Chapter 5 Conidiophore Initiation and Conidiogenesis 5.1

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Chapter 5

Conidiophore initiation and conidiogenesis

5.1 INTRODUCTION

In this chapter are described the processes of conidiophore initiation and conidiogenesis in some Australian cercosporoid fungi. The involvement of wall layers of the conidiophore mother cell in conidiophore initiation is examined, and the events involved in conidiogenesis are then considered in terms of the following successive developmental steps proposed by Minter et al. (1982).

(a) Conidiophore initiation. Ultrastructural studies showed that the production of conidiophores from stroma cells in Cercospora beticola can be enteroblastic or holoblastic (Pons et al., 1985). In enteroblastic initiation, the outer, opaque layer of the conidiophore wall was continuous with the middle wall layer of the stroma cell. The outer layer of the generative cell either stopped abruptly or else merged imperceptibly with the wall of the conidiophore. The latter type of conidiophore initiation resembled that from vegetative hyphae in Pleiochaeta setosa (Kirchn.) Hughes (Harvey, 1974).

(b) Conidium ontogeny, described as 'the ways in which conidial cell walls are produced' (Minter et al., 1982). The accepted view that conidium initiation is holoblastic in the cercosporoid fungi (Ellis, 1971) was supported by the results of Pons et al. (1985).

(c) Conidium delimitation, described as 'the ways in which delimiting septa are produced' (Minter et al., 1982). Every conidium is delimited by a septum prior to abscission, but the exact structure of the septum, the stage of development at which it is formed (relative to the stage of expansion of the conidium) and the timing of the plugging of the septal pore by Woronin bodies (which interrupt its cytoplasmic connection with the conidiogenous cell) can vary. The septum of C. beticola is five-layered, comprising a central electron-translucent layer bordered on each side by an electron-opaque layer and then another electron- translucent layer (Pons et al., 1985). The middle layer, which has been referred to as the septal membrane (Griffiths & Swart, 1973) or septal-plate (Gay & Martin, 1971), terminates before reaching the outer layer of the cell wall. The other two layers are continuous with the inner and middle layers of the conidiogenous cell and the conidium (Pons et al., 1985).

(d) Conidium secession, described as 'the ways in which conidia become detached' (Minter et al., 1982). Conidium secession can be schizolytic or rhexolytic. Schizolytic secession involves the circumscissile rupture of the periclinal wall in the region of the conidium-delimiting septum, followed by centripetal splitting of the septum (Cole & Samson, 1979). The upper half of the septum becomes the hilum of the conidium, while the lower half forms the scar at the apex of the conidiogenous cell. Schizolytic secession was demonstrated in C. beticola (Pons et al., 1985). Rhexolitic secession, on the other hand, involves rupture of the periclinal wall some distance below the septum, which remains intact. It has not been demonstrated in cercosporoid fungi.

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(e) Proliferation, described as 'the ways in which conidiogenous cells are modified to produce more than one conidium or a new conidiogenous cell' (Minter et al., 1982). The formation of a new conidiogenous locus in blastic conidiogenesis can involve proliferation of either the inner layer of the conidiophore wall (enteroblastic), all layers (holoblastic) or neither layer, as in phialides (Hawksworth et al., 1983). Proliferation by means of endohyphae has been described in the Spilocaea state of Venturia inaequalis (Corlett et al., 1976), Acrogenospora sphaerocephala (Hammill, 1972) and other dematiaceous fungi (Wang, 1990).

Whereas Cole & Samson (1979) apparently regarded sympodial proliferation as holoblastic (in contrast with the enteroblastic proliferations that produce new conidiogenous loci in phialides and annellides), enteroblastic sympodial proliferation is now a well recognised phenomenon (Madelin, 1979; Minter et al., 1982; Sutton & Pascoe, 1987). It occurs when the outer layer of the conidiogenous cell wall is too inflexible to be blown out in the course of proliferation, and is instead ruptured by the emerging inner layer. This is particularly likely to happen when growth is slow or intermittent, as may occur under natural conditions, and in conidiophores with 'brown' outer wall layers (Luttrell, 1979).

Ellis (1971) described both Cercospora and Pseudocercospora as exhibiting sympodial proliferation, with Pseudocercospora often also proliferating percurrently in young conidiophores. Pons & Sutton (1988) described both genera as exhibiting enteroblastic sympodial proliferation, and also mentioned Pseudocercospora proliferating percurrently. Their description of Pseudocercospora was presumably intended to also include holoblastic sympodial proliferation, because they described the type of the genus, P. vitis, and four other species of Pseudocercospora as proliferating in this manner.

Enteroblastic sympodial proliferation has been demonstrated in Cercospora beticola (Pons et al., 1985). The thickened scar at the apex of the conidiogenous cell was displaced laterally by the emerging cell, which had its origins in the middle and inner wall layers of the conidiogenous cell. Pseudocercospora correae was attributed with enteroblastic sympodial, holoblastic sympodial and percurrent proliferation (Sutton et al., 1987). Percurrent proliferations (referred to by Minter et al. (1982) as enteroblastic percurrent regeneration) were equated by Sutton et al. (1987) with those described by Deighton (1976) as 'falsely percurrent' or 'pseudopercurrent'. Deighton (1976) described the conidiogenous cells of Pseudocercospora as 'sometimes pseudo-percurrent on one and the same conidiophore that is also denticulate, proliferating through the apex and either displacing the old apical scar into a lateral position or, more usually, completely rupturing the old apical scar, to produce annellations...' Deighton (1976) noted that the resulting annellations (which he termed pseudo-annellations) were difficult to detect, and that the clearest examples were found in the type specimens of P. helleri (Earle) Deighton and P. colocasiae Deighton. It is apparent from Deighton's descriptions and illustrations of these species that he uses the term pseudo-percurrent to describe enteroblastic proliferation either straight through the apical scar on the conidiogenous cell or at its periphery, in which case the scar may remain intact but is pushed aside by the emerging proliferation and often then lies flat against the side of the conidiophore.

In some fungi, 'pseudo-percurrent proliferations' (Deighton, 1976) or, to use the alternative terminology, 'enteroblastic percurrent regenerations' (Minter et al., 1982) have been shown by ultrastructural

104 examination to result from the production of endohyphae and their extension through or beside the apical scar on the conidiogenous cell (Hammill, 1972; Wang, 1990).

The production of endohyphae has frequently been linked with the regeneration of hyphae which were aged, damaged, mutated or otherwise grown under adverse conditions. Such endohyphae were mostly of small diameter, at times branched, and in several cases sporulated within the cell lumen. They were generally reported to have arisen from the septa separating damaged from healthy cells. Hughes (1971) dismissed all such regenerations, including those at conidiophore apices, as simply repair mechanisms, and 'not concerned with the normal production of a succession of reproductive structures'. Evidence from TEM studies has indicated, however, that the regeneration of stromatal and conidiogenous cells by endohyphae may be the normal course of events in certain dematiaceous hyphomycetes. Such regeneration leads to the production of annellate conidiophores to which Wang (1990) applied the term annellophores, a term originally introduced by Hughes (1953), distinguishing them from annellides which produce a plurality of conidia from a meristematic region on the conidiogenous cell. Wang further suggested that Hughes' (1953) Section III readily encompassed fungi that produce annellophores, but not annellidic fungi, which should be excluded. Her suggestions concerning the place of annellophores in Hughes' scheme rested on the percurrent nature of the endoconidiophores.

5.2 METHODS

Specimens were mounted in lactic acid and warmed to dispel air bubbles in preparation for examination under Nomarski interference optics and bright field optics.

Freshly collected specimens were prepared for ultrastructural examination. A list of the specimens examined by transmission electron microscopy and the method of their preparation are given in Appendices C and E, respectively. The specimens examined by scanning electron microscopy, and the method of their preparation, are given in Appendices D and F, respectively.

5.3 OBSERVATIONS

5.3.1 Conidiophore initiation

With two exceptions, only holoblastic conidiophore initiation was seen in the specimens studied by transmission electron microscopy. Enteroblastic conidiophore initiation was, however, observed in some sporodochial conidiophores of P. platylobii (Fig. 5.4). Each conidiophore of P. platylobii had a bulbous base which was originally the stroma cell from which the conidiophore had developed. The dark outer layer of the basal cell wall was not always continuous with the upper part of the conidiophore, which in such cases had developed by extension of the inner wall layers of the basal cell. The outer wall layer of the basal cell then merged imperceptibly with the outer wall of the conidiophore.

Similarly, one of the two conidiophores on an external hypha of a Pseudocercospora growing on Eucalyptus sp appears to have been initiated enteroblastically and the other either holoblastically or else enteroblastically, but with a more subtle mergence of old and new outer wall layers (Fig. 3.54).

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5.3.2 Conidium ontogeny

Conidium ontogeny was consistently holoblastic, as seen in ultra-thin sections through conidiophores with persistent, attached conidia in Pseudocercospora platylobii (Fig. 5.1), P. pultenaeae (Fig. 5.2) and Pseudocercospora sp. on Eucalyptus macrorhyncha (Fig. 5.3).

5.3.3 Conidium delimitation

In most of the species under study, delimitation occurred when the conidium initial was ca 15-50 µm long, and preceded the septation of the conidium. There is inconclusive evidence, however, of delayed conidium delimitation in several species of Pseudocercospora, in particular the Pseudocercospora from the E. morrisbyi, E. exserta and other Queensland eucalypts, and from E macrorhyncha. Although attached conidia were rarely found in specimens of this fungus, several were observed to be internally 1-3 septate prior to delimitation. Ultrathin sections were obtained through abscission septa of three species of Pseudocercospora typified by persistent conidia. The structure of the abscission septum in Pseudocercospora sp. on E. macrorhyncha (Fig. 5.3) was not particularly clear. However, in P. pultenaeae (Fig. 5.2), a thin, well-defined electron-lucent septal membrane was bordered by irregularly electron dense layers which in turn graded into outer electron-lucent layers. The septal membrane terminated in the inner, electron-lucent layer of the conidiophore wall. The irregularly electron-opaque septal layer extended alongside the membrane into the inner layer of the conidiophore wall, from where it spread longitudinally a short distance in each direction.

The septal membrane in Pseudocercospora platylobii (Fig. 5.1) was again electron-lucent. The layers bordering it resembled those in P. pultenaeae except that the pigmentation was less marked. The septum of P. platylobii was less than half the thickness of that of P. pultenaeae, and the thickness of the conidiophore walls in the two species differed accordingly.

5.3.4 Conidium secession

A scanning electron micrograph of a seceding conidium of Pseudocercospora pultenaeae illustrates an intermediate stage of schizolytic secession (Fig.5.5). The outer wall layer of the conidiophore has ruptured in line with the septum (circumscissile rupture), and the septum, still intact in the region of the pore, is in the process of splitting centripetally along the septal plate. A similar stage was seen by light optics in P. kennediicola and the Pseudocercospora on E. macrorhyncha. Each cell involved in secession retained half of the conidium-delimiting septum, and equivalent wall remnants fringed the scars on the two separated cells. Woronin bodies were associated with abscission septa (5.1, 5.3) and plugged the septal pores of scars on detached conidia and on conidiogenous cells (Figs 4.20, 4.23, 4.25, 4.27).

5.3.5 Conidiogenous cell proliferation

Proliferation was usually sympodial, each apical scar being left either on a lateral, protuberant peg as in the Pseudocercospora on Eucalyptus morrisbyi (Fig. 3.25), E. exserta (Fig. 3.52) and Solanum spp. (Fig. 5.10), on a sloping shoulder as in Pseudocercospora pultenaeae (Fig. 5.6) and the Pseudocercospora spp.

106 on Eucalyptus ?globulus (Fig. 5.11) or flat against the side of the conidiophore (Fig. 3.54, 5.13). When the conidiophore apex was markedly attenuated, proliferation occurred just below the attenuation, which was left as a peg-shaped projection which retained its original orientation, or nearly so (Fig. 3.52, 5.10). However, when the attenuation was less marked or even absent, proliferations emerged close to the previously formed scar, and the scars were left on shoulder-like projections which were broader and less protruberant than the pegs (Figs 5.6, 5.11).

Both enteroblastic and holoblastic sympodial proliferation were observed, sometimes on the one specimen. Light microscope and SEM studies demonstrated enteroblastic sympodial proliferation in the following fungi: Pseudocercospora loranthi, P. correae, P. kennediicola, P. pultenaeae, the Pseudocercospora species on E. exserta, E. macrorhyncha and E. morrisbyi and the Stenella on Platylobium formosum. It will be shown later in this chapter that enteroblastic sympodial proliferation can be achieved in more than one way, and more specifically that in P. loranthi and several other species a mechanism exists other than that involving the breach of the outer layer of the conidiophore wall by the extension through it of the more plastic inner layer.

When a proliferating conidiogenous cell emerged at the periphery of a scar, it lifted the scar to one side and grew on past it (Figs 3.54, 5.11), typically maintaining the original direction of growth of the conidiogenous cell. The scar was usually left lying flat against the side of the proliferation, still partly attached to the rim of the ruptured outer layer of the conidiophore wall, which was visible as a pseudoannellation. This situation was detected by light optics in Pseudocercospora correae, P. loranthi, Cercosporidium duboisiae and the Pseudocercospora on Solanum, and in scanning electron micrographs of external conidiophores of Pseudocercospora kennediicola (Fig. 3.29) and a Pseudocercospora on Eucalyptus sp. (VPRI 17444) (Fig. 3.54). Proliferations of this type could be distinguished from lateral enteroblastic sympodial proliferations partly because they more closely followed the original direction of growth of the conidiogenous cell. The displaced scar was often not visible, either because it had fallen off or because the specimen was unfavourably oriented. In addition, annellations were often difficult to see by light optics, particularly when the conidiophore wall was pale. Although pseudo-annellations were usually more ragged, they could be mistaken for true annellations if displaced scars were not in view.

The difficulty in distinguishing the outlines of annellations or pseudoannellations, particularly on pale conidiophores, may have caused them to be overlooked both in the present and earlier studies. Nevertheless, many sympodial proliferations observed during the present study were almost certainly holoblastic. So far, three different methods of proliferation have been mentioned as operating in the specimens under study. They are enteroblastic sympodial, holoblastic sympodial and pseudo-percurrent proliferation. The latter was typified by 'semi-axial'4 emergence of the inner wall layer of the conidiogenous cell, and can be regarded as a particular type of enteroblastic sympodial proliferation.

4A term borrowed from Luttrell (1963), who used it to describe germ tubes emerging from a point adjacent to the hilum of a Helminthosporium conidium. Its application to the equivalent position on a proliferating conidiogenous cell is useful in this context.

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Two, and sometimes all three, of these proliferative mechanisms were noted in single specimens of some species.

A fourth method of proliferation, involving endohyphae, was particularly apparent in Pseudocercospora loranthi, P. kennediicola and P. platylobii. Endohyphae were also transected in several conidiophores of the Pseudocercospora on Eucalyptus macrorhyncha and of Cercosporidium daviesiae. Endohyphal proliferations were usually not distinguishable from normal enteroblastic proliferations by means of light microscope or SEM examination, and they could usually be detected with certainty only with the TEM. They were, however, seen in epigenous conidiophores of P. kennediicola and in conidiophores of P. loranthi and C. davieisae, by light optics.

Although endohyphae have been referred to here as proliferative, their other function, regeneration, is not ignored. It is reasonable to expect that proliferation, as defined by Minter et al., 1982, will be detected as a succession of similar events occurring in healthy conidiophores under conditions favourable to conidiogenesis and culminating each time in conidium production. Regeneration, on the other hand, can be expected to usually occur as an isolated event following conidiophore deterioration or senescence.

Of the four separate collections of P. loranthi studied with the TEM, two were made in June (early winter), one in November (spring) and the other in February (late summer). In south-eastern Australia, at least, this pathogen generally sporulates most vigorously in mid-summer. Several individual mistletoe plants on which P. loranthi was sporing vigorously in summer were free of lesions in some winters, but in general sporulating lesions can be found throughout the year. The June collection may be regarded as somewhat out of season, even though there was active sporulation. The small February collection, comprising only two lesions, was in poor condition. The November collection, however, comprised leaves bearing small lesions, some very immature (visible as minute white flecks), and others a little larger and just starting to sporulate. Viewed by TEM, the fungus appeared to be in good condition, and the method of proliferation could therefore be reasonably expected to be 'normal'. As it happened, all four specimens had endohyphae within many conidiophores and stromatic cells, and no method of proliferation other than by endohyphae was identified in these specimens.

Electron micrographs suggest that each proliferation in P. loranthi involved the production of a new conidiogenous cell in the form of an endohypha (Figs 5.14, 5.19). Each endohypha was generated from the innermost electron-lucent wall layer present in the conidiophore (or its subtending stroma cell) at the time. Thus, the first endohypha was generated from the inner wall layer of the parent cell. Where successive endohyphae were formed, each was generated from the wall of the last-formed endohypha. Endohyphae initiated in conidiophores were usually generated low down (Fig. 5.19), but at times were generated closer to the apex (Fig. 5.14). Endohyphae were often free of the walls of the parent conidiophores (Figs 5.14, 5.19), in which case they were surrounded by degenerate cytoplasm. At times, however, they rejoined the conidiophore wall or lined it (Fig. 5.19). Endohyphae often became septate (Figs 5.14, 5.18), and it was common for an endohypha to lay down a septum where it passed through a septum in the parent conidiophore, or immediately afterwards (Figs 5.16, 5.17). The cytoplasm of cells

108 occupied by endohyphae was invariably degraded, electron-opaque, and lacking clearly defined organelles.

Most endohyphae grew to the apex of the intact parent conidiophore before breaching the conidiophore wall. The parent conidiophore evidently snapped off near its base some time later. The apical portion of one conidiophore appears to have been pushed off by an extending endohypha (Fig. 5.19), although this could be an artefact of preparation. After several successive proliferations, the most recently formed endohyphal conidiophores were found nested in the bases of old conidiophores. A conidiophore in Fig. 5.14 has undergone four successive endohyphal proliferations - the bases of two broken conidiophores envelop an intact, thick-walled conidiophore within which two more endohyphae have developed. Despite their common occurrence, endohyphae were rarely detected in conidiophores of P. loranthi by light optics (although they were visible in the holotype).

Endohyphae were also evident in sections through a vigorously sporulating epigenous fructification of P. kennediicola (Fig. 5.15). Two conidiophores arising from a common conidiophore mother cell were occupied by endohyphae that had originated in the mother cell. One endohypha was well-separated from the walls of its parent conidiophore, while the other was in close contact. The remnants of the septum which originally separated one conidiophore from the basal cell can still be seen, ruptured and pushed aside by the endohypha which had grown straight through it. Similar septum remnants are associated with the endohypha seen in the conidiophore on the left in the same section. This endohypha is unusual in having remained aseptate after passing through the conidiophore septum. Endohyphae were commonly seen by light optics in epigenous conidiophores of P. kennediicola, but not in hypogenous conidiophores from the same specimens. Unfortunately, no hypogenous fructifications were examined in ultrathin section.

Endohyphae were also seen in sections through an epigenous stroma of Pseudocercospora platylobii (VPRI 17432). Most cells in the upper layers of the stroma were occupied by endohyphae, and many conidiophores contained endohyphae which had formed either in the stroma or low in the conidiophores. The base of one endohypha was nested within two old conidiophore bases. Several endohyphae were transected, also, in conidiophores of the Pseudocercospora on E. macrorhyncha.

Ultrathin sections through emerging endohyphae were obtained only for P. loranthi. Emergence was close to, but not through, the apical scar on the conidiogenous cell (Figs 5.7, 5.8). In one transect, the endohypha had emerged so close to the periphery of the scar that it is difficult to know whether the outer conidiophore wall was breached in the process (Fig. 5.8). It is significant, however, that in the other transect all layers of the periclinal wall were breached by the emerging endohypha, ca 0.6 µm below the broken end of the existing conidiophore wall (Fig. 5.7). The conidiophore wall was not pushed out of position by the emerging endohypha, which strongly suggests that egress was aided by enzymatic dissolution or softening of the wall. Although proliferations in Figs 5.7 and 5.8 are endohyphal, the appearance of the scars is puzzling in that there is little or no visible half-septum supporting the external thickening. In Fig. 5.7, the Woronin body should be blocking the pore in the half-septum. The alignment of the electron-opaque flecks in the pocket of material between the scar and the endohypha suggests the

109 presence of an ill-defined, narrow, inner wall layer. However, the trapped material does not look like the degraded cytoplasm surrounding endohyphae in Figs. 5.14, 5.15, 5.17 and 5.19, and it could possibly be the remnants of lysed wall material. The emerging endohypha has, after all, chemically dissolved or softened the wall of the conidiophore very close by. A similarly puzzling profile is seen in a section through a scar on a conidiogenous cell of Pseudocercospora pultenaeae (Fig. 4.29), where the half- septum is separated from the scar deposit in the pore region.

While protected by the parent conidiophore, endohyphae always had electron-lucent walls consisting of only one layer, in this way resembling the inner wall of the conidiophore from which they formed (Fig. 5.17). As an endohypha emerged, the outer portion of its wall became slightly electron-dense, a feature which became more marked with maturity. The mature, exposed wall of the endohypha, now a conidiogenous cell, appeared two-layered, with a slightly rough surface (Fig. 5.17).

Endohyphae commonly contained well-defined clusters of concentric bodies (Figs 5.14-5.19). The occurrence of these organelles in the cercosporoid fungi is discussed in Chapter 7.

It is relevant to the present discussion that some naturally-produced conidia of P. loranthi germinated basally by means of endohyphae, either on the host surface in the natural situation, or after collection and incubation in the laboratory (Figs 5.16, 8.1). These are discussed in Chapter 8.2.1.

5.3.6 Regeneration

Regeneration, 'the way in which a no-longer functional conidiogenous cell is replaced' (Minter et al., 1982), is largely indistinguishable from proliferation in fungi such as P. loranthi and P. kennediicola which form intraconidiophoral endohyphae. I have already stated that regeneration can be expected to occur as an isolated, irregularly occurring event related to conidiophore deterioration or senescence. The endohypha in Fig. 5.17 falls into this category. It originated from endohyphae ramifying within the underlying, mostly collapsed, stroma cells. It has grown through the lumen of a collapsed conidiophore, and maintained its direction of growth by emerging through the bend in the conidiophore wall.

5.4 DISCUSSION AND CONCLUSIONS

The enteroblastic initiation of some conidiophores in P. platylobii was similar to that reported for some conidiophores of C. beticola by Pons et al. (1985), and for Pleiochaeta setosa by Harvey (1974). In each case the discontinuous outer wall of the conidiophore mother cell merged subtly with the outer wall of the conidiophore. The enteroblastic conidiophore initiation observed in a Pseudocercospora from eucalypt, on the other hand, appeared to involve an abrupt breach of the outer hyphal wall layer by an emerging inner layer in one instance, and by either a more subtle enteroblastic or a holoblastic initiation in the other.

The involvement of the outer wall layer of the conidiophore mother cell in conidiophore initiation varies in P. platylobii and possibly P. eucalyptorum, as well as in C. beticola (Pons et al., 1985). Further studies may demonstrate similar variation in other cercosporoid fungi. The variation observed in this study and in C. beticola (Pons et al., 1985) may simply reflect differing degrees of plasticity of the outer layer of the

110 conidiogenous cell wall, as suggested by Madelin (1979) in his explanation of variation in cell wall involvement in enteroblastic conidium initiation. However, the transect through a conidiophore of C. beticola (arrowed in Fig. 1A of Pons et al. (1985)), which was interpreted as enteroblastic conidiophore initiation in which the outer layer of the stroma cell wall was abruptly breached, more closely resembles transects through endohyphae obtained in the present study. The abruptly-terminating outer wall layer appears to be the base of the previous conidiophore, rather than wall of the stroma cell from which the conidiophore was produced. Endohyphal proliferation is discussed later in this chapter.

In summary, the degree of involvement of the wall layers of the conidiophore mother cell in conidiophore initiation is variable between and within taxa in the cercosporoid fungi. If cell wall characteristics such as flexibility and extensibility were influenced by the age of the cell wall and the environment, as suggested by Madelin (1979) for the allied process of conidium initiation, variation in wall involvement in conidiophore initiation could not be regarded as a reliable taxonomic criterion.

In most species under study, the holoblastic nature of conidium ontogeny was clearly evident. Proliferation, whether holoblastic or enteroblastic, resulted in well-defined conidiogenous cells, each with a pigmented outer wall layer and one or more pale inner layers. All of these layers were continuous with those of the next -formed conidium. According to Minter et al. (1982), this is consistent with holoblastic conidium ontogeny.

Exactly the same argument applies to proliferation by means of endohyphae, even though the pale, thin walls of the endohyphae had, at most, gained only a slight pigmentation by the time the new conidium was initiated. No matter how little the endohypha had developed in wall thickness and pigmentation, the fact remains that conidium initiation involved all wall layers present at the conidiogenous locus, and ontogeny was once again holoblastic, according to the definitions proposed by Minter et al. (1982).

Conidium-delimiting septa, in the three species in which they were transected in ultra-thin section prior to abscission, were typically five-layered and generally resembled the septa seen in hyphae, conidiophores and stromatic cells. Their basic structure was also similar to that of septa in Saccharomyces ludwigii (Gay & Martin, 1971), but contrasted with those of the basidiomycete Rhizoctonia solani Kuehn which, apart from having the central swelling typical of the dolipore septum, were also composed of a greater number of layers (Bracker & Butler, 1963). All septal membranes observed in this study were electron- lucent, as were those of Cercospora beticola (Pons et al., 1985). In contrast, septal membranes in two species of Seimatosporium were shown by Griffiths & Swart (1973) to differ in pigmentation, one of several factors leading the authors to conclude that the species were not congeneric.

The schizolytic secession demonstrated in this study accorded with the findings of Pons et al. (1985) in relation to C. beticola.

Because proliferation and regeneration can often not be distinguished in the cercosporoid fungi, they are considered together in the following discussion.

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Any description of proliferation should cover two separate aspects - firstly, the position at which the proliferation occurs, and secondly, which wall layers (if any) of the conidiogenous cell contribute to the proliferation. It has already been demonstrated by light optics that proliferation in P. correae can be enteroblastic sympodial, holoblastic sympodial or enteroblastic percurrent (Sutton et al., 1987). That these three modes of proliferation were all found at one time or another in the cercosporoid fungi studied here was therefore not unexpected, as most of the fungi fit into the broadly defined genus Pseudocercospora as described by Pons & Sutton (1988). These authors stated, in reference to the different methods of proliferation found in Pseudocercospora, that 'the absence of one of these features in some species would provide sufficient reason to separate them into a different generic taxon'. However, Madelin's (1979) suggestions regarding the influence of cell age and the environment on wall layer involvement in blastic conidium initiation raise the possibility that wall layer involvement in proliferation might be similarly influenced. In support of the view of Pons & Sutton (1988), however, the capacity of a fungus to adapt to this type of variation in wall characteristics could be genetically determined. Nevertheless, the probable influence of cell age and the environment on the mode of proliferation obtaining in any given circumstance means that it is difficult to be sure that a 'missing' feature would not appear under other circumstances. At the very least, a large number of samples would have to be examined before generic separations could be based on this character. This contrasts with tretic conidiogenesis, a stable and specific method of enteroblastic cell initiation involving the inner wall layer of the conidiogenous cell blowing out through a pore dissolved in the outer layer(s), characteristic of genera such as Drechslera.

Accounts of endohyphal proliferation in Acrogenospora sphaerocephala (Hammill, 1972), Berksleasmium concinnum, Monodictus paradoxa and Sporidesmium folliculatum (Wang, 1990) have been referred to in the introduction. P. loranthi, P. kennediicola and P. platylobii showed similar behaviour in the present study. In these three species endohyphae were common in stromatic cells as well as in conidiophores, as was also the case in A. sphaerocephala (Hammill, 1972). Although this study presents ultrastructural evidence of endohypha initiation from the inner walls of conidiogenous cells, some of which were previously-formed endohyphae, initiation commonly occurred in the subtending stroma cells. These can be regarded as the basal cells of the conidiophores (they are sometimes, but not invariably, separated from the stem of the conidiophore by a septum). The question which has to be addressed is whether the conidiophoral endohyphae observed in the cercosporoid fungi are involved in proliferation or regeneration, and whether the two functions can even be distinguished.

According to Minter et al. (1982), proliferation is concerned with modification of the conidiogenous cell to produce more than one conidium or a new conidiogenous cell, whereas regeneration is concerned with the production of new conidiophores to replace conidiophores which are no longer functional because of senescence or mechanical damage.

There is little doubt that the endohyphae observed in the conidiophores of P. loranthi, P. kennediicola, and P. phebalii were directly concerned with reproduction. In this way they differ from those endohyphae (usually thin-diameter, and often produced from septa) which are concerned with the

112 regeneration of damaged mycelial hyphae (Buller, 1933; Miller & Anderson, 1961; Lowry & Sussman, 1966; Chan & Stephen, 1967; Calonge, 1968). They also differ markedly from those endohyphae observed to sporulate, often abnormally, within their parent hyphae (Miller & Anderson, 1961; Kendrick & Molnar, 1965). However, as both regeneration and proliferation are ultimately concerned with reproduction (Minter et al., 1982), the two processes cannot be distinguished on this basis.

Again according to the definitions of Minter et al. (1982), the distinction between proliferation and regeneration rests largely on two criteria: (a) the position of initiation of the endohypha (it needs to be initiated within the conidiogenous cell to qualify as proliferative); and (b) the physiological condition of the conidiophore involved (regeneration is concerned with senescent or damaged conidiophores, proliferation with living conidiophores capable of generating more conidia).

The initiation of endohyphae in stroma cells is consistent with Minter et al.'s (1982) concept of regeneration, but not proliferation. Endohyphae initiated in conidiophores, and more specifically in conidiogenous cells, are consistent with the concepts both of regeneration (if the cell were already senescing) and proliferation (if it were not). If a conidiogenous cell is able to support the initiation and development of an endohypha (which will also go on to sporulate), it is clearly fully functional. On this basis, endohyphae initiated within conidiogenous cells in these fungi must be regarded as proliferative.

Although conidiophores occupied by endohyphae which had their origin in stroma cells could have been moribund at the time of their occupation, the second and third generation endohyphae in Fig. 5.14 must have originated in living conidiogenous cells. Regardless of their site of origin, all these endohyphae are structurally similar and involved in perpetuating the reproductive function of their respective conidiophores. Proliferation was certainly occurring in these fungi, and probably regeneration also. It is difficult to distinguish regeneration from proliferation in profiles such as these. However, Minter et al.'s (1982) definition of proliferation ('the ways in which conidiogenous cells are modified') itself needs modification if endohyphae initiated other than in the conidiogenous cell are to be accepted as proliferative.

Proliferation in the Spilocea state of Venturia inaequalis was shown ultrastructurally to involve the formation of a new, two-layered inner wall consisting of an outer electron-opaque layer and an inner electron-lucent layer 'adjacent to the plasmalemma around the entire annellide or conidiogenous cell' (Corlett et al., 1976). Furthermore, a completely new two-layered wall was formed for every percurrent proliferation, so that 'an annellide which has produced a number of conidia has a multi-layered or compound wall consisting of alternate dense and transparent layers.' (Corlett et al., 1976). In addition, the living stromatic cell beneath a dead annellide would often proliferate into the annellide. Corlett et al. comment that 'It can only be assumed that the proliferation eventually functions as a new annellide'. The endohyphal mode of proliferation described in Spilocea is exactly the same as that observed in this study and by Hammill (1972) and Wang (1990).

The production of endohyphae results in the degradation of the cytoplasm of the cell in which they are initiated, and of living cells through which they subsequently grow. Endohyphae possibly also absorb

113 nutrients from the cytoplasm of moribund cells which they occupy. Benhamou & Ouellete (1987) believed that endohyphae in the aerial mycelium of Ascocalyx abietina were responsible for the death of the parent cells.

As already mentioned, endohyphae were seen in many epigenous conidiophores of P. kennediicola viewed by light microscopy. Epigenous conidiophores of this fungus are often considerably longer than their hypogenous equivalents, and it is the longer conidiophores which have one to three endohyphal proliferations. The hypogenous conidiophores appear to have proliferated pseudopercurrently, but whether endohyphae are involved can be determined only by TEM studies.

Endohyphae also occurred in epigenous conidiophores of P. platylobii, but no hypogenous conidiophores were transected. Epigenous conidiophores of another specimen of P. platylobii, this time on P. obtusangulum, contained no endohyphae. The two specimens were collected at different places in different seasons, so that several factors could have contributed to the presence of endohyphae in one specimen and not in the other.

Three pieces of evidence in particular support the view that at least some endohyphae observed in this study were functioning in a proliferative rather than regenerative capacity.

The first, already mentioned, is the production of a succession of endohyphae within conidiogenous cells (Fig. 5.14), which is in accord with Minter et al.'s (1982) definition of proliferation.

The second comes from Hammill's (1972) work on Acrogenospora sphaerocephala in which conidiophoral endohyphae were shown to be active in two-week-old cultures. Hammill's observations support the notion that the character 'proliferation by endohyphae' is genetically set in A. sphaerocephala, and does not require the fluctuating conditions of a natural environment or extended periods of inactivity for its expression. Admittedly, Hammill's cultures were kept at room temperature, so some fluctuation in temperature and humidity would have occurred.

The third piece of evidence is the number (as many as four) of successive endohyphae (some initiated low in the conidiophore or in a stroma cell) present in some conidiophores. This adds weight to the hypothesis that endohyphae can form in response to stimuli which occur more frequently and are less extreme than cell injury or death.

I suggest that regeneration by endohyphae may have sufficiently favoured sporulation and overall survival of fungi such as P. loranthi that it has also become their normal means of proliferation. In other words, that this method of regeneration became incorporated into the genome with the result that the mechanism is triggered not only by the advent of conditions favouring growth after a period of inactivity or after mechanical damage, but also by conditions favouring the production of a rapid succession of conidiogenous cells from healthy conidiophores or conidiogenous cells.

If the above argument is accepted, the definition of proliferation (Minter et al., 1982) will require modification. Consider the following example: an endohypha is produced within a single-celled

114 conidiophore which is also, by definition, the conidiogenous cell. The endohypha, if involved in conidiogenesis almost immediately after emergence, is, according to the above argument, proliferative. If, however, the same conidiophore becomes a little longer, and a septum is laid down to support the cell walls, then an endohypha produced at exactly the same locus will be (according to Minter et al.'s definition) regenerative, even if it, also, is involved in conidiogenesis immediately after emergence. It may, therefore, be no more than an accident of timing which determines the supposed function of the endohypha in each case.

I propose that the definition of proliferation be modified to read 'the ways in which conidiogenous cells (or, in the case of endohyphal proliferation, also their subtending cells) are modified to produce more than one conidium or a new conidiogenous cell'.

The results of this study indicate that in cercosporoid fungi such as P. loranthi, proliferation mimics regeneration and endohyphae are involved in both. I suggest that P. loranthi and the fungi described by Hammill (1972) and Wang (1990) are adapted to regenerate from stromata after periods of inactivity in response to favourable environmental conditions. Regeneration is achieved by new cell formation in the form of endohyphae, often initially in the stroma cells but also in the lower cells of the conidiophores or the conidiogenous cells themselves. It always results in the formation of new conidiogenous cells within the old conidiophores. Regeneration by endohyphae is probably of considerable advantage to stroma- forming cercosporoid fungi and the fungi mentioned by Hammill (1972) and Wang (1990), nearly all of which form stromata in relatively persis tent substrates. Because these fungi are presumably subject to considerable environmental fluctuations, resulting in intermittent growth, it is important that they can respond rapidly and efficiently to conditions favouring sporulation. By growing within pre-existing cells, endohyphae are protected and also provided with an immediate source of nutrients. Thin-walled endohyphae within conidiophores are able to reach conidiophore apices (a favourable height for conidium dissemination) without needing to invest the larger amounts of energy required for the production of completely new, thick-walled conidiophores from the stroma.

Wang (1990) argued that fungi proliferating by endohyphae belong in Hughes' (1953) Section III, whereas those producing true annellidic proliferations should be placed elsewhere. As mentioned earlier, her argument depends on the true percurrent nature of endohyphal proliferations, as specified by Hughes. The endohyphae in Acrogenospora sphaerocephala emerged either through the centre of the apical scar, rupturing it, or semi-axially. In semi-axial proliferation, the scar is lifted like a hinged lid, intact and still attached to the conidiogenous cell wall on one side. Enteroblastic semi-axial emergence is closer to sympodial than to percurrent proliferation. However, neither the conidiogenous cell wall nor the apical scar is breached, and there can be a superficial resemblance to either mode of proliferation depending on whether the conidiophore becomes geniculate or maintains its original direction of growth (as in pseudo- percurrency). Cole & Samson (1979) presented a diagrammatic interpretation of endohyphal proliferation in A. sphaerocephala, with reference to Hammill's (1972) description. They showed the same inner wall layer contributing to the formation of successive conidia, which is incorrect (Beckett, Heath & McLaughlin, 1974). Minter et al. (1982) presented a similar diagram which was apparently based on Cole

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& Samson's (1979) account. In fact, neither wall layer involved in conidium formation is involved in the formation of the next conidium (as illustrated by Wang (1990)).

Endohyphae in the species described by Wang (1990) appear to have all emerged directly through the apical scar, and thereby fulfil the requirements of Hughes' Section III. However, the endohyphae of P. loranthi can proliferate laterally, through all wall layers of the conidiogenous cell, and possibly also semi- axially. This means that their emergence is certainly not consistently percurrent (through the apical scar), and may often or always be sympodial (depending partly on whether semiaxial emergence is regarded as sympodial). Acrogenospora sphaerocephala (Hammill, 1972) exhibited only semi-axial and percurrent, but not lateral (sympodial), proliferation.

If P. loranthi does not fit into Hughes'(1953) Section III, there is no category within his scheme suited to fungi proliferating by endohyphae. Although P. loranthi could be placed in Section II, it would be artificial to separate the endohyphal taxa on the basis of the exact position of emergence, given that endohyphal proliferation as such is the strong linking character. Endohyphal proliferation is unique, and attempts to fit it into Hughes' categories are meaningless.

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Beilharz, Vyrna Caldwell

Title: Cercosporoid fungi on Australian native plants

Date: 1994-05

Citation: Beilharz, V. C. (1994). Cercosporoid fungi on Australian native plants. PhD thesis, Department of Agriculture, The University of Melbourne.

Publication Status: Unpublished

Persistent Link: http://hdl.handle.net/11343/39430

File Description: Chapter 5

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