The genus Arthroderma contains predominantly geophilic dermatophytes (naturally occuring in soil). Some species, especially those from Trichophyton terrestre complex, cause human and animal dermatomycosis. In the past, the species boundaries were determined mainly on the basis of biological species concept using in vitro mating experiments. But these nearly 70-years-old findings have not been tested by means of modern taxonomic methods. In total 194 species of the genus Arthroderma (including all available ex-type strains) originating predominantly in USA, Canada and Europe were studied in this thesis. They were mostly isolated from soil (n = 77), animals (n = 50), human clinical material (n = 41) and cave sediment (n = 9). The main goal of the thesis was to elucidate the species boundaries between species A. insingulare, A. lenticulare and A. quadrifidum, that were classified into the T. terrestre complex because of their seemingly identical asexual stage. Further, this work aimed to resolve the relationship between Arthroderma species using the multigene phylogeny and clarify which species are clinically relevant. A multigene phylogeny of the genus Arthroderma was based on the sequences of the ITS rDNA region, β-tubulin (TUB2) and translation elongation factor 1α (TEF1α) genes. The genus...

Sweet potato is a popular food security crop in South Africa and has a considerable commercial value. Fusarium wilt (FW), caused by the fungal pathogen Fusarium oxysporum formae speciales (f. sp.) batatas, has been reported worldwide and is widespread in sweet potato production areas in South Africa. Preliminary molecular identification of South African isolates from diseased sweet potato plants indicated that there are other formae speciales besides F. oxysporum f. sp. batatas associated with FW. The objectives of the study were to conduct a field survey and to characterise the isolates of the Fusarium oxysporum species complex (FOSC) using phylogenetic analyses, morphological characterisation and DNA barcoding. Phylogenetic analyses revealed two other formae speciales, namely F. oxysporum f. sp. tuberosi and F. oxysporum f. sp. vanillae that were associated with FW. This study has contributed in understanding and knowledge of FOSC associated with FW of sweet potato in South Africa. / Life and Consumer Sciences / M. Sc. (Life Sciences)

В данной работе исследуется ряд недостаточно изученных ранее аспектов биологии семейства Peloridiidae: поведение, внутривидовая коммуникация, трофические связи с растениями и тонкие детали морфологии. Полученная информация используется для создания новой филогенетической гипотезы о родственных связях Peloridiidae и для критической оценки существующих гипотез. Кормовые растения семейства систематически изучены в Австралии, Новой Зеландии и Чили. Peloridiidae в целом, как выяснилось, не привязаны к отдельным таксонам мохообразных, хотя они регулярно встречались на представителях Dicranaceae, Hypopterygiaceae, Polytrichaceae и Sphagnaceae. Однако, трофическая специфичность отдельных родов и видов может сильно различаться. Впервые изучены вибрационные сигналы четырех видов пелоридиид. Черты сигналов различались внутри семейства: сигналы южноамериканских и австралийских видов были похожи, в то время как новозеландские виды от них отличались. Впервые представлена подробная информация о морфологии антенн, кончика хоботка, лапок, скульптуры тегменов, брюшка и покровных желез для 21 вида пелоридиид и некоторых внешних групп. Эти находки были организованы в матрицу из 93 признаков и проанализированы филогенетически с помощью методов минимизации изменений. В результате монофилетические Peloridiidae оказались сестринской группой монофилетических Auchenorrhyncha, с неплохим статистическим подтверждением. Эта группировка обсуждается в свете других работ и данных литературы. Внутренняя структура семейства в настоящей работе оказалась довольно похожей на данные литературы, за важным исключением рода Peloridium, который оказался на самом нижнем ответвлении филогенетического древа. Когда к анализу были привлечены три дополнительных признака (биоакустические и поведенческие), позиция Peloridium изменилась. Этот результат иллюстрирует потенциал интегративных методов, хотя и требует осторожного к себе отношения из-за сложностей гомологизации поведенческих признаков. / Einige wenig bekannte Aspekte der Biologie der Peloridiidae (Verhalten, intraspezifische Kommunikation, Wirtspflanzenassoziationen, Feinmorphologie usw.) wurden untersucht. Die gewonnenen Informationen wurden benutzt, um eine phylogenetische Hypothese zu Verhältnissen der Peloridiidae untereinander und zu anderen Hemiptera aufzustellen. Wirtspflanzen der Peloridiidae wurden in Australien, Chile und Neuseeland systematisch besammelt. Peloridiidae als Familie scheinen keine Präferenz für ein Taxon der Bryophyta zu haben, auch wenn sie eine Affinität zu einigen Gruppen der Moose zeigen. Unterschiedliche Arten und Gattungen der Peloridiidae können sich aber in ihrer Selektivität unterscheiden. Vibrationssignale von vier Arten der Peloridiidae wurden zum ersten Mal untersucht. Einige Eigenschaften dieser Signale variierten zwischen den Arten: australische und südamerikanische Vertreter waren einander ähnlich, die neuseeländischen unterschieden sich aber von ihnen beiden. Detaillierte Informationen zu feinen morphologischen Merkmalen der Antennen, Labiumspitze, Tarsen, Integumentaldrüsen usw. in 21 Arten der Peloridiidae und einigen Außengruppen wurden zum ersten Mal präsentiert. Die erarbeiteten Merkmale wurden zur Herstellung einer phylogenetischen Hypothese herangezogen. Die Peloridiidae erwiesen sich da als ein Monophylum, dessen Schwestergruppe die Auchenorrhyncha waren. Dieses Verhältnis wird anhand von Daten aus Literatur und anderen Untersuchungen diskutiert und etwas in Frage gestellt. Die intrafamiliären Verhältnisse der Peloridiidae sind nach der erarbeiteten phylogenetischen Hypothese denen aus Literatur bekannten ähnlich; der wichtige Unterschied ist die basale Position der Gattung Peloridium. In einer Analyse mit zusätzlichen drei verhaltensökologischen Merkmalen ändert Peloridium aber seine Position, was das Potential der integrativen Ansätze illustriert, aber vorsichtig interpretiert werden muss, da solche Merkmale schwer zu homologisieren sind. / Some insufficiently studied aspects of Peloridiidae biology such as behavior, intraspecific communication, host plant preferences and fine morphology were investigated. The newly acquired information was used for production of a phylogenetic hypothesis on Peloridiidae relationships and critical evaluation of the existing ones. Host plants of Peloridiidae were studied systematically in Australia, Chile and New Zealand. Peloridiidae as a whole were found not to be bound to particular bryophyte taxa, although they regularly occurred in species of Dicranaceae, Hypopterygiaceae, Polytrichaceae and Sphagnaceae. Still, different species and genera could vary in their host plant specificity. Vibrational signals of four Peloridiidae species were studied for the first time. Features of these signals varied between species, the Australian and South American ones being similar to each other and the New Zealand species different from both of them. Detailed information on fine morphology of antennae, genae, labium tip, tegminal sculpture, tarsi, abdominal sculpture and integumental glands in 21 Peloridiidae species and some sister groups was presented for the first time. The findings were formalized as a matrix of 93 characters and analyzed phylogenetically with methods of maximum parsimony. A monophyletic Peloridiidae resulted, with significant support of the sister-group relationship to Auchenorrhyncha. This relationship was discussed on the background of other studies and literature data. The intrafamiliar structure of Peloridiidae in the present study was quite similar to previously published works, with the major exception of the genus Peloridium branching off most basally in the phylogenetic tree. Three additional bioacoustic and behavioral characters, when integrated into the matrix, change the position of Peloridium and demonstrate the potential of integrative approaches, although this result must be treated with care due to complicated homologization of behavioral traits.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014