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; Caterina Villari

doi:10.3390/f12040479

Forests (Apr 2021)

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,
Caterina Villari

Affiliations

Chiara Aglietti: Department of Agriculture, Food, Environment and Forestry (DAGRI), University of Florence, Piazzale delle Cascine 28, I-50144 Firenze, Italy
Colton D. Meinecke: Warnell School of Forestry & Natural Resources, University of Georgia, Athens, GA 30602-2152, USA
Luisa Ghelardini: Department of Agriculture, Food, Environment and Forestry (DAGRI), University of Florence, Piazzale delle Cascine 28, I-50144 Firenze, Italy
Irene Barnes: Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 002, South Africa
Ariska van der Nest: Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 002, South Africa
Caterina Villari: Warnell School of Forestry & Natural Resources, University of Georgia, Athens, GA 30602-2152, USA

DOI: https://doi.org/10.3390/f12040479
Journal volume & issue: Vol. 12, no. 4
p. 479

Abstract

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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 (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.

Published in Forests

ISSN: 1999-4907 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Botany: Plant ecology
Website: http://www.mdpi.com/journal/forests/

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