First Report of Pythium dissotocum Causing Pythium Root Rot on Hydroponically Grown Lettuce in Connecticut

Abstract

In August 2016, lettuce (Lactuca sativa L.) plants presented root rot, severe wilting, and high incidence of mortality in New Haven County, CT. The plants were grown in a hydroponic deep-water culture system in a commercial greenhouse. Mature plants were stunted, the lower leaves were chlorotic, the roots were necrotic, and some plants collapsed. Lettuce roots were collected from several symptomatic plants, washed three times with sterile deionized water, blotted dry, and plated on PARP-V8 selective medium (0.005 g of pimarcin, 0.25 mg of ampicillin, 0.01 g of rifampicin, 0.10 g of pentachloronitrobenzene, 200 ml of clarified V8 juice, and 800 ml of distilled water) (Moorman et al. 2002). The plates were incubated in the dark at 21°C. Mycelium resembling the morphology of Pythium species was observed in all plates after 3 days. The isolate was identified as Pythium dissotocum based on the morphology of sexual and asexual structures following van der Plaats-Niterink’s key (van der Plaats-Niterink 1981). Colonies grown on V8-juice agar had filamentous sporangia with a dendroid structure, the oogonia were 23 μm in diameter and subglobose, the antheridia developed on unbranched stalks and were sessile, and the oospores were aplerotic. DNA was extracted from mycelial mats using a DNeasy Plant Mini Kit (Qiagen, U.S.A.), and the internal transcribed spacer (ITS) region was amplified using universal primers ITS1 and ITS4 (White et al. 1990). BLAST nucleotide analyses against publicly available Pythium sequences in GenBank confirmed that the isolates obtained were P. dissotocum with 97% query coverage and 99% identity match to GenBank accession number KM061701.1. The nucleotide sequence has been assigned the GenBank accession number MG993546. To confirm Koch’s postulates, 14-day-old lettuce seedlings, cultivar Rex (n = 25), were inoculated with 1 × 10⁵ zoospores of the isolate of P. dissotocum produced on V8 broth. After 7 days, symptoms appeared as reduced growth, necrotic roots, and chlorotic leaves. Control plants were symptomless and healthy. Roots were collected from each infected lettuce plant, and four root fragments were plated on PARP-V8 selective medium. Mycelial growth resembling Pythium was observed in all plates from inoculated plants, and no growth was observed from roots of noninoculated plants (n = 25, 1 plate per plant). The putative P. dissotocum was reisolated from the plates; DNA was extracted from mycelial mats using the same methods described above and was confirmed to be Pythium using primers ITS1 and ITS4. The amplicon was the same size as for the P. dissotocum recovered from the original plants. This is the first report of P. dissotocum being detected on lettuce in Connecticut. P. dissotocum reduces crop yield and was first reported in 1986 as a root pathogen on hydroponic lettuce in Arizona (Stanghellini and Kronland 1986). There are currently no registered synthetic-chemical fungicides in the United States for the suppression of Pythium species in hydroponic lettuce (Utkhede et al. 2009). Prevention of this disease is recommended by controlling temperature and salt levels in the nutrient solution, sanitizing surfaces, and applying preventative biological fungicides registered for Pythium root rot.