Functional anatomy of haustoria formed by Rhinanthus minor: linking evidence from histology and isotope tracing

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 (15)N 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 (15)N 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.     

Genetic variation changes the interactions between the parasitic plant-ecosystem engineer Rhinanthus and its hosts

Within-species genetic variation is a potent factor influencing between-species interactions and community-level structure. Species of the hemi-parasitic plant genus Rhinanthus act as ecosystem engineers, significantly altering above- and below-ground community structure in grasslands. Here, we show the importance of genotypic variation within a single host species (barley-Hordeum vulgare), and population-level variation among two species of parasite (Rhinanthus minor and Rhinanthus angustifolius) on the outcome of parasite infection for both partners. We measured host fitness (number of seeds) and calculated parasite virulence as the difference in seed set between infected and uninfected hosts (the inverse of host tolerance). Virulence was determined by genetic variation within the host species and among the parasite species, but R. angustifolius was consistently more virulent than R. minor. The most tolerant host had the lowest inherent fitness and did not gain a fitness advantage over other infected hosts. We measured parasite size as a proxy for transmission ability (ability to infect further hosts) and host resistance. Parasite size depended on the specific combination of host genotype, parasite species and parasite population, and no species was consistently larger. We demonstrate that the outcome of infection by Rhinanthus depends not only on the host species, but also on the underlying genetics of both host and parasite. Thus, genetic variations within host and parasite are probably essential components of the ecosystem-altering effects of Rhinanthus.     

Suppression of host photosynthesis by the parasitic plant Rhinanthus minor

BACKGROUND AND AIMS: Parasitism is well understood to have wide-ranging deleterious effects on host performance in species thus far characterized. Photosynthetic performance reductions have been noted in the Striga-Zea mays association; however, no such information exists for facultative hemiparasitic plants and their hosts, nor are the effects of host species understood. METHODS: Chlorophyll fluorimetry was used to study the effects of parasitism by the hemiparasite Rhinanthus minor on the grass Phleum bertolinii and the forb Plantago lanceolata, and the effects of host species on the photosynthetic apparatus of R. minor. KEY RESULTS: Parasitism by Rhinanthus led to a significant decrease in the host, and total (host + parasite) biomass in Phleum; however, in Plantago, no significant repression of growth was noted. Maximum quantum yield (F(v)/F(m)) was reduced in parasitized Plantago, relative to control plants, but not in Phleum. F(v)/F(m) was significantly lower in R. minor parasitizing Phleum than Plantago, suggesting Phleum to be a superior host to Plantago for R. minor. Steady-state quantum yield (Phi(PSII)) was significantly depressed in parasitized Phleum, but only at low irradiances in Plantago. Phi(PSII) was very low for R. minor grown on Plantago, but not Phleum. CONCLUSIONS: Shown here is the first evidence of the suppression of host photosynthesis by a facultative hemiparasitic plant, which has significant effects on total biomass production. Host identity is a significant factor in parasite success, with the forb Plantago lanceolata exhibiting apparent chemical as well as previously identified physical defences to parasitism. It is proposed that the electron transport rate (as denoted by Phi(PSII)) represents the limiting factor for biomass accumulation in this system, and that Plantago is able to suppress the growth of Rhinanthus by suppressing the electron transport rate.     

An anatomical study of the haustoria of Rhinanthus minor attached to roots of different hosts

Roots of a range of potential hosts responded differently when Rhinanthus minor attempted to form haustoria. Roots of Fabaceae show the weakest reaction as apart from slight lignification, no reaction was observed at the interface between the endophyte and the cortical tissue of the host root. Grass roots react with strong lignification of all cells within the stele with the exception of a small number of phloem cells whilst the endodermis fully enters the tertiary stage. In the case of Phleum bertolonii the cortical cells also become lignified. The lignification is even observed in the host root tissue in a distance of about 1 mm from the haustorium (both apically and basipetally). In the case of Leucanthemum vulgare, strong suberisation can be observed in the cell walls of the interface between endophyte (tip of the sucker) and host. Plantago lanceolata exhibits the strongest reactions against the haustorial tissues. Cells of the interface between the endophyte and the host cortex are completely destroyed, as well as a few cell layers outside the central xylem cylinder, even in some distance from the haustorium. Thus, host xylem is completely isolated from the haustorium in this case. Extraction of sap from xylem vessels is likely to be drastically impaired in such a situation.     

Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare

Using the facultative root hemiparasite Rhinanthus minor and its host Hordeum vulgare several aspects of water relations have been measured in this parasitic association. Extraction of xylem sap by the parasite from the host's roots is facilitated by con siderably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another root hemiparasite, Melampyrum arvense, parasitizing various grasses in the field, showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values and that of Rhinanthus seminal roots showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition, and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host's roots. This water uptake by the parasitized host, however, due to a parasite-induced reduction in the host's growth, was decreased by 22% as compared to non-parasitized barley. The overall changes in growth-related water deposition in the host and parasite pointed to decreased shoot growth and relatively favoured root growth in the host and to strongly favoured shoot growth in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitizing one barley plant.     

Does legume nitrogen fixation underpin host quality for the hemiparasitic plant Rhinanthus minor?

The high quality of leguminous hosts for the parasitic plantRhinanthus minor (in terms of growth and fecundity), comparedwith forbs (non-leguminous dicots) has long been assumed tobe a function of the legume's ability to fix atmospheric nitrogen(N) from the air and the potential for direct transfer of compatibleamino compounds to the parasite. Using associations betweenRhinanthus minor and Vicia faba (Fabaceae) that receive N eitherexclusively via symbiotic associations with rhizobia supplyingorganic N fixed from N₂ or exclusively through the supply ofinorganic nitrate to the substrate, the underlying reasons forthe quality of legumes as hosts for this parasite are unravelled.It is shown that sole dependence of the host, V. faba, on Nfixation results in lower growth of the attached parasite thanwhen the host is grown in a substrate supplied exclusively withinorganic N. In contrast, the host plants themselves achieveda similar biomass irrespective of their N source. The physiologicalbasis for this is investigated in terms of N and abscisic acid(ABA) partitioning, haustorial penetration, and xylem sap aminoacid profiles. It is concluded that legume N fixation does notunderpin the quality of legumes as hosts for Rhinanthus butrather the well-developed haustorium formed by the parasite,coupled with the lack of defensive response of the host tissuesto the invading haustorium and the presence of sufficient nitrogenouscompounds in the xylem sap accessible to the parasite haustoria,would appear to be the primary factors influencing host qualityof the legumes. Key words: ABA, haustorium, legume, nitrogen fixation, nodules, parasitic plant Received 14 November 2007; Revised 7 January 2008 Accepted 8 January 2008     

Correlations between Haustoria Formation and Parasitic Development in Orthocarpus purpurascens (Scrophulariaceae)

Three lines of evidence correlate the parasitic performane ofOrthocarpus purpuruscens Benth. with numbers of haustoria produced:(i) the pattern of variation in numbers of haustoria producedin agar culture with different chemical stimuli correspondsclosely to the variation pattern of parasite vigour producedby a range of host plants; (ii) the progeny of plants demonstratingvigorous growth with hosts produce significantly more haustoriathan progeny from parents exhibiting weak parasitic development;(iii) conversely, seedlings that produce high numbers of haustoriain agar culture grow significantly better when transplantedwith hosts than do seedlings with low numbers of haustoria.Haustoria-forming potential is heritable, but highly influencedby environmental factors. Potential number of haustoria is aproduct of the concentration and/or quality of haustoria inducingstimuli, and the parasite's individual ability to respond. Intra-populationdifferences in parasitic development appear to be largely dueto the quantity rather than the quality of substrates receivedfrom host plants. haustoria, Orthocarpus purpurarcens, parasitic development     

Water Relations of the Hemiparasite Rhinanthus serotinus before and after Attachment

Growth of the hemiparasite Rhinanthus serotinus (Schönh.) Oborny was greatly stimulated after attachment of the parasite to the roots of the host plant, Hordeum vulgare L. Before attachment the hydrostatic pressure in the xylem, determined by the pressure bomb technique, was found to be lower in Rhinanthus than in the host. It increased after the formation of haustoria between host and parasite. Apparently, the water transport to Rhinanthus was facilitated. The hydrostatic pressure remained lower than that of the host, accounting for the flow of water and solutes in the direction of the parasite and indicating that there exists a resistance to water transport in the haustoria. Water and solutes were absorbed by the cells, which increased in size. The turgor pressure of the parasite rose steeply, but the osmotic potential was hardly affected.     

Interactions between arbuscular mycorrhizal fungi and the hemiparasitic angiosperm Rhinanthus minor during co-infection of a host

The outcome of dual infection of the grass Lolium perenne L. by arbuscular mycorrhizal (AM) fungi and the parasitic angiosperm Rhinanthus minor L. was investigated in a glasshouse study. Colonization of L. perenne roots by AM fungi was significantly reduced by the presence of R. minor , as was host growth which fell by 44–51%. It was concluded that these two responses were linked, with AM colonization declining in response to the reduction in availability of host carbon. Parasite growth and reproductive output rose by 58% and 47% respectively when the hosts were mycorrhizal. These trends were unrelated to the attachment success of the parasite, but were accompanied by a significant increase in the formation of secondary haustoria. The benefits afforded the parasite when the hosts were mycorrhizal were attributed to increased carbon and nutrient flux resulting from alternations in sink strength. Host responses to parasitism and mycorrhizal colonization were not affected by the interaction between the two symbionts. However, the suggestion is made that the interaction between the AM fungi and parasite might have long-term ecological implications for the host species via its impact on parasite fecundity.     

Abscisic acid (ABA) flows from Hordeum vulgare to the hemiparasite Rhinanthus minor and the influence of infection on host and parasite abscisic acid relations

Using the facultative root hemiparasite Rhinanthus minor and Hordeum vulgare as a host, the flows, depositions, and metabolism of abscisic acid (ABA) within the host, within the parasite, and between host and parasite have been studied. When the plants were supplied with 5 mM NO(3)(-), there were weak or no effects of parasitism on ABA flows, biosynthesis, and ABA degradation in barley. However, ABA deposition was significantly affected in the leaf laminae (3-fold) and in the leaf sheath (2.4-fold), but not in roots. Dramatic changes in ABA flows, metabolism, and deposition on a per plant basis, however, have been observed in Rhinanthus. Biosynthesis in the roots was 12-fold higher after attachment, resulting in 14-fold higher ABA flows in the xylem. A large portion of this ABA was metabolized, a small portion was deposited. Phloem flows of ABA were increased 13-fold after attachment. The concentrations of ABA in tissues and transport fluids were higher in attached Rhinanthus by an order of magnitude than in host tissues and xylem sap. The same tendency was also found in a comparison between single Rhinanthus and unparasitized barley. As compared with 5 mM NO(3)(-), lower NO(3)(-) or 1 mM NH(4)(+) supply doubled the ABA concentrations in barley leaf laminae, while having only small or non-significant effects in the other organs. The possible function of ABA for the parasite is discussed.     

Development and Structure of the Haustorium of the Parasite Rhamphicarpa fistulosa (Scrophulariaceae)

Rhamphicarpa fistulosa (Hochst.) Benth. (Scrophulariaceae), a parasite of African cereals, develops secondary haustoria which penetrate the roots of the host plant. Light and electron microscopy have been used to study the structure and development of haustoria in this species, which, until now, have not been well characterized. Haustoria are initiated in the hypodermis of the parasite roots. A meristematic strand is developed between the parasite root stele and the host-parasite interface. From this strand, cells differentiate into xylem elements after penetration of the host root. Xylem differentiation follows an acropetal pattern. Mature haustoria are characterized by a continuous xylem bridge between water conducting elements of parasite and host. A detailed study of the hostparasite interface revealed the presence of collapsed and compressed host cells at the lateral interface (between parasite cells and host cortex), whereas the central interface between parasite cells and the host stele is almost devoid of host cell remnants. Implications of these observations for the penetration mechanisms are discussed.     

Parasite establishment and host extinction in model communities

Studies of host–parasite dynamics usually consider one, or at most two, host species, neglecting the possible effects of other species on the focal hosts and vice versa. To explore the interaction of community structure with host–parasite dynamics, we model the invasion of stable communities of varying size by a parasite. The communities are generated with random interaction coefficients and connectance 0.5. Each community is invaded by parasites with different values of virulence (disease-induced host mortality rate), specificity and transmission rate. The result of each invasion is determined by numerically simulating the dynamics of the community. We classify the outcomes by whether the parasite successfully establishes in the focal host population(s), and, if so, by the proportion of host and non-host species that go extinct as a result of the parasite's introduction. We discuss how the structure of the community and the interaction between hosts and other species affect several important processes of disease ecology: the density threshold for parasite invasion, extinction cascades caused by the parasite, and the frequency of extinctions of hosts and non-hosts. In our simulated communities, non-host species went extinct more frequently than hosts, suggesting the importance of the community context of disease. In some cases, the parasite's invasion induced regular population cycles in the previously stable community.     

Modelling the arms race in avian brood parasitism

In brood parasitism, interactions between a parasite and its host lead to a co-evolutionary process called an arms race, in which evolutionary progress on one side provokes a further response on the other side. The host evolves defensive means to reduce the impact of parasitism, while the parasite evolves means to counter the host's defence. To gain insights into the co-evolutionary process of the arms race, a model is developed and analysed, in which the host's defence and the parasite's counterdefence are assumed to be genetically determined. First, the effect of parasite counterdefence on host defence is analysed. I show that parasite counterdefence can critically affect the establishment of host defence, giving rise to three situations in the equilibrium state: The host shows (1) no defence, (2) an intermediate level of defence or (3) perfect defence. Based on these results, the evolution of parasite counterdefence is considered in connection with host defence. It is suggested that the parasite can evolve counterdefence to a certain degree, but once it has established counterdefence beyond this, the host gives up its defence against parasitism provided the defence entails some cost to perform. Dynamic aspects of selection pressure are crucial for these results. Based on these results, I propose a hypothetical evolutionary sequence in the arms race, along which interactions between the host and parasite proceed.     

Host selectivity,haustorial anatomy and impact of the invasive parasite Parentucellia viscosa on floodplain vegetative communities in Japan

The parasitic plant Parentucellia viscosa has been introduced recently to the Japanese floodplain. Because of its parasitic nature and high fecundity, P. viscosa may well become a major plant invader with a significant impact on floodplain ecosystems. Thus, a knowledge of the host selectivity of P. viscosa will not only provide basic biological information, but will also contribute to the understanding of floodplain vegetation conservation. We evaluated the host selectivity by comparing the observed numbers of haustoria with those expected from the relative below‐ground biomass. In addition, we examined the haustorial anatomy to determine whether haustoria are functional, and compared the above‐ground biomass of three functional plant groups (grasses, legumes and nonleguminous forbs) in intact and parasite removal quadrats. We found that haustoria were nonrandomly distributed amongst host species, suggesting host preference for Poaceae and Fabaceae. In addition, haustoria attached to certain forbs did not penetrate into the stele. The above‐ground biomass of grasses and legumes was increased significantly by the removal of P. viscosa, but the biomass of forbs did not change significantly. These results suggest that host preference depresses the performance of Poaceae and Fabaceae, thus affecting the competitive relationships among plants, meaning that P. viscosa may pose a serious threat to indigenous endangered legumes and grasses. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 69–78.     

Parasite–grass–forb interactions and rock–paper– scissor dynamics: predicting the effects of the parasitic plant Rhinanthus minor on host plant communities

1.  Parasitic plants affect the growth, reproduction and metabolism of their hosts and may also influence the outcome of competitive interactions between host species and, consequently, the structure of entire host communities.
2.  We investigate the effect of the root hemiparasitic plant Rhinanthus minor on plant community dynamics using a spatial theoretical model. The model is parameterized with data from pairwise interaction experiments under two nutrient levels between the hemiparasite and three grass species ( Cynosurus cristatus , Festuca rubra and Phleum bertolonii ) and three forb species ( Leucanthemum vulgare , Plantago lanceolata and Ranunculus acris ).
3.  Relative interaction coefficients were intransitive, with the dynamics of the system conforming to a rock–paper–scissors game. Stable deterministic dynamics emerge from parameters obtained under low-nutrient conditions. Under high-nutrient conditions, the dynamics are unstable, but are stabilized in spatially explicit models. The outcomes are sensitive to initial spatial pattern and frequency.
4.   Synthesis . This study supports the idea that hemiparasite populations may form 'shifting clouds' in natural populations and explains seemingly unpredictable shifts in host community structure following introduction of hemiparasites. Management of plant communities using hemiparasites needs to take these complex dynamics into account.     

The Regulation of the Water Potential Gradient in the Host and Parasite Relationship betweenSorghum bicolorandStriga hermonthica

A glasshouse experiment was carried out to investigate the factorscontrolling the abstraction of xylem fluid from its host bythe parasiteStriga hermonthica(Scrophulariaceae).Strigahad amean daily transpiration rate far exceeding that of its hostsorghum (Sorghum bicolor), with infestation byStrigaalso shownto lower the transpiration rate of the host. Stopping the host'stranspiration was shown to decrease the transpiration rate ofthe parasite. Stopping the parasite's transpiration only gavean initial increase in the host's transpiration rate which wasnot sustained. The parasite had a lower water potential thanits host, values being -0.42 MPa and -0.23 MPa, respectively,and an accompanying higher osmotic pressure of 0.68 MPa against0.51 MPa for sorghum. Modifying the water potential gradientby bagging both partners together showed that the differentialin osmotic pressure and water potential was largely maintainedby the parasite's higher rate of transpiration. A favourablewater potential gradient towards the parasite still existedfollowing the cessation of transpiration, this being generatedby the haustorial resistance to hydraulic conductivity whichwas found to be some 1.5–4.5 times greater than that offeredby the parasite shoot. Both the high rate of transpiration andthe increased resistance across the haustoria would appear tobe necessary means to facilitate the diversion of host resourcesto the parasite.Copyright 1997 Annals of Botany Company Striga hermonthica; sorghum; water relations; haustorium; root parasite     

Attachment of bean rust cell wall material to host and non-host plant tissue

A preparation of bean rust (Uromyces phaseoli) germ tube walls, consisting of short, filamentous particles, was labeled with fluorescein iso-thiocyanate. Freeze sections of host and non-host tissue were incubated in the labeled preparation. Maximum staining was observed in host plant tissue (Phaseolus vulgaris), in which bean rust regularly forms haustoria. In tissue of the non-host plantsVigna sinensis andPhaseolus lunatus, where fewer haustoria were formed, staining was only weak. However, no staining was observed in the non-host tissue ofPhaseolus aureus, Helianthus annuus, Brassica oleracea andHordeum vulgare in which the infection hypha did not form haustoria. This would appear to indicate that formation of haustoria is induced by a specific attachment of the hyphal wall to the host wall. The possibility that elicitors attach in a similar way, is discussed.Abbreviations FITC Fluorescein iso-thiocyanate - cv cultivar     

Mobility of boron-polyol complexes in the hemiparasitic association between Rhinanthus minor and Hordeum vulgare: the effects of nitrogen nutrition

Boron (B) is an essential nutrient required for plant growth and physiological processes. Long-distance B transport is facilitated by the formation of B–polyol complexes. We investigated B uptake and distribution in response to differing levels of exogenous nitrogen supply in the hemiparasitic association between Rhinanthus minor and Hordeum vulgare (barley) and in unparasitised barley and single Rhinanthus plants. In this system, the polyol mannitol is the major assimilate in Rhinanthus , whereas polyols are not detectable in barley. Furthermore, previous studies have shown that the accumulation of polyols within Rhinanthus is negatively affected by the application of exogenous nitrogen. Within the association, the strongest accumulation of B was detected in lateral buds and inflorescences of Rhinanthus , consistent with the greatest B demand in strong sink organs supplied through the phloem that contain high concentrations of mannitol. In the host, the strongest B accumulation was found in xylem-supported leaf lamellae. Roots and sheaths did not accumulate substantial amounts of B, while re-circulation of B through the phloem vessels accounted for only 10% (unparasitised) and 8% (parasitised) of the xylem sap-imported B in the mannitol-free barley hosts. In contrast, 53% (attached) and 39% (in the absence of a host) of the xylem sap-imported B was re-circulated in the phloem in the mannitol-rich Rhinanthus . We therefore present the first quantitative uptake and flow models of long-distance B transport in polyol-rich and polyol-free plants. Our findings are consistent with a close relationship between B re-translocation and mannitol concentrations in phloem vessels.     

Contents and flows of assimilates (mannitol and sucrose) in the hemiparasiticRhinanthus minor/Hordeum vulgare association

Using the facultative root hemiparasiteRhinanthus minor andHordeum vulgare as a host, the flows and partitioning of mannitol in the parasite, and of sucrose in the host have been studied during the period of 41 to 54 days after planting, i.e, about 30 to 43 days after successful attachment of the parasite to the host. The biosynthesis of mannitol inRhinanthus shoots increased 16-fold by parasitism, resulting in a 15-fold higher mannitol flow in the phloem and a 10-fold higher deposition in the shoot. Under reduced nitrogen supply and with ammonium as the only N-form the concentrations of mannitol tended to be increased by approximately 2-fold. Xylem flows of mannitol were increased 10-fold after attachment. No mannitol was found in barley roots even in the direct vicinity of the haustoria. Compared to unparasitized barley, the net biosynthesis and deposition in the shoot and the phloem flow was decreased substantially. No sucrose has been detected in barley xylem sap and consequently there was no indication of a sucrose transfer from the host to the parasite. A possible involvement of mannitol in the abscisic acid relations of the parasite is discussed.     

Induced Systemic Resistance Against Sphaerotheca fuliginea in Cucumber: Efficiency of Tobacco Necrosis Virus (TNV) and Copper Sulphate (CuSO4) in Eliciting Defence Reactions

Plants of Cucumis sativus were eithe preinoculated with TNV or treated with drops of a 5% solution of CuSO₄ on the cotyledons, and 5 days later challenge-inoculated with Sphaerotheca fuliginea on the first true leaf. The induced systemic resistance was assessed by evaluating the percentage of conidial germination, the length of the hyphae derived from single conidia, the number of cells with lignified walls and that of cells containing haustoria. The number of necrotic cells was also recorded. Compared with controls, in TNV-preinoculated plants conidial germination was lower, hyphal length shorter and the number of haustoria much reduced. The majority of haustoria was found in cells with lignified walls. Pretreatment with CuSO₄, although inducing a slight increase of cell wall lignification, did not appreciably reduce the infection process. The number of necrotic cells was very low in all cases. These results suggest that, in this host/pathogen interaction, hypersensitive cell death has no part in the induced defence reaction, although a major role is played by the lignification process.