A LAMP at the end of the tunnel: A rapid, field deployable assay for the kauri dieback pathogen, Phytophthora agathidicida


Autoři: Richard C. Winkworth aff001;  Briana C. W. Nelson aff002;  Stanley E. Bellgard aff003;  Chantal M. Probst aff003;  Patricia A. McLenachan aff002;  Peter J. Lockhart aff001
Působiště autorů: Bio-Protection Research Centre, Massey University, Palmerston North, New Zealand aff001;  School of Fundamental Sciences, Massey University, Palmerston North, New Zealand aff002;  Manaaki Whenua–Landcare Research, Auckland, New Zealand aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224007

Souhrn

The root rot causing oomycete, Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14–20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection of P. agathidicida that targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of other Phytophthora isolates and detected as little as 1 fg of total P. agathidicida DNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended soil bioassay and LAMP testing of DNA extracted from baits. In these comparisons, P. agathidicida was detected more frequently using the LAMP test. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the extended soil bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions about P. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid LAMP bioassay also has the potential to empower local communities to evaluate the pathogen status of local kauri stands, providing information for disease management and conservation initiatives.

Klíčová slova:

DNA extraction – DNA isolation – Forests – Mitochondria – Phytophthora – Polymerase chain reaction – Sequence databases – Oomycetes


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