Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2007. / Submitted to the African Centre for Crop Improvement. Full text also available online. Scroll down for electronic link.

Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2008. / Full text also available online. Scroll down for electronic link.

Made available in DSpace on 2014-10-09T12:42:20Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:41Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:42:20Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:41Z (GMT). No. of bitstreams: 0 / A candidíase vaginal (CV) é uma doença causada por fungos do gênero Candida spp. que acomete milhares de mulheres no mundo. Estima-se que 75% das mulheres sofrerão CV pelo menos uma vez durante a vida fértil, 40 a 50% terão o segundo episódio e 5 a 8% sofrerão a forma recorrente, caracterizada pela ocorrência de quatro ou mais episódios durante um ano. O tratamento ainda permanece um desafio para as candidíases complicadas. A terapia fotodinâmica (TFD) é uma modalidade terapêutica que utiliza substâncias fotossensibilizadoras (FS) e uma fonte de luz, que juntas, produzem espécies reativas de oxigênio, tóxicas para micro-organismos e inofensivas para a célula animal hospedeira. O objetivo deste trabalho foi avaliar os efeitos antifúngicos e anti-inflamatórios da TFD mediada por azul de metileno (AM) e laser de emissao vermelha (LEV) no tratamento da CV em modelo animal. Fêmeas de camundongos BALB/c, com 6 a 10 semanas foram estrogenizadas e, 72h após, receberam via intravaginal 20μL de suspensão contendo 107 UFC/mL de C. albicans ATCC 90028. Cinco dias após, a TFD foi aplicada na vagina das fêmeas, utilizando AM 1000μM e laser (100mW, 660nm, energia de 36J por 6 min ou duas aplicações de 18J por 3min, com intervalo de 24h entre sessões). Após 0, 24 e 96h foram feitas lavagens vaginais para recuperação fúngica, cultivo microbiológico, eutanásia para remoção das vaginas e estudo histológico. Lâminas coradas por hematoxilina e eosina foram utilizadas para contagem da área de células inflamatórias (ACI), utilizando o programa ImageJ. Os resultados mostraram que TFD in vivo reduziu a carga fúngica em aproximadamente 1,6 log UFC/mL e, quando aplicada em duas sessões de 18J por 3min, diminuiu a ACI. A TFD mediada por LEV e AM 1000μM mostra-se como alternativa promissora para o desenvolvimento de novas modalidades terapêuticas para vaginites fúngicas. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Among fungal pathogens responsible for opportunistic infections, species of the genus Candida have a major role (Mitchell, 1998). Various Candida species cause superficial infections which can be cured by the currently available antifungal arsenal (Noble and Johnson, 2007). However, species of the genus Candida are also responsible for life-threatening systemic infections, particularly in immunocompromised patients with weakened immune system. Among Candida species, C. albicans, which can also be a commensal of the skin and the gastrointestinal and genitourinary tracts, is responsible for the majority of Candida bloodstream infections. However, there is an increasing incidence of infections caused by C. glabrata because it is less susceptible to azoles. Other medically important Candida species include C. parapsilosis, C. tropicalis and C. dubliniensis. The problem has been further worsened by the emergence of many drug resistant isolates which pose a major hurdle during a given treatment regimen. Therefore, there is a dire need to identify novel drug targets and the current study focuses on one such protein found in C. albicans and related Candida species. CaGAL102 does not encode a functional galactose epimerase CaGAL102 was previously identified in the lab as a paralog of CaGAL10. CaGAL10 endoes a functional UDP-galactose 4-epimerase and it can complement a Scgal10 null strain. Further, work on the Gal10 protein in the encapsulated yeast Cryptococcus neoformans identified two Gal10 paralogs in the genome, Uge1 and Uge2 with distinct functions (Moyrand et al., 2008). A similar scenario is found in S. pombe in which two Gal10 sequence homologs have been annotated. In the light of these observations, we wanted to test if CaGAL102 also encodes a functional ScGAL10 homolog. We found that CaGAL102 could not complement Scgal10 null strain though there was a strong conservation in the cofactor and the catalytic motif in both the proteins. We found after a careful literature review that Gal10 belongs to a family of proteins called the short chain dehydratase/reductase family (SDR) (Jornvall et al., 1995), members of which are characterised by the presence of glycine rich cofactor binding motif at the N-terminus and an YXXXK catalytic motif. Proteins belonging to the SDR family have a residue level identity of 15-30% indicating early duplication and divergence. Based on our literature survey we carried out a BLAST search in the NCBI protein database using CaGal102 as the bait protein. We found that CaGal102 is 32% identical at the protein level to dTDP-glucose 4,6 dehydratase (RmlB), another member of the SDR family. RmlB is the second enzyme of the rhamnose biosynthetic pathway which gives rise to dTDP-rhamnose. This pathway is involved in cell wall biosynthesis in bacteria and it has been shown that rmlB is essential for growth of Mycobacterium smegmatis (Li et al, 2006). Interestingly rhamnose is not present in the cell wall of C. albicans. Biochemical characterisation of CaCaGal102 A plant homolog of RmlB is found in A. thaliana which uses UDP-glucose as the substrate (Oka et al., 2007). Based on our alignment data we identified many critical residues in CaGal102. Most importantly we identified that lysine at position 159 lies in the YXXXK motif and could be important for activity. We therefore, mutated the lysine at position 159 to alanine. In order to find out the biochemical function of CaGal102 in vitro, we cloned expressed and purified recombinant wild type and catalytic mutant proteins from E. coli and used the purified proteins for our assays. We found that CaGal102 uses UDP-glucose as the preferred substrate. To further substantiate our data, we reintegrated the wild type or the mutant alleles in the native locus of CaGAL102 and checked for the rescue of morphology defects like filamentation and sensitivity to cell wall damaging agents. We also found that the Cagal102∆/∆ strain is avirulent in a mouse model of systemic infection. We have also carried out infection studies with the null mutant and the wild type and the catalytic mutant reintegrant strains. Our observation suggests that reintegrating one copy of the wild type allele rescues the virulence defect. Interestingly the strain harbouring one copy of the mutant allele behaves like the null mutant in a mouse model of systemic infection. We have also identified sequence homologs of CaGal102 in related Candida species. It is plausible to think that the homologs in related species also have similar effects and hence targeting this protein by a small molecule could help in treating candidiasis caused by related species. CaGAL102 is involved in cell wall architecture in C. albicans To elucidate the role of CaGal102 in C. albicans we generated a knockout out strain and studied various mutant phenotypes. The most striking observation was that the cells of the null mutant were filamentous as compared to the wild type control when grown in normal rich media. Further the cells were sensitive to various cell wall damaging agents and also to hygromycin B. We reasoned that lack of CaGal102 causes perturbation in the cell wall architecture rendering the cells sensitive to various cell wall damaging agents. To further strengthen this hypothesis, we decided to study the genetic interaction of CaGAL102 with genes known to be involved in cell wall biosynthesis in C. albicans. One of the candidate genes we chose for our study was GAL10, deletion of which in C. albicans renders the cells sensitive to various cell wall damaging agents. Loss of function of UGE1 in C. neoformans impaired biosynthesis of a cell wall component, galactoxylomannan. We found that cells lacking both Gal102 and Gal10 adhered to nylon membranes poorly as compared to single mutants or the wild type control. The second gene we chose was a P-type ATPase, PMR1 deletion of which causes increased sensitivity to cell wall damaging agents and hyper-activation of the cell wall integrity pathway similar to Cagal102∆/∆ strain. We found that cells lacking both Pmr1 and Gal102 were more sensitive to hygromycin B as compared to the single mutants. This confirmed our idea that CaGal102 is a novel gene involved in cell wall biogenesis in C. albicans. REFERENCES: Mitchell, A.P. (1998) Dimorphism and virulence in Candida albicans. Curr Opin Microbiol, 1, 687-692. Noble, S.M. and Johnson, A.D. (2007) Genetics of Candida albicans, a diploid human fungal pathogen. Annu Rev Genet, 41, 193-211. Moyrand, F., Lafontaine, I., Fontaine, T. and Janbon, G. (2008) UGE1 and UGE2 regulate the UDP-glucose/UDP-galactose equilibrium in Cryptococcus neoformans. Eukaryot Cell, 7: 2069-2077. Jornvall Hans, Persson Bengt, Krook Maria,‟ Atrian Silvia, Gonzalez-Duarte Roser, Jeffery Jonathan, and Ghosh Debashis (1995). Short-Chain Dehydrogenases Reductases (SDR). Biochemistry, 34: 6004-13. Li, W., Xin, Y., McNeil, M.R. and Ma, Y. (2006) rmlB and rmlC genes are essential for growth of mycobacteria. Biochem Biophys Res Commun, 342: 170-178. Oka, T., Nemoto, T. and Jigami, Y. (2007) Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. J Biol Chem, 282: 5389-5403.

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is a major foliar disease on wheat. The pathosystem involves three pairs of necrotrophic effector (NE) and host sensitivity (S) gene interactions, namely Ptr ToxA-Tsn1, Ptr ToxB-Tsc2 and Ptr ToxC-Tsc1. Additionally, genetic factors conferring race-nonspecific resistance have been identified. The objectives of this study were to map tan spot resistance QTL and investigate the role of NE-S interactions in disease in durum using association and bi-parental mapping. Evaluation of a worldwide collection of durum accessions allowed identifying highly resistant nineteen lines to multiple Ptr races. Association mapping revealed genomic regions on chromosomes 1A, 2B and 3B significantly associated with resistance to tan spot, which likely correspond to Tsc1, Tsc2 and racenonspecific resistance. Using a bi-parental population derived from Ben and PI 41025, we found that ToxA-Tsn1 interaction plays no significant role in disease, instead a major race-nonspecific QTL on chromosome 5A was identified.

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

Please read the abstract in the section 00front of this document / Dissertation (MSc (Agric) Plant Pathology)--University of Pretoria, 2003. / Microbiology and Plant Pathology / unrestricted

Gray leaf spot (GLS) disease of maize (Zea mays L.), caused by the fungus Cercospora zeae-maydis, causes significant corn yield losses in Virginia and other mid-Atlantic states. A new greenhouse assay method with filter paper discs of C. zeaemaydis mycelia has been developed to evaluate corn germplasm for resistance to GLS. Mycelial inoculum obtained from cultures of mycelia in liquid malt media was pipetted at 100 ul samples onto each filter paper disc which was then adhered to the lower leaf surface by transparent tape. The inoculated corn seedlings were placed in a moist plastic chamber with high relative humidity provided by a humidifier. The first macroscopic symptoms induced by this inoculation method appeared 3 days after inoculation. This new inoculation method with mycelial discs was used on five corn genotypes (VA14, B68, PA875 , B73, and M017) to screen resistance to GLS disease. With this inoculation method, resistant and susceptible inbreds were easily differentiated based on lesion type. Resistant inbreds including VA14, B68, and P A875 were characterized by water-soaked appearance or small chlorotic flecks while susceptible inbreds like B73 and M017 were characterized by more extensive necrosis. Necrotic area under the mycelial disc was a good indicator for disease severity. However, the percent leaf area under discs affected by mycelia which reflected the total host responses was not appropriate to indicate disease severity. The effects of plant physiological factors on the expression of resistance to GLS was also investigated. Placing mycelial discs on lower leaf surfaces induced more responses than placing on upper leaf surfaces. Inoculation of lower older leaves induced more severe lesions than inoculation of upper leaves. The effect of cercosporin was investigated by inoculating corn seedlings with cercosporin-producing mycelia and with non-cercosporin containing mycelia. The former induced much more severe host response than the latter. Conidiation of C. zeae-maydis was examined with the mycelial inoculation method in the greenhouse. Conidiophores were found emerging from stomata as early as 15 days after inoculation in B73 and M017 and limited only to necrotic tissue. No conidiation was observed in resistant genotypes VA14, B68 and PA875. / Master of Science

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Grey mould, caused by Botrytis cinerea, is the most important foliar disease of rooibos seedlings. Although the disease is primarily controlled with applications of fungicides, the improvement of cultural methods of disease management should lessen this dependence on chemical control. Such improvements would, however, not be possible without knowledge of the inoculum sources and dispersal of the pathogen. The aim of this study was to investigate the inoculum ecology of B. cinerea in rooibos nurseries in order to identify primary sources of inoculum and to improve the environmentally friendly management of the disease. The study was conducted in four nurseries over two production seasons (March to July 2003 and 2004). Levels of airborne inoculum of B. cinerea were monitored on a monthly basis inside and around the nurseries with spore traps. Samples of plant material and organic debris were taken in the corresponding areas to determine the incidence of plant material infected by the pathogen and the incidences of grey mould in the nurseries were recorded. Low numbers of B. cinerea colonies were observed on the spore traps. Similar levels of airborne inoculum were observed inside and around the nurseries. The incidence of plant material yielding B. cinerea was higher outside the nurseries than inside, indicating the importance of such materials as potential sources of inoculum. Since patterns of airborne inoculum observed in this study confirmed reports of the local dispersal of B. cinerea, the removal of possible hosts outside the nurseries could aid in the management of grey mould in rooibos nurseries. Resistance to dicarboximide fungicides is a genetically stable trait in B. cinerea, and therefore has the potential to be used as a phenotypic marker. This marker can be used to gain knowledge on the dispersal of B. cinerea inoculum inside and outside rooibos nurseries. Isolates of B. cinerea collected from the air and from plant material in and around four rooibos nurseries were assessed for resistance to iprodione at 1 and 3 μg/ml a.i. Some of the isolates showed resistance to iprodione at 1 μg/ml a.i. However, none of the isolates showed resistance at 3 μg/ml a.i. iprodione. The initial incidence of dicarboximide-resistance at the nurseries was slightly higher than expected. As the season progressed, the incidence of iprodione-resistant isolates decreased towards May, after which an increase was observed towards July. A relatively high percentage of isolates collected outside the nurseries was found to be dicarboximide-resistant. Two of the nurseries had a significant higher incidence of resistant isolates on plant material collected inside, than on plant material collected outside the nursery. However, when looking at resistance levels of airborne isolates, no significant differences were found in the incidence of resistant isolates sampled inside and outside the four nurseries. The data indicated the importance of organic debris and seed-borne infections in the survival and dispersal of dicarboximide-resistant isolates of the pathogen. With the current emphasis on organic agriculture the knowledge gained in this study presents valuable possibilities of improving the cultural management of grey mould in rooibos nurseries. / AFRIKAANSE OPSOMMING: Vaalvrot, veroorsaak deur Botrytis cinerea, is die belangrikste bo-grondse siekte van rooibossaailinge. Alhoewel die beheer van die siekte hoofsaaklik op die gebruik van fungisiede berus, behoort die verbetering van verbouingspraktyke hierdie afhanklikheid van chemiese beheer te verminder. Sulke verbeteringe sal egter slegs moontlik wees indien voldoende kennis van die inokulumbronne en verspreiding van die patogeen beskikbaar is. Die doel van hierdie ondersoek was om die inokulum ekologie van B. cinerea in rooibos kwekerye te ondersoek sodat primêre inokulumbronne opgespoor en omgewingsvriendelike siektebestuurspraktyke verbeter kan word. Die ondersoek is in vier kwekerye oor twee produksie seisoene (Maart tot Julie 2003 en 2004) uitgevoer. Vlakke van luggedraagde inokulum van B. cinerea is op ’n maandelikse basis met behulp van spoorvangers binne en buite die kwekerye gemonitor. Monsters van plantmateriaal en organiese materiaal is in ooreenstemmende areas geneem om die voorkoms van B. cinerea geïnfekteerde plantmateriaal vas te stel en die voorkoms van vaalvrot in die kwekerye is aangeteken. Min B. cinerea kolonies is op die spoorvangers waargeneem. Soortgelyke vlakke van luggedraagde inokulum is binne en buite die kwekerye waargeneem. Die hoër voorkoms van B. cinerea geïnfekteerde plantmateriaal buite die kwekerye as binne, dui op die belang van sulke materiaal as potensiële inokulumbronne. Aangesien die patrone van luggedraagde inokulum, soos waargeneem in hierdie ondersoek, ander berigte van B. cinerea se beperkte verspreidingsvermoë bevestig, kan die verwydering van moontlike alternatiewe gashere buite die kwekerye die bestuur van die siekte binne die kwekerye verbeter. Weerstand teen dikarboksimied fungisiede is ’n geneties-stabiele kenmerk in B. cinerea en het daarom potensiaal om as ’n fenotipiese merker gebruik te word. Hierdie merker kan gebruik word om kennis aangaande die verspreiding van B. cinerea in en om rooibos kwekerye in te samel. Botrytis cinerea isolate in lug en op plantmateriaal in en om vier rooibos kwekerye is gedurende 2003 en 2004 versamel. Die isolate is vir weerstandbiedendheid teen iprodioon by konsentrasies van 1 en 3 μg/ml aktiewe bestandeel (a.b.) getoets. Isolate met weerstand teen 1 μg/ml a.b. iprodioon is waargeneem, maar nie teen 3 μg/ml nie. Die aanvanklike voorkoms van dikarboksimiedweerstand by die kwekerye was hoër as verwag. Hierdie vlak het egter gedaal met die verloop van die seisoen tot in Mei, waarna ’n toename tot in Julie waargeneem is. Die persentasie dikarboksimied-weerstandbiedende isolate buite die kwekerye was relatief hoog. In twee van die kwekerye was die voorkoms van weerstandbiedende isolate op plantmateriaal in die kwekerye betekenisvol hoër as op plantmateriaal buite die kwekerye. Daar was egter geen betekenisvolle verskille in die voorkoms van luggedraagde weerstandbiedende isolate nie, ongeag van die kwekery of posisie. Die data dui op die belang van organiese materiaal en saadgedraagde infeksies in die oorlewing en verspreiding van dikarboksimied-weerstandbiedende isolate van die patogeen. Met die huidige klem op organiese landbou bied die inligting wat in hierdie ondersoek versamel is moontlike praktyke wat geïmplementeer kan word om die beheer van vaalvrot in kwekerye met behulp van verbouingspraktyke te verbeter.