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Project Title: Molecular methods for virus detection in fruit plants

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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 Strawberry crinkle virus (SCV), the transfer from strawberry to the herbaceous hosts Physalis pubescens and Nicotiana occidentalis was made by aphids (Chaetosiphon fragaefolii). Uninfected aphids, reared on healthy plants of Fragaria vesca subsp. semperflorens or Fragaria vesca clone UC-5, were transferred to infected plants and reared for a minimum of 14 days. Aphids were then transferred in groups of 10-20 to the herbaceous test plants to allow transmission of the virus. Transmission between the herbaceous plants, for experimental and maintenance purposes, was by sap inoculation using an extract of 2-5 g plant material in 5-10 ml buffer (225 mM phosphate pH 7.8, containing 10 g/litre Celite and 5 g/litre sodium sulphite). Plants of Physalis pubescens were held in the dark for 24 h before inoculation to increase susceptibility.
Virus isolates were collected from commercial and experimental fields in the UK, mainly in Kent, but also in East Anglia (Table 1). Presence of SCV in these plants was confirmed by grafting onto UC-5. SCV isolates were maintained in the original host plants and/ or in UC-5 indicator plants in an insect-proofed tunnel.

Table 1. Summary of the SCV isolates.

Isolate name

Place of collection

Date of collection


East Anglia



East Anglia














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.

Confirmation of infection with SCV in commercial strawberry plants was by leaf grafting to F. vesca UC-5 and /or by symptoms in P. pubescens.

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.

Double-budding field assay for Apple stem grooving virus (ASGV)

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.

Enzyme-linked immunosorbent assay (ELISA)
Assays for Apple stem grooving virus (ASGV) were conducted with antiserum provided by Loewe Phytodiagnostica. Reagents were diluted according to the supplier’s recommendations and used in a ‘Simultan-Cocktail-ELISA’. Samples were extracted (at a dilution of approximately 0.1 g plant material in 5 ml buffer) in phosphate-buffered saline containing 0.5 ml Tween 20, 20 g polyvinyl pyrrolidone,

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.

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