Detached leaf assay for determining early detection of P. infestans on leaves

A detached leaf experiment was performed to evaluate the earliest time point when P. infestans was detected by ITS1-LAMP. Potato plants were grown in a greenhouse in plastic pots filled with equal proportions of sterile mixture consist loamy soil, sand and composted farmyard manure. The plants were maintained at a mean daily temperature of 22 °C under a 12-h day photoperiod. A suspension of P. infestans sporangia were made consisting of 10⁴ sporangia/ml (Appel et al. 2001). The leaves of the 6-week-old potato plant was placed in a closed plastic tray with holder consist of wet blotter paper (ddH₂O) on its base to maintain the moisture. Prior inoculation, a suspension of sporangia (10⁴ sporangia/ml) was prepared and incubated it at 4 °C for 1 h. Mock inoculation was performed with sterile ddH₂O. The tray was incubated at 18 °C for 124 h in a BOD incubator. Samples were drawn at every 24 h post inoculation and subjected to total DNA isolation and ITS1-LAMP assay. The amplified LAMP products were detected as described above.