1. The difficulties in separating the individual members of the Uromyces pisi group are outlined. 2. A review of the literature on rust fungi is given in which it is brought out that the major criteria for delimiting the species are (i) dimensions, wall thickness, number of germ pores and their distribution, for uredospores, (ii) dimensions, thickness of wall and pore cap, and wall sculpturing of the teleutospores, (iii) host specialisation, and (iv) length of life cycle. 3, 76 herbarium specimens along with the locally occurring member of the U. pisi group were examined from these stand-points, and the relative desoxy-ribose nucleic acid (D.F. A) content per nucleus of U. pisi. U. punctatus and U. loti was determined. 4. It was found that the species of the group could be separated by statistically analysing the spore measurements. Only U. jordianus could be separated by the number of germ pores of its uredospores, and both spore types of all the Species had approximately the same wall thickness. It is shown that the sculpturing of the teleutospore wall varies within each species and shows a gradation from a weakly punctate, to a definitely punctate-striate condition. A review of the literature on host specialisation and life cycle is given for each species, and a host list and synonym list has been compiled for each. From this review it is pointed out that, (1) it is difficult to separate certain of the species of the U. pisi group by host reaction and (ii) biological races exist in some of the heteroecious species. 6. Inoculation experiments were carried out with the locally occurring U. punctatue on Astragalus danicus, and it was found that the rust could infect 9 other species of Astragalus and one of Medicago. Attempts to geminate the teleutospores by various chemical stimulants failed with the exception of one teleutospore. Observation and experiment showed that U. punctatus could overwinter by means of uredospores. Inoculation experiments were carried out on the aecidial host, but no infection ensued after four years. It is concluded that U.punetatus at St, Andrew has lost the ability to infect the aecidial host and that this is due to, (i) the absence of the aecidia host over a period of years leading to adaptation to the uredo host, and (ii) various climatic factors. 7. Inoculation experiments carried out with the local specimens of U. pisi on Lathyrus pratensis and U. loti on Lotus corniculatus, showed that these rusts were narrowly specialised on their resptective hosts. Other closely related rusts which exist in the Hemi- form and are highly specialised with regard to host selection, are described and it is suggested that these rusts have probably arisen from specialised races of the heteroecious members of the group. 9. The ecology and geographical distribution of the group is discussed. The number of leguminous rusts in Britain is less than the number on the Continent. With the exception of U. pisi, heteroecism in the group is confined to Central and S. Europe. Outside this region the heteroecious species adopt the Hemi- form, and can overwinter in the uredo stage. In this condition they 'become more specialised. The number of possible British hosts is greater than the actual number parasitised, except. U. loti and U. laburni where the actual numbers are the same as the possible numbers of hosts. In considering these points it is suggested that the absence of the aecidial hosts from the region outside central S. Europe, has led to the species adopting the Hemi-form and becoming more specialised. Also in this region the climate is not favourable to the development of the aecidium. 10. It is suggested on this evidence, that the members of the group probably have a common ancestor.

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Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-03Bitstream added on 2014-06-13T20:56:24Z : No. of bitstreams: 1 baldissera_bl_me_rcla.pdf: 1165282 bytes, checksum: e54d30574e495ef3d4a7a7c98bd8cf32 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Agaricus blazei é uma das espécies de fungo do grupo basidiomiceto nativo do Brasil, popularmente conhecido como cogumelo-do-sol®. O Brasil é considerado um grande fornecedor desse fungo para o mercado internacional cujos padrões de qualidade são bastante restritivos em relação à concentração de metais pesados, principalmente cádmio. Esse fungo possui propriedades medicinais e seus cogumelos são consumidos por grande número de pessoas que procuram melhorar a qualidade de vida. O trabalho objetivou estudar o acúmulo de cádmio, cromo e chumbo por A. blazei. Foram realizados 3 experimentos, utilizando 2 tipos de composto e 3 tipos de solo para cobertura. O fungo foi cultivado em sacos de plástico preto contendo no fundo o composto inoculado, tendo como cobertura diferentes tipos de solo. O solo e o composto foram analisados para quantificação de Cd, Cr, Pb por espectrometria de absorção atômica, propriedades físicas e químicas (Instituto Campineiro de Análise de Solo e Adubo ICASA). Além das quantias de metais encontradas no solo e no composto foram adicionados 66 mg de cloreto de cádmio, 132 mg de sulfato de cromo e 12 mg de nitrato de chumbo ao solo de cobertura. Após a frutificação, os basidiocarpos foram analisados para quantificar proteínas, aminoácidos, metais pesados e identificação de modificações protéicas por espectroscopia de infravermelho (FTIR). O resultado das análises demonstrou que havia acúmulo de metais pesados nos basidiocarpos, ocorrendo maior percentual nos contaminados com cádmio.Além disso, detectou-se nos basidiocarpos contaminados menor teor de proteínas. Através de FTIR não foi possível detectar alterações nas proteínas nos basidiocarpos analisados. / Agaricus blazei is a basidiomycete fungus native from Brazil and popularly known as Cogumelo-do-sol® the sun-mushroom. Brazil is a supplier of this edible mushroom to international markets to which quality standards are very restrictive regarding the concentration of heavy metals, especially cadmium. This fungus exhibits medicinal properties and its fruiting bodies are consumed by a large number of people that seek to improve life quality. The objective of this work was to study the accumulation of cadmium, chromium and lead by A. blazei. Three experiments were made using two composts and three types of covering soil. The fungus was cultivated in black plastic bags contend in the deep inoculated compost and as covering different types of soils. The soil and the compost were analyzed to determine the amount of heavy metals: Cd, Cr and Pb by atomic absorption spectrophotometer, and physical and chemical properties (ICASA). Besides the metal found in the soil and compost, 66 mg of cadmium chloride, 132 mg of chromium sulfate and 12 mg of lead nitrate were also added to the covering soil. After fructification, the fruiting bodies were analyzed to measure the amount of raw proteins, amino acids, heavy metals and proteinic modifications for Spectroscopy of Infra Red (FTIR). The analyses of results showed accumulation of heavy metals in fruiting bodies, occurring a higher percentage in fruiting bodies contaminated of cadmium. Furthermore detected in contaminated fruiting bodies a less drift of raw proteins. Through FTIR was not possible to detect alteration in proteins of fruiting bodies analyzed.

Resumo: Fungos do grupo das leveduras negras têm revelado grande potencial em degradar compostos aromáticos como únicas fontes de carbono, capacidade de sobreviver sob atmosferas contendo hidrocarbonetos e habitar nichos com baixa concentração de nutrientes. Apesar do potencial de aplicação destes nas áreas ambiental e industrial, sua ecologia é pouco conhecida e seu isolamento na natureza ainda é difícil. A técnica de flotação em óleo (IWATSU et al., 1981) é aplicada com sucesso para recuperação de leveduras negras provenientes da natureza. No entanto, os mecanismos envolvidos neste método são desconhecidos. Assim, neste trabalho, testaram-se três hipóteses de seleção da técnica: hidrofobicidade celular, assimilação de hidrocarbonetos e oligotrofismo. A metodologia foi aplicada em amostras de solo de "landfarming" da Refinaria do Planalto Paulista (REPLAN). Cepas isoladas e padrão foram submetidas aos testes para verificação das hipóteses formuladas. A hidrofobicidade celular das cepas foi verificada mediante quantificação da concentração celular da suspensão na fase oleosa após agitação com hidrocarboneto. Os testes de assimilação de hidrocarbonetos e oligotrofismo foram realizados mediante análises de curvas de crescimento obtidas por leituras de absorbância. Os resultados obtidos mostraram que o solo de "landfarming" é um local propício para o estudo de leveduras negras, uma vez que 107 cepas foram recuperadas e a análise molecular revelou uma espécie nova: Cladophialophora immunda. O teste de hidrofobicidade indicou que, a hidrofobicidade celular pode não ser o principal fator seletivo, pois espécies hidrofóbicas e hidrofílicas foram recuperadas pela técnica em estudo. A maioria das cepas de leveduras negras testadas apresentou crescimento em meio contendo hidrocarbonetos como fontes únicas de carbono,...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Black yeast fungi have shown great potential to degrade aromatic hydrocarbons as single carbon source, ability to survive under atmospheres with hydrocarbons and to live in niches poor in nutrient. Despite the application potential of these fungi in the environmental and industrial areas, the knowledge about their ecology is incomplete and their isolation from nature is still difficult. The oil flotation technique (IWATSU et al., 1981) has been succeed on the black yeast strains recovery from nature, however, mechanisms involved in this method are still unknown. The present study tests three hypothesis on its selective mechanisms: cell surface hydrophobicity, hydrocarbon assimilation and oligotrophism. The methodology was applied in landfarming soil samples from Planalto Paulísta Refinery (REPLAN). Six isolates and control strains were submitted to experiments to verify the hypothesis done. Hydrophobicity was studied through quantification of oil-phase cell concentration after shaking the cell suspension with hydrocarbon. Hydrocarbon assimilation and oligotrophism were verified by analyzing the growth curves obtained with absorbance data. The results showed that landfarming soil has great potential for black yeast isolation, as 107 strains were recovered and a new species - Cladophialophora immunda- was revealed by molecular analyzes. Hydrophobicity test showed that cell hydrophobicity could not be the main selective factor, since hydrophobic and hydrophillic strains were recovered. Growth in media with hydrocarbons as sole carbon source was observed in the majority of the tested black yeast strains, indicating that hydrocarbon assimilation or tolerance to these substances could be the selective factors of the method. Exophiala xenobiotica was able to grow in the poor media tested, what suggests that this substrate with the addition of hydrocarbons could be used for the isolation...(Complete abstract, click electronic access below) / Orientador: Derlene Attili de Angelis / Coorientador: Dejanira de Francechi de Angelis / Banca: Rosely Ana Piccolo Grandi / Banca: Fernando Carlos Pagnocca / Mestre

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Previous taxonomic studies culminated in the current differentiation of the mitosporic fungal genera Drechslera, Curvularia, Bipolaris and Exserohilum. The circumscription of Drechslera and Exserohilum are generally accepted and supported by the association with the teleomorphic ascomycetous genera Pyrenophora and Setosphaeria respectively. The separation of Curvularia and Bipolaris, both anamorphs of Cochliobolus, has been disputed. Many highly similar species have been described that share features of more than one genus. In this study 45 strains representing 14 species of Bipolaris, Curvularia and Exserohilum were characterised with regard to spore morphology, growth characteristics in culture and host preferences. Nucleic acid-based methods were used to evaluate the status of current morphological species. Integration of all this information represents a new approach to the taxonomy of this group. A list of Bipolaris, Curvularia and Exserohilum species known to occur in South Africa is provided and confrrmed their host relations presently known from the literature and includes eight new records. The stability of various spore dimensions and physiological reactions of cultures was analysed by using First Order Inductive Logic (FOIL) and a Decision Tree (DT) algorithms. Both tools proved spore shape to be more consistent than dimensions, and that colony diameter on days three to five, provided measurements with the best diagnostic value. Colony growth rates can be diagnostic within the long-spored species (fusoid to cylindrical spores) but not between the short-spored (clavate to navicular spores) species of the Bipolaris-complex. Differences in colony diameters from five of 25 culture-based tests enabled FOIL to differentiate between species with 79% certainty. Partial sequencing of the 28S rRNA encoding gene differentiated between Exserohilum species and a second group including Bipolaris and Curvularia species. The molecular marker type, Inter-simple sequence repeats (ISSR) using four anchored primers DBD-(AC)7, BDB-(CAC) 5, DHB-(CGA) 5 and VHV -(GT) 7G, were used with the similarity coefficients of Dice and Pearson and the clustering method of Ward to reveal various levels of intra-specific variability. ISSR data indicated greater similarity between Cochliobolus anamorph species with three to four septate conidia than the species with multiseptated conidia. All short-spored species investigated are plurivorous while the long-spored species seem to have a definite graminicolous host preference. Based on these observations it is proposed that the genera Curvularia and Bipolaris be retained as distinct taxa but amended: plurivorous mitosporic Cochliobolus species with short, clavate or navicular spores should be assigned to Curvularia and mitosporic Cochliobolus species with fusoid to cylindrical spores with gramicolous host preferences should be assigned to Bipolaris. The study confrrmed Exserohilum to be a well-defmed genus. Exserohilum inaequale was found to be synonymous with C. cymbopogonis. Two species, separated only on spore length, E. longirostratum and E. rostratum should be retained since they are also distinct in physiology and ISSR patterns. Integration of physiological traits, spore dimensions, host relationships and ISSR fingerprint patterns can therefore enhance the taxonomy of the Bipolaris complex. / Thesis (PhD (Botany))--University of Pretoria, 2005. / Plant Science / unrestricted

Due to the rapid development of tick resistance to synthetic chemical acaricides and the potential risk posed by these chemicals to non-target species, efforts are intensifying towards the development of entomopathogenic fungi as an environmentally benign alternative tick control strategy. The main objective of this study is the development of formulations and delivery systems for control of ticks with entomopathogenic fungi. Screening of entomopathogenic fungal isolates is the first step towards the development of mycoacaricides. Twelve isolates of Metarhizium anisopliae (Metschnik.) Sorok. and three isolates of Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) were evaluated for pathogenicity against unfed Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) adults under laboratory conditions. Out of the 15 isolates screened, 4 isolates were virulent against R. pulchellus, inducing significantly (F value = 11.86; DF = 14, 75; P < 0.0001) higher tick mortalities; i.e. ICIPE 78 (76.1 ± 5.9%), ICIPE 69 (62.6 ± 5%), ICIPE 62 (49.8 ± 5.8%) and ICIPE 60 (49.6 ± 7.7%), four weeks post-treatment compared to the others. The results of this study demonstrate that R. pulchellus ticks were susceptible to some M. anisopliae isolates. Since the process of strain selection is an important step in the development of insect pathogens for biological control, an appropriate bioassay technique for infecting different life stages of a host is of paramount importance. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using R. pulchellus as a model. In a preliminary experiment, an oil-based formulation of M. anisopliae titred at 109 conidia ml-1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality) , although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 x 104 ± 1.1 x 103 conidia ml-1) after spraying by Burgerjon’s spray tower compared to 1 x 106 conidia ml-1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC50 values following contamination of nymphs (LC50 = 1.4 x 107 conidia ml-1) and adults (LC50 = 6.7 x 107 conidia ml-1) in SS were significantly lower compared to SP; nymphs (LC50 = 5.7 x 108 conidia ml-1) and adults (LC50 = 5.3 x 109 conidia ml-1) and SW; nymphs (LC50 = 5 x 108 conidia ml-1) and adults (LC50 > 1010 conidia ml-1). Although the LC50 value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at two weeks post-treatment) and (23.3% at three weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8% respectively) and SW (0.0 and 0.0). Results show that among the modes of contamination tested, SP was the most appropriate for contaminating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil. The integrated use of semiochemicals and entomopathogenic fungus (hypocreales) may optimise the performance of a fungus as a biocontrol agent against ticks. Initially, experiments were conducted to evaluate the attraction of the hunter tick Amblyomma variegatum Fabriscius (Acari: Ixodidae) to semiochemicals. In one of the experiments, the simultaneous release of 1-octen-3-ol and AAAP together with CO2 from a trap in simulated field plots attracted up to 94 ± 6% of adult ticks from a distance of 6 m, and up to 24.0 ± 5.1% from 8 m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP and 1-octen-3-ol resulted in high levels of contamination of the ticks attracted to the traps. However, 48 hr after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation lost the lowest number of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in the rainy and dry seasons. The emulsifiable formulation gave the highest relative reduction in tick numbers in both seasons: 54.7 and 46.5% in the rainy and dry seasons, respectively, followed by the oil formulation (32 and 23.8%) and the powder formulation (38.0 and 24.4%). Following the high attraction and contamination of A. variegatum under semi-field conditions, experiments were done to evaluate the efficacy of M. anisopliae-treated semiochemical-baited traps for control of A. variegatum under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot. An emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil [CHEF cooking oil, Premier Oil Mills LTD] and 1% Tween 80) titrated at 109 conidia ml-1- was applied in a semiochemical-baited traps (900 cm2) which were placed at 5 spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45o) in order to cover different sections of the plot. In the control plots, traps baited with semiochemicals only were used. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1 ± 5.2%) than in the control plots (85.6% ± 3%) (P < 0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8 ± 2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for two weeks; while only 3.3 ± 0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly, low cost product to control these economically important ticks. / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Cryphonectria canker of Eucalyptus, caused by Cryphonectria cubensis, is considered to be one of the most important fungal diseases affecting Forestry in South Africa. This disease also occurs in other tropical and sub-tropical regions of the world, including South America, Australia and South East Asia. Due to the commercial importance of C. cubensis, several recent studies in South Africa have focused on the elucidation of population diversity and phylogenetic relationships of the fungus from diverse geographic origins. These studies have resulted in more effective tree breeding programmes, and the identification of the possible origin for C. cubensis in South Africa. In this thesis, various aspects of the biology of C. cubensis in South Africa and Colombia are addressed. These studies have focused on the elucidation of the diversity of populations of the fungus occurring in Colombia and South Africa, as well as determination of the phylogenetic relationships of C. cubensis from Colombia. The sexual reproductive system of the fungus from Colombia has also been investigated . . In an investigation into the phylogenetic relationships of C. cubensis isolates from Colombia (Chapter IT), it was found that these isolates are most closely related to other South American isolates. This finding suggests that properties displayed by populations in other South American countries can be extrapolated to the Colombian population, and vice versa. The Colombian isolates were distinct from C. cubensis found in South East Asia, implying a more distant relatedness between these isolates. A study of homothallism and the possibility of sexual outcrossing in Colombian C. cubensis isolates (Chapter III), revealed that the fungus is homothallic. This was shown by allowing single ascospore isolates to reproduce sexually on Eucalyptus twigs, followed by genetic analysis of progeny using DNA fingerprinting. The DNA fuigerprinting profiles of these progeny were identical, indicating that no outcrossing had occurred. The sexual event was, therefore, due to self-fertilisation. In contrast, when progeny from naturally occurring perithecia were analysed genetically using vegetative compatibility groups (VCGs) and DNA fingerprinting, results suggested that oucrossing had occurred, but only to a limited extent. C. cubensis in Colombia is therefore homothallic, but can outcross. Presumably, the same is true for other populations of the fungus in South America. The genotypic diversity of the Colombian population of C. cubensis was investigated using VCGs and RAPDs. Results of this study indicated that the genotypic diversity of this population was similar to diversities previously found for other South American populations of C. cubensis. However, the phenotypic (VCG) and genetic (RAPD) data for the Colombian population were significantly different. The estimation of genotypic diversity based on RAPDs was significantly higher than the same figure for VCG data The reason for the difference in obtained values is attributed to the low level of sensitivity ofVCGs to detect genetic differences between isolates. In Chapter V, a novel technique for obtaining polymorphic, micro satellite-like DNA markers from fungi is described. The technique is based on the identification and characterisation of polymorphic DNA fragments originating from a random amplification of micro satellite sequences using the polymerase chain reaction (PCR). Sequence data from these fragments were used to construct specific primers to amplify polymorphic loci from genomic DNA. The technique has a high success rate in comparison to traditional techniques for the isolation of polymorphic markers. It was also shown that markers obtained with the new technique can be used to differentiate C. cubensis isolates originating in South Africa from those originating in other countries. The last chapter (VI) of this thesis represents the first intensive molecular population diversity study of C. cubensis in South Africa Using polymorphic markers from an earlier study (Chapter V), it was possible to assess the molecular variation of the South African population, and to compare this with phenotypic data obtained previously. It was found that molecular and phenotypic data yield different estimations of population diversity. An estimation of the gametic disequilibrium of the South African C. cubensis population revealed that the tested alleles were randomly associated. Such a situation is expected for populations that preferentially reproduce sexually, and consequently outcross. These figures are, however, only an indirect indication that outcrossing occurs in the South African population. Future studies on the South African C. cubensis population would need to be based on a larger number of markers, and should also include a greater number of isolates of the fungus. Knowledge gained through the studies presented in this thesis will hopefully aid to develop more effective control strategies against Cryphonectria canker of eucalypts. However, new questions about the biology of C. cubensis have emerged that need urgent attention. Data from this thesis might, therefore, prove important to future studies of the fungus. / Dissertation (MSc (Agric))--University of Pretoria, 2000. / Genetics / unrestricted

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