玉蜀黍黑粉菌环介导等温扩增检测体系的建立及应用

 本文根据玉蜀黍黑粉菌(Ustilago maydis )的UmPep1、UmPit2和UmSee1基因各设计4套环介导等温扩增(LAMP)引物,从中筛选出1套引物对LAMP反应体系进行3因素(Bst DNA聚合酶浓度、Mg²⁺浓度、内外引物浓度比)3水平的优化试验。并对优化的U. maydis LAMP反应体系进行特异性、灵敏度及田间检测可行性试验。特异性试验表明,该方法能特异性检测U. maydis,而与其他病原菌的DNA没有交叉反应;灵敏度试验表明,该反应体系的最低检出限为44 fg·μL^-1 pEasy-Pep质粒DNA,制作的标准曲线可对U. maydis进行定量分析。该方法也适用于在U. maydis侵染前或侵染早期对田间样品进行检测,对现场采集的172份田间样品进行检测,其中140个样品显示为阳性。本研究所建立的LAMP体系具有特异性好、灵敏度高、重复性好的特点,并能在45 min内完成对田间样品的检测,是快速、定量检测U. maydis的有效手段。     

Survey and examination of the potential alternative hosts of Villosiclava virens,the pathogen of rice false smut,in China

Rice false smut is caused by ascomycete Villosiclava virens, whose potential alternative hosts have been assumed previously. Here its potential alternative hosts were surveyed and identified from 2008 to 2013 in the main rice-growing regions in China. Two common weeds in paddy fields, Digitaria sanguinalis Scop. and Echinochloa crusgalli(L.) Beauv., were found to present the similar symptoms to smut diseases in a few individuals in 2012 and 2013 in Zhejiang and Sichuan provinces of China, respectively. After the examinations of the spore morphology, their infection and extension mode in hosts, pathogen cell wall components, and molecular identification, the two pathogens were identified to be the Basidiomycetes, Ustilago syntherismae and Ustilago trichophora, respectively. So far there has been no alternative host of V. virens to be identified in China. These suggest that the alternative hosts of V. virens, if they do exist, are not possible to play an important role in the pathogen life cycle and the disease epidemics.     

茭白黑粉菌不同培养形态及能谱分析

将茭白灰茭内的黑粉菌厚垣孢子放置在PSA培养基上培养,结果表明:茭白黑粉菌能够在PSA培养基上完成其生活史,但形成菌丝体及厚垣孢子需要较长的时间;X-EDS分析发现不同培养形态之间的原子比值存在差异,其中采集的与培养的厚垣孢子差异明显.     

玉米瘤黑粉病菌生物学特性研究

对影响玉蜀黍黑粉菌冬孢子萌发和双核菌丝生长的生物学特性进行研究。结果表明,冬孢子萌发最适温度为30℃,pH为3,碳源为蔗糖、麦芽糖和葡萄糖,氮源为半胱氨酸、天冬氨酸和甘氨酸;双核菌丝生长的最适温度为30℃,pH为8,碳源为葡萄糖和蔗糖,氮源为天冬氨酸、半胱氨酸和甘氨酸。光照条件对冬孢子萌发和菌丝生长的影响不显著。     

利用斑茅cDNA芯片研究甘蔗受黑穗病菌侵染后基因差异表达

为了解甘蔗抗黑穗病的分子机制,利用斑茅干旱胁迫cDNA芯片检测了甘蔗受黑穗病菌胁迫后的基因差异表达。经杂交,在cDNA芯片的3 860个模板中,有效差异表达(Ratio值≥2.0或≤0.5)的基因为101个,其中上调55个,下调46个。部分基因的定量PCR验证表明,芯片杂交结果可靠。上调表达基因经测序、冗余序列剔除,一共获得36个unique ESTs。已知功能的22个上调表达基因,涉及多条生理代谢途径,如光合作用、离子转运和核酸代谢途径;以及多种分子水平的进程,如基因转录、蛋白质合成与修饰以及细胞信号转导等。另外,还检测到14个未知功能基因。结果表明,甘蔗对黑穗病的抗性具有复杂的机制。本研究为解释甘蔗受黑穗病菌胁迫后的基因差异表达及其网络构建奠定了基础,还可以为今后系统研究甘蔗对生物与非生物胁迫的响应机制提供技术支持。     

吡虫啉·戊唑醇种子处理悬浮剂对燕麦红叶病的田间防效

通过田间对比试验,测试了吡虫啉.戊唑醇种子处理悬浮剂对燕麦红叶病的防治效果,结果表明,采用(120 mL高巧+42 mL立克秀)/100 kg种子(商品量)拌种包衣,可显著提高皮燕麦的出苗率,促进幼苗生长,对皮燕麦、裸燕麦红叶病的防效分别达84.97%和47.87%,皮燕麦、裸燕麦的增产率分别达60.43%和48.96%。     

不同海拔高度对茭白生长及孕茭的影响

对在海拔高度200～1 000 m 地区栽培的单季茭的生长和孕茭特性进行研究。结果表明：高海拔地区栽培的茭白萌芽期、出叶和株高增速峰值均比低海拔处明显延迟。高海拔处茭白萌芽较低海拔处需要更多5 ℃以上的有效积温;各海拔处茭白出叶速率和株高增速与有效积温间呈正相关。各海拔高度孕茭先后顺序为：410 m ＞ 650 m ＞ 1 008 m ＞ 815 m。各海拔处茭白的孕茭期均发生在气温适宜孕茭的时间段内。因此,海拔高度对茭白生长和孕茭的影响主要是通过不同海拔高度的温度差异来实现的,高温抑制茭白黑粉菌的正常生长及代谢可能是茭白夏季高温条件下难以孕茭的重要原因。     

青稞坚黑穗病LAMP快速检测技术的建立

由大麦坚黑粉菌(Ustilgo hordei)引起的坚黑穗病广泛分布于我国青稞主产区,病原侵染后形成病穗从而造成产量损失。基于前期初步建立的青稞黑穗病快速检测体系,通过对种子洗涤沉淀物的总基因组DNA进行提取,并进行环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测人工模拟及实际种子带菌情况,以期建立完善的青稞坚黑穗病快速检测技术。结果表明,裂解液快速提取法可以对样品总DNA进行有效提取,完善后的LAMP快速检测技术可以有效开展青稞种子携带大麦坚黑粉菌的检测。     

Changes in soluble and cell wall-bound hydroxycinnamic and hydroxybenzoic acids in sugarcane cultivars inoculated with <Emphasis Type="Italic">Sporisorium scitamineum</Emphasis> sporidia

The accumulation of soluble and cell wall-bound phenolics in the sugarcane stems of young plants from highly resistant cv. My 5514 and susceptible cv. B 42231, inoculated or not inoculated with smut sporidia, was studied. The ratio of inoculated to uninoculated plants of some cell wall-bound phenolics, such as ferulic, caffeic, and syringic acids increased for the resistant cv. My 5514, whereas it was maintained more or less constantly for the susceptible cv. B 42231. The highest increase of this ratio in the resistant cv. My 5514 corresponded to both caffeic and syringic acids. This could result in a better capacity to cv. My 5514 for an increase in the frequency of bridges between lignin fragments through ester-ether linkages for reinforcing the cell wall and major resistance to the disease. This reinforcement of the cell wall could provide an effective barrier to pathogen entry and spread. Soluble sub-fractions of all phenolics detected showed non-stable patterns. Caffeic acid, that regulates phenylalanine ammonia-lyase activity in sugarcane, showed a significant decrease in its titre at 24 h in the resistant cultivar, principally in the free soluble fraction, whilst the susceptible cultivar enhanced it. We hypothesise that the pathway of hydroxybenzoic acids is only activated once the level of p-coumaric acid justifies the accumulation of hydroxycinnamic acids required for reinforcing the cell wall after inoculation.     

茭白黑粉菌脉冲电泳分析方法

采用脉冲电泳法对茭白黑粉菌染色体进行核型分析。分析结果表明,茭白黑粉菌脉冲电泳样品处理方法包括原生质体的制备、电泳参数的设置等,对试验结果有一定影响,在制备电泳样品时,包埋的原生质体或孢子的浓度不能太低,蛋白酶K的浓度不能过高,低熔点琼脂糖的浓度不能太低,此外还应确保电泳槽水平,并且电泳结束后及时取出凝胶。