Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.
There are three major clonal lineages of Phytophthora ramorum present in North America and Europe named NA1, NA2, and EU1. Twenty-three isolates representing all three lineages were evaluated for phenotype including (i) aggressiveness on detached Rhododendron leaves and (ii) growth rate at minimum, optimum, and maximum temperatures. Closely related species P. foliorum and P. hibernalis were included in phenotypic tests since these species are encountered in nursery surveys for P. ramorum. Isolates from the NA2 and EU1 lineages were the most aggressive and isolates from the NA1 group were the least aggressive. The NA1 lineage of P. ramorum was the most variable in aggressiveness and growth rate. The variability in the NA1 lineage was due to the presence of non-wild type (nwt) isolates. There was no significant difference in growth rate among NA1 wild type (wt), NA2, and EU1 lineages at any temperature tested. The difference between wt and nwt P. ramorum isolates is discussed.
Five commercially available biological control products were tested in vitro with seven isolates of Phytophthora ramorum from North American (NA1, NA2), and European (EU1) populations. The in vitro tests included dual culture methods and detached leaf assays on wounded Rhododendron and Camellia leaves. Variability in response to biocontrol agents among isolates of P. ramorum from North American and European populations was examined. In dual culture tests, both Bacillus subtilis products (Companion † and Serenade † ) resulted in better inhibition of the NA1 group than NA2 and EU1. Actinovate † (Streptomyces lydicus) was the least effective of the three bacterial biocontrol agents and there was no difference in percent inhibition among P. ramorum lineages. Two products containing Trichoderma spp. were tested: Plant Helper † (T. atroviride) caused 100% inhibition of all lineages of P. ramorum, while SoilGard TM (T. virens) was only about 30% effective. There was great variability among P. ramorum isolates in their response to biocontrol agents. All treatments reduced P. ramorum lesion size on both Rhododendron and Camellia. Combined treatments of Actinovate † with one other BCA did not perform as well as either treatment used individually. Best results were obtained with Serenade † on Rhododendron and Camellia foliage, especially against the NA1 group. Lack of a linear relationship between percent inhibition of P. ramorum by BCAs in vitro and foliar treatments on detached Rhododendron and Camellia leaves indicates that in vitro testing is a poor predictor of BCA performance on plant material.
Summary
A set of quantitative hierarchical real‐time PCR assays was developed for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and of the entire Heterobasidion annosum complex. These assays enable specific and accurate detection and quantification of the target species from DNA extracted on airborne collected spores. Heterobasidion‐specific TaqMan™ real‐time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96‐ or 384‐well plate format arrays for high‐throughput testing of large numbers of samples against multiple targets. Assays were validated for (i) specificity, (ii) sensitivity and (iii) repeatability. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and a hundred ITS gene region copies or one conidia count equivalent. Precision or repeatability of each assay revealed a mean coefficient of variation of 5.9%. These molecular tools are now available for rapid and reliable monitoring of one of the most significant pathogen species complex of temperate northern coniferous forest around the world.
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