Archivio Journal of Plant Pathology

Abstract:

A foliar disease of holly (Ilex aquifolium), observed in November 2009 in a natural ecosystem in the region of Thessaloniki (northern Greece), consisted of circular or irregularly shaped necrotic spots with yellow border, 3-7 mm in diameter, present on ca. 30% of the leaves. Small pieces of tissue at the margin of the spots were excised, surface-disinfected (1% sodium hypochlorite for 1 min) and plated on potato dextrose agar. Two single-spore cultures in potato carrot agar, gave rise to initially white colonies turning grayish-black due to abundant sporulation. Conidiophores were green to brown, short, septate, branched or unbranched. Conidia, produced in single or more often branched chains, were obpyriform, with a conical or cylindrical beak, ovoid or ellipsoidal, measured 7.7-27.4x5.6-15.0 μm (average 16.3x8.8 μm), and showed 1 to 5 transverse and 0 to 3 longitudinal septa. These morphological traits and measurements tally with those of Alternaria alternata (Fr.) Keissl. (Simmons, 2007). The ITS1-5.8S-ITS2 region of the two single-spore isolates, amplified with primers ITS1 and ITS4 and sequenced (GenBank accession Nos. JQ809323, JQ809324), revealed 100% homology with the sequence of various A. alternata isolates (e.g. JQ070079). Eight 3-year-old holly plants were sprayed with a 106 conidia/ml spore suspension, covered with a polyethylene bag for 48 h and placed in a greenhouse at 23±2°C together with sterile distilled water-sprayed controls. Leaf spots like those observed in the field developed on all inoculated plants but not on controls. A. alternata was reisolated from artificially inoculated leaves. Holly infections by A. alternata have previously been reported from Poland (Orlikowski and Szkuta, 2004), the USA and Canada (Sinclair et al., 1987) but, to our knowledge, never from Greece.