First report of Hakea sericea leaf infection caused by Pestalotiopsis funerea in Portugal

Hakea sericea (Proteaceae) is native to south-eastern Australia and has been considered as an invader of natural habitats. In northern Portugal, dense stands are rapidly spreading after forest fires (Fig. 1a). In May 2003, unusual leaf spots were observed on naturally growing plants. Infected plants exhibit reddish leaves bearing black, 1-3 mm circular lesions (Fig. 1b,c). Leaf sections containing necrotic lesions were plated onto potato dextrose agar (PDA) and eight fungal isolates were obtained. Pure cultures exhibit pinkish mycelium, bearing compact acervuli that contained black slimy spore masses (Fig. 2a). Microscopic observation revealed typical Pestalotiopsis sp. 5-celled spores (3 coloured median and 2 hyaline end cells) with 3-4 apical and 1 basal appendages (Fig. 2b,c).

Genomic DNA from fungi isolates was purified using DNeasy^® Plant Mini Kit (Qiagen), and used in thermocyclic amplifications using Ready-To-Go^TM PCR Beads (Amersham Biosciences) with ITS5 and ITS4 internal transcribed spacer (ITS) universal primers (White et al., 1990). The amplified sequences (599 bp) were analysed together with other Pestalotiopsis sequences (Jeewon et al., 2002) using the programs ClustalX, GeneDoc and Phylip. ITS sequences from all fungal isolates were identical to each other and only four base pair different (99.3% similarity) from Pestalotiopsis funerea (Fig. 3). To confirm the pathogenicity of P. funerea on H. sericea, leaf-wounded and non-wounded 6 week-old in vitro plants were sprayed with a suspension containing 10⁵ spores per ml or with sterile water. After 6 days, only leaf-wounded plants infected by P. funerea exhibited lesions identical to those observed in field plants, bearing fungus spores identical to those from original isolates.

embers of Pestalotiopsis genus have been described as pathogenic for Proteaceae species (Taylor et al., 2001). Countries where H. sericea invasion has become a problem, its biological control has been successfully achieved using Colletotrichum gloeosporioides (Richardson & Manders, 1985). The identification of naturally occurring pathogenic fungi in portuguese H. sericea could allow the design of an integrated control strategy for this invader.