Genetic diversity and gene expression analysis of Phytophthora pluvialis, a foliar pathogen of conifers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics, Massey University, Manawatū, New Zealand

Abstract

Phytophthora pluvialis is the causal agent of red needle cast on Pinus radiata in New Zealand. It was first isolated in 2008 but had previously been recovered from tanoak (Notholithocarpus densiflorus) and Douglas fir (Pseudotsuga menziesii) trees in Oregon, USA in 2002. Phytophthora pluvialis was subsequently described as a new species in 2013 and classified as a clade 3 Phytophthora species. The aims of this study were to (1) gain a better understanding of the genetic diversity and population structure of P. pluvialis and (2) examine gene expression profiles of P. pluvialis from naturally infected P. radiata seedlings. Studying the genetic diversity and population structure of P. pluvialis provided insight into the mode of reproduction of this pathogen and helped determine if P. pluvialis was introduced into New Zealand. This information is also important for the development of management strategies for P. pluvialis. Twenty-seven single nucleotide polymorphism (SNP) markers were designed to genotype a total of 360 isolates of P. pluvialis collected from New Zealand and the USA. The genotypic data showed that the population in New Zealand has lower diversity than the USA population. A minimum spanning network (MSN) showed two unique clusters in the New Zealand population, suggesting there may have been two separate introductions of P. pluvialis. For the second study, samples were collected from 45 P. radiata grafted plants that were part of a field trial, with the aim of identifying genes that are highly expressed and may be important for virulence. Interestingly, Phytophthora kernoviae was found in more of the samples than P. pluvialis. Needle samples were collected, RNA was extracted and sequenced, and the normalised reads that mapped to the genome of P. pluvialis were compared to those from P. pluvialis grown in culture. Differentially expressed genes (DEGs) of P. pluvialis that showed higher expression in the field trial included potential orthologs of sugar transporter, GH12 and effector genes with known pathogenicity functions in other species. This is the first study to examine the genetic diversity of P. pluvialis in New Zealand and the USA., and to examine the gene expression of a Phytophthora forest pathogen in the field. The results from these studies provide useful tools for forest disease management. The SNP markers can be used to monitor the population of P. pluvialis in New Zealand. The highly expressed genes can be used to help identify resistance genes in P. radiata that can be incorporated into future breeding programs.