LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach

Abstract

Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.

Figure 1. Images of Calonectria pseudonaviculata and C. henricotiae.

(A,B) Infection of C. pseudonaviculata on a boxwood landscape in New Jersey (images courtesy of Richard Buckley). (C,D) Scanning electronic images of C. henricotiae conidiophores and cylindrical conidia.

Figure 2. LAMP detection of Calonectria henricotiae and C. pseudonaviculata isolates using three different primers sets.

Note the different patterns of ladder-like fragments from each set. Lanes: M: DNA marker; 1–9: C. pseudonaviculata and C. henricotiae isolates; 10–12: negative controls.