Quantification of <i>Phytophthora capsici</i> 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, 10^1.5, 10², 10^2.5, 10³, 10^3.5, 10⁴, 10^4.5, 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.