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

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.

Fungal endophytic communities on twigs of fast and slow growing Scots pine (Pinus sylvestris L.) in northern Spain.

Most plant species harbour a diverse community of endophytic, but their role is still unknown in most cases, including ecologically and economically important tree species. This study describes the culturable fungal endophytic community of Pinus sylvestris L. twigs in northern Spain and its relationship with diametric growth of the host. In all, 360 twig samples were collected from 30 Scots pines in fifteen stands. Isolates were obtained from all twig samples and 43 fungal taxa were identified by morphogrouping and subsequent ITS rDNA sequencing. All isolates were Ascomycetes, being Dothideomycetes and Sordariomycetes the most abundant classes. Half of the species were host generalists while the others were conifer or pine specialists. We found three new endophytic species for the Pinaceae: Biscogniauxia mediterranea, Phaeomoniella effusa and Plectania milleri, and additional six new species for P. sylvestris: Daldinia fissa, Hypocrea viridescens, Nigrospora oryzae, Ophiostoma nigrocarpum, Penicillium melinii and Penicillium polonicum. The endophytic community of fast and slow growing trees showed differences in species composition, abundance and evenness, but not in diversity. Phoma herbarum was associated to fast growing trees and Hypocrea lixii to those growing slow. Our results support the hypothesis that some endophytic species may affect growth of P. sylvestris.