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De focus van dit project was het ontwikkelen van een lerende omgeving binnen het tuinbouwcluster van Greenport West Holland. Met het actieonderzoek werd de kennisstroom verrijkt en is een aanpak ontwikkeld waarmee ook andere bedrijven binnen de sector aan de slag kunnen om een leercultuur te ontwikkelen.

De focus van dit project was het ontwikkelen van een lerende omgeving binnen het tuinbouwcluster van Greenport West Holland. Met het actieonderzoek werd de kennisstroom verrijkt en is een aanpak ontwikkeld waarmee ook andere bedrijven binnen de sector aan de slag kunnen om een leercultuur te ontwikkelen.

Met de twee workshoptemplates kan er binnen andere bedrijven een vergelijkbaar actieonderzoek worden uitgevoerd. De workshoptemplates moeten worden aangepast aan de context en de vraag van de bedrijven. Op deze manier kan het ontwikkelen van een actieve leercultuur worden bevorderd bij andere bedrijven in de sector. Workshoptemplate 1 biedt een format om de vraagarticulatie van een bedrijf te onderzoeken. Workshoptemplate 2 biedt een format voor het ontwerpen van een leerarrangement.

Der Konsum der industrialisierten Länder zeigt seit Jahren einen steigenden Trend und geht mit diversen negativ zu betrachtenden Auswirkungen einher, wie beispielsweise steigenden Emissionen, Ressourcenverknappung, Überkonsum und einer Reduktion der Biodiversität und Artenvielfalt (Brunner, 2014). Deshalb ist eine Entwicklung zu nachhaltigerem Konsum und nachhaltigeren Kaufentscheidungen seitens Konsumenten unabdingbar. Labels und Zertifzierungssysteme sollen den Konsumenten helfen verantwortungsvolle Kaufentscheidungen zu treffen (von Meyer-Höfer, 2016), jedoch steigt die Anzahl der Labels stetig an, was zu einem sogenannten «Labeldschungel» geführt hat (Sander, Heim & Kohnle, 2016). Dies resultiert in einer Überforderung der Konsumenten, welche aufgrund des Überangebotes kaum mehr seriöse Labels herausfiltern können (Sander et al., 2016). Aufgrund dessen lancierte die Forschungsgruppe Geography of Food der ZHAW das Projekt «MyFoodChoice», welches eine Weiterentwicklung der klassischen Labels in Richtung einer individualisierten Einkaufentscheidungshilfe anstrebt. Das aus dem Projekt resultierende Instrument soll als Einkaufshilfe für verschiedene Produktgruppen dienen und aufzeigen, worauf die Konsumenten achten sollten, um gemäss ihren eigenen moralischen Werten einzukaufen. Die vorliegende Arbeit untersucht im Rahmen dieses Projekts die moralischen Bedenken (Moral Concerns) anhand der Produktgruppe Tomaten. Ausserdem werden Umwelteinstellungen analysiert und untersucht, ob bei diesen und den Moral Concerns geschlechts-, alters- und bildungsspezifische Unterschiede bestehen. Zusätzlich wird nach einer Möglichkeit gesucht, die moralischen Bedenken in konkrete Kaufempfehlungen zu übersetzen.

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the At CHR12/ 23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato ( Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated Sl CHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of Sl CHR1 show reduced growth in all developmental stages of tomato. This confirms that Sl CHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non- GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.

Marktgericht produceren begint bij de smaak. Maar van welke smaak houdt de consument? Wageningen UR Glastuinbouw ontwikkelde een meetmodel voor de smaak van tomaten, paprika's en meloenen en werkt nu aan de aardbei. Bovendien brengt het de smaak van consumententypen in kaart. "Als je de smaakvoorkeur van consumenten kent, dan kun je pas écht produceren voor de markt."

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2015. Tomato spotted wilt virus (TSWV) bem como outras espécies de tospovírus (Família Bunyaviridae) é responsável por perdas substanciais na produção de vegetais ao redor do mundo. Tospovírus são transmitidos de maneira circulativa e propagativa por tripes (Ordem Thysanoptera). Como para outros patógenos, ações contra tospovírus exigem um olhar holístico, envolvendo uma combinação de táticas culturais, fitosanitárias, químicas e biológicas, quando apropriadas, além do uso de cultivares resistentes. Entretanto, o controle de doenças causadas por tospovírus tem se mostrado difícil, dado o grande espectro de hospedeiros desses vírus e a grande eficiência dos tripes vetores em transmiti-los. Além disso, a dependência e o aumento no uso de inseticidas de baixo custo tem exacerbado a presença de tospovírus pela forte pressão de seleção sobre os tripes vetores que se tornam resistentes a diferentes inseticidas. Para solucionar de forma simples todas as questões financeiras e ambientais associadas ao uso abusivo de inseticidas para o controle de tripes, esforços têm aumentado para a obtenção de cultivares geneticamente resistentes como um componente integral das estratégias de controle de doenças. Até o momento existem duas fontes de resistência disponíveis para o melhoramento genético de hortaliças à TSWV. Uma dessas fontes é o cluster genético Sw-5, o qual foi encontrado em Solanum peruvianum L., uma espécie de tomate selvagem do Peru, e tem sido introduzido em cultivares de S. lycopersicum L. (tomate comercial). Após mapeamento gênico, pelo menos cinco parálogos compõem o cluster genético Sw-5 em S. peruvianum, os quais foram nomeados de Sw-5a a Sw-5e. Esses parálogos codificam receptores do tipo NB-LRR, uma classe de proteínas citoplasmática que ativam resistência após reconhecimento direto ou indireto de patógenos que tenham ultrapassado as primeiras barreiras de defesa do sistema imune vegetal. Transformação de plantas de tabaco com os parálogos Sw-a e Sw-5b revelou que somente o último ativa resistência contra isolados de TSWV. Com a disponibilidade do genoma do tomate, genes ortólogos também foram mapeados em S. lycopersicum. Esta tese inicia-se com uma descrição detalhada do sistema imune vegetal e descobertas anteriores sobre o cluster genético Sw-5 (Capítulo 1). Como parte da introdução, os problemas causados por tospovírus e suas características são descritas. Tendo essas informações como ponto de partida, esta tese focou no entendimento de pontos-chaves da resistência mediada pela proteína Sw-5b contra tospovírus com uma atenção especial nos eventos moleculares e celulares envolvidos atrás desse mecanismo de resistência. Análises funcionais foram realizadas para esclarecer as delimitações genéticas entre os ortólogos funcionais e não funcionais do cluster Sw-5 no reconhecimento de tospovírus e ativação de resistência. Já que transformantes de N. tabacum com o gene Sw-5b feitos anteriormente não apresentaram resposta hipersensitiva (HR) após inoculação com TSWV, plantas de N. benthamiana foram transformadas com o gene Sw-5b buscando linhas transgênicas que poderiam responder com HR e assim facilitar a identificação do determinante de avirulência (Avr) de TSWV (Capítulo 2). Enquanto N. tabacum foi transformada com o gene Sw-5b sob controle de seus próprios elementos regulatórios, N. benthamiana foi transformada com o gene Sw-5b sob controle do promotor 35S do Cauliflower mosaic virus (CaMV). De forma interessante, as plantas transformadas de N. benthamiana apresentaram forte HR após inoculação de suas folhas com TSWV e outras quatro espécies de tospovírus: Alstroemeria necrotic streak virus (ANSV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV) (Capítulos 2 e 7). Para identificação do Avr, os genes de TSWV foram clonados e expressos individualmente em folhas de N. benthamiana expressando Sw-5b e em isolinhas de tomate resistentes (contendo Sw-5) para monitoramento de HR. Como resultado, HR foi induzida após expressão da proteína não-estrutural NSM do isolado BR-01 de TSWV que induz resistência, mas não do isolado GRAU de TSWV que quebra resistência (Capítulo 2). Foco sobre a proteína NSM seguiu no capítulo 3. Versões truncadas dessa proteína foram transientemente co-expressas com Sw-5b em folhas de N. benthamiana (wild type). Tais versões truncadas excluíam domínios anteriormente associados com formação de túbulos, movimento viral célula-célula e sistêmico. Proteínas NSM truncadas faltando até 50 aminoácidos (aa) de ambos N e C terminais (NSM inteira contém 301 aa) ainda induziram HR, sugerindo que funções associadas ao movimento viral e comportamento como Avr são características independentes para NSM. No capítulo 4 experimentos foram realizados para caracterizar uma nova espécie de tospovírus observado em plantas de feijão que apresentavam sintomas de mosaico necrótico em São Paulo, Brasil (2006). Microscopia eletrônica de folhas sintomáticas revelou partículas pleiomórficas agrupadas em vesículas. Devido ao fato de feijão ser um hospedeiro não usual e pelo resultado negativo em testes de ELISA para outras espécies já conhecidas por circular no Brasil, testes biológicos, sorológicos e moleculares foram realizados para caracterização de uma provável nova espécie de tospovírus. Este vírus apresentou um estreito espectro de hospedeiros, infectando sistemicamente três de vintes espécies de plantas indicadoras e apresentou propriedades sorológicas únicas quando comparado com outras espécies de tospovírus encontradas no Brasil. O sequenciamento do genoma deste tospovírus revelou uma nova espécie que, junto com Soybean vein necrosis-associated virus (SVNaV), representa uma nova linhagem evolutiva de tospovírus circulando no continente americano. Este tospovírus foi tentativamente chamado Bean necrotic mosaic virus (BeNMV). Já que a proteína Sw-5b reconhece pelo menos cinco espécies de tospovírus de origem “americana”, a proteína NSM do BeNMV também foi testada para monitoramento de HR através de sua co-expressão com Sw- 5b em folhas de N. benthamiana. O resultado, entretanto, mostrou que a proteína NSM do BeNMV não é um Avr cognato da proteína Sw-5b. No capítulo 5 é mostrado que a proteína Sw-5b induz HR dependentemente e independentemente da presença de NSM. A forma independente foi observada através da coexpressão da proteína Sw-5b com supressores de silenciamento gênico (p19 e NSS), os quais aumentaram a acumulação celular da proteína Sw-5b, induzindo auto-HR. Esta observação permitiu o screening de indução de HR e reconhecimento de NSM como eventos independentes para as outras proteínas Sw-5. Enquanto Sw-5a induziu auto-HR, ela não foi capaz de reconhecer NSM como Avr. De forma diferente, o ortólogo mais conservado das proteínas Sw- 5a e Sw-5b de S. lycopersicum susceptível a TSWV, nomeado Sw-5aS, não induziu qualquer tipo de HR. Através da co-expressão dos domínios individuais de Sw-5b, CC, NB-ARC, LRR ou de versões combinadas (CC-NB-ARC e NB-ARC-LRR) com NSM e com o supressor de silenciamento gênico p19, o papel desses domínios na indução de HR e no reconhecimento de NSM foram determinados. Enquanto NB-ARC foi suficiente para induzir auto-HR, NB-ARC-LRR induziu tanto auto quanto HR dependente de NSM, indicando que o domínio LRR especifica o reconhecimento do Avr. Já o domínio CC suprimiu HR induzida por NB-ARC em cis e trans, apontando para uma função regulatória de CC. A superexpressão do domínio NB-ARC de Sw- 5aS não resultou em HR, similar ao resultado da proteína Sw-5aS completa. Após alinhamento dos domínios NB-ARC, três variações de aminoácidos (aa) foram encontradas entre as proteínas Sw-5a, Sw-5b e Sw-5aS, os quais foram revertidos no último. Quando a glutamina da posição 599 foi convertida em uma arginina (Q599R), igual a proteína Sw-5b nesta posição, o domínio NB-ARC da proteína Sw-5aS tornou-se funcional para indução de HR. Modelagem deste domínio revelou que esta mutação encontra-se fora do domínio de ligação de ADP/ATP, o qual é importante para o switching entre os estados on e off dos domínios NB-ARC. Finalmente, a fusão do domínio LRR da proteína Sw-5b na variante Q599R do domínio NBARC de Sw-5aS resultou em auto-HR e HR dependente de NSM. Os construtos codificando os domínios da proteína Sw-5b também foram usados para estudos subcelulares no capítulo 6. Para este propósito, todas as proteínas estavam fusionadas a Green Fluorescence Protein (GFP) na porção N-terminal para tornar possível a visualização por microscopia confocal em tecido folhear. Enquanto a proteína Sw-5b inteira, os domínios CC, NB-ARC e LRR e o combinado CC-NB-ARC apresentaram distribuição nucleocitoplasmática, NB-ARC-LRR localizou-se somente no citoplasma, sugerindo que CC sinaliza o importe nuclear. Os domínios NB-ARC e LRR também foram investigados para uma possível interação direta com NSM através da técnica Bimolecular Fluorescence Complementation (BiFC). Enquanto interação direta não foi observada entre LRR e NSM, o domínio NB-ARC aparentou interagir com NSM. As especificidades para o reconhecimento de NSM como Avr são discutidas no capítulo 7, levando em consideração aspectos evolutivos de tomates e tospovírus. Um modelo da ativação da proteína Sw-5b é postulado no texto tendo como base a dissecção desta proteína e os ensaios de localização subcelular, além de sua putativa interação direta com NSM. Tomato spotted wilt virus (TSWV) along with other tospovirus species (family Bunyaviridae) is responsible for substantial losses in crop production around the world. Tospoviruses are transmitted in a propagative and circulative manner by thrips vectors (Order Thysanoptera). As for other pathogens, disease management against tospoviruses pursues a holistic approach involving a combination of cultural, phytosanitary, chemical, and biological tactics, when suitable in addition to the use of resistant crops. Nevertheless, successful control has proven difficult given the broad host range of these viruses and their effective spread by thrips vectors. More importantly, increased reliance on the use of low-cost insecticides has exacerbated tospovirus spread by causing thrips resistance. To ease the financial and environmental constraints associated with insecticide abuse to control thrips, efforts have been increased to obtain genetically resistant cultivars as an integral component of disease management strategies. So far there are two resistance sources available for commercial breeding of vegetables against TSWV. One of these sources is the Sw-5 gene cluster, which has been found in Solanum peruvianum L., a wild species of tomato from Peru, and has been introgressed in S. lycopersicum L. cultivars (commercial tomatoes). After gene mapping, it has been reported that at least five paralogs compose the Sw-5 gene cluster in S. peruvianum, which were named Sw- 5a to Sw-5e. These paralogs encode NB-LRR receptors, a class of cytoplasmic proteins that activate disease resistance upon direct or indirect recognition of pathogens that have overcome the first lines of defense of the plant immune system. Transformation of tobacco plants with the paralogs Sw-5a and Sw-5b, revealed that only the latter triggers resistance against TSWV isolates. With the availability of the tomato genome, highly conserved orthologs have been mapped in S. lycopersicum as well. This thesis started off with a detailed description of the plant immune system and previous findings about the Sw-5 gene cluster (Chapter 1). As part of this introduction, the problems caused by tospoviruses, to which Sw-5b locus confers resistance and the characteristics of these viruses have been described. With this knowledge as a starting point, this thesis focused on unraveling the key features of Sw-5b-mediated resistance against TSWV with special attention to the molecular and cellular events underlying the resistance mechanism. Functional analyses have been performed towards clarification of the genetic delimitations between functional and non-functional Sw-5 orthologs considering tospovirus recognition and resistance triggering. Since earlier made N. tabacum transformants containing Sw-5b did not show a hypersensitive response (HR) upon challenging with TSWV, N. benthamiana have been transformed with Sw-5b aiming to obtain transgenic lines that would respond with a visual HR and thereby facilitate the identification of the avirulence determinant (Avr) from TSWV (Chapter 2). While N. tabacum plants were transformed with Sw-5b gene under control of its own regulatory elements, the N. benthamiana plants were transformed with the Sw-5b gene under control of the Cauliflower mosaic virus (CaMV) 35S promoter. Interestingly, the Sw-5btransformed N. benthamiana plants presented a strong HR upon challenging with TSWV and four other tospoviruses: Alstroemeria necrotic streak virus (ANSV), Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV) (Chapter 2 and 7). To identify the Avr, TSWV genes were cloned and expressed individually in leaves of Sw-5b-transformed N. benthamiana plants and Sw-5-resistant tomato isolines for HR monitoring. As a result, HR was triggered upon expression of the non-structural protein NSM from the resistance-inducing TSWV isolate BR-01, but not from the resistance-breaking TSWV isolate GRAU (Chapter 2). The research on NSM was continued in Chapter 3, in which truncated versions of this protein were transiently co-expressed with Sw-5b in wild type N. benthamiana leaves. These truncations lacked domains previously associated with tubule formation, cell-tocell and systemic viral movement. Truncated NSM proteins lacking up to 50 amino acids (aa) from either the N- or C-terminus (full NSM is 301 aa in size) still triggered Sw-5b-mediated HR, suggesting that viral movement functions of NSM and its fate as Avr are independent from each other. Chapter 4 described experiments to characterize a new tospovirus collected from bean plants showing necrotic mosaic symptoms during a field survey in São Paulo, Brazil (2006). Electron microscopy of symptomatic leaves revealed pleomorphic particles packed in vesicles. Due to its unusual natural host for a “Brazilian” tospovirus and being negative in ELISA for tospovirus species known to be circulating in Brazil, biological, serological, and molecular tests were performed to further characterize this putatively new tospovirus species. The virus appeared to have a narrow host-range, systemically infecting only three out of twenty test-plants of various species and presented unique serological properties when compared to other known tospovirus species. The genome sequencing of this tospovirus revealed a completely new species, which, together with Soybean vein necrosis-associated virus (SVNaV), represents a second evolutionary lineage of tospoviruses circulating in the American continent. This new tospovirus was tentatively named Bean necrotic mosaic virus (BeNMV). Since the Sw-5b protein recognizes at least five tospovirus species of “American” origin, the NSM protein of BeNMV was also tested for HR-triggering through co-expression with Sw-5b in N. benthamiana leaves. The outcome, however, indicated that the NSM protein of BeNMV is not a cognate Avr of the Sw-5b protein. In Chapter 5 it is shown that the Sw-5b protein triggers both NSM-dependent and - independent HR. The latter was achieved by co-expression of Sw-5b with RNA silencing suppressors (p19 and NSS), which increased the Sw-5b cellular accumulation and lead to auto- HR. This observation allowed the screening of HR-triggering and NSM-recognition as uncoupled events for other Sw-5 proteins as well. While Sw-5a could trigger auto-HR, it lacked the ability to recognize NSM as Avr. On the other hand, the highest conserved ortholog of Sw-5a and Sw-5b from susceptible S. lycopersicum, named here as Sw-5aS, lacked both auto-HR and NSMdependent HR. By co-expression of the individual Sw-5b domains CC, NB-ARC, LRR or combined versions (CC-NB-ARC and NB-ARC-LRR) with NSM and the silencing suppressor p19, the role of these domains in HR-triggering and NSM-recognition was determined. While NBARC was sufficient for auto-HR, NB-ARC-LRR triggered both auto- and NSM-dependent HR, indicating that LRR specifies the Avr recognition. The CC domain suppressed HR triggered by NB-ARC in cis and trans, pointing towards a regulatory function for CC. The overexpression of the NB-ARC domain from Sw-5aS did not result in HR, similar to the outcome with the full-length Sw-5aS protein. After alignment of the NB-ARC domain, three mismatches were found between Sw-5a, Sw-5b, and Sw-5aS which were reverted in the latter. When the glutamine at position 599 was converted into an arginine (Q599R), to mimic the situation in the Sw-5b protein at this position, the Sw-5aS NB-ARC domain became functional for auto-HR triggering. Modeling of this domain revealed that this mutation was outside of the ADP/ATP binding site, which is important for the switching between “on” and “off” states of NB-ARC domains. Finally, the fusion of the LRR domain of Sw-5b to the Q599R variant of the Sw-5aS NB-ARC domain resulted in both auto- and NSM-dependent HR. The constructs encoding the various Sw-5b domains were used for subcellular studies in Chapter 6. To this end, all constructs encoded proteins were fused with Green Fluorescence Protein (GFP) at their N-terminus, to enable easy visualization by confocal microscopy in leaf tissue. Whereas the full-length Sw-5b protein, the individual domains CC, NB-ARC, and LRR and the combined CC-NB-ARC version showed a nucleocytoplasmic distribution, NB-ARC-LRR localized only in the cytoplasm, suggesting that CC signalizes nuclear import. The subdomains NB-ARC and LRR were also investigated for a possible direct interaction with NSM using Bimolecular Fluorescence Complementation (BiFC). While for LRR interaction with NSM was not observed, the NB-ARC domain seemed to directly bind to NSM. The specificities for NSM recognition have been discussed in Chapter 7 taking into consideration evolutionary aspects of tomatoes and tospoviruses. A model for Sw-5b activation is postulated throughout the text based on the dissection of this protein in HR-triggering and subcellular localization assays and on its putative direct interaction with NSM.

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.

Tomato production is threatened by climate change. High temperatures lead to a decrease of fruit set which correlates with a decrease of pollen fertility. The low viability of tomato pollen under heat stress was previously shown to be associated with alterations in specific metabolites. In this thesis, we used untargeted metabolomics approaches to broaden the identification of metabolites affected by heat stress. We assessed the suitability of pollen isolation methods for metabolomics analysis and considered the pitfalls for our further analysis. We explored the developmental metabolomes of pollen and anthers of different tomato genotypes under control and high temperature conditions and identified that microsporogenesis is a critical developmental stage for the production of mature and fertile pollen grain under heat stress. Several metabolites were putatively associated with tolerance to high temperature such as specific flavonoids, polyamines and alkaloids. These metabolites can be further used as markers in breeding programs to develop new genotypes tolerant to high temperatures.

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

De glastuinbouwsector is een miljarden business en daardoor van grote economische waarde. Keerzijde is dat de sector veel fossiele energie gebruikt, waarbij grote hoeveelheden CO2 worden uitgestoten. Duurzaamheid komt daardoor steeds nadrukkelijker op de agenda van de tuinbouw te staan. Een belangrijke stap in het verlagen van de CO2 voetafdruk van belichte teeltsystemen kan de toepassing van led zijn in plaats van de huidige standaard SON-T belichting.

De toepassing van LED-belichting in de glastuinbouw staat nog in de kinderschoenen. Enkele bedrijven zetten al LED’s in, vooral rode en blauwe. Over hoe de verschillende lichtkleuren precies uitwerken op de fysiologie van de plant, is nog maar weinig bekend. Een onderzoek van Wageningen UR Glastuinbouw laat zien dat blauwe en groene LED’s kunnen worden gebruikt als stuurlicht. Een grotere kasproef moet inzicht geven hoe dit praktisch toe te passen is.

Global warming already has and will significantly impact crop productivity and yield in the near future. In order to meet the forecasted requirements of the future agricultural production, a proper assessment of crops environmental stress tolerance needs to be designed and implemented, from the laboratory to field. Genetic variation in the ability of tomatoes to set fruit under high temperature conditions has made selection for heat tolerance possible and multiple opportunities for improvement exist, as tolerance to high temperatures is a multi-genic character involving a complex network of chaperones and other protective proteins acting together to defend the cells from heat injury. Breeding programs involved in the development of heat tolerant cultivars should identify and make use of such tolerance traits already available in collected or wild germplasm. The goal of this thesis was to characterize the response to high temperatures in meiotic tomato anthers with contrasting responses to heat and to identify genes that could be related to thermo-tolerance mechanisms during gamete development. Several molecular tools such as transcriptomic profiling by cDNA-AFLP and microarray analysis, RT-PCR or in situ RNA hybridisation were used to achieve this goal. The second chapter reviews the effects of heat stress on reproductive flower development, candidate tolerance pathways and methods for production of heat tolerant crops. The third chapter provides a general overview of the expression changes occurring in the developing anthers of a sensitive tomato genotype following exposure to a (short and) moderate high temperature stress (MHS). Using a combination of cDNA-AFLP, RT-PCR, and in situ RNA hybridisation, we characterized and verified the general transcriptional response to heat of tomato plants. Our results revealed that approximately 1% of the examined transcript-derived fragments exhibit alterations in expression pattern and the majority of these were down-regulated The putative functions associated with the genes identified by cDNA-AFLP indicated involvement of heat shock, metabolism, antioxidant and developmental processes. Based upon the observed transcriptional changes in response to MHS and on literature sources, we identified a number of candidate transcripts to be involved in heat-tolerance. The spatial expression of several such candidate genes was further examined using in situ RNA hybridisation and this showed that the investigated genes are expressed in the tapetum or/and in developing microspores. Furthermore, the expression of several candidate genes has been quantified by RT-PCR in additional genotypes with different degrees of heat tolerance. The results suggested a correlation between gene expression levels, pollen germination rates and tolerance to heat (Chapter 4). In the fourth chapter we proceeded to profile the response to heat of meiotic anthers in a tolerant and a sensitive tomato genotype and investigated the expression of the identified candidate genes in several pairs of contrasting genotypes. Using microarray analysis (for an extensive overview of the meiotic response to heat) and RT-PCR, we were able to clearly distinguish differential responses of the tolerant genotype. After 2h of moderate heat stress, the heat-tolerant genotype exhibits fewer transcriptional changes than the heat-sensitive genotype. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression soon after MHS. Moreover, the heat-tolerant genotype also shows a different level of constitutive gene expression profiles when compared to the heat-sensitive genotype indicating a difference in genetic adaptation with regards to increased temperatures. The putative functions associated with the genes identified by microarray profiling indicate involvement of heat shock, antioxidant, metabolic, and cell development pathways. Based upon the observed differences in response to MHS we selected a number of candidate transcripts involved in heat-tolerance and confirmed their expression pattern in different tomato genotypes with contrasting responses to heat. The results suggested that the candidate genes are involved in the activation of protection mechanisms in the tomato anthers during moderate heat stress and, could therefore contribute to normal growth and development of the male gametophyte and implicitly a successful fruit set under adverse temperatures. In the fifth chapter we tested the hypothesis that heat tolerance is associated with maintenance of organ identity, fertility and lower ABA levels during heat stress (for several tomato genotypes) and analysed the dynamics of ABA accumulation under temperature stress in several tomato genotypes with contrasting responses to heat. Furthermore, pollen germination tests were performed and additional physiological aspects of anther development for each genotype were analysed as well. The general trend observed was the accumulation of lower relative levels of ABA at the end of the experimental period compared to the initial stages in more tolerant genotypes and of higher levels in the sensitive genotypes. We concluded from these results that the morphological changes in the floral tissues and the overall changes in ABA levels are correlated with the molecular responses under increased temperature in the genotypes analysed. Whether these correlations are causally related is not clear; therefore more research is needed to resolve these issues. The sixth chapter examines our analysis of the heat stress response in meiotic tomato anthers in a broader scientific context. I discuss the different aspects of our results and present several candidate genes involved in plant thermo-tolerance. In addition, I also discuss the potential involvement of plant growth regulators in plants´ responses to heat stress and suggest various potential follow-up experimental strategies.

Jonge tomaten- en paprikaplanten, maar ook een aantal andere gewassen, houden soms opeens op met groeien. Koploosheid was lange tijd een lastig maar zeer slecht begrepen fenomeen. De laatste jaren is het inzicht in een stroomversnelling gekomen. In ieder geval is het aantal mogelijke oorzaken drastisch ingeperkt.

Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance. A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium. After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs. In Chapter 2 , we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role. Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function. In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species. In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein. In Chapter 5 , we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution. In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber. In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T a A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2. Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.

Wageningen UR Greenhouse horticulture searched in the “The New Crop” project for the crop structure that best contributes to the goal of energy-efficient production and therefore energy saving, by removing 33, 44 or 55% of the leaves at a young stage. On October 10th 2014 the experiment started with topped plants of the tomato variety Brioso grafted on Maxifort. Dry matter production was lowest in the very open crop, however partitioning to the fruits was highest in that treatment. Therefore, this crop produced in the winter under assimilation lighting most. In summer, the standard treatment was the best and the production in this treatment was highest. The plants in the very open treatment were shorter and had smaller leaves than the plants of the standard treatment. This is might be due to a different red: far red ratio of light in the crop. Leaf picking at a young stage contributes to better distribution of assimilates to the fruits. This can be used as a crop management measure as the plant in winter develops too much leaves. The project was funded by the Dutch energy transition program “Kas als Energiebron”.

Marktgericht produceren begint bij de smaak. Maar van welke smaak houdt de consument? Wageningen UR Glastuinbouw ontwikkelde een meetmodel voor de smaak van tomaten, paprika's en meloenen en werkt nu aan de aardbei. Bovendien brengt het de smaak van consumententypen in kaart. "Als je de smaakvoorkeur van consumenten kent, dan kun je pas écht produceren voor de markt."

Samen met een goede fotosynthese staat voldoende vruchtzetting op de gedeelde eerste plaats bij het welslagen van de teelt van vruchtgroenten. Het zou echter veel gemakkelijker zijn als die bevruchting helemaal niet nodig was. Bij verscheidene gewassen is parthenocarpie inmiddels gewoon, maar bij een belangrijk deel blijft het zaak de juiste omstandigheden voor vruchtzetting te creëren.

Energy savings through a greenhouse cover of insulation glass requires a large investment. In the search for a cheaper alternative by a consortium of companies consisting of VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems a Glass-Film-greenhouse cover, better known as the 2SaveEnergy greenhouse concept was realized in summer 2014. The combination of clear glass with a diffuse ETFE film and a double screen mounted at a distance of only a few centimetres, in the year 2015, resulted in a low energy consumption and a better than expected good tomato production. During cultivation, the principles of the new cultivation methods were used. With respect to the common practice, the energy consumption was more than 50% lower at a minimum equal production. Energiebesparing door een isolerend kasdek van isolatieglas vergt een grote investering. In de zoektocht naar een goedkoper alternatief is door een consortium van bedrijven bestaande uit VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems een Glas-FilmKasdek, beter bekend als het 2SaveEnergy concept ontwikkeld en in de zomer van 2014 ook gerealiseerd. De combinatie van helder glas met een diffuse ETFE film en een dubbel scherm welke op slechts enkele centimeters afstand van elkaar gemonteerd is heeft in het teeltjaar 2015 tot een laag energiegebruik en een prima tomaten productie geleid. Tijdens de teelt zijn de principes van het nieuwe telen zoveel mogelijk aangehouden. Ten opzichte van de gangbare praktijk was het energiegebruik meer dan 50% lager bij een minimaal gelijke productie.

The main, most important function of roots belonging to horticultural crops is the uptake of water and nutrients. Healthy roots are essential for a healthy plant. After all, if the uptake of water and nutrients is not functioning properly, then other aspects also leave a lot to be desired

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the At CHR12/ 23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato ( Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated Sl CHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of Sl CHR1 show reduced growth in all developmental stages of tomato. This confirms that Sl CHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non- GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.

Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited. In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies. In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance. Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress. To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects. The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control. Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

The supply of additional CO2 in a greenhouse will be restricted in the future. The concentration in outside air has risen above 400 ppm. This may open the possibility to blow this air through the canopy to increase growth. In this project, the vertical CO2 concentration was measured in a vertical plane within to the canopy under different combinations of window opening, the activation of vertical fans and with or without dosing of additional CO2. For a Freesia and a tomato crop the result was that without CO2 dosing it was possible to maintain a concentration of over 350 ppm in the canopy at 5-10 cm distance from the leaf surface when the ventilation windows were open. Since, this is below outside concentration, additional supply of outside air may be an advantage. When extra CO2 was supplied, a reduction in window opening and the use of a screen increased the concentration between the canopy. The vertical distribution of CO2 within the canopy was never a problem. It can be concluded that the crop resistance to take up CO2 for a tomato and freesia crop is small and with respect to the other CO2 resistances, the crop resistance can be neglected. A positive effect of the use of vertical fans or the use of high pressure misting in the tomato greenhouse was not found, due to the strategy to keep the ventilation windows wide open. The concentration at 5-10 cm distance from the leaf is not necessarily the same concentration around the stomata because of boundary layer resistance. The effect of the boundary layer resitance on CO2 uptake is described in the report of Plant Dynamics called “Effecten van grenslaagweerstand op de fotosynthese bij tomaat en Freesia”. Het verhogen van de CO2 concentratie in een kas wordt door allerlei oorzaken moeilijker en draagt ook niet bij aan de energie neutrale kas. Omdat de CO2 concentratie in de buitenlucht boven een waarde van 400 ppm is gekomen loont het wellicht om deze lucht door het gewas heen te blazen. De vraag is dan wel hoe deze lucht zich mengt binnen het bladpakket. Gaat dat vanzelf omdat CO2 zwaarder is dan lucht of moet er geblazen worden met een ventilator? In dit onderzoek is gemeten hoe de verticale verdeling van CO2 in een gewas wordt bij verschillende behandelingen zoals wel of niet doseren, ramen open of dicht en met verticale ventilatoren kaslucht door het gewas stuwen. Daarbij bleek dat zonder dosering van CO2 bij voldoende raamopening er bij freesia en tomaat ook onderin het gewas een concentratie van 350 ppm kon worden gehandhaafd. Omdat dit beneden de buitenwaarde is, zou toevoeren van extra buitenlucht met een ventilator zinvol kunnen zijn. Als er wel gedoseerd werd nam de concentratie in de kas toe wanneer de ramen of een scherm meer gesloten werden. De verticale verschillen in CO2 concentratie in het gewas waren erg klein, ook wanneer geen verticale ventilator werd gebruikt. Er kan dus geconcludeerd worden dat voor zowel een tomaten gewas als fresia gewas de gewasweerstand voor CO2 gering was en te verwaarlozen in ten opzichte van de overige weerstanden voor opname van CO2. De CO2 concentraties zijn gemeten op 5-10 cm afstand van het blad. Dat zegt op zich nog niets over de concentratie vlak bij het huidmondje omdat zich daar omheen een laagje stilstaande lucht kan bevinden waarin de CO2 onvoldoende snel getransporteerd kan worden en dus uitputting op kan treden. Aan deze zogenaamde grenslaagweerstand van de lucht dichter bij het blad wordt aandacht besteedt in een apart rapport van Plant Dynamics met de titel “Effecten van grenslaagweerstand op de fotosynthese bij tomaat en Freesia”.

Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited. In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies. In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance. Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress. To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects. The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control. Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

The basic process for crop growth and production is photosynthesis. Measuring crop photosynthesis is therefore important to monitor the status of the crop and whether the greenhouse climate is set to the needs of the crop. In this project, two monitoring systems for crop photosynthesis were developed and tested. (1) The crop photosynthesis monitor is a soft sensor that can calculate the CO2 uptake of an entire crop. The basis for these calculations are the balance between CO2 supply and CO2 loss via ventilation and crop photosynthesis. By measuring the CO2 concentration and humidity inside and outside the greenhouse, the crop photosynthesis can be calculated. (2) The CropObserver is a fluorescence sensor that measures the light use efficiency of photosynthesis of a large crop area (3 x 3 m2). The crop receives light pulses from a laser in the top of the greenhouse, the sensor measures the fluorescence signal of the crop. Both sensors were tested in a tomato crop in 2014 with promising results. The sensors functioned without problems and delivered patterns of daily photosynthesis which matched the reference measurements reasonably well up to well.

Wageningen UR Greenhouse horticulture searched in the “The New Crop” project for the crop structure that best contributes to the goal of energy-efficient production and therefore energy saving, by removing 33, 44 or 55% of the leaves at a young stage. On October 10th 2014 the experiment started with topped plants of the tomato variety Brioso grafted on Maxifort. Dry matter production was lowest in the very open crop, however partitioning to the fruits was highest in that treatment. Therefore, this crop produced in the winter under assimilation lighting most. In summer, the standard treatment was the best and the production in this treatment was highest. The plants in the very open treatment were shorter and had smaller leaves than the plants of the standard treatment. This is might be due to a different red: far red ratio of light in the crop. Leaf picking at a young stage contributes to better distribution of assimilates to the fruits. This can be used as a crop management measure as the plant in winter develops too much leaves. The project was funded by the Dutch energy transition program “Kas als Energiebron”. In het project “Het Nieuwe Gewas” is Wageningen UR Glastuinbouw in opdracht van Kas als Energiebron op zoek gegaan naar de gewasopbouw die optimaal bijdraagt aan de doelstelling van energie-efficiënt en dus energiezuinig produceren, door 33, 44 of 55 % van de bladeren in een jong stadium weg te nemen. Op 10 oktober 2014 is het experiment gestart met getopte planten van het tomatenras Brioso geënt op Maxifort. De biomassa productie gemeten in kg droge stof was bij het zeer open gewas het laagste, maar de verdeling hiervan over vruchten en plant was bij het zeer open gewas meer naar de vruchten. Hierdoor produceerde dit gewas in de winter onder assimilatie belichting het meeste. In de zomer was de standaard behandeling het beste en werd de productie in deze behandeling het hoogste. De planten in de zeer open behandeling bleven korter met kleinere bladeren, dan de planten van de standaard behandeling. Dit is mogelijk te verklaren uit een veranderde rood:verrood verhouding van het licht in het gewas. Bladplukken in een jong stadium draagt bij aan het beter sturen van assimilaten naar de vruchten. Dit kan als gewasmanagement maatregel worden gebruikt als de plant in de winter te veel blad ontwikkelt.

The basic process for crop growth and production is photosynthesis. Measuring crop photosynthesis is therefore important to monitor the status of the crop and whether the greenhouse climate is set to the needs of the crop. In this project, two monitoring systems for crop photosynthesis were developed and tested. (1) The crop photosynthesis monitor is a soft sensor that can calculate the CO2 uptake of an entire crop. The basis for these calculations are the balance between CO2 supply and CO2 loss via ventilation and crop photosynthesis. By measuring the CO2 concentration and humidity inside and outside the greenhouse, the crop photosynthesis can be calculated. (2) The CropObserver is a fluorescence sensor that measures the light use efficiency of photosynthesis of a large crop area (3 x 3 m2). The crop receives light pulses from a laser in the top of the greenhouse, the sensor measures the fluorescence signal of the crop. Both sensors were tested in a tomato crop in 2014 with promising results. The sensors functioned without problems and delivered patterns of daily photosynthesis which matched the reference measurements reasonably well up to well. Het basisproces voor de groei en productie van een gewas is de fotosynthese. Het meten van de gewasfotosynthese is dus van belang om goed te monitoren hoe het gewas er voor staat en of het kasklimaat is ingesteld naar de behoefte van het gewas. In dit project zijn twee meetsystemen ontwikkeld en getest om de gewasfotosynthese te meten. (1) De gewasfotosynthesemonitor is een soft sensor die de CO2 opname van een hele kas met gewas kan berekenen. Dit doet deze monitor op basis van de balans tussen enerzijds CO2 dosering en anderzijds verlies van CO2 door ventilatie en gewasfotosynthese. Door de CO2 concentratie en de luchtvochtigheid binnen en buiten de kas te meten kan de gewasfotosynthese berekend worden. (2) De nieuwe fluorescentiesensor die ontwikkeld is, de CropObserver, meet de efficiëntie van de lichtbenutting als maat voor de fotosynthese van een groot oppervlakte gewas (3 x 3 m2). Het gewas wordt belicht met korte laserlichtpulsen bovenin de kas, de sensor meet het terugkomende fluorescentiesignaal. Beide nieuwe sensoren zijn begin 2014 getest in een tomatengewas. De eerste testen waren veelbelovend. De sensoren hebben zonder problemen gefunctioneerd en leverden een dagverloop van de fotosynthese op, dat redelijk tot goed overeen kwam met referentiemetingen.

Dehumidification of a greenhouse by ventilation increases the energy input with 8-10 m3/m2.year of natural gas to compensate the heat losses. This study shows different methods to reduce those energy losses. A 25% reduction in ventilation is obtained by using heat exchangers connected to plastic distribution ducts or by using the Ventilationjet system. The sensible heat from the exhaust air can be used to heat the incoming outside air to greenhouse temperature. Heat exchangers with 100% efficiency to do that are available. The latent heat included in the water vapour leaving the greenhouse can be recovered by means of a condensating wall. A good working priciple is the Dewpoint Heat Exchanger in which outside air is wettened to reach the lowest possible temperature and then used to cool down the greenhouse air far below the temperature where condensation begins. The recovered latent heat must be stored in a water tank to use it in the heating pipes. Another approach is to skip ventilation and use a set of a cold and warm heat exchanger connected to a heat pump or to use a salt water absorber. The absorber opens the possibility to use solar or wind energy to dehumidify the greenhouse and collect 100% of the sensible and latent heat. Ontvochtigen van een kas door ventilatie kost voor een tomatenteelt al gauw 8-10 m3/m2.jaar aardgas om de ontstane warmteverliezen weer te compenseren. Uit deze studie blijkt dat er verschillende methoden voorhanden zijn of nog ontwikkeld kunnen worden om daarop te besparen. De hoeveelheid ventilatie kan met 25% gereduceerd worden met Ventilationjets of met luchtbehandelingskasten met slurven. Uit de luchtstroom die naar buiten verdwijnt kan de voelbare warmte worden teruggewonnen en gebruikt om de binnenstromende buitenlucht op te warmen. De latente warmte, opgesloten in de waterdamp, kan worden teruggewonnen en opgeslagen in een waterbassin of een aquifer door deze te laten condenseren op een koud oppervlak. Daardoor kan deze energie worden ingezet om de kas te verwarmen via de buizen. Een veelbelovende techniek voor condensatie is de dauwpuntskoeling waarbij buitenlucht door bevochtiging extra koud wordt gemaakt en daarna langs de kaslucht wordt geleid. Dat systeem kan eenvoudig in een gevel worden ingebouwd. Een heel andere manier van ontvochtigen waarbij geen ventilatie nodig is, is het gebruik van de Next Generation techniek of door een zoutabsorber. De laatste biedt zelfs perspectieven op het gebruik van wind of zon als energiebron voor het ontvochtigen.

Energy savings through a greenhouse cover of insulation glass requires a large investment. In the search for a cheaper alternative by a consortium of companies consisting of VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems a Glass-Film-greenhouse cover, better known as the 2SaveEnergy greenhouse concept was realized in summer 2014. The combination of clear glass with a diffuse ETFE film and a double screen mounted at a distance of only a few centimetres, in the year 2015, resulted in a low energy consumption and a better than expected good tomato production. During cultivation, the principles of the new cultivation methods were used. With respect to the common practice, the energy consumption was more than 50% lower at a minimum equal production. Die Realisierung von Energieeinsparungen mithilfe von Gewächshausdächern aus Isolierglas erfordert meist hohe Investitionen. Auf der Suche nach einer kostengünstigeren Alternative hat ein Konsortium der Unternehmen VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe und Boal Systems das Gewächshausdachkonzept ‘2SaveEnergy’ entwickelt und im Sommer 2014 realisiert. Die Kombination von Klarglas und diffuser ETFE-Folie als Bedachung mit einem doppelten Energieschirm mit einem Zwischenraumvon nur wenigen Zentimetern führte im Versuchsjahr 2015 zu einer guten Tomatenproduktion bei geringem Energieverbrauch. Die Produktion selbst erfolgte weitestmöglich nach den Grundsätzen der ‘Neuen Kulturverfahren’. Im Vergleich zur üblichen Praxis war der Energieverbrauch bei mindestens gleicher Produktion 50% geringer.

Energy savings through a greenhouse cover of insulation glass requires a large investment. In the search for a cheaper alternative by a consortium of companies consisting of VDH Plastic Greenhouses, Van der Valk Horti Systems, AGC Chemicals Europe en Boal Systems a Glass-Film-greenhouse cover, better known as the 2SaveEnergy greenhouse concept was realized in summer 2014. The combination of clear glass with a diffuse ETFE film and a double screen mounted at a distance of only a few centimetres, in the year 2015, resulted in a low energy consumption and a better than expected good tomato production. During cultivation, the principles of the new cultivation methods were used. With respect to the common practice, the energy consumption was more than 50% lower at a minimum equal production.