Chilvers Martin I, du Toit Lindsey J, Akamatsu Hajime, Peever Tobin L
Postdoctoral Research Associate.
Associate Scientist.
Plant Dis. 2007 May;91(5):599-608. doi: 10.1094/PDIS-91-5-0599.
A real-time fluorescent polymerase chain reaction (PCR) assay was developed using SYBR Green chemistry to quantify the Botrytis spp. associated with onion (Allium cepa) seed that are also able to induce neck rot of onion bulbs, i.e., B. aclada, B. allii, and B. byssoidea. The nuclear ribosomal intergenic spacer (IGS) regions of target and nontarget Botrytis spp. were sequenced, aligned, and used to design a primer pair specific to B. aclada, B. allii, and B. byssoidea. Primers and amplification parameters were optimized to avoid amplifying the related species B. cinerea, B. porri, and B. squamosa, as well as Sclerotinia sclerotiorum and isolates of 15 other fungal species commonly found associated with onion seed. The primers reliably detected 10 fg of genomic DNA per PCR reaction extracted from pure cultures of B. aclada and B. allii. Conventional assays of surface-disinfested and nondisinfested seed on an agar medium were used to determine the incidence of neck rot Botrytis spp. associated with each of 23 commercial onion seed lots, and the real-time PCR assay was used to determine the quantity of DNA of neck rot Botrytis spp. in each seed lot. A linear relationship could not be found between the incidence of seed infected with the neck rot Botrytis spp. using the conventional agar seed assays and the quantity of DNA of the neck rot Botrytis spp. detected by the real-time PCR assay. However, the real-time PCR assay appeared to be more sensitive than the conventional agar assay, allowing detection of neck rot Botrytis spp. in 5 of the 23 seed lots that tested negative using the conventional agar seed assay.
利用SYBR Green化学技术开发了一种实时荧光聚合酶链反应（PCR）检测方法，用于定量分析与洋葱（Allium cepa）种子相关的、也能够引发洋葱鳞茎颈部腐烂的葡萄孢属真菌，即黑葡萄孢、葱葡萄孢和棉絮状葡萄孢。对目标和非目标葡萄孢属真菌的核糖体基因间隔区（IGS）进行了测序、比对，并用于设计一对特异于黑葡萄孢、葱葡萄孢和棉絮状葡萄孢的引物。对引物和扩增参数进行了优化，以避免扩增相关物种灰葡萄孢、葱柄葡萄孢和鳞茎葡萄孢，以及核盘菌和其他15种常见于洋葱种子上的真菌分离物。这些引物在每个PCR反应中能够可靠地检测到从黑葡萄孢和葱葡萄孢纯培养物中提取的10 fg基因组DNA。采用在琼脂培养基上对表面消毒和未消毒种子进行常规检测的方法，来确定与23个商业洋葱种子批次中每一批相关的颈部腐烂葡萄孢属真菌的发生率，并用实时PCR检测方法来测定每个种子批次中颈部腐烂葡萄孢属真菌的DNA含量。在使用常规琼脂种子检测方法检测到的感染颈部腐烂葡萄孢属真菌的种子发生率与通过实时PCR检测方法检测到的颈部腐烂葡萄孢属真菌的DNA含量之间未发现线性关系。然而，实时PCR检测方法似乎比常规琼脂检测方法更灵敏，能够在23个种子批次中的5个批次中检测到颈部腐烂葡萄孢属真菌，而这些批次使用常规琼脂种子检测方法检测为阴性。
