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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The fish egg microbiome : diversity and activity against the oomycete pathogen Saprolegnia
Liu, Y. - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): Irene de Bruijn. - Wageningen : Wageningen University - ISBN 9789462577671 - 169
salmon - fish eggs - marine microorganisms - microbial diversity - bioinformatics - genomics - saprolegnia - oomycota - fish diseases - suppression - fungal antagonists - zalm - visseneieren - mariene micro-organismen - microbiële diversiteit - bio-informatica - genomica - saprolegnia - oömycota - visziekten - onderdrukking - schimmelantagonisten

Y. Liu

Prof. dr. F. Govers (promotor); Prof. dr. J.M. Raaijmakers (promotor); Dr. I. de Bruijn (co-promotor); Wageningen University, 13 June 2016, 170 pp.

The fish egg microbiome: diversity and activity against the oomycete pathogen Saprolegnia

Emerging oomycete pathogens increasingly threaten biodiversity and food security. This thesis describes the study of the microbiome of Atlantic salmon (Salmo salar L.) eggs and analyses of the effects of infections by the oomycete pathogen Saprolegnia on the microbial architecture. A low incidence of Saprolegniosis was correlated with a relatively high abundance and richness of specific commensal Actinobacteria. Among the bacterial community, the isolates Frondihabitans sp. 762G35 (Microbacteriaceae) and Pseudomonas sp. H6 significantly inhibited hyphal attachment of Saprolegnia diclina to live salmon eggs. Chemical profiling showed that these two isolates produce furancarboxylic acid-derived metabolites and a lipopeptide viscosin-like biosurfactant, respectively, which inhibited hyphal growth of S. diclina in vitro. Among the fungal community, the fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride. Both a quantitative and qualitative difference in the Trichoderma population between Saprolegnia-infected and healthy salmon eggs was observed, which suggested that mycoparasitic Trichoderma species could play a role in Saprolegnia suppression in aquaculture. This research provides a scientific framework for studying the diversity and dynamics of microbial communities to mitigate emerging diseases. The Frondihabitans, Pseudomonas and Trichoderma isolates, and/or their bioactive metabolites, are proposed as effective candidates to control Saprolegniosis.

Influence of adjuvants on the deposition of mancozeb
Schepers, H.T.A.M. ; Evenhuis, A. ; Topper, C.G. - \ 2014
Wageningen : Wageningen UR (vegIMPACT report 5) - 14
solanum tuberosum - aardappelen - plantenziekten - oömycota - phytophthora infestans - fungiciden - mancozeb - hulpstoffen - depositie - indonesië - potproeven - nederland - potatoes - plant diseases - oomycota - fungicides - adjuvants - deposition - indonesia - pot experimentation - netherlands
Late Blight demonstrations December 2013-February 2014
Schepers, H.T.A.M. ; Gunadi, N. ; Putter, H. de; Wustman, R. ; Moekasan, T.K. ; Laksminiwati, P. ; Karjadi, A.K. - \ 2014
Wageningen : Wageningen UR (vegIMPACT report 4) - 16
solanum tuberosum - aardappelen - solanum lycopersicum - tomaten - plantenziekten - phytophthora infestans - oömycota - gewasbescherming - fungiciden - experimenteel veldonderzoek - demonstratiebedrijven, landbouw - indonesië - potatoes - tomatoes - plant diseases - oomycota - plant protection - fungicides - field experimentation - demonstration farms - indonesia
Late blight caused by Phytophthora infestans is one of the most important diseases worldwide. Also in Indonesia control of late blight is very important in potato and tomato, especially in the rainy season. In order to learn more about the important factors that determine late blight control - such as product choice, application frequency, spray volume and use of adjuvants - two demo plots were laid out in the potato growing regions of Garut and Pangalengan. The treatments in the demo-plots consisted of different fungicide application strategies.
Black shank of tobacco in the former Dutch East Indies, caused by Phytophthora nicotianae
Zadoks, J.C. - \ 2014
Leiden, The Netherlands : Sidestone Press - ISBN 9789088902833 - 206
nicotiana tabacum - tabak - phytophthora nicotianae - oömycota - plantenziekten - nederlands indië - tobacco - oomycota - plant diseases - netherlands east indies
Jacob van Breda de Haan is known as the author of the name Phytophthora nicotianae n.sp., the causal agent of ‘black shank’, an important disease of tobacco. Who was he? Where did he work? What did he publish? He published in Dutch, 1896, in a Dutch colonial report series. Next question: what more on tobacco diseases was written in obscure, colonial Dutch documents? Another scientist, Thung Tjeng Hiang, better known as the first Wageningen professor of plant virology, presented two original papers in Dutch on ‘black shank’ with the word ‘epidemiologie’ in their title, 1931 and 1938. Therewith Thung was an early bird in plant disease epidemiology. The foundational paper by van Breda de Haan and two important papers by Thung are presented here in English translation. Both authors worked in the former Dutch East Indies, present Indonesia, the first on the island of Sumatera, the latter on that of Java. Both were in the service of tobacco planters; they had to solve immediate problems as fast as possible. In a pioneer situation, van Breda de Haan was confronted with a sudden seedling disease which devastated the tobacco seedlings in the seed beds and which, yes, could lead to ‘black shank’ in adult plants. Thung, working in a well-organized environment, had to prevent ‘black shank’ in the tobacco plantations. Both authors were successful in controlling disease by means of a combination of ecological intervention and chemical treatment. Whereas van Breda de Haan could only dream of genetic control, Thung could incorporate the use of a fairly resistant cultivar in his recommendations. The 1896 paper has epidemiological observations scattered throughout, without using the word epidemiology. The 1931 and 1938 papers are probably ‘firsts’ in the Dutch phytopathological literature having epidemiology in their title, one an early study in quantitative, comparative epidemiology and the other an early version of landscape epidemiology. The three papers are preceded by a sketch of tobacco cultivation in the former Dutch East Indies, describing the position of the two authors in the tobacco scene; they are followed by a long-due biography of a forgotten plant pathologist, Jacob van Breda de Haan.
Understanding the role of L-type lectin receptor kinases in Phytophthora resistance
Wang, Y. - \ 2014
Wageningen University. Promotor(en): Francine Govers, co-promotor(en): W. Shan; Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571327 - 214
phytophthora - phytophthora capsici - oömycota - plantenziekteverwekkende schimmels - plant-microbe interacties - arabidopsis - transgene planten - genexpressie - receptoren - kinasen - genen - ziekteresistentie - immuniteit - phytophthora - phytophthora capsici - oomycota - plant pathogenic fungi - plant-microbe interactions - arabidopsis - transgenic plants - gene expression - receptors - kinases - genes - disease resistance - immunity

Abstract

Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens.

Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens.

In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance.

We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns.

Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance.

In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer.

Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species.

In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.

Phytophthora infestans RXLR effector AVR1 and its host target Sec5
Du, Y. - \ 2014
Wageningen University. Promotor(en): Francine Govers, co-promotor(en): Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571310 - 188
phytophthora infestans - oömycota - plantenziekteverwekkende schimmels - virulentie - genen - plant-microbe interacties - ziekteresistentie - verdedigingsmechanismen - vatbaarheid - uitschakelen van genexpressie - phytophthora infestans - oomycota - plant pathogenic fungi - virulence - genes - plant-microbe interactions - disease resistance - defence mechanisms - susceptibility - gene silencing

Summary

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating potato diseases worldwide. To successfully colonize its host, P. infestans secretes a plethora of RXLR effectors that translocate into host cells to modulate plant defense. The RXLR effectors form the largest and most diverse effector family in oomycete plant pathogens, and include several that were demonstrated to trigger host resistance mediated by intracellular host immune receptors. Chapter 1 is a summary focussing on the molecular mechanisms underlying host–pathogen interactions. It introduces the multi-layered innate immune system of plants, as well as the strategies that pathogens exploit to circumvent and suppress host defense. Furthermore, it highlights the importance of vesicle-trafficking during plant defense.

The central subject of this thesis is AVR1, one of the race-specific avirulence (AVR) factors of P. infestans. AVR1 triggers plant resistance mediated by its corresponding potato Nucleotide-binding Leucine-rich repeat (NLR) resistance protein R1. P. infestans isolates that are avirulent on R1-containing potato cultivars always contain AVR1, while virulent isolates lack AVR1 but contain a related gene that we baptized as AVR1-like. AVR1 has all hallmarks of a typical RXLR effector; it contains a signal peptide, an RXLR domain and a C-terminal effector domain that contains two W motifs and one Y motif. In addition, it has, at the very end a stretch of 38 amino acids in length that we named the Tail (T)-region. AVR1-like, or in short A-L, shares high sequence similarity with AVR1. However, due to a premature stop codon the 38 amino acid T-region is missing.

Chapter 2 explores the conserved motifs and regions in the C-terminal effector domain of AVR1 that are required to trigger R1-mediated hypersensitive response (HR). Various truncated and chimeric constructs of AVR1 and A-L were generated and assayed for their ability to elicit R1-mediated HR. Results show that the T-region of AVR1 plays an important role in HR activation. Furthermore, we revealed that R1 recognizes two epitopes in AVR1, one located in the C-terminal region containing the conserved W and Y motifs, and one comprised by the T region.

In Chapter 3 the subcellular localization of AVR1 and R1 was investigated. Both were demonstrated to be nucleocytoplasmic proteins. We artificially modified the nucleocytoplasmic partitioning of AVR1 and R1 using nuclear localization and export signals (NLS/NES), and studied the effect on R1-AVR1 recognition. This revealed that nuclear localization of both AVR1 and R1 is important to induce R1-mediated immunity. In addition, we showed that AVR1-mediated suppression of CRN2-induced cell death is dependent on cytosolic localization of AVR1.

In Chapter 4, we investigated how AVR1 modulates host defense. In a yeast two-hybrid screening we identified the exocyst subunit Sec5 as a host target for AVR1. Interaction between AVR1 and Sec5 was confirmed in planta by co-immunoprecipitation and bimolecular fluorescent complementation. Although A-L shares high sequence similarity with AVR1, we found that it is not able to interact with Sec5. Sec5 was shown to be required for proper plant defense against P. infestans. The role of Sec5 in plant response upon pathogen attack was further supported by its role in callose deposition and in secretion of the pathogenesis-related protein PR-1, which indicates that Sec5 plays a crucial role in vesicle trafficking during host defense. AVR1 is able to suppress callose deposition while A-L is not, which suggests that P. infestans manipulates host vesicle trafficking by secretion of AVR1 to target Sec5. Overall, our findings unravelled a novel strategy that oomycete pathogens exploit in order to modulate host defense.

In Chapter 5 we further analysed the potential virulence activities of AVR1 and A-L. Both AVR1 and A-L were able to promote P. infestans colonization, indicating that both are genuine P. infestans virulence factors. Moreover, AVR1 was found to suppress not only callose deposition, but also Sec5-dependent cell death induced by the P. infestans elicitors INF1 and CRN2. In contrast, A-L was neither able to suppress Sec5-dependent nor Sec5-independent cell death. The conserved C-terminal motifs and regions required for virulence activity of AVR1 were investigated using AVR1 truncated constructs. In addition, the conserved C-terminal motifs and regions of AVR1 required for Sec5 interaction were studied by Y2H assays. Although the T-region of AVR1 was found to be sufficient to facilitate P. infestans colonization and suppression of CRN2-induced cell death, it could not fully accommodate the interaction of AVR1 with Sec5. Instead, both the Y motif and the T-region of AVR1 appear to be required for Sec5 targeting.

Next to Sec5, the role of other exocyst subunits in Phytophthora resistance was studied (Chapter 6). The evolutionary relationships of exocyst subunits from three Solanaceous plants, i.e. Nicotiana benthamiana, tomato and potato, were investigated in comparison to their Arabidopsis orthologs. Virus-induced gene silencing in N. benthamiana of the majority of the exocyst subunit genes (exo84s were not yet included) showed that, except for some Exo70 members, all other tested exocyst subunits are required for plant defense against P. infestans and callose deposition. In addition, all of the analysed exocyst subunit gene-silenced tomato plants showed gain of susceptibility to both P. infestans and Phytophthora capsici.

In Chapter 7, our findings obtained in this thesis on the mechanisms of AVR1-triggered host immunity and susceptibility are discussed in a broader perspective with emphasis on the current developments in the field of effector biology.

R gene stacking by trans- and cisgenesis to achieve durable late blight resistance in potato
Zhu, S. - \ 2014
Wageningen University. Promotor(en): Evert Jacobsen; Richard Visser, co-promotor(en): Jack Vossen. - Wageningen : Wageningen University - ISBN 9789461735706 - 164
solanum tuberosum - aardappelen - phytophthora infestans - oömycota - plantenziekteverwekkende schimmels - ziekteresistentie - genen - cisgenese - transgene planten - plantenveredeling - genetische modificatie - solanum tuberosum - potatoes - phytophthora infestans - oomycota - plant pathogenic fungi - disease resistance - genes - cisgenesis - transgenic plants - plant breeding - genetic engineering

Among the many diseases of potato (Solanum tuberosum L.), which is the third food crop in the world after wheat and rice, late blight caused by the oomycete pathogen Phytophthora infestans, is one of the most serious diseases. In the last century, major resistance (R) genes were introgressed mainly from the wild species Solanum demissum into the cultivated potato Solanum tuberosum. However, introgression of late blight resistance genes by interspecific crosses followed by backcrosses, proved to be associated with linkage drag problems. The desired R gene is then closely linked with one or more unfavorable genes. Moreover, the obtained resistance in the varieties could be easily overcome by fast evolving virulence among P.infestans isolates. The introduction of the A2 mating type from Mexico to Europe resulted in genetically more diverse and complex P.infestans offspring, since initially only the A1 mating type existed. Therefore, new strategies for breeding varieties with durable and broad spectrum resistance needed to be developed.

Previous research indicated that varieties containing single major R genes did not show durable resistance. Therefore, the potato breeding and research community abandoned the introgression of major R genes and started breeding for horizontal resistance by combining multiple partial resistance genes. This quantitative resistance breeding approach was also not successful because the levels of resistance were too low, breeding was too complicated and the spectrum was not as broad as anticipated. Nowadays, the introgression of major R genes regained interest and two ways of resistance breeding can be distinguished: 1. molecular marker assisted resistance breeding or 2. genetic modification (GM) of existing varieties with cloned major R genes.

In this thesis, the time-saving GM approach has been investigated to achieve durable resistance against potato late blight in existing varieties by stacking of major R genes via transgenesis and cisgenesis (Chapters 2, 3, 4). These R genes are so called cisgenes and are unmodified copies of genes from the same or crossable species, harboring their own promoter and terminator sequences.

The main difference between cisgenesis and transgenesis is the resulting (end) product. The end products for the latter case are transformants, which contain transgenes, that can come from a very different species, such as the selection marker gene nptII coding for antibiotic resistance from bacteria. However, the end products of cisgenesis, called cisformants, only harbor cisgenes (which are natural genes from the same or crossable species). These cisformants are selected by PCR for the presence of R gene(s) and for the absence of vector backbone sequences. In our study, functionality of the individual R genes, in trans- and cisformants containing stacked R genes, was determined by detached leaf assays (DLA) using avirulent isolates and by agro-infiltration with Avr genes matching every single R gene. Their foliar resistance was also tested in the field, and their resistance in tubers was tested in the lab.

In order to ensure durability, an accurate and robust system must be available to monitor virulence in P.infestans populations. Differential sets with plants containing single R genes are important and developed in many crops in order to facilitate both resistance breeding and genetic research on pathogen populations in different locations worldwide. The existing conventional differential potato set of Mastenbroek was updated and a start was made to develop a GM differential set with cloned R genes in individual transformants of cv Desiree (Chapter 5).

In Chapter 2, R genes with broad and complementary resistance spectrum were selected as a first step for R gene stacking. Selection for these R genes was performed using DLA with 44 selected late blight isolates. Out of four R genes (Rpi-sto1, Rpi-vnt1.1, Rpi-blb3, and R3a), three were selected for stacking experiments, Rpi-sto1 from S. stoloniferum, Rpi-vnt1.1 from S. venturii and Rpi-blb3 from S. bulbocastanum. Cv Desiree transformants containing these three single R genes conferred resistance to 40, 43 and 37 out of 44 isolates, respectively. The R3a containing transformant conferred resistance to only five out of 44 isolates. These three broad spectrum R genes were then combined in one binary vector pBINPLUS containing nptII as kanamycin resistance marker. Transformants containing nptII and the three R genes showed foliar resistance in DLA against two isolates PIC99189 (avrsto1, Avrvnt1, avrblb3) and EC1 (Avrsto1, avrvnt1, Avrblb3). Furthermore, the functions of these three individualR genes were confirmed using the cross reacting Avr genes from the pathogen, since no isolates were available to distinguish the function of each R gene individually due to the broad resistance spectrum. The resistance spectrum of transformants containing the three R genes Rpi-sto1, Rpi-vnt1.1 and Rpi-blb3 showed after DLA the expected sum of resistance spectrum from all three individual R genes and no indications for epistatic effects were observed (Chapter 2). These triple R genes containing transformants showed also full resistance in the field after inoculation with IPO-C (Avrsto1, Avrvnt1, avrblb3) both in 2011 and 2012. Furthermore, these three R genes were inherited to the next generation as a cluster and retained their functionality after crossing. Generally, resistance in tubers of these plants showed also the summed spectrum of all individual R genes in both generations, as was the case in the foliar resistance test. It was remarkable that transgenic Desiree plants, harboring Rpi-sto1 or Rpi-blb3,showed increased resistance in tubers, while their functional homologs Rpi-blb1 and R2, did not show resistance in tubers of conventionally bred materials. The integration of T-DNA borders and vector backbone sequences was also investigated. Around 45% of the triple R gene containing transformants harbored one or two T-DNA copies, without the integration of T-DNA borders and vector backbone (Chapter 3).

The introduction of multiple R genes was also applied to produce cisformants, plants containing only cisgenes. Three approaches were taken: 1) two cisgenes were introduced through one marker free transformation vector, 2) two cisgenes were introduced through two separate marker free vectors by co-transformation, 3) co-transformation of two vectors, one only containing nptII, and the other one is a marker free transformation vector harboring three cisgenes. This co-transformation was followed by sexual crossing to remove selection marker nptII. All three approaches were successful in the production of cisformants. The first approach produced a high percentage (73%) of cisformants but, in contrast to transgenic plants, the percentage of plants showing full resistance in DLA was relatively low (42%). The second approach produced only 4% of cisformants with stacked R genes, due to the high incidence of vector backbone sequence integration from two vectors used for co-transformation. All transformants obtained by the third approach showed full late blight resistance, which was very efficient compared to the first two approaches. This must be due to the use of the nptII selection marker. After crossing, the integration of both T-DNAs appeared to be unlinked in all tested transformants. Therefore, cisformants with active R genes could be obtained. The resistance level in tubers of cisformants was more frequently sufficient in plants with integration of two or more T-DNA copies, as it was also observed in the triple R gene transformants (Chapter 3). Not only the R genes from cisformants obtained using the third approach but also the cisformants from the first approach showed clustered inheritance in a crossing population, while the R genes segregated independently in the crossing population from a cisformant obtained using the second approach (Chapter 4).

The potato late blight differential set is used to characterize the virulence of P.infestans isolates, consisting of eleven plants which are expected to represent eleven different late blight R genes. Most differential plants were found to be susceptible to current late blight isolates, with the exception of the MaR8 and MaR9 plants. It had already been described that additional R genes were present in some members of this differential set. In Chapter 5, all eleven differential plants were tested for a hypersensitive reaction towards seven Avr genes. Only in three differential plants (MaR1, MaR2 and MaR4) no additional R genes were found, while for example MaR3,MaR8 andMaR9 contained multiple R genes. The conventional differential set was extended with F1 and BC1 segregants harboring a reduced number of these R genes and potentially containing only one R gene (R3a, R3b, R8 or R9, respectively) and with plants containing recently cloned R genes (Rpi-blb3, Rpi-sto1, Rpi-blb1, Rpi-pta1, Rpi-blb2, Rpi-vnt1.1 and Rpi-chc1). A disadvantage of the (extended) conventional differential set is that their genetic background is different which is complicating the use of this set. Moreover, for none of the extended differential plants it can be ruled out that different additional R genes are present. Therefore, a GM differential set consisting of ten transformants of cv Desiree, each harboring a single R gene was compiled. This GM differential set is more reliable for characterization of P.infestans isolates and for the functional test of individual R genes, due to the isogenic background. As a proof of concept, the conventional and the GM differential sets were compared using recently collected isolates from Dutch fields in detached leaf assays. It was found that plants containing Rpi-blb3, Rpi-blb1, Rpi-chc1, R8, R9, Rpi-vnt1.1 and Rpi-blb2 showed a broader resistance spectrum as compared to R1, R3a, R3b andR4. Furthermore, the application of the GM differential set to monitor virulence towards the different R genes in local late blight populations using trap fields was investigated. The extended conventional and the GM differential sets are on continuously growing lists, which can be in the future updated with better performing, genetically more isogenic plants harboring novel R genes, or when new R genes are transformed into cv Desiree.

In the general discussion (chapter 6), related topics from different experimental chapters are discussed simultaneously, some additional experimental data are provided and a broader view on the research area is given.

In summary, five main conclusions can be drawn from this work: 1. broad spectrum resistance in leaf and tuber with stable inheritance can be achieved by gene stacking via transgenesis and cisgenesis; 2. The frequency of cisformants with sufficient resistance at foliage and tuber level is lower than in transformants; 3. Avr genes are highly needed to test for functionality of all stacked R genes in trans- or cisformants; 4. the GM differential set can be used to accurately characterize P.infestans isolates and to assess the employability of certain R genes in particular geographic locations; and 5. genetic transformation is a unique way to improve existing susceptible potato varieties such as the cvs Bintje and Russet Burbank which are grown at relatively large areas worldwide.

Phytophthora infestans, een dynamische ziekteverwekker
Govers, F. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 128 - 132.
phytophthora infestans - aardappelen - ziektebestrijding - moleculaire genetica - genetische modificatie - phytophthora - genetisch bepaalde resistentie - oömycota - resistentieveredeling - gastheer-pathogeen interacties - potatoes - disease control - molecular genetics - genetic engineering - genetic resistance - oomycota - resistance breeding - host pathogen interactions
Samenvatting van de inaugurele rede van Francine Govers op 11 juni 2009. Dit artikel beschrijft de stand van zaken in het onderzoek aan oömyceten en in het bijzonder aan Phytophthora infestans, de veroorzaker van de aardappelziekte. Er wordt ingegaan op ziektebestrijding en resistentieveredeling, de diversiteit van Phytophthora en zijn gastheren en de genetische blauwdruk: het DNA, de genen, de effectoren en de resistentiegenen.
Dynamische ziekteverwekkers; wat we (willen) weten over oömyceten
Govers, F. - \ 2009
Wageningen, the Netherlands : Wageningen University - ISBN 9789085852780 - 32
plantenziekteverwekkers - phytophthora - dna - plantenziektekunde - oömycota - openbare redes - plant pathogens - plant pathology - oomycota - public speeches
Inaugurele rede bij de aanvaarding van het ambt van persoonlijk hoogleraar bij het Laboratorium voor Fytopathologie aan Wageningen Universiteit op 11 juni 2009: "Dynamische ziekteverwekkers : Wat we (willen) weten over oömyceten". In deze rede over oömyceten wordt ingegaan op de plaats van deze ziekteverwekker in de evolutie. Daarna op de oömoceet Phytophthora infestans, de verwekker van de aardappelziekte, ziektebestrijding en resistentieveredeling, de diversiteit van Phytophthora en zijn gastheren en de genetische blauwdruk: het DNA, de genen, de effectoren en de resistentiegenen
Phytophthora vindt altijd wel een weg om resistentie te omzeilen (interview met F. Govers)
Hanse, L. ; Govers, F. - \ 2009
Aardappelwereld 63 (2009)7. - ISSN 0169-653X - p. 4 - 5.
akkerbouw - aardappelen - phytophthora - phytophthora infestans - plantenziekteverwekkende schimmels - gewasbescherming - solanum tuberosum - oömycota - arable farming - potatoes - phytophthora - phytophthora infestans - plant pathogenic fungi - plant protection - solanum tuberosum - oomycota
Zeven vragen aan de Professor Govers over de meest gevreesde oömyceet in aardappelland
Interactions between biosurfactant-producing Pseudomonas and Phytophthora species
Tran, H. - \ 2007
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jos Raaijmakers. - [S.l.] : S.n. - ISBN 9789085047445 - 136
plantenziekteverwekkende schimmels - phytophthora - biologische bestrijding - pseudomonas - oppervlaktespanningsverlagende stoffen - gastheer parasiet relaties - oömycota - plant pathogenic fungi - phytophthora - biological control - pseudomonas - surfactants - host parasite relationships - oomycota
Fluorescent Pseudomonas bacteria produce a wide variety of antimicrobial metabolites, including soap-like compounds referred to as biosurfactants. The results of this thesis showed that biosurfactant-producing Pseudomonas bacteria are effective in controlling Phytophthora foot rot disease of black pepper in Vietnam and promote root and shoot development of the ‘King of Spices’. Biosurfactant-producing P. fluorescens strain SS101 was also effective in controlling tomato late blight caused by Phytophthora infestans. Current and future studies focus on how to implement these biocontrol agents in an integrated management practice to control Phytophthora diseases. The broad-spectrum activity of these antagonistic Pseudomonas bacteria and their biosurfactants also provide new opportunities to apply these agents for the protection and growth promotion of other crops.
Footprints of evolution: the dynamics of effector genes in the Phytophthora genome
Jiang, R.H.Y. - \ 2006
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Francine Govers. - [S.l.] : S.n. - ISBN 908504359X - 231
phytophthora - plantenziekteverwekkers - genen - virulentie - genoomanalyse - gastheer parasiet relaties - genexpressie - evolutie - co-evolutie - oömycota - genexpressieanalyse - phytophthora - plant pathogens - genes - virulence - genome analysis - host parasite relationships - gene expression - evolution - coevolution - oomycota - genomics
Activiteit van cyclische lipopeptide surfactants tegen pathogene Oomyceten
Raaijmakers, J.M. ; Tran Thi Thu Ha, H. ; Boer, M. de; Geerds, C.F. ; Waard, P. de; Beek, T.A. van; Souza, J.T. ; Ficke, A. - \ 2005
Gewasbescherming 36 (2005)1. - ISSN 0166-6495 - p. 23 - 24.
biologische bestrijding - pseudomonas fluorescens - schimmelantagonisten - oömycota - biological control - fungal antagonists - oomycota
Biologische buffering van substraatsystemen ten aanzien van wortelpathogenen
Postma, J. ; Kerssies, A. - \ 2000
Unknown Publisher - 26
pythium aphanidermatum - komkommers - rozen - phytophthora - biologische bestrijding - cultuur zonder grond - oömycota - cucumbers - roses - biological control - soilless culture - oomycota
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