Ultrastructure of lesions produced by Cercospora beticola in leaves of Beta vulgaris

Phytiological

Plant Patholou

(1979)

15, 13-26

Ultrastructure of lesions produced by Cercospora beficola in leaves of Beta vulgaris M. P. STEINKAMP, BSDF,

Crops Research Laboratory,

Fort Collins,

Colorado

State University,

CO 80523

S. S. MARTIN, L. L. HOEFERT and E. G. RUPPEL USDA,

SEA-AR,

Fort Collins,

CO 80523

(Accefited for publkation

and Salines, December

CA 93915,

U.S.A.

1978)

The fungus Ccrcospora beticola Sacc. incites a leaf spot disease of sugarbeet (Beta vulgaris L.). Lesion formation was studied by electron microscopy. One week after inoculation, the fungus was established in the intercellular spaces of the mesophyll and degenerative changes had occurred in cells near infection sites. Visible necrotic spots were produced as these. lesions enlarged. During the degenerative sequence cell membrane systems and organelles were disrupted; affected cells quickly collapsed and became necrotic. After 3 weeks a boundary zone with two regions separated the central necrotic tissue from apparently unaffected, healthy tissue outside the lesion. Two types of cell wall apposition occurred, one a localized callosetype in the lesion center, and the second a generalized wall thickening in the inner boundary zone. Electron-dense material occluded intercellular spaces in the outer boundary zone. During most phases of the disease, hyphae bridged the intercellular spaces or were attached to and followed the contours of host cell walls. Only late in the disease, long after host cells were necrotic, were hyphae observed within largely disintegrated host cell walls.

INTRODUCTION The deuteromycete fungus Cercosfiorabeticola Sacc. incites a leaf spotting disease of sugarbeet, Beta vulgaris L. (Chenopodiaceae). The uncontrolled disease is of potentially great economic importance in sugarbeet-growing areas where warm conditions and high humidity prevail during the growing season. Small necrotic lesions are produced at sites of fungal penetration into the leaf. Although leaf tissue outside lesions often appears healthy, entire leaves senesce and die as a result of heavy or multiple infections. Replacement of lost leaves occurs at the expense of actual or potential stored reserves, resulting not only in a decreased root yield, but also in a decreased sucrose content of roots at harvest [7, 461. Microscopic studies of this host-pathogen relationship have dealt with mechanisms of, and factors affecting, fungal germination and penetrations into the leaf 0048-4059/79/0400

13 + 22$02.00/O

@ 1979 Academic

Press Inc.

(London)

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M. P. Steinkamp

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[2O, 35, 36, 37, 451. Several studies have included observations of cellular and subcellular effects of the fungus after penetration [IO, 13, 15, 20, 32, 35, 43, 451. Michalikova [32] and Feindt [20] have made ultrastructural studies of some aspects of C. b&cola infection in sugarbeet, but there have been no comprehensive developmental studies, by either light (LM) or electron (EM) microscopy. Studies of other Cercospora-host combinations [S, 15, 25, 26, 29, 30, 331 have been equally limited. Our study was undertaken to extend and clarify the morphological observations of C. beticola infection in sugarbeet, including the location of the fungus within the lesion, its association with host cells, and its effects on host cell ultrastructure during lesion formation. This study also forms the basis for other investigations, now being conducted, concerning the physiological and biochemical interactions of this host-pathogen combination. MATERIALS

AND

METHODS

Cercospora culture Single-spore isolates of C. beticola were cultured on potato-dextrose agar and sporulation was induced on sugarbeet leaf extract agar as described by Ruppel [39]. Sugarbeet inoculation

and sampling

Sugarbeets, B. vulgaris “SP5822-0”, with intermediate resistance to cercospora leaf spot infection [40], were grown from seed in steamed soil in a glasshouse. At approximately 8 weeks of age, plants were inoculated with C. beticola by atomizing a conidial suspension (35 000 conidia per ml) on the foliage until leaves were throughly wetted. Plants were placed in a mist chamber for 48 to 72 h at 100% r.h. and 30 “C with 16 h of fluorescent light and then returned to the glasshouse. Because the fungus does not sporulate under glasshouse conditions, sporulation in leaf lesions was induced by returning plants 4 weeks after inoculation to the mist chamber for 3 days. Samples for electron microscopy were taken at four visually different stages of lesion development: (1) at 5 and 6 days after inoculation, when lesions were barely detectable with a hand lens and appeared as tiny, very slightly chlorotic depressions in the leaf surface; (2) at 10 to 13 days after inoculation, when enlarging lesions had reached about 1 mm in diameter and had definite brown necrotic centers, but no definite boundary zone; (3) at 32 days after inoculation, when lesions had ceased enlarging and frequently had red boundary areas surrounding holonecrotic, non-sporulating centers (because no further macroscopic changes were noted in lesions older than these, these lesions are called “mature”); (4) at 32 days after inoculation, when the fungus in mature lesions had been stimulated to sporulate. Tissue sambling

and processing

Samples of leaf tissue that included a single lesion were cut with a 5 mm cork borer or leather punch and placed in a drop of fixative. Each disc was then retrimmed and cut into square or wedge-shaped pieces 1 mm or less in width at the outer edge. Tissue pieces were transferred to fresh fixative containing 3% acrolein and 3% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.25, evacuated 15 to 45 min to remove air from the intercellular spaces, and fixed 1 to 2 h at room temperature

Lesions produced

by Cercospora beticola

15

[,?,?I. After three washes in the same buffer, samples were postfixed 2 to 3 h at 4 “C in 2% osmium tetroxide in O-1 M cacodylate buffer, washed with three changes of cold glass-distilled water, and soaked overnight at 4 “C in 0-5Oh aqueous uranyl acetate. After three additional washes in glass-distilled water, samples were dehydrated in an acetone series, passed through propylene oxide and embedded in Epon 812. Sections 0.5 to 1 pm thick were cut for LM monitoring of thin sections and stained with azure B [23]. Monitor sections of lesions 11 days after inoculation were stained with aniline blue and examined by fluorescence microscopy [la]. Paradermal sections were stained with Millonig’s lead [34] or uranyl acetate and lead, and examined with an with an (EM). RESULTS

Observationsof healthy mesophyllcells Healthy mesophyll cells of B. vulgaris have the usual complement of organelles typical of other such plant cells (Plate 1). Cell organelles are found primarily within a narrow band of peripheral cytoplasm that surrounds a large central vacuole. The nucleus is not lobed and contains a nucleolus and condensed chromatin. Chloroplasts occur singly and contain starch and small plastoglobuli. Observations5 to 6 days after inoculation Lesions at this stage consisted of tiny, slightly chlorotic depressions in the leaf surface. Tissue outside these depressions seemed to be healthy. Although gross necrosis of host tissue was not apparent in the depressions, collapsed cells with electron-dense necrotic cytoplasm were found (Plate 2). Other cells showing various degrees of collapse and distintegration were interspersed with the necrotic cells throughout the lesion. Small amounts of fungal hyphae were found in the lesions, especially in areas having the more severe degenerative changes. Hyphae bridged the intercellular spaces or were attached to host cell walls. Cell walls did not appear to have been penetrated by the fungus, although some cell walls (especially those of necrotic cells) appeared to have been altered or partially dissolved. Infrequently, cells with broken cell walls were seen and cellular debris was found in the intercellular spaces (Plate 8). Interspersed among necrotic cells were other cells showing slight to severe degenerative changes, with some areas of individual cells appearing more affected than others. Often slightly affected cells were partly collapsed. Although membrane systems within these cells generally were intact and organelles were recognizable, some abnormalities were apparent. The plasmalemma, tonoplast and organelle bounding membranes usually were intact. Many small vesicles, often with fibrillar contents, were present in the cytoplasm and some swelling of the endoplasmic reticulum (ER) occurred (Plate 2). Other cells contained abundant rough ER (Plate 4) or dictyosomes. Large vacuoles also were found in the cytoplasm (Plates 2 and 4). Nuclei occasionally were lobed, and somewhat vesiculate cytoplasmic areas were found between the nuclear lobes (Plates 3 and 4). Some nuclei had electron-lucent regions (“holes”) containing fibrillar material. Mitochondria also had occasional “holes”, and often showed thickened bounding membranes and swollen cristae (Plate 5). Microbodies were numerous and large (Plate 5). Chloroplast morphology was little altered in slightly affected cells; a slight increase in the electron-density of

M. P. Steinkamp

16

et al.

the chloroplast matrix and some grouping or stacking of the chloroplasts was noted. Occasionally, organelles in contact had thickening or proliferation of membranes at the interface (Plate 5). A layer of moderately electron-dense middle lamella-like material sometimes was found in the angles of adjacent cells, surrounding relatively healthy,cells between the plasmalemma and cell wall, between adjacent “healthy” cells or between ,“healthy” and necrotic cells (Plate 2). Increasingly degenerative changes involved the organelles and membrane systems of ‘other cells within these lesions. Cells in later or more advanced stages of collapse and necrosis had contents scattered throughout the cell interior. The plasmalemma and tonoplast were identifiable only in localized areas of the cell. More or larger plastoglobuli were present in some chloroplasts (Plate 6), but increases in starch were not apparent. Other chloroplasts were deformed, with outpocketings of stroma material and disruption of the bounding membrane (Plate 8). Mitochondria appeared swollen or disintegrated (Plate 8). Only fragments of other membrane systems and organelles remained. Large, irregular vesicles, perhaps representing swollen ER or dictyosome vesicles, were common throughout the cell interior. Cell wall appositions sometimes were found in these cells where they bordered intercellular spaces containing fungal hyphae (Plates 7 and 8). These appositions usually had a diffuse character with much electron-dense material in the form of droplets, vesicles and membranes in a rather sparse electron-lucent matrix. In addition, the boundaries between the appositions and cytoplasmic remains often were irregular and poorly defined. Bodies of unknown origin, usually with electron-dense granular cores surrounded by irregular less-dense areas, were found in the vacuolar region of some cells at all stages of degeneration (Plate 6). Observations

IO and 13 days after inoculation

Lesions had enlarged to about 1 mm in diameter and consisted of a central, visibly necrotic area which graded into visually healthy tissue at the periphery. Degenerative changes were observed as a continuum between slight ultrastructural modifications in cells at the periphery to macroscopic necrosis of cells at the lesion center. No distinct boundary area was present between healthy cells outside the lesion and degenerating cells within the lesion. Fungal hyphae were present in the intercellular spaces or attached to the outside of host cell walls throughout the area in which changes were found, but fewer hyphae were seen at the periphery of the forming lesions than in the central, more necrotic portion. Cells at the lesion periphery, when viewed by LM, appeared to be healthy and unaffected by the fungus. When these cells were examined with the EM, however, many structural modifications were found (Plates 9 and 10). Cells had somewhat irregular shapes. The ground cytoplasm was extremely electron-dense, obscuring the plasmalemma, tonoplast and organelle bounding membranes (Plate 10). Ribosomes could not be distinguished in this dense cytoplasm. Nuclei, while not otherwise altered, often were quite irregular in outline. Chloroplast shape also was somewhat irregular, and in many, an increased number and size of plastoglobuli were apparent (Plates 9 and 10). Dictyosomes and ER were swollen and contained fibrillar material in an electron-lucent matrix (Plate IO). Mitochondria also had a swollen appearance and contained thin, elongated or otherwise distorted cristae. Cytoplasmic vacuoles

Abbreviations used in plates: A, cell wall apposition; B, bacteria; C, chloroplast; CL, chloroplast lamellae; CV, cytoplasmic vacuole; CW, cell wall; D, dictyosome; Db, cell debris; ER, endoplasmic reticulum; F, fungal hypha; G, globular membrane debris; H “holes”; IM, intercellular material; IS, intercellular space; L, cell wall loop; M, mitochondrion; Ma, matrix material; Mb, microbody; N, nucleus; NC, necrotic cell; NL, nuclear lobe; P, plasmalemma remnants; Pg, plastoglobuli; PI, plastid; S, starch grain, T; tonoplast remnants; V, vacuole; VB, vacuolar body; Vs, vesicle; WT, cell wall thickening. PLATE 1. Healthy mesophyll cell. Chloroplasts (C) have starch grains and small plastoglobuli. A large central vacuole (V) is found. Arrowheads indicate the small amounts of material usually found in the angles of adjacent cells. x 4600. PLATE 2. Five-day lesion. A collapsed necrotic cell (NC) is adjacent to an essentially healthy cell. In the healthy cell, cell membranes and organelles are intact, although vesicles with fibrillar contents (Vs) and slightly swollen ER are found; a large cytoplasmic vacuole (CV) also is present. Arrowheads indicate a thick layer of middle lamella-like material found between the cell walls. x 12 000. PLATE 3. Five-day lesion. Nuclear “holes” (H) have fibrillar contents. Arrowheads :;how indentation of nuclear lobe. x 14 400. PLATE 4. Five-day lesion. Rough ER (ER) is abundant in cytoplasm. Vesiculate cyto,?lasm extends deeply into the nucleus between nuclear lobes (NL). A portion of a cytoplasmic .vacuole (CV) is present. x 18 000. PLATE 5. Five-day lesion. Mitochondria (M) have swollen cristae and a thickening or proliferation of bounding membranes at interfaces with other organelles (arrows). “Holes” :H) are present in one mitochondrion and in the plastid (PI). A large microbody (Mb) is present. x 33 600. PLATE 6. Six-day lesion. The tonoplast has fragmented, allowing mixture of cytoplasmic and vacuolar contents. The chloroplast has enlarged plastoglobuli (Pg). Electron-dense bodies (VB) are in the vacuolar region. x 17 600. PLATE 7. Six-day lesion. Portion of a relatively diffuse cell wall apposition containing electron-dense membranes and droplets. x 16 800. PLATE 8. Six-day lesion. Fungal hypha (F) and cell debris (Db) in the intercellular space [IS). The fungus abuts a necrotic cell wall. In the adjacent degenerated cell the chloroplast bounding membrane has disintegrated around the outpocketings of stroma (arrowheads), the plasmalemma (P) has degenerated into a series of droplets, mitochondria (M) are disintegrated and a diffuse cell wall apposition (A) is near the intercellular space. x 18 500. PLATE 9. Thirteen-day lesion. Although the cell is from the “healthy” lesion margin and organelles and membranes appear intact, the ground cytoplasm has increased density, cytoplasmic vacuoles (CV) are present, and chloroplasts have enlarged plastoglobuli but no unusual accumulation of starch. Some electron-dense material (IM) has accumulated in the intercellular space. A small apposition (A) is near a fungal hypha (F) : the hypha is in the intercellular space and is appressed to the wall of the adjacent cell. x 4400. PLATE 10. Thirteen-day lesion. Enlarged portion of a cell similar to that in Plate 9. The IIR has fibrillar contents (arrows), abnormal cristae occur in swollen mitochondria (M) and ribosomes are obscured by the dense ground cytoplasm. Chloroplasts have large plastoglobuli (Pg), but little starch (S). x 13 500. (T) is fragmented although organrlles are PLATE 11. Thirteen-day lesion. The tonoplast basically intact. ER and dictyosomes (D) are abundant. x 13 500. PLATE 12. Thirteen-day lesion. The plasmalemma has disintegrated into a series of electron-dense droplets (P) that line the cell wall and apposition (A). The apposition cont.ains much electron-dense material and is opposite a fungal hypha (F). An electron-dense mass of material (IM) confluent xvith the host cell IvaIl projects into the intercellular spar< near the fungus. x 14 400. PLATE 13. Thirteen-day lesion. A highly collapsed cell in which the plasmalemma (P) has fragmented or disintegrated into electron-dense droplets. Organelles have lost their bounding membranes and, except for the chloroplast lamcllar system (CL) and associated plastoglobuli (Pg), are not identifiable. x 9600. PLYL.E 14. l‘hirteen-day lesion. Small electron-lucrnt crystal from a cell similar to that sho~vn in Plate 13. x 18 500.

PLATE 15. Thirteen-day lesion. Starch grains (S) and plastoglobuli have been retained in association with internal chloroplast membranes after loss of the chloroplast bounding membrane and ground substance. Electron-dense globular material (G) may represent the product of the breakdown of the chloroplast bounding membrane. x 15 608. PLATE 16. Thirteen-day lesion. Although the nuclear membrane has ruptured, the nucleus (N) has retained some integrity and identity. Other organelle bounding membranes also have been lost or fragmented. x 14 700. PLATE 17. Thirteen-day lesion. Portion of necrotic cytoplasm that once material presumably dissolved during specimen preparation. x 16 800. PLATE 18. Thirteen-day lesion. Collapsed cell (NC) filled cytoplasm. Starch grains (S) are the only identifiable cytoplasmic not broken despite extensive bending into tight loops (L). Fungal the disproportionately large intercellular space. x 4000.

held

crystallized

with amorphous necrotic remains. Cell walls are hyphae (F) arc present in

PLATE 19. Thirty-two-day lesion. Cells from the outer boundary zone adjacent to healthy leaf tissue appear metabolically active with many mitochondria (M) and dictyosomm (D). Membrane systems and organelles are intact and appear healthy. The intercellular spaces are occluded with granular electron-dense material (IM). x 17 600. PLATE 20. Thirty-two-day lesion. Another view of the outer boundary zone. The intercellular space is occluded with electron-dense material (IM). Chloroplasts contain plastoglobuli that are similar in size and number to those of healthy material. x 14 000. PLATE 2 1. Thirty-two-day lesion. Collapsed necrotic cell from the inner boundary zone. The cytoplasm has organelle remnants with negative image membranes (arrows). Appositional wall thickenings (WT) are present. Moderately large amounts of intercellular material (IM) have accumulated in the intercellular space. x 13 200. PLATE 22. Thirty-two-day lesion. Collapsed necrotic cell from the inner boundary zone near the necrotic center of the lesion. The entire cell wall has become layered with irregularly electron-dense material (WT). Another mass of electron-dense material (IM) extends from the outer portion of the cell wall into the intercellular space. x 10 500. Healthy fungal tissue (F) is present in the intercellular PLATE 23. Thirty-two-day lesion. space (IS) of the necrotic area of the lesion. Large cell wall appositions (A) have formed in cells opposite the fungus, Cell walls adjacent to the fungus do not appear altered, although a granular matrix (Ma) is present between the hypha and cell wall. x 11 600. Portion of a cell from the central necrotic area of the PLATE 24. Thirty-two-day lesion. lesion. Remnants of the nucleus (N) and chloroplasts (C) with starch grains and negative image membranes are shown. The cell wall (CW) appears somewhat degraded. x 11 600. PLATE 25. Thirty-two-day lesion, sporulating. Healthy fungal tissue (F) is in the intercellular space (IS) between two necrotic cells (NC) and abuts a mass of intercellular material (IM). One necrotic cell (*) is almost devoid of cytoplasmit remains, whereas the other has typically necrotic cytoplasm. Bacteria (B) also are in the intercellular space. x 14 000. PLATE 26. Thirty-two-day lesion, sporulating. A fungal hypha (F) is within a largely disintegrated host cell. A cell wall apposition (A) and starch grains (S) are present in the adjacent necrotic cell. x 15 500.

PLATES

1, 2, 3, 4 and 5

_LI

I’LATEII

11, 12, 13, 14, 15 and

16

.

-

1

PLATES 21, 22, 23, 24, 25 and

26

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or vesicles of various sizes and with electron-lucent contents were found in some localized areas of the cytoplasm (Plate 9). Wall appositions occurred in some areas of the cells between the plasmalemma and cell wall (Plate 9). Small amounts of granular, electron-dense material were found in the intercellular spaces, especially in the angles of adjoining cells (Plate 9). More degenerative changes, some of which were apparent with the LM, occurred in cells located just interior to or interspersed with those near the periphery of the lesion. These cells had highly irregular and sinuous contours indicative of partial collapse. The tonoplast had fragmented or disappeared and the cell contents were scattered throughout the cell interior (Plate 11). Thus, the central vacuole had largely disappeared from these cells. Apart from the tonoplast (and in some cells the plasmalemma), membranes usually appeared intact. Dictyosomes and ER were abundant in some of these cells (Plate 11). Further degenerative changes were found in cells nearer the lesion center (Plates 12 to 16). Cells were largely collapsed (Plate 13). In addition to the tonoplast, many other membranes were broken or otherwise disintegrated. The plasmalemma was found only as occasional fragments of intact membrane (Plate 13), or, more commonly, as a series of small osmiophilic droplets bordering the cell wall in the usual location of the plasmalemma (Plates 12 and 13). Membranes bounding organelles also had ruptured or dissolved, leaving only remnants of organelles and fragments of membranes scattered through the cell interior (Plate 13). The internal membranes of the chloroplasts appeared least affected and frequently remained more or less intact even after their limiting membranes and ground substance had been lost (Plates 13 and 15). Plastoglobuli and starch grains usually remained associated with the internal chloroplast membrane system. Aggregates of small globules with electron-lucent centers and electron-dense boundaries often were adjacent to the chloroplast remnants (Plate 15). Although the nuclear envelope had fragmented, the nucleoplasm retained some coherence and identity (Plate 16). Wall appositions sometimes were seen between the cell wall and remnants of the plasmalemma, especially in areas where fungal hyphae were near or attached to the cell wall (Plate 12). In monitor sections stained with aniline blue, these regions fluoresced brightly under U.V. excitation. Although sometimes containing electron-dense material and having fungal hyphae attached to them, cell walls appeared intact (Plate 12). Accumulations of intercellular electron-dense material occasionally were found confluent with host cell walls (Plate 12). These masses were located in the angles between adjoining cells and in small spaces between adjacent cells, in addition to simply projecting into the intercellular spaces. Fungal hyphae sometimes were located nearby, but were rarely directly associated or in contact with the intercellular material (Plate 12). Cells nearer the lesion center than those just described had the same general appearance, but were even more collapsed, and had more amorphous and electrondense cell contents. Small electron-lucent crystals occasionally were noted in these cells (Plate 14), but no large areas of crystallized material were seen. Completely collapsed and necrotic cells comprised the central area of the lesion (Plate 18). Fungal hyphae frequently occurred in the intercellular spaces or attached to cell walls (Plate 18). Although cell walls often were so bent that they formed tight 3

M. P. Steinkamp

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loops, breaks in the celI wall were rarely seen. No hyphae were found within the cells, although cell walls sometimes had collapsed around, and thereby enclosed, a hypha. Most cytoplasm was excluded from the cell wall loops, and that remaining in the cells was confined to their central, less collapsed areas; this cytoplasm was extremely electron-dense and amorphous. Starch grains, negative-image membranes apparently derived from chloroplast lamellae and, occasionally, amorphous, electrondense nuclear material were the only recognizable organellar remnants in the necrotic cytoplasm. Plastoglobuli, which had been prominent until late in the degenerative sequence, could not be identified in totally necrotic cells. Cytoplasm of the necrotic cells frequently contained areas that apparently had held crystallized material (Plate 17). These sometimes extensive areas appeared empty; the material probably was dissolved during specimen preparation. Fungal hyphae generally had a healthy appearance throughout the developing lesion. Observations

32 days a&r

inoculation

At approximately 3 weeks after inoculation, lesions reached their maximum size; little if any additional enlargement of the typically collapsed necrotic area occurred until much later when the entire leaf, if heavily infected, gradually senesced. Unlike younger developing lesions, in 32-day lesions there was a distinct boundary area between the relatively healthy tissue surrounding a lesion and the grey-brown tissue comprising the lesion proper. The boundary zone tissue was green to wine-red in color. Tissue outside the boundary zone appeared healthy and no ultrastructural modifications associated with the disease were found in these cells. The boundary zone is, ultrastructurally, a highly modified area of the leaf mesophyll, and cells adjoining healthy leaf tissue differ from those adjoining the necrotic area. Thus, two areas can be distinguished. Cells in the “outer” boundary zone adjoining healthy leaf tissue were not collapsed, and were generally similar in size and structure to other healthy, mature mesophyll cells. No meristematic activity was noted. Although not necessarily collapsed, “inner” boundary zone cells near the border with the necrotic area were necrotic and contained, around the remains of the central vacuole, cytoplasmic remnants with negative image membranes (Plate 21). Others of these cells were completely collapsed and had typical amorphous cytoplasm. However, many of these border area cells had cell wall thickenings or appositions between the cytoplasmic remains and the inner surfaces of the cell walls (Plates 21 and 22), but these appositions appeared structurally different from the appositions described earlier. Appositions in boundary zone cells often were thinner in cross-sectional dimensions, more evenly and extensively deposited along the cell wall, and contained larger amounts of electron-dense material. In addition, larger amounts of electron-dense material were found in the intercellular spaces of inner boundary zone tissue than were found in the central lesion area; this material also was found attached as blebs to cell walls or in the angles where two cells abut (Plate 21). Fungal hyphae were found only in this inner portion of the boundary zone. Outer boundary zone cells appeared relatively healthy and metabolically active with large amounts of cytoplasm containing rough ER and many mitochondria, chloroplasts and dictyosomes (Plate 19). Chloroplasts in these cells sometimes contained many or larger-than-usual plastoglobuli, but no unusual accumulations of starch.

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No cell wall appositions or thickenings were present in these cells. The most prominent feature of this boundary zone region was the very large amount of intercellular material, which often filled and obliterated the intercellular spaces (Plates 19 and 20). The granular, electron-dense material was similar to the more localized masses found in developing and mature lesions and in the inner boundary zone. In the healthy portion of the leaf outside the boundary zone, the intercellular material progressively and rapidly diminished until it was no longer found. Cells in the central necrotic region of mature lesions differed little in appearance from necrotic cells in developing lesions. Cells were collapsed and filled with dense amorphous cytoplasm containing, as recognizable organelle remnants, only negative image membranes, usually from chloroplasts, occasional starch grains and amorphous nucleoplasm (Plate 24). Wall appositions sometimes were prominent in the necrotic cells, and frequently were found adjacent to apparently healthy fungal hyphae (Plate 23). A few necrotic cells had degraded areas of the cell wall (Plate 24); hyphae, however, were not necessarily found in close proximity to these areas. Observationsof sporulating lesions 32 days afir inoculation Lesions containing sporulating fungi were similar to mature 32-day lesions; no further enlargement had occurred and a boundary zone separated each lesion from surrounding healthy tissue. The necrotic centers of these lesions appeared greyish-white with many small, darkly pigmented spots (pseudostromata) scattered throughout the center. Conidiophores bearing conidia projected above the lesion surface, apparently extending from the pigmented spots. Necrotic tissue from sporulating lesions was thinner, softer and more fragile than the dry and brittle necrotic tissue of nonsporulating lesions. Necrotic host cells of sporulating lesions were more extensively degraded than those of non-sporulating lesions. Although starch grains often were recognizable, most organelle and negative-image membrane vestiges had disappeared, leaving the necrotic cytoplasm as an irregular, electron-dense, granulate mass (Plate 26). Although some cells contained large amounts of this material, many cells were almost devoid of cytoplasmic remains (Plate 25). Cell wall ultrastructure varied greatly; areas appearing intact and relatively unaltered were found near areas having either localized erosion (Plate 25) or extensive disintegration (Plate 26). Cell wall appositions occasionally were noted and appeared intact, regardless of the condition of the adjacent cytoplasm or cell wall (Plate 26). The granular, electron-dense material found in the intercellular spaces appeared morphologically similar to that found in other stages of the disease (Plate 25). This material did not appear to have any significant morphological effect on the fungus and vice versa. Healthy-appearing fungal tissue was abundant in the sporulating lesion, either as long filamentous strands or as groups of rounded cells. The filamentous hyphae were ramified throughout the lesion, usually in the intercellular spaces and following the contours of the necrotic cells (Plate 25), but also growing through fractured or disintegrated cell walls and within necrotic cell interiors (Plate 26). The rounded groups of fungal cells formed compact tissue pads (pseudostromata) which were large enough to fill the intercellular space and sometimes displace adjacent necrotic host cells. These pseudostromata comprised the darkly pigmented areas from which the

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M. P. Steinkamp

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conidiophores bearing conidia rose above the jesion surface. The fungal cells were embedded in an electron-dense granular matrrx, and appeared metabolically active with large amounts of cytoplasm containing many mitochondria, ribosomes, and extensive ER. Some necrotic or senescent fungal cells without contents or with small amounts of condensed, electron-dense, amorphous cytoplasm were noted, but these were rare. Bacteria sometimes were observed in tissue samples from sporulating lesions (Plate 25). These secondary invaders usually were found in the intercellular spaces, but occasionally were seen within necrotic cells. They appeared not to be involved in the disease or degenerative sequence.

DISCUSSION

The degenerative process within the leaf begins with the entrance of the pathogen through a stomate and its subsequent limited ramification through the intercellular spaces of the mesophyll. Under favourable conditions, within 5 days after inoculation, fungal hyphae are present in the intercellular spaces and sometimes are appressed or attached in places to host cell walls. Although there is no macroscopic necrosis in the earliest detectable lesions, a few necrotic cells are interspersed with other cells in various stages of degeneration and collapse. Intercellular material is present in small amounts. As lesion development continues, the amount of necrotic tissue gradually increases until the lesion reaches maturity. Mature lesions differ from developing lesions in several ways. Mature lesions have ceased enlarging, and there is no longer a continuum between healthy and necrotic cells. Instead, all cells within the central portion of the lesion are necrotic, and fungal hyphae are found throughout that area. Furthermore, the mature lesion possessesa clearly defined, sometimes colored, “boundary zone” that separates the central necrotic tissue from the healthy leaf tissue beyond. Two boundary zone regions have been distinguished. The “inner” region is characterized by necrotic cells which may or may not be collapsed, but which often have a generalized thickening of their cell walls. Fungal hyphae often extend from the necrotic central area of the lesion into this boundary region. This region may correspond to either of two “cicatrice” regions described by Cunningham [1.5] as the “mass of dead collapsed cells attached to the phellem layer”, or the phellem layer in which cells are pictured as irregularly shaped, and are described in general with other “cicatrice” cells as having slightly thickened walls. In the “outer” boundary zone region, no hyphae are found and the intercellular spaces are largely occluded with electron-dense granular material. Cells surrounded by this material appear healthy and metabolically active, but not meristematic. Cunningham ascribed occlusion of the intercellular spaces to renewed meristematic activity of the mesophyll; the cells were illustrated and described as large and irregularly shaped, with some cell wall thickening. Where the intercellular material does not entirely fill the intercellar spaces, cell walls, by light microscopy, could appear thickened. Sole1 & Minz [43] in a LM study also found no evidence of a cicatricial barrier such as that described by Cunningham. Therefore, Cunningham’s term “cicatrice” for this region appears to be inaccurate and a more neutral term such as “boundary zone” should be used until definitive

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Cercospora betida

21

studies of the development of this region are made. It is noteworthy that no “cicatrite” was found in other Cercospora infections studied by Cunningham [X5]. Electron-dense granular material is a prominent constituent of the intercellular spaces of the outer boundary zone. The material does not fluoresce in aniline bluestained sections; therefore, it probably is not callose. Its EM appearance does not resemble the intercellular fibrillar material, consisting primarily of lignin and suberin, that is found around virus-induced local lesions in leaves of Gomphrena globosa [4]. Instead, its EM appearance and frequent location in the angles of adjacent cells suggest similarity to pectic material of the middle lamella. Intercellular pectic material in the forms of “warts” has been described in several plants and plant tissues and organs [ 14, 281. Occlusion of intercellular spaces by pectic material also has been reported [14], and is a feature of collenchyma tissue [17]. The appearance of the pectic “warts” resembles that of the intercellular material in the lesions produced by C. beticola. The function of these materials is unknown, either in healthy plants [143 or in Cercospora-infected sugarbeet leaves. The material does not appear harmful to the fungus, as seemingly healthy hyphae are found in contact with it. It does not appear to be degraded by the fungus. Although some of the material is found throughout lesion development, by far the largest amounts occur in the outer boundary zone. Here the material might help in wound healing by sealing off healthy from necrotic tissue, or it could act as a physical barrier to the fungus [3.5] or its products. However, Schlosser [II], referring to Cunningham’s description of a “wound periderm”, discounted such a zone as a resistance mechanism against fungal infection. The fungus could be self-limiting, but evidence indicates that the plant is involved in fungal limitation; sugarbeet genotypes selected for their field resistance to the disease have fewer and smaller lesions than susceptible varieties (Ruppel, unpublished), and sugarbeet produces two flavonoids that in vitro can limit fungal growth and thus are potential phytoalexins [27, 311. Depending on the sugarbeet variety, the boundary zone sometimes is colored from pale pink to deep wine-red as a result of betacyanin accumulation. These highly water-soluble pigments are present in the cell vacuole, and they are known to accumulate in response to wounding [48], although their function around the wound site is unknown. Examination of leaf samples prepared from a deep maroon, betacyanin-containing sugarbeet variety showed, as expected, that these compounds mostly are removed during tissue processing; thus, they appear unrelated to the intercellular material that also accumulates primarily in the outer region of the boundary zone. The intercellular material also is found in boundary zones in which no betacyanin accumulation can be seen. Cell wall apposition is a general term used to describe the paramural growth or deposit of materials onto an existing cell wall surface [9]. The occurrence, structure, histochemistry and possible functions of appositions in wound healing or as defense mechanisms against pathogens have been reviewed [I, 2, 9, 42, 471. Two types of cell wall appositions were found in lesions produced by C. beticola, one in the central area of the lesions and the second in the inner region of the boundary zone. The first type of apposition, more typical of those encountered in other hostpathogen interactions [1, 91, was localized but irregular in shape, and was comprised of electron-lucent, flocculent material that often contained electron-dense

22

M. P. Steinkamp ef al.

droplets or membranes. In developing lesions at 11 days after inoculation, these appositions in aniline blue-stained monitor sections appeared as frothy, fluorescing regions appressed to the inner cell wall; they did not autofluoresce in azure B. Thus, we assume that a glucan [19], probably callose [16, 181, is an important constituent of these appositions. They were found in some cells at all stages of lesion development. In the earliest stages, appositions occurred in degenerating cells where they were infrequent, relatively small, and structurally diffuse. In older, more advanced lesions, larger and structurally more defined and compacted appositions were found in proportionally more cells. In such lesions, appositions were present in cells at all degenerative stages, including necrotic cells. Appositions persisted in sporulating lesions, even though most other cellular components had been degraded beyond recognition. Callose-type appositions frequently were juxtaposed with fungal hyphae, but numerous examples were seen of appositions without nearby hyphae, and of hyphae appressed to host cell walls without adjacent appositions. The unseen fungus or apposition could, of course, be nearby but out of the plane of section. Thus, the precise relationship between the fungus and production of appositions is unclear, but the fungus or its products evidently can induce appositions in cells. Appositions increase from the earliest developing lesions to mature lesions, suggesting either that the host response is not rapid or that elicitation of the response increases as the fungus ramifies and the lesion develops. The second type of apposition was common in cells of the inner boundary zone near the necrotic area of the lesion, but was not found in the outer boundary zone. These appositions were comprised of compact, electron-dense material spread in rather even layers around much of the inner surface of the cell walls. They can be called appositions in the definitive sense [9] because they are located between the cell wall and protoplast. We have not yet investigated the chemical nature of these wall thickenings. Cunningham [15] did not report callose-type appositions in tissue he studied, but noted the presence in the boundary region of slight cell wall thickenings, which he attributed to lignification and suberization. Sole1 & Minz [43], however, found no evidence of suberization of cell walls. This discrepancy could be due to differences in the age of lesions examined, which was not specified in either case. As we have noted, these wall changes were not seen in lesions less than about 3 weeks of age. Cell collapse is an interesting feature of this host-pathogen interaction. Early lesions are detected in part by leaf surface depressions resulting from this collapse. Within lesions, cells may be partly collapsed, although their cytoplasm appears almost normal ultrastructurally; cell collapse increases progressively with the degenerative sequence. This early collapse of entire cells is quite different from plasmolysis, or from collapse of the protoplast (false plasmolysis) as described by Hanchey & Wheeler [21]. Obviously, host cell wall changes must occur to allow such dramatic collapse, but the nature of the changes is not shown by EM. C. beticola in culture produces moderate amounts of pectinolytic enzymes [44], and thus, presumably, has the in vivo ability to attack cell walls [3, 61, but only occasionally have we observed small, localized areas of wall degradation, usually in already necrotic cells. It is not clear whether these changes are direct effects of the fungus or are associated with

Lesions produced

by Cercospora beticola

!a

host cell autolysis. Collapsed cells filled with necrotic cytoplasm are noted in other local lesion diseases [e.g. 241, and collapse of cells in several Cercs.$oru-host combinations has been shown [15, 25, 331. The densely staining substance described in the latter studies and in sugarbeet cells affected by C. betkola [X5] is the degenerating cytoplasm of cells in which the tonoplast has been lost, or the necrotic cytoplasmic remains we have described. That C. beticola has an intercellular growth phase in sugarbeet has been recognized widely [13, 32, 35, 431, but it usually has been assumed that this fungus is basically an intracellular parasite, and that intracellularity is an essential aspect of the disease process that results in cell death [IO, 32, 381. However, several observations suggest that intracellularity is not significant or necessary either to production of disease symptoms or to completion of the fungal life cycle. For example, the fungal mycelium remains viable for at least 10 weeks in mature sugarbeet lesions, where it is completely surrounded by necrotic tissue and perhaps walled off from healthy tissue by the boundary zone. The life cycle can be completed by sporulation at any time given proper temperature and humidity. Furthermore, C. beticola has been reported “growing” on dead leaves of several “non-host” plants [45]. Most importantly, we have found no evidence of the active penetration or even significant alteration of a living host cell wall. In developing or mature, non-sporulating lesions, the extensive collapse of cells around fungal hyphae creates relatively large, rounded intercellular spaces, and this can make it appear (especially by LM) that hyphae are within cells. The accumulation of rounded masses of intercellular material can further confuse a viewer. However, EM examination always shows that such hyphae are in fact in the intercellular spaces. We have observed the fungus within cells only in highly degraded necrotic tissue, found mostly in sporulating lesions. We therefore believe that the occasional fungal presence within a host cell probably results from its passive entrance through a broken or degraded cell wall. Latham [29], in an early study of cercospora leaf spot of cowpea, reported that only after cells reached a holonecrotic condition did the fungus become intracellular. The fungal “haustorium” Latham illustrated inside a cell may actually have been a cell wall apposition. In an ultrastructural study of sugarbeet leaves infected by C. beticola, Michalikova reported an increased number and size of starch grains in chloroplasts at 3 days after inoculation [32]. Later, after the pathogen presumably penetrated the cells (but for which no ultrastructural evidence was presented), convolution and disruption of the chloroplast membrane and lamellae were described. We found occasional apparent increases in starch content of chloroplasts of cells in some early developing lesions, but we are not convinced that the starch content was outside the normal range of variability found in healthy cells. Other changes similar to those described by Michalikova were found in chloroplasts of more heavily damaged cells, but we found no evidence that the pathogen entered the cell before these changes took place. We also found a consistent and impressive increase in the size or number of plastoglobuli in chloroplasts of cells at most stages of the degenerative process. This was not described by Michalikova, and could be due to varietal differences or to lack of tissue preservation in her material. Increases in the size and number of plastoglobuli have been observed in chloroplasts both from other diseased tissue [II, 12, 241 and in naturally senescing tissue [II]. Often these increases were associated with thylakoid membrane

M. P. Steinkamp

24

et al.

degeneration within the chloroplasts [11]. In C. beticolu infected sugarbeet, however, the chloroplast lamellae remain apparent for some time following loss of the chloroplast bounding membranes and stroma. We emphasize that chloroplast effects are only a small part of concurrent degenerative effects in sugarbeet cells, and that cells showing early effects ultimately pass through the degenerative sequence leading to necrosis. Several observations corroborating our description of the Cercospora-sugarbeet interaction have been reported by Feindt [2&J, who made a comparative ultrastructural study of lesions from relatively resistant or susceptible sugarbeet varieties. Unfortunately, he gave the age of lesions studied by EM only as “Zwischen dem 4-8 d p. i. . . .“, a period during which many developmental changes can occur. Feindt described and discussed the presence of extracellular material (our “intercellular material”), callose-type appositions and appositional cell wall thickenings. He noted the collapse of cells in forming lesions and that the fungus remains in the intercellular spaces. His description of effects on host cell organelles was limited, but some effects similar to those we have described can be seen in his illustrations. Effects of C. beticola on sugarbeet are produced while the pathogen is in the intercellular spaces throughout the period of symptom development. Thus, host-pathogen interchanges must occur across cell walls of both organisms. C. beticola produces, in culture, at least two toxins capable of inducing necrotic lesions when applied exogenously to leaves of sugarbeet [5], and the host responds to fungal presence by accumulation of phytoalexin-like compounds in and around lesions [27, 311. Biochemical and ultrastructural studies of the effects of these compounds are in progress. Preliminary findings suggest that the fungus-produced toxins may effect some of the degenerative changes seen in the lesions described here. We thank Dorothy Donoghue for excellent technical assistance, and Dr Penelope Hanchey for loan of equipment. This cooperative investigation of USDA, SEA-AR, the Colorado State University Experiment Station and the Beet Sugar Development Foundation is published with the approval of the Director, Colorado State University Experiment Station, as Scientific Series Paper No. 2383. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that also may be suitable. REFERENCES 1. AIST, J. R. (1976). Papillae and related wound plugs of plant cells. Annual Review of Phytopatholo~ 14, 145-163. 2. &ST, J. R. (1977). Mechanically induced wall appositions of plant cells can prevent penetration by a parasitic fungus. ScicncG 197, 568-570. 3. ALBERSHEIM, P. & ~DERSON-PROW-Y, A. J. (1975). Carbohydrates, proteins, cell surfaces, and the biochemistry of pathogenesis. Ann& &vi& of Plant Physiology k, 31-52. 4. A~PIANO, A., PENNAZIO, S.. D’Aoosrmo, G. & REDOLFI, P. (1977). Fine structure of necrotic local lesions induced by tomato bushy stunt virus in Gotnihre; globosa leaves. Physiological Plant Pathology 11, 327-332. 5. BALIS, C. & P+yrm, M. G. (1971). Triglycerides and cercosporin from Cercospora beticola: Fungal growth and cercosporin production. Phytofiathologv 61, 1477-1484.

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produced

by Cercospora

betkola

25

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