Development of a Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Dothistroma septosporum

Colin L Myrholm,Renate Heinzelmann,Nicolas Feau,Richard C Winkworth,Richard C Hamelin,Tod D Ramsfield,Bradley D Tomm,Rebecca Mcdougal

doi:10.3390/f12030362

Abstract

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

Highlights

Dothistroma needle blight, a disease caused by the pathogens Dothistroma septosporum and Dothistroma pini, results in significant damage to pines in both natural and plantation settings globally [1]
The identification of D. septosporum in Alberta and the potential for eastward spread through the boreal forest have led to research into population genomics [4], host range [3], and, as we describe here, rapid diagnostics for D. septosporum, the pathogen associated with the disease in western Canada
Have been described by Ioos et al [5], Langrell [6], and Schneider et al [7]. These diagnostics are sensitive and able to detect the pathogen within infected tissue they must be performed within a well-equipped laboratory

Summary

Introduction

Dothistroma needle blight, a disease caused by the pathogens Dothistroma septosporum and Dothistroma pini, results in significant damage to pines in both natural and plantation settings globally [1]. Polymerase chain reaction (PCR)-based diagnostics for detecting D. septosporum DNA have been described by Ioos et al [5], Langrell [6], and Schneider et al [7]. These diagnostics are sensitive and able to detect the pathogen within infected tissue they must be performed within a well-equipped laboratory. LAMP assays have been developed for point-of-care diagnostics in medicine [9], agriculture [10], and important forest pathogens [11]. Portable instruments such as the Genie® III developed by OptiGene (Horsham, West Sussex, UK) or the BioRanger instrument developed by Diagenetix Inc

Methods

Results

Discussion

Conclusion