Genetic evidence for sexual reproduction and multiple infections of Norway spruce cones by the rust fungus Thekopsora areolata

Rust fungi are obligate parasites, of plants, with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid, and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry‐spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilized mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction, and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.     

Characterization of protein kinase PsSRPKL,a novel pathogenicity factor in the wheat stripe rust fungus

As in other eukaryotes, protein kinases (PKs) are generally evolutionarily conserved and play major regulatory roles in plant pathogenic fungi. Many PKs have been proven to be important for pathogenesis in model fungal plant pathogens, but little is currently known about their roles in the pathogenesis of cereal rust fungi, devastating pathogens in agriculture worldwide. Here, we report on an in planta highly induced PK gene PsSRPKL from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), one of the most important cereal rust fungi. PsSRPKL belongs to a group of PKs that are evolutionarily specific to cereal rust fungi. It shows a high level of intraspecies polymorphism in the kinase domains and directed green fluorescent protein chimers to plant nuclei. Overexpression of PsSRPKL in fission yeast induces aberrant cell morphology and a decreased resistance to environmental stresses. Most importantly, PsSRPKL is proven to be an important pathogenicity factor responsible for fungal growth and responses to environmental stresses, therefore contributing significantly to Pst virulence in wheat. We hypothesize that cereal rust fungi have developed specific PKs as pathogenicity factors for adaptation to their host species during evolution. Thus, our findings provide significant insights into pathogenicity and virulence evolution in cereal rust fungi.     

Life cycle and life strategy features of Puccinia glechomatis (Uredinales) favorable for extending the natural range of distribution

The objective of this study was to find features in microcyclic rust fungi (Uredinales) on wild host plants favorable for extension of the natural range of distribution. Puccinia glechomatis, a leptosporic rust fungus and its herbal host Glechoma hederacea (Lamiaceae), both natives to Eurasia and introduced in North America, were used for this study. Although the host has been known from North America since the beginning of the nineteenth century, the rust fungus was first observed there only in recent years. Favorable features were identified by studying the life cycle of the rust, including nuclear conditions and seasonal characteristics as well as its spread in North America. The life cycle was studied macroscopically by inoculation experiments, by various light microscope techniques, and by scanning electron microscopy. The spread of the pathogen and its host were reconstructed by evaluating host plant herbarium specimens and databases, literature, and field study data. The studies on P. glechomatis show that, generally for microcyclic rust fungi, establishment and potential for spread are based on several favorable features of both the host (e.g., synanthropic occurence and dispersal, genetic stability, regeneration of vegetative plant parts) and the rust fungus (asexual reproduction/genetic stability, homothallism, propagation with host plant, formation of both leptospores and thick-walled teliospores).     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.     

The haustorium of the root parasite Triphysaria (Scrophulariaceae), with special reference to xylem bridge ultrastructure

Haustoria of Triphysaria pusilla and T. versicolor subsp. faucibarbata from a natural habitat were analyzed by light and electron microscopy. Secretory trichomes (root hairs) participate in securing the haustorium to the surface of the host root. The keel-shaped intrusive part of the secondary haustorium penetrates to the depth of the vascular tissue of the host. Some of the epidermal interface cells differentiate into xylem elements. A significant number of haustoria do not differentiate further, but in most haustoria one to five of the epidermal xylem elements terminate a similar number of xylem strands. The strands mostly consist of vessel members and they connect host xylem or occasionally host parenchyma to the plate xylem adjacent to the stele of the parasite root. Each strand of this xylem bridge is accompanied by highly protoplasmic parenchyma cells with supposed transfer cell function. Increased surface area of the plasmalemma occurs in these cells as it does in interface parenchyma cells. Graniferous tracheary elements are restricted to the haustorium and occur most frequently in the plate xylem. The plate xylem is also accompanied by highly protoplasmic parenchyma cells. Hyphae of mycorrhizal fungi of the host root occasionally penetrate into the distal part of the xylem bridge. We combine structural observations and physiological facts into a hypothesis for translocation of water and nutrients between host and parasite. Some evolutionary aspects related to endogeny/exogeny of haustoria are discussed, and it is argued that the Triphysaria haustorium represents a greatly advanced and/or reduced condition within Scrophulariaceae.     

How to find a needle in a haystack – host plant finding of the weevil Ceratapion onopordi

The weevil Ceratapion onopordi Kirby (Coleoptera: Apionidae) shows a mutualistic interaction with the rust fungus Puccinia punctiformis (Str.) Röhl (Pucciniaceae). One of the weevil’s host plants, the thistle Cirsium arvense (L.) Scop. (Asteraceae), is also the host of the rust fungus. It has been argued that weevils prefer rust‐infected thistle shoots for egg deposition and consequently should be able to detect such shoots. Olfactory, visual, and gustatory orientation was tested using a four‐chamber olfactometer, a visual testing arena, and feeding choice tests. Whereas the weevils used olfactory cues to find their host plants, visual orientation does not seem to be important. Rust‐infected thistle shoots were not preferred over healthy shoots in any of the tests. We conclude that host plants infected with rust fungi, which are rather rare in the field, appear to be found more or less by chance.     

RELATION OF DWARFMISTLETOE (ARCEUTHOBIUM) TO THE XYLEM TISSUE OF CONIFERS. II. EFFECT OF THE PARASITE ON THE XYLEM ANATOMY OF THE HOST12

Srivastava , L. M., and K. Esau , (U. California, Davis.) Relation of dwarfmistletoe (Arceuthobium) to the xylem tissue of conifers. II. Effect of the parasite on the xylem anatomy of the host. Amer. Jour. Bot. (48(3): 209–215. Illus. 1961.—The changes in the xylem anatomy induced by dwarfmistletoe infection were studied in 7 coniferous species. The most pronounced abnormalities are observed in the shape and size of the infected rays. Because of the presence of parasite tissue, the rays assume a hypertrophied appearance; moreover, they fuse to form large composite rays. The union of rays involves intrusive growth of ray cells and displacement of fusiform initials. Some division of fusiform initials also occurs. Rays may increase in number and they may contain more host cells than normal rays. Axial tracheids in infected host woods differ more or less strongly from those of noninfected woods. They may be shorter, wider, and more irregular in shape than the axial tracheids in healthy wood. The samples of xylem from infected pines had a larger number of resin canals than those from healthy trees. Resin canals were also found in infected Tsuga, which normally lacks these structures.     

Boron uptake by ectomycorrhizas of silver birch

Boron (B) is an essential micronutrient for plants but it is thought not to be essential for fungi. We studied whether the extraradical mycelia of Paxillus involutus in symbiosis with silver birch (Betula pendula) take up B and transport it to the host plant. We grew mycorrhizal plants in flat microcosms with a partitioning wall, below which there was only extraradical mycelium. A boric acid solution enriched in ¹⁰B was applied to these mycelia. Increased ¹⁰B/¹¹B isotope ratios were subsequently measured in birch leaves, stems, and roots plus mycorrhizas in the upper compartment. Boron was therefore taken up by the mycorrhizal mycelia and transported to the host plant in this species combination.     

Volatiles modulate the development of plant pathogenic rust fungi

Rust fungi are obligate biotrophic pathogens that differentiate a series of specialized cells to establish infection. One of these cells, the haustorium, which serves to absorb nutrients from living host cells, normally develops only in planta. Here, we show that the rust fungus Uromyces fabae (Pers.) Schroet. stimulates volatile emission of its host, broad bean (Vicia faba L.). Volatiles were identified and shown to be perceived by the fungus in in vitro assays that excluded the host. Three of them, nonanal, decanal, and hexenyl acetate promoted the development of haustoria on artificial membranes. In contrast, the terpenoid farnesyl acetate suppressed this differentiation. In assays using whole plants, farnesyl acetate reduced rust disease not only on broad bean but also on several cereals and legumes including soybean. This natural substance was effective against all rusts tested when directly applied to the host. This demonstrated that farnesyl acetate may serve as a powerful novel tool to combat rust fungi including Phakopsora pachyrhizi that currently threatens the production of soybeans world-wide.     

Bipolar incompatibility system of an ectomycorrhizal basidiomycete, Rhizopogon rubescens

The mating systems of most ectomycorrhizal fungi have not been elucidated because of two reasons. One is the difficulty of obtaining homokaryotic isolates for mating tests caused by the low germination rate of basidiospores, and another is the difficulty of checking dikaryotization caused by the absence or inconsistent production of clamp connections on heterokaryotic mycelia under laboratory conditions. Basidiospore germination of a few ectomycorrhizal fungi has been induced by living roots of their host plants. Based on this information, we examined methods to obtain homokaryotic isolates of Rhizopogon rubescens using its host plant, Pinus thunbergii. The basidiospores of R. rubescens appeared to germinate well on an agar plate, on which axenic pine seedlings were grown in advance to induce germination, even when the seedlings were removed from the plate at the time of spore inoculation. To enhance the production rate of clamp connections on the heterokaryotic mycelia of R. rubescens, the culture medium composition was modified. The pH of the medium was critical for the production of clamp connections, and the optimal pH was higher for the production of clamp connections than for mycelial growth. These findings made it possible to conduct mating tests, and we found that the mating system of R. rubescens is bipolar with a multiallelic mating type factor.     

Tympanosporium parasiticum gen. et sp. nov., a hyperparasitic Hyphomycete on Tubercularia vulgaris with pleomorphic conidiogenesis

Tympanosporium parasiticum forms annellated conidiogenous cells with short cylindric (tympaniform) conidia in white sporodochia on Tubercularia vulgaris Tode ex Fr. On agar media the conidia form short necks and blow out narrow secondary conidia in a phialidic manner producing very restricted yeast-like colonies. Only in the vicinity of the host hyphae (without tropic growth or hyphal connections) mycelia develop which form phialidic or annellate conidiogenous cells with narrowly cylindrical conidia in the agar, and sporodochia with short truncate conidia like those on the natural substrate in the aerial mycelium. No other host fungus was found to support this kind of growth. The mode of parasitism is compared with that of other hyperparasitic fungi.For Tympanosporium parasiticum the name Oospora candidula Sacc. was incorrectly used by Grove (1885) and the name Oospora filamentosa was proposed by Arnaud but never published.     

Tests for Parasite-mediated Frequency-dependent Selection in Natural Populations of an Asexual Plant Species

Genetic variation in plant populations for resistance to pathogens and herbivores might be maintained by parasite-mediated negative frequency-dependent selection (FDS). But it is difficult to observe the time-lagged oscillations between host and parasite genotypes that should result from FDS. To evaluate the potential for FDS, we tested for local adaptation of parasites to common clones, the role of host genetic diversity in resistance to parasites, and genetic correlations among fitness, parasitism, and the frequency of host clones. We studied three populations of Arabis holboellii, a short-lived apomictic (asexual by seed) plant attacked by rust fungi and insect herbivores. To estimate clone frequency, we used polymorphic allozyme markers on 200 individuals in each population in 1990 and in 2000. We also recorded levels of parasitism and host fitness (fruit production). Only the rust fungi showed evidence for local host adaptation; they usually increased in incidence as a function of clone frequency, and they tracked temporal change in clone frequency. In further support of FDS, parasitism was lower in populations with higher genetic diversity. However, total parasitism (herbivory and disease combined) decreased as host clone frequency and fitness increased. Thus, although the highly virulent rust pathogen showed potential for driving the cycles that result from FDS, this apparently does not occur in the populations studied because the host clones were also attacked by herbivores.Co-ordinating editor: J.F. Stuefer     

Additions to the rust fungi of South Africa

This paper presents new species, combinations, national reports and host records for the South African rust fungi (Uredinales/Pucciniales). Endophyllum mpenjatiense on cf. Hibiscus sp. (Malvaceae), Phakopsora combretorum (anamorph Uredo combreticola) on the new host Combretum apiculatum (Combretaceae) and Uredo sekhukhunensis on Ziziphus mucronata (Rhamnaceae) are described as new species. Dietelia cardiospermi and E. metalasiae are proposed as new combinations to replace Aecidium cardiospermi on Cardiospermum halicacabum (Sapindaceae) and A. metalasiae on Metalasia spp. (Asteraceae), respectively. Four species are new records for South Africa: Crossopsora antidesmae-dioicae on Antidesma venosum (Euphorbiaceae), Phakopsora ziziphi-vulgaris on Z. mucronata, and Uromyces cypericola and Puccinia subcoronata, both on a new host, Cyperus albostriatus (Cyperaceae). The record of P. subcoronata is the first one from outside the New World. Puccinia scirpi is reported as a possible addition to the South African rust fungi. New host records and observations are presented for Pucciniastrum agrimoniae that is recorded on two new host genera and species, Cliffortia odorata and Leucosidea sericea (Rosaceae), Uromyces cypericola whose urediniospores are described for the first time, Phakopsora stratosa in that spermogonia and Uredo-like aecia were discovered, and for Sphaerophragmium dalbergiae in that characters of the urediniospores are re-evaluated. A lectotype is selected for Aecidium garckeanum and spermogonia are reported for this rust for the first time. The rust fungi of Ehrharta (Poaceae) are discussed and critically evaluated in the light of spore morphology and host species.     

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.     

Coping with an antagonist: the impact of a phytopathogenic fungus on the development and behaviour of two species of alpine leaf beetle

Herbivorous insects and phytopathogenic fungi often share their host plants. This creates a network of direct and indirect interactions, with far‐reaching consequences for the ecology and evolution of all three parties. In the Alps, the leaf beetles Oreina elongata and Oreina cacaliae (Coleoptera: Chrysomelidae), and the rust fungus Uromyces cacaliae (Uredinales: Pucciniaceae) are found on the same host plant, Adenostyles alliariae (Asterales: Asteraceae). We compare the impact of rust infection on these two closely‐related beetle species, one of which, O. cacaliae, is a specialist on A. alliariae, while the other, O. elongata, moves repeatedly between Adenostyles and an alternative host, Cirsium spinosissimum. Larval performance, feeding preference, oviposition choice and dispersal behaviour were studied in field and laboratory experiments. When reared on rust‐infected leaves, larvae of both beetle species had lower growth rates, lower maximum weights and longer development times. Larvae and adults discriminated among diets in feeding trials, showing a preference for discs cut from healthy leaves over those bearing a patch of sporulating rust, those from elsewhere on an infected leaf, and those from an upper leaf on an infected plant. Females of the two species differed in behaviour: in O. cacaliae they favoured healthy leaves for larviposition, while in O. elongata they showed no significant preference during oviposition. In the field, larvae and adults of both species dispersed more rapidly when placed on infected host plants. The results demonstrate that rust infection reduces the quality of the plant as a host for both Oreina species, and they combine the ability to detect systemic infection with the evolution of evasive behaviours. For these beetles, competition with a rust clearly increases the difficulty of survival in the harsh conditions of alpine environments, and may have a profound impact on the evolution of their life history traits and host plant use.     

Grapevine xylem response to fungi involved in trunk diseases

Grapevine trunk diseases (GTD), caused by a wide range of different fungi, are responsible for decline and productivity losses in vines at all growth stages. Grapevine responses to fungal attack include morphological and physiochemical defence mechanisms in the vascular system to reduce fungal infections. However, the extent to which these responses could control further spread by GTD‐fungi in the xylem vessels is poorly known. This study shows the formation of tyloses inside xylem vessels of diseased grapevines, as well as extracellular ligninolytic activities [lignin peroxidase, manganese peroxidase (MnP) and/or laccase] exhibited by some GTD‐fungi isolated here from symptomatic grapevines. In particular, Botryosphaeriaceae spp. and Phaeoacremonium minimum showed all three lignin‐degrading enzymatic activities. We also examined whether selected vine phenolic compounds, often located in the vascular system in response to fungal infection, could affect the lignin‐degrading activity from those GTD‐fungi as well as fungal colonisation. We found that phenolic compounds appeared to inhibit MnP activity, in addition to reducing fungal growth by causing anomalies in the hyphae morphology. Our results support that affected grapevines can initiate the tylosis formation in order to constrain fungi in the xylem vessels, while highlight the complementary action of the phenolic compounds to inhibit the fungi growth and colonisation. Phenolic compounds are therefore likely to have important role in alternative strategies for preventing trunk diseases.     

The rust transferred proteins—a new family of effector proteins exhibiting protease inhibitor function

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.     

Thermal dissipation probe measurements of sap flow in the xylem of trees documenting dynamic relations to variable transpiration given by instantaneous weather changes and the activities of a mistletoe xylem parasite

The thermal dissipation probe was described in the early 1930s for the demonstration of a volume and mass flow of sap in the conductive elements of the xylem in trees. It was subsequently developed further and is now widely used in physiological ecology including measurements in the field. Thermal dissipation demonstrates the occurrence of sap flow and allows determination of its velocity. Here we report simultaneous continuous measurements of sap flow using the thermal dissipation technique and of transpiration by infrared gas analysis for diurnal and annual cycles in a deciduous and an evergreen oak tree, Quercus robur L. and Quercus turneri Willd., respectively, in a deciduous and an evergreen conifer, Larix decidua Mill. and Pinus griffithii McClell., respectively, and the host/mistletoe consortium of the deciduous linden Tilia mandschurica Rupr. & Max. and the evergreen Viscum album L. We show (1) that in diurnal cycles sap flow closely follows dynamic changes of the rate of transpiration elicited by daily fluctuations of weather parameters (sunshine, cloudiness, air temperature and humidity), (2) that in annual cycles sap flow reflects autumnal yellowing and shedding of leaves of the deciduous trees. We report for the first time comparative measurements of sap flow towards mistletoe shoots and host branches in a parasite/host consortium. This demonstrates (3) that mistletoes maintain considerably larger sap flow rates in their xylem conduits than the adjacent host branches dragging the transpiration stream of their host towards their own shoots. We also show (4) that even after the deciduous host has shed its leaves and itself does not transpire any more the evergreen mistletoe towards its shoots can maintain the persistence of a continuous sap flow via the stem and branches of the host as long as frost does not prevent that. The work presented underlines the contention that transpiration is the driving force for sap flow with continuous files of water in the xylem. It shows for the first time that mistletoes direct the flow of water through host roots and stems towards its own shoots by not only performing stronger transpiration as it is known from the literature but also by maintaining larger sap flow rates in the xylem conduits of its stems.     

The effect and transmission of one isolate of the rust Puccinia minussensis on five clones of Lactuca sibirica

In a greenhouse experiment, one isolate of the systemic rust fungus Puccinia minussensis was applied to the host clone from which it was collected and to four other clones of the host Lactuca sibirica. The plants were grown in fertilized potting compost (N+) to promote growth and in peat (N-) to hamper growth, for three growing periods during one year. The results show that the expression of host plant resistance could not be determined visually, but there were differences in effects on the clones. The rust isolate was found to produce a significantly higher percentage of diseased shoots on clone A (the clone it was taken from). Furthermore, the rust also had the strongest effect on both biomass and shoot production on clone A compared to the other four clones. The data suggest that the rust isolate is highly adapted to the clone from which it originated. We suggest that selection in this system has not favoured a benign pathogen and that similar patterns are likely to occur for plants that (i) rarely establish by seeds; (ii) have strong lateral growth; and (iii) may persist for long periods once established.     

Uromyces vankyorum sp. nov. and Uromyces atriplicis, putative vicariant rust species of Patagonian and Australian semi-desert biomes

Uromyces vankyorum (Basidiomycota, Uredinales) growing on Atriplex lampa in the cool Patagonian dwarf shrub semi-desert is described as a new species and a new member of the Argentinean rust mycota. The fungus is morphologically similar to Uromyces atriplicis on Atriplex vesicaria from halophytic shrublands of South Australia. U. atriplicis seems to be derived from U. vankyorum as indicated by its shortened life cycle lacking the urediniospore stage. The species are interpreted as trans-pacific vicariants and represent one of the rare examples for the austral South American-Australasian element among rust fungi. The disjunct distribution of rust fungi between austral South America, Australia and New Zealand is reviewed and discussed. Available data suggest migration of rust fungi along Gondwanan land bridges rather than long-distance dispersal of spores by air currents. Part 198 of the series “Studies in Heterobasidiomycetous Fungi” of the Botanical Institute, University of Tübingen