Thesis (M.S.)--University of Wisconsin--Madison, 1980. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 95-102).

Thesis (Ph. D.)--University of Wisconsin--Madison,1983. / Typescript. Vita. Description based on print version record. Includes bibliographical references (leaves 146-159).

Thesis (Ph. D.)--University of Wisconsin-Madison, 1976. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

In total, 801 samples of potato leafroll disease were collected and tested for potato leafroll virus (PLRV) and beet western yellows virus (BWYV) in 1986, 1987, and 1988 using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and virus-specific monoclonal antibodies. The samples represented 32 cultivars and originated in eight Canadian provinces and 12 American states. None of the samples tested positive for BWYV, whereas 772 (96.4%) tested positive for PLRV. Neither PLRV nor BWYV could be recovered, with aphid transfers to indicator hosts, from 28 of the 29 samples that tested negative for both viruses. PLRV was recovered from one sample that originally tested negative by TAS-ELISA; the indicator plant tested positive for PLRV by TAS-ELISA. Nucleic acid spot hybridization (NASH) using random primed and cloned cDNA probes was compared with double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and TAS-ELISA, and aphid transmission tests for detection and identification of PLRV and BWYV in 165 potato leafroll disease samples. All of the samples tested negative for BWYV with each of the assay procedures. PLRV was detected in all of the samples with TAS-ELISA, NASH with a cloned cDNA probe for PLRV, and with aphid transmission to ground cherry (Physalis pubescens). Both DAS-ELISA and NASH using random primed cDNA produced one false-negative result. Shepherd's purse (Capsella bursa-pastoris) was a host for 72% (119/165) of the PLRV isolates. The susceptibility of potato to BWYV was tested by inoculating Russet Burbank with three isolates of BWYV from Canada and four from the United States. Two of the isolates were in a mixed infection with PLRV. None of the isolates were transmitted by Myzus persicae to virus-free potato plants, either by themselves or in association with PLRV. Common weeds were surveyed in the potato-producing areas of British Columbia for PLRV and BWYV. In total, 10,098 weed samples, representing 98 species in 22 plant families, were collected and tested by TAS-ELISA from 1986 to 1989. BWYV was detected in 1% of the samples; the hosts were: chickweed, common groundsel, heart-podded hoary cress, hedge mustard, little western bittercress, prickly lettuce, shepherd's purse, and wild radish. PLRV was detected in three volunteer potato plants, two samples of shepherd's purse, and one black nightshade plant. The low incidence of PLRV in plants other than potato indicates that weeds are of minor importance in the epidemiology of potato leafroll disease in British Columbia. / Land and Food Systems, Faculty of / Graduate

No description available.

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Pathology, 1985. / Includes bibliographical references (leaves 119-127).

Thesis (Ph. D.)--University of Idaho, 2006. / Abstract. "May 2006." Includes bibliographical references. Also available online in PDF format.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Presently, the observed variation in symptoms of Potato leafroll virus (PLRV) infection in potato cultivars in South Africa cannot be reconciled with PLRV symptoms obtained 10-15 years ago, even if the different interactions between the pathogen and the cultivar are taken into account. In an effort to analyze this variation, mutations in the coat protein (CP) gene of South African isolates of PLRV were assessed. The CP gene of PLRV isolates from different areas within South Africa was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. Significant sequence variation in the CP gene was found within the analyzed South African isolates of PLRV. Phylogenetic analysis revealed two major clades with most South African isolates and an Australian and North American isolate grouped together and the remainder grouped with isolates from diverse countries worldwide. The deduced amino acid sequences from representatives of these two clades indicated differences in CP threedimensional structure. In an effort to produce recombinant PLRV CP for the production of antibodies specific for South African isolates of PLRV for use in enzyme-linked immunosorbent assay (ELISA), the CP gene of a South African isolate of PLRV was subcloned into a bacterial expression vector (pET14-b). Expression of full length recombinant PLRV CP was attempted in Escherichia coli strains BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS and Rosetta-2(DE3)pLysS. As this was not successful, the PLRV CP gene was subcloned in another expression vector (pGEX) for expression as an N-terminal fusion protein with glutathione-S-transferase (GST) in E. coli strains BL21(DE3)pLysS and Rosetta-2(DE3)pLysS. The recombinant GST-PLRV CP fusion protein was purified and used for antibody production in rabbits. Using western blots, the effectiveness of antibodies produced to recombinant GST-PLRV CP fusion protein was assessed for PLRV recognition. It was found that antibodies to the recombinant GST-PLRV CP fusion protein were more effective for the detection of GST than PLRV CP and that production of antibodies to the cleaved PLRV CP product would be necessary if antibodies are required for ELISA applications. / AFRIKAANSE OPSOMMING: Huidiglik kan die waargeneemde simptome van infeksie met aartappelrolbladvirus (Potato leafroll virus, PLRV) in aartappelkultivars in Suid-Afrika nie vereenselwig word met PLRV simptome wat 10-15 jaar gelede verkry was nie, selfs al word die verskillende interaksies tussen die patogeen en kultivar in ag geneem. In ‘n poging om hierdie variasie te analiseer, was mutasies in die mantelproteïen (CP) geen van Suid-Afrikaanse isolate van PLRV bepaal. Die CP geen van PLRV isolate van verskillende areas in Suid-Afrika was ge-amplifiseer met behulp van die tru transkripsie-polimerase ketting reaksie (RT-PCR), gekloneer en die nukleotiedvolgorde bepaal. Noemenswaardige nukleotied variasie is in die CP gene van die ge-analiseerde Suid-Afrikaanse isolate van PLRV gevind. Filogenetiese analises het gedui op twee hoof klades met die meeste van die Suid-Afrikaanse isolate wat saam met ‘n Australiese en Noord-Amerikaanse isolaat gegroepeer en die res wat met isolate van verskillende lande wêreldwyd gegroepeer. Die afgeleide aminosuurvolgordes van verteenwoordigers van bogenoemde twee klades het gedui op verskille in die CP driedimensionele struktuur. In ‘n poging om rekombinante PLRV CP te produseer vir die produksie van antiliggame spesifiek teen Suid-Afrikaanse isolate van PLRV om in “enzyme-linked immunosorbent assay” (ELISA) te gebruik, was die CP geen van ‘n Suid-Afrikaanse isolaat van PLRV gesubkloneer in ‘n bakteriële ekspressie vektor (pET14-b). Daar was gepoog om vollengte rekombinante PLRV CP in die Escherichia coli rasse BL21(DE3)pLysS, Rosetta-gami B(DE3)pLysS en Rosetta- 2(DE3)pLysS te produseer. Aangesien dit nie suksesvol was nie, was die PLRV CP gesubkloneer in ‘n ander ekspressie vektor (pGEX) sodat die proteïen as ‘n N-terminale fusie proteïen met “glutathione-S-transferase” (GST) in E. coli rasse BL21(DE3)pLysS en Rosetta- 2(DE3)pLysS geproduseer kon word. Die rekombinante GST-PLRV CP fusie proteïen was gesuiwer en gebruik vir antiliggaam produksie in konyne. Die effektiwiteit van die antiliggame wat teen rekombinante GST-PLRV CP fusie proteïen geproduseer was vir PLRV herkenning is deur middel van “western blots” geanaliseer. Dit was gevind dat antiliggame teen die rekombinante GST-PLRV CP fusie proteïen meer effektief was vir die herkenning van GST as PLRV CP. Gevolglik sal dit nodig wees om antiliggame teen die gesnyde PLRV CP produk te maak vir gebruik in ELISA.

Complementary DNA (cDNA) clones representing approximately 5800 nucleotides of potato leafroll virus (PLRV) genomic RNA were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) that could encode a 23 kDa protein was identified and further characterized. Comparison of the deduced amino acid sequence with the coat protein amino acid sequence of the PAV strain of barley yellow dwarf luteovirus (BYDV-PAV) showed significant similarity. This observation together with its size and internal location within the genome suggested that this gene encoded the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including a 17 kDa ORF within the ORF encoding the 23 kDa coat protein, and termination of the latter with an amber codon which is immediately followed by a large ORF in the same reading frame. Three PLRV coat protein gene sequences were used to transform tobacco and the potato cultivars 'Desiree' and 'Russet Burbank' via Agrobacterium tumefaciens mediated gene transfers. One construct possessed 12 nucleotides of the untranslated leader sequence 5' to the putative coat protein gene start codon. The other construct, which was also inserted in the reverse orientation to produce negative-sense RNA, had 192 nucleotides from this leader sequence. When these sequences were introduced as chimaeric genes under the control of a duplicated cauliflower mosaic virus 35S (CaMV) promoter, transcription levels were high. Both positive-sense transcripts produced potato leafroll coat protein which accumulated to maximum levels of approximately 0.5% and 0.01% of total leaf protein in tobacco and potato, respectively. Results show that significant levels of inoculum concentration-independent sustained resistance were obtained with each construct, resulting in PLRV titres as low as 1% of the level observed in untransformed plants, as determined by enzyme-linked immunosorbent assays. Virus transmission from PLRV-inoculated transgenic 'Russet Burbank' was reduced substantially and was correlated with virus titre. The pattern and level of protection were the same for constructs producing positive- and negative-sense RNA, suggesting a similar mechanism of resistance. Virus levels were negatively correlated to transcript levels within the transgenic plants. This resistance will have practical applications for the control, of PLRV. Elucidation of the mechanism of resistance may also help understand the mechanisms of virus infection. / Land and Food Systems, Faculty of / Graduate

Potato leafroll virus (PLRV) causes yield and quality losses in potato. PLRV is identified by plant symptoms and serological tests such as an enzyme-linked immunosorbent assay (ELISA). A similar serological test, direct tissue blotting assay (DTBA), was used to detect and monitor PLRV movement in field-inoculated Russet Burbank plants and plant tissues from Russet Burbank and Russet Norkotah seed tubers submitted by growers for winter certification tests. DTBA was as accurate as ELISA and easier to use for detecting tuber-perpetuated PLRV in stems and petioles of plants grown from grower-submitted seed tubers. ELISA detected twice as many PLRV positives as DTBA in leaflet tests. DTBA detected PLRV in tuber tissue but results matched ELISA in only 74% or less of the samples. Results of DTBA tuber tests were sometimes difficult to interpret while stem and petiole results were distinct and unambiguous. As inoculations were delayed later in the season and as plants matured, PLRV infection levels decreased sharply, most often within a two week period in early July. In same-age plants inoculated 43 days after planting but 18 days apart, early inoculation produced higher PLRV levels. Conversely, when same-age plants were inoculated 62 days after planting but 19 days apart, late inoculation produced higher PLRV levels. This discrepancy is not fully understood, but larger tuber size at the later inoculation probably produced a stronger sink for source-to-sink translocation of nutrients and phloem-limited viruses. Results of DTBA winter grow-out tests of summer-infected tubers approximated those of ELISA and visual inspections. Indirect DTBA testing of tubers utilizing stem and petiole tissues from winter growout plants detected more PLRV than directly testing tuber tissue 21 days post inoculation in summer. DTBA detected current season (primary) PLRV less reliably than secondary (tuber-borne) PLRV, similar to reported ELISA results. PLRV infection increased tuber numbers but decreased size. Size reduction was most evident in plants infected early in the season. Average tuber size in healthy plots was always larger than the average tuber size in infected plots. Within an infected plant, small tubers tended to be infected less often than large tubers. / Graduation date: 1993