Studies on Cuscuta reflexa ROXB. V. The level of endogenous hormones in the parasite,Cuscuta reflexa,and its host,Vicia faba L., and a suggested role in the transfer of nutrients from host to parasite

Summary The mechanism of parasitism of Cuscuta, especially the absorption of nutrients from its host, is not clear. As it might be connected with the function of plant hormones, the endogenous levels of all hormone groups in the parasite, Cuscuta reflexa, and its host, Vicia faba, were investigated. Since the content of auxins, gibberellins and cytokinins is higher in the host than in the parasite, there is no indication that any of these phytohormones is involved in the absorption of nutrients by Cuscuta. However, the content of growth inhibitors, especially free abscisic acid, is much higher in Cuscuta than in the host. There is a gradient of abscisic acid with a maximum in the basal, haustoria-bearing stem region in which the transfer of nutrients from host to parasite occurs. The high content of abscisic acid within the parasite may be a causal connection with the parasitic absorption of nutrients from host sieve tubes.This publication is respectfully dedicated to the academician Prof. Dr. A. L. Kursanov, Moscow, on the occasion of the 80th anniversary of his birthday.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Utilizzazione Di Afidi Per Il Prelevamento Della Linfa in « Cuscuta Epithymum »

Abstract

Use of Aphids for sampling the cell sap of Cuscuta epithymum. — A variety of Myzus persicae that feeds specifically on Cuscuta epithymum has been employed in a study of the translocation of nutrilites from the host, Trifolium repens, to the parasite, C. epithymum. If the host was exposed to an atmosphere containing C¹⁴,O₂, the Aphids feeding on the Cuscuta filaments became labelled very rapidly and to considerable extent. The analysis of the distribution of radioactivity in the Cuscuta filaments and the Aphids revealed that extensive degradation of the compounds and randomization of the label occurred in the insects.     

Interaction of a host plant and its holoparasite: effects of previous selection by the parasite

If parasites decrease the fitness of their hosts one could expect selection for host traits (e.g. resistance and tolerance) that decrease the negative effects of parasitic infection. To study selection caused by parasitism, we used a novel study system: we grew host plants (Urtica dioica) that originated from previously parasitized and unparasitized natural populations (four of each) with or without a holoparasitic plant (Cuscuta europaea). Infectivity of the parasite (i.e. qualitative resistance of the host) did not differ between the two host types. Parasites grown with hosts from parasitized populations had lower performance than parasites grown with hosts from unparasitized populations, indicating host resistance in terms of parasite’s performance (i.e. quantitative resistance). However, our results suggest that the tolerance of parasitic infection was lower in hosts from parasitized populations compared with hosts from unparasitized populations as indicated by the lower above‐ground vegetative biomass of the infected host plants from previously parasitized populations.     

Metabolite changes with induction of Cuscuta haustorium and translocation from host plants

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramatically due to the absorption of specific host plant metabolites such as pinitol. Cuscuta seedlings attached to pinitol rich host plants contained more pinitol and showed different profiles from those attached to plants having less or lacking pinitol.     

First report of parasite dodder (Cuscuta campestris) on jujube trees (Ziziphus jujuba) from Iran

Jujube (Zizyphus jujuba Mill.) as a naturally host for dodder (Cuscuta campestris Yunck.) is newly recorded from Southern Khorasan province of Iran. This is the first report of C. campestris, a stem parasite plant, naturally infecting jujube trees worldwide.     

Modelling of the flows and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: II. Flows between host and parasite and within the parasitized host

A recently developed empirically based modelling technique wasused to quantify uptake, flow and utilization of C and N inLupinus albus L., uninfected and parasitized by Cuscuta reflexaRoxb. plants over a 12 d period during flowering and early fruitsetting of the host. The modelling combined data on molar C:Nratios in host phloem and pressure-induced xylem sap, net incrementsof C and N in host and parasite plant parts and respiratorylosses of C. The modelling of the solute transfer between hostand Cuscuta was achieved by assuming non-specific intake fromthe xylem. The models predicted that Cuscuta derived 99.5% ofits carbon and 93.6% of its nitrogen demand from the host phloem.The overriding sink strength of the parasite diverted most ofthe basipetally translocated host assimilates and massivelycompeted with the host root and inhibited fruit setting. Carbonincorporation in Cuscuta consumed 56%, respiration 24% and secretionby extrafloral nectaries 1.8% of the current host photosynthate.Root respiration was inhibited by 59% and carbon was mobilizedfrom host root and leaves. Competition by the parasite for Nwas even more severe and Cuscuta incorporated nitrogen equalling223% of current fixation, but N₂ fixation of the host was severelyrestricted to 37%. Withdrawal of N from host phloem led to severelosses of N from leaves and the root and marked decreases inN concentration. It required massive xylem-to-phloem transferof N, because the xylem as the major supply route for N wasnot exploited substantially by Cuscuta. The results are discussedin relation to likely causes for parasite-induced pathogeniceffects, suggesting that Cuscuta affected the host adverselyby depriving it mainly of its nitrogen, but that causal to incipientnitrogen deficiency and restricted N2 fixation was the superiorsink potential of Cuscuta, which prevented adequate supply ofassimilates to the nodulated root. The dominating sink potentialof Cuscuta is compared with the similarly strong sink competitionexerted by fruits at the stage of seed filling in annual plants. Key words: Cuscuta reflexa, Lupinus albus, parasitism, carbon, nitrogen, phloem, xylem, transport     

Significance of Cuscutain,a cysteine protease from <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>, in host-parasite interactions

Background  

Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified.     

Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells

Summary.  The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia (Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscuta spp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress. Received June 12, 2002; accepted August 26, 2002; published online March 11, 2003     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Phenolics in Parasites and Hosts

the contents of total phenolics in three parasitic angiosperms, Cuscuta species, Orobanche aegyptiaca and Dendrophthoe falcata and their respective hosts, were colorimetrieally determined. A biochemical comparison was made of the phenolics on the basis of the ability of alcoholic extracts of the tissues to inhibit amylose phosphorylase in vitro. High concentration of phenolics seemed to be a general feature of parasitic angiosperms. An increase in the concentration of the phenolics occurred in the tissues of infected hosts, in comparison with controls. the phenolics of Orobanche and mistletoe had inhibitory activity against amylose phosphorylase, but those of Cuscuta developed the inhibitory ability only when growing on hosts which themselves possessed inhibitory phenolics. the inhibitory activity of host phenolics was sometimes altered as a result of infection by parasite. Although the hosts often exerted some influence on the concentration and the inhibitory activity of phenolics in the parasites, there was no direct relationship between host and parasite phenolics. the sum of the phenolics in the tissues of parasite and the infected bost generally exceeded the phenolics in the tissues of the control host. The content of phenolics and their inhibitory activity did not appear to be directly related to the resistance of a host or to the extent of its susceptibility to parasite infection.     

In Vitro Studies of Floral Induction on Stem Apices of Cuscuta reflexa Roxb. --A Short-day Plant

Stem tips of Cuscuta reflexa, cultured on modified White's medium,were subjected to different light and dark conditions. The culturesflowered when maintained either in continuous darkness or exposedto 14 hours of daily dark period. Thus Cuscuta reflexa behavesas a typically short-day plant. The presence of 5 per cent.sucrose in the medium completely obviates the requirement forhigh-intensity light exposures, otherwise essential for SDP.It appears that the bud itself is sensitive to photoinduction.In spite of the presence of natural tissue-bridge between thehost and the parasite, provided by the haustorial connexions,there is no transportation of flower-forming substance fromone plant to another. The flowering periods of host and parasiteare independent of each other.     

Involvement of ABA in reduced photosynthesis and stomatal conductance in <Emphasis Type="Italic">Cuscuta campestris — Mikania micrantha</Emphasis> association

We investigated whether changes in abscisic acid (ABA) content in leaves of Mikania micrantha infected by the holoparasite Cuscuta campestris at five growth stages, influenced the host stomatal conductance (g_s), transpiration rate (E) and net photosynthetic rate (P_N). C. campestris infection caused a negative effect on g_s, E and P_N of the host plants. ABA content in host leaves infected by C. campestris was significantly lower at 6 d after parasitization (DAP) and significantly higher at 13 and 33 DAP, relative to uninfected controls. In the parasite, ABA content was lowest at 13 DAP and then sharply increased to the maximum at 26 DAP. Moreover, the ABA content in the parasite was always lower than in the infected host leaves. The results suggest that an increase in host ABA concentration contributes to reduced host g_s, E and P_N in the holoparasitic C. campestris — M. micrantha association.     

The carbon canopy economy of the association between cowpea and the parasitic angiosperm Striga gesnerioides

The association between the parasite Striga gesnerioides and cowpea (Vigna unguiculata) was investigated using measurements of growth and gas exchange together with calculations of the carbon budget of the association. Striga gesnerioides has a very low photosynthetic capacity coupled with high rates of respiration. Even at photosynthetic light saturation shoots exhibit no net carbon gain. Thus S. gesnerioides is highly dependent on its host for carbon as well as for water and inorganic solutes. It is estimated that 70% of the carbon transferred from host to parasite is used in parasite respiration. Infected cowpea had a lower photosynthetic capacity, at times less than half that of uninfected plants. Infection with S. gesnerioides reduced the growth of cowpea by 75%. Calculations indicate that the loss of carbon from the host by export to the parasite is more important than reduced photosynthetic capacity of the host in accounting for the observed growth reductions.     

Defense response of resistant host Impatiens balsamina to the parasitic angiosperm Cuscuta japonica

The response of the stem of a resistant host (Impatiens baslamina) to infection by the parasitic flowering plant Cuscuta japonica was studied with light and electron microscopy. The intra- and interfascicular cambial cells in the host stem first reacted to the penetrating upper haustorium by dividing, and the differentiation of the host xylem (vascular) tissues proceeded toward interfascicular areas from vascular bundles. When the host vascular tissue was invaded by the endophyte (haustorial portion in the host stem), the host xylem was displaced, and host vessels became occluded with parenchyma cells, resulting in tyloses. As the parasitism progressed, areas of the host stem penetrated by the endophyte became swollen via secondary growth and cell division in the parenchymatous cortex, pith, and interfascicular areas. During this intrusion by the endophyte, darkly stained necrotic reactions were detected at the interface between the host tissue and the invading endophyte. The results suggested that in the host tissues penetrated by the parasite, the formation of secondary tissue and swellings caused by active cell division of ground tissue and host vessel occlusion by tyloses constitute the host structural defense against the parasite.     

Selective mineral transport barriers at Cuscuta-host infection sites

The uptake of inorganic nutrients by rootless parasitic plants, which depend on host connections for all nutrient supplies, is largely uncharted. Using X-ray fluorescence spectroscopy (XRF), we analyzed the element composition of macro- and micronutrients at infection sites of the parasitic angiosperm Cuscuta reflexa growing on hosts of the genus Pelargonium. Imaging methods combining XRF with 2-D or 3-D (confocal) microscopy show that most of the measured elements are present at similar concentrations in the parasite compared to the host. However, calcium and strontium levels drop pronouncedly at the host/parasite interface, and manganese appears to accumulate in the host tissue surrounding the interface. Chlorine is present in the haustorium at similar levels as in the host tissue but is decreased in the stem of the parasite. Thus, our observations indicate a restricted uptake of calcium, strontium, manganese and chlorine by the parasite. Xylem-mobile dyes, which can probe for xylem connectivity between host and parasite, provided evidence for an interspecies xylem flow, which in theory would be expected to carry all of the elements indiscriminately. We thus conclude that inorganic nutrient uptake by the parasite Cuscuta is regulated by specific selective barriers whose existence has evaded detection until now.     

Growth but Not Photosynthesis Response of a Host Plant to Infection by a Holoparasitic Plant Depends on Nitrogen Supply

Parasitic plants can adversely influence the growth of their hosts by removing resources and by affecting photosynthesis. Such negative effects depend on resource availability. However, at varied resource levels, to what extent the negative effects on growth are attributed to the effects on photosynthesis has not been well elucidated. Here, we examined the influence of nitrogen supply on the growth and photosynthesis responses of the host plant Mikania micrantha to infection by the holoparasite Cuscuta campestris by focusing on the interaction of nitrogen and infection. Mikania micrantha plants fertilized at 0.2, 1 and 5 mM nitrate were grown with and without C. campestris infection. We observed that the infection significantly reduced M. micrantha growth at each nitrate fertilization and more severely at low than at high nitrate. Such alleviation at high nitrate was largely attributed to a stronger influence of infection on root biomass at low than at high nitrate fertilization. However, although C. campestris altered allometry and inhibited host photosynthesis, the magnitude of the effects was independent of nitrate fertilizations. The infection reduced light saturation point, net photosynthesis at saturating irradiances, apparent quantum yield, CO₂ saturated rate of photosynthesis, carboxylation efficiency, the maximum carboxylation rate of Rubisco, and maximum light-saturated rate of electron transport_, and increased light compensation point in host leaves similarly across nitrate levels, corresponding to a similar magnitude of negative effects of the parasite on host leaf soluble protein and Rubisco concentrations, photosynthetic nitrogen use efficiency and stomatal conductance across nitrate concentrations. Thus, the more severe inhibition in host growth at low than at high nitrate supplies cannot be attributed to a greater parasite-induced reduction in host photosynthesis, but the result of a higher proportion of host resources transferred to the parasite at low than at high nitrate levels.     

Constraints on host use by a parasitic plant

Consumers do not always utilize all suitable hosts. Understanding why parasitic plants do not always parasitize potentially suitable hosts requires a better understanding of the constraints that limit host use by parasitic plants. In Texas salt marshes, the parasitic plant Cuscuta indecora rarely parasitizes three hosts that support vigorous growth in the greenhouse. We identified three constraints on host use by C. indecora. First, a mismatch between the phenology of C. indecora and some suitable hosts meant that these hosts were not abundant when C. indecora was growing most vigorously, and therefore were underutilized. Second, C. indecora preferentially parasitized tall plants versus short ones, causing relatively short species to be underutilized. Third, C. indecora overwinters in some perennial hosts but has to reinfect annual hosts each year, causing annuals and perennials that do not support overwintering to be underutilized. In combination, these constraints, which reflect the general lack of mobility of parasitic plants relative to herbivores, remove half of the potential host species from the actual diet of C. indecora, and therefore likely represent a major limitation on the success of this parasite. Similar constraints are likely to limit the realized host range of many parasitic plants and select for generalized diet preferences.     

Modelling the flow and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: I. Methods for estimating net flows

Nodulated Lupinus albus L. was grown on quartz sand in the greenhouseand supplied with a N-free culture solution. Half the plantswere infected with Cuscuta reflexa Roxb. at 33 DAS. An empiricallybased modelling technique was developed to quantitatively depictuptake, flow and utilization of C and N in the host plant andbetween host and parasite over a 12 d period. The modellingincorporated C: N ratios of solutes in phloem and pressure-inducedxylem sap, net increments of C and N and respiratory lossesof C. For assessing the transfer of solutes from host phloemto Cuscuta it was not possible to use the C: N ratio of phloemsap close to the site of parasite attachment, a procedure whichwould have assumed non-specific withdrawal of phloem-borne solutes,since this would have implied unimpeded mass flow from hostto parasite. The relative intake of C and N by the parasiteby specific withdrawal of nitrogenous and carbonaceous solutesfrom the phloem was obtained independently by assuming thatxylem intake occurred non-specifically. Xylem import was thusobtained (a) from transpiration and tissue water increment ofCuscuta and the concentrations of N and C in xylem sap and (b)from the Ca²⁺ increment of Cuscuta and the ratios Ca: N andCa: C in lupin xylem sap, assuming that Ca²⁺ intake occurredsolely via xylem. By subtracting net xylem import from totaluptake of C and N by Cuscuta the methods resulted in comparableratios of C: N intake from the phloem. The average ratio (53.4)was smaller than the C:N ratio in host phloem (85.6) indicatingspecific withdrawal of solutes with a distinct preference forN. Using this ratio, modelling of flows of C and N was possibleand showed that Cuscuta abstracted C and N mainly from the hostphloem, but xylem supply was nutrient-dependent and amountedto 6.4% of the N but only 0.5% of the C demand. The resultsindicated that Cuscuta exerted a very strong sink and competedefficiently with the root, the major sink of L. albus, by attracting81% of the current photosynthate and more N (223%) than wascurrently fixed. The massive demand of the parasite led to lossesparticularly of N from leaves and the root and apart from causingcarbon losses it appeared to induce a sink-dependent stimulationof photosynthesis. In contrast, nitrogen fixation in the Cuscuta-infectedlupin was inhibited to 37% of the control. Key words: Cuscuta reflexa, Lupinus albus, carbon, nitrogen, phloem, xylem, transport, parasites, modelling     

Sink-stimulated photosynthesis, increased transpiration and increased demand-dependent stimulation of nitrate uptake: nitrogen and carbon relations in the parasitic association Cuscuta reflexa-Coleus blumei

When parasitizing Coleus blumei Benth., grown in quartz sandculture and fed with 0.2, 1 or 5 mM nitrate, the biomass productionof Cuscuta reflexa Roxb. was inhibited to a similar extent asthat of the host supplied limiting concentrations of nitrate.In the presence of Cuscuta the growth and dry matter increaseof the host plant was severely inhibited. However, dry matterproduction of host plus parasite was only slightly less thanor at 0.2 mM nitrate almost the same as that of uninfected Coleusplants. Under all conditions of nitrate nutrition, parasitismby Cuscuta led to a substantial increase in photosynthesis inhost leaves under light-saturating conditions and in transpiration.Particularly with 0.2 mM and mM