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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16290 |
| Date | 04 1900 |
| Creators | Cornelissen, Stephanie |
| Contributors | Botha, A., Wolfaardt, G.M., University of Stellenbosch. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xi, 85 leaves : ill. |
| Rights | University of Stellenbosch |
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