A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed
A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed
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DOI：
10.1094/phyto-10-11-0280
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发表时间：
2012-04-01
期刊：
影响因子：
3.2
通讯作者：
Klosterman, Steven J.
中科院分区：
文献类型：
--
作者：
Duressa, Dechassa;Rauscher, Gilda;Klosterman, Steven J.
大丽花黄萎病是一种土壤传播的真菌，它会导致加州中部沿海地区多种作物的黄萎病。尽管在该地区种植的新鲜和加工商业生产的菠菜作物没有显示黄萎病症状，但在美国或欧洲生产的菠菜种子通常感染了大丽花弧菌。种植受感染的种子增加了土壤的接种密度，并可能引入外来菌株，导致生菜和其他与菠菜轮作的作物流行黄萎病。一种灵敏、快速、可靠的定量菠菜种子中大丽花弧菌的方法，将有助于识别高感染批次，减少其种植，并最大限度地减少外来菌株通过菠菜种子的传播。本研究优化了实时定量聚合酶链反应（qPCR）方法，并将其应用于菠菜种质资源和15批商品菠菜种子中大丽花病菌的检测和定量。该试验使用了先前报道的大丽草特异性引物对（VertBt-F和VertBt-R）和用于研磨坚韧菠菜种子以提取DNA的分析研磨机。该方法能够可靠地定量菠菜种子中的大丽花弧菌，灵敏度限约为每100个感染种子的千分之一（一个种子批次感染1.3%）。在同一种子批次的重复样品和不同的实时PCR仪器中，定量结果具有很高的重复性。当在商业种子批次上测试时，病原体DNA含量对应于定量循环值>= 31，对应于种子侵染百分比< 1.3%。该方法可用于定量评价大丽花病原菌的侵染程度，其结果可用于指导种子处理的决策。
Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of approximate to a parts per thousand 1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of >= 31 corresponded with a percent seed infection of < 1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.