First report of Phytophthora hedraiandra on Viburnum tinus in Italy

Laurustinus (Viburnum tinus) is an ornamental shrub widely used in private gardens and public parks for background, screen planting, or clipped and unclipped hedging. It is commonly present in natural wood stands in the Mediterranean area. In spring 2004, several cases of decline and mortality of laurustinus plants were observed in nurseries located in central Italy, particularly in Pistoia province (Tuscany, Italy). Symptoms were characterized by collar and root rot. Affected plants showed wilting of leaves and shoots. A Phytophthora spp. was isolated from infected tissues and soil removed from the root zone of plants showing symptoms. Isolations were made by direct plating of tissue fragments cut from the margin of collar necrosis, and from soil by baiting with rhododendron leaves. The selective medium PARBhy (Vettraino et al., 2001) was used. The species isolated was identified as Phytophthora hedraindra, based on cultural and morphological features (De Cock & Lévesque, 2004). Six single hyphal tip isolates, obtained from different diseased and dead plants, were analysed for morphological features. They produced smooth distinct papillate, ovoid to obpyriform caducous sporangia (length/width average 1·3), with a short stalk (av. 2 µm), terminal chlamydospores, and were homothallic with smooth, globose oogonia (av. 30 µm in diameter), mostly paragynous sessile or short-stalked antheridia (av. 11·5 µm long), occasionally amphigynous, oospores commonly aplerotic (av. 32 µm in diameter). Identification was confirmed by sequencing the ITS region of rDNA of all the six isolates and comparing the sequences (overall length of 792 bp) with those of Phytophthora species present in the NCBI database. The sequences of four isolates displayed 100% homology with sequence AY707987 (P. hedraiandra isolated from Viburnum spp. in the Netherlands), whilst the sequences of the other two isolates had 99% homology. The sequences of four isolates were submitted to GenBank: accession numbers AM049389 and AM049390 were given to the ITS sequences of isolates ISPaVe 1866 and ISPaVe 1867, respectively (which displayed 100% homology with AY707987), and AM049391 and AM049388 to the ITS sequences of isolates ISPaVe 1868 and ISPaVe 1865 (which differed by one and two bases, respectively, from AY707987). All the P. hedraiandra ITS sequences displayed common single base pair substitutions at positions 74, 100, 101 and 686 in comparison to P. cactorum ITS sequences. Pathogenicity tests were conducted by soil infection on 3-month-old potted laurustinus plants using the isolates ISPaVe 1865, 1866, 1867 and 1868. Sporulation was induced by 24 h flooding of the soil. One month after inoculation, symptoms were produced similar to those observed on naturally infected plants. Controls remained healthy; P. hedraiandra was reisolated from infected collar and roots. This is the first report of P. hedraiandra as agent of collar and root rot of V. tinus in Italy.