Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples

Abstract

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization. Symptoms closely resembling those due to F. circinatum can be instead associated with C. pinea. Since F. circinatum is a quarantine organism, subjected to provisional emergency measures, its report immediately causes serious economic implications, while C. pinea, even if now emerging, is not regulated in the EU nor in the USA. For this reason, a reliable and accurate diagnostic tool able to distinguish between the two organisms was considered a priority. In this study, we developed and standardized a duplex real-time PCR assay allowing the simultaneous recognition of C. pinea and F. circinatum DNA in pine tissue in a reasonably short time and for amounts as small as 0.06 pg/μl. The molecular assay is, therefore, able to detect the infection even before symptoms have fully developed. The test was challenged with a very large set of strains (110 different isolates) collected in different regions of the world and host trees, and gave reliable results. The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls. In addition, the duplex real-time PCR assay presented here is the first DNA-based method designed to detect C. pinea, which is becoming an increasing threat to pine stands both in North America and in Europe.

Fast and reliable molecular methods to detect fungal pathogens in woody plants

“…The use of a multiplex assay is particularly important to distinguish pathogens that cause similar symptoms. A new PCR assay has been developed to also distinguish Fusarium circinatum and Caliciopsis pinea that cause comparable symptoms on P. radiata in the initial stages of colonization (Luchi et al 2018). All four European species of Heterobasidion attacking conifers may be simultaneously detected in two reactions (Ioos et al 2019b).…”

Sampling and Detection Strategies for the Pine Pitch Canker (PPC) Disease Pathogen Fusarium circinatum in Europe

“…Lamarche et al [91] proposed an alternative assay also targeting the IGS region, which was validated with seeds, woody tissue and insects. Another locus in the F. circinatum genome was targeted by Luchi et al [92], who used the Tef1 gene to develop a duplex real-time PCR test for the simultaneous detection of the pine pitch canker pathogen and Caliciopsis pinea in pine woody tissues. This test may be useful since both pathogens share the same ecological niche on Monterey pine causing similar bleeding canker symptoms, especially at the early stage of colonization.…”

Detection and quantification of the air inoculum of Caliciopsis pinea in a plantation of Pinus radiata in Italy

“…Cankers length ranged around 1-3 cm. The pathogen was detected by isolation on PDA (Luchi et al 2015) and molecular techniques (Luchi et al 2018).…”

“…The primer set Cpin-F (5'-ATTGTCGTCTGA ATTCTGATGCA-3') and Cpin-R (5'-TTCATC GATGCCAGAACCAA-3') and the TaqMan ® MGB probe (Cpin-Pr) (5'-VIC-AATAAAACTTT CAACAACGGATC-MGBNFQ-3') described by Luchi et al (2018) were used to detect and quantify C. pinea by real-time quantitative PCR (qPCR). DNA extracts from spore trap samples were assayed in MicroAmp Fast 96-well Reaction Plate (0.1 mL) closed with Optical Adhesive, and by using the StepOnePlus TM Real Time PCR System (Applied Biosystems, Life Science, Foster City, CA, USA).…”