Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR.

Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

Pathogenicity and Host Range of Pythium kashmirense-A Soil-Borne Oomycete Recently Discovered in the UK.

During a survey of oomycetes in ornamental plants carried out at the University of Aberdeen in 2014-2015, Pythium kashmirense was isolated from a specimen of Viburnum plicatum 'Lanarth', the first report of this oomycete in the UK (and in Europe). Pathogenicity of a Py. kashmirense isolate was examined using a range of plant species. Inoculations were carried out under controlled conditions in the absence of other Pythium and Phytophthora species, on Glycine max (soya bean), Phaseolus vulgaris (common bean), Lupinus angustifolius (blue lupin), Cucumis sativa (cucumber) and Viburnum opulus. The majority of inoculations caused pre-emergence damping-off, as well as seed rot and root rot. In the in vitro assays, germination rates (%) of soya bean and blue lupin seeds were less than 50%; in the in vivo inoculations on plants, over 50% of soya bean, blue lupin and common bean plants died; in contrast, cucumber plants showed lower susceptibility in pathogenicity tests, with an approximately 80% germination rate in in vitro tests, and 25% dead plants in the in planta inoculations. Inoculations carried out on root systems of Viburnum opulus caused severe necrosis and root rot. Little research was previously conducted on pathogenicity of Py. kashmirense and its relationship with losses in crop yield and quality. The present study showed varying virulence on the different plant species tested after inoculation with Py. kashmirense. Despite the lack of clear host specialization, infection by Py. kashmirense decreased seedling survival and health of plants in a range of important agricultural and ornamental plant species.

Application of Real-Time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock.

Numerous Phytophthora and Pythium disease outbreaks have occurred in Europe following inadvertent introduction of contaminated ornamental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diversity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses. Oomycete DNA was quantified using real-time PCR of environmental DNA from the plants using three loci: ITS, trnM-trnP-trnM and atp9-nad9. At least one oomycete species was isolated from 89.9% of plants using classical techniques. In total, 10 Phytophthora spp., 17 Pythium spp. and 5 Phytopythium spp. were isolated. Oomycetes were isolated from 86% of asymptomatic plants, but real-time PCR demonstrated that oomycetes were associated with all plants tested. More oomycete DNA occurred in composts in comparison with roots and filters from baiting water (a mean of 7.91 ng g-1, 6.55 × 10-1 ng g-1 and 5.62 × 10-1 ng g-1 of oomycete DNA detected in compost with ITS, trnM and atp9 probes, respectively); the ITS probe detected the highest quantities of oomycete DNA. No significant differences were found in quantities of oomycete DNA detected using real-time PCR in plants purchased online or from traditional retailers.