Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes
severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood
for infection by other pathogens. Knowledge about the epidemiology and especially inoculum
sources of this disease is imperative for subsequent development of management strategies.
Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through
infected propagation material in South Africa. However, the infection pathways and inoculum
sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen
in various media is by means of isolation onto artificial growth media. This has proven to be
problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to
identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it
can be identified. The aim of this study was (i) to develop a protocol for the molecular detection
of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test
different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from
nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora.
A protocol was developed and validated for the molecular detection of Pa.
chlamydospora in grapevine wood. Firstly, several previously published protocols were used to
develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of
potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and
Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic
DNA from grapevine wood. The protocol was validated using various grapevine material from 3
different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3
different nurseries, including grapevines that were subjected to hot water treatment. The basal
end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial
medium and molecular detection. The identity of PCR products obtained from a subset of
samples, that only tested positive for Pa. chlamydospora based on molecular detection, was
confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular
detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the
molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1%
of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora
was not isolated from hot water treated samples. The results confirm the importance of hot water
treatment for proactive management of Petri disease in grapevine nurseries. However, Pa.
chlamydospora DNA was molecularly detected in hot water treated samples in frequencies
similar to that detected in non-hot water treated samples. As expected, the DNA in hot water
treated plants was not destroyed and could be detected by the developed molecular detection
protocol. This is an important consideration when using molecular detection for disease
diagnosis or pathogen detection and shows that these methods should be used in conjunction
with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10
to 15 times cheaper than commercial DNA extraction kits.
Preliminary studies showed that the aforementioned molecular detection technique was
not specific and sensitive enough for detection of Pa. chlamydospora in soil and water
(unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa.
chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood.
Rootstock cane sections and soil samples were taking from the mother blocks from several
nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage
and grafting. Scion and rootstock cuttings were also collected during grafting and soil were
collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive
enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from
wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the
presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence
analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands
were Pa. chlamydospora specific, except for five bands obtained from callusing media and one
band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular
detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane
sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings
collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12-
hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the
callusing medium samples. These media should therefore be considered as potential inoculum
sources or infection points of the pathogen during the nursery stages. The results furthermore
confirmed previous findings that Pa. chlamydospora is mainly distributed through infected
rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother
plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or
chemical amendments in the hydration water and callusing medium and wound protection from
soil borne infections. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte
wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak
verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie
en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling
van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel
van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne
in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing
van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige
groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig
groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur
ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n
protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en
(ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond,
onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid-
Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora.
‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa.
chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as
grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie
protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers
(Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora
genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie
wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter
99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke
is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel
geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige
groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte
van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van
restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer
sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as
negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9%
van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief
getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie
geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig
hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in
wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in
warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde
monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie
vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing
protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing
gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die
metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA
ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële
DNA ekstraksie pakkette.
Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie
spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie
(ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die
opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium
en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van
verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters
versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel
gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis
geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese
DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die
verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe
Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA
volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa.
chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een
band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is
daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van
die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die
12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die
kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne
of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook
verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde
onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die
patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante
insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling),
toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en
wondbeskerming teen grondgedraagde infeksies.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20940 |
| Date | 04 1900 |
| Creators | Retief, Estianne |
| Contributors | Fourie, P.H., Mcleod, A., Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 63 leaves : ill. |
| Rights | Stellenbosch University |
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