Multitrophic Interactions in a Plant – Pathogen – Vector - Antagonist System

Apple proliferation disease causes considerable economic damage to apple production, as the fruits of infected apple trees are smaller, inedible, and thus impossible to market. Typical symptoms are enlarged stipules and 'witches broom' caused by proliferation of axillary buds. These morphological and physiological changes in the plant are caused by the cell wall-less phloem-restricted bacterium 'Candidatus Phytoplasma mali'. Phytoplasmas are mainly transmitted by phloem-feeding insects. The phytoplasma is acquired when the insect feeds on the phloem of infected trees. After multiplication in the vector’s body, the phytoplasma can then be transmitted through the insect's saliva and infect healthy trees. In Germany, the summer apple psyllid Cacopsylla picta is the only known vector of the phytoplasma 'Ca. P. mali'. The univoltine species changes its host plants twice during its nearly one year-long life: the nymphs develop on young apple shoots in spring/summer before the winged adults hatch. These so-called emigrants briefly remain on the apple trees before migrating to conifers as their overwintering hosts, to spend the rest of the year and the entire winter. The next spring, the overwintered adults (remigrants) migrate back to the apple orchards for mating and oviposition. All phloem-feeding life stages, i.e. nymphs but also the adult migratory stages (emigrants and remigrants) can acquire and transmit 'Ca. P. mali'. Since phytoplasma-infected trees cannot be cured so far, vector control is considered the most important measure to reduce phytoplasmosis. Therefore, the vector insect C. picta had the central role in my studies, in which I investigated different aspects of the interaction between plant, phytoplasma (pathogen) and vector (herbivore). Additional studies with a vector antagonist further involved another trophic level, that of natural enemies, allowing the investigation of the multitrophic interaction in the plant - pathogen - vector – antagonist system. To study the interaction between the phytoplasma and its host plant, I analyzed the effects of 'Ca. P. mali' infection on the phloem composition of apple trees. The chemical analyses revealed that the phloem metabolite composition of infected apple trees differed significantly from that of non-infected apple trees. The relative amount of all sugar and sugar alcohols was significantly increased in ‘Ca. P. mali’ infected plants, whereas amino acid and other organic acid content was similar to non-infected plants. Hence, through this increase in sugars and sugar alcohols but unchanged amounts of amino acids, the ratio of carbohydrates: amino acids shifted in the phloem sap of infected plants. Thus, I was able to show that ‘Ca. P. mali’ infection altered the chemical composition of the phloem, the nutritional medium used by phytoplasma and vector alike. Therefore, I investigated how these changes in the phloem composition affected the behavior of the vector C. picta, since the acceptance of a plant as a suitable host for feeding or oviposition is a crucial individual decision with potentially serious implication for insect population dynamics. Overwintered females indeed preferred non-infected plants for oviposition and thus as food source for the next generation. Using electropenetrography (EPG) for the first time for measuring the feeding behavior of C. picta, I was able to show that plant-mediated effects of 'Ca. P. mali' infection also had an influence on its probing behavior. The release of watery saliva into the phloem, and thus the first phloem contact, occurred significantly later on infected apple trees. However, this behavioral change of the vector could not be attributed solely to gustatory stimuli or changes in nutrient availability due to the altered phloem composition, as the duration and frequency of actual phloem ingestion remained unaffected. Nevertheless, EPG recordings revealed that nymphs ingested phloem significantly more frequently during their development than adult stages. Therefore, I conclude that phloem ingestion is a demand-regulated process. This is also supported by the observation that remigrant females, the stage responsible for egg production with consequently increased nutritional demands, ingested significantly more phloem than the female stage which does not produce or lay eggs (emigrants). Apart from potential effects on vector behavior, pathogen infection may also have direct or indirect effects on vector fitness. Therefore, I investigated how the 'Ca. P. mali' infection of the host plant indirectly, as well as the infection of the insect itself directly affects C. picta’s fitness with regard to its susceptibility to a fungal antagonist. However, I could not detect any effect of neither the infection of the plant nor of the insect on its susceptibility to the entomopathogenic fungus Pandora sp. nov. inedit. This newly discovered and hitherto undescribed entomopathogenic fungus significantly reduced the mean survival time of the psyllids and soon grew out of the cadavers of infected insects to produce new spores for further dispersal. In infection bioassays, I was also able to show that Pandora sp. nov. inedit. could successfully kill several phloem-feeding species of the insect order Hemiptera. In addition to C. picta, other economically important vector insects such as Cacopsylla pyri, Cacopsylla pruni and Trioza apicalis could be infected under laboratory conditions. Hence, the natural potential of this entomopathogenic fungus became evident to be used in biological vector control strategies. These promising results can now be followed up by the development of suitable formulations for field studies to further investigate the control success for vector insects in their natural environment and thus in interaction with pathogens and plants. The knowledge gained in this thesis on the multitrophic interaction within this plant-pathogen-antagonist system will contribute to a better understanding of the epidemiology of the phytoplasma 'Ca. P. mali' and, in particular, might be used to develop innovative and selective vector control approaches to reduce the spread of apple proliferation disease