Phytophthora ramorum in Canada: evidence for migration within North America and from Europe.

Phytophthora ramorum, the cause of sudden oak death on oak and ramorum blight on woody ornamentals, has been reported in ornamental nurseries on the West Coast of North America from British Columbia to California. Long-distance migration of P. ramorum has occurred via the nursery trade, and shipments of host plants are known to have crossed the U.S.-Canadian border. We investigated the genotypic diversity of P. ramorum in Canadian nurseries and compared the Canadian population with U.S. and European nursery isolates for evidence of migration among populations. All three of the P. ramorum clonal lineages were found in Canada but, unexpectedly, the most common was the NA2 lineage. The NA1 clonal lineage, which has been the most common lineage in U.S. nurseries, was found relatively infrequently in Canada, and these isolates may have been the result of migration from the United States to Canada. The EU1 lineage was observed almost every year and shared multilocus genotypes with isolates from Europe and the United States. Estimation of migration rates between Europe and North America indicated that migration was higher from Europe to North America than vice versa, and that unidirectional migration from Europe to North America was more likely than bidirectional migration.

Alnus glutinosa Threatened by Alder Phytophthora: A Histological Study of Roots.

Alder dieback remains a major problem in European alder stands and its spread continues to threaten their existence. The causal agent of this disease is the so-called alder Phytophthora species complex, which includes the hybrid Phytophthora ×alni and its parental species P. uniformis and P. ×multiformis. Little is known about the survival of these Phytophthora species in alder. The aim of our investigations was to find out whether, and if so where, the pathogen survives. The subject of these studies was alder roots. Therefore, artificial infection studies and histological studies with P. ×alni and P. uniformis were carried out on seedlings of black alder (Alnus glutinosa). These histological studies revealed oogonia and oospores of P. ×alni and P. uniformis in different parts of the root tissue.

Unravelling hybridization in Phytophthora using phylogenomics and genome size estimation.

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum.

Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

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.

First Detection of Phytophthora ramorum Mating Type A2 in Europe.

Since its original isolation in 1993, Phytophthora ramorum has become an important pathogen. Initially, it was determined to be the causal agent of a twig blight of Rhododendron spp. in Germany and the Netherlands (3). Around the same period, symptoms and mortality on oak (Quercus spp.) and tanoak (Lithocarpus densiflorus) were associated with P. ramorum in California (2), where the disease was named sudden oak death. Subsequently, P. ramorum has been detected on a wide range of forest trees and shrub species in the United States. In Europe, the pathogen has spread to many countries, primarily on nursery plants of Rhododendron and Viburnum spp., and recently, on Camellia japonica, Kalmia latifolia, Pieris formosa var. forrestii, P. japonica, Leucothoe sp., Syringa vulgaris, and Taxus baccata. P. ramorum has not been observed in European forests. P. ramorum is heterothallic, and initial in vitro mating studies on agar media suggested that only the A1 mating type occurred in Europe, while only the A2 mating type was present in the United States (4). However, an isolate collected in 2002 in Belgium (1) appears to be the A2 mating type. This isolate (CBS 110901, Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands) originated from an imported V. bodnantense plant at an ornamental nursery. A hyphal tip culture (BBA 26/02) of this isolate produced no oogonia on carrot piece agar after 6 weeks in pairing tests with other Phytophthora species of mating type A2. When paired with mating type A1 of P. cambivora, P. cinnamomi, P. cryptogea, and P. drechsleri, however, oogonia were observed in all pairings within 6 weeks. The number of oogonia was low in all pairings but was highest in pairings with P. cryptogea. No oospores were produced after 6 weeks between P. ramorum isolates BBA 26/02 and BBA 9/95 (from the holotype, mating type A1), but gametangia were observed when these isolates were paired on Rhododendron sp. twigs. Normal oogonia were produced on the outgrowing mycelium when pieces from these twigs were placed on carrot piece agar. The shape and size of the oogonia produced on carrot piece agar after pairing with P. cryptogea and on Rhododendron sp. twigs after pairing with P. ramorum BBA 9/95 were similar (24 to 34 µm, mean 29.6 µm and 25 to 33 µm, mean 30.6 µm, respectively). To our knowledge, this is the first observation of P. ramorum mating type A2 in Europe. References: (1) D. De Merlier et al. Plant Dis. 87:203, 2003. (2) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (3) S. Werres et al. Mycol. Res. 105:1166, 2001. (4) S. Werres and B. Zielke. J. Plant Dis. Prot. 110:129, 2003.

Development of a lab-on-a-chip device for diagnosis of plant pathogens.

A lab-on-a-chip system for rapid nucleic acid-based analysis was developed that can be applied for diagnosis of selected Phytophthora species as a first example for use in plant pathology. All necessary polymerase chain reaction process (PCR) and hybridization steps can be performed consecutively within a single chip consisting of two components, an inflexible and a flexible one, with integrated microchannels and microchambers. Data from the microarray is collected from a simple electrical measurement that is based on elementary silver deposition by enzymatical catalyzation. Temperatures in the PCR and in the hybridization zone are managed by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply for use on site. The specificity of the lab-on-a-chip system could be demonstrated for the tested five Phytophthora species. The two Pythium species gave signals below the threshold. The results of the electrical detection of the microarray correspond to the values obtained with the control method (optical grey scale analysis).

Characterisation of European and North American Phytophthora ramorum isolates due to their morphology and mating behaviour in vitro with heterothallic Phytophthora species.

Vegetative growth rate, and size of sporangia, chlamydospores and oospores from 94 P. ramorum isolates were measured and the isolates were paired in vitro with four different heterothallic Phytophthora species isolated from infected nursery plants in Germany. P. ramorum isolates originated from different European countries and from Canada and the USA. 66 of the 67 European isolates were determined as mating type A1; only one isolate was of mating type A2. Of the 27 North American isolates tested, seven (all from nurseries) were determined to be the A1 mating type and 17 to be the A2 mating type. Three isolates did not produce gametangia during the incubation period. Discriminant analysis of all data allowed a grouping based on the vegetative growth rate. The two groups corresponded with the mating type no matter whether the isolates originated from Europe or North America. The A1 isolates were much more homogeneous in their morphology than the A2 isolates. They grew faster, had larger chlamydospores and did not produce gametangia with P. cambivora. Within the A2 group, the single European isolate of mating type A2 (BBA 16/02) and three US isolates showed intermediate characters and were classified with the discriminant function into that of the opposite mating type. The morphological characters and the mating behaviour of the isolates will be discussed.