A fast and specific assay for the surveillance of the invasive forest pathogen Heterobasidion irregulare based on loop-mediated isothermal amplification (LAMP).

The North American species H. irregulare Garbel. & Otrosina was accidentally introduced in central Italy during the World War II. The pathogen has become invasive and is currently distributed in pine and oak stands over about 105 km of coast around Rome, often in association with significant mortality of Italian stone pine (Pinus pinea L.) trees. Based on its observed and potential impact, H. irregulare is recommended for regulation in Europe by the European and Mediterranean Plant Protection Organisation (EPPO). In the frame of the EU Project EMPHASIS (grant agreement 634179), a fast and specific molecular diagnostic tool based on Loop-mediated isothermal AMPlification (LAMP) coupled with two different DNA extraction methods was developed and optimized for the detection of H. irregulare. The LAMP assay was successfully validated both in controlled conditions and in the field on different samples, including fruiting bodies, infected plants, wood colonized by the pathogen, and airspora collected by using woody spore traps. The detection of the pathogen in airspora was optimized both in terms of methods and in terms of length of incubation period of traps. The limit of detection of this assay is approximately 20 picograms of target DNA with a detection time of about 40 minutes. This molecular diagnostic tool may be used for the surveillance in the ports of entry of wood imported from North America and for the monitoring of pine forests, especially those located in a buffer zone surrounding the infested area.