A new host and phenotypic variation of Phytophthora hedraiandra in Spain

The oomycete Phytophthora hedraiandra De Cock & Man in’t Veld is first reported in Spain on the ornamental plant Rhododendron catawbiense Michx., as well as on leaves of Viburnum tinus L. in nurseries. The identification of these isolates was carried out by examining their morphological and cultural features, and by comparing the sequence of the nuclear rDNA ITS region and the mitochondrial cox1 gene with those published in GenBank database. The phenotypes of the isolates fitted the species description, but a higher intraspecific variation was noticed in respect to their sporangial size, colony pattern and radial growth rates. Due to the similarities between P. cactorum (Lebert et Cohn) and P. hedraiandra, the taxonomy and host ranges of the P. cactorum/P. hedraiandra complex in ornamental nurseries and natural ecosystems need reviewing. Additional key words: ornamental plant disease, phenotypic variation, plant pathogen.

identif ication together with several other putative P. hedraiandra isolates collected from V. tinus in Girona, and compared with other previously identified isolates collected in Mallorca, Spain (Table 1).For this purpose, they were grown in 90 mm diam.Petri dishes on corn meal agar (CMA, Sigma), potato dextrose agar (PDA, Sigma), carrot agar (CA; Brasier, 1967) and malt extract agar (MEA; 48 g of malt extract plus 15 g of agar dissolved in 1,000 ml of distilled water) and their colony pattern and radial growth rates estimated after 4 and 7 days.The presence of sporangia and other asexual structures on all agar media was checked over two weeks.To induce the formation of sporangia, three 12 mm diam.disks were taken from the edge of a seven-day-old colony grown on CA and placed in a 60 mm diam Petri dish previously flooded with 10 ml of soil extract (filtered extract of 50 g of oak forest soil in 1,000 ml of distilled water, autoclaved at 121ºC for 15 min).The dishes were kept for 48-72 h at 20ºC under continuous white light.The shape, papilla formation, and caducity of sporangia were recorded.The presence of gametangia on CA was checked after 10 days growth at 20ºC in darkness.
Mycelial DNA was extracted from pure cultures grown in sterile pea broth, and checked for quality as previously described (Belbahri et al., 2006).Ribosomal DNA ITS amplifications were completed using the previously described universal primers ITS4 and ITS6 that target conserved regions in the 18S and 28S rDNA genes for amplification of the internal transcribed spacer region 1 (ITS1), the 5.8S rRNA gene and the internal transcribed spacer region 2 (ITS2) (Cooke et al., 2000).A partial sequence of the cytochrome c oxidase subunit I gene (COX1) was amplif ied with primers COXF4N and COXR4N developed by Kroon et al. (2004).PCR product purification and DNA sequencing were performed according to Belbahri et al. (2006).The sequences obtained were registered in GenBank (Table 1).
The culture characteristics of P. hedraiandra were clearly distinct from those of other homothallic Phytophthora species examined [e.g.P. cactorum (Lebert et Cohn) Shröter and P. citricola Sawada], but showed some intraspecific variation in their colony pattern and radial growth rates (Fig. 1).It is noteworthy that isolates collected from V. tinus leaves had lower radial growth rates, especially on MEA and PDA.Colony patterns  1), were confirmed by sequencing of the PCR amplified mitochondrial cox1 gene (DQ643973) and the ITS region of the rDNA (DQ643972).Sequence comparisons between the ITS region and the cox1 gene of Spanish isolates (this study) and of isolates available in GenBank did not show any polymorphism in these two DNA regions.
To assess the pathogenicity of P. hedraiandra on R. catawbiense, five leaves were placed on a metal grid in moist chambers consisting of a transparent plastic box lined on the bottom with sterile paper towels.The lower surface of four out of the five leaves was inoculated by placing a single 100 µl drop of a ca. 10 4 ml -1 zoospore suspension near the centre of the midrib.The remaining leaf was used as a control by replacing the inoculum with a drop of sterilized de-ionized water.The box was incubated at 20°C under cool white light.All leaves except the control formed large lesions and the pathogen could be re-isolated when leaf tissue was plated onto P 5 ARP medium.
The emergence of P. hedraiandra diseases is similar to that of the early stages of the invasive P. ramorum.Likewise, it is spreading worldwide within nurseries through the international trade of Viburnum and Rhododendron; although P. ramorum was noteworthy due to the extensive damage it was causing to Californian oak trees.Maybe P. hedraiandra has remained unnoticed until now because of its morphological similarities with P. cactorum, which seems to have a wide host range (Erwin and Ribeiro, 1996).This latter fact adds further uncertainties on the actual chronology and pathology of P. hedraiandra, that is, whether it has been recently introduced or misidentified as P. cactorum.More doubts on its origin have risen after a single isolation of P. hedraiandra within an extensive survey of soils in natural ecosystems in Poland (Belbahri, unpublished), which suggests a recent introduction of the pathogen.In this study some evidence of phenotypic variation has been found in the small collection of isolates from Spain, which is a little surprising considering that P. hedraiandra is homothallic and thus a sexually inbreeding species.Further research is being carried out to determine its potential host range.

Table 1 .
Origin of the isolates of P. hedraiandra used in this study with their ITS and COX1 Genbank accession numbers In contrast, isolate P11935 formed large sporangia.The identity of the P. hedraiandra isolate P11935 from R. catawbiense, along with those of several new isolates recovered from leaves and stem lesions on V. tinus in December 2005 (Table Figure 1.Average colony radial growth rates of six isolates of Phytophthora hedraiandra at 20°C in four agar media (CA, carrot agar; MEA, malt extract agar; PDA, potato dextrose agar, and CMA, cornmeal agar).Values averaged from measurement of two replicates.

Table 2 .
Dimensions of sporangia and sexual structures of P. hedraiandra isolates grown on carrot agar (CA)