- Email: [email protected]
Compatibility of Alectra vogelii with different leguminous host species
J.PlantPlrysiol. Vol. 135.pp. 737-745(1990)
Compatibility of Alectra vogelii with different leguminous host speCIes J. H. VISSER), INGE DORR *, and R. KOLLMANN 2
2
) Dept. of Botany, University of Stell en bosch, 7600 Stellenbosch, RSA Botanisches Institut, Universitat Kiel, D-2300 Kiel, BRD
2
Received July 24, 1989 . Accepted November 6, 1989
Summary The ability of the root parasite Alectra vogelii Benth. to attach to and penetrate two closely related nonhost species, Pisum sativum and Vicia faba, was light- and electron microscopically compared to the development of this parasite on one of its more common host species, Vigna unguiculata. Twelve days after infection, by transferring a germinating seed of the parasite directly onto the host roots, a firmly established parasite developed on V. unguiculata. In this compatible host, parasitic cells grew inter- and intracellularly up to the host's vascular tissue. With the other two less compatible host species, penetration generally stopped in the host cortex and was intercellularly, possibly due to an inability of the penetrating parasitic cells to digest the host cell walls. Encapsulation and subsequent starvation of the parasite resulted in its death soon afterwards.
Key words: Alectra vogelii, compatibility, host, parasite. Introduction Most angiospermous root parasites belonging to the Scrophulariaceae exhibit a well-defined, if not specific, host range {Visser, 1982). It has been well-documented that the fabacean host, Vigna unguiculata, is commonly attacked by Alectra vogelii throughout large parts of the African continent (Visser, 1978). Host specifity has, in some cases, been attributed to the presence of root exudates that may induce the germination of the seed of these parasites. Such compounds have been considered part of a mechanism by which the parasite recognises its host and their absence has been interpreted as a mechanism of resistance in the case of Striga asiatica (Williams, 1959; Maiti et aI., 1984). Plants producing compounds that promote the germination of the parasite's seed without being parasitised themselves have been termed false hosts. Such plants have been recommended as catch crops in order to decrease the population of parasite seed in the soil (Parker, 1965). >+ To whom correspondence and reprint requests should be addressed.
© 1990 by Gustav Fischer Verlag, Stuttgart
The presence of haustorial initiation factors, first recovered from gum tragacanth, has been claimed by Nickrent et al. (1979) to be implicated in the host specifity of Agalinis and Striga. Attachment as well as penetration is claimed by these authors to be indiscriminate of a host, providing haustorial hairs have been initiated; they demonstrated this for three hosts (Avena sativa, Sorghum vulgare and Zea mays) and five non-hosts (Glycine max., Gossypium hirsutum, Phaseolus vulgaris, Phaseolus limensis and Pisum sativum). The formation of a mechanical barrier by the deposition of lignified or otherwise impenetrable substances has been held responsible for the inability of Orobanche crenata (Panchenko & Antonova, 1974) and Striga asiatica (Saunders, 1933) to penetrate their respective hosts. Subsequently, Maiti et a1. (1984) presented evidence that mechanical resistance seems to be an important, but not necessarily the only, mechanism of resistance to S. astattca. Mechanical mechanisms have also been observed with the penetration of pathogenic fungi into their hosts (Hijwegen, 1963; Ride, 1975). Enzymatic degradation of cell walls would appear to be another prerequisite for the successful penetration of host tissue by an invading organism. With pathogenic microorganisms, inductively produced microbial enzymes are held
738
J. H. VISSEJI., INGE D<:iRR, and R. KOLLMANN
to be responsible for the degradation of host cell walls preceding penetration (Bateman and Basham, 1976). Inability to degrade cell walls has been attributed to either a lack of specific enzymes or some form of enzyme inactivation by phenolic substances and their oxidation products (Pati! and Dimond, 1967) or tannins and related materials (Bateman and Millar, 1966). In the case of parasitic angiosperms Kuijt (1969, 1983) suspected similar mechanisms, resulting in the formation of an isolation layer. Nagar et a!. (1984) could actually demonstrate differences in enzyme activity between susceptible and non-susceptible hosts. An important question that needs to be considered in the case of parasitic angiosperms is the resistance of the cell wall of the pathogen to its own enzyme attack. In reaction to the penetration of Cuscuta lupuliformis Krock into the incompatible Gossypium hirsutum L., Capdepon et a!. (1985) described the formation of a layer of suberized cells acting as a defensive system against the penetrating haustorial cells. These authors compared this resistance to the process of abscission rather than to a wound reaction and suspected a phytohormonal stimulus emanating from the host tissue. Subsequently, Wellenkamp (1986) and Kollmann and Dorr (1987) demonstrated a defensive reaction in Hibiscus rosa-sinensis attacked by Cuscuta odorata. Resistance due to induced immunity to penetrating fungi by the production of phytoalexins is a well-documented phenomenon (Kuc, 1976) that may also be implicated in resistance mechanisms against invading angiospermous parasites, although no evidence exists as yet. The first visible sign of the successful penetration of A. vogelii into its host V. unguiculata is accompanied by the development of lateral roots, as previously documented and described by Visser et a!. (1977). This phenomenon has been interpreted as an induced enlargement of the adsorption surface of the host whereby the parasite stands to benefit the most. The present investigation was undertaken to establish the ability of some taxonomically related fabacean non-host species, namely Pisum sativum and Vicia Jaba, to serve as hosts for A. vogelii in order to gain an understanding of the mechanisms involved in the compatibility of root parasites with their hosts. P. sativum and V. Jaba were selected as alternative hosts for A. vogelii because they are taxonomically closely related to the reported host, V. unguiculata. P. sativum and V. Jaba have never been reported as hosts for A . vogelii, and quite likely, have never been exposed to this parasite in their evolution, as they are of temperate origin in contrast to the subtropical and tropical origin of V. unguiculata. Usually they are not cultivated during the time of the year that A. vogelii encounters optimal temperatures for its development. In this paper, incompatibility between host and parasite is considered the ability of the host to withstand both the penetration of the parasite into its tissue and subsequent establishment of a permanent connection between the vascular systems of host and parasite.
Materials and Methods One of the most common hosts, Vigna unguiculata Walp. cv. Saunders Upright and two closely related, but apparently non-sus-
ceptible species, namely Pisum sativum L. cv. Kleine RheinHinderin and Viciafaba L. cv. Staygreen, were cultivated at 28°C in a growth room in boxes constructed in such a way as to facilitate the observation of the root system (Visser et aI., 1984). When the host plants reached an age of 2 - 3 weeks their roots were well-developed and well-suited for infection with parasite seeds. Seed of A. vogelii growing on V. unguiculata was harvested during May 1987 from infected fields at Mkuzi Farm, White River, South Africa, cleaned and stored at room temperature in closed glass containers until required. Two weeks before infecting the host plants a small amount of seed was placed on moistened filter paper, sealed in a petri dish and conditioned at 28°C. One day before infecting the roots of the host plants a few drops of 1 ppm solution of strigol analogue GR7 (Visser and Johnson, 1982) were added to the conditioned seed (which finally resulted in a germination percentage in excess of 90%). On the day of infection the stimulated, but still ungerminated seed, was placed on the host roots at the desired position. After regular inspections the developing haustoria were selected at the appropriate stage, dissected out, fixed in paraformaldehyde/glutaraldehyde, followed by osmium tetroxide, stained with uranyl acetate and then dehydrated in an ascending series of ethyl alcohol and finally embedded in Spurr's resin. For light microscopy 0.5 -1 JLm sections were taken, stained with crystal violet and photographed with a Zeiss Photomikroskop II. Scanning electron microscopy was done with the aid of a Leitz AMR 1000 after the preparations had been critical point-dried and then gold-coated. Transmission electron microscopy was carried out on a Siemens Elmiskop 10l. Germination tests were carried out to test for the presence of stimulants of A. vogelii seed by exposing conditioned seed to the solution in which the different host plants had been growing, according to Visser (1975).
Results and Discussion Within 12 days after the germinating seed had been placed in contact with the root of the compatible host, Vigna unguiculata, almost 100 % of the seedlings of Alectra vogelii penetrated and induced lateral root development of the host root (Plate I A). At this age the plumule might still be contained in the seed, but might also already have broken out of the testa following differentiation of the first leaves (Plate IB). This development would appear to signify a functional attachment and uninterrupted flow of nutrients from the host into the parasite. Thickening of the hypocotyl just above the point of entry into the host root is another sign of the successful attachment. In section (Plate IB), the thickened hypocotyl is seen to contain the hyaline body, which has been described previously for Alectra orobanchoides (Visser et al., 1984). At this stage strands of parasite cells ramify throughout the very much thickened host root and single cells have already taken up contact with the host xylem. With Pisum sativum and Vicia Jaba at the point of contact between the radicle tip of the parasite seedling and the host's roots, a reddish-brown discolouration and eventual necrosis of the host epidermal cells resulted. Only about five percent of the seedlings developing on the roots of these hosts did manage to take up contact with the host's conducting elements, in which case, however, a very much retarded development of the parasite was observed. It is significant that apparently once contact with the host conducting system could be established, the parasite could continue its develop-
Compatibility of Alectra with hosts
739
B
, )f
.,),1
O,1mm Plate I: The development of Alectra vogelii on the compatible host Vigna unguiculata, 12 days after infection. A: Successful penetration is characterised by the development of lateral host roots (LR) at the point of infection. SC: Seed Coat, HY: Hypocotyl, HR: Host Root. B: Structural features reveal the intimate contact between main host root (HR) and parasite tissue. Plumule development (PL) has started and the hyaline body (HB) has already differentiated. HX: Host Xylem Vessels, HY: Hypocotyl, LR: Lateral Host Root.
ment. These results are in good accordance to those of Jacob and Ihl (1987) and Ihl et al. (1988), who localized the reasons for incompatibility between Cuscuta re/lexa and Lycopersicon esculentum in the peripheral cell layers of the host. By par-
tially removing the cortex, a formation of functional haustoria followed by normal Cuscuta growth was possible. Pisum and Vida produce germination stimulants for A. vogelii as established in germination tests with conditioned
740
J. H . VISSER, INGE DORR, and R. KOLLMANN
Plate II: The development of Alectra vogelii on Pisum sativum (A, B) and Vicia /aba (C, D) 12 days after infection. Haustoria! hairs develop at the seedling parasite's root tip (A). Penetration into host tissue is restricted to the cortica! cell layers of the host Pisum (B), respectively, superficially on Vicia (D). The plumule (PL) is scarcely developed and still enclosed by the seed coat. HR: Host Root, HY: Hypocotyl.
seed {results not shown}. With the experimental technique used in this investigation conditions were perhaps much more optimal than would be encountered under natural conditions. Attachment to and penetration into the roots of P. sativum and V. faba is portrayed in Plate II. Haustorial hairs, in the sense of Riopel and Baird {1987}, are a prominent feature of the attachment of A. vogelii to both these hosts (Plate II A, C). This is the first time that these structures are reported for this particular root parasite. Whether haustorial hairs are in-
volved in the attachment to the compatible V. unguiculata is not clear as they have so far neither been observed nor could their formation be induced by substances such as xenognosin that result in their formation in Agalinis purpurea and Striga asiatica {Riopel and Baird, 1987}. At this stage, 12 days after infection of the non-hosts, almost no differentiation of the plumule has taken place and the endosperm and other storage tissue of the seed are completely depleted of reserve material.
Compatibility of Alectra with hosts
741
He ~~'.:"." " -"'"
Plate III: Structural features of penetration into the compatible Vigna unguiculata (A, B), the resistant Pisum sativum (C, D) and Vicia /aba (E, F). A: Inter- and intracellular penetration of susceptible host (HC) by plasma dense parasite cells (PC) 5 days after infection. B: Lateral host root development (LR) and disruption of host xylem continuity (HX) by parasitic cells (PC) 9 days after infection. C: Merely intercellular development of parasite tissue (PC) takes place within the incompatible host root as a result of the apparent inability to digest the walls of the host cells (HC). D: Cell death of the parasite (PC) 12 days after infection. E: Increased cell division in the host cortex in front of an attempted penetration by the parasite (PC). F: Encapsulation (arrow) of penetrating parasite cells (PC) effectively isolate them from the host tissue.
Penetration of the parasite into the tissue of the P. sativum host seems to progress to a limited extent only, almost reaching the stele (Plate II B). The penetrating cells of the parasite characteristically have a less dense cytoplasm. On V. /aba, penetration is restricted to the superficial cell layers, with a
pronounced densely staining isolation layer around the invading cells of the parasite (Plate II D). The mechanism of penetration into the compatible host, V. unguiculata, and the induction of lateral roots are depicted in Plate III A and B where intracellular invasion of host cells
742
]. H. VISSER, INGE DORR, and R. KOLLMANN
Plate IV: Ultrastructural features of penetration into the compatible Vigna unguiculata (A), and resistant Pisum sativum (B) and Vicia faba (C). A: An extremely plasma dense penetrating front cell of Alectra vogelii (PC) grows intracellularly within a host cortex cell (HC). A conspicuous feature of the compatible system is the high density of cellular organelles including mitochondria, microbodies and a large nucleus (NU). B: In the more resistant host tissue (HC) of Pisum sativum the cytoplasm of the advancing cell of the parasite (PC) becomes depleted of cellular organelles although partial dissolution of host cell wall material is still apparent in places (arrow). C: In the even more resistant host tissue (HC) of Vicia faba the parasite cells (PC) become encapsulated, possibly due to the inability to break down wall material of the host and deposition of electron dense material. Almost complete depletion of organelles of the cytoplasm of the parasite cells is characteristic.
Compatibility of Alectra with hosts
743
8
)
I
c
5.um Plate v: Ultrastructural features of penetration into the compatible Vigna unguiculata (A), and resistant Pisum sativum (B) and Viaa /aha (C). A: Within the compatible system the high structural density of the parasitic cell (PC) is striking. While the A/cctra cell wall stays unaffected the cell wall of the host (HC) is broken down in several places (arrows). B: The almost intact host cell wall (HW) in the more resistant Pisum sativum (HC) restricts the advance of the parasite (PC). Cellular content of the parasite cell decreases. C: The parasite cell (PC) becomes completely isolated from the host (HC) by the deposition of an as yet unknown electron dense material. The protoplast becomes disintegrated very soon.
744
J. H. VISSER, INGE DORR, and R. KOLLMANN
as well as the intercellular growth of the penetrating parasite cells with dense plasma are discernable 5 days after infection. Very soon after penetration has commenced lateral root development is induced, as manifested in areas of increased cell division after 9 days (Plate III B). Penetration in P. sativum would appear to be mainly restricted to intercellular growth in the cortical layers of the root. In this process superficial layers expand outwards and compression of deeper layers of host cells results as the parasite cells enlarge (Plate III C). The cytoplasm of the parasite cells also appears to be significantly less dense than is the case in V. unguiculata. Subsequent lysis of the invading parasite results, as portrayed in Plate III D. Increased host cell division in front of the penetrating parasite cells has been observed in the case of invasion of V. /aba roots, as evidenced in Plate III E. Encapsulation of the invading pathogen by the host is a pronounced feature with this host (Plate III F), where parasite cell division appears to continue for some time. Here, too, indication for the compression of indigestible host cell wall and deposition of densely staining material is apparent. It can thus be concluded that the parasite cells invading P. sativum and V. /aba roots encounter some degree of resistance to penetration, possibly due to an inability to break down host cell walls. Breakdown of the middle lamella would not seem to be much of an obstacle, as can be deduced from the extensive intercellular development, especially in the case of P. sativum. At the subcellular level convincing confirmation could be found for the preceding light microscopical observations (Plate IV A). An enlarged nucleus and high cytoplasmic density as a result of abundant ribosomes, large numbers of mitochondria, Golgi apparatus, rough endoplasmic reticulum and the presence of microbodies all confirm high metabolic activity in the front cells of the parasite invading the compatible V. unguiculata. When invading P. sativum (Plate IV B) the parasite cells are less dense in cytoplasm with significantly fewer cell organelles, indicating decreased metabolic activity. There is some evidence for the partial digestion of the host cell wall in the case of P. sativum (Plate IVB). Encapsulation and the subsequent starvation of the penetrating parasite cells invading V. [aba is portrayed in Plate IV C, where evidence for the presence of undigested wall is also to be found. As the plasma dense front cells of the parasite advance intercellularly through the cortex of the compatible host, V. unguiculata, the host cell wall is gradually broken down (Plate V A). The mechanism of breakdown is still largely obscure, but enzymatic involvement appears to play a major role. The host cell wall appears to be broken down at various points, which facilitates the penetration into the neighbouring host cell. Plate VB very clearly shows the relatively intact nature of the cell wall of the partially incompatible P. sativum. The integrity of both walls demonstrates the difficulty that the parasite encounters in breaking down host cell walls. Encapsulation of the foreign parasitic cell in the case of V. /aba (Plate V C) results in a densely staining deposit of unknown chemical nature around the foreign parasitic cell. In this way the penetrating cells are effectively isolated from the
potential host and for survival are only dependent on what little own reserves the parasite seed carries. This, in turn, results in lysis and subsequent death of the parasite.
Conclusions Certainly the major fact that emerges from this investigation is the realisation of different grades of susceptibility of potential host plants to penetration by the root parasite, A. vogelii. The susceptible host, represented by V. unguiculata, is invaded by the parasite without any appreciable resistance. On the other hand, two hosts have been identified that resist the invasion of foreign cells to varying degrees, from the inability of host cell wall degradation to encapsulation. The mechanism of this resistance would thus appear to be based on chemical/biochemical principles and could indicate future ways of introducing resistance into susceptible hosts. Acknowledgements Financial support to jHV by the Foundation for Research and Development is gratefully acknowledged. The help of Brigitte Friedrich with the scanning electron microscopy and Margarethe Rasch for invaluable assistance is greatly appreciated.
References BATEMAN, D. F. and H. G. BASHAM: Degradation of plant cell walls and membranes by microbial enzymes. In: PIRSON, A. and M. ZIMMERMANN (eds.): Encyclopedia of Plant Physiology, Vol. 4 (eds. R. HEITEFUSS and P. H. WILLIAMS), 316-355, Berlin, Heidelberg, New York, Springer (1976). BATEMAN, D. F. and R. L. MILLAR: Pectic enzymes in tissue degradation. Ann. Rev. Phytopath. 4, 119-146 (1966). CAPDEPON, M., A. FER, and P. OZENDA: Sur un systeme inedit de rejet d'un parasite: exemple de la Cuscute sur Cotonnier (C lupuIi/ormis Krock. sur Gossypium hirsutum L.). C.R. Acad. Sc. Paris 300,227 -232 (1985).
HI]WEGEN, T.: Lignification, a possible mechanism of native resistance against pathogens. Neth. J. Plant Pathology 69, 314-317 (1963).
IHL, B., N. TUTAKHIL, A. HAGEN, and F. JACOB: Studien an Cuscuta reflexa Roxb. VII. Zum Abwehrmechanismus von Lycopersicon esculentum Mill. Flora 181, 383 -393 (1988). JACOB, F. and B. IHL: Specific interaction between Cuscuta and its hosts. In: GREUTER, W., B. ZIMMER, and H.-D. BEHNKE (eds.): XIV. International Botanical Congress-Abstracts, p. 370, Berlin (1987).
KOLLMANN, R. and I. DORR: Parasitische Bliitenpflanzen. Naturwissenschaften 74, 12-21 (1987). Kuc, J. A.: Phytoalexins. In: PIRSON, A. and M. ZIMMERMANN (eds.): Encyclopedia of Plant Physiology, Vol. 4 (eds. R. HEITEFUSS and P. H. WILLIAMS), 632-652, Berlin, Heidelberg, New York, Springer (1976). KUI]T, J.: The biology of parasitic flowering plants. Univ. of California Press (1969). - Tissue compatibility and the haustoria of parasitic angiosperms. In: MOORE, R. (ed.): Vegetative compatibility in plants, pp. 1-12, Baylor University Press, Waco, Texas 76798 (1983).
Compatibility of Alectra with hosts
MAm, R. K., K. V. RAMAlAH, S. S. BISEN, and V. L. CHIDLEY: A comparative study of the haustorial development of Striga asiatica (L.) Kuntze on Sorghum cultivars. Ann. Bot. 54, 447 -457 (1984). NAGAR, R. M., M. SINGH, and G. G. SANWAL: Cell wall degrading enzymes in Cuscuta reflexa and its hosts. J. Expt. Bot. 35, 1104-1112 (1984). NICKRENT, D . L., L. J. MUSSELMAN, J. L. RIoPEL, and R. E. EPLEE: Haustorial initiation and non-host penetration in witchweed (Striga asiatica). Ann. Bot. 43, 233-236 (1979). PARKER, The Striga problem - a review. PANS 11, 99-111 (1965). PANCHENKO, A. Y. and T. S. ANTONOVA: Characteristics of the protective reaction of varieties of sunflower to penetration by broomrape. Seliskokhyosaisvennaya biologya 94, 554 - 558 (1974). PAUL, S. S. and A. E. DIMOND: Depression of polygalacturonase by oxidation products of polyphenols. Phytopath. 57, 492 - 496 (1967). RIDE, R. P.: Lignification in wounded wheat leaves in response to fungi and its possible role in resistance. Physiol. Plant Pathology 5, 124-134 (1975). RIOPEL, J. L. and W. V. BAIRD: Morphogenesis of the early development of primary haustoria in Striga asiatica. In: MUSSELMAN, L. J. (ed.): Parasitic weeds in agriculture. Vol. 1. Striga. CRC Press, Boca Raton, Florida, U.S.A. (1987).
c.:
745
SAUNDERS, A. R.: Studies in phanerogamic parasitism with particular reference to Striga lutea Lour. South African Dept. Agr. Bulletin 128 (1933). VISSER, J. H.: Germination stimulants of Alectra vogelii Benth. seed. Z . Pflanzenphysiol. 74, 464-469 (1975). - The biology of Alectra vogelii Benth., an angiospermous root I?arasite. Beitdige zur chemischen Kommunikation in Bio- and Okosystemen, pp. 279-294, Witzenhausen (1978). - South African parasitic flowering plants. Juta & Co. Cape Town (1982). VISSER, J. H., 1. DORR, and R. KOLLMANN: On the parasitism of Alectra vogelii Benth. (Scrophulariaceae). 1. Early development of the primary haustorium and initiation of the stem. Z . pflanzenphysiol. 84, 213-222 (1977). - - - The «hyaline body~ of the root parasite Alectra oroban· choides Benth. (Scrophulariaceae) - its anatomy, ultrastructure and histochemistry. Protoplasma 121, 146-156 (1984). VISSER, J. H. and A. JOHNSON: The effect of certain strigol analogues on the seed germination of Alectra. S. Afr. J. Bot. 1, 75-76 (1982). WELLENKAMP, 5.: Strukturelle Grundlagen der Kompatibilitat - Inkompatibilitat bei interspezifischen Zellkontakten. Diplomarbeit, Botanisches Institut der Universitat, Kiel (1986). WILLIAMS, C. N.: Resistance of Sorghum to witchweed. Nature 184, 1511 (1959).
Compatibility of Alectra vogelii with different leguminous host speCIes J. H. VISSER), INGE DORR *, and R. KOLLMANN 2
2
) Dept. of Botany, University of Stell en bosch, 7600 Stellenbosch, RSA Botanisches Institut, Universitat Kiel, D-2300 Kiel, BRD
2
Received July 24, 1989 . Accepted November 6, 1989
Summary The ability of the root parasite Alectra vogelii Benth. to attach to and penetrate two closely related nonhost species, Pisum sativum and Vicia faba, was light- and electron microscopically compared to the development of this parasite on one of its more common host species, Vigna unguiculata. Twelve days after infection, by transferring a germinating seed of the parasite directly onto the host roots, a firmly established parasite developed on V. unguiculata. In this compatible host, parasitic cells grew inter- and intracellularly up to the host's vascular tissue. With the other two less compatible host species, penetration generally stopped in the host cortex and was intercellularly, possibly due to an inability of the penetrating parasitic cells to digest the host cell walls. Encapsulation and subsequent starvation of the parasite resulted in its death soon afterwards.
Key words: Alectra vogelii, compatibility, host, parasite. Introduction Most angiospermous root parasites belonging to the Scrophulariaceae exhibit a well-defined, if not specific, host range {Visser, 1982). It has been well-documented that the fabacean host, Vigna unguiculata, is commonly attacked by Alectra vogelii throughout large parts of the African continent (Visser, 1978). Host specifity has, in some cases, been attributed to the presence of root exudates that may induce the germination of the seed of these parasites. Such compounds have been considered part of a mechanism by which the parasite recognises its host and their absence has been interpreted as a mechanism of resistance in the case of Striga asiatica (Williams, 1959; Maiti et aI., 1984). Plants producing compounds that promote the germination of the parasite's seed without being parasitised themselves have been termed false hosts. Such plants have been recommended as catch crops in order to decrease the population of parasite seed in the soil (Parker, 1965). >+ To whom correspondence and reprint requests should be addressed.
© 1990 by Gustav Fischer Verlag, Stuttgart
The presence of haustorial initiation factors, first recovered from gum tragacanth, has been claimed by Nickrent et al. (1979) to be implicated in the host specifity of Agalinis and Striga. Attachment as well as penetration is claimed by these authors to be indiscriminate of a host, providing haustorial hairs have been initiated; they demonstrated this for three hosts (Avena sativa, Sorghum vulgare and Zea mays) and five non-hosts (Glycine max., Gossypium hirsutum, Phaseolus vulgaris, Phaseolus limensis and Pisum sativum). The formation of a mechanical barrier by the deposition of lignified or otherwise impenetrable substances has been held responsible for the inability of Orobanche crenata (Panchenko & Antonova, 1974) and Striga asiatica (Saunders, 1933) to penetrate their respective hosts. Subsequently, Maiti et a1. (1984) presented evidence that mechanical resistance seems to be an important, but not necessarily the only, mechanism of resistance to S. astattca. Mechanical mechanisms have also been observed with the penetration of pathogenic fungi into their hosts (Hijwegen, 1963; Ride, 1975). Enzymatic degradation of cell walls would appear to be another prerequisite for the successful penetration of host tissue by an invading organism. With pathogenic microorganisms, inductively produced microbial enzymes are held
738
J. H. VISSEJI., INGE D<:iRR, and R. KOLLMANN
to be responsible for the degradation of host cell walls preceding penetration (Bateman and Basham, 1976). Inability to degrade cell walls has been attributed to either a lack of specific enzymes or some form of enzyme inactivation by phenolic substances and their oxidation products (Pati! and Dimond, 1967) or tannins and related materials (Bateman and Millar, 1966). In the case of parasitic angiosperms Kuijt (1969, 1983) suspected similar mechanisms, resulting in the formation of an isolation layer. Nagar et a!. (1984) could actually demonstrate differences in enzyme activity between susceptible and non-susceptible hosts. An important question that needs to be considered in the case of parasitic angiosperms is the resistance of the cell wall of the pathogen to its own enzyme attack. In reaction to the penetration of Cuscuta lupuliformis Krock into the incompatible Gossypium hirsutum L., Capdepon et a!. (1985) described the formation of a layer of suberized cells acting as a defensive system against the penetrating haustorial cells. These authors compared this resistance to the process of abscission rather than to a wound reaction and suspected a phytohormonal stimulus emanating from the host tissue. Subsequently, Wellenkamp (1986) and Kollmann and Dorr (1987) demonstrated a defensive reaction in Hibiscus rosa-sinensis attacked by Cuscuta odorata. Resistance due to induced immunity to penetrating fungi by the production of phytoalexins is a well-documented phenomenon (Kuc, 1976) that may also be implicated in resistance mechanisms against invading angiospermous parasites, although no evidence exists as yet. The first visible sign of the successful penetration of A. vogelii into its host V. unguiculata is accompanied by the development of lateral roots, as previously documented and described by Visser et a!. (1977). This phenomenon has been interpreted as an induced enlargement of the adsorption surface of the host whereby the parasite stands to benefit the most. The present investigation was undertaken to establish the ability of some taxonomically related fabacean non-host species, namely Pisum sativum and Vicia Jaba, to serve as hosts for A. vogelii in order to gain an understanding of the mechanisms involved in the compatibility of root parasites with their hosts. P. sativum and V. Jaba were selected as alternative hosts for A. vogelii because they are taxonomically closely related to the reported host, V. unguiculata. P. sativum and V. Jaba have never been reported as hosts for A . vogelii, and quite likely, have never been exposed to this parasite in their evolution, as they are of temperate origin in contrast to the subtropical and tropical origin of V. unguiculata. Usually they are not cultivated during the time of the year that A. vogelii encounters optimal temperatures for its development. In this paper, incompatibility between host and parasite is considered the ability of the host to withstand both the penetration of the parasite into its tissue and subsequent establishment of a permanent connection between the vascular systems of host and parasite.
Materials and Methods One of the most common hosts, Vigna unguiculata Walp. cv. Saunders Upright and two closely related, but apparently non-sus-
ceptible species, namely Pisum sativum L. cv. Kleine RheinHinderin and Viciafaba L. cv. Staygreen, were cultivated at 28°C in a growth room in boxes constructed in such a way as to facilitate the observation of the root system (Visser et aI., 1984). When the host plants reached an age of 2 - 3 weeks their roots were well-developed and well-suited for infection with parasite seeds. Seed of A. vogelii growing on V. unguiculata was harvested during May 1987 from infected fields at Mkuzi Farm, White River, South Africa, cleaned and stored at room temperature in closed glass containers until required. Two weeks before infecting the host plants a small amount of seed was placed on moistened filter paper, sealed in a petri dish and conditioned at 28°C. One day before infecting the roots of the host plants a few drops of 1 ppm solution of strigol analogue GR7 (Visser and Johnson, 1982) were added to the conditioned seed (which finally resulted in a germination percentage in excess of 90%). On the day of infection the stimulated, but still ungerminated seed, was placed on the host roots at the desired position. After regular inspections the developing haustoria were selected at the appropriate stage, dissected out, fixed in paraformaldehyde/glutaraldehyde, followed by osmium tetroxide, stained with uranyl acetate and then dehydrated in an ascending series of ethyl alcohol and finally embedded in Spurr's resin. For light microscopy 0.5 -1 JLm sections were taken, stained with crystal violet and photographed with a Zeiss Photomikroskop II. Scanning electron microscopy was done with the aid of a Leitz AMR 1000 after the preparations had been critical point-dried and then gold-coated. Transmission electron microscopy was carried out on a Siemens Elmiskop 10l. Germination tests were carried out to test for the presence of stimulants of A. vogelii seed by exposing conditioned seed to the solution in which the different host plants had been growing, according to Visser (1975).
Results and Discussion Within 12 days after the germinating seed had been placed in contact with the root of the compatible host, Vigna unguiculata, almost 100 % of the seedlings of Alectra vogelii penetrated and induced lateral root development of the host root (Plate I A). At this age the plumule might still be contained in the seed, but might also already have broken out of the testa following differentiation of the first leaves (Plate IB). This development would appear to signify a functional attachment and uninterrupted flow of nutrients from the host into the parasite. Thickening of the hypocotyl just above the point of entry into the host root is another sign of the successful attachment. In section (Plate IB), the thickened hypocotyl is seen to contain the hyaline body, which has been described previously for Alectra orobanchoides (Visser et al., 1984). At this stage strands of parasite cells ramify throughout the very much thickened host root and single cells have already taken up contact with the host xylem. With Pisum sativum and Vicia Jaba at the point of contact between the radicle tip of the parasite seedling and the host's roots, a reddish-brown discolouration and eventual necrosis of the host epidermal cells resulted. Only about five percent of the seedlings developing on the roots of these hosts did manage to take up contact with the host's conducting elements, in which case, however, a very much retarded development of the parasite was observed. It is significant that apparently once contact with the host conducting system could be established, the parasite could continue its develop-
Compatibility of Alectra with hosts
739
B
, )f
.,),1
O,1mm Plate I: The development of Alectra vogelii on the compatible host Vigna unguiculata, 12 days after infection. A: Successful penetration is characterised by the development of lateral host roots (LR) at the point of infection. SC: Seed Coat, HY: Hypocotyl, HR: Host Root. B: Structural features reveal the intimate contact between main host root (HR) and parasite tissue. Plumule development (PL) has started and the hyaline body (HB) has already differentiated. HX: Host Xylem Vessels, HY: Hypocotyl, LR: Lateral Host Root.
ment. These results are in good accordance to those of Jacob and Ihl (1987) and Ihl et al. (1988), who localized the reasons for incompatibility between Cuscuta re/lexa and Lycopersicon esculentum in the peripheral cell layers of the host. By par-
tially removing the cortex, a formation of functional haustoria followed by normal Cuscuta growth was possible. Pisum and Vida produce germination stimulants for A. vogelii as established in germination tests with conditioned
740
J. H . VISSER, INGE DORR, and R. KOLLMANN
Plate II: The development of Alectra vogelii on Pisum sativum (A, B) and Vicia /aba (C, D) 12 days after infection. Haustoria! hairs develop at the seedling parasite's root tip (A). Penetration into host tissue is restricted to the cortica! cell layers of the host Pisum (B), respectively, superficially on Vicia (D). The plumule (PL) is scarcely developed and still enclosed by the seed coat. HR: Host Root, HY: Hypocotyl.
seed {results not shown}. With the experimental technique used in this investigation conditions were perhaps much more optimal than would be encountered under natural conditions. Attachment to and penetration into the roots of P. sativum and V. faba is portrayed in Plate II. Haustorial hairs, in the sense of Riopel and Baird {1987}, are a prominent feature of the attachment of A. vogelii to both these hosts (Plate II A, C). This is the first time that these structures are reported for this particular root parasite. Whether haustorial hairs are in-
volved in the attachment to the compatible V. unguiculata is not clear as they have so far neither been observed nor could their formation be induced by substances such as xenognosin that result in their formation in Agalinis purpurea and Striga asiatica {Riopel and Baird, 1987}. At this stage, 12 days after infection of the non-hosts, almost no differentiation of the plumule has taken place and the endosperm and other storage tissue of the seed are completely depleted of reserve material.
Compatibility of Alectra with hosts
741
He ~~'.:"." " -"'"
Plate III: Structural features of penetration into the compatible Vigna unguiculata (A, B), the resistant Pisum sativum (C, D) and Vicia /aba (E, F). A: Inter- and intracellular penetration of susceptible host (HC) by plasma dense parasite cells (PC) 5 days after infection. B: Lateral host root development (LR) and disruption of host xylem continuity (HX) by parasitic cells (PC) 9 days after infection. C: Merely intercellular development of parasite tissue (PC) takes place within the incompatible host root as a result of the apparent inability to digest the walls of the host cells (HC). D: Cell death of the parasite (PC) 12 days after infection. E: Increased cell division in the host cortex in front of an attempted penetration by the parasite (PC). F: Encapsulation (arrow) of penetrating parasite cells (PC) effectively isolate them from the host tissue.
Penetration of the parasite into the tissue of the P. sativum host seems to progress to a limited extent only, almost reaching the stele (Plate II B). The penetrating cells of the parasite characteristically have a less dense cytoplasm. On V. /aba, penetration is restricted to the superficial cell layers, with a
pronounced densely staining isolation layer around the invading cells of the parasite (Plate II D). The mechanism of penetration into the compatible host, V. unguiculata, and the induction of lateral roots are depicted in Plate III A and B where intracellular invasion of host cells
742
]. H. VISSER, INGE DORR, and R. KOLLMANN
Plate IV: Ultrastructural features of penetration into the compatible Vigna unguiculata (A), and resistant Pisum sativum (B) and Vicia faba (C). A: An extremely plasma dense penetrating front cell of Alectra vogelii (PC) grows intracellularly within a host cortex cell (HC). A conspicuous feature of the compatible system is the high density of cellular organelles including mitochondria, microbodies and a large nucleus (NU). B: In the more resistant host tissue (HC) of Pisum sativum the cytoplasm of the advancing cell of the parasite (PC) becomes depleted of cellular organelles although partial dissolution of host cell wall material is still apparent in places (arrow). C: In the even more resistant host tissue (HC) of Vicia faba the parasite cells (PC) become encapsulated, possibly due to the inability to break down wall material of the host and deposition of electron dense material. Almost complete depletion of organelles of the cytoplasm of the parasite cells is characteristic.
Compatibility of Alectra with hosts
743
8
)
I
c
5.um Plate v: Ultrastructural features of penetration into the compatible Vigna unguiculata (A), and resistant Pisum sativum (B) and Viaa /aha (C). A: Within the compatible system the high structural density of the parasitic cell (PC) is striking. While the A/cctra cell wall stays unaffected the cell wall of the host (HC) is broken down in several places (arrows). B: The almost intact host cell wall (HW) in the more resistant Pisum sativum (HC) restricts the advance of the parasite (PC). Cellular content of the parasite cell decreases. C: The parasite cell (PC) becomes completely isolated from the host (HC) by the deposition of an as yet unknown electron dense material. The protoplast becomes disintegrated very soon.
744
J. H. VISSER, INGE DORR, and R. KOLLMANN
as well as the intercellular growth of the penetrating parasite cells with dense plasma are discernable 5 days after infection. Very soon after penetration has commenced lateral root development is induced, as manifested in areas of increased cell division after 9 days (Plate III B). Penetration in P. sativum would appear to be mainly restricted to intercellular growth in the cortical layers of the root. In this process superficial layers expand outwards and compression of deeper layers of host cells results as the parasite cells enlarge (Plate III C). The cytoplasm of the parasite cells also appears to be significantly less dense than is the case in V. unguiculata. Subsequent lysis of the invading parasite results, as portrayed in Plate III D. Increased host cell division in front of the penetrating parasite cells has been observed in the case of invasion of V. /aba roots, as evidenced in Plate III E. Encapsulation of the invading pathogen by the host is a pronounced feature with this host (Plate III F), where parasite cell division appears to continue for some time. Here, too, indication for the compression of indigestible host cell wall and deposition of densely staining material is apparent. It can thus be concluded that the parasite cells invading P. sativum and V. /aba roots encounter some degree of resistance to penetration, possibly due to an inability to break down host cell walls. Breakdown of the middle lamella would not seem to be much of an obstacle, as can be deduced from the extensive intercellular development, especially in the case of P. sativum. At the subcellular level convincing confirmation could be found for the preceding light microscopical observations (Plate IV A). An enlarged nucleus and high cytoplasmic density as a result of abundant ribosomes, large numbers of mitochondria, Golgi apparatus, rough endoplasmic reticulum and the presence of microbodies all confirm high metabolic activity in the front cells of the parasite invading the compatible V. unguiculata. When invading P. sativum (Plate IV B) the parasite cells are less dense in cytoplasm with significantly fewer cell organelles, indicating decreased metabolic activity. There is some evidence for the partial digestion of the host cell wall in the case of P. sativum (Plate IVB). Encapsulation and the subsequent starvation of the penetrating parasite cells invading V. [aba is portrayed in Plate IV C, where evidence for the presence of undigested wall is also to be found. As the plasma dense front cells of the parasite advance intercellularly through the cortex of the compatible host, V. unguiculata, the host cell wall is gradually broken down (Plate V A). The mechanism of breakdown is still largely obscure, but enzymatic involvement appears to play a major role. The host cell wall appears to be broken down at various points, which facilitates the penetration into the neighbouring host cell. Plate VB very clearly shows the relatively intact nature of the cell wall of the partially incompatible P. sativum. The integrity of both walls demonstrates the difficulty that the parasite encounters in breaking down host cell walls. Encapsulation of the foreign parasitic cell in the case of V. /aba (Plate V C) results in a densely staining deposit of unknown chemical nature around the foreign parasitic cell. In this way the penetrating cells are effectively isolated from the
potential host and for survival are only dependent on what little own reserves the parasite seed carries. This, in turn, results in lysis and subsequent death of the parasite.
Conclusions Certainly the major fact that emerges from this investigation is the realisation of different grades of susceptibility of potential host plants to penetration by the root parasite, A. vogelii. The susceptible host, represented by V. unguiculata, is invaded by the parasite without any appreciable resistance. On the other hand, two hosts have been identified that resist the invasion of foreign cells to varying degrees, from the inability of host cell wall degradation to encapsulation. The mechanism of this resistance would thus appear to be based on chemical/biochemical principles and could indicate future ways of introducing resistance into susceptible hosts. Acknowledgements Financial support to jHV by the Foundation for Research and Development is gratefully acknowledged. The help of Brigitte Friedrich with the scanning electron microscopy and Margarethe Rasch for invaluable assistance is greatly appreciated.
References BATEMAN, D. F. and H. G. BASHAM: Degradation of plant cell walls and membranes by microbial enzymes. In: PIRSON, A. and M. ZIMMERMANN (eds.): Encyclopedia of Plant Physiology, Vol. 4 (eds. R. HEITEFUSS and P. H. WILLIAMS), 316-355, Berlin, Heidelberg, New York, Springer (1976). BATEMAN, D. F. and R. L. MILLAR: Pectic enzymes in tissue degradation. Ann. Rev. Phytopath. 4, 119-146 (1966). CAPDEPON, M., A. FER, and P. OZENDA: Sur un systeme inedit de rejet d'un parasite: exemple de la Cuscute sur Cotonnier (C lupuIi/ormis Krock. sur Gossypium hirsutum L.). C.R. Acad. Sc. Paris 300,227 -232 (1985).
HI]WEGEN, T.: Lignification, a possible mechanism of native resistance against pathogens. Neth. J. Plant Pathology 69, 314-317 (1963).
IHL, B., N. TUTAKHIL, A. HAGEN, and F. JACOB: Studien an Cuscuta reflexa Roxb. VII. Zum Abwehrmechanismus von Lycopersicon esculentum Mill. Flora 181, 383 -393 (1988). JACOB, F. and B. IHL: Specific interaction between Cuscuta and its hosts. In: GREUTER, W., B. ZIMMER, and H.-D. BEHNKE (eds.): XIV. International Botanical Congress-Abstracts, p. 370, Berlin (1987).
KOLLMANN, R. and I. DORR: Parasitische Bliitenpflanzen. Naturwissenschaften 74, 12-21 (1987). Kuc, J. A.: Phytoalexins. In: PIRSON, A. and M. ZIMMERMANN (eds.): Encyclopedia of Plant Physiology, Vol. 4 (eds. R. HEITEFUSS and P. H. WILLIAMS), 632-652, Berlin, Heidelberg, New York, Springer (1976). KUI]T, J.: The biology of parasitic flowering plants. Univ. of California Press (1969). - Tissue compatibility and the haustoria of parasitic angiosperms. In: MOORE, R. (ed.): Vegetative compatibility in plants, pp. 1-12, Baylor University Press, Waco, Texas 76798 (1983).
Compatibility of Alectra with hosts
MAm, R. K., K. V. RAMAlAH, S. S. BISEN, and V. L. CHIDLEY: A comparative study of the haustorial development of Striga asiatica (L.) Kuntze on Sorghum cultivars. Ann. Bot. 54, 447 -457 (1984). NAGAR, R. M., M. SINGH, and G. G. SANWAL: Cell wall degrading enzymes in Cuscuta reflexa and its hosts. J. Expt. Bot. 35, 1104-1112 (1984). NICKRENT, D . L., L. J. MUSSELMAN, J. L. RIoPEL, and R. E. EPLEE: Haustorial initiation and non-host penetration in witchweed (Striga asiatica). Ann. Bot. 43, 233-236 (1979). PARKER, The Striga problem - a review. PANS 11, 99-111 (1965). PANCHENKO, A. Y. and T. S. ANTONOVA: Characteristics of the protective reaction of varieties of sunflower to penetration by broomrape. Seliskokhyosaisvennaya biologya 94, 554 - 558 (1974). PAUL, S. S. and A. E. DIMOND: Depression of polygalacturonase by oxidation products of polyphenols. Phytopath. 57, 492 - 496 (1967). RIDE, R. P.: Lignification in wounded wheat leaves in response to fungi and its possible role in resistance. Physiol. Plant Pathology 5, 124-134 (1975). RIOPEL, J. L. and W. V. BAIRD: Morphogenesis of the early development of primary haustoria in Striga asiatica. In: MUSSELMAN, L. J. (ed.): Parasitic weeds in agriculture. Vol. 1. Striga. CRC Press, Boca Raton, Florida, U.S.A. (1987).
c.:
745
SAUNDERS, A. R.: Studies in phanerogamic parasitism with particular reference to Striga lutea Lour. South African Dept. Agr. Bulletin 128 (1933). VISSER, J. H.: Germination stimulants of Alectra vogelii Benth. seed. Z . Pflanzenphysiol. 74, 464-469 (1975). - The biology of Alectra vogelii Benth., an angiospermous root I?arasite. Beitdige zur chemischen Kommunikation in Bio- and Okosystemen, pp. 279-294, Witzenhausen (1978). - South African parasitic flowering plants. Juta & Co. Cape Town (1982). VISSER, J. H., 1. DORR, and R. KOLLMANN: On the parasitism of Alectra vogelii Benth. (Scrophulariaceae). 1. Early development of the primary haustorium and initiation of the stem. Z . pflanzenphysiol. 84, 213-222 (1977). - - - The «hyaline body~ of the root parasite Alectra oroban· choides Benth. (Scrophulariaceae) - its anatomy, ultrastructure and histochemistry. Protoplasma 121, 146-156 (1984). VISSER, J. H. and A. JOHNSON: The effect of certain strigol analogues on the seed germination of Alectra. S. Afr. J. Bot. 1, 75-76 (1982). WELLENKAMP, 5.: Strukturelle Grundlagen der Kompatibilitat - Inkompatibilitat bei interspezifischen Zellkontakten. Diplomarbeit, Botanisches Institut der Universitat, Kiel (1986). WILLIAMS, C. N.: Resistance of Sorghum to witchweed. Nature 184, 1511 (1959).