Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma
pini and D. septosporum on Needles by Probe-Based LAMP Assays
Chiara Aglietti, Colton D. Meinecke, Luisa Ghelardini, Irene Barnes, Ariska van der Nest and Caterina Villari
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 (β-tub2) gene for the two Dothistroma species
and the elongation factor (EF-1α) 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.