Physiological Plant Pathology (1977) 11, 171-178

Ultrastructure of penetration by Welminthosporium maydis

Department of Plant Pathology, Unioersity of Kentucky, Lexington, Kentucky SO506, U.S.A.

(Acceptedfor publication April 1977)

Penetration by Helminthosporium maydis race T into susceptible leaves from Texas male sterile and resistant ones from normal plants was studied with four different corn inbred lines. Six hours after inoculation, appressoria-like structures were present but were rare, and no evi- dence of penetration beneath such structures was found. All penetrations seen 6 h after inoculation took place between epidermal cells, and at least two-thirds of these occurred between cells adjacent to stomates. Breaks in the cuticle above subsidiary cells of the stomata1 apparatus may account for the high frequency of penetration in this area. Eight to 10 h after inoculation, miniature infection cushions had formed at initial points of penetration, and from these, hyphae radiated out beneath the cuticle of epidermal cells. Haustoria-like branches produced by these subcuticular hyphae appeared to function in secondary infec- tion. Hyphae of the fungus were seen occasionally within stomata1 openings (apparent stomata1 penetration), but these were consistently associated with penetrations which had occurred between nearby epidermal cells. No differences were observed at any stage of penetra- tion or initial colonization between Texas male sterile and normal plants of the same inbred or among the four inbreds studied. These results suggest that resistance to this pathogen develops only after infection has occurred and that the selective pathotoxin produced by race T does not play an important rBle in the infection process.

INTRODUCTION Southern corn leaf blight caused by Helminthosporium maydis Nisik. and Miyak. on <ea mays L. has been the subject of extensive investigation since the costly outbreak of this disease in 1970. Two races of H. maydis, designated 0 and T, have been recognized. Corn cultivars that carry Texas male sterile (Tms) cytoplasm are much more susceptible to race T than those with normal (N) cytoplasm. Cultivars with Tms cytoplasm are also much more sensitive than N cultivars to a pathotoxin produced by isolates of race T but not by those of race 0 [8]. This selective toxicity a.ppears to account, at least in part, for the high virulence of race T on Tms plants.

Since the extensive literature on physiological effects and possible mode of action of the pathotoxin produced by H. maydis (Hm T-toxin) has been reviewed several times [8, 12, 141, only a brief summary of this work will be given here. The discovery that low concentrations of Hm T-toxin caused swelling and loss of respira- tory control in mitochondria isolated from Tms plants but not in those from N plants suggested that mitochondria were the site of action of this toxin [IO]. This hypothesis was attractive because an effect on a cytoplasmic organelle would account for the cytoplasmic inheritance of disease reaction [SJ. Failure to detect early effects of Hm T-toxin on respiration or on mitochondria in situ coupled with evidence

172 H. Wheeler

of rapid selective effects on K + fluxes and electrochemical potentials led to the alternative hypothesis that the initial effect of the toxin was on cell permeability [Z]. To date neither hypothesis has been ruled out, and it should be noted that they are not mutually exclusive. Selective effects of Hm T-toxin on root growth, pollen germination, photosynthesis, dark CO, fixation and transpiration also have been reported [12, J4]. These results suggest that Hm T-toxin acts, directly or indirectly, at many different sites.

Compared to the extensive work on physiological changes associated with southern corn leaf blight, relatively little is known about the cytological aspects of this disease. In a histological study, presumably using a race 0 isolate of H. maydis since the work was done before race T was recognized, Jennings & Ullstrup [9] found that spore germination and penetration were similar on resistant and susceptible leaves. The same results, no significant differences in spore germination, germ tube production, appressorial formation or penetration, were obtained when Tms and N leaves were inoculated with H. maydis race T (W. K. Wynn, personal communication). White et al. [IS] reported that the first detectable ultrastructural change, rupture of the tonoplast, occurred 6 h after inoculation of Tms leaves with spores of H. maydis race T. Later the chloroplasts became spherical, and 24 h after inoculation plasma membranes were ruptured and chloroplasts and mito- chondria were disorganized. Whether similar changes occurred in resistant N leaves was not determined.

In scanning electron micrographs, Blanchard [13] observed that hyphae of H. maydis growing on the surface of corn leaves appeared to be coated with an extracellular mucilaginous sheath. The discovery that hyphae of this pathogen ramify extensively beneath the cuticle of epidermal cell walls [17] led to the sugges- tion that structures which appeared to be fungal sheaths in scanning electron micro- graphs might actually represent the host cuticle which ruptured during specimen preparation. Blanchard [5] also reported the deposition of an uncharacterized intercellular substance termed “matrix” in resistant N but not in susceptible Tms leaves 48 h after inoculation with race T. Brotzman et al. [S] have presented cyto- chemical evidence that this matrix material, which appears fibrous in electron micrographs, is proteinaceous.

Two investigations [Z, 151 failed to detect ultrastructural changes in Tms or N roots exposed for up to 2 h to Hm T-toxin at a concentration that caused 90% inhibition of root growth of Tms seedlings. Longer exposures (up to 8 h) resulted in a general and gradual disorganization of cellular ultrastructure in Tms but not in N roots. The only rapid effect of T-toxin at this concentration was on uptake of uranyl salts into vacuoles which was strongly inhibited by exposures to toxin for 30 min [15]. Other investigators [1] using a toxin preparation, calculated to be 5 times more concentrated, observed a loss of density in the matrix of mitochondria in cells of Tms but not N roots exposed to T-toxin for 15 min.

Lack of information at the ultrastructural level about penetration and other early events in pathogenesis caused by H. maydis represents a serious gap in our knowledge of southern corn leaf blight. The present investigation was undertaken to close this gap and thereby make southern corn leaf blight more valuable as a model for foliar diseases. An abstract of some of the results has been published [I7].

Ultrastructure of penetration by Helminthosporium maydis

MATERIALS AND METHODS

173

Spores of H. maydis race T from the high toxin producer, isolate F [16], were washed 3 times by centrifugation and finally resuspended at a concentration of 104 spores/ ml in sterile deionized water to which one drop of Tween 40 per each 50 ml had been added. Small drops (c. 5 ~1) of this spore suspension were placed on the upper surface of first leaves of corn plants grown for 13 to 15 days at 26 “C under a 16 h photoperiod of 8800 lux. In one test, first leaves of intact plants were inoculated, whereas, in two additional tests, detached first leaves were used. Entirely similar results were obtained in all three tests.

Four pairs of inbred corn lines, W64A, B37, MO1 7 and 33-16, with one member of each pair carrying Tms and the other N cytoplasm were studied. To guard against the possibility that some of these pairs were not well matched, three different lots of W64A and two of 33-16 seeds were used. In a preliminary test, tissue samples were taken 2, 4, 6, 8 and 10 h after inoculation, fixed in glutaraldehyde followed by osmium tetroxide and processed for electron microscopy as in previous work [11]. No evidence of infection could be found in the 2 or 4 h samples, whereas in those taken 8 and 10 h after inoculation, the fungus had ramified extensively beneath the cuticle of epidermal cells and had penetrated up to halfway through the leaf. Since the objective was to investigate the earliest stages in the infection process, work was concentrated on samples taken 6 h after inoculation in which penetrations had occurred but were rare. In each of three separate tests, at least one point of penetration in at least two, and in most cases three leaves of each Tms and N inbred line, was examined in samples taken 6 h after inoculation. One leaf from each inbred line was examined in each of two tests at 8 and 10 h after inoculation. Single control leaves of each inbred line were examined 6 and 10 h after they had received droplets of water. Whenever possible, 5 to 20 different sections through each infection structure were examined. No attempt was made to obtain complete sets of serial .sections.

RESULTS .Pre#enetration observations

Hyphae of H. maydis were found, for the most part, growing on or just above the surface of the leaves 6 h after inoculation. None of these superficial hyphae had Twell-defined coats or sheaths but occasionally diffuse, poorly defined extracellular :material was present [Plate 1 (d)]. Hyphal tips were found close to or in contact -with the host cuticle but no evidence of cuticular dissolution or changes in the host cell walls was seen [Plate 1 (a) and (d)].

Structures which resembled appressoria were rare [Plate 1 (b) and (e)]. A total of eight, five on Tms and three on N leaves, were found in material fixed 6 h after inoculation. In all cases, the upper surfaces of such structures were coated with extracellular material, but there was no evidence that this served as a muci- l.aginous sheath to cement the structure to the leaf surface. As Plate 1 (b) and (e) shows, this extracellular substance was greatly reduced or absent in the area of contact between the fungus and the host cell wall. Although poorly defined, the ruptured cuticle appeared partially to encase the lower part of the fungal structures [Plate l(b) and (e)].

14

174 H, Wheeler

Evidence of a host response in the form of a small papilla [Plate l(e) arrow] was seen beneath only two of eight appressoria-like structures in material fixed 6 h after inoculation. Larger papillae were found 8 and 10 h after inoculation [Plate l(c) and (f)] but these also were rare. The largest papilla seen [Plate l(f)] developed beneath a structure which cannot be considered an appressorium because it is clearly beneath the host cuticle. Plate 1 (f) probably represents a cross-section of a subcuticular hypha such as those shown in Plate 7. No evidence of successful penetration beneath the appressoria-like structures shown in Plate 1 (b) and (e) was ever found in Tms or N leaves.

Primary penetration

All successful penetrations in leaves fixed 6 h after inoculation took place between epidermal cells. Most occurred near stomates, either between a guard cell and a subsidiary cell or between a subsidiary cell and the adjoining epidermal cell. Penetra- tions in this area accounted for 23 of 32 on Tms and 20 of 28 on N leaves. These figures indicate that approximately two-thirds of all penetrations occurred between cells adjacent to the stomata1 apparatus. Actually many more, and perhaps all, penetrations occurred in this area because stomates above or below the point of penetration would not be detected unless a large number of sections was examined.

Typical examples of early stages in penetration, which were entirely similar on Tms and N leaves, are shown in Plate 2. Ten different sections were obtained through the infection structure on an N leaf shown in Plate 2(a). These indicated that the fungus was contained within the cell wall between the two cells and had penetrated only slightly farther than shown in Plate 2(a). Nevertheless, the sub- sidiary cell adjacent to the infection hypha appeared dead, or at least severely dis- rupted, and its cell wall was grossly swollen and distorted in ‘areas well removed from the fungus. Very similar effects are apparent in Plate 3 which shows a more advanced stage of penetration of a Tms leaf. Here the fungus, which appears as two cells that are actually different sections through a single hypha, has penetrated into the stomata1 cavity. These observations indicate that cell wall swelling and cellular disruption prior to invasion of the protoplast are early responses to infection in both Tms and N leaves. It should be noted, however, that these effects are localized in cells adjacent to the invading fungus (Plates 2 and 3).

Two of the most advanced stages of infection seen on N and Tms leaves 6 h after inoculation are shown in Plates 4 and 5. In both cases, the epidermal cells on each side of the infection hyphae appear moribund. They contain no recog- nizable organelles and their densely stained cytoplasmic contents have coagulated along the cell walls adjacent to the fungus. There is, however, no evidence of gross swelling or distortion of cell walls such as that seen where penetrations occurred between guard and subsidiary cells (Plates 2 and 3). Additional sections showed that both of the infection hyphae (Plates 4 and 5) had penetrated the first two layers of mesophyll cells. They also revealed that the short branch visible on the infection hypha in Plate 4 was forming along or within a cell wall horizontal to and slightly above the plane of the section. Thus, both penetration and initial growth of the fungus were mostly, if not entireIy, intercehular.

l’LA.rEs 1 to 10. Penetration of resistant corn leaves carrying normal (N) cytoplasm and susceptible ones carrying Texas male sterile (Tms) cytoplasm by Helminthosporiun maydis raw T.

PLATE 1. Prepenetration structures formed by H. muydis on N (a to c) and Tms (d to f) leaves of the corn inbred W64A. (a, b, d, e) 6 h, (c) 8 h and (f) 10 h after inoculation. All x 22 000.

hA1.E 2. Penetration betwren guard and subsidiary cells G h after inoculation. (a) 837 (N), x 4600. (b) B37 (Tms), x 11 000.

PLATE 3. Penetration into the substomatal cavity of a W64A (Tms) leaf. x 5800.

PLATE 4. Penetration between epidermal cells of a 33-36 (Iv) leaf 6 h after inoculation. x 3000.

I’LAIX 5. Penetration between epidermal cells of a 33316 (Tms) leaf 6 h after inoculation. x 4200.

PLATE 6. (a) Detail of penetration between a guard and subsidiary cell of B37 (Ii) showing typical loosening and folding of the cuticle. x 14 000. (b) Break in the cuticle above the

junction of a guard and subsidiary cell of a non-inoculated leaf. x 4200.

PLATE 7. Hyphae of H. maydis ramifying beneath the cuticle (arrows) of epidermal cell walls 8 h after inoculation. (a) On a leaf of W64A (N), x 15 000. (b) On a leaf of LY64A (Tms), x 12 000.

PLATE 8. Haustoria-like structures produced by subcuticular hyphae of H. maydis 10 h after inoculation. Arrows point to the cuticle. (a) On B37 (N), x 22 000. (b) On W64A (‘l-m), x 20 000.

PLATE 9. (a) Apparent stomata1 penetration on a leaf of W64A (N) 10 h after inoculation. (1,) A different section through the stomate shown in (a) with evidence of penetration between two neighboring epidermal cells. Both x 4100.

Pr-ArE 10. Typical example of hyphae of H. maydis within stomata1 openings 10 h after inoculation. Fungal structures within the stomata1 opening are thought to be extensions of a hypha which penetrated between a subsidiary and an adjacent epidermal cell. On a leaf of B37 (Tms). x 3800.

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PLATE 1

PLATE 2

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PLATE 4

PLATE 5

PLATE 6

PLATE 7

PLATE 8

PLATE 9

PLATE 10

Ultrastructure of penetration by Helminthosporium maydis

Nature of cuticular#-netration

175

Despite an extensive search, stages in the infection process which might provide clear evidence of the nature of cuticular penetration were not found. Hyphal tips, close to or in contact with the cuticle [Plate 1 (a) and (d)] were common, but no evidence of any enzymatic or mechanical effect on the cuticle was seen. A total of 60 different penetrations were studied in leaves 6 h after inoculation, but in all cases cuticular penetration had already occurred. Failure to find stages in penetration intermediate to those shown in Plates 1 and 2 suggests that cuticular penetration, by whatever means, occurs very rapidly.

In a few cases, infection hyphae were sharply constricted in the area of passage through the cuticle [Plate 2(b)]. Th is suggests that penetration occurred through a small hole produced, perhaps, by enzymatic activity. If so, this activity must have been sharply localized for the cuticle to retain sufficient toughness to restrict lateral expansion of the fungus. In the vast majority of cases, infection hyphae showed no evidence of cuticular constriction [Plates 2(a) and 6(a)]. Instead the cuticle often appeared to be loosely folded and not firmly cemented to the epidermal cell wall in the area adjacent to the infection hypha [Plate 6(a)].

Breaks in the cuticular membrane, not associated with points of penetration by the pathogen, were found once on a non-inoculated control leaf [Plate 6(b)] and twice in non-infected areas of inoculated leaves. In all three cases, the breaks occurred at the junction of a guard and a subsidiary cell, and the cuticle had separated from the cell wall surface as shown in Plate 6(b). Such breaks, if present prior to fixation, would provide avenues of entrance for the fungus and their location would account for the fact that most penetrations occur adjacent to stomates. Such a mode of entrance would also account for failure to detect intermediate stages in cuticular penetration and the loosely folded, separated appearance of the cuticle at points of penetration [Plate 6(a)]. d

Secondary penetration

In Tms and N leaves fixed 8 or 10 h after inoculation, hyphae of the pathogen were most abundant in epidermal cell walls. The cell walls surrounding these subcuti- cular hyphae were swollen and nearly electron transparent (Plate 7). One such hypha was found which spanned three epidermal cells without penetrating the cell lumen or rupturing the cuticle. This gives some indication of how extensively this pathogen ramifies beneath the cuticle of epidermal cell walls.

In five cases, three on N and two on Tms leaves, it was possible to trace sub- cuticular hyphae to their origin. In all cases, they appeared to represent branches of the fungus which had radiated out from miniature infection cushions formed, presumably, at the primary point of penetration [Plate 7(b)]. Haustoria-like structures produced by subcuticular hyphae were seen occasionally in leaves fixed 8 h after inoculation but most often in those fixed after 10 h. These structures, which were encased by material continuous with and of the same appearance as the swollen host cell walls, appeared to function in secondary penetration and spread of the pathogen (Plate 8).

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Th.9 question of stomata1 penetration

H. Wheeler

On the basis of light microscopy, Jennings & Ullstrup [9] reported that 15 to 20% of all penetrations by H. maydis were stomatal. In this study, approximately 16% (8 of 47) penetrations observed on leaves fixed 8 or 10 h after inoculation appeared to be stomatal. A typical example of an apparent early stage in stomata1 penetration [Plate 9(a)] was the first of 16 sections through this structure. The last of these [Plate 9(b)] shows that Plate 9(a) cannot be an initial stage of stomata1 penetration since the fungus protrudes into the substomatal cavity. More important, a second penetration apparently occurred between two epidermal cells adjacent to the stomate and a projection of the hypha in the substomatal cavity in the direction of this second point of penetration suggests that the two may be connected [Plate 9@)1.

The situation shown in Plate 10 occurred in seven of the eight cases in which apparent stomata1 penetration was observed. In the single exceptional case, only two sections through the apparent penetration structure were obtained. Additional sections through the infection structures shown in Plate 10 demonstrated that three fungal structures within and just below the stomata1 opening were different sections through a single hypha. Furthermore, this hypha was very close to, and quite possibly an extension of, the one which had penetrated between the subsidiary cell and its neighbour. From these observations it is possible, if not probable, that fungal hyphae seen in stomata1 openings are emerging rather than penetrating.

DISCUSSION In this study, no differences were observed at any stage of penetration or initial colonization by H. maydis among four different pairs of inbred lines. These results, which confirm previous light microscopic investigations, indicate that differences in disease reactions between Tms and N plants of the same inbred or among different inbred lines develop only after the pathogen has become established in leaf tissues. They also support the suggestion, based on genetic data [ZO], that the selective pathotoxin produced by H. maydis does not play an important role during early stages in the infection process.

Effects on cell walls, swelling, distortion and changes in staining properties, similar to those seen in this study (Plates 2, 3 and 7), have been observed during penetration by a number of other pathogens [I3]. Presumably, these changes reflect the activity of cell wall degrading enzymes produced by the plant or the pathogen. Cultures of H. maydis race T have been reported to produce extracellular pectolytic, cellulolytic and xylanase enzymes [4] and one or more of these could account for initial changes in cell walls. Since lethal effects of pectolytic enzymes are well documented [3], such enzyme activity may also be responsible for the mori- bund appearance of cells adjacent to points of penetration (Plates 2 to 6 ). The possibility that degenerative changes in cells adjacent to penetrating hyphae are caused by Hm T-toxin appears remote; first, because they are similar in both toxin- sensitive Tms and toxin-resistant N leaves, and second, because they do not resemble the effects observed when tissues are exposed to T-toxin in the absence of the fungus [Am.

Ultrastructure of penetration by Helminfhosporium maydis 177

A tendency for penetration to occur between cells adjacent to stomata, similar to that observed in this study with H. maydis race T, has been reported for H. victoriae, i-f. sorokinianun and several other foliar pathogens [19]. Measurements made from silicone-rubber replicas of the upper surface of corn leaves indicated that the stomata1 apparatus (guard and subsidiary cells) occupies approximately 6% of the leaf surface area. Since at least two-thirds, and probably more, of all penetrations occurred adjacent to stomates, infection is obviously not a completely random process. Changes in turgor, especially in thinwalled subsidiary cells, during opening and closing of stomates, would result in repeated flexing of the cuticular membrane and could cause breaks similar to that shown in Plate 6(b). Such localized breaks would also account for the observation that when detached barley leaves inoculated with Erysiphe graminis were placed in water or salt solutions, infections of subsidiary cells increased sharply to as high as 80 to 90% of total infections [7]. Although the hypothesis that breaks in the cuticle, caused by stomata1 movements and thus localized above subsidiary cells, provide ready avenues of entrance for the pathogen and thus account for the frequency of penetration adjacent to stomates, it should be kept in mind that few breaks were seen and these could have occurred during fi.xation or processing.

Although the question of stomata1 penetration cannot be answered conclusively, several lines of evidence indicate that it occurs rarely, if at all, under the conditions of these experiments. First, no evidence of stomata1 penetration was found among 60 penetrations observed in material fixed 6 h after inoculation. Second, evidence af a second closely adjacent penetration was found in all but one case of apparent stomata1 penetration in leaves fixed 8 or 10 h after inoculation. Finally, no evidence of closely adjacent penetrations was found unless one of the two was apparently stomatal.

The technical assistance of D. J. Politis in the initial stages of this investigation is greatfully acknowledged. Kentucky Agricultural Experiment Station Journal Series No. 77-l l-29. Supported in part by Grant 216-15-21 from CSRS, USDA.

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