Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum
Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has
been identified as the leading cause of harvest and storage losses. This pathogen is of
world-wide importance and causes Fusarium basal rot of onions (Allium cepa),
affecting all onion growth stages. No information is available on the evolution,
genetic diversity, molecular detection and inoculum sources of the South African
Focep population.
Similar to what is the case for South Africa, limited information is available
on Focep in other regions of the world. World-wide, four vegetative compatibility
groups (VCGs) and two single-member VCGs (SMVs) have been identified among
two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep
suggested by VCG analyses was confirmed through molecular analyses of isolates
from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported
for Focep isolates from Welsh onion (Allium fistulosum).
The development of sustainable management strategies of Focep is dependent
on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high
throughput molecular methods can be developed for identifying the most virulent and
widespread Focep genotypes and (iii) the role of seedlings and seeds as primary
inoculum sources, and the Focep genotypes associated with these growth stages.
Therefore, the three main aims of the current study were to investigate the
aforementioned three aspects.
In the first aim of the study, the genetic diversity and evolution of Focep was
investigated using a collection of 79 F. oxysporum isolates from South Africa (27
Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG
analyses revealed the presence of six VCGs, four among the Colorado Focep isolates
(VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated
isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs
in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible
(HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation
factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The
phylogeny divided the Focep isolates into two main clades, of which one contained
the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The
second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and
non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were
highly virulent toward onion bulbs, the ancestral clade contained isolates that were
mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with
an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs
within the same isolate for some isolates, suggested possible exchange of genetic
material between isolates.
The second aim of the study was to develop molecular methods for identifying
the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and
sequence-characterized amplified region (SCAR) markers. These techniques were
first developed using the F. oxysporum isolates from the first aim, and were then used
to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum
isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA
primers provided two diagnostic amplicons for VCG 0425, but attempts to develop
SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon
amplified polymorphism (IRAP) fingerprinting method enabled the
developed of a multiplex IR-SCAR polymerase chain reaction method that detected
the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR
marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65
Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South
Africa associated with mature onion bulbs, since 63 of the Focep isolates had the
molecular characteristics of VCG 0425.
The third aim of the study was to determine whether seed and seedling
transplants are inoculum sources of Focep, and whether the same genotype (VCG
0425) that dominated on mature bulbs could be detected from these sources. Focep
isolates were obtained from seven of the 13 investigated onion seed lots, as well as
from onion seedling transplants that were collected from all five onion nurseries in the
Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the
seedborne nature of Focep was confirmed by showing that a green fluorescent protein
labelled Focep transformant could be transmitted from infected soil to onion seed via
the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are
inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are
different from those in mature bulbs and were not dominated by VCG 0425.
Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately
virulent, as compared to the mostly highly virulent isolates from mature bulbs. / AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum
Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep)
geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie
patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium
cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting
beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en
inokulumbronne van die Focep-populasie nie.
Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting
beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe
versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder
twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van
Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van
isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir
Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium
fistulosum) berig.
Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis
van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre
metodes ontwikkel kan word vir die identifisering van die mees virulente en
wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre
inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer
word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie
aspekte te bestudeer.
Om die eerste doel van die studie te bereik is die genetiese diversiteit en
evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F.
oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit
Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe
aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424)
en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426).
VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook
geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is
ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en
mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate
in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en
0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het
die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In
teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle
bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die
inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde
(IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne
dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese
materiaal tussen isolate gedui.
Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir
die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te
maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area
(NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate
van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie
van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in
Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS
(RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer,
maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te
onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon
geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n
multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die
VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het.
Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F.
oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat
VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word,
aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad
het. Die derde doel van die studie was om vas te stel of saad en saailinge
inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse
bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry
van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf
uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer
as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was.
Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is
bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende
proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die
uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as
inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil
egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was
die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die
meestal hoogs virulente isolate uit volwasse bolle.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4499 |
| Date | 03 1900 |
| Creators | Southwood, Michael J. |
| Contributors | McLeod, A., Viljoen, A., Stellenbosch University. Faculty of Agrisciences. Dept. of Plant Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 160 p. : ill. (some col.), map |
| Rights | Stellenbosch University |
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