Quantification of Phytophthora capsici Oospores in Soil by Sieving-Centrifugation and Real-Time Polymerase Chain Reaction

Abstract

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 101, 102, 103, 104, or 105 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 – 0.03X2 (R2 = 0.98), in which Ŷ = log10 of number of oospores recovered from the soil and X = log10 of number of oospores incorporated into the soil. The oospores were germinated after treatment with 0.1% KMnO4 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 101, 101.5, 102, 102.5, 103, 103.5, 104, 104.5, and 105 oospores per ml of water. The relationship between the DNA quantities and number of P. capsici oospores was Ŷ = –3.57 – 0.54X + 0.30X2 (R2 = 0.93), in which Ŷ = log10 (nanogram of P. capsici DNA) and X = log10 (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 + 0.32X2 (R2 = 0.955, P < 0.05), in which Ŷ = log10 (DNA quantity of P. capsici oospores recovered from the soil) and X = log10 (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.