Introduction
The relationship between a parasite and its host is important ecologically and widely discussed in animal and plant pathology and physiology. Most research on parasite–host relationships in plants has concentrated on host responses to infections by parasites (Solomon, James, Alphonsus, & Nkiruka, 2015; Streicker, Fenton, & Pedersen, 2013). In contrast, the interactions between plant hosts and plant parasites, especially the effects of hosts on parasites in different habitats and varying site conditions, have rarely been studied. However, such parasite–host relationships, including a possible feedback system between the host and parasite, are of central interest because they can strongly affect the growth and survival of the higher plants serve as hosts.
Mistletoes are well-known hemiparasitic plants that maintain their own carbon assimilation by photosynthesis and can infect many tree species in various ecosystem types worldwide, making them an important and relevant species in parasitism research (Glatzel & Geils, 2009; Zuber, 2004).Viscum album ssp. austriacum (Santalaceae), pine mistletoe, is the most widely distributed species across the European continent (M Dobbertin & Rigling, 2006; Zuber, 2004). Pine mistletoe survival and development in forest ecosystems mainly rely on water and mineral resources obtained from the xylem sap of the host tree (M Dobbertin & Rigling, 2006; Rigling, Eilmann, Koechli, & Dobbertin, 2010). If water availability is high and nutrients are not limited, pine mistletoes and their hosts co-exist for years without major restrictions for the host tree (Solomon et al., 2015; Zuber, 2004). However, if water is limited during dry periods, the high-water consumption and low water-use efficiency of pine mistletoes may exacerbate drought stress in the host tree, with negative consequences on the host’s physiology and growth performance (M Dobbertin & Rigling, 2006; Rigling et al., 2010; Zweifel, Bangerter, Rigling, & Sterck, 2012). As a consequence, pine mistletoe infection leads to a reduction of branching and of branch and needle growth(Rigling et al., 2010), resulting in an increased risk of mortality for the host tree (M Dobbertin & Rigling, 2006). This contribution of pine mistletoe to drought-induced forest decline processes has been demonstrated in several xeric forest ecosystems in Spain (Galiano, Martínez‐Vilalta, & Lloret, 2011; Sangüesa-Barreda, Linares, & Camarero, 2012, 2013; M. Scalon, Haridasan, & Franco, 2013) and inner-Alpine regions in Switzerland and Italy (M Dobbertin & Rigling, 2006; Rigling et al., 2010; Vacchiano, Garbarino, Mondino, & Motta, 2012).
Along with the negative effects of mistletoes on the host water balance, pine mistletoes have also been found to affect the carbon balance of the host in a variety of ways (Glatzel & Geils, 2009; Q. Le, K. U. Tennakoon, F. Metali, L. B. Lim, & J. F. Bolin, 2016; M. C. Scalon & Wright, 2015). High water use of mistletoes and thus considerable water loss from the whole host–parasite system may induce closure of the stomata in host trees to save water (Rigling et al., 2010; Zweifel et al., 2012), resulting in lower photosynthesis rates of the trees (M Dobbertin & Rigling, 2006; Yan et al., 2016). Therefore, pine mistletoe can indirectly reduce the host’s ability to acquire carbon resources, especially under drought-stress conditions (Sangüesa-Barreda et al., 2013; Yan et al., 2016).
Mistletoes perform photosynthesis at a rate similar to that of the host (Lüttge et al., 1998; M. C. Scalon & Wright, 2017). In some studies, however, it has been reported that mistletoes are able to additionally acquire organic carbon from the host in the form of xylem-mobile organic acids and amino acids (Escher, Eiblmeier, Hetzger, & Rennenberg, 2004b; Těšitel, Plavcová, & Cameron, 2010). Richter, Popp, Mensen, Stewart, and Willert (1995) estimated that mistletoe leaves take up over 50% of its required heterotrophic carbon from its host. Nevertheless, according to Smith and Gledhill (1983), the haustorium of V. album grows only within the host’s xylem and does not connect to the host’s phloem. This means that there should be only acropetal carbon transport from the host xylem to the mistletoe via the transpiration stream, with no basipetal carbon flow from the mistletoe to the host, even under strong carbon limitation of the host (Glatzel & Geils, 2009; M. C. Scalon & Wright, 2015). Hence, it remains unclear whether mistletoes can directly absorb carbon resources from host tissues in considerable amounts, in addition to their own photosynthetic activities. By utilizing the stable 13C isotope tracer technique, it is possible to determine the direction and quantity of carbon assimilate flow between mistletoe and host, and also to assess how this process depends on carbon and water availability.
Most studies on mistletoe–host relationships have been conducted by comparing trees infected by mistletoes with non-infected trees growing under the same conditions (M. Dobbertin et al., 2010; M Dobbertin & Rigling, 2006; Rigling et al., 2010; Yan et al., 2016). Whether the host’s carbon resource availability, which is strongly associated with its growth conditions (e.g. soil water moisture), affects the mistletoe–host relationship has only been investigated in a few studies, and these studies were only focused on the response of hosts to mistletoe infection (Q. Le, K. Tennakoon, F. Metali, L. Lim, & J. Bolin, 2016; Sangüesa-Barreda et al., 2013; Zweifel et al., 2012). It is still unclear if the carbon dynamics in the mistletoe and in its host, as well as the potential exchange of assimilates between the two, changes in response to the local water availability of both the host tree and the mistletoe.
To address these unresolved questions, we conducted two separate experiments under the umbrella of a whole-tree 13C-pulse labeling experiment with mature Scots pine (Pinus sylvestris ) trees infected by pine mistletoe (V. album ). Host trees whose crowns were exposed to13CO2 were growing either in naturally dry conditions (~600 mm precipitation per year) or in irrigated areas (+700 mm per year, applied during the growing season) for 15 years in the Swiss Pfynwald forest ecosystem experimental platform (Joseph et al., 2020; Schaub, Haeni, Hug, Gessler, & Rigling, 2016).
In a wrapping experiment (Exp. 1), we shielded mistletoe clusters with gas-tight plastic foil and darkened them with aluminum foil before the whole-tree labeling to prevent 13C assimilation by these clusters. We investigated the 13C values in both wrapped and non-wrapped mistletoes, as well as in their host twigs, to test the hypothesis (H1 ) thatV. album takes up carbon resources from its host via the haustorium. Any signal in the wrapped mistletoes (shielding from 13CO2 and light exclusion) would originate from the host and we assumed the contribution of the host (if any) to be higher in irrigated vs drought-stressed trees due to increased assimilation rates in irrigated trees (Schonbeck et al., 2021)).
To change source–sink carbon and water relationships, we performed a tissue removal experiment (Exp. 2). We girdled pine branches infected with mistletoes of drought-stressed and irrigated host trees to restrict the phloem carbon translocation between the remaining tree and the girdled branch (Andersen, Nikolov, Nikolova, Matyssek, & Haberle, 2005; De Schepper & Steppe, 2013), while keeping a constant water and nutrient flow. Beyond the girdling point, we then removed all pine needles or all mistletoe tissues (including stem and leaves) from the girdled pine branches to manipulate source-sink relationships and water relations locally on the branch level. Through Exp. 2, we aimed to test the hypothesis (H2a ) that local changes in source–sink relationships by reducing assimilate ability (i.e. host needle removal), would decrease the mistletoes’ carbon level due to lower amounts of carbon obtained from the host (conditional H1 is supported). An alternative hypothesis (H2b ) is that needle removal increases the mistletoes’ carbon level due to increased carbon assimilation by the hemiparasite itself as a result of decreased competition for water with the host. This effect would be more pronounced under the dry control conditions. Finally, we hypothesize (H3 ) that mistletoes do not provide any carbon to the host, even when the host is carbon limited due to needle removal.