葡萄植原体黄化病及其检疫技术
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摘要
野生和栽培葡萄上的植原体病害由于引起叶片黄化而被称为葡萄黄化病。本研究称其为葡萄植原体黄化病(GPY),以便与生理性黄化症和由个别病毒引起的黄化症相区别。本研究对与GPY相关的检疫、检测和鉴定技术进行了系统研究,主要的研究结果如下:
①休眠期葡萄植株中植原体的浓度很低,而且分布不均匀。植原体主要存在于根系和尚未完全木栓化的枝条中。
②不同植原体检测引物的灵敏度不同,可能相差上千倍。
③Eppendorf管人工培养液饲养法该方法是筛选植原体介体昆虫的高效方法。
④首次发现自然感染了葡萄上的一种曾被误称为“Vergilungskrankheit”植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin。
⑤对嵌套引物P1/P7(U5/P7)-U5/U3的巢式PCR检测植原体时扩增出的0.36 kb的额外片段进行了研究。证明了该额外片段源于植原体16SrRNA基因上存在的一个与U5引物部分互补的位点。
⑥尝试结果表明:经过恰当设计,AFLP技术也可以用于植原体的分类和分型。
⑦研究认为把“由S. titanus传播”作为区分ALY植原体和FD植原体的条件之一是不合适的。ALY植原体也是FD植原体,即属于FD-C类型的FD植原体。
⑧对胶东葡萄产区GPY的调查结果表明:该地区尚未发生GPY;尚不存在已知GPY的传播介体。但有已知GPY的野生寄主植物分布。
⑨对GPY进行的有害生物风险分析结果表明:应当将FD植原体、ALY植原体、澳大利植原体候选种以及它们的传播介体列为中国检疫性有害生物。
⑩对葡萄金黄化病和S. titanus在中国的潜在分布区分析表明:渤海湾葡萄产区的胶东半岛和辽东半岛、甘肃、云南高原葡萄产区是二者的适生区域。
11制定了SN/T×××—2006《葡萄金黄化病植原体检疫鉴定方法》的检验检疫行业标准。同时为标准配套设计了FD植原体检测鉴定试剂盒。
Plant disorder occurred on wild and cultivated grapevines caused by phytoplasmas infection is usually called grapevine yellows. The typical symptom of the disease is shown as yellow leaves on the infected plant. In order to distinguish the disease from the yellow symptoms caused by several viruses and nutrition deficiency, this disease is referred as grapevine phytoplasma yellows (GPY) in this research. Comprehensive systematic research including quarantine, risk assessment, detection and identification was conducted on GPYs in this research. The results are as follows:
①Phytoplasma concentration inside dormant grapevine plants is extremely low, and its distribution is uneven. phytoplasmas mainly persist inside root and shoot tissues which are not completely suberized.
②Different primer pairs have different detect sensitivities, the differences among them are possibly over thousand-fold.
③The method of incubating leafhopper like insects inside Eppendorf tube is a highly effective method for the screening of phytoplasma insect vectors.
④Leafhopper Aprodes makarovi Zachvatkin naturally infected by so-called“Vergilungskrankheit”phytoplasma (AY group) was detected for the first time.
⑤Analysis of an extra 0.36 kb fragment amplified by P1/P7 (U5/P7) nested U5/U3 primers in grapevine yellow (Stolbur) phytoplasma detection revealed that this extra fragment located on phytoplasma 16SrRNA gene with a partial compliment site of U5 primer.
⑥The results show that AFLP technique can be used in the classification and typing of phytoplasmas when properly designed.
⑦It is unreasonable using“dissemination by S. titanus”as one of the preconditions to distinguish ALY phytoplasma from FD phytoplasma. ALY phytoplasma is actually FD phytoplasma, i.e. FD-C type of FD phytoplasma.
⑧No GPY was observed or detected during a survey of GPY in Jiaodong peninsula viticulture region. No known insect vector of GPY was found while wild host plants of GPY was found in this region.
①休眠期葡萄植株中植原体的浓度很低,而且分布不均匀。植原体主要存在于根系和尚未完全木栓化的枝条中。
②不同植原体检测引物的灵敏度不同,可能相差上千倍。
③Eppendorf管人工培养液饲养法该方法是筛选植原体介体昆虫的高效方法。
④首次发现自然感染了葡萄上的一种曾被误称为“Vergilungskrankheit”植原体(AY组)的草地脊冠叶蝉Aprodes makarovi Zachvatkin。
⑤对嵌套引物P1/P7(U5/P7)-U5/U3的巢式PCR检测植原体时扩增出的0.36 kb的额外片段进行了研究。证明了该额外片段源于植原体16SrRNA基因上存在的一个与U5引物部分互补的位点。
⑥尝试结果表明:经过恰当设计,AFLP技术也可以用于植原体的分类和分型。
⑦研究认为把“由S. titanus传播”作为区分ALY植原体和FD植原体的条件之一是不合适的。ALY植原体也是FD植原体,即属于FD-C类型的FD植原体。
⑧对胶东葡萄产区GPY的调查结果表明:该地区尚未发生GPY;尚不存在已知GPY的传播介体。但有已知GPY的野生寄主植物分布。
⑨对GPY进行的有害生物风险分析结果表明:应当将FD植原体、ALY植原体、澳大利植原体候选种以及它们的传播介体列为中国检疫性有害生物。
⑩对葡萄金黄化病和S. titanus在中国的潜在分布区分析表明:渤海湾葡萄产区的胶东半岛和辽东半岛、甘肃、云南高原葡萄产区是二者的适生区域。
11制定了SN/T×××—2006《葡萄金黄化病植原体检疫鉴定方法》的检验检疫行业标准。同时为标准配套设计了FD植原体检测鉴定试剂盒。
Plant disorder occurred on wild and cultivated grapevines caused by phytoplasmas infection is usually called grapevine yellows. The typical symptom of the disease is shown as yellow leaves on the infected plant. In order to distinguish the disease from the yellow symptoms caused by several viruses and nutrition deficiency, this disease is referred as grapevine phytoplasma yellows (GPY) in this research. Comprehensive systematic research including quarantine, risk assessment, detection and identification was conducted on GPYs in this research. The results are as follows:
①Phytoplasma concentration inside dormant grapevine plants is extremely low, and its distribution is uneven. phytoplasmas mainly persist inside root and shoot tissues which are not completely suberized.
②Different primer pairs have different detect sensitivities, the differences among them are possibly over thousand-fold.
③The method of incubating leafhopper like insects inside Eppendorf tube is a highly effective method for the screening of phytoplasma insect vectors.
④Leafhopper Aprodes makarovi Zachvatkin naturally infected by so-called“Vergilungskrankheit”phytoplasma (AY group) was detected for the first time.
⑤Analysis of an extra 0.36 kb fragment amplified by P1/P7 (U5/P7) nested U5/U3 primers in grapevine yellow (Stolbur) phytoplasma detection revealed that this extra fragment located on phytoplasma 16SrRNA gene with a partial compliment site of U5 primer.
⑥The results show that AFLP technique can be used in the classification and typing of phytoplasmas when properly designed.
⑦It is unreasonable using“dissemination by S. titanus”as one of the preconditions to distinguish ALY phytoplasma from FD phytoplasma. ALY phytoplasma is actually FD phytoplasma, i.e. FD-C type of FD phytoplasma.
⑧No GPY was observed or detected during a survey of GPY in Jiaodong peninsula viticulture region. No known insect vector of GPY was found while wild host plants of GPY was found in this region.
引文
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