Fig 1.
Schematic diagram comparing the extended bioassay and hybrid LAMP bioassay workflows and timelines.
Fig 2.
Multiple sequence alignment for 13 representative Phytophthora species plus Pythium ultimum for a section of the mitochondrial genome containing the P. agathidicida LAMP assay target.
LAMP primer binding sites are indicated by grey outlines; F3 and B3 are binding sites for the external primer pair (i.e., PTAF3 and PTAB3), F2/F1 and B2/B1 form the binding sites for the internal primers (i.e., PTAFIP and PTABIP, respectively), and LF and LB are binding sites for the loop primers (i.e., PTALAF and PTALB).
Table 1.
Primer sequences for the P. agathidicida loop-mediated isothermal amplification (LAMP) assay.
Fig 3.
Results of the P. agathidicida LAMP assay.
A. Endpoint visualisation of LAMP products using SYBR Safe (Invitrogen) following electrophoresis on a 1% TAE agarose gel. Lane A, 2 pg PCR amplification products from ICMP 18244; lane B, 2 pg total DNA isolate ICMP 18244; lane C, 2 pg total DNA isolate ICMP18410; lane D, 5 ng total bait DNA from Waitakere Ranges Regional Park sample HTHF 1018; lane E, 5 ng total bait DNA from Waitakere sample Ranges Regional Park HTHF 1020; lane F, 5 ng total bait DNA from Waipoua Forest Sanctuary sample HTHF 1072; lane G, 5 ng total bait DNA from Waipoua Forest Sanctuary sample HTHF 1081; lane H, no DNA control; lane L, 1 kb plus DNA ladder. B. Real-time visualisation of LAMP products using raw fluorescence data. Samples labelled as for A.
Table 2.
Phytophthora isolates used in specificity testing and results of testing with the P. agathidicida LAMP assay for these isolates.
Table 3.
Comparison of Phytophthora agathidicida detections from field collected soil samples using the extended soil bioassay and hybrid LAMP bioassay assay.