Rhodococcus rhodochrous is an aerobic, non- pathogenic ,gram-positive bacterium that is often used in industries as a biocatalyst.R. rhodochrous DAP 96253 is capable of exhibiting contact-independent inhibition of selected fungal pathogens.The use of R. rhodochrous as a potential biocontrol agent against plant and animal fungi was examined.The fungi tested were Botrytis cinerea,Pseudogymnoascus destructans,Aspergillus flavus, Fusarium oxysporum’Cigar Tip’ , Rhizopus stolonifer’D1’ ,and other species isolated from berries.Each species was studied to establish the effect of dose (g/cells) and time of exposure to R. rhodochrous.Antifungal inhibition tests were done with the use of dosing,agar diffusion, frozen fermentation paste and exposed slides.Inhibition was observed with B.cinerea,P.destructans,A.flavus and D1,and reduced sporulation was observed with Cigar Tip. The results varied amongst the type of tests used on each target species.

Fungos patogênicos, como Cryptococcus neoformans e Candida albicans, são uma das mais frequentes causas de infecções oportunistas em todo o mundo, sendo capazes de sobreviver, proliferar e escapar de macrófagos, células de primeira linha da resposta imune de hospedeiros mamíferos. Macrófagos geralmente expõem o patógeno intracelular a um ambiente tóxico, caracterizado por pH ácido, presença de espécies reativas, como também de peptídeos antimicrobianos. Neste contexto, há uma competição entre o patógeno intracelular e o hospedeiro mamífero por nutrientes essenciais, como por exemplo, metais de transição. Cobre é um metal de transição essencial para a vida aeróbica, porém pode ser tóxico em altas concentrações e, devido a isto, diversos organismos desenvolveram mecanismos regulatórios para controlar suas concentrações. O objetivo deste trabalho foi avaliar a modulação da homeostase de cobre durante a interação entre macrófagos e células de C. neoformans ou C. albicans. Neste trabalho, foi possível demonstrar que a presença de cobre contribui para uma menor atividade fungicida de macrófagos infectados. Também, o pré-carregamento das células fúngicas com cobre alterou a sensibilidade de ambas leveduras patogênicas à atividade anti-fúngica. Além disso, foi demonstrado que a expressão de transportadores de cobre (CTR1 e ATP7A) e proteínas ligadoras de cobre (ceruloplasmina e metalotioneína 1) de macrófagos foram moduladas em resposta à infecção por estes fungos patogênicos. Entretanto, essa regulação pode envolver mecanismos mais complexos, como estratégias do patógeno para subverter a ação antimicrobiana de macrófagos. / Pathogenic yeasts, such as Cryptococcus neoformans and Candida albicans, are one of the most frequent causes of the opportunistic infections worldwide, being able to survive, proliferate and escape from macrophages, the first line cells engaged in the immune defense of the mammalian host. Macrophages generally expose the intracellular pathogen to a toxic environment, which is characterized by acid pH, presence of reactive species, as well the presence of antimicrobial peptides. In this context, there is a competition between intracellular pathogen and mammalian host for essential nutrients, such as transition metals. Copper is an essential transition metal for aerobic life, but can be toxic at high concentrations and, therefore, many organisms have evolved highly regulated mechanisms for controlling its concentration. In this work, we aim is evaluate a modulation of copper homeostasis during interaction between macrophages and C. neoformans or C. albicans cells. This work demonstrated that the presence of copper resulted in a lower fungicidal activity of infected macrophages. Also, the pre-loading of fungal cells with copper can alter the sensitivity of both pathogenic yeasts to an antifungal activity. In addition, we showed that the expression of macrophage copper transporters (CTR1 and ATP7A) and copper-binding proteins (ceruloplasmin and metallothionein 1) are modulated in response infection by pathogenic fungi. However, this regulation may involve more complex mechanisms, such as strategies of pathogen to subvert the antimicrobial action of macrophages.

Fungos patogênicos, como Cryptococcus neoformans e Candida albicans, são uma das mais frequentes causas de infecções oportunistas em todo o mundo, sendo capazes de sobreviver, proliferar e escapar de macrófagos, células de primeira linha da resposta imune de hospedeiros mamíferos. Macrófagos geralmente expõem o patógeno intracelular a um ambiente tóxico, caracterizado por pH ácido, presença de espécies reativas, como também de peptídeos antimicrobianos. Neste contexto, há uma competição entre o patógeno intracelular e o hospedeiro mamífero por nutrientes essenciais, como por exemplo, metais de transição. Cobre é um metal de transição essencial para a vida aeróbica, porém pode ser tóxico em altas concentrações e, devido a isto, diversos organismos desenvolveram mecanismos regulatórios para controlar suas concentrações. O objetivo deste trabalho foi avaliar a modulação da homeostase de cobre durante a interação entre macrófagos e células de C. neoformans ou C. albicans. Neste trabalho, foi possível demonstrar que a presença de cobre contribui para uma menor atividade fungicida de macrófagos infectados. Também, o pré-carregamento das células fúngicas com cobre alterou a sensibilidade de ambas leveduras patogênicas à atividade anti-fúngica. Além disso, foi demonstrado que a expressão de transportadores de cobre (CTR1 e ATP7A) e proteínas ligadoras de cobre (ceruloplasmina e metalotioneína 1) de macrófagos foram moduladas em resposta à infecção por estes fungos patogênicos. Entretanto, essa regulação pode envolver mecanismos mais complexos, como estratégias do patógeno para subverter a ação antimicrobiana de macrófagos. / Pathogenic yeasts, such as Cryptococcus neoformans and Candida albicans, are one of the most frequent causes of the opportunistic infections worldwide, being able to survive, proliferate and escape from macrophages, the first line cells engaged in the immune defense of the mammalian host. Macrophages generally expose the intracellular pathogen to a toxic environment, which is characterized by acid pH, presence of reactive species, as well the presence of antimicrobial peptides. In this context, there is a competition between intracellular pathogen and mammalian host for essential nutrients, such as transition metals. Copper is an essential transition metal for aerobic life, but can be toxic at high concentrations and, therefore, many organisms have evolved highly regulated mechanisms for controlling its concentration. In this work, we aim is evaluate a modulation of copper homeostasis during interaction between macrophages and C. neoformans or C. albicans cells. This work demonstrated that the presence of copper resulted in a lower fungicidal activity of infected macrophages. Also, the pre-loading of fungal cells with copper can alter the sensitivity of both pathogenic yeasts to an antifungal activity. In addition, we showed that the expression of macrophage copper transporters (CTR1 and ATP7A) and copper-binding proteins (ceruloplasmin and metallothionein 1) are modulated in response infection by pathogenic fungi. However, this regulation may involve more complex mechanisms, such as strategies of pathogen to subvert the antimicrobial action of macrophages.

Fungos patogênicos, como Cryptococcus neoformans e Candida albicans, são uma das mais frequentes causas de infecções oportunistas em todo o mundo, sendo capazes de sobreviver, proliferar e escapar de macrófagos, células de primeira linha da resposta imune de hospedeiros mamíferos. Macrófagos geralmente expõem o patógeno intracelular a um ambiente tóxico, caracterizado por pH ácido, presença de espécies reativas, como também de peptídeos antimicrobianos. Neste contexto, há uma competição entre o patógeno intracelular e o hospedeiro mamífero por nutrientes essenciais, como por exemplo, metais de transição. Cobre é um metal de transição essencial para a vida aeróbica, porém pode ser tóxico em altas concentrações e, devido a isto, diversos organismos desenvolveram mecanismos regulatórios para controlar suas concentrações. O objetivo deste trabalho foi avaliar a modulação da homeostase de cobre durante a interação entre macrófagos e células de C. neoformans ou C. albicans. Neste trabalho, foi possível demonstrar que a presença de cobre contribui para uma menor atividade fungicida de macrófagos infectados. Também, o pré-carregamento das células fúngicas com cobre alterou a sensibilidade de ambas leveduras patogênicas à atividade anti-fúngica. Além disso, foi demonstrado que a expressão de transportadores de cobre (CTR1 e ATP7A) e proteínas ligadoras de cobre (ceruloplasmina e metalotioneína 1) de macrófagos foram moduladas em resposta à infecção por estes fungos patogênicos. Entretanto, essa regulação pode envolver mecanismos mais complexos, como estratégias do patógeno para subverter a ação antimicrobiana de macrófagos. / Pathogenic yeasts, such as Cryptococcus neoformans and Candida albicans, are one of the most frequent causes of the opportunistic infections worldwide, being able to survive, proliferate and escape from macrophages, the first line cells engaged in the immune defense of the mammalian host. Macrophages generally expose the intracellular pathogen to a toxic environment, which is characterized by acid pH, presence of reactive species, as well the presence of antimicrobial peptides. In this context, there is a competition between intracellular pathogen and mammalian host for essential nutrients, such as transition metals. Copper is an essential transition metal for aerobic life, but can be toxic at high concentrations and, therefore, many organisms have evolved highly regulated mechanisms for controlling its concentration. In this work, we aim is evaluate a modulation of copper homeostasis during interaction between macrophages and C. neoformans or C. albicans cells. This work demonstrated that the presence of copper resulted in a lower fungicidal activity of infected macrophages. Also, the pre-loading of fungal cells with copper can alter the sensitivity of both pathogenic yeasts to an antifungal activity. In addition, we showed that the expression of macrophage copper transporters (CTR1 and ATP7A) and copper-binding proteins (ceruloplasmin and metallothionein 1) are modulated in response infection by pathogenic fungi. However, this regulation may involve more complex mechanisms, such as strategies of pathogen to subvert the antimicrobial action of macrophages.

Human fungal infections among severely immunocompromised individuals have increased dramatically over the last 30 years and that coincidence with an expanding patient numbers of bone marrow and solid organ transplantation and those receiving aggressive cytotoxic chemotherapy for neoplastic diseases or immunosuppressive drugs. In recent years, many of opportunistic fungi have emerged as serious human pathogens and causing life-threatening infections of humans such as Fusarium species. Due to lack of a highly accurate diagnostic test for tracking the pathogenic Fusarium species, fusariosis is frequently misdiagnosed as aspergillosis. Delays in identification and differentiation of Fusarium spp. from other causative agents of hyalohyphomycetes associated with high morbidity and mortality rate among immunocompromised patients. This research aimed to develop a highly specific monoclonal antibody (mAb) using hybridoma technology to produce a highly genus-specific murine mAb ED7 that could be used for tracking and early detecting circulating Fusarium species antigens from other opportunistic pathogens. At present, a very little is known about the pathogenicity and interaction of human pathogenic F. solani and cells of the innate immune system like alveolar macrophages (AMØ), the residential innate immune cells of alveoli. For this reason, F. solani was genetically transformed with GFP gene and a model of immunoassay was developed to investigate the interactions of F. solani with AMØ that would allow studying the fungal pathogenicity, visualising and quantification of the pathogen during the process of macrophage phagocytosis. In addition, this model can be used to evaluate the effect of a mAb on fungal uptake by AMØ. Habitates providing direct human exposure to infectious propargules are largely unknown, but there is growing evidence that plumbing systems are sources of human pathogenic strains in the F. solani and F. oxysporum species complexes, the most common groups infecting humans. Using mAb ED7 specific to the Fusarium species, this work demonstrates how the mAb can be used as a powerful immunodiagnostic tool for accurately tracking the Fusarium species antigens in sink drain biofilms and water system samples containing mixed populations of human opportunistic filamentous and yeasts pathogenic fungi across a University campus and a tertiary care hospital. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of culturable yeasts and moulds that were recovered using mycological culture, while translation elongation factor (TEF)-1 analysis of Fusarium isolates included FSSC 1-a, FOSC 33 and FDSC ET-gr, the most common clinical pathotypes in each group. The mAb ED7 is, therefore, suitable to be carried forward for use in diagnostic assays, such as the lateral flow device.

Schizolobium parahybum is native to Ecuador, South America, where plantations of this tree is being established. Development of these plantations has been unsuccessful due to a serious die-back disease. A survey on the role fungal pathogens might play in disease development were conducted by isolating possible pathogenic fungi from streaks in the xylem and from machete wounds from dying trees. The primary aim of this study was to identify the isolated fungi and to determine their pathogenicity to S. parahybum through a series of greenhouse trials. Ceratocystis fimbriata, C. moniliformis, Thielaviopsis basicola, Graphium penicillioides, Ophiostoma quercus and a Pesotum species were identified as possible pathogens of S. parahybum. Inoculation trials conducted with these fungi revealed that C. fimbriata and C. moniliformis were able to cause significant lesions on young S. parahybum trees under greenhouse conditions. These fungi were, however, not consistently isolated from diseased trees and the lesions in greenhouse trials were not consistently produced. Graphium penicillioides, O. quercus and the Pesotum species were unable to cause any notable lesions. Thielaviopsis basicola caused lesions that differed significantly from the control inoculations of S. parahybum. These results were intriguing, as T. basicola is not a known tree pathogen, but is predominantly known to cause disease of agricultural crops such as groundnuts and chicory. The ability of T. basicola to cause lesions on S. parahybum initiated the second part of the thesis that dealt with a population diversity study of this pathogen. Comparison of T. basicola isolates from Ecuador and other parts of the world with isolates from groundnut and chicory in South Africa revealed that the Ecuador isolates did not originate from S. parahybum but from the carrots that was used to isolate them. Although T. basicola from carrots were able to cause lesions on S. parahybum, we did not investigate this phenomenon further as this fell beyond the scope of this thesis. Seven polymorphic primers were developed for T. basicola using the ISSR-PCR technique. These primers were used to determine the population diversity of T. basicola from groundnuts and chicory populations in South Africa. Results showed a low diversity for both populations and suggest that T. basicola was introduced to South Africa. The markers were also used to compare isolates from the groundnut and chicory populations with T. basicola isolates from other hosts and geographical regions. This indicated that T. basicola may be native to Europe from where it possibly spread to other countries. / Dissertation (MSc)--University of Pretoria, 2007. / Microbiology and Plant Pathology / MSc / Unrestricted

Due to consumer resistance to the use of synthetic chemicals in agriculture, the aim of this research was to develop an antifungal extract from the leaves of Melianthus comosus that could be marketed as an organic fungicide by Healthtech Laboratories (Pty) Ltd. Ten solvents of varying polarities (hexane, carbon tetrachloride, diethyl ether, dichloromethane, chloroform, acetone, ethanol, ethyl acetate, methanol and water) were used to extract the dried and powdered leaves of Melianthus comosus. The antifungal activity of each extract was tested against 10 plant pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Penicillium janthinelum, Penicillium expansum, Colletotrichum gloeosporioides, Trichoderma harzianum, Pythium ultimum, Phytophthora nicotiana, Aspergillus niger, and Aspergillus parasiticus). The acetone extract had the highest antifungal activity (average MIC of 0.10 mg/ml against 8 pathogens), followed by the ethanol extract with an average MIC of 0.22 mg/ml against 8 pathogens. Bioautography was used to determine the number of antifungal compounds in the extracts and to locate the active compounds on bioautograms, to facilitate bioassay guided isolation. The main active compound against all organisms was present in the acetone extract and was of intermediate polarity with an Rf value of 0.33 in BEA. There was another compound that was non-polar with an Rf value of 0.86 that had moderate antifungal activity against Fusarium oxysporum. The inactive compounds were generally of high polarity (4) and some were also non-polar (5). Enrichment procedures removing inactive compounds were developed to increase the concentration of the active compounds. Acetone was the best primary extractant for enrichment based on antifungal activity. Two pathways were used to enrich the acetone extract. Pathway 1 included the use of water to extract the highly polar inactive compounds from the plant material. The water-“washed” marc was dried and “washed” (extracted once) separately with hexane or dichloromethane or ethyl acetate. The respective marcs were again dried and extracted with acetone. All extracts were tested for antifungal activity. The acetone extract following the water and dichloromethane “washes” was the most active (average MIC of 0.088 mg/ml against all 10 fungal organisms) and was termed “HT01”. In pathway 2, a solvent-solvent extraction method was used. The plant material was first extracted with acetone and the acetone extract dried. The dried extract was dissolved in a 1:1 mixture of ethyl acetate and water in a separating funnel. The ethyl acetate fraction was extracted with water several times to remove all the very polar inactive compounds. The water and the ethyl acetate fractions were both dried and used for phytochemical analysis and bioassays. The water fraction was relatively inactive against all organisms. The ethyl acetate fraction was termed “HT02” and had an average MIC of 0.066 mg/ml against all 10 fungal organisms. Work was continued on HT02 including a field trial and cytotoxicity assays as it was the most active extract. The HT02 extract was used for a field trial (done in collaboration with the Healthtech Laboratories) on the plant Symphytum officinale (Comfrey) infected by rust. When the in vitro activity of HT02 was compared with six commonly used fungicides, it had the most activity against Penicillium expansum and the second highest activity against Fusarium oxysporum. After nearly two months plants treated with HT02 (0.2 mg/ml) had c. 50 infected leaves. Plants treated with a commercial fungicide “Bravo 500” (containing 1.5 mg/ml chlorothalonil) had c. 250 infected leaves and untreated plants had extensive infection. Moreover, plants treated with HT02 had flourished when compared to the other treatments. The HT02 extract was more effective against the particular fungus at a six times lower concentration than “Bravo 500”. The cytotoxicity of HT02 was determined using the brine shrimp and cell line toxicity assays. The LC50 of HT02 was 4.5 mg/ml against brine shrimp larvae and 0.0445 mg/ml against Vero cell lines. The toxic principles present in the extract were tentatively identified on TLC using a 20% solution of antimony-III-chloride in ethanol and Kedde’s reagent as cardiac glycosides of Rf values: 0, 0.08, 0.23, 0.34 and 0.58 in EMW solvent system. Lead precipitation and vacuum column chromatography were used to remove the cardiac glycosides from the extract, hence reducing the cytotoxicity of the extract. Lead precipitation removed all five cardiac glycosides but the antifungal activity of the extract was reduced four-fold. Fractionation of HT02 by means of column chromatography gave nine fractions. The antifungal fractions (F3 with MIC values of 0.04 and 0.08 mg/ml against Fusarium oxysporum and Penicillium janthinellum respectively, and F2 with MIC values of 0.08 and 0.16 mg/ml against Fusarium oxysporum and Penicillium janthinellum) were combined and showed no apparent cytotoxicity against the Vero cell lines. The inactive fractions were combined and had an LC50 of 0.0861 mg/ml against the Vero cell lines. To isolate the main antifungal compound, gradient vacuum liquid chromatography and gravity assisted column chromatography was used in the bioassay-guided fractionation of the acetone extract. The major active compound of Rf 0.33 in BEA, 0.71 in CEF and 0.44 in hexane: ethyl acetate (7:3, v/v) was isolated and identified as the triterpene 3-hydroxy-12-oleanen-28-oic acid (oleanolic acid). The compound had high antifungal activity with MIC values ranging from 7.8 to 15.6 ìg/ml against fungal pathogens used. This compound has been isolated from the root bark of Melianthus comosus and other plants but this is the first description of its antifungal activity against plant pathogenic fungi. The compound had no apparent cytotoxicity on Vero cell lines Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Angeh, IE 2006, Potentising and application of an extract of Melanthus Comosus against plant fungal pathogens, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02232012-172854 / > E506/gm / Dissertation (MSc)--University of Pretoria, 2006. / Paraclinical Sciences / unrestricted

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Many barriers can limit restoration success. In the first chapter of this thesis, the barrier of strong seed dormancy is addressed. While dormancy benefits the species' long-term survival, it can present a challenge within a restoration scenario where rapid establishment is required. Soaking seeds in gibberellic acid (GA3) can overcome dormancy. An easier and potentially more effective method to apply this hormone is to coat seeds with a GA3-impregnated polymer, which provides a slow release of the hormone. Seed dormancy can also be mitigated by creating a favorable microsite with increased soil moisture. We compared the emergence and establishment of penstemon seeds that were coated with GA3 to uncoated seeds planted in shallow drill rows versus deep, U-shaped furrows. These treatments were evaluated in fall and spring plantings at three field sites in the Great Basin Region of the United States. Overall, coating with GA3 improved the emergence and establishment of Palmer's penstemon (Penstemon palmeri; p < 0.01) and thickleaf penstemon (P. pachyphyllus; p < 0.001) but did not improve the emergence or establishment of firecracker penstemon (P. eatonii; p = 1). Between planting seasons, fewer seedlings emerged or established from spring than from fall planting (p < 0.001). Emergence and establishment were higher for all species in deep furrows than in shallow drill rows (p < 0.001). These results indicate that GA3 seed coating and deep, U-shaped furrows may improve the restoration success of some native forbs. Land managers could use these techniques to restore native forbs in dry, disturbed areas. The second chapter of this thesis addresses another barrier to successful restoration, specifically pathogenesis from soil and seed-borne fungus. Survival and growth of native seeds and seedlings can be limited by soil and seed-borne pathogens. Fungicides can combat fungal pathogens, but in some studies, fungicide treatments were ineffective at improving seedling emergence. These studies cite dry conditions leading to low fungal presence as the cause of the ineffectiveness of fungicide treatments for some years and sites. This study tested if fungicide treatment effectiveness is indeed related to the amount of fungus in the soil. We analyzed the emergence and biomass of uncoated, blank-coated, and fungicide-coated bluebunch wheatgrass (Pseudoroegneria spicata) across five soil fungal levels. For both percent emergence and total biomass, uncoated seed performed best in autoclaved soil and declined with increasing level of fungus, but the level of fungus did not impact fungicide-coated seed. When grown in autoclaved, untreated, or low fungal soils, percent emergence and total biomass from fungicide-coated seeds was not different from uncoated seeds. However, in medium and high fungal soils, the percent emergence and total biomass from fungicide-coated seeds were more than two times greater than uncoated seeds (p < 0.05). These results indicate fungicide seed coatings can be effective at increasing restoration success for bluebunch wheatgrass, but the effectiveness of this treatment depends on the microbial environment of the planting site.

The potential of Pandora neoaphidis (Remaudiére & Hennebert) Humber as a fungal pathogen for the control of the tobacco aphid, Myzus nicotianae Blackman, on tobacco was evaluated in a 4-year study between 1992 and 1995. The objectives of this study included determination of the seasonal incidence of P. neoaphidis in populations of tobacco aphid on tobacco and non solanaceous host plants, within plant distribution of the pathogen in aphids on tobacco, influence of tobacco cultivars and cultural practices on the incidence of the pathogen, methods of artificially introducing the pathogen into tobacco aphid populations and their potential in controlling aphids relative to chemical control, virulence of the Virginia isolate of the pathogen to tobacco aphid and green peach aphid, M. persicae (Sulzer), from different geographic locations in the eastern United States, and influence of temperature and type of substrate on the developmental morphology of the pathogen. Infections as high as 91% occurred in aphid populations on tobacco under favorable weather. The pathogen survived at moderate levels in the red morph of tobacco aphid on non solanaceous hosts during fall, parts of winter and spring. P. neoaphidis infections in aphids tended to increase towards the upper leaf positions. Incidence of the pathogen in aphid populations varied widely on various cultivars and types of tobacco. Planting date, topping of tobacco, and stage at which tobacco was topped did not influence the incidence of P. neoaphidis in tobacco aphids. Artificial introduction of P. neoaphidis successfully established infections in tobacco aphids, but failed to prevent the build up of aphid populations. P. neoaphidis was equally virulent to the red and green morphs of the tobacco aphid, and the green peach aphid. The developmental morphology of P. neoaphidis was influenced by temperature and was similar on the surfaces of living substrates, tobacco aphid and tobacco leaf, but different on the inert surface of the coverslip. / Ph. D.