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Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten
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.
The role of strigolactones and the fungal microbiome in rice during drought adaptation
Andreo Jimenez, Beatriz - \ 2017
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789463437028 - 205
drought resistance - drought - abiotic injuries - rice - oryza sativa - plant-microbe interactions - nutrient uptake - defence mechanisms - hormones - fungi - genes - droogteresistentie - droogte - abiotische beschadigingen - rijst - plant-microbe interacties - voedingsstoffenopname (planten) - verdedigingsmechanismen - hormonen - schimmels - genen
Rice is the most important food crop in the world, feeding over half the world’s population. However, rice water use efficiency, defined by units of yield produced per unit of water used, is the lowest of all crops. The aim of this thesis was to study the effect of plant hormones and the root microbiome on drought tolerance in rice. The new plant hormone, strigolactone, was shown to be upregulated under drought and to regulate drought tolerance in interaction with the drought-hormone abscisic acid. Using a large collection of rice genotypes grown in the field, we showed that the composition of the root associated fungal microbiome is determined by the rice genotype and can contribute to drought tolerance.
Adapting to change : on the mechanism of type I-E CRISPR-Cas defence
Künne, Tim A. - \ 2017
University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - Wageningen : Wageningen University - ISBN 9789463436649 - 239
immunity - defence mechanisms - rna - bacteria - escherichia coli - analytical methods - priming - immuniteit - verdedigingsmechanismen - bacteriën - analytische methoden - zaadbevochtiging
Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery.
Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times.
Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis.
Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders.
In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.
In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming.
Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.
Nieuwe methoden in plantversterking tegen ondergrondse ziekten en plagen : gebruik van lokaal aanwezige antagonisten uit groeisubstraat en plant
Wurff, Andre van der; Streminska, M.A. ; Boer, F.A. de; Bruyant, Ewen ; Cuesta Arenas, Y. - \ 2016
Bleiswijk : Wageningen University & Research, BU Glastuinbouw (Rapport GTB 1427) - 42
kasgewassen - kassen - glastuinbouw - gewasbescherming - plantenziekteverwekkers - antagonisten - bacteriën - verdediging - verdedigingsmechanismen - endofyten - pythium ultimum - meloidogyne - rhizobium rhizogenes - fusarium oxysporum - fusarium - micro-organismen - proteïnaseremmers - bèta-glucanase - chitinase - greenhouse crops - greenhouses - greenhouse horticulture - plant protection - plant pathogens - antagonists - bacteria - defence - defence mechanisms - endophytes - microorganisms - proteinase inhibitors - beta-glucanase
Within this project, two new methods of the control of pathogens were investigated. New methods are: a. use of local bacteria that are isolated from soils or growing substrates; and b. bacteria that are present within the plant. By using local antagonists, already present in growing substrates or within plants in the greenhouse, the chance is higher that antagonist can be successfully used against local pathogens. Bacteria that were isolated from soil of growers were assessed on their antagonistic potential in lab trials against Pythium ultimum, Meloidogyne spp. and Rhizobium rhizogenes and Fusarium solani and F. oxysporum. Finally, the effect of antagonists against Pythium and Meloidogyne was evaluated in pot trials in the greenhouse. All antagonists diminished brown colourization symptoms in stems caused by Pythium. Alcaligenues sp., Bacillus sp. en two unidentified species diminished root damage and Alcaligenues sp. as well as Bacillus also reduced also the number of offspring of Meloidogyne spp. within the roots. The use of local microorganisms offers a sustainable-, new solution to control pathogens. In this study, it was shown that Proteinase inhibitor 2 (PINII), Glucanase (LeGluB) and Chitinase (LeChi3) can be used in tomato to investigate the influence of antagonists or endophytes on the plant defence.
Volatile-mediated interactions in the rhizosphere
Cordovez da Cunha, Viviane - \ 2016
University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): V.J. Carrion. - Wageningen : Wageningen University - ISBN 9789462579019 - 219
rhizosphere bacteria - rhizosphere fungi - microbial interactions - volatile compounds - suppressive soils - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - growth stimulators - biological control - defence mechanisms - genomics - transcriptomics - rizosfeerbacteriën - rizosfeerschimmels - microbiële interacties - vluchtige verbindingen - ziektewerende gronden - groeistimulatoren - biologische bestrijding - verdedigingsmechanismen - genomica - transcriptomica
Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions.
To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.
In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance.
Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack.
In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.
The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis
Chong Aguirre, Pablo A. - \ 2016
University. Promotor(en): Gert Kema; Pedro Crous. - Wageningen : Wageningen University - ISBN 9789462578791 - 289
pseudocercospora - plant pathogenic fungi - fungicides - pesticide resistance - defence mechanisms - genetic diversity - genetic mapping - sensitivity - musa - bananas - fungal diseases - disease control - plantenziekteverwekkende schimmels - fungiciden - resistentie tegen pesticiden - verdedigingsmechanismen - genetische diversiteit - genetische kartering - gevoeligheid - bananen - schimmelziekten - ziektebestrijding
Pseudocercospora fijiensis causes black Sigatoka disease of banana. It is one of the most damaging threats of the crop requiring excessive fungicide applications for disease control as the major export “Cavendish” clones are highly susceptible. The consequence of this practice is the reduced efficacy of disease management strategies due to increasing levels of fungicide resistance. In this thesis the history and current practices of black Sigatoka disease management as well as the underlying mechanisms of fungicide resistance to a major group of fungicides are described. We discovered that both target site mutations and promotor insertions are crucial for modulating sensitivity. The more insertions, the higher the expression of the gene and the more resistant the strain. Using this information, we advocate modern monitoring techniques and improved disease control strategies as well as the urgent need for innovative banana breeding to develop resistant varieties for a sustainable global banana production.
Plant-mediated insect interactions on a perennial plant : consequences for community dynamics
Stam, J.M. - \ 2016
University. Promotor(en): Marcel Dicke, co-promotor(en): Erik Poelman. - Wageningen : Wageningen University - ISBN 9789462578647 - 254 p.
016-3976 - perennials - brassica oleracea - defence mechanisms - glucosinolates - insect pests - herbivory - plutella xylostella - mamestra brassicae - pieris rapae - herbivore induced plant volatiles - animal communities - population dynamics - overblijvende planten - verdedigingsmechanismen - glucosinolaten - insectenplagen - herbivorie - herbivoor-geinduceerde plantengeuren - diergemeenschappen - populatiedynamica
Plants interact with many organisms around them, and one of the most important groups that a plant has to deal with, are the herbivores. Insects represent the most diverse group of herbivores and have many different ways of using the plant as a food source. Plants can, however, defend themselves against those herbivores, either by constitutive defences, or by traits that are induced upon herbivory. These traits, such as the formation of more trichomes or the production of secondary metabolites, can deter an insect herbivore in its decision to eat from the plant, can be toxic, or otherwise hamper the insect to feed, grow or reproduce. The way a plant responds to herbivory is very specific, depending on the feeding mode or the species of the attacking insect. Furthermore, plant responses to dual herbivory differ from the sum of responses to each herbivore alone. Also the time and order at which multiple insects arrive on a plant, influence the plant’s response. Finally, plant species or populations can show different responses to herbivory. Altogether these factors result in a plant phenotype that the attacking herbivore has to deal with. In addition to the attacker, also subsequently arriving insects will be affected by a change in plant phenotype. Because plants and insects can respond to each other in a continuous chain of interactions, an herbivore early in the season can indirectly affect the later-season community composition through the induced plant response. However, we know only little about the consequences of a dual-herbivore induced plant phenotype for subsequent feeders, and ultimately, the effects on the assembly and dynamics of an insect community as a whole.
The aim of this thesis project was to study the consequences of feeding by multiple insects from the same plant, not only to a subsequent herbivore, but also to the dynamics of a whole insect community over the course of a growing season, and beyond. Furthermore, I studied how the order of herbivore arrival and timing of arrival affected both a next herbivore’s choice and performance in a greenhouse setting, as well as the development of the whole insect community in the field. In addition, I studied how plant populations vary in induced responses, have specific plant-mediated interactions among insect herbivores, and how long these induced responses influence the insect community and plant fitness. Finally, I identified non-additive effects of the history of insect attacks and plant ontogeny to the future insect community.
In the first chapter of this thesis, I introduce the study system. For this project I used several populations of the wild cabbage plant, Brassica oleracea. This is an herbaceous perennial plant that flowers from the second growing season onwards, and supports a large and diverse above-ground arthropod community of more than thirty different species. The plant belongs to the family of Brassicaceae, which is known for the biosynthesis of a group of secondary metabolites, the glucosinolates. These metabolites may deter insects, although some insect species use it as a feeding cue. Two specialist insect herbivores from different feeding guilds, the caterpillar of the diamondback moth Plutella xylostella and the cabbage aphid Brevicoryne brassicae, were used to study their effect as early-season inducer of plant responses either alone or in combination. The caterpillar of the generalist cabbage moth, Mamestra brassicae, was used in bioassays to assess the effects of the induced plant phenotype by single or dual herbivory. Furthermore, in three chapters (Chapters 4, 6 and 7) I have closely studied the composition of the naturally occurring insect community throughout the season for one or two years in a common-garden field setting. In the last of these three chapters, I used the caterpillars of the specialist cabbage white, Pieris rapae, to induce plants at different moments of their ontogeny, while excluding the insect community for varying periods of time by a net or exposing plants to their natural insect community.
In an elaborate literature review, I and my collaborators concluded in chapter 2 that plant responses to dual herbivory evoke different plant responses than the sum of each herbivore alone. This has consequences at all levels from arthropod community assembly to the choice and performance of individual insects. The mechanisms of plant responses to dual herbivory are found in gene expression, hormone production and other molecular processes within the plant. All these aspects of interactions between insects and plants occur and are connected at different time scales.
To follow up on the question how timing plays a role in dual herbivory, we varied the time between, as well as the order of arrival of aphids and/or caterpillars on a plant. We observed that both affected the preference and performance of a subsequently feeding caterpillar (Chapter 3). Mamestra brassicae performed better on plants with a longer time interval between the first and second feeder. Also in a field setting (Chapter 4), the order of herbivore arrival early in the season affected the insect community composition later in the season in two different years, likely through a chain of indirect insect interactions. In this field study, the plant population influenced the outcome of early-season herbivory to later community dynamics. In chapter 5 we found that three plant populations in response to simultaneous aphid and caterpillar attack differed in the expression of two genes that are important for the regulation of herbivore-induced responses. Also, the production of one of two important plant hormones, salicylic acid, responded differently to single or dual herbivory in a unique pattern for each of the plant populations. These different plant responses subsequently negatively affected a next caterpillar on the same plant; M. brassicae growth was impaired on plants which had been fed upon by both aphids and caterpillars, in comparison to control plants. These field and greenhouse studies thus show the implications of dual herbivory beyond effects in the plant; it affects subsequent herbivory, and through a chain of plant-mediated insect interactions, the dynamics of a whole insect community.
In the sixth chapter we show that variation in insect community dynamics can last beyond the moment that the insects were present, even across years. In this field study, the naturally occurring carnivore community influenced the carnivore community composition a year later. Importantly, the herbivore community affected plant fitness across years (but not within years). We propose that such legacy effects are mediated by plant traits, which vary upon insect induction in the first year, and affect the insect community in the next year.
Finally, the history of all insect attacks to a plant up until that moment shape the future insect community by influencing the colonisation of insect species on the plant. Moreover, also plant ontogeny plays a role in shaping the insect community; plant-mediated responses to herbivory at different plant ages resulted in different insect colonisation rates. The most important conclusion from this last data chapter (Chapter 7) is that the two processes, insect community history and plant ontogeny, are non-additive and affect the colonisation of insect species in the same (synergistic) or opposite (antagonistic) direction.
By framing my study results in a time line from minutes to months to years, I show in the general discussion (Chapter 8) that the consequences of dual herbivory for subsequently arriving insects are connected at different time scales. Plant responses to herbivory can occur within hours to days, which affect herbivore choices and performance in the following days and weeks. In their turn, variation of a few days in arrival time of insects may change how plants respond and prioritize their responses to insects throughout the rest of the season. The insects that subsequently arrive on a dual-herbivore induced plant may change the plant phenotype even further and through a chain of insect-plant interactions, the effects on the insects and the plant can last throughout the season, and even across seasons. Furthermore, various factors such as the species of the attacking insect and its feeding guild, the timing after previous attack and the plant age at which herbivory occurs, as well as the genotypic background of the plant, all affect the outcomes of dynamic insect-plant interactions.
The results presented in this thesis thus contribute to the knowledge and interpretation of plant interactions with multiple herbivores. As plants are seldom attacked by a single herbivore, this implies that we have to take into account that multiple herbivory is not the same as the additive effects of single herbivores, and that this has long-lasting consequences for the insect community and the plant. To further understand how plants and insects have adapted to such a dynamic environment, I suggest future research to focus even more on the kinetics of plant physiological responses to dual attack, and to aim at answering the question of how predictable insect communities on a plant really are.
Host-plant resistance to western flower thrips in Arabidopsis
Thoen, Manus P.M. - \ 2016
University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462578807 - 191
arabidopsis thaliana - host plants - insect pests - frankliniella occidentalis - defence mechanisms - pest resistance - genomics - genome analysis - host-seeking behaviour - optical tracking - data analysis - insect plant relations - waardplanten - insectenplagen - verdedigingsmechanismen - plaagresistentie - genomica - genoomanalyse - gedrag bij zoeken van een gastheer - optisch sporen - gegevensanalyse - insect-plant relaties
Western flower thrips is a pest on a large variety of vegetable, fruit and ornamental crops. The damage these minute slender insects cause in agriculture through feeding and the transmission of tospoviruses requires a sustainable solution. Host-plant resistance is a cornerstone of Integrated Pest Management (IPM). Plants have many natural defense compounds and morphological features that aid in the protection against herbivorous insects. However, the molecular and physiological aspects that control host-plant resistance to thrips are largely unknown.
A novel and powerful tool to study host-plant resistance to insects in natural populations is genome-wide association (GWA) mapping. GWA mapping provides a comprehensive untargeted approach to explore the whole array of plant defense mechanisms. The development of high-throughput phenotyping (HTP) systems is a necessity when large plant panels need to be screened for host-plant resistance to insects. An automated video-tracking platform that could screen large plant panels for host-plant resistance to thrips, and dissect host-plant resistance to thrips in component traits related to thrips behavior, was developed. This phenotyping platform allows the screening for host-plant resistance against thrips in a parallel two-choice setup using EthoVision tracking software. The platform was used to establish host-plant preference of thrips with a large plant population of 345 wild Arabidopsis accessions (the Arabidopsis HapMap population) and the method was optimized with two extreme accessions from this population that differed in resistance to thrips. This method can be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. EthoAnalysis, a novel software package was developed to improve the analyses of insect behavior. There were several benefits from using EthoAnalysis to analyze EthoVision data. The detailed event statistics that could be extracted from EthoAnalysis allows researchers to distinguish detailed differences in moving and feeding behavior of thrips. The potential of this additional information is discussed in the light of quantitative genetic studies.
Stress resistance was studied in the HapMap population on a total of 15 different biotic and abiotic stresses ranging from biotic stresses like insects and nematodes, to abiotic stresses like drought and salt. A multi-trait GWA study to unravel the genetic architecture underlying plant responses to the different stresses was performed. A genetic network in this study revealed little correlation between the plant responses to the different insect herbivores studied (aphids, whiteflies, thrips and caterpillars). For thrips resistance a weak positive correlation with resistance to drought stress and Botrytis, and a negative correlation with resistance to parasitic plants were observed. One of the surprising outcomes of this study was the absence of shared major QTLs for host-plant resistance and abiotic stress tolerance mechanisms. RESISTANCE METHYLATED GENE 1 (RMG1) was one of the candidate genes in this multi-trait GWA study that could be controlling shared resistance mechanisms against many different stresses in Arabidopsis. RMG1 is a nucleotide-binding site Leucine-rich repeat (NB-LRR) disease resistance protein and its potential relation to several resistance/tolerance traits was successfully demonstrated with T-DNA insertion lines.
The 15 stresses were used in a comparison with a metabolomics dataset on this Arabidopsis HapMap population. It was discovered that levels of certain aliphatic glucosinolates correlated positively with the levels of resistance to thrips. This correlation was further investigated with the screening of a RIL (Recombinant Inbred Line) population for resistance to thrips, several knockout mutants and the analysis of co-localization of GWA mapping results between glucosinolates genes and thrips resistance. In a GWA analysis, the C4 alkenyl glucosinolates that correlated the strongest with thrips resistance mapped to the genomic regions containing genes known to regulate the biosynthesis of these compounds. However, thrips resistance did not co-localize with any of the GSL genes, unless a correction for population stratification was omitted. Additional screening of a Cvi x Ler RIL population showed a QTL for thrips resistance on chromosome 2, but no co-localization with any known glucosinolate genes, nor with thrips resistance loci identified by GWA mapping. Knock-out mutants and overexpressors of glucosinolate synthesis genes could also not confirm a causal link between glucosinolates and resistance to thrips. It is possible that the crucial factors that control resistance to thrips may not have been present in sufficient quantities or in the right combinations in the mutants, RILs and NIL screened in this study. Alternatively, the correlation between thrips feeding damage and glucosinolate profiles could be based on independent geographical clines. More research should be conducted to assess which of these explanations is correct.
In the general discussion, the results from this thesis are discussed in a broader perspective. Some prototypes of new phenotyping platforms that could further aid screening for resistance to thrips in the future are presented. Natural variation in host-plant resistance to thrips is compared to the variation in host-plant resistance to aphids and caterpillars. The geographic distribution of host-plant resistance to thrips is not evident in the other insects, in line with the distribution of glucosinolate profiles and other climate factors. The chapter concludes with some suggestions for future research in the field of host-plant resistance to thrips.
Plant responses to multiple herbivory : phenotypic changes and their ecological consequences
Li, Yehua - \ 2016
University. Promotor(en): Marcel Dicke, co-promotor(en): Rieta Gols. - Wageningen : Wageningen University - ISBN 9789462578043 - 165
brassica oleracea - brevicoryne brassicae - aphidoidea - caterpillars - insect pests - pest resistance - defence mechanisms - phenotypes - insect plant relations - parasitoids - natural enemies - herbivore induced plant volatiles - plant-herbivore interactions - genetic variation - rupsen - insectenplagen - plaagresistentie - verdedigingsmechanismen - fenotypen - insect-plant relaties - parasitoïden - natuurlijke vijanden - herbivoor-geinduceerde plantengeuren - plant-herbivoor relaties - genetische variatie
This thesis explores whether aphid-infestation interferes with the plant response to chewing herbivores and whether this impacts performance and behaviour of individual chewing insect herbivores and their natural enemies, as well as the entire insect community. I investigated this using three wild cabbage populations (Brassica oleracea) that are known to differ in inducible secondary chemistry, to reveal whether patterns were consistent.
A literature review on recent developments in the field of plant interactions with multiple herbivores (Chapter 2) addressed how plant traits mediate interactions with various species of the associated insect community and their dynamics. In addition, the mechanisms underlying phenotypic changes in response to different herbivores were discussed from the expression of defence-related genes, phytohormones and secondary metabolites in plants to their effects on the performance and behaviour of individual insects as well as the entire insect community. In Chapter 3, I investigated the effects of early-season infestation by the aphid Brevicoryne brassicae on the composition and dynamics of the entire insect community throughout the season in a garden experiment replicated in two consecutive years. Aphid infestation in the early season only affected a subset of the community, i.e. the natural enemies of aphids, but not the chewing herbivores and their natural enemies. Moreover, the effects were only significant in the first half (June & July), but waned in the second half of the season (August & September). The effect of aphid infestation on the community of natural enemies also varied among the cabbage populations. Chapter 4 investigated the effects of aphid infestation on plant direct defences against chewing herbivores in laboratory experiments by comparing the performance of chewing herbivores and their parasitoids on aphid-infested and aphid-free plants. The performance of the specialist herbivore Plutella xylostella and its parasitoid Diadegma semiclausum was better on plants infested with aphids than on aphid-free plants, whereas the performance of the generalist herbivore Mamestra brassicae and its parasitoid Microplitis mediator was not affected by aphid infestation. These results suggest that aphid induced changes in plant traits may differentially affect the performance of leaf-chewing herbivore species attacking the same host plant, and also varied among the cabbage populations. Chapter 5 examined the effects of B. brassicae aphid infestation on plant indirect defences against chewing herbivores. In a two-choice olfactometer bioassay, preference behaviour for volatiles emitted by plants infested with hosts alone and those emitted by plants infested with aphids and hosts was compared for D. semiclausum and M mediator, larval endoparasitoids of caterpillars of P. xylostella and M. brassicae, respectively. In addition, the headspace volatiles emitted by host-infested and dually-infested plants were collected and analyzed. Co-infestation with aphids differentially affected volatile-mediated foraging behaviour of the two parasitoid species in an infestation period-dependent manner. Diadegma semiclausum preferred dually infested plants over host-infested plants when aphids infested the plants for a short time period, i.e. 7 days, but the volatile preference of D. semiclausum was reversed when aphid infestation was extended to 14 days. In contrast, M. mediator consistently preferred volatiles emitted by the dually-infested plants over those emitted by host-infested plants. The patterns of preference behaviour of the two wasp species were consistent across the three cabbage populations. Interestingly, the emission rate of most volatile compounds was reduced in plants dually-infested with caterpillars and aphids compared to singly-infested with caterpillars. This study showed that aphid infestation increased plant indirect defences against caterpillars, but depended on the aphid infestation period and specific caterpillar-parasitoid association. We hypothesized a negative interference of aphid infestation on plant defences against chewing herbivores based on previously reported SA-JA antagonism. In Chapter 6, we assessed the activation of SA and JA signaling pathways in plants infested by both aphids (B. brassicae) and various caterpillar species (P. xylostella, M. brassicae and Pieris brassicae) in different time sequences by quantifying transcription levels of the SA- and JA-responsive marker genes, PR-1 and LOX respectively. The results did not provide support for SA-JA antagonism. Compared to single infestation with each of the herbivore species, dual infestation with aphid and caterpillars had no interactive effects on the transcription levels of the SA- and JA-responsive maker genes, regardless of the temporal sequence of aphid and caterpillar attack, or the identity of the attacking caterpillar species.
The findings of this thesis contribute to our understanding of plant responses to herbivory by insect species belonging to different feeding guilds and their ecological effects on other associated community members. Aphid infestation may interfere with plant direct and indirect defences against leaf-chewing herbivores at the individual species level, but the effects are species-specific and also depend on the infestation period of aphids. Early-season aphid infestation may further affect the composition of the insect community, but the effect is smaller influencing only a subset of the community compared to early infestation by chewing herbivores. The molecular mechanism underlying plant responses to both phloem-feeding and leaf-chewing herbivores are complex and require the investigation of a range of genes involved in JA- and SA-mediated defence signal transduction. Plant interact with multiple herbivores at different levels of biological organization ranging from the subcellular level to the individual and the community level, and an integrated multidisciplinary approach is required to investigate plant-insect interactions.
Plants under dual attack : consequences for plant chemistry and parasitoid behavior
Ponzio, C.A.M. - \ 2016
University. Promotor(en): Marcel Dicke, co-promotor(en): Rieta Gols. - Wageningen : Wageningen University - ISBN 9789462577718 - 191 p.
016-3954 - brassica nigra - insect pests - pieris brassicae - parasitoids - parasitoid wasps - cotesia glomerata - defence mechanisms - phytochemistry - herbivore induced plant volatiles - insect plant relations - animal behaviour - multitrophic interactions - plant pathogenic bacteria - xanthomonas campestris - insectenplagen - parasitoïden - sluipwespen - verdedigingsmechanismen - fytochemie - herbivoor-geinduceerde plantengeuren - insect-plant relaties - diergedrag - multitrofe interacties - plantenziekteverwekkende bacteriën
Though immobile, plants are members of complex environments, and are under constant threat from a wide range of attackers, which includes organisms such as insect herbivores or plant pathogens. Plants have developed sophisticated defenses against these attackers, and include chemical responses such as the production of and emission of volatile compounds, which can be used by natural enemies of the herbivores to locate herbivore-infested plants. While the production and use of induced volatiles by foraging natural enemies has been well studied in a single attacker/natural enemy combination, in natural situations it is common for a plant to be challenged by multiple attackers. However there is little knowledge about what happens during multiple attack, especially when one of the secondary attackers is a plant pathogen.
The aim of this thesis was to explore how dual attack modifies plant chemistry, and how changes in the emitted volatile blends then affect the behavior of foraging parasitoid, with a strong focus on the effects of non-host herbivore density and plant pathogen challenge. This study focused on the system consisting of black mustard plants (Brassica nigra), the large cabbage white butterfly, Pieris brassicae, and its larval parasitoid, Cotesia glomerata. Butterfly eggs, Brevicoryne brassicae aphids and the plant pathogen Xanthomonas campestris were used as secondary attackers. The results presented in the thesis showed that C. glomerata wasps are attracted to host-infested plants, irrespective of the presence and identity of the non-host attacker. However, when the responses of several parasitoid species to volatiles of plants infested with Pieris and/or aphids were compared, the wasp species were not equally affected and aphid infestation altered, in a density-dependent manner, the foraging behavior of all three species. In terms of volatiles, while differences in induced blends could be seen between individual attackers, these effects disappeared when plants were subjected to dual attack with caterpillars, and in the case of infestation with different aphid densities, non-linear volatile responses were revealed. Furthermore, the effects of dual attack by aphids and caterpillars were present at the level of leaf chemistry. Single and dual herbivory, as well as aphid density, led to metabolome-wide effects, driven mainly by changes in glucosinolates, sugars and antioxidant-related metabolites. The effect of pathogen challenge was further assessed by comparing the effects of virulent and avirulent pathovars, and results showed that both virulence and disease severity strongly affected the induced plant volatile blend. Remarkably, C.glomerata wasps were strongly attracted to volatiles of all the pathogen-challenged treatments, even in the absence of hosts.
The data presented in this thesis contribute to our understanding of how dual attack affects the chemistry of B. nigra plants, and modifies plant interactions with the natural enemies of attacking herbivores. This work reveals that plant pathogen challenge can affect volatile-mediated tritrophic interactions, and shows that focusing only on general species-specific effects of dual attack is too simplistic of an approach. The outcomes of this thesis contribute to our understanding of how plants modulate their defense responses against multiple attackers.
Unraveling molecular mechanisms underlying plant defense in response to dual insect attack : studying density-dependent effects
Kroes, A. - \ 2016
University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462577756 - 265 p.
016-3953 - arabidopsis thaliana - insect pests - herbivory - pest resistance - defence mechanisms - insect plant relations - molecular plant pathology - density - insectenplagen - herbivorie - plaagresistentie - verdedigingsmechanismen - insect-plant relaties - moleculaire plantenziektekunde - dichtheid
In the field, plants suffer from attack by herbivorous insects. Plants have numerous adaptations to defend against herbivory. Not only do these defense responses reduce performance of the feeding herbivore, they also result in the attraction of natural enemies of herbivores.
The majority of studies investigating plant-insect interactions addressed mainly the effects of attack by a single herbivore species on induced plant defenses. However, because plants are members of complex communities, plants are exposed to different insect attackers at the same time. Moreover, attacks by different herbivores interact at different levels of biological organization, ranging from the level of gene expression, phytohormone production and biochemical changes up to the individual level. Effects of plant responses to feeding by two or more herbivore species simultaneously might cascade through the community and thereby affect insect community composition.
The induction of plant defense responses is regulated by a network of signaling pathways that mainly involve the phytohormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET). The signaling pathways of the two phytohormones SA and JA interact antagonistically, whereas JA and ET signaling pathways can interact both synergistically and antagonistically in regulating plant defense responses. In general, JA-mediated signaling underlies defense responses against leaf-chewing herbivores, such as caterpillars, whereas phloem-feeding insects, such as aphids, mainly induce SA-regulated defenses.
When caterpillars and aphids simultaneously feed on the same host plant, crosstalk between phytohormonal signaling pathways may affect the regulation of plant defenses. Consequently, multiple insect herbivores feeding on plants interact indirectly through plant-mediated effects. Studies investigating molecular mechanisms underlying interference by multiple attacking insects with induced plant defenses will benefit studies on the ecological consequences of induced plant responses.
The aim of this thesis was to elucidate molecular mechanisms that underlie plant-mediated interactions between attacking herbivores from different feeding guilds, namely Brevicoryne brassicae aphids and Plutella xylostella caterpillars.
Because herbivore density affects the regulation of plant defense responses, it may also influence the outcome of multiple insect-plant interactions. To study if modulation of induced plant defenses in response to dual insect attack depends on insect density, plants were infested with two densities of aphids.
Responses of Arabidopsis thaliana plants to simultaneous feeding by aphids and caterpillars were investigated by combining analyses of phytohormone levels, defense gene expression, volatile emission, insect performance and behavioral responses of parasitoids. To better predict consequences of interactions between plants and multiple insect attackers for herbivore communities, the regulation of defense responses against aphids and caterpillars was also studied in the ecological model plant wild Brassica oleracea.
Transcriptomic changes of plants during multiple insect attack and their consequences for the plant’s interactions with members of the associated insect community take place at different time scales. Direct correlation of transcriptomic responses with community development is, therefore, challenging. However, detailed knowledge of subcellular mechanisms can provide tools to address this challenge.
One of the objectives of this thesis, therefore, was to investigate the involvement of phytohormonal signaling pathways and their interactions during defense responses against caterpillars or aphids at different densities, when feeding alone or simultaneously on the model plant A. thaliana. The studies show that aphids at different densities interfere in contrasting ways with caterpillar-induced defenses, which required both SA- and JA-signal-transduction pathways. Transcriptional analysis revealed that expression of the SA transcription factor gene WRKY70 was differentially affected upon infestation by aphids at low or high densities. Interestingly, the expression data indicated that a lower expression level of WRKY70 led to significantly higher MYC2 expression through SA-JA crosstalk. Based on these findings, it is proposed that by down-regulating WRKY70 expression, the plant activates JA-dependent defenses which could lead to a higher resistance against aphids and caterpillars.
Plutella xylostella caterpillars also influenced plant defense responses when feeding simultaneously with aphids. Caterpillar feeding affected aphid-induced defenses which had negative consequences for aphid performance. Induction of both ET- and JA-mediated defense responses is required for this interference. Moreover, aphid density also played an important role in the modulation by P. xylostella of aphid-induced defenses: P. xylostella caterpillars induced changes in levels of JA and its biologically active from, JA-Ile, only when feeding simultaneously with aphids at a high density.
To study the overall effect of dual herbivory on induced plant defenses, not only interference with induced direct defense, but also with induced indirect defenses was addressed in A. thaliana. We found a significant preference of the aphid parasitoid Diaeretiella rapae for volatiles from aphid-infested A. thaliana wild-type plants and ein2-1 (ET-insensitive) mutants. Interestingly, simultaneous feeding by P. xylostella caterpillars on wild-type plants increased D. rapae’s preference for odors from aphid-infested plants. However, upon disruption of the ET-signaling pathway, D. rapae did not distinguish between ein2-1 mutants infested by aphids or by both aphids and caterpillars. This showed that intact ET signaling is needed for caterpillar modulation of the attraction of D. rapae parasitoids.
On the other hand, attraction of the caterpillar parasitoid Diadegma semiclausum to volatiles emitted by A. thaliana plants simultaneously infested by caterpillars and aphids was influenced by the density of the feeding aphids. Biosynthesis and emission of the terpene (E,E)-α-farnesene could be linked to the observed preference of D. semiclausum parasitoids for the HIPV blend emitted by plants dually infested by caterpillars and aphids at a high density, compared to dually infested plants with a low aphid density.
Transcriptomic changes in the response of A. thaliana wild-type plants to simultaneous feeding by P. xylostella caterpillars and B. brassicae aphids compared to plants infested by P. xylostella caterpillars alone were assessed using a microarray analysis. I particularly addressed the question whether the transcriptomic response to simultaneously attacking aphids and caterpillars was dependent on aphid density and time since initiation of herbivory. The data show that in response to simultaneous feeding by P. xylostella caterpillars and B. brassicae aphids the number of differentially expressed genes was higher compared to plants on which caterpillars had been feeding alone. Additionally, specific genes were differentially expressed in response to aphids feeding at low or high density. Cluster analysis showed that the pattern of gene expression over the different time points in response to dual infestation was also affected by the density of the attacking aphids. These results suggest that insects attacking at a high density cause an acceleration in plant responses compared to insects attacking at low density.
As a next step in the study of multiple interacting herbivores, I studied whether plant responses to dual herbivory have consequences for the performance of a subsequently arriving herbivore, Mamestra brassicae caterpillars. The ecological consequences of plant responses to dual herbivory cascading into a chain of interactions affecting other community members have remained unstudied so far. We used wild B. oleracea plants to evaluate dual herbivore-induced plant adaptations for subsequent herbivory. We found that simultaneous feeding by P. xylostella and B. brassicae resulted in different plant defense-related gene expression and differences in plant hormone levels compared to single herbivory, and this had a negative effect on subsequently arriving M. brassicae caterpillars. Differential induction of JA-regulated transcriptional responses to dual insect attack was observed which could have mediated a decrease in M. brassicae performance. The induction of plant defense signaling also affected both P. xylostella and B. brassicae performance. This study further helps to understand herbivore community build-up in the context of plant-mediated species interactions.
Altogether, findings from this thesis reveal a molecular basis underlying plant responses against multiple herbivory and provide insight in plant-mediated interactions between aphids and caterpillars feeding on plants growing in the field or used in agriculture.
Plant verdedigt zich vaak te goed tegen lichtstress : vrije radicalen, schadelijk maar ook nuttig
Kaiser, M.E. ; Heuvelink, E. ; Kierkels, T. - \ 2016
Onder Glas 13 (2016)3. - p. 12 - 13.
horticulture - greenhouse horticulture - pot plants - anthurium - light intensity - defence mechanisms - adverse effects - shade plants - antioxidants - tuinbouw - glastuinbouw - potplanten - lichtsterkte - verdedigingsmechanismen - nadelige gevolgen - schaduwplanten - antioxidanten
Een plant heeft te maken met voortdurend wisselende lichtniveaus die soms zodanig hoog kunnen oplopen dat ze schade veroorzaken. Er bestaat een uitgebreid stelsel van verdedigingslinies op alle niveaus: blad, cel, celorganen en moleculair niveau. Soms is de voorbereiding op mogelijke lichtschade zo goed dat het productie kost.
Omgevings- en gewasconditie essentieel bij bestrijding meeldauw : Telers wachten met smart op effectieve beheerstrategieën.
Hofland-Zijlstra, Jantineke - \ 2016
horticulture - greenhouse horticulture - cut flowers - pot plants - vegetables - gerbera - tomatoes - kalanchoe - fungal diseases - mildews - environmental factors - defence mechanisms
geJantineke hofland bij de jonge gerberaplanten in een klimaatkast: "In de startblokken voor een nieuwe proef met meeldauw"
Arriving at the right time : a temporal perspective on above-belowground herbivore interactions
Wang, Minggang - \ 2016
University. Promotor(en): Wim van der Putten, co-promotor(en): T.M. Bezemer; A. Biere. - Wageningen : Wageningen University - ISBN 9789462578142 - 174 p.
herbivores - aboveground belowground interactions - herbivory - defence mechanisms - roots - leaves - mycorrhizas - population dynamics - soil biology - herbivoren - boven- en ondergrondse interacties - herbivorie - verdedigingsmechanismen - wortels - bladeren - mycorrhizae - populatiedynamica - bodembiologie
Regulation of cucumber (Cucumis sativus) induced defence against the two-spotted spider mite (Tetranychus urticae
He, J. - \ 2016
University. Promotor(en): Harro Bouwmeester; Marcel Dicke, co-promotor(en): Iris Kappers. - Wageningen : Wageningen University - ISBN 9789462576810 - 211 p.
cucumis sativus - cucumbers - induced resistance - plant pests - tetranychus urticae - mites - defence mechanisms - herbivore induced plant volatiles - herbivory - metabolomics - terpenoids - genomics - komkommers - geïnduceerde resistentie - plantenplagen - mijten - verdedigingsmechanismen - herbivoor-geinduceerde plantengeuren - herbivorie - metabolomica - terpenen - genomica
Plants have evolved mechanisms to combat herbivory. These mechanisms can be classified as direct defences which have a negative influence on the herbivores and indirect defence that attracts natural enemies of the attacking herbivores. Both direct and indirect defences can be constantly present or induced upon attack. This study, using cucumber (Cucumis sativus) and the two-spotted spider mite (Tetranychus urticae) as model, aimed to reveal the molecular mechanisms underlying the induced defence during herbivory, with emphasis on transcriptional changes and the involved TFs, the enzymatic function of the genes associated with volatile biosynthesis, and their promoters which regulate their expression.
Supplementary data: Combined biotic and abiotic stress resistance in tomato
Kissoudis, C. ; Chowdhury, Rawnaq ; Heusden, A.W. van; Wiel, C.C.M. van de; Finkers, H.J. ; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Abiotic and biotic stress factors are the major constrains for the realization of crop yield potential. As climate change progresses, the spread and intensity of abiotic as well as biotic stressors is expected to increase, with increased probability of crops being exposed to both types of stress. Shielding crops from combinatorial stress requires a better understanding of the plant’s response and its genetic architecture. In this study, we evaluated resistance to salt stress, powdery mildew and to both stresses combined in tomato, using the S. habrochaites LYC4 introgression line (IL) population. The IL population segregated for both salt stress tolerance and powdery mildew resistance. Using SNP array marker data, QTLs were identified for salt tolerance as well as Na+ and Cl- accumulation. Salt stress increased the susceptibility of the population to powdery mildew in an additive manner. Phenotypic variation for disease resistance was reduced under combined stress as indicated by the coefficient of variation (CV). No correlation was found between disease resistance and Na+ and Cl- accumulation under combined stress Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance. These findings increase our understanding of the genetic regulation of responses to abiotic and biotic stress combinations and can provide leads to more efficiently breeding for tomatoes and other crops with a high level of disease resistance while maintaining their performance in combination with abiotic stress.
Supplementary data: Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and mechanism of resistance
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Stress conditions in agricultural ecosystems can occur in variable intensities. Different resistance mechanisms to abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100 and 150mM NaCl) on powdery mildew (PM) resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as isogenic lines carrying the PM resistance genes Ol-1 (associated with slow Hypersensitivity Response; HR), ol-2 (a mlo mutant associated with papilla formation) and Ol-4 (a R gene associated with fast HR). PM resistance was affected by salt stress in a genotype and stress intensity dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied with accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced disease symptoms. Na+ and Cl- accumulation in the leaves was linearly related to the decreased pathogen growth under severe stress, suggesting a more direct role for the salt in suppressing PM growth. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress of the variety Moneymaker (MM) and the NIL Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes as well as of the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant’s performance under abiotic and biotic stress combination.
Supplementary data: Hormone signalling regulation of tomato response to combined biotic and abiotic stress
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Plant hormones are paramount to plant adaptation to changing environmental conditions and interactions with microorganisms. There is currently limited knowledge on their significance in the response to stress combination. Using near isogenic lines (NILs) that carry the Ol-1, ol-2 and Ol-4 gene for resistance to tomato powdery mildew caused by Oidium neolycopersici, this study focused on the responses of these NILs to powdery mildew and salt stress combination. In these NILs, marker genes for monitoring hormonal pathways showed differential expression pattern upon powdery mildew infection. Further by crossing these NILs with tomato mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) the cross-talk among hormonal pathways was further investigated. Among the mutants, epinastic resulted in increased susceptibility of NIL-Ol-1 and breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition, effects that were more pronounced under combination with salt stress. On the other hand notabilis, resulting in H2O2 overproduction greatly reduced susceptibility of NIL-Ol-1 under combined stress accompanied however by heightened sensitivity to salt stress. Callose deposition reduction led to partial resistance breakdown in NIL-ol-2 which was reversed under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. We discuss the critical role that hormone signalling appears to have for the outcome of combined stress and powdery mildew in terms of resistance and plant fitness integrating observations from physiological, histochemical and gene expression analyses. These significant insights obtained extend our understanding of hormonal regulation of combined stress responses and can aid in narrowing down targets for improving crop performance under stress combinations.
Supplementary data: Roles and contribution of tomato WRKY genes to salt stress and powdery mildew resistance
Kissoudis, C. ; Gao, D. ; Pramanik, Dewi ; Birhanu, Mengistu ; Wiel, C.C.M. van de; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
WRKY is a transcription factor family unique to plants with diverse functions in defense pathways, abiotic stress tolerance and developmental programs. Family members are characterized by the conserved WRKY domain and significant sequence variation in the remainder of the protein, which is translated into distinct functions even for closely related genes. We utilized the extensive functional characterization of the Arabidopsis thaliana WRKY family to identify tomato homologues of Arabidopsis WRKY genes that are involved in defense responses (AtWRKY 11, 29, 48, 70 and 72). In total 13 tomato WRKY homologues were identified for these genes, of which 9 were successfully over-expressed, and 12 stably silenced via RNAi in transgenic tomato lines. The transgenic lines were evaluated for their response to salt stress, powdery mildew resistance and the combination of these stresses. Lines overexpressing SlWRKY11 and SlWRKY23, and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and improved salt tolerance. For SlWRKY11 and SlWRKY23 overexpression (OE) lines, this was accompanied by a moderate increase in oxidative stress tolerance. The SlWRKY6-OE line showed strongly improved salt stress tolerance, but a growth penalty under control conditions. Exceptional phenotypes were observed for the SlWRKY10-OE line (stunted growth) and the RNAi line SlWRKY23-RNAi (necrotic symptoms), but these phenotypes were partly restored to normal under salt stress. Both these lines exhibited increased resistance to powdery mildew, but this was compromised when the plants were put under salt-stress as well. Important functions for tomato WRKY genes were revealed in both the abiotic and biotic stress response and several genes should be further explored to elucidate their downstream regulatory functions that lead to increased stress tolerance.
Genetic variation in plant chemistry : consequences for plant-insect interactions
Geem, Moniek van - \ 2016
University. Promotor(en): Wim van der Putten; J.A. Harvey, co-promotor(en): Rieta Gols. - Wageningen : Wageningen University - ISBN 9789462576681 - 141 p.
phytochemistry - plant composition - genetic variation - insect plant relations - interactions - defence mechanisms - soil biology - fytochemie - plantensamenstelling - genetische variatie - insect-plant relaties - interacties - verdedigingsmechanismen - bodembiologie
Plants form the basis of many food webs and are consumed by a wide variety of organisms, including herbivorous insects. Over the course of evolution, plants have evolved mechanisms to defend themselves against herbivory, whereas herbivorous insects have evolved counter-mechanisms to overcome these defences (a.k.a. co-evolutionary arms races). Plant-insect interactions are not restricted to plants and their herbivores (bi-trophic interactions), but also involve natural enemies of the herbivores such as parasitoids and predators (tri-trophic interactions). Plant quality can affect the quality of the host or prey for parasitoids and predators, respectively. In addition, other plant traits are important in providing shelter, alternative food sources, or chemical cues that can be used for host/prey location. Moreover, as plants reside in both soil and air, they mediate interactions between organisms above- and belowground through changes in plant quality. Plant quality is determined by secondary metabolites and morphological traits that may negatively affect the performance of insects, as well as by primary metabolites that plants produce in order to grow, develop and reproduce, which also provide essential nutrients for insects.
Natural plant populations often exhibit genetic variation in various plant traits that include, amongst others, primary and secondary chemistry. Genetic variation in plant defence traits, such as the production of secondary metabolites, can be under selection pressure from a suite of biotic and abiotic factors that vary in space and time. Herbivorous insects may encounter a wide range of plant metabolites because the total concentrations of primary and secondary metabolites and the concentrations of individual compounds vary between genetically different plants. Also as a consequence of genetic variation, plants can respond differently to herbivory in terms of induced defence chemistry and re-allocation of metabolites.
The main aim of this thesis was to study how genetic variation in plant chemistry affects (multi)trophic interactions between wild cabbage plants and associated insects, both above- and belowground. As a model system I used five naturally occurring populations of wild cabbage (Brassica oleracea) located in the Dorset area in the UK. These populations have been shown to genetically differ in their defence chemistry profiles even though they are located in relatively close proximity to each other. Wild cabbages belong to the Brassicaceae, a plant family that is characterized by the production of glucosinolates, a group of secondary metabolites. Together with the enzyme myrosinase they form the chemical defence system of Brassicaceous plants including wild cabbage. Glucosinolates and myrosinases are stored separately in plant tissues but upon tissue damage they come into contact with each other upon which the glucosinolates are hydrolysed into potentially toxic break down products. The wild cabbage populations used in this thesis differ in their total glucosinolate concentrations as well as in the expression of individual glucosinolates.
In chapter 1 I describe plant-insect interactions in a multi-trophic framework, including both the above- and belowground compartments. Genetic variation in plant traits is introduced as the main topic of this thesis, and I present the main aim and outline of my work.
In chapter 2 I discuss how aboveground-belowground interactions influence the evolution and maintenance of genetic variation in plant defence chemistry. I review literature on AG-BG interactions as selection pressures for genetic variation, discuss hypotheses about plant mediation of AG-BG interactions, identify gaps in our knowledge such as the influence of spatial-temporal variation in AG-BG interactions, and in the end present new data on genetic variation in secondary chemistry of wild cabbage and related species.
The co-evolutionary arms race between plants and insects has resulted in adaptations in herbivores to cope with plant defence traits. Some insect herbivore species concentrate or sequester secondary metabolites from their food plant and use them in defence against their own enemies. In chapter 3 I studied whether sequestration of glucosinolates by a specialist herbivore is an effective defence mechanism against a generalist predatory bug. I used the sequestering herbivore Athalia rosae as one prey species, and the non-sequestering herbivore Pieris rapae as the control prey species. I compared the performance of the predatory stink bug Podisus maculiventris on these two prey species. As an extra factor, the two prey species were each reared on three different wild cabbage populations to test if plant population would have an effect on the predator through the sequestering herbivore. I found no consistent effect of plant population on the performance of the predator, and prey species only marginally affected its performance. Based on the results I suggest that in some trophic interactions sequestration is not an effective defence mechanism but merely an alternative way of harmlessly dealing with plant secondary metabolites.
In addition to aboveground plant-insect interactions, belowground interactions were considered as well. To test whether the performance of the belowground specialist herbivore Delia radicum, of which the larvae feed on root tissues, was influenced by population-related variation in defence chemistry, I reared this species on the five wild cabbage populations (chapter 4). Chemical analyses of root tissues revealed that there were differences amongst the populations in plant primary (amino acids and sugars) and secondary (glucosinolates) chemistry, but this did not affect the performance of the root herbivore, suggesting that D. radicum is well adapted to a wide range of total concentrations and concentrations of individual metabolites.
Whereas in chapters 3 and 4 I only focused on one compartment (aboveground and belowground respectively), in chapter 5 I included both compartments in one experiment. I studied the effect of belowground herbivory by larvae of the root fly D. radicum on the performance of an aboveground multi-trophic food chain, and whether this effect differed among three wild cabbage populations. I found that belowground herbivory differentially affected the performances of a specialist aboveground herbivore, the diamondback moth Plutella xylostella, and its parasitoid, Cotesia vestalis, with the parasitoid being more affected than the herbivore. Their performance also differed between the wild cabbage populations, often in interaction with the presence/absence of the belowground herbivore. For both the above- and belowground herbivore I found correlations between performance and plant chemistry, which differed between the insect species and also between males and females.
In chapter 6 I discuss the results of my experiments in relation to other studies. I finish with a general conclusion about my work and provide some ideas for future studies that could contribute to our knowledge in the field of (multi)trophic above-belowground interactions with regard to genetic variation in plant chemistry.
In my thesis I show that genetic variation in plant chemistry can affect the outcome of above-belowground plant-insect interactions. Herbivores and higher trophic levels were differently affected by the wild cabbage populations, and this difference was also influenced by the location of herbivory (i.e. aboveground or belowground). In both chapter 4 and chapter 5 I found no strong, unidirectional links between plant chemistry and insect performance, suggesting that other metabolites may have played a role in the observed differential effects of the wild cabbage populations. I also show that sequestration of plant allelochemicals in some herbivores is an alternative way of harmlessly dealing with plant secondary metabolites instead of an effective defence mechanism against predators (chapter 3).