Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Damping-off of rooibos (Aspalathus linearis), which is an important indigenous crop
in South Africa, causes serious losses in rooibos nurseries and is caused by a complex of
pathogens of which oomycetes, mainly Pythium, are an important component. The
management of damping-off in organic rooibos nurseries is problematic, since phenylamide
fungicides may not be used. Therefore, alternative management strategies such as rotation
crops, compost and biological control agents, must be investigated. The management of
damping-off requires knowledge, which currently is lacking, of the Pythium species involved,
and their pathogenicity towards rooibos and two nursery rotation crops (lupin and oats).
Pythium species identification can be difficult since the genus is complex and consists of
more than 120 species. Species identification is, however, greatly facilitated by analyses of
the internal transcribed spacer (ITS) regions. These regions, have also been used to divide the
genus into 11 phylogenetic clades (A to K), with some clades, such as clade G, still being
poorly characterised.
The first aim of the study was to characterize 12 Pythium clade G isolates that were
obtained from damped-off rooibos seedlings, along with six known clade G species.
Subsequently, oligonucleotides were designed for differentiating two rooibos associated
groups that may represent new taxons, for future use in DNA macro-array analyses.
Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions
(ITS, -tubulin and, COX1 and COX2 [cytochrome c oxidase subunits I and II]) identified
five sub-clades within Pythium clade G. The rooibos isolates formed two groups, Rooibos
group I (RB I) and II (RB II) that clustered into two groups within sub-clade 1 with good
support (64%-89% bootstrap, 1.00 probability). The Pythium RB I isolates had P. iwayamai
as its nearest neighbour, and may represent a new species. The Pythium RB II isolates had P.
canariense and P. violae as their closest relatives and may, along with other isolates
contained in the RB II sub-clade, represent several new species. Morphological analyses of
the rooibos isolates were inconclusive, since the isolates all contained similar morphological characteristics that did not correspond to the description of known Pythium species. The
Pythium RB I and II isolates were all non-pathogenic toward rooibos, lupin and oats
seedlings. For each of the two rooibos groups, one newly developed oligonucleotide was able
to differentiate the isolates from clade G reference isolates using DNA macro-array analyses.
The second aim of the study was to determine the oomycetes species associated with
rooibos in nurseries and in a native rooibos site, and their pathogenicity towards rooibos and
two nursery rotation crops (lupin and oats). Since some isolates were shown to be nonpathogenic,
another aim was to determine whether these isolates, along with the previously
characterised non-pathogenic Pythium RB I and RB II isolates, could suppress pathogenic
oomycetes. Characterisation of isolates from 19 nurseries and one native rooibos site revealed
the presence of five Pythium species (P. acanthicum, P. irregulare, P. mamillatum, P.
myriotylum, and P. pyrilobum) and Phytophthora cinnamomi. In nurseries, P. irregulare was
the most common species (81%) followed by P. myriotylum (14%). Similarly, P. irregulare
was also the most prevalent species (57%) in native rooibos, but P. pyrilobum (26%) was
second most prevalent. Pathogenicity studies on rooibos showed that all species, except P.
acanthicum, were highly virulent causing 100% damping-off. On lupin, P. acanthicum was
also the only non-pathogenic species, with the other species being less virulent on lupin than
on rooibos. Only P. irregulare, P. myriotylum, and P. pyrilobum were pathogenic towards
oats, and were also less virulent on oats than on rooibos. On lupin and oats, not all off the
isolates from a specific species was pathogenic. Non-pathogenic Pythium species (P.
acanthicum, Pythium RB I and II) was only effective at suppressing disease on the less
susceptible crops of lupin and oats, but not on rooibos.
The third aim of the study was to investigate the management of rooibos damping-off
using two composts (A and B), and composts combined with non-pathogenic Pythium
species. Evaluation of the suppression by composts of Ph. cinnamomi and 29 Pythium
isolates, which represented the four pathogenic Pythium rooibos species, showed that both
composts were able to suppress some, but not all of the pathogenic Pythium isolates. Both
composts were very effective at, and the highest percentage control was achieved, with
suppression of Ph. cinnamomi. Most isolates of P. mamillatum and P. pyrilobum were
suppressed by composts, whereas most P. irregulare (> 62%) and P. myriotylum (>50%)
isolates were not suppressed. Non-pathogenic Pythium species combined with either of the
two composts were able to significantly reduce damping-off caused by P. irregulare or a
combination of pathogenic species (P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, and Ph. cinnamomi), compared to than when only the pathogens were present. In
the absence of non-pathogenic species, neither of the composts was able to suppress the
aforementioned pathogenic isolates.
This study has improved our knowledge of the oomycete species that are involved in
rooibos damping-off, and has identified possible management strategies for use in organic
nurseries. Several oomycete species are involved in causing damping-off and their
differential virulence, and responses to being suppressed by composts, will require the use of
integrated management strategies. Management strategies that showed promise include the
combined use of compost and non-pathogenic Pythium taxons. The use of oats, which is
susceptible to fewer oomycete isolates than rooibos, could also be valuable as a rotation crop.
Altogether, knowledge obtained in this study can be used to (i) optimize integrated
management strategies for organic nurseries, (ii) elucidate the mechanisms involved in
disease suppression and (ii) develop molecular techniques, such as DNA macro-arrays and
quantitative PCR (qPCR) for the rapid assessment of the species involved, and the
quantification of inoculum in nursery soils. / AFRIKAANSE OPSOMMING: Omvalsiekte van rooibos (Aspalathus linearis), wat ‘n belangrike inheemse gewas in
Suid-Afrika is, veroorsaak ernstige verliese in rooiboskwekerye, en word deur ‘n kompleks
van patogene veroorsaak, waarvan oömysete, hoofsaaklik Pythium, ’n belangrike komponent
is. Die bestuur van omvalsiekte in organiese rooiboskwekerye is problematies, aangesien
fenielamied fungisiedes nie gebruik mag word nie. Alternatiewe bestuurstrategieë, soos
rotasie-gewasse, kompos en biologiese beheer-agente, moet dus ondersoek word. Die bestuur
van omvalsiekte vereis kennis, wat tans ontbreek, naamlik die Pythium spesies wat betrokke
is, hul patogenisiteit teenoor rooibos, en twee kwekery rotasie-gewasse (lupiene en hawer).
Pythium spesie-identifikasie kan moeilik wees aangesien die genus kompleks is en uit meer
as 120 spesies bestaan. Spesie-identifikasie word egter grootliks vergemaklik deur analise
van die interne getranskribeerde spasieerder (ITS) areas. Hierdie areas is ook gebruik om die
genus in 11 filogenetiese “clades” (A tot K) te verdeel, met sommige “clades”, soos “clade”
G, wat steeds swak gekarakteriseer is.
Die eerste doelwit van die studie was om 12 Pythium “clade” G isolate te
karakteriseer, wat vanaf omvalsiekte rooibossaailinge verkry is, tesame met ses bekende
“clade” G spesies. Gevolglik is oligonukleotiede ontwerp ten einde twee rooibosgeassosieerde
groepe, wat nuwe taksons kan verteenwoordig, te onderskei, en vir toekomstige
gebruik in DNS makro-“array” analise. Filogenetiese analise van die ITS area en ‘n
gekombineerde filogenie van vier geen-areas (ITS, -tubulien en, COX1 en COX2 [sitokroom
c oksidase sub-eenhede I en II]) het vyf sub-“clades” binne Pythium “clade” G geïdentifiseer.
Die rooibos isolate het twee groepe gevorm, Rooibos groep I (RB I) en II (RB II) wat twee
groepe binne sub-“clade” 1 gevorm het, met goeie ondersteuning (64%-89% “bootstrap”,
1.00 waarskynlikheid). Die Pythium RB I isolate het P. iwayamai as sy naaste verwant, en
mag ‘n nuwe spesie verteenwoordig. Die Pythium RB II isolate het P. canariense en P.
violae as hul naaste verwante en mag, tesame met ander isolate wat in die RB II sub-“clade”
ingesluit word, verskeie nuwe spesies verteenwoordig. Morfologiese analise van die rooibos-isolate was onbeslis, aangesien die isolate almal soortgelyke morfologiese kenmerke bevat
het, wat nie met die beskrywing van bekende Pythium spesies ooreengestem het nie. Die
Pythium RB I en II isolate was almal nie-patogenies teenoor rooibos-, lupien- en
hawersaailinge. Vir elk van die twee rooibosgroepe, was een nuut-ontwikkelde
oligonukleotied in staat om die isolate van “clade” G verwysingsisolate te differensieer, deur
die gebruik van DNS makro-“array” analise.
Die tweede doelwit van die studie was om die oömysete spesies wat met rooibos in
kwekerye en in ‘n inheemse rooibos-area geassosieer word, te bepaal, en hul patogenisiteit
teenoor rooibos en twee kwekery rotasie-gewasse (lupien en hawer). Aangesien van die
isolate nie-patogenies was, was ’n ander doelwit om te bepaal of hierdie isolate, tesame met
die voorheen gekarakteriseerde nie-patogeniese Pythium RB I en RB II isolate, patogeniese
oömysete kan onderdruk. Karakterisering van isolate van 19 kwekerye en een inheemse
rooibos-area, het op die teenwoordigheid van vyf Pythium spesies (P. acanthicum, P.
irregulare, P. mamillatum, P. myriotylum, en P. pyrilobum) en Phytophthora cinnamomi
gedui. P. irregulare was die mees algemene spesie (81%) in kwekerye, gevolg deur P.
myriotylum (14%). Soortgelyk was P. irregulare ook die mees algemene spesie (57%) in
inheemse rooibos, maar P. pyrilobum (26%) was tweede mees algemeen.
Patogenisiteitstudies op rooibos het getoon dat alle spesies, behalwe P. acanthicum, hoogs
virulent was en 100% omvalsiekte veroorsaak het. Op lupien was P. acanthicum ook die
enigste nie-patogeniese spesie, terwyl die ander spesies minder virulent op lupien as op
rooibos was. Slegs P. irregulare, P. myriotylum en P. pyrilobum was patogenies teenoor
hawer, en was ook minder virulent op hawer as op rooibos. Op lupien en hawer was nie alle
isolate van ‘n spesifieke spesie patogenies nie. Nie-patogeniese Pythium spesies (P.
acanthicum, Pythium RB I en II) was slegs effektief om siekte op die minder vatbare
gewasse, lupien en hawer, te onderdruk, maar nie op rooibos nie.
Die derde doelwit van die studie was om die bestuur van rooibos omvalsiekte te
ondersoek, deur die gebruik van twee tipes kompos (A en B), en kompos gekombineer met
nie-patogeniese Pythium spesies. Evaluasie van die onderdrukking deur kompos van Ph.
cinnamomi en 29 Pythium isolate, wat die vier patogeniese Pythium rooibosspesies
verteenwoordig het, het getoon dat beide tipes kompos in staat was om sommige, maar nie al
die patogeniese Pythium isolate, te onderdruk nie. Beide tipes kompos was baie effektief, en
die hoogste persentasie beheer was met die onderdrukking van Ph. cinnamomi verkry.
Meeste isolate van P. mamillatum en P. pyrilobum is deur kompos onderdruk, terwyl meeste P. irregulare (> 62%) en P. myriotylum (>50%) isolate nie onderdruk is nie. Nie-patogeniese
Pythium spesies, in kombinasie met enige van die twee tipes kompos, was in staat om
betekenisvol omvalsiekte veroorsaak deur P. irregulare, of in ’n kombinasie met patogeniese
spesies (P. irregulare, P. mamillatum, P. myriotylum, P. pyrilobum, en Ph. cinnamomi), te
verminder, in vergelyking met wanneer slegs die patogene aanwesig was. In die afwesigheid
van nie-patogeniese spesies, was nie een van die tipes kompos in staat om die
voorafgenoemde patogeniese isolate te onderdruk nie.
Hierdie studie het ons kennis rakende die oömysete spesies betrokke in rooibos
omvalsiekte verbeter, en het moontlike bestuurstrategieë geïdentifiseer wat in organiese
kwekerye gebruik kan word. Verskeie oömysete spesies is betrokke in die oorsaak van
omvalsiekte, en hul verskille in virulensie, en reaksies op onderdrukking deur kompos, sal die
gebruik van geïntegreerde bestuurstrategieë vereis. Bestuurstrategieë wat belofte toon, sluit
die gekombineerde gebruik van kompos en nie-patogeniese Pythium taksons in. Die gebruik
van hawer, wat vir minder oömysete isolate as rooibos vatbaar is, kan ook waardevol as ‘n
rotasie-gewas wees. Tesame, kan kennis wat in die studie opgedoen is gebruik word om (i)
geïntegreerde bestuurstrategieë vir organiese kwekerye te optimaliseer, (ii) die meganismes
betrokke in siekte-onderdrukking te bepaal, en (iii) molekulêre tegnieke, soos DNS makro-
“arrays” en kwantitatiewe PKR (qPKR) te ontwikkel vir die vinnige bepaling van die spesies
betrokke, en die kwantifisering van inokulum in kwekery-gronde.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/20406 |
| Date | 03 1900 |
| Creators | Bahramisharif, Amirhossein |
| Contributors | McLeod, Adele, Lamprecht, Sandra C., 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 | 145 pages : illustrations |
| Rights | Stellenbosch University |
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