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Characterization and detection of Puccinia horiana on chrysanthemum for resistance breeding and sustainable control

Mathias De Backer UGent (2012)
abstract
Cultivation of chrysanthemum (Chrysanthemum x morifolium) originates more than 2000 years ago in Asia. Chrysanthemum is currently one of the most important ornamental crops in different parts of the world, where it is either grown as cut flowers or as potted plants. A major threat in commercial chrysanthemum production is chrysanthemum white rust, caused by the quarantine fungus Puccinia horiana, which can cause devastating damage in susceptible cultivars if not controlled properly. Outbreaks of the disease are mainly prevented by fungicide treatments, but due to the development of fungicide resistant strains and the decreasing social acceptance of the excessive use of fungicides, more sustainable control strategies are required. However, a lack of knowledge about the spread and the pathogenic diversity of the pathogen as well as the complex segregation in chrysanthemum hamper the deployment of alternative control strategies such as a guided fungicide treatment and resistance breeding. In this context, the objectives of this thesis were to determine the phenotypic and genotypic diversity of the pathogen, to develop and apply a molecular detection technique for P. horiana spores in air samples and to study the segregation of molecular markers and the resistance to the pathogen in chrysanthemum. In the first part of this dissertation the focus was on the phenotypic diversity of a worldwide collection of P. horiana isolates. In this part, emphasis was placed on the identification and characterization of pathotypes. Using an optimized bioassay, 22 isolates originating in different continents were screened on a specific set of 36 cultivars. All isolates showed different interaction phenotype profiles and infected between 4 and 19 differential cultivars. Based on the Person analysis of these profiles, the presence of a minimum of seven resistance genes and corresponding avirulence genes was demonstrated in this pathosystems, which illustrates its complexity. A selection of 17 isolates was also tested for fungicide resistance to two strobilurins and three triazoles. Most isolates were highly resistant to one or several triazole fungicides, while resistance to strobilurins was considerably lower. Only two isolates were still susceptible to all fungicides included in the test. In the second part, the biology of the fungus was studied in more detail using molecular techniques. CRoPS™ technology, based on next generation sequencing of AFLP fragments, was used to develop 32 SNP markers and one SSR marker in P. horiana. These markers were used to genotype a worldwide collection of 45 isolates. In most cases, phylogenetic clustering of isolates was related to the geographic origin. In cases where the isolates were collected over multiple years and in several parts of the region, this indicates the establishment of the pathogen in such regions. Nevertheless, evidence for migration was also observed, including migration between Europe and South America and between South-East Asia and Europe. A strong indication of recombination between different genotypes was observed, disproving the presumed clonal propagation of the pathogen. The correlation between genotype and pathotype was poor, although a molecular marker to one important pathotype group could be developed. In the second part the development of protocols for the molecular detection of P. horiana basidiospores in air samples using two types of spore traps is described. Optimization of the techniques resulted in a limit of detection of a single spore and a limit of quantification of 10 spores. The capture efficiency of the two spore traps differed. Using these techniques, the presence of the pathogen was surveyed during five field trials. These showed that sporulation of the pathogen especially occurs at night and is related to a high relative humidity and especially to rainfall events. Large volumes of spores can be released and these can easily be dispersed over the distance of a commercial field. A third and final part of this thesis is dedicated to the segregation of resistance to P. horiana and the inheritance of AFLP makers in chrysanthemum. The segregation of resistance to an isolate mixture of P. horiana in the progeny of 20 crosses between resistant and susceptible parents suggest that preferential chromosome pairing occurs in chrysanthemum. However, it was not possible to determine the exact number of resistance genes in the resistant parents. A more detailed study on the inheritance of resistance to different pathotypes of P. horiana in a single cross demonstrated that resistance to different pathotypes is located on different loci each containing a cluster of R genes. The segregation of 182 AFLP markers confirmed the hypothesis of preferential chromosome pairing observed earlier. Some of the AFLP markers also cosegregated with resistance to specific pathotypes of P. horiana. The association between several of these markers also confirmed the presence of separate clusters of R genes. The insights obtained during this research offer a basis for resistance breeding to particular pathotypes of P. horiana. The bioassay developed for pathotype screening is also suitable for high throughput resistance screening of candidate cultivars. By using a combination of isolates that can overcome specific resistance genes, cultivars resistant to all known pathotypes can be developed. The segregating population of progeny from one of the crosses can be used for the identification and characterization of loci that are associated with resistance to P. horiana in chrysanthemum. In the future this could offer the possibility to select resistant cultivars using marker-assisted selection. Using the optimized spore detection protocol, the biology and epidemiology of the pathogen can be studied more in detail. This technique can be developed into a warning system giving the opportunity to decrease the use of fungicides. In combination with a well thought-out use of fungicides this can help prevent the development of fungicide resistance. Using the SNP markers developed for the characterization of P. horiana, the introduction, spread and local survival of P. horiana in a particular region can be analyzed. The markers can also be used to identify higher risk exotic genotypes among intercepted isolates. Together, implementation of the knowledge obtained during this research can form the basis for a more sustainable control of P. horiana.
De teelt van chrysant (Chrysanthemum x morifolium) vindt zijn oorsprong meer dan 2000 jaar geleden in Azië. Tegenwoordig is chrysant één van de belangrijkste sierteeltgewassen in verschillende werelddelen, waar ze wordt geteeld als snijbloem of als potplant. Een belangrijke bedreiging voor de commerciële chrysantenteelt is Japanse roest, die veroorzaakt wordt door de quarantaine schimmel Puccinia horiana en die enorme schade kan aanrichten in vatbare cultivars indien de ziekte niet naar behoren wordt beheerst. Uitbraken van de schimmel worden hoofdzakelijk vermeden door middel van preventieve fungicidenbehandelingen, maar door het verschijnen van fungicidenresistente stammen en een afname van het sociaal draagvlak voor het overmatig gebruik van fungiciden, zijn duurzamere controlestrategieën vereist. Een gebrek aan kennis over de verspreiding en de pathotypische diversiteit van P. horiana evenals de complexe overerving bij chrysant staan een goede ontplooiing van duurzamere controle strategieën zoals begeleide fungicidenbehandeling en resistentieveredeling echter in de weg. In het kader hiervan waren de objectieven van deze scriptie om de fenotypische en genotypische diversiteit van de pathogeen te bepalen, om een moleculaire techniek voor de detectie van sporen van de schimmel in luchtstalen te ontwikkelen en toe te passen en om de overerving van moleculaire merkers en resistentie in chrysant diepgaander te bestuderen. In het eerste deel van deze scriptie lag de focus op de fenotypische diversiteit van een wereldwijde collectie isolaten van P. horiana. Hierbij lag de nadruk op het identificeren en karakteriseren van pathotypes. Met behulp van een geoptimaliseerde biotoets werden 22 isolaten getoetst op een specifieke set van 36 cultivars. Alle isolaten hadden een uniek interactieprofiel en infecteerden tussen 4 en 19 differentiële cultivars. Op basis van de Person analyse van deze profielen werd de aanwezigheid van minimum zeven resistentiegenen en overeenkomstige avirulentiegenen in dit pathosysteem aangetoond, wat de complexiteit ervan illustreert. Een selectie van 17 isolaten werd ook getest op fungicidenresistentie tegen twee strobilurines en 3 triazolen. De meeste isolaten vertoonden een hoge resistentie tegen één of meerdere triazolen, terwijl de resistentie tegen strobilurines aanzienlijk lager was. Slechts twee isolaten waren nog vatbaar voor alle geteste fungiciden. In het tweede deel werd de biologie van de schimmel meer in detail bestudeerd met behulp van moleculaire technieken. Door middel van de CRoPS™ technologie, gebaseerd op next generation sequencing van AFLP fragmenten, werden 32 SNP merkers en één SSR merker in P. horiana ontwikkeld. Deze merkers werden vervolgens gebruikt voor de genotypering van een wereldwijde collectie van 45 isolaten. In de meeste gevallen was de fylogenetische clustering van isolaten gerelateerd aan de geografische oorspong. Voor die gevallen waarin de isolaten werden verzameld in verschillende jaren en in verschillende delen van een regio, geeft dit aan dat de pathogeen zich lokaal heeft gevestigd. Er werden echter ook verschillende gevallen van migratie van de pathogeen vastgesteld, onder andere tussen Europa en Zuid-Amerika en tussen Zuid-Oost Azië en Europa. Een sterke indicatie van recombinatie tussen verschillende genotypes werd waargenomen, wat de veronderstelde klonale vermeerdering van de pathogeen weerlegt. De correlatie tussen genotype en pathotype was beperkt, hoewel een moleculaire merker voor één belangrijke pathotype groep kon worden ontwikkeld. In het tweede deel wordt ook de ontwikkeling van protocols voor de moleculaire detectie van P. horiana basidiosporen in twee types luchtstalen beschreven. Optimalisatie van de technieken resulteerde in een detectie limiet van één enkele spore en een kwantificeringslimiet van 10 sporen. De vangstefficiëntie van de twee sporenvallen verschilde significant. Met behulp van de geoptimaliseerde technieken werd de aanwezigheid van de pathogeen gedurende vijf veldexperimenten opgevolgd. Deze toonden aan dat sporulatie van de pathogeen vooral ’s nachts plaats grijpt, dat sporulatie sterk gerelateerd is met hoge luchtvochtigheid en in het bijzonder met regenval. Grote volumes sporen kunnen worden vrijgesteld en deze kunnen gemakkelijk over de afstand van een commercieel veld worden verspreid. Een derde en laatste deel beschrijft de segregatie van roestresistentie en de overerving van AFLP-merkers bij chrysant. De segregatie van resistentie tegen een mengisolaat van P. horiana in 20 kruisingen tussen resistente en vatbare ouders suggereert dat preferentiële chromosoom paring de norm is in chrysant. Het was echter niet steeds mogelijk om het exacte aantal resistentiegenen in de ouders te bepalen. Een meer gedetailleerde studie van de overerving van resistentie tegen verschillende pathotypes van P. horiana in één enkele kruising toonde aan dat resistentie tegen verschillende pathotypes op verschillende loci met elk een cluster van R genen liggen. De uitsplitsing van 182 AFLP merkers bevestigende de hypothese van preferentiële chromosoomparing die eerder was waargenomen. Enkele van deze merkers vertoonden ook cosegregatie met resistentie tegen specifieke pathotypes van P. horiana. De associatie tussen verschillende van deze merkers bevestigt eveneens het voorkomen van afzonderlijke clusters van R genen. De inzichten die tijdens deze scriptie werden verworven bieden de basis voor een gerichte resistentieveredeling tegen P. horiana. De biotoets die ontwikkeld werd voor de pathotypescreening leent zich ook voor de resistentiescreening van kandidaat cultivars. Door gebruik te maken van een combinatie van isolaten die specifieke resistentiegenen doorbreken, kunnen cultivars die resistent zijn tegen alle gekende pathotypes worden ontwikkeld. De segregerende populatie van nakomelingen van één van de kruisingen kan worden gebruikt voor de identificatie en karakterisering van loci die geassocieerd zijn met resistentie tegen P. horiana in chrysant. Dit biedt de mogelijkheid om in de toekomst resistente cultivars te selecteren met behulp van merker-geassisteerde selectie. Het geoptimaliseerde protocol voor detectie van sporen in luchtstalen leent zich perfect voor het verder bestuderen van de epidemiologie van de pathogeen. In de toekomst kan deze techniek worden gebruikt voor het ontwikkelen van een waarschuwingssysteem waardoor het gebruik van fungiciden kan worden beperkt. In combinatie met een doordacht gebruik van fungiciden moet dit de verdere ontwikkeling van fungicidenresistentie helpen tegen gaan. Door gebruik te maken van de SNP-merkers voor de karakterisering van onderschepte isolaten kan de introductie, verspreiding en lokale overleving van P. horiana worden geanalyseerd. De merkers kunnen ook worden gebruikt voor de identificatie van exotische genotypes met een verhoogd risico in onderschepte isolaten. Samengevat kan implementatie van de kennis die tijdens dit onderzoek werd vergaard een basis vormen voor een duurzamere controle van P. horiana.
Please use this url to cite or link to this publication:
author
promoter
UGent and Kurt Heungens
organization
alternative title
Karakterisering en detectie van Puccinia horiana op chrysant in het kader van resistentieveredeling en duurzame controle
year
type
dissertation (monograph)
subject
keyword
Chrysanthemum white rust, Chrysanthemum, Puccinia, breeding, bioassay, fungicide resistance, genotyping, detection
pages
217 pages
publisher
Ghent University. Faculty of Bioscience Engineering
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Bio-ingenieurswetenschappen (A0.030)
defense date
2012-08-21 16:00
ISBN
9789059895409
language
English
UGent publication?
yes
classification
D1
additional info
dissertation in part contains copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
2973078
handle
http://hdl.handle.net/1854/LU-2973078
date created
2012-08-20 16:08:12
date last changed
2012-09-10 09:52:01
@phdthesis{2973078,
  abstract     = {Cultivation of chrysanthemum (Chrysanthemum x morifolium) originates more than 2000 years ago in Asia. Chrysanthemum is currently one of the most important ornamental crops in different parts of the world, where it is either grown as cut flowers or as potted plants. A major threat in commercial chrysanthemum production is chrysanthemum white rust, caused by the quarantine fungus Puccinia horiana, which can cause devastating damage in susceptible cultivars if not controlled properly. Outbreaks of the disease are mainly prevented by fungicide treatments, but due to the development of fungicide resistant strains and the decreasing social acceptance of the excessive use of fungicides, more sustainable control strategies are required. However, a lack of knowledge about the spread and the pathogenic diversity of the pathogen as well as the complex segregation in chrysanthemum hamper the deployment of alternative control strategies such as a guided fungicide treatment and resistance breeding. In this context, the objectives of this thesis were to determine the phenotypic and genotypic diversity of the pathogen, to develop and apply a molecular detection technique for P. horiana spores in air samples and to study the segregation of molecular markers and the resistance to the pathogen in chrysanthemum. In the first part of this dissertation the focus was on the phenotypic diversity of a worldwide collection of P. horiana isolates. In this part, emphasis was placed on the identification and characterization of pathotypes. Using an optimized bioassay, 22 isolates originating in different continents were screened on a specific set of 36 cultivars. All isolates showed different interaction phenotype profiles and infected between 4 and 19 differential cultivars. Based on the Person analysis of these profiles, the presence of a minimum of seven resistance genes and corresponding avirulence genes was demonstrated in this pathosystems, which illustrates its complexity. A selection of 17 isolates was also tested for fungicide resistance to two strobilurins and three triazoles. Most isolates were highly resistant to one or several triazole fungicides, while resistance to strobilurins was considerably lower. Only two isolates were still susceptible to all fungicides included in the test. In the second part, the biology of the fungus was studied in more detail using molecular techniques. CRoPS{\texttrademark} technology, based on next generation sequencing of AFLP fragments, was used to develop 32 SNP markers and one SSR marker in P. horiana. These markers were used to genotype a worldwide collection of 45 isolates. In most cases, phylogenetic clustering of isolates was related to the geographic origin. In cases where the isolates were collected over multiple years and in several parts of the region, this indicates the establishment of the pathogen in such regions. Nevertheless, evidence for migration was also observed, including migration between Europe and South America and between South-East Asia and Europe. A strong indication of recombination between different genotypes was observed, disproving the presumed clonal propagation of the pathogen. The correlation between genotype and pathotype was poor, although a molecular marker to one important pathotype group could be developed. In the second part the development of protocols for the molecular detection of P. horiana basidiospores in air samples using two types of spore traps is described. Optimization of the techniques resulted in a limit of detection of a single spore and a limit of quantification of 10 spores. The capture efficiency of the two spore traps differed. Using these techniques, the presence of the pathogen was surveyed during five field trials. These showed that sporulation of the pathogen especially occurs at night and is related to a high relative humidity and especially to rainfall events. Large volumes of spores can be released and these can easily be dispersed over the distance of a commercial field. A third and final part of this thesis is dedicated to the segregation of resistance to P. horiana and the inheritance of AFLP makers in chrysanthemum. The segregation of resistance to an isolate mixture of P. horiana in the progeny of 20 crosses between resistant and susceptible parents suggest that preferential chromosome pairing occurs in chrysanthemum. However, it was not possible to determine the exact number of resistance genes in the resistant parents. A more detailed study on the inheritance of resistance to different pathotypes of P. horiana in a single cross demonstrated that resistance to different pathotypes is located on different loci each containing a cluster of R genes. The segregation of 182 AFLP markers confirmed the hypothesis of preferential chromosome pairing observed earlier. Some of the AFLP markers also cosegregated with resistance to specific pathotypes of P. horiana. The association between several of these markers also confirmed the presence of separate clusters of R genes. The insights obtained during this research offer a basis for resistance breeding to particular pathotypes of P. horiana. The bioassay developed for pathotype screening is also suitable for high throughput resistance screening of candidate cultivars. By using a combination of isolates that can overcome specific resistance genes, cultivars resistant to all known pathotypes can be developed. The segregating population of progeny from one of the crosses can be used for the identification and characterization of loci that are associated with resistance to P. horiana in chrysanthemum. In the future this could offer the possibility to select resistant cultivars using marker-assisted selection. Using the optimized spore detection protocol, the biology and epidemiology of the pathogen can be studied more in detail. This technique can be developed into a warning system giving the opportunity to decrease the use of fungicides. In combination with a well thought-out use of fungicides this can help prevent the development of fungicide resistance. Using the SNP markers developed for the characterization of P. horiana, the introduction, spread and local survival of P. horiana in a particular region can be analyzed. The markers can also be used to identify higher risk exotic genotypes among intercepted isolates. Together, implementation of the knowledge obtained during this research can form the basis for a more sustainable control of P. horiana.},
  author       = {De Backer, Mathias},
  isbn         = {9789059895409},
  keyword      = {Chrysanthemum white rust,Chrysanthemum,Puccinia,breeding,bioassay,fungicide resistance,genotyping,detection},
  language     = {eng},
  pages        = {217},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Characterization and detection of Puccinia horiana on chrysanthemum for resistance breeding and sustainable control},
  year         = {2012},
}

Chicago
De Backer, Mathias. 2012. “Characterization and Detection of Puccinia Horiana on Chrysanthemum for Resistance Breeding and Sustainable Control”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
De Backer, Mathias. (2012). Characterization and detection of Puccinia horiana on chrysanthemum for resistance breeding and sustainable control. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
De Backer M. Characterization and detection of Puccinia horiana on chrysanthemum for resistance breeding and sustainable control. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2012.
MLA
De Backer, Mathias. “Characterization and Detection of Puccinia Horiana on Chrysanthemum for Resistance Breeding and Sustainable Control.” 2012 : n. pag. Print.