Influence of primary and secondary plant metabolites on the migration and feeding behavior of Cacopsylla pruni, the vector of European Stone Fruit Yellows

Der Pflaumenblattsauger, Cacopsylla pruni ist eine univoltine Insektenart, welche sich mit ihren spezialisierten Mundwerkzeugen stechend-saugend vom Phloem ihrer Wirtspflanzen ernährt. Während ihres Lebens alternieren Pflaumenblattsauger zwischen laubtragenden Prunus-Bäumen und immergrünen Koniferen. Zu Beginn des Frühjahrs fliegen die adulten Blattsauger in Steinobstanlagen, wo sie ihre Eier bevorzugt auf bestimmte Prunus-Arten ablegen. Nach der Paarung und der Eiablage sterben die Individuen der alten Generation (Remigrants). Die nächste Generation entwickelt sich von April bis Juni. Die jungen Adulten (Emigrants) bleiben noch einige Tage auf den Steinobstbäumen, bevor sie auf Nadelbäume in höheren Lagen abwandern. Dort verbleiben sie bis zum nächsten Frühjahr, in welchem sie zwecks Reproduktion wieder zurück zum Steinobst wandern. Das Verbreitungsgebiet des Pflaumenblattsaugers erstreckt sich über Europa und angrenzende Gebiete. Von Relevanz für den Obstanbau ist C. pruni hauptsächlich wegen seiner Fähigkeit, das Phytoplasma ‘Candidatus Phytoplasma prunorum’ zu übertragen. Dabei handelt es sich um ein zellwandloses Bakterium, welches in seinen Wirtspflanzen ausschließlich im Phloem verbreitet ist. Wenn die Pflaumenblattsauger an infizierten Prunus-Bäumen saugen, nehmen sie die Phytoplasmen aus dem Phloem auf. Nachdem sich die Bakterien in den Insekten vermehrt haben, können sie über den Speichel der Blattsauger auf gesunde Prunus-Bäume übertragen werden. ‘Ca. P. prunorum’ ruft die sog. Europäische Steinobstvergilbung (European Stone Fruit Yellows, ESFY) hervor. Dabei handelt es sich um eine der bedeutendsten Pflanzenkrankheiten im Europäischen Obstanbau, welche zu massiven Ernteausfällen und wirtschaftlichen Einbußen führt. Von den typischen Symptomen, wie dem verfrühten Austrieb, dem chlorotischen Blattrollen und der Notreifung der Früchte, sind vor allem für den Anbau kultivierte Sorten von Pfirsichen (Prunus persica), Aprikosen (Prunus armeniaca) und Japanischen Pflaumen (Prunus salicina) betroffen. In diesen Arten führt die Infektion innerhalb weniger Jahre zum Absterben der Bäume. Heimische Arten wie Schlehen (Prunus spinosa) und wilde Pflaumen (Prunus cerasifera, Prunus insititia) zeigen meist keine schwerwiegenden Symptome, ebenso die meisten kultivierten Sorten von Pflaumen (Prunus domestica). Bis heute gibt es keine Maßnahmen zur Bekämpfung von Phytoplasmosen. Eine Alternative stellt die Regulation der Vektorinsekten dar. Mit Hilfe sogenannter Infochemikalien könnte das Verhalten der Blattsauger so manipuliert werden, dass diese aus den Steinobstanlagen ferngehalten werden. Dadurch kann die Anzahl der Neuinfektionen mit ESFY gemindert werden. Da bisher nur wenig über die Biologie und Ökologie des Pflaumenblattsaugers bekannt ist, habe ich in der vorliegenden Arbeit den Einfluss von pflanzenbürtigen Duft- und Inhaltsstoffen auf das Verhalten und die Fitness von C. pruni untersucht. Anhand von Feldstudien zum Vorkommen von C. pruni in verschiedenen Prunus-Arten identifizierte ich welche Wirtspflanzen von C. pruni bevorzugt besiedelt werden. Dadurch konnte ich P. insititia als eine der bevorzugten Prunus-Arten einstufen. Im Gegensatz dazu wurden nur wenige Pflaumenblattsauger auf P. persica Bäumen gefunden. In den Studien zur Wirts-Präferenz von C. pruni der vorliegenden Arbeit wurden die Duft- und Inhaltsstoffe dieser beiden Prunus-Arten und Koniferen verglichen, um den Einfluss von olfaktorischen und gustatorischen Reizen auf das Verhalten der Pflaumenblattsauger zu beurteilen. Mit der Aufzeichnung von Elektroantennogrammen konnte ich zeigen, dass Pflaumenblattsauger Weibchen sowohl volatile Substanzen aus dem Duft von Prunus-Bäumen als auch typische Nadelbaumdüfte wahrnehmen können. An Hand von Olfaktometerversuchen mit C. pruni konnte ich jedoch die Bevorzugung bestimmter Wirtspflanzen nicht allein auf olfaktorische Reize zurückführen. Daher untersuchte ich ebenfalls den Einfluss der Pflanzeninhaltstoffe auf C. pruni. In einer Entwicklungsstudie konnte ich beweisen, dass sich C. pruni Nymphen besser auf der präferierten wilden Pflaumenart P. insititia, als auf der weniger bevorzugten kultivierten Pfirsichsorte P. persica cv. South Haven entwickeln können. Die  Entwicklungsunterschiede korrelieren mit den Ergebnissen meiner Untersuchung zur Zusammensetzung des Phloemsaftes beider Prunus-Arten. Für die Präferenz von bestimmten Wirtspflanzen scheinen gustatorische Reize für C. pruni wichtiger zu sein als olfaktorische Signale. Dass die Zusammensetzung des Pflanzensaftes eine wichtige Rolle für C. pruni spielt, konnte ich durch weitere Entwicklungsstudien an Koniferen bestätigen. Es zeigte sich, dass sich C. pruni Nymphen nicht auf Nadelbäumen entwickeln können, auch wenn sie Pflanzensaft von Koniferen aufnehmen. Adulte C. pruni hingegen überleben signifikant länger auf Nadelbäumen als ohne Nahrungsquelle. Woraus ich schließe, dass sie die immergrünen Nadelbäume als Wasser- und Nährstoffquellen im Winter benötigen und daher auf den Wirtswechsel angewiesen sind. Des Weiteren wurde in der vorliegenden Arbeit untersucht, ob sich eine Infektion mit ‘Ca. P. prunorum’ auf die Interaktion zwischen den Vektorinsekten und ihren Wirtspflanzen auswirkt. Zu diesem Zweck wurde das Saugverhalten der Nymphen an ESFY-infizierten und nichtinfizierten P. insititia und P. persica Bäumen mittels Elektropenetrographie untersucht. Dabei zeigte sich, dass sich die Infektion der Prunus-Pflanzen nur auf die mittlere Dauer der Aufnahme von Xylemsaft auswirkt. C. pruni-Nymphen saugten durchschnittlich weniger am Xylem von infizierten Prunus-Bäumen. Die durchschnittliche Dauer der Saugaktivität im Phloem der Wirtspflanzen wurde nicht durch die Infektion beeinflusst. Zusätzlich analysierte ich den Inhalt des Phloems. Dabei war es nicht möglich, dessen chemische Zusammensetzung auf Grund der ‘Ca. P. prunorum’ Infektion zu unterscheiden. In Übereinstimmung mit diesen Ergebnissen wirkte sich die Infektion der Wirtspflanzen nicht auf die Entwicklungsgeschwindigkeit von C. pruni-Nymphen aus.The plum psyllid Cacopsylla pruni is a univoltine herbivore, specialized on Prunus and coniferous tree species. During their lifetime, plum psyllids are alternating twice between their deciduous and evergreen hosts. For reproduction, C. pruni adults migrate to stone fruit orchards in spring, where they lay their eggs exclusively on several Prunus species. Adults of the old generation die after mating and oviposition. Young adults emerge from egg to adults during April, May and June. After several days the young adults (called emigrants) emigrate to conifers in higher regions until they remigrate (remigrants) to Prunus orchards in the next spring. Plum psyllids transmit the Phytoplasma ‘Candidatus Phytoplasma prunorum’ and are therefore of significant importance for fruit growers. In host plants, the wall-less bacterium is restricted to the phloem and causes the European Stone Fruit Yellows (ESFY). Psyllids acquire the bacteria during feeding on the phloem of infected Prunus trees. After multiplication of the phytoplasma inside the vector, plum psyllids transmit the disease to non-infected Prunus trees by salivary excretion during feeding. C. pruni is distributed all over Europe and bordering areas. ESFY is one of the most serious plant diseases in European fruit production, causing severe plant damage leading toa poor harvest and high economic losses. Peaches (Prunus persica), apricots (Prunus armeniaca) and Japanese plums (Prunus salicina) are worst affected by typical symptoms, such as reduced dormancy, chlorotic leaf roll and premature ripening of the fruits. Trees of these species suffer severely from the infections, decline and finally die. Commonly indigenous Prunus species, such as blackthorn (Prunus spinosa) und wild plums (Prunus cerasifera, Prunus insititia) show more tolerance towards ESFY infections. Likewise, most of the cultivated varieties of European plums (Prunus domestica) do not develop severe symptoms.To date no effective control agents or cures for phytoplasma diseases are available. The control of vector insects is an alternative strategy. Psyllid behavior could be manipulated with infochemicals and prevent C. pruni from feeding and oviposition on stone fruit crops and thus reduce the number of new infections. In this thesis I investigated the impact of plant-borne volatiles and phloem chemistry on the behavior of C. pruni as yet barely anything is known about the biology and chemical ecology of plum psyllids. The field monitoring presented in this thesis proved a preference of C. pruni for some Prunus species over others. P. insititia was identified as a favored host of C. pruni, in contrast very low numbers of plum psyllids were detected on P. insititia trees, which was therefore categorized as a non-preferred host for C. pruni. In the studies of this thesis, I compared the volatile organic compounds and the phloem sap composition of these two Prunus species and Conifers, to identify signals that were important for host plant preference of C. pruni. I demonstrated the detection of volatile compounds characteristic for Prunus trees as well as characteristic coniferous host volatiles of female plum psyllid antenna by electroantennography. Olfactometer tests revealed that this preference is not only based on olfactory cues. Additionally, gustatory cues seem to play a major role in host acceptance and preference. C. pruni nymphs showed greater development success on preferred wild plum species (P. insititia) compared to nymphs on non-preferred peach trees (P. persica). Next to effects on psyllid development, I detected differences in the phloem composition of both plant species. My research on the feeding behavior of plum psyllids on coniferous diets revealed that although C. pruni nymphs showed feeding on conifer needles, they are not able to develop on conifers. In contrast, adult plum psyllids survived longer on spruce (Picea abies) and silver fir (Abies alba) than without food supply. I concluded that adult C. pruni need evergreen tree species as resource of water and nutrition during overwintering. Furthermore, I investigated the impact of ‘Ca. P. prunorum’ infections of Prunus trees on the interaction between vector insects and their host plants. For this purpose, I recorded the feeding behaviour of C. pruni nymphs on infected and non-infected P. insititia and P. persica trees by electropenetrography. Interestingly, the average duration each nymph spend with the ingestion of xylem was shorter on infected than on non-infected Prunus trees. I found no influence on the average duration of phloem phases per nymph due to the infection status of both Prunus species. Chemical analysis of the phloem centrifugates showed that the chemical composition of trees infected with ‘Ca. P. prunorum’ was indistinguishable from the composition of non-infected Prunus trees. In accordance, the development of C. pruni nymphs was not influenced by the infection of host plants.The knowledge obtained in this thesis is essential for the development of innovative and selective control strategies against C. pruni based on semiochemicals, such as push-pull and attractand-kill strategies. Further breeding programs of resistant Prunus cultivars should take the findings of this work into account

Communication between undamaged plants can elicit changes in volatile emissions from neighbouring plants, thereby altering their susceptibility to aphids

Plant volatiles play an important role in intra- and interspecific plant communication, inducing direct and indirect defenses against insect pests. However, it remains unknown whether volatile interactions between undamaged cultivars alter host plant volatile emissions and their perception by insect pests. Here, we tested the effects of exposure of a spring barley, Hordeum vulgare L., cultivar, Salome, to volatiles from other cultivars: Fairytale and Anakin. We found that exposing Salome to Fairytale induced a significantly higher emission of trans-beta-ocimene and two unidentified compounds compared when exposed to Anakin. Aphids were repelled at a higher concentration of trans-beta-ocimene. Salome exposure to Fairytale had significant repulsive effects on aphid olfactory preference, yet not when Salome was exposed to Anakin. We demonstrate that volatile interactions between specific undamaged plants can induce changes in volatile emission by receiver plants enhancing certain compounds, which can disrupt aphid olfactory preferences. Our results highlight the significant roles of volatiles in plant-plant interactions, affecting plant-insect interactions in suppressing insect pests. This has important implications for crop protection and sustainable agriculture.The airborne interactions between certain undamaged spring barley (Hordeum vulgare L.) cultivars induce changes in volatile emission in receiving plants by enhancing the production of certain ideal volatile organic compounds. These changes trigger strong repellent effects on aphids (Rhopalosiphum padi L.), suggesting the significant roles of volatile-mediated plant-plant interactions in the development of integrated pest management for sustainable agriculture practices

Mating still disrupted: Future elevated CO2 concentrations are likely to not interfere with Lobesia botrana and Eupoecilia ambiguella mating disruption in vineyards in the near future

The successful, area-wide application of the mating disruption (MD) technique, an insect sex pheromone-based biotechnological pest control method, against the European grapevine moth Lobesia botrana and the European grape berry moth Eupoecilia ambiguella, has led to drastic reductions in insecticide application in vineyards. However, since insect pheromone perception and emission can be affected by abiotic conditions, the future success of MD may be affected by climate change. At the same time, politics and society are calling for drastic and sustainable reductions in pesticide application, making highly specific, efficient, and environmentally friendly pest control techniques like MD more important than ever. To anticipate whether climate change factors will interfere with the MD of L. botrana and E. ambiguella in vineyards, we conducted field experiments in the Geisenheim VineyardFACE (Free-Air Carbon dioxide Enrichment) facility. The insects were raised at ambient or elevated temperatures in the lab and male moths were released in cages installed in the VineyardFACE facility. Trap recapture rates obtained by pheromone lures or female moths under elevated or ambient CO2 in areas with and without MD were evaluated. Our results did not indicate a reduced efficacy of L. botrana or E. ambiguella MD at elevated CO2 concentrations, irrespective of the temperature the moths were raised under. From a practical point of view-and especially from an ecological one-our results are good news. They indicate that MD will not be negatively affected by future elevated CO2 concentrations

Altered volatile emission of pear trees under elevated atmospheric CO2 levels has no relevance to pear psyllid host choice

The impact of climate change drivers on cultivated plants and pest insects has come into research focus. One of the most significant drivers is atmospheric carbon dioxide, which is converted into primary plant metabolites by photosynthesis. Increased atmospheric CO2 concentrations therefore affect plant chemistry. The chemical composition of non-volatile and volatile organic compounds of plants is used by insects to locate and identify suitable host plants for feeding and reproduction. We investigated whether elevated CO2 concentrations in the atmosphere affect the plant-pest interaction in a fruit crop of high economic importance in Europe. Therefore, potted pear trees were cultivated under specified CO2 conditions in a Free-Air Carbon dioxide Enrichment (FACE) facility at Geisenheim University in Germany for up to 14 weeks, beginning from bud swelling. We compared emitted volatiles from these pear trees cultivated for 7 and 14 weeks under two different CO2 levels (ambient: ca. 400 ppm and elevated: ca. 450 ppm CO2) and their impact on pest insect behavior. In total, we detected and analyzed 76 VOCs from pear trees. While we did not detect an overall change in VOC compositions, the relative release of single compounds changed in response to CO2 increase. Differences in VOC release were inconsistent over time (phenology stages) and between study years, indicating interactions with other climate parameters, such as temperature. Even though insect-plant interaction can rely on specific volatile compounds and specific mixtures of compounds, respectively, the changes of VOC patterns in our field study did not impact the host choice behavior of C. pyri females. In olfactometer trials, 64% and 60% of the females preferred the odor of pear trees cultivated under elevated CO2 for 7 and 14 weeks, respectively, over the odor from pear trees cultivated under ambient CO2. In binary-choice oviposition assays, C. pyri females laid most eggs on pears during April 2020; on average, 51.9 (+/- 51.3) eggs were laid on pears cultivated under eCO(2) and 60.3 (+/- 48.7) eggs on aCO(2.

Convergence and molecular evolution of floral fragrance after independent transitions to self-fertilization

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution. 1 The transition from outcrossing to self -fertilization is common in plant evolution 2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards. 3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases. 2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self -fertilization in Capsella . 4-9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of b-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation

Alterations in the odor profile of plants in cultivar mixtures affect aphid host-location behavior

The effect of cultivar mixtures on aphid control is attributed to the masking or alteration of host-preferred cultivar odor cues. However, the underlying physiological mechanism remains unclear. This study assessed alterations in the volatile emissions of wheat cultivars grown together (Florence-Aurora and Forment; Florence-Aurora and Montcada) and the consequences for the olfactory preference of aphids. Volatile organic compounds were collected from wheat plants grown in a laboratory under mixed or monoculture conditions and subsequently analyzed. The odor profiles of Florence-Aurora and Montcada were indistinguishable from each other. However, the odors of Florence-Aurora and Forment grown in monocultures differed significantly from those emitted by their mixture. The Florence-Aurora and Forment mixture induced plant physiological responses that affected the emission of single volatile compounds and, consequently, altered volatile organic compound ratios. English grain aphids (Sitobion avenae) were less attracted to the odors of Florence-Aurora and Forment when grown as a mixture than the combination of the odors from Florence-Aurora and Forment monocultures. Moreover, aphids preferred clean air over the odor from the Florence-Aurora and Forment mixture but preferred the odor from the Florence-Aurora and Montcada mixture over clean air. This study highlights the beneficial effects of intraspecific plant diversity on aphid control by altering plant odors in response to plant-plant interactions. The emission of less attractive odor cues consequently affects plant-aphid interactions; hence, less attractive odors are likely to impair aphid host-locating behavior. This effect was exclusive to certain cultivar mixtures, which supports the "right neighbor" concept

Alterations in the odor profile of plants in cultivar mixtures affect aphid host-location behavior

The effect of cultivar mixtures on aphid control is attributed to the masking or alteration of host-preferred cultivar odor cues. However, the underlying physiological mechanism remains unclear. This study assessed alterations in the volatile emissions of wheat cultivars grown together (Florence-Aurora and Forment; Florence-Aurora and Montcada) and the consequences for the olfactory preference of aphids. Volatile organic compounds were collected from wheat plants grown in a laboratory under mixed or monoculture conditions and subsequently analyzed. The odor profiles of Florence-Aurora and Montcada were indistinguishable from each other. However, the odors of Florence-Aurora and Forment grown in monocultures differed significantly from those emitted by their mixture. The Florence-Aurora and Forment mixture induced plant physiological responses that affected the emission of single volatile compounds and, consequently, altered volatile organic compound ratios. English grain aphids (Sitobion avenae) were less attracted to the odors of Florence-Aurora and Forment when grown as a mixture than the combination of the odors from Florence-Aurora and Forment monocultures. Moreover, aphids preferred clean air over the odor from the Florence-Aurora and Forment mixture but preferred the odor from the Florence-Aurora and Montcada mixture over clean air. This study highlights the beneficial effects of intraspecific plant diversity on aphid control by altering plant odors in response to plant-plant interactions. The emission of less attractive odor cues consequently affects plant-aphid interactions; hence, less attractive odors are likely to impair aphid host-locating behavior. This effect was exclusive to certain cultivar mixtures, which supports the “right neighbor” concept

Unraveling the Host Plant Alternation of Cacopsylla pruni – Adults but Not Nymphs Can Survive on Conifers Due to Phloem/Xylem Composition

Plant sap feeding insects like psyllids are known to be vectors of phloem dwelling bacteria (‘Candidatus Phytoplasma’ and ‘Ca. Liberibacter’), plant pathogens which cause severe diseases and economically important crop damage. Some univoltine psyllid species have a particular life cycle, within one generation they alternate two times between different host plant species. The plum psyllid Cacopsylla pruni, the vector of European Stone Fruit Yellows (ESFY), one of the most serious pests in European fruit production, migrates to stone fruit orchards (Prunus spp.) for mating and oviposition in early spring. The young adults of the new generation leave the Prunus trees in summer and emigrate to their overwintering hosts like spruce and other conifers. Very little is known about the factors responsible for the regulation of migration, reasons for host alternation, and the behavior of psyllids during their phase of life on conifers. Because insect feeding behavior and host acceptance is driven by different biotic factors, such as olfactory and gustatory cues as well as mechanical barriers, we carried out electrical penetration graph (EPG) recordings and survival bioassays with C. pruni on different conifer species as potential overwintering hosts and analyzed the chemical composition of the respective plant saps. We are the first to show that migrating psyllids do feed on overwintering hosts and that nymphs are able to ingest phloem and xylem sap of coniferous trees, but cannot develop on conifer diet. Analyses of plant saps reveal qualitative differences in the chemical composition between coniferous trees and Prunus as well as within conifer species. These differences are discussed with regard to nutritional needs of psyllid nymphs for proper development, overwintering needs of adults and restriction of ‘Ca. P. prunorum’ to Prunus phloem