During the spring of 2014, spinach (Spinacia oleracea L.) plants of the cv. Crocodile (Rijk Zwaan, De Lier, The Netherlands), grown in a clay loam soil under commercial greenhouse conditions near Salerno (southern Italy), showed stunting, extensive chlorosis, and root rot. Plants were irrigated by overhead sprinklers using well water. Symptoms first developed 20 days after sowing, at air temperatures of 23 to 30°C, and 35% of plants (approximately 15 million plants in 10 ha) were affected. Roots were severely affected, appeared water-soaked and brown, and were characterized by a soft rot. Eventually, affected plants wilted and collapsed. Fifty fragments, each 1 mm2, were excised from symptomatic roots of 10 plants, dipped in a solution containing 1% sodium hypochlorite, rinsed in sterilized water, dried on sterilized paper towel, and plated on both potato dextrose agar (PDA) and the medium BNPRA, which is semi-selective for oomycetes (3). After 5 days of incubation under constant fluorescent light at 22 ± 1°C, 80% of the root sections developed oomycete colonies. One representative isolate, grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup; 15 g agar; 0.5 g CaCO3; 1 liter distilled water) and observed with a light microscope, showed aseptate hyphae 3.3 to 6.5 (mean 5.5) μm wide. Oogonia were globose, smooth, and 22.2 to 31.0 (average 26.3) μm in diameter. Antheridia were barrel-shaped, while oospores were globose and 17.3 to 22.6 (mean 20.9) μm in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of this isolate was amplified using ITS1/ITS4 primers and sequenced. BLAST analysis (1) of the 647-bp segment showed 100% homology with ITS sequences of Pythium aphanidermatum in GenBank (Accession Nos. KJ755088.1, KJ162355.1, KF840479, and KF561235.1). The nucleotide sequence for the Italian spinach isolate was assigned No. KM111256. Pathogenicity tests were performed twice on 20-day-old spinach plants of the cv. Merlo (L'Ortolano, Cesena, Italy), grown in 2-liter pots in a steam-disinfested organic peat substrate (black peat, pH 6.5 to 6.8, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) moistened to field capacity, and infested with wheat and hemp kernels colonized with isolate Py 1-14 of P. aphanidermatum at 1 g/liter. Five plants were transplanted into each of four pots filled with infested peat, while the same number of plants was grown in non-infested substrate as a control. Plants were kept in two growth chambers, each with 12 h of light per day at 20 or 30°C, and were irrigated daily to maintain the potting medium at field capacity. Symptoms first developed 5 days after the spinach was transplanted into infested potting medium in the growth chamber maintained at 30°C. After 10 days, all plants in this growth chamber were dead, while only 5% of the plants growing in infested potting medium in the 20°C growth chamber were affected. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in infested medium. No fungi were re-isolated from the asymptomatic control plants grown in non-infested substrate. To our knowledge, this is the first report of P. aphanidermatum causing root rot on S. oleracea in Italy. The same pathogen has been reported to cause root rot of spinach in other countries, including the United States (2). The disease is, at present, limited to the affected greenhouses observed in this study.
References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. L. Bates and M. E. Stanghellini. Plant Dis. 68:989, 1984. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, FL., 2002.