# Phytophthora pluvialis maintenance, spore production and detached needle assays

## TL;DR

This paper outlines a method to study how Phytophthora pluvialis infects pine trees, using spore production and detached needle assays to assess virulence and infection mechanisms.

## Contribution

The paper introduces a reproducible protocol for spore production and detached needle assays to study P. pluvialis pathogenicity in pine needles.

## Key findings

- Detached needle assays enable high-throughput screening of pine needles with different genetic backgrounds.
- qPCR was used to assess gene expression of the candidate effector protein PpR01 during infection.
- Combining assays with RNA sequencing and metabolomics can reveal infection mechanisms in detail.

## Abstract

Phytophthora pluvialis is an oomycete that primarily infects Pinus radiata and Pseudotsuga menziesii causing the destructive foliar disease red needle cast (RNC). Recent observations show that P. pluvialis can also infect western hemlock inducing resinous cankers. High-throughput and reproducible infection assays are integral to find key information on tree health and oomycete pathogenicity. In this protocol, we describe the propagation and spore induction of P. pluvialis, followed by detached needle assays for verification and quantification of virulence of P. pluvialis in P. radiata needles. These needle assays can be employed for high-throughput screening of tree needles with diverse genetic backgrounds. In downstream analysis, Quantitative PCR (qPCR) was utilized to assess relative gene expression, as exemplified by candidate RxLR effector protein PpR01. Additional techniques like RNA sequencing, metabolomics, and proteomics can be combined with needle assays and can offer comprehensive insights into P. pluvialis infection mechanisms.