| Summary: | Thesis (MSc)--Stellenbosch University, 2013. === ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world.
Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly
detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules
expressed by almost all organisms as part of their non-specific defence system. These
peptides can offer protection against a wide variety of bacterial and fungal pathogens in
plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are
considered to be perfect candidates to confer resistance to this phytopathogen. The current
study intends to explore the in planta activity of AMPs against the grapevine pathogen aster
yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression.
The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were
selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S
expression vectors containing four different AMP-encoding sequences were therefore
constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in
planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used.
To allow assumptions about AMP efficacy in this transient expression system, attempts were
made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv
"Chardonnay‟ material. Additionally, transmission experiments were carried out to infect
Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia
fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using
universal primers described by Gundersen and Lee (1996). For quantification of AYp
infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the
SYBR Green-based system.
In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-,
three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp
infection could however be detected and plantlets displayed a "recovery phenotype‟. To
examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant,
leaf and the corresponding node material from five canes were screened by a nested-PCR
procedure. It can be concluded, that AYp was found predominantly in the nodes when
compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be
detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of
VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and
N. benthamiana plants with AYp was successfully achieved by insect vector transmission
experiments. Transient expression assays were conducted on AYp-infected N. benthamiana
material. Quantification of phytoplasma in this material showed a decrease of AYp in both
the AMP treatment groups and the control groups.
This study optimized a qPCR procedure to detect and quantify AYp in infected plant
material. The Agrobacterium-mediated transient expression system used during this study
was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This
study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera
cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp
in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of
AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine. === AFRIKAANSE OPSOMMING: Fitoplasma siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan
die onlangse ontdekking van fitoplasma in Suid-Afrikaanse wingerde baie nadelige gevolge
vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat
in amper al organismes as deel van hulle nie-spesifieke verdedegingsstelsel tot uitdruk kom.
Hierdie peptiede kan beskerming aanbied teen ʼn wye verskeidenheid van bakteriële en
swampatogene in plante. As gevolg van die feit dat fitoplasmas geen buitenste membraan en
selwand het nie, word AMPe oorweeg as middle om weerstand te verleen teen hierdie
fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die
wingerdstok patogeen aster geel fitoplasma (AYp) deur middle van Agrobakteriumbemiddelde
tydelike uitdrukkingssiteme, te ondersoek.
Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer van aartappel en wingerd plante) is
gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-viruse 35S
uitdrukkingsvektore met vier verskillende AMP-kodering rye, is dus ontwikkel. As ʼn
aternatiewe method om die moontlike effek van Vv-AMP1 op AYp in planta in ag te neem,
is oorplantings van die Vv-AMP1 transgeniese Vitis vinifera cv "Sultana‟ plantmateriaal
gebruik. Om voorsiening te maak vir AMPe se doeltreffenheid in hierdie tydelike
uitdrukkingsvektore, is pogings aangewend om die ruimlike verspreiding en patogeen
konsentrasie van AYp in V. vinifera cv "Chardonnay‟ te beskryf. Addisioneel is transmissie
eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana te besmet met
AYp deur die insekvektor, Mgenia fuscovaria. Plantmateriaal is getoets vir AYp deur van ʼn
PCR protokol gebruik te maak met universele inleiers (grondlae) soos beskyf deur
Grundersen en Lee (1996). Vir kwantifiseering van die AYp infeksie, is n semi-kwantitatiewe
qPCR protokol geoptimaliseer, met hulp van die SYBR Groen-gebaseerde stelsel.
In geheel is 86 V. vinifera cv "Chardonnay‟ planties getoets vir AYp infeksie – twee-, drie-,
vier-, sewe- en elf weke na die bekendstelling aan die in vitro voorwaardes. Geen AYp
infeksie kon egter opgespoor word en die plante het “herstel fenotipe‟ vertoon. Om die
verspreiding van AYp in stingelknope van ʼn besemtte V. vinifera cv "Cardonnay‟ plant, blaar
en ooreenstemmende stingelknope uit vyf stingels te ondersoek, is hulle getoets deur ʼn PCR
protokol. Daar kon afgelei word dat AYp hoofsaaklik in die stingelknop in vergelyking met die blaarmaterial laat in die season, gevind word. Dit is hoogs oonwaarskynlik om fitoplasma
infeksies in blaarmaterial te vind, as in die ooreenstemmende stingelknop daar geen AYp
oopgespoor kon word nie. As gevolge daarvan dat die AYp-geinfekteerde wingerdmateriaal
nie in vitro gegroei kon word nie, kon die effek van VvAMP-1 transgeniese wingerd teen
AYp nie getoets word nie. Infeksies van C. roseus en N. benthamiana plante met AYp is
suksesvol bereik deur transmissie eksperiemente. met ʼn insekvektor. Tydellike
uitdrukkingvektore toetse is uitgevoer op die AYp besmette N. benthamiana material.
Kwantifisering van fitoplasma in hierdie material het die afname van AYp in altwee, die
AMP behandelings groepe en die kontrole groepe getoon.
Hierdie studie het ʼn qPCR geoptimaliseer om besmette plantmaterial met AYp op te spoor
en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in
hierdie studie gebruik is, was nie vertroubaar genoeg, want geen beduidelike effek van die
AMPe op AYp konsentrasie kon waargeneen word nie. Hierdie studie het bewys dat AYp nie
vasgestel is en in stand gehou kan word deur in vitro aankweeking van V. vinifera cv
"Chardonnay‟ material nie, en weefselkulture kan dus ʼn manier wees om AYp in hierdie
kultivar uit te roei. Tot kennis, is hierdie studie die eerste studie om die ruimtelike
verspreiding van AYp in stingelknope van ʼn besmette V. vinifera cv "Chardonnay‟
wingerstok te rapporteur. === Winetech and DAAD
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