Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The concentrations of copper (Cu) in vineyard soils of the Western Cape range from 0.1 to 20 ppm. However, more than 160 tons of the fungicide copper oxychloride are annually being sprayed on these vineyards. This has raised concerns that Cu may accumulate in these soils, resulting in a negative impact on the soil biological processes, especially since the soils in the Western Cape are slightly acidic, making Cu more mobile and available for soil organisms than would have been the case in alkaline soils. The goal of the initial part of this study was therefore to identify those soil microbial communities indigenous to the Western Cape, which are most susceptible to Cu-induced stress as a result of the addition of copper oxychloride. These potential bioindicators of Cu-induced stress were first searched for in uncultivated agricultural soil from Nietvoorbij experimental farm. Consequently, a series of soil microcosms was prepared by adding various concentrations of Cu as a component of copper oxychloride, to each of eight aliquots of soil: 0 (control), 10, 20, 30, 40, 50, 100, 500 and 1000 ppm. The resulting concentrations of exchangeable Cu in these microcosms were found to be 2 (control), 12,23,34,42,59, 126,516 and 1112 ppm. Selected microbial communities in each microcosm were subsequently monitored over a period of 245 days. It was found that the culturable microbial numbers did not provide a reliable indication of the effect of Cu on community integrity. However, analyses of terminal-restriction fragment length polymorphism (T-RFLP) community fingerprints and especially analyses of the whole community metabolic profiles, revealed that shifts in the soil microbial communities took place as the Cu concentration increased. Direct counts of soil protozoa also revealed that the addition of Cu to the soil impacted negatively on the numbers of these eukaryotes. To confirm these findings in other soil ecosystems, the impact of copper oxychloride on whole community metabolic profiles and protozoan numbers were investigated in soils from Koopmanskloof commercial farm and Nietvoorbij experimental farm. These potential bioindicators were subsequently monitored in a series of soil microcosms prepared for each soil type by adding the estimated amounts of 0 (control), 30, 100 and 1000 ppm Cu as a component of copper oxychloride to the soil. The results confirmed the fmdings that elevated levels of copper impact negatively on the metabolic potential and protozoan numbers of soil. Consequently, it was decided to investigate a combination of protozoan counts and metabolic profiling as a potential bioindicator for Cu-induced stress in soil. Data collected from all the microcosms containing exchangeable Cu concentrations ranging from 1 ppm to 1112 ppm was used to construct a dendrogram using carbon source utilization profiles in combination with protozoan counts. It was found that the microcosms grouped into clusters, which correlated with the concentration of exchangeable Cu in the soil. Under the experimental conditions used in this study, the combination of protozoan counts and metabolic profiling seemed to be a reliable indicator of Cu-induced stress. However, this bioindicator must be further investigated in other soil types using other types of stress inducing pollutants. In addition to the above fmdings it was also found that the numbers of soil protozoa was particularly susceptible to Cu-induced stress in soils with a low soil pH. This is in agreement with the fmdings of others on the bio-availability of heavy metals in low pH soils. In these soils, nutrient cycling as a result of protozoan activity, may therefore be particularly susceptible to the negative impact of copper to the soil. / AFRIKAANSE OPSOMMING: Die konsentrasies van koper (Cu) in wingerdgronde van die Wes-Kaap wissel tussen 0.1 en 20 dpm. Meer as 160 ton van die fungisied koper-oksichloried word egter jaarliks op dié wingerde gespuit, wat kommer laat ontstaan het oor die moontlike akkumulasie van Cu in dié grond en die gevaar van 'n negatiewe impak op die biologiese prosesse in die grond. Die gevaar word vererger deur die feit dat die Wes-Kaapse grond effens suur is, wat Cu meer mobiel en beskikbaar maak vir grondorganismes as wat die geval sou wees in alkaliese grond. Die eerste doelstelling van hierdie studie was dus om die mikrobiese gemeenskappe in die grond, wat inheems is aan die Wes-Kaap, te identifiseer wat die meeste vatbaar is vir Cu-geïnduseerde stres as gevolg van die toevoeging van koper-oksichloried. Hierdie potensiële bioindikatore van Cu-geïnduseerde stres is eerstens gesoek in onbewerkte landbougrond van die Nietvoorbij-proefplaas. 'n Reeks grondmikrokosmosse is gevolglik berei deur verskillende konsentrasies Cu, as 'n komponent van koperoksichloried, by elk van agt hoeveelhede grond te voeg naamlik 0 (kontrole), 10,20, 30, 40, 50, 100, 500 en 1000 dpm. Die gevolglike konsentrasies van uitruilbare Cu in hierdie mikrokosmosse was 2 (kontrole), 12, 23, 34, 42, 59, 126, 516 en 1112 dpm. Geselekteerde mikrobiese gemeenskappe in elke mikrokosmos is vervolgens oor 'n tydperk van 245 dae bestudeer. Daar is gevind dat die kweekbare mikrobiese tellings nie 'n betroubare aanduiding kon gee van die uitwerking van Cu op gemeenskapsintegriteit nie. Die ontledings van terminale-restriksie fragment lengte polymorfisme (T-RFLP) gemeenskapsvingerafdrukke en veral van die metaboliese profiele van die totale gemeenskap, het getoon dat verskuiwings in die grondmikrobiese gemeenskappe plaasgevind het met 'n toename in Cu-konsentrasies. Direkte tellings van grondprotosoë het ook aangedui dat die toevoeging van Cu tot die grond 'n negatiewe uitwerking op die getalle van hierdie eukariote gehad het. Om dié resultate te bevestig, is die impak van koper-oksichloried op die metaboliese profiele van totale gemeenskappe en protosoë-getalle in ander grond-ekosisteme vervolgens bestudeer deur grond van die kommersiële plaas Koopmanskloof en die Nietvoorbij-proefplaas te gebruik. Dié potensiële bioindikatore is vervolgens bestudeer in 'n reeks grondmikrokosmosse, wat vir elke grondtipe voorberei is deur die toevoeging van beraamde hoeveelhede van 0 (kontrole), 30, 100 en 1000 dpm Cu as 'n komponent van koper-oksichloried. Die resultate het die bevindings bevestig dat verhoogde vlakke van Cu 'n negatiewe uitwerking het op die metaboliese potensiaal en op die protosoëgetalle in die grond. Daar is gevolglik besluit om 'n kombinasie van protosoë-tellings en metaboliese profiele te ondersoek as 'n potensiële bioindikator van Cu-geïnduseerde stres in grond. Data van al die mikrokosmosse wat uitruilbare Cu bevat, wisselend van 1 dpm tot 1112 dpm, is gebruik om 'n dendrogram te konstrueer wat koolstofbronbenuttingsprofiele in kombinasie met protosoë tellings gebruik. Daar is gevind dat die mikrokosmosse groepe vorm wat korrelleer met die konsentrasie uitruilbare Cu in die grond. Onder die eksperimentele kondisies wat in dié studie gebruik is, wil dit voorkom of die kombinasie van protosoë-tellings en metaboliese profiele 'n betroubare indikator van Cugeïnduseerde stres is. Hierdie bioindikator moet egter verder in ander grondtipes en met ander tipes stres-induserende besoedeling ondersoek word. By bogenoemde bevindings is daar ook gevind dat die getalle grondprotosoë besonder gevoelig is vir Cu-geïnduseerde stres in grond met In lae pH. Dit is in ooreenstemming met die bevindings van andere met betrekking tot die bio-beskikbaarheid van swaar metale in grond met 'n lae pH. In dié tipe grond mag nutriëntsiklering as gevolg van protosoë aktiwiteit besonder gevoelig wees vir die negatiewe uitwerking van koper in die grond.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Numerous workers studied the impact of pollutants and agricultural chemicals, containing heavy metals such as copper (Cu), on soil microbes. It was found that elevated soil Cu levels do have a detrimental effect on soil bacterial populations however the filamentous fungi seemed to be less affected. Most of these studies were conducted in soils containing already relatively high Cu levels and the effect of this heavy metal on the nonfilamentous fungi (i.e. yeasts) was never investigated. The aim of this study was therefore to determine the impact of elevated Cu levels on filamentous fungi and yeasts occurring in soils containing relatively low natural Cu levels. A synthetic selective medium containing glucose as carbon source, thymine as nitrogen source, vitamins, minerals and chloramphenicol as anti-bacterial agent (TMV-agar), was used to enumerate ascomycetous and basidiomycetous Cu resistant yeasts in a sample of virgin soil containing ~ 2ppm Cu. Media that were used to enumerate Cu resistant filamentous fungi were malt extract agar, malt extract agar with streptomycin sulfate, maltyeast- extract-peptone agar with chloramphenicol and streptomycin sulfate, benomyl–dichloran-streptomycin medium for the enumeration of hymenomycetous fungi and two selective media for the isolation of mucoralean fungi. Cu resistant fungi able to grow on all of the above mentioned solid media supplemented with 32 ppm Cu occurred in the soil sample. To obtain an indication of the level of Cu tolerance of fungi present in this soil sample, a number of fungal isolates were screened for the ability to grow on a series of agar plates, prepared from glucose-glutamate-yeast extract agar, containing increasing concentrations of Cu. It was found that filamentous fungi and yeasts that were able to grow on this agar medium containing up to 100 ppm Cu were present in the soil. A series of soil microcosms was subsequently prepared from the soil sample by experimentally contaminating the soil with increasing amounts of copper oxychloride, were after fungal populations in the microcosms, including Cu resistant fungi, were monitored using plate counts. At the end of the incubation period, after 245 days, fungal biomass in the microcosms was compared by determining the concentrations of the fungal sterol, ergosterol, inthe soil. Generally, Cu had little impact on the numbers of filamentous fungal colony forming units on the plates, as well as on the ergosterol content of the soil. The numbers of filamentous fungi in the soil, including the Mucorales and hymenomycetes, seemed to be less affected by the addition of copper oxychloride than the numbers of soil yeasts able to grow on TMV-agar. The focus of the next chapter was on the response of yeasts in different soils to elevated levels of Cu in the soil. TMV-agar was used to enumerate yeasts in soil microcosms prepared from four different soil samples, which were experimentally treated with copper oxychloride resulting in Cu concentrations of up to 1000 ppm. The selective medium supplemented with 32 ppm Cu was used to enumerate Cu resistant yeasts in the microcosms. The results showed that the addition of Cu at concentrations ≥ ~1000 ppm did not have a significant effect on total yeast numbers in the soil. Furthermore, it was found that Cu resistant yeasts were present in all the soil samples regardless of the amount of Cu that the soil was challenged with. At the end of the incubation period, yeasts in the microcosms with zero and ~1000 ppm additional Cu were enumerated, isolated and identified using sequence analyses of the D1/D2 600-650bp region of the large subunit of ribosomal DNA. Hymenomycetous species dominated in the control soil, while higher numbers of the urediniomycetous species were found in the soil that received Cu. These observations suggest that urediniomycetous yeasts may play an important role in re-establishing overall microbial activity in soils following perturbations such as the addition of Cu-based fungicides. / AFRIKAANSE OPSOMMING: Vele navorsers het al die impak van besoedelingstowwe en landbouchemikalieë wat swaarmetale soos koper (Cu) bevat, op grond-mikrobes bestudeer. Dit is gevind dat verhoogde Cu vlakke ‘n nadelige effek het op grond-bakteriese populasies, maar dat die filamentagtige fungi geneig is om minder geaffekteer te word. Meeste van hierdie studies is gedoen met gronde wat alreeds relatief hoë Cu vlakke bevat het en die effek van hierdie swaarmetaal op die nie-filamentagtige fungi (d.i. giste) is nooit ondersoek nie. Die doel van hierdie studie was dus om die impak van verhoogde Cu vlakke op filamentagtige fungi en giste in gronde, wat natuurlike lae vlakke van Cu bevat, te bepaal. ‘n Sintetiese selektiewe medium wat glukose as koolstofbron, timien as stikstofbron, vitamiene, minerale asook chloramfenikol as anti-bakteriese agent bevat (TMV-agar), is gebruik om askomisete en basidiomisete Cu weerstandbiedende giste in ‘n monster ongeskonde grond, bevattende ~ 2dpm Cu, te tel. Media wat gebruik is om Cu weerstandbiedende filamentagtige fungi te tel, was mout-ekstrak agar, moutekstrak agar met streptomisiensulfaat, benomiel-dichloran-streptomisien medium vir die tel van hiemenomiseetagtige fungi en twee media vir die isolasie van mukoraliese fungi. Cu-weerstandbiedende fungi wat op al die bogenoemde media, aangevul met 32 dpm Cu, kon groei, het in die grondmonster voorgekom. Om die mate van Cu-weerstandbiedendheid van fungi wat in die grondmonster voorkom, te bepaal, is ‘n getal fungus-isolate op agarplate, voorberei met glukose-glutamaat-gis ekstrak agar, bevattende verhoogde konsentrasies Cu, nagegaan. Daar is gevind dat daar filamentagtige fungi en giste in die grond voorkom wat die vermoë het om op media bevattende 100 dpm Cu te groei. ‘n Reeks grond mikrokosmosse is dus voorberei vanaf die grondmonster deur om dit eksperimenteel te kontamineer met verhoogde hoeveelhede koper oksichloried, waarna die fungus-populasies asook die Cu-weerstandbiedende fungi in die mikrokosmos gemoniteer is deur middel van plaattellings. Aan die einde van die inkubasie periode, 245 dae, is die fungus biomassa in al die mikrokosmosse bereken deur die konsentrasie van die fungus sterool ergosterool te bepaal en dit met mekaar te vergelyk. Oor die algemeen het Cu min impak ten opsigte van diegetal filamentagtige fungi kolonie vormende eenhede die plate, asook op die ergosterool inhoud van die grond gehad. Dit wil voorkom of die getal filamentagtige fungi in die grond, insluitende die Mucorales en die hymenomisete, minder geaffekteer is deur die toediening van koperoksichloried as die aantal grondgiste wat op die TMV-agar kan groei. Die fokus van die volgende hoofstuk was dus op die reaksie wat giste in verskillende grondtipes gehad het op verhoogde Cu in die grond. TMV-agar is gebruik om die getal giste te bepaal in die grond mikrokosmosse van die vier verskillende grondmonsters, wat voorberei is deur om dit eksperimenteel met koper oksikloried te kontamineer tot en met Cu konsentrasies van 1000 dpm. Die selektiewe medium wat gesupplementeer is met 32 dpm Cu, is gebruik om Cu weerstandbiedende giste in die mikrokosmosse te bepaal. Die resultate toon dat die toevoeging van Cu by konsentrasies ≥ ~1000 dpm nie enige beduidende effek op die totale gis getalle gehad het nie. Daar is ook gevind dat daar Cu weerstandbiedende giste in die grond monsters voorkom gekom het ten spyte van die hoeveelheid Cu wat tot die grond toegevoeg is. Aan die einde van die inkubasie periode is die giste wat die die mikrokosmosse bevattende nul en ~1000 dpm Cu getel, geïsoleer en geïdentifiseer deur gebruik te maak van DNA volgorde bepaling van die D1/D2 600-650 bp areas geleë in die groter subeenheid van die ribosonale DNA. Hymenomisete spesies het in die grond kontrole gedomineer, terwyl hoër getalle uredinomisete spesies in die grond met addisionele Cu gevind is. Die resultate dui daarop dat uredinomisete giste dalk ‘n belangrike rol kan speel in die hervestiging van die oorwegende mikrobiese aktiwiteit in grond na skoktoestande soos die aanwending van Cu-gebaseerde fungisiede.