Validation of sampling, isolation and identification technique for Phytophthora infestans: Species and mating type

Abstract

Importance of the work: Sample damage and misleading Phytophthora infestans
identification are frequent, which could be addressed by the development of suitable
validation sampling, isolation and identification techniques.
Objectives: To compare several methods for the sampling, isolation and identification of
P. infestans using molecular and conventional approaches.
Materials and Methods: Infected potato leaves were used as the source of isolates
and for culturing. Whatman paper, DNA extraction kit and V-8 agar medium were used
for sampling, DNA isolation and culturing, respectively. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) used specific marker and
restriction enzymes for species and mating type identification.
Results: The Whatman paper could be an alternative for DNA sampling due to being
more affordable, available and simpler to use than a Flinders Technology Agreement
Card. Marker O8-1F/2R showed high sensitivity for species identification among the
four markers tested. A 245 bp of PCR product followed by sequencing, blasting and
constructing a phylogenetic tree showed high sensitivity in the levels of species, genus
and class. The combination pairing method, molecular marker PCR-RFLP (W16-1F/
2R fb. BsuRI) and sequencing constituted the best technique for mating type identification,
while the SmaI enzyme could be an alternative.
Main finding: Whatman paper could solve the long distance sampling issue.
Marker O-1F/2R fb. sequencing was the most accurate marker for species identification,
whereas a combination of W16-1F/2R fb. BsuRI or SmaI, sequencing and a pairing
test produced better results in mating type determination.