Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs.

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

Diagnostic Values and Utility of Immunological, Morphological, and Molecular Methods for In Planta Detection of Phytophthora ramorum.

ABSTRACT 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.

Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences.

A molecular phylogenetic analysis of the genus Phytophthora was performed, 113 isolates from 48 Phytophthora species were included in this analysis. Phylogenetic analyses were performed on regions of mitochondrial (cytochrome c oxidase subunit 1; NADH dehydrogenase subunit 1) and nuclear gene sequences (translation elongation factor 1alpha; beta-tubulin) and comparisons made to test for incongruence between the mitochondrial and nuclear data sets. The genus Phytophthora was confirmed to be monophyletic. In addition, results confirm that the classical taxonomic grouping as described by [Waterhouse (1963)] does not reflect true phylogenetic relations. Phytophthora species were redistributed into 8 clades, providing a more accurate representation of phylogenetic relationships within the genus Phytophthora. The evolution and transition of morphological, pathogenic, and reproductive traits was inferred from the cladogram generated in this study. Mating system was inferred to be a homoplasious trait, with at least eight independent transitions from homothallism to heterothallism observed.

Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis).

ABSTRACT 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.