• The root parasite Rhinanthus minor feeds on the xylem of a diverse range of species. Grasses and legumes are the best hosts, while on forbs R. minor typically shows poorer growth. It has been hypothesized that host quality is linked to the expression of defences against the parasite seen in forb roots, but never in grasses. The efficacy of these defence mechanisms in preventing resource loss has not, however, been measured directly.
• Here we combine histological characterization of haustoria formed on Cynosurus cristatus (a grass), Leucanthemum vulgare and Plantago lanceolata (forbs) with 15N tracers supplied to the host to quantify the efficacy of these defence responses.
• Rhinanthus minor penetrated only the xylem of C. cristatus, abstracting an average of 17% of the 15N tracer taken up, but only 2.5 and 0.2%, respectively, when attached to L. vulgare and P. lanceolata.
• For the first time, this study has established that the resistance mechanisms of the forbs are effective in preventing the parasite from directly accessing their xylem solutes.
Rhinanthus minor is a root hemiparasitic plant with a wide host range extending to over 20 species (Gibson & Watkinson, 1989). Parasitic plants have evolved specialist mechanisms to enable them to steal resources (nonstructural C and mineral nutrients) from their hosts (Musselman & Press, 1995). Their ability to access a host's vascular tissue and withdraw resources through the transfer organ known as the haustorium is a crucial adaptation. Depending on the species of parasitic plant, the haustorium interconnects either the host and parasite xylem (Dorr, 1997; Gurney et al., 2003), phloem (Riopel & Timko, 1995; Hibberd et al., 1999) or interfacial parenchyma (Tennakoon & Cameron, 2006; Pate et al., 1990), or a combination of the three. The fully differentiated, mature haustorium formed by R. minor attacks the host's xylem: it first surrounds the host root and crushes the outer cortex, forming a penetration peg or endophyte that is forced into the host's stele, directly penetrating the vascular system. The penetration peg is, in turn, connected to the xylem elements in the parasite's root by secondary xylem (Cameron, 2004). The secondary xylem is located centrally in the haustorium and is surrounded by the hyaline body, a region of the haustorium rich in nuclei and believed to be functional during solute abstraction and processing (Riopel & Timko, 1995).
Rhinanthus minor is primarily a xylem feeder (Seel & Press, 1993); its direct connection to the xylem of its compatible hosts allows it to withdraw water (containing dissolved minerals and carbon) from hosts by cohesion. This solute transfer is driven by the elevated transpiration of the parasite pulling the host's xylem sap through the xylem continuum and into the parasite (Press et al., 1990; Stewart & Press, 1990). Solutes dissolved within the xylem sap therefore move into the parasite by mass flow. The parasite maintains a sink strength that exceeds that of the host, the resulting reduction in the water potential (ψ) of the parasite tissues is below that of the host tissues and generates a water potential gradient promoting mass flow to the parasite. In the Striga–Sorghum system, the water potential of each partner plant has been shown to be −0.42 MPa (parasite) and −0.23 MPa (host) (Ackroyd & Graves, 1997). Root hemiparasites such as Striga (Press et al., 1987b) and Rhinanthus (Jiang et al., 2003) can sustain high transpiration rates, as they have stomata that are relatively insensitive to water loss. For example, Striga's stomata close only as a result of severe drought stress, and this is probably the result of ‘direct loss of cell turgor in the guard cells’ (Graves, 1995). Furthermore, the low parasite water potential may also be mediated by the accumulation of osmotically active compounds such as sugars and sugar alcohols (Hodgeson, 1973), particularly mannitol (Jiang et al., 2005).
Hemiparasites were thought to be largely self-sufficient for C; however, transport of C has been observed from parasite to host (Govier et al., 1967; Hodgeson, 1973), indicating that the hemiparasites, such as R. minor, that can photosynthesize also acquire C from their hosts. Additionally Press et al. (1987a) and Tennakoon & Pate (1996) used natural abundance stable isotope-mixing models to show that parasitic plants could access up to 35% of their host's nonstructural C. This is somewhat intuitive as there is potential for large fluxes of amino and organic acids transported in the host xylem to reach the parasite through the haustorium (Raven, 1983). As well as fixed C, the parasite may also acquire significant amounts of nitrogen along with the C in such organic and amino acids. The parasite is therefore also reliant on hosts for mineral nutrients such as N and phosphorus (Seel et al., 1993; Davies et al., 1997; Seel & Jeschke, 1999). Jiang et al. (2003, 2004) provide further evidence for mineral flow from host to parasite by using the assumption of mass flow to model resource translocation from host to parasite, predicting that the parasite can access up to 20% of its host's translocatable resources.
Rhinanthus minor is a generalist, and will form haustoria on most roots, but not all potential hosts are equally beneficial to the parasite. Grasses and legumes are the best hosts for R. minor, while forbs (perennial, nonleguminous dicots) are the poorest, with the parasite often achieving a lower biomass than when growing without a host; an association with a forb may even result in the death of the parasite (Seel et al., 1993; Hwangbo, 2000; Cameron, 2004; Cameron et al., 2006). Furthermore, parasite success is positively correlated with host damage, as the growth of the grasses is restricted severely while that of the forbs is unaffected (Hwangbo, 2000; Cameron et al., 2006). Previous studies have suggested that this difference in host quality (and therefore in parasite-induced host damage) is caused by the defence reactions enacted by the forbs, but not by the grasses, preventing the parasite from accessing xylem resources. Cameron et al. (2006) and Rümer et al. (in press) have observed hypersensitive cell death at the host–parasite interface with Plantago lanceolata and encapsulation of the haustoria with lignin by Leucanthemum vulgare, while the grasses did not show signs of any defence responses. Cameron et al. (2006) hypothesized that these responses effectively sealed off the vascular system of the host, preventing access by the parasite haustorium, thus preventing resource loss and underpinning the poor performance of R. minor on many species of forb and the absence of parasite-induced host damage in these species. It is important to elucidate fully the mechanisms underpinning the variable effects of R. minor, as it has been used as a restoration tool to promote diversity in fertilized grasslands (Westbury, 2004), but can be a pest in some agro-ecosystems.
Surprisingly no study has investigated whether the defence responses exhibited by the forbs actually prevent R. minor from abstracting its host's xylem sap. This study combines histological analysis of the haustorium with 15N-labelled potassium nitrate as a tracer to investigate the hypothesis that the resistance responses of the P. lanceolata and L. vulgare prevent mass flow of solutes to R. minor.
Materials and Methods
Plant material and histology
Three individuals of 2-wk-old Cynosurus cristatus L., Leucanthemum vulgare Lam. and Plantago lanceolata L. seedlings (hosts) were grown for a further 4 wk in 15-cm-diameter pots containing 50 : 50 sand : John Innes No. 3 compost (one plant per pot). Rhinanthus minor L. seeds were surface-sterilized for 5 min in 3% sodium hypochlorite solution, washed in distilled water and preconditioned on moist filter paper at 4°C until germination (approx. 8 wk). Four seedlings of R. minor were transplanted into the pots. Host plants were thus 6 wk old at the at the point of parasite introduction, and were well established before R. minor attached to their roots. Plants of this age were used so that it would be possible to separate the root ball of the host from the substrate at harvest for 15N analysis. The parasites were subsequently reduced to one per host when the first parasite showed morphological changes associated with attachment (Klaren & Janssen, 1978). Twelve weeks after attachment of the parasite, roots were removed and washed clean of the sand/soil substrate, and sections of root were removed from the attachment points of primary haustoria. Host root pieces with haustoria attached were fixed in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1 m phosphate buffer (pH 7.2) and placed under vacuum for 10 min to allow the fixative to penetrate the tissue. The tissues were then washed in 0.1 m phosphate buffer (pH 7.2) and dehydrated through a graduated ethanol series. Tissue was embedded in LR-white resin (TAAB, Reading, UK), and haustoria growing on each of the nine host species were sectioned to 1 µm. Sections were floated onto polysine microscope slides (TAAB) and the sections were fixed to the slides on an 80°C hot plate for 5 min. Sections were stained for structure using 0.5% Toluidine blue in boric acid for 1 min at 80°C, and mounted using histomount (TAAB) and a glass cover slip. Haustoria sections were examined using a Leitz Laborlux 5 microscope (Leica, Milton Keynes, UK) in bright-field mode, and images were captured using a Polaroid DMC Ie CCD digital camera (Datacell, Wokingham, UK).
Six replicate pots of each host species were set up as detailed above, and the pots were suspended over aerated trays of water to allow host roots to grow out of the bottom of the pot. An air gap was left as a hydraulic switch to prevent saturation of the pots. Water was supplied to the soil daily and the pots were randomized. The hosts together with the parasites were then grown for a further 12 wk (after parasite attachment) in a glasshouse (temperature maximum 26°C; minimum 15°C), after which they were given no water for 48 h (aside from what they could withdraw from the water tray) to prevent water leakage into the 15N source once it was introduced.
The pots containing the host–parasite associations were removed from the aerated trays of water and the roots protruding from the bottom of the pots were inspected for haustoria. None was found, so all pots were entered into the experimental set up. The pots were suspended over open Petri dishes, half were filled with 50 ml 10 mm 15N-labelled (54 at %) potassium nitrate (K15NO3) with the remainder containing 50 ml 10 mm unlabelled potassium nitrate as an unlabelled control for natural 15N abundance (Fig. 1). An air gap of approx. 5 mm was left to act as a hydraulic switch to prevent capillary rise of potassium nitrate into the substrate. The junction between pots and Petri dishes was then sealed with three layers of Parafilm to prevent evaporative losses. The parasite thus had access to the labelled or unlabelled potassium nitrate only via direct tapping of the host root and through remobilization of any labelled N from the host tissue (Fig. 1). The labelling period was 24 h. Pots were arranged in a randomized block design (with host species as the blocking factor) and unlabelled controls were paired with treatment pots.
After 24 h of labelling, the whole system was harvested and roots were blotted on dry filter paper (Whatman No. 1). The roots were separated into two parts, those in the K15NO3 and the remaining root ball. The host shoots and roots and the parasite were dried at 80°C for 2 d, and weighed. The shoots, parasite and roots not in the K15NO3 were ground in a ball mill (Retsch, Haan, Germany), and 1 mg material was weighed into ultraclean tin capsules. Samples were analysed for N and 15N content by continuous-flow isotope ratio mass spectrometry (CFIRMS) using a Europa Scientific Tracermass Stable Isotope Analyser with Roboprep sample converter (Europa Scientific, Crewe, UK) (Schmidt & Scrimgeour, 2001).
The amount of 15N (mg) in the different parts of the system was calculated using Eqn 1, and the distribution of 15N as a percentage of total uptake present in each part of the system was calculated using Eqn 2.
Mt = MN(E/100)(Eqn 1)
where Mt = mass of 15N in the tissue, either in host shoots, in host roots or in the parasite (mg 15N); MN = mass of N (mg); E= enrichment, the proportion of N which is 15N-labelled (above background). Thus E = ATL − ATC, where ATL = at %15N in labelled material; ATC = at %15N in control material (natural abundance).
F = (Mtp/Tp) × 100(Eqn 2)
where F = fraction of the total 15N pool transferred to the parasite (%); Tp = total association pool (mg 15N); Mtp = mass of tracer in the parasite.
Statistical analysis was performed using minitab (ver. 13); details of each statistical test are given in figure legends and in the text where appropriate.
Figure 2 shows photomicroscope images of the structure of mature haustoria, and schematic diagrams of the ontogeny of haustoria formed on the three target species based on additional tissue sections reported by Cameron et al. (2006). The haustorium is most developed on the grass C. cristatus, where it contained a hyaline body and secondary xylem, crushed the host root cortex and penetrated the host xylem (Fig. 2a–d; Table 1). The haustoria formed on L. vulgare and P. lanceolata are poorly differentiated, lacking either developed secondary xylem or a hyaline body (Fig. 2e–l; Table 1). Furthermore, the parasite never penetrated the xylem of both L. vulgare and P. lanceolata, and this vascular tissue was thickened and/or occluded with darkly staining material (Fig. 2h,l).
Table 1. Summary of the structural attributes of haustoria formed by Rhinanthus minor on three potential host species
Individuals of R. minor attached to the grass C. cristatus achieved a significantly larger biomass than those attached to either L. vulgare or P. lanceolata (Fig. 3; one-way anova, df = 2,8, F = 7.5, P = 0.023).
Amounts of 15N uptake
The parasites attached to C. cristatus sequestered significantly more of the 15N tracer than those attached to either L. vulgare or P. lanceolata (Fig. 4a; one-way anova, df = 2,8, F = 8.07, P = 0.020). There were no significant differences between the amount of 15N present in either roots (Fig. 4b; one-way anova, df = 2,8, F = 0.89, P = 0.459) or shoots (Fig. 4c; one-way anova, df = 2,8, F = 1.99, P = 0.217) of the three host species. There were no significant differences between the concentration of 15N in L. vulgare and P. lanceolata tissues; however, C. cristatus tissues were significantly less concentrated in 15N than either L. vulgare or P. lanceolata (Fig. 5a; one-way anova, df = 2,8, F = 7.58, P = 0.023).
Transfer of 15N tracer to the parasite as percentage of uptake
Rhinanthus minor attached to C. cristatus sequestered a significantly greater percentage of the total amount of 15N taken up by the host than when attached to either L. vulgare or P. lanceolata (Fig. 5b; one-way anova, df = 2,8, F = 16.15, P = 0.004). The parasite obtained 17% of the 15N resource taken up by C. cristatus, compared with only 2.5% of the 15N obtained by L. vulgare and 0.2% obtained by P. lanceolata.
Relationship between 15N uptake and parasite biomass
There was a highly significant positive relationship between the amounts of 15N abstracted by the parasite and the biomass it achieved in the experiment (regression, R2 = 0.945, P < 0.001) (Fig. 6).
This study is the first to quantify the extent to which the resistance responses of forbs can constrain the abstraction of their xylem solutes by the hemiparasitic plant R. minor using a stable isotope tracer.
Along with earlier studies (Seel et al., 1993; Hwangbo, 2000) we have shown that R. minor achieves the lowest biomass when growing on the two forb species studied, L. vulgare and P. lanceolata, compared with the grass C. cristatus, on which the parasite achieved the highest biomass. Previous studies (Table 1) have shown differences in the degree of development of the haustorium formed on the three host species investigated in this study, and differences in the cellular-level responses of host roots to the invading haustoria formed by R. minor. Haustoria formed on L. vulgare and P. lanceolata never develop the hyaline body (Table 1). Furthermore, these haustoria never develop secondary xylem connections with the host (although secondary xylem has been reported in the haustoria formed on P. lanceolata, this never develops into the penetration peg; Rümer et al., in press); and oscula – the conduits between the host and parasite vasculature (Dorr, 1997) – have never been observed in either forb species. In contrast, the haustoria formed on C. cristatus are fully differentiated and display all these key features (Table 1).
The response of the host tissue at the site of the invading parasite differs between the three species. First, the penetration peg formed by R. minor on L. vulgare is encapsulated by lignin, preventing direct access to the host's xylem; second, there is hypersensitive cell death of the host root at the point of parasite contact in the P. lanceolata–R. minor association, thus physically separating host and parasite (Cameron et al., 2006). By contrast, C. cristatus shows no morphological change in response to the parasite (Cameron et al., 2006). The resistance responses observed in the two forbs, combined with poor haustorial development, are shown in the present study to restrict access of the parasite to the host xylem stream, and explain the poor growth of the parasite when attached to the forbs.
We have shown that significantly more of the host's xylem solutes can be transferred to the parasite when growing on the grass than either forb species (with average rate functions over the 24-h labelling period of 650, 163 and 12 nmol h−1 15N transferred, respectively). These differences in the amount of 15N abstracted by the parasite cannot be explained by differing uptake of the tracer by the three host species, as there was no significant difference in the amounts of 15N present in either the roots or shoots of any host species. This trend is also reflected in the percentage of total 15N assimilated by hosts that was translocated to the parasite. Furthermore, the tissues of both forbs were more concentrated in 15N than those of the grass C. cristatus, with the latter species supplying more 15N to the parasite.
As R. minor maintains vascular continuity with a compatible host, these fluxes of the isotope tracer represent a proxy for the extent of mass flow of solutes from host to parasite. Jiang et al. (2003, 2004) used the assumption of mass flow of solutes from host to parasite through the xylem continuum to model the partitioning of resources between the graminoid host Hordeum vulgare and R. minor. Fluxes of N, phosphate, potassium and water were shown to be in the order of 18, 22, 20 and 17% of host uptake, respectively (Jiang et al., 2003, 2004). In the case of N, the modelled value of 18% of host uptake is strikingly similar to the 17% of host uptake that has been shown in the current study to be abstracted from C. cristatus, another graminoid host for R. minor. It is not possible to exclude the role of parasite size controlling sink strength, hence the rate of solute transfer. However, the morphological studies indicate that the defences of L. vulgare and P. lanceolata will prevent R. minor from accessing their xylem solutes directly, and this in turn is likely to be the factor controlling the rates of solute transfer to the parasite and its subsequent growth. From this we infer that the biomass of the parasite is controlled by the morphology of the host–parasite interface, and conclude that the defence responses seen in L. vulgare and P. lanceolata are extremely effective at restricting parasite development and thus minimizing parasite-induced host damage.
The parasites attached to the forbs received little of the 15N tracer, but some of the labelled N was sequestered when R. minor attached to these species, and warrants further consideration. This uptake could be caused by labelled compounds leaking out from host roots, although the labelling period was short (24 h), minimizing the opportunity of exudation from the roots. Second, the resistance mechanisms proposed by Cameron et al. (2006) may not be entirely effective if some apoplastic resource transfer occurs via interfacial parenchyma, or if some xylem transfer occurs before the host is able to seal off its vascular system from the parasite; however, this hypothesis remains to be proven. Moreover, only a very small amount of resources is translocated from the two forbs to R. minor, thus they represent poor host species, indeed R. minor attached to L. vulgare and P. lanceolata achieves a lower biomass than when growing without a host (Cameron et al., 2006); in the latter case, the parasite often dies before it is able to complete its life cycle (Cameron, 2004). This suggests that the parasite is not able to sequester enough resources from the association to overcome the cost of competition shown to be associated with growing in close proximity with the host (Keith et al., 2004).
We would like to thank Drs Jonathan Leake, Gareth Phoenix and Mike Pilkington (University of Sheffield, UK), Professor David Robinson (University of Aberdeen, UK) and Dr Louis Irving (Massey University, New Zealand) for useful discussions. We also thank David Hadwen and Janet Woo (Aberdeen) for technical assistance, Dot MacKinnon (Aberdeen) for help with analytical chemistry, and Dr Charlie Scrimgeour (SCRI, Dundee, UK) for analysing the samples for 15N content. We acknowledge financial support provided by the NERC (NER/S/A/2001/05959).