102 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. 103 (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