Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Streptomyces spp. are responsible for a large proportion of the world-wide quality deterioration of potatoes causing a potato tuber disease called cornmon scab. Determining the genetic diversity of the Streptomyces spp., especially the main pathogen, S. scabies, has been a prerequisite for the ultimate control of common scab. Techniques responsible for the classification and determination of genetic diversity have improved with advances in DNA technology. Analysis of South African (S.A.) S. scabies isolates has been focusing on the organisms' morphology, physiology, pathogenicity and melanin production, but the classification of S. scabies using DNA techniques has not yet been explored. In this study various DNA techniques were screened for optimal use in determining the genetic diversity within and among isolates of S. scabies. Bacteria had been sampled from the main potato producing regions in S.A. and a few other regions. The techniques explored included RAPDs, AFLPs, RAMS, Rep-PCR, 16S rDNA sequencing and ITS analysis. The first three techniques had to be abandoned due to non-reproducibility between the same isolate extracted on separate occasions and ITS analysis was abandoned due to sequencing difficulties. Of the three Rep-PCR techniques tested (BOX, ERIC and REP), BOX was selected because it produced the clearest and most reproducible results. BOX-PCR and 16S rDNA sequencing were therefore ultimately selected as the methods to analyse the genetic diversity of the S. scabies isolates. Information concerning the pathogenicity of the isolates was supplied by the Vegetable and Ornamental Plant Research Institute of the Agricultural Research Council (VOPI, ARC, Roodeplaat). A brief analysis of the pathogenicity prediction of the isolates in this study was explored with the PCR technique. Presence of the necJ gene was previously shown to be an indication of the pathogenicity within the Streptomyces spp. group. PCR analysis is based on the amplification of a O.72kb fragment (necl) in pathogenic isolates which was absent in non-pathogenic isolates. However, in this study the test for pathogenicity lacked specificity and sensitivity and some of the problems experienced included non-reproducibility between PCR reactions and the presence of the pathogenic fragment in the nonpathogenic isolates (as designated by VOPI, ARC). These observations led to the conclusion that this technique is not an ultimate test for pathogenicity of S. scabies isolates in a South African context. The genetic distances and similarity matrices of the Rep-PCR results were calculated using Nei's genetic distance calculation (Nei M, 1975). Clusters from these matrices were constructed using the unweighted pair group average (UPGMA) with the PAUP4 package. The clusters for the 16S rDNA sequences were formed with the Neighbor Joining (NJ) method and the PAUP4 package. The NJ trees do not take small sequencing differences into account, therefore a Parsimony Network had to be constructed. The trees obtained with the 16S rDNA sequencing techniques grouped most S. scabies isolates into one major group with a 100% bootstrap robustness of this group. More genetic diversity was illustrated by the BOX-PCR technique and the isolates were generally grouped according to their different regions of origin. However, the bootstrap values were low, indicating a lack of robustness regarding the BOXPCR clustering. This was not unexpected as the number of data points employed in the BOX technique is very limited. Both techniques revealed unexpected grouping of a few isolates. Their isolated positions could be attributed to possible misclassification or to the fact that they could be genetically different S. scabies isolates. Streptomyces spp. (other than S. scabies) displayed enough differences to place them in their own distinct groups using both techniques. Comparison of the cluster results obtained in this study did not correlate to the data supplied by the VOPI, ARC (morphology, physiology, pathogenicity and melanin production) which revealed differences between the S. scabies isolates within their respective regions. The lack of diversity displayed by the 16S rDNA technique can be attributed to the fact that only a limited section of the genome is involved making it inappropriate for intra-species genetic diversity analysis. The BOX technique takes various loci within the genome but is still not ideal for a thorough genetic diversity analysis. This study represents the first attempt to determine the genetic diversity of S. scabies in S.A. on DNA level. / AFRIKAANSE OPSOMMING: Streptomyces spp. is verantwoordelik vir 'n _groot deel van die wereld afname in aartappel kwaliteit as gevolg van die aartappelknol siekte bruinskurf. Die bepaling van die genetiese diversiteit tussen die Streptomyces spp., varal die hoof patogeen in die groep, S. scabies, is 'n vooreiste vir die uiteindelike beheer van bruinskurf. Tegnieke verantwoordelik vir die klassifikasie en bepaling van genetiese diversiteit het verbeter met vooruitgang in DNA tegnologie. Analise van Suid Afrika (S.A.) se S. scabies isolate konsentreer op die organisme se morfologie, fisiologie, patogenisiteit en malanien produksie, maar die klassifikasie van S. scabies met die behulp van DNA tegnieke is nog nie uitgevoer me. In hierdie studie is verskeie DNA tegnieke ondersoek vir optimale bepaling van genetiese diversiteit binne en tussen S. scabies isolate van S.A Bakteriee is verkry van die hoof aartappel-produserende areas in S.A. en ook van 'n paar ander areas. Die tegnieke wat in die studie gebruik is, het RAPDs, AFLPs, Rep-PKR, 16S rDNA volgordebepaling en ITS analise ingesluit. Die eersgenoemde drie tegnieke is uitgesluit as gevolg van nie-herhalende resultate tussen dieselfde isolaat geisoleer op verskillende geleenthede. ITS analise is uitgesluit as gevolg van probleme met volgordebepaling. Rep- PKR en 16S rDNA volgordebepaling is uiteindelik gekies as die mees geskikte metodes vir die analise van genetiese diversiteit tussen S. scabies isolate in hierdie studie omdat albei skynbare herhaalbare resultate gelewer het. Inligting met betrekking tot die patogenisiteit van die isolate is voorsien deur die Groente en Sierplant Instituut van die Landbou Navorsinsraad (YOPI, LNR). 'n Vinnige analise van die patogenisiteits voorspelling van die isolate is uitgevoer met die PKR tegniek. Dit is voorheen aangetoon dat die teenwoordiheid van die necJ geen dui op die patogenisiteit in die Streptomyces sp _groep. PKR analise het 'n O.72kb fragment (necl) in patogeniese isolate geamplifiseer wat nie teenwoordig was in niepatogeniese isolate nie. Hierdie toets vir patogenisiteit soos gebruik in hierdie studie was egter onspesifiek en onsensitief en sommige van die probleme wat ondervind is sluit in nie-herhaalbaarheid tussen PKR reaksies en die teenwoordigheid van die patogeniese fragment in die nie-patogeniese isolaat (soos beskryf deur YOPI, LNR). Uit hierdie waarnemings word afgelei dat die tegniek nie 'n geskikte toets vir patogenisiteit van S. scabies isolate in 'n S.A konteks is nie. Die genetiese afstande en ooreenkomstige matrikse van die Rep-PCR resultate is bereken met die genetiese afstand bepaling van Nei (Nei M, 1975). Groepe is gevorm met die "unweighted pair group average" (UPGMA) en PAUP4 pakket. Die "Neighbor Joining" (NJ) groepe is gevorm met die 16S rDNA volgordebepaling data mbv die PAUP4 pakket. Die NJ groepering neem nie klein volgorde verskille in ag nie en gevolglik moes'n "Parsimony Network" opgestel word. Die groepering met die 16S rDNA volgordebepaling het meeste van die isolate in een hoof groep geplaas met 'n 100% "bootstrap" waarde. Meer genetiese diversiteit is met die BOX-PCR tegniek gevind en isolate was oor die algemeen gegroepeer vol gens van hul oorsprong. Die "bootstrap" waardes vir die BOX tegniek was baie laag. Dit was nie onverwags nie, want die hoeveelheid data punte was beperk met die BOX tegniek. Albei tegnieke het 'n aantal afwykende isolate vertoon. Hul ge-isoleerde posisies kan toegeskryf word aan moontlike misklassifikasies van die isolaat. Die moontlikheid dat daar wel genetiese verkille tussen die isolate is, kan egter nie uitgesluit word nie. Streptomyces spp. (uitgesluit S. scabies) het genoeg variasie vertoon om hulle in hul eie groepe met die gebruik van beide tegnieke te plaas. Vergelyking tussen die groepe in die studie stem nie ooreen met die data verkry vanaf YOPl, LNR (morfologie, fisiologie, patogenesiteit en melanien produksie) nie wat verskille tussen S. scabies isolate binne 'n sekere gebied vertoon. Die gebrek aan diversiteit soos vertoon deur die 16S rDNA tegniek kan toegeskryf word aan die feit dat slegs 'n beperkte gedeelte van die genoom ondersoek word, wat dit ongeskik vir intra-species genetiese diversiteit analise maak. Die BOX tegniek neem verskeie loci in die genoom in ag, maar is steeds nie ideaal vir deeglike genetiese diversiteit analise nie. Hierdie studie verteenwoordig die eerste poging om die genetiese diversiteit van S. scabies in S.A. op DNA vlak te bepaal.

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

Sweetpotato, Ipomoea batatas (L.) Lamarck was introduced into Lesotho in 1992 in the hope that it would help alleviate poverty levels. Efforts are being made to learn the potential constraints to optimal production of this crop. Insect pests especially the sweetpotato weevils, Cylas species are a major production constraint worldwide. Hence the main objective of the study was to predict if these pests have potential to establish themselves in Lesotho. Two Cylas species, C. formicarius and C. puncticollis are present in South Africa and the latter is found in the northern Free State and the Eastern Cape both of which border the central and southern lowlands of Lesotho respectively. Cylas puncticollis was chosen as the subject of this study because of its potential spread into Lesotho. Thermal requirements (lower development threshold and degree-days) of this pest were calculated in the laboratory by studying the effects of temperature on its development and survival at six constant temperatures (16°C, 19°C, 24°C, 26°C, 31°C and 36°C). The photoperiod was maintained at 12L:12D for all temperatures, but RH was not controlled. Thermal requirements (r and k) of this pest species were estimated for all the immature stages and for the total life-cycle using the linear regression method. The estimated lower temperature threshold (r) of the total development of the pest lies between 8°C and 12°C and the thermal constant (k) between 360°D and 380°D. The thermal needs of this pest obtained from the laboratory work were used to predict the potential for its establishment in Lesotho as well as determining the possible areas of distribution if it invades Lesotho. Actual soil temperatures to which the pest would be exposed to in Lesotho were recorded for a year. Both the calculated thermal needs of the pest and the field-recorded temperatures were used in the degree-day model to predict potential establishment of this pest. The second approach, climate matching in Geographical Information System (GIS) used the bio-climatic profile of C. puncticollis calculated from the known areas of its distribution in both South Africa and Swaziland. The bio-climatic profiles of the two countries were matched to the climatic conditions of Lesotho to predict the potential for its establishment. The two approaches, linear degree-day model and climate matching approach revealed that Cylas puncticollis is a potential pest in Lesotho. The former predicted the occurrence of this pest throughout the whole country with a maximum of eight generations per year being possible in the lowlands. Fewer generations (two to three) were predicted for the highlands and foothills agro-ecological zones, which are colder than the lowlands. The climate matching approach also confirmed the prediction although according to this method a patchy distribution of the pest was predicted. A survey was then carried out in Lesotho, first to determine if Cylas species were already present in Lesotho, secondly to identify any other pests of sweetpotato and lastly to determine other possible production constraints other than insect pests. The survey was conducted in the form of questionnaire and field sampling. Cylas species were neither documented by the farmers who were interviewed nor by the field sampling. Numerous common pests of sweetpotato were recorded during the sampling survey. These included the following leaf-feeding pests: Bedellia somnulentella Zeller, Acraea acerata Hewitson, Agrius convolvuli Linnaeus and locusts and grasshoppers. The root pests that were recorded were mole-rats, Blosyrus sp. and millipedes (Narceus sp.). According to the sampling carried out in Lesotho there were no insect pests that could be rated as major pests as yet. Sweetpotato farmers did not consider insect pests as an important production constraint for optimal yield of the crop. The major constraint was found to be lack of planting material, which contributed towards a slow adoption of the crop throughout the country. / Dissertation (MSc (Entomology))--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

The main objectives of this study were to examine the nature of the basic-applied research continuum, to evaluate projected costs and benefits of the research continuum for a specific example, namely, the development of multiple disease resistant potato cultivars, and to examine the changing roles and interactions between public research institutions and private industry. Given the existing budgetary constraints and the increasing demands for accountability that research administrators and policy makers face, it seems necessary for decision makers to give adequate consideration to the existing interdependency of basic and applied research in determining the most appropriate levels of research to fund. The establishment of of an adequate balance of both basic and applied research is important in any attempts to maximize the returns from the research continuum while at the same time developing and maintaining new biotechnologies. The projected rate of return for potato disease resistant research was calculated at 34 percent which falls within the range given by similar studies. With the advent of the new biotechnologies such as genetic engineering, and the increased competition for the limited research dollars, there has been an evolving new relationship between universities and private industry in which universities are seeking more private funding and industry demanding more control of technologies developed through their funding. Separate but interdependent roles of both private companies and universities seems necessary for the achievement of desirable and a adequate maintenance of the research continuum. / M.S.

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Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Infection of potatoes by viral pathogens causes reduced crop yield and subsequent economic loss. In South Africa Potato virus Y (PVY) and Potato leafroll virus (PLRV) are the two most destructive viruses infecting potatoes. Several other viral pathogens exist, including Potato virus X (PVX), Potato virus M (PVM), Potato virus A (PVA), Potato virus S (PVS), Potato mop-top virus (PMTV), Tomato spotted wilt virus (TSWV) and Potato spindle tuber viroid (PSTVd). Although the aforementioned pathogens are found infecting potatoes around the world, there are no published information pertaining to the prevalence of these viral agents in South Africa. Currently, the occurrence of PLRV infection in potatoes of South Africa has reached epidemic proportions. A previous phylogenetic investigation undertaken in our laboratory of South African PLRV isolates, using coat protein (CP) gene sequences, found large variation between native South African PLRV isolates and most other isolates from elsewhere in the world; with their nearest relatives being single isolates from Australia and North America. In this study the incidence of PVX, PVM, PVA, PVS, PMTV, TSWV and PSTVd was investigated. A large number of potato plant and tuber samples was collected and infected samples were identified with reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the CP gene or the whole genome in the case of PSTVd. The amplified nucleic acid segments were sequenced, aligned with international reference sequences and analysed phylogenetically to determine their relative relationships with these reference sequences. The CP genes of PLRV isolates were sequenced and phylogenetically investigated to determine how these new isolates compared relative to the previous findings from our laboratory. In addition, the complete genomes of two PLRV isolates were sequenced and phylogenetically investigated as a preliminary study to investigate the apparent increase of pathogenicity of certain variants of South African PLRV. Results obtained showed that only PVX and PVS were present in the samples collected and the incidences of these viruses were very low (2.0 and 1.1% respectively). The phylogenetic analyses of the CP genes, indicated that the PVX and PVS variants isolated in this study, were part of the dominant types of variants found worldwide. From the analyses of the PLRV CP and whole genome sequences, it was determined that many of the PLRV variants found in South Africa, are genetically distinctly different from those around the world. This warrants further investigation into the increased pathogenicity experienced with South African PLRV. / AFRIKAANSE OPSOMMING: Infeksie van aartappels deur virale patogene veroorsaak verlaagde opbrengs en gevolglike ekonomiese verlies. In Suid-Afrika is Aartappelvirus Y (PVY) en Aartappelrolblad virus (PLRV) die twee mees vernietigende virusse wat aartappels infekteer. Verskeie ander virale patogene, insluitend Aartappelvirus X (PVX), Aartappelvirus M (PVM), Aartappelvirus A (PVA), Aartappelvirus S (PVS), Aartappel "moptop" virus (PMTV), Kromnekvirus (TSWV) en Aartappel "spindle tuber" viroïed (PSTVd) kom ook wêreldwyd in aartappels voor. Alhoewel hierdie virusse aartappels wêreldwyd besmet, is daar geen gepubliseerde inligting met betrekking tot die voorkoms van hierdie virusse of die viroïed in Suid-Afrika nie. Tans het die voorkoms van PLRV infeksie in aartappels in Suid-Afrika epidemiese proporsies bereik. In 'n vorige filogenetiese ondersoek van die mantelproteïen (MP) nukleotiedvolgordes van Suid Afrikaanse PLRV isolate in ons laboratorium, is groot variasie tussen hierdie inheemse isolate en die meeste ander isolate van elders in die wêreld bevind. Die Suid Afrikaanse PLRV variante betree 'n unieke intermediêre posisie tussen die internasionale isolate en enkele isolate van Australië en Amerika. In hierdie studie is die voorkoms van PVX, PVM, PVA, PVS, PMTV, TSWV en PSTVd ondersoek. Groot aantal aartappelplant en -knol monsters is versamel en infeksie is getoets met tru-transkripsie polimerase kettingreaksie (RT-PCR) amplifisering van die MP geen, of die hele genoom in die geval van PSTVd. Die nukleïensuurvolgordes is bepaal en vergelyk met internasionale verwysingsvolgordes. Die relatiewe verhoudings tussen die bepaalde volgordes en die verwysingsvolgordes is geanaliseer met filogeneties ontledings. Die MP gene van PLRV isolate se volgordes is bepaal en filogeneties ontleed om hierdie nuwe isolate te vergelyk relatief tot vorige bevindinge in ons laboratorium. Die volledige genome van twee PLRV isolate se volgordes is bepaal en filogeneties ontleed as 'n voorlopige studie om die oënskynlike toename in patogenisiteit van Suid-Afrikaanse PLRV te ondersoek. Resultate het getoon dat slegs PVX en PVS teenwoordig was in die monsters wat versamel is en dat die voorkoms van hierdie virusse baie laag was (2.0% en 1.1% onderskeidelik). Die filogenetiese ontleding van die MP gene het aangedui dat die Suid Afrikaanse variante van PVX en PVS, geisoleer in hierdie studie, van die dominante tipes is wat mees gereeld internationaal voorkom. Uit die ontleding van die PLRV MP en heelgenoom volgordes, is vasgestel dat baie van die PLRV variante wat in Suid-Afrika aangetref word, geneties meer verskillend is as die van regoor die wêreld. Dus, regverdig dit, verdere ondersoek van die verhoogde patogenisiteit van Suid Afrikaanse PLRV variante.

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Three pages of addenda pasted inside back cover. Bibliography: leaves 166-189. Rhizoctonia solani is a complex species comprising morphologically basidiomycetous imperfect fungi. This study aimed to determine genetic diversity within R. solani AG-3 causing rhizoctonia disease of potato in South Australia. For this purpose, pectic zymogram, PCR, DNA fingerprinting and RFLP techniques were used in conjunction with traditional plant pathology procedures.