The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real-time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real-time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi-copy rDNA gene. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real-time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real-time PCR methods showed that the TaqMan method was more sensitive.

In this study, six methods for the detection of Phytophthora ramorum in planta were compared using naturally infested rhododendron plant material. The methods included two immunological methods, one an enzyme-linked immunosorbent assay (ELISA) and the other using a lateral flow format (LFD). Three molecular tests based on the polymerase chain reaction (PCR) using TaqMan chemistry also were assessed, including two assays designed for specific detection of P. ramorum and one designed for genus-level detection of Phytophthora. Isolation followed by morphological identification also was assessed. The diagnostic values of each of the methods, evaluated based on diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value, were calculated based upon the test results from 148 field samples. The "gold standard" used for the calculations was the final diagnosis, which was based on either a positive PCR result or successful isolation of P. ramorum. The Phytophthora spp. TaqMan PCR, ELISA, and LFD had higher sensitivities than the P. ramorum-specific methods, which make them useful as prescreening methods, where positive results must be confirmed by PCR or isolation. The article discusses practical advantages and disadvantages of each of the methods and how they are valuable in the diagnostic process, according to the circumstances of use (that is, diagnosis or surveillance) and in relation to the prevalence of P. ramorum infestation in the population to be tested.

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real-time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus-specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena ⁄ M. laxa ⁄ Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No crossreactions were observed. The assay was found to have a detection limit of 0AE6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real-time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.

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