Molecular Phylogenomics and Population Structure of Phytophthora pluvialis

Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir−tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.

Contrasting the Pathogen Loads in Co-Existing Populations of Phytophthora pluvialis and Nothophaeocryptopus gaeumannii in Douglas Fir Plantations in New Zealand and the Pacific Northwest United States

The emergence of Phytophthora pluvialis as a foliar pathogen of Douglas fir in New Zealand and the Pacific Northwest United States has raised questions about its interaction with the widespread Swiss needle cast (SNC) disease. During Spring 2017, we repeatedly sampled 30 trees along an environmental gradient in each region and 292 additional trees in a longitudinal transect to assess the P. pluvialis epidemic and the association between P. pluvialis and Nothophaeocryptopus gaeumannii, which are causal agents of SNC. Both pathogens were consistently more abundant in the host’s exotic environment in New Zealand. In both areas, the two pathogens co-exist in different spatial scales for regions and needles. The relative abundance of both pathogens was negatively correlated in the Pacific Northwest, where both presumably have co-existed for longer. Our findings confirmed the interaction of P. pluvialis and N. gaeumannii as foliar pathogens of Douglas fir and suggest a within-site spatial variation in the Pacific Northwest.

Validating outsourced high throughput automated qPCR for increased research outputs from forest pathology trials

Needle diseases of Pinus radiata caused by Phytophthora pluvialis and Phythophthora kernoviae have been increasingly recognised since the discovery of red needle cast in 2008. There is a need for rapid diagnostic screening of numerous samples, but sample processing time, equipment and staff availability limit the throughput and utilisation of diagnostic qPCR analysis in the research environment. Automated and high-throughput capable DNA extraction and real-time PCR provides the opportunity to expand the capacity of research trial analysis and a potential alternative to laborious isolation and plating but must be thoroughly validated before results can be used with confidence. The use of a high-throughput format for qPCR assays targeting Phytophthora pluvialis and Phythophthora kernoviae was validated on a robotic platform, proving to be consistently more sensitive than isolation, achieving qPCR detection down to 1% diluted inoculated material for Phytophthora kernoviae and 10% for Phytophthora pluvialis. Plating results yielded a 60% detection rate of Phythophthora pluvialis in inoculated needle fragments, whereas qPCR yielded a 100% detection on the same material. High throughout automated qPCR can therefore be utilised with confidence in forest pathology research trial analyses in future.

Phytophthora pluvialis Studies on Douglas-fir Require Swiss Needle Cast Suppression

Phytophthora pluvialis is associated with early defoliation and shoot dieback in Douglas-fir in Oregon and New Zealand. In 2013, P. pluvialis was described from mixed tanoak-Douglas-fir forests in the Pacific Northwest and concurrently recognized as the main causal agent of red needle cast (RNC) in New Zealand radiata pine plantations. Little is known about its infection cycle and impact on host physiology. P. pluvialis studies in Douglas-fir are challenging due to the ubiquitous presence of the endophyte Phaeocryptopus gaeumannii, which produces similar symptoms and premature defoliation with persistent needle wetness, known as Swiss needle cast (SNC). Nonetheless, our study showed P. pluvialis infection in the presence of SNC. Exclusive expression of P. pluvialis is difficult to achieve as both diseases are promoted by high humidity. Here we established a ‘dry leaf’ strategy to suppress SNC when inoculating Douglas-fir needles for RNC studies. Sheltering plants along with drip irrigation to avoid needle wetness during the P. gaeumannii sporulation period suppressed its development in the new season flush. The diminished endophyte inoculum enabled bias-reduced studies of P. pluvialis impacts on Douglas-fir without the confounding effects of stomatal blockage and premature defoliation caused by P. gaeumannii.