Pathogen Detection Strategies: PCR and Sequencing

We do not know what is causing the sword fern die-off.  Mountain beaver and drought may be contributing causes.   But the spatial pattern of the die-off strongly suggests a pathogen is the driving cause:

1. The die-off, unlike drought and mountain beavers, is limited to one region of the forest
2. This region is growing approximately radially, as would infection

Phytophthora is a plausible candidate, but repeated WSU and Ribeiro lab studies have been unable to provide confirmation (see earlier blog entries for full reports).  New approaches may be needed, among which are PCR, and next-generation sequencing (NGS).  

Concerning PCR (polymerase chain reaction), Paul Talbert suggests (email, 6/8/2016)

Not to discourage the sequencing idea, which is unbiased with respect to pathogens, but I wonder if a simple PCR assay for Phytophthora would be a simpler first step (although we don't a Phytophthora sample have a positive control). The genomes of at least 8 species of Phytophthora have been sequenced, including P. cinnamomi (see attached for  six species, plus P. infestans and P.ramorum were sequenced several years ago).  Genome sequences of six Phytophthora species associated with forests in New Zealand.

Jenny Glass of the WSU Puyallup Plant diagnostics lab used the  agdia ImmunoStrip for Phytophthora.  If I understand her, and Marianne Elliott's subsequent lab techniques, PCR was not otherwise employed.  

Speculatively, and with no guarantee of success, we may wish to consider both PCR (with appropriate primers) and unbiased NGS sequencing of affected and unaffected samples.   Marianne provides this poster as a good starting point:  

 Detection of Phytophthora in plants by the Polymerase Chain Reaction

NGS sequencing has proved successful in at least one somewhat-related field study:

Unfortunately, the genome of Western Sword Fern, polystichum munitum,  has not yet been sequenced: