Laboratory protocol for enhancing sporulation and inoculum potential of Phytophthora megakarya for detached cocoa pod inoculation assay

Abstract

Phytophthora megakarya (Pmeg) causes a severe form of Phytophthora pod rot (Ppr) of cocoa in Africa. Selecting cocoa varieties resistant to Ppr is important for managing this disease. However, Pmeg does not sporulate well on agar media to facilitate laboratory screening for resistant cocoa materials. Zoospores are the main propagule of Pmeg causing plant infection. This study developed a method to obtain high amounts of zoospores and identified an inoculum concentration adequate to cause Ppr infection of detached cocoa pods for resistance screening. Oatmeal agar (OMA) and V-8 juice agar media diluted to half the recommended concentration induced rapid (within 2–3 days) sporulation. A similar observation was made for cocoa-based media, i.e. ripe cocoa husk agar and dry cocoa leaf agar (DCLA). The optimum temperature for Pmeg sporulation was 23 °C. The highest zoospore abundance of 5.9 × 10⁴ zoospores mL⁻¹ was achieved on half-strength OMA followed by 4.2 × 10⁴ zoospores mL⁻¹ on DCLA. More spores were produced under continuous light than under alternate 12 h darkness and 12 h light. Cultures of Pmeg should be grown on half-strength OMA or DCLA medium at 23 °C under light for rapid and abundant sporulation. A concentration of 0.9 × 10⁴ zoospores mL⁻¹ showed high efficiency in distinguishing resistant and susceptible cocoa genotypes. Symptoms on resistant pods were smaller than on susceptible pods and the zoospore concentration is recommended for a detached pod assay.