Hema Kasinathan Thesis May 1 2012.pdf - Atrium - University of ...

When the cotyledons were fully expanded, individual seedlings were transplanted intopipette tips / Falcon tubes containing autoclaved, non-calcareous sand at pH 6.5(Hutcheson sand, Hutcheson Sand Mixes, Huntsville, ON) for the root hair infection trialand tall plastic containers (Conetainers, 164 mL, Stuewe Sons Inc. Corvallis, OR) for thesymptom development trial. The plants were fertilized with 15: 15: 18 N:P:K andammonium sulphate. A stock solution was prepared using 40 g of N:P:K and 20 g ofammonium sulphate in 1 L of water. A 5-mL aliquot of stock solution was added to 1 Lof deionized water and the pH was adjusted before watering as described below. A 14-hrphotoperiod with a light intensity of 200–250 µmolm -2 s -1 and 65% relative humidity wasmaintained throughout the trial. The transplanted seedlings were maintained at 20°C for 5days, and then they were inoculated and moved to the respective temperature treatments.2.3.3 Inoculum preparationTo prepare the spore suspension, resting spores of P. brassicae pathotype 6 wereextracted from clubbed roots of cabbage collected from the Muck Crops ResearchStation, Holland Marsh, Ontario in 2008 and stored at -20°C until required. A 3-g sampleof clubbed roots was macerated in 100 mL of sterile deionized water in a commercialblender. The resulting suspension was filtered through eight layers of cheesecloth. Theconcentration of resting spores in the filtered solution was estimated using ahaemocytometer and diluted with sterile deionized water to attain the desiredconcentration of 1 x 10 6 spores mL -1 . The inoculum was prepared on the day ofinoculation. After inoculation, the plants were moved to growth cabinets set at therespective temperature treatments. For both studies, 10-day-old seedlings were inoculatedon 25 May, 2010 and the repetition of both studies was inoculated on 07 October, 2010.37