Project Title: Molecular methods for virus detection in fruit plants

Virus isolation and maintenance

Extracts were made by grinding the leaves or bark of fruit trees, infected with apple stem pitting or apple stem grooving virus, (about 0.1g/2ml of buffer) with a pestle and mortar. The buffer for initial extraction and for passage between herbaceous plants was: 0.025 M sodium/potassium phosphate pH 7.5 containing 0.2 g/litre sodium diethyl dithiocarbamate (DIECA), 0.2 g/litre sodium thioglycollate, 1.5 g/litre polyethylene glycol (mol. wt 6000). Herbaceous host plants were dusted with carborundum (300 mesh), the leaves wiped with the plant extract and the excess inoculum rinsed off the leaves. Herbaceous plants were kept in a glasshouse compartment with supplementary lighting during winter to provide 16 h daylength. Apple stem grooving virus was maintained in Chenopodium quinoa and Apple stem pitting virus in Nicotiana occidentalis (37B).

For library construction, plants were inoculated with SCV isolate G90/11 and control plants for providing RNA were inoculated at the same time with a mixture of buffer and water. The healthy and infected plants were grown in a randomised design in the glasshouse.

Care of trees in glasshouse double-budding test

MM106 rootstocks (7-9 mm) from F P Matthews Ltd were potted in forestry liners at the end of February. The liners were 250 mm deep, diameter 64 mm (650 ml), from Stuewe & Sons, 2290 SE Kiger Island Drive, Corvallis, Oregon, USA. The compost was 9/1 peat/loam containing 4 kg/m³ Osmacote Plus. Rootstocks were budded in mid April and cut back to the top bud a month later. The pots were irrigated with drippers that were controlled with a timer to deliver 65 ml water twice daily. The plants were given Maxicrop Triple drenches in July and August and sprayed with Nimrod for mildew and an acaricide as needed. The plants were assessed for bud take and symptoms before discarding in September.

400 M26 were planted in double rows, in March, in soil treated with chloropicrin (200 litres per hectare), at a spacing of 1.0 m between rows and 0.5 m within rows. The rootstocks were budded in August, each with a test bud about 10 cm above soil level and a bud of the Virginia Crab indicator 15 cm above the ground. The buds were placed on the north side of the rootstock. Buds from each tree under test were grafted to three replicate rootstocks. Five positive controls, grafted with buds from trees known to be infected with ASGV and five negative controls that lacked infector buds, were included after each run of 10 tests. Grafting tape was left in place for 6 wk and bud-take recorded one to two months later. The rootstocks were cut back to the top scion bud during the winter and allowed to grow for three seasons before being cut down for symptom assessment. The trunks were cut at about 30 cm above the graft union and 5 cm below it. The shoots were then autoclaved at 121 ^oC for 15 min and plunged into warm water. The bark was then easily removed for observation of the condition of the wood and union.

2 g ovalbumin, per litre. 100 l each of sample and alkaline phosphatase-labelled antibody was incubated in each well of a 96 well microtitre plate, overnight at 6 ^oC. The plate was developed with 4-nitrophenyl phosphate substrate for 1 hour and the reaction monitored at 405 nm. Each sample was duplicated and the plates each included five negative samples (10 wells) and a positive control. Samples were considered infected if the ELISA values were greater than the negative mean value plus three times the standard deviation.