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Overexpression and evaluation of an antimicrobial peptide from Heuchera sanguinea (Hs-AFP1) for inhibition of fungal pathogens in transgenic tabaccoDe Beer, Abre 04 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Seed germination is the most vulnerable time in a plant's life cycle, since the thick protective
seed coat ruptures and the moist and humid soil environment not only favours seed
germination, but also the growth and development of plant pathogens. Infection of plant seeds
during germination, however, is the exception rather than the rule. Plant seeds have
- - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuse
into the surrounding environment to form a protective layer around the seed. This
protective layer prevents fungal and bacterial pathogens from infecting the young seedling.
Over the last decade, scientists have studied the defense mechanisms of different
seeds in an effort to understand and ultimately to introduce and/or manipulate these
mechanisms in plants as part of the plant's endogenous disease resistance to pathogens.
Various chemical compounds, peptides and proteins that showed strong in vitro activities
against various fungi were isolated in these efforts. The mere demonstration of in vitro activity
alone, however, is not sufficient to assign a defense role to these antifungal agents. Typically,
mutant plants that have lost the ability to produce the antifungal agent, or mutants that are
overproducing the agent, have been used to correlate the mutant phenotype to either a
decline or increase in disease resistance respectively. Genetic transformation and the
subsequent development of transgenic plants have made an unprecedented impact in this
regard, specifically in understanding the role of specific defense-related proteins and their
interaction with plant pathogens.
In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plant
defensin, was evaluated in a heterologous in planta environment as a defense protein with
potential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1
against Botrytis cinerea showed antifungal activities of 88% growth inhibition at a
concentration of 8 J,lg/ml of the purified peptide, while inducing a characteristic
hyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with a
construct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. The
peptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, an
antimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenic
plant systems, two additional constructs were prepared and transformed into tobacco to
anticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putative
peptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and the
antifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A control
construct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2
signal peptide, was also prepared and transformed into tobacco to normalize the background
antifungal activity as a result of the exoglucanase in the fusion construct lines. Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1,
resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgene
expression was confirmed for the THs and THE transgenic lines. The translation of these
transcripts into proteins was also confirmed with Western blot analysis. Moreover, the
heterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance to
B. cinerea in the THs lines in comparison with the untransformed tobacco controls. An
increase of up to 42% in disease resistance was observed in an in planta detached leaf
assay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitative
fungal growth assay. This assay confirmed the results obtained with the disease resistance
assay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. The
incubation of B. cinerea in the presence of crude protein extracts from THs lines resulted in
hyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity.
From these analyses it was clear that the heterologously expressed Hs-AFP1 was
quite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did not
increase the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All the
analyses performed showed the THE lines to be reduced in their ability to inhibit fungal
infection in comparison to the THs line. Also, microscopic analysis of the effects of the crude
THE extracts on B. cinerea growth showed no hyperbranching activity, again confirming the
loss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, in
which sarcotoxin 1A was fused to a reporter gene and also lost activity.
Although integration of the Mj-EXG1 expression cassette was confirmed, no mRNA
levels could be detected with Northern blot or RT-PCR analysis of the TME lines. These lines
also did not show any in vitro antifungal activities, probably indicating post-transcriptional
gene silencing. This silencing was overcome in the fusion constructs that were expressed in
the THE plant lines. These lines also showed EXG1 protein activity, as measured by
~-glucosidase assays. Although the THE lines did not serve the functions originally
envisaged, they fortuitously showed that a fusion strategy might stabilize glucanase
expression in a transgenic environment. A variety of glucanases have been shown to be
prone to gene silencing when overexpressed in a plant environment and the yeast glucanase
can now be added to that list if it is not present as a fusion protein.
Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems and
provided valuable information on the stability of small peptides in a heterologous environment.
The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this study
merits further investigations into the use of this peptide in the engineering of disease-resistant
crops. / AFRIKAANSE OPSOMMING: Saadontkieming is die mees vatbare tyd vir siekteontwikkeling gedurende 'n plant se
lewenssiklus. Die saadhuid bars en die vogtige grondkondisies bevoordeel nie net
saadontkieming nie, maar ook die groei en ontwikkeling van plantpatogene. Infeksie van
plantsade tydens ontkieming is egter die uitsondering eerder as die reël. Plantsade besit
komplekse -veraeaigingsfueganfsmes-reen moontlike - patoqeeninteksies. Die meqanismes
sluit die produksie van antifungiese agense, wat tydens saadontkieming na die omliggende
omgewing diffundeer om 'n beskermende sone om die ontkiemende saad te vorm, in. Die
gevolglike antifungiese sone beskerm die saad teen infeksie deur bakterieë en swamme.
Gedurende die laaste dekade het navorsers baie aandag aan die bestudering van
plantsaadverdedigingsmeganismes gegee. Dié kennis word gebruik om die verdedigingsmeganismes
beter te verstaan, asook om dié meganismes te manipuleer en/of oor te dra aan
plantspesies met inherente swak weerstandsmeganismes wat gereeld aan
plantpatogeeninfeksies onderhewig is. Navorsing op plantsade het tot die isolasie van
verskeie chemiese agense, peptiede en proteïene, wat sterk in vitro aktiwiteite teen 'n wye
reeks swampatogene vertoon, gelei. Die vermoë van dié agense om swamme in 'n in vitro
omgewing te inhibeer, is alleen egter nie 'n bewys dat hulle 'n rol in plantverdeging speel nie.
Studies waar mutante gebruik word, is gewens om addisionele bewys te lewer dat die
substanse 'n rol in plantverdediging vervul. Sodanige mutante sluit plantlyne, waarin die geen
van belang gemuteer is of ooruitgedruk word om so die rol van die geen in 'n in planta
omgewing te bepaal in. In hierdie toepassings het genetiese transformasie en die daarstelling
van transgeniese plante 'n ongeëwenaarde bydrae gelewer.
In dié studie is die antifungiese peptied, Hs-AFP1, wat aan die peptiedgroep van plant-
"defensins" behoort en van Heuchera sanguine a afkomstig is, in 'n heteroloë in planta
omgewing geëvalueer as 'n verdedigingspeptied met die potensiaal om in die generering van
transgeniese siektebestande gewasse gebruik te word. Die antifungiese aktiwiteit van
Hs-AFP1 is teen Botrytis cinerea in 'n in vitro reaksie geëvalueer, waar die toediening van
8 ,",g/mlgesuiwerde Hs-AFP1 peptied aanleiding gegee het tot 'n 88% afname in hifegroei van
B. cinerea. Hipervertakkings van swamhifes, 'n kenmerkende eienskap van Hs-AFP1
aktiwiteit, kon duidelik waargeneem word. Tabakplante is voorts getransformeer met 'n
konstruk, pFAJ3068, wat die koderende geen van Hs-AFP1 onder die sterk konstitutiewe
CaMV 35S promotor bevat het. Die peptied is met behulp van die seinpeptied wat afkomstig
is van die Mirabilis jalapa antimikrobiese peptied, Mj-AMP2, na die apoplastiese omgewing
geteiken. Voorheen is gerapporteer dat transgeniese peptiede in die heteroloë omgewing
soms onstabiel is. Dit het gelei tot die generering van twee addisionele konstrukte om die
moontlikheid van peptiedonstabiliteit te ondervang. 'n Stabiliseringskonstruk, pHs-EXG1, bestaande uit In versmelting tussen Hs-AFP1 en In antifungiese eksoglukanase van
Saccharomyces cerevisiae, gekodeer deur EXG1, is in tabakplante getransformeer. In
Kontrolekonstruk, pMj-EXG1, met die EXG1-geen saam met die Mj-AMP2-seinpeptied, is ook
voorberei en in tabakplante getransformeer. Dit is gebruik om die antifungiese aktiwiteit van
die eksoglukanase in die antifungiese aktiwiteitstoetse van die stabiliseringskonstruk te
kwantifiseer en te normaliseer.
Tabak is suksesvol met pFAJ3068, pHs-EXG1 en pMj-EXG1 getransformeer, wat
onderskeidelik gelei het tot die sogenaamde THs, THE en TME transgeniese tabaklyne.
Transgeentranskripsie en -translasie in die THs en THE tabaklyne is onderskeidelik deur
Noordelike- en Westelike-kladanalises bevestig. Die aktiewe uitdrukking van Hs-AFP1 het die
vermoë van tabakplante om B. cinerea infeksies te weerstaan, met tot 42% verhoog in
vergelyking met ongetransformeerde kontrole tabakplante tydens 'n in planta
siekteweerstandstoets. Totale proteïenekstrakte van THs tabaklyne is voorts ook in In in vitro
inhibisietoets geëvalueer, wat gelei het tot resultate wat goed met dié van die in planta toetse
ooreenstem. Die totale proteïenekstrakte het swamgroei met 40% geïnhibeer en die
kenmerkende hipervertakking van Hs-AFP1-aktiwiteit is ook mikroskopies waargeneem.
Resultate wat verkry is vanaf al die analises wat op die transgeniese THs tabaklyne
uitgevoer is, het aangedui dat Hs-AFP1 baie stabiel in die heteroloë tabakomgewing is en
peptiedstabiliteit was dus nie In probleem, soos verwag is nie. Die fusie tussen Hs-AFP1 en
EXG1 het dus nie die stabiliteit van die reeds stabiele Hs-AFP1 peptied verder verbeter nie,
maar het wel tot die verlies van Hs-AFP1 aktiwiteit gelei. Die antifungiese analises van die
THE tabaklyne het verder bevestig dat dié lyne selfs swakker inhibisie van B. cinereainfeksies
tot gevolg gehad het, as ongetransformeerde tabakplante. Mikroskopiese analises
van totale THE proteïenekstrakte het voorts ook geen kenmerkende hipervertakkings in die
swamhifes vertoon nie, wat alles daarop dui dat die Hs-AFP1-deel van die fusieproteïen as
gevolg van die fusie met EXG1 geïnaktiveer is. Dié resultaat is in lyn met vorige navorsing,
wat getoon het dat In ander peptied, sarcotoxin 1A, sy antifungiese aktiwiteit verloor indien dit
met In verklikkergeen versmelt word.
Alhoewel integrasie van die pMj-EXG1-konstruk in die TME-tabaklyne bevestig is, kon
geen mRNA met Noordelike-klad- of trutranskriptase-PKR (RT-PKR)-analises waargeneem
word nie. Die TME plant het ook geen antifungiese aktiwiteit in in vitro toetse getoon nie en dit
het geblyk dat die pMj-EXG1-konstruk aan geenafskakeling in die heteroloë tabakomgewing
onderworpe was. Dié afskakelingseffek is egter in die THE plante oorkom, aangesien
laasgenoemde sterk EXG1 proteïenaktiwiteit met J3-glukosidase aktiwiteitstoetse vertoon het.
Alhoewel die THE plante nie die stabiliteit van Hs-AFP1 verbeter het nie, het dit onwerwags
tot die stabilisering van EXG1 in In heteroloë omgewing gelei. Versmeltingstegnologie kan
dus moontlik gebruik word as 'n strategie om ander glukanases, wat bekend is vir
geenafskakeling in transgeniese omgewings, heteroloog uit te druk. In die geheel gesien, het dié studie getoon dat Hs-AFP1 'n onbetwiste rol in
plantverdedigingsmeganismes speel en daar is voorts ook meer kennis oor die stabiliteit van
peptiede in 'n heteraloë plantomgewing ingewin. Die positiewe resultate t.o.v. die verhoogde
siekteweerstand in die transgeniese THs plantlyne regverdig ook die verdere bestudering van
dié peptied om transgeniese siekteweerstand in gewasse te bewerkstellig.
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Characterisation and management of trunk disease-causing pathogens on table grapevinesBester, Wilma 04 1900 (has links)
Thesis (MScAgric)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and
Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and
dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence
and symptomatology of these pathogens on table grapes were largely unknown. A survey was
therefore conducted to determine the status and distribution of these pathogens and associated
symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in
the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and
Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah
vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly
selected plants in each vineyard. These sections were dissected and isolations were made from
each of the various symptom types observed: brown or black vascular streaking, brown internal
necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as
well as asymptomatic wood. Fungal isolates were identified using molecular and morphological
techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by
Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa
(3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B.
dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of
these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis
can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas
sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found
predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not
isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated
Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only
in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated
from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara,
Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes,
which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal
pathogens, which has serious implications for disease diagnosis and management.
Protection of wounds against infection by any of these trunk disease pathogens is the
most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous
research on the effectiveness of chemical pruning wound protectants has mostly focused on the
control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa.
chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the
pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were
therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or
pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B.
rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid
were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml;
20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese
chloride and flusilazole were the most effective fungicides with EC50 values for the different
species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These
fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and
were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results
from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl,
tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in
pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The
bioassay findings were, however, inconclusive due to low and varied re-isolation data of the
inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can
nonetheless be identified as fungicides to be evaluated as pruning wound protectants in
additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other
grapevine trunk disease pathogens. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en
Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë
terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op
wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus
grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar
tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met
hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en
Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die
kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004
geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke
wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is,
naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose,
waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout.
Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en
molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur
Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa
(3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B.
dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie
patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat
siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is
geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die
winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola
slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en
Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in
Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva
in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella,
Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer
vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie
resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene
veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes.
Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees
doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing
aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op
die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa.
chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die
patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus
getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese
Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en
B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was
oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium
asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees
effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36
μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz
mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa.
chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc
wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die
effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria
spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was
egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool,
prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat
verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies
asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.
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Comparative studies on genetic variability and fungicide resistance in Tapesia yallundaeNtushelo, Khayalethu 12 1900 (has links)
Thesis (MScAgric)--Stellenbosch University, 1998. / ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species
of Ramulispora are associated with this disease, of which Tapesia yallundae and T.
acuformis. are common. This thesis investigates the broader subjects of genetic
variability, reproductive dynamics and fungicide resistance in Tapesia yallundae.
Each of the chapters treats specific but related topics. T. yallundae, which is the only
species thus far reported from South Africa, has been associated with yield losses of
up to 50%. To enable the implementation of more accurate and effective control
measures, understanding the dynamics of reproduction and the genetics of the
pathogen is of utmost importance. Of the many plant disease control measures such
as cultural practices, sanitation, biological control, etc., fungicide application is the
most commonly resorted to measure in eyespot control. This thesis investigates the
broader subjects of genetic variability, reproductive dynamics and fungicide
resistance of Tapesia yallzll7dae.
Fungicide application, however, is not without problems. The pathogen can
build up resistance to fungicides. The most commonly used fungicides in eyespot
control include the benzimidazole carbendazim, triazoles such as flusilazole,
tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol,
and the imidazole, prochloraz. Cases of resistance to the groups listed above have
been reported. Frequent monitoring for resistance is thus crucial to prevent wastage
of fungicide and unnecessary impregnantation of the environment with potentially
ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15
fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole,
propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated
that to some triazoles, such as fenbuconazole, a high level of resistance was already
present in field populations.
In a sexually reproducing fungus such as T. yallundae, knowledge pertaining
to its ability to pass resistance factors to offspring is equally important. Mating
studies were, therefore, also conducted with parental strains that showed signs of
triazole resistance. Three generations were subsequently tested for resistance to five
triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and
flutriafol. Results of this study showed variable sensitivity in progeny, which
indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the
foundation for the long-term understanding of the population dynamics of the fungus.
The ability of a heterothallic ascomycete population to reproduce sexually is
dependent on the availability of its two mating types, MATI-I and MATI-2, their
distribution, and female fertility amongst other factors. In the UK. the teleomorph is
commonly observed in the field, which is in contrast to the situation in South Africa,
where it has only been induced in the laboratory. A comparative study between the
South African and the UK. populations was therefore undertaken. Isolates
representative of the two populations were mated with tester strains as both sperm
recipients and as sperm donors. This allowed the percentage of hermaphrodites to be
determined. No difference in terms of female fertility was observed between the
South African and the UK. populations, with both populations showing low effective
population numbers. These data suggested, therefore, that the teleomorph would also
occur more frequently in South Africa if the climate was more indusive to its
development.
The overall results of this study indicated that eyes pot could still be controlled
by means of fungicide application in South Africa. Although a shift in sensitivity was
observed towards fenbuconazole and flusilazole, no resistance was detected towards
carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field,
coupled by the monocyclic nature of the pathogen and sensible fungicide regimes.
The absence of T. acujormis makes the disease situation less complicated in terms of
fungicide application and management. Continuous surveys will have to be
conducted, however, to monitor this situation in future. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek die genetiese variasie, reproduksie dinamika en fungisied
weerstand in Tapesia yallundae. Elke hoofstuk handel oor spesifieke maar verwante
onderwerpe. Oogvlek is 'n belangrike siekte van lentekoring (Triticum aestivum L.).
Vier spesies van Ramulispora word geassosieer met die siekte, waarvan Tapesia
yallundae en T. acuformis mees algemeen voorkom. T. yallundae, wat tans die enigste
spesie is wat in Suid-Afrika aangeteken is, het al verliese van tot 50% veroorsaak. Om
meer akkurate en effektiewe beheermaatreels te implementeer, is dit noodsaaklik om die
oorlewingsdinamika van die patogeen te verstaan. Van al die siektebeheermaatreels soos
kulturele praktyke, sanitasie, biologiese beheer ens., bly fungisiedbehandeling die mees
algemene maatreel vir die beheer van oogvlek.
Fungisiedtoediening het egter ook verskeie probleme. Die patogeen kan
weerstand opbou teen die fungisied. Die mees algemene fungisiedes wat vir
oogvlekbeheer aangewend word sluit onder meer die benzimidasool karbendazim in,
triasole soos flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol,
fenbukonasool, triadimenol, en die imidasool, prochloraz. Weerstand is egter reeds teen
hierdie middels bekend. Gedurige monitering vir weerstand is dus krities om die
vermorsing van fungisied en besoedeling van die omgewing met oneffektiewe middels te
beperk. In hoofstuk 2 van hierdie manuskrip word 300 isolate van T. yallundae van 15
lande geevalueer vir weerstand teenoor karbendazim, flusilasool, tebukonasool,
propikonasool, bromukonasool, flutriafol en fenbukonasool. Resultate dui daarop dat
teen sommige van hierdie triasole, soos bv. fenbukonasool, daar reeds 'n hoe vlak van
weerstand teenwoordig was in veldpopulasies.
In 'n seksueel reproduserende fungus soos T. yalluJ1dae, is dit noodsaaklik om te
bepaal wat sy vermoe is om weerstandbiedenheid aan die nageslag oor te dra. Om die
rede is paringstudies ook op ouers wat tekens van weerstand teenoor triasole getoon het
uitgevoer. Drie generasies was gevolglik getoets vir weerstand teenoor vyf triasole,
naamlik flusilasool, tebuconasool, propikonasool, brumukonasool en flutriafol. Resultate
van die studie het 'n variasie in sensitiwiteit van die nageslag getoon, wat op 'n
kwantitatiewe oorerwing van weerstand teen £riasole dui. Alhoewel die teleomorf nog nie in lande in Suid-Afrika opgemerk is nie, Ie hierdie kennis die fondament vir die
langtermyn vertolking van die populasie dinamika van hierdie fungus.
Die vermoe van 'n heterotalliese askomiseet populasie om seksueel voort te plant
is afhanklik van die beskikbaarheid van sy twee paringstipes, MATI-I en MATl-2, hul
verpreiding, vroulike vrugbaarheid en ander faktore. Alhoewel die teleomorf algemeen
in lande in die Verenigde Koninkryk opgemerk word, is dit in kontras met die situasie in
Suid-Afrika, waar hierdie stadium nog slegs in die laboratorium gelnduseer kon word. 'n
Studie is dus onderneem om die Suid-Afrikaanse en V.K. populasies met mekaar te
vergelyk. Isolate van die twee populasies is dus gepaar met paringsisolate as beide sperm
ontvangers en sperm donors. Hierdie prosedure het dit moontlik gemaak om die
persentasie hermafrodiete te bepaal. Geen verskille in vroulike fertiliteit is tussen die
Suid-Afrikaanse en V.K. populasies bespeur nie, en beide populasies het ook 'n lae
effektiewe populasie getal getoon. Hierdie data het dus voorgestel dat die teleomorf ook
meer algemeen in Suid-Afrika sou voorkom as die klimaat meer geskik was vir teleomorf
vormmg.
Die resultate van hierdie studie het tot die slotsom gelei dat oogvlek steeds deur
fungisiedbehandeling in Suid-Afrika beheer kan word. Alhoewel daar 'n merkbare
verskuiwing in sensitiwiteit teenoor fenbukonasool en flusilasool was, was geen
weerstand teenoor karbendazim waargeneem nie. Laasgenoemde kan dalk toegeskryf
word aan die afwesigheid van die teleomorf in die veld, gekombineer met die
monosikliese natuur van die patogeen en gebruik van alternerende fungisiedes. Die
afwesigheid van T. acuformis maak die plaaslike siektetoestand minder gekompliseerd in
terme van fungisied aanwending en bestuur. Voortdurende opnames sal egter uitgevoer
moet word om hierdie situasie ook in die toekoms te monitor.
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