Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

Abstract

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii , causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F . circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F . circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F . circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

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

“…Although a test is sometimes very efficient in the initial conditions of development, it is conceivable to imagine that the performance of a qPCR or PCR detection protocol may vary if the conditions of use differ from those of the development team (Grosdidier et al 2017). In a study focused on the transferability of molecular test detection, in the pine pathogen Fusarium circinatum, Ioos et al (2019a) showed that changes in reagents, equipment, and qPCR fluorescence data processing software could have major effects on the reliability of the results (false positives and false negatives). Much of the variance produced in qPCR results (expressed as Ct values) is not simply due to reagent changes.…”

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

“…A recent collaborative study joined 23 partners to assess the transferability and performance of nine different protocols (conventional PCR and real-time PCR) using a wide panel of F. circinatum and other Fusarium spp. DNA [89].…”

“…Since the test requires the simultaneous amplification of two unrelated but specific regions of the F. circinatum genome, it has been suggested that it brings additional reliability. The conventional PCR tests described above are easy to set up and need less expensive equipment than qPCR methods, but require an additional electrophoresis step, and thus, more time to obtain a result [89].…”

“…In addition to existing tests targeting known taxonomic markers, several other F. circinatum-specific regions have been unraveled by in silico analysis, and these putative genes should be used for the development of new detection tools, either by PCR, real-time PCR or LAMP analysis [94]. Inclusivity (i.e., ability to target all F. circinatum strains) and specificity (i.e., ability not to cross-react with non-target species) of the currently available molecular detection tools targeting F. circinatum have been recently assessed and compared [89]. Overall, the study demonstrated that current diagnostic protocols for the detection of F. circinatum is not easily transferable, and that end-users should perform validation tests, especially under modified conditions, such as different reagents and/or equipment, prior to use.…”

A worldwide perspective of the legislation and regulations governing sentinel plants

“…Since Roman times the history of Europe has been characterised by an intense trade across continents that has resulted in the introduction of many non-native tree species, such as Sweet chestnut from western Asia (Adua 1999), and also of pests of woody plants (Santini et al 2018). PRA and PEQ are worth to be applied, even where are not compulsory, to strengthen a prevention to pest introduction based only on diagnostic procedures (Ioos et al 2019). PEQ can be implemented in a variety of ways, ranging from secure quarantine greenhouses to surveyed open-field plantations, depending on country and perceived risk.…”