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Evolutionary theory of plant defences against herbivores predicts a trade-off between direct (anti-herbivore traits) and indirect defences (attraction of carnivores) when carnivore fitness is reduced. Such a trade-off is expected in plant species that kill herbivore eggs by exhibiting a hypersensitive response (HR)-like necrosis, which should then negatively affect carnivores. We used the black mustard ( Brassica nigra ) to investigate how this potentially lethal direct trait affects preferences and/or performances of specialist cabbage white butterflies ( Pieris spp.), and their natural enemies, tiny egg parasitoid wasps ( Trichogramma spp.). Both within and between black mustard populations, we observed variation in the expression of Pieris egg-induced HR. Butterfly eggs on plants with HR-like necrosis suffered lower hatching rates and higher parasitism than eggs that did not induce the trait. In addition, Trichogramma wasps were attracted to volatiles of egg-induced plants that also expressed HR, and this attraction depended on the Trichogramma strain used. Consequently, HR did not have a negative effect on egg parasitoid survival. We conclude that even within a system where plants deploy lethal direct defences, such defences may still act with indirect defences in a synergistic manner to reduce herbivore pressure.

This chapter describes the components (elevated CO2, temperature and drought) of climate change and their direct and indirect effects on plant-insect interactions. The genetic resources (such as wild relatives and traditional, locally adapted landraces) important for increasing pest/disease resistance in crop plants are described. Some strategies for introduction of pest and disease resistance are presented, which include traditional and biotechnological (genetic modification) approaches.

Despite its high economic value in many countries (especially in developing regions of the tropics and sub-tropics), okra has received little attention with respect to its source of origin and genetic diversity, particularly at the molecular level. Phenotypic description (morphology, pod characteristics and seed germination) and AFLP (amplified fragment length polymorphism) analysis were performed on Greek and international genotypes. Whereas morphological descriptors did not separate the accessions according to their geographical origin, AFLP analysis revealed a low level (12%) of polymorphism and distinct geographical groupings. Greek germplasm separated into three distinct groups with no overlap between them on the basis of molecular markers. A higher degree of genetic heterogeneity was found (UPGMA analysis) among the accessions of the Boyiatiou group than in the Pylaias group, whereas the occurrence of some common phylogenetic characteristics made separation on the basis of morphology alone difficult. The results from AFLP markers indicate that Greek germplasm constitutes a significant pool of variation with respect to morphological parameters, pod characteristics and seed germinability. Moreover, differences in seed germination among phenotypes may relate to their geographical origin (mainland or islands).

Downy mildew (Bremia lactucae) in lettuce (Lactuca sativa) is a devastating foliar disease causing high losses in lettuce cultivation. The wild lettuce and nonhost species, Lactuca saligna, is absolute resistant to downy mildew and cross-fertile with L. sativa, albeit with a low success rate and occasional reduced fertility and/or vitality in later inbred generations. This exceptional availability of hybrid plant offsprings creates a unique opportunity to study nonhost resistance by a genetic approach. The L. saligna nonhost resistance genes might be more durable than the classical monogenic race-specific R genes that are mainly used in lettuce breeding. The identification of genes conferring nonhost resistance is a crucial step in its understanding and usage in breeding. In this thesis the quantitative resistances of three backcross introgression lines (BILs), carrying an individual 30 to 50 cM long introgression segment from L. saligna in a L. sativa background, were fine mapped. Disease evaluation of sub-BILs with smaller introgression segments revealed that the resistance of all three BILs was explained by 17 sub-QTLs with a smaller and plant stage dependent effect, some segments reducing, others even promoting downy mildew infection. Further the potential of stacking quantitative resistances of eight BILs per combinations of two was tested under field conditions. Only three out of ten double-combinations resulted in an increased resistance level compared to their parental individual lines, from which one had additive and two had epistatic interactions between the introgressions. As the studies on individual QTL effects of BILs did not reveal potential genetic interactions that could explain the complete resistance of L. saligna, a novel approach was set out to search for indications of epistatic interactions. ‘Selective genotyping’ was applied on the phenotypic disease extremes of large F2 offsprings, in which multi-locus interactions between L. saligna alleles are still prevalent. In a kind of bulked segregant analysis approach four major resistance regions were identified. Preliminary results showed epistatic interactions between the regions on Chromosome 6 and 1 and between Chromosome 6 and 7. During the development of sub-BILs, a digenic hybrid incompatibility was observed: plants carrying a L. saligna segment on Chromosome 6 always required a L. saligna segment on Chromosome 4. Segregation analysis suggested a prezygotic reproductive barrier by non-transmission of one specific hybrid gametophyte (male and female). In cooperation with the research group of Guido van den Ackerveken of University Utrecht a lettuce germplasm screening was conducted for candidate downy mildew effector proteins, which interact with resistance genes in lettuce and trigger a defence response. One of two responsive effector proteins, ‘BLG01’, triggered a hypersensitive cell death response in most tested L. saligna accessions and its response was mapped on Chromosome 9. Despite the complex interactions between resistance QTLs, this thesis research has delivered many insights that are important steps forward towards understanding the incompatible interaction between B. lactucae and L. saligna and its future application in resistance breeding.

Key message In a stacking study of eight resistance QTLs in lettuce against downy mildew, only three out of ten double combinations showed an increased resistance effect under field conditions. Abstract Complete race nonspecific resistance to lettuce downy mildew, as observed for the nonhost wild lettuce species Lactuca saligna, is desired in lettuce cultivation. Genetic dissection of L. saligna’s complete resistance has revealed several quantitative loci (QTL) for resistance with field infection reductions of 30–50 %. To test the effect of stacking these QTL, we analyzed interactions between homozygous L. saligna CGN05271 chromosome segments introgressed into the genetic background of L. sativa cv. Olof. Eight different backcross inbred lines (BILs) with single introgressions of 30–70 cM and selected predominately for quantitative resistance in field situations were intercrossed. Ten developed homozygous lines with stacked introgression segments (double combinations) were evaluated for resistance in the field. Seven double combinations showed a similar infection as the individual most resistant parental BIL, revealing epistatic interactions with ‘less-than-additive’ effects. Three double combinations showed an increased resistance level compared to their parental BILs and their interactions were additive, ‘less-than-additive’ epistatic and ‘more-than-additive’ epistatic, respectively. The additive interaction reduced field infection by 73 %. The double combination with a ‘morethan-additive’ epistatic effect, derived from a combination between a susceptible and a resistant BIL with 0 and 30 % infection reduction, respectively, showed an average field infection reduction of 52 %. For the latter line, an attempt to genetically dissect its underlying epistatic loci by substitution mapping did not result in smaller mapping intervals as none of the 22 substitution lines reached a similar high resistance level. Implications for breeding and the inheritance of L. saligna’s complete resistance are discussed.

Three regions with quantitative resistance to downy mildew of non-host and wild lettuce species, Lactuca saligna , disintegrate into seventeen sub-QTLs with plant-stage-dependent effects, reducing or even promoting the infection. Previous studies on the genetic dissection of the complete resistance of wild lettuce, Lactuca saligna, to downy mildew revealed 15 introgression regions that conferred plant stage dependent quantitative resistances (QTLs). Three backcross inbred lines (BILs), carrying an individual 30-50 cM long introgression segment from L. saligna in a cultivated lettuce, L. sativa, background, reduced infection by 60-70 % at young plant stage and by 30-50 % at adult plant stage in field situations. We studied these three quantitative resistances in order to narrow down their mapping interval and determine their number of loci, either single or multiple. We performed recombinant screenings and developed near isogenic lines (NILs) with smaller overlapping L. saligna introgressions (substitution mapping). In segregating introgression line populations, recombination was suppressed up to 17-fold compared to the original L. saligna × L. sativa F 2 population. Recombination suppression depended on the chromosome region and was stronger suppressed at the smallest introgression lengths. Disease evaluation of the NILs revealed that the resistance of all three BILs was not explained by a single locus but by multiple sub-QTLs. The 17 L. saligna-derived sub-QTLs had a smaller and plant stage dependent resistance effect, some segments reducing; others even promoting downy mildew infection. Implications for lettuce breeding are outlined.

Upon herbivore attack, plants activate an indirect defense, that is, the release of a complex mixture of volatiles that attract natural enemies of the herbivore. When plants are simultaneously exposed to two herbivore species belonging to different feeding guilds, one herbivore may interfere with the indirect plant defense induced by the other herbivore. However, little is understood about the mechanisms underlying such interference. Here, we address the effect of herbivory by the phloem-feeding whitefly Bemisia tabaci on the induced indirect defense of Arabidopsis thaliana plants to Plutella xylostella caterpillars, that is, the attraction of the parasitoid wasp Diadegma semiclausum. Assays with various Arabidopsis mutants reveal that B. tabaci infestation interferes with indirect plant defense induced by P. xylostella, and that intact jasmonic acid and ethylene signaling are required for such interference caused by B. tabaci. Chemical analysis of plant volatiles showed that the composition of the blend emitted in response to the caterpillars was significantly altered by co-infestation with whiteflies. Moreover, whitefly infestation also had a considerable effect on the transcriptomic response of the plant to the caterpillars. Understanding the mechanisms underlying a plant's responses to multiple attackers will be important for the development of crop protection strategies in a multi-attacker context.

Aphids are insects that cause direct damage to crops by the removal of phloem sap, but more importantly they spread devastating viruses. Aphids use their sophisticated mouthpart (i.e. stylet) to feed from the phloem sieve elements of the host plant. To identify genes that affect host plant resistance to aphids, we previously screened an Arabidopsis thaliana activation tag mutant collection. In such mutants, tagged genes are overexpressed by a strong 35S enhancer adjacent to the natural promoter, resulting in a dominant gain-of-function phenotype. We previously identified several of these mutants on which the aphid Myzus persicae showed a reduced population development compared with wild type. In the present study we show that the gene responsible for the phenotype of one of the mutants is At5g65040 and named this gene Increased Resistance to Myzus persicae 1 (IRM1). Overexpression of the cloned IRM1 gene conferred a phenotype identical to that of the original mutant. Conversely, an IRM1 knockout mutant promoted aphid population development compared to the wild type. We performed Electrical Penetration Graph analysis to investigate how probing and feeding behaviour of aphids was affected on plants that either overexpressed IRM1 or contained a knockout mutation in this gene. The EPG results indicated that the aphids encounter resistance factors while reaching for the phloem on the overexpressing line. This resistance mechanism also affected other aphid species and is suggested to be of mechanical nature. Interestingly, genetic variation for IRM1 expression in response to aphid attack was observed. Upon aphid attack the expression of IRM1 was initially (after 6 hours) induced in ecotype Wassilewskija followed by suppression. In Columbia-0, IRM1 expression was already suppressed six hours after the start of the infestation. The resistance conferred by the overexpression of IRM1 in A. thaliana trades off with plant growth.

1. Plants face threats from a variety of herbivorous insects and can use induced responses to defend themselves against these attackers. Induced responses are mediated by signal transduction involving phytohormones, such as jasmonic acid (JA) and salicylic acid (SA). Cross-talk between signal transduction pathways triggered by attackers with contrasting feeding styles allows plants to fine-tune defences. A central question in this emerging field is to understand how responses to single attackers interfere with responses to other attackers, especially by integratively addressing molecular and ecological aspects. 2. We examined the plant-mediated interactions between the leaf-chewing Pieris brassicae and the phloem-sucking Brevicoryne brassicae, and their respective parasitoids Cotesia glomerata and Diaeretiella rapae, when feeding simultaneously, sequentially or in isolation on the brassicaceous ecological model plant Brassica oleracea. We analysed the underlying defence mechanisms in the plant. Levels of the phytohormones JA and SA transcriptional responses of a number of selected defence-related genes and secondary plant compounds were quantified at different time points during the single and multiple infestations. 3. The caterpillars developed faster and reached a larger body mass on plants previously attacked by aphids. Aphids initially developed faster on plants with caterpillars, although the moment of moulting to adults was independent of the presence of caterpillars. Both parasitoid species performed better under multiple-infestation scenarios than in single-herbivore situations. 4. On plants attacked by aphids, the JA levels were tenfold lower than on undamaged plants or plants with caterpillars. Additionally, the low transcript levels of LOX and MYC, genes coding for a JA biosynthesis-related enzyme and a transcription factor, respectively, in aphid-infested plants, suggest that the facilitation of the caterpillar performance was mediated by interference in signal transduction. Levels of carbon and nitrogen and secondary plant compounds (glucosinolates) did not differ significantly between treatments, suggesting that these compounds did not mediate the facilitation. 5. Our data show that the leaf chewer and phloem feeder asymmetrically interact not via competition as would be expected from interspecific herbivores but instead via facilitation; the phloem feeder attenuated JA-related plant defences, thus facilitating the growth and development of the leaf chewers. In linear bitrophic systems, interactions between JA and SA signalling pathways have been proposed to allow plants to fine-tune their defences, but if facilitation frequently occurs in interspecific interguild interactions among herbivores this may represent an important constraint for plant defences. Such a constraint might be reduced if, as in our model system, parasitoids also benefit from interactions between interguild hosts and nonhosts, but parasitoids are rarely considered in model molecular systems to assess the impact of herbivore-induced plant defences.

The cabbage whitefly [Aleyrodes proletella L. (Hemiptera: Aleyrodidae)] is becoming a serious pest in Brassica oleracea L. (Brassicaceae) crops. However, almost nothing is known about the interaction of this insect with its host plants. Previous studies have shown differences in the natural occurrence of adults, eggs, and nymphs on the closely related B. oleracea cultivars Christmas Drumhead and Riviera grown in the field. In this study, we aimed to identify the nature of these differences and to gain insight into the resistance mechanisms against A. proletella. We used no-choice experiments on field- and greenhouse-grown plants to show that the differences between the two cultivars are mainly based on antibiosis (traits that reduce herbivore performance) and not on antixenosis (traits that deter herbivory). This was further supported by laboratory choice experiments that indicated little or no discrimination between the two cultivars based on plant volatiles. We showed that resistance is dependent on plant age, that is, resistance increased during plant development, and is mainly independent of environmental factors. Analysis of probing behaviour revealed that the resistance trait affects A. proletella at the phloem level and that morphological differences between the two cultivars are most likely not involved. We suggest that compounds present in the phloem reduce sap ingestion by the whitefly and that this explains the observed resistance

The jasmonic acid (JA) pathway is the main signal-transduction pathway induced by insect folivory. Mutant plants affected in the jasmonate pathway (18:0 and/or 16:0-oxylipin routes) were studied to assess the effects of JA and its oxylipin intermediates 12-oxophytodienoic acid (OPDA) and dinor-OPDA (dnOPDA) on interconnected signal-transduction pathways that underlie induced defenses in Arabidopsis. Our data show that the oxylipin jasmonates dnOPDA, OPDA and JA have different roles in defense signaling induced after feeding by the chewing-biting caterpillar Pieris rapae. Jasmonic acid, and not OPDA or dnOPDA, is the major signaling compound required for the induction of the defense-related genes LOX2 (Lipoxygenase 2), OPR3 (12-Oxophytodienoate reductase 3), ACX1 (Acyl-CoA oxidase 1) and PAL1 (Phenylalanine ammonia-lyase 1). Monitoring PAL1 transcript levels clearly showed that accumulation of JA upon P. rapae feeding results in the induction of the salicylic acid pathway. Furthermore, JA is the major signaling compound required for the P. rapae-induced expression of the defense-related gene HPL1 (Hydroperoxide lyase 1). The jasmonate dnOPDA influences the induction of the HPL-branch as well, yet its effect is antagonistic to the effect of JA. Our data show that these jasmonates may be used to fine-tune Arabidopsis’ herbivore-induced responses in terms of the HPL-branch from the oxylipin pathway.

Plants show phenotypic changes when challenged with herbivorous insects. The mechanisms underlying these changes include the activation of transcriptional responses, which are dependent on the attacking insect. Most transcriptomic studies on crucifer–insect interactions have focused on the model plant Arabidopsis thaliana, a species that faces low herbivore pressure in nature. Here, we study the transcriptional responses of plants from a wild black mustard (Brassica nigra) population to herbivores of different feeding guilds using an A. thaliana-based whole-genome microarray that has previously been shown to be suitable for transcriptomic analyses in Brassica. Transcriptional responses of B. nigra after infestation with either Pieris rapae caterpillars or Brevicoryne brassicae aphids are analyzed and compared. Additionally, the insect-induced expression changes of some individual genes are analyzed through quantitative real-time polymerase chain reaction. The results show that feeding by both insect species results in the accumulation of transcripts encoding proteins involved in the detoxification of reactive oxygen species, defensive proteins and glucosinolates and this is correlated with experimental evidence in the literature on such biochemical effects. Although genes encoding proteins involved in similar processes are regulated by both insects, there was little overlap in the induction or repression of individual genes. Furthermore, P. rapae and B. brassicae seem to affect different phytohormone signaling pathways. In conclusion, our results indicate that B. nigra activates several defense-related genes in response to P. rapae or B. brassicae feeding, but that the response is dependent on the attacking insect species.

1. Arthropod communities are structured by complex interactions between bottom-up (resource-based) and top-down (natural enemy-based) forces. Their relative importance in shaping arthropod communities, however, continues to be under debate. Bottom-up and top-down forces can be affected by intraspecific plant variation, for example by differences in concentrations of secondary metabolites that affect herbivore abundance through plant quality (bottom-up) or attract natural enemies of these herbivores (top-down). 2. Our objective was to investigate whether herbivore abundance is more strongly affected by plant-mediated bottom-up or top-down forces. 3. We used a model system of four cultivars of Brassica oleracea that show a high degree of variation in several plant traits, resistance to herbivores and attraction of natural enemies. During two field seasons, we recorded the abundance of several herbivorous and carnivorous insect species. To assess the relative importance of bottom-up and top-down forces, we quantified chemical and morphological traits of the cultivars (bottom-up) and assessed parasitization of herbivores and predator oviposition on plants inoculated with a controlled number of herbivores (top-down). 4. We show that intraspecific variation in plant chemistry and morphology consistently affects the abundance of insect herbivores and their natural enemies, resulting in cascading effects on tritrophic interactions in the associated insect community. Foliar profiles of glucosinolates and leaf toughness appeared most important for these effects. Brassica oleracea cultivars that harboured the largest numbers of herbivores also harboured the largest numbers of natural enemies. Differences in the fraction of herbivores parasitized and in predator oviposition on plants inoculated with a controlled number of herbivores could not explain the differences in natural abundance of herbivores. 5. Although abundance of herbivores is most likely influenced by a combination of bottom-up and top-down forces, it appears that in the tritrophic system investigated, bottom-up forces (plant chemistry and morphology) were more important for herbivore abundance than plant-mediated top-down forces (attraction and arrestment of natural enemies).

Background Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. Results In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. Conclusions This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.