# Improved isolation and PCR detection of Phytophthora agathidicida oospores from soils

## TL;DR

A new PCR method detects Phytophthora agathidicida oospores in soil more effectively than traditional methods, aiding in the protection of kauri forests.

## Contribution

A PCR-based method for direct detection of P. agathidicida oospores in soil, eliminating the need for baiting and improving sensitivity.

## Key findings

- The new method detected P. agathidicida in 69% of soil samples compared to 11% with existing methods.
- The detection limit was 1 femtogram of P. agathidicida DNA using endpoint PCR.

## Abstract

Phytophthora species are eukaryotic microorganisms
responsible for severe dieback and root rot in plants worldwide, impacting
crops, forests, and other important ecosystems. In New Zealand, P.
agathidicida leads to fatal dieback in kauri (Agathis
australis), long-lived endemic trees of significant cultural
and ecological importance. A critical aspect of the P.
agathidicida lifecycle is the production of
oospores—thick-walled spores essential for long-term survival in
soil, dispersal, and disease inoculation. However, their heterogeneous
distribution in soils, robust structure, and dormant state make them
challenging to detect using soil baiting or DNA-based methods. Soil baiting
is the basis of most current testing for P. agathidicida,
but baiting-based methods have low sensitivity, are slow, and require
specialised facilities. To address these challenges, we developed and
validated a PCR-based method for detecting P. agathidicida
oospores directly from soil. Our approach includes a technique for
separating oospores from soil, improved oospore lysis and DNA extraction,
and a primer pair that targets a repeat region of the P.
agathidicida genome with high sensitivity and specificity. The
primers amplified the target product in all tested P.
agathidicida isolates without cross-reactivity against eight
non-target Phytophthora species. The detection limit was 1
femtogram of P. agathidicida DNA via endpoint PCR.
Performance assessment against 65 soil samples from kauri forests revealed
P. agathidicida in 69% of samples compared to only 11%
detected by existing methods. By eliminating the need for baiting, our assay
enhances the speed, accuracy, and accessibility of testing, thereby
facilitating more comprehensive monitoring and improved disease
management.

Phytophthora species are notorious plant pathogens
responsible for severe dieback and root rot diseases, significantly
impacting crops, forests, and irreplaceable natural ecosystems. Rapid and
accurate detection of these pathogens is essential for effective disease
management. In New Zealand, P. agathidicida threatens the
country’s endemic kauri forests. In this study, we developed and
validated a PCR-based method for detecting P. agathidicida
oospores in soil. Oospores are long-lived, thick-walled spores that serve as
key propagules for survival in soil and the spread of disease. Their robust
structure and dormant state make them particularly challenging to detect
using traditional soil baiting techniques or DNA-based methods. Our method
is fast, accurate, and requires minimal equipment, enabling local testing
and thereby empowering communities and enhancing surveillance efforts.
Although developed for P. agathidicida, this method could
be adapted for other plant pathogens, potentially improving disease
management across various agricultural and ecological contexts.