Pythium species associated with rooibos, and the influence of management practices on disease development

Bahramisharif, Amirhossein (2012-03)

Thesis (MSc)--Stellenbosch University, 2012.

Thesis

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.

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