First report of root rot of hydroponically grown lettuce (Lactuca sativa) caused by a Pythium species from the cluster B2a species complex in Cyprus

Abstract

Although widely adopted by growers worldwide, hydroponic cultivation of lettuce is relatively new in Cyprus. Currently, ∼32 ha of lettuce are cultivated in the region, with only a few commercial trials using hydroponics. During March and April 2016, ∼23% of lettuce plants cv. Verde Degli Ortolani grown hydroponically (deep flow technique) in Polemidia, Limassol, showed severe stunting and poor growth. Older leaves wilted during the warmer period of the day and over the next 2 weeks the outer leaves became chlorotic and yellow. Small feeder roots had brown lesions, and subsequently the entire root system became necrotic. Diseased roots of 10 plants were excised, surface-disinfested, and plated on PARP-V8 medium selective for oomycetes (Jeffers and Martin 1986). Root isolations consistently yielded pure cultures of Pythium species. After 48 h incubation at 25°C, isolates produced white, circular, flat colonies with little aerial mycelia. Sections from the margin of colonies were transferred to 9 cm dia. plates containing 15 ml sterile water, which was incubated for at least 24 h at 25°C. Microscopic examination revealed filamentous and slightly inflated sporangia forming dendroid structures. Zoospores formed in vesicles and were up to 8 μm in diameter. The isolates failed to produce sexual structures during 30 days of incubation. Based on these morphological characteristics, the isolates could not be identified to the species level. Therefore, genomic DNA was extracted from six isolates, and the internal transcribed spacer (ITS) region of rDNA was partially amplified and sequenced using universal primers ITS4 and ITS5. A representative sequence was deposited in GenBank (accession no. KX518318). BLAST analysis revealed a 99% similarity to a P. dissotocum strain CBS 166.68 sequence (AY598634). This species belongs to the Pythium cluster B2a species complex including P. coloratum, P. diclinum, P. dissotocum, and P. lutarium (Robideau et al. 2011), which are indistinguishable based on ITS and cytochrome c oxidase subunit I (COI) sequences. To confirm pathogenicity under hydroponic conditions, six different Pythium isolates, of which the sequences were similar than accession AY598634, were used in pathogenicity experiments. Two-week-old lettuce plants cv. Nogal, started in a nursery in peat pellets, were placed in 14-liter plastic containers containing a full-based nutrient solution (Christoulaki et al. 2014) in growth chambers at 25 ± 1°C and 16-h photoperiod. Three containers were used per isolate, each containing four plants. Five days after transplanting, one intact 48-h old Pythium culture growing on a V-8 agar plate (9 cm dia.) was added to each container and removed 48 h later. Control plants were inoculated with sterile V-8 agar plates. After 3 weeks, older leaves of inoculated plants exhibited chlorosis, and feeder roots and taproots showed discrete brownish lesions. Control plants did not show any symptoms. Pythium was consistently reisolated from symptomatic roots but not from the control plants, fulfilling Koch’s postulates. The pathogenicity experiments were repeated once. P. dissotocum, which belongs to Pythium cluster B2a, has long been reported to infect lettuce plants in hydroponic culture (Stanghellini and Kronland 1986). To our knowledge, this is the first report of root rot of lettuce caused by a Pythium spp. belonging to Pythium cluster B2a species complex in Cyprus.