Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA) toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid- Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar, goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is. In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal. Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol. Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

Dissertation (PhD(Agric))--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The codling moth, Cydia pomonella (L.) has been a major pest of pome fruits since before the turn of the last century. However, despite its high economic profile little is known about the bionomics and . behaviour of this pest in apple orchards in South Africa, information required for the development of a sustainable integrated management programme. In field trials there was contingency between the time of year and the upper and lower half of the tree. First generation moths laid significantly more eggs in the bottom half of the tree while second and third generation moths laid significantly more eggs in the top half of the tree. The preferred oviposition sites on Granny Smith (GS) and Golden Delicious (GD) cultivars, in order of preference, were leaves, fruit and wood. More eggs were laid on the fruit ofGS spurs (35.6 %) than on those ofGD spurs (10.7 %). On fruit spurs there was a significant increase in the number of eggs on GD leaves and GS fruit over the season, whereas the number of eggs on GS leaves and GD fruit remained constant. On branches there was an increase in the number of eggs on GD and GS leaves, but not on the fruit or wood. The preferred oviposition site on the fruit was the fovea of the stalk insertion and the rounded cheek area surrounding the fovea. The distribution within different fruit bearing classes (1 - 4 fruit per spur) was random only for one fruit per spur, while on the other spur classes clustering occurred. In laboratory studies of the embryonic and immature stages there was a linear relationship between rate of development and constant temperatures of 15, 17,20,25 and 30·C ± l·C. The lower threshold temperatures for embryonic, larval and pupal development were 11.1, 7.9, 9.9°C respectively. The degree-days required to complete embryonic, larval and pupal development were 80.5, 345, and 279 respectively. The response of the different stages to constant temperatures was similar to that under fluctuating temperatures. At temperatures below 16°C or abouve 27°C moths did not mate and few eggs were laid. Moth longevity decreased with increasing temperature. There was seasonal variation in longevity and oviposition at constant and fluctuating temperatures. Summer adults produced significantly more eggs than spring adults at constant temperatures. At a constant temperature of2S·C and fluctuating temperatures there were five distinct larval instars. The similarity between the mean head capsule width and ranges for each instar reared on fruit of different stages of development at fluctuating temperatures indicates that fruit development and temperature have little influence on mean head capsule width. From sleeve-cage studies in the orchard there was no significant difference in the fecundity of spring and summer moths. In the beginning of October spring moths produced significantly fewer eggs than in November. Egg mortality increased from 8.2 %in spring to 21.2 %in summer. Failure of 1st instar larvae to penetrate the fruit ranged from 4.9 % to 19.5 %, while mortality oflarvae from egg hatch to emergence from the fruit ranged from 29.7 % to 42.9 %. Mortality of 5th instar larvae after emerging from the apples ranged from 0 % to 8.7 % and pupal mortality from 0 % to 3.5 %. On large 27-year old trees more overwintering larvaewere found on Golden Delicious (13.9) than on Granny Smith trees (5.7), with over 70 % oflarvae being found in pruning wounds on both cultivars. On small 7-year old Golden Delicious and Granny Smith trees the mean number oflarvae was 0.5 and 2.0 per tree. A combined mating disruption and insecticide control programme reduced codling moth resistant populations to levels requiring a minimum to no insecticide intervention for several seasons. The efficacy of a pheromone based strategy, number of pheromone treatments, number of dispenserslha and level of insecticide intervention required are strongly influenced by prevailing weather conditions. Fruit infestation in orchards under a mating disruption programme and under an insecticide programme were greater along the borders compared to the interior. The presence of horticultural mineral oil on the leaves and branches did not have a detrimental effect on oviposition nor was there any significant ovicidal effect. A significant ovicidal effect was obtained when applied after oviposition. In field trials, insecticides with lower levels of efficacy than the primary insecticide, azinphos-methyl, provided acceptable control when successfully incorporated into a spray programme which followed a policy of alternation of insecticides across generations. The least variation in the number of degree-days between biofix and first egg hatch of the spring flight was when the second trap catch (Biofix 2) was used as the biofix. A biofix based on the first evening when the temperature reached or exceeded 1TC at 18:00 after first trap catch also showed less variation than when the biofix was based on first trap catch. The mean number of degree-days accumulated between Biofix 2 and first egg hatch was found to be 139.1 ° D. The number of degreedays between the first and second flight biofixes varied between 531.2 and 488.87°D with a mean of 508.1°D. / AFRIKAANSE OPSOMMING: Kodlingmot (Cydia pomonella) is reeds sedert die vorige eeu 'n sleutelplaag van kemvrugte. Ten spyte van die hoë ekonomiese profiel, is daar min kennis betreffende die bionomie en gedrag van hierdie plaag in appelboorde in Suid-Afrika. Hierdie inligting is noodsaaklik vir die ontwikkeling van 'n volhoubare geïntegreerde bestuursprogram. Tydens veldproewe is 'n korrelasie tussen die tyd van die jaar en eierlegging in die boonste of onderste helfte van bome gevind. Eerste-generasie motte het betekenisvol meer eiers in die onderste helfte van die bome gelê, terwyl tweede- en derde-generasie motte meer eiers in die boonste helfte van die bome gelê het. In toenemende volgorde was die voorkeur eierleggingsposisies op Granny Smith (GS) en Golden Delicious (GD) appels die blare, vrugte en hout. Meer eiers is op vrugte van GS spore (35.6%) as op dié van GD spore (10.7%) gelê. Op vrugspore was daar 'n betekenisvolle toename in aantal eiers op GD blare en GS vrugte deur die seisoen, terwyl die getal eiers op GS blare en GD vrugte konstant gebly het. Op takke was daar 'n toename in aantal eiers op GD en GS blare, maar nie op vrugte of hout nie. Die voorkeur eierleggingsposisie op vrugte was die fovea van die steelaanhegting en die wang rondom die fovea. Die verspreiding tussen verskillende vrugdraende klasse (1 - 4 vrugte per spoor) was slegs in die een-Vrug-per-spoorklas ewekansig, terwyl daar in die ander spoorklasse groepering voorgekom het. In laboratoriumstudies van embrioniese en onvolwasse stadia is 'n lineêre verband tussen ontwikkelingskoers en konstante temperature van 15, 17,20,25 en 30°C±I°C gevind. Die onderste drempel-temperature vir embrioniese, larwale en papie-ontwikkeling was onderskeidelik II. 1°C, 7.f?C en 9.9°C. Graaddae benodig vir voltooiing van embrioniese, larwale en papie-ontwikkeling was onderskeidelik 80.5,345 en 279. Die respons van verskillende stadia by konstante temperature het ooreengestem met hul respons onder wisselende temperature. Motte het nie gepaar nie en min eiers is gelê by temperature onder 16°C of bo 27°C. Die lewensverwagting van motte het afgeneem met toename in temperatuur. Seisoenale variasie in . lewensverwagting en eierlegging het voorgekom by konstante sowel as wisselende temperature. By konstante temperature het somer-volwassenes betekenisvol meer eiers as lente-volwassenes geproduseer. By 'n konstante temperatuur van 25°C, sowel as by wisselende temperature, het vyf duidelik onderskeibare larwale instars voorgekom. Die ooreenkoms tussen die gemiddelde kopkapsulewydte en wydte-reeks vir elke instar wat op vrugte van verskillende stadiums van ontwikkeling by wisselende temperature geteel is, dui daarop dat vrugontwikkeling en temperatuur weinig invloed op gemiddelde kopkapsule-wydte het. Tydens mou-hok studies in die boord is geen betekenisvolle verskil in die fekunditeit van lente- en somer-motte waargeneem nie. Vroeg in Oktober het lente-motte betekenisvol meer eiers as in November geproduseer. Eiermortaliteit het van 8.2% in die lente tot 21.2% in die somer toegeneem. Faling van 1ste instar larwes om vrugte te penetreer het van 4.9% tot 19.5% gewissel, terwyl mortaliteit van larwes vanaf uitbroei tot uitkoms uit die vrug van 29.7% tot 42.9% gewissel het. Mortaliteit van Sde instar larwes na uitkoms uit die vrug het van 0% tot 8.7% gewissel, en papie-mortaliteit van 0% tot 3.5%. Op groot, 27-jaar oue bome is meer oorwinterende larwes op Golden Delicious (13.9) as op Granny Smith (5.7) gevind, en meer as 70% van die larwes op beide kultivars is op snoeiwonde gevind. Op klein, 7-jaar oue bome was die gemiddelde aantallarwes op Golden Delicious en Granny Smithbome 0.5 en 2.0 onderskeidelik. 'n Gekombineerde paringsontwrigting- en insekdoder beheerprogram het weerstandbiedende kodlingmot-populasies verminder tot 'n vlak waar minimum tot geen insekdoder-toedienings vir verskeie seisoene gemaak is. Die effektiwiteit van 'n feromoon-gebaseerde strategie, aantal feromoonbehandelings, aantal vrystellers/ha en vlak van insekdoder-toediening word sterk deur heersende weersomstandighede beïnvloed. Die rande het hoër vruginfestasie as die middel getoon in boorde onder paringsontwrigting sowel as boorde onder insekdoder-programme. Die teenwoordigheid van minerale olie op blare en takke het geen nadelige effek op eierlegging gehad nie en dit het geen betekenisvolle eierdodende effek gehad nie. Indien die olie ná eierlegging toegedien is, is daar wel 'n betekenisvolle eierdodende effek waargeneem. Tydens veldproewe het insekdoders met laer effektiwiteit as die primêre insekdoder, azinfos-metiel, aanvaarbare beheer verskaf indien dit suksesvol geïnkorporeer is in 'n spuitprogram deur 'n beleid van afwisseling van insekdoders oor generasies. Die kleinste variasie tussen die aantal graaddae tussen biofix en eerste uitbroei van eiers is gevind indien die tweede lokvalvangs as biofix gebruik is. 'n Biofix gebaseer op die eerste aand na die eerste lokval vangste wat die temperatuur 17°Cofhoër was teen 18:00, het ook 'n kleiner variasie getoon as die eerste lokvalvangs. Die aantal graaddae tussen die tweede en derde vlug biofix het tussen 531.2 en 488.87°D gewissel, met 'n gemiddelde van 508.1°D.

Thesis (MScAgric)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Characterizing the genetic structure of a pest population can provide an understanding of the factors influencing its evolution and assist in its ultimate control. The aim of the present study was to characterize the genetic structure of woolly apple aphid Eriosoma lanigerum (Hausmann) populations in the Western Cape Province in South Africa. Since this economically important apple pest has not previously been characterized at molecular level, it was necessary to evaluate methods for determining the genetic structure of E. lanigerum populations. Two different molecular techniques were evaluated viz. random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). This study represents the first application of the latter technique to members of the Aphididae. Aphids were sampled from four regions in the Western Cape in South Africa viz. Elgin, Ceres, Vyeboom and Villiersdorp. A spatially nested sampling design was used to establish the distribution of the genetic variance of aphids. A total of 192 individuals from 13 farms were analysed. Ten RAPD primers were chosen for analysis from an initial assay of 25 after fragment reproducibility had been confirmed. For AFLP analysis three different rare-cutting restriction enzymes were evaluated for AFLP analysis, viz. EcoRI, SseI and MluI. The latter yielded the best results in combination with the frequent-cutting enzyme MseI. Twenty-five AFLP selective primer pairs were evaluated, out of which five were chosen for analysis of the total population. Two hundred and fifty AFLP fragments and 47 RAPD fragments were scored for analysis. Both analyses indicated that a low level of genetic variation was apparent in E. lanigerum populations and that no differentiation resulted from geographic isolation. From RAPD analyses it was deduced that all variation could be attributed to differences between individuals. AFLP analysis indicated that, whereas genetic differences in E. lanigerum populations between orchards were negligible, a significant portion of genetic variation could be attributed to differences between farms and individuals within farms. Therefore, AFLP analysis allowed for finer discrimination of the genetic structure of E. lanigerum populations than RAPD analysis and is recommended for studies of other aphid species. The fact that most of the genetic variation present in E. lanigerum populations could be found on small spatial scales indicated that sampling individuals over a wide geographic area was an ineffective way of detecting the genetic diversity present in E. lanigerum populations. The low level of variation in populations is most likely due to the exclusive occurrence of parthenogenetic reproduction, founder effects (including distribution of infested plant material from a limited source) and selective factors such as the use of resistant rootstocks or pesticides. Furthermore, the low level of variation found indicated that the possibility of controlling E. lanigerum in the Western Cape using host plant resistance is favourable. Thus, plant breeders developing resistance to E. lanigerum can expect plant entries to be exposed to most of the genetic diversity present in Western Cape populations, regardless of location. / AFRIKAANSE OPSOMMING: Die bepaling van die genetiese struktuur van 'n landboukundige plaagpopulasie kan lei tot begrip van die faktore wat die populasie beïnvloed en kan uiteindelike beheer vergemaklik. Die doel van die huidige studie was om die genetiese struktuur van die appelbloedluis Eriosoma lanigerum (Hausmann) in die Wes-Kaap Provinsie van Suid-Afrika te bepaal. Aangesien hierdie belangrike appelplaag nie van tevore op molekulêre vlak bestudeer is nie, was dit nodig om metodes vir die bepaling van die genetiese struktuur van E. lanigerum populasies te evalueer. Twee molekulêre tegnieke is geëvalueer, nl. lukraak geamplifiseerde polimorfiese ONS (RAPD) en geamplifiseerde fragment-lengte polimorfismes (AFLP). Hierdie studie is die eerste om laasgenoemde tegniek te gebruik om lede van die Aphididae te bestudeer. Plantluise is verkry van vier verskillende gebiede in die Wes-Kaap Provinsie van Suid-Afrika nl. Elgin, Ceres, Vyeboom en Villiersdorp. 'n Hierargiese sisteem is gebruik om die verspreiding van die genetiese variasie van plantluise te bepaal. In totaal is 192 individue van 13 plase geanaliseer. Tien RAPD inleiers is gekies uit 'n analise van 25 verskillende inleiers nadat fragment reproduseerbaarheid bevestig is. Drie verskillende restriksie ensieme is geëvalueer vir AFLP analise nl. EcoRI, SseI en Mlul. Die beste resultate is verkry toe MluI saam met MseI gebruik is. Vyf-en-twintig AFLP selektiewe inleier pare is geëvalueer waarvan vyf gekies is vir analise van die totale populasie. Twee-honderd-en-vyftig AFLP fragmente en 47 RAPD fragmente is gedokumenteer vir analise. Beide RAPD en AFLP analises het getoon dat daar 'n lae vlak van genetiese variasie in E. lanigerum populasies is en dat geen differensiasie as gevolg van geografiese isolasie ontstaan het nie. Uit RAPD analise is daar afgelei dat al die variasie toegeskryf kon word aan verskille tussen individue. AFLP het aangetoon dat alhoewel verskille in E. lanigerum populasies tussen boorde laag was, kon 'n hoë persentasie van die variasie toegeskryf word aan verskille tussen plase en individue binne plase. AFLP analise het meer insig in die genetiese struktuur van E. lanigerum populasies verskaf, en word dus aanbeveel vir studies van ander plantluise. Omdat meeste van die genetiese variasie oor klein geografiese afstande verkry word, is steekproefueming oor groot gebiede 'n ondoeltreffende manier om die genetiese variasie binne 'n monster te meet. Die lae vlak van genetiese variasie is waarskynlik te wyte aan partenogenetiese vermeerdering, stigter gevolge (insluitend verspreiding van geïnfesteerde plantmateriaal vanaf 'n beperkte bron), sowel as selektiewe faktore soos die gebruik van bestande onderstokke en insekdoders. Verder dui die lae vlak van variasie aan dat die moontlikheid vir beheer deur gasheerplantbestandheid goed is in die Wes-Kaap. Planttelers kan verseker wees dat hulle plante blootgestel sal wees aan meeste van die genetiese variasie in die Wes-Kaap appelbloedluis populasies ongeag hulle ligging.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The commercial cultivation of Proteaceae is an important industry in the Western Cape, however, farmers are challenged with arthropod infestation which compels them to solely rely on chemical pesticides. Past studies in South Africa have shown that Proteaceae comprise a rich and diverse arthropod fauna. However, as most of these studies were conducted on wild Proteaceae, they may not be representative of cultivated proteas. Moreover, most of these species remained unidentified due to lack of identification expertise. These past studies, however, form a useful baseline for arthropod studies in proteas, e.g. the feeding guilds found in proteas. The aim of this research was to conduct an intensive and extensive survey of the arthropod-fauna associated with commercially-cultivated proteas across an entire year. Specifically, this survey was designed to document the composition of the arthropod fauna (creating a comprehensive reference collection for pest management purposes) and to assess whether the arthropod fauna differed between seasons and pesticide treatments. Infructescences, inflorescences and foliage of mainly commercial Proteaceae were sampled for arthropods seasonally for a period of twelve months by collection of plant material and direct searching. Seven commercial protea blocks, and a wild protea block (remnant patch of fynbos vegetation), were used as the sampling sites, and two sprayed blocks were used for assessing pesticide efficacy. Individual arthropods were identified as far as possible, with 37% identified to species level. A species accumulation curve showed that rare (minor) arthropod species made up of 70% of arthropods occurring in cultivated proteas. More than 8 700 individuals from more than 140 species and about 80 families were collected and identified, revealing that cultivated proteas have a rich and diverse insect fauna. These arthropods represent the full range of plant-feeding guilds: leaf miners, leaf chewers, flower bud borers, sap suckers and seed feeders. Flower visitors/free living guild was the most abundant (72%) and speciose (25%). In addition to phytophages, there was a large suite of insect predators and parasitoids. A large number of the arthropods were endemic to the Cape Floristic Region (CFR) and some (7.86%) have a pest status, in that they cause significant damage to the protea plants (for example, 60% of Safari sunset cultivar (Leucadendron salignum x L. laureolum) new flush stems and leaves were affected by Epichoristodes acerbella (Tortricidae). Capys alphaeus (Lycaenidae) and Phyllocnistis sp. (Phyllocnistidae) appear to be specialist pests, as they attack mainly Protea cynaroides and Susara cultivar (Protea magnifica x P. susannae) respectively. Arthropod abundance did not differ significantly between seasons, although significant seasonal effects were observed in species richness when the protea cultivars were examined separately. Pesticide application did not affect arthropod abundance, but did decrease species richness in sprayed blocks. Pesticides appeared to negatively affect minor (rare) species disproportionately, probably due to their lack of prior exposure to pesticides and hence sensitivity. Due to this inefficacy of pesticides in cultivated proteas, an increasing emphasis on the importance of non-chemical control measures, and our improved knowledge of the predatory and parasitic species in this system, integrated pest management strategies deserve greater research attention. Monitoring and use of threshold values for arthropod pests were suggested here, as well as the use of biological, cultural, physical and chemical (optimal use) control. For instance, in cultural control, polycropping and intercropping in proteas to increase plant diversity in the monocultures to promote a higher density of predators and parasitoids can be used. Certain flowering plants are known to provide greater temporal and spatial distribution of nectar and pollen sources, which can increase parasitoid reproductive potential and abundance of alternative hosts/prey when the pest species are scarce or at an inappropriate stage. / AFRIKAANSE OPSOMMING: Die kommersiële verbouing van Proteaceae (proteas) is 'n belangrike bedryf in die Wes-Kaap. Menige plantasie wemel egter van artropodes, wat boere noop om slegs van chemiese plaagdoders gebruik te maak. Vorige studies in Suid-Afrika toon dat proteas die gasheerplant vir 'n ryke en diverse artropodefauna is. Aangesien die meeste van hierdie studies egter op wilde proteas uitgevoer is, weerspieël dit moontlik nie die stand van sake met verboude proteas nie. Weens 'n gebrek aan kundigheid om die artropodes te eien word baie van die spesies boonop nooit uitgeken nie. Dié studies voorsien egter 'n nuttige grondlyn vir 'n ondersoek na die artropodes op proteas, veral vir die bestudering van die gilde wat van die protea leef (“the feeding guild”). Hierdie navorsing het ten doel om 'n intensiewe en omvattende opname te maak van die artropodefauna wat oor die tydperk van 'n jaar op kommersieel verboude proteas voorkom. Die opname is meer bepaald ontwerp om die samestelling van die artropodefauna te bestudeer (deur 'n omvattende verwysingsversameling vir plaagbestuurdoeleindes te skep), en om vas te stel of seisoene en plaagbehandelings enige beduidende uitwerking op die artropodefauna het. Oor 'n tydperk van 12 maande is seisoenale monsters van die vrug- en bloeistadia, saadkoppe en blare van hoofsaaklik kommersiële proteas gesoek en ingesamel. Sewe kommersiële proteablokke sowel as 'n blok wilde proteas het as proefpersele gedien, en twee bespuite blokke is gebruik om die doeltreffendheid van plaagdoder te beoordeel. Individuele artropodes is so noukeurig moontlik uitgeken – 37% tot op spesievlak. Volgens 'n spesieakkumulasiekurwe maak seldsame (kleiner) artropodespesies sowat 70% van die artropodes uit wat op verboude proteas voorkom. Die meer as 8 700 individue van meer as 140 spesies en sowat 80 families wat ingesamel en uitgeken is, toon die rykheid en diversiteit van die artropodefauna op verboude proteas. Hierdie artropodes verteenwoordig die volle reeks plantvreterspesies – van blaardelwers en blaarkouers tot blomknopboorders, sapsuiers en saadvreters. Blombesoeker-/vrylewende spesies was die volopste (72%) en mees divers (25%). Buiten plantvreters was daar ook 'n groot aantal roofinsekte en parasitoïede. Baie van die artropodes was inheems, en sommige (7,86%) het boonop plaagstatus, aangesien hulle beduidende skade aan die proteaplant aanrig. [By ongeveer 60% van die Safari Sunset-kultivar (Leucadendron salignum x L. laureolum) is nuwe stamme en blare byvoorbeeld deur die Epichoristodes acerbella (Tortricidae) aangetas.] Capys alphaeus (Lycaenidae) en Phyllocnistis sp. (Phyllocnistidae) blyk spesialisplae te wees wat onderskeidelik hoofsaaklik die Protea cynaroides en die Susarakultivar (Protea magnifica x P. susannae) in die visier het. Artropodegetalle het nie juis tussen seisoene gewissel nie, hoewel 'n afsonderlike ondersoek van die proteakultivars 'n beduidende seisoenale uitwerking op spesierykheid aan die lig gebring het. Eweneens het die toediening van plaagdoder nie die artropodegetalle verminder nie, maar wel spesierykheid op die bespuite blokke verswak. Plaagdoders blyk besonder negatiewe uitwerking op kleiner (seldsame) spesies te hê – waarskynlik omdat dié spesies nie voorheen aan plaagdoders blootgestel was nie, en dus gevoelig is daarvoor. Weens die oënskynlike ondoeltreffendheid van plaagdoders op verboude proteas, verg 'n toenemende klem op die belang van niechemiese beheermaatreëls, 'n behoefte aan meer kennis van die roof- en parasitiese spesies in die stelsel, en die vraag na geïntegreerde plaagbeheerstrategieë, meer navorsing. Die studie moniteer en gebruik drempelwaardes vir artropodeplae, sowel as biologiese, kulturele, fisiese én chemiese (‘optimalegebruik’-) plaagbeheer. Met kulturele beheer kan poli- en interverbouing van proteas byvoorbeeld gebruik word om plantdiversiteit in die monokulture te verbeter, ten einde só 'n hoër digtheid van roofspesies en parasitoïede in die hand te werk. Sekere blomplante bied kenmerkend 'n wyer tyd- en ruimtelike verspreiding van nektar- en stuifmeelbronne, wat parasitoïede se voortplantingsvermoë en die getalle van alternatiewe gashere/prooi kan verbeter wanneer die plaagspesies skaars is of in 'n ontoepaslike stadium verkeer.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs. / AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

Thesis (PhD(Agric))--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The purpose of this study was to characterize the race and vegetative compatibility of Fusarium oxysporum f. sp. melonis (FOM) isolates collected in the major melon producing areas, to report on their geographical distribution, and their possible relatedness to isolates from other countries. Seventy two FOM isolates obtained from 30 fields in 17 melon producing regions were race-typed using the differential cultivars Topmark (susceptible to all races), Doublon (Fomi), CM 17187 (Fom2) and Perlita (Fom3) and grouped by means of vegetative compatibility. All isolates belonged to vegetative compatibility group 0134, indicating a high degree of genetic homogeneity among the South African FOM population. Fifty four isolates were identified as race 0, eight as race 1, and 10 as race 2. Race 0 occurred in 15 of the regions whereas race 1 was sporadically recovered. Race 2, on the other hand, was obtained only from four fields located in one geographical region. Perlita plants (carrying the gene Fom3) inoculated with local isolates ofrace 0 and race 2 and reference isolates of race 0 became stunted, their leaves turned yellow, and became thickened and brittle. These results suggested that Fom3 in Perlita confers a tolerant reaction compared to the resistant reaction of gene FornI in Doublon. The disease reaction of cultivar Perlita to FOM was therefore reinvestigated. Twenty isolates, including the four FOM races (0, 1, 2, and 1,2) obtained from different countries, were used. The differential cultivars were included to verify virulence of the isolates. Perlita plants inoculated with three isolates of race 2 remained asymptomatic. The remaining race 2 and 0 isolates, induced severe stunting of Perlita plants, but mean percentage stunting values did not differ significantly (P = 0.05) and ranged between 25.1 and 50.0. Leaves of stunted plants were chlorotic, thickened and brittle. Disease reaction of Perlita was verified at a lower inoculum concentration with two race 2 (pipette method) and two race 0 isolates (root dip method). Results proved that Fom3 does not confer similar resistance towards race 0 and some race 2 isolates as FornI in Doublon. Cultivars possessing Fom3, should therefore be considered tolerant to FOM races 0 and 2. The ability of a nit mutant isolate, generated from FOM race 0 which belongs to VCG 0134, to change its virulence during infection of melon plants, was investigated under quarantine. Seedlings of melon cultivars Imperial 45 and Early Sweet (no resistance genes), Amber (Fom2) and Fiata (FomI, Fom2) were consecutively grown in two cement troughs in a gauzehouse. Each planting was terminated when plants had advanced Fusarium wilt or after the fruit were harvested. In the first planting, Imperial 45 seedlings were transplanted and artificially inoculated with the nil mutant isolate. In the consecutive plantings, seeds were sown in the infested soil to enable natural infection. For each crop, representative plants showing Fusarium wilt were selected for isolation. All F. oxysporum isolates recovered were single-spored and their nit mutant and VCG status verified. Virulence of the labelled isolates was determined using differential cultivars. In trough A, all plants of the susceptible cultivars Imperial 45 and Early Sweet crops showed Fusarium wilt. The labelled isolates recovered from the selected plants were all designated race O. In the first crop (planting No.5) of the resistant cultivar Amber, 6.7% of the plants developed Fusarium wilt. In the second Amber crop the disease incidence increased to 56.6%, and to 81.8% in the final crop. Contrary to the susceptible cultivars, only race 2 isolates were obtained from the symptomatic Amber plants. Similar data were found with the susceptible cultivar Imperial 45 and the resistant cultivar Amber in trough B. Planting of Fiata caused a dramatic reduction in Fusarium wilt incidence in trough B. However, 1.2% of plants were affected by Fusarium wilt in the first Fiata crop (planting No.6), whereas 4% of the plants were symptomatic in the final planting. From these symptomatic Fiata plants only race 1,2 isolates were obtained. These findings, and the fact that the symptomatic plants represented a substantial proportion of the first Amber (approximately 7-15%) and Fiata (approximately 2%) crops, provedthat changes in the race structure of this fungal pathogen occurred rapidly when confronted with a resistant cultivar. The potential of RAPD analysis to differentiate between the isolates displaying virulence changes was evaluated. Four F. oxysporum f. sp. niveum isolates were included as an outgroup. A histopathological study was conducted to verify whether these isolates retain their ability to behave as true vascular pathogens. The three primers used clearly distinguished the 12 FOM isolates from the four F. oxysporum f. sp. niveum isolates. However, the primers showed a highly conserved and characteristic banding pattern for the FOM isolates which represented three physiological races (race 0, race 2, race 1,2), indicating that RAPD analysis cannot detect race-specific groupings in FOM. Disease reactions on the three differential cultivars confirmed the virulence of FOM isolates. The histopathological data furthermore proved that the two FOM races (race 2, race 1,2), which derived from the race 0 parent isolate, retained their ability to behave as true vascular pathogens. / AFRIKAANSE OPSOMMING: DIE KARAKTERISERING EN PATOGENESITEIT VAN SUID-AFRIKAANSE ISOLATE VAN FUSARIUMOXYSPORUMF. SP.MELONIS Die doel van die studie was om Fusarium oxysporum f. sp. melonis (FOM) isolate wat in die hoof spanspekproduserende gebiede versamel is, volgens ras en vegetatiewe verenigbaarheid te karakteriseer, en hul geografiese verspreiding en verwantskap met isolate van ander lande aan te dui. Twee en sewentig FOM isolate afkomstig vanaf 30 landerye wat 17 spanspekproduserende areas verteenwoordig, is gebruik. Die differensiële kultivars Topmark (vatbaar vir alle rasse), Doublon (Forni), CM 17187 (Fom2) en Perlita (Fond) is gebruik om die rasbepalings te doen asook om die vegetatiewe verenigbare groepe (VVG) te bepaal. Al die isolate is as VVG 0134 geklassifiseer, wat 'n hoë mate van genetiese homogenesiteit binne die Suid-Afrikaanse populasie aandui. Vier en vyftig isolate is as ras 0, agt as ras 1 en 10 as ras 2 geklassifiseer. Ras 0 is vanaf 15 gebiede afkomstig, terwyl ras 1 sporadies voorgekom het. Ras 2 is vanuit vier landerye binne dieselfde geografiese gebied verkry. Plante van die kultivar Perlita wat met plaaslike isolate van ras 0 en 2, asook verwysings-isolate van ras 0 geïnokuleer is, het verdwerg voorgekom. Die blare van die plante het vergeel, verdik en bros voorgekom. Hierdie siekte reaksie het aangedui dat Fond in Perlita toleransie bewerkstellig in teenstelling met die weerstandbiedende reaksie van geen Fomi in Doublon. Die siekte reaksie van Perlita teenoor FOM is dus verder ondesoek. Hiervoor is 20 isolate wat al vier FOM rasse insluit (0, 1, 2, en 1,2), en van verskillende wêrelddele afkomstig is, gebruik. Die virulensie van die isolate is met die differensiële kultivars bevestig. Drie van die ras 2 isolate het geen siektesimptome op Perlita veroorsaak nie. Die ander ras 2 isolate, en al die ras 0 isolate, het egter die Perlita plante aansienlik verdwerg en die blare vergeel en verdik. Laasgenoemde groep isolate het 'n gemiddelde verdwergingsindeks van tussen 25.1% en 50.0% veroorsaak. Die siekte reaksie by Perlita is verder bevestig deur plante teen 'n laer inokulumdigtheid van twee ras 2 (pipet metode), en twee ras 0 (wortel-doop metode) isolate, te inokuleer. Uit die resultate was dit duidelik dat die weerstand wat Fom3 teenoor ras 0 en sommige ras 2 isolate verskaf, van FornI verskil. Kultivars wat oor die weerstandsgeen Fom3 beskik moet dus as tolerant beskou word. 'n Ondersoek is geloods na die vermoë van 'n nil mutant isolaat, genereer vanaf die wilde ras 0 isolaat van FOM (VVG 0134), om onder kwarantyn sy virulensie gedurende infeksie van spanspekplante te verander. Saailinge van die spanspekkultivars Early Sweet (geen weerstandsgene), Amber (Fom2) en Fiata (FornI, Fom2) is opeenvolgens in twee sement trêe in 'n gaashuis verbou. Die afsonderlike aanplantings is beëindig sodra gevorderde Fusarium-verwelksimptome verkry is, of nadat vrugte ge-oes is. Vir die eerste aanplanting is oorgeplante Imperial 45 saailinge kunsmatig met die nil mutant isolaat geïnokuleer. Tydens die opeenvolgende aanplantings is saad direk in die besmette grond gesaai ten einde natuurlike infeksie te verkry. Met elke aanplanting is isolasies gedoen vanaf verteenwoordigende plante wat Fusarium-verwelksimptome getoon het. Alle F. oxysporum isolate wat verkry is, is ge-enkelspoor en hul nit mutant status en VVG is bevestig. Virulensie van die gemerkte isolate is bepaal deur inokulasie van die differensiële kultivars. Alle plante van die vatbare Imperial 45 en Early Sweet kultivars wat in trog A geplant is, het Fusarium-verwelksimptome getoon. Die gemerkte isolate wat vanaf die verteenwoordigende plante verkry is, is almal as ras 0 geklassifiseer. Tydens die eerste aanplanting van die weerstandbiedende kultivar, Amber (aanplanting No.5), het 6.7% van die plante Fusarium-verwe1ksimptome ontwikkel. Tydens die tweede en derde aanplanting van Amber het die frekwensie van siektevoorkoms verhoog na 56.6% en 81.8 %, onderskeidelik. In teenstelling met die vatbare kultivars, is slegs ras 2 vanuit die Amber plante met siektesimptome verkry. Soortgelyke resultate is met Imperial 45 en Amber in trog B verkry. Aanplanting van kultivar Fiata het egter 'n dramatiese verlaging in die voorkoms van Fusarium-verwelk bewerkstellig. Tydens die eerste Fiata aanplanting (aanplanting No.6) het 1.2% plante Fusarium-verwelksimptome ontwikkel, en 4% tydens die laaste aanplanting. Vanaf hierdie plante is slegs ras 1,2 isolate verkry. Hierdie bevindings, en die feit dat 'n aansienlike hoeveelheid van die Amber (ongeveer 7-15%) en Fiata plante (ongeveer 2%) siektesimptome getoon het, bewys dat FOM vinnig van virulensie verander wanneer die patogeen 'n weerstanbiedende kultivar infekteer. Die vermoë van RAPD analise om tussen isolate wat in virulensie verander het, te onderskei, is ondersoek. Vier isolate van F. oxysporum f. sp. niveum is as 'n buite-groep ingesluit. Om te bevestig dat die isolate wat van ras verander het wel egte vaskulêre patogene is, is 'n histopatologiese ondersoek gedoen. Die drie inleiers wat gebruik is, het die 12 FOM isolate duidelik van die vier F. oxysporum f. sp. niveum isolate onderskei. Die 12 FOM isolate wat drie fiosologiese rasse (ras 0, ras 2, ras 1,2) verteenwoordig het, is egter saam gegroepeer, wat aandui dat hierdie metode nie tussen rasse van FOM kan onderskei nie. Inokulasiestudies met die differensiële kultivars het die virulensie van die isolate bevestig. Die histopatologiese ondersoek het verder bewys dat beide FOM rasse (ras 2, ras 1,2) wat vanaf die wilde tipe ras ° isolaat ontstaan het, hul vermoë behou het om as egte vaskulêre patogene op te tree.

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.

Capsicum baccatum var. pendulum was first grown in the Makana District in 2005. Extremely little was known about best practices for cultivation or the insects and diseases associated with the crop in this area. The study was conducted during the second year of production, November 2005 and November 2006, in an attempt to identify the composition and phenology of insects occurring on C. baccatum. In the more rural parts of the Eastern Cape, and more particularly in Grahamstown, there are very few industries. With the advent of this new agricultural venture, a processing factory has been opened in Grahamstown creating more than 600 seasonal jobs in the factory and 1000 seasonal jobs on farms for local people. This business enterprise has not only brought about the creation of jobs, but also training and skills development and empowerment, generating much-needed income in this area. An extensive literature review yielded limited information on insect pests associated with Capsicum. Data from a pilot sampling trial undertaken were statistically analyzed to establish the number of plants to be scouted per site and the most effective scouting techniques to use. Based on the data available and insects collected during the pilot sampling trial, a surveillance programme was designed. Five different types of monitoring traps were placed in each of the eight study sites. Collection of trap catches and scouting of fifteen individual plants per site was undertaken on a weekly basis over the 52-week study period. The most commonly occurring potential insect pests were African Bollworm Helicoverpa armigera (Hübner), False Codling Moth Thaumatotibia leucotreta (= Cryptophlebia leucotreta) (Meyrick), Mediterranean Fruit Fly Ceratitis capitata (Wiedemann) and several species of thrips. Population densities of these pests and their phenology on Capsicum were determined. Statistical analyses established the efficacy of the monitoring traps for each pest, tested for differences among and between study sites, calculated an estimate of the number of pods damaged and a measure of plant damage.The results show that the majority of damage caused to the Capsicum baccatum cropping system was due to Mediterranean Fruit Fly populations. It was established that, although African Bollworm and False Codling Moth were present during the study period, their numbers were negligible and only nominal damage was caused by these pests. Damage caused by thrips species was apparent but not quantifiable. Intervention strategies using an Integrated Pest Management approach, are discussed.

Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.