Pavón C F, Babadoost M, Lambert K N
Department of Crop Sciences, University of Illinois, Urbana, 61801.
Plant Dis. 2008 Jan;92(1):143-149. doi: 10.1094/PDIS-92-1-0143.
A procedure was developed to quantify Phytophthora capsici oospores in soil by combining a sieving-centrifugation method and a real-time quantitative polymerase chain reaction (QPCR) assay. Five soil samples representing three different soil textures were infested with oospores of P. capsici to produce 10, 10, 10, 10, or 10 spores per 10 g of air-dried soil. Each 10-g sample of infested soil was suspended in 400 ml of water and then passed through 106-, 63-, and 38-μm metal sieves. The filtrate was then passed through a 20-μm mesh filter. Materials caught on the filter were washed with water into two 50-ml centrifuge tubes and spun for 4 min (900 × g). The pellet was suspended in 30 ml of 1.6 M sucrose solution and centrifuged for 45 s (190 × g). The supernatant was passed through the 20-μm mesh filter. The sucrose extraction process of oospores was repeated five times to maximize oospore extraction. Materials caught on the 20-μm mesh filter were washed with water into a 50-ml tube and spun for 4 min (900 × g). The pellet was suspended in 1 ml of water, and the number of oospores was determined with a haemocytometer. The relationship between number of oospores recovered from the soil and number of oospores incorporated into the soil was Ŷ = -0.95 + 1.31X - 0.03X (R = 0.98), in which Ŷ = log of number of oospores recovered from the soil and X = log of number of oospores incorporated into the soil. The oospores were germinated after treatment with 0.1% KMnO solution for 10 min to induce germination. On the basis of the detection of ribosomal DNA, a QPCR method for P. capsici oospores was developed. PCR inhibitors were eliminated by extracting oospores from the soil by sieving-centrifugation. DNA was extracted and quantified from P. capsici oospores with suspensions of 10, 10, 10, 10, 10, 10, 10, 10, and 10 oospores per ml of water. The relationship between the DNA quantities and number of P. capsici oospores was Ŷ = -3.57 - 0.54X + 0.30X (R = 0.93), in which Ŷ = log (nanogram of P. capsici DNA) and X = log (number of oospores). The relationship between the quantity of DNA of P. capsici oospores recovered from the soil and the number of oospores incorporated into the soil was determined by Ŷ = -3.53 - 0.73X + 0.32X (R = 0.955, P < 0.05), in which Ŷ = log (DNA quantity of P. capsici oospores recovered from the soil) and X = log (number of P. capsici oospores incorporated into the soil). Utilizing the sieving-centrifugation and QPCR methods, oospores of P. capsici were quantified in soil samples collected from commercial fields.
通过结合筛分-离心法和实时定量聚合酶链反应（QPCR）分析，开发了一种定量土壤中辣椒疫霉卵孢子的方法。用辣椒疫霉卵孢子侵染代表三种不同土壤质地的五个土壤样品，使每10克风干土壤中含有10、10、10、10或10个孢子。将每个10克受侵染土壤样品悬浮于400毫升水中，然后依次通过106微米、63微米和38微米的金属筛。滤液再通过一个20微米的筛网过滤器。过滤器上截留的物质用水冲洗到两个50毫升离心管中，离心4分钟（900×g）。沉淀悬浮于30毫升1.6M蔗糖溶液中，再离心45秒（190×g）。上清液通过20微米筛网过滤器。卵孢子的蔗糖提取过程重复五次以最大限度地提取卵孢子。20微米筛网过滤器上截留的物质用水冲洗到一个50毫升管中，离心4分钟（900×g）。沉淀悬浮于1毫升水中，用血球计数板测定卵孢子数量。从土壤中回收的卵孢子数量与加入土壤中的卵孢子数量之间的关系为Ŷ = -0.95 + 1.31X - 0.03X（R = 0.98），其中Ŷ为从土壤中回收的卵孢子数量的对数，X为加入土壤中的卵孢子数量的对数。用0.1%高锰酸钾溶液处理卵孢子10分钟以诱导萌发。基于核糖体DNA的检测，开发了一种针对辣椒疫霉卵孢子的QPCR方法。通过筛分-离心从土壤中提取卵孢子以消除PCR抑制剂。从每毫升水中含有10、10、10、10、10、10、10、10和10个卵孢子的悬浮液中提取辣椒疫霉卵孢子的DNA并进行定量。辣椒疫霉DNA量与卵孢子数量之间的关系为Ŷ = -3.57 - 0.54X + 0.30X（R = 0.93），其中Ŷ为辣椒疫霉DNA（纳克）的对数，X为卵孢子数量的对数。从土壤中回收的辣椒疫霉卵孢子DNA量与加入土壤中的卵孢子数量之间的关系通过Ŷ = -3.53 - 0.73X + 0.32X（R = 0.955，P < 0.05）确定，其中Ŷ为从土壤中回收的辣椒疫霉卵孢子DNA量的对数，X为加入土壤中的辣椒疫霉卵孢子数量的对数。利用筛分-离心和QPCR方法对从商业田地采集的土壤样品中的辣椒疫霉卵孢子进行了定量。
