- Email: [email protected]
Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita
Physiological
Plant
Pathology
(1972)
2, 227-234
Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita RONALD
E.
and JOHN
PAULSON
M. WEBSTER
Department of Biological Sciences, Simon I;mrer Burnaby, Vancouver, B.C., Canada (Accepted for
The
publication
January
University,
1972)
hypersensitive
reaction in roots of Lycofxrsicon esculentum L., cv. Nematex, infected by was studied by light and electron microscopy. An increase in cytoplasmic density characterized by increased numbers of ribosomes, proliferation of endoplasmic reticulum and increased stainability of the cytoplasmic ground substance was the first symptom of the resistance response to the nematode larvae. Concomitant with this change was a disappearance of dense osmiophilic inclusions from the vacuoles of hypersensitive cells. There followed a general loss in distinctness of the cell membranes resulting in the disappearance of mitochondria and Golgi bodies. The fibrillar structure of the nucleoplasm disappeared and numerous electron-dense inclusions appeared in the nucleoplasm. Organized arrays of ribosomes appeared on the outer membrane of the nuclear envelope. The plastid stroma lost its granular texture although large starch grains persisted. Changes in cell structure were restricted to cells close to the nematode, but the products of cell breakdown moved away from this region through intercellular spaces. Giant cell production was generally suppressed by the hypersensitive reaction. Heat-treatment of Nematex roots inhibited the hypersensitive reaction and thus enabled giant cell formation. Treatment of roots with the growth substance kinetin did not inhibit the hypersensitive reaction although it appeared to enhance giant cell formation.
Meloidopne
incognita
INTRODUCTION The hypersensitive reaction (HR) in plants is a cell response that is initiated by a wide variety of pathogens [S] including plant-parasitic nematodes. The plant response involves a rapid disorganization of cell structure and function in the area of the infection site. This incompatible host-parasite interaction is thought to restrict the spread of the pathogen. Structural changes that occur during the development of the HR have been described mainly from light microscope observations. However, a recent study of the ultrastructure of the HR of tobacco leaf cells observed 7 h after infection with Erwinia amylovora or with Pseudomonas pisi indicated that considerable disorganization of cell membranes and cytoplasm had occurred by the time that the earliest symptoms of hypersensitivity were visible in the leaf tissue [4]. Similarly, in a study of rust-infected cowpea leaves, Heath and Heath [.5] demonstrated signs of incompatibility at the level of cell ultrastructure before any such evidence was observed with the light microscope. Numerous reports have described light microscope observations of the HR of plant roots to nematode parasites. The root-knot nematode, Meloidopyne incognita, infecting roots of Lycopersicon esculentum (cv. Hawaii 5229), caused cell death around
228
R. E. Paulson
and J. M. Webster
the nematode after 24 h and death of the nematode occurred within 96 h [JZ]. In contrast the HR of flue-cured tobacco resistant to M. incognita acrita was induced only after the susceptible response namely, giant cell formation, had taken place [II]. The citrus nematode, Tylenchulus semifienetrans, on citrus root-stock initiated a HR consisting of collapse of the cytoplasm and the appearance of stainable material between the cell walls [15]. Observations of the soybean cyst nematode, Heterodera glycines, on resistant soybean indicate that once migrating larvae become sedentary, cells around the head become disorganized and necrotic thus preventing giant cell formation [13]. Plant-parasitic nematodes generally move more rapidly and extensively through host roots than do most bacterial or fungal pathogens. However, the mechanism by which plant hypersensitivity immobilizes parasitic nematodes and prevents their development is unclear. A study of the changes in cell ultrastructure immediately following an attack on Nematex (a cv. resistant to M. incognita) tomato roots by M. incognita and by M. hapla was made in order to ascertain the progressive changes occurring in cells. By associating these changes with known functions of the cell organelles, it was hoped to gain a further understanding of the mechanism of initiation of hypersensitivity and the way in which hypersensitive cells inhibit the movement and development of the nematode pathogen. The HR of Nematex tomato roots to M. incognita is modified by high temperatures [I] and by the application of kinetin [2]. The extent to which these factors modify the ultrastructure of host cells and the development of the HR was studied in order to gain a further understanding of the physiological and ultrastructural basis for the HR.
MATERIALS
AND
METHODS
Freshly hatched, surface-sterilized larvae from Meloidogyne incognita (Kofoid and White) Chitwood or from M. hapla Chitwood egg sacs were applied under aseptic conditions in a small drop of water to 1 cm long radicles of tomato (Lycopersicon esculentum L. var. Nematex) seedlings. The nematode larvae were surface sterilized for 10 min in 0.1 o/o Hibitane [IO] and the seeds were sterilized with 0.5% mercuric chloride in acidified 70% ethanol and washed with sterile water several times prior to use. The seedlings were washed 8 h after larval application, to remove the larvae that had not penetrated the root, and transferred to filter paper moistened with Hoagland’s nutrient solution. Samples of nematode-infected roots for microscopic examination were removed at 4, 8, 12, 24, 36, 72 and 96 h after application of the larvae to the seedlings. In the second set of experiments seedlings were heat-treated by germinating on moist filter paper at 33 “C until the radicles were 1 cm long, then surface-sterilized nematode larvae were applied at room temperature and the plants were returned to 33 “C prior to subsequent sampling of the roots for microscopic examination. In the third set of experiments kinetin-treated seedlings were germinated in the usual fashion but with the addition of 08 PM or 1.2 PM-kinetin to the nutrient solution, the nematode larvae were applied in the usual way and then the seedlings were maintained on kinetin supplemented nutrient.
Root-knot nematode in tomato
229
Pieces of root were processed for light and electron microscopic examination as described previously [9]. Sections for light microscopy were stained with 0.5% toluidine blue in 0.5 M-phosphate buffer; those sections for electron microscopy were stained with uranyl acetate (10 min) and lead citrate (5 min). RESULTS
Most larvae in the roots of the resistant tomato seedlings were associated with some cellular breakdown of plant tissue. By 12 h after inoculation larvae had penetrated the root tip and the outer cortex behind the root tip and only those cells adjacent to the larvae showed structural changes. After 24 to 36 h most larvae were located in or near the provascular tissue 2 to 3 mm behind the root tip. Cell disruption associated with the larvae was more extensive than after 12 h, and some small root-galls resulting from hypertrophied cortical cells were visible. The changes in cell ultrastructure as the hypersensitive reaction progressed were similar in cortical, provascular and root-apical cells. However, the changes in structure of the cytoplasm and organelles were more readily observed in the provascular cells and in the root apex than in the more highly vacuolated cortical tissues. Most micrographs therefore describe changes observed in the undifferentiated cells just behind the region of cell division in the root tip. The earliest indications of the HR were observed about 12 h after the application of the larvae to the roots. The cytoplasm of hypersensitive cells was seen under the light microscope to have an increased affinity for stains whilst under the electron microscope the cytoplasm of these cells appeared more electron-dense than that of adjacent, normal cells (Plates 1 to 3). The increased affinity of the cytoplasm for stains could be accounted for by increased numbers of ribosomes, a proliferation of the endoplasmic reticulum and an increased stainability of some component in the cytoplasmic ground substance (Plate 3). Mechanical disruption of some cell walls was caused by nematode movement between and through the cells. Other cell walls close to the nematode separated in the region of the middle lamella without any noticeable effects on the cytoplasm within the cells (Plate 5) and the region of separation between these walls either appeared empty or contained some fibrous material. Spaces between other cells up to three or four cells distant from the site of the nematode became filled with electron-dense material within 24 h of exposure to the nematodes (Plates 2, 6 and 7). This electron-dense material is probably composed of cellular debris and nematode secretions and/or excretions. The cytoplasm and organelles of the adjacent cells were normal in appearance (Plates 6 and 7). Most root tip cells, the outer cells of the vascular cylinder and the innermost cortical cells normally contained small accretions of an electron-dense osmiophilic material within their vacuoles (Plates 2 and 8). This material occurred in a highly condensed state in the cell vacuoles of non-infected resistant and susceptible varieties in similar amounts, but was more diffuse or was absent within cells undergoing the HR (compare the normal and hypersensitive cells in Plate 8). Fibrous material in the vacuoles of hypersensitive cells (Plate 8) may be the remains of the more condensed material characteristic of unaffected cells. In some apparently normal cells adjacent to obviously hypersensitive cells this condensed osmiophilic material in
230
R. E. Paulson
and J. M. Webster
the vacuoles appeared to be breaking down (Plate 9). This feature may be the earliest recognizable stage of the HR. Breakdown of these electron-dense vacuolar contents was observed at about the time that the cytoplasm showed an increased electron density. Vacuoles lost their regular outline and appeared to fuse with adjacent vacuoles as the HR progressed (Plates 2, 8 and 10). Resolution of the tonoplast became increasingly difficult as cytoplasmic density increased. Thus, although vacuole-like regions could be identified in hypersensitive cells (Plates 8 and 10) unequivocal statements as to the integrity of their limiting membranes were impossible in all but the earliest stages of hypersensitivity (Plate 4). The general loss in distinctness of cell membranes that accompanied the increase in density of the cytoplasm makes it difficult to identify changes in the structure of mitochondria and Golgi bodies. Both these organelles are readily identifiable in normal cells (Plates 7 and 8) and neither can be identified in hypersensitive cells (Plates 10 and 11). In some early stages of the HR mitochondria may be very indistinct when nuclei and plastids can still be readily identified (Plate 12). In unaffected cells the endoplasmic reticulum was present in the form of short isolated segments of parallel membranes usually associated with ribosomes (Plates 7 and 8). In hypersensitive cells, however, it was often replaced by long, branched forms that were more distinct owing to the contrast between the increased electron density of the cytoplasm and the less dense intralamellar space of the endoplasmic reticulum (Plates 3 and 12). Even in the most advanced stages of the HR this intralamellar space remained less dense than the adjacent cytoplasm (Plate 11). Changes in the fine structure of the nuclei involved a rapid and general disappearance of the fibrillar component from the nucleoplasm (Plate 12). Subsequently there appeared numerous dense granules within the nucleoplasm (Plates 11 and 13). The nucleolus appeared to occupy a larger proportion of the nuclear volume in hypersensitive cells than in normal cells. Granular and fibrillar regions within the nucleolus became increasingly difficult to identify (cf. Plates 8 and 13) and eventually could not be discerned (Plate 11). The double nature of the nuclear envelope became less readily identified (cf. Plates 8 and 12) and during later stages of hypersensitivity this envelope appeared to fragment (Plate 13). The nuclear membrane fragments were characterized by numerous attached ribosomes (Plate 13), a feature rarely seen in plant cells and only occasionally observed, but to a much lesser extent, in cells adjacent to hypersensitive cells (Plate 8). List of abbreviations
used in the plates
Ch = Chromatin. CW = Cell wall. DI = Dense reticulum. FR = Fibrillar region of the nucleolus. region of the nucleolus. HC = Hypersensitive NM = Nuclear N = Nucleus. Ne = Nematode. L = Plasmalemma. T = Tonoplast. V = Vacuole.
inclusion in the vacuoles. ER = Endoplasmic G = Golgi. GC = Giant cell. GR = Granular cell. M = Mitochondrion. Mt = Microtubule. membrane. Nu = Nucleolus. P = Plastid. X = Xylem.
Legends to plates Unless otherwise stated all figures show portions of the nematode resistant examined at different intervals after exposure to M. incognita larvae.
tomato
root
(cv.
Nematex)
PLATE 1. Electron micrograph of portions of three cortical cells 12 h after addition of the nematode larvae to the roots, two of which (A and B) show the early stages of the hypersensitive reaction. Note the increased electron-density of the cytoplasm and nucleoplasm in the hypersensitive cells. ( x 4300.) PLATE 2. Electron micrograph showing a nematode and at least three hypersensitive cells and adjacent normal cells after 24 h. In the hypersensitive cells the cytoplasm and nuclei are conspicuously electron-dense and fusion of vacuoles has occurred. Note the presence of electron dense inclusions in the vacuoles of normal cells (arrows) and their absence from the vacuoles of the hypersensitive cells. ( x 3100.) PLATE 3. A portion of two hypersensitive (cells with electron-dense cytoplasm) contrasted with a more electron-light normal cell below. Note in the hypersensitive cells the electronlight channels of the endoplasmic reticulum (arrows). Cell walls, tonoplast and plasmalemma appear unaltered. ( x 11,200.) PLATE 4. A higher magnification plasmalemma and tonoplast and normal. ( x 38,600.) PLATE separated
5. A portion although the
of two cytoplasm
of the region the fact that
indicated in Plate 3. Note the presence the cytoplasm is more electron-dense
cells adjacent to a nematode after 24 h. of both ceils is normal in appearance.
PLATE 6. Electron micrograph of a hypersensitive occurs between the walls of adjacent cells (arrows). PLATE 7. A higher magnification between two cells is not associated ( x 14,400.)
cell (HC) ( x 3400.)
after
The cell ( x 4800.) 24 h.
walls
Cellular
of the than have debris
of normal cells similar to those in Plate 6. Cellular with any cytoplasmic disorganization in the adjacent
debris cells.
PLATE 8. Part of a hypersensitive cell (HC) and normal adjacent cells (below) after 24 h. Vacuoles in the unaffected cells contain electron-dense material in a condensed state (arrows) whereas vacuoles in the hypersensitive cell do not contain this material or they have it in a highly dispersed, fibrous form. An array of ribosomes is evident adjacent to the outer membrane of the nuclear envelope (*) in one of the normal cells. ( x 12,600.) PLATE 9. Partially dispersed electron-dense the initial stages of the hypersensitive reaction. is much more advanced. ( x 11,200.) PLATE 10. Hypersensitive plast and other cell membranes of the extensive hypersensitive
material in vacuoles after 24 h may indicate The hypersensitive reaction in the cell on the left
(HC) and adjacent normal cells (above) after 48 h. The tonoand the vacuolar inclusions in the normal cells show no effects reaction in the adjacent cells. ( x 9840.)
PLATE 11. A portion of a hypersensitive cell after 48 h showing disorganized cytoplasm. The finely granular texture of the cytoplasm and the nucleoplasm is obscure. Extensive channels of endoplasmic reticulum (arrows), indistinct plastids and part of a nucleus are shown. (x 14,500.) PLATE 12. An early stage in the hypersensitive surrounding the organelles and abnormal membrane structure of plastids and nuclei appears unaltered. endoplasmic reticulum in the darkened cytoplasm. PLATE 13. A disorganized nucleus The nuclear envelope has fragmented contains abnormal, densely stained
reaction showing indistinct membranes formations along the walls. The internal Note the electron-light channels of thr ( x 10,000.)
and abnormal plastids and become invested inclusions (arrows).
in a hypersensitive cell after 48 h. with ribosomes (*). The nucleus Compare with Plates 8 and 12.
( x 6500.) PLATE 14. Light micrograph of a M. Note the absence of a hypersensitive reaction the vacuoles of adjacent cells. ( x 165.)
ha@ and
larva with associated giant cells after 48 h. the abundance of electron-dense inclusions in
PLATE 15. An electron micrograph of the cells in Plate in the giant cell as well as in the adjacent cells. ( x 3850.)
14.
Note
the vacuolar
inclusions
PLATE 16. Light micrograph of M. incognita larvae in a heat-treated resistant root after 48 h. Note the absence of a hypersensitive reaction and an early stage of giant cell formation (multinucleate cell). ( x 960.) PLATE
hypersensitive formation.
17.
The same material reaction in the cells ( x 520.)
PLATE 18. M. incognita larvae extensive hypersensitive reaction
as shown adjacent
in Plate 16. In this instance there is a localized to the giant cells but it has not retarded giant cell
in a resistant root treated with 0.8 FM-kinetin. After and abnormal xylem differentiation has occurred.
2 days an (x 135.)
PLATE 19. Tissue similar to that shown in Plate 18. A few giant cells have developed without initiating an extensive hypersensitive reaction. Note the extensive cell division which has occurred adjacent to the giant cells. ( x 495.)
Root-knot
nematode
Meloidogyne
in tomato
231
hapla on Nematex roots
When Meloidopyne ha&a, to which Nematex roots are susceptible, were placed on the roots galling and giant cell formation were evident after 48 h and the HR did not occur. Light microscope sections showed giant cell development, and only the occasional resistant reaction. In regions of the root where most cells contained conspicuous amounts of the electron-dense vacuolar inclusions this material was ununaltered by the presence of M. hapla, and it was observed within the giant cells and in adjacent unmodified cells (Plates 14 and 15). This feature is in marked contrast to the breakdown of the electron-dense materials in the HR to M. incognita. Heat treatment of Nematex roots
The HR caused by M. incognita in Nematex roots was absent or much less extensive at 33 “C (Plate 16) than at lower temperatures (Plate 2). Most larvae did not induce a significant HR in plants treated at 33 “C but induced giant cells typical of the response of susceptible varieties of tomato. A few larvae induced a very localized HR (Plate 17) that did not prevent giant cell formation. The changes observed during breakdown of these few cells were similar to those observed during the HR of cells of plants that had not been heat treated. Kinetin
treatment of Nematex roots
Kinetin treatment caused stunting of the roots, increased galling and development of numerous lateral roots, but it did not significantly decrease the extent or intensity of the HR. The progress of development of the reaction was similar to that already described. In addition to the extensive HR many kinetin treated roots showed giant cell development, and an abnormal differentiation of xylem elements associated with multiple lateral root development (Plates 18 and 19). DISCUSSION Some of the abnormal cell ultrastructure observed in the nematode resistant roots was due merely to physical breakage of walls and membranes caused by nematode movements and was not related to the changes in cell structure referred to as the HR. Cells damaged by passage of the nematode could be identified by their broken cell walls and the accumulation of cellular debris around the cell periphery. It is difficult to define precisely the time of initiation of the HR in terms of specific changes in cell ultrastructure. While some of the larvae initiated a slight HR within 8 to 12 h of inoculation onto the seedlings others were observed in a similar state only after 24 to 36 h. This variation was due partially to the variability in time taken for the larvae to penetrate the root and partially to the fact that the larvae likely do not elicit an extensive HR while they are migrating through the root tissue, but do so only after becoming more sedentary whilst attempting to establish a feeding site. Typically, therefore, the HR is centrally located in the root. Some 2 to 3 days after the application of the larvae to the roots the HR was localized around the larvae, and cells adjacent to the hypersensitive cells showed no abnormalities in their ultrastructure that would indicate a graded response away from the infection site. The lack of such a graded response suggests that the stimulus initiating the HR does not pass readily from cell to cell and, in fact, is induced only by penetration
232
R. E. Paulson
and
J. M. Webster
of and secretion into a cell by the nematode stylet. The necessity of direct stimulation by the stylet would explain the presence of the relatively few hypersensitive cells along the path of nematode migration through the root tissue. Progressive breakdown of cell membranes and cytoplasm occurs during the HR of tobacco leaves to bacteria [4]. It has been suggested that changes in the permeability of vacuolar membranes and subsequent movement of toxic materials out of the vacuole cause the HR of plants to bacteria [6], and changes in the permeability of plant cell membranes are known to be induced by fungal toxins [la] and by fungal enzymes [S]. The disappearance of the electron-dense inclusions from the vacuoles of hypersensitive cells suggests that a change in the permeability of the tonoplast to the contents of the vacuole or to the enzymes necessary to dissolve the inclusions may have occurred. The rapid increase in electron density of the cytoplasm during the HR of Nematex to M. incognita is not seen in the reaction of tobacco leaves to bacteria [4] or in the reaction of cowpea leaves to rusts [5]. The increased electron-density of the cytoplasm of tomato roots may be accounted for by the presence in it of material normally found in the vacuole, the release of toxic products from the vacuole or the result of an interaction of the cytoplasm and nematode secretions. The fact that the electron-dense vacuolar inclusions disappeared and that the cytoplasm increased in electron density before any changes were seen in cell walls, membranes or organelles provides visual evidence that changes in permeability of the tonoplast may be the first changes that lead to general disorganization of the cell during the HR. An apparent increase in the number of ribosomes and a change in the form of the endoplasmic reticulum from short isolated segments associated with ribosomes to long smooth forms without ribosomes were the only changes in the structure of hypersensitive cells that were not obviously symptoms of cellular breakdown. These changes, which suggest increased protein synthesis, were not seen in adjacent apparently unaffected cells and were considered to be some of the first indications of the hypersensitive reaction. These features were followed closely by the loss of membrane distinctness and by the disappearance of mitochondria, features more closely indicative of cellular breakdown. Heterodera g&Gzes infection of resistant soybean roots [3] and Meloidogyne javanica infection of resistant peach root-stock [7] induced a susceptible response, in the form In contrast, of giant cell production, before a resistant response was initiated. M. incognita in Nematex, appears to activate resistance mechanisms resulting in a HR within 8 to 12 h. The stimulus that initiates hypersensitivity is likely a component of the nematode oesophageal gland secretions that acts sufficiently rapidly to prevent giant cell formation, a process which normally takes 24 to 36 h [9]. M. hapla larvae usually do not initiate any of the symptoms of hypersensitivity characteristic of M. incognita in Nematex roots. Apparently the secretions from M. hapla which initiate giant cell formation do not contain sufficient amounts of whatever component activates the resistance mechanism. The fact that a few cells do show a HR suggests that there is some differential sensitivity of Nematex root cells to this hypothetical component. Temperatures of about 33 “C drastically decreased the number of cells manifesting the HR but did not appear to cause any changes in the ultrastructure of
Root-knot
nematode
in tomato
233
cells unaffected by nematodes. The fact that this higher temperature did not preclude giant cell formation and did not result in any observable modifications in cell ultrastructure makes it tempting to postulate the inactivation of some enzyme system responsible for initiating the development of the HR. On the basis of observations of unsectioned Nematex roots infected by M. incognita, Dropkin [I] reported that 0.8 PM-kinetin significantly decreased the HR. However, our light and electron-microscopy observations indicate that while the kinetin-treated roots were stunted, swollen and had extensive lateral roots the HR was not significantly reduced. The fact that more giant cells developed in the kinetin-treated Nematex roots than in those without kinetin suggests that the kinetin favours development of giant cells while not significantly inhibiting the HR. Perhaps the excess kinetin suppresses the HR in the few cells that are close enough to the nematode to be stimulated into giant cell formation. However, the exogenous kinetin is not able to suppress completely the trigger which causes the HR. The hypersensitive cells, while not providing the nematode with sufficient nutrients for development, provide a sufficient stimulus to prevent it from seeking a more compatible site for development. The absence of structural changes in the cells adjacent to the hypersensitive region makes it unlikely that physical restrictions, analogous with the wall thickenings or the formation of a wood periderm observed by Van Gundy and Kirkpatrick [15], prevent the nematode from leaving the hypersensitive region. Second stage larvae removed from hypersensitive roots 2 days after inoculation are capable of subsequently infecting susceptible roots which indicates the absence of a toxic factor in the hypersensitive roots. Observations of the ultrastructural changes in the developing hypersensitive cells suggest that the HR may be initiated by the loss of some materials from the vacuoles to the cytoplasm which causes the deterioration of cytoplasmic organization and so effectively prevents giant cell formation. Subsequent death of the nematode larvae is likely due to the absence of the nutritive giant cells rather than to the toxic effects of the hypersensitive cells. This work was completed while the senior author was Research Council of Canada Scholarship, and the other grant (No. A4679) from the National Research Council seeds were kindly supplied by Dr V. H. Dropkin of Pathology, University of Missouri.
supported by a National author held an operating of Canada. The Nematex the Department of Plant
REFERENCES
V. H. (1969). The necroticreaction of tomatoesand other hosts resistantto Meloidogym: Reversalby temperature. PhytopathoZogV 59, 1632-1637. DROPKIN, V. H., HELGESON, J. P. & UPPER, C. D. (1969). The hypersensitivityreaction of tomatoes resistant to Meloidogyne incognita: Reversal by cytokinins. Journal of Nmatology 1,
1. DROPKIN, 2.
55-61. 3. ENDO, B. Y. (1965). Histological responses of resistant and susceptible soybean varieties and backcross progeny to entry and development of Heterodera g&&m. Phytofiathology 55, 375-381. 4. GOODMAN, R. N. & PLURAD, S. B. (1971). Ultrastructural changes in tobacco undergoing the hypersensitive reaction caused by plant pathogenic bacteria. PhysiologkaJ Plant Pathology 1, 11-15.
234
R. E. Paulson
and J. M. Webster
5. HEATH, M. C. & HEATH, I. B. (1971). Ultrastructure of an immune and a susceptible reaction of cowpea leaves to rust infection. Physiological Plant Pathology 1, 277-237. 6. KLEMENT, Z. & GOODMAN, R. N. (1967). The hypersensitive reaction to infection by bacterial plant pathogens. Annual Review of Phytopatholopy 5, 17-44. 7. MALO, S. E. (1968). Nature of resistance of “Okinawa” and “Nemaguard” peach to the root-knot nematode Meloidqyw jauanica. Proceedings of the American Society for Horticultural Science 90, 39-46. 8. MOUNT, M. S., BATEMAN, D. F. & BASHAM, H. G. (1970). Induction of electrolyte loss, tissue maceration and cellular death of potato tissue by an endopolygalacturonate trans-eliminase. Phytofiathologv 60, 924-93 1. 9. PAULSON, R. E. & WEBSTER, J. M. (1970). Giant cell formation in tomato roots caused by Meloidogpe incognita and Meloidogyne hapla (Nematoda) infection. A light and electron microscope study. Canadian Journal of Botany 48, 271-276. 10. PEACOCK, F. C. (1959). The development of a technique for studying the host/parasite relationship of the root-knot nematode Meloidogyne incognita under controlled conditions. Nematologica 4, 43-55. 11. POWELL, N. T. (1962). Histological basis of resistance to root-knot nematodes in flue-cured tobacco. Phytopathologu 52, 25. 12. RIGGS, R. D. & WINSTEAD, N. N. (1959). Studies on resistance in tomato to root-knot nematodes and on the occurrence of pathogenic biotypes. Phytofiathology 49, 716-724. 13. Ross, J. P. (1958). Host-parasite relationship of the soybean cyst nematode in resistant soybean roots. Phytopathologv 48, 578-579. 14. SAMADDAR, K. R. & SCHEPFER, R. P. (1971). Early effects of Helminthosporium victoriae toxin on plasma membranes and counteraction by chemical treatments. Physiological Plant Pathology 1, 319-328. 15. VAN GUNDY, S. D. & KIRKPATRICK, J. D. (1964). Nature of resistance in certain citrus rootstocks to citrus nematode. Phytopathology 54, 419-427.
Plant
Pathology
(1972)
2, 227-234
Ultrastructure of the hypersensitive reaction in roots of tomato, Lycopersicon esculentum L., to infection by the root-knot nematode, Meloidogyne incognita RONALD
E.
and JOHN
PAULSON
M. WEBSTER
Department of Biological Sciences, Simon I;mrer Burnaby, Vancouver, B.C., Canada (Accepted for
The
publication
January
University,
1972)
hypersensitive
reaction in roots of Lycofxrsicon esculentum L., cv. Nematex, infected by was studied by light and electron microscopy. An increase in cytoplasmic density characterized by increased numbers of ribosomes, proliferation of endoplasmic reticulum and increased stainability of the cytoplasmic ground substance was the first symptom of the resistance response to the nematode larvae. Concomitant with this change was a disappearance of dense osmiophilic inclusions from the vacuoles of hypersensitive cells. There followed a general loss in distinctness of the cell membranes resulting in the disappearance of mitochondria and Golgi bodies. The fibrillar structure of the nucleoplasm disappeared and numerous electron-dense inclusions appeared in the nucleoplasm. Organized arrays of ribosomes appeared on the outer membrane of the nuclear envelope. The plastid stroma lost its granular texture although large starch grains persisted. Changes in cell structure were restricted to cells close to the nematode, but the products of cell breakdown moved away from this region through intercellular spaces. Giant cell production was generally suppressed by the hypersensitive reaction. Heat-treatment of Nematex roots inhibited the hypersensitive reaction and thus enabled giant cell formation. Treatment of roots with the growth substance kinetin did not inhibit the hypersensitive reaction although it appeared to enhance giant cell formation.
Meloidopne
incognita
INTRODUCTION The hypersensitive reaction (HR) in plants is a cell response that is initiated by a wide variety of pathogens [S] including plant-parasitic nematodes. The plant response involves a rapid disorganization of cell structure and function in the area of the infection site. This incompatible host-parasite interaction is thought to restrict the spread of the pathogen. Structural changes that occur during the development of the HR have been described mainly from light microscope observations. However, a recent study of the ultrastructure of the HR of tobacco leaf cells observed 7 h after infection with Erwinia amylovora or with Pseudomonas pisi indicated that considerable disorganization of cell membranes and cytoplasm had occurred by the time that the earliest symptoms of hypersensitivity were visible in the leaf tissue [4]. Similarly, in a study of rust-infected cowpea leaves, Heath and Heath [.5] demonstrated signs of incompatibility at the level of cell ultrastructure before any such evidence was observed with the light microscope. Numerous reports have described light microscope observations of the HR of plant roots to nematode parasites. The root-knot nematode, Meloidopyne incognita, infecting roots of Lycopersicon esculentum (cv. Hawaii 5229), caused cell death around
228
R. E. Paulson
and J. M. Webster
the nematode after 24 h and death of the nematode occurred within 96 h [JZ]. In contrast the HR of flue-cured tobacco resistant to M. incognita acrita was induced only after the susceptible response namely, giant cell formation, had taken place [II]. The citrus nematode, Tylenchulus semifienetrans, on citrus root-stock initiated a HR consisting of collapse of the cytoplasm and the appearance of stainable material between the cell walls [15]. Observations of the soybean cyst nematode, Heterodera glycines, on resistant soybean indicate that once migrating larvae become sedentary, cells around the head become disorganized and necrotic thus preventing giant cell formation [13]. Plant-parasitic nematodes generally move more rapidly and extensively through host roots than do most bacterial or fungal pathogens. However, the mechanism by which plant hypersensitivity immobilizes parasitic nematodes and prevents their development is unclear. A study of the changes in cell ultrastructure immediately following an attack on Nematex (a cv. resistant to M. incognita) tomato roots by M. incognita and by M. hapla was made in order to ascertain the progressive changes occurring in cells. By associating these changes with known functions of the cell organelles, it was hoped to gain a further understanding of the mechanism of initiation of hypersensitivity and the way in which hypersensitive cells inhibit the movement and development of the nematode pathogen. The HR of Nematex tomato roots to M. incognita is modified by high temperatures [I] and by the application of kinetin [2]. The extent to which these factors modify the ultrastructure of host cells and the development of the HR was studied in order to gain a further understanding of the physiological and ultrastructural basis for the HR.
MATERIALS
AND
METHODS
Freshly hatched, surface-sterilized larvae from Meloidogyne incognita (Kofoid and White) Chitwood or from M. hapla Chitwood egg sacs were applied under aseptic conditions in a small drop of water to 1 cm long radicles of tomato (Lycopersicon esculentum L. var. Nematex) seedlings. The nematode larvae were surface sterilized for 10 min in 0.1 o/o Hibitane [IO] and the seeds were sterilized with 0.5% mercuric chloride in acidified 70% ethanol and washed with sterile water several times prior to use. The seedlings were washed 8 h after larval application, to remove the larvae that had not penetrated the root, and transferred to filter paper moistened with Hoagland’s nutrient solution. Samples of nematode-infected roots for microscopic examination were removed at 4, 8, 12, 24, 36, 72 and 96 h after application of the larvae to the seedlings. In the second set of experiments seedlings were heat-treated by germinating on moist filter paper at 33 “C until the radicles were 1 cm long, then surface-sterilized nematode larvae were applied at room temperature and the plants were returned to 33 “C prior to subsequent sampling of the roots for microscopic examination. In the third set of experiments kinetin-treated seedlings were germinated in the usual fashion but with the addition of 08 PM or 1.2 PM-kinetin to the nutrient solution, the nematode larvae were applied in the usual way and then the seedlings were maintained on kinetin supplemented nutrient.
Root-knot nematode in tomato
229
Pieces of root were processed for light and electron microscopic examination as described previously [9]. Sections for light microscopy were stained with 0.5% toluidine blue in 0.5 M-phosphate buffer; those sections for electron microscopy were stained with uranyl acetate (10 min) and lead citrate (5 min). RESULTS
Most larvae in the roots of the resistant tomato seedlings were associated with some cellular breakdown of plant tissue. By 12 h after inoculation larvae had penetrated the root tip and the outer cortex behind the root tip and only those cells adjacent to the larvae showed structural changes. After 24 to 36 h most larvae were located in or near the provascular tissue 2 to 3 mm behind the root tip. Cell disruption associated with the larvae was more extensive than after 12 h, and some small root-galls resulting from hypertrophied cortical cells were visible. The changes in cell ultrastructure as the hypersensitive reaction progressed were similar in cortical, provascular and root-apical cells. However, the changes in structure of the cytoplasm and organelles were more readily observed in the provascular cells and in the root apex than in the more highly vacuolated cortical tissues. Most micrographs therefore describe changes observed in the undifferentiated cells just behind the region of cell division in the root tip. The earliest indications of the HR were observed about 12 h after the application of the larvae to the roots. The cytoplasm of hypersensitive cells was seen under the light microscope to have an increased affinity for stains whilst under the electron microscope the cytoplasm of these cells appeared more electron-dense than that of adjacent, normal cells (Plates 1 to 3). The increased affinity of the cytoplasm for stains could be accounted for by increased numbers of ribosomes, a proliferation of the endoplasmic reticulum and an increased stainability of some component in the cytoplasmic ground substance (Plate 3). Mechanical disruption of some cell walls was caused by nematode movement between and through the cells. Other cell walls close to the nematode separated in the region of the middle lamella without any noticeable effects on the cytoplasm within the cells (Plate 5) and the region of separation between these walls either appeared empty or contained some fibrous material. Spaces between other cells up to three or four cells distant from the site of the nematode became filled with electron-dense material within 24 h of exposure to the nematodes (Plates 2, 6 and 7). This electron-dense material is probably composed of cellular debris and nematode secretions and/or excretions. The cytoplasm and organelles of the adjacent cells were normal in appearance (Plates 6 and 7). Most root tip cells, the outer cells of the vascular cylinder and the innermost cortical cells normally contained small accretions of an electron-dense osmiophilic material within their vacuoles (Plates 2 and 8). This material occurred in a highly condensed state in the cell vacuoles of non-infected resistant and susceptible varieties in similar amounts, but was more diffuse or was absent within cells undergoing the HR (compare the normal and hypersensitive cells in Plate 8). Fibrous material in the vacuoles of hypersensitive cells (Plate 8) may be the remains of the more condensed material characteristic of unaffected cells. In some apparently normal cells adjacent to obviously hypersensitive cells this condensed osmiophilic material in
230
R. E. Paulson
and J. M. Webster
the vacuoles appeared to be breaking down (Plate 9). This feature may be the earliest recognizable stage of the HR. Breakdown of these electron-dense vacuolar contents was observed at about the time that the cytoplasm showed an increased electron density. Vacuoles lost their regular outline and appeared to fuse with adjacent vacuoles as the HR progressed (Plates 2, 8 and 10). Resolution of the tonoplast became increasingly difficult as cytoplasmic density increased. Thus, although vacuole-like regions could be identified in hypersensitive cells (Plates 8 and 10) unequivocal statements as to the integrity of their limiting membranes were impossible in all but the earliest stages of hypersensitivity (Plate 4). The general loss in distinctness of cell membranes that accompanied the increase in density of the cytoplasm makes it difficult to identify changes in the structure of mitochondria and Golgi bodies. Both these organelles are readily identifiable in normal cells (Plates 7 and 8) and neither can be identified in hypersensitive cells (Plates 10 and 11). In some early stages of the HR mitochondria may be very indistinct when nuclei and plastids can still be readily identified (Plate 12). In unaffected cells the endoplasmic reticulum was present in the form of short isolated segments of parallel membranes usually associated with ribosomes (Plates 7 and 8). In hypersensitive cells, however, it was often replaced by long, branched forms that were more distinct owing to the contrast between the increased electron density of the cytoplasm and the less dense intralamellar space of the endoplasmic reticulum (Plates 3 and 12). Even in the most advanced stages of the HR this intralamellar space remained less dense than the adjacent cytoplasm (Plate 11). Changes in the fine structure of the nuclei involved a rapid and general disappearance of the fibrillar component from the nucleoplasm (Plate 12). Subsequently there appeared numerous dense granules within the nucleoplasm (Plates 11 and 13). The nucleolus appeared to occupy a larger proportion of the nuclear volume in hypersensitive cells than in normal cells. Granular and fibrillar regions within the nucleolus became increasingly difficult to identify (cf. Plates 8 and 13) and eventually could not be discerned (Plate 11). The double nature of the nuclear envelope became less readily identified (cf. Plates 8 and 12) and during later stages of hypersensitivity this envelope appeared to fragment (Plate 13). The nuclear membrane fragments were characterized by numerous attached ribosomes (Plate 13), a feature rarely seen in plant cells and only occasionally observed, but to a much lesser extent, in cells adjacent to hypersensitive cells (Plate 8). List of abbreviations
used in the plates
Ch = Chromatin. CW = Cell wall. DI = Dense reticulum. FR = Fibrillar region of the nucleolus. region of the nucleolus. HC = Hypersensitive NM = Nuclear N = Nucleus. Ne = Nematode. L = Plasmalemma. T = Tonoplast. V = Vacuole.
inclusion in the vacuoles. ER = Endoplasmic G = Golgi. GC = Giant cell. GR = Granular cell. M = Mitochondrion. Mt = Microtubule. membrane. Nu = Nucleolus. P = Plastid. X = Xylem.
Legends to plates Unless otherwise stated all figures show portions of the nematode resistant examined at different intervals after exposure to M. incognita larvae.
tomato
root
(cv.
Nematex)
PLATE 1. Electron micrograph of portions of three cortical cells 12 h after addition of the nematode larvae to the roots, two of which (A and B) show the early stages of the hypersensitive reaction. Note the increased electron-density of the cytoplasm and nucleoplasm in the hypersensitive cells. ( x 4300.) PLATE 2. Electron micrograph showing a nematode and at least three hypersensitive cells and adjacent normal cells after 24 h. In the hypersensitive cells the cytoplasm and nuclei are conspicuously electron-dense and fusion of vacuoles has occurred. Note the presence of electron dense inclusions in the vacuoles of normal cells (arrows) and their absence from the vacuoles of the hypersensitive cells. ( x 3100.) PLATE 3. A portion of two hypersensitive (cells with electron-dense cytoplasm) contrasted with a more electron-light normal cell below. Note in the hypersensitive cells the electronlight channels of the endoplasmic reticulum (arrows). Cell walls, tonoplast and plasmalemma appear unaltered. ( x 11,200.) PLATE 4. A higher magnification plasmalemma and tonoplast and normal. ( x 38,600.) PLATE separated
5. A portion although the
of two cytoplasm
of the region the fact that
indicated in Plate 3. Note the presence the cytoplasm is more electron-dense
cells adjacent to a nematode after 24 h. of both ceils is normal in appearance.
PLATE 6. Electron micrograph of a hypersensitive occurs between the walls of adjacent cells (arrows). PLATE 7. A higher magnification between two cells is not associated ( x 14,400.)
cell (HC) ( x 3400.)
after
The cell ( x 4800.) 24 h.
walls
Cellular
of the than have debris
of normal cells similar to those in Plate 6. Cellular with any cytoplasmic disorganization in the adjacent
debris cells.
PLATE 8. Part of a hypersensitive cell (HC) and normal adjacent cells (below) after 24 h. Vacuoles in the unaffected cells contain electron-dense material in a condensed state (arrows) whereas vacuoles in the hypersensitive cell do not contain this material or they have it in a highly dispersed, fibrous form. An array of ribosomes is evident adjacent to the outer membrane of the nuclear envelope (*) in one of the normal cells. ( x 12,600.) PLATE 9. Partially dispersed electron-dense the initial stages of the hypersensitive reaction. is much more advanced. ( x 11,200.) PLATE 10. Hypersensitive plast and other cell membranes of the extensive hypersensitive
material in vacuoles after 24 h may indicate The hypersensitive reaction in the cell on the left
(HC) and adjacent normal cells (above) after 48 h. The tonoand the vacuolar inclusions in the normal cells show no effects reaction in the adjacent cells. ( x 9840.)
PLATE 11. A portion of a hypersensitive cell after 48 h showing disorganized cytoplasm. The finely granular texture of the cytoplasm and the nucleoplasm is obscure. Extensive channels of endoplasmic reticulum (arrows), indistinct plastids and part of a nucleus are shown. (x 14,500.) PLATE 12. An early stage in the hypersensitive surrounding the organelles and abnormal membrane structure of plastids and nuclei appears unaltered. endoplasmic reticulum in the darkened cytoplasm. PLATE 13. A disorganized nucleus The nuclear envelope has fragmented contains abnormal, densely stained
reaction showing indistinct membranes formations along the walls. The internal Note the electron-light channels of thr ( x 10,000.)
and abnormal plastids and become invested inclusions (arrows).
in a hypersensitive cell after 48 h. with ribosomes (*). The nucleus Compare with Plates 8 and 12.
( x 6500.) PLATE 14. Light micrograph of a M. Note the absence of a hypersensitive reaction the vacuoles of adjacent cells. ( x 165.)
ha@ and
larva with associated giant cells after 48 h. the abundance of electron-dense inclusions in
PLATE 15. An electron micrograph of the cells in Plate in the giant cell as well as in the adjacent cells. ( x 3850.)
14.
Note
the vacuolar
inclusions
PLATE 16. Light micrograph of M. incognita larvae in a heat-treated resistant root after 48 h. Note the absence of a hypersensitive reaction and an early stage of giant cell formation (multinucleate cell). ( x 960.) PLATE
hypersensitive formation.
17.
The same material reaction in the cells ( x 520.)
PLATE 18. M. incognita larvae extensive hypersensitive reaction
as shown adjacent
in Plate 16. In this instance there is a localized to the giant cells but it has not retarded giant cell
in a resistant root treated with 0.8 FM-kinetin. After and abnormal xylem differentiation has occurred.
2 days an (x 135.)
PLATE 19. Tissue similar to that shown in Plate 18. A few giant cells have developed without initiating an extensive hypersensitive reaction. Note the extensive cell division which has occurred adjacent to the giant cells. ( x 495.)
Root-knot
nematode
Meloidogyne
in tomato
231
hapla on Nematex roots
When Meloidopyne ha&a, to which Nematex roots are susceptible, were placed on the roots galling and giant cell formation were evident after 48 h and the HR did not occur. Light microscope sections showed giant cell development, and only the occasional resistant reaction. In regions of the root where most cells contained conspicuous amounts of the electron-dense vacuolar inclusions this material was ununaltered by the presence of M. hapla, and it was observed within the giant cells and in adjacent unmodified cells (Plates 14 and 15). This feature is in marked contrast to the breakdown of the electron-dense materials in the HR to M. incognita. Heat treatment of Nematex roots
The HR caused by M. incognita in Nematex roots was absent or much less extensive at 33 “C (Plate 16) than at lower temperatures (Plate 2). Most larvae did not induce a significant HR in plants treated at 33 “C but induced giant cells typical of the response of susceptible varieties of tomato. A few larvae induced a very localized HR (Plate 17) that did not prevent giant cell formation. The changes observed during breakdown of these few cells were similar to those observed during the HR of cells of plants that had not been heat treated. Kinetin
treatment of Nematex roots
Kinetin treatment caused stunting of the roots, increased galling and development of numerous lateral roots, but it did not significantly decrease the extent or intensity of the HR. The progress of development of the reaction was similar to that already described. In addition to the extensive HR many kinetin treated roots showed giant cell development, and an abnormal differentiation of xylem elements associated with multiple lateral root development (Plates 18 and 19). DISCUSSION Some of the abnormal cell ultrastructure observed in the nematode resistant roots was due merely to physical breakage of walls and membranes caused by nematode movements and was not related to the changes in cell structure referred to as the HR. Cells damaged by passage of the nematode could be identified by their broken cell walls and the accumulation of cellular debris around the cell periphery. It is difficult to define precisely the time of initiation of the HR in terms of specific changes in cell ultrastructure. While some of the larvae initiated a slight HR within 8 to 12 h of inoculation onto the seedlings others were observed in a similar state only after 24 to 36 h. This variation was due partially to the variability in time taken for the larvae to penetrate the root and partially to the fact that the larvae likely do not elicit an extensive HR while they are migrating through the root tissue, but do so only after becoming more sedentary whilst attempting to establish a feeding site. Typically, therefore, the HR is centrally located in the root. Some 2 to 3 days after the application of the larvae to the roots the HR was localized around the larvae, and cells adjacent to the hypersensitive cells showed no abnormalities in their ultrastructure that would indicate a graded response away from the infection site. The lack of such a graded response suggests that the stimulus initiating the HR does not pass readily from cell to cell and, in fact, is induced only by penetration
232
R. E. Paulson
and
J. M. Webster
of and secretion into a cell by the nematode stylet. The necessity of direct stimulation by the stylet would explain the presence of the relatively few hypersensitive cells along the path of nematode migration through the root tissue. Progressive breakdown of cell membranes and cytoplasm occurs during the HR of tobacco leaves to bacteria [4]. It has been suggested that changes in the permeability of vacuolar membranes and subsequent movement of toxic materials out of the vacuole cause the HR of plants to bacteria [6], and changes in the permeability of plant cell membranes are known to be induced by fungal toxins [la] and by fungal enzymes [S]. The disappearance of the electron-dense inclusions from the vacuoles of hypersensitive cells suggests that a change in the permeability of the tonoplast to the contents of the vacuole or to the enzymes necessary to dissolve the inclusions may have occurred. The rapid increase in electron density of the cytoplasm during the HR of Nematex to M. incognita is not seen in the reaction of tobacco leaves to bacteria [4] or in the reaction of cowpea leaves to rusts [5]. The increased electron-density of the cytoplasm of tomato roots may be accounted for by the presence in it of material normally found in the vacuole, the release of toxic products from the vacuole or the result of an interaction of the cytoplasm and nematode secretions. The fact that the electron-dense vacuolar inclusions disappeared and that the cytoplasm increased in electron density before any changes were seen in cell walls, membranes or organelles provides visual evidence that changes in permeability of the tonoplast may be the first changes that lead to general disorganization of the cell during the HR. An apparent increase in the number of ribosomes and a change in the form of the endoplasmic reticulum from short isolated segments associated with ribosomes to long smooth forms without ribosomes were the only changes in the structure of hypersensitive cells that were not obviously symptoms of cellular breakdown. These changes, which suggest increased protein synthesis, were not seen in adjacent apparently unaffected cells and were considered to be some of the first indications of the hypersensitive reaction. These features were followed closely by the loss of membrane distinctness and by the disappearance of mitochondria, features more closely indicative of cellular breakdown. Heterodera g&Gzes infection of resistant soybean roots [3] and Meloidogyne javanica infection of resistant peach root-stock [7] induced a susceptible response, in the form In contrast, of giant cell production, before a resistant response was initiated. M. incognita in Nematex, appears to activate resistance mechanisms resulting in a HR within 8 to 12 h. The stimulus that initiates hypersensitivity is likely a component of the nematode oesophageal gland secretions that acts sufficiently rapidly to prevent giant cell formation, a process which normally takes 24 to 36 h [9]. M. hapla larvae usually do not initiate any of the symptoms of hypersensitivity characteristic of M. incognita in Nematex roots. Apparently the secretions from M. hapla which initiate giant cell formation do not contain sufficient amounts of whatever component activates the resistance mechanism. The fact that a few cells do show a HR suggests that there is some differential sensitivity of Nematex root cells to this hypothetical component. Temperatures of about 33 “C drastically decreased the number of cells manifesting the HR but did not appear to cause any changes in the ultrastructure of
Root-knot
nematode
in tomato
233
cells unaffected by nematodes. The fact that this higher temperature did not preclude giant cell formation and did not result in any observable modifications in cell ultrastructure makes it tempting to postulate the inactivation of some enzyme system responsible for initiating the development of the HR. On the basis of observations of unsectioned Nematex roots infected by M. incognita, Dropkin [I] reported that 0.8 PM-kinetin significantly decreased the HR. However, our light and electron-microscopy observations indicate that while the kinetin-treated roots were stunted, swollen and had extensive lateral roots the HR was not significantly reduced. The fact that more giant cells developed in the kinetin-treated Nematex roots than in those without kinetin suggests that the kinetin favours development of giant cells while not significantly inhibiting the HR. Perhaps the excess kinetin suppresses the HR in the few cells that are close enough to the nematode to be stimulated into giant cell formation. However, the exogenous kinetin is not able to suppress completely the trigger which causes the HR. The hypersensitive cells, while not providing the nematode with sufficient nutrients for development, provide a sufficient stimulus to prevent it from seeking a more compatible site for development. The absence of structural changes in the cells adjacent to the hypersensitive region makes it unlikely that physical restrictions, analogous with the wall thickenings or the formation of a wood periderm observed by Van Gundy and Kirkpatrick [15], prevent the nematode from leaving the hypersensitive region. Second stage larvae removed from hypersensitive roots 2 days after inoculation are capable of subsequently infecting susceptible roots which indicates the absence of a toxic factor in the hypersensitive roots. Observations of the ultrastructural changes in the developing hypersensitive cells suggest that the HR may be initiated by the loss of some materials from the vacuoles to the cytoplasm which causes the deterioration of cytoplasmic organization and so effectively prevents giant cell formation. Subsequent death of the nematode larvae is likely due to the absence of the nutritive giant cells rather than to the toxic effects of the hypersensitive cells. This work was completed while the senior author was Research Council of Canada Scholarship, and the other grant (No. A4679) from the National Research Council seeds were kindly supplied by Dr V. H. Dropkin of Pathology, University of Missouri.
supported by a National author held an operating of Canada. The Nematex the Department of Plant
REFERENCES
V. H. (1969). The necroticreaction of tomatoesand other hosts resistantto Meloidogym: Reversalby temperature. PhytopathoZogV 59, 1632-1637. DROPKIN, V. H., HELGESON, J. P. & UPPER, C. D. (1969). The hypersensitivityreaction of tomatoes resistant to Meloidogyne incognita: Reversal by cytokinins. Journal of Nmatology 1,
1. DROPKIN, 2.
55-61. 3. ENDO, B. Y. (1965). Histological responses of resistant and susceptible soybean varieties and backcross progeny to entry and development of Heterodera g&&m. Phytofiathology 55, 375-381. 4. GOODMAN, R. N. & PLURAD, S. B. (1971). Ultrastructural changes in tobacco undergoing the hypersensitive reaction caused by plant pathogenic bacteria. PhysiologkaJ Plant Pathology 1, 11-15.
234
R. E. Paulson
and J. M. Webster
5. HEATH, M. C. & HEATH, I. B. (1971). Ultrastructure of an immune and a susceptible reaction of cowpea leaves to rust infection. Physiological Plant Pathology 1, 277-237. 6. KLEMENT, Z. & GOODMAN, R. N. (1967). The hypersensitive reaction to infection by bacterial plant pathogens. Annual Review of Phytopatholopy 5, 17-44. 7. MALO, S. E. (1968). Nature of resistance of “Okinawa” and “Nemaguard” peach to the root-knot nematode Meloidqyw jauanica. Proceedings of the American Society for Horticultural Science 90, 39-46. 8. MOUNT, M. S., BATEMAN, D. F. & BASHAM, H. G. (1970). Induction of electrolyte loss, tissue maceration and cellular death of potato tissue by an endopolygalacturonate trans-eliminase. Phytofiathologv 60, 924-93 1. 9. PAULSON, R. E. & WEBSTER, J. M. (1970). Giant cell formation in tomato roots caused by Meloidogpe incognita and Meloidogyne hapla (Nematoda) infection. A light and electron microscope study. Canadian Journal of Botany 48, 271-276. 10. PEACOCK, F. C. (1959). The development of a technique for studying the host/parasite relationship of the root-knot nematode Meloidogyne incognita under controlled conditions. Nematologica 4, 43-55. 11. POWELL, N. T. (1962). Histological basis of resistance to root-knot nematodes in flue-cured tobacco. Phytopathologu 52, 25. 12. RIGGS, R. D. & WINSTEAD, N. N. (1959). Studies on resistance in tomato to root-knot nematodes and on the occurrence of pathogenic biotypes. Phytofiathology 49, 716-724. 13. Ross, J. P. (1958). Host-parasite relationship of the soybean cyst nematode in resistant soybean roots. Phytopathologv 48, 578-579. 14. SAMADDAR, K. R. & SCHEPFER, R. P. (1971). Early effects of Helminthosporium victoriae toxin on plasma membranes and counteraction by chemical treatments. Physiological Plant Pathology 1, 319-328. 15. VAN GUNDY, S. D. & KIRKPATRICK, J. D. (1964). Nature of resistance in certain citrus rootstocks to citrus nematode. Phytopathology 54, 419-427.