A simple, fast and cheap isolation and multiplication technique of Phytophthora infestans

Abstract

ABSTRACT: In this study, we present a method for isolation and propagation of Phytophthora infestans, a challenging-to-isolate phytopathogenic oomycete. The procedure initiated with naturally infected potato leaves, which underwent a 30-minute sanitization under running water. Subsequently, healthy potato tubers were meticulously washed with a neutral detergent, sterilized using alcohol, and then flamed. Slices 4 mm thick were carefully cut out of the potato tubers. Slices were used to cover 2 x 4 mm sterilized leaf pieces cut from the border of two-day old young P. infestans lesions, within sterilized empty Petri dishes. The sealed plates were then transferred to BOD growth chambers set at 18 ºC in complete darkness for 5 days. At the end of this incubation period, the development of a sparse, white mycelium was observed on top of the potato slices. Using a Drigalski loop, the visible mycelium was carefully transferred to rye agar medium in Petri dishes. To avoid contamination, care was taken not no touch potato slices. After mycelium reaching the Petri dish border, and with aid of a Neubauer chamber, the inoculum density per plate was of 1.79 x 104 sporangia mL-1. Sporangia germination rate ranged from 69 to 78%. This isolation technique simplify in vitro production of P. infestans, enhancing the possibility for research with this important pathogen.