During July 2012, symptoms of root rot were observed on bell pepper (Capsicum annuum) grown in 2,000 m2 of commercial greenhouses near Cuneo in northern Italy. Symptoms first developed 30 to 40 days after transplanting, when greenhouse temperatures ranged from 25 to 30°C, and 10% of the plants were affected. Affected plants were stunted with leaf chlorosis, reduced growth, and sudden wilting. Roots were severely affected with a brown discoloration, water-soaking, and soft rot. Eventually, affected plants collapsed. Tissue fragments of 1 mm2 were excised from symptomatic roots, dipped in a 1% sodium hypochlorite solution, and placed on potato dextrose agar (PDA) and an agar medium selective for oomycetes (3). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. An isolate grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup, 15 g agar, 0.5 g CaCO3, and 1 liter distilled water) showed aseptate hyphae that were 3.5 to 6.3 μm (avg. 5.2 μm) wide. Oogonia were globose, smooth, and 24.3 to 29.0 (avg. 25.1) μm in diameter. Antheridia were barrel-shaped, while oospores were globose, and 17.3 to 23.5 μm (avg. 21.2 μm) in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The ITS region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 781-bp segment (GenBank Accession KF840479) showed 100% homology with the ITS sequence of an isolate of Pythium aphanidermatum in GenBank (AY598622.2). Pathogenicity tests were performed twice on 30-day-old plants of C. annuum cv. Cuneo grown in 2-L pots (4 plants/pot), containing a steam-disinfested, organic peat substrate (70% black peat and 30% white peat, pH 5.5 to 6.0, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) that was infested with wheat and hemp kernels colonized by the isolate of P. aphanidermatum, at a rate of 1 g colonized kernels/liter potting medium. The inoculum was prepared by autoclaving at 121°C for 30 min a mixture of wheat-hemp kernels (2:1 v/v) in a 1-liter flask, to which the bell pepper isolate of P. aphanidermatum was added in the form of colonized agar medium selective for oomycetes plugs. Before use, the inoculated flask was incubated for 10 days at 22°C in the dark. Four plants/pot were transplanted into each of four pots filled with the infested medium/growth chamber, while the same number of plants were grown in non-infested substrate in pots in each growth chamber. Plants were kept in two growth chambers, one set at 20°C and the other at 28°C. Symptoms first developed 7 days after inoculation. After 30 days, 50% of inoculated plants showed brown roots and died in the growth chamber set at 28°C, while only 10% of the plants were symptomatic at 20°C. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in the infested soil by using the same protocol as the original isolations, while no fungal colonies were obtained from asymptomatic roots of the non-inoculated control plants. To our knowledge, this is the first report of the presence of P. aphanidermatum on C. annuum in Italy. The same disease was reported in the United States (2). The importance of the disease, although limited in distribution at present to the greenhouses surveyed in northern Italy, could increase in areas where sweet pepper is grown intensively. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. O. Chellemi et al. Plant Dis. 84:1271, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Boca Raton, FL, 2002.
First Report of Pythium aphanidermatum Crown and Root Rot of Industrial Hemp in the United States
