First Report of Sydowia polyspora on Aleppo Pine (Pinus halepensis) in Italy

Pinus halepensis Mill. is a pine native to the Mediterranean region that is generally located from sea level up to an altitude of 200 meters. In July 2012, extensive leaf yellowing and scorching were observed on the foliage of two specimens of P. halepensis in a public park of Alassio municipality, Liguria region (northern Italy). Diseased needles showed chlorotic and reddish brown colored areas that were randomly distributed among healthy needles. In order to isolate the potential pathogen, diseased needle tissues were surface sterilized for 1 min in 1% NaOCl and plated onto potato dextrose agar (PDA) amended with 100 ppm of streptomycin sulfate. After 7 to 10 days, a brown to dark green mycelium slowly developed on the PDA. From the mycelium, single cell conidia averaging 7.0 (4.7 to 12.2) × 2.9 (2.4 to 5.4) μm (n = 50) were produced. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS1f/ITS4 and sequenced. A BLAST (1) search yielded 100% of maximum identity with Sydowia polyspora (Bref. & Tav.) E. Müller for ITS1f and ITS4. Pathogenicity was confirmed by inoculating 15 3-year-old plants (approximately 5 months after bud break) of P. halepensis grown singly in 8-cm diameter pots and maintained in greenhouse. Twelve of 15 plants were wounded by gently rubbing needles together. Five non-inoculated plants, with wounded and non-wounded branches, were kept as controls. Inoculations were carried out at 16.5 to 18.5°C. A suspension of S. polyspora conidia was made by adding 1 to 2 ml sterile water to the spore mass on 1-month-old PDA cultures in 9-cm petri dishes. Inoculum (10 7 spores/ml) was applied on the needles with a soft paint brush. After inoculation plants were covered with polythene bags for 5 days, kept at temperatures ranging from 5.5 to 26.4°C (average 14.5°C), and watered as needed. The inoculation trial was repeated once. All inoculated plants, both wounded and non-wounded, developed leaf yellowing and browning since the fifth day after inoculation in both inoculation tests, while control plants remained symptomless. After 15 days from first symptom appearance, S. polyspora was re-isolated from the needles of inoculated plants according to the procedure already described. S. polyspora is associated with current season needle necrosis (CSNN) on various conifer species. P. halepensis was reported in Spain to be a susceptible host (2) and S. polyspora has been isolated from infected tissues of fir (Abies spp.) (3). To our knowledge, this is the first report of S. polyspora on P. halepensis in Italy. Control of the pathogen with fungicides was ineffective on fir species, while application of very high rates of calcium chloride during shoot elongation was able to reduce the severity of CSNN (3). In forest areas, municipality gardens, and parks, effective management strategies have not yet been developed. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) L. Botella et al. Fungal Diversity 40:1, 2010. (3) V. Talgø et al. Fungal Biol. 114:545, 2010.