Temporary Translocation of Entire Mistletoe Plants to Understand the Mechanistic Basis of Animal Foraging Decisions.

Fruiting mistletoes present a model system for understanding decisions made by foraging animals when locating food. Where, when, and how animals find food is central to many ecological questions, relating to the basis of individual foraging decisions and the extent to which these decisions are innate or acquired. Ecologists have paid particular attention to frugivores, quantifying their preference for fruits with specific shapes, colors, or scents, which, over evolutionary time, confer selection for suites of traits in their favored plants whose seeds they disperse. This work outlines a novel experimental approach to manipulating food plant occurrence and measuring the response of wild, free-living animals, ideally suited to studying the evolutionary origin and ecological maintenance of seed dispersal. This "cut and paste" protocol involves removing an entire fruiting mistletoe plant from its host and either returning it to its original location or moving it to a novel location, affixing it to a 'pseudo-host' of the same or different tree species. By counting visits to the mistletoe and noting the duration, species, and behaviors, a series of comparisons can discern the most important factors affecting foraging decisions and the consequences for both plant and animal. Here, the protocol is illustrated with a case study to determine between-guild differences in mistletoe frugivory. The experimental approach teases apart the mechanistic basis of search image formation and refinement, spatial learning, interspecific differences in foraging strategies, and how these changes modify seed dispersal effectiveness. Finally, potential modifications are considered with respect to addressing other questions on foraging ecology, plant-animal interactions, and coevolution.

Macroscale analysis of mistletoe host ranges in the Andean-Patagonian forest.

The number of host species infected by a mistletoe (host range) is critical in that it influences prevalence, virulence and overall distribution of the parasite; however, macroecological analyses of this life history feature are lacking for many regions. The Andean-Patagonian forest, found along the southern Andes from 35 °S to Tierra del Fuego at 55 °S, contains 12 mistletoe species in three families (Loranthaceae, Misodendraceae and Santalaceae). By tabulating herbarium records, the host ranges and geographical distributions of these mistletoes were explored. Our results show that these parasites occur on 43 plant species in 24 families but with varying degrees of specificity. All Misodendrum species and Desmaria mutabilis (Loranthaceae) are specialists that use Nothofagus as their primary hosts. Tristerix and Notanthera (Loranthaceae) and Antidaphne and Lepidoceras (Santalaceae) are generalists parasitizing more than six host species from several genera and families. Although many of the mistletoe species are sympatric, there is low overlap in host use. Our data show that in the southern South American bioregion, generalist mistletoes have smaller geographic ranges than specialists. This contrast with a previous hypothesis that predicted mistletoes with large geographic ranges would also have large host ranges, and conversely, less diverse regions would have more specialised mistletoes.

The hidden costs of living in a transformed habitat: Ecological and evolutionary consequences in a tripartite mutualistic system with a keystone mistletoe.

Land use change is one of the most important anthropogenic drivers of biodiversity loss. Nevertheless, the ecological and evolutionary consequences of habitat transformation remain less understood than those from habitat fragmentation. Transformed habitats are structurally simpler, altering species composition and their ecological interactions, potentially compromising gene flow and genetic diversity. We focused on a tripartite mutualistic system composed of a mistletoe (Tristerix corymbosus), its pollinator (Sephanoides sephaniodes) and its seed disperser (Dromiciops gliroides) to assess changes in their ecological and evolutionary dynamics as a result of habitat transformation. We used eight microsatellite markers to compare genetic diversity, relatedness and gene flow among five mistletoe groups inhabiting native and transformed habitats (abandoned Eucalyptus globulus plantations). We found that these groups were genetically structured, with greater allelic richness and genetic diversity in their native habitat. Also, we found higher relatedness among mistletoe individuals in transformed habitats, which varied as a function of the geographic distance among plants, probably as a result of larger resource availability, which influenced mutualist visitation rates. We did not find differences in the current migration patterns, which suggests that Tristerix corymbosus may be resilient to habitat transformation. Yet, its highly specialized interactions along with changes in its spatial configuration depict a more complex scenario, which probably impose a cost in terms of lower genetic diversity and increased relatedness that might compromise its long-term viability.

Parasitic Mistletoes of the Genera Scurrula and Viscum: From Bench to Bedside.

The mistletoes, stem hemiparasites of Asia and Europe, have been used as medicinal herbs for many years and possess sophisticated systems to obtain nutrients from their host plants. Although knowledge about ethnomedicinal uses of mistletoes is prevalent in Asia, systematic scientific study of these plants is still lacking, unlike its European counterparts. This review aims to evaluate the literature on Scurrula and Viscum mistletoes. Both mistletoes were found to have anticancer, antimicrobial, antioxidant and antihypertensive properties. Plants from the genus Scurrula were found to inhibit cancer growth due to presence of phytoconstituents such as quercetin and fatty acid chains. Similar to plants from the genus Viscum, Scurrula also possesses TNFα activity to strengthen the immune system to combat cancer. In line with its anticancer activity, both mistletoes are rich in antioxidants that confer protection against cancer as well as neurodegeneration. Extracts from plants of both genera showed evidence of vasodilation and thus, antihypertensive effects. Other therapeutic effects such as weight loss, postpartum and gastrointestinal healing from different plants of the genus Scurrula are documented. As the therapeutic effects of plants from Scurrula are still in exploration stage, there is currently no known clinical trial on these plants. However, there are few on-going clinical trials for Viscum album that demonstrate the functionalities of these mistletoes. Future work required for exploring the benefits of these plants and ways to develop both parasitic plants as a source of pharmacological drug are explained in this article.

Fleshing out facilitation - reframing interaction networks beyond top-down versus bottom-up.

803 I. 803 II. 804 III. 804 IV. 805 V. 805 VI. 806 References 807 SUMMARY: Rather than direct plant-plant interactions, research on the community-scale influence of mistletoes reveals hitherto unappreciated roles of animals in mediating facilitation. Lacking roots and reliant upon animal vectors, mistletoes represent model systems with which to understand mechanisms underlying interaction networks. In addition to direct effects on nutrient dynamics via enriched litter-fall, mistletoes are visited by pollinators, seed dispersers and natural enemies, complementing increased heterogeneity in nutrient returns reallocated from infected hosts with increased external inputs. These amplified bottom-up effects are coupled with top-down influences of insectivores attracted to infected hosts and stands by increased availability of favoured prey. Simultaneously influencing nutrient dynamics and plant-plant interactions from below and above, visiting animals help explain variation in the context dependence of facilitation.

Hemiparasite--host plant interactions in a fragmented landscape assessed via imaging spectroscopy and LiDAR.

Species interactions are susceptible to anthropogenic changes in ecosystems, but this has been poorly investigated in a spatially explicit manner in the case of plant parasitism, such as the omnipresent hemiparasitic mistletoe-host plant interactions. Analyzing such interactions at a large spatial scale may advance our understanding of parasitism patterns over complex landscapes. Combining high-resolution airborne imaging spectroscopy and LiDAR, we studied hemiparasite incidence within and among tree host stands to examine the prevalence and spatial distribution of hemiparasite load in ecosystems. Specifically, we aimed to assess: (1) detection accuracy of mistletoes on their oak hosts; (2) hemiparasitism prevalence within host tree canopies depending on tree height, and (3) spatial variation in hemiparasitism across fragmented woodlands, in a low-diversity mediterranean oak woodland in California, USA. We identified mistletoe infestations with 55-96% accuracy, and detected significant differences in remote-sensed spectra between oak trees with and without mistletoe infestation. We also found that host canopy height had little influence on infestation degree, whereas landscape-level variation showed consistent; non-random patterns: isolated host trees had twice the infestation load than did trees located at the core of forest fragments. Overall, we found that canopy exposure (i.e., lower canopy density or proximity to forest edge) is more important than canopy height for mistletoe infestation, and that by changing landscape structure, parasitic prevalence increased with woodland fragmentation. We conclude that reducing fragmentation in oak woodlands will minimize anthropogenic impact on mistletoe infestation at the landscape level. We argue that advanced remote sensing technology can provide baselines to quantitatively analyze and monitor parasite-host trajectories in light of global environmental change, and that this is a promising approach to be further tested in other temperate and tropical forests.

Temporal dynamic of parasite-mediated linkages between the forest canopy and soil processes and the microbial community.

Parasitic plants are important drivers of community and ecosystem properties. In this study, we identify different mechanisms by which mistletoe (Viscum album subsp. austriacum) can affect soil chemical and biological properties at different temporal stages of parasitism. We quantified the effect of parasitism on host growth and the number of frugivorous mutualists visiting the host canopy. Then we collected, identified, and weighed the organic matter input underneath tree canopies and analyzed its nutrient content. Simultaneously, we analyzed soil samples under tree canopies and examined the chemical properties, microbial abundance, and functional evenness of heterotrophic microbial communities. Mistletoe increased the amount, quality, and diversity of organic matter input beneath the host canopy, directly through its nutrient-rich litter and indirectly through a reduction in host litterfall and an increase in bird-derived debris. All these effects gave rise to enriched hotspots able to support larger and more functionally even soil microbial communities beneath parasitized hosts, the effects of which were accentuated after host death. We conclude that mistletoe, together with the biotic interactions it mediates, plays a key role in intensifying soil resource availability, regulating the functional evenness, abundance, and spatial distribution of soil microbial communities.

Leaf morphophysiology of a Neotropical mistletoe is shaped by seasonal patterns of host leaf phenology.

Several mistletoe species are able to grow and reproduce on both deciduous and evergreen hosts, suggesting a degree of plasticity in their ability to cope with differences in intrinsic host functions. The aim of this study was to investigate the influence of host phenology on mistletoe water relations and leaf gas exchange. Mistletoe Passovia ovata parasitizing evergreen (Miconia albicans) hosts and P. ovata parasitizing deciduous (Byrsonima verbascifolia) hosts were sampled in a Neotropical savanna. Photosynthetic parameters, diurnal cycles of stomatal conductance, pre-dawn and midday leaf water potential, and stomatal anatomical traits were measured during the peak of the dry and wet seasons, respectively. P. ovata showed distinct water-use strategies that were dependent on host phenology. For P. ovata parasitizing the deciduous host, water use efficiency (WUE; ratio of photosynthetic rate to transpirational water loss) was 2-fold lower in the dry season than in the wet season; in contrast, WUE was maintained at the same level during the wet and dry seasons in P. ovata parasitizing the evergreen host. Generally, mistletoe and host diurnal cycles of stomatal conductance were linked, although there were clear differences in leaf water potential, with mistletoe showing anisohydric behaviour and the host showing isohydric behaviour. Compared to mistletoes attached to evergreen hosts, those parasitizing deciduous hosts had a 1.4-fold lower stomatal density and 1.2-fold wider stomata on both leaf surfaces, suggesting that the latter suffered less intense drought stress. This is the first study to show morphophysiological differences in the same mistletoe species parasitizing hosts of different phenological groups. Our results provide evidence that phenotypical plasticity (anatomical and physiological) might be essential to favour the use of a greater range of hosts.

Reproductive biology and nectar production of the Mexican endemic Psittacanthus auriculatus (Loranthaceae), a hummingbird-pollinated mistletoe.

Many mistletoe species produce 'bird'-pollinated flowers; however, the reproductive biology of the majority of these species has not been studied. Psittacanthus auriculatus is a Mexican endemic mistletoe, most common in open, dry mesquite grassland. Knowledge of the reproductive biology of P. auriculatus is essential for understanding species formation and diversification of Psittacanthus mistletoes, but it is currently poorly understood. Thus, we studied floral biology and phenology, nectar production and breeding system and pollination of this species. The hermaphroditic red-pink flowers open from the middle to the tip and petals are curly, but remain partially fused forming a floral tube of ca. 20-mm long. Flowers are partially protandrous, produce large amounts of nectar, last 2 days, and stigma receptivity is highest during the second day. We recorded hummingbirds (Cynanthus latirostris, Hylocharis leucotis, Amazilia beryllina, A. violiceps, Calothorax lucifer, Archilochus colubris) and less commonly butterflies (Agraulis vanillae, Anteos clorinde, Papilio multicaudatus, Phocides urania, Phoebis sennae) as floral visitors. P. auriculatus flowers are self-compatible. However, this mistletoe is an obligate animal-pollinated species, as the sensitive stigma avoids self-pollination. Under natural conditions, reproductive success was higher than in manually selfed or cross-pollinated flowers, likely due to the traplining foraging behaviour of hummingbirds. We suggest that the apparent efficient foraging behaviour of hummingbirds maintains gene flow among P. auriculatus, promoting outcrossing.

Disproportionate Declines in Ground-Foraging Insectivorous Birds after Mistletoe Removal.

Insectivorous birds have been recognized as disproportionately sensitive to land-use intensification and habitat loss, with those species feeding primarily on the ground exhibiting some of the most dramatic declines. Altered litter inputs and availability of epigeic arthropods have been suggested to underlie reduced abundances and shrinking distributions but direct evidence is lacking. I used a patch-scale removal experiment in southern Australia to evaluate whether ground-feeding insectivores are especially vulnerable to altered litter-fall. Building on work demonstrating the importance of mistletoe litter to nutrient dynamics, litter was reduced by removing mistletoe (Loranthaceae) from one set of eucalypt woodlands, responses of birds three years after mistletoe removal compared with otherwise similar control woodlands containing mistletoe. Despite not feeding on mistletoes directly, insectivores exhibited the greatest response to mistletoe removal. Among woodland residents, ground-foraging insectivores showed the most dramatic response; treatment woodlands losing an average of 37.4% of their pre-treatment species richness. Once these 19 species of ground-foraging insectivores were excluded, remaining woodland species showed no significant effect of mistletoe removal. This response reflects greater initial losses in treatment woodlands during the study (which coincided with a severe drought) and double the number of species returning to control woodlands (where mistletoe numbers and litter were not manipulated) post-drought. These findings support the productivity-based explanation of declining insectivores, suggesting diminished litter-fall reduced habitat quality for these birds via decreased availability of their preferred prey. In addition to altered prey availability, interactions between litter-fall and epigeic arthropods exemplify the importance of below-ground / above-ground linkages driving ecosystem function.

Modeling occupancy of hosts by mistletoe seeds after accounting for imperfect detectability.

The detection of an organism in a given site is widely used as a state variable in many metapopulation and epidemiological studies. However, failure to detect the species does not necessarily mean that it is absent. Assessing detectability is important for occupancy (presence-absence) surveys; and identifying the factors reducing detectability may help improve survey precision and efficiency. A method was used to estimate the occupancy status of host trees colonized by mistletoe seeds of Psittacanthus plagiophyllus as a function of host covariates: host size and presence of mistletoe infections on the same or on the nearest neighboring host (the cashew tree Anacardium occidentale). The technique also evaluated the effect of taking detectability into account for estimating host occupancy by mistletoe seeds. Individual host trees were surveyed for presence of mistletoe seeds with the aid of two or three observers to estimate detectability and occupancy. Detectability was, on average, 17% higher in focal-host trees with infected neighbors, while decreased about 23 to 50% from smallest to largest hosts. The presence of mistletoe plants in the sample tree had negligible effect on detectability. Failure to detect hosts as occupied decreased occupancy by 2.5% on average, with maximum of 10% for large and isolated hosts. The method presented in this study has potential for use with metapopulation studies of mistletoes, especially those focusing on the seed stage, but also as improvement of accuracy in occupancy models estimates often used for metapopulation dynamics of tree-dwelling plants in general.

Thermogenesis-triggered seed dispersal in dwarf mistletoe.

Lodgepole pine dwarf mistletoe (DM), Arceuthobium americanum, is a parasitic flowering plant and forest pathogen in North America. Seed dispersal in DM occurs by explosive discharge. Notably, slight warming of ripe DM fruit in the laboratory can trigger explosions. Previously, we showed that alternative oxidase, a protein involved in endogenous heat production (thermogenesis) in plants, is present in DM fruit. These observations have led us to investigate if thermogenesis induces discharge. Here, infrared thermographs reveal that ripe DM fruits display an anomalous increase in surface temperature by an average of 2.1±0.8 °C over an average time of 103±29 s (n=9, 95% confidence interval) before dehiscence. Furthermore, both non-isothermal and isothermal modulated differential scanning calorimetry consistently show an exothermic event (~1 J g(-1)) in the non-reversible heat flow just prior to discharge. These results support thermogenesis-triggered seed discharge, never before observed in any plant.

Spatial distribution of oak mistletoe as it relates to habits of oak woodland frugivores.

This study addresses the underlying spatial distribution of oak mistletoe, Phoradendron villosum, a hemi-parasitic plant that provides a continuous supply of berries for frugivorous birds overwintering the oak savanna habitat of California's outer coast range. As the winter community of birds consuming oak mistletoe varies from group-living territorial species to birds that roam in flocks, we asked if mistletoe volume was spatially autocorrelated at the scale of persistent territories or whether the patterns predicted by long-term territory use by western bluebirds are overcome by seed dispersal by more mobile bird species. The abundance of mistletoe was mapped on trees within a 700 ha study site in Carmel Valley, California. Spatial autocorrelation of mistletoe volume was analyzed using the variogram method and spatial distribution of oak mistletoe trees was analyzed using Ripley's K and O-ring statistics. On a separate set of 45 trees, mistletoe volume was highly correlated with the volume of female, fruit-bearing plants, indicating that overall mistletoe volume is a good predictor of fruit availability. Variogram analysis showed that mistletoe volume was spatially autocorrelated up to approximately 250 m, a distance consistent with persistent territoriality of western bluebirds and philopatry of sons, which often breed next door to their parents and are more likely to remain home when their parents have abundant mistletoe. Using Ripley's K and O-ring analyses, we showed that mistletoe trees were aggregated for distances up to 558 m, but for distances between 558 to 724 m the O-ring analysis deviated from Ripley's K in showing repulsion rather than aggregation. While trees with mistletoe were aggregated at larger distances, mistletoe was spatially correlated at a smaller distance, consistent with what is expected based on persistent group territoriality of western bluebirds in winter and the extreme philopatry of their sons.

Generalist birds govern the seed dispersal of a parasitic plant with strong recruitment constraints.

Mistletoes constitute instructive study cases with which to address the role of generalist consumers in the study of plant-animal interactions. Their ranges of safe sites for recruitment are among the most restricted of any plant; therefore, frugivores specializing in mistletoe have been considered almost indispensable for the seed dispersal of these parasitic plants. However, the absence of such specialists in numerous regions inhabited by many mistletoe species raises the question of whether unspecialized vectors may successfully disperse mistletoe seeds to narrowly defined safe sites. Using the European mistletoe Viscum album subsp. austriacum as a study case, we recorded a broad range of 11 bird species that disperse mistletoe seeds. For these species, we studied the mistletoe-visitation rate and feeding behavior to estimate the quantity component of dispersal effectiveness, and the post-foraging microhabitat use, seed handling, and recruitment probabilities of different microhabitats as a measure of the quality component of effectiveness. Both endozoochory and ectozoochory are valid dispersal mechanisms, as the seeds do not need to be ingested to germinate, increasing seed-dispersal versatility. Thrushes were the most effective dispersers, although they were rather inefficient, whereas small birds (both frugivores and non-frugivores) offered low-quantity but high-quality services for depositing seeds directly upon safe sites. As birds behave similarly on parasitized and non-parasitized hosts, and vectors have broad home ranges, reinfection within patches and the colonization of new patches are ensured by an ample assemblage of generalist birds. Thus, a parasitic plant requiring precision in seed dispersal can rely on unspecialized dispersers.

Node-by-node disassembly of a mutualistic interaction web driven by species introductions.

Interaction webs summarize the diverse interactions among species in communities. The addition or loss of particular species and the alteration of key interactions can lead to the disassembly of the entire interaction web, although the nontrophic effects of species loss on interaction webs are poorly understood. We took advantage of ongoing invasions by a suite of exotic species to examine their impact in terms of the disassembly of an interaction web in Patagonia, Argentina. We found that the reduction of one species (a host of a keystone mistletoe species) resulted in diverse indirect effects that led to the disassembly of an interaction web through the loss of the mistletoe, two key seed-dispersers (a marsupial and a bird), and a pollinator (hummingbird). Our results demonstrate that the gains and losses of species are both consequences and drivers of global change that can lead to underappreciated cascading coextinctions through the disruption of mutualisms.

Metapopulation dynamics of the mistletoe and its host in savanna areas with different fire occurrence.

Mistletoes are aerial hemiparasitic plants which occupy patches of favorable habitat (host trees) surrounded by unfavorable habitat and may be possibly modeled as a metapopulation. A metapopulation is defined as a subdivided population that persists due to the balance between colonization and extinction in discrete habitat patches. Our aim was to evaluate the dynamics of the mistletoe Psittacanthus robustus and its host Vochysia thyrsoidea in three Brazilian savanna areas using a metapopulation approach. We also evaluated how the differences in terms of fire occurrence affected the dynamic of those populations (two areas burned during the study and one was fire protected). We monitored the populations at six-month intervals. P. robustus population structure and dynamics met the expected criteria for a metapopulation: i) the suitable habitats for the mistletoe occur in discrete patches; (ii) local populations went extinct during the study and (iii) colonization of previously non-occupied patches occurred. The ratio of occupied patches decreased in all areas with time. Local mistletoe populations went extinct due to two different causes: patch extinction in area with no fire and fire killing in the burned areas. In a burned area, the largest decrease of occupied patch ratios occurred due to a fire event that killed the parasites without, however, killing the host trees. The greatest mortality of V. thyrsoidea occurred in the area without fire. In this area, all the dead trees supported mistletoe individuals and no mortality was observed for parasite-free trees. Because P. robustus is a fire sensitive species and V. thyrsoidea is fire tolerant, P. robustus seems to increase host mortality, but its effect is lessened by periodic burning that reduces the parasite loads.

Neighborhood effects on seed dispersal by frugivores: testing theory with a mistletoe-marsupial system in Patagonia.

The outcome of the dispersal process in zoochorous plants is largely determined by the behavior of frugivorous animals. Recent simulation studies have found that fruit removal rates and mean dispersal distances are strongly affected by fruiting plant neighborhoods. We empirically tested the effects of conspecific fruiting plant neighborhoods, crop sizes, and plant accessibility on fruit removal rates and seed dispersal distances of a mistletoe species exclusively dispersed by an arboreal marsupial in Northern Patagonia. Moreover, in this study, we overcome technical limitations in the empirical estimation of seed dispersal by using a novel 15N stable isotope enrichment technique together with Bayesian mixing models that allowed us to identify dispersed seeds from focal plants without the need of extensive genotyping. We found that, as predicted by theory, plants in denser neighborhoods had greater fruit removal and shorter mean dispersal distances than more isolated plants. Furthermore, the probability of dispersing seeds farther away decreased with neighborhood density. Larger crop sizes resulted in larger fruit removal rates and smaller probabilities of longer distance dispersal. The interplay between frugivore behavioral decisions and the spatial distribution of plants could have important consequences for plant spatial dynamics.

Mistletoe-induced crown degradation in Scots pine in a xeric environment.

Increasing Scots pine (Pinus sylvestris L.) mortality has been recently observed in the dry inner valleys of the European Alps. Besides drought, infection with pine mistletoe (Viscum album ssp. austriacum) seems to play an important role in the mortality dynamics of Scots pines, but how mistletoes promote pine decline remains unclear. To verify whether pine mistletoe infection weakens the host via crown degradation, as observed for dwarf mistletoes, we studied the negative effects of pine mistletoe infestation on the photosynthetic tissues and branch growth of pairs of infested and non-infested branches. Pine mistletoe infection leads to crown degradation in its host by reducing the length, the radial increment, the ramification, the needle length and the number of needle years of the infested branches. This massive loss in photosynthetic tissue results in a reduction in primary production and a subsequent decrease in carbohydrate availability. The significant reduction in needle length due to mistletoe infection is an indication for a lower water and nutrient availability in infested branches. Thus, mistletoe infection might lead to a decrease in the availability of water and carbohydrates, the two most important growth factors, which are already shortened due to the chronic drought situation in the area. Therefore, pine mistletoe increases the risk of drought-induced mortality of its host when growing in a xeric environment.

A simple parametric method for reducing sample sizes in gut passage time trials.

Seed dispersal is now regularly analyzed using spatially explicit models, relying in part on frugivore gut passage times to produce model outputs. In determining species-specific gut passage times, there is a trade-off in sample size between minimizing collection effort and maintaining statistical reliability. Here we demonstrate that a two-parameter lognormal parametric distribution reliably fits empirical gut passage time distributions and is easily parameterized using relatively small data sets of approximately 30 defecations. We suggest this approach as a statistically reliable substitute for larger empirical gut passage data sets in seed dispersal modeling, and also as a way of using published gut passage data sets to parameterize new models.

Antifungal effects and mechanism of action of viscotoxin A3.

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine-rich peptides of approximately 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal-derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion-channel-like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3-induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.