissible cytoplasmic hypovirulent element in hypovirulent isolate EplPN of S.sclerotiorum.     

深层发酵核盘菌多糖的研究

核盘菌多糖是由核盘菌（Ｓｃｌｅｒｏｔｉｎｉａ ｓｃｌｅｒｏｔｉｏｒｕｍ）通过深层发酵产生的一种胞外多糖。本文通过单因子试验和正交试验对其摇瓶发酵条件进行了优选，使ＳＳＧ的产率达到１２．０３ｇ／Ｌ，碳源转化率达４０．１％；纸层析和红外光谱分析表明，ＳＳＧ是一种由β－键连接的葡聚糖，其水溶液具有良好的增粘性和假塑性。