Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the
slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating
impact on vineyards worldwide, reducing growth and yield, eventually killing the
grapevine. The causal organism of Eutypa dieback was first described as Eutypa
armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987
this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul
(anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two
species that are capable of infecting grapevines are responsible for Eutypa dieback.
Consequently, the molecular identification and characterisation of Eutypa dieback was
used to delineate the species occurring on infected grapevines in South Africa. This
involved the molecular analyses of three molecular markers, namely, the internal
transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon,
and the -tubulin gene. The results obtained revealed the presence of a second species,
namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on
infected grapevines.
Also co-habiting with these pathogens were related fungi form the Diatrypaceae family,
Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart.
Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca,
and E. vitis revealed that all were pathogenic to grapevine. Several species of
Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also
pathogenic to grapevine. The symptoms in grapevine commonly associated with
Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback
which prompted the need for the development of a detection method that can correctly
identify the presence of multiple pathogens.
A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a
rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa
disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify
and label the regions of DNA that are used as pathogen specific probes. Consequently,
membrane immobilised species-specific oligonucleotides synthesised from the ITS, -
tubulin and LSU molecular data were evaluated during the application of this diagnostic
method to detect Eutypa species. It was found that the species-specific oligonucleotides,
designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca.
The application of the RDBH method for the detection of these Eutypa species, based on
-tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a
RDBH method, utilising species-specific oligonucleotides designed from elongation
factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria
dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels)
Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels)
Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.)
Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the
detection of Diplodia seriata De Not.
In addition to the above-mentioned shortcomings, the RDBH was not amenable to the
detection of pathogens directly from field or environmental samples, but required
preparation of DNA from pure cultures. The method, however, allows for the
identification of multiple pathogens in a single assay. As DNA extraction methods are
amended, improved and honed to obtain DNA from environmental samples, so would it
increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om
wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak.
Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat
dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan
dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. &
Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987
word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul
(anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten
minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te
veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om
te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak.
Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne
getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU
rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n
tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in
geïnfekteerde plante voorkom.
Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae
familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis
(Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met
verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop
dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in
houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne
simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie
maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan
om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige
infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om
Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en
betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as
patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en
merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke
oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n
membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species.
Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die
opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH
was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon
susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not.,
Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips,
Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and
Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips
op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie.
Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie
metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie
en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter
identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes
aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die
bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21757 |
| Date | 12 1900 |
| Creators | Safodien, Sieyaam |
| Contributors | Botha, A., Smit, W.A., Crous, P.W., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | iii, 101 leaves : ill. |
| Rights | Stellenbosch University |
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