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Research Project: Investigation on Evolution of Virulence in the Sudden Oak Death Pathogen, Phytophthora Ramorum

Location: Crops Pathology and Genetics Research

Project Number: 2032-22000-016-02-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2014
End Date: Dec 31, 2018

It is not well understood how phenotypic diversity is generated within invasive populations that exhibit paucity of genetic variation. Phytophthora ramorum, the causal agent of “sudden oak death”, is a recent example of an exotic pathogen displaying extensive phenotypic variation within a clonal lineage. Phenotypes of North American P. ramorum isolates have been associated with their originating host species, yet no host-driven population subdivision was observed. Isolates from oaks are on average less virulent to several host species and more likely to show an irregular growth phenotype on Petri dish than isolates originating from California bay laurel. We have described the phenomenon of host-induced phenotypic diversification (HIPD) in P. ramorum: stable phenotypic changes of the pathogen inside host plants occurring at a rate which is host species dependent. HIPD can be reproduced experimentally: bay laurel isolates inoculated and re-isolated from oak hosts display phenotypes resembling to those of isolates from naturally infected oaks. Furthermore, experimental studies on growth rate revealed re-isolates from oaks evolved significantly faster than those from bay laurels implicating participation of HIPD in adaptation. In this project, we will characterize molecular signatures associated with HIPD through experimental evolution and population genomics approaches and (2) search for host factors that induce HIPD. Exotic fungal and oomycete pathogens have been devastating both our agricultural and natural ecosystems. Studying the molecular mechanism underlying diversification of pathogens can potentially pave the way to predict the direction and rate of evolution, and find means to counteract it.

In order to identify molecular signatures associated with HIPD, we will examine clones of P. ramorum from diverse host species. We already have generated these clones in our APHIS funded research project at the National Ornamentals Research Site at Dominican University of California, where an original isolate from camellia was inoculated and recovered from seedlings of oaks, bay laurels, viburnums and Rhododendrons, 1, 2 and 6 months after inoculum. These isolates will be subjected to next generation sequencing (NGS)-based methods to uncover DNA signatures associated with HIPD and host-dependent rates of HIPD. We will also measure difference in epigenetic status by means of NGS combined with chromatin immunoprecipitation (ChIP-Seq). RNA sequencing (RNA-Seq) will be used to analyze changes in transcriptomes of re-isolates growing axenically on solid medium. Differentially expressed genes, DNA polymorphisms, and differences in epigenetic patterns as well as colony phenotypes will be cross-examined to establish association between genetics, epigenetics and HIPD. We have previously shown that isolates from oaks can evolve significantly faster than those from foliar hosts. This phenomenon will be used to screen host factors that trigger HIPD. Candidates for host factors include inhibitory compounds found in oak cambium. Certain soluble phenolic compounds found in phloem of coast live oak are associated with field resistance to P. ramorum. In particular, tyrosol and ellagic acid were shown to have strong inhibitory effects in vitro. We will include each of these phenolics as well as total methanol extract from phloem samples in the V8 growth medium and growth rate of a bay laurel isolate in race tubes (40 cm-long glass tubes) will be monitored for over 18 weeks. When changes in growth rate are detected, the pathogen will be recovered from the race tube and its molecular signature will be cross-examined with those associated with HIPD.