School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemStudies of barley yellow dwarf virus infection in ryegrass (Lolium spp.)( 1989)Measurements were made of the effect of barley yellow dwarf virus (BYDV) infection on the early growth of four commercial cultivars of ryegrass (Lolium spp.) under two different temperatures (24C and 16C). At 24C, BYDV infection was associated with reduced root dry weight (30-40%) in all cultivars; the effect of infection on shoot dry weight and leaf area was variable. At 16C, the effect of BYDV infection was variable, being associated with increases in both leaf area and root and shoot dry weight in one cultivar (Grasslands Arild) and decreases in another (Victorian). In two other cultivars, both leaf area and root and shoot dry weight were not significantly affected (p > 0.05) by infection with BYDV. There was no evidence of visible symptoms associated with BYDV infection. At 240C, reductions in root dry weight associated with BYDV infection were not reflected in reductions in the relative growth rates of the roots. Up to at least 28 days after inoculation (46-50 days after germination) reductions in root dry weight were associated with both aphid-feeding damage and virus infection. Experiments with the cultivar Victorian showed that shoot dry weight was not significantly affected (p > 0.05) by feeding with either viruliferous (BYDV) or non-viruliferous aphids (Rhopalosiphum padi L.). At 16C, changes in root and shoot dry weight were associated with changes in the respective relative growth rates. Both visual scoring and changes in plant dry weight were found to be unreliable criteria for assessing disease severity. Two assay systems, an enzyme-linked immunosorbent assay (ELISA) and a nucleic acid, or complementary DNA (cDNA) probe, were developed for the detection of BYDV in ryegrass. The conditions for detecting BYDV with the ELISA (the "double sandwich" variant) were optimized. Exposure of the virus to extracts of ryegrass leaf tissue for 30 min or more resulted in reductions in absorbance, with the amount of the reduction dependent on the antigen (virus) incubation temperature. The optimization of conditions for the immunoglobulin (IgG) used for coating microtitre plates (coat IgG), the IgG - alkaline phosphatase conjugate (conjugate IgG) and the antigen (virus), in conjunction with the use of polyethylene glycol - 6 000 in the preparation of the conjugate buffer, gave a rapid and reliable ELISA procedure for the consistent detection of BYDV at levels of 2 ng/ml. Both ELISA and cDNA probe techniques were used to study the incidence of BYDV isolates in perennial ryegrass at three sites (Balmoral, Mininera and Hamilton) in south-western Victoria. BYDV was detected in perennial ryegrass cultivars from the three sites, with different levels of incidence of BYDV isolates between cultivars. While the PAV-related isolate was prevalent at the three sites, the incidence of the MAV- and RPVrelated isolates varied with the site and the cultivar. One difficulty with the ELISA was that absorbance values did not fall in to distinct infected and uninfected groups within the cultivars, and at Mininera, the incidence of BYDV varied significantly between the various positive - negative ELISA thresholds that were used. At Balmoral, the incidence of BYDV determined with each threshold was compared with that determined by cDNA probing using a Spearman correlation test. A threshold of double the mean negative (uninfected ryegrass) control showed the highest correlation with the cDNA probe for a number of replicate plots at the site. The incidence of the PAV-related isolate was compared for the two cultivars Ellett and Victorian, and significant (p < 0.01) reductions in its incidence in cv. Ellett compared with cv. Victorian at the three sites, suggested that cv. Ellett is resistant to the PAVrelated isolate of BYDV. The comparative resistance of cv. Ellett to at least this isolate of BYDV indicated the feasibility of breeding and selecting for resistance to other isolates of BYDV in perennial ryegrass. In attempts to quantify resistance in perennial ryegrass, the ELISA was further developed to allow measurement of BYDV titre in ryegrass shoot tissue. In twenty selections of cv. Victorian, the performance, as measured by changes in both root and shoot dry weights, of individual selections to infection with BYDV, did not significantly (p < 0.05) correlate with virus titre measurements. Experiments with twenty selections of cv. Ellett indicated that its resistance to BYDV that was e' ident at the three field sites was not associated with resistance to aphid (Rhopalosiphum path) feeding, and consequently, transmission of the virus. Attempts to inoculate cv. Ellett with either the PAV- or RPVrelated isolates were of limited success, and it was speculated that its resistance to the virus may be due to reductions in the level of virus replication.