Institut für Phytomedizin

Permanent URI for this collectionhttps://hohpublica.uni-hohenheim.de/handle/123456789/14

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Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches
(2022) Jeckel, Adriana Moriguchi; Beran, Franziska; Züst, Tobias; Younkin, Gordon; Petschenka, Georg; Pokharel, Prayan; Dreisbach, Domenic; Ganal-Vonarburg, Stephanie Christine; Robert, Christelle Aurélie Maud
Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.
Morphological and chemical plant properties mediate host plant selection of whiteflies (Hemiptera: Aleyrodidae)
(2021) Stoll, Nina Sara; Zebitz, Claus P. W.
Whiteflies are among the most important pests causing severe damage to numerous cultivated and ornamental plants worldwide. The present dissertation comprises four studies and contributes to the knowledge of the host plant selection process by whiteflies. In the first study, host preferences were determined in dual choice tests for Aleyrodes proletella (L.), Bemisia tabaci (Genn.), and Trialeurodes vaporariorum (Westw.) on several host plants. On the one hand, this study extends the knowledge on the food spectrum of these economically important pests; on the other hand, the results highlight the host adaptation of whiteflies. The second study elucidated potential sources of host plant resistance against A. proletella, B. tabaci, and T. vaporariorum by recording their probing and feeding behaviour on two host plants each using the electrical penetration graph (EPG) method. It is concluded that whiteflies decide upon host plant acceptance by evaluation of multiple plant factors located in epidermal and/or mesophyll tissues of leaves as well as in the phloem sap of plants. Moreover, epicuticular leaf waxes are a key factor in the host selection process of A. proletella. It is hypothesized that constituents of the leaf surface wax act as stimulants promoting leaf penetration and phloem accession. The findings of this study shed light on the whitefly-host adaptation. The goal of the third study was to identify the role of epicuticular leaf waxes of several Brassica cultivars in the host selection process of A. proletella. For this purpose, dual choice tests were carried out on both waxy and dewaxed plant leaves as well as on Parafilm® treated with different leaf wax extracts. Also, life-history traits were monitored on waxy and dewaxed leaves, and the feeding activity of A. proletella was recorded on Parafilm® with and without leaf wax extracts. Scanning electron microscopy (SEM) imaging was used to visualize epicuticular leaf waxes on the plant surface. Finally, it was proved that leaf surface waxes of host plants promote feeding and act as phagostimulants. Although the wax compounds mediating host plant selection remain unknown, these findings offer breeding potential for resistant crop cultivars. In the fourth study, the influence of free phloem amino acids on the host plant selection of T. vaporariorum was investigated. Via liquid chromatography-mass spectrometry (LC-MS), the amino acid profiles in the phloem sap of six vegetable crops varying in their host plant attractiveness were analysed. Subsequently, stepwise multiple regressions of the relative amino acid compositions and the pre-determined host plant preferences were performed. To verify the contribution of single amino acids on host choice, dual choice tests on sucrose media with and without added single amino acids were carried out. Single amino acids play an active role in phagostimulation, whereas some amino acids exert strong inhibitory effects. This indicates that the dominant presence of such amino acids might reduce phloem sap uptake, thus contributing to host plant resistance towards T. vaporariorum. Overall, this research compared the host selection process of three whitefly species to identify their underlying mechanisms. It is hypothesized that the observed host selection strategies are the result of evolutionary adaptations between whiteflies and their host plants. Depending on the occupied ecological niche, species-specific host plant ranges of varying complexity were formed. Accordingly, the host selection process of the more specialised species A. proletella is particularly efficient by consideration of characteristic leaf surface wax stimuli. In contrast, host selection of the extreme generalists B. tabaci and T. vaporariorum is regulated by simple gustatory stimuli in order to take advantage of the host diversity they are offered. The findings of this research provide the basis for new approaches to optimizing breeding programs for whitefly resistant crops.
Physiological and ecological implications of sequestered cardenolides in the milkweed bugs (Heteroptera: Lygaeinae)
(2022) Pokharel, Prayan; Petschenka, Georg
Milkweed bugs (Heteroptera: Lygaeinae) have a predilection for toxic plants, and possess a distinctive black and red coloration. Although many milkweed bugs are generalist seed predators, they commonly feed on plants in the family Apocynaceae (milkweed) which often contain toxic cardenolides. Cardenolides inhibit the ubiquitous Na+/K+-ATPase, an essential animal enzyme mediating essential physiological functions. Milkweed bugs possess pronounced insensitivity towards cardenolides due to a few amino acid substitutions in the Na+/K+-ATPase (i.e., target site insensitivity) and sequester cardenolides for protection against their predators. The overarching question remains whether chemical defenses, in aposematic individuals sequestering toxins, incur physiological costs, such as effects on growth or other fitness parameters like longevity and fecundity, production of color pigments, and handling oxidative stresses, and/or ecological costs, such as universality of toxin defense. Using an artificial diet, I raised larvae of four milkweed bug species (Oncopeltus fasciatus, Caenocoris nerii, Spilostethus pandurus and Arocatus longiceps) and a closely related pyrrhocorid bug species (Pyrrhocoris apterus) on three increasing dietary doses of cardenolides, and assessed the increase in growth by recording the mass until adult. Additionally, I investigated the life-history parameters only in O. fasciatus. To understand if milkweed bugs exhibit honest signaling, using same artificial diet treatment, the color intensity of O. fasciatus was measured by taking photographs in each larval stage until adulthood. To understand if toxin sequestration in milkweed bugs imposes oxidative stress, biomarkers of oxidative stress was measured through biochemical assays for lipid peroxidation (malondialdehyde, or MDA), superoxide dismutase (SOD), and total glutathione content (GSH). To understand why protection against certain predators is not observed in all bug species although they feed on seeds containing cardenolides, I tested if the outcome of predator- prey interaction was mediated by the structural variation within the same class of compound or by the insect species. For this purpose, I raised two milkweed bug species (Lygaeus equestris and Horvathiolus superbus) on the seeds of two phylogenetically unrelated host plants (Ranunculaceae: Adonis vernalis and Plantaginaceae: Digitalis purpurea) from which the bugs sequestered cardenolides, and carried out predation assays with lacewing larvae. The amount of toxins sequestered by the milkweed bugs was estimated using high performance liquid chromatography. My research revealed that dietary plant toxins increased growth in the sequestering specialists (O. fasciatus and C. nerii) but not in the sequestering generalist, S. pandurus, despite all possessing toxin-resistant Na+/K+-ATPases. Under exposure to the dietary toxins, the growth of A. longiceps nymphs (resistant and non-sequestering) was unaffected, while that of P. apterus (non-resistant and non-sequestering) was impaired. In addition, O. fasciatus nymphs developed to adults faster and lived longer as adults under toxin exposure when compared to individuals raised on the control diet, but produced significantly fewer offspring unless being transferred to a toxin-free diet after reaching adulthood. Furthermore, I showed that O. fasciatus raised on the high and medium levels of dietary cardenolides had significantly lower levels of GSH. Bugs with more GSH levels had brighter warning signals but these signals were not related to sequestration. Besides physiological aspects, the chance of milkweed bugs surviving a predator attack strongly depended on the structural differences of sequestered toxins. Overall, I found that cardenolide consumption exerts a positive effect on overall fitness in milkweed bugs, a conclusion in disagreement with current theory predicting costs of sequestration. Oxidative state may be a fundamental aspect where costs lie in aposematic individuals sequestering toxins, and the effect of plant-toxin sequestration on predators is affected by the structural variation of defensive compounds and therefore depends on the ecological context, i.e., host-plant use. My dissertation provides insight into the implications of physiology and ecology on sequestering aposematic insects, giving us a better understanding of plant-insect-predator interactions.
Role of benzoic acid and lettucenin A in the defense response of lettuce against soil-borne pathogens
(2021) Windisch, Saskia; Walter, Anja; Moradtalab, Narges; Walker, Frank; Höglinger, Birgit; El-Hasan, Abbas; Ludewig, Uwe; Neumann, Günter; Grosch, Rita
Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.
Sequestration of plant toxins in milkweed bugs (Heteroptera: Lygaeinae) : physiological implications and mechanisms
(2023) Espinosa del Alba, Laura; Petschenka, Georg
Insect herbivores and plants together are a crucial component of terrestrial macro-biodiversity. Within the realm of plant-insect interactions, phytophagy by insects triggered an “arms-race” dynamic resulting in escalatory adaptation and counter-adaptation over time. This coevolution led to complex phenomena such as sequestration of plant toxins by specialized insects, with the main aim to deter predators. Although sequestration is an extensively reported phenomenon, many physiological aspects and underlying mechanisms remain largely unexplored. Milkweed bugs (Heteroptera: Lygaeinae) constitute a versatile model ideally suited for studying both areas due to their particular evolutionary history. They are primarily associated with plant species in the Apocynaceae which commonly produce cardenolides, but remarkably some milkweed bug species secondarily evolved novel associations with phylogenetically disparate plant families supplying new sources of chemically related or unrelated toxins. Using as model milkweed bugs the cardenolide specialist Oncopeltus fasciatus and Spilostethus saxatilis, a species that shifted to sequestration of the chemically unrelated colchicoids, the present thesis first aimed to develop a new artificial diet that allowed the incorporation of the desired types and amounts of toxins without impairing insect performance. Taking a simplified approach, an artificial diet presented in a pill form and made of 100% organic sunflower meal was established. Despite the fact that the new diet has remarkable energy and nutrient differences with sunflower seeds (the laboratory diet), no differences in terms of insect performance were found between the two diets in O. fasciatus and S. saxatilis. Moreover, the new diet presented an acceptable concentration accuracy and shelf-life for short-term toxin feeding assays. Once established, the new diet was used to investigate the effects of cardenolides and colchicoids on several life-history traits of S. saxatilis, and to compare them with the effect of cardenolides in O. fasciatus. Although both classes of toxins have different molecular targets (cardenolides: Na+/K+-ATPase; colchicoids: tubulin), S. saxatilis was able to sequester them at a cost-free level. In fact, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis after dietary exposure to cardenolides and colchicoids, respectively. Among cardenolides, labriformin is especially toxic for milkweed-specialist Na+/K+-ATPases in vitro. Nevertheless, it was shown to have no costs in terms of growth and fertility at the whole organism level for O. fasciatus. This finding might be an example of reciprocal evolution between milkweed plants and its herbivores, where highly toxic cardenolides specifically targeted to insect specialists are counteracted by tolerance, detoxification, and sequestration strategies. To assess the role of sequestration beyond normal physiological conditions, O. fasciatus and S. saxatilis were orally infected with the bacterium Pseudomonas entomophila. Neither cardenolides nor colchicoids provided a higher resistance or tolerance. Regarding mechanisms of sequestration, the other overarching research topic of the present thesis, both in vivo (whole animal) and in vitro (isolated digestive tracts) approaches showed no reciprocal competition for the same transport mechanism between chemically related and unrelated toxins. Furthermore, the digestive tract of milkweed bugs did not seem to be a critical mediator as it is for other non-sequestering and sequestering species. The time course of sequestration for the model species was resolved from three days to one hour, and the higher levels of colchicoids detected in S. saxatilis compared to the level of cardenolides in O. fasciatus might indicate an early acquisition of defenses with the shift from cardenolide to colchicoid-containing plants. Finally, a hint to preadaptation mechanisms to resist novel toxins was documented in Spilostethus pandurus, a species that belongs to the same genus as S. saxatilis, thereby providing a basis for future investigations.

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