Dissertationen zum Thema „Cassava – Diseases and pests – South Africa“

Studies presented in this dissertation highlight the importance of fungal pathogens in forestry nurseries in South Africa. Both Acacia meamsii seedlings and Eucalyptus hybrid cuttings are shown to be affected by important nursery pathogens. Chapter one presents an evaluation of the potential importance of pathogens to Eucalyptus hedge plants maintained in hydroponics. Hydroponics is a new technology being used in South African forest nurseries, which allows for the rapid establishment of Eucalyptus hedge plants. However, no information is available on pathogens affecting Eucalyptus in hydroponics. By applying information on pathogens of other hydroponic crops, several potentially important pathogens were identified and these reside in the genera Phytophthora, Pythium and Fusarium. Possible disease symptoms in Eucalyptus caused by these pathogens include wilting, stem cankers and root rots. Implementation of appropriate control measures that include cultural, biological and chemical practices could prevent and/or reduce disease impact in hydroponics. Chapter two presents the results of a survey of the roots of Eucalyptus hedge plants grown in an ebb and flow hydroponic system. An interesting result of the survey was the discovery of Cylindrocladium pauciramosum in the hydroponic system. This is the first report of the pathogen in a hydroponic system. Other important pathogens in the genera Phytophthora and Pythium were also isolated. Two Pythium species, namely P. dissotocum and P. helicoids, found in the roots and nutrient solution are new to Eucalyptus. Several Fusarium species were also isolated of which two, namely F. nygamai and F. lateritium, are also new to Eucalyptus. Chapter three of this dissertation presents the results of a survey of Eucalyptus cuttings conducted at four forestry nurseries in KwaZulu-Natal, South Africa. Several well-known Eucalyptus nursery pathogens were isolated. Cylindrocladium pauciramosum was identified as the dominant pathogen on Eucalyptus cuttings. This was confirmed based on morphological characteristics and DNA sequence comparisons. Pathogenicity tests conducted using a spore suspension of C. pauciramosum indicated that this pathogen is capable of infecting most commercial Eucalyptus clones used in South Africa. Chapter four considers a serious disease of Acacia mearnsii seedlings caused by an unidentified species of Cylindrocladium. Cylindrocladium pauciramosum was isolated from A. mearnsii seedlings showing girdling and stem canker symptoms. The pathogen was identified based on morphological characteristics and DNA sequence comparisons. Pathogenicity tests with Acacia seedlings confirmed the susceptibility of this tree to C. pauciramosum infection. This dissertation clearly indicates that Cylindrocladium pauciramosum is an important nursery pathogen in South African forestry nurseries. This pathogen has already been shown to be limiting during production of planting stock. I hope to have highlighted the importance of C. pauciramosum and other nursery pathogens in forestry nurseries in South Africa. This study will also hopefully provide information to forestry nursery managers and help them improve production.
Dissertation (MSc)--University of Pretoria, 2004.
Microbiology and Plant Pathology
Unrestricted

Leaf diseases are an integral part of banana production. While currently not a cause for major concern in South Africa, many of these diseases van reach epidemic proportions and cause severe crop loss. To determine the present status of leaf diseases in South Africa, a survey was conducted in the five banana-growing regions of the country. The study indicated the following: Yello Sigatoka, caused by Mycosphaerella musicola was the most prevalent disease and occurred in all five the regions. Mycosphaerella speckle and Cordana leaf spot, caused by M. musae and Cornana musae respectively, were present in four regions. Cladosporium speckle, caused by Cladosporium musae, was found only in the Levubu area. Various other fungi, mainly saprobes and endophytes, were also isolated. The most commonly encountered species included Alternaria alternate, Colletrichum gloeosporioides, Nigrospora oryzae, N. sacchari, N. Spaerica, Pestalotiopsis sp., Phoma glomerata, Selenophoma asterina and S. juncea. Following morphological identification of the pathogenic species, monoconidial isolates were established from representative isolates of each and their virulence confirmed in artificial inoculation studies. The identity of M. musciola and Cladosporium musae was verified molecularly by means of species-specific primers and/or sequencing of the ITS region. Validation of the identity of Cladosporium musae constitutes the first report of Cladosporium speckle on banana in South Africa. Sequence data of the ITS region of isolates from Mycospaerella speckle lesions indicated that the symptoms are caused by two species, M. musae and one closely related to M. colombiensis, the latter previously described only from lesions on leaves of Eucalyptus urophylla in Colombia.
Dissertation (MSc (Microbiology))--University of Pretoria, 2002.
Microbiology and Plant Pathology
unrestricted

Thesis (MSc)--Stellenbosch University, 2000.
Full text to be digitised and attached to bibliographic record.
ENGLISH ABSTRACT: The cultivation of citrus in the Vaalharts region is a fairly recent development. With the introduction of this crop, an insect pest complex has also developed in this region. The presence of these pests was studied in eleven orchards, planted with three citrus cultivars and of varying ages, distributed in the 300 square kilometer cultivation area. Each orchard was inspected for the presence of pests by making use of two weekly sampling techniques. Ten of the most important insect pests of citrus in the Vaalharts region are briefly described by refering to their general appearance, life cycles, feeding and pest status and economic threshold. For every pest various control options, including operational systems, crop cultivation, biological and chemical control, are discussed and, where applicable, illustrated by means of graphic presentations. The seasonal presence of the cirtrus pests in the Vaalharts region is tabulated and discussed individually. With these details as background, an insect pest management programme for citrus in the Vaalharts region is compiled.
AFRIKAANSE OPSOMMING: Die verbouing van sitrus in die Vaalhartsgebied is 'n redelik onlangse ontwikkeling. Gepaard met die nuwe gewas het daar ook 'n insekplaagkompleks in die gebied ontstaan. Die voorkoms van die plae is in elt .boorde, beplant met drie sitruskultivars en van verskillende ouderdomme, verspreid in die 300 vierkante kilometer verbouingsareaal, bestudeer. Elk van die boorde is weekliks ondersoek vir die aanwesigheid van plae deur van twee moniteringstegnieke gebruik te maak. Die tien belangrikste insekplae van sitrus in die Vaalhartsgebied word kortliks beskryf deur na hulle algemene voorkoms, lewenssiklus, voeding en plaagstatus en ekonomiese drempelwaardes asook die moniteringsmetodes wat gebruik is, te verwys. Vir elke plaag word beheeropsies, wat operasionele stelsels, gewasverbouing, bloloqlese en chemiese beheer insluit, bespreek wat, waar toepaslik, aan die hand van grafiese voorstellings gemustreer word. Die seisoenale aanwesigheid van die sitrusplae word in 'n tabel aangedui en individueel bespreek. Met die gegewens as agtergrond is 'n insekplaagbestuurprogram vir sitrus in die Vaalhartsgebied opgestel.

The South African Citrus Thrips, Scirtothrips aurantii Faure (Thysanoptera: Thripidae) has been a serious pest of the citrus industry of Southern Africa for over 70 years. It is indigenous to Africa and has no recorded parasitoids and, in most citrus-growing regions, predators are not economically effective. Firstly, in this study, the general ecology of thrips was reviewed along with the recorded history of S. aurantii and its control. Host plant relationships of S. aurantii were then examined and wild hosts were not found to be important in promoting citrus thrips outbreaks in the orchard after flowering. In addition, Scirtothrips dorsalis Hood, which attacks citrus in Asia, was collected for the first time in Africa, but from Castor Oil plant. It is therefore a potential pest of citrus here. Notes on its appearance compared to that of S. aurantii were prepared. Weekly sampling of S. aurantii adults was carried out from June 1984 to May 1990. Population fluctuations were then correlated with phenology of the citrus trees and the direct and indirect effects of weather. Temperature and rainfall were not found to be significantly directly correlated with thrips numbers recorded. However, the indirect effects of rainfall were important in promoting a large winter thrips population in certain years. Relatively high rainfall during March and April compared to that of the previous January and February stimulated atypical flushing of the citrus trees during autumn and the setting of out-of-season fruit. S. aurantii then exploited this unusual food source and high numbers were subsequently recorded in the following winter; as well as in spring. Thus the mild winter climate alone could not suppress thrips numbers at Letaba. Dispersal/Emergence traps, which are used in the U.S.A. for the monitoring of Scirtothrips citri, were evaluated over 24 months, and were effective in recording population peaks of S. aurantii. The emergence rate of adults in relation to second instar larvae trapped was 43.7%. 35.7% of adults caught after emerging from the soil were males and 64.3 were females. In contrast, yellow traps had recorded 59.1% males and 40.9% females over the same period. The yellow traps were subsequently found to be biased towards male catches when young fruit and/or soft flush was present on the citrus trees. Relative inhibition of female flight activity during times of food abundance is known in other thrips species. During times of food scarcity, the bias in the yellow trap results disappeared. At Letaba Estates, availability of food rather than the direct effects of weather was seen to be the most important factor in governing S. aurantii numbers. Because of the importance of soft flush in the promotion of S. aurantii population increase, it is recommended that more attention should be given to the control of thrips on, as well as conducting surveys for, this food source. As the climate at Letaba is so favourable for citrus thrips, even during winter, it is further recommended that trapping for S. aurantii is carried out throughout the year. Furthermore, males and females should be recorded separately in trapping results as the sex ratio gives an indication of the phenological state of the tree.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Grapevine virus E (GVE) is a newly identified virus that has been detected in an established vineyard in South Africa. This virus is a member of the genus Vitivirus, family Flexiviridae. Members of this genus are known to infecte grapevine and are associated with various disease complexes, such as the Rugose wood complex (RWC) and Shiraz disease (SD). However, the role and impact of GVE in South African vineyards are still unknown. It is important to study these viruses to determine how they infect and the possible impact they may have on vine health. The accurate and early detection of grapevine viruses is the first important step in disease management. In this study, reverse transcription-polymerase chain reaction (RT-PCR), double antibody sandwich enzyme linked immunesorbent assay (DAS-ELISA) and quantitative (q)RT-PCR were used for the detection of GVE in the vineyard (Vitis vinifera cv Merlot) where GVE was first identified in South Africa. Reverse transcription-PCR was used for detection and determining the incidence of GVE. The incidence was as low as 3% in the vineyard surveyed. All the GVE positive plants were co-infected with GLRaV-3 and no disease association could therefore be made. Evaluation of the Bioreba Grapevine virus A (GVA) DAS-ELISA kit showed that it did not detect GVE. No cross-reactivity occurred with epitopes of GVE, confirming this kit to be a valid and specific assay for GVA infection. The relative virus titer of GVE was calculated over the growing season of 2010/2011, using qRT-PCR. No fluctuation in virus titer was observed during that growing season. Transmission experiments were performed in an attempt to transfer GVE from grapevine to an alternative host. Three different transmission buffers as well as nine different herbaceous plant species, that have shown to be susceptible to several plant viruses in previous studies, were evaluated. In these experiments, GVE could not be transmitted to any of the herbaceous species. To further characterize GVE, chimeric clones were constructed with GVA. The ORF2 and ORF5 of GVE were cloned into previously constructed GVA ORF2 and ORF5 deletion mutants. Construction of the chimeric clones, 35S-GVA-GR5-ΔORF2-GVE-ORF2 and 35S-GVA-118-ΔORF5-GVE-ORF5 were successful and they were evaluated for their infectivity in N. benthamiana. The 35S-GVA-GR5-ΔORF2-GVE-ORF2 chimera was able to infect and replicate in these plants and disease symptoms such as yellowing of veins and leaf curling were observed. Virus, derived from this vector, was detected by TPIA, RT-PCR and DAS-ELISA. The 35S-GVA-118-ΔORF5-GVE-ORF5 chimeric vector was not able to infect N. benthamiana as no disease symptoms were observed in any of the infiltrated plants and virus was not detected with serological analysis and RT-PCR. This study was aimed at further characterizing the recently identified virus GVE. Here, insight is given into the prevalence of this virus in the vineyard where it was first identified and attempts to biologically characterize GVE were made.
AFRIKAANSE OPSOMMING: Grapevine virus E (GVE) is „n nuut geïndetifiseerde virus wat onlangs in „n gevestigde wingerd in Suid Afrika opgespoor is. Hierdie virus vorm deel van die genus Vitivirus, familie Betaflexiviridae. Spesies in hierdie genus is bekend vir wingerdinfeksies en word met „n verskeidenheid wingerd siektes geassosieer, soos bv. Rugose wood complex (RWC) en Shiraz siekte (SD). Die rol en impak van GVE is nog onbekend. Dit is dus belangrik om die virus te bestudeer om te bepaal hoe dit infekteer en of dit enige impak het op wingerd gesondheid. Akkurate en vroeë opsporing van virusse is die eerste belangrike stap vir virussiekte beheer. In hierdie studie word tru-transkripsie (TT) – polimerase ketting reaksie (PKR), dubbel teenliggaam (DAS) -ensiem gekoppelde immuno-absorberende analise (ELISA) en qTT-PKR gebruik vir die opsporing van GVE in die wingerd (Vitis vinifera cv Merlot) waar dit vroeër in Suid Afrika geïdentifiseer was. Vir opsporing en bepaling van verspreiding is TT-PKR gebruik. Daar is bepaal dat 3% van die wingerd met GVE geïnfekteer is. Al die GVE-positiewe stokke het ook positief getoets vir GLRaV-3 en geen assosiasie met siekte simptome kon gemaak word nie. Evaluering van die Bioreba GVA DAS-ELISA met GVE positiewe stokke het nie GVE opgespoor nie. Geen kruisreaktiwiteit het plaasgevind met epitope van GVE nie en dus is die DAS-ELISA ʼn betroubare toets vir GVA infeksie. Die relatiewe virus titer van GVE was ook bepaal oor die groeiseisoen van 2010/2011 deur qTT-PKR te gebruik. Geen fluktuasie in virus titer gedurende die groeiseisoen is waargeneem nie. Transmissie eksperimente is gedoen om GVE vanaf wingerd na ʼn alternatiewe gasheer oor te dra. Drie verskillende transmissie buffers en tien verskillende sagteplant spesies, wat voorheen vatbaarheid vir plantvirusse getoon het, is gebruik. In die transmissie eksperimente kon GVE nie na enige van die sagteplante oorgedra word nie. Om GVE verder te karakteriseer is hibried-virusse met GVA gemaak. Die leesraam (ORF) 2 en ORF5 van GVE gekloneer in GVA ORF2 en -ORF5 delesie konstrukte, 35S-GVA-GR5-ΔORF2 en 35S-GVA-118-ΔORF5, onderskeidelik (Blignaut, 2009; Du Preez, 2010). Klonering van die hibried konstrukte, 35S-GVA-GR5-ΔORF2-GVE-ORF2 en 35S-GVA-118-ΔORF5-GVE-ORF5, was suksesvol en is in N. benthamiana geëvalueer. Virus afkomstig van die 35S-GVA-GR5-ΔORF2-GVE-ORF2 hibried konstruk, kon plante suksesvol infekteer en kon repliseer binne hierdie plante. Siektesimptome soos vergeling van die are en rolblaar is ook waargeneem in plante geïnfekteer met hierdie hibried konstruk. Plante is getoets met weefsel afdruk immuno analise (TPIA), TT-PKR en DAS-ELISA en is positief gevind vir virus afkomstig van hierdie konstruk. Die 35S-GVA-118-ΔORF5-GVE-ORF5 hibried kon nie N. benthamiana infekteer nie en geen siektesimptome is waargeneem in enige van die plante geïnfiltreer met hierdie konstruk. Serologiese analise en TT-PKR het ook nie virus in die N. benthamiana plante opgespoor nie. Die doel van hierdie studie was om GVE te karakteriseer. In hierdie studie word insig gegee oor die verspreiding van hierdie virus in Suid Afrika en pogings is gemaak om GVE biologies te karakteriseer.

Thesis (MScAgric)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Cydia pomonella (codling moth), Thaumatotibia leucotreta (False codling moth), Thaumatotibia batrachopa (Macadamia nut borer), Grapholita molesta (Oriental fruit moth), Cryptophlebia peltastica (Litchi moth), Epichoristodes acerbella (Pear leafroller/Carnation worm) and Lozotaenia capensana (Apple leafroller) are the most economically important tortricids affecting various crops in South Africa. The correct identification of these species, especially of the larval stage, is of great importance in pest management. Using available literature, augmented by additional morphological studies, an interactive identification key (Lucid key) for larval and adult stages of the seven species was developed. The colour and markings of the head, characteristics of the prothoracic and anal shields, the position of the prespiracular setae (L-group) relative to the spiracle on the prothoracic segment, the position of the spiracle on the eighth abdominal segment and L-group on the ninth abdominal segment, as well as the presence or absence of the anal comb are key characteristics for larval identification. For adult identification, wing pattern and genitalia are the most important features. However, the use of genitalia for moth identification might be difficult for the lay user, as the dissection and mounting of these structures requires certain skills and specialized equipment. Thus, genitalia have not been included in the Lucid Key. Differences in the morphological characteristics of most pupae were so minute that this stage was also not included in the Lucid key. However, the pupae of E. acerbella and L. capensana are easily distinguished from those of the other species by the presence of acremaster. This study also included the first morphological description of the pupa of L. capensana, which can be distinguished from that of E. acerbella by various features of the cremaster, antennae, spiracle shape, number of setae on abdominal segments A5-7, the size of spines on A3-7, and the presence/absence of spines on A9. A previous study by Timm (2005) indicated that geographically isolated populations of T. leucotreta tend to be genetically distinct. This raised the question of whether speciation/subspeciation has occurred or is occurring. Male moth genitalia are thought to evolve rapidly and are often the only features that can reliably distinguish similar species. Hence, variation in the shape of the valvae of T. leucotreta was used to determine whether divergence has occurred between populations of T. leucotreta. Elliptical Fourier analysis was used to analyze the valvar variation in three different populations. Although some variation in valvar shape was detected among mean population values for certain traits, no clear pattern emerged. Principle component analysis also showed no distinct clustering of valvae shape among populations, providing no evidence for divergence in male genitalia and therefore no morphological evidence of incipient speciation.
AFRIKAANSE OPSOMMING: Cydia pomonella (Kodlingmot), Thaumatotibia leucotreta (Valskodlingmot), T. batrachopa (Makadamianeutboorder), Grapholita molesta (Oosterse vrugtemot), Cryptophlebia peltastica (Lietsjiemot), Epichoristodes acerbella (Peerbladroller/Angelierrusper) en Lozotaenia capensana (Appelbladroller) is die mees ekonomies belangrike tortrisiede van die vrugtebedryf in Suid-Afrika. Die juiste identifikasie van hierdie spesies, veral van hulle larwale stadium, is van groot belang by plaagbestuur. Deur gebruik te maak van beskikbare literatuur, aangevul deur bykomstige morfologiese studies, is ‗n interaktiewe uitkenningssleutel (―Lucid key‖) vir die larwale en volwasse stadia van die sewe spesies ontwikkel. Die kleur en tekening van die kop, kenmerke van die prothorakale en anale skild, die ligging van die prespirakulêre setae (L-groep) relatief tot die spiraculum op die prothorakale segment, die ligging van die spirakulum op die agste abdominale segment en L-groep op die negende abdominale segment, asook die aan- of afwesigheid van die anale kam is sleutel kenmerke vir larwale uitkenning. Vir die volwassenes is die vlerktekening en genitalia die mees belangrike kenmerke. Die gebruik van die genitalia vir motuitkenning kan egter vir die leek gebruiker moeilik wees omdat die disseksie en montering van hierdie strukture bepaalde vaardighede en gespesialiseerde toerusting vereis. Vir die rede is die genitalia nie in die Lucid-sleutel ingesluit nie. Verskille in die morfologiese kenmerke van meeste papies is klein en die stadium is gevolglik ook nie in die sleutel ingesluit nie. Die papies van E. acerbella en L. capensana kan egter maklik van die ander spesies onderskei word deur die aanwesigheid van ‗n cremaster. Hierdie studie sluit ook die eerste morfologiese beskrywing van die papie van L. capensana in, wat van dié van E. acerbella onderskei kan word deur gebruik te maak van kenmerke van die cremaster, antennae, spirakulêre vorm, aantal setae op abdominale segmente A5-7, die grootte van stekels op A3-7, en die aan- of afwesigheid van stekels op A9. ‗n Vroeëre studie (Timm 2005) het aangedui dat geografies geïsoleerde bevolkings van T. leucotreta neig om geneties verskillend te wees. Dit het die vraag laat ontstaan of spesiasie/subspesiasie moontlik plaasgevind het of steeds plaasvind. Manlike mot genitalië word geag om vinnig te ontwikkel en is dikwels die enigste kenmerke wat betroubaar tussen soortgelyke spesies kan onderskei. Dus is die variasie in die vorm van die valvae van T. leucotreta gebruik om te bepaal of divergensie wel tussen bevolkings van T. leucotreta plaasgevind het. Elliptiese Fourier ontleding is gebruik om die valvae se variasie by drie verskillende bevolkings te ontleed. Alhoewel enkele variasie in die vorm van die valvae bespeur is by die gemiddelde bevolkingswaardes vir bepaalde eienskappe, kon geen duidelike patroon bespeur word nie. Hoofkomponentontleding het ook geen duidelike groepering van valvae se vorm tussen bevolkings getoon nie, wat geen bewys lewer van divergensie in die manlike genitalia en dus geen morfologiese bewys van beginnende spesiasie.

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.

Thesis (MScAgric)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Lucerne is the most important pasture and fodder crop in the winter rainfall area of South Africa. Various pests are known to cause damage to this crop. The use of the sweep net for monitoring pests is a cheap, easy and quick technique. If the sweep net is suitable for the lucerne pests in South Africa, potential pest status can be determined easily and quickly and the necessary precautionary measures taken to prevent crop losses. From a managerial point of view, it is also important to know the composition of the insect community in order to follow practices in which the number of beneficial insects can be increased and the injurious insects decreased. Therefore a study was done to quantify the use of the sweep net as a survey technique for monitoring pests on established lucerne stands. Insect diversity was also determined to obtain information on the insect families and guilds on lucerne. The redlegged earth mite, due to its importance as a pest, and the Anystis mite, important as a predator, were also included. The sweep net proved to be suitable for the sampling of the main lucerne pests. If a 29 cm diameter sweep net is swiped once per pace for six long paces, twelve systematically chosen sampling units are recommended for the lucerne earth flea and aphids. It is not necessary to differentiate amongst the three aphid species, or between the winged and unwinged aphids. Actual counts should be used instead of absence-presence data. Instead of counting all the insects in a sample, sub-samples can be taken. Operational characteristic curves can be used to determine the risk involved in the decision not to intervene, for example by spraying or grazing. Recommendations for monitoring and the accuracy of control decisions for the redlegged earth mite, Sitona weevil and lucerne butterfly can only be made after threshold values have been determined. The pea aphid, bluegreen aphid and lucerne earth flea showed peaks in their population levels during spring. Peak numbers of the spotted alfalfa aphid occurred during late summer and autumn. The Sitona weevil and lucerne butterfly numbers reached peak levels during late spring and early summer. For all pests population levels were dramatically reduced after grazing or cutting of the plantings. Therefore, these cultivation practices provided good control. The herbivores made up more than 85% of the insect community in lucerne. The largest herbivorous families, in terms of the number of individuals per family, were the Aphididae and Sminthuridae. These two families contain the main lucerne pests, the pea aphid, bluegreen aphid, spotted alfalfa aphid and the lucerne earth flea. The largest predatory family was the Anystidae, represented by the Anystis mite, the most important predator of the red legged earth mite and lucerne earth flea. Another well represented predatory family was the Coccinellidae, containing natural enemies of the aphids. The dryland plantings had a higher percentage of predators than the irrigated lucerne. The most important parasitaids were those in the superfamily Chalcidoidea and in the family Braconidae. The main detritivores were springtails in the suborder Arthropleona, insects in the families Mycetophilidae on irrigated lucerne, and Mycetophagidae on dryland lucerne. The most abundant visitors were in the families Chironomidae, Drosophilidae and Tephritidae. The dryland plantings had a lower percentage of visitors than the irrigated plantings. The number of insect families, as well as the number of individuals per family, was lower at the dryland plantings than at the irrigated plantings. The vast majority of insect families found on lucerne were collected during the one-year sampling period. A lower diversity was found where grazing was more severe, and there was a negative relationship between diversity and evenness.
AFRIKAANSE OPSOMMING: Lusern is die belangrikste wei- en voergewas 10 die winterreëngebied van Suid- Afrika. Hierdie gewas word deur 'n verskeidenheid plae aangeval. Die gebruik van die swaainet vir die monitering van plae is 'n goedkoop, maklike en vinnige tegniek. lndien die swaainet geskik is vir die betrokke plae in Suid-Afrika, kan potensiële plaagstatus van die plae dus maklik en vinnig bepaal word en die nodige voorsorgmaatreëls getref word om verliese te voorkom. Vanuit 'n bestuursoogpunt is dit ook belangrik om te weet wat die samestelling van die insekgemeenskap is sodat praktyke gevolg kan word waardeur die getal voordelige insekte verhoog en nadelige insekte verlaag word. Gevolglik is 'n studie uitgevoer om die gebruik van die swaainet te kwantifiseer as 'n monsternemingsmetode vir die monitering van plae op gevestigde lusernstande. Insekdiversiteit is ook bepaal ten einde inligting te bekom oor die insekfamilies en -gildes op lusern. Die lusernerdvlooi en swartsandmyt, vanweë hul belang as plae, en die Anystis-roofmyt, vanweë sy belang as predator, is ook ingesluit. Die swaainet blyk geskik te wees vir die monitering van die. vernaamste lusernplae. Wanneer 'n 29 cm deursnee swaainet vir ses lang treë een keer per tree geswaai word, word 12 sistematies gekose steekproefnemingseenhede vir die lusernerdvlooi en plantluise aanbeveel. Daar hoef nie onderskeid tussen die plantluisspesies en tussen gevleuelde en ongevleuelde plantluise getref te word nie. Daar moet gebruik gemaak word van werklike insektellings en nie van aanwesigheid-afwesigheid data nie. In plaas van om al die insekte in 'n monster te tel, kan submonsters geneem word. Operasionele karakteristieke kurwes kan gebruik word om die risiko verbonde aan die besluit om nie op te tree, deur byvoorbeeld te spuit of bewei nie, te bepaal. Vir die swartsandmyt, Sitona-snuitkewer en lusernskoenlapper moet drempelwaardes eers vasgestel word voordat aanbevelings vir monitering en die akkuraatheid van besluite rakende beheer, gegee kan word. Vir die ertjieluis, blougroenluis en lusernerdvlooi het die bevolkingsvlakke 'n piek in die lente bereik. Die gevlekte lusernluis se piekgetalle was hoofsaaklik in die laat somer en herfs. Die Sitona-snuitkewer en lusernskoenlapper het piekgetalle gehad in die laat lente en vroeë somer. Vir al die plae het bevolkingspieke drasties afgeneem nadat die aanplantings bewei of gesny is. Hierdie verbouingspraktyke blyk dus goeie beheer te verskaf. Die herbivore op lusern het meer as 85% van die insekgemeenskap beslaan. Die grootste herbivoorfamilies, in terme van aantal individue per familie, was die Aphididae en Sminthuridae. Hierdie twee families bevat die vernaamste lusernplae, naamlik die ertjieluis, blougroenluis, gevlekte lusernluis en lusernerdvlooi. Die grootste predatoriese familie was die Anystidae, wat verteenwoordig is deur die Anystis-roofmyt. 'n belangrike predator van die swartsandmyt en lusernerdvlooi. Nog 'n predatoriese familie wat goed verteenwoordig was, was die Coccinellidae, natuurlike vyande van plantluise. Die droëland aanplantings het 'n hoër persentasie predatore gehad as die besproeide lusern. Die belangrikste parasitoïede aanwesig was dié in die superfamilie Chalcidoidea en familie Braconidae. Die vernaamste detritivore was erdvlooie in die suborde Arthropleona, insekte in die families Mycetophilidae by besproeide lusern, en Mycetophagidae by droëland lusern. Die volopste besoekers was lede van die families Chironomidae, Drosophilidae en Tephritidae. Die droëland aanplantings het 'n laer persentasie besoekers gehad as die besproeide lusern. Die aantal insekfamilies, asook die aantal individue per familie, was laer by die droëland aanplantings as by die besproeide aanplantings. Die oorgrote meerderheid insekfamilies wat op lusern voorkom, is gedurende die een jaar opnameperiode waargeneem. 'n Laer insekdiversiteit is gevind waar beweiding strawwer was, en daar was 'n negatiewe verband tussen diversiteit en gelykmatigheid.

False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. Major concerns such as progressive insecticidal resistance, the negative impact of insecticides on the environment, as well as the influence of consumers opposed to chemical residues on fruit, created opportunities for biological control methods such as Sterile Insect Technology (SIT). This technology is now established in the Western and Eastern Cape provinces of South Africa as an effective, sustainable alternative to conventional FCM control methods. Due to the prevalence of the pest in all citrus producing areas of South Africa, potential for SIT to expand is enormous. Success of an SIT programme is highly dependent on efficient application of the technology to achieve its objectives in a timeous manner. The aim of this study was to advance the application of SIT for control of FCM on citrus in South Africa, by investigating the effect of certain critical stages in the process. The effect of long-distance transportation on fitness of irradiated FCM was determined, showing reduced performance with cold-immobilized transport. A significant decrease in flight ability and longevity of irradiated FCM was found, although critically, realized fecundity was not affected. The effect of two different insecticides in the pyrethroid and organophosphate chemical groups were investigated for their residual effect on mortality of released irradiated FCM, to determine if these pest control programmes could be integrated. Both chlorpyrifos and tau-fluvalinate were effective in killing irradiated FCM for a number of days after application, after which degradation of the active ingredient rendered it harmless. This effect was found to be similar for irradiated and non-irradiated males, consequently ratios of sterile : wild male FCM should be retained regardless of whether sprays are applied or not. The modes for release of sterile FCM in an SIT programme were investigated. Efficacy of ground and aerial release platforms were tested by evaluating the recovery of released irradiated male FCM in these orchards. More irradiated FCM were recovered in orchards released from the ground compared to air. However, an economic analysis of both methods shows application of irradiated insects over a large geographical area is more cost-effective by air. Depending on the terrain and size of the target area, a combination of both methods is ideal for application of SIT for control of FCM in citrus. Development of application technology for advance of the programme is discussed and recommendations for future research and development are offered.

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of citrus in South Africa and challenging to manage due to its inconspicuous nature. An effective method currently being employed for the area-wide suppression of the FCM is the Sterile Insect Technique (SIT) and the effective dispersal of sterile moths is very important for success with SIT. This study was conducted in the Addo area of the Sundays River Valley (Eastern Cape) where the programme is commercially used. In this study, sterile male moths were released in different orchards on a citrus farm, and in nearby veld at different times of the year, and their dispersal was monitored through the use of pheromone traps. Various climatic factors were monitored. This provided insight into the local dispersal of sterile male FCM adults in response to abiotic cues (particularly climatic factors). The movement of the FCM in four citrus cultivars, namely lemons, navel and Valencia oranges and mandarins and in the nearby veld (open field), was determined at six different stages of the year. Results clearly indicated that sterile FCM movement is concentrated within citrus orchards, as very few moths were trapped beyond 30 m from the release point, particularly in navel and Valencia orchards. Of the climatic factors measured, minimum and maximum temperatures had the most significant influence on FCM dispersal, and based on the results, various recommendations are made for the releases of sterile FCM in an area-wide SIT management programmes on citrus. A better understanding of the dispersal capabilities of the FCM in an agricultural system, under different conditions and at different times of the year, is invaluable not only in improving release strategies in an SIT programme but in planning future control strategies against the FCM.

Two ant species, Pheidole megacephala (Fabricius) and Anoplolepis custodiens (Smith) reach pest status in citrus orchards through precipitating outbreaks of homopterous pests. However, predacious ants, including these two ant species, play an important role in pest suppression in agroecosystems and are therefore beneficial to these systems. If A. custodiens and P. megacephala are important natural control agents in citrus, using ant bands to break the mutualism between the ants and the Homoptera as a method of ant control is preferable to poisoning. Ant communities were sampled by pitfall trapping in three experimental subunits of 2-, 4-, 15- and 30-year-old citrus orchards, in the Grahamstown area of the Eastern Cape. In one subunit in each orchard, populations of P. megacephala and A. custodiens were suppressed by poison applications. In a second subunit, trees were banded with trunk barriers so that ants were prevented from foraging in the trees and a third subunit served as the untreated control. Bait pupae of bollworm, false codling moth and fruit fly were planted in bait trays in all of the subunits to investigate predation on these citrus pests in the relative absence of predacious ants and where they were excluded from the trees. Pheidole megacephala dominated exclusively in all of the plots. Community composition did not change dramatically with increasing age of the trees, but species diversity and species abundance did. Rank-abundance curves showed that community diversity was highest in the 2-year-old plots and lowest in the 30-year-old plots. The Simpson and Shannon-Wiener diversity indices and their evenness measures indicated that diversity and equitability were highest in the poisoned subunits and lowest in the banded subunits. Principle component analysis revealed that the poisoned subunits were similar and distinct in species composition, that there was significant monthly variation in species composition and that community stability increases with an increase in orchard age. The presence of P. megacephala was significantly negatively correlated (rs = -0.293; p < 0.001) with pest pupal survival. Pupal survival was significantly higher for bollworm (p < 0.001), FCM (p < 0.001) and fruit fly (p < 0.001) in the poisoned subunits, than in the banded and control subunits. There was a general trend for survivorship to increase with an increase in the age of the trees. A significant difference (p < 0.001) was found between the months in which the trials were carried out. Pupal survival was significantly lower (p < 0.001) for FCM than for bollworm and fruit fly. In citrus orchards, ant communities are organised by ecological processes and interactions and are influenced by methods of ant control. Ant bands are preferable to poisoning as a method of ant control, so that beneficial species are left on the ground to prey on pests that pupate in the soil. Maintaining high ant species diversity in citrus orchards would be beneficial as predation on the pupae was more effective where ant species diversity was higher.

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.

Thesis (MScAgric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Soilborne diseases of grapevines represent a complex problem with limited information available, both locally and internationally. Previous research in South Africa indicated that Phytophthora and Pythium spp. were the most widespread and devastating pathogens in grapevine nurseries and vineyards in the Western Cape province. The local grapevine industry is currently expanding; new cultivars, methods and agricultural chemicals are being used which can affect soilborne pathogens. It has therefore become necessary to reassess the status of soilborne pathogens in nurseries, since information in this regard is crucial for the development of disease management practices for the expanding local grapevine industry. Soilborne fungal genera associated with roots and crowns of declining nursery grapevines were assessed in surveys conducted at three different grapevine nurseries in the Western Cape province. Cylindrocarpon, Fusarium, Pythium, and Rhizoctonia spp. were consistently isolated from roots and crowns of declining nursery grapevines. Cylindrocladiella spp. and Phytophthora cinnamomi were infrequently isolated from diseased roots, crowns and soil whereas Pythium spp. were abundant in most of the soils. Results suggest that the status of soilborne fungal pathogens in grapevine nurseries in the Western Cape province has changed over the last 30 years. The DNA phylogeny and pathogenicity of the isolates of Cylindrocladiella were determined. Four species of Cylindrocladiella occur on grapevines in South Africa, namely C. lageniformis, C. parva, C. peruviana, as well as a new species, described in this study as C. viticola, which forms part of the C. infestans species complex. Pathogenicity trials were inconclusive. Ten Fusarium spp. were isolated from roots and crowns of declining nursery grapevines, namely F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum and F. solani. The dominant species was F. oxysporum, followed by F. proliferatum and F. solani. In pathogenicity trials F. oxysporum and F. solani significantly reduced root volume, root dry mass, length of new shoots, stem diameter and number of leaves, but increased the percentage of chlorotic leaves and root rot severity. Fusarium proliferatum also caused a significant reduction in new shoot growth, number of leaves and increased root rot severity compared to the controls. Fusarium so/ani seems to be more virulent than F. oxysporum, followed by F. pro/iferatum. This is the first report of F. oxysporum, F. pro/iferatum and F. so/ani as pathogens of grapevines in South Africa, and the first report of F. proliferatum as a pathogen of grapevines in the world. Phytophthora cinnamomi was isolated at low frequencies from declined grapevines, although present in the rhizosphere soil. It is possible that the extensive use of downy mildew chemicals in grapevine nurseries may protect grapevines from infection by P. cinnamomi. The effect of chemicals used to combat downy mildew on Phytophthora root rot of nursery grapevines was evaluated in a glasshouse. There was very little discernable effect of the chemicals tested relative to the control plants for the parameters measured and it was concluded that the inoculation technique needed refinement. However, plants treated with phosphorous acid tended to be taller and have more leaves, greater stem diameter and root volume than controls or plants treated with the other chemicals. The data obtained in this study are not conclusive, but indicated certain trends that more glasshouse trials and field trials would resolve. Results presented in this thesis indicate that a major shift has occurred in the status of soilborne fungi associated with roots and crowns of grapevines in nurseries in the Western Cape since the 1970s when Phytophthora and Pythium were predominant. The prevalence and role of soilborne fungi need to be determined so that new appropriate disease management strategies can be developed to limit losses in grapevine nurseries and ensure the sustainable production of healthy plants for the grapevine industry.
AFRIKAANSE OPSOMMING: 'N ONDERSOEK NA GRONDGEDRAAGDE SWAMME GEASSOSIEER MET WORTELS EN KRONE VAN WINGERD IN KWEKERYE Grondgedraagde siektes van wingerd is 'n komplekse probleem waaroor min inligting, beide plaaslik en internasionaal, beskikbaar is. Vorige navorsing in Suid-Afrika het aangedui dat swamme van die genera Phytophthora en Pythium die mees algemene en vernietigende grondgedraagde patogene in kwekerye en wingerde in die Wes-Kaap provinsie is. Die plaaslike wingerdbedryf brei huidiglik uit; nuwe kultivars, metodes en landbouchemikalieë word gebruik wat 'n invloed kan hê op grondgedraagde patogene. Gevolglik het dit noodsaaklik geword om die status van grondgedraagde patogene in wingerdkwekerye weer te bepaal, aangesien inligting in hierdie verband noodsaaklik is vir die ontwikkeling van siekte bestuurspraktyke vir die ontwikkelende plaaslike wingerdbedryf. Grondgedraagde swamgenera geassosieer met wortels en krone van terugsterwende wingerd in kwekerye is bepaal in opnames wat by drie verskillende wingerdkwekerye in die Wes-Kaap provinsie uitgevoer is. Cylindrocarpon, Fusarium, Pythium, en Rhizoctonia spp. is konstant vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, Cylindrocladiella spp. en Phytophthora cinnamomi is ongereeld vanuit siek wortels, krone en grond geïsoleer, terwyl Pythium spp. algemeen in meeste gronde voorgekom het. Resultate dui daarop dat die status van grondgedraagde swampatogene in wingerdkwekerye in die Wes- Kaap provinsie oor die laaste 30 jaar verander het. Die DNA filogenie en patogenisiteit van die isolate van Cylindrocladiella is bepaal. Vier spesies van Cylindrocladiella kom voor op wingerd in Suid-Afrika, naamlik C. lageniformis, C. parva, C. peruviana, sowel as 'n nuwe spesie, wat in hierdie studie as C. viticola aangedui is en wat deel is van die C. infestans spesie kompleks. Patogenisiteits proewe was onvoldoende om die patogeniese status van die swam me te bepaal. Tien Fusarium spp. is vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, naamlik F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum en F. solani. Die dominante spesies was F. oxysporum, gevolg deur F. proliferatum en F. solani. In pathogenisteitsproewe het F. oxysporum en F. solani gelei tot 'n betekenisvolle laer wortelvolume, droë massa van wortels, lengte en droë massa van nuwe groei en aantal blare, maar het die persentasie chlorotiese blare en graad van wortelvrot verhoog. Fusarium proliferatum het ook gelei tot 'n betekenisvolle afname in lengte en massa van nuwe groei, aantal blare en 'n verhoogde graad van wortelvrot in vergelyking met die kontrole behandelings. Dit wil voorkom asof Fusarium solani meer virulent is as F. oxysporum, gevolg deur F. proliferatum. Hierdie is die eerste aanmelding van F. oxysporum, F. proliferatum en F. solani as patogene van wingerd in Suid-Afrika, en die eerste aanmelding van F. proliferatum as 'n patogeen van wingerd in die wêreld. Phytophthora cinnamomi is konstant teen lae frekwensies vanuit terugsterwende wingerd in kwekerye geïsoleer, alhoewel dit in risosfeer gronde teenwoordig was. Dit is moontlik dat die ekstensiewe gebruik van chemikalieë teen donsskimmel in wingerdkwekerye die wingerdplante kan beskerm teen infeksie deur P. cinnamomi. Die effek van chemikalieë wat gebruik word teen donsskimmel op Phytophthora wortelverrotting van wingerd in kwekerye, is 'n glashuis geëvalueer. Die chemikalieë wat gestoets is, het vir die gemete parameters, tot baie min onderskeibare effek gelei relatief tot die kontrole plante, en daar is afgelei dat die inokulasie tegniek verbetering benodig. Plante wat met fosforiensuur behandel is, het egter geneig om langer te wees met meer blare, 'n groter stamdeursnee en wortelvolume as kontrole plante of plante behandel met ander chemikalieë. Data verkry vanuit die hierdie studie was onvoldoende, maar sekere neigings is aangedui wat deur verdere glashuis- en veldproewe verklaar sal word. Resultate wat in hierdie tesis weergegee is, het aangedui dat 'n algehele verskuiwing in die status van grondgedraagde swamme geassosieer met wortels en krone van wingerd in kwekerye vanaf die 1970s, toe Phytophthora en Pythium die dominante genera was, plaasgevind het. Die voorkoms en rol van grondgedraagde swamme moet bepaal word, sodat nuwe voldoende siektebestuurspraktyke ontwikkel kan word om verliese in wingerdkwekerye te beperk en sodoende die volhoubare produksie van gesonde plante vir die wingerdbedryf te verseker.

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) (African bollworm) is a typical noctuid with a very catholic taste in food plants and whose larvae feed on a wide range of cultivated and wild plants. It has been identified as the most polyphagous and injurious pest in South Africa. Helicoverpa armigera is also a key pest of cotton in many parts of the world. This key pest requires extensive control as it adversely effects yield and has built up resistance to synthetic pyrethroid insecticides. Cotton is an important crop produced by commercial and small-scale farmers in South Africa. The local demand for cotton has not been exceeded yet, but to satisfy a demanding market, pest control costs play an important role in cotton production. The threat of an insect pest that has already shown resistance prompted the present study to investigate the possibility of resistance to Bt-cotton. Genetically engineered or Bt-cotton was introduced commercially in 1996 in South Africa. All Bt-cotton plants contain one or more foreign genes derived from the soil-dwelling bacterium, Bacillus thuringiensis (Berliner), which produces protein crystals. These crystals were isolated and transferred into the genome of a cotton plant resulting in the plant producing it’s own protein insecticide. In 1998, Monsanto (Pty) Ltd requested research into the geographic susceptibility of H. armigera to the insecticidal proteins in Bt-cotton in SA. Laboratory reared and field sampled populations of H. armigera were exposed to a diet mixed with various baseline concentrations of the Bt-gene Cry1Ac freeze dried protein. This study also determined the performance of H. armigera and Spodoptera littoralis (Boisduval) on different Bt-cotton field cultivars containing different Cry-protein genes. Results obtained indicated a significant difference in susceptibility in two field populations of H. armigera to the Bt-protein Cry1Ac, even though the LD50,s in the 2003 season did not indicate resistance. Bt-cotton cultivar 15985 BX controlled H. armigera and S. littoralis larvae, the best followed in descending order by cultivar 15985 X, 15985 B and DP50 B. Results on H. armigera also indicated that the Cry-proteins in the plant parts of the different cultivars did not diminish as the season progressed. The Bt-cotton cultivars induced retarded growth of larvae, due to either a repellent effect or lack of feeding by larvae. Widespread adoption of Bt-cotton by South African farmers led to regional declines in bollworm populations, reduced insecticide use, and increased yields. Genetically modified crops therefore contribute to a cost effective, sustainable, productive and efficient form of agriculture, with a resultant positive impact on the environment. As the market for commercial Bt-cotton in South Africa expands, it is recommended that a monitoring programme for potential resistant genes in H. armigera should be implemented at least every 2 - 3 years. This will ensure that effective resistance management strategies are utilised. Coupled with this are the Biosafety Risks regarding the effect of new proteins expressed in transgenic plants, which require further studies.

Thesis (MSc (Botany and Zoology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT:Chapter 1 - Mealybugs are tiny, soft-bodied insects which constitute the second largest scale insect family Pseudococcidae (Downie & Gullan 2004). The family comprises approximately 2000 species in 300 genera (Ben-Dov 1994), of which 20 species are pests of cultivated plants in South Africa (Annecke & Moran 1982). In South Africa, approximately 109 species of mealybugs have been recorded from 50 genera (Millar 2002). Chapter 2 - The effect of constant temperatures on the development, survival and fecundity of the oleander mealybug, Paracoccus burnerae on citrus was determined. Developmental time, rate of development, fecundity and survival were investigated at five constant temperatures and a 16L: 8D light: darkness regime. The rate of development increased linearly with an increase in temperature for the egg, 1st nymphal and pupal stages as well as the entire biological cycle (egg – adult), but was nonlinear for the 2nd and 3rd nymphal stages. Survival decreased with an increase in temperature. P. burnerae required 666.7 degree-days above a lower threshold of 8.7°C to complete one generation. The highest mean number of 68 eggs per female was reached at 22°C. A sex ratio of 0.52:0.48 (male:female) was obtained from the life table. The net reproductive rate (Ro) was >1 at all five temperatures, an indication that it is capable of increasing its population numbers despite the high mortality experienced in the 1st and 2nd nymphal stages. Chapter 3 - The oleander mealybug, Paracoccus burnerae (Brain) is a pest of citrus in South Africa. This study was carried out to determine the effect of temperature on development rate of P. burnerae and to investigate whether development rate is the reason why P. burnerae is out competing the citrus mealybug, Planococcus citri (Risso), in the Eastern and Western Cape Provinces of South Africa. The influence of temperature on life history traits of P. burnerae was determined at 20, 22, 25 and 27°C and compared with corresponding data for P. citri. The rate of development increased linearly with an increase in rearing temperature in the embryonic, first nymphal and pupal stages but reached a climax at 26.13 and 28.6°C in the second nymphal stage of both species, respectively. P. citri exhibited lower developmental thresholds except in first instar, shorter degree-days and higher developmental rates than P. burnerae. Results of the current study indicated that the dominance of oleander mealybug over the citrus mealybug is neither linked to developmental rates nor sum of effective temperatures. Chapter 4 - The importance of Paracoccus burnerae has risen over the years to an extent where it is now regarded as a quarantine pest for citrus fruit from South Africa. The field biology of P. burnerae on citrus in the Western Cape Province of South Africa was studied through periodic sampling of leaves from twigs enclosed in sleeve cages. The species composition and abundance of natural enemies was investigated. Both adult and immature stages attained maximum population peaks in March and P. burnerae had four generations. The highest level of mortality was experienced in the immature stages. Climate and an unidentified fungus were the key mortality factors. The level of abundance of the two observed predators, the harlequin beetle, Harmonia axyridis and the green lacewing, Chrysoperla sp. was relatively low. Although parasitism occurred in some cages, the level was low ranging between 1.62 to 9.43%. If biocontrol is the preferred method of controlling P. burnerae, suitable candidate parasitoids for inoculative biocontrol are Acerophagus sp., Leptomastix sp. and Microterys nietneri. The oleander mealybug does not share the same parasitoids with Planococcus citri, Pseudococcus calceolariae and Pseudococcus longispinus except the parasitoid Coccophagus sp. The most popular species of parasitoids used in the biolological control of mealybugs, Anagyrus sp. and Coccixenoides sp. were insignificant in the case of P. burnerae. Chapetr 5 - Biological control programs of mealybug species have relied on sprouting potatoes, pumpkins and butternut for rearing of both mealybugs and their natural enemies. In this study, the suitability of sprouting potatoes, butternuts and citrus as mass rearing substrates for the oleander mealybug, Paracoccus burnerae was investigated. Developmental times, rate and fecundity on each substrate were determined and compared at three different temperatures. The developmental time on sprouting potatoes was shorter than on citrus. P. burnerae was unable to complete its life cycle on butternut. The rate of development increased linearly with an increase in temperature on both sprouting potatoes and citrus. P. burnerae required 666.7 degree-days on citrus and 434.8 degree-days on sprouting potatoes above lower developmental thresholds of 7.6°C and 10.4°C respectively to complete one generation. The mean number of eggs per female was higher on sprouting potatoes (121.3) than on citrus (68), but declined with an increase in temperature from 22 to 27°C. Despite the shorter shelf life, sprouting potatoes are the preferred host for mass rearing of the oleander mealybug. Chapter 6 - general conclusions Chapter 7 - Researchers often present impressive results of their studies on the biology of the Coccoidea without mentioning the problems they came across and had to solve. In this paper the practical problems encountered during a study of the biology of the oleander mealybug, Paracoccus burnerae (Brain), an endemic pest of citrus in South Africa, are discussed.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.

The study examined the larval parasitoids of Cryptophlebia leucotreta (Meyrick) on citrus in South Africa and aimed to improve the existing rearing techniques of C. leucotreta with a view to mass rearing of biological control agents. The biological characteristics of Agathis bishopi Nixon (Hymenoptera: Braconidae) were studied, with an emphasis on parasitism rates in the field, host stage preference, developmental rate, life span and offspring sex ratios. Two larval parasitoids, A. bishopi and Apophua leucotretae (Wilkinson) (Hymenoptera: Ichneumonidae), and an egg parasitoid, Trichogrammatoidea cryptophlebiae Nagaraja (Hymenoptera: Trichogrammatidae), were recorded on C. leucotreta on citrus. A. bishopi was the more abundant of the larval parasitoids and exhibited density dependent parasitism. The highest parasitism rates were observed in December with up to 38% in Sundays River Valley and 34% in Gamtoos River Valley, at a time when the highest false codling moth infestations were observed. Agathis bishopi was recorded only in the Eastern Cape Province. The sex ratio of A. bishopi was biased towards females throughout the study (77% in Gamtoos River Valley and 72% in Sundays River Valley). Agathis bishopi is a solitary, koinobiont, larval-pupal endoparasitoid. The species showed a preference for 1st and 2"d instar hosts. Females regulate the sex of their progeny according to the size and larval stage of the host, ovipositing unfertilised eggs in younger, smaller larvae (1st instars) and fertilised eggs in older, larger larvae (2nd instars). The developmental rate of A. bishopi is in synchrony with that of the moth and adults emerge when adult moths that have escaped parasitism emerge. Agathis bishopi and T. cryptophlebiae compliment each other because they have different niches and strategies of attack. Integrating A. bishopi and T. cryptophlebiae into the management of citrus orchards has potential to suppress false codling moth. Larger rearing containers seemed ideal for large-scale rearing of false codling moth. A higher percentage of adults was obtained from larvae reared in larger containers than in smaller ones. The width of the sponges used as stoppers prevented escape of the larvae. Media prepared in larger containers are easier and simpler to prepare than in smaller ones, thus eliminating many precautions otherwise necessary to prevent contamination. Moth production was greatly reduced by the high concentration of Sporekill used for egg decontamination.

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.

Thesis (MScAgric)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Various aspects of the biology and ecology of woolly apple aphid, Eriosoma lanigerum, were investigated, including initial galling damage caused by E. lanigerum to the roots of apple trees, the possible relationship between E. lanigerum and Xiphinema and Pratylenchus nematodes and the effectiveness of Biostart 2000® and Furfural® as possible control agents of E. lanigerum in the orchard. Preliminary root damage by first instar E. lanigerum feeding was characterized by the mechanical injury of endodermis and parenchyma tissues. Damage by second, third and fourth instar E. lanigerum was similar, but the symptoms were more pronounced. Damage caused by adults included a pronounced swelling at infected areas of the root. Cell walls hardened until the root was radially strengthened with sclerenchyma tissue and nonconducting xylem vessels while the cuticle expanded greatly through the growth of corklike cambium tissue. There was no direct relationship between the population dynamics of E. lanigerum and those of Xiphinema and Pratylenchus nematodes. The occurrence of E. lanigerum appeared to be seasonal while P. penetrans and Xiphinema numbers fluctuated erratically. Undamaged root nitrogen levels seemed to correspond with the normal root growth cycle. Nitrogen levels from galled roots were significantly lower than those of undamaged roots, probably due to E. lanigerum feeding. Soils rich in fine sand and clay sustained higher populations of E. lanigerum and Xiphinema than sandy soils. The number of E. lanigerum found in soil samples correlated well with the damage index allocated to the samples. The numbers of Xiphinema found in soil samples also correlated well with the damage index allocated to the samples according to suspected Xiphinema damage symptoms. Both Biostart 2000® and Furfural® were effective as control agents of woolly apple aphid. Furfural'Ï, a chemical waste product of the sugarcane industry, was however not as effective as Biostart 2000®, a product that includes an activator and three bacterial species, Bacillus laterosporus, B. chitinosporus and B. licheniformis. The bacteria in the Biostart 2000® treated pots could replicate themselves under suitable conditions while Furfural® dilutes with each watering. Biostart 2000® is also easier to prepare than Furfural® since the components of Biostart 2000® readily mix to form a paste easily thinned by water, whereas Furfural® is an oily substance that does not easily disperse in water. Root damage was initiated soon after E. lanigerum started feeding, however there was no apparent relationship between E. lanigerum and the nematode species. The most promising, environmentally friendly control measure was Biostart 2000®.
AFRIKAANSE OPSOMMING: Verskeie aspekte van biologie en die ekologie van die appel bloedluis, Eriosoma lanigerum, was ondersoek insluitende aanvanklike galvorming veroorsaak deur E. lanigerum op wortels van appelbome, die moontlike verwantskap tussen E. lanigerum en Xiphinema en Pratylenchus nematodes en die effektiwiteit van Biostart 2000® en Furfural® as moontlike beheeragente van E. lanigerum in die boord. Aanvanklike wortelskade deur eerste ins tar E. lanigerum voeding was gekenmerk deur die meganiese beskadiging van endodermale en parenchiem weefsel. Skade veroorsaak deur tweede, derde en vierde instar E. lanigerum was soortgelyk alhoewel die simptome meer beklemtoond was. Skade deur volwassenes het 'n meer duidelike swelsel by geïnfekteerde wortelareas ingesluit. Selwande het verhard totdat die wortel radiaalsgewys versterk was met skierenchiem weefsel en nie-geleidende xileemvate terwyl die kutikula grootliks toegeneem het deur die groei van kurkagtige kambiumweefsel. Daar was geen direkte verwantskap tussen die bevolkingsdinamika van E. lanigerum en dié van Xiphinema en Pratylenchus nematodes nie. Die voorkoms van E. lanigerum was seisoenaal terwyl P. penetrans en Xiphinema se getalle onvoorspelbaar gefluktueer het. Onbeskadigde wortel stikstofvlakke het ooreengestem met die normale wortel groeisiklus. Stikstof vlakke van galwortels was noemenswaardig laer as dié van onbeskadigde wortels, heel waarskynlik as gevolg van voeding deur E. lanigerum. Grond ryk aan fyn sand en klei het groter bevolkings van E. lanigerum en Xiphinema onderhou as sanderige gronde. Die aantal E. lanigerum in grondmonsters het goed ooreengestem met die skade indeks wat aan die monsters toegeken was. Die aantal Xiphinema in grondmonsters het ook goed ooreengestem met die beskadigingsindeks wat aan die monsters toegeken is weens vermoedelike Xiphinema skade simptome. Beide Biostart 2000® en Furfural® was effektief as beheeragente van die appelbloedluis. Furfural'", 'n afvalproduk van die suikerriet industrie, was egter minder effektief as Biostart 2000®, 'n produk bestaande uit 'n aktiveerder en drie bakterie spesies, Bacillus laterosporus, B. chitinosporus en B. licheniformis. Die bakterië in die Biostart 2000® behandelde potte kon vermeerder onder gunstige toestande terwyl Furfural® na elke besproeiing verdun het. Biostart 2000® is ook makliker om aan te maak as Furfural® aangesien die bestanddele van Biostart 2000® geredelik meng tot 'n wateroplosbare pasta, terwyl Furfural® 'n olierige vloeistofis wat moeilik 'n waterige suspensie vorm. Wortelskade het plaasgevind kort nadat E. lanigerum begin voed het, alhoewel daar geen duidelike verwantskap tussen E. lanigerum en nematode spesies voorgekom het nie. Die mees belowende omgewingsvriendelike beheermaatreël was Biostart 2000®.

Cereal crop productivity is hampered when these plants are infested by phloem feeding aphids. A great deal of research has been carried out with the direct aim of a clearer understanding of the mechanism involved in the interaction between aphids and their host plants. Research has directly or indirectly been geared towards enhanced plant productivity and achieving sustainable agriculture. Barley (Hordeum vulgare L.) is an important small grain crop in South Africa, whose crop performance is negatively affected by fluctuations in weather patterns as well as by agricultural pests. One of the insect pests infesting barley is the Russian wheat aphid, Diuraphis noxia Kurdjumov (RWA), of which the two South African biotypes, codenamed RWASA1 and RWASA2, were studied in this thesis. During dry spells, RWA infestation becomes a more serious threat to barley productivity. Resistant plants have been used to combat RWA infestation of small grains. In South Africa, 27 RWA-resistant wheat cultivars are currently used in commercial cultivation, but no resistant barley lines have, unfortunately, been developed, in spite of this grain’s significant economic importance. This informed the study in this thesis, and this interest particularly focussed on three RWA-resistant lines developed by the USDA, testing their performance against South African RWA biotypes, for possible adaptation to South Africa. The aim was thus to examine the plant-aphid interactions, aphid breeding rates, plant damage and sustainability, evidence of resistance or tolerance and finally potential performance under elevated CO2 (a very real climate change threat). Two major avenues of research were undertaken. The first aspect involved examination of structural and functional damage caused by RWASA1 and RWASA2 on the three resistant and a non-resistant line. Aphid population growth and damage symptoms (chlorosis and leaf roll) of infestation by these aphid biotypes were evaluated. This was followed by a structural and functional approach in which the effects of feeding on the transport systems (phloem and xylem) of barley were investigated. Fluorescence microscopy techniques (using aniline blue fluorochrome, a specific stain for callose and 5,6-CFDA, a phloem-mobile probe) were applied to investigate the feeding-related damage caused by the aphids, through an examination of wound callose formation and related to this, the resultant reduction in phloem transport capacity. Transmission electron microscopy (TEM) techniques provided evidence of the extent of the feeding-related cell damage. The second aspect involved a study of the effect of changing CO2 concentrations ([CO2]) on the resistant and susceptible barley cultivars to feeding by the two RWA biotypes. Leaves of plants grown at ambient and two elevated levels of [CO2] were analysed to investigate the effect of changing [CO2] on biomass, leaf nitrogen content and C:N ratio of control (uninfested) and infested plants. The population growth studies showed that the populations of the two RWA biotypes as well as bird cherry-oat aphid (BCA, Rhopalosiphum padi L.) increased substantially on the four barley lines. BCA was included here, as it had been the subject of several previous studies. RWASA2 bred faster than RWASA1 on all lines. The breeding rates of the two RWA biotypes were both suppressed and at near-equivalent levels on the three resistant lines, compared to the non-resistant PUMA. This suggests that the resistant lines possessed an antibiosis resistance mechanism against the feeding aphids. Feeding by the aphids manifested in morphological damage symptoms of chlorosis and leaf roll. The two biotypes inflicted severe chlorosis and leaf roll on the non-resistant PUMA. In the resistant plants, leaf rolling was more severe because of RWASA2 feeding compared to RWASA1 feeding. In contrast, chlorosis symptoms were more severe during RWASA1 feeding than was the case with RWASA2 feeding. Investigation of the effect of aphid feeding on the plants showed that callose was deposited within 24h and that this increased with longer feeding exposure. Wound callose distribution is more extensive in the non-resistant PUMA than in the resistant plants. RWASA2 feeding on the resistant lines caused deposition of more callose than was evident with RWASA1 feeding. During long-term feeding, it was evident that variation in the intensity and amount of wound callose was visible in the longitudinal and transverse veins of the resistant plants. Of the three STARS plants, STARS-9301B had the least callose. Interestingly, wound callose occurred in both resistant and non-resistant plants, in sharp contrast to what has been reported on resistant wheat cultivars that were developed in South Africa. The relative reduction in the wound callose deposited in the resistant line, when compared to the non-resistant lines, suggests the presence of a mechanism in the resistant lines, which may prevent excessive callose formation. Alternatively, the mechanism may stimulate callose breakdown. RWASA2 feeding on the resistant lines deposited more wound callose than RWASA1 feeding. This evidence supports the hypothesis that RWASA2 is a resistance breaking and more aggressive feeder than RWASA1 is; and further underscores the urgent need for development of RWA-resistant barley cultivars. The ultrastructural investigation of the feeding damage showed that the two biotypes caused extensive vascular damage in non-resistant plants. There was extensive and severe cell disruption and often obliteration of cell structure of the vascular parenchyma, xylem and phloem elements. In sharp contrast, among the resistant plants, feeding-related cell damage appeared to be substantially reduced compared to the non-resistant PUMA. Low frequency of damaged cells indicated that majority of the cell components of the vascular tissues were intact and presumed functional. There was evidence of salivary material lining the secondary walls of the vascular tissue, which resulted in severe damage. Within xylem vessels, saliva material impregnated half-bordered pit pairs between the vessels and adjacent xylem parenchyma. This is believed to prevent solute exchange through this interface, thereby inducing leaf stress and vi leaf roll. A notable finding is that RWASA2 effectively induced more cell damage to vascular tissues in the resistant lines than did RWASA1. In general the experimental evidence (see Chapter 5) suggests that the resistant lines are possibly more tolerant (or able to cope with) to RWA feeding. Evidence for this is the reduction of wound callose and at the TEM level, a comparatively less obvious cell damage in the resistant lines, which suggests that they possess antibiosis and tolerance capacity. The apparent reduction of feeding-related cell damage from the TEM study confirmed the disruptive action of the feeding aphids in experiments using the phloem-mobile probe, 5,6-CF. Results showed that feeding by RWASA1 and RWASA2 reducedthe transport functionality of the phloem in all cases, but that RWASA2 feeding caused a more obvious reduction in the rate and distance that 5,6-carboxyfluorescein was transported, than did RWASA1. Investigation of the effect of changing [CO2] on the barley cultivars showed that in the absence of aphids and under elevated CO2 conditions, the plants grew more vigorously. In this series of experiments, the infested plants suffered significant reduction in biomass under ambient (as was expected) and under the two elevated CO2 regimes. Biomass loss was greater at elevated CO2 than under ambient [CO2]. The infested nonresistant PUMA plants showed a more significant biomass loss than did the resistant cultivars. Clearly, the benefits derived from elevated CO2 enrichment was thus redirected to the now-advantaged aphids. Uninfested vii plants showed an increase in leaf nitrogen under the experimental conditions. However, feeding aphids depleted leaf nitrogen content and this was more apparent on plants exposed to RWASA2 than was the case with RWASA1. The end result of this was that C:N ratio of infested plants were higher than uninfested plants. Clearly, the faster breeding rates of the aphids at elevated CO2 caused depletion of N and a resultant deficiency exacerbated chlorosis as well as leaf rolling due to the higher aphid population density under elevated CO2 than at ambient. By 28 days after infestation (DAI), majority of the plants exposed to enriched CO2 treatments had died. A major finding here was thus that although this study demonstrated that elevated CO2 resulted in an increase in biomass, this was detrimentally offset in plants infested by the aphids, with a decline in biomass and loss of functionality leading to plant death at 28DAI. The overriding conclusion from this study is a clear signal that the twin effects of CO2 enrichment (a feature of current climate change) and aphid infestations may precipitate potential grain shortages. A disastrous food security threat looms.

Thesis (PhD(Agric))--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The apple industry in South Africa currently relies entirely on chemical fungicides to control apple scab, caused by Venturia inaequalis. In this dissertation, alterative management strategies against V. inaequalis were tested for the first time in South Africa. New information on the behaviour of the sexual winter phase of V. inaequalis in different climatic conditions was found and sources of asexual inoculum overwintering in apple orchards were identified. The effect of leaf shredding on fruit and leaf scab incidence and severity was tested against a non-shredded, non-sprayed negative control, a positive control that followed a commercial fungicide programme and a combined treatment of a commercial fungicide programme with leaf shredding, from 2010 to 2013. Reductions in fruit and leaf scab incidence and severity in the leaf shredding treatment were significantly lower compared to the negative control. Quantitative real-time polymerase chain reaction (qPCR) of airborne ascospores trapped using volumetric spore traps was used to measure the reduction in airborne ascospores in the shredded plots, and confirmed the efficacy of shredding found by comparing scab incidence and severity on fruit and leaves. Shredding twice during leaf-drop increased the efficacy of the treatment. Results indicate that leaf shredding should be integrated into scab management strategies in future. However, practical considerations unique to South African orchards, e.g. timing of leaf shredding relative to leaf-drop and orchard layouts, need to be addressed. Pseudothecial densities (PD, number of pseudothecia per fertile lesion) and ascal densities (AD, number of asci per pseudothecium) were compared between in Koue Bokkeveld (KB), a cold winter region, and Elgin (EL), a warm winter region experiencing climate warming, in 2012 and 2013. Scabbed leaves were detached during leaf-drop and overwintered in their region of origin and in the other region. The PD in leaves collected in KB and overwintered in KB was significantly higher than for leaves collected in EL and overwintered in EL, and leaves collected in KB and overwintered in EL. These results agreed with what was expected, as temperature during pseudothecial formation (i.e. the first four weeks after leaf-drop) was significantly lower in KB than in EL. However, the PD for leaves collected in EL and overwintered in EL did not differ significantly from EL leaves overwintered in KB. AD values in all treatments did not differ significantly from one another. Results suggest that factors other than temperature may be involved in controlling PD, e.g. the EL population may include strains not present in the KB population, with higher optimal temperatures for pseudothecial formation. Apple buds and pygmy apples were collected and tested for presence, number and viability of conidia in 2010, 2011 and 2012. Pygmy apples are small, late season fruit that remain attached to the tree throughout winter, especially in regions with warmer winters where trees do not experience sufficient chilling to complete dormancy. High conidial numbers were found on outer bud tissue and low numbers on inner bud tissue, but viable conidia were only found on inner bud tissue, using microscopy, and generally in orchards with high scab levels in the previous season. Molecular methods using PCR-RFLP and qPCR confirmed the presence of high amounts of V. inaequalis DNA in outer bud tissues, although calculated conidial amounts were higher than data obtained when using microscopy, which could indicate presence of mycelia not detected during microscopic examination. Higher numbers of conidia with higher percentage viability were found on pygmy apples, which are a more likely source of asexual inoculum in South African apple orchards than the low number of viable conidia on inner bud tissue.
AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse appelbedryf is tans afhanklik van chemiese swamdoders vir die beheer van die appelskurf patogeen, Venturia inaequalis. In hierdie proefskrif is alternatiewe bestuurstrategiëe vir die eerste keer in Suid-Afrika ondersoek. Nuwe inligting te opsigte van die gedrag van die geslagtelike winterfase van V. inaequalis, is onder verskillende klimaatstoestande ingewin en bronne van die oorwinterende ongeslagtelike inokulum in appelboorde, is identifiseer. Die invloed van blaarversnippering op die voorkoms en erns van appelskurf op vrugte en blare, is vanaf 2010 tot 2013 ondersoek en met ʼn negatiewe kontrole (onversnipperde blare sonder spuitprogram), ʼn positiewe kontrole (ʼn kommersiële swamdoderspuitprogram is gevolg) en gekombineerde behandelings (kommersiële swamdoderspuitprogram en blaarversnippering) vergelyk. Daar was ʼn betekenisvolle verskil in die voorkoms en erns van skurf op vrugte en blare met blaarversnippering teenoor die negatiewe kontrole. Kwantitatiewe intydse polimerase kettingvermeerderingsreaksie (kPKR) van luggedraagde askospore, vasgevang in volumetriese lokvalle, is gebruik om die afname van luggedraagde askospore in versnipperde behandelings te meet. Die doeltreffendheid van versnippering as behandeling, is bevestig deur die voorkoms van appelskurf te vergelyk met die ernstigheidsgraad daarvan op vrugte en blare. Die uitvoer van blaarversnippering twee keer gedurende die blaarvalperiode het die effektiwiteit van hierdie behandeling verhoog. Hiervan kan dus afgelei word dat blaarversnippering voordelig sal wees vir die bestuur van appelskurf en in toekomstige bestuurspraktyke ingesluit moet word. Praktiese oorwegings, uniek aan Suid-Afrikaanse boorde, soos boorduitleg en die tydsberekening van blaarversnippering teenoor blaarval, moet egter in ag geneem word. Pseudothesiale digtheid (PD; die aantal pseudothesia per vrugbare letsel) en askale digtheid (AD; die aantal aski per pseudothesium) is gedurende 2012 en 2013 vir die Koue Bokkeveld (KB), 'n koue winterstreek, en warm winterstreek Elgin (EL), 'n winterstreek wat klimaatsverwarming ervaar, vergelyk. Blare, met skurf, is gedurende blaarval gepluk en oorwinter in hul gebied van oorsprong, asook in die ander klimaatstreek. Blare wat in KB versamel is en in KB oorwinter het, se PD was aansienlik hoër as dié wat in EL versamel is en in EL oorwinter het, sowel as dié wat in KB versamel is en in EL oorwinter het. Hierdie resultate stem ooreen met wat verwag is, om rede die temperatuur gedurende pseudothesiale vorming, d.w.s. die eerste vier weke na blaarval, aansienlik laer in KB as in EL was. Die PD van blare wat in EL versamel en daar oorwinter het, het egter nie betekenisvol verskil van blare wat in KB oorwinter het nie. Die AD-waardes tussen behandelings verskil nie noemenswaardig nie en word as onbeduidend beskou. Die verkrygde resultate dui aan dat daar ander faktore as temperatuur betrokke is by die beheer van PD, bv. die EL-skurfpopulasie, waar die warmer klimaat meer optimaal is vir pseudothesiale vorming, rasse wat nie in die KB-bevolking teenwoordig is nie, mag insluit. Appelknoppe en dwerg-appels is gedurende 2010, 2011 en 2012 versamel en vir die teenwoordigheid, aantal en lewensvatbaarheid van konidiospore getoets. Dwergappels is klein laatseisoen appeltjies wat reg deur die winter aan die boom bly hang; veral in die streke met warmer winters waar die bome nie die nodige koue ervaar om dormansie te voltooi nie. Met behulp van mikroskopie is ʼn hoë aantal spore op die buitenste knopweefsel en lae getalle in die binneweefsel bespeur; maar lewensvatbare spore is net in die binneweefsel van knoppe waargeneem, wat hoofsaaklik afkomstig is van boorde wat hoë vlakke van appelskurf in die vorige seisoen ervaar het. Molekulêre tegnieke, PKR-RFLP en kPKR, is gebruik vir bepaling van V. inequalis DNA hoeveelhede op die buitenste knopweefsel. Hoër getalle konidiospore is met die molekulêre analise gevind, as dié verkry met mikroskopiese ondersoek en dui op die moontlike teenwoordigheid van miselium wat nie met visuele waarneming sigbaar was nie. Meer konidiospore met 'n hoër vlak van lewensvatbaarheid is op dwerg-apples gevind en dit is moontlik 'n meer waarskynlike bron van ongeslagtelike inokulum in Suid-Afrikaanse appelboorde, as die lae getalle van lewensvatbare konidiospore op die binneweefsel van die appelknoppe.

The interaction between farmers, agricultural scientists and extension workers is sometimes overlooked in agricultural entomology. In an attempt to respond to this reality this study examines some foundation of this interaction in relation to the pest management practices of subsistence and small-scale farmers and also highlights the problems that might arise in the implementation of IPM. Problems involving pests occurrence; language barriers; beliefs, knowledge and perception about insects, and visual literacy are examined. The thesis has a two-fold focus, firstly the study of pests on cabbages of subsistence farmers in Grahamstown and secondly a broader focus on other aspects such as cultural entomology, perception of insects and visual literacy specifically in relation to Xhosa speaking people in the Eastern Cape. The most important crop for emergent farmers in the Eastern Cape are cabbages, which have a variety of pests of which diamondback moths and are the most important. Traditional pest management practices tend to influence the development of IPM programmes adopted by these farmers. Eastern Cape farmers apply periodic cropping systems, which had an effect on the population densities of diamondback moth (DBM), other lepidopteran pests and their parasitoids. Considering the maximum population densities of DBM, which were 0.2 - 2.9 larvae/plant, there were no major pest problems. The availability of parasitoids, even in highly disturbed and patchy environments, showed good potential for biological control. Since some extension officers cannot speak the local farmers’ language, a dictionary of insect names was formulated in their language (isiXhosa) to assist communication. Response-frequency distribution analysis showed that the dictionary is essentially complete. The literal translations of some names show that isiXhosa speakers often relate insects to people, or to their habitat or classify them according to their behaviour. Farmers from eight sites in the Eastern Cape were interviewed regarding their knowledge and perception of insect pests and their control thereof. To some extent, farmers still rely on cultural control and have beliefs about insects that reflected both reality and superstition. There is no difference between the Ciskei and Transkei regions regarding insect-related beliefs. Farmers generally lack an understanding of insect ecology. There is a need for farmers to be taught about insects to assist with the implementation of IPM. Leftover pesticides from commercial farms or detergents are sometimes used to manage the pests. When training illiterate or semi-literate farmers, it is important to understand their media literacy so as to design useful graphic and object training media. Generally farmers showed that they either understand graphic or object media depending on the features of the insects being looked at. These findings are discussed with regard to the potential development of IPM training material for subsistence and small-scale farmers in a community.

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.

Thesis (MScAgric)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en (ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond, onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid- Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora. ‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers (Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter 99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9% van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële DNA ekstraksie pakkette. Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie (ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa. chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die 12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling), toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en wondbeskerming teen grondgedraagde infeksies.

Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.

This thesis has two focuses. The first problem facing the olive industry in the Eastern Cape is the growers’ perceptions of both what the industry will provide them and what a pest management program might entail. The second focus is the biology of olive pests in the Eastern Cape in terms of understanding their populations and their natural enemies on private farms, with future hopes of understanding how Integrated Pest Management strategies can be developed for this crop. Eastern Cape private farmers, small-scale farmers and workers from agricultural training institutions were interviewed regarding the history and cultivation of the local olive crop. Only one commercially viable olive grove was identified; other groves were small, experimental pilot ventures. The introduction of olives to small-scale farmers and agricultural training schools was generally a top-down initiative that led to a lack of sense of ownership and the trees being neglected. Other problems included poor human capital; poor financial capital; lack of adequate support; lack of knowledge transfer and stability; lack of communication and evaluation procedures of the project; miscommunication; and finally, olive pests. Apart from hesitancy to plant at a commercial scale, the main problem facing private farmers (Varnam Farm, Hewlands Farm and Springvale Farm) was pests. Therefore an investigation of pests from private farms was conducted ranging from collection of cultivated and wild olive fruit and flea beetle larvae for parasitism, trapping systems both for fruit flies and olive flea beetle adults. A survey of olive fruits yielded larval fruit flies of the families Tephritidae (Bactrocera oleae (Rossi), B. biguttula (Bezzi) and Ceratitis capitata (Wiedemann)) and Drosophilidae (Drosophila melanogaster (Meigen)) from wild olives (O. europaea cuspidata (Wall. ex G. Don) Cif.) but none from cultivated olives (O. e. europaea L.). Braconid wasps (Opiinae and Braconinae) were reared only from fruits containing B. oleae and B. biguttula. This suggests that B. oleae is not of economic significance in the Eastern Cape, perhaps because it is controlled to a significant level by natural enemies, but B. biguttula may be a potential economic pest. A survey of adult fruit flies using ChamP traps baited with ammonium bicarbonate and spiroketal capsules and Sensus trap baited with methyl eugenol and Questlure confirmed the relative importance of B. biguttula over B. oleae. ChamP traps were over 50 times better than Sensus traps for mass trapping of B. biguttula but both were ineffective for trapping B. oleae and C. capitata. Six indigenous flea beetles of the genus Argopistes Motschulsky (Chrysomelidae: Alticinae) were found, three described by Bryant in 1922 and 1944 and three new species. Their morphology was investigated by scanning electron microscopy and mutivariate morphometric analysis. The leaf-mining larvae are pests of wild and cultivated olives in South Africa and threaten the local olive industry. At Springvale Farm, A. oleae Bryant and A. sexvittatus Bryant preferred the upper parts of trees, near new leaves. Pseudophanomeris inopinatus (Blkb.) (Braconidae) was reared from 23 Argopistes larvae. The beetle larvae might not be controlled to a significant level by natural enemies because the rate of parasitism was low. The olive flea beetles showed no attraction to traps containing various volatile compounds as baits. The lace bug, Plerochila australis Distant (Tingidae), was sometimes a pest. It showed a preference for the underside of leaves on the lower parts of the trees. A moth, Palpita unionalis Hübner (Crambidae), was reared in very low numbers and without parasitoids. A twig-boring beetle larva, chalcidoid parasitoids and seed wasps of the families Eurytomidae, Ormyridae and Eupelmidae were also recorded.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Microsatellites were used to differentiate Leaf (Puccinia triticina Eriks.) and Yellow rust (Puccinia striiformis Westend. f. sp. tritici Eriks.) pathotypes. There was sufficient diversity in the Leaf rust microsatellite markers to differentiate the pathotypes and create a phylogenetic tree of Leaf rust. Three of the microsatellite markers were sufficient to differentiate all the Leaf rust pathotypes. Sufficient diversity in the Yellow rust microsatellite markers was also observed which made it possible to differentiate the pathotypes. Only three pathotypes were used so no phylogenetic inference was made. Two microsatellite markers were sufficient to differentiate all the yellow rust pathotypes. Microsatellite and Amplified Fragment Length Polymorphisms (AFLP) markers were used to differentiate Stem rust (Puccinia graminis f. sp. tritici Eriks. and Henn.) pathotypes, and the data was combined for phylogenetic analysis. AFLP bands unique to each Stem rust pathotype were converted to Sequence Characterised Amplified Region (SCAR) markers. A single specific SCAR marker was created for UVPgt52. A second SCAR marker amplified four of the eight pathotypes. None of the other SCAR markers were specific. A 270 basepair fragment of the ITS1 region of the rDNA gene of all the Puccinia spp. was also sequenced in order to develop pathotype specific primers that could be used in a Real Time-PCR to determine relative levels of pathogen inoculum in a sample. Unfortunately insufficient diversity in the sequences of the ITS1 region of the rDNA gene did not allow unique primers to be designed for each pathotype making it impossible to proceed with the relative quantification using Real Time-PCR. Following marker development ninety one field isolates were collected from eleven sites in the Overberg and Swartland regions during 2008 and 2009. In the field isolates, four different Leaf rust pathotypes were identifiable. UVPgt13 and UVPgt10 were most prevalent. The most prevalent Stem rust pathotypes were UVPgt50, UVPgt52, UVPgt54 and UVPgt57. Only 6E16A- was identifiable in the Yellow rust isolates. There were no apparent patterns in the distribution of Leaf, Stem or Yellow rust. Leaf and Stem rust were widely distributed, while Yellow rust was confined to three sites in the central South Cape, the only sites where climatic conditions were favourable for its development during the sampling period. The low levels of diversity found in the rust population when compared to international populations are probably due to the relatively small population size, the lack of a host for sexual reproduction, the small sample size, the effective monoculture and the strong selective pressure created by artificial control methods.
AFRIKAANSE OPSOMMING: Mikrosatellietmerkers is gebruik om Blaar- (Puccinia triticina Eriks.) en Geelroes-( Puccinia striiformis Westend. f. sp. tritici Eriks.) patotipes te onderskei. Daar was genoeg diversiteit in die Blaarroesmerkers om verskillende patotipes te kon onderskei en om „n filogenetiese-boom te kon saamstel. Met drie van die mikrosatellietmerkers was dit moontlik om al die Blaarroespatotipes te kon onderskei. Daar was genoeg diversiteit in die Geelroesmerkers om al die patotipes te kon skei en met twee van die mikrosatellietmerkers kon al drie Geelroespatotipes van mekaar onderskei word. Mikrosatelliet- en ge-Amplifiseerde-Fragment-Lengte-Polimorfismes (AFLP) is gebruik om die Stamroes- (Puccinia graminis f. sp. tritici Eriks. and Henn.) patotipes te skei. AFLP-fragmente uniek aan „n spesifieke patotipe is omgeskakel na Volgorde-Spesifieke-ge-Amplifiseerde-Streek (SCAR) merkers. „n Spesifieke SCAR-merker is gemaak vir UVPgt52. „n Tweede SCAR-merker het vier van die patotipes geidentifiseer. Nie een van die ander SCAR-merkers was spesifiek t.o.v. „n spesifieke patotipe nie. Die volgorde van „n 270 basispaar fragment van die ITS1-streek van die rDNS-geen van al die Puccinia spp. is bepaal om patotipe spesifieke inleiers te kon ontwerp. Hierdie inleiers kan gebruik word om „n Intydse-Polimerase-Ketting-Reaksie (RT-PCR) te ontwerp om sodoende die relatiewe vlakke van die patogeen besmetting in „n monster te bepaal. Daar was nie genoeg diversiteit in die bepaalde volgordes om die spes1fieke inleiers te kon identifiseer nie en dus is RT-PCR laat vaar. Na die ontwikkeling van die merkers was een-en-negentig veldmonsters ingesamel afkomstig van elf lokaliteite in die Overberg en Swartland gedurende 2008 en 2009. Vier Blaarroespatotipes was uitkenbaar. Blaarroespatotipes UVPrt10 en UVPrt13 was die mees algemeenste. UVPgt50, UVPgt52, UVPgt54 en UVPgt57 was die mees algemene Stamroespatotipes. Net 6E16A- is geidentifiseer by die Geelroes-isolate. Daar was geen patroon in die verspreiding van Blaar-, Stam- of Geelroes patotipes. Blaar- en Stamroes was die wydste versprei, maar Geelroes het net by drie lokale in die sentrale Suid-Kaap voorgekom. Die lokaliteite is die enigste waar die weersomstandighede gunstig was vir Geelroes ontwikkeling gedurende die periode van monsterneming. Die lae vlakke van diversiteit wat in die roespopulasie gevind was is in teenstelling met internasionale populasies. Dit mag moontlik wees as gevolg van die relatief beperkte populasie grootte, die afwesigheid van „n gasheer vir seksuele voortplanting, die beperkte hoeveelheid monsters wat ingesamel is en die sterk selektiewe druk weens kunsmatige beheer.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: A survey was conducted in the Western Cape Province during the 1999/2000 and 2000/2001 seasons on mealybugs occurring in vineyards. P/anococcus ficus (Signoret) was the dominant mealybug in vineyards during this time. During this study P. ficus was recorded for the first time on roots of grapevines, which has far reaching implications for the control of this important vine leafroll virus vector as control actions were focused on above ground control. Other mealybugs presently recorded in local vineyards included Pseudococcus /ongispinus (Targioni) and Ferrisia ma/vastra (McDaniel). Pseudococcus viburni (Maskell) and Ps. so/ani Ferris were found on weeds in vineyards. Natural enemies of P. ficus recorded most frequently were species of Nephus predatory beetles, and the parasitaids Coccidoxenoides peregrinus (Timberlake), Anagyrus sp. and Leptomastix dacty/opii (Howard). Developmental studies on P. ficus and C. peregrinus indicated that the intrinsic rate of increase (rm) was similar, peaking at 25°C (rm = 0.169 for P. ficus; rm = 0.149 for C. peregrinus). The net replacement rate (Ra) was higher for P. ficus than for C. peregrinus at all five temperatures tested. The Ra for P. ficus reached a maximum at 21°C (308.87) and C. peregrinus at 25°C for C. peregrinus (69.94). The lower and upper thresholds for development of P. ficus were estimated at 16.59 and 35.61°C respectively. The lower threshold for development of C. peregrinus was 8.85°C. These parameters indicated that both insects were well adapted to temperatures in the Western Cape Province. The lower minimum threshold temperature of C. peregrinus in relation to that of P. ficus suggests that C. peregrinus should be more active during winter and early spring than P. ficus. A central systematic presence-absence sampling system was developed for P. ficus. Monitoring three different plant parts on the vine indicated that new growth areas on vines adjacent to the main stem could serve as an early warning system for pending P. ficus bunch infestations. Intervention should be planned when 2 % of the stems are infested with P. ficus when using this system. Seasonal population studies of P. ficus and its natural enemies showed that stem infestation by P. ficus reached peak levels during January in Robertson and Stellenbosch and during February in the Hex River Valley. Vine mealybugs colonised new growth early in the season, followed by the leaves and eventually the bunches towards the end of the season. High stem infestations early in the season resulted in high bunch infestation levels at harvest. A density dependent relationship was evident between P. ficus populations and parasitoid populations, suggesting that the parasitoids played a mayor role in the biological control of P. ficus populations. Biological control was however only achieved towards the end of the season when damage to the crop had already occurred. Mass releases of C. peregrinus on P. ficus populations were done in order to augment biological control as an alternative to chemical control. Between five and six releases of 20 000 C. peregrinus per release were done at monthly intervals in three grapegrowing areas. Mass released C. peregrinus controlled P. ficus adequately in the Hex River Valley. Control of P. ficus using this approach was no worse than using chemical control in Robertson and Stellenbosch. C. peregrinus is commercially available and can therefore be used as an alternative to chemical control by producers. Degree day estimation was used to predict development of P. ficus populations. This information was used as an input in a P. ficus pest management model. Data acquired from P. ficus and ant monitoring were used as components to construct a decision chart. This chart can be used by producers to optimise the control of P. ficus populations using either chemical control or mass releases of C. peregrinus.
AFRIKAANSE OPSOMMING: "n Studie is gedurende die 1999/2000 en 2000/2001 seisoene gedoen met die doelom die witluisspesies wat in wingerde voorkom, te identifiseer. Planococcus ficus (Signoret) is tans die dominante witluisspesie in wingerde in die Wes Kaap Provinsie. P. ficus kolonies is op wingerdwortels gevind. Dié bevinding kan verreikende gevolge hê vir die beheer van dié plaag as "n belangrike rolbladvirus vektor aangesien beheer tot dusver gefokus het op bogrondse gedeeltes. Ander witluisspesies wat in wingerde gevind is, sluit in Pseudococcus /ongispinus (Targioni) en Ferrisia malvastra (McDaniel). Pseudococcus vibumi (Maskell) en Ps. so/ani Ferris is op onkruide in wingerde gevind. Dominante natuurlike vyande van P. ficus sluit predatoriese kewertjies van verskeie Nephus spp. en die parasitoïede Coccidoxenoides peregrinus (Timberlake), Anagyrus sp. en Leptomastix dacty/opii (Howard) in. Ontwikkelingstudies op P. ficus en C. peregrinus het aangetoon dat die inhirente voortplantingstempo (rm) soortgelyk was vir beide insekte met "n maksimum by 25°C (0.169 vir P. ficus, 0.149 vir C. peregrinus). Die netto vervangingstempo (Ra) was in vergelyking met C. peregrinus hoër vir P. ficus by al vyf temperature getoets. Die Ra van P. ficus het "n maksimum bereik teen 21°C (308.87) en die van e. peregrinus by 25°C (69.94). Die teoretiese hoër en laer drempels vir ontwikkeling van P. ficus was onderskeidelik 16.59 en 35.61 oe. Die teoretiese laer drempelwaarde van ontwikkeling vir e. peregrinus was 8.85°e. Hierdie parameters dui aan dat beide insekte goed aangepas is by temperature in die Wes Kaap Provinsie. Die laer minimum drempel vir ontwikkeling van C. peregrinus in verhouding tot P. ficus impliseer dat C. peregrinus in die winter en vroeë lente meer aktief sal wees as P. ficus. 'n Sentrale sistematiese aan-afwesig moniteringsisteem met bekende vlakke van steekproefnemingsfout is ontwikkel in kommersiële wingerde vir P. ficus. Monitering van drie verskillende dele op die wingerdstok het aangedui dat die nuwe groei areas kan dien as 'n vroeë waarskuwing vir latere P. ficus trosinfestasies. Dié sisteem sal produsente in staat stelom te bepaal wanneer optrede noodsaaklik is. Daar word voorgestel dat optrede noodsaaklik is by 'n P. ficus besmettingsvlak van 2 % op die nuwe groei areas op stokke. Stambesmetting deur P. ficus het in Januarie piekvlakke bereik in Stellenbosch en Robertson, en in Februarie in die Hex Rivier Vallei. P. ficus koloniseer nuwe groei vroeg in die seisoen waarna blare en trosse aan die einde van die seisoen gekoloniseer word. Dié data dui aan dat P. ficus besmetting op nuwe groei vroeg in die seisoen 'n aanduiding kan gee van hoë trosbesmetting aan die einde van die seisoen. 'n Digtheidsafhanklike verwantskap was waarneembaar tussen P. ficus plaagpopulasies en parasitoïed populasies. Dié verwantskap dui aan dat parasitoïede die belangrikste rol speel in biologiese beheer van P. ficus populasies. Biologiese beheer van witluis is egter eers aan die einde van die seisoen bereik toe die oes reeds beskadig was. Massavrylatings van C. peregrinus is in P. ficus besmette blokke gedoen om biologiese beheer aan te help en sodoende as alternatief tot chemiese beheer te dien. Tussen vyf en ses vrylatings met 20 000 C. peregrinus is een keer per maand gedurende die seisoen gedoen. Die vrygelate C. peregrinus het P. ficus populasies voldoende beheer in die Hex Rivier Vallei. Beheer van P. ficus deur massavrylatings van C. peregrinus was soortgelyk as chemiese beheer in Robertson en Stellenbosch. C. peregrinus is kommersieel beskikbaar en kan om hierdie rede as alternatief tot chemiese beheer gebruik word. Graaddag bepaling is gebruik om die ontwikkeling van P. ficus populasies te voorspel. Hierdie inligting is gebruik as 'n verdere hulpmiddel in die P. ficus plaagbeheermodel. Inligting verkry vanuit P. ficus en mier monitering is gebruik as komponente in die opstel van 'n besluitnemingstabel. Hierdie tabel kan gebruik word deur produsente om beheer van P. ficus plaagpopulasies te optimaliseer deur chemiese beheer of massavrylatings van C. peregrinus.

Thesis (MSc)--Stellenbosch University, 2003.
Full text to be digitised and attached to bibliographic record.
ENGLISH ABSTRACT: The losses incurred to by the South African hardwood industry because of damage caused by the larvae of the Australian eucalyptus borers Phoracantha semipunctata (Fabricius) and P. recurva Newman (Coleoptera: Cerambycidae) were countered by the introduction of various biological control agents. Megalyra fasciipennis Westwood (Hymenoptera: Ichneumonidae), restricted to the southwestern Cape for nearly 91 years after its probable establishment in 1910, is a specialist pupal parasitoid achieving a parasitism level of up to 52.5 %. It has an activity peak in early spring, which coincides with the pupation of a large percentage of its hosts that had overwintered as larvae. The average length of the ovipositor of M. fasciipennis (42.71 ± 5.33 mm S.D.) was longer than the average tunnel length (31.34 ± 11.85 mm S.D.) to the pupal chamber of Phoracantha spp, within the log despite variations in the thicknesses of the eucalypt stems. Stem thickness therefore did not adversely affect the level of parasitism. Megalyra fasciipennis adults are diurnal, with activity largely determined by temperature. Over 70 % were active between 25°C and 34 °C, the minimum threshold for activity being 16°C. Optimum temperature for oviposition was 30 °C. Males became active before the females and maximum oviposition occurred between 10hOOand 15hOO. In 1993, a host specific egg parasitoid, Avetianella longoi Siscaro (Hymenoptera: Encyrtidae), was introduced for the control of Phoracantha spp. A total of 7791 A. longoi adults and 80 parasitised eggs were released around Cape Town between 1993 and 1995 before establishment was confirmed. Dispersal was monitored annually and was determined to occur at a rate of 50 km/year. By 1998 A. longoi had dispersed 300 km north of Cape Town to Lutzville and 270 km east to Riversdal. Subsequent to a satellite release in Knysna during 1994, it has been established 40 km from this release site, at Plettenberg Bay. The parasitoid has also bridged a LO km expanse of ocean to establish on Robben Island, immediately off the west coast of Cape Town. Avetianella longoi has a preference for the eggs of P. semipunctata, which is the most likely cause for the decline in the population of P. semipunctata. However, P. recurva remains relatively unaffected. Average parasitism of Phoracantha spp. eggs by A. longoi was 59.4 %. An undescribed Cleonymus sp. (Hymenoptera: Pteromalidae) of unknown origin (the genus being widely distributed on several continents), was discovered in the Cape Peninsula parasitising late instar larvae of P. semipunctata and P. recurva. This ectoparasitoid lays its eggs (mean number per larva = 20.3 ± 15.2 S.D.) through the bark into the host chamber after the host has been paralysed. The host is entirely consumed and pupation takes place in the chamber with wasps emerging in the ratio of 1 male: 3 females. Although uncommon in the field, mass rearing of these wasps in culture was easily accomplished and a consignment was released in the Tzaneen district in 1993, where it was confirmed to have become established in 1996. Bark thickness constraints on the effectiveness of this parasitoid as a biological control agent because it's short ovipositor restricts the wasp to certain eucalypt species or trees with thin bark. The introduction into South Africa in 1995 and attempted establishment of the larval parasitoids, Syngaster lepidus Brullé (Hymenoptera: Braconidae), Jarra phoracantha Marsh & Austin (Hymenoptera: Braconidae) and J maculipennis Marsh & Austin proved unsuccessful in the Western Cape. However, the former two species were established in the Tzaneen district and their recruitment for release in the Western Cape should be considered. The present guild of biological control agents has been insufficient to give the required control. In the absence of biological control agents, intraspecific competition amongst host larvae is the major mortality factor. Although high levels of mortality are achieved as a result of parasitism despite the biological constraints of the parasitoids (e.g. the narrow activity peak of A. longoi and the restriction of Cleonymus sp. to thin barked eucalypts), their combined parasitism has succeeded in reducing the competition between host larvae, resulting in fewer yet larger host beetles emerging. The introduction of additional viable agents to assist in the biocontrol of Phoracantha spp. is required.
AFRIKAANSE OPSOMMING: Die verliese wat die Suid-Afrikaanse hardehoutbedryf ly as gevolg van skade veroorsaak deur die bloekomboorders Phoracantha semipunctata en P. recurva (Coleoptera: Cerambycidae), is bekamp deur die invoer van verskeie biologiese beheeragente. Megalyra fasciipennis Westwood (Hymenoptera: Ichneumonidae), beperk tot die Suidwes-Kaap vir byna 91 jaar nadat dit waarskynlik in 1910 daar gevestig is, is In spesialis papieparasitoïd wat In parasitismevlak van tot 52.5% bereik. Dit het In aktiwiteitspiek in die vroeë lente wat saamval met die papievorming van baie gasheerlarwes wat oorwinter het. Die gemiddelde lengte van die eierboor van M. fasciipennis (42.71 ± 5.33 mm S.A.) was langer as die gemiddelde tonnellengte (31.34 ± 11.85 mm S.A.) na die papieholte van die gasheer binne in die hout, ten spyte van die variasie in die dikte van die bloekomstamme. Stamdikte het dus nie In nadelige uitwerking op die vlak van parasitisme nie. Volwassenes van M.fasciipennis is bedags aktief en aktiwiteit word hoofsaaklik deur temperatuur bepaal. Meer as 70% was tussen 25°C en 34 °C aktief, met 16°C as die minimum drumpel vir aktiwiteit. Mannetjies het voor die wyfies aktief geword en maksimum eierlegging het tussen 10hOOen 15hOO plaasgevind. In 1993 is die gasheerspesifieke eierparasitoïd Avetianella longoi Siscaro (Hymenoptera: Encyrtidae) vir die beheer van Phoracantha spp. ingevoer. Van 1993 tot 1995 is 7791 volwassenes van A. longoi en 80 geparasiteerde eiers rondom Kaapstad vrygelaat en dis vasgestel dat die spesies gevestig het. Die verspreiding daarvan is jaarliks gemonitor en dis vasgestel dat dit teen 50 km per jaar plaasvind. Teen 1998 het dit versprei tot 300km noord van Kaapstad na Lutzville en 270 km oos na Riversdal. Na 'n satelliet-loslating by Knysna in 1994 het dit 40 km verder by Plettenbergbaai gevestig. Die parasitoïd het ook 10 km van die oseaan oorgesteek om op Robbeneiland, wes van Kaapstad te vestig. Avetianella longoi gee voorkeur aan die eiers van P. semipunctata en dis waarskynlik die rede vir die afname in die getalle van hierdie spesies, maar P.recurva word relatief min beïnvloed. Die gemiddelde graad van parasitisme van Phoracantha spp. was 59.4%. Dit is gevind dat 'n onbeskryfde Cleonymus sp. (Hymenoptera: Pteromalidae) van onbekende oorsprong (die genus kom wyd verspreid in verskeie vastelande voor) die laat instar larwes van P. semipunctata en P. recurva parasiteer. Hierdie ektoparasitoïd lê sy eiers (gemiddeld 20.3 ± 15.2 S.A.) in die gasheerholte nadat die gasheer eers verlam is. Die gasheer word heeltemalopgevreet en pupering vind plaas in die holte plaas. Volwassenes kom uit in verhoudingvan drie mannetjies tot een wyfie. Alhoewel skaars in die natuur, kan hierdie wesp maklik in massa geteel word. 'n Besending is in die Tzaneen distrik vrygestel en in 1996 is vasgestel dat hulle gevestig het. Basdikte is 'n beperkende faktor in die gebruik van hierdie parasitoïd as effektiewe beheeragent vir biologiese beheer omdat die kort lengte van die eierboor die wesp sal beperk tot bloekomsoorte met dun bas. Die invoer na Suid-Afrika in 1995 en vestiging van die larwale parasitoïde Syngaster lepidus Brullé (Hymenoptera: Braconidae), J. phoracantha Marsh & Austin (Hymenoptera: Braconidae) en J. maculipennis Marsh & Austin was onsuksesvol in dieWes-Kaap. Die twee spesies is egter in die distrik Tzaneen gevestig en versameling met die oog op loslating in die Wes-Kaap behoort oorweeg te word. Die huidige gilde van biologiese beheer-agente is onvoldoende om die vereiste graad van beheer te verskaf. In die afwesigheid van biologiese beheer-agente is intraspesifieke kompetisie tussen gasheerlarwes die belangrikste mortaliteitfaktor. Alhoewel hoë vlakke van mortaliteit as gevolg van parasitisme bereik word, ten spyte van die biologiese beperkings van die parasitoïde (bv. die kort aktiwiteitspiek van A. longoi en die beperking van Cleonymus tot bloekoms met dun bas), het die gekombineerde parasitisme daarin geslaag om die kompetisie tussen gasheerlarwes te verlaag, met die gevolg dat minder maar groter gasheerkewers verskyn het. Dit is dus nodig dat addisionele organismes gevestig word om by te dra tot die biologiese beheer van Phoracantha spp.

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.

Thesis (MSc (Plant Pathology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Esca is a disease affecting grapevines and is potentially devastating as there are economic losses due to a decrease in yield, wine quality and berry quality. Vineyards also need to be replaced earlier and therefore esca has a great impact on the wine, table grape and raisin industries. The disease is known to affect vineyards worldwide and has been studied extensively in Europe, but not in South Africa. Esca diseased grapevines were observed for the first time prior to 1981 in South African vineyards. The disease is primarily caused by Phaeoacremonium aleophilum, Phaeomoniella chlamydospora (both causing brown and black wood streaking) and white rot basidiomycete species such as Fomitiporia mediterranea which cause wood rot in the trunks and arms of generally older grapevines. Species of the Botryosphaeriaceae and Phomopsis (mainly Phomopsis viticola) and Eutypa lata have also been isolated from esca diseased vines, but their association with esca is unclear. Some of the symptoms associated with the disease on most grapevine cultivars include ‘tiger-stripe’ foliar symptoms, apoplexy and berry symptoms such as shriveling, discoloration and ‘black measles’. These external symptoms as well as internal symptoms are thought to be a result of toxin and enzyme production by the fungi involved. Symptom expression is erratic and varies from year to year making investigations into the causal fungi and the toxins and enzymes secreted in planta difficult. Vines with internal or external symptoms of esca were sampled in this study from table and wine grape cultivars in 37 towns in the Western Cape, Northern Cape and Limpopo provinces. The majority of sampled vines were over ten years of age, but vines as young as two to three years were also found to be infected. The external symptoms included dieback, tiger striped leaves, berry symptoms (shriveling, insufficient colouring and black spots) and apoplexy. These symptoms resembled those found on grapevines in Europe, Australia and the USA. The internal symptoms found were also similar to European symptoms and included white rot, black and brown wood streaking, brown necrosis within white rot, sectorial brown necrosis and central brown/ red/ black margin. The fungi mostly isolated from the white rot were the basidiomycetes. Black and brown wood streaking was primarily caused by Phaeomoniella chlamydospora. Brown necrosis within the white rot was caused by Phaeomoniella chlamydospora and less frequently by Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae and Pleurostomophora richardsiae. The sectorial brown necrosis and the central/ brown/ red/ black margin were dominated by Phaeomoniella chlamydospora. The fruiting bodies of the basidiomycetes were found on only a few grapevines. The fungal species associated with the internal wood symptoms were characterized on cultural growth patterns, morphology as well as phylogenetic inference. The gene areas sequenced included the internal transcribed spacers and the 5.8S rRNA gene for the basidiomycetes and Phomopsis isolates, the partial b-tubulin gene for Phaeoacremonium isolates and the partial translation elongation-1a gene for the Botryosphaeriaceae isolates. The basidiomycete isolates fell into ten taxa within the Hymenochaetales of which two could be linked to known genera, namely Fomitiporia and Phellinus. The ten basidiomycete taxa do not correspond to any published sequences. Eutypa lata, Diaporthe ambigua, Diplodia seriata, Neofusicoccum australe, Neofusicoccum parvum, Phomopsis viticola, Phomopsis sp. 1, Phaeomoniella chlamydospora and six species of Phaeoacremonium including P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae and P. sicilianum were also isolated of which the latter three are reported for the first time in South Africa. To understand the role of the basidiomycetes in the complex, toxin and enzyme analyses was determined for these fungi. Selected basidiomycete isolates were grown up in liquid broth and extractions performed to test for the presence of 4-hydroxy-benzaldehyde. All of the basidiomycete isolates were able to produce this toxin which is known to be phytotoxic. The basidiomycetes were then tested for the presence of certain wood degrading enzymes. All of the taxa were able to produce manganese peroxidase. Laccase was produced by all taxa, except Taxon 8. Lignin peroxidase was produced by Taxa 1, 2, 7, Fomitiporia sp. and the Phellinus sp. All the basidiomycete isolates were able to produce cellulose and none were able to produce xylanase. These enzyme tests showed that the basidiomycetes produce a wide variety of enzymes which are able to degrade cellulase and lignin which are both structural components of wood. Given the wide distribution of esca in the grape growing regions investigated in South Africa and the diverse amount of species found, this disease must surely be seen as a limiting factor to the productive lifespan of vineyards and quality of produce. Preventative measures such as sanitation and pruning wound protection contribute to the management of the disease, but many questions still remain about the synergy of the causal fungi, epidemiology and management of esca.
AFRIKAANSE OPSOMMING: Esca is ‘n wingerd siekte wat potensieel skade kan aanrig as gevolg van ekonomiese verliese weens verlaagde opbrengs, wyn kwaliteit en vrug kwaliteit. Wingerde moet ook vroeër vervang word en daarom het esca ’n groot impak op die wyn, tafeldryf en rosyne industrieë. Esca word wêreldwyd gevind op wingerd en is al intensief nagevors in Europa, maar nog nie in Suid-Afrika. Esca is vir die eerste keer in die 1980’s in Suid-Afrikaanse wingerde gerapporteer. Die primêre veroorsaakende organismes van esca is Phaeoacremonium aleophilum, Phaeomoniella chlamydospora wat bruin en swart vaatweefsel verkleuring veroorsaak en basidiomycete spesies soos Fomitiporia mediterranea wat wit verotting veroorsaak in die stam en arms van ouer wingerd. Spesies van die Botryosphaeriaceae en Phomopsis (hoofsaaklik Phomopsis viticola) en Eutypa lata is ook al vanaf esca simptome geïsoleer, maar hul assosiasie met die siekte is nie duidelik nie. Algemene simptome wat voorkom op die meeste wingerd kultivars met esca sluit in ‘tiger-stripe’ blaar simptome, apopleksie en vrug simptome soos verdroging, verkleuring en spikkels (black measles). Interne en eksterne simptome kan wees as gevolg van toksiene en ensiem produksie van die swamme wat betrokke is by esca. Eksterne simptoom uitdrukking is wisselvallig en varieer van jaar tot jaar. Dit bemoelik die bestudering van die swamme en die toksiene en ensieme wat afgeskei word in planta. Wingerd monsters met eksterne en interne simptome is versamel van tafel en wyndruif kultivars in 37 dorpe in die Wes-Kaap, Noord-Kaap en Limpopo provinsies. Die meerderheid monsters was ouer as tien jaar maar wingerde wat twee tot drie jaar oud was, was ook gevind. Die eksterne simptome wat op hierdie kultivars gevind is het terugsterwing, ‘tiger striped’ blare, vrug simptome (verkrimping en onvoldoende verkleuring) en apopleksie ingesluit. Hierdie simptome stem ooreen met soortgelyke simptome gevind op wingerd in Europa, Australië en die VSA. Interne simptome was ooreenstemmend met simptome wat gevind word in Europa. Die interne simptome het wit verotting, bruin en swart streepvorming, bruin nekrose met wit verotting, sektoriale bruin nekrose en sentrale bruin/ rooi/ swart kante ingesluit. Basidiomycete swamme is meestal uit die wit verotting gedeeltes geïsoleer. Swart en bruin hout streepvorming was meestal deur Phaeomoniella chlamydospora veroorsaak. Bruin nekrose binne die wit verotting was meestal deur Phaeomoniella chlamydospora veroorsaak en in ‘n mindere mate deur Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae en Pleurostomophora richardsiae. Phaeomoniella chlamydospora was die hoof veroorsakende organisme van sektoriale bruin nekrose en die sentrale bruin/ rooi/ swart kante. Vrugliggame van die basiodiomycete is op enkele wingerde gevind. Swam soorte wat geassosieer word met die interne hout simptome was verder gekarakteriseer op kultuur groei, morfologiese eienskappe, en filogenetiese analise. Die geen areas waarvan die basis paar volgorde bepaal was sluit in die interne getranskribeerde spasies en die 5.8S rRNA geen vir die basidiomycete en Phomopsis isolate, die gedeeltelike btubulien geen vir Phaeoacremonium isolate en die gedeeltelike translasie velenging-1a geen vir die Botryosphaericeae isolate. Die basidiomycete isolate was versprei oor tien taksons binne die Hymenochaetales waarvan twee genusse gekoppel kon word aan die genera Fomitiporia en Phellinus. Die tien basidiomycete taksons kom nie ooreen met enige gepubliseerde DNS volgordes. Eutypa lata, Phomopsis viticola, Phomopsis sp. 1, Diaporthe ambigua, Diplodia seriata, Neofusicoccum parvum, Neofusicoccum australe, Phaeomoniella chlamydospora en ses spesies van Phaeoacremonium insluitend P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae en P. sicilianum is ook geïsoleer. Hierdie is die eerste keer dat P. iranianum, P. mortoniae en P. sicilianum in Suid-Afrika gerapporteer word. Om die rol wat die basidiomycete in die siekte-kompleks speel beter te verstaan is toksien en ensiem analises uitgevoer. Geselekteerde basidiomycete isolate is gekweek in vloeibare groei medium en ekstraksies uitgevoer om te toets vir die teenwoordigheid van 4- hydroxy-benzaldehyde. Al die basidiomycete isolate kon 4-hydroxy-benzaldehyde, wat bekend is om fitotoksies te wees, produseer. Die basidiomycete isolate was verder getoets vir die produksie van spesifieke hout afbrekende ensieme. Al die basidiomycete taksons kon mangaan-peroksidase produseer. Lakkase was geproduseer deur al die taksons, uitsluitend Takson 8. Lignien-peroksidase was geproduseer deur Taksons 1, 2, 7, Fomitiporia sp. en die Phellinus sp. Al die basidiomycete isolate kon sellulose produseer, maar geen kon xilanase produseer. Die ensiem analises het gewys dat die basidiomycete wat moontlik betrokke is by esca ‘n wye reeks van ensieme kan produseer wat sellulose en lignien kan degradeer. Sellulose en lignien is beide strukturele komponente van hout. Weens die wye verspeiding van esca geaffekteerde wingerde in Suid Afrika en die wye reeks van spesies wat betrokke is by die siekte kompleks moet esca sekerlik gesien word as een van die beperkende faktore op die produktiewe leeftyd van wingerde en die kwaliteit van druiwe wat geproduseer word. Sanitasie en snoeiwond beskerming is voorkomende maatreëls wat ingestel kan word om die effek en verspreiding van esca te beperk maar daar is nog baie vrae wat antwoorde benodig oor die sinergie van die veroorsakende swamme, epidemiologie en bestuur van esca.

Thesis (PhD (Agric) (Conservation Ecology and Entomology))--University of Stellenbosch, 2006.
A study was performed to develop a generic pest monitoring system for sampling the main table grape pests in vineyards in the Hex River Valley, Western Cape Province of South Africa. The presence of phytophagous and predatory mites on cover crop plants was also investigated as this may contribute to biological control of the phytophagous mites in vines. Life table studies for Epichoristodes acerbella (Walker), an important phytosanitary pest, were conducted to determine whether or not this pest was sensitive to high temperatures. Information gained from the latter can also be used for breeding purposes in the possible future development of a sterile insect technique (SIT) programme to control this pest. The sampling system consisted of inspecting 20 plots of five vines per plot per one to two hectares. The top fork of each of the five vines per plot was examined for Planococcus ficus (Signoret) to a distance of within 30 cm of the stem, as well as the distal 15 cm of one cane per vine for the presence of P. ficus and damage caused by Phlyctinus callosus Boh. One bunch per vine was examined for insect damage or presence, and one leaf per vine for the presence of leaf infesting arthropods, such as Tetranychus urticae Koch, P. ficus and Frankliniella occidentalis (Pergande). Corrugated cardboard bands, tied around the stem of one vine per plot, were used to monitor activity of P. callosus. Blue sticky traps, at a density of four to five traps per one to two hectares, were used to monitor activity of F. occidentalis. Pheromone traps, at a density of one trap per one to two hectares, were used to monitor activity of P. ficus, E. acerbella and Helicoverpa armigera (Hübner). All the above-mentioned inspections were done at two-weekly intervals, except traps for E. acerbella and H. armigera, which were inspected weekly. In each of the rows in which the sample plots were situated, one leaf of each of the cover crop plant species was examined for the presence of phytophagous mites and their predators. The abundance and distribution of cover crop plants were determined using a co-ordinate sampling system. Cover crop sampling was done at monthly intervals. The current threshold for P. ficus is 2% stem infestation, which is reached when more than 65 males per pheromone trap are recorded. Counting mealybugs on the sticky pads in the pheromone traps is time consuming. However, the number of grid blocks on the sticky pad with males present can be counted. When P. ficus males are found in 27 blocks on the sticky pad, stem inspections should commence. Due to the spatial association between P. ficus bunch and stem infestation, stem infestation could give an indication of where bunch infestation could be expected. The use of blue sticky traps for predicting halo spot damage, caused by F. occidentalis, is not recommended. The presence of thrips on the vine leaves could not give an indication of where to expect bunch damage, since thrips on the leaves and halo spot damage were not spatially associated. A suitable sampling method for F. occidentalis still needs to be developed. The monitoring system described here can only provide information on the infestation status of the vineyard. For E. acerbella, H. armigera and P. callosus, the traps and cardboard bands could be used to identify vineyards where these pests are present and therefore, where phytosanitary problems may arise. The presence of P. callosus under the bands was spatially associated with P. callosus damage and could be used as an indicator of the latter. The presence of drosophilid flies in the bunches could not be used as an indicator of the presence of E. acerbella in the bunches. If 5% bunch damage is used as an economic threshold for E. acerbella and P. callosus, there will be a good chance of not under spraying if control measures are applied at 1% bunch damage. Epichoristodes acerbella favoured more moderate constant temperatures, with constant temperatures of 28°C and above being unfavourable for development. The economic threshold for Tetranychus urticae Koch is six mites per leaf, or if presence-absence sampling is used, 11 to 29% leaf infestation. Three important predatory mites, that kept T. urticae under control, were found in the Hex River Valley, namely Euseius addoensis (Van der Merwe & Ryke), Neoseiulus californicus (McGregor) and an undescribed phytoseiid in the genus Typhlodromus. Various cover crop plants served as hosts for T. urticae and predatory mites. The presence of these plants created suitable conditions for the survival of these mites and may have influenced their presence on the vine leaves. In the case of phytosanitary pests, both field and pack shed inspections can be used to conclude with a 99% degree of certainty that infestation levels in the pack shed will be 10% or less, since similar results for both methods were obtained. However, more than 20 plots will have to be inspected.

Maize contributes substantially to food security in the Eastern Cape province of South Africa. It is a staple food to many of the province’s rural and urban inhabitants. Insect pests are one of the factors that hamper its productivity and as a result, deprive farmers of good yields. The adverse effects of insecticides and the high cost associated with them and the cost of transgenic seeds are some of the challenges faced by small-scale farmers in rural areas. Alternative control methods which include the use of indigenous techniques to control pests are now sought. A study to identify and document ethnobiological means used by rural farmers to manage insect pests of maize was conducted in the rural areas of the Eastern Cape Province. A total of 217 participants were interviewed on the matter, using semi-structured but detailed questionnaires. Rural farmers due to their linkage to agriculture activities and the fact that they are considered as custodians of agricultural indigenous knowledge were selected as respondents. Only maize producing and IsiXhosa speaking people were chosen to contribute. Main focus was on the demography of respondents, crop production activities and insect pest control. Pretesting of the questionnaire in order to assess the appropriateness of questions and comprehension by both farmers and enumerators was done. Data was analysed using descriptive statistics. Fifty five percent (55 %) of the respondents were females and the highest number of participants was from the Chris Hani District Municipality. Majority of the people were unemployed or pensioners. Most had only attended primary school and the mean age was 59 years. Apart from maize, respondents were cultivating other crops such as cabbage, Swiss chard, potatoes etc. Stalk borers followed by cutworms were the main pests of maize in these areas. Respondents used mainly insecticides, followed by alternative substances, which also included cultural control methods such as planting date manipulation. Few respondents used plants as control agents for insect pests. Some people did not control pests even though they were a problem in their fields. The most used plant was Chenopodium ambrosiodes L, while the most used substance was Madubula (a detergent). The most used insecticide was carbaryl from the carbamite family. Respondents listed different preparation techniques for all the control methods mentioned. These techniques revealed different times of preparation, quantities of ingredients, amounts applied on plants, modes of application and intervals of application. Rural farmers in the study areas used different atypical methods which may play a significant role in pest management today. Some of the products may have a positive influence on agriculture, while some are dangerous to humans and environmental health. Further research which will investigate their potential use in pest control needs to be done.

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.

Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant.
AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ’n lid van die genus Ampelovirus in die familie Closteroviridae en word beskou as die hoof bydraende faktor van wingerd-rolbladsiekte wêreldwyd. ’n Metagenomiese studie het bewys dat daar ’n nuwe variant van GLRaV-3 bestaan wat nog nie voorheen in Suid Afrika opgespoor kon word met die huidige opsporingsmetodes nie. Hierdie nuwe variant was naaste verwant aan ’n Nieu-Seelandse isolaat, NZ-1. In hierdie studie is die genoomvolgorde van twee isolate, GH11 en GH30, van hierdie nuwe GLRaV-3 variant groep bepaal. Hierdie twee isolate was minder as 70% identies aan ander GLRaV-3 variante, wat daarop dui dat hulle as variante van ’n nuwe virus-ras beskou behoort te word. Ons beveel aan dat hierdie GLRaV-3-verwante virus geklassifiseer word saam met die NZ-1 isolaat en ander isolate uit Kalifornië, as groep VI van GLRaV-3. Hierdie studie het ook verdere bewyse verskaf dat volgende-generasie volgordebepalingstegnologie ’n waardevolle benadering is om nuwe virusse en variante te identifiseer, deurdat die huidige studie gewys het dat die voorlopige volgorde, wat gegenereer is deur bioinformatika-instrumente, 98% identies was aan die GH11 volgorde wat met Sanger volgordebepaling verkry was. Hierdie studie het ook gevind dat die industrie-standaard ELISA, nog steeds ’n effektiewe GLRaV-3 diagnostiese metode is en wel infeksies, veroorsaak deur al die variant-groepe, sal kan identifiseer. Die ELISA toets is egter nie in staat om te onderskei tussen GLRaV-3 variant-groepe nie. In hierdie studie is ’n variant-identifiseerbare in-tyd tru-transkripsie polimerase ketting reaksie (PKR) ontwerp wat GLRaV-3 variant-groepe I, II, III en VI kan identifiseer deur middel van ’n enkele inleier-stel wat die GLRaV-3 Hsp70h-geen teiken. As hoë-resolusie smeltingskurwe-analise bygevoeg word by die in-tyd tru-transkripsie PKR, is dit moontlik om te onderskei tussen variant-groepe op grond van drie smeltingspunt intervalle. Die tru-transkripsie hoë-resolusie smeltingskurwe-toets verskaf meer sensitiewe en geoutomatiseerde metodes om GLRaV-3 variant-groepe te identifiseer en te onderskei. ’n Veelvuldige tru-transkripsie PKR is ook ontwerp om tussen variante wat tans in Suid-Afrika aangetref word, te onderskei en te dien as ’n valideringsmetode vir die in-tyd tru-transkripsie hoë-resolusie smeltingskurwe-toets. Sensitiewe en akkurate toetse, soos bogenoemde, is noodsaaklik vir die bestudering van die verspreiding en impak van die verskillende GLRaV-3 variante in wingerd. Hierdie metodes kan gebruik word om kennis ten opsigte van rolblad patogenese te verbreed en om by te dra tot epidemiologiese studies wat ondersoek hoe hierdie variant-groepe versprei, of daar ’n assosiasie bestaan tussen ’n spesifieke variant en siekte-simptome en of daar ’n verskil is in die witluisvektor oordragseffektiwitiet vir elke GLRaV-3 variant.

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.

Sunflower production in South Africa has increased four fold since the 1970's. This study was done to elucidate the pest status of Heliothis armigera (Hiibner) on sunflower. Field studies were undertaken at Warmbaths, Brits and Delmas during the summer seasons of 1988/89 to 1990/91. The infestations at the latter two areas were negligible throughout the study period and their statistics have not been included in this thesis. Plant development and oviposition by natural H. armigera populations were found to be correlated as, regardless of planting date, oviposition started at six to seven weeks after planting; when the flowering stage began. A peak in egg numbers was reached by the tenth week with an average for the three seasons at two eggs per plant. The eggs were laid singly on the bracts and bases of the flower buds. A peak in larval numbers was reached at the thirteenth week after planting with the average for the three seasons of 0,4 larvae per plant. The preferred feeding sites were on, between and under the bracts from where the larvae burrow into the pithy tissue of the receptacle. Only six percent of the larvae were found feeding directly on the achenes. Eggs and larvae collected were reared to determine the degree of parasitism. Overall, 19% of the eggs were parasitised; 18% by Telenomus ullyetti Nixon (Scelionidae) and one percent by Trichogrammatoidea lutea (Trichogrammatidae). Larval parasitism at Warmbaths was 23% in 1988/89, 27% in 1989/90 and 34% in 1990/91. Of the parasitised larvae, 44% succumbed to Palexorista prob. laxa (Tachinidae). The remainder were unidentified Braconidae and Ichneumonidae. Predators, such as chrysopids, were observed during the study but their effect on egg and larvae numbers was not studied in detail. A polyhedral virus occurred late in the seasons and caused mortality of the larvae. The low numbers of H. armigera on sunflower, the slight damage to the crop and the reasonably high rate of parasitism, all seem to indicate that H. armigera is not an economica1ly important pest of sunflower and that additional control methods are not justified.

Tecoma stans (L.) Juss. Ex Kunth (Bignoniaceae) also known as yellow bells, has a native distribution from Northern Argentina, central America, Mexico and the Southern USA. In many warm climatic regions of the world, T. stans is commonly planted as an ornamental plant because of its yellow flowers, hence the name yellow bells, and pinnate foliage. As a result, this evergreen shrub has wide distribution in the tropical and subtropical parts of the western hemisphere. As is the case in many other parts of the world, T. stans was introduced into South Africa as an ornamental plant, but escaped cultivation and now invades roadsides, urban open spaces, watercourses, rocky sites in subtropical and tropical areas of five South African provinces; Gauteng, Mpumalanga, Limpopo, KwaZulu-Natal, Eastern Cape and neighboring countries. Tecoma stans has the potential of extending its range because its seeds are easily dispersed by wind. The purpose of this work was to carry out pre-release efficacy studies to determine the host specificity and suitability of Pseudonapomyza sp. (Diptera: Agromyzidae), a leaf-mining fly, as a biological control agent of T. stans. Available information suggests that the fly was brought to South Africa (SA) from Argentina in 2005. In that year a worker collected adult root feeding fleabeetles from T. stans and their eggs by collecting soil around the plants in the Argentinian Province of Jujuy, at San Pedro (24°12’592”S, 64°51’328”W). The soil was brought to the SA quarantine laboratory of the Agricultural Research Council, Plant Protection Research Institute (Weeds Division), Pretoria, and placed in a cage containing T. stans plants for flea-beetle larvae to emerge from the eggs. The Pseudonapomyza sp. flies which emerged from that soil were reared to produce a colony of flies used in the study reported here. The feeding behavior of Pseudonapomyza sp. adults is initiated by females which use their ovipositor to puncture holes in the leaf mesophyll and then they feed on the sap oozing from the holes. Since males have no means of puncturing the leaves, they feed from holes made by females. Eggs are laid singly into the tubular leaf punctures. Soon after hatching, the larva feeds on the leaf mesophyll tissue. As the larva feeds within the leaf it creates mines which eventually coalesce to form large blotches. The damaged leaf area reduces the photosynthetic potential of the plant especially when damaged leaves dry and fall off the plants. The potential of Pseudonapomyza sp. as a biocontrol agent is enhanced by the fact that it has a high level of fecundity and a short life cycle. As a result, its populations can build up rapidly to exert a significant impact on T. stans. Host-specificity tests undertaken on 35 plant species in 12 plant families showed that out of the 35 plant species tested, the fly was able to develop on T. stans only. Although Pseudonapomyza sp. adults fed on T. capensis, a South African indigenous ornamental shrub, no larval mines were observed on this plant. This suggests two possibilities; either females of Pseudonapomyza sp. do not oviposit on T. capensis or oviposition takes place but larvae cannot feed and develop on this plant. These studies indicate that this fly is sufficiently host-specific, and can be released against T. stans without posing any threat to either commercial or indigenous plant species grown in South Africa. Experimental designs simulating high populations of Pseudonapomyza sp. showed that the impact of leaf mining fly on T. stans can cause approximately 56 percent aboveground biomass reduction. Other concurrent studies have also showed that low and high density fly infestations can cause 23 percent and 48 percent belowground biomass reductions, respectively. Based on the available information, it appears that Pseudonapomyza sp. may have the potential to reduce the invasive capacity of T. stans in the affected areas. In order to exert more herbivore pressure on T. stans, it is suggested that agents belonging to other feeding guilds, such as root-, stem- and seed-feeding insects, be considered for release to complement the leaf-feeding of Pseudonapomyza sp. An application to release this fly in SA has been submitted to one of the two regulatory authorities.

In the KwaZulu-Natal (KZN) Midlands North region of South Africa, the importance and increased prevalence of endemic scarabaeids, particularly Hypopholis sommeri Burmeister and Schizonycha affinis Boheman (Coleoptera: Melolonthinae), as soil pests of sugarcane, and a need for their control was established. The development of a mycoinsecticide offers an environmentally friendly alternative to chemical insecticides. The identification of a diversity of white grub species, in two Scarabaeidae subfamilies, representing seven genera were collected in sugarcane as a pest complex. Hypopholis sommeri and S. affinis were the most prevalent species. The increased seasonal abundances, diversity and highly aggregated nature of these scarabaeid species in summer months, suggested that targeting and control strategies for these pests should be considered in this season. Increased rainfall, relative humidity and soil temperatures were linked to the increased occurrence of scarab adults and neonate grubs. Beauveria brongniartii (Saccardo) Petch epizootics were recorded at two sites in the KZN Midlands North on H. sommeri. Seventeen different fluorescently-labelled microsatellite PCR primers were used to target 78 isolates of Beauveria sp. DNA. Microsatellite data resolved two distinct clusters of Beauveria isolates which represented the Beauveria bassiana senso stricto (Balsamo) Vuillemin and B. brongniartii species groups. These groupings were supported by two gene regions, the nuclear ribosomal Internal Transcribed Spacer (ITS) and the nuclear B locus (Bloc) gene of which 23 exemplar Beauveria isolates were represented and sequenced. When microsatellite data were analysed, 26 haplotypes among 58 isolates of B. brongniartii were distinguished. Relatively low levels of genetic diversity were detected in B. brongniartii and isolates were shown to be closely related. There was no genetic differentiation between the two sites, Harden Heights and Canema in the KZN Midlands North. High gene flow from swarming H. sommeri beetles is the proposed mechanism for this lack of genetic differentiation between populations. Microsatellite analyses also showed that B. brongniartii conidia were being cycled from arboreal to subterranean habitats in the environment by H. sommeri beetles. This was the first record of this species of fungus causing epizootics on the larvae and adults of H. sommeri in South Africa. The virulence of 21 isolates of Beauveria brongniartii and two isolates of B. bassiana were evaluated against the adults and larvae of S. affinis and the adults of H. sommeri and Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae). Despite being closely-related, B. brongniartii isolates varied significantly in their virulence towards different hosts and highlighted the host specific nature of B. brongniartii towards S. affinis when compared to B. bassiana. Adults of S. affinis were significantly more susceptible to B. brongniartii isolates than the second (L2) or third instar (L3) grubs. The median lethal time (LT₅₀) of the most virulent B. brongniartii isolate (C13) against S. affinis adults was 7.8 days and probit analysis estimated a median lethal concentration (LC₅₀) of 4.4×10⁷ conidia/ml⁻¹. When L2 grubs were treated with a concentration of 1.0×10⁸ conidia/ml⁻¹, B. brongniartii isolates HHWG1, HHB39A and C17 caused mortality in L2 grubs within 18.4-19.8 days (LT₅₀). Beauveria brongniartii isolate HHWG1 was tested against the L3 grubs of S. affinis at four different concentrations. At the lowest concentration (1×10⁶ conidia/ml⁻¹), the LT₅₀ was 25.8 days, and at the highest concentration (1×10⁹ conidia/ml⁻¹) the LT₅₀ dropped to 15.1 days. The persistence of B. bassiana isolate 4222 formulated on rice and wheat bran and buried at eight field sites in the KZN Midlands North was evaluated by plating out a suspension of treated soil onto a selective medium. All eight field sites showed a significant decline in B. bassiana CFUs per gram of soil over time, with few conidia still present in the samples after a year. Greater declines in CFUs were observed at some sites but there were no significant differences observed in the persistence of conidia formulated on rice or wheat bran as carriers. Overall, poor persistence of B. bassiana isolate 4222 was attributed to suboptimum temperatures, rainfall, which rapidly degraded the nutritive carriers, attenuated fungal genotype and the action of antagonistic soil microbes. Growers’ perceptions of white grubs as pests and the feasibility of a mycoinsecticide market were evaluated by means of a semi-structured questionnaire. The study showed that the reduced feasibility of application, general lack of potential demand for a product, high cost factors and most importantly, the lack of pest perception, were factors which would negatively affect the adoption of a granular mycoinsecticide. Growers however exhibited a positive attitude towards mycoinsecticides, and showed all the relevant attributes for successful technology adoption. It is recommended that because B. brongniartii epizootics were recorded on target pests which indicated good host specificity, dispersal ability and persistence of the fungus in the intended environment of application; that a mycoinsecticide based on this fungal species be developed. What will likely increase adoption and success of a mycoinsecticide is collaboration between various industries partners to increases market potential in other crops such as Acacia mearnsii De Wild (Fabales: Fabaceae).

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: South Africa is ranked eighth in the world as far as international wine production is concerned and in terms of area under bearing vines South Africa is ranked 12th. In 2011 the wine industry contributed R4 204.4 million to the South African economy in state revenue from wine products. The importance of viticulture to the economy of South Africa forces the industry to limit the effect of all disease causing pathogens in order to keep their competitive edge. Aster yellows (AY) phytoplasma 16SrI-B subgroup was reported for the first time in grapevine (Vitis vinifera L. (Vitaceae)) in South Africa in 2006. Worldwide phytoplasma diseases of grapevine cause serious damage ranging from lower yields to the death of vines. The lack of knowledge about the epidemiology of AY disease makes it difficult to determine the impact of the disease on the South African wine industry. The aim of this study was to conduct surveys in disease-affected vineyards in the Vredendal region to determine the incidence and spatial distribution of the disease in a variety of cultivars. The field surveys based on visual symptoms of AY disease were confirmed by polymerase chain reaction (PCR). A survey was also conducted in and around AY-infected vineyards in search of possible alternative host plants of the phytoplasma. Spatial distribution of AY-affected vines were analysed using the PATCHY spatial analysis package. A rapid decline of AY-affected Chardonnay eventually leading to the death of vines was observed, confirming the sensitivity of Chardonnay towards grapevine yellows infections. Symptomless AY infections occurred and AY could not be detected in all symptomatic vines, which indicate uneven distribution of AY in individual vines. Molecular analyses using PCR-RFLP showed that all vines sampled in the Vredendal vicinity contained AY phytoplasma only. No phytoplasmas were present in any weeds or other possible host plants tested. Although the mean yearly disease incidences of Chardonnay (29.95%) and Chenin blanc (16.64%) were higher than Pinotage (5.80%) over the four-year survey period, there was no statistically significant difference between the disease incidences of these three cultivars. The mean yearly disease incidence showed a trend over time and the disease incidence of the first year was significantly lower than that of the other years. Chardonnay showed a cumulative disease incidence of 37.77% at the end of the 4-year study which means that Chardonnay vineyards can be 100% AY infected in ten years’ time. Spatial distribution patterns of AY-infected vines were mostly non-random with clustering of disease affected vines along and across vine rows. With the exception of one vineyard, aggregation of AY-affected vines mostly occurred on the edge of vineyards adjacent to infected vineyards. This epidemiological study gives an indication of the sensitivity of the different cultivars towards AY, the tempo of spreading and the future impact of the disease on the South African wine industry. It also contributes valuable information towards the development of a management strategy for grapevine yellows disease in South African vineyards.
AFRIKAANSE OPSOMMING: Suid- Afrika is op agtste op die wêreld ranglys wat internasionale produksie van wyn aan betref, en in terme van oppervlakte onder wingerd, is Suid-Afrika 12de. In 2011 het die wynbedryf R4 204.4 miljoen tot die Suid-Afrikaanse ekonomie bygedra in staats inkomste uit wyn produkte. Die belangrikheid van wingerd tot die ekonomie van Suid-Afrika dwing die bedryf om die effek van alle siekteveroorsakende patogene te beperk, om sodoende hul kompeterende voordeel te behou. Aster vergeling (AY) fitoplasma 16SrI-B subgroep is vir die eerste keer in 2006 in wingerd (Vitis vinifera L. (Vitaceae)) in Suid-Afrika waargeneem. Fitoplasma siektes van wingerd veroorsaak wêreldwyd ernstige skade wat wissel van laer opbrengste tot die afsterf van wingerdstokke. Die gebrek aan kennis oor die epidemiologie van astervergeling siekte maak dit moeilik om die impak van die siekte op die Suid-Afrikaanse wynbedryf te bepaal. Die doel van hierdie studie was om ‘n opname te maak in siekte geaffekteerde wingerde in die Vredendal omgewing om sodoende siekte voorkoms en verspreidingspatrone van die siekte in 'n verskeidenheid van kultivars te bepaal. Die veld opnames, gebaseer op visuele simptome van aster vergeling siekte, was bevestig deur polimerase kettingreaksie (PKR). ‘n Opname is ook in en om aster vergeling geaffekteerde wingerde uitgevoer, op soek na moontlike alternatiewe gasheer plante van die fitoplasma. Verspreidingspatrone van astervergeling geaffekteerde wingerde is ontleed met behulp van die PATCHY ruimtelike analise pakket. 'n Vinnige agteruitgang van AY geaffekteerde Chardonnay, wat uiteindelik gelei het tot die afsterf van wingerde, is waargeneem, wat die sensitiwiteit van Chardonnay teenoor wingerdvergeling infeksie bevestig. Simptoomlose astervergeling fitoplasma infeksies kom voor en astervergeling fitoplasma kon nie opgespoor word in alle simptomatiese wingerdstokke nie, wat op oneweredige verspreiding van AY fitoplasma in individuele wingerdstokke dui. Molekulêre ontledings met behulp van PKR-RFLP het getoon dat alle wingerdstokke, wat in die Vredendal omgewing getoets is, slegs astervergeling fitoplasma bevat. Geen fitoplasmas was teenwoordig in enige onkruide of ander moontlike gasheer plante. Hoewel die gemiddelde jaarlikse siekte voorkoms van Chardonnay (29,95%) en Chenin Blanc (16,64%) oor die vier-jaar opname periode hoër was as dié van Pinotage (5,80%), was daar geen statisties beduidende verskil tussen die siekte voorkoms van hierdie drie kultivars nie. Die gemiddelde jaarlikse siekte voorkoms het 'n tendens oor tyd getoon, en die siekte voorkoms van die eerste jaar was betekenisvol laer as dié van die ander jare. Chardonnay het ‘n kumulatiewe siekte voorkoms van 37.77% aan die einde van die 4-jaar studie getoon, wat beteken dat Chardonnay wingerde binne 10 jaar 100% besmet kan wees met AY. Verspreidingspatrone van AY geaffekteerde wingerdstokke was meestal nie-ewekansig met bondeling van geaffekteerde wingerdstokke in en oor wingerd rye. Bondeling van AY geaffekteerde wingerdstokke het, met die uitsondering van een wingerd, meestal op die kant van wingerde aanliggend aan besmette wingerde, voorgekom. Die epidemiologiese studie gee 'n aanduiding van die sensitiwiteit van die verskillende kultivars ten opsigte van AY, die tempo van die verspreiding en die toekomstige impak van die siekte op die Suid-Afrikaanse wynbedryf. Dit dra ook waardevolle inligting by tot die ontwikkeling van 'n strategie vir die bestuur van wingerdvergeling siekte in Suid-Afrikaanse wingerde.

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 (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Very little is known about the diversity of fungi associated with Afromontane forests of the Cape Floristic Region (CFR) of South Africa. The ophiostomatoid fungi include many species, some known as pathogens in the CFR, while others are well-known saprophytes important in wood degradation. This study focused on the biodiversity and ecology of tree-associated ophiostomatoid fungi (Ophiostomatales) in the CFR. In addition to this, mites and subcortical beetles associated with the CFR trees were collected, regardless of whether they were associated with ophiostomatoid fungi or not. A relatively high diversity of ophiostomatoid fungi were collected from native trees, ten of which were newly described here. Three further fungal species, two of which are probably new to science, were also collected from exotic Pinus species growing in these forests. Four Ophiostomatales species (including three newly described species) were associated with subcortical beetles on Rapanea melanophloeos and Olea capensis ssp. macrocarpa. These were Sporothrix pallida, Sporothrix aemuluphilus, Raffaelea scabbardiae and Raffaelea rapaneae, associated with the beetles Lanurgus sp. 1, Ctonoxylon sp. 1, Xyleborinus aemuluphilus and a Platypodinae species. This represents a first study to explore the associations between subcortical beetles and ophiostomatoid fungi on native trees in the CFR. In addition to fungi associated with subcortical beetles, several members of the Ophiostomatales associated with wounds on Rapanea melanophloes trees were also collected. These included Ophiostoma stenoceras, Sporothrix reniformis, S. rapaneae, S. lunateae and S. noisomeae. All but O. stenoceras were new to science, and were formally described here. All of these wound-associated species from R. melanophloeos belong to the Sporothrix schenckii – O. stenoceras complex, except for S. noisomeae that was provisionally placed in the S. lignivora complex. Besides fungal taxa collected from wounds on Rapanea melanophloeos, other fungi were also collected from wounds on other host trees species. Three more previously undescribed ophiostomatoid fungal species were collected from this niche. They included Sporothix capensis collected from O. capensis ssp. macrocarpa, Graphilbum roseus collected from many different, unrelated host trees and Graphium ilexiense (Microascales), isolated from wounds on Ilex mitis. The latter represented the first isolation of an ophiostomatoid fungus from this host tree species. Two possibly new fungal species (Sporothrix sp. 1, Ceratocystiopsis sp. 1) and Ophiostoma ips, associated with three bark beetles (Orthotomicus erosus, Hylurgus ligniperda and Hylastes angustatus), were collected from Pinus. Several fungal species were collected from both native trees and non-native trees. These included Sporothrix fusiforme from Brabejum stellatifolium and Acacia mearnsii, O. quercus and O. pluriannulatum-like fungus from several native trees and from A. mearnsii. This suggests a possibility for host shifting of some of these fungi between native and non-native hosts or even between different native hosts. Eight non-ophiostomatoid fungi associated subcortical beetles taxa were found also to infest native trees in the Afromontane forests and in total more than 4500 beetle individuals were collected. Some species of ophiostomatoid fungi collected in this study were found to be associated with other arthropods such as mites. Four phoretic mites species associated with ophiostomatoid fungi (Dendrolaelaps quadrisetus, Histiogaster sp. 3, Elattoma sp. 1 & 2) were collected. In addition, sixteen species of tree wound-associated mites were collected from 12 native trees. Of these, nine were associated with several ophiostomatoid fungi (Graphilbum roseus, O. pluriannulatum-like, O. quercus) that were isolated from several different host trees. This suggests that they may aid in the transport of these fungi from one host species to another. The possible consequences of transfers of Ophiostomatales species between hosts were tested using pathogenicity tests, which highlighted that some fungi are pathogenic on several different trees. Transfers seemed most likely in fungal species isolated from wounds, especially those associated with mites, because the mites may aid in the vectoring of these. When phoretic mites were tested for their specificity to their vector beetles, they proved to be highly specific. Although some of the fungi associated with these mites and their sub-cortical beetles were also pathogenic, it is less likely for these fungi to be transferred to other host tree species due to the high specificity of their arthropod associates. This study represents one of a few studies that focused on ophiostomatoid fungi, subcortical beetles and mites associated with trees in the Afromontane forests of South Africa. Although we collected a high diversity of Ophiostomatales members, many more still await discovery. It is recommended that future studies focus on the complex inter-organismal interactions in many of the systems uncovered in this study.
AFRIKAANSE OPSOMMING: Baie min is bekend oor die diversiteit van fungi wat met die Afromontane woude van die Kaapse Floristiese Streek (KFS) van Suid Afrika geassosieer is. Die ophiostomatoïde fungi sluit baie spesies in, sommiges bekend as patogene in die KFS, terwyl ander bekende en belangrike saprofiete in houtdegradasie is. Hierdie studie het op die biodiversiteit en ekologie van die boom-geassosieerde ophiostomatoïde fungi (Ophiostomatales) in die KFS gefokus. Daarbenewens is myte en subkortikale kewers wat met die KFS bome geassosieer word ook versamel, ongeag of hulle geassosieerd was met ophiostomatoïde fungi of nie. „n Relatief hoë diversiteit van ophiostomatoïde fungi is van inheemse bome versamel, tien waarvan hier nuut beskryf is. Drie verdere fungi spesies, twee waarvan ook waarskynlik nuut is tot die wetenskap, is ook vanaf Pinus spesies versamel wat in hierdie woude gegroei het. Vier Ophiostomatales spesies (insluitend drie nuut beskryfde spesies) wat met subkortikale kewers op Rapanea melanophloeos en Olea capensis L. ssp. macrocarpa geassosieer is, is ook versamel. Hulle was Sporothrix pallida, Sporothrix aemuluphilus, Raffaelea scabbardiae en Raffaelea rapaneae, geassosieer met die kewers Lanurgus sp. 1, Ctonoxylon sp. 1, Xyleborinus aemuluphilus en „n Platypodinae spesie. Hierdie verteenwoordig die eerste studie wat die assosiasies tussen subkortikale kewers en ophiostomatoïde fungi op inheemse bome in die KFS ondersoek. Addisioneel tot fungi geassosieer met die subkortikale kewers, is verskeie lede van die Ophiostomatales vanaf wonde op Rapanea melanophloes bome versamel. Hulle sluit in Ophiostoma stenoceras, Sporothrix reniformis, S. rapaneae, S. lunateae en S. noisomeae. Almal behalwe O. stenoceras was nuut tot die wetenskap, en is hier formeel beskryf. Al hierdie wond-geassosieerde spesies vanaf R. melanophloeos behoort aan die Sporothrix schenckii – O. stenoceras kompleks, behalwe vir S. noisomeae wat voorlopig in die S. lignivora kompleks geplaas is. Benewens fungi taxa wat van die wonde op Rapanea melanophloes versamel is, is ander fungi ook vanaf die wonde op ander gasheer boom spesies versamel. Drie verdere ophiostomatoïde fungus spesies is in hierdie nis versamel. Hulle sluit in Sporothix capensis wat vanaf O. capensis ssp. macrocarpa versamel is, Graphilbum roseus wat vanaf baie verskillende, onverwante gasheer bome versamel is en Graphium ilexiense (Microascales), wat vanaf wonde op Ilex mitis versamel is. Laasgenoemde verteenwoordig die eerste isolasie van „n ophiostomatoïde fungus vanaf hierdie gasheer boom spesie. Twee moontlik nuwe fungus spesies (Sporothrix sp. 1, Ceratocystiopsis sp. 1) en Ophiostoma ips, geassosieer met drie baskewers (Orthotomicus erosus, Hylurgus ligniperda en Hylastes angustatus) is vanaf Pinus versamel. Verskeie fungi spesies is van beide inheemse en nie-inheemse bome versamel. Hulle het Sporothrix fusiforme vanaf Brabejum stellatifolium en Acacia mearnsii, O. quercus en O. pluriannulatum-like fungus vanaf verskeie inheemse bome en vanaf A. mearnsii ingesluit. Dit suggereer die moontlikheid van gasheer-skuiwing van sommige van hierdie fungi tussen inheemse en uitheemse gashere of selfs tussen verskillende inheemse gashere. Agt nie- ophiostomatoïde geassosieerde subkortikale kewers was ook versamel en in totaal is meer as 4500 kewer indiwidue versamel. Sommige ophiostomatoïde fungus spesies wat in hierdie studie versamel is, was met ander geleedpotiges soos myte geassosieer. Vier foretiese myt spesies wat met ophiostomatoïde fungi geassosieer is (Dendrolaelaps quadrisetus, Histiogaster sp. 3, Elattoma sp. 1 & 2), is versamel. Nege addisioneële myt spesies was met verskeie ophiostomatoïde spesies vanaf verskeie boomspesies geassosieer (Graphilbum roseus, O. pluriannulatum-like, O. quercus). Dit suggereer dat myte die vervoer van hierdie fungi van een gasheer spesie na die ander mag bewerkstellig. Die moontlike gevolge van die oordrag van Ophiostomatales spesies tussen gashere is getoets deur patogeniteitstoetse. Dit het beklemtoon dat sommige fungi patogenies is op verskeie onverwante boomspesies. Oordraag van spesies is mees waarskynlik in fungi spesies wat vanaf wonde geisoleer is, veral dié wat met myte geassosieer is, want die myte mag hierdie fungi help vervoer. Toe foretiese myte getoets is vir hulle spesifisiteit tot hulle vektore, is hulle hoogs spesifiek bevind. Alhoewel sommige fungi wat met hierdie myte en hulle geassosieerde kewers geassosieer word wel patogenies is, is dit minder waarskylik dat hulle na ander gasheer bome sal verskuif as gevolg van die hoë spesifisiteit van hulle geleedpotige assosiate. Hierdie studie verteenwoordig een van net enkele studies gefokus op ophiostomatoïde fungi, subkortikale kewers en myte wat met bome van die Afromontane woude van Suid-Afrika geassosieer is. Alhoewel ons „n hoë diversiteit van Ophiotomatale lede versamel het, wag baie meer fungi spesies waarskynlik nog op ontdekking. Daar word voorgestel dat toekomstige studies fokus op die komplekse inter-organismiese interaksies in baie van die sisteme wat in hierdie studie blootgelê is.

The common maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), a well known pest of stored-maize grain in most parts of the world, was identified as one of the major constraints of harvested maize grains in the Eastern Cape Province, South Africa. The use of plants or their products is one of the recent methods being investigated for insect pest control worldwide. Therefore, the main aim of the present study was to identify available plants in the Eastern Cape that could be used to combat the problem of Sitophilus zeamais in stored-maize grains. For the realization of the aims of this research, the following studies were carried out; a preliminary survey was conducted to obtain baseline information on the farmers’ knowledge and experience of indigenous insect pest control methods in the Eastern Cape. Also, studies on the insecticidal potential of the essential oils of some plants were investigated against the maize weevil. The quality parameters of maize grains treated with the essential oils was also studied and, using a rat model, the toxicity of the essential oils was investigated. The outcome from this study revealed that there is awareness amongst the farmers in the Eastern Cape on the use of plants or their products to control insect pests. Unfortunately, such methods are currently being neglected and the knowledge of their application was found to be eroding. Among the various essential oils screened were those from Mentha longifolia L. and Tagetes minuta L. which evoked an appreciable level of contact, fumigant and repellent toxicity on the maize weevil. Further work done to determine the effects of the oils on maize stored over a period of three months revealed that the two oils had no adverse effect on the proximate compositions and some quality parameters of the stored maize. However, the toxicological study conducted on rats showed that the oils at tested concentrations exhibited some level of toxicity. It is, therefore, suggested that the essential oils of M. longifolia and T. minuta should not be used to treat maize grains intended for human consumption.

Thesis (PhDAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Barley leaf scald, caused by Rhynchosporium secalis, is the most important disease of barley (Hordeum vulgare) in the Western Cape province of South Africa. The disease was first reported from South Africa in 1937. The present study is the first attempt to characterise the South African R. secalis population. Topics such as pathogenesisrelated proteins, virulence spectra, variability of pathotypes, sources of variation, host resistance, breeding strategies, molecular characterisation and fungicide sensitivity are summarised in Part 1 of this dissertation. In succeeding Parts the focus is on the characteristics of the local R. secalis population regarding virulence spectrum, DNA polymorphisms, in vitro as well as in vivo fungicide sensitivity. These aspects are treated as separate entities, leading to some duplication which is unavoidable. In Part 2 the virulence spectra of 50 R. secalis isolates from a population in the. Western Cape province were determined. Twenty-one races were detected using 17 differential barley cultivars. The two most prevalent races, namely races 4 and 7 had three and four virulence genes respectively. Both race 4 and 7 were virulent on the most susceptible cultivars, namely West China, Steudelli, C.I.8618 and C.I.2226. Considering the resistance genes reported for cultivars Atlas 46, Turk, and C.I.3515 which showed no susceptible cultivar-pathogen interaction, it would appear that the Rh- Rh3-Rh4 complex is primarily involved in conferring resistance to the local R. secalis isolates. A total of 20 races (47 isolates) characterised in Part 2 were selected for further characterisation by means of DNA fingerprinting. In Part 3 an anonymous multilocus DNA probe was used to characterise the genotypic structure of these isolates by means of RFLP analysis. No correlation between any particular fingerprint pattern, race, district, field or lesion was found. The two most prevalent races, 4 and 7, did not share the same genotypes, even when isolated from the same field or lesion. The genotypic diversity of the isolates studied was 46.5% of the theoretical maximum diversity. The high level of genotypic variation observed in the South African R. secalis population resembled the genotypic diversity observed in other cereal pathogens with known sexual structures. Although no teleomorph has yet been observed, these data suggest that sexual recombination may operate within the local population of R. secalis. In South Africa barley scald is primarily controlled by means of fungicides. The continued use of fungicides on cereal crops results in the build-up of fungicide resistance in the population, which could lower the efficacy of these compounds. These aspects were investigated in Part 4, where isolates (collected during 1993 to 1995) were evaluated in vitro for sensitivity to triadimenol, tebuconazole, flusilazole and propiconazole. The sensitivity fluctuated but in 1995 isolates were significantly less sensitive towards triadimenol than in the previous two years. In a second experiment, isolates collected from two fields with a 5-6 year-history of triadimenol seed treatments and tebuconazole applications, were evaluated for their fungicide sensitivity. A significant positive correlation was observed between tebuconazole and triadimenol sensitivity among,R. secalis populations from these fields. However, such a correlation was not found within the R. secalis population collected during 1993-1995 where shorter crop rotation patterns and a range of fungicides was applied. In a third experiment, the fungicide sensitivity of local R. secalis isolates was evaluated towards two new triazole fungicides, namely bromuconazole and triticonazole. Correlation coefficients observed between these new triazoles and those previously applied in South Africa were not significantly positive. The lack of significant cross-resistance has important practical implications regarding the management of fungicide resistance. In Part 5, isolates with different minimum inhibitory concentration (MIC) towards tebuconazole in vitro (1, 3 and 10 ug/ml) were compared in vivo. The aim of this study was to determine how MIC values would influence virulence (leaf area affected) and sporulation. Results indicated that all isolates were equally fit to induce lesions and sporulate in the absence of tebuconazole. Thus no fitness cost was associated with the degree of tebuconazole sensitivity in the present study. All R. secalis isolates were able to induce lesions on tebuconazole treated leaves, but differed significantly with respect to the percentage leaf area affected. Isolates, least sensitive (MIC = 10 ug/rnl) towards tebuconazole were more adapted on tebuconazole treated leaves, being able to repeatedly cause larger lesions than sensitive R. secalis isolates (MIC = 1 ug/rnl), Sporulation was not significantly different between isolates on lesions of untreated or tebuconazole treated leaves. Larger leaf areas affected and adequate sporulation suggest that a less sensitive population would result in more disease in tebuconazole treated fields. In conclusion, this study revealed the variability associated with the South African R. secalis population regarding virulence spectrum and genotypic structure. The data in this study suggest that it is likely that the local population will easily adapt to newly introduced, single gene resistance. For more durable resistance, higher levels of quantitative resistance should be introduced. This type of resistance is, however, more difficult to identify and incorporate than single gene resistance. Consequently, barley scald control will remain dependent on the efficacy of fungicide applications. Furthermore, the lack of cross-resistance and low frequency of resistant isolates indicates a low risk for the development of fungicide resistance in the local R. secalis population. Other factors such as current crop rotation practices and the range of fungicides being ~pplied also contribute to this low risk level. However, the status of these factors can change over time. The in vivo tebuconazole sensitivity study has indicated that a resistant field population of R. secalis may be able to build-up. It is, therefore, necessary to monitor the fungicide sensitivity of R. secalis isolates at timely intervals with view to successful barley cultivation in the future.
AFRIKAANSE OPSOMMING: Blaarvlek op gars (Hordeum vulgare), veroorsaak deur Rhynchosporium secalis, is die belangrikste siekte van gars in die Wes-Kaap provinsie van Suid-Afrika. Die voorkoms van R. secalis op gars is in Suid-Afrika vir die eertse keer in 1937 gerapporteer. Hierdie studie is die eerste poging tot karakterisering van die plaaslike R. secalis-populasie. Aspekte soos proteïene betrokke by patogenese, virulensiespektra, variabiliteit van patotipes, bronne van variasie, gasheerweerstand, teeltprogramme, molekulêre karakterisering en swamdodersensitiwiteit word in Deel I van die tesis opgesom. In die daaropvolgende gedeelte is die fokus op die karakterisering van die R. secalis-populasie en behels DNA karakterisering, virulensiespektrum, en swamdodersensitiwiteit in vitro asook in vivo. .. In Deel 2 is die virulensiespektra van 50 R. secalis isolate van 'n populasie in die. Wes-Kaap geëvalueer teenoor 17 differensiëel weerstandbiedende gars kultivars en hieruit is 21 rasse geïdentifiseer. Die twee mees algemene rasse (rasse 4 en 7), met onderskeidelik drie en vier virulensie gene, het virulent vertoon teenoor die mees vatbare kultivars soos West China, Steudelli, C.I.8618 en C.I.2226. Geen vatbare kultivar-patogeen interaksies is met kultivars Atlas 46, Turk en C.I.3515, wat al drie die Rh-Rh3-Rh4 kompleks dra, gevind nie. Dit wil dus voorkom asof hierdie genekompleks effektiewe gasheerweerstand teen die plaaslike R. secalis isolate kan bied. 'n Totaal van 20 rasse (47 isolate), gekarakteriseer in Deel 2, is geselekteer vir verdere karakterisering met behulp van DNA bandpatrone. In Deel 3 is 'n anonieme multilokus DNA peiler gebruik om deur middel van RFLP analise die genotipiese struktuur van hierdie R. secalis-isolate te bepaal. Geen assosiasie is gevind tussen DNA bandpatroon en ras, distrik, garsland of letsel nie. Die twee rasse (4 en 7) wat mees algemeen voorkom, het nie dieselfde bandpatroon vertoon nie, ook nie dié afkomstig vanuit dieselfde garsland of letsel nie. Die genotipiese diversiteit van isolate was 46.5% van die teoretiese maksimum diversiteit. Die hoë vlak van variasie waargeneem in die R. secalis populasie is soortgelyk aan variasie waargeneem in ander graanpatogene wat oor 'n geslagtelike stadium in die lewenssiklus beskik. Alhoewel geen geslagtelike stadium tot dusver geidentifiseer is nie, dui die vlak van variasie daarop dat geslagtelike rekombinasie moontlik wel plaasvind binne die plaaslike R. secalis populasie. In Suid-Afrika word blaarvlek op gars primêr deur swamdoders beheer. Die toenemende gebruik van swamdoders op graangewasse veroorsaak moontlik 'n opbou van swamdoderweerstand in die populasie. Dit kan die effektiwiteit van swamdoders verlaag. Hierdie veronderstelling is in Deel 4 ondersoek, waar die sensitiwiteit van isolate in vitro teenoor triadimenol, tebukonasool, flusilasool en propikonasool geëvalueer is. Die triasooi sensitiwiteit van R. secalis isolate wat gedurende die 1993- 1995 seisoen versamel is het gewissel en slegs vir triadimenol was daar 'n tendens na meer weerstandbiedenheid. 'n Swamdoder-evaluasie is in 'n aparte eksperiment op isolate gedoen wat versamel is vanaf twee garslande met 'n 5-6 jaar geskiedenis van triadimenol saadbehandelings en tebukonasool bespuitings. 'n Betekenisvolle positiewe korrelasie is waaJ~geneem tussen tebukonasool en triadimenol sensitiwiteit in R. secalis isolate afkomstig vanaf hierdie twee garslande. 'n Soortgelyke korrelasie is egter nie gevind in die populasie wat gedurende die 1993-1995 seisoene versamel IS me. Laasgenoemde kan moontlik toegeskryf word aan korter wisselboupatrone en die toediening van 'n verskeidenheid van swamdoders. In 'n derde eksperiment is die sensitiwiteit van plaaslike R. secalis isolate teenoor twee nuwe triasole, naamlik bromukonasool en tritikonasool getoets. Die korrelasie waargeneem tussen die twee nuwe triasole en triasooi swamdoders reeds voorheen in gebruik in die Wes-Kaap was me betekenisvol positief me. Die gebrek aan betekenisvolle kruisweerstandbiedendheid het belangrike praktiese implikasies vir die bestuur van swamdoder -weerstandbiedendheid. In Deel 5 is isolate met wisselende minimum inhiberende konsentrasies (MIKs) teenoor tebukonasool in vitro (1, 3 en 10 ug/ml) en in vivo vergelyk. Die doel van hierdie studie was om te bepaal hoe wisselende MIK-waardes virulensie (blaaroppervlakte geïnfekteer) en sporulasie sal beïnvloed. Resultate dui daarop dat alle R. secalis isolate in hierdie studie ewe fiks was om, in die afwesigheid van tebukonasool, letsels te induseer en te sporuleer. Die bevinding is dat die verlies in fiksheid nie geassosieer is met die mate van tebukonasool weerstand nie. Alle R. secalis isolate het die vermoë gehad om letsels op tebukonasool-behandelde blare te veroorsaak maar het betekenisvol verskil ten opsigte van die blaaroppervlakte geaffekteer. Isolate wat minder sensitief (MIK = 10 ug/rnl) teenoor tebukonasool in vitro is, het meer aangepastheid op tebukonasool-behandelde blare getoon. Gevolglik het hierdie isolate herhaaldelik meer letsels veroorsaak as sensitiewe isolate (MIK = 1 ug/ml), Sporulasie het nie betekenisvol verskil tussen isolate vanaf letsels op ondehandelde of tebukonsoolbehandelde blare nie. Hierdie resultate dui egter daarop dat 'n minder sensitiewe populasie tot meer siektevoorkoms in tebukonasool-bespuite lande kan lei. Die studie het die veranderlike karakter van die Suid-Afrikaanse R. secalispopulasie aangaande virulensiespektrum en genotipiese struktuur blootgelê. Dit is dus baie moontlik dat die R. secalis-populasie maklik sal aanpas by teelmateriaal met nuwe enkelgeen-weerstand. Vir volgehoue gasheerweerstand is dit egter nodig dat hoër vlakke van kwantitatiewe weerstand ingeteel moet word. In die praktyk is hierdie tipe weerstand egter baie moeiliker om te identifiseer en by nuwe teelmateriaal in te sluit as in die geval van enkelgeen-weerstand, Dit bring mee dat blaarvlekbeheer afhanklik bly van swamdodertoedienings as beheermaatreël. Die resultate van hierdie studie dui daarop dat daar tans 'n lae risiko vir die ontwikkeling van swamdoderweerstand in die plaaslike populasie is, as gevolg van die afwesigheid van kruisweerstandbiedendheid en die lae voorkoms van weerstandbiediende isolate. Ander faktore soos die wisselboustelsels wat toegepas word en die verskeidenheid van swamdoders toegedien dra ook daartoe by. Ten spyte hiervan kan die status van hierdie faktore egter oor tyd verander. Die in vivo tebukonasool studie het daarop gedui dat 'n weerstandbiedende veldpopulasie van R. secalis die potensiaal het om te vermeerder. Gevolglik is die tydige monitering van swamdodersenisitiwiteit van R. secalis isolate noodsaaklik om 'n volhoubare garsproduksie te verseker.

Thesis (MScAgric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Botrytis cinerea Pers.: Fr., a pathogen of grapevine (Vitis vinifera L.), moves mainly through conidia by air currents in vineyards which are deposited intermittently on the surfaces of leaves, inflorescences and bunches. Little is known about the relationship between the inoculum dosage in air and incidence of Botrytis bunch rot, and how the relationship is influenced by environmental and host factors. To better understand this relationship, information is needed on the period over which conidia have accumulated, the time they are able to survive and remain infectious, time of symptom expression in relation to conidium arrival at the infection court and host surface wetness. The aims of this study were (i) to estimate the amount of viable B. cinerea occurring in air in vineyards, and at different positions on leaves, inflorescences and bunches of grape at different phenological stages, (ii) to determine the relationships between the number of B. cinerea colonies recorded on spore traps placed in the bunch zone of vines and the incidence of B. cinerea recorded from the different tissues, and (iii) to compare the efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum with those inoculated with dry, airborne conidia. Different techniques were used to detect viable Botrytis cinerea in air currents and on plant material obtained from table (cultivars Dauphine and Waltham Cross in Paarl- and Worcester-district) and wine grape (cultivars Chardonnay, Sauvignon Blanc and Merlot in Stellenbosch- and Malmesbury district) vineyards in the Western Cape province during 2001-02 and 2002-03. For four consecutive days during prebloorn, bloom, pea-size, bunch closure, veraison and harvest, sets of Petri dishes with freshly prepared Kerssies' B. cinerea selective medium (spore traps) were left overnight in the bunch zone of vines. Plant material was collected from the vines on the fourth day. Leaves, infloresence and bunches were treated with paraquat to terminate host resistance and to promote the development of the pathogen on the tissues. The B. cinerea inoculum dosage in air, and the incidence at which the pathogen was detected at various positions on leaves and in bunches normally differed between vineyards. However, the various tests revealed that the pathogen generally occurred in a consistent pattern in air in the bunch zone of vines, on leaves and in bunches from all vineyards. The inoculum dosage in air in the bunch zone of the vine was generally highest during prebioom or during bloom, it decreased at pea size and mostly remained at a very low level at the later growth stages. The estimations of viable B. cinerea residing naturally on leaves and in bunches, showed that their amounts depicted levels occurring in air in the bunch zone of the vine. Necrotic leaves occurring early season in vineyards were identified as an important source of secondary inoculum for dispersal to the developing bunches. Latent infections at the various positions in bunches were few at véraison and harvest. However, due to the necrotrophic ability of the pathogen, extensive berry rot (due to berry-to-berry contact) and thus severe bunch rot developed from a single berry that become symptomatic at the base of the pedicel/berry attachment zone. The B. cinerea occupation pattern explains why Botrytis bunch rot develops mostly from the inner bunch and why disease management strategies should concentrate on the bloom to pre-bunch closure stage and on inhibiting B. cinerea development in the inner bunch during the early part of the season. Thus, to effectively reduce B. cinerea in grapevine, preventative applications are recommended to reduce two primary infection events: (a) between budding and pre-bloom to counteract primary leaf infection; (b) during late bloom or early pea size stage, to reduce the amount of the pathogen on leaves and infloresences and to prevent colonisation of floral debris. A third spray can be applied at bunch closure to reduce the amount of B. cinerea at various positions of the inner bunch, especially for cultivars with tight bunches. The efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum was compared with those inoculated with dry, airborne conidia. Shoots were obtained during late bloom from a vineyard (wine grape cultivar Merlot) in the Stellenbosch region. The shoots were divided into two main groups. One group of shoots was left uninoculated, the other shoots were inoculated by dusting with dry B. cinerea conidia in a settling tower. Before inoculation, equal numbers of shoots in each main group was sprayed with fenhexamid, or left unsprayed. Following inoculation and incubation, shoots of each treatment were divided in two equal groups. The one lot of shoots were rinsed in water. The other lot of shoots were immersed in paraquat solution to terminate host resistance and to promote the development of the pathogen from the tissues. For both uninoculated and inoculated shoots, irrespective of fungicide treatment, leaves remained asymptomatic at both the blade and petiole position for the water rinse treatment. No symptom of B. cinerea decay developed at any of the positions on leaves from shoots sprayed with fenhexamid. Spraying of shoots with fenhexamid completely suppressed B. cinerea infection and symptom expression on both uninoculated and inoculated inflorescens. For inoculated shoots, B. cinerea developed from approximately 50% of the laterals in the water rinse treatment. However, inflorescences rinsed in water remained asymptomatic. The laboratory studies showed that fungicides, if applied properly to shoots and bunches under controlled conditions, effectively reduced the amount of B. cinerea at the various positions on leaves and inflorescence, and prevented infection and symptom expression at bloom. However, these goals are not achieved in vineyards where the fungicides are applied by conventional spraying methods. Therefore, more work is needed to evaluate fungicide application techniques by conventional spraying methods for proper fungicide coverage, and the reduction of B. cinerea in bunches.
AFRIKAANSE OPSOMMING: Botrytis cinerea Pers.: Fr., 'n patogeen van druiwe (Vilis vinifera L.), beweeg hoofsaaklik deur middel van konidia in lugstrome deur die wingerd, en word dan afwisselend op die oppervlakte van die blare, bloeiwyses en trosse gedeponeer. Daar is nog min bekend oor die verhouding tussen die hoeveelheid inokulum in die lug en die voorkoms van Botrytis op die trosse, en hoe die verhouding deur omgewings- en gasheerfaktore beïnvloed word. Ten einde hierdie interaksie beter te verstaan, word inligting benodig oor die tydperk waarin die konidia akkumuleer, die tyd wat hulle oorleef en virulent bly, en die tyd van simptoom-uitdrukking in verhouding tot die verspreiding van die konidia by die infeksie-setel en benatbaarheid van die gasheer-oppervlakte. Die doel van hierdie studie was (i) om die hoeveelheid lewensvatbare B. cinerea wat in die lug voorkom, asook by verskeie posisies op blare, bloeiwyses en trosse by verskillende fenologiese stadiums te kwantifiseer, (ii) om die verhouding tussen die aantal aangetekende B. cinerea kolonies op spoorvangers wat in die trossone van die wingerd geplaas is, en die voorkoms van B. cinerea, aangeteken van verskeie weefsels, te bepaal, en (iii) om die effektiwiteit van fenhexamid op blare en bloeiwyses wat natuurlike B. cinerea inokulum dra, te vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Verskillende tegnieke is gebruik om lewensvatbare Botrytis cinerea in lugstrome en op plantmateriaal van tafeldruiwe (kultivars Dauphine en Waltham Cross In Paarl- en Worcester-distrik) en wyndruiwe (kultivar Chardonnay, Sauvignon Blanc en Merlot in Stellenbosch- en Malmesbury distrik) in wingerde van die Wes-Kaap provinsie gedurende 2001-02 en 2002-03 te kwantifiseer. Petri bakkies met vars voorbereide Kerssies medium, selektief vir B. cinerea (spoorvangers), is vir vier agtereenvolgende dae gedurende vóórblom, blom, ertjiekorrel, trostoemaak, kleurbreek en oes, oornag in die trossone van wingerdstokke in betrokke wingerde, gelaat. Plantmateriaal is op die vierde dag versamel. Blare, bloeiwyses en trosse is met paraquat behandel ten einde die gasheerweerstand af te breek en ontwikkeling van die patogeen op die weefsel te bevorder. B. cinerea inokulum in die lug, en die frekwensie waarby die patogeen op verskeie posisies op blare en in die trosse voorgekom het, het normaalweg tussen wingerde verskil. Die verskeie toetse het getoon dat die patogeen normaalweg in 'n vaste patroon in die lug en die trossones van wingerde, asook op blare en in trosse van alle wingerde voorkom. Die inokulumkonsentrasie in die lug in die trossones van wingerdstokke was normaalweg die hoogste gedurende vóórblom of gedurende blom. Die inokulumdruk het by ertjiekorrel verminder en meestal by 'n 'n baie lae vlak tydens die latere groeistadia gebly. Die bepaling van lewensvatbare B. cinerea wat natuurlik op blare en in trosse gedeponeer is, het getoon dat hul hoeveelhede ooreenstem met vlakke wat in die lug in die trossone van die wingerd voorkom. Nekrotiese blare vroeg in die seisoen is 'n belangrike bron van sekondêre inokulum en speel dus 'n belangrike rol by die verspreiding van Botrytis tussen die ontwikkelende trosse. Latente infeksies by die verskeie posisies in trosse was laag by kleurbreek en oes. Weens die saprofitiese vermoëns van die patogeen, kan uitgebreide korrelvrot (a.g.v. korrel-tot-korrel kontak) en dus ernstige trosvrot, ontwikkel. 'n Enkele korrel kan by die basis van die pedisel/korrel vashegtingsone simptomaties raak, en vandaar na aangrensende korrels versprei. Die B. cinerea kolonisasiepatroon verduidelik waarom Botrytis trosvrot meestal vanaf die binneste tros ontwikkel en waarom siektebeheerstrategieë op die vóórblom- tot blomstadium gekonsentreer moet word, en op die inhibering van B. cinerea ontwikkeling in die binneste tros gedurende die vroeë stadia van die seisoen. Dus, om B. cinerea effektief tydens die twee primêre infeksie stadiums in wingerde te verminder, kan voorkomende toedienings aanbeveel word: (a) tussen knopvorming en vóórblom om primêre blaarinfeksie te verhoed; (b) gedurende láátblom en vroeë ertjiekorrel om die hoeveelheid inokulum op die blare en bloeiwyses te verminder, en die kolonisasie van blomdebris te voorkom. 'n Derde toediening kan tydens trostoemaak aangewend word om B. cinerea by verskeie posisies in die binneste tros te verminder, veral by kultivars met digte trosse. Die effektiwitiet van fenhexamid op blare en bloeiwyses waarop natuurlike B. cinerea inokulum voorkom is vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Lote is vanaf 'n wingerd (wyndruif kultivar Merlot) in die Stellenbosch distrik tydens láátblom verkry en in twee hoofgroepe verdeel. Die een groep lote is geïnokuleer deur droë B. cinerea konidia in 'n afsettingstoring te strooi, terwyl die ander groep nie geïnokuleer is nie. Vóór inokulasie, is die helfte van die lote in elke groep met fenhexamid behandel, terwyl die ander helfte onbehandeld gelaat is. Ná inokulasie en inkubasie, is lote van elke behandeling verder in twee eweredige groepe verdeel. Die een groep lote is in water gespoel, terwyl die ander groep lote in 'n paraquatoplossing gedompel is om die gasheerweerstand te verwyder, en die ontwikkeling van die patogeen vanuit die weefsels te bevorder. Vir die waterspoelbehandeling van beide ongeïnokuleerde en geïnokuleerde lote, ongeag van die fungisiedbehandeling, het die blare asimptomaties by beide die bladoppervlakte en blaarsteelposisie gebly. Geen simptome van B. cinerea verrotting het by emge van die blaarposisies van die lote, met fenhexamid gespuit, ontwikkel nie. Die spuit van die lote met fenhexamid het die B. cinerea infeksie en die simptoomontwikkeling op beide die ongeïnokuleerde en geïnokuleerde bloeiwyses heeltemalonderdruk. By die geïnokuleerde lote, het B. cinerea vanaf ongeveer 50% van die laterale in die waterspoelbehandeling ontwikkel, alhoewel, bloeiwyses wat in water afgespoel is, heeltemal asimptomaties gebly het. Laboratoriumstudies het getoon dat fungisiedes, indien korrek toegedien op lote en trosse onder gekontroleerde toestande, tot effektiewe vermindering van B. cinerea getalle by die verskillende posisies op blare en bloeiwyses lei, en infeksie en simptoomuitdrukking tydens blom voorkom. Weens die feit dat die doelwitte nie behaal kan word in wingerde waar die fungisiede deur konvensionele spuitmetodes toegedien is nie, moet meer studies gedoen word om fungisied toedieningstegnieke, by konvensionele spuitmetodes, VIr deeglike fungisiedbedekking en die vermindering van B. cinerea in trosse, te evalueer.

Thesis (PhD)--Stellenbosch University, 2012.
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 20 years. In previous studies nonrecombinant strains of PVY, PVY N and PVY O, were detected in South African potatoes. In a recent study the occurrence of non-recombinant strains of PVY in South African potatoes was shown to have decreased while infection by more virulent recombinant strains, PVY NTN and PVY N-W, had increased dramatically. Infection of potato plants with PVY may cause stunted growth and mosaic or necrotic leaf symptoms which in turn can lead to a significant reduction in yield. Highly virulent recombinant PVY isolates as well as some of the non-recombinant strains may cause potato tuber necrotic ringspot disease (PTNRD) which may result in losses of 10% to total crop failure. For this reason investigation of infection by local recombinant isolates on local cultivars was important. To this end a representative number of isolates were selected for whole genome sequencing based on the relative occurrence of the various isolates in South Africa. A number of these sequenced isolates were subsequently used to infect local cultivars of potato in order to investigate the influence of genetic variation within the viral genome on symptom expression. In this study 27 South African isolates of PVY were sequenced through overlapping RT-PCR fragments. Seven of these isolates, six PVY NTN and one PVY N-W, were used to mechanically infect four local cultivars of potatoes under greenhouse conditions. The infected plants were monitored to establish the rate of systemic spread using a highly sensitive qRT-PCR and resulting tubers were visually screened for PTNRD. Highly variable recombinant isolates appear to be less virulent than the more conserved recombinant isolates possibly indicating molecular determinants for pathogenicity. For this reason the amino acid sequences of the South African isolates were compared to those of international isolates and scrutinized for variation and substitutions. Some South African isolates displayed amino acid substitutions unique to the specific isolate, making them unlike those found internationally. Substitution rates throughout the amino acid sequences differed greatly, with some isolates displaying hardly any changes whilst others varied a great deal from overseas isolates. Certain regions, many of which had specific functions, were more conserved than others. This study further investigated the recombination events within the PVY genome using reticulate phylogenetic analysis, molecular dating and network construction techniques. Unlike existing approaches, the one described in this study neither assumes an underlying strictly bifurcating species tree nor assumes prior knowledge of processes underlying deviations between individual gene trees. Through the use of the resulting robust time calibrated phylogeny, the patterns of diversification and recombination in PVY may be placed in the historical context of human cultivation of potatoes. Through the use of these techniques the study aimed to test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. From these analyses it can be deduced that recombinant strains of PVY were imported into South Africa.
AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengs verliese in die Suid-Afrikaanse aartappelbedryf. Die voorkoms van die virus het grootliks toegeneem oor die afgelope 20 jaar. In vorige studies is nie-rekombinante rasse van PVY, PVY N en PVY O, gedokumenteer in Suid-Afrikaanse aartappels. 'n Onlangse studie het gevind dat die voorkoms van nie-rekombinante rasse van PVY in Suid- Afrikaanse aartappels aansienlik gedaal het terwyl infeksie deur virulente rekombinante rasse, PVY NTN en PVY N-W, dramaties toegeneem het. Infeksie van aartappelplante met PVY kan vertraagde groei en mosaïek- of nekrotiese blaarsimptome veroorsaak wat kan lei tot aansienlike vermindering in opbrengs. Hoogs virulente rekombinante PVY isolate, sowel as sommige nie-rekombinante rasse, kan aartappel nekrotiese ring simptome (PTNRD) veroorsaak wat verliese van 10% tot totale misoes tot gevolg kan hê. Om hierdie rede was die ondersoek van infeksie deur plaaslike rekombinante isolate op plaaslike kultivare belangrik. Vir hierdie doel is 'n verteenwoordigende aantal isolate gekies, gebaseer op die relatiewe voorkoms daarvan in Suid-Afrika, vir heelgenoom-volgordebepaling. Van die isolate is vervolgens gebruik om plaaslike kultivare te besmet ten einde die invloed van genetiese variasie binne die virale genoom op simptoom uitdrukking te ondersoek. In hierdie studie is 27 heelgenoomvolgordes van Suid-Afrikaanse PVY isolate bepaal deur oorvleuelende RT-PCR fragmente. Sewe van hierdie isolate, ses PVY NTN en een PVY N-W, is gebruik om vier plaaslike aartappel kultivare, gegroei onder kweekhuis kondisies, meganies te infekteer. Die geïnfekteerde plante is gemonitor om die tempo van sistemiese verspreiding vas te stel deur middel van 'n hoogs sensitiewe qRTPCR en knolle is visueel inspekteer vir PTNRD. Hoogs variante rekombinante isolate blyk om minder virulent te wees as die meer bewaarde rekombinante isolate wat dui op molekulêre determinante van patogenisiteit. Om hierdie rede is die aminosuurvolgordes van die Suid-Afrikaanse isolate vergelyk met die van internasionale isolate en ondersoek vir variasie en substitusies. Sommige Suid-Afrikaanse isolate vertoon aminosuur substitusies wat uniek is tot die spesifieke isolaat en maak hul dus anders as internasionale isolate. Die aantal aminosuursubstitusies in die volgordes verskil grootliks. In vergelyking met internasionale isolate toon sommige isolate skaars enige veranderinge terwyl ander ‘n aantal verskille toon. Sekere gebiede, waarvan baie spesifieke funksies het, was meer gekonserveerd as ander. Hierdie studie ondersoek ook rekombinasie gebeure binne die PVY genoom deur retikulêre filogenetiese analise, molekulêre datering en netwerk konstruksie tegnieke. In teenstelling met bestaande benaderinge, aanvaar die tegniek wat hier beskryf word nie ‘n streng bifurkeerende filogenie, wat onderliggende verdeel, of enige voorafgaande kennis van die prosesse onderliggend aan afwykings tussen individuele filogenieë nie. ‘n Robuuste, tyd gekalibreer filogenie kan diversifikasie patrone en rekombinasie van PVY plaas in die historiese konteks van menslike verbouing van aartappels. Deur gebruik te maak van hierdie tegnieke poog die studie om te toets of diversifikasie en rekombinasie van PVY rasse plaasgevind het binne die tydsbestek van die inburgering en moderne verbouing van aartappels. Van hierdie ontledinge word afgelei dat rekombinante rasse van PVY wat in Suid-Afrika voorkom, ingevoer is.

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 (MScAgric (Conservation Ecology and Entomology)--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: The obscure mealybug, Pseudococcus viburni (Signoret) (Pseudococcidae), is one of the common and serious pests of apples and pears in South Africa. The management of this pest in South Africa is dominated by the use of insecticides, while research into using natural enemies for biological control of mealybugs is still ongoing. Increasing concern over the environmental impact, pesticide residues in fruits, resistance, and expense associated with frequent use of insecticides make it necessary to investigate alternative biological control methods, such as the use of entomopathogenic nematodes, for the control of mealybugs. Entomopathogenic nematodes have proven comparable or even superior to chemicals in controlling certain insect pests, without residue problems or a harmful effect on the environment. An important aspect of using endemic nematodes includes the identification of species of nematodes and their symbiotic bacterial cells. A study was carried out to describe a new species of Steinernema, which was recovered during a previous survey in citrus orchards in three provinces of South Africa. Morphometrics, morphology, crossbreeding, drawings, light microscopy and Scanning Electron Microscopy (SEM) photographs were used to describe the new species. A cryopreservation method has been simplified and optimised for the long-term storage of Steinernema khoisanae (SF87) and Heterorhabditis zealandica (J34). Different cryoprotectants used included 15% glycerol, 8% ethylene glycol and 8% dimethyl sulfoxide (DMSO), in which S. khoisanae was incubated at room temperature for periods of two, three, four and five days, followed by a methanol wash. An optimum survival rate of 69% was obtained for S. khoisanae after a four-day incubation period in 15% glycerol. This technique has been used for the cryopreservation of H. zealandica, with a 78% survival rate. The thawed nematodes of both species were able to infect Galleria mellonella larvae after 42 days of cryopreservation (-196ºC) and were able to complete their life cycles.
AFRIKAANSE OPSOMMING: Die ligrooswitluis, Pseudococcus viburni (Signoret) (Pseudococcidae), is een van die algemene en ernstige peste van appels en pere in Suid-Afrika. Die bestuur van hierdie pes word tans in Suid-Afrika deur die gebruik van insekdoders gedomineer terwyl navorsing oor die gebruik van natuurlike vyande vir die beheer van P. viburni nog aan die gang is. Die verhoogde kommer oor die omgewing, residue in vrugte, weerstand, en die koste verbonde aan die gereelde gebruik van chemiese middels maak dit nodig om alternatiewe biologiese metodes van beheer, soos die gebruik van entomopatogeniese nematodes vir die beheer van witluis, te ondersoek. In ander lande is reeds aangetoon dat entomopatogeniese nematodes onder sekere omstandighede en vir sekere insekte gelykwaardige of selfs beter beheer kan gee as chemiese middels. ʼn Belangrike aspek van die gebruik van endemiese nematodes vir die beheer van insekte sluit die korrekte identifikasie van die spesies met hul geassosieerde bakteriese simbionte in. ʼn Nuwe spesie van Steinernema is uit ʼn vorige opname van entomopatogeniese nematodes in sitrusboorde in drie provinsies van Suid-Afrika geïsoleer. Morfometrie, morfologie, kruisteling, ligmikroskoop en SEM fotografie is gebruik om ʼn nuwe spesies te beskryf. ʼn Kriopreserveringsmetode is ontwikkel en ge-optimaliseer vir die langtermyn bewaring van Steinernema khoisanae (SF87) en Heterorhabditis zealandica (J34). Verskillende kriobeskermingsmiddels insluitend 15% gliserol, 8% dimetiel sulfokied (DMSO) en 8% etileen glikol, waarin S. khoisanae vir periodes van twee, drie, vier, en vyf dae geïnkubeer is, is teen kamertemperatuur, getoets, gevolg deur ʼn metanolbad. Optimum oorlewing van 69% is verkry vir S. khoisanae nadat die infektiewe larwes (IJ) vir vier dae in 15% gliserol gehou is. Hierdie tegniek is ook toegepas op H. zealandica, met 78% oorlewing van die IJ. Die ontvriesde nematodes van beide spesies was in staat om Galleria mellonella larwes suksesvol te infekteer en hulle lewensiklus te voltooi nadat hulle vir 45 dae onder kriopreservering gehou is teen -196ºC.

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: The Cape Floristic Region (CFR) of South Africa is well-recognised for exceptionally high plant species diversity and endemism. However, little attention has been bestowed on the pests and pathogens in this region, even though these may greatly influence plant distribution and evolution. In this study we identify various arthropods and fungi as pests and diseasecausing organisms of the ecologically and economically important CFR-endemic tree taxa of Virgilia. We isolated, identified and determined the pathogenicity of key fungal taxa from diseased Virgilia trees throughout the CFR. In addition we evaluated the role of possible pest arthropod taxa, including bark beetles, phoretic mites, larvae of a cerambycid beetle and larvae of the endemic Leto venus (ghost moth), in the death of Virgilia trees. Key fungal taxa were identified by comparisons of the internal transcribed spacer rDNA regions of the isolated taxa with those available on GenBank. Pathogenicity of the most commonly encountered fungal taxa was determined both in the field and under greenhouse conditions. Five different disease symptoms were observed on Virgilia trees throughout the CFR. At Table Mountain, Virgilia oroboides subsp. oroboides showed symptoms of: (1) several small cankers on stems, seemingly caused by a Fusarium acuminatum-like fungus, (2) a root rot disease caused by Armillaria mellea and (3) small bracket fungi on stems associated with Schizophyllum commune. Virgilia oroboides from the Harold Porter National Botanical Garden was diagnosed with a root disease consistently associated with an un-described Phomopsis species. Virgilia oroboides subsp. ferruginea and V. divaricata from Knysna and the Tsitsikamma area often showed symptoms of rapid wilting and death. The Virgilia stems were damaged by the tunnelling larvae of the ghost moth and those of an unidentified cerambycid beetle. Galleries and the surrounding wood tissues often housed the ophiostomatoid fungi Ceratocystis tsitsikammensis and Ophiostoma plurianulatum. These seem to originate from nitidulid beetles found feeding on gum exudate. Pathogenicity trials confirmed the virulence of the undescribed Phomopsis species, the F. acuminatum-like fungus, S. commune and C. tsitsikammensis to Virgilia. All four morpho-species of bark beetles found in this study, together with phoretic mites on two of the beetle morphospecies, were only collected from dead and dying Virgilia hosts and were classified as secondary pests. Both beetle taxa and mites commonly carried spores of various Geosmithia spp. These are not pathogenic to Virgilia trees, but may be an important food source for the bark beetles, as it dominated the fungal community in galleries. The phoretic mites were unable to feed on their Geosmithia associates, but have been observed to feed on dead bark beetle larvae within galleries. This suggests that the relationship of bark beetles, mites and their associated Geosmithia species in this system is complex and in need of further study. Our results show that natural populations of Virgilia play host to numerous destructive pathogens, some of which are non-native (e.g. A. mellea) and a cause for special concern. Additionally, the isolation of the undescribed Phomopsis species and A. mellea from botanical gardens, with A. mellea now spreading to natural areas, calls for stricter control over the movement of organic material from these areas.
AFRIKAANSE OPSOMMING: Die Kaapse Floristiese Streek (KFS) van Suid-Afrika is bekend vir buitengewoon hoë plantspesie-diversiteit en endemisme. Min aandag is egter tot dusver geskenk aan die peste en patogene in hierdie streek, al mag hulle plantverspreiding en evolusie dramaties beinvloed. In hierdie studie identifiseer ons verskeie geleedpotige diere en fungi as peste en organismes wat siektes veroorsaak in die ekologies en ekonomies belangrike, KFS-endemiese boom genus Virgilia. Ons het die sleutel fungi vanaf Virgilia oor die hele KFS geisoleer, geidentifiseer en hulle patogeniteit bepaal. Addisioneel het ons ook die rol van moontlike pes geleedpotiges, insluitende baskewers, cerambycid kewerlarwes en die endemiese Leto venus (spookmot) in die dood van Virgilia bome geevalueer. Sleutel fungi taksa is geidentifiseer deur die interne getranskribeerde spasieerder rDNS streke van die geisoleerde taksa met die wat op GenBank beskikbaar was te vergelyk. Patogenisiteit van die mees algemeen geisoleerde fungi taxa is beide in die veld en onder glashuis-toestande bepaal. Vyf verskillende siekte simptome is by Virgilia bome regdeur die KFS waargeneem. By Tafelberg het Virgilia oroboides subsp. oroboides simptome getoon van: (1) verskeie klein kankers op stamme, blykbaar veroorsaak deur ‘n Fusarium acuminatum-agtige fungus, (2) ‘n wortelvrot siekte veroorsaak deur Armillaria mellea en (3) klein rakswamme op stamme geassosieer met Schizophyllum commune. Virgilia oroboides in die Harold Porter Nationale Botaniese Tuin is gediagnoseer met ‘n wortelvrot siekte wat altyd met ‘n onbeskryfde Phomopsis spesie geassosieer is. Virgilia oroboides subsp. ferruginea and V. divaricata van Knysna en die Tsitsikamma area het dikwels simptome getoon van vinnige verwelking en dood. Die Virgilia stamme is deur die tonnelende larwes van die spookmot en dié van ‘n ongeidentifiseerde cerambycid kewer beskadig. Galerye en die omringende houtweefsel het dikwels die ophiostomatoid fungi Ceratocystis tsitsikammensis en Ophiostoma plurianulatum gehuisves. Dit lyk asof hierdie fungi van nitidulid kewers afkomstig is wat op die gomuitskeidings gevoed het. Patogeniteitsproewe het die kwaadaardigheid van die onbeskryfde Phomopsis spesie, die F. acuminatum-agtige fungus, S. commune en C. tsitsikammensis teenoor Virgilia bevestig. Al vier morfo-spesies baskewer wat in hierdie studie gevind is, sowel as die foretiese myte op twee van die kewer morfo-spesies, is slegs van dooie of sterwende Virgilia gashere versamel, en is as sekondêre peste geklassifiseer. Beide kewerspesies en myt taksa het algemeen spore van verskeie Geosmithia spesies (Geosmithia pallida, G. flava, G. microcorthyli, G. sp. 1 en G. sp. 2) gedra. Die Geosmithia spesies is nie patogenies teenoor Virgilia bome nie, maar mag ‘n belangrike voedselbron vir die baskewers wees, aangesien dit die fungus-gemeenskap in die galarye gedomineer het. Die foretiese myte was nie instaat om op Geosmithia-assosiate te voed nie, maar is waargeneem om op dooie baskewer larwes te voed binne die galerye. Dit stel voor dat die verhouding van die baskewers, myte en hulle geassosieerde Geosmithia spesies in die sisteem kompleks is, en verdere studie benodig. Ons resultate dui aan dat natuurlike populasies van Virgilia gashere is vir verskeie destruktiewe patogene, sommige waarvan nieinheems (bv. A. mellea) wat ‘n bron van groot kommer is. Verder noodsaak die isolasie van die Phomopsis spesie en A. mellea, wat beide wortelvrot siektes in botaniese tuine veroorsaak, strenger kontrole oor die verskuiwing van organiese materiaal uit hierdie areas, veral gegewe dat A. mellea reeds na natuurlike areas versprei het.
The Centre of Excellence In Tree Health Biotechnology for a bursary and funding the research conducted in this study