Studies on the luteoviruses potato leaf roll virus, beet western yellows virus and tomato yellow top virus: a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the University of Canterbury [Lincoln College]

Abstract

Overseas, beet western yellows virus (BWYV) has been implicated in the leaf roll disease of potatoes, considered to be caused by potato leaf roll virus (PLRV). A project was initiated to determine if BWYV was infecting potatoes in New Zealand, either as part of a potato leaf roll (PLR) complex, or as a separate disease, perhaps without causing symptoms. Both indicator plants and ELISA, using polyclonal antisera and monoclonal antibodies (MAbs), were evaluated for the detection of BWYV and PLRV. Crambe abyssinica was shown to be a differential host for BWYV, being resistant to PLRV. Capsella bursa-pastoris was also a good indicator for BWYV but may be susceptible to PLRV. Tomato, Datura stramonium and D. tatula were differential hosts for PLRV, being resistant to BWYV. In DASELISA, cross-reactions between PLRV or TYTV and BWYV polyclonal antisera occurred. BWYV MAbs were more specific, but there was still a weak reaction with PLRV and TYTV, both with purified preparations and sap from infected plants. The etiology of the PLR disease was investigated by ELISA and by aphid transmission to indicators. ELISA results suggested BWYV could be present at low concentrations in PLR-diseased plants, as a mixed infection with PLRV, the major component. Attempts to isolate BWYV from PLR-diseased plants by aphid transmission to crambe or shepherd's purse were not successful. It was concluded that BWYV was not present in PLR-diseased potatoes and PLRV particles cross-reacting with BWYV antibodies were responsible for weak reactions with PLR-diseased plants in ELISA. The ability of BWYV to infect potatoes was investigated by experimental inoculation, and field trials during two seasons, to detect natural infection by BWYV. Attempts to infect six potato cultivars with BWYV ex turnips were unsuccessful. Results from 1984-85 suggested a low incidence of BWYV (5-15%) in Ilam Hardy potatoes, based on ELISA tests of 36 plants with the PLRV/BWYV 023 and BWYV 048 antisera, and aphid transmission to crambe and shepherd's purse. These results were not confirmed in 1985-86; nil infection of 100 Ilam Hardy and 100 Rua potato plants was detected, using more specific, more sensitive antibody probes (BWYV MAbs) and aphid transmission to crambe and shepherd's purse. During the springs of 1984-85 and 1985-86, numbers of Myzus persicae flying were small. Thus the inoculation pressure was low. Incidences of BWYV in crambe bait plants during November and December were 0-43% in 1984-85 and 25-65% in 1985-86. M. persicae was the main aphid colonizing potatoes. The propagation and purification of BWYV and PLRV were investigated to improve virus yields. Crambe roots were a better source of BWYV than crambe shoots, containing 10-20 times more virus, based on quantitative ELISA data and purification experiments. However, physalis shoots were a better source of PLRV than physalis roots. In purifying PLRV, raising the pH of extracts from 6.0 to 7.0 after incubation with enzymes and before chloroform-butanol clarification greatly increased virus yields. Double-stranded RNA analysis of BWYV, PLRV and TYTV was attempted, in order to differentiate them. Two species, with molecular weights of 3.8 x 10⁶ and 1.8 x 10⁶ daltons, were resolved by gel electrophoresis for BWYV from infected crambe shoots (40 g). However, no detectable amounts of dsRNA were extracted from PLRV or TYTV-infected physalis.