Development of quantification bioassays for live propagules of Phytophthora sojae in soil

Abstract

To estimate inoculum density of Phytophthora sojae in natural soils to predict disease risk and to study the ecology of P. sojae in the soil, we developed two bioassays to quantify zoospores and another for oospores of P. sojae in soil based on inoculation of Mollisol (the most conducive soil type for this disease according to our disease survey) with a known density of zoospores or oospores of enhanced green fluorescent protein (EGFP)-labeled P. sojae Eps597-3 (pathotype 1a3c7). A linear regression equation, \( y_{ 1} = 1.0 9 2 7x_{ 1} + 7. 3 6 5 2 \) (Eq. 1), and an index regression equation, \( {\text{y}}_{ 2} \, \;{ = }\; 1 8 6 2. 9 {\text{e}}^{{0.00 9 5 {\text{x}}_{ 2} }} \) (Eq. 2), were established to quantify the zoospores in the soil, where x1 is the concentration of zoospores counted directly using a light microscope and y1 is the concentration of zoospores added to a soil suspension, x2 is the number of zoospores at the margin of 18 leaf discs, and y2 is the number of zoospores added to 10 mL of a soil suspension. Another linear regression equation, \( y_{ 3} = 3. 3 8 3 6x_{ 3} + 20 7.0 5 \) (Eq. 3), was determined to quantify the oospores in the soil, where x3 is the number of zoosporangia at the margin of 18 leaf discs, and y3 is the number of oospores added to 10 mL of a soil suspension. These three equations provided high reproducibility and accuracy. To verify their applicability, two other isolates with different pathotypes (3a3b678 and 1a1d23a3c578) and two other soil types (Albic luvisols and Aquic cambosols) were tested. Pathotype and soil type did not markedly affect the parameters in the three equations, suggesting that these three equations are accurate enough to serve as universal quantification bioassays of live zoospores and oospores of P. sojae isolates of any pathotype in any soil. Equations (1) and (2) can be used only to estimate zoospores, Eq. (2) for zoospores, and Eq. (3) for oospores of P. sojae in natural or inoculated soil. This method can be finished within 2–3 h for Eq. (1), 4–5 h for Eq. (2), and 7–10 days for Eq. (3).