Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling

Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.

Unraveling the genetics of Botrytis cinerea resistance in Gerbera hybrida
Fu, Yiqian - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Paul Arens; Jaap van Tuyl. - Wageningen : Wageningen University - ISBN 9789463431811 - 159
gerbera - plant pathogenic fungi - botrytis cinerea - disease resistance - genetic mapping - transcriptomics - quantitative trait loci - plant breeding - plantenziekteverwekkende schimmels - ziekteresistentie - genetische kartering - transcriptomica - loci voor kwantitatief kenmerk - plantenveredeling

Gerbera hybrida is one of the top five cut flowers. It is well-known to people for its variation in flower color and patterning. Gerbera breeding at the moment is done using conventional methods which are based on a phenotypic selection. This has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance. Gerbera gray mold, promoted by high humidity during the production in greenhouses or by an accumulation of condensate during transportation, is a considerable threat to the gerbera production. Gerbera gray mold is caused by Botrytis cinerea and plant resistance to B. cinerea is considered to be a polygenic trait that needs the contribution of multiple loci, and on top of that is highly affected by the environment. Conventional breeding might be inefficient for improving Botrytis resistance in gerbera.

In this study, the transcriptomes of four parents of two gerbera populations were sequenced using Illumina paired-end sequencing. Transcriptome data provides a resource for genetic dissection and an insight to explore gene functions for this ornamental crop. To identify the QTL regions leading to the phenotypic variation in Botrytis resistance, and establishing a relationship between marker genotype and phenotypic variation for marker assisted selection (MAS), genetic linkage maps were constructed with SNP markers in the two F1 segregating populations. A total of 20 QTLs were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflects the complex mechanism of Botrytis disease response. Narrowing down the QTL region and identifying the causal gene(s) underlying a QTL could maximize the effective use of MAS in breeding. Homologs of known functional genes involved in Botrytis resistance from other species were obtained in gerbera and SNP markers identified and mapped. Twenty-nine candidate genes were mapped and seven candidate genes could be mapped on both populations. Seven candidate genes were located in the vicinity of the QTLs detected. The co-localization of QTLs with CGs gives an indication that these candidate genes could probably be involved in resistance to Botrytis and provide a more precise possibility to use MAS in gerbera breeding in the future. A tobacco rattle virus (TRV) based gene silencing system which was used to inspect the function of two candidate genes. The two CGs are the homologs of the genes responsible for Botrytis resistance in tomato and both mapped in QTL regions related to Botrytis resistance in gerbera ray floret test. Silencing the two genes by VIGS, showed smaller lesion sizes upon Botrytis infection on gerbera ray florets compared with the controls.

The entire research went from the generation of four parental transcriptome data sets to development of SNP markers (Chapter 2), construction of genetic maps and to mapping QTLs for Botrytis resistance (Chapter 3). This was further on combined with candidate gene searching in other crops, querying and mapping homologous genes (Chapter 4) and characterizing the candidate genes which co-localized with QTLs (Chapter 5). The whole process not only helped us to unravel the genetics of Botrytis resistance in gerbera and develop genetic tools for gerbera improvement, but also could serve as guidance for developing marker-assisted selection for other ornamental plants from the beginning.

Susceptibility genes : an additional source for improved resistance
Sun, Kaile - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Evert Jacobsen; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789463431415 - 174
solanum tuberosum - potatoes - solanum lycopersicum - tomatoes - genes - susceptibility - plant pathogenic fungi - phytophthora infestans - disease resistance - plant breeding - aardappelen - tomaten - genen - vatbaarheid - plantenziekteverwekkende schimmels - ziekteresistentie - plantenveredeling

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Unravelling aspects of spatial and temporal distribution of Verticillium dahliae in olive, maple and ash trees and improvement of detection methods
Keykhasaber, Mojtaba - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit, co-promotor(en): Jelle Hiemstra. - Wageningen : Wageningen University - ISBN 9789463430142 - 163
olea europaea - olives - acer - fraxinus - plant pathogenic fungi - verticillium dahliae - distribution - virulence - detection - olijven - plantenziekteverwekkende schimmels - distributie - virulentie - detectie

Vascular wilts caused by xylem-colonizing pathogens are among the most devastating plant diseases that affect a wide range of plant species worldwide. Information on the distribution of V. dahliae in infected trees helps to design an appropriate and efficient sampling method for reliable detection of the pathogen in diseased trees. In Chapter 3, the distribution of V. dahliae in young twigs and leaves of infected olive trees is studied by real-time quantification of V. dahliae DNA. Analysis of twig and leaf samples collected from different sides of the crown of infected olive trees showed a non-uniform distribution of the fungus within infected parts of diseased olive trees. It was demonstrated that testing of combined samples comprising subsamples from at least 5 twigs from different sides of the tree, or 5-10 random leaves, can reliably detect the pathogen. V. dahliae isolates that infect olive trees can be classified as defoliating (D) isolates that are highly virulent, or non-defoliating (ND) isolates that are generally less aggressive. Discrimination of these pathotypes is important in order to predict the severity of disease, and decide on appropriate disease management strategies. This is particularly important due to the alarming spread of highly virulent isolates of the D pathotype worldwide. In Chapter 4, a novel method is designed for accurate discrimination and sensitive detection of D and ND isolates of V. dahliae. Through comparative genomics of multiple D and ND isolates of V. dahliae a region was identified that is present in all sequenced ND isolates, while absent from all D isolates. Based on this presence-absence polymorphism, a set of primers was designed spanning this region that was able to generate differentially sized amplicons for isolates that belong to the different pathotypes. Additionally, a nested-PCR assay was designed to increase the sensitivity and improve detection of D and ND isolates in planta. In Chapter 5, the relation of the dynamics in pathogen distribution in infected plants to the differences in extent and severity of disease caused by D and ND isolates in resistant and susceptible olive genotypes is studied. To this end, the distribution of a D (V117) and a ND (V4) isolate of V. dahliae in root-inoculated young plants of a susceptible (Picual) and a partially resistant cultivar (Frantoio) of olive and its relationship to the disease progression was investigated using real-time PCR. The amount of pathogen DNA detected in the two cultivars correlated with their susceptibility to Verticillium wilt, with lower quantities of V4 and V117 DNA detected in ‘Frantoio’ than in ‘Picual’. Also quantities of pathogen DNA in V117-inoculated plants were higher than quantities of pathogen DNA in V4-inoculated plants. The distribution patterns of D and ND isolates in the lower, middle and top parts of tested olive cultivars showed that differences in symptom severity were related to amounts of the pathogen in lower and middle parts of the trees, since colonization of the pathogen in top parts of the stem of inoculated plants was minor and was not significantly different between treatments. Moreover, microscopic analysis of infection and colonization processes of V. dahliae in olive plants inoculated with GFP-labelled isolates revealed that colonization of the above ground tissues of infected olive plants is by means of conidia transported upward with the xylem sap stream. In Chapter 6 we investigated the spatial and temporal distribution of V. dahliae in relation to disease progression and recovery in stem-inoculated maple and ash trees. These species differ strongly in vascular anatomy with maple having a diffuse porous xylem anatomy whereas ash has a ring porous xylem anatomy. Results showed that that differences in the xylem anatomy of ash and maple did not significantly affect the speed and extent of the upward spread of the pathogen in stem-inoculated trees. Nevertheless, the xylem of ash trees is much less supportive for growth and survival of V. dahliae than that of maple trees, as in the year after inoculation disease incidence and also quantities of V. dahliae detected in maple trees were significantly higher than in ash trees. Moreover, V. dahliae could not be reisolated at all from ash trees that had recovered from disease. However, it could be detected by PCR in some cases in the xylem formed in the year of inoculation, never in the xylem formed in the year after inoculation. Nevertheless, V. dahliae easily could be detected in the wood of diseased ash and maple trees in the year after inoculation. Notably, despite the presence of a layer of terminal parenchyma cells between growth rings, in ash trees showing disease symptoms in the year after inoculation V. dahliae was present in the xylem of the new growth ring. It was also observed that in stem-inoculated trees V. dahliae can move downward from the point of inoculation into the root collar, which may provide an avenue for infection of new growth rings in ash trees.

Fungicide evaluation to rate efficacy to control leaf late blight for the EuroBlight table Results 2006-2015
Evenhuis, A. ; Bain, R. ; Hausladen, H. ; Nielsen, B.J. ; Berg, W. van den; Schepers, H.T.A.M. - \ 2016
Lelystad : Applied Plant Research - 46 p.
solanum tuberosum - potatoes - plant pathogenic fungi - oomycetes - phytophthora infestans - fungicides - protocols - field experimentation - europe - aardappelen - plantenziekteverwekkende schimmels - oömyceten - fungiciden - protocollen - experimenteel veldonderzoek - europa
The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis
Chong Aguirre, Pablo A. - \ 2016
University. Promotor(en): Gert Kema; Pedro Crous. - Wageningen : Wageningen University - ISBN 9789462578791 - 289
pseudocercospora - plant pathogenic fungi - fungicides - pesticide resistance - defence mechanisms - genetic diversity - genetic mapping - sensitivity - musa - bananas - fungal diseases - disease control - plantenziekteverwekkende schimmels - fungiciden - resistentie tegen pesticiden - verdedigingsmechanismen - genetische diversiteit - genetische kartering - gevoeligheid - bananen - schimmelziekten - ziektebestrijding

Pseudocercospora fijiensis causes black Sigatoka disease of banana. It is one of the most damaging threats of the crop requiring excessive fungicide applications for disease control as the major export “Cavendish” clones are highly susceptible. The consequence of this practice is the reduced efficacy of disease management strategies due to increasing levels of fungicide resistance. In this thesis the history and current practices of black Sigatoka disease management as well as the underlying mechanisms of fungicide resistance to a major group of fungicides are described. We discovered that both target site mutations and promotor insertions are crucial for modulating sensitivity. The more insertions, the higher the expression of the gene and the more resistant the strain. Using this information, we advocate modern monitoring techniques and improved disease control strategies as well as the urgent need for innovative banana breeding to develop resistant varieties for a sustainable global banana production.

Het bodemschimmelschema : vernieuwd schema, 2016
Lamers, J.G. ; Rozen, K. van; Hanse, B. - \ 2016
Lelystad : Praktijkonderzoek Plant & Omgeving, onderdeel van Wageningen UR, Business Unit PPO-agv - 76 p.
veldgewassen - bodemschimmels - plantenziekteverwekkende schimmels - akkerbouw - tuinbouw - gewasopbrengst - waardplanten - nederland - field crops - soil fungi - plant pathogenic fungi - arable farming - horticulture - crop yield - host plants - netherlands
Deze studie over Nederlandse bodemschimmels heeft geleidt tot een eerste exemplaar van een overzichtelijk schema met gewassen in relatie tot schadepotentieel en vermeerdering. Achtergrondinformatie over waardplanten, vermeerdering en schade is verzameld en beschreven van vijftien schimmels in 40 landbouwgewassen. Per ziekte is aangegeven op welke grondsoort deze (hoofdzakelijk) voorkomt en welke substantiële schade deze kan veroorzaken. Deze informatie biedt nieuwe mogelijkheden om beter te anticiperen op de grilligheid en incidentie van bodemgebonden schimmels. Het rapport vloeit voort uit de financiering door PT en PA.
Role of MLO genes in susceptibility to powdery mildew in apple and grapevine
Pessina, Stefano - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; M. Malnoy; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576209 - 222 p.
malus domestica - apples - vitis vinifera - grapes - plant pathogenic fungi - podosphaera leucotricha - erysiphe necator - disease resistance - susceptibility - genes - gene expression - gene knock-out - resistance breeding - appels - druiven - plantenziekteverwekkende schimmels - ziekteresistentie - vatbaarheid - genen - genexpressie - inactivering van genen - resistentieveredeling

Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority.

PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not.

Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection.

Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control.

Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other.

Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity.

In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.

Ecogenomics of plant resistance to biotic and abiotic stresses
Davila Olivas, N.H. - \ 2016
University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576575 - 259 p.
016-3932 - arabidopsis thaliana - defence mechanisms - drought resistance - insect pests - plant pathogenic fungi - stress - stress response - transcriptomics - genomics - genetic mapping - verdedigingsmechanismen - droogteresistentie - insectenplagen - plantenziekteverwekkende schimmels - stressreactie - transcriptomica - genomica - genetische kartering
Summary

In natural and agricultural ecosystems, plants are exposed to a wide diversity of abiotic and biotic stresses such as drought, salinity, pathogens and insect herbivores. Under natural conditions, these stresses do not occur in isolation but commonly occur simultaneously. However, plants have developed sophisticated mechanisms to survive and reproduce under suboptimal conditions. Genetic screenings and molecular genetic assays have shed light on the molecular players that provide resistance to single biotic and abiotic stresses. Induced defenses are attacker specific and phytohormones play an essential role in tailoring these defense responses. Because phytohormones display antagonistic and synergistic interactions, the question emerges how plants elicit an effective defense response when exposed to conflicting signals under multiple attack. Recent studies have shed light on this issue by studying the effects of combinations of stresses at the phenotypic, transcriptomic and genetic level. These studies have concluded that the responses to combined stresses can often not be predicted based on information about responses to the single stress situations or the phytohormones involved. Thus, combined stresses are starting to be regarded as a different state of stress in the plant. Studying the effects of combinations of stresses is relevant since they are more representative of the type of stresses experienced by plants in natural conditions.

In a coordinated effort, responses of Arabidopsis thaliana to a range of abiotic and biotic stresses and stress combinations have been explored at the genetic, phenotypic, and transcriptional level. For this purpose we used an ecogenomic approach in which we integrated the assessment of phenotypic variation and Genome-Wide Association (GWA) analysis for a large number of A. thaliana accessions with an in-depth transcriptional analysis. The focus of this thesis is especially on (but not limited to) three stresses, i.e. drought, herbivory by Pieris rapae caterpillars, and infection by the necrotrophic fungal pathogen Botrytis cinerea. These stresses were chosen because the responses of A. thaliana to these three stresses are highly divergent but at the same time regulated by the plant hormones JA and/or ABA. Consequently, analysis of responses to combinatorial stresses is likely to yield information on signaling nodes that are involved in tailoring the plant’s adaptive response to combinations of these stresses. Responses of A. thaliana to other biotic and abiotic stresses are included in an integrative study (Chapter 6).

We first investigated (Chapter 2) the extent of natural variation in the response to one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips, Myzus persicae aphids) and two combined stresses (drought plus P. rapae, and B. cinerea plus P. rapae). Using 308 A. thaliana accessions originating from Europe, the native range of the species, we focused on the eco-evolutionary context of stress responses. We analyzed how the response to stress is influenced by geographical origin, genetic relatedness and life-cycle strategy, i.e. summer versus winter annual. We identified heritable genetic variation for responses to the different stresses. We found that winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars and to the combined stresses of drought followed by P. rapae and infection by the fungus B. cinerea followed by herbivory by P. rapae. Furthermore, we found differential responses to drought along a longitudinal gradient.

We further investigated, using A. thaliana accession Col-0, how phenotypic and whole-genome transcriptional responses to one stress are altered by a preceding or co-occurring stress (Chapters 3 and 4). The whole-transcriptomic profile of A. thaliana triggered by single and combined abiotic (drought) and biotic (herbivory by caterpillars of P. rapae, infection by B. cinerea) stresses was analyzed by RNA sequencing (RNA-seq). Comparative analysis of plant gene expression triggered by single and double stresses revealed a complex transcriptional reprogramming. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis highlighted biological processes specifically affected by single and double stresses (Chapters 3). For example, ethylene (ET) biosynthetic genes were induced at 12 h by B. cinerea alone or drought followed by B. cinerea inoculation. This induction was delayed when plants were pretreated with P. rapae by inducing ET biosynthetic genes only 18 hours post inoculation. Other processes affected by combined stresses include wound response, systemic acquired resistance (SAR), water deprivation and ABA response, and camalexin biosynthesis.

In Chapter 4, we focused on the stress imposed by P. rapae herbivory alone or in combination with prior exposure to drought or infection with B. cinerea. We found that pre-exposure to drought stress or B. cinerea infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite the larger reduction in plant biomass observed for plants exposed to drought plus P. rapae feeding compared to P. rapae feeding alone, this did not affect weight gain of this specialist caterpillar.

In Chapter 5, we used univariate GWA to (1) understand the genetic architecture of resistance to the different stresses and (2) identify regions of the genome and possible candidate genes associated with variation in resistance to those stresses. In Chapter 5 a subset of the stresses addressed in Chapter 1 (i.e. drought, herbivory by P. rapae and P. xylostella, and the combined stresses drought plus P. rapae and B. cinerea plus P. rapae) were investigated. Results from GWA were integrated with expression data generated in Chapters 3 and 4 or available from the literature. We identified differences in genetic architecture and QTLs underlying variation in resistance to (1) P. rapae andP. xylostella and (2) resistance to P. rapae and combined stresses drought plus P. rapae and B. cinerea plus P. rapae. Furthermore, several of the QTLs identified contained genes that were differentially expressed in response to the relevant stress. For example, for P. xylostella one of the QTLs contained only two genes encoding cysteine proteases (CP1 and CP2). The expression data indicated that these genes were induced by P. rapae and P. xylostella herbivory.

In Chapter 6, the genetic architecture underlying plant resistance to 11 single stresses and some of their combinations was investigated. First, the genetic commonality underlying responses to different stresses was investigated by means of genetic correlations,, revealing that stresses that share phytohormonal signaling pathways also share part of their genetic architecture. For instance, a strong negative genetic correlation was observed between SA and JA inducers. Furthermore, multi-trait GWA identified candidate genes influencing the response to more than one stress. For example, a functional RMG1 gene seems to be associated with susceptibility to herbivory by P. rapae and osmotic stress since loss of function mutants in RMG1 displayed higher resistance to both stresses. Finally, multi-trait GWA was used to identify QTLs with contrasting and with similar effects on the response to (a) biotic or abiotic stresses and (b) belowground or aboveground stresses.

Finally, In Chapter 7, I discuss the feasibility of obtaining plants that are resistant to multiple stresses from the point of view of genetic trade-offs and experimental limitations. The ecogenomic approach for gene discovery taken in this thesis is discussed, and recommendations are especially given on the use of herbivorous insects in quantitative genetic studies of stress resistance. Furthermore, alternatives to the use of insects in quantitative genetic studies of stress resistance are discussed and proposed. Finally, I discuss the feasibility of using an ecogenomic approach to study stress responses in other plant species than the model plant of molecular genetics, A. thaliana.

A wealth of candidate genes was generated by taking an ecogenomic approach, in particular transcriptome analysis and GWA analysis. Functional characterization of these genes is a next challenge, especially in the context of multiple stress situations. These genes constitute a rich source of potential factors important for resistance to abiotic, biotic and combined stresses that in the future may be applied for crop improvement.

Screening of the COGEM lists of non-pathogenic bacteria and fungi for postharvest diseases and plant pathogens
Vlugt, R.A.A. van der; Verbeek, M. ; Molhoek, W. ; Stevens, L. - \ 2015
Wageningen : Wageningen UR, Business Unit Biointeractions and Plant Heath (CGM onderzoeksrapport 2016-06) - 109 p.
plantenziekteverwekkers - plantenziekten - bederf na de oogst - plantenziekteverwekkende bacteriën - plantenziekteverwekkende schimmels - screenen - plant pathogens - plant diseases - postharvest decay - plant pathogenic bacteria - plant pathogenic fungi - screening
Duurzame beheersing van echte meeldauw
Hofland-Zijlstra, J.D. ; Breeuwsma, S.J. ; Noordam, Marianne - \ 2015
Bleiswijk : Wageningen UR Glastuinbouw (Rapport GTB 1385) - 44 p.
plantenziekteverwekkende schimmels - erysiphales - podosphaera pannosa - oidium - meeldauw - glastuinbouw - kasgewassen - ziekteresistentie - verbeteraars - fungiciden - geïntegreerde bestrijding - nederland - plant pathogenic fungi - mildews - greenhouse horticulture - greenhouse crops - disease resistance - amendments - fungicides - integrated control - netherlands
In this study Wageningen UR Greenhouse Horticulture developed together with LTO Glaskracht Nederland and a group of growers, advisors and producers a sustainable control strategy for mildew. Several green products with a systemic or a contact mode of action were tested on their contribution to a resilient plant system. Three systemic green products were able to stimulate hormonal signalling pathways against biotrophic organisms and effective combinations were possible with biological green products. This research shows the first proof-ofprinciple that in a young plant system with smart combining of green products infection of mildew spores can be prevented. Follow-up research in multiple cropping systems with longer cultivation periods have to indicate what the impact is on the disease development over a longer period of time under more commercial growing conditions. For the development of (early) indicators of plant resilience, it is clear that multiple measuring methods are needed at the same time, during a certain cultivation phase, because the germination and infection is dependent of several climate and plant factors.
Identification and functional characterization of putative (a)virulence factors in the fungal wheat pathogen Zymoseptoria tritici
Mirzadi Gohari, A. - \ 2015
University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462575912 - 159
triticum aestivum - wheat - plant pathogenic fungi - mycosphaerella graminicola - virulence factors - genetic analysis - pathogenesis - bioinformatics - tarwe - plantenziekteverwekkende schimmels - virulente factoren - genetische analyse - pathogenese - bio-informatica

Zymoseptoria tritici (Desm.) Quaedvlieg & Crous (previously known as Mycosphaerella graminicola) is the causal agent of septoria tritici blotch (STB), which is a devastating foliar wheat disease worldwide. It is responsible for significant yield losses occurring annually in all major wheat-growing areas and threatens global food security. Z. tritici is a hemi-biotrophic fungal pathogen that, after stomatal penetration, establishes a stealthy biotrophic and symptomless relation with its host plant that is followed by a sudden switch to a necrotrophic growth phase coinciding with chlorosis that eventually develops in large necrotic blotches containing many pycnidia producing asexual splash-borne conidia. Under natural conditions - once competent mating partners are present and conditions are conducive- pseudothecia are formed producing airborne ascospores. Disease management of STB is primarily achieved through fungicide applications and growing commercial cultivars carrying Stb resistance genes. However, the efficacy of both strategies is limited as strains resistant to fungicides frequently develop and progressively dominate natural populations, which hampers disease management; also the deployed Stb genes are often overcome by existing or newly developed isolates of the fungus. Hence, there is a need for discovery research to better understand the molecular basis of the host-pathogen interaction that enables breeders to identify and deploy new Stb genes, which will eventually contribute to more sustainable disease control.

Chapter 1 introduces the subject of the thesis and describes various aspects of the lifestyle of Z. tritici with emphasis on dissecting the various stages and physiological processes during pathogenesis on wheat. In addition, it includes a short summary and discussion of the current understanding of the role of (a)virulence factors in the Z. tritici–wheat pathosystem.

Chapter 2 describes new gateway technology-driven molecular tools comprising 22 entry constructs facilitating rapid construction of binary vectors for functional analyses of fungal genes. The entry vectors for single, double or triple gene deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, these entry vectors contain the genes encoding green fluorescent (GFP) or red fluorescent (RFP) protein in combination with the three selection markers, which enables simultaneous tagging of gene deletion mutants for microscopic analyses. The functionality of these entry vectors was validated in Z. tritici and described in Chapters 3, 4 and 5.

Chapter 3 describes the functional characterization of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans. ZtWor1 is up-regulated during initiation of colonization and fructification, and regulates expression of candidate effector genes, including one that was discovered after comparative proteome analysis of Z. tritici wild-type and ΔZtWor1 strains. Cell fusion and anastomosis occurred frequently in ΔZtWor1 strains, which is reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression profiling of ΔZtWor1, ΔMgGpb1 and ΔMgTpk2 (the catalytic subunit of protein kinase A) strains, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenicity of many fungal plant pathogens.

Chapter 4 describes combined bioinformatics and expression profiling studies during pathogenesis in order to discover candidate effectors of  Z. tritici important for virulence. In addition, a genetic approach was followed to map quantitative trait loci (QTLs) in Z. tritici carrying putative effectors. Functional analysis of two top effector candidates, small-secreted proteins SSP15 and SSP18, which were selected based on their expression profile in planta, showed that they are dispensable for virulence of Z. tritici. These analyses suggest that generally adopted criteria for effector discovery, such as protein size, number of cysteine residues and up-regulated expression during pathogenesis, should be taken with caution and cannot be applied to every pathosystem, as they likely represent only a subset of effector genes.

Chapter 5 describes the functional characterization of ZtCpx1 and ZtCpx2 encoding a secreted and a cytoplasmic catalase-peroxidase (CP) in Z. tritici, respectively. Gene replacement of ZtCpx1 resulted in mutant strains that were sensitive to exogenously added H2O2 and in planta phenotyping showed they are significantly less virulent compared to wild-type. All mutant phenotypes could be restored to wild-type by complementation with the wild-type allele of ZtCpx1 driven by its native promoter. Additionally, functional analysis of ZtCpx2 confirmed that this gene encodes a secreted CP and is, however, dispensable for virulence of Z. tritici on wheat. However, we showed that both genes act synergistically, as the generated double knock-out strain showed a significantly stronger reduction in virulence than the individual single knock-out strains. Hence, both genes are required by Z. tritici for successful infection and colonization of wheat.

In Chapter 6 I discuss and summarize the genetic approaches used in this study, reflect on the major findings and bottlenecks encountered, and propose new strategies to identify effectors of Z. tritici in the future.

Studies on global transcriptional regulator EBR1 and genome-wide gene expression in the fungal plant pathogen Fusarium graminearum
Zhao, C. - \ 2015
University. Promotor(en): Pierre de Wit; D. Tang, co-promotor(en): Theo van der Lee. - Wageningen : Wageningen University - ISBN 9789462575998 - 167
plant pathogenic fungi - gibberella zeae - transcription factors - gene expression - gene mapping - genomics - plantenziekteverwekkende schimmels - transcriptiefactoren - genexpressie - genkartering - genomica

Abstract of PhD thesis

Fusarium graminearum is a destructive plant pathogen that causes Fusarium head blight (FHB) on many crops, such as wheat, barley, rye and oat. In the first part of this thesis, we studied a transcription factor EBR1 that is required for radial growth and virulence in F. graminearum. Mutant ebr1 shows reduced apical dominance of the hyphal tip and loses its ability to penetrate the rachis of the spikelets. Subcellular localization analysis showed that EBR1 protein is exclusively localized in the nucleus of both conidia and hyphae. In the second part of thesis, by using RNA-Seq data, we revised 655 incorrectly predicted gene models and identified 231 genes with two or more alternative splice variants in F. graminearum. Furthermore, we analyzed the genome-wide gene expression pattern and found that genes locate in non-conserved regions of chromosomes showed relatively lower expression level. We further provided evidence showing that the non-conserved regions are full of gene relocations in F. graminearum.

T0-bespuiting op de kaart door gele roest
Timmer, R.D. - \ 2015
Akker magazine 11 (2015)3. - ISSN 1875-9688 - p. 12 - 13.
akkerbouw - graangewassen - tarwe - gewasbescherming - plantenziekteverwekkende schimmels - puccinia striiformis - chemische bestrijding - rassenkeuze (gewassen) - septoria - arable farming - grain crops - wheat - plant protection - plant pathogenic fungi - chemical control - choice of varieties
Afgelopen seizoen hield een nieuwe, agressieve gele-roestvariant (Puccinia striiformis) flink huis in de tarwe. Veel telers zagen de bui al hangen en kozen voor een extra fungicidebespuiting op T0. Na weer een zachte winter rijst de vraag: wordt zo’n vroege ziektebestrijding een gangbare aanpak?
Integrale aanpak enige optie beheersing Leucocoprinus : plooipaddestoel remt potplanten in groei
Velden, P. van; Hofland-Zijlstra, J.D. - \ 2015
Onder Glas 12 (2015)1. - p. 28 - 29.
glastuinbouw - potplanten - plantenziekteverwekkende schimmels - agaricaceae - ziektepreventie - groeivertraging - bedrijfshygiëne - in groei belemmeren - proeven - methodologie - greenhouse horticulture - pot plants - plant pathogenic fungi - disease prevention - growth retardation - industrial hygiene - dwarfing - trials - methodology
Leucocoprinus is een hardnekkige schimmel die niet alleen het eindproduct ontsiert, maar indirect ook groeiremming geeft. Eigenlijk hebben potplantentelers maar één schimmelbestrijdingsmiddel dat nog werkt. Meer toekomst bieden plantversterkende middelen op basis van schimmels en bacteriën, gecombineerd met een streng hygiëneprotocol.
Liesje Mommer schijnt licht op de wortelwereld
Kleis, R. ; Mommer, L. - \ 2015
Resource: weekblad voor Wageningen UR 10 (2015)4. - ISSN 1874-3625 - p. 9 - 9.
plantenecologie - wortelsystemen - biodiversiteit - soortendiversiteit - stofwisselingsstoornissen - gewasbescherming - plantenziekteverwekkende schimmels - plant ecology - root systems - biodiversity - species diversity - metabolic disorders - plant protection - plant pathogenic fungi
Nieuwe persoonlijk hoogleraar Plantenecologie. Hoe meer soorten, hoe minder schimmels.
Sexual development of Botrytis species
Terhem, R.B. - \ 2015
University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - Wageningen : Wageningen University - ISBN 9789462574144 - 188
botrytis - plantenziekteverwekkende schimmels - geslachtsontwikkeling - fylogenie - genomica - transcriptomica - paarsystemen - schimmelmorfologie - nieuwe soorten - plant pathogenic fungi - sexual development - phylogeny - genomics - transcriptomics - mating systems - fungal morphology - new species

Sexual Development of Botrytis Species

PhD Thesis

Razak bin Terhem

The fruiting bodies of species in the genus Botrytis are called apothecia. Apothecia are ascomas with an open cup shape on top of a stipe. Currently there is little information on processes occurring during apothecium development in Botrytis species. The aims of the research described in this thesis were to study the mechanisms involved in apothecium development of Botrytis cinerea, and to describe the morphology of Botrytis species and their fruiting bodies. Chapter 2 describes a genome-wide transcriptome analysis of different stages of apothecium development and a study on the function of MAT genes in apothecium development of B. cinerea. Functional analyses by targeted knockout mutagenesis revealed that the MAT1-1-1 gene and the MAT1-2-1 gene are both required for the initiation of sexual development. By contrast, mutants in the MAT1-1-5 gene and the MAT1-2-4 resulted in normal development of stipes which, however, were defective in the formation of an apothecial disk, asci and ascospores. Chapter 3 describes the functional analysis of three hydrophobin genes in sclerotium and apothecium development of B. cinerea. All three genes contribute to sclerotium and apothecium development. Chapter 4 describes the structure of the MAT1-1 and MAT1-2 locus in Botrytis elliptica and the morphology of apothecia of B. elliptica. Chapter 5 provides a morphological and phylogenetic description of Botrytis deweyae, the only species within the genus that behaves as an endophyte and in certain conditions is able to cause disease on Hemerocallis plants. Chapter 6 discusses the results presented in this thesis and puts them in a broader perspective. A model of processes and mechanisms involved in apothecium development is proposed.

Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - tomatoes - proteomics

Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.

Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.

In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.

In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.

In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.

In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.

Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.

Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.

Restyling Alternaria
Woudenberg, J.H.C. - \ 2015
University. Promotor(en): Pedro Crous; Pierre de Wit, co-promotor(en): J.Z. Groenewald. - Wageningen : Wageningen University - ISBN 9789462574106 - 250
alternaria - taxonomie - fylogenie - moleculaire taxonomie - plantenziekteverwekkende schimmels - taxonomy - phylogeny - molecular taxonomy - plant pathogenic fungi

The omnipresent dematiaceous hyphomycete genus Alternaria is associated with a wide variety of substrates including seeds, plants, agricultural products, humans, soil and even the atmosphere. It includes saprophytic, endophytic and pathogenic species, among which multiple plant pathogens, post-harvest pathogens, and human pathogens (causative agents of phaeohyphomycosis and hypersensitivity reactions). Molecular studies reveal that the Alternaria complex comprises nine genera. Within this complex several genera are non-monophyletic and Alternaria species cluster into multiple distinct species clades, which are not always correlated with species-groups based on morphological characteristics. The most commonly reported species in literature and type species of the genus Alternaria, A. alternata, also comprises one such species-group. The small-spored Alternaria species within this group are mainly described based on morphology and / or host-specificity, but are difficult to distinguish based on molecular techniques alone. As A. alternata is considered as one of the most prolific producers of fungal allergens and is reported as pathogen on over 100 host plants, correct species identification is of utmost importance. The research presented in this thesis discusses the taxonomic status of Alternaria and its related genera, with a further focus on the two biggest and most important species complexes; the large-spored A. porri and small-spored A. alternata species complexes. With the phylogenies and classifications presented in this thesis, more robust and understandable taxonomy and nomenclature in Alternaria and allied genera within the Alternaria complex are created.

Chapter 1 gives a general introduction to the genus Alternaria and related genera. The history of the genus and its economic importance as plant pathogen, post-harvest pathogen, causative agent of phaeohyphomycosis and common allergen causing hypersensitivity reactions are summarized. The introduction of the morphological species complexes, based on characters of the conidia, the pattern of chain formation, and the nature of the apical extensions of conidia are treated. Molecular studies recognise seven Alternaria species-groups within the Alternaria complex. Besides Alternaria, eight other genera are assigned to the Alternaria complex based on molecular and morphological studies.

Chapter 2 focusses on the relationship of Alternaria and its closely related genera within the broader Alternaria complex. The phylogenetic lineages within the Alternaria complex are delineated based on nucleotide sequence data of parts of the 18S nrDNA (SSU), 28S nrDNA (LSU), the internal transcribed spacer regions 1 and 2 and intervening 5.8S nrDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1) gene regions. The phylogenetic data reveal a Stemphylium clade sister to Embellisia annulata and a big Alternaria clade. The Alternaria clade contains six monotypic lineages and 24 internal clades, which are treated as sections of Alternaria. In order to create a stable phylogenetic taxonomy, and supported by i) a well-supported phylogenetic node in multiple analyses, ii) a high-similarity of clades within Alternaria based on SSU, LSU and ITS data, and iii) variation in the clade order between the different gene phylogenies, 13 genera are placed into synonymy with Alternaria. Embellisia annulata is synonymized with Dendryphiella salina, and together with D. arenariae placed in the new genus Paradendryphiella. The sexual genera Clathrospora and Comoclathris, with asexual forms linked to Alternaria, cluster within the Pleosporaceae, as does Alternaria, but outside Alternaria s. str. The genus Alternariaster, described to accommodate Alternaria helianthi, clusters within the Leptosphaeriaceae.

Chapter 3 describes the reappraisal of the genus Alternariaster. Alternaria helianthi, the causal agent of leaf spot on Helianthus annuus (sunflower) was segregated from Alternaria based on conidial morphology, and placed in the new genus Alternariaster. A multi-gene phylogeny of parts of the ITS, LSU, RPB2 and GAPDH gene regions placed a fungal pathogen associated with leaf spot on Bidens sulphurea (yellow cosmos) in Brazil in close relation with Al. helianthi. Based on the close phylogenetic relation to Al. helianthi, but distinct morphological and pathogenicity characters, the fungal pathogen associated with leaf spot on B. sulphurea is newly described as Al. bidentis.

Chapter 4 treats the Alternaria species which form the largest section of Alternaria, sect. Porri. This section contains almost all Alternaria species with medium to large conidia with long beaks, some of which are important plant pathogens. A multi-gene phylogeny on parts of the ITS, GAPDH, RPB2, TEF1 and Alternaria major allergen (Alt a 1) gene regions, supplemented with morphological and cultural studies, forms the basis for species recognition in this section. The polyphasic data reveal 63 species in sect. Porri, of which 10 are newly described, and 27 names are synonymized.

Chapter 5 treats the small-spored Alternaria species, which reside in sect. Alternaria. A lot of confusion around the naming of species within this section exists, since the naming is mostly based on morphology and host-specificity, although the molecular variation is minimal. Whole genome sequencing, combined with transcriptome profiling and multi-gene sequencing of nine gene regions, SSU, LSU, ITS, GAPDH, RPB2, TEF1, Alt a 1, endopolygalacturonase (endoPG) and an anonymous gene region (OPA10-2), is used to create a clear and stable species classification in this section. The nine sequenced Alternaria genomes range in size from 32.0 - 39.1 Mb. The number of repetitive sequences varies significantly, with a relative low percentage of repeats within sect. Alternaria. The genome identity within sect. Alternaria is high, compared to the genome identity for isolates from other sections to the A. alternata reference genome. Similarly, a relative low percentage of single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences between isolates from sect. Alternaria, compared to the percentage of SNP’s found in isolates from different sections compared to the A. alternata reference genome. A set of core proteins was extracted from the genome and transcriptome data, and primers were designed on two eukaryotic orthologous group (KOG) protein loci with a relatively low degree of conservation within section Alternaria. The phylogenies from these two gene regions, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria better than the phylogenies based on the nine commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies are synonymized under A. alternata. The subclades that are formed by these isolates are incongruent between the different gene regions sequenced; no two genes show the same groupings for any of the over 100 isolates. A sequence-based identification guide is provided for the species which are now recognized in sect. Alternaria. None of the genes sequenced in this study can distinguish all of the species recognized here on its own.

Chapter 6 investigates the molecular diversity of indoor Alternaria isolates in the USA, with the help of a phylogeographic / population genetic approach. Isolates collected throughout the USA were identified using ITS, GAPDH and endoPG gene sequencing, followed by genotyping and population genetic inference of the sect. Alternaria isolates and 37 reference isolates, using five microsatellite markers. Phylogenetic analyses revealed that 98 % (153 isolates) of the indoor isolates consisted of species from Alternaria sect. Alternaria. The remaining 2 % (three isolates) represented one sect. Infectoriae and two sect. Pseudoulocladium isolates. From the 153 isolates that belonged to sect. Alternaria, one could be assigned to A. burnsii, 15 to the A. arborescens species complex and the remaining 137 isolates were identified as A. alternata. Based on the microsatellite data, no specific indoor population could be distinguished. Population assignment analyses of the A. alternata isolates suggested that subpopulations did not exist within the sample, which we thus divided into four artificial subpopulations to represent four quadrants of the USA. Genotypic diversity was extremely high for all quadrants and a test for linkage disequilibrium suggested that A. alternata has a cryptic sexual cycle. The SouthWest-USA population displayed the highest level of uniqueness, based on private alleles. Intriguingly, the highest amount of gene flow, between SouthWest-USA and SouthEast-USA, correlated with the west-to-east movement of the antitrade winds. This suggests that indoor A. alternata isolates, although extremely diverse, have a continental distribution and high levels of gene flow over the continent.

Chapter 7 discusses the data presented in this thesis. The implications of the performed studies are placed in a broader context, with a focus on the relation between morphology and the new species classification based on molecular tools and the use of genome data in contrast to multi-gene data.

Duurzaam Bodemleven GoeddoorGrond
Oele, C. ; Os, G.J. van - \ 2015
gewasbescherming - bollenstreek - plantenziekteverwekkende schimmels - rhizoctonia - pythium - pratylenchus penetrans - agrarische bedrijfsvoering - bodem - plant protection - plant pathogenic fungi - farm management - soil
Een gebiedsgerichte aanpak van drie bodemgerelateerde ziekten, namelijk Rhizoctonia solani, Pythium en Pratylenchus penetrans.
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