Pros and cons of virus infections in plants

~ An ecological perspective ~

Een wetenschappelijke proeve op het gebied van de Natuurwetenschappen, Wiskunde en Informatica

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen

op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het College van Decanen

in het openbaar te verdedigen op donderdag 28 mei 2009 om 12:00 uur precies

door

Maria Antonia Renée van Mölken geboren op 7 december 1979

te Sittard

Promotor: Prof. dr. J.C.J.M. de Kroon

Manuscriptcommissie:

Prof. dr. T.C.J. Turlings (University of Neuchâtel, Switserland) Dr. A. Biere (Netherlands Institute of Ecology, Heteren) Prof. dr. J.M. van Groenendael

ISBN: 978-90-9024202-6

Veur Mam

- On ne voit bien qu'avec le cœur. L'essentiel est invisible pour les yeux.-

Antoine de Saint-Exupéry

Chapter 1: General introduction.

Chapter 2: Clonal integration beyond resource sharing: implications for defence signalling and disease transmission in clonal plant networks.

Chapter 3: Virulence in clonal plants: conflicting selection pressures at work?

Chapter 4: Highways for internal virus spread: patterns of viral disease development in the stoloniferous

herb Trifolium repens.

Chapter 5: Can plant viruses maintain genotypic diversity in their hosts?

Chapter 6: Effects of shading on viral disease development in a clonal herb.

Chapter 7: Multiple stressors: shading and virus infection in Arabidopsis thaliana.

Chapter 8: Volatile emissions by virus-infected plants repel herbivores.

Chapter 9: Summarizing discussion

References

Nederlandse samenvatting

Dankwoord

List of publications

Curriculum Vitae

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LONG-DISTANCE TRAFFICKING IN CLONAL PLANT NETWORKS

Clonal growth often involves the formation of horizontal stems (rhizomes or stolons), which expand laterally from the parent plant. These stems produce genetically identical and functionally autonomous offspring individuals, called ramets. Internodes physically connect these ramets, and through branching and nodal stem rooting clonal plant individuals can develop into extensive net-work-like structures of various dimensions with multiple access points to soil-based resources. Vascular connections between ramets allow for the internal long-distance transport of resources following source-sink gradients. Sharing resources such as carbohydrates (Chapman et al., 1992), water (de Kroon et al., 1996) and mineral nutrients (Abrahamson et al., 1991) promotes growth and survival in a wide range of stressful and heterogeneous environments (van Groenendael & de Kroon, 1990; Stuefer et al., 1994, 1996).

Physiological integration in ramet networks of clonal plants also allows for the internal distribution of non-resource substances, for instance the fast and reliable spread of systemic defence signals after local herbivore attacks (Gómez & Stuefer, 2006). In spite of its evident advantages, however, clonal integration bears strong risks. It has long been suggested that physiological integration between ramets may facilitate the internal spread of potentially harmful toxins and pathogens (Cook, 1985; Silander, 1985; Eriksson & Jerling, 1990, Stuefer et al., 2004 and references therein), but empirical evidence for this contention remains scarce. Interconnected ramets of clonal plants may be especially vulnerable for systemic pathogen infection, as physical connections between ramets could serve as internal highways allowing pathogens to spread among connected ramets and to disperse rapidly within clonal ramet populations (Cook, 1985; Eriksson & Jerling, 1990).

Apart from the inherent risks of vascular connections for pathogen in-fection, the potentially very long life span of many clonal plant individuals may increase the likelihood of infections in this group of plants. It is conceivable that the majority of clonal plants become infected by single or multiple patho-gens (Silander, 1985, and references therein) at some point in their lifetime. Single and multiple infections are very likely to take place if host plants grow in a pathogen prone environment for extended periods of time (Van Baalen & Sabelis, 1995). After successful infection, viruses and other systemic patho-gens may readily infect successive ramet generations of clonal plants, thereby establishing very long lasting, and possibly life-long infections (Silander, 1985; Stuefer et al., 2004; van Mölken & Stuefer, 2008).

VIRUS INFECTIONS

Viruses represent an important group of plant pathogens. Studies on the inci-dence of virus infections, on the severity of disease symptoms and on patterns of viral spread in natural system are rare. However, several studies report high virus incidence in natural plant populations. MacClement and Richards (1956) for instance analysed virus presence in various plant species originating from different habitats and reported that 45% of all species were infected by vi-ruses, and infection levels varied between 10-100% of all individuals of a given species. In another study, 28% of the weed species tested in a melon field were infected with Cucumber Mosaic Virus (cited by Friess & Maillet, 1996), with infection rates ranging from 1-90% of individuals. These results provide some evidence for the generally-accepted notion that virus infections of may be a very general and wide-spread feature of many natural plant communities (Balachandran et al., 1997).

Viruses are responsible for numerous plant diseases with effects rang-ing from very mild to lethal (Gibbs & Harrison, 1976; Hull, 2004). Viruses can dramatically decrease host performance and fitness and cause enormous eco-nomic losses in a wide variety of commercially important plants (Hull & Davies, 1992; Bosque-Pérez et al., 1998; Strange & Scott, 2005). A broad range of qualitatively and quantitatively different virus effects are reported: many vi-ruses cause a decrease in photosynthetic activity (Chia & He, 1999; Fu-nayama & Terashima, 1999; Sampol et al., 2003), while others alter resource allocation patterns to different organs and functions (García-Guzmán & Bur-don, 1997) or affect hormone levels of their hosts (Clarke et al., 1999). Virus infection may lead to a deformation of leaves (Gibbs & Harrison, 1976), re-duced growth rates (Jones, 1992; Potter, 1993; Piqueras, 1999) and changes in the growth form of plants (Piqueras, 1999).

In general, pathogens are believed to exert strong selection pressure on plants (Jarosz & Davelos, 1995) and to have the potential of shaping plant populations and communities (Dobson & Crawley, 1994; Godfree et al., 2007; Bradley et al., 2008). However, the ecological relevance of these effects has received limited attention, and the interface between virology and ecology is a newly emerging field. Recently, efforts have been made to evaluate the signifi-cance of pathogen infections for ecological and evolutionary processes. For instance, the effects of virus infection on competitive strength and plant sur-vival in ecological relevant settings have been investigated (Malmstrom et al., 2006).

GENOTYPIC VARIATION

While the ecological aspects of plant-virus interactions are beginning to be unravelled on the level of host plant species, the effects of virus infection may vary between genetically distinct host individuals. Genotypic diversity in itself is essential for the maintenance of species in natural environments (Hedrick et al., 1976; Gillespie & Turelli, 1989; Vellend, 2006) and is the primary substrate for natural selection to act on (Fisher, 1958; Endler, 1986). Genotype-specific responses to environmental conditions (for instance virus prone environments) enable the expression of different morphological or physiological phenotypes in diverse environments (Conover & Schultz, 1995; Via et al., 1995; Zhi-votovsky et al., 1996; Pigliucci, 2005; Fordyce, 2006). Phenotypic plasticity enables organisms to operate successfully in a range of environments. Diseases have been identified as a possible key factor for the mainte-nance of genotypic variation in plant populations (Haldane, 1949; Burdon, 1987; Kirchner & Roy, 2001; Summers et al., 2003; Bradley et al., 2008). As a result of genotype-specific responses to viral infections (GxE interactions), viruses may exert variable selection on their hosts. Viruses may thus play a crucial, yet largely unknown role (Malmstrom et al., 2005) in shaping micro-evolutionary processes and affecting patterns of genotypic diversity in plant populations (Gilbert, 2002; Burdon et al., 2006).

MULTIPLE STRESSORS

Misfortunes never come singly. Under natural conditions plants are continu-ously exposed to a multitude of abiotic and biotic stresses to which they need to produce appropriate responses in order to survive. These responses can be energetically costly and resource investments in one response may influence the expression of functional responses to other stressors (Izaguirre et al., 2006). Similarly, interconnected physiological processes and shared molecular pathways may prevent the concurrent expression of plant responses to multi-ple stress factors. Due to such ecological and physiological trade-offs plants have to prioritize their actions against multiple challenges. Although simultane-ous stress by abiotic and/or biotic factors is probably a universal aspect of natural systems, plant responses to multiple challenges have only rarely been studied in an integrative way (Izaguirre et al., 2006). Many stoloniferous plants grow predominantly in a horizontal direction and can hence easily be shaded by neighbouring plants with vertically growing stems. In addition, owing to the long life span of individual genotypes, clonal plants face a high risk of virus infection (Van Baalen & Sabelis, 1995; Stuefer

et al., 2004; Van Mölken & Stuefer, 2008). For these reasons virus infections may often coincide with shading events in clonal plants. As a consequence of the possible trade-offs mentioned above, investment in shade-avoidance re-sponses may be realized at the expense of defence expression, and vice versa, which could potentially lead to serious consequences for plant perform-ance. On the other hand, it is conceivable that low light conditions decrease the effects of virus infections on photosynthesis, resulting in a weakened virus effect on plant growth and performance (Balchandran et al., 1997). Apart from abiotic stresses, plants may also face herbivore attacks in combination with virus infections. The likelihood of these two stress factors coinciding may be very high since many plant viruses are transmitted by her-bivorous insect vectors. Apart from the close association between viruses and their herbivore vectors, other plant-feeding insects are prone to consume virus-infected plants as well. An increasing body of literature provides insight in the activation of inducible defence pathways when plants suffer from simultaneous pathogen and herbivore attacks. Cross-talk between these pathways allows plants to regulate and prioritize the defence against biotic stressors. The out-come of the combined activation of defence pathways can be mutually syner-gistic or antagonistic, depending on the exact interaction (Koornneef & Piet-erse, 2008). However, the ecological functioning and implications of this triple interaction between plants, viruses and herbivores remains largely elusive.

OUTLINE AND AIMS OF THIS THESIS

The aim of this thesis is to increase insight in some of the ecological aspects of virus infections in plants. More specifically, I was interested in the conse-quences of virus infection on plant performance, the intra-specific variation therein, and role of other abiotic and biotic stressors. I used the clonal herb Trifolium repens L. (white clover) as host plant and White clover mosaic virus (WClMV) for virus infections in most of the experiments. In chapter 7, I used Arabidopsis thaliana as host plant and Turnip crinkle virus (TCV) for virus in-fection. Fungus gnats from the genus Bradysia spec. were used as herbivores in chapter 8. This thesis starts (chapter 2) with a general exploration of the proc-esses and phenomena related to non-resource sharing in clonal plants. The ecological implications of non-resource sharing are outlined for the transport of defence signals and distribution of viral diseases. We predict that viruses can cause long-term infections in clonal host plants. Clonal plants may provide viruses a storage possibility from which they may easily infect neighbouring

plants. The potentially long lifespan of clonal plants is likely to affect patterns of virulence evolution of viral pathogens (chapter 3). In a short note we dis-cussed two major theories of virulence evolution in the context of long-lived clonal plant - pathogen interactions. We suggest that increased pathogen viru-lence may be selected for in pathogens which infect clonally propagating plants. In chapter 4 I investigated the basic assumption that clonal integration allows for the internal spread of systemic pathogens by analyzing the patterns of viral disease development in groups of interconnected ramets. Building on these results, I investigated the consequences of systemic virus infections for plant performance (chapter 5). Besides the effects of virus infection on fitness-related traits I analyzed whether these effects vary between distinct plant genotypes and discussed the possible role of viruses for the maintenance of genotypic diversity in their hosts. One should realize that plants are bound to face virus infections in a range of environmental conditions. Therefore, I have tested the effects of virus infections on plant performance in different light con-ditions (chapters 6 & 7). The photosynthetic rate and genotype-specific plant responses to WClMV infection were analyzed in light and shade conditions (chapter 6) and I observed whether shading effects on virus infection was re-lated to virus amounts in the host plant (chapter 7). Besides changes in the abiotic environment, biotic factors are likely to influence the outcome of plant-virus interactions. I examined the interaction between herbivore attack and virus infection in a number of experiments (chapter 8). Virus-infected plants were infested with fungus gnat larvae and the combined effects of these stressors on plant performance were recorded. Furthermore, I explored whether virus-infected and non-infected plants differed in their attractiveness for adult fungus gnats. Possible positive effects of virus infections in plants were identified. The ecological and evolutionary relevance of the main results are outlined in the summarizing discussion (chapter 9).

ABSTRACT

Resource sharing between ramets of clonal plants is a well-known phenome-non, which allows stoloniferous and rhizomatous species to internally translo-cate water, mineral nutrients and carbohydrates from sites of high supply to sites of high demand. The mechanisms and implications of resource integra-tion in clonal plants have extensively been studied in the past. Vascular ramet connections are likely to provide an excellent means to share substances other than resources, such as systemic defence signals and pathogens. The aim of this paper is to propose the idea that physical ramet connections of clonal plants can be used (1) to transmit signals, which enable members of clonal plant networks to share information about their biotic and abiotic envi-ronments, and (2) to facilitate the internal distribution of systemic pathogens in clonal plant networks and populations. We will focus on possible mechanisms as well as on potential ecological and evolutionary implications of clonal inte-gration beyond resource sharing. More specifically, we will explore the role of physiological integration in clonal plant networks for the systemic transmission of direct and indirect defence signals after localized herbivore attack. We pro-pose that sharing defence induction signals among ramets may be the basis for an efficient early warning system, and it may allow for effective indirect de-fence signalling to herbivore enemies through a systemic release of volatiles from entire clonal fragments. In addition, we will examine the role of clonal in-tegration for the internal spread of systemic pathogens and pathogen defence signals within clonal plants. Clonal plants may use developmental mechanisms such as increased flowering and clone fragmentation, but also specific bio-chemical defence strategies to fight pathogens. We propose that clonal plant networks can act as stores and vectors of diseases in plant populations and communities and that clonal life histories favour the evolution of pathogens with a low virulence.

KEYWORDS: physiological integration; clonal growth; chemical information transfer; induced systemic resistance; systemic pathogen spread; defence signalling.

INTRODUCTION

Many clonal plant species form horizontal stems (rhizomes or stolons), which expand laterally from the parent plant and grow roots and new shoots at each of their nodes. This type of clonal growth leads to the formation of networks of physically interconnected, genetically identical, and functionally autonomous offspring individuals, called ramets. Physical connections between ramets al-low for the internal transport of substances such as water, carbohydrates and mineral nutrients between different parts of the clonal network. This phenome-non, termed physiological integration or resource sharing, has received rather broad attention in the last few decades, resulting in an extensive body of litera-ture dealing with the physiological mechanisms as well as the ecological impli-cations of resource integration in clonal plants. Since the pioneering work of Qureshi and Spanner (1971, 1973), and the seminal review by Pitelka and Ashmun (1985), clonal integration has attracted considerable attention from (eco)-physiologists (e.g. Chapman et al., 1991, 1992; Kemball & Marshall, 1995), ecologists (e.g. Shumway, 1995; Alpert, 1991, 1996; Evans, 1991, 1992; Stuefer, et al., 1994, 1996) and theoretical biologists (Caraco & Kelly, 1991; Oborny et al., 2000, 2001; Magori et al., 2003). These and numerous other studies have helped to elucidate the mechanisms, preconditions and constraints of carbohydrate, water and mineral nutrient sharing in clonal plants. They have also provided clear evidence for the ecological importance of clonal integration for enabling plant species to provide their (clonal) offspring with post-natal care, for avoiding the vulnerable life-cycle processes of seed germination and seedling establishment, and for allowing an efficient resource extraction from heterogeneous environments and the provisioning of internal support to damaged or resource-stressed ramets (Pitelka & Ashmun, 1985; Marshall, 1990; Marshall & Price, 1997). While it is clear that physical connections between ramets can be used to transport resources within clonal plants, much less is known about the pos-sible function of stolon and rhizome connections for the internal distribution of non-resource substances and agents, such as plant hormones, defence sig-nals, toxins, pathogens, and others. In spite of early suggestions that clonal plant networks may be used for sharing substances other than water, carbohy-drates and mineral nutrients (Cook, 1978; Pitelka & Ashmun, 1985), the mechanisms, dynamics and implications of clonal integration beyond resource sharing has never been considered in any comprehensive way so far. None-theless, it seems likely that vascular ramet connections are an efficient and suitable means to distribute information among interlinked members of a clonal

network, and that clonal fragments bear an inherent risk of intrusion and possi-ble rapid internal spread of systemic diseases. Since any form of clonal inte-gration (i.e., irrespective of what substance or agent is shared among con-nected ramets) makes use of the vascular system for long-distance transport within clonal plant networks, non-resource integration is likely to follow the same (or very similar) principles as ordinary resource integration, including known constraints and limits on clone-internal movement of substances along vascular pathways. Irrespective of their similarity with respect to mechanisms and basic principles, however, resource and non-resource sharing are likely to have very different implications for plant functioning, and for plant responses to the biotic and abiotic environment. The existing knowledge and broad experimental experience with re-source sharing in clonal plants can serve as an excellent basis for studying and predicting aspects of information sharing and disease spread in clonal plants. Plant traits and environmental features which are known to be of major importance for the dynamics and the extent of resource integration can also be expected to play a prominent role in non-resource sharing. Architectural and physiological features, such as the branching structure, the average distance between connected ramets, vascular sectoriality, source-sink dynamics, plant-internal water potential gradients, as well as life-history traits, such as the lon-gevity of ramets and ramet connections, are all likely to affect processes and phenomena that rely on resource as well as non-resource integration. Sectori-ality for instance, can prevent resources and anything else transported in the vascular system from reaching all parts of a plant (Watson & Casper, 1984; Vuorisalo & Hutchings, 1996; Stuefer, 1996). In clonal plants sectoriality can effectively isolate ramets or larger clone parts in terms of physiological integra-tion (Hay & Sackville-Hamilton, 1996; Price et al., 1996). Sectoriality can be regarded as “a structural constraint on the transport of resources in both the phloem and xylem that results in their distribution to restricted regions of the plant” (Marshall & Price, 1997). In addition, aspects of the (biotic and abiotic) environment can promote and constrain physiological integration in clonal plants. Spatial habitat hetero-geneity (e.g. in the availability of resources or other abiotic and biotic factors) has often been shown to promote or constrain the exchange of resources be-tween interconnected clone parts of clonal plants (Stuefer & Hutchings, 1994; Evans, 1991, 1992; Shumway, 1995). The longevity and transport capacity of connecting stolon or rhizome internodes is an example for plant-internal fea-tures that strongly affect the spatio-temporal degree and the quantitative amount of physiological integration and related processes in clonal plants

(Stuefer et al., 1998; Oborny et al., 2001). These and other plant-internal and external features can thus be expected to critically affect non-resource integra-tion and its implications in clonal plants. The aims of this paper are (1) to explore processes and phenomena which rely on the transmission of signals and other non-resource agents and substances through physical connections between ramets of a clonal plant, (2) to draw attention to possible ecological implications of non-resource integra-tion, and (3) to put forward expectations, predictions and hypotheses which can be tested in future research initiatives. Due to the scarcity of specific back-ground information, most of our predictions will be speculative in nature. We will focus on two main topics, namely the role of clonal integration (a) for dis-tributing defence induction signals in response to herbivory and (b) on mecha-nisms and implications of the systemic spread of pathogens and pathogen defence signals in clonal plant networks.

PLANT HERBIVORE DEFENCE

Plants have developed a broad array of mechanisms to cope with herbivores. Plant defence traits can be constitutive or inducible (i.e. plastic), and defence strategies may be direct or indirect. Constitutive defence traits are always ex-pressed, even at times and in environments where they are not needed(Wittstock & Gershenzon, 2002). Plastic defence traits are only expressed af-ter initial damage or they may be induced by external signals such as volatiles (Bruin et al., 1991; Karban & Baldwin, 1997; Dicke & Hilker, 2003). Defence mechanisms can have direct or indirect effects on herbivores. Direct defences consist of inducible changes in tissue quality, plant palatability and toxicity, or in plastic alterations of anatomical and morphological traits that reduce the herbivores’ preference for, or performance on the host plant (Karban & Bald-win 1997). Many plants can also make use of indirect defences against herbi-vores (e.g. damaged plants may release specific info-chemicals to attract the natural enemies of the herbivore; Karban & Baldwin, 1997; Takabayashi & Dicke, 1996). Inducible defences can either be exclusively expressed at the site of damage or they can also be activated in other, undamaged parts of the plant. The latter phenomenon, usually termed induced systemic resistance (ISR) is a common defence mechanism of plants against herbivores (Agrawal et al., 1999; Tollrian & Harvell, 1999). The complex signalling cascade leading to the local induction, systemic spread of the signal and non-local activation of de-fence traits is not yet fully understood (Roda & Baldwin, 2003). Nevertheless it

seems clear that the jasmonic acid pathway and phloem-transmitted signals play a key role in the systemic induction of defence genes after herbivore damage (e.g. proteinase inhibitor genes in Solanaceae; Stratmann, 2003; Thaler et al., 2002ab). ISR, though mechanistically not fully understood, has been described for numerous plant-herbivore systems (Karban & Baldwin, 1997; Agrawal et al., 1999; Tollrian & Harvell, 1999).

EARLY WARNING SYSTEM?

Herbivory triggers defensive responses in host plants showing systemic induc-ible resistance. Herbivore feeding (and in some cases also mechanical leaf damage) elicits a specific response, which leads to the production of a de-fence induction signal at the site of attack (Karban & Baldwin, 1997). This alerting signal then travels through vascular pathways (i.e. phloem vessels) from the site of damage to other parts of the plant. As a consequence undam-aged plant parts will also activate their defensive machinery. Though never tested explicitly, stolon and rhizome connections between ramets of clonal plants are most likely to act as pathways for systemic defence induction sig-nals (see Haukioja et al.,1990, Haukioja, 1991). If herbivores attack one or a few ramets of a clonal plant, a warning sig-nal may be produced at the site of damage and sent to other uninfested ramets through physiological clonal integration. This may allow undamaged ramets to receive a defence induction signal even if they are rather distant from the site of first herbivore attack, and to activate direct or indirect defence mechanisms before the herbivores actually arrive. ISR in clonal plants can be seen as a preemptive defence strategy of connected, uninfested ramets against impending herbivore attacks. Such a spatial alerting strategy is bene-ficial whenever the induction signal spreads faster than the herbivore, and if initial herbivore attack is a reliable cue for future damage in connected ramets of a clonal plant (Karban et al., 1999). Due to the potentially large size of clonal plant networks, they may constitute an ideal system to study the com-plex space-time relationships between benefits and costs of ISR on one hand, and cue reliability and the spatio-temporal dynamics of herbivore spread in relation to the speed and extent of systemic signal transduction, on the other hand. The rate and spatial dimensions of internal signal transmission can be expected to depend crucially on architectural plant characteristics such as av-erage ramet distance, vascular sectoriality, source-sink relationships, and on the transport capacity of connecting internodes between ramets. In most cases herbivore-caused damage of a given ramet is likely to

entail a considerable risk of attack for adjacent ramets. However, the risk of attack by an herbivore present on a connected sibling ramet should decreasewith increasing distance from the point of initial damage. In other words, the information content of the systemic warning signal is very likely to decrease with increasing distance between sender and receiver ramets in a clonal plant network. The rate of decrease (i.e., the exact decay profile of spatial autocor-relation in attack risk) depends on the behaviour, mobility and population size of the herbivore, the size and architecture (e.g. average ramet distances) of the clonal plant under attack, and on general canopy characteristics (e.g. pres-ence of alternative host plants). To be beneficial in the long run, the physio-logical and ecological costs of inducible defences should be equalled or out-weighed by benefits in terms of enhanced plant performance and fitness (Karban & Baldwin, 1997; Heil, 2001, 2002; Cipollini et al., 2003). This implies that uninfested ramets of a clonal plant should only respond to the systemic signal and switch on their inducible defences if the danger of being attacked is high enough to justify the costs of inducing and temporarily expressing de-fence traits (Karban et al., 1999). Any mismatch in time or space between sys-temic defence induction and herbivore attack is likely to lead to a costly misal-location of resources. Examples for such mismatches include cases in which herbivores disperse faster than the induction signal and cases in which the herbivore does not spread to adjacent ramets (no attack of systemically in-duced ramets). Intermediate levels of spatio-temporal spread of the induction signal may be the most appropriate response of many clonal plant networks to local-ized herbivore attack. If the spatial scale of the systemic defence induction is too local (e.g. induction occurs only in nearest neighbour ramets) or if the in-duction does not spread fast enough within connected ramets, the herbivores may be able to disperse to surrounding ramets before systemic induction has occurred there. If, however, the scale of systemic induction is too large, distant ramets of a clonal fragment may be induced even though the risk of being reached by the herbivore is rather low. A graded response of ramets according to their distance to the site of attack, possibly mediated by a decrease in in-duction over space and time, may also allow optimizing the cost-benefit bal-ance of systemically induced defence mechanisms in response to local herbi-vore damage. No data are currently available to confirm or to reject this and other predictions about the mechanisms and implications of ISR in clonal plant networks. Although the same induction and defence mechanisms are expected to operate in clonal and non-clonal plants, sharing ISR elicitors between inte-

grated ramets of clonal plants could have important population-level conse-quences for plant performance, plant-plant and plant-herbivore interactions. The systemic induction of direct defence traits in clonal ramet populations could act as an effective spatial information and early warning system for spa-tially scattered network members in case of local herbivore attack. Such an early warning system would be particularly beneficial for clonal plants by con-ferring integrated ramets faster and better protection from herbivore damage (through reduced palatability or other direct defences) than their surrounding, un-induced competitors. This effect may be enforced by the selectivity of her-bivores which may be discouraged from feeding on the induced parts of a clonal fragment and move preferentially to neighbouring host plants for feed-ing. In herbivore-prone environments the ability of clonal fragments to share ISR signals over considerable distances may critically affect competitive rela-tions between clonal and non-clonal plants. Physical inter-ramet connections may not only reduce, but can also in-crease the risk and intensity of future herbivore damage. In many clonal plant species, ramets are connected by aboveground structures (i.e. stolon inter-nodes). They can be used as bridges by foraging herbivores, guiding them to uninfested ramets. This negative effect of physical integration should be strongest if clonal plants with a sparse growth habit (relatively long inter-ramet distances) are attacked by specialist herbivores with limited mobility. Except for enhancing protection through ISR, clonal connections between ramets might thus also increase the chance of being located by herbivores in specific cases.

Resource flows and transport pathways within clonal plant networks allow for, but also constrain the distribution of ISR signals among intercon-nected ramets. Resource flows in the xylem and in the phloem are largely gov-erned by plant-internal source-sink relationships and water potential gradients, which are a function of environmental factors (such as heterogeneity in re-source availability) and of the developmental relations between connected ramets and branches (Marshall, 1990). Transport of resources and signals can be severely constrained by the physical construction of the vascular system (sectoriality) and/or by predominantly unidirectional flows of organic resources in several clonal species (Marshall and Price, 1997). In addition, herbivore at-tacks themselves, may change the source-sink relationships and resource movement patterns within interconnected groups of ramets (Haukioja, 1991; Honkanen & Haukioja, 1998; Honkanen et al., 1998), thereby affecting the di-rection and/or speed of information sharing within the network. Generally speaking, the effects of induced systemic resistance in clonal

plants should be strongest in plants with large-scale phloem integration among connected ramets (e.g. Trifolium repens; Chapman et al., 1992; Marshall & Price, 1997; Stuefer et al., 1996) and little directional constraints on carbohy-drate movement (e.g. Potentilla spp.; Stuefer et al., 1994; van Kleunen & Stue-fer, 1999; Hydrocotyle bonariensis; Evans, 1991, 1992). Strongly sectorial spe-cies, such as Glechoma hederacea, Trifolium repens (Price et al., 1992ab; Price et al., 1996; Hay & Sackville-Hamilton, 1996; Lötscher & Hay, 1997) and possibly most other species with monopodial stem growth and so-called leaf ramets (i.e. ramets that consist of only one leaf, an axillary meristem, and a root system) are likely to be constrained in the internal transmission of signals. Sectoriality, however, may not strongly affect the effectiveness of ISR in clonal plants, if herbivore damage and defence induction occurs not on a single but on several adjacent ramets. Due to the basic differences in vasculature, mono-cotyledonous species are less likely to be constrained by sectoriality than di-cots (Stuefer, 1996). Based on knowledge of clone-internal resource sharing, we can expect that young ramets and growing parts of the clonal network in general will be most likely to receive induction signals shortly after an attack has happened, because they usually represent strong sinks for carbohydrates (Marshall, 1990). Environmental heterogeneity, such as partial shading of a clonal plant network, may enhance the transmission rate and distance of ISR signals by promoting the transport of phloem based resources to stressed clone parts. Environmental heterogeneity imposing source-sink gradients is also likely to block or constrain the dispersal of defence signals from ramets that act as strong resource sinks. Whether herbivores can make use of this fact (e.g. by preferentially feeding on shaded ramets in habitats with small-scale heteroge-neity in light conditions) is unknown to date.

MASSIVE PERFUME ATTRACTION

Many plant species emit a specific blend of gaseous substances (volatiles) when they are damaged by herbivores. The volatile mixture and concentration profile of constituent compounds can be species-specific and may differ for different herbivores. In various systems damage-induced volatiles attract natu-ral enemies of the herbivores (such as carnivores or parasitoids) that are causing the damage through feeding (Pichersky & Gershenzon, 2002; Dicke et al., 1993a). Strong evidence from the scientific literature and the successful applica-tion of such triangular interactions for the biological control of herbivores

(Karban & Baldwin, 1997, and studies quoted there) confirm the effectiveness of this indirect defence mechanism in several plant-animal systems. Experi-mental studies have shown that recruiting volatiles may not only be emitted from the site of damage, but systemic induction of volatile emission can lead to the production and release of info-chemicals from undamaged parts of a plant (Dicke et al., 1993b; Takabayashi & Dicke, 1997; Dicke & Dijkman, 2001). In the case of large clonal plant networks such as formed by many stoloniferous and rhizomatous species, a systemic emission of volatiles could lead to a sig-nificant amplification (in terms of air volume containing the info-chemical) of the indirect defence signal, and a highly increased chance of attracting natural enemies of the herbivore that caused the defence induction (see Fig. 1). The release of volatiles from numerous ramets after systemic induction would facilitate the attraction of predators and parasitoids. However, the spatial de-coupling of herbivore position and volatile release due to the sys-temic transmission of induction signals among scattered ramets of a clonal network may re-duce the information content of the volatile sig-nal, thereby potentially jeopardizing the ability of predators and parasitoids to locate their prey. This may occur if infested and uninfested ramets emit volatiles in similar concentrations after an herbivore attack. According to Dicke (1994/1995), however, the emission of volatiles from uninfested plant parts may be weaker than the emission from the site of wounding. If so, the strength of the volatile signal produced by interconnected ramets may decrease with increasing distance from the attacked ramet, thereby creating a concentration gradient which can facilitate the predator or parasitoids to locate the herbivores among infested and uninfested ramets of a clonal plant. Experimen-tal studies are needed to clarify the existence and action of indirect defence signalling from clonal plant networks to enemies of their herbi-vores.

A. Clonal plant network

Figure 1. Emission of volatiles after initial herbivory in one ramet (black). (A) The attacked ramet sends a signal to induce volatile emission in adjacent ramets to attract the natural enemies of the herbivores. To help predators locate their prey, there should be a concentration gradient in relation to the distance from the attack point (fading coloured arrows). (B) In non-clonal plants, only the attacked individual can systemically induce volatiles in other parts of the plant. Only very few and partly con-troversial studies (Dicke & Bruin, 2001 and studies mentioned therein) have reported volatile emission of neighboring plants after contact with volatiles from infested plants.

B. Non-clonal plants

The clonal architecture can also affect the total volume and concentra-tion of emitted volatiles. One can expect that species with a spatially scattered distribution of ramets (“guerrilla”-type of clonal growth) produce bigger and less concentrated volatile emissions than species with an aggregated ramet distribution (“phalanx”-type). In both cases, however, clonal plants are likely to release a bigger amount of volatiles than infested individual of comparable non-clonal plants or non-integrated ramets of clonal species. Indirect defences of this type could provide clonal plants with an increased protection, which could result in an enhanced competitive strength of clonal versus non-clonal plants in herbaceous canopies. This prediction can be tested in experimental studies by comparing volatile emissions and carnivore attraction between integrated and non-integrated ramets of clonal plants, or by competition experiments be-tween clonal and non-clonal plants grown in the presence of herbivores and a natural enemy that can perceive volatile signals. Such studies would also al-low for the measurement of volatile concentration gradients around attacked ramets, and the assessment of the foraging precision of attracted enemies. The beneficial effects of a massive emission of volatiles from intercon-nected ramets of clonal plants may also depend on whether or not neighbour-ing plants are able to perceive and respond to these info-chemicals (Bruin & Dicke, 2001). Benefits to neighbours can arise from the direct perception of info-chemicals, or they may stem from an increased protection from herbivory by attracted carnivores or parasitoids. The net effect of information sharing with unconnected (and possibly unrelated) neighbours can be positive (through increased protection of a larger area) or negative (through increased performance and competitive strength of the neighbours). In other words eavesdropping (in the sense of an activation of defensive phenotypes after being exposed to info-chemicals produced by other damaged plants; Karban et al., 2000; Dolch & Tscharntke, 2000; Karban & Baldwin, 1997) as well as mechanisms of group selection could favour or disfavour the massive emis-sion of defence signals from clonal plant networks, depending on the competi-tive relationship and spatial arrangement of clonal fragments, genets and other species in natural populations and communities. These unresolved questions call for specific studies into the proximate mechanisms and ultimate implica-tions of multi-trophic interactions between clonal plants, herbivores and their enemies. It should be noted, that the information content, reliability and specificity of gaseous info-chemicals released to the air by damaged plants or ramets is probably lower than that of internally transmitted direct defence signals. The effectiveness of an alerting system based on volatile emissions is hence likely

to be lower than the early warning system based on the systemic induction of direct defence traits in interconnected ramets of clonal fragments. In most of the studies reporting plant-plant communication, the activation is thought to be mediated by airborne info-chemicals (Karban & Baldwin, 1997 and studies therein, Dicke & Bruin, 2001 and studies therein, Karban et al., 2003) and also in some cases by root-exudates (Chamberlain et al., 2001; Dicke & Dijkman, 2001; Guerrieri et al., 2002). However, in these cases of “external warning” the distance between infested and uninfested plants should be small in order to perceive the warning signal (both via air and soil) and activate their defensive phenotype. Normally in these studies the distance between infested and clean plants is about 15 cm or less (e.g. 15 cm for volatiles perception in Karban et al., 2000, less than 15 cm for root exudates perception in Chamberlain et al., 2001 and Guerrieri et al., 2002). Another factor that could interfere in the per-ception of the volatiles emitted by infested plants is the direction of the wind. In clonal networks, the members would be able to overcome these constraints by the systemic transmission of the warning signal through physiological integra-tion between ramets.

SYSTEMIC PATHOGENS: THE DARK SIDE OF NETWORK INTEGRATION?

In spite of all obvious advantages, resource and information sharing has its risks. Modern, man-made information networks are well known for their vulner-ability to viruses and worms. In direct analogy, populations of interconnected ramets of clonal plants may be especially susceptible to infections by systemic diseases, as physical links between clone members can be (ab-) used as in-ternal dispersal highways, enabling pathogens to spread among connected ramets and to disperse rapidly within clonal ramet populations (Cook, 1985; Eriksson & Jerling, 1990). This risk might create selection pressures against communication and resource integration in clonal plants, and/or it may prompt a co-evolutionary arms race between networks and intruders (such as seen in electronic information networks). Although interconnected ramets of clonal plants can be functionally independent, they may not have independent risks of pathogen infection. After an initial infection, pathogens can trace uninfected ramets by following vascular connections through the use of the plant internal transport system for water and carbohydrates. Specialist pathogens may espe-cially benefit from internal spread, because they can successfully locate and infect genetically identical hosts without the help of external vectors. From this perspective, clonal plants seem ideal hosts for systemic pathogens. In non-

clonal plants, infections by internal disease transmission are restricted to one individual (Fig. 2). Pathogen effects on host plants are extremely diverse, ranging from lethal or severely damaging effects, to symptomless infections, and positive impacts on plant growth and performance. A wide range of qualitatively and quantitatively different pathogen actions is described in the literature: many pathogens cause a decrease in photosynthetic activity (Chia & He, 1999; Fu-nayama & Terashima, 1999; Sampol et al., 2003), while others affect hormone levels of their hosts (Pan & Clay, 2002) or alter resource allocation patterns to different organs and functions (García-Guzmán & Burdon, 1997). Pathogen infection may lead to a deformation of leaves (Gibbs & Harrison, 1976), re-duced growth rates (Jones, 1992; Piqueras, 1999; Potter, 1993) and changes in the growth form of plants (Piqueras, 1999; Wennström & Ericson, 1992). In some cases, pathogens can effectively castrate (impede sexual reproduction) the host plant (García-Guzmán & Burdon, 1997; Groppe et al., 1999; Pan & Clay, 2002). In rather rare cases, host plants may actually benefit from patho-gen infections in terms of increased biomass production (Pan & Clay, 2002; Groppe et al., 1999) or enhanced levels of allelopathy (Mattner & Parbery, 2001). However, plants are by no means defenceless against patho-gens. On the contrary, they have evolved an impressive array of mechanisms and strategies to toler-ate, avoid or fight pathogens. In the following sections, we will focus on systemic pathogen spread and vari-ous defence mechanisms that (clonal) host plants may exhibit in response to disease infection and pathogen spread. We will concentrate on possi-ble ecological implications of clonality in relation to systemic pathogens. RACE AGAINST TIME: SYSTEMIC SPREAD OF PATHOGENS AND DEFENCE SIGNALS

Pathogens can either be systemic or non-systemic. Systemic pathogens are able to move away from the initial site of infection and can contaminate other

Figure 2. Systemic spread of a pathogen in clonal and non-clonal plants. In clonal plants (A) the pathogen can spread through many potential individuals. The spread of a pathogen in a non- clonal plant (B) is restricted to one individual. Black filling indicates the infected plant parts.

A

B

parts of the plant. In most cases they live perennially in the host plant. In con-trast, non-systemic pathogens are restricted to the initial site of infection. They are often annuals that re-infect their host plants every year (Wennström, 1999). In the following sections we will focus exclusively on systemic patho-gens, which can spread through the vascular system of their host plants. Fungi, for example, may grow along vascular vessels or sporulate directly into the xylem sap. Viruses can be transported in the phloem. They can directly be entered into the vascular system by feeding aphids or other animal vectors, or they can use plasmodesmata to enter and exit the phloem. Once inside the phloem, viruses usually follow plant-internal source-sink flows (Cheng et al., 2000; Leisner & Turgeon, 1993; Thompson & Schulz, 1999), thereby predomi-nantly ending up at sites with high sink strengths (e.g. young, developing plant parts, resource deficient and damaged plant parts or ramets). In analogy, pathogens and pathogen propagules present in the xylem sap of plants are likely to move along water potential gradients from sites of water uptake to sites of high water loss through transpiration (Marshall, 1990; Stuefer, 1996).

Plants have developed different defence strategies to cope with their pathogens. Pathogen defence mechanisms can act on morphological, devel-opmental and biochemical levels of the plant. An infected host plant may, for instance, escape the pathogen by a (partial or full) developmental switch from vegetative growth to flowering. Korves and Bergelson (2003) have recently shown that the time to flowering can be significantly shortened by pathogen infection in Arabidopsis. Several studies have shown that clonal plants may be able to escape their systemic pathogens by fast vegetative growth (Frantzen, 1994; García-Guzmán & Burdon, 1997; Wennström & Ericson, 1992) and by clone fragmentation (McCrea & Abrahamson, 1985; Kelly, 1995). It is still unknown however, whether the timing and the extent of ramet isolation through clone fragmentation is a pathogen-inducible trait in clonal plant species. It is known for several groups of clonal plants (e.g. pseudo-annuals, Jerling, 1988; tussock-forming grasses, Wilhalm, 1995) that the con-nections between ramets are short-lived and that clones fragment into individ-ual ramets or small ramet groups as part of their regular development. These so-called genet splitters (as opposed to the group of genet integrators) exhibit spontaneous clonal fragmentation. Their ramets do not stay interconnected for extended periods of time, but become physiologically and physically independ-ent after a short offspring production and establishment phase (Eriksson & Jerling,1990; Piqueras & Klimes, 1998; Piqueras, 1999; Verburg & During, 1998). This seems counterintuitive because of the apparently low costs of maintaining connections and the broad evidence for positive effects of clonal

integration on plant performance and fitness. It has been suggested that genet splitters may give up physical ramet connections to spread the risk of mortality (e.g. generated for instance by pathogens; Eriksson & Jerling, 1990) among independent ramets. According to this hypothesis, genet splitters could have lost their ability for prolonged integration due to strong past and/or current se-lection pressures created by systemic pathogens. This idea proved difficult to verify as most clonal species are either obligate splitters or integrators, and genetic variation for clone fragmentation is usually small or absent in most clonal species. Direct experimentation with pathogens, intact and artificially severed clonal fragments may provide more insight into this topic. Plants can also defend themselves against pathogens by a mechanism known as post-transcriptional gene silencing (PTGS). PTGS is an effective defence mechanism targeted specifically at viruses, which protects plant cells by degrading the nucleic acid of RNA viruses (Waterhouse et al., 2001). PTGS can spread through the plant by an unknown signal that is capable of travelling both between cells (through plasmodesmata) and through the phloem. In-fected plant cells can use this system to send a warning message to unin-fected parts of the plant. These parts can then prepare their virus degradation machinery, in order to stop the infection (Waterhouse et al., 2001, and refer-ences therein). This mechanism is induced whenever a pathogen carrying an avirulence (Avr) gene challenges a host plant with the matching resistance (R) gene. A so-called hypersensitive response is usually induced after infection. This process is mediated by salicylic acid (SA) and is commonly referred to as systemic acquired resistance (SAR; Gozzo, 2003). Through this mechanism, the host plant may temporarily exhibit a stronger resistance to following chal-lenges by the same or in some cases also by other pathogens. SAR is active against viruses, bacteria and fungi (Conrath et al., 2002; Gozzo, 2003; Maleck & Dietrich, 2003). PTGS and SAR can systemically protect plants against invading patho-gens. Both defence mechanisms may be of considerable importance in clonal plant networks, because they can internally spread to many (or all) functional individuals on a clonal fragment. In analogy to the early warning system against herbivores (see above) PTGS and SAR may be effective means to save connected sibling ramets from getting infected. The costs-benefit balance of PTGS and SAR will most likely depend on the effectiveness of protection against further pathogen damage, which is in turn a function of the relative speed with which pathogens and pathogen defence signals can travel within clonal plants.

The spatio-temporal extent and the dynamics of internal spread of sys-

temic pathogens and appropriate defence mechanisms (SAR, PTGS, fragmen-tation) in clonal plants are largely unknown. Both the speed and the spatial extent of this spread may be system-specific and depend strongly on environ-mental conditions and on source-sink relationships between connected ramets. The outcome of the race between pathogens and specific defence signals may vary according to circumstances at the time and place of infection. In the absence of experimental data, any prediction as to whether systemic pathogens or systemic defence signals may win this race against time seems futile and excessively speculative. However, if pathogens could on average spread faster than the defence mechanism, clonality would be a major disad-vantage for plants, and systemic pathogens should exert selection pressures against the prolonged maintenance of physical inter-ramet connections. If, on the other hand, the defence mechanisms were usually effective, clonal growth and physiological integration can be a great benefit for plants, because it al-lows for an effective protection of spatially scattered, yet functionally independ-ent individuals. In non-clonal plants systemic defence mechanisms are re-stricted to a single individual (Fig. 3).

CLONAL PLANTS:STORES AND VECTORS FOR DISEASES?

The presence of systemic pathogens in clonal plants may have serious reper-cussions on the population and community level, because clonal plant net-works could serve as vectors for diseases and provide ideal long-term storage space for pathogens. The survival chances and the longevity of clonal plant genets are generally expected to be higher than those of non-clonal plants. Clonal plants can persist as long as the rate of clonal proliferation by initiating new meristems is higher or equal than the rate at which old plant parts die off (Thomas, 2002). Clonal plants can circumvent senescence and avoid the de-velopmentally programmed death of the genetic individual by repeated rejuve-nation from newly activated meristems (i.e. by spontaneous self-cloning). Therefore, clonal genets can be extremely long-lived (Oinonen, 1967; Kemper-man & Barnes, 1976; Cook, 1985; Steinger et al., 1996). Systemic pathogens that can persist in plants during the whole lifetime of the host may be pre-served in clonal plants for very long, potentially endless periods of time. Spe-cialist pathogens (which can only infect few species), could especially benefit from storage in clonal plants, since the need to transmit to new, maybe difficult to find, hosts diminishes. In general the availability of suitable host can be ex-pected to be higher for generalist pathogens, therefore storage in clonal plants

may be less necessary for these pathogens. Non-clonal plants are less likely to play such a long-term storage role, because their lifetime is generally more restricted than that of clonal plant individuals. Clonal plants could function as spatial vectors for pathogens in natural plant populations and communities. The spread of systemic diseases within populations and communities may be significantly facilitated by the presence of clonal plant networks, because they allow pathogens to move between plants in the absence of suitable external vectors, and without the production of specialized dispersal units. A pathogen that can persist in a clonal plant can use its host as a long-term basis and spatial vector to spread to other plants within the system. Non-clonal plants are more likely to die of senescence be-fore the pathogen can spread to other plants. From this perspective, clonal plant networks may represent spatio-temporal stepping-stones facilitating the spread of systemic diseases within populations and communities. Specialist as well as generalist pathogens are likely to use clonal plants as vectors and stor-age medium. However, generalist pathogens probably use clonal plants in par-ticular as spatial vectors. Because they can spread to many host species, clonal plants may provide a perfect starting-point for generalist pathogens to (re-) infect surrounding plants. The presence of generalist pathogens in long-lived, spatially extensive clonal networks may pose a greater threat to neighbouring plants, because they are more likely to infect individuals of other

Figure 3. Race between the pathogen and the defence signals, in clonal and non- clonal plants. After infec-tion by a pathogen (indicated with the left black arrows), PTGS and/ or SAR are induced. If these defence mechanisms (indicated by the grey cross) can be established faster than the infection, the defence signals will spread through many potential individuals in clonal plants (A), thereby protecting them from further infec-tions. Whereas in non- clonal (B) plants only one individual is protected by these defence mechanisms and other individuals are still susceptible to infection. On the other hand if the pathogen wins, many potential individuals are infected in clonal plants (A1), whereas only one individual is infected in non- clonal plants (B1). Black filling indicates the infected plant parts; grey filling indicates the plant parts protected by SAR and/ or PTGS. Arrows indicate (possible) infection sites.

A1

B1

A

B

genets and species in a plant community. Specialist pathogens may benefit predominantly from clonal plants as temporal vectors. Clonal plants could give specialist pathogens the time to “wait” for suitable host species that may not be present at all times. This notion of clonal plants as possible stores and vec-tors of diseases would predict that, in the long run and under comparable envi-ronmental conditions, populations with a high frequency of clonal plants might accumulate more resident pathogens and therefore suffer from higher disease loads than populations with a lower presence of clonal species. Specific data to test this prediction are not currently available. High levels of virulence are likely to preclude systemic pathogens from using clonal host plants as long-term storage space and spatio-temporal vec-tors. Highly virulent pathogens are likely to kill or seriously damage the host plant in short periods of time, thereby creating the need to spread quickly be-tween hosts (Lively, 2001). Highly virulent, systemic pathogens should be able to rapidly eradicate entire clonal networks. To be ecologically and evolutionar-ily feasible, however, high levels of virulence must be coupled to very fast and efficient between-plant dispersal (Day, 2003), and is therefore expected to be more common for generalist than for specialist pathogens. To date we do not have any compelling evidence for the existence or common occurrence of highly virulent killer-pathogens in clonal plants. If they existed, they should cause very strong selection pressures against clonality. We suggest that clonal plant life histories selectively favour pathogens with a low virulence. The bene-fits conferred to the pathogen by the potentially unrestricted lifetime of clonal host plants might strongly select against high levels of pathogen virulence. This expectation is in concordance with general dispersion-virulence models that predict a positive relation between transmission rates and pathogen viru-lence (Day, 2003; Lipsitch & Moxon, 1997; Lively, 2001). In specific cases clonal plants can actually benefit from pathogen infec-tions. Groppe et al. (1999) have shown that the internal concentration of the endophytic fungus Epichloë bromicola is positively correlated with the vegeta-tive vigour of the clonal host plant Bromus erectus. Although infected plants showed a significant increase in vegetative growth and performance, endo-phyte infection also had strongly negative impacts on sexual reproduction. Seed output of the host plant was negatively correlated with fungal concentra-tion. Pan & Clay (2002) reported a similar pathogen-mediated trade-off be-tween vegetative growth and sexual reproduction in their system. Epichloëglyceria infections enhance stolon production and accumulation of clonal growth biomass of the clonal host plant Glyceria striata, and at the same time E. glyceria effectively castrates its host. By doing so, the fungal endophyte

blocks the possible escape route for the host to dispose of the pathogen by flowering and sexual reproduction. In terms of vegetative growth and competi-tive ability of the host plant these fungi-plant associations can be considered mutualistic: the fungal endophyte enhances host performance and the host plant provides a suitable environment for the pathogen. In terms of life-history evolution of these pathogens, the lack of sexual reproduction of the host in combination with the virtual absence of genet senescence in clonal plants re-moves the necessity to disperse after successfully infecting a host. A very low pressure to disperse might eventually lead to the evolution of low virulence in these specific clonal plant pathogens, possibly generating a basis for the evo-lution of (partially) mutualistic plant-pathogen systems (Clay, 1990).

CONCLUSION

Based on the information and arguments provided above, we conclude that sharing substances and agents other than resources between ramets of clonal plants may have far-reaching consequences for the functioning of plant indi-viduals, populations and communities, as well as for interactions between clonal plants on one hand and pathogens, herbivores and the natural enemies of herbivores on the other hand. We are currently only at the beginning of re-search that will hopefully elucidate the various ecological roles, proximate mechanisms and ultimate implications of clonal integration beyond resource sharing. Future studies may shed light on these complex yet fascinating inter-actions.

ACKNOWLEDGEMENTS. We thank Hans de Kroon for comments on a previous version of the manuscript. This work was partially funded by the Netherlands Organisation for Scientific Research (NWO), VIDI-016.021.002 grant to JFS.

KEYWORDS: virulence; clonal plants; escape; sexual reproduction; pathogen transmission; multiple infections.

The evolution of pathogen virulence has received considerable attention dur-ing the last decades (van Baalen & Sabelis, 1995; Frank, 1996; Day & Proulx, 2004). In this note we will discuss two major theories of virulence evolution in the context of clonal plant-pathogen interactions and argue that they can lead to contradictory predictions when applied to very long-lived hosts such as clonally propagating plants. We propose that clonal plants and their pathogens may be especially suitable systems for empirically testing hypotheses of viru-lence evolution. A generally accepted theory of virulence evolution is based on the trade-off between pathogen transmission and within-host pathogen reproduction (Frank, 1996; Lipsitch & Moxon, 1997; Day, 2003; Andre & Hochberg, 2005), the latter being positively correlated with pathogen virulence (see Table 1 for definitions). Pathogens can benefit for a long time from hosts with low intrinsic mortality rates, provided that they exploit them prudently (low virulence), while severe host exploitation (high virulence) shortens the time during which a pathogen can benefit from the host. This effect can be seen as a mortality costof virulence (Day & Proulx, 2004). As a consequence, high host longevity should select for lower levels of virulence. Conversely, increased virulence should be favoured in short-lived hosts (Gandon et al., 2001, and references therein; Day & Proulx 2004), due to the necessity to spread quickly to new hosts and due to the lower mortality costs of virulence (Ewald, 1994; Day & Proulx, 2004). Depending on the specific host-pathogen interaction a whole range of evolutionary stable strategies can be expected. The optimal strategy balances the above-mentioned costs and benefits resulting in the general pre-diction that pathogens evolve towards intermediate levels of virulence (Gandon et al., 2001, and references therein; Day, 2003). This theory, how-ever, is based on single infection events. Predictions for virulence evolution change when multiple infections (for a definition, see Table 1) are taken into account, since pathogens have to deal with both the host and one or more other pathogens simultaneously (van Baalen & Sabelis, 1995). The general prediction is that in multiply infected hosts pathogens evolve towards higher levels of virulence than in the case of single infections (Nowak & May, 1994; May & Nowak, 1995; Brown et al., 2002). Prudent host exploitation, facilitating host longevity, becomes less beneficial for a pathogen if other pathogens simultaneously decrease host lon-gevity (van Baalen & Sabelis, 1995). In addition, within-host competition among pathogens is expected to result in increased host exploitation rates and therefore result in increased overall levels of virulence (van Baalen & Sabelis, 1995; Frank 1996). Van Baalen & Sabelis, (1995) proposed that pathogen

virulence should also increase in anticipation of multiple infections if the prob-ability of multiple infections is high. Clearly, pathogens in multiply infected hosts face a whole array of evolutionary forces that determine the final optimal strategy in each individual case. Both of these opposing selection pressures on pathogen virulence may act on clonal plants, which can be extremely long-lived (Oinonen, 1967; Kemperman & Barnes, 1976; Cook, 1985; Steinger et al., 1996) and which spread their mortality risks by partial or full independence of ramets (Eriksson, 1993). In principle, individuals of clonal plants can persist as long as the rate of vegetative propagation equals or exceeds the rate at which old parts die off (Thomas, 2002). This gives systemic pathogens the possibility to establish a virtually unlimited, lifetime infection in clonal species (Stuefer et al., 2004). The extreme longevity of genetic individuals and their multiple representations in populations should select for decreased levels of pathogen virulence for the following two reasons. First, the trade-off between pathogen transmission and reproduction should impose selection pressures for decreased levels of patho-gen virulence in clonal plants. Second, clonal propagation and high longevity is likely to buffer spatio-temporal variability in the genetic make-up of popula-tions. In other words, clonal growth enhances the probability of finding a sus-ceptible host in the vicinity of the initially infected host. Host features which assist local pathogen transmission are expected to select for lower levels of virulence (Boots & Mealor, 2007). The nature and magnitude of this effect is likely to depend on the spatial positioning of ramets (i.e., clonal growth form), the genetic diversity of the host plant population, and on the spatio-temporal dynamics of pathogen dispersal. In contrast to selection for decreased levels of virulence, the longevity of clonal plants is likely to increase the chance for multiple infections and should therefore select for increased pathogen virulence. Van Baalen and Sabelis (1995) state that “If the force of infection is high, and hosts remain exposed long enough, multiple infections are bound to occur”. Thus, it seems that both

Table 1. Glossary of used terms.

Virulence: The fitness reduction of a host caused by pathogen infection(based on Brown et al., 2006).

Pathogen: An organism which can infect and cause disease in a host (Brownet al., 2006).

Multiple-infections: Occurs when a host is infected by more than one pathogen,belonging to the same or different pathogen species.

high and low pathogen virulence can be selected for in clonal plants and in other long-lived organisms. However, current models on virulence evolution do not explain how these conflicting selection pressures are resolved and what levels of pathogen virulence can be expected to result from opposing selection forces. When considering both above-mentioned theories, one may conclude that pathogens of clonal plants ought to evolve towards higher levels of viru-lence, as multiple infection events are bound to occur during the lifetime of a clonal plant and the hypothesized trade-off between pathogen transmission and pathogen replication is based on single infection events only. Exposure to increasingly virulent pathogens should strongly select for escape strategies of the host plant. Since most clonal plants can reproduce both vegetatively and sexually, a switch to flowering and fruit set may represent a way for clonal plants to evade pathogens. It has been proposed that sexual reproduction should be favoured over asexual propagation in infected plants (Hamilton et al., 1990). Therefore we expect infected clonal plants to show increased sex-ual reproduction through more rapid and intensive flower production, a faster developmental switch to flowering and increased flower or seed production. Empirical data to confirm or reject these hypotheses are not available to date. To our knowledge there is no evidence for highly virulent pathogens in clonal plants, neither for increased flowering after pathogen infection. Based on this lack of evidence for increased levels of virulence one could conclude that selection favours low virulent pathogens in clonal plants. For pathogens this would imply that the benefits of potential long-term infections outweigh the costs of possible multiple infections. However, the lack of evidence for clonal growth promoting the evolution of increasingly virulent pathogens can only suggest, but does not necessarily prove that clonality selects for decreased levels of virulence. To our knowledge, no comparative studies of virulence lev-els between short-lived and long-lived plants, infected with the same patho-gen, have been conducted so far. Neither did we find any studies that analyze in detail the effect of pathogen infection on the timing and extent of sexual re-production in clonal plants. Future research on these topics, empirical studies on multiple infections, and modelling work applying virulence evolution theo-ries for long-lived organisms are hence called for. In this context clonal plants could be essential since they provide ideal systems to test these hypotheses. Ultimately, the question remains what virulence levels can be expected in pathogens of clonal plants. Not only are the pathogens themselves sub-jected to conflicting selections pressures, their host in return may have to cope with a range of pathogen virulence levels as well. This interaction between

pathogen and host combined with the contrasting selection pressures on viru-lence mentioned above, will make it even more difficult to define one compre-hensive prediction on expected pathogen virulence in clonal plants. Difficult, but all the more intriguing.

ACKNOWLEDGEMENTS. The authors thank Hans de Kroon and two anonymous referees for their comments on a previous version of this manuscript.

ABSTRACT

Resource sharing between connected ramets can be advantageous for clonal plants since it facilitates growth and survival in a variety of environments. How-ever, widespread vascular integration bears the risks of enhanced internal pathogen spread, which may pose a serious threat to clonal plants. Pathogens can have major negative effects on plant fitness and internal pathogen spread may be one of the most prominent disadvantage of clonal growth. In this paper we analyzed patterns of internal virus spread in ramet groups of the stolonifer-ous herb Trifolium repens and investigated the effect of leaf ontogeny on intra-clonal disease development. To assess the dynamics of internal pathogen spread we inoculated single leaves of T. repens with White clover mosaic virus(WClMV) and analyzed the infection status of ramets at different positions and distances from the point of infection, and in leaves from different developmen-tal stages. WClMV could infect all the young, developing plant parts. All ramets positioned on basal branches or on the main stolon became infected. The pat-tern of plant-internal virus spread was not affected by heterogeneous light con-ditions. Leaf ontogeny strongly affected disease development, i.e. fully mature leaves on the main stolon remained virus free while younger ramets became readily infected with WClMV. We discuss possible ecological implications of intra-clonal virus spread and identify potential hazards associated with this phenomenon. Despite the well-described advantages of physiological integra-tion, our data suggest that clonal integration may lead to negative selection pressures on clonal growth in pathogen-prone environments.

KEYWORDS: ecology; age-related resistance; disease; pathogen; systemic spread; White clover mosaic virus.

INTRODUCTION

Stoloniferous plants grow by the production of physically linked and functio-nally autonomous offspring individuals, called ramets, which are connected by the internodes of horizontally extending stems, thereby forming network-like structures of variable dimensions. Many studies have shown that sharing re-sources such as water (De Kroon et al., 1996), carbohydrates (Chapman et al., 1992) and mineral nutrients (Abrahamson et al., 1991) among ramets con-siderably enhances growth and survival in a broad variety of environments (van Groenendael & De Kroon, 1990; Stuefer et al., 1994, 1996). However, in contrast to its obvious benefits surprisingly little is known about potential risks and costs of physiological integration in ramet networks of clonal plants. It has been suggested that physical connections between ramets are likely to facili-tate the internal spread of potentially harmful toxins and pathogens (Cook, 1985; Silander, 1985; Eriksson & Jerling, 1990, Stuefer et al., 2004, and refer-ences therein), creating potentially powerful selection pressures against clonal growth. So far, however, this assertion has not been tested explicitly. Systemic infections of ramet populations may pose a serious threat to wild and cultivated clonal plants (Simmonds, 1979; Silander, 1985; Cook, 1985). Virus infections can have detrimental effects on plant performance and fitness (Hull & Davies, 1992; Strange & Scott, 2005). It is conceivable that upon infection of a single leaf the whole network of interconnected ramets be-come rapidly virus-infected through vascular long-distance trafficking (Silander, 1985; Lough & Lucas, 2006). Viruses and other systemic pathogens may readily infect successive ramet generations of clonal plants, thereby es-tablishing very long lasting, potentially life-time infections (Silander, 1985; van Mölken & Stuefer, 2008). The internal spread of fungal pathogens (Wennström & Ericson, 1992; Piqueras, 1999) and their effects on clonal growth and archi-tecture have been studied to some degree (D’Hertefeldt & van der Putten, 1998; Piqueras, 1999; Pan & Clay, 2002). However, despite of predictions that under natural conditions the majority of clonal plants may be infected by single or multiple viral pathogens (Silander, 1985, and references therein), and the long-standing notion of vascular links serving as highways for virus movement (Gilbertson & Lucas, 1996; Thomspon & Schulz, 1999), the patterns and dy-namics of viral disease development in these plants has never explicitly been investigated. The systemic spread of viruses within plants relies mainly on phloem-mediated long-distance transport (Leisner et al., 1992; Gilbertson & Lucas, 1996; Andrianifahanana et al., 1997; Oparka & Santa Cruz, 2000; van Bel,

2003) and follows plant-internal source sink gradients (Santa Cruz, 1999; Thompson & Schulz, 1999; Cheng et al., 2000). In analogy to carbohydrate transport (Marshall, 1990; Chapman et al., 1992), virus movement in ramet networks of clonal plants should be directed predominantly toward sites with high sink strengths, such as stressed plant parts, or young and developing tissues. Viral disease development within clonal plant networks should thus be constrained by directional sap flows (Marshall, 1990) and vascular sectoriality (Price et al., 1996; Orians, 2005). In addition, virus infections in mature plant parts may be limited (Garcia-Ruiz & Murphy, 2001) and patterns of disease spread may depend on leaf ontogeny. Clonal integration is considered advantageous in spatially heterogene-ous environments. Source ramets can take up locally abundant resources such as light, water and nutrients and send them to sink ramets through vas-cular connections. This type of resource integration promotes the growth and establishment of clonal offspring (Pitelka & Ashmun, 1985) and effectively buff-ers clonal plants against small-scale habitat heterogeneity (Stuefer, 1996; Hutchings & Wijesinghe, 1997). The efficiency of resource integration between connected ramets depends largely on source-sink relationships and on the degree of intra-clonal transport (Stuefer et al., 1998). Enhanced resource ex-change between individual members of a clonal plant network hence can be expected to promote internal virus spread. For instance, developing, shaded or damaged plant parts can be nurtured by carbohydrate transport towards these parts, which may imply ameliorated virus import.

The aim of this study is to investigate patterns of viral disease develop-ment in ramet groups of the stoloniferous herb Trifolium repens. More specifi-cally, we carried out two experiments to investigate (i) whether the virus can spread from the inoculated ramet to all its connected sibling ramets and whether this intra-clonal virus spread is altered by partial shading and (ii) how leaf ontogeny influences patterns of virus spread.

MATERIALS AND METHODS

Study organisms - The stoloniferous herb Trifolium repens L. was used for both experiments. T. repens propagates vegetatively through the formation of ramets on horizontal stems (stolons) that expand laterally from the parent plant. Each individual ramet consists of a single leaf and an internode and the ramets can develop roots and branches on their nodes. We used a single genotype for the first experiment (B35) and five genotypes (A120, B35, C50, C79 and D39) for the second experiment. All genotypes have been collected in

the field (riverine grasslands) and grown under common garden and green-house conditions for three years before we conducted this experiment (see Weijschedé et al., 2006 for details).

White clover mosaic virus (WClMV; necrosis strain, originally isolated from T. repens) was obtained from the Deutsche Sammlung von Mikroorganis-men und Zellkulturen GmbH (Braunschweig, Germany). This virus is a mem-ber of the genus Potexvirus and is transmitted mechanically between hosts. WClMV is generally believed not to be transmitted by insect vectors such as aphids (Johnson, 1942; Bancroft et al., 1960; Tapio, 1970; but see Goth, 1962).

Patterns of disease spread - In March 2006, 28 cuttings consisting of six ramets each, were allowed to root in water and then planted on sterilized pot-ting soil in plastic trays (15x23x5 cm). These cuttings (subsequently referred to as plants) were grown in a greenhouse with a 16 h light and 8 h dark period at 20/18 ºC. High pressure sodium lamps (Hortilux-Schréder, 600W) were switched on automatically whenever the irradiance dropped below 250 mol m-2 s-1. Under homogeneous conditions, photo-assimilates are predominantly transported acropetally from older (source sites) to younger (sink sites) ramets (Marshall, 1990; Kemball & Marshall, 1995). T. repens leaves transition from source to sink leaves once their leaf area is fully expanded (Carlson, 1966).The source-sink dynamics can be experimentally manipulated (Evans, 1991; Stuefer & Hutchings, 1994; Shumway, 1995) by partial shading (Marshall, 1990; Chapman et al., 1992; Stuefer et al., 1996; Gómez & Stuefer, 2006) or disruption of phloem flows (van Kleunen & Stuefer, 1999). We simulated a heterogeneous environment by creating a carbon sink in the oldest part of the main stolon (subsequently referred to as the base of the main stolon). Shade cages were placed over the first two oldest ramets on the main stolon of individual, including the branches that originated from these ramets (Fig. 1). The cages were covered with shade cloth which reduced the light intensity (PPFD) by approximately 55% and caused a slight reduction in the R:FR ratio of transmitted light (from 1.8 to 1.6 under greenhouse condi-tions). To disrupt the phloem stream, girdling was applied (referred to as steam-girdling from now on; Stuefer, 1995; van Kleunen & Stuefer, 1999) to the stolon internode between the fourth and fifth ramet counted from the base of the plants (Fig. 1). With this technique phloem transport is disabled between adjacent ramets, while the xylem remains functional. Shading and steam girdling were applied in a factorial design. Steam

girdling was applied three days before the plants were planted. The plants were shaded and inoculated with WClMV two days after they had been planted. The leaves from ramets three and four of all plants were mechanically inoculated with with cell sap prepared by grinding WClMV-infected plant mate-rial in inoculation buffer (50 mM Na2HPO4 buffer, 1 mM EDTA, set to pH 7.0 with HCL). 10 μl virus suspension was rubbed on each leaf by hand. Each plant received 100 ml half-strength Hoagland solution at two days post-inoculation (dpi). At 6 dpi all plants received 50 ml half-strength Hoagland solution, and they were inoculated with 50 μl Rhizobium solution. All plants were moved to a climate chamber with a 16 h light and 8 h dark period at 20/18 ºC, irradiance was 250 mol m-2 s-1, at 15 dpi. Water was given regu-larly. The leaf material for testing the presence of WClMV was harvested at 23 dpi. All plant material was stored at -20 ºC between harvest and further analy-sis. To test for WClMV presence with DAS-ELISA we collected the following leaves from ramets on the main stolon (Fig. 1). The leaf from one of the first two ramets at the base of the stolon was analyzed to study basipetal virus transport (Fig. 1A). As the duration of the experiment exceeded the average life span of mature leaves, most of the oldest two leaves on the main stolon had died by the end of the experiment and could not be used for DAS-ELISA testing. In such cases we analyzed WClMV presence in leaves from branches which originated from ramets one and two. Leaf material from ramet five or six

Figure 1. Schematic drawing of a Trifolium repens plant with target ramets for sampling indicated with capital letters. Ramet numbers are shown at the top of the drawing (r1-r11); r1 and r11 refer to the oldest and youngest ramets, respectively. Arrows indicate growing stolon tips; dashed lines indicate the newly formed ramets, i.e. ramets that were not present at the moment of inoculation. The inoculated leaves are encircled (ramets three & four) and the location where steam girdling was applied is indicated by a black triangle (between ramets four & five). The grey rectangle indicates the place were shading was applied. Leaves were collected from (A) ramets one and two, or from the branches of these ramets, (B) ramets five and six, and (C) ramet nine, for virus detec-tion by DAS-ELISA.

was sampled to investigate acropetal virus transport to ramets from the point of inoculation and beyond the place at which steam girdling was applied (Fig. 1B). These leaves were already present at the start of the experiment. Leaf material from ramet nine was sampled to analyze acropetal virus transport beyond the point of steam girdling and towards the younger, newly developing parts on the main stolon (Fig. 1C). Fresh leaves of T. repens stock plants were used as a negative control.

Leaf ontogeny - In December 2007 we started the experiment with apical cut-tings consisting of six ramets each. Seven cuttings per genotype were indi-vidually planted in plastic pots (6.25x6.25x7.5 cm) filled with potting soil. These cuttings (subsequently referred to as plants) were placed in a growth chamber with a 16 h light and 8 h dark period at 20/18 ºC. All plants received 50 ml half-strength Hoagland solution every week. For each genotype, six plants were assigned to the virus treatment and one plant to the control treatment. The plants were allowed to grow for 28 days before inoculation. All plants in the virus treatment were inoculated with WClMV on the sixth and seventh youngest ramets. Inoculation was performed as described above. Control plants were mock-inoculated with inoculation buffer only. Based on previous results (data not shown) we assumed that virus in-fection can start to spread systemically from 7 dpi onwards in our system. To analyse whether WClMV spread depends on leaf ontogeny we recorded the developmental stage (according to Carlson, 1966; 10 stages from 0.1 when the leaf first becomes visible to 1.0 when the leaf is fully unfolded) of each ramet which was situated between the inoculated ramets and the apex at 7 dpi. All ramets were individually harvested at 11 dpi and stored at -20 ºC. Ramets from different developmental stages (Carlson, 1966) were pooled together in four groups: stage 1.0, fully expanded mature leaves; stages 0.7-0.9, partially expanded leaves; stages 0.4-0.6, closed leaflets; stages 0.1-0.3 freshly emerged leaves. We selected a ramet from every group for each genotype to analyse WClMV presence. Leaves from the non-infected plants were used as a negative control. We then calculated the average ELISA absorbance (see below) per genotype for each ontogeny group resulting in five replicates (one for each genotype) per group. To allow for comparisons with the first experiment the control values were set to the same value as in the first experiment and all other values were corrected accordingly.

ELISA testing - WClMV presence was tested by DAS-ELISA (based on Clark

& Adams 1977). This method is based on the specific binding of WClMV anti-bodies to WClMV particles in the test sample. Alkaline phosphatase pre-attached to the WClMV antibodies converts the enzymatic substrate (p-nitrophenyl phosphate), resulting in yellowing of the samples which contain WClMV. The absorbance was measured at 405 nm with a Wallac VICTORtm2 1420 spectrophotometer (WALLAC Oy, Turku, Finland). Plants were consid-ered infected if the absorbance of the test sample was at least twice as high as that of the negative controls (Dijkstra & de Jager, 1998).

Statistical analysis - To analyse whether shading and steam girdling can af-fect the relative amount of virus particles in the infected ramets we performed a two-way ANOVA on the infected plant samples. This implies that all samples with absorbance values lower than 0.4 were excluded from this analysis (Fig. 2a-d). For the basal leaves we thus analyzed both the effects of steam-girdling and shading on the relative amount of virus particles (i.e. absorbance). For the acropetal leaves (ramet 9) we analyzed the effect of shading on the relative amount of virus particles, but not the effect of steam-girdling since the virus was not transported acropetally in these treatments (see results section). To test whether leaf ontogeny has an effect on the relative amount of virus particles (i.e. absorbance) we performed a one-way ANOVA with devel-opmental stage as fixed factor. All statistical tests were carried out with SAS version 9.1 (SAS Institute Inc., Cary, USA).

RESULTS

Infection status - DAS-ELISA testing showed that the negative controls had values of 0.198 ± 0.010 (mean ± SE) in the first experiment. Consequently, all samples (Fig. 2a-d) with an absorbance higher than 0.4 represent leaves in-fected with White clover mosaic virus (WClMV). The absorbance values in the second experiment were corrected relative to the controls in the first experi-ment (see materials and methods). All infected plants used for the analysis contained WClMV in at least one of their leaves, showing that each replicate was inoculated successfully. Treatment effects are given in Table 1.

Disease pattern - First, we studied virus spread in the acropetal direction on the main stolon. Virus presence was analyzed in the mature leaves of ramets five and six, which were present at the time of inoculation, and in the young, newly formed leaf of ramet nine which was produced during the experiment (Fig. 1). Figures 2a-b show that the clonal offspring becomes infected with

Figure 2a-d. Virus presence in different parts of the plants, given for each of the four treatments. (A) WClMV- treatment. (B) Shade treatment. (C) Steam-girdling treatment. (D) Shade & steam-girdling treatment. Leaves were considered infected when the absorbance at 405nm was twice as high as the negative controls. All bars that exceed the horizontal line at 0.400 are hence considered representing WClMV-infected leaves. The grey rectangle indicates shaded plant parts and the black triangles indicate the point of steam-girdling. Numbers one through six on the x-axes indicate the different replicates. The direction of transport is given on the lower x-axis. Basipetal: basipetal transport to ramets one and two (r1 & r2), bars that represent ramets on the main stolon are marked “ ” all other basipetal ramets originated from the branches. Acropetal: acropetal transport to ramets five and six (r5 & r6), or to ramet nine (r9).

WClMV. This was true for all replicates. However, when steam-girdling was applied, WClMV could not be detected in the leaf of ramet nine anymore (Fig. 2c-d). The mature ramets, e.g. five or six, did not become virus infected in any of the treatments (Fig. 2a-d) except for one replicate (Fig. 2b). Analysis of virus spread in the basipetal direction revealed a similar pat-tern. In all treatments (Fig. 2a-d), the newly produced young leaves on the basal branches, e.g. the branches which originated on the first two ramets of the main stolon were infected with WClMV. In spite of basal shading, WClMV did not spread into the mature leaves (formed pre-inoculation) on the main stolon, e.g. leaves of ramets one or two (Fig. 2a-b).

Leaf ontogeny - The developmental stage of the leaves had a significant ef-fect (F = 4.9, p = 0.0109) on the relative amount of virus particles (i.e. absorb-ance) present in leaves. Ramets with fully mature leaves (stage 1.0; Fig. 3) did not become infected with WClMV. However, virus infection can readily spread towards ramets with younger leaves (stages 0.9 – 0.1; Fig. 3).

DISCUSSION

Although benefits of clonal integration are well-studied, the possible hazards associated with systemic pathogen spread between interconnected ramets are largely unknown (Wennström and Ericson, 1992; Stuefer et al., 2004). Here we have shown that White clover mosaic virus (WClMV) is transported sys-temically within groups of interconnected ramets of Trifolium repens. We ob-served spatial variation in infection status within the clonal plant networks and show that young developing ramets can become readily infected through sys-temic spread.

Parameters df MS F df MS F

Shade 1 0.0195 5.21* 1 0.0006 0.21

Steam 1 0.0006 0.17 - - -

Shade*steam 1 0.0109 2.91 - - -

Error 15 0.0037 10 0.0027

Basipetal: ramets 1 & 2 Acropetal: ramet 9

Table 1. Statistical analysis of the effects of steam-girdling and shading on the accu-mulation of WClMV. We used a two-way ANOVA to analyze the effect of steam-girdling and shading on the relative amount of virus particles (i.e. absorbance) in basal leaves. Since steam-girdling fully disabled acropetal virus transport we used a one-way ANOVA to test for effect of shading on virus accumulation in newly formed leaves (ramet 9).

Patterns of systemic virus spread - Physiological integration between groups of ramets allows for virus spread both in basipetal and acropetal direc-tion. This spread is impeded by steam girdling, confirming that internal virus spread is based on phloem transport (Leisner et al., 1992; Oparka & Santa Cruz, 2000). Young ramets on the main stolon as well as on the basipetal branches became infected with WClMV, indicating that young plant parts are especially vulnerable for virus infections. This is consistent with previous re-ports of sink-directed virus transport in non-clonal plants (Thompson & Schulz, 1999; Cheng et al., 2000). The direction of virus spread did not change when we created a heterogeneous environ-ment through partial shading: clonal offspring always became infected. However, the amount of virus accumulation in ramets from the basipetal branches was de-creased by partial shading (Table 1). Virus replication depends largely on the activity of the host tissue (Hull, 2004) and may there-fore decrease in shaded and hence less actively growing plant parts. For this reason, accumula-tion patterns for other systemic pathogens, such as fungi, whose replication does not depend on host growth, are likely to differ from viral accumulation patterns in shaded or otherwise stressed plant parts. Age-dependent resistance - Fully mature leaves remained uninfected, whereas the younger leaves all became virus infected (Fig. 3), showing that leaf ontogeny is of great importance for patterns of disease development (Silander, 1985; Develey-Rivière & Galiana, 2007) and that virus import is re-stricted to the developing plant parts. Our data are consistent with other stud-ies (Garcia-Ruiz & Murphy 2001) that show restricted virus movement in ma-ture plants and support the suggestion of Leisner & Turgeon (1993) that vi-

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Developmental stages

Figure 3. Virus presence in leaves from different devel-opmental stages. Ramets from different developmental stages (Carlson, 1966) were pooled together in four groups: stage 1.0, fully expanded mature leaves; stages 0.7-0.9, partially expanded leaves; stages 0.4-0.6, closed leaflets; stages 0.1-0.3 freshly emerged leaves. Leaves were considered infected when the absorbance at 405nm was twice as high as the negative controls. All points that exceed the horizontal line at 0.400 are hence considered representing WClMV-infected leaves.

ruses can move through plant parts without causing a local infection. Our study shows that age-related resistance can cause spatial variation in infection status within a clonal plant, corroborating early predictions about intra-clonal heterogeneity in viral disease expression (Silander 1985). Age-dependent virus movement could stem from a lack of phloem-based transport towards mature source tissues, e.g. these tissues do not func-tion as strong enough sinks. However, Gómez & Stuefer (2006) have recently shown that mature leaves can express phloem-based, induced systemic resis-tance to herbivory when shaded, indicating that these leaves have the poten-tial to function as sinks. In mature tissue, virus transfer from leaf cells to the phloem, and vice versa, can be restricted by the passage through plasmodes-mata (Lucas & Wolf, 1999 Santa Cruz, 1999, Lough & Lucas, 2006). However, newly formed leaves may become directly infected via the phloem (Gilbertson & Lucas, 1996).

Ecological implications - Our results clearly demonstrate that physiological integration facilitates systemic virus spread between interconnected ramets of a clonal plant. Both distance and direction between interconnected ramets im-pose no restrictions on the spatial pattern of disease development after local WClMV infection. Our results therefore support the notion that viral spread can represent a negative side-effect of stoloniferous growth. Enhanced viral infec-tion of young offspring ramets is likely to cause long-term restrictions on growth and development of clonal host plants as the production of new meris-tems is of great importance for the growth and survival of T. repens (Beinhart, 1963; Huber & During 2000). Our data show that internal virus transport is not slower than the rate of clonal expansion, which implies that T. repens is unable to escape virus infec-tion by rapid vegetative growth as shown for fungal infections (Wennström & Ericson, 1992; Frantzen, 1994) and subsequent ramet disconnection (Kelly, 1995; Stuefer et al., 2004; Hay & Kelly, 2008). If clonal plants cannot escape viral infections, they may largely depend on their defence systems to cope with viruses. Defence mechanisms such as RNA silencing can protect plants through the specific degradation of viral RNA (Voinnet, 2001). It is conceivable that RNA silencing is activated in clonal plants which may enable ramet net-works to overcome viral infections in the longer term. Stuefer et al. (2004) suggested that clonal plants could provide long term storage space for viruses and simultaneously serve as disease vectors in plant communities by repeatedly causing inter- and intra-specific infections of susceptible hosts in their neighbourhoods. Our data support the prediction that

clonal plants may serve as excellent highways for virus infections. A spatially dispersed, guerrilla-like growth form (long internodes between ramets and pro-fuse branching) of infected host plants should facilitate virus distribution throughout the community. Clonal plants are expected to serve as stepping-stones for virus infections within populations and communities. Systems with high frequencies of clonal plants may therefore be especially vulnerable to virus infections (Stuefer et al. 2004). These potential costs in terms of virus infection may ultimately lead to negative selection pressures on clonal integra-tion (D’Hertefeldt & van der Putten, 1998).

ACKNOWLEDGEMENTS. We thank Dick Verduin, Dick Lohuis and Stephan Win-ter for their advice on the ELISA testing. We are grateful to Sara Gómez, Hans de Kroon and Eric Visser for their useful comments on a previous version of this manuscript. This work was partially funded by the Netherlands Organisa-tion for Scientific Research (NWO), VIDI-016.021.002 grant to JFS.

ABSTRACT

Genotype by environment (GxE) interactions are important for the long-term persistence of plant species in heterogeneous environments. Disease has of-ten been suggested as a key factor for the maintenance of genotypic diversity in plant populations. However, empirical evidence for this contention is scarce. We analyzed the effects of White clover mosaic virus (WClMV) on the perform-ance of natural genotypes of white clover (T. repens) and examined genotype-specific plant responses to WClMV infection. WClMV had an overall negative effect on plant performance. However, these effects differed greatly between host genotypes. Moreover, the relative fitness and associated ranking of geno-types changed significantly between virus-free and virus-prone environments. The GxE interactions reported in this study emphasize the potential impor-tance of plant viruses for shaping ecological and micro-evolutionary processes in host populations by promoting genotypic diversity.

KEYWORDS: disease; genotypic variation; GxE interactions; host-parasite inter-action; plasticity; Trifolium repens; virus; WClMV.

INTRODUCTION

Genotypic diversity is essential for the long-term maintenance of species in natural environments (Hedrick et al., 1976; Gillespie & Turelli, 1989; Vellend, 2006). It is the primary substrate for natural selection to act on (Fisher, 1958; Endler, 1986), and genotypic diversity can profoundly impact on ecological processes at the population, community and ecosystem level (Hughes et al., 2008). Genotypes often differ in their responses to environmental conditions. Such GxE interactions represent qualitative or quantitative variation in pheno-typic plasticity (Via et al., 1995; Pigliucci, 2005; Conover & Schultz, 1995; Zhi-votovsky et al., 1996; Fordyce et al., 2006) and are commonly visualized by non-parallel reaction norms (Conover & Schultz, 1995; Sultan, 2007). Geno-type-specific responses to environmental variation are of primary importance for the coexistence of genotypes (Silander, 1985; Gillespie & Turelli, 1989), as they fuel micro-evolutionary processes in natural environments with spatio-temporally complex selection regimes (Sultan, 2000; Fordyce et al., 2006). Disease has repeatedly been proposed as a key factor for the mainte-nance of genotypic diversity in plant populations (Haldane, 1949; Burdon, 1987; Kirchner & Roy, 2001; Summers et al., 2003; Bradley et al., 2008). Pathogens are believed to exert strong selection pressure on plants (Jarosz & Davelos, 1995) and they can profoundly affect the structure, diversity and functioning of plant populations (Dobson & Crawley, 1994; Godfree et al., 2007; Bradley et al., 2008). Viruses may play a crucial role (Malmstrom et al.,2005) in shaping micro-evolutionary processes and genotypic diversity in plants (Gilbert, 2002; Burdon et al., 2006). Plant viruses are virtually ubiquitous in the field and they can strongly decrease host performance and fitness (Hull & Davies, 1992; Bosque-Pérez et al., 1998; Strange & Scott, 2005). Variable selection, caused by genotype-specific responses to viral infections (GxE inter-actions), is likely to counteract selection forces which tend to depress host plant diversity. Three conditions should be met if viruses are to preserve genotypic di-versity in their host plants via GxE interactions (Mitchell-Olds, 1992). First, there should be genotypic variation in components determining plant fitness. Second, the ranking of genotypes in terms of performance and fitness should change between different patches of the environment, preventing a single genotype from dominating multiple environments. Third, plant populations should experience environmental heterogeneity in the sense of variation in virus incidence. In this study we aim at showing that plant-virus interactions meet all of these conditions and may hence be a common, yet underappreci-

ated player influencing patterns and dynamics of genotypic diversity in wild plants. Variation in virus incidence has clearly been demonstrated for our sys-tem by other studies. Sherwood (1997) has shown that the incidence of White clover mosaic virus (WClMV) fluctuates considerably in populations of the stoloniferous herb Trifolium repens. The same study reports that 30 % of all plants were infected by WClMV and infection levels varied from 1 % to 96 % between different sites. In another study, 1 % of the plants were found infected with WClMV at one site, while infection rates ranged from 9-46 % at another site (Coutts & Jones, 2002). We experimentally investigated the first and sec-ond condition proposed by Mitchell-Olds (1992) by testing the following spe-cific hypotheses: (i) Genotypes of T. repens vary significantly with respect to fitness-related traits, (ii) virus infection has negative effects on fitness-related traits and plant performance, and (iii) the ranking of genotypes changes in re-sponse to WClMV infection. In order to test these hypotheses, we examined the growth and perform-ance of genetically distinct individuals of T. repens in virus-free and virus-prone environments, and we evaluated GxE interactions in terms of genotype-specific plant responses to WClMV infection. We report strong negative effects of virus infection on plant performance. Substantial GxE interactions resulted in significant shifts in the relative fitness of host genotypes grown in virus-free and virus-prone environments, respectively.

MATERIALS AND METHODS

Study organisms - The stoloniferous herb Trifolium repens L. was used for this study. T. repens can propagate vegetatively through the production of ge-netically identical offspring (ramets) which develop at the nodes of horizontally growing stems (stolons), or by sexual reproduction. Each individual ramet con-sists of a single leaf, an internode and meristems which can develop into roots, branches and flowers. Clonal growth represented by the total number of clonal offspring as well as the total biomass can be considered as closely fitness-related traits (Sackville-Hamilton et al., 1987). In this case fitness is defined as “the rate of change in number of units (ramets) carrying a certain allele or al-lele complex” (Wikberg, 1995). We used eleven genotypes, which had been collected from natural field populations (riverine grasslands) and grown under common garden and greenhouse conditions for two years before we con-ducted this experiment (see Weijschedé et al., 2006 for details). White clover mosaic virus (WClMV; necrosis strain, originally isolated

from T. repens) was obtained from the Deutsche Sammlung von Mikroorganis-men und Zellkulturen GmbH (Braunschweig, Germany). This virus is a mem-ber of the genus Potexvirus and is transmitted mechanically between hosts. WClMV is not transmitted by insect vectors such as aphids (Tapio, 1970).

Experimental design - In April 2005 we started the experiment with rooted, apical cuttings consisting of six ramets each. 14 Cuttings per genotype were individually planted in plastic trays (15x23x5 cm) filled with SERAMIS clay granules (Masterfoods GMbH, Verden, Germany). These cuttings (subsequently referred to as plants) were grown in a greenhouse with a 16 hours light and 8 hours dark period at 19/18 ºC. High pressure sodium lamps (Hortilux-Schréder 600W, Monster, Netherlands) were switched on automati-cally whenever the irradiance dropped below 250 mol m-2 s-1. Stolons that grew out of the trays were bent back to facilitate root formation. At 19 and 32 days after transplanting the cuttings, each tray received 50 ml half-strength Hoagland nutrient solution. All plants were nodulated with rhizobium bacteria. Seven replicates of each genotype were randomly assigned to the con-trol treatment (no virus infection) and to the infection treatment (experimental virus infection), respectively. Ten days after planting, all plants in the virus treatment were inoculated with WClMV on the third and fourth youngest ramets. Inoculation was performed mechanically with cell sap prepared by grinding calcium-chloride dried WClMV-infected plant material in inoculation buffer (50 mM Na2HPO4 buffer, 1 mM EDTA, set to pH 7.0 with HCl). Leaves on the third and fourth ramets were dusted with carborundum (500 mesh), and 10 μl virus suspension was rubbed on each leaf by hand. Control plants were mock-inoculated with inoculation buffer only. All plants were re-inoculated on the third and fourth youngest ramets (newly formed) after 17 days with fresh WClMV infected material (Phaseolus vulgaris leaves infected with WClMV ob-tained from DSMZ), using the same procedure as described above All plant material was harvested 50 days after the first inoculation, and the fourth youngest leaf was sampled for ELISA testing. The length of the pri-mary stolon was measured and the number of ramets on the primary stolon, number of ramets on the branches, number of branches and the number of flowers were counted. All plant material was dried at 70 ºC for 72 h and dry weights of stolons, leaves, flowers and roots were measured separately. We used the total number of ramets and the total biomass of plants to calculate relative fitness values for genotypes within the control and virus treatment. The relative fitness was calculated as mean genotypic trait value divided by the overall mean (i.e., mean of all genotypes) for the same trait within the control

or the virus treatment, respectively.

ELISA testing - Qualitative analysis of WClMV presence was tested by DAS-ELISA (based on Clark & Adams, 1977) on the ninth oldest ramet on the pri-mary stolon. WClMV proved to be present in all tested leaf samples, showing that virus application was successful (data not shown).

Data analysis - We performed a two-way analysis of variance (ANOVA) to determine the effects of WClMV on development, growth and flowering of T.repens and to analyze genotype x virus interactions for relative fitness, using genotype and virus infection as main factors. Genotype was regarded as a random factor. The effect of WClMV on flowering was analyzed only for those four genotypes that produced flowers (e.g. A15, B51, D129 and D134). All tests were carried out with SAS, version 9.1 (SAS Institute Inc., Cary, USA).

RESULTS

Virus effects on plant performance - WClMV infection had a clear negative effect on plant growth and development (Table 1a, virus effects). The total number of ramets was reduced by 25%, and WClMV caused a 17% decrease in the branching probability of primary stolons. WClMV infection caused a re-duction in the biomass of roots (28%) and leaves (32%), as well as in the total plant biomass (30%), but had no significant effect on stolon biomass or propor-tional biomass allocation to plant organs (Table 1b). WClMV infection did not change any of the recorded flowering traits (Table 1c).

Genotype x Environment interactions - Genotypic variation was strong and significant with respect to all traits (Table 1a-c, genotype effects). More impor-tantly, however, genotypes differed greatly in their response to WClMV infec-tion (Table 1a, genotype x virus interaction). In several genotypes (e.g. A120, C79 and D39) WClMV caused a dramatic decrease in the length of branches, while other genotypes (e.g. B35, B122 and D134) showed no response to the virus treatment (Fig. 1a). Similar patterns were recorded for the percentage of branches on the primary stolon (Fig. 1b) and for the total number of vegetative offspring produced during the experiment (Fig. 1c). With the exception of biomass allocation to leaves, all biomass produc-tion and allocation traits showed genotypic variation in the effect of WClMV infection (Table 1b). Total biomass values (Fig. 1d) decreased strongly for some genotypes (e.g., A120, C79 and D39), while they remained equal for

others. Average biomass per ramet (Fig. 1e) and percentage biomass alloca-tion to the stolons (Fig. 1f) decreased in some infected genotypes, and in-creased in others.

*Deg

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l no.

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3910

5662

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8496

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Erro

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Tabl

e 1a

-c. S

tatis

tical

ana

lysi

s of

the

effe

cts

of W

ClM

V, g

enot

ype

and

thei

r in

tera

ctio

n (A

NO

VA)

on (

a) d

iffer

ent d

evel

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enta

l and

arc

hite

ctur

al

traits

, (b)

abs

olut

e bi

omas

s of

diff

eren

t pla

nt p

arts

and

bio

mas

s al

loca

tion

(% b

iom

ass)

to v

ario

us p

lant

par

ts, a

nd (

c) d

iffer

ent f

low

erin

g tra

its. P

r. st

olon

= th

e pr

imar

y st

olon

.

a b c

Figure 1a-f. Genotypic variation in the effect of WClMV infection for (a) length of the branches, (b) percentage of branches on the primary stolon, (c) total number of ramets, (d) total biomass, (e) average biomass per ramet, and (f) biomass allocation to the stolons, e.g. % biomass stolons. All traits show a significant genotype x virus interaction (two-way ANOVA, *= p < 0.05, **= p < 0.01 and ***= p < 0.001), e.g. the genotypes are differently affected by the virus infection.

Length branches

0

10

20

30

40

50

60

Control Virus

Leng

th (c

m)

Percentage branches pr. stolon

0

20

40

60

80

100

Control Virus

Perc

enta

ge

Total number of ramets

0

20

40

60

80

100

Control Virus

Num

ber

Total biomass

0.0

0.5

1.0

1.5

Control Virus

Biom

ass

(g)

Biomass per ramet

0

2

4

6

8

Control Virus

Biom

ass

(mg)

% Biomass stolons

0

10

20

30

40

Control Virus

Perc

enta

ge

a

fe

dc

b**

**

*

**

****

A120 A15 B35 B51 B122 C50C61 C79 D39 D129 D134

The flowering probability of primary stolons showed a non-significant trend to differ between genotypes after infection with WClMV (Table 1c). There was no significant interaction effect between virus infection and genotype for any of the flowering traits.

Relative fitness - The relative fitness in terms of total number of ramets and total biomass shows a strong genotype-by-virus interaction (Table 2, genotype x virus interaction) leading to significant shifts in genotype ranking between the two experimental environments (Fig. 2a-b). The highest ranking genotypes in the control environment, such as genotypes A120 and D39, consistently occu-pied lower ranks in the virus-prone environment (Fig. 2a-b).

Relative fitness

df* MS† df* MS† F‡ p df* MS† F‡ p df* MS† F‡ p

Total no. ramets 130 0.1477 10 1.8000 12.19 <0.0001 1 0.0013 0.00 0.9523 10 0.3342 2.26 0.0179

Total biomass 130 0.2220 10 2.7417 12.35 <0.0001 1 0.0000 0.00 0.9947 10 0.9259 4.17 <0.0001

Error Genotype Virus Genotype*virus

Table 2. Statistical analysis (ANOVA) of the relative fitness of the different genotypes in both treatments.

*Degrees of freedom; †Mean square; ‡F-statistics

Total number of ramets

0.0

0.5

1.0

1.5

2.0

Control Virus

Rel

ativ

e fit

ness

Total biomass

0.0

0.5

1.0

1.5

2.0

Control Virus

Rel

ativ

e fit

ness

a b* ***

A120 A15 B35 B51 B122 C50C61 C79 D39 D129 D134

Figure 2a-b. GxE interactions for relative fitness of the different genotypes, represented as reaction norm plots. Relative fitness of each genotype is calculated relative to the mean fitness within an environ-ment (e.g. control or virus), where fitness is expressed by both the total number of ramets (a) and total biomass (b). Relative fitness of genotypes differs significantly between the virus free and virus prone environments (two-way ANOVA, *= p < 0.05, **= p < 0.01 and ***= p < 0.001).

DISCUSSION

GxE interactions can sustain genotypic diversity in natural environments, thereby promoting long-term coexistence (Gillespie & Turelli, 1989) and en-hancing system stability (Thompson, 1991). Here we demonstrate that virus infections can substantially shift the ranking of plant genotypes with respect to relative fitness (in terms of total number of ramets and total biomass) between virus-free and virus-prone environments. Based on our findings and on general predictions from evolutionary theory we suggest that viruses may play an im-portant yet unrecognized role for the long-term maintenance of genotypic di-versity in their host populations through variable selection and GxE interac-tions. For pathogen-caused GxE interactions to occur, infections should sig-nificantly affect plant performance and fitness. In our study WClMV compro-mised biomass accumulation, retarded vegetative propagation and curtailed the spatial expansion capabilities of infected as compared to non-infected plants. These findings are in accordance with other studies reporting negative effects of virus infection on plant performance (Jones,1992; Funayama et al., 1997; Dudas et al., 1998; Godfree et al., 2007). The effects of virus infection on host plants showed conspicuous levels of genotypic variation for most development and growth-related traits recorded in this experiment. Consequently, the genotypes which performed best in the virus-free environment did not occupy high ranks in the virus-prone environ-ment, and vice versa. This suggests that virus infections can cause significant alterations in genotype frequencies within host populations that depend mainly on vegetative reproduction for growth. The observed GxE interactions indicate genotypic dissimilarities in host plant sensitivity to viral infection, which may be caused by variation in virulence levels. Virulence can be understood as patho-gen-caused reduction of host fitness (Brown et al., 2006) and is mainly a func-tion of the activity of the host tissue (Hull, 2004) and the degree of host resis-tance. Pathogen virulence can vary considerably between host genotypes (Godfree et al., 2007). Fast-growing and hence larger genotypes are likely to experience higher virulence levels than slow-growing, smaller genotypes (Morrison, 1996) owing to their superior metabolic activity which promotes vi-rus replication. Mitchell-Olds (1992) postulated three conditions for the maintenance of genotypic variation through GxE interactions. The first condition demands genotypic variation in fitness: here we demonstrated strong genotypic variation in closely fitness-related traits such as clonal offspring production and total

plant biomass. These results are consistent with other studies showing geno-typic variation for many fitness-associated traits in T. repens (Turkington, 1989; Weijschedé et al., 2006). The second condition requires genotype fit-ness to vary between environments: the performance of genotypes differed greatly between virus-free and virus-prone environments in our study, resulting in a marked shift in the ranking of genotypes between these environments. Our results are in agreement with Pagan et al. (2008) who show that different accessions of Arabidopsis thaliana vary in their response on growth invest-ment to infection with Cucumber mosaic virus. The third condition requires environmental heterogeneity in virus prevalence which has been clearly dem-onstrated by others (Sherwood, 1997; Norton & Johnstone,1998; Coutts & Jones, 2002) for the plant-virus system used in this study. They show that vi-rus incidence shows considerable fluctuations both within and between popu-lations of T. repens. Marked heterogeneity in disease incidence has also been described for other plant viruses (Bosque-Pérez et al., 1998; Godfree et al., 2007). We hence conclude that viruses are excellent candidates for maintain-ing genotypic variation in their hosts, and that their virtual omnipresence in nature may render them prime biotic agents counteracting declines of geno-typic diversity in natural plant populations. The shift in relative fitness between genotypes indicates the existence of trade-offs between plant performance in virus-free and virus-prone sites. As a result genotypes successful in the control environment perform relatively much worse in virus-prone environments. This suggests the potential for WClMV to provoke differential selection on T. repens genotypes, which may lead to nega-tive frequency-dependent selection in host populations. Such negative fre-quency-dependent selection occurs when common genotypes as compared to less common genotypes suffer from a fitness disadvantage in virus-prone envi-ronments (Haldane, 1949; Brunet & Mundt, 2000; Rueffler et al., 2006). Although there is ample evidence of significant negative effects of virus infection on plant vigour, there is surprisingly little information about their po-tential role as selective agents. The hypothesis that pathogens can maintain genotypic variation in their hosts has been often proposed, but hardly ever studied empirically. Our data suggest that virus infections may be excellent candidates for promoting genotypic diversity in their host plants and call for empirical studies that analyze virus-induced frequency dependent selection. The apparent negative effects on plant performance, significant GxE interac-tion and evident repercussions for relative fitness reported in this study clearly stress the significance of virus infections for ecological and evolutionary proc-esses and identify viruses as possible key factors for driving population dy-

namics and selection in the wild.

ACKNOWLEDGEMENTS. We thank Arjen Biere, Hans de Kroon, Eric Visser for useful comments on a previous version of the manuscript. This work was par-tially funded by the Netherlands Organisation for Scientific Research (NWO), VIDI-016.021.002 grant to JFS.

ABSTRACT

Plants are constantly challenged by multiple stresses and their ability to cope with them is of major importance for their growth and survival in an ever-changing environment. Due to trade-offs, plants have to prioritize environ-mental stress factors and express functional traits accordingly. While inde-pendent effects of various abiotic and biotic stress factors on plant perform-ance and fitness have been well described, their combined effects have re-ceived far less attention. Here we analyze the combined effects of plant infec-tions with White clover mosaic virus and shading on the performance and de-velopment of different genotypes of the stoloniferous herb Trifolium repens,and we analyze the effects of these factors on traits associated with photosyn-thesis. Virus infection and shading separately caused negative effects on plant performance. However, virus infection did not alter maximum photosynthetic rates of their hosts, while a clear reduction was caused by shading. The nega-tive effects of virus infection on plant performance, as well as genotypic varia-tion in these effects were far less pronounced in light-limiting than in high light conditions. Based on these findings we suggest that the negative effects of virus infection strongly depend on the environmental conditions, which may facilitate the maintenance of genotypic variation in the host plant. Our results support the notion that virus aggressiveness can vary strongly between habi-tats and seasons and help explain the observed spatial and temporal variation in virus effects.

Keywords: disease; multiple stress; photosynthesis; shading; Trifolium re-pens; White clover mosaic virus.

INTRODUCTION

Under natural conditions, plants are continuously exposed to a multitude of abiotic and biotic stresses and they have to produce appropriate responses to these challenges in order to survive, grow, and reproduce. Plant responses to stress can be energetically costly, and resource investments in a particular response may compromise functional responses to other challenges. Like-wise, shared molecular pathways and coupled physiological processes may preclude the simultaneous expression of plant features directed towards multi-ple stress factors. Natural selection acts to optimize plant performance and fitness over longer periods of times, conditioning plants to prioritize their action against multiple challenges and to balance investments accordingly. Conse-quently, the priority given to a certain stress factor may provide insight into the frequency and severity of environmental factors in the evolutionary history of plants. Although simultaneous stress by abiotic and/or biotic factors is proba-bly a universal aspect of natural systems, plant responses to multiple chal-lenges have only rarely been studied in an integrative way (Izaguirre et al., 2006). Variation in light levels is one of the best-studied abiotic factors influenc-ing plant performance and fitness. Light availability can be considered of pri-mary importance, since plant growth depends crucially on the resources gen-erated by photosynthesis (Lambers et al., 1998; Roberts & Paul, 2006; Frank-lin, 2008). Under natural conditions light levels show enormous variation in space and in time resulting from a multitude of factors such as spatio-temporal differences in plant structure and density, environmental perturbations on dif-ferent spatial scales, grazing, and others. In order to cope with changes in lo-cal light climate plants from well-lit environments display a wide array of re-sponses, commonly referred to as the shade-avoidance syndrome (Schlichting & Smith, 2002; Franklin, 2008). The negative effects of light limitation (Schmitt & Wulff, 1993; Schmitt et al., 1999) may be particularly severe for plants with a limited ability to avoid shading, such as stoloniferous species which lack verti-cally growing stems and hence depend on petiole elongation for enhancing their light harvesting capacity in the presence of competitors (Thompson, 1995; Leeflang et al., 1998; Vermeulen et al., 2008). Viruses are virtually ubiquitous in the field and they cause innumerable plant diseases with effects ranging from very mild to lethal (Gibbs & Harrison, 1976; Hull, 2004). Viruses can dramatically decrease host performance and fitness and are responsible for substantial economic losses in a wide variety of commercially important plants (Hull & Davies, 1992; Bosque-Pérez et al.,

1998; Strange & Scott, 2005). The wide distribution of viral infections in natural plant populations suggests that many plants are simultaneously exposed to both, variation in light availability as well as infections by one or more viruses. Moreover, virus infections are well-known to exert negative effects on the pho-tosynthetic capacity of many plants by causing decreases in the net photosyn-thetic rate (Zhou et al., 2004; Guo et al., 2005), chlorophyll content (Funuyama-Noguchi, 2001; Guo et al., 2005; Wilhelmova et al., 2005), and Rubisco activ-ity (Zhou et al., 2004; Guo et al., 2005,) Most stoloniferous plants invest mainly in vegetative propagation through horizontal expansion and can hence easily be shaded by neighboring plants with vertically growing stems. In addition, clonal plants face a high risk of virus infection owing to the long life span of individual genotypes (Van Baalen & Sabelis, 1995; Stuefer et al., 2004; Van Mölken & Stuefer, 2008). Viruses utilize plant resources for reproduction (Hull, 2004) and light limitation reduces photosynthetic rates (Lambers et al., 1998), both resulting in dimin-ished resource availability. Consequently, investment in shade-avoidance re-sponses may be realized at the expense of defense expression, and vice versa, potentially leading to serious consequences for plant performance. On the other hand, Balchandran et al. (1997) proposed that low light and high nu-trient availability might decrease the effects of virus infections on photosynthe-sis, resulting in a weakened virus effect on plant growth and performance un-der light limiting conditions. The reaction of plants to multiple stressors may show intra-species variation, since genotype-specific responses to environmental conditions en-ables organisms to operate successfully in a range of environments. Fine-grained, variable selection can lead to significant genotype-by-environment interactions which prevents individual genotypes from performing best under a broad range of environmental conditions. Genotypic diversity represents quali-tative or quantitative variation in phenotypic plant plasticity, i.e. in the ability of genotypes to express different morphological or physiological phenotypes in diverse environments (Via et al., 1995; Pigliucci, 2005; Conover & Schultz, 1995; Zhivotovsky et al., 1996; Fordyce et al., 2006). The aim of this study is to investigate the combined effects of White clo-ver mosaic virus and shading on the performance of the stoloniferous herb Trifolium repens. More specifically, we tested the following hypotheses. (i) Shading and virus-infection have independent negative effects on fitness-related traits and on photosynthetic capacity; (ii) the environmental light condi-tions have strong impact on viral disease development (iii) the separate and combined effects of virus infection and shading vary among different plant

genotypes. In order to test these hypotheses, we examined the performance and photosynthetic capacity of genetically distinct individuals of T. repens in virus-free and virus-prone environments, under high and low light conditions, re-spectively. Furthermore, we evaluated GxE interactions in terms of genotype-specific plant responses to WClMV infection and shading.

MATERIALS AND METHODS

The stoloniferous herb Trifolium repens L. was used for this study. T. repenspropagates vegetatively through genetically identical offspring individuals (ramets), which develop at the nodes of horizontally growing stems (stolons). Each individual ramet consists of a single leaf, an internode and meristems which can develop into roots, branches and flowers. The performance and fitness of long-lived, clonally growing plants can be estimated by the number of ramets and the total biomass produced in a given period of time (Sackville-Hamilton et al., 1987; Wikberg, 1995). We used six genotypes which had been collected from natural field populations (riverine grasslands) and grown under garden and greenhouse conditions for two years before we conducted this experiment (see Weijschedé et al., 2006 for details). White clover mosaic virus (WClMV) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Ger-many). This virus belongs to the potex group and is transmitted mechanically between hosts (Tapio, 1970). Plants in the virus treatment were mechanically inoculated with infected cell sap, prepared by grinding fresh leaves of WClMV-infected Phaseolus vulgaris var. rentegevers plants in inoculation buffer (50 mM Na2HPO4 buffer, 1 mM EDTA, pH 7.0). Leaves were dusted with car-borundum (500 mesh) and 10 μl virus suspension was rubbed on each leaf by hand. Control plants were mock-inoculated with inoculation buffer only. Plants in the shade treatment were placed individually under cages cov-ered with shade cloth that reduced the light intensity (PPFD) by approximately 55 % and caused a slight reduction in the R:FR ratio of transmitted light (from 1.8 to 1.6 under greenhouse conditions).

Experimental setup - Twenty rooted, apical cuttings per genotype consisting of six ramets were individually planted in plastic trays (12x16x5 cm) filled with a 2:1 mixture of potting soil and sand. These cuttings (subsequently referred to as plants) were grown in a greenhouse with a 16 hours light and 8 hours dark period at 19/18 ºC. High pressure sodium lamps (Hortilux-Schréder 600W,

Monster, Netherlands) switched on automatically whenever irradiance levels dropped below 250 mol m-2 s-1. Stolons that grew out of the trays were bent back to facilitate root formation. All plants showed proper nodulation. Shading and WClMV were applied in a fully factorial design with 4 treat-ments and five randomly assigned replicates per genotype and per treatment. Three days after planting, all plants in the virus treatment were inoculated with WClMV on the third-youngest and fourth-youngest ramet. Inoculation was re-peated after 18 days. Shading was applied six days post inoculation. The photosynthetic capacity (Pmax) was measured on the newly formed third-youngest leaf of each plant in the fifth week post inoculation. CO2 uptake was measured with a portable infrared gas analyzer (LiCor 6400, Lincoln Ne-braska, USA), after the leaves were given time to acclimate. Optimal light and CO2 levels were determined with a different set of plants and set to a PPFD of 800 μmol m-2 s-1 light and 800 μmol mol-1 CO2. The flow rate was kept con-stant at 350 μmol s-1 (equal to 500 ml min-1), the temperature inside the leaf chamber was 23 °C, and the relative air humidity ranged between 70 and 80%. All plant material was harvested at 39-40 days after the first inoculation. To analyze the leaf chlorophyll content (based on Wintermans & Mots, 1965), six leaf disks (diameter 5 mm) were cut into smaller pieces and placed to-gether in dark plastic tubes containing 3 ml ethanol (96 %) and CaCO3. All tubes were placed on ice during sampling and stored over night at 4 C. Ab-sorption was measured at 410, 649, 665 and 710 nm. Chlorophyll and carote-noid concentrations were calculated according to Lichtenthaler and Wellburn (1983). One fully expanded leaf of approximately the same developmental stage was sampled from each plant and its surface was measured separately. These leaves were then dried at 70 C for 48h, and weighed individually to allow cal-culating specific leaf areas. To analyze the nutrient concentration we grinded the leaves individually (Retsch® mm300, Haan, Germany) and nitrogen and carbon concentrations were measured with a CNS analyzer (type NA1500; Carlo Erba Instruments, Milan Italy). The number of ramets on the primary stolon, number of ramets on the branches, number of branches and the num-ber of flowers were counted. Dry weights of stolons, leaves, flowers and roots were measured separately.

ELISA testing - Qualitative analysis of WClMV presence was tested by DAS-ELISA (based on Clark & Adams, 1977). This method is based on the specific binding of WClMV antibodies to WClMV particles in the test sample. Alkaline

phosphatase pre-attached to the WClMV antibodies converts the enzymatic substrate (p-nitrophenyl phosphate), resulting in yellowing of the samples which contain WClMV. The absorbance was measured at 405 nm with a Wal-lac VICTORtm2 1420 spectrophotometer (WALLAC Oy, Turku, Finland). Fresh leaves of T. repens stock plants were used as a negative control. Plants were considered infected if the absorbance of the test sample was at least twice as high as that of the negative controls (Dijkstra & de Jager, 1998).

Data analysis - To determine the effects of WClMV and shading on perform-ance and photosynthesis-related traits we performed a three-way analysis of variance, using genotype, virus and shade as main fixed factors. To determine the effect of virus infection within shading treatments we sliced the virus by shade interaction using the SLICE option in the LSMEANS statement of PROC GLM. All tests were carried out with SAS 9.1 (SAS Institute Inc., Cary, USA).

RESULTS

WClMV was present in the tested leaf samples (data not shown), as confirmed by DAS-ELISA, showing that the virus was successfully applied to our experi-mental plants

Virus effects on plant performance - WClMV infection had a negative effect on plant growth and development, independent of shade (Table 1, virus ef-fects). WClMV infection reduced the total number by 10 %. The number of ramets on the primary stolon, number of flowers and branching probability of primary stolons remained unaffected. The biomass of roots (9 %) and stolons (12 %), as well as a total plant biomass (7 %) were decreased in WClMV infected plants (Table 1), while the dry weight of leaves and flowers showed no response to infection (Table 1). Proportional biomass allocation to stolons decreased by 5 % with virus infec-tion, while allocation to leaves increased by 3 %. Biomass allocation to roots and flowers was not affected by WClMV infection (Table 1). Remarkably, we found no effect of virus infection on photosynthetic ca-pacity or any other photosynthesis-related trait such as specific leaf area (SLA), chlorophyll and carotenoid concentrations, nitrogen content, and C/N ratio (Table 1). We observed a 2 % increase in carbohydrate content due to virus infection (Table 1).

Trai

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No. r

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961

0.01

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Gen

otyp

e (g

en)

Shad

eVi

rus

Viru

s * s

hade

* ge

nSh

ade

* gen

Viru

s * g

enVi

rus

* sha

de

Tabl

e 1.

Sta

tistic

al a

naly

sis

of th

e ef

fect

s of

TC

V, s

hadi

ng, g

enot

ype

and

thei

r int

erac

tions

(AN

OV

A) o

n a

num

ber o

f pla

nt d

evel

opm

enta

l and

gro

wth

trai

ts, p

hoto

synt

hetic

ca

paci

ty a

nd o

ther

pho

tosy

nthe

sis

rela

ted

traits

. Pr.

stol

on =

prim

ary

stol

on.

Shading effects on plant performance - As expected, shading had a clear and strongly negative effect on plant performance and development, inde-pendent of virus infection (Table 1, shade effects). Shading decreased the to-tal number of ramets as well as the number of ramets on the primary stolon (56 % and 10 % respectively). The number of flowers (91 %) and the branch-ing probability (17 %) sharply declined as a consequence of shading (Table 1). The accumulation of root, leaf, stolon, and flower biomass (68 %, 55 %, 68 % and 91 %, respectively) as well as total biomass production (61 %) were strongly reduced by shading (Table 1). The biomass allocation towards roots (18 %), stolons (16%) and flowers (73 %), dropped with shading (Table 1). Low light levels caused a significant increase of 18 % in the biomass allocation

Traits

df F p F p df F p F p

No. ramets pr. stolon 1 0.34 0.5607 0.56 0.4547 11 2.79 0.0034 3.41 0.0005

No. ramets branches 1 7.50 0.0074 1.19 0.2782 11 12.51 < 0.0001 1.53 0.1321

Total no. ramets 1 7.57 0.0071 1.12 0.2916 11 12.29 < 0.0001 1.51 0.1394

No. flowers 1 0.01 0.9143 0.24 0.6285 5 8.12 < 0.0001 0.25 0.9383

% Branches pr. stolon 1 0.19 0.6655 4.62 0.0342 11 7.26 < 0.0001 3.17 0.0011

Biomass roots (g) 1 5.07 0.0266 0.76 0.3864 11 8.83 < 0.0001 1.10 0.3675

Biomass stolons (g) 1 15.61 0.0001 0.71 0.4021 11 8.86 < 0.0001 0.52 0.8870

Biomass leaves (g) 1 2.56 0.1130 0.08 0.7717 11 9.15 < 0.0001 2.33 0.0140

Biomass flowers (g) 1 0.12 0.7328 0.11 0.7389 5 8.56 < 0.0001 0.25 0.9383

Total biomass (g) 1 5.70 0.0189 0.26 0.6080 11 6.26 < 0.0001 1.03 0.4279

% Biomass roots 1 0.05 0.8254 3.04 0.0843 11 6.80 < 0.0001 4.27 < 0.0001

% Biomass stolons 1 8.33 0.0048 4.70 0.0327 11 18.17 < 0.0001 30.83 < 0.0001

% Biomass leaves 1 1.98 0.1623 3.79 0.0546 11 8.79 < 0.0001 12.37 < 0.0001

% Biomass flowers 1 0.96 0.3324 0.46 0.5013 5 9.67 < 0.0001 1.68 0.1586

SLA 1 0.14 0.7140 0.03 0.8652 11 1.40 0.1842 2.48 0.0088

Chlorophyll a 1 0.01 0.9352 0.19 0.6610 11 2.41 0.0110 1.05 0.4118

Chlorophyll b 1 0.24 0.6275 0.01 0.9155 11 4.14 < 0.0001 0.81 0.6296

Carotenoid 1 0.19 0.6643 1.84 0.1781 11 1.65 0.0969 1.51 0.1396

Photosynthesis 1 2.00 0.1610 0.09 0.7643 11 1.46 0.1582 1.08 0.3836

% Nitrogen 1 0.00 0.9657 0.52 0.4710 11 1.29 0.2403 1.51 0.1411

% Carbohydrates 1 0.25 0.6178 8.74 0.0039 11 0.59 0.8368 1.53 0.1324

C/ N ratio 1 0.00 0.9808 0.07 0.7909 11 21.13 0.3453 2.07 0.0294

Virus Virus * genotype

ShadeLightShadeLight

Table 2. Statistical analysis of the effects of WClMV, and the interaction with genotype within each level of light (SLICE option, ANOVA).

to leaves (Table 1). Shading negatively affected photosynthetic capacity (29 % reduction) and most of the photosynthesis-related traits (Table 1). SLA increased with 31 % under shaded conditions, while the chlorophyll content decreased by 11 % on average (Table 1). Carotenoid content remained unaffected. We observed a decrease in nitrogen and carbohydrate content while the C/N ratio showed an increase due to shading (10 %; Table 1).

Dealing with two stress factors - To our surprise we found only a few and rather weak interaction effects between the virus and shading treatments. With the exception of a decrease in stolon biomass, virus infection did not alter plant responses to reduction in light availability (Table 1, virus x shade effects). A detailed analysis of viral infection effects within each of the two light treat-ments revealed strong differences in plant responses to infection between the shade and the light treatment, respectively (Table 2). For example, total ramet and biomass production decreased significantly by virus infection under high-light conditions (Fig. 1a-b). Shading canceled out virus effects on these traits. The same was true for the number of ramets on side branches, root and stolon biomass (Table 2). The reverse pattern was observed for branching frequen-cies of the primary stolon, biomass allocation towards leaves, and for leaf car-bohydrate content (Table 2). These traits were only affected by virus infection if plants were simultaneous exposed to shading. Biomass allocation to the stolons was decreased by WClMV under both light conditions (Fig. 1c). Photo-synthetic rate, specific leaf area and C/N ratio remained unaffected by virus infection, regardless of the light availability (Fig. 1d-f).

Genotype x Environment interactions - Genotypic variation was strong and significant with respect to all traits, except for leaf carbohydrate content (Table 1, genotype effects). Genotypes did not differ in their response to WClMV in-fection, except for SLA (Table 1, virus x genotype interaction). This greatly contrasts with the strong interaction between genotypes and shading found in many of the developmental and growth-related traits (Table 1, shade x geno-type interaction). Genotypes did not respond differently to shading with respect to photosynthetic capacity and other photosynthesis-related traits (Table 1). We found no significant three-way interactions between genotype, virus infection and shading (Table 1, virus x shade x genotype interaction). By in-specting interactions between virus infection and host genotypes within each light condition (Table 2; genotype x virus interaction) we found all growth and developmental traits to show genotypic variation for WClMV infection under

high light conditions. Many of these virus x genotype interactions disappeared in the shade treatment (Table 2). Genotypic variation for virus effects on the total number of ramets (Fig. 2a) and on total biomass (Fig. 2b) was only de-tected under high light conditions. Genotypes differed in the effect of virus in-fection on biomass allocation towards the solons, independent of light treat-ments (Fig. 2c). We did not observe a genotype x virus interaction for photo-synthesis (Fig. 2d). However, genotypic mean values for chlorophyll content

0

150

300

450

Tota

l num

ber o

f ram

ets

0

2

4

6

Tota

l bio

mas

s (g

)

0

10

20

30

40

Light Shade

Per

cent

age

biom

ass

stol

ons

0

10

20

30

Pm

ax (μ

mol

m-2

s-1

)

0

20

40

60

80

SLA

(m2 k

g-1)

0

5

10

15

Light Shade

C/ N

ratio

a

b

c

d

e

f

**

*

**

ns

ns

ns

ns

ns

ns

ns

ns

*

Control Virus

Figure 1a-f. Effects (means ± SE) of WClMV within each level of light on (a) total number of ramets, (b) total biomass, (c) percentage biomass allocated to stolons, (d) maximum photo-synthetic rate (Pmax), (e) specific leaf area (SLA), and (f) C/N ratio (ns= not significant, *= p < 0.05, **= p < 0.01 and ***= p < 0.001).

Figure 2a-f. Genotypic variation in the effect of WClMV infection for (a) total number of ramets, (b) total biomass, (c) percentage biomass allocated to stolons, (d) maximum photosynthetic rate (Pmax), (e) specific leaf area (SLA), and (f) C/N ratio. Virus x genotype interactions are given for the light (left-hand column) and shade (right-hand column) treatments, separately (ns= not significant, *= p < 0.05, **= p < 0.01 and ***= p < 0.001).

0

150

300

450

Tota

l num

ber o

f ram

ets

0

2

4

6

Tota

l bio

mas

s (g

)

0

10

20

30

40

Per

cent

age

biom

ass

stol

ons

0

10

20

30

Phot

osyn

thet

ic ra

te(

mol

m-2

s-1)

0

20

40

60

80

SLA

(m2

kg-1

)

0

5

10

15

C/ N

ratio

A120 A15 B35 C50 C61 D134

a

b

c

d

e

f

Light

Control Virus

Shade

Control Virus

***

***

***

ns

***

ns

ns

ns

ns

ns

*

**

0

150

300

450

0

2

4

6

0

10

20

30

40

0

10

20

30

0

20

40

60

80

0

5

10

15

a

b

c

d

e

f

(Table 2) differed between virus infected and healthy plants grown under well- lit conditions. Conversely, we observed significant genotypic variation in the effect of virus infection on SLA (Fig. 2e) and C/N ratio (Fig. 2f) if plants were grown under shaded conditions (Table 2).

DISCUSSION

In natural environments, plants are bound to face multiple abiotic and biotic stresses at the same time. Light limitation (Bazzaz & Carlson, 1982; Walters et al., 1993; Valladares et al., 2007; Schmitt & Wulff, 1993; Schmitt et al., 1999) and viral infections (Jones, 1992; Funayama et al., 1997; Dudas et al., 1998; Godfree et al., 2007) are very common factors in the field, and each of them can considerably affect plant performance and prompt specific plant re-sponses. Here we report that the negative effects of virus infection on plant per-formance declined in light-limiting conditions, and that virus infection had no detectable effect on the maximum photosynthetic rate and photosynthesis-related traits of their host plants. Genotypic variation in the effects of virus in-fection was more pronounced in high light than in shaded conditions, implying that the vulnerability of plants to virus infection is environment-dependent. The relative disadvantage of susceptible genotypes is hence likely to disappear under shaded conditions, possibly promoting genotypic diversity through vari-able environment-dependent selection. This study provides evidence that WClMV compromised biomass accu-mulation and retarded vegetative propagation of infected plants, corroborating earlier findings of negative effects of virus infection on plant performance (Jones, 1992; Funayama et al., 1997; Dudas et al., 1998; Godfree et al., 2007; van Mölken & Stuefer, submitted). As to be expected shading led to very strong alterations of nearly all traits recorded in this experiment. In contrast to several other studies (Funayama & Terashima, 1999; Funuyama-Noguchi, 2001; Zhou et al., 2004; Guo et al., 2005; Wilhelmova et al., 2005), however, we found no effect of WClMV infection on the photosynthetic capacity of leaves. This suggests that the photosynthetic rate of the plants was not altered by virus infection. Furthermore, photosynthesis-related traits such as the spe-cific leaf area, and the chlorophyll and carotenoid contents were unaffected by virus infection. Shading did strongly depressed maximum photosynthetic rates, and the specific leaf area clearly increased under light-limited conditions. These findings suggest that WClMV did not affect plant performance via pho-tosynthesis or closely photosynthesis-related traits.

The observed reduction in plant performance by WClMV infection must hence originate from other processes than those studied in this experiment. Resources utilized for virus replication can translate into growth reduction of the host plant (Sacristan et al., 2005). Virus-induced changes in hormone bal-ances (Clarke et al., 1999) may also play a role in determining disease symp-toms. Plant defense mechanisms targeted at combating virus infections may also be energetically costly (Heil, 2001). It becomes increasingly clear that in addition to resource-mediated effects, viruses can disrupt a multitude of plant functions through very specific effects on molecular and developmental path-ways (Culver & Padmanabhan, 2007), thereby adding significantly to the po-tential complexity of plant virus interactions and their ecological and evolution-ary consequences. We found no significant interaction between shading and virus infection, however a detailed analysis of interaction effects between shading and virus infection revealed that viral disease effects on various plant traits disappeared under shaded conditions. Fitness-related traits such as clonal offspring pro-duction and biomass accumulation were clearly reduced by virus-infection un-der high-light conditions, but remained unaffected when host plants were shaded. These results point at a strong environment dependence of virus in-fection on plant performance, which is likely to alter the rank order of compet-ing genotypes in different environments. Shading may thus promote the growth and survival of plants in virus-prone environments, thereby acting to maintain susceptible genotypes within host populations. These results are in analogy with other studies reporting reduced effects of virus infection under low-light conditions (Balachandran et al., 1997; Funayama & Terashima, 1999). With the exception of stolon biomass, the effects of virus infection on host plants showed no genotypic variation for any of the traits measured in this experiment, implying that genotypes responded uniformly to virus infection. Shading, by contrast, prompted genotypes to respond in different ways as shown by conspicuous levels of genotypic variation for all developmental and growth-related traits. Strikingly, all growth and development-related traits showed clear genotypic variation for virus effects, but many of these interac-tions vanished under shaded conditions. We conclude that genotypic variation in response to virus infection depends largely on host growth conditions. Plant growth rates could play an important role in determining the sever-ity of WClMV infection, as viral replication and disease development is mainly a function of the activity of the host tissue (Hull, 2004) and the degree of host resistance. Fast-growing plants may experience higher virulence levels than

slow-growing, plants (Morrison, 1996) due to their superior metabolic activity which promotes virus replication. In our experiment growth rates were heavily depressed under shaded conditions which may explain the discrepancy in vi-rus effects between high light and shaded conditions. The results of this study contrast a significant body of work reporting negative effects of virus infection on maximum photosynthetic rates and other photosynthesis-related traits (Funayama & Terashima, 1999; Funuyama-Noguchi, 2001; Zhou et al., 2004; Guo et al., 2005; Wilhelmova et al., 2005). Although there is ample evidence of profound negative effects of shading and virus infection on plant vigor, surprisingly little information is available on the interaction between these stressors. Environmental conditions, such as light availability influence the development of virus infections, supporting the notion that virus aggressiveness may vary between habitats and seasons. Our results emphasize that the effect of simultaneous stresses cannot be extrapolated directly from the effects of individual stress factors alone and call for explicit studies analyzing the integrative effects of multiple stressors on plant perform-ance and fitness.

ACKNOWLEDGEMENTS. We thank Hans de Kroon for comments on a previous version of the manuscript. This work was partially funded by the Netherlands Organisation for Scientific Research (NWO), VIDI-016.021.002 grant to JFS.

ABSTRACT

Plants are constantly challenged by multiple stresses and their ability to cope with them is of major importance for their growth and survival in natural envi-ronments. Due to trade-offs, plants have to prioritize environmental stress fac-tors and express functional traits accordingly. Here we analyze the combined effects of Turnip crinkle virus (TCV) and shading on the performance and de-velopment of Arabidopsis thaliana and attempt to link the results to virus accu-mulation. TCV infection and shading separately caused conspicuous negative effects on plant performance. We found a strong interaction between these two stress factors and the negative effects of virus infection on plant perform-ance, were far less pronounced in light-limiting than in high light conditions. However, there was no clear difference in virus accumulation between plants from different light treatments. Based on these findings we suggest that the negative effects of virus infection strongly depend on the environmental condi-tions. Our results support the notion that virus aggressiveness can vary strongly between habitats and seasons and help explain the observed spatial and temporal variation in virus effects.

Keywords: Arabidopsis thaliana; disease; multiple stress; shading; Turnip crinkle virus; virus accumulation.

INTRODUCTION

Plants can invest their resources into the three main functions of growth, re-production and defense. Since these investments are energetically costly, in-vestment in one function may hence compromise one or several other func-tions. Shared molecular pathways and coupled physiological processes may prohibit or hamper the simultaneous expression of traits coupled with growth, reproduction and defense. In natural systems, plants are constantly challenged by a multitude of different abiotic and biotic stresses which requires plants to prioritize their responses and balance their investments to maximize perform-ance and fitness (Izaguirre et al., 2006). Although concurrent stress by abiotic and biotic agents is a general feature of natural systems, plant responses to multiple challenges have not often been studied in an integrative way. One of the best-understood environmental factors influencing plant per-formance is variation in light levels. The amount and spectral composition of irradiation can be regarded of crucial importance for plant growth (Lambers et al., 1998; Roberts & Paul, 2006; Franklin, 2008), Under natural conditions light levels can vary immensely in space and in time, owing to spatiotemporal varia-tion in plant structure and density, environmental perturbations on different scales, grazing, and other factors. Plants from well-lit environments display a wide array of responses, commonly referred to as the shade-avoidance syn-drome to buffer changes in local light availability (Schlichting & Smith, 2002; Franklin, 2008). Viruses can cause numerous plant diseases with effects ranging from very mild to lethal (Gibbs & Harrison, 1976; Hull, 2004) and Viral infections can be considered very common under field conditions. Host performance and fit-ness can strongly decrease due to viral infections resulting in substantial eco-nomic losses in a wide variety of commercially important plants (Hull & Davies, 1992; Bosque-Pérez et al., 1998; Strange & Scott, 2005). The wide distribution of viral infections in natural plant populations suggests that many plants are simultaneously exposed to both, variation in light availability as well as infec-tions by one or more viruses. Both light limitation and virus infections result in reduced resource avail-ability. Viruses utilize plant resources for reproduction (Hull, 2004) and low light availability reduces photosynthetic rates (Lambers et al., 1998). Conse-quently, investment in defense expression may be realized at the expense of investment in shade-avoidance responses, and vice versa, potentially impair-ing plant growth and performance. However, shading may as well reduce virus replication, as this process depends on the activity of the host tissue (Hull,

2004), which in turn is suppressed by shading. This interaction would result in a weakened virus effect on plant performance when infections coincide with the exposure of host plants to low light availabilities (chapter 6). In this study we examine the combined effects of Turnip crinkle virusand shading on the performance of Arabidopsis thaliana. In particular, we tested the hypotheses that (i) shading and virus-infection have independent negative effects on plant performance, and (ii) that light conditions have a strong impact on viral disease development. To test these hypotheses we in-fected Arabidopsis thaliana plants with Turnip crinkle virus and placed them under three different light regimes. We measured various plant traits in an at-tempt to link them to virus accumulation in different light conditions.

MATERIALS AND METHODS

Twenty day old Arabidopsis thaliana ecotype Columbia (col-0) seedlings were individually planted in pots (5x5x5.5 cm) filled with potting soil (Special custom mix™ compost with Intercept® 5GR; The Scotts Company, Suffolk, UK). These pots were part of a bigger tray which contained 24 pots in total. We planted a total of 78 seedlings in 4 trays. We covered the trays with plastic for another 4 days to maintain high air humidity. The plants were grown in a climate cham-ber with a 10 hours light and 16 hours dark period at continuous 20 ºC. Virus infection and two levels of shading were applied in a fully factorial design with six treatments and thirteen randomly assigned replicates per treatment. Five days after planting, all plants in the shade treatment were placed under cages covered with either one or four layers of white shade cloth, reduc-ing the light intensity (PPFD) by approximately 30 % and 60 %, respectively. Nine days after planting, the plants in the virus treatment were mechanically inoculated with Turnip crinkle virus (TCV). Two randomly selected leaves per plant were dusted with carborundum (500 mesh) and 5 μl virus suspension (0.95 μg ml-1 in Tris-HCl buffer, pH 7.3) were rubbed on each leaf by hand. Control plants were mock-inoculated with buffer only. All plants were rinsed with H2O after inoculation. Five days later the first symptoms were observed in the virus-infected plants. Eighteen days post inoculation, leaves were counted and the length and width of the longest leaf was measured. We randomly harvested three plants per treatment for RNA extraction. The plants were cut at the base (between shoot and roots), put individually in 15 ml plastic tubes and instantaneously frozen in liquid nitrogen. The material was stored at -80 °C until further proc-essing. All plants were harvested in the same way, dried to constant mass for

72h at 60 °C and weight to measure total leaf dry mass. To analyze virus accumulation in plants from different light conditions, total nucleic acids were extracted from leaf material following a protocol adapted from White and Kaper (1989). Three sam-ples from the low light conditions did not yield suffi-cient amounts of RNA for northern blotting. North-ern blotting was performed with a Hybond N+ (Amersham Biosciences) membrane, according to the manufacturer’s protocol, and UV cross-linked. Probes complementary to TCV-RNA were end-labeled with [�-32P] ATP with the use of T4 polynu-cleotide kinase (New England BioLabs). Hybridiza-tion was performed in 5 ml of 7 % SDS in 0.25 M sodium phosphate, 0.5 M EDTA buffer pH 7.2, and the hybridization temperature was set at 65 °C. Membranes were washed twice at hybridization temperature with 2x SSC, 0.1 % SDS for 20 min, and 1x 0.1 % SDS for 15 min. The membrane was exposed to Phosphor Imager plates. To determine the effects of TCV and shad-ing on plant performance we performed a two-way analysis of variance, using virus and shade as main fixed factors. All tests were carried out with SAS 9.1 (SAS Institute Inc., Cary, USA). RESULTS

Plant growth was strongly reduced by TCV infec-tion (Table 1, virus effects). The number of leaves as well as the length and width of the longest leaf showed a conspicuous decrease due to the virus treatment (28 %, 39 % and 16 % respectively). TCV infection had a strong negative effect on plant growth, resulting in a marked decline in total leaf bio-mass (Fig. 1a) and biomass per leaf (39 %). Shading had profound effects on plant performance (Table 1, shade effects). The number of leaves, as well as the length and width of the longest leaf decreased sharply under low as compared to high light conditions (33 %,

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29 % and 28 %). We observed a similar pattern for the total leaf biomass (Fig. 1b) and the mean biomass per leaf (63 %). Virus infection and shading showed a significant interaction, indicating that the effects of viral disease on plant performance vary with changing light conditions (Table 1; Fig. 2). The size difference between non-infected and vi-rus-infected plants was much more pronounced in the high light as compared

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Figure 1a-b. Main effects (means ± SE) of (a) TCV infection and (b) different light conditions on total leaf bio-mass. Virus infection and light level both affect total leaf biomass (two-way ANOVA, *= p < 0.05, **= p < 0.01 and ***= p < 0.001).

High light Low light Intermediate light

Control

Virus

Figure 2. Plant size of virus-infected and non-infected (control) plants at 18 days post inoculation, when grown under different light regimes.

*** ***

to the low light environment (Fig. 2, plants at 18 days post inoculation), imply-ing that severity of viral infection was dependent on light conditions experi-enced by the host plants. A more detailed analysis of viral infection effects within each of the three light treatments revealed strong and consistent differ-ences in plant responses to infection between the different light levels. The effects of virus infection on the number of leaves, the length of the longest leaf varied with changing light conditions (Fig. 3a-b). The reduction in leaf width caused by virus infection was independent of the light availability (Fig. 3c). The negative effect of virus infection on total leaf biomass decreased with light availability (Fig. 3d). In high light conditions, TCV caused a decrease the total leaf biomass of 66 %, while the reduction in total leaf biomass caused by TCV in low light conditions was 33 %. Our Northern blot analysis revealed that control plants remained free of virus infection throughout the experiment, since all these plants showed no radioactivity and hence did not display a dark coloring on the membrane (Fig. 4). In contrast, we detected TCV in all the virus-infected plants tested (Fig. 4,

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Figure 3a-d. The combined effects (means ± SE) of virus infection and light availability on (a) number of leaves, (b) length longest leaf, (c) width longest leaf, and (d) total leaf biomass. All traits except for width longest leaf show a significant virus x light interaction (two-way ANOVA, ns= not significant,*= p < 0.05, **= p < 0.01 and ***= p < 0.001).

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dark lanes). While the intensity of the color correlates with virus content, we observed no clear difference in virus accumulation between the different light treatments (Fig. 4).

DISCUSSION

In nature plants are bound to face abiotic and biotic stresses at the same time. Light limitation and viral infections may often coincide. Each of them can con-siderably affect plant performance and prompt specific plant responses (Bazzaz & Carlson, 1982; Jones, 1992; Schmitt & Wulff, 1993; Walters et al., 1993; Funayama et al., 1997; Schmitt et al., 1999; Dudas et al., 1998; Godfree et al., 2007; Valladares et al., 2007).Their integrative effects on plants, how-ever, are not well understood (Balachandran et al., 1997). Here we report that light limiting conditions greatly decrease the negative effects of virus infection on plant performance, implying that the vulnerability of plants to virus infection is strongly environment-dependent. In this study we show that plant development and growth are strongly compromised by TCV, supporting earlier findings of negative effects of virus infection on plant performance (Jones, 1992; Funayama et al., 1997; Dudas et al., 1998; Godfree et al., 2007; van Mölken & Stuefer, submitted; chapter 6).As expected, plant performance declined with light limitation, which had a very strong negative effect on all traits recorded in this experiment. We observed a

C= Control T= TCV HL= High Light IL= Intermediate light LL= Low light

C C C C C C C C T T T T T T T

HL HL HL IL IL IL LL LL HL HL HL IL IL IL LL

Dilution series (μg)7.5 5.0 2.5 1.25 0.7

Figure 4. Virus accumulation in plants grown under different light regimes. Dark lanes indicate virus presence. The intensity of the dark colour relates to the concentration of virus particles.

significant interaction between shading and virus infection. Detailed analysis of the interaction effects between shading and virus infection revealed that the effects of viral disease on most traits (except leaf width) sharply declined with shading. The symptoms of TCV infection became more pronounced with in-creasing light levels, resulting in a progressive discoloration of leaves, and in retarded plant growth, suggesting that the severity of virus infection is strongly environment-dependent. This finding supports the notion that under heteroge-neous field conditions shading may improve relative fitness and survival chances of susceptible plants in virus-prone environments. The decline of virus effects under shaded conditions may originate from decreased growth rates of host plant when grown in light-limited environments.Viral replication and disease development is mainly a function of host resis-tance and the activity of the host tissue (Hull, 2004). Fast-growing plants may experience higher virulence levels than slow-growing plants (Morrison, 1996) as their superior metabolic activity promotes virus replication. In our experi-ment, increasing levels of shading resulted in enhanced depression of growth rates. Surprisingly, the decrease in growth rate did not result in a different amount of virus particles in plant tissues between plants grown under various light conditions, suggesting that virus replication was not retarded by light limi-tation. In contrast, the development of systemic disease symptoms did seem to depend on the light availability, and was much more pronounced under high-light conditions. Handford & Carr (2007) found that symptom development of three other viruses in A. thaliana changed with varying light conditions and, consistent with our results, they did not observe a change in virus accumula-tion under different light regimes. Their results suggest that symptom develop-ment is influenced by carbohydrate metabolism. This could mean that the con-sequences of virus infection in terms of leaf coloring are much more important for growth reduction than virus replication per se. While there is no doubt that both shading and virus infection can have profound negative effects on plant vigor, surprisingly little information is avail-able on the interaction between these stress factors. Environmental conditions such as light can greatly influence the development of virus infections. Our results show that this interaction does not necessarily depend on virus replica-tion alone, but that symptom development plays a much more important role. The fact that light limitation can reduce the negative effects of virus infection on plant performance supports the notion that virus aggressiveness can vary between habitats and seasons, and it shows that plant responses to simulta-neously acting stress factors cannot be reliably extrapolated from studies on the effects of individual stress factors. Further research on the integrative ef-

fects of multiple abiotic and/ or biotic factors may reveal unexpected conse-quences for plant performance and fitness.

ACKNOWLEDGEMENTS. We thank Hans de Kroon for comments on a previous version of the manuscript. This work was funded by grants awarded to TvM by the European Molecular Biology Organisation (EMBO; short-term fellowship), the Royal Botanical Society of the Netherlands (KNBV; Stipendium Bottelier) and the Netherlands Society for Plant Biotechnology and Tissue Culture (NVPW; travel grant).

KEYWORDS: virus; vaccination; herbivores; ecology; interactions.

In natural environments plants are bound to face simultaneous exposure to pathogens and herbivores both of which can dramatically impair their perform-ance and fitness (Hull & Davies, 1992; Strange & Scott, 2005; Schoonhoven et al., 2005). Recent studies on plant responses to multiple biotic stress factors (Paul et al., 2000; Pieterse & Dicke, 2007) show that pathogen-infected plants frequently exhibit increased susceptibility to herbivores (Koornneef & Pieterse, 2008), thereby further adding to the paradigm that pathogens have predomi-nantly negative effects on plants. However, the ecological implications of pathogen infections for plant-herbivore interactions remain largely elusive (Paul et al., 2000; Pieterse & Dicke, 2007). Here we provide evidence that vi-rus infections can positively affect plant performance by protecting host plants from damage by root-feeding insects. We show that virus-infected plants emit a volatile blend that repels potentially damaging saprophagous insects. This indicates that virus infections can provide ecological benefits to their hosts by mitigating herbivore damage. Hence, virus-infected plants may be conferred with a fitness advantage if protection from herbivory outweighs biomass losses through virus infection. Ecological benefits associated with virus infections are likely to affect co-evolutionary trajectories, as they may weaken selection for plant resistance and pathogen virulence. Based on our results we propose that viruses qualify as key candidates for plant vaccination against insect herbi-vores.

The notion that virus infections confer ecological benefits on host plants origi-nated from the observation that infected plants can occasionally perform better than non-infected plants. While studying the competitive strength of virus-infected plants we observed that White Clover (Trifolium repens L.) plants in-fected with White clover mosaic virus (WClMV) performed significantly better than non-infected plants in terms of vegetative offspring (i.e. total number of ramets; F= 36.45, p <0.0001) and total biomass production (F=36.02, p<0.0001; Fig. 1a-b). This positive effect of virus infection on plant growth was in stark contrast to our previous findings and many other studies showing negative effects of virus infection on host plant performance and fitness (Jones, 1992; Funayama et al., 1997; Dudas et al., 1998; Godfree et al., 2007). A closer examination revealed that the substrate of both virus-infected and uninfected plants was heavily infested with larvae of fungus gnats (Bradysia sp.). Fungus gnats are pervasive greenhouse pests and common in nature (Menzel et al., 2006; Kjaerandsen et al., 2007; Vilkamaa et al., 2007), causing substantial damage in indoor crop systems (Harris et al., 1996). Since the plants in our experiment were under simultaneous attack by fungus gnat

larvae and WClMV, the observed positive effect of virus infection on plant per-formance was taken as an indication of the virus conferring ecological benefits on its host. This led us to investigate the potential of plant viruses for “vaccinating” their host plants against insect feeding. Such vaccination effects have previously been reported for plants exposed to subsequent attacks by two different herbivores (Kessler & Baldwin, 2004; Dicke & Sabelis, 1998), but have not been described for pathogens. To investigate the vaccination poten-tial of plant viruses we tested the following hypotheses: (i) virus infection en-hances the host plant’s resistance against larval feeding, resulting in a stronger growth depression in non-infected as compared to virus-infected plants; (ii) adult female fungus gnats are repelled by virus-infected plants. This hypothesis requires virus infection altering the composition of the plants’ vola-tile blend, thereby making them less attractive for egg-laying herbivores.

To test the first hypothesis we applied both fungus gnat larvae and WClMV to T. repens plants in a fully factorial design. Plants were grown in sterilized clay grains and plants of the same treatment were put into mesh cages to avoid contamination with fungus gnats from elsewhere in the greenhouse. We planted 16 rooted cuttings of T. repens for each of the four treatments; 7 days later we applied the virus by mechanical inoculation. We allowed the virus in-fection to establish for another 5 days before adding the first fungus gnat lar-vae. A total of 50 larvae (3rd and 4th instars) were applied at 4 different time points to ensure a continuous herbivore pressure. At 36 days post inoculation we harvested all plants and analyzed their growth and development. WClMV infection and fungus gnat infestation caused a decrease in total biomass pro-duction of 27.6 % (F= 22.00, p <0.0001) and 53.3 % (F= 124.36, p <0.0001), respectively, as compared to either non-infected or non-infested control plants.

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Figure 1. Virus infection leads to an increase in plant performance. (a) Mean number of ramets and (b) mean total biomass produced by non-infected plants and virus-infected plants, respectively. Error bars indicate standard errors, *= p < 0.05, **= p < 0.01 and ***= p < 0.001.

The total number of ramets was significantly reduced by fungus gnat infesta-tion (41.7 %; F=125.04, p <0.0001), but remained unaffected by virus infection (F=2.72, p= 0.1041). This study showed that both factors severely impaired plant performance, and that fungus gnat infestation caused the strongest re-duction in plant growth, indicating that these primarily saprophagous insects can also be very herbivorous. Contrary to our prediction, however, virus-infected plants did not perform better than non-infected plants when infested with fungus gnats. There was no significant interaction between virus infection and fungus gnat infestation with respect to the total number of ramets (F=0.04, p= 0.8419; Fig. 2a). Although the absolute reduction in biomass caused by fungus gnat infestation was less severe in virus-infected plants as compared to non-infected plants (F= 0.24, p= 0.0272; Fig. 2b), this interaction did not result in a superior performance of virus-infected plants over non-infected plants. Direct effects on herbivore feeding are hence unlikely to be of importance for

the hypothesized plant vaccination by viruses. Alternatively, virus infection al-tering the composition of the volatile blend emitted by host plants could result in selective oviposition by adult fungus gnats, as insects can effectively dis-criminate between different bouquets of volatile organic compounds (VOCs; Dicke & Sabelis, 1988; Turlings et al., 1990; De Moraes et al., 1998; Kessler & Baldwin, 2001). A preference of adult female fungus gnats for non-infected plants over virus-infected plants could account for the benefits of virus infec-tion in our first study. To test this hypothesis we determined the effect of virus infection on the volatile blend and subsequent preference of adult fungus gnats. First we analyzed the headspace of plants that were either virus-inoculated or mock inoculated 4 days prior to VOC sampling. The sampling

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Figure 2. Virus-infected plants do not show a superior performance over non-infected (Control) plants in the presence of fungus gnat larvae. (a) Mean number of ramets and (b) mean total biomass produced by non-infected and virus-infected plants, in the absence or presence of fungus gnat larvae, respectively. Error bars indicate standard errors, ns= not significant, *= p < 0.05, **= p < 0.01 and ***= p < 0.001.

was repeated 13 times. We selected a total of 24 compounds known for their involvement in plant defence for further analysis. We used a canonical dis-criminant analysis to determine whether the control and virus-infected plants can be distinguished on the basis of their volatile blends. This analysis pro-vides a ranking of compounds according to their power for discriminating VOC blends emitted from virus-infected and virus-free plants, respectively. High-ranking compounds correlate most closely with the first canonical axis and are hence most important for differentiating between the two groups. Control and infected plants emitted significantly different volatile blends ( = 0.9231; p=<0.0001; Fig. 3a), thereby confirming the notion that virus infection resulted in markedly altered VOC blends. Strikingly, -Caryophyllene, which was most important for the discrimination between the two groups (rank 1 for total ca-nonical structure; table 1), was exclusively detected in the headspace of virus-infected plants and was not emitted by control plants (Fig. 3b). Recent evi-dence shows that -caryophyllene serves as an indirect defence signal in her-bivore-induced maize plants and leads to the attraction of entomopathogenic nematodes (Rasmann et al., 2005) . Our results provide novel insight in the complex interplay between viruses and their host and they may be of great importance for identifying the mechanisms underlying plant vaccination by vi-ruses.

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Figure 3. Difference in volatile bled between virus-infected and non-infected plants and the preference of fungus gnats for either one of these two types of plants. (a) Results of a canonical discrimination analysis between volatile blends emitted by non-infected and virus-infected plants, respectively. Non-infected and virus-infected plants can be dis-criminated on the basis of their volatile blends, and virus infection alters the emission of volatile compounds. The analysis is based on 24 selected volatile compounds. (b) Emis-sion of -caryophyllene increases with virus infection, and this compound not emitted by non-infected plants. Error bars indicate standard errors, *= p < 0.05, **= p < 0.01 and ***= p < 0.001.

To find out whether adult fungus gnats prefer healthy over virus-infected plants, we carried out choice tests with adult females of Bradysia. We placed WClMV-infected and non-infected plants in jars at the upwind end of a glass Y-tube olfactometer (Takabayashi & Dicke, 1992), and established a constant air flow towards the lower part of the tube. For each comparison we individually released 8-10 adult female fungus gnats at the downwind end and recorded their preference for either virus-infected or uninfected plants in seven replicate trials. The preference of fungus gnats was expressed as a percentage choice for either one of the two plant groups. The adults clearly preferred non-infected plants (67%) over virus-infected plants (33%), confirming that fungus gnatadults discriminate between the two plant groups based on the different emis-sions of VOCs (Fig. 4, F= 12.56, p=0.0122).

While the mechanisms behind this interaction effects await further clarification, we hypothesize that virus-induced plant vaccination depends at least partly on the emission of -caryophyllene. Jasmonic acid (JA) may play an important role in this proc-ess, since increased JA levels are known to stimulate the biosynthesis of -caryophyllene (Thaler et al., 2002b; Ari-mura et al., 2008), and JA activity increases in response to WClMV infection in Phaseo-lus vulgaris, reaching a plateau within five days post inoculation (Clarke et al., 2000). Virus infections are often associated with salicylic-acid (SA) mediated defences and hypersensitive response, which in turn may negatively interfere with JA dependent defence (Koornneef & Pieterse, 2008). However, we did not observe WClMV-infection to result in a hypersensitive response in T. repens, suggesting that SA mediated defence may not play a prominent role in our system, as has been observed in other host plants (Clarke et al., 1998). The possible importance of JA is emphasised by the fact that Arabidopsis mutants deficient in JA production are preferred over wild-type plants by Bradysia adults (McConn et al., 1997). This preference resulted in a mortality of ~80 % of the mutants plants, but application of exogenous methyl-jasmonate protected the plants from Bradysia damage and reduced mortality rates to ~12 %. These studies show that WClMV infection can ac-

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Figure 4. Fungus gnats show a preference for non-infected (Control) as compared to virus-infected plants. Bars give the mean percentage of adult female fungus gnats which preferred non-infected or the virus-infected plants, as determined in dual choice tests. Error bars indicate standard errors, *= p < 0.05, **= p < 0.01 and ***= p < 0.001.

count for increased JA levels in host plants and that enhanced JA concentra-tions can lead to a better protection from fungus gnats. Our results suggest a key role for -caryophyllene in the JA mediated repellence of herbivorous in-sects. The analysis of plant-vaccination by viruses has led to new insights and hypotheses regarding the complex and ecologically relevant tripartite interac-tion between plants, herbivores and viruses. We propose that while viruses

Compound RI TCS Rank

F p

-Caryophyllene 1412 0.513 1 0 ± 0 420 ± 144 11.39 0.0027

1-Hexadecanol 1882 0.201 2 4872 ± 2421 97496 ± 92185 1.66 0.2113

2-Nonanone 1091 0.174 3 116 ± 51 196 ± 77 0.30 0.5907

Benzonitrile 978 0.174 4 682 ± 203 963 ± 252 4.59 0.0435

3-Hexen-1-ol-acetate (mix of E and Z ) 1008 0.139 5 8331 ± 3114 13154 ± 6282 1.13 0.2983

Pentanoic acid 880 0.116 6 717 ± 224 946 ± 334 0.21 0.6501

Dimethyldisulfide 738 0.098 7 449 ± 88 515 ± 105 0.06 0.8115

Acetic acid 569 0.094 8 4632 ± 1839 5985 ± 2255 0.48 0.4948

Ethylbenzene 857 0.072 9 17019 ± 6521 20452 ± 7242 0.02 0.8811

Methyl-cyclohexane 720 0.046 10 1324 ± 454 1473 ± 470 0.00 0.9995

2-Ethyl-1-hexanol 1032 0.041 11 24998 ± 8140 27530 ± 9620 0.00 0.9777

Menthol isomer 1174 0.034 12 1916 ± 857 2091 ± 592 0.14 0.7137

Acetic acid butyl ester 804 0.030 13 1127 ± 487 1234 ± 556 0.00 0.9980

Propyl-cyclohexane 921 -0.002 14 132 ± 37 132 ± 48 0.04 0.8413

-Pinene 927 -0.023 15 1344 ± 406 1282 ± 353 0.24 0.6276

2-Propanol-1-butoxy 938 -0.056 16 4234 ± 1438 3678 ± 1440 0.21 0.6481

Limonene 1027 -0.133 17 6171 ± 1742 4424 ± 2004 0.75 0.3961

1-Butanol 658 -0.169 18 5022 ± 2166 2945 ± 1197 1.30 0.2663

2-Hexenal (E ) 841 -0.182 19 1446 ± 552 880 ± 292 0.40 0.5339

Phenylethyl alcohol 1106 -0.196 20 357 ± 113 218 ± 86 1.33 0.2606

Ethyl-cyclohexane 823 -0.211 21 787 ± 312 426 ± 135 2.22 0.1508

2-Methyl-1,3-butadiene 522 -0.218 22 3058 ± 1119 1684 ± 573 3.03 0.0956

-Copaene 1371 -0.228 23 420 ± 157 209 ± 96 2.74 0.1120

2- -pinene 970 -0.229 24 626 ± 240 316 ± 121 3.77 0.0650

Control Virus

ANOVAMean peak area ml-1 sampled ± SE

Table 1. Volatile compounds emitted by virus-infected and non-infected plants. The linear retention indices (RI) used for identification of the compounds are given in the first column. The total canonical structure (TCS) pro-vides correlation coefficients between each compound and the first canonical axis, indicating the relative power of each compound for discriminating between volatile blends emitted by virus-infected and non-infected plants. The compounds are ranked according to their TCS values. Mean peak areas ml-1 sampled air are given for each compound in the control and the virus treatment, respectively. The last two columns give results of univariate ANOVA tests, comparing the concentrations of each compound in the control and virus treatment. Significant differences are indicated in bold face. -Caryophyllene is most important compound for discriminating between virus-infected and non-infected plants (highest TCS and rank) and the emission of this compound was altered significantly by virus-infection.

may harm plants and utilize their resources, they may also confer ecological benefits to their hosts. Significant fitness advantages may accrue for virus-infected plants if protection from herbivory outweighs performance losses by virus infection. Such ecological benefits of virus infections are likely to weaken selection for host resistance and they may allow for the long-term persistence of mild viruses under field conditions. The results of this study emphasize the potential importance of virus infections for plant-herbivore interactions and suggest that mild virus infections may be an excellent means for plant vaccina-tion against herbivores. The notion of viruses having exclusively negative ef-fects on host plants needs to be revised, taking into account the complex biotic interactions occurring in nature.

MATERIALS AND METHODS

Study organisms and experimental conditions - Trifolium repens L. (white clover) and White clover mosaic virus (WClMV) were used for all experiments. T. repens produces clonal offspring (ramets) which develop at the nodes of horizontally growing stems (stolons). The number of ramets and the total plant biomass can be considered closely fitness-related traits (Sackville-Hamilton, 1987). Except for the competition experiment we performed all experiments with cuttings from a single genotype. All experiments (unless stated differently) were carried out in a greenhouse with a 16/8 hours light/dark period, at 19/18 ºC. White clover mosaic virus (WClMV; necrosis strain, originally isolated from T. repens) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Plants in the virus treat-ments were mechanically inoculated with infected cell sap, prepared by grind-ing WClMV-infected plant material in inoculation buffer (50 mM Na2HPO4

buffer, 1 mM EDTA, set to pH 7.0 with HCl). Leaves were dusted with car-borundum (500 mesh), and 10 μl virus suspension was rubbed on each leaf by hand. Control plants were mock-inoculated with inoculation buffer only. Analy-sis of WClMV presence was tested by DAS-ELISA (Clark & Adams, 1977). Adult fungus gnats (Bradysia sp.) were collected from the greenhouse and maintained in the laboratory under controlled conditions. Adult female fungus gnats lay eggs into the soil and the emerging larvae feed on organic soil mat-ter and young roots. Fungus gnats are pervasive greenhouse pests and com-mon in nature (Menzel et al., 2006; Kjaerandsen et al., 2007; Vilkamaa et al., 2007). The collected adults were placed in 1 l plastic pots containing moist potting soil mixed with rabbit food (2:1/ v:v; Hope farms, Woerden, the Nether-lands) and allowed to propagate. Naive fungus gnats were used for all experi-

ments.

Plant performance experiments - To analyse virus effects on inter-genotypic competition, two rooted cuttings of eight different genotypes were individually planted in various combinations in a total of 120 trays (16x12x5 cm; four plants per tray). Plants in half of the trays were inoculated with WClMV. All plants were harvested 30 days post inoculation (dpi). The total number of ramets was counted, and total dry weight was measured. To study combined effects of fungus gnats and virus infection, 64 cut-tings were individually planted in plastic trays (16x12x5 cm) filled with sterilized clay grains (SERAMIS; Masterfoods GmbH, Germany) and placed in a climate chamber with a 16/8 h light/dark period at 20/18 ºC. Plants of the same treat-ment were put into mesh cages to avoid contamination with fungus gnats from elsewhere in the greenhouse. Fungus gnats and WClMV were applied in a factorial design. Cuttings assigned to the infection treatment were mechani-cally inoculated 7 days after planting. We allowed the virus infection to estab-lish for another 5 days before adding the first fungus gnat larvae. A total of 50 larvae (3rd and 4th instars) were applied at 4 different time points to ensure a continuous herbivore pressure. 20 Fungus gnat larvae were applied to each tray in the fungus gnat treatment on 6 and 13 dpi. Another 5 larvae per tray were added at 20 and 31 dpi. At 36 days post inoculation we harvested all plants, counted the total number of ramets was counted and measured total plant biomass.

Volatile measurements - To analyse volatile production of virus infected and non-infected plants we planted individual cuttings of 4 ramets each in plastic pots (7x7x8 cm). Cuttings in the virus treatment were inoculated with WClMV 4 days prior to the analysis. Each replicate consisted of 4 independent cuttings grouped together during the measurements; each cutting was used once. The sampling was repeated 13 times per treatment. VOC collection, desorption and identification were performed according to protocol (Soler et al., 2007) with the following modifications. The glass collection chambers containing the plants were constantly supplied at the top with 150 ml of pressurized air (Hoekloos, NL). The headspace was collected for 80 min in steel traps filled with 150mg Tenax TA and 150 mg Carbopack B, and analyzed after direct desorption with a GC-MS system. We measured five replicates per treatment and two background VOC profiles from pots filled with substrate in parallel sessions. The whole procedure was repeated with an additional eight repli-cates per treatment and one background VOC profile. Volatiles were desorbed

from the traps using an automated thermodesorption unit (model Unity, Mar-kes, Llantrisant, United Kingdom) at 200 °C for 12 min and focused on a cold Tenax trap (–10 °C). After 1 min of dry purging, trapped volatiles were intro-duced into the GC-MS (model Trace, ThermoFinnigan, Austin, Texas) by heat-ing the cold trap for 3 min to 270 °C (split ratio 1:4; column 30 m x 0.32 mm ID RTX-5 Silms; film thickness 0.33 m). The volatiles were detected by the MS operating at 70 eV in EI mode. Mass spectra were acquired in full scan mode (33-300 AMU, 0.4 scan sec-1). Compounds were identified by their mass spec-tra28 using deconvolution software (AMDIS) in combination with Nist 98 and Wiley 7th edition spectral libraries and by comparing their linear retention indi-ces (RI). Additionally, mass spectra and/or linear retention indices of chroma-tographic peaks were compared with values reported in the literature. Further confirmation of compound identification was obtained by interpolating retention indices of homologous series and by comparing analytical data with those of reference substances. The integrated signals generated by the AMDIS soft-ware from the MS-chromatograms were used for comparing treatments.

Choice tests - To determine whether adult fungus gnats prefer healthy over virus-infected plants, we carried out choice tests with adult females of Bra-dysia. We planted individual cuttings consisting of 4 ramets each in plastic pots (7x7x8 cm). A second set of replicates was planted in pots (5x7x5 cm). Cuttings in the virus treatment were inoculated with WClMV 18 and 3 days prior to the analysis for the first and second set of replicates, respectively. To allow for sufficient volatile emission, we placed 3 pots with plants of the same treatment together. For each treatment the cuttings were placed in a 5 l glass cuvette at the upwind end of one of the two arms of a closed-system Y-tube olfactometer (Takabayashi & Dicke, 1992), and established a constant air flow towards the lower part of the tube. 4 l min-1 of air (filtered through activated charcoal) was driven trough each olfactometer arm and pulled towards the base of the olfactometer by the laboratory vacuum system at 8 l min-1. After an acclimation period of 30 min, an individual female fungus gnat was placed at the downwind end of the olfactometer and observed for a maximum of 10 min. The choice was recorded when the insect remained in the far end of one of the arms for more than 20 s, or if it remained in the middle part (5 cm upwind of the junction) of one of the arms for more than 50 s. Six fungus gnats which did not make a choice within 10 min were excluded from subsequent analyses. We recorded the choice of 8-10 females per replicate and replaced the plants for each new replicate. The preference of fungus gnats was expressed as a percentage choice for either one of the two plant groups. The experiments

were carried out at 24 °C and repeated seven times in total on two separate days. The results from both trials were very similar and hence pooled for final analysis.

Statistical analysis - We performed a one-way analysis of variance (ANOVA) to determine the effects of WClMV infection on plant performance in the com-petition experiment. Main and interaction effects of WClMV and fungus gnats on plant growth were analyzed by two-way ANOVA. A subset of 24 volatile compounds was selected for further analysis, based on their possible role in plant-insect interactions. Similarities in the volatile blends between virus-infected and non-infected plants were analyzed by canonical discriminant analysis (CDA; Soler et al., 2007). CDA allowed for the identification of com-pounds that discriminate most clearly between treatments. This analysis pro-vides a ranking of compounds according to their power for discriminating VOC blends emitted from virus-infected and virus-free plants, respectively. High-ranking compounds correlate most closely with the first canonical axis and are hence most important for differentiating between the two groups. Simple corre-lation coefficients (total canonical structure) were used to quantify the associa-tion between compounds and the canonical axis. The probabilities of misclas-sification were estimated with the CROSS-VALIDATE option in SAS procedure DISCRIM. This approach reclassifies every data point as if it were a new ob-servation, thereby reconstructing the discriminant function without that obser-vation. Cohen’s (kappa) coefficient was calculated to assess the significance of classification agreement. A two-way ANOVA was carried out to test for the effect of virus infection on peak areas of each of the 24 selected compounds. Virus infection was used as main factor and sampling time as block effect. As-sumptions of ANOVA were checked for each variable prior to analysis. The effect of virus infection on fungus gnat preference was analyzed with repeated measures ANOVA, regarding each replicate as an independent subject. Per-centages were arcsin transformed. All tests were carried out with SAS, version 9.1 (SAS Institute Inc., Cary, USA).

ACKNOWLEDGEMENTS. We are grateful to Marcel Dicke, Mike Hay, Martin Heil, Hans de Kroon and Eric Visser for useful comments on a previous version of the manuscript. We thank Annemiek Smit-Tiekstra and Finy van Mölken for their help with the practical work. This work was partially funded by the Nether-lands Organisation for Scientific Research (NWO), VIDI-016.021.002 grant to JFS.

A DARK SIDE OF CLONAL INTEGRATION

Sharing resources between interconnected ramets facilitates the growth and survival of clonal plants in a variety of environments (van Groenendael & de Kroon, 1990; Stuefer et al., 1994, 1996). Furthermore, Gómez & Stuefer (2006) have shown that clonal integration allows for long-distance transport of internal defence signals in response to local herbivory. Although benefits of clonal integration are well-studied, the possible hazards associated with sys-temic pathogen spread between interconnected ramets are largely unknown (Wennström & Ericson, 1992; chapter 2). As argued in this thesis, clonal plants may provide long- term storage space for viruses, and at the same time they could serve as disease vectors in plant communities by causing inter- and intra-specific infections of susceptible hosts in their neighbourhoods (chapter 2). These processes may promote viral spread and may potentially lead to increased levels of pathogen virulence in clonal plants (chapter 3). In chapter 4 I have investigated the patterns and dynamics of viral dis-ease development in ramet networks of Trifolium repens, showing that all juve-nile ramets on the main stolon and on the basal branches become systemi-cally infected with White clover mosaic virus (WClMV). However, mature leaves on the main stolon remained virus-free, showing that patterns of dis-ease development depend on leaf age (Silander, 1985; Develey-Rivière & Ga-liana, 2007) and that virus import is restricted to developing plant parts. The results presented in this chapter show that such age-related resistance can cause considerable spatial variation in infection status within a clonal plant, and they confirm early predictions about intra-clonal heterogeneity in viral dis-ease expression (Silander, 1985). In chapters 5 & 6 I analyzed the effects of virus infection on the performance and development of T. repens. It became clear that WClMV infection can have a strong negative impact on clonal off-spring production (total number of ramets) and plant growth (total biomass). Besides a decrease in vegetative propagation, the architecture of the clonal network was notably affected by virus infection, as shown by a strong de-crease in branching probability (chapter 5). In chapter 6 I found qualitatively similar, but quantitatively smaller effects of WClMV infection on plant perform-ance than in chapter 5. The results from these chapters clearly show that physiological integra-tion facilitates systemic virus spread between interconnected ramets of a clonal plant, demonstrating the potential for a negative side-effect of stolonifer-ous growth. As a result of severe changes in development, growth and archi-tecture, virus-infected plants consisted of less ramets than non-infected plants,

possibly leading to negative implications for their resource capturing capaci-ties. Virus-infected plants may be considered spatially handicapped in exploit-ing fine-grained heterogeneous environments, and viral spread within clonal plant networks may thus have serious repercussions for the competitive ability of host plants.

GENOTYPE-SPECIFIC REPONSES

The apparent negative effects of virus infection on plant growth and develop-ment as described in chapters 5 & 6 may very well differ between genetically distinct host individuals. Genotypic diversity is essential for the long-term main-tenance of plant species in heterogeneous environments, and pathogens have often been proposed to preserve genotypic variation of their hosts (Haldane, 1949; Burdon, 1987; Kirchner & Roy, 2001; Summers et al., 2003; Bradley et al., 2008). However, this hypothesis has hardly ever been tested empirically. In chapter 5 I focused on the intra-specific variation in virus effects and explored the potential of virus infections for maintaining genotypic variation in their host. The effects of WClMV infection on T. repens showed conspicuous levels of genotypic variation for nearly all developmental and growth-related traits. As a result, those genotypes which performed best in the virus-free environment did not occupy the same ranks in the virus-prone environment, and vice versa. I found similar results for a subset of the same genotypes in chapter 6, showing considerable genotypic variation for virus effects under high light conditions. The shift in relative fitness between genotypes (chapter 5) indicates the exis-tence of trade-offs between plant performance in virus-free and virus-prone sites, implying that successful genotypes in the control environment sustain considerable losses in relative fitness in virus-prone environments. Because WClMV can alter the performance ranking of genotypes, virus infections are likely to cause profound alterations in genotype frequencies within host popu-lations. The potential of viruses to alter rank relationship between genotypes in terms of relative fitness may have important implications for the dynamics and outcome of intra-specific competition. The results from chapters 5 & 6 imply that virus infections may alleviate asymmetric competition effects, allowing subordinate plants in virus-free environments to co-exist with, and potentially prevail over more dominant, competitively stronger plants in case of virus in-fection. Virus-induced alterations in the competitive ability of their hosts may influence the structure and composition of host populations (Dobson & Craw-ley, 1994) and invasiveness of plants (Malmstrom et al., 2006). Most impor-

tantly however, the results from chapters 5 & 6 suggest that virus infections can be of considerable importance for maintaining genotypic diversity in their hosts. As a result of genotype-specific responses to viral infections (GxE inter-actions), viruses may exert variable selection on their hosts. In combination with the virtual omnipresence of virus infections in nature it seems conceivable that viruses play a substantial role as selective agents, acting to maintain within-species diversity in natural plant populations. The clear and strongly negative effects of viral infections on plant performance, and their plausible impact on genotypic variation in host populations reported in this study, stress the significance of viruses for ecological and evolutionary processes. Viruses may hence play an pivotal yet largely under-estimated role in shaping popula-tion dynamics and micro-evolutionary processes.

MULTIPLE STRESSORS AT WORK

Under natural conditions plants are very likely to face multiple stressors simul-taneously. Pathogen attacks can easily coincide with abiotic stress such as light limitation or biotic stress factors such as herbivore pressure. Since re-sources are limited plants have to prioritize environmental challenges and re-spond to them accordingly. As a result of resource-mediated or other trade-offs plants may not be able to produce fully functional responses to multiple stress factors (Izaguirre et al., 2006). The existence of such trade-offs implies that the combined effects of multiple stressors are difficult to predict based on their individual effects. Due to the limited ability for height growth and the long life span of stolo-niferous plants, shading and virus attacks may readily coincide under natural conditions. I have investigated the consequences of these combined stressors for plant performance in chapters 6 & 7 and found that under shaded condi-tions, the effects of virus infection on plant performance diminished. This is true for the main effects of virus infection on plant performance (chapters 6 & 7) as well as for the variation in these effects between host genotypes (chapter 6). In other words, the severity of virus infection and the genotypic variation in response to infection strongly depends on the environmental condi-tions experienced by the host plants. These results were consistent for the two different systems used in chapter 6 (T. repens – WClMV) and chapter 7(Arabidopsis thaliana – Turnip crinkle virus), respectively. The strong decrease in virus effects caused by shading is not related to systemic virus spread, since the direction of plant-internal virus spread was not affected by heteroge-neous light conditions (chapter 4). However, shading may reduce virus repli-

cation which depends on the activity of the host tissue (Hull, 2004). Support from this notion comes from chapter 4, where I show that virus accumulation in T. repens is lower in shaded parts as compared to non-shaded parts. How-ever, for A. thaliana, I found no clear difference in virus accumulation between shaded and non-shaded plants (chapter 7), indicating that the mechanisms behind the effects of shading on viral disease development may differ between species, even though the outcome is very similar. Plants do not only face virus infections under abiotic stress conditions, but they may also simultaneously suffer from virus infection and herbivore feeding. The likelihood of these two stress factors coinciding is very high, since many plant viruses are transmitted by herbivorous insect (Hull, 2004). The re-sponses of plants to simultaneous attack of herbivores and viruses may be evolutionary conditioned to suit to the most common or most severe forms of attack. I conducted a set of experiments to analyze the complex interplay be-tween viruses, herbivores and plants (chapter 8). In an experiment which was set up to study interactions between in-fected and uninfected host plants, I observed that virus-infected plants per-formed remarkably better than non-infected plants. This unexpected and coun-terintuitive, positive effect of virus infection on plant performance was in stark contrast to my previous findings (chapters 5 & 6) and to many other studies showing mild to strongly negative effects of virus infection on host plants (Gibbs & Harrison, 1976; Hull, 2004). Since this experiment was performed during an outbreak of the root feeding herbivore Bradysia spec. (fungus gnats) in the greenhouse, I hypothesized that these organisms could have been an important factor causing the unexpected result. A subsequent experiment, however, provided no evidence that virus infected plants performed better in response to fungus gnat infestations than uninfected plants. This suggested the absence of direct effects of virus infection on fungus gnat feeding. In other words, the positive effect of virus infection on plant performance in the pres-ence of herbivores was thus not due to larval preferences, but may have been caused by selective oviposition of adult fungus gnats on virus-free plants. To test this assumption I compared the volatile blends emitted by virus-infected and non-infected plants and found that virus infection indeed resulted in marked alterations of volatile blends. When given the choice, adult fungus gnats clearly preferred non-infected plants over virus-infected plants, indicating that fungus gnat adults are able to discriminate between the two plant groups. If this preference results in differential oviposition patterns, it can be assumed to easily translate into increased herbivore feeding pressures on non-infected as compared to virus-infected plants. These results suggest that viruses do not

only harm plants by utilizing their resources, but that they can provide ecologi-cal benefits to their hosts by contributing to their defence against herbivorous insects. Considering multiple biotic interactions, virus infections may thus have considerable benefits for their hosts.

ECOLOGICAL & EVOLUTIONARY IMPLICATIONS

The results presented in this thesis reveal a fascinating and complex interplay between plant viruses and their hosts. Although it is evident that virus infection can have major negative repercussions for plant growth and development, this thesis clearly demonstrates that these effects can be neutral or even positive as well, depending on biotic and abiotic circumstances (Fig. 1). This strong environmental influence on virus effects raises the question of what wider im-plications viral infections can have for host plant ecology and evolution, and how infections may shape plant growth and survival strategies. Integrating the results from this thesis leads to the following predictions. It is generally as-sumed that virus strains capable of infecting and exploiting either the most common genotypes or those host genotypes which possess the most preva-lent resistance alleles are conferred with an evolutionary advantage and should therefore be selected (Kirchner & Roy, 2001). Consequently, the most common genotypes should suffer most strongly in virus-prone environments, thereby granting rare genotypes a relative fitness advantage (Haldane, 1949; Brunet & Mundt, 2000; Rueffler et al., 2006), and boosting their frequency in populations. Over time previously rare genotypes will become more and more common and virus strains able to infect them will positively be selected for. Such virus-induced, negative frequency-dependent selection in host popula-tions should lead to oscillations in genotype frequencies over longer periods of time. Evidently, the results presented in this thesis merely reflect a snapshot in the dynamics of micro-evolutionary processes, and it is highly unlikely that interaction between WClMV and T. repens has reached a stable equilibrium. Nevertheless, the results from this thesis shed some new light on possible mechanisms behind the maintenance of genotypes which are sensitive to virus infections. Firstly, chapters 6 & 7 clearly show that the severe effects of virus infections on host plants, and associated genotypic variation in viral effects on their hosts declined strongly under shaded conditions. This means that those genotypes which show a high sensitivity to viral infections under high light con-ditions perform equally well as non-sensitive genotypes when they grow in light-limited environments. Shaded micro-sites may thus provide refuges for

sensitive genotypes by safeguarding them from negative impacts of virus in-fections. Any possible positive association between sensitivity to virus infection and shade tolerance may cause natural selection to strongly favour non-sensitive, well defended genotypes under high light conditions and sensitive,

shade-adapted genotypes under low light conditions. A second explanation for the existence of virus-sensitive genotypes in natural plant populations stems from possible ecological benefits of virus infec-tion as described in chapter 8. I have shown that virus infection can lead to altered emissions of volatile organic compounds (VOCs), which results in the repellence of insect herbivores. For these experiments I have used a single, susceptible genotype (chapter 5). I speculate that viral disease severity is re-lated to differential emissions of VOCs. Since host genotypes respond differ-ently to virus infection, it seems conceivable that the virus-induced emission of VOCs varies between different genotypes. If true, this implies that sensitive genotypes would show a change in VOC emission upon infection (as reported in chapter 8), whereas non-sensitive genotypes may show VOC emissions

Figure 1. Schematic representation of the interplay between plants, viruses and other features of the abiotic and biotic environment. Virus-infected (black) and non-infected plants (white) are drawn in the right and left-hand panel, respectively. The effect of virus infection on plant performance, as indicated by plant size, can be nega-tive (compare C1 and V1) if host plants grow in high-light conditions and are not challenged by other biotic stresses. Positive effects of virus infection on plant performance (compare C2 and V2) can be expected if host plants emit volatiles (curved lines) which repel adult egg-laying insects, thereby resulting in decreased numbers of root herbivores. Under low-light conditions and in the absence of herbivores (compare C3 and V3), virus infection does not alter plant performance. Icon size represents plant performance, the grey background indi-cates low-light conditions, adult insect herbivores are drawn in the upper part of the figure and root-herbivores are drawn as soil-dwelling larvae. The components in this figure are not drawn to scale.

C1 C2 C3 V3 V2 V1

comparable to non-infected plants. Ultimately, this would confer sensitive genotypes with an ecological benefit as compared to non-sensitive genotypes when facing simultaneous virus infection and herbivore attacks. The eventual outcomes of the interactions described above may depend on disease sever-ity, which is mainly a function of pathogen virulence. Highly virulent viruses may strongly harm or even kill their host plants irrespective of the genotypic make-up or environmental conditions of the host. In this case the negative ef-fects of virus infections are likely to outweigh the ecological benefits in terms of herbivore repellence. Since the effects of viruses on wild plants are generally believed to be mild I propose that the complex interactions leading to ecologi-cal benefits of virus infection may play an important role for micro-evolutionary processes and for the maintenance of genotypic diversity in natural plant populations. The notion of plant viruses having exclusively negative (or no) effects on their hosts deserves to be reconsidered in the light of complex eco-logical interactions.

FINAL REMARKS

While the research areas of ecology and virology have a long standing history in scientific research, the interdisciplinary field between them is only beginning to emerge. As a result, ecological aspects and evolutionary implications of vi-rus infections in plants have not been studied very intensively in the past. This thesis provides novel insight into complex and intriguing ecological features associated with plant virus interactions, identifying original research questions and proposing testable hypotheses. It hence creates a basis for future re-search into the exciting interplay between plants and their viral pathogens in the context of multiple biotic and abiotic interactions.

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NEDERLANDSE SAMENVATTING

De meeste experimenten die in dit proefschrift staan beschreven zijn uitge-voerd met witte klaver (Trifolium repens). Witte klaver is een kruipende plant die, net als aardbei, groeit door middel van horizontale uitlopers en wordt ook wel een klonale plant genoemd. Op de knopen van deze uitlopers kunnen bla-deren, bloemen en wortels, maar ook nieuwe zijtakken worden gevormd. Elke knoop kan dus in principe zelf met de bladeren koolhydraten produceren en met de wortels water en voedingsstoffen opnemen uit de bodem en wordt doorgaans een ramet genoemd. De verschillende ramets van één plant kun-nen ruimtelijk ver uit elkaar liggen, maar zolang de ramets door middel van de uitlopers met elkaar verbonden blijven, kunnen ze onderling stoffen uitwisse-len. Deze uitwisseling van stoffen gebeurt via het vatenstelsel en kan heel gunstig zijn voor de plant. Ramets die bijvoorbeeld op een droog stukje grond terecht komen en zelf geen water kunnen opnemen krijgen water van andere ramets die dat wèl tot hun beschikking hebben. Hetzelfde geldt voor ramets in de schaduw die koolhydraten kunnen krijgen van ramets die in het licht staan. Door deze uitwisseling van stoffen groeit de plant in zijn geheel veel beter dan wanneer die uitwisseling niet mogelijk was.

De uitwisseling van stoffen tussen de ramets kan echter ook gevaarlijk zijn. Het zou kunnen dat ziekteverwekkers dezelfde kanalen gebruiken om zich binnen de plant van de ene ramet naar de andere te verspreiden. Als de-ze plant niet afsterft maar steeds nieuwe ramets blijft maken die verbonden zijn met de moederplant kan zo’n ziekteverwekker dus mogelijkerwijs voor al-tijd in de plant blijven zitten. Dit betekent dat klonale planten wellicht als op-slagplaats voor ziekteverwekkers dienen. Vanuit deze opslagplaatsen zou de ziekteverwekker zich naar andere planten in de omgeving kunnen verplaatsen om deze te infecteren (hoofdstuk 2). Deze processen zouden de verspreiding van bijvoorbeeld ziekteverwekkers sterk kunnen bevorderen. In hoofdstuk 3ga ik na hoe klonale planten van invloed kunnen zijn op de agressiviteit van ziekteverwekkers.

In hoofdstuk 4 heb ik onderzocht hoe een virus (Witte klaver mosaic virus; WClMV) zich daadwerkelijk door witte klaver kan verspreiden. Uit dit onderzoek bleek dat het virus inderdaad alle nieuwe gevormde ramets kan infecteren, ongeacht waar deze ramets zich in de plant bevinden. Echter de ramets die op het moment van infectie al volgroeid waren bleven virusvrij. Dat wil zeggen dat er binnen een klonale plant variatie ontstaat met betrekking tot de ontwikkeling van de ziekte. Dat de verspreiding van het virus effect kan hebben op het welzijn van de plant laat ik zien in hoofdstuk 5 & 6. De resulta-

ten van dit onderzoek laten zien dat geïnfecteerde planten aanzienlijk minder nieuwe ramets produceren dan gezonde planten en dat ook het gewicht en dus de groei van deze planten sterk afneemt. Daarnaast maken geïnfecteerde planten ook veel minder zijtakken dan gezonde planten. In hoofdstuk 6 zien we dezelfde effecten, maar dan iets minder sterk. Uit deze hoofdstukken blijkt duidelijk dat de verbinding tussen ramets ook gevaarlijk kan zijn en nadelig is wanneer de plant wordt blootgesteld aan ziekteverwekkers. Doordat een zieke plant minder ramets en zijtakken maakt heeft deze ook minder mogelijkheden om grondstoffen (zoals water en koolhydraten) op te nemen. Dit zou nog extra nadelig kunnen zijn wanneer deze planten met andere soorten voor grondstof-fen moeten concurreren. GENOTYPISCHE VARIATIE

Hoewel alle witte klaver planten tot dezelfde soort behoren zijn niet alle indivi-duen gelijk. Afzonderlijke planten kunnen genetisch heel verschillend zijn waardoor ze er vaak anders uitzien, maar ook anders kunnen reageren op hun omgeving. Individuele planten die genetisch van elkaar verschillen noemen we genotypen, wat betekend dat deze individuen een “typisch” of te wel verschil-lend genoom hebben. Vaak wordt ervan uitgegaan dat een ziekte in planten op alle genotypen (individuen) een vergelijkbaar effect heeft. Echter in hoofd-stuk 5 laat ik zien dat nagenoeg alle negatieve effecten van virus infectie zo-als ze hierboven beschreven staan, sterk kunnen variëren tussen verschillen-de individuen van witte klaver. Met andere woorden, niet elke afzonderlijke plant heeft evenveel last van het virus; sommige planten hebben nergens last van terwijl andere nauwelijks meer groeien en bijna geen nieuwe ramets meer kunnen maken. Dit betekent dat individuen die het beste groeien in een omge-ving zonder virus, niet per sé de beste zijn als ze besmet worden met een vi-rus. Andersom geldt hetzelfde, als een individu dat niet zo goed groeit ten op-zichte van andere planten wanneer er geen virus aanwezig is, zou het kunnen dat dit individu het beste kan groeien als er wel een virus aanwezig is. In een omgeving zonder virus zullen zulke slecht groeiende individuen waarschijnlijk weggeconcurreerd worden door de grote sterke individuen. Echter wanneer alle planten besmet zijn met het virus kunnen deze individuen wel de concur-rentiestrijd winnen als ze minder last ondervinden van het virus. Het virus kan dus de rangschikking van individuen binnen een populatie veranderen (hoofdstuk5 & 6) en daarmee ervoor zorgen dat sommige individuen kunnen blijven bestaan. Hierdoor zou het virus een belangrijke rol kunnen spelen bij de samenstelling van populaties van planten.

MEERDERE STRESSFACTOREN TEGELIJKERTIJD

Het is heel waarschijnlijk dat planten in natuurlijke omstandigheden met meer-dere stressfactoren tegelijkertijd te maken hebben. Zieke planten kunnen mak-kelijk blootgesteld worden aan planteneters zoals rupsen of last hebben van een tekort aan licht omdat ze in de schaduw komen te staan. Aangezien de beschikbare energie voor planten beperkt is, moeten ze als het ware “beslissen” aan welke stressfactor ze de meeste energie besteden. Wanneer ze bijvoorbeeld energie besteden aan het ontgroeien van de schaduw, dan gaat dit wellicht ten koste van de afweer tegen ziekteverwekkers en vice ver-sa. Aangezien planten die uitlopers vormen vaak maar heel weinig in de hoogte kunnen groeien en tegelijkertijd heel erg lang kunnen leven, is het waarschijnlijk dat ze planten regelmatig gelijktijdig te kampen hebben met schaduw en virusinfecties. In hoofdstuk 6 & 7 heb ik onderzocht hoe de com-binatie van deze twee stressfactoren de groei van planten beïnvloedt. Uit dit onderzoek bleek dat het negatieve effect van virus infectie op plantengroei verdwijnt als de planten een tekort aan licht hebben. Ook het verschil in virus-effect tussen individuele planten was niet meer zichtbaar wanneer deze plan-ten een tekort aan licht hadden (hoofdstuk 6). Deze resultaten heb ik gevon-den voor twee verschillende plant-virus combinaties, namelijk witte klaver met Witte klaver mosaic virus (hoofdstuk 6) en zandraket met Turnip crinkle virus(hoofdstuk 7). Planten hebben niet alleen te maken met schaduw als stressfactor, maar kunnen ook simultaan blootgesteld worden aan virus infecties en plan-teneters (herbivoren). Het is heel waarschijnlijk dat een plant tegelijkertijd te kampen heeft met deze twee stressfactoren, aangezien veel virussen door herbivoren verspreid worden. De reactie van de plant op de gelijktijdige bloot-stelling aan deze twee stressfactoren is waarschijnlijk zo geëvolueerd dat de plant de meest voorkomende en/of ergste stressfactor kan overleven. In hoofdstuk 8 heb ik een reeks experimenten gedaan om de ingewikkelde wis-selwerking tussen virussen, herbivoren en planten te onderzoeken. Het eerste idee om dit te onderzoeken kwam van een experiment waar-in ik virusgeïnfecteerde en gezonde planten met elkaar heb laten concurreren en de zieke planten groter werden dan de gezonde planten. Dit onverwachte resultaat was sterk in tegenspraak met de negatieve effecten van virusinfectie op plantengroei zoals ik die eerder had gevonden (hoofdstuk 5 & 6) en ande-re onderzoeken waaruit blijkt dat virus infecties meestal negatieve effecten hebben op de groei van hun gastheer. Echter tijdens mijn experiment hadden

we in de kassen te maken met een varenrouwmuggen uitbraak. Deze muggen leggen eitjes in de grond waaruit kleine larven komen die zich vervolgens voe-den met plantenwortels. Ik had het idee dat de aanwezigheid van deze varen-rouwmuggen iets te maken had met het onverwachte resultaat en heb daarom een experiment ingezet om dat te onderzoeken. Echter, met dit experiment kon ik niet bewijzen dat zieke planten in aanwezigheid van de larven het beter doen dan gezonde planten. Hieruit blijkt dat er geen direct effect is van virusin-fectie op het eetgedrag van de larven. Met andere woorden, het positieve ef-fect van virusinfectie op plantengroei was niet veroorzaakt door selectief eet-gedrag van de larven. Een tweede verklaring voor het positieve effect zou kun-nen zijn dat de volwassen varenrouwmuggen hun eitjes bij voorkeur bij gezon-de planten leggen en de zieke planten ontwijken. Hiervoor zouden de varen-rouwmuggen een onderscheid moeten kunnen maken tussen de zieke en ge-zonde planten. Ik heb daarom onderzocht of zieke planten andere vluchtige stoffen uitstoten dan gezonde planten, en dit blijkt inderdaad het geval te zijn. Met andere woorden, het virus zorgt ervoor dat geïnfecteerde planten anders gaan ruiken dan de gezonde planten. Wanneer vrouwelijke varenrouwmuggen mochten kiezen tussen zieke en gezonde planten bleek dat de vrouwtjes een duidelijke voorkeur hadden voor gezonde planten! Als deze voorkeur zich ver-taald in verschillende hoeveelheden eitjes die respectievelijk in de buurt van zieke en gezonde planten worden gelegd, dan heeft dit heel waarschijnlijk tot resultaat dat de gezonde planten veel meer worden aangevreten door de lar-ven (want daar zijn er meer) dan de zieke planten. Deze resultaten suggereren dat virussen niet alleen maar slecht zijn voor hun gastheren, maar ook voorde-len kunnen hebben in de aanwezigheid van herbivoren. Ten slotte eindigt dit proefschrift met een samenvattende discussie (hoofdstuk 9) van de belang-rijkste resultaten en de voor en nadelen van virusinfecties in planten.

DANKWOORD

Heel graag wil ik IEDEREEN bedanken die op welke manier dan ook een bi-jdrage heeft geleverd aan dit proefschrift! Het was zonder jullie hulp en inzet simpelweg onmogelijk geweest dit boekje te schrijven. Allereerst wil ik jou, Josef bedanken omdat je mij de mogelijkheid hebt gegeven te promoveren. Dank! Jouw enthousiasme en vertrouwen zijn lang de peilers geweest waarop ik mijn werk heb gebouwd. Dank voor je nooit aflatende interesse in mijn werk en de ruimte die je me daarin hebt gegeven om mezelf te ontwikkelen zonder dat je me het gevoel gaf dat ik er alleen voor stond. Je sloot niets op voorhand uit en liet mij mijn eigen weg vinden. In tijden dat ik mij inhoudelijk geïsoleerd voelde was het geweldig dat jouw deur altijd openstond! Je hebt me qua werk altijd enorm gestimuleerd en ik heb ongelofe-lijk veel van jou geleerd! Natuurlijk ook heel erg bedankt voor het samen-voegen van de referenties! Ik heb erg genoten van onze “core”-uitjes met Coco, en ook van de vele gesprekken die we over zeer uiteenlopende onder-werpen hebben gehad en natuurlijk de safari’s. Je goede humeur en gevoel voor humor (dat zijn twee verschillende dingen hè, ;) zijn onbetaalbaar! Hans, bedankt voor het bewaken van de “grote lijn” en het becommen-tariëren van mijn stukken. Een van de dingen die ik altijd in jou gewaardeerd heb is je vermogen om rustig te luisteren. Als ik het weer eens ergens mee oneens was of heftig reageerde bleef je altijd kalm. Dat je op die momenten oor voor mij had en mij mijn problemen liet vertellen waardeer ik nog steeds. Tijdens mijn studie Biologie zijn er een aantal mensen geweest die een onuitwisbare indruk op mij hebben gemaakt en die ik hier wil bedanken. Titti, dank voor de vele gesprekken en voor al je hulp. Je hebt me erg gestimuleerd om het onderzoek in te gaan en daar ben ik blij om! Koen, mijn eerste stage was bij jou. Je hebt mij de principes van onderzoek bijgebracht en bent een van de beste docenten die ik gehad heb! Dank voor alles (inclusief de over-tuiging dat ik nooit een “Mac” wil ;)! Het ga je goed. Linda, ik ben heel erg blij dat mijn tweede stage bij jou was. Ik heb ongelofelijk veel geleerd over ecolo-gie en mijn eerste artikel hebben wij samen geschreven! Dank voor je onvoor-waardelijke steun en je motiverende woorden en in het bijzonder voor je geloof in mij! Gelukkig hebben we nog steeds contact ook al woon je dan in het verre Noorden ;).

Frans, ik heb je al eerder bedankt voor alles wat je voor me hebt ge-daan, maar hier mag je niet ontbreken. Mijn liefde voor de Biologie ontstond in jouw klaslokaal op de Groenewald en heeft ervoor gezorgd dat ik hier ben gaan studeren. De belangrijkste reden om je hier te noemen heeft echter niets

met Biologie te maken. Jij hebt me iets gegeven wat van onschatbare waarde is. Dankzij jou weet ik, voel ik dat er altijd hoop is, hoe uitzichtloos het ook lijkt. Die overtuiging is de afgelopen jaren erg waardevol gebleken! Natuurlijk mis ik je nog steeds en dat zal altijd wel blijven, maar de gedachte aan een boom met blauwe bladeren kan nog steeds een glimlach toveren op mijn gezicht! Dan zijn er natuurlijk de mensen van -Experimentele Planten Ecologie-. Mijn dank gaat uit naar jullie allemaal! Ik heb me ontzettend thuis gevoeld op de afdeling en ben altijd met heel veel plezier naar mijn werk gegaan, dank daarvoor en natuurlijk in het bijzonder voor de bijdrage die ieder van jullie aan dit proefschrift heeft geleverd. Zonder jullie had het niet gekund! Eric, jou wil ik heel erg bedanken voor de vele discussies die we tijdens en buiten de werkbe-sprekingen om hebben gevoerd (dat kan dus ook met een “6” ;). Natuurlijk ook voor het lezen van mijn manuscripten en alle interesse die je voor mijn werk hebt gehad! Jouw mening heeft voor mij een bijzondere waarde omdat je zo eerlijk en direct bent. Ik vond het heel fijn dat ik mee mocht naar Binn en ga daar zeker nog eens terug! Op persoonlijk vlak heb ik erg genoten van die paar keer dat we fossielen zijn gaan zoeken, ik kan nog steeds de namen niet onthouden, maar ik ga graag mee naar Antwerpen om wat zand te verschep-pen! In het bijzonder bedankt voor alle off-topic gesprekken en voor het lenen van je schouder die ene keer! Heidi, bedankt voor je constructieve bijdrage aan de werkbesprekingen en je scherpe opmerkingen over allerlei onderwerp. Ik vond het altijd leuk om met jou te praten. Corien, ik heb een grote bewon-dering voor jou! De manier waarop je je bureau “zichtbaar” kon houden is voor mij nog steeds onverklaarbaar (en onmogelijk). Ik vond het erg leuk om over films te praten en heb je oprechte interesse in mij altijd heel erg gewaardeerd! Ik kom snel nog eens bij je langs! Jelmer, dank voor alle gesprekken en alle gelach! Ik vond het heel leuk in Estland (samen met Bart en Josef) en je mag altijd langskomen voor een snoepje! Xin, thank you for all your questions and our nice talks when I moved to the “room”. Good luck with finishing your thesis! Liesje, bedankt voor de gezelligheid! Eelke, dankjewel voor alle gesprekken. Jullie beiden succes met de VENIs! Ronald, we hebben eigenlijk niet echt samengewerkt, maar ik vind het nog steeds erg gezellig om met je te kletsen als we elkaar tegenkomen! Werner Harry en Julia het was kort, maar leuk. Ik heb ook veel hulp gekregen van Annemiek, Hannie en Gerard. Annemiek, be-dankt dat je me geholpen hebt bij het praktische werk. Hannie, waar moet ik beginnen! Je bent een ontzettend lief mens. Dankjewel voor al je hulp, vooral bij het opslurpen van die varenrouwmuggen! Jouw inzet is ongelooflijk! Ik kijk met plezier terug op al onze gesprekken en wil je in het bijzonder bedanken voor je lieve zorgen de afgelopen tijd. Mede dankzij jou gaat het steeds beter!

Voor de zekerheid zeg ik het hier nog maar eens: dit is geen afscheid! Mijn beste Gerard, ook bij jou weet ik niet waar ik moet beginnen. We kennen el-kaar al lang en naar mijn gevoel ook goed. Ik heb heel erg genoten van de vele jaren waarin ik mee mocht doen met de cursus biodiversiteit. Het is fan-tastisch mooi om te zien hoezeer je daarbij betrokken bent en hoe je steeds opnieuw weer probeert het onderwijs te verbeteren. Ook mij heb je veel bijge-bracht en nooit was je ongeduldig! En dat terwijl ik altijd zoveel vergat… Dank voor je onvoorwaardelijke steun en goede zorgen! Je had altijd tijd voor mij en probeerde een antwoord te vinden op al mijn vragen, óók als ik die al eerder gesteld had! Ik hoop dat we nog vele gesprekken over poëzie en andere draaglijke en ondraaglijke zaken zullen hebben, want die zijn voor mij bijzon-der waardevol! José A, bedankt voor alle hulp. José B, dank voor alles wat je de afgelopen jaren voor me hebt gedaan! Nagenoeg al mijn experimenten zijn in de kassen uitgevoerd en ik ben grote dank verschuldigd aan de mensen daar voor alle hulp die ze mij gegeven hebben, ik hoop dat het jullie allemaal goed gaat en wens jullie het beste toe. Gerard, je stond altijd klaar om mij te helpen en we konden altijd over alles praten. In het bijzonder wil ik je bedanken voor je lieve zorgen de afgelopen tijd en de steun die je mij als mens daarin hebt gegeven! Harry, dankjewel voor al je hulp en geniet van die Carnaval hè! Walter, bedankt voor je interesse in mijn werk en in mij als persoon. De vele gesprekken zijn erg waardevol geweest, ook als het verdrietige zaken betrof en natuurlijk ook als we hard konden lachen! Ik zal maar niks zeggen van die appeltaart… Yvette,je bent en blijft een bijzonder mens! Jij ook bedankt voor je zorgen en je inter-esse in mij en alle gezelligheid!. Ik heb veel geleerd over vrouwenbillen en dat had ik zelf nooit gedacht ;). Gaan we binnenkort nog eens naar de film? Dan de mensen van Aquatische Ecologie, bedankt allemaal! Dries be-dank voor het installeren en de-installeren van vele programma’s! Radju, I en-joyed all our conversations about life and things that really matter! It is a good thing to know you always come back :-). Iain and Pete, you M&Ms make a great couple and I really enjoyed having you around. Thanks for the turtle-faces and daily jokes from the book. Hope to see you again some day! Ook mensen van andere afdelingen wil ik graag bedanken: Richard, joamer dat se weg bis gegange! Ich houw geer noch get mit dich same gedoan! Missjien seen veer os in Aelse. Mieke, dankjewel voor al je hulp als ik weer eens iets niet wist! Ik vond het heel leuk om met jou over van alles en nog wat te praten. Dat geldt ook voor Else! Jullie twee ook bedankt voor alle steun de afgelopen jaren, ik waardeer dat zeer! Peter: auf wieder sehn! Marian, dank voor je depc. Jan, je hebt me gered met de pipet! Dank ook voor alle andere dingen waar-

mee je me hebt geholpen. Dank ook aan Tom en Janny, voor alles. Tom, je unieke kijk op de wereld werkt erg relativerend en ik vind het fijn dat je met ons meedenkt. Ik zou zeggen: “soek die geluck”, dat doen wij ook! Rinus, je hebt gelijk wat die investering in mensen betreft, bedankt! Jelle, dankjewel voor je hulp bij de stikstof metingen, Liesbeth bedankt voor de microscopie en alle moeite die je daarvoor hebt gedaan! Geert-Jan, heel erg bedankt voor al je hulp bij de elektronenmicroscoop!!! Ik vond het geweldig dat je altijd tijd voor mij hebt gemaakt en dat je allerlei dingen al voor mij klaar had liggen als ik weer eens langs kwam. Niels bedankt voor alle hulp en gezelligheid! Gelukkig waren er ook een aantal studenten die mij hebben geholpen bij dit proefschrift, Sietse, Ruud en Reinier, jullie zijn stuk voor stuk uniek en ik heb enorm veel van jullie geleerd. Nu weet ik dat onderzoek ook sexy-plexy kan zijn ;) Dank jullie allemaal en alle goeds!

In de afgelopen jaren hebben we met een groep mensen de seminars voor het IWWR georganiseerd en ik heb dat altijd erg leuk gevonden om te doen! Lisette, Maaike, Anneke, Katharina, Marjolijn, Aafke, Christian en Peter bedankt, voor alle gezellige vergaderingen! Succes met alles en hopelijk tot ziens. Lisette en Maaike, ik heb ongelofelijk veel met jullie gelachen, bedankt voor alle gesprekken over zin en onzin! Peter, ik wil jou hier nog extra be-danken voor de lunches en voor alle gesprekken over wetenschap en publi-ceren! Geniet van je nieuwe huis en tot snel! Op deze plek wil ik ook Mike Jet-ten, Maurice Martens en Martijn Frieling bedanken voor hun steun bij het op-zetten en uitvoeren van de seminars. Martijn, het was gezellig met jou na afloop van de seminars te praten, bedankt voor je interesse. Maurice, we heb-ben elkaar al in 1998 leren kennen toen je nog bij het secretariaat Biologie werkzaam was en later bij de cursus biodiversiteit ik vond het erg leuk om met jou te praten over van alles en nog wat (al dan niet in het “plat”). Veul geluk in Mestreech! Ik ben erg dankbaar voor alle hulp die ik van mensen buiten de “uni” heb gekregen en ben erg blij met de plezierige samenwerking in verschillende labs! Stephan Winter (DSMZ, DE), thank you for answering my thousand questions about the ELISA testing! Tony Lough (BioJoule, NZ), thanks for pro-viding me with the WClMV-GFP construct and your help on other topics. Dick Verduin and Dick Lohuis (WUR) bedankt voor alle hulp en alle werk voor de vergunningen en het aan de praat krijgen van het GFP- construct! Helaas is het niet gelukt, maar ik heb desalniettemin heel veel van jullie geleerd. Tomáš Koubek (Charles University, CZ), thanks for visiting and for the book! I’ll do my best to never grow up and expect the same from you :-). My sincere gratitude goes to all the people in the Sainsbury lab. Special thanks to David Baulcombe

(John Innes Centre, UK) for hosting me. Jagger Harvey, thanks for sharing the “totally hot” experiment with me and all the things you’ve thought me. I very much enjoyed your company and you are right, it is cool when ecology and virology meet! Good luck in Kenya! Ook de mensen van fytopathologie (UU) wil ik hartelijk bedanken voor hun hulp! Corné, ik vond het heel fijn dat ik bij jou op het lab experimenten mocht doen en dat je zo openstaat voor nieuwe din-gen! Bedankt ook voor je altijd stimulerende instelling! Antonio, I really liked working with you!!! Your humor and knowledge make a perfect combination and I especially enjoyed all our chats. Thanks for taking care of “that ecolo-gist”! I hope I will see you again soon and wish you well. De afgelopen jaren heb ik een aantal hele fijne mensen op het NIOO in Heteren leren kennen. Allereerst Arjen, je was al vroeg bij mijn project betrokken en ik heb een enorme bewondering voor je positieve manier van denken en je vermogen om kritisch te zijn zonder dat het persoonlijk wordt! Dank voor al je hulp. Nicole, uiteindelijk wist ik je dan toch te overtuigen dat we vluchtige stoffen moesten meten in geïnfecteerde klaver planten. Gelukkig! Ik heb steeds met heel veel plezier met je samengewerkt en kan erg met je lachen. Dankjewel voor al je hulp bij het analyseren van de resultaten en het corrigeren van dat stuk. Erg belangrijk voor mij is ook je steun de afgelopen tijd! Roxi, I enjoyed all our chats and hope we can cooperate in future! Here is my kiss for you. Wim, be-dankt voor de plek. Tim en Ciska, bedankt voor het lenen van, en hulp met de netten, helaas is het niet gelukt. Also thanks to Martin Heil for commenting on my paper and stimulating discussions. Ook bij entomologie (WUR) heb ik met veel plezier enkele experimenten uitgevoerd. Marcel, hartelijk bedankt voor je gastvrijheid, je bereidheid mijn stuk van commentaar te voorzien en de discus-sies over vervolgonderzoek! ¡Hola Tjeerd! Allereerst dankjewel voor je hulp bij mijn proefjes en de tijd die je daarvoor hebt vrijgemaakt! Daarnaast vind ik het erg leuk om met je bij te kletsen over van alles en nog wat! Wanneer gaan we nou dat biertje in Arnhem pakken? I am very grateful to the people at AgResearch (NZ) for hosting me and for making my time in New Zealand an unforgettable experience! First of all thank you Mike, for all your efforts and help and your great hospitality! It was really nice talking to you and having dinner with you and Sue! Thanks for your support and see you soon. Dear Jim, it was a pleasure to work with you, thank you so much for all your help and for providing me with all the necessary plants and pots and sand. Dear Pip, thank you as well for all your help and nice discussions and of course for taking us to the horse races, which I proba-bly never will forget. I am very grateful for the support the two of you gave me and hope we can continue our collaboration in the future! My thanks also go to

Li for help with the experiment. Derreck, thanks for everything, especially for helping us with the practical things; still miss those yellow helmets! Dan gaat er natuurlijk een enorme dank uit naar mijn vrienden! Al-lereerst Rolf, Cécile en Wilbert, we zien elkaar maar heel zelden, maar ik heb een warme herinnering aan de tijd die we samen doorgebracht hebben! Cheryl, fijn dat we nog steeds contact hebben en kunnen filosoferen over het leven! Dat we op dezelfde dag geboren zijn zegt eigenlijk al alles :-). Lieve Rob, je bent na al die jaren nog steeds een van mijn allerbeste vrienden. Vliegeren aan het strand, picknicken aan de Waal, tot diep in de nacht kletsen, eten bij Jo, apfelkorn op het bankje enz. die dingen zijn voor mij onbetaalbaar! Dankjewel dat ik je altijd en voor alles kan bereiken. Ik ben heel erg blij met jou als paranimf aan mijn zijde! Tweety-pak of niet. Juut, we kennen elkaar al lang en hebben samen heel wat lief en leed gedeeld. Je kan dwars door me heen kijken en ik vind het heerlijk dat we open en eerlijk tegen elkaar kunnen zijn. Je bent een geweldige vriendin! Veel geluk met Paul en Igor en bedankt voor je altijd onvoorwaardelijke steun!!! Mark en Anneke ik heb erg genoten van onze etentjes samen, al dan niet met spelletjes (huifkarren!), en vind het altijd erg leuk om met jullie te praten. Natuurlijk ook super bedankt dat ik bij jullie thuis mocht werken, dat heeft ervoor gezorgd dat dit boekje nog op tijd is afge-komen. Jullie steun is onmisbaar. Veel geluk in Helsinki! Mark (MOTU ;) jou wil ik hier nog extra bedanken voor onze lange vriendschap en omdat ik al mijn lief en leed met jou kan delen. Ik zal onze squash-potjes missen! Geniet van je reis, ik hoop dat je veel nieuwe dingen ontdekt net als “Bastiaan” in het boek. Als je terug bent doen we weer een LOTR marathon. Dear Coco-chica, thanks for being my paranimf! I very much enjoyed your company in the lab and greenhouse and want to thank you for all the fun we had together the past years! I really liked our core-uitjes and long conversations. You have become one of my best friends and your support and criticism are invaluable to me. We have shared a lot of happiness and sadness on all kind of topics and above all, you have always been there for me. I am very happy that we still keep in touch despite that big ocean between us even though it occasionally means going to bed in the middle of the night ;). Thank you for your friendship, it means the world to me! Lieve Emile en Diana, bedankt voor alle gezelligheid en alle steun de afgelopen jaren! Fijn en gezellig dat jullie weer in de buurt (donut?) zijn! Beste André, jij hebt tijdens mijn studie en het grootste deel van dit pro-motieonderzoek aan mijn zijde gestaan. Ik wil je heel erg bedanken voor alle steun en relativerende woorden! Je kritische kijk op de wetenschap en werk in het algemeen konden bepaalde dingen nogal eens in perspectief zetten. Een perspectief dat ik zonder jou nooit gezien zou hebben. Ik denk met plezier

terug aan de tijd die we samen hadden, aan alle reizen en aan alle lief en leed dat we gedeeld hebben. Je bent een bijzonder mens en ik wens je niets dan goeds! Op deze plek wil ik ook Ad en Carla bedanken voor hun interesse in mijn werk en mij als persoon en voor alle tijd die we gedeeld hebben. Ik ben blij dat ik jullie heb leren kennen. Huub en Maaike, Jurgen en Sherilda jullie ook bedankt voor alles! Sherilda, jou wil ik in het bijzonder bedanken voor de vele gesprekken die we hebben gehad en je steun in moeilijke tijden! Lieve Tim en Myrca, jullie zijn twee geweldige kinderen en ik mis jullie ontzettend! Ik wens jullie allemaal veel goeds. Bei der Familie Stuefer bedanke ich mich sehr herzlich dafür, dass sie mich so gastfreundlich aufgenommen haben. Insbesondere Frau Klara Stuefer möchte ich meinen aufrichtigsten Dank aussprechen für ihre liebevolle Sorge und ihre Freundlichkeit. Ich hoffe, dass wir uns bald wiedersehen! (meneer Dinjens bedankt!) Lieve Daniel en Miriam, ik vind het heerlijk als jullie er zijn! Bedankt voor alle grapjes, duimpjes worstelen en lego op de computer. Door jullie zie ik de wereld op een heel andere manier en dat is onbetaalbaar! Dikke kus voor de kietelmonsters!

Mijn dank gaat ook uit naar mijn familie. Jullie zijn er altijd voor mij ge-weest en als we samen zijn voelt dat aan als een thuiskomst! Dit is dus waar ik de afgelopen jaren aan heb gewerkt, maar aangezien ik jullie er nooit van heb kunnen overtuigen dat ik aan het werk was moeten jullie dit maar beschouwen als mijn afstudeerproject ;). Jullie allemaal heel hartelijk bedankt voor alle gezelligheid en genegenheid! Tante Mie, u bent een heel bijzonder mens en ik ben blij dat we in sommige opzichten zo op elkaar lijken! Bedankt voor alle steun en liefde de afgelopen jaren. Ome Maj, dat van die Willemsbrug moet ik nog opzoeken, Tante Marga bedankt voor je interesse in mij en je humor! Nonc Henri, dankjewel voor je gezelligheid enne wanneer spelen die “rode duivels” nou weer eens een E.K.? Cissy, bedankt voor je interesse in mij en je steun en liefde! De koffie staat nog steeds klaar :-). Martijn, Kristian, Patrick, Stephanie, Nathalie en Philippe ik hoop dat we elkaar snel weer zien, bedankt voor alle gezelligheid als we samen zijn! Oh, en Patrick wanneer kom je nog eens op buitenlandmissie ;). Van dit hele boekje zou niets zijn geworden zonder de onvoorwaarde-lijke steun en liefde en het grote vertrouwen dat jullie Pap, Mam en Bart in mij hebben. Daarom heel hartelijk bedankt voor alles wat jullie voor mij gedaan hebben en nog steeds doen! Ik vind het heel erg fijn dat ik altijd bij jullie terecht kan en dat jullie mij liefhebben voor wie ik ben. Ook hartelijk bedankt voor het feest! In het bijzonder wil ik jou Mam bedanken. Jij en ik hebben een heel bi-jzondere band. Bedankt dat ik alles tegen je kan zeggen en dat je eerlijk tegen

me bent zonder dat je daarbij mij als persoon kwetst. Ik heb heel erg veel be-wondering voor je zachtheid en oneindige optimisme die van jou zo’n lief mens maken. Wat de totstandkoming van dit boekje aangaat heb je een heel bijzon-dere bijdrage geleverd. Al van jongs af aan heb je mij geleerd dat je als mens niet alles hoeft te kunnen, maar dat je hard je best moet doen om datgene wat je kan zo goed mogelijk te doen. Die overtuiging alleen, heeft ervoor gezorgd dat ik bij alle tegenslagen die ik de afgelopen jaren moest verwerken (zo’n boekje komt er niet vanzelf) als persoon nooit geraakt ben. Daardoor heb ik de kracht gehad om steeds opnieuw weer de draad op te pakken en elke keer weer mijn uiterste best te doen. Dit boekje is voor jou. Lieve Sepp, ik heb jou op twee manieren leren kennen en daarom sta je ook twee keer in dit dankwoord! Dat had ik zelf nooit gedacht, maar ik ben ver-schrikkelijk blij dat het wel zo is. Wij zijn aan een heel bijzondere reis begon-nen en je hebt me landschappen en steden laten zien die ik eerder nooit gezien heb en waarvan ik in veel gevallen niet eens wist dat ze bestonden. Ik hoop dat we nog heel lang samen zullen reizen en nooit zullen aankomen want ik vind het heerlijk om samen met jou het leven te ontdekken en daarvan te genieten. Bedankt voor je onvoorwaardelijke steun en liefde en weet dat die wederzijds zijn! “Tra un fiore colto e l'altro donato l'inesprimibile nulla.” Sd’a, gg.

PUBLISHED PAPERS

Van Mölken T & Stuefer JF (2008). Virulence in clonal plants: conflict-ing selection pressures at work? Evolutionary Ecology 22: 467-470.

Van Mölken T, Jorritsma-Wienk LD, Van Hoek PHW & De Kroon H (2005). Only seed size matters for germination in different popu-lations of the dimorphic Tragopogon pratensis subsp. pratensis(Asteraceae). American Journal of Botany 92: 432–437.

Stuefer JF, Gómez S & Van Mölken T (2004). Clonal integration be-yond resource sharing: implications for defence signalling and disease transmission in clonal plant networks. Evolutionary Ecol-ogy 18: 647–667.

SUBMITTED PAPERS

Van Mölken T & Stuefer JF (submitted). Highways for internal virus spread: patterns of viral disease development in the stoloniferous herb Trifolium repens.

Van Mölken T & Stuefer JF (under review). The potential of plant vi-ruses to promote genotypic diversity via GxE interactions.

Van Mölken T, De Caluwe H, Van Dam NM, Hordijk C, Leon-Reyes A, Snoeren TAL & Stuefer JF (submitted). Volatile emissions by virus-infected plants repel herbivores.

TAMARA VAN MÖLKEN

Tamara van Mölken was born on December 7, 1979 in Sittard, the Netherlands. In 1998 she ob-tained her Athenaeum degree at the Groenewald in Stein. In the same year she started her under-graduate studies in Biology at the Radboud Uni-versity Nijmegen. During her first MSc thesis she studied the origin of the single integument in Nico-tiana tabacum. During her second MSc thesis she studied the difference in germination behaviour and subsequent seedling growth between the di-morphic seeds of Tragopogon pratensis ssp. prat-ensis in relation to population characteristics. At that time Tamara got interested in phytopathology and wrote an essays titled: “General virus features and specific information on the main viruses infecting Trifolium species”. In March 2003 she graduated in Biology at the Radboud University Nijmegen. Her PhD project on pros and cons of virus infections in plants, presented in this thesis, was started in October 2003 at the department of Experimental Plant Ecology. In September 2008 she started a short post-doctoral project at AgResearch in New Zealand, in which she investigated how plant virus infections affect herbivore consumption of white clover.

Trifolium repens

White clover mosaic virus Bradysia sp.