Abstract:
Needle blights are serious needle fungal diseases affecting pines both in natural and productive
forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini
and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics
since they are regulated species in many countries and may require differential management
measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species
are hard to distinguish using morphological characteristics. The symptoms can also be confused
with those caused by insects or abiotic agents. DNA-based detection is therefore recommended.
However, the specific PCR assays that have been produced to date for the differential diagnosis
of these pathogens can be applied only in a well-furnished laboratory and the procedure takes
a relatively long execution time. Surveillance and forest protection would benefit from a faster
diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires
less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP
assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this
work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each
assay, targeting the beta tubulin ( -tub2) gene for the two Dothistroma species and the elongation
factor (EF-1 ) region for L. acicola. Each reaction detected its respective pathogen rapidly and with
high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected
pine needles. These qualities and the compatibility with inexpensive portable instrumentation
position these LAMP assays as an effective method for routine phytosanitary control of plant material
in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.
