150 Chapter 7 Concentric Bodies 7.1 INTRODUCTION The term 'concentric body' applies to a distinctive spherical or ellipsoidal organelle, visible only at the ultrastructural level and of unknown function, initially found in a number of lichenized ascomycetes (Brown & Wilson, 1968; Griffiths & Greenwood, 1972) and more recently in certain non-lichenized ascomycetes (Griffiths & Greenwood, 1972; Granett, 1974; Beilharz, 1985) and anamorphs of ascomycetes including the cercosporoid fungus Cercospora beticola (Pons et al., 1984). These authors pointed out that although C. beticola has no known teleomorph, several other Cercospora species have been connected with Mycosphaerella sexual states, and it is likely that C. beticola has, or at least had at some time in the past, a similar connection with a member of the Dothideales. Other members of the Dothideales known to contain concentric bodies are Venturia inaequalis (Cke) Wint. (both anamorph and teleomorph) (Granett, 1974), Rhopographus Nitschke (Griffiths & Greenwood, 1972) and Hysterographium Cda (Bellemère, 1973). By 1984, concentric bodies had been found in only a few orders of Ascomycetes (Dothideales, Ostropales, Helotiales, Lecanorales, Verrucariales, Caliciales, Teloschistales and Peltigerales), all of which contain some lichen-forming taxa, and the last two of which contain only lichen- forming taxa (Pons et al., 1984). The connection between concentric bodies and ascomycetes is so firmly established that the occurrence of concentric bodies in fungi whose sexual state is unknown can now be taken as a reliable indication that these fungi have ascomycetous connections. Examples of such fungi are Ampelomyces quisqualis Ces., a coelomycete parasitic on members of the Erysiphales (Hashioka & Nakai, 1980), and the pycnothyrial coelomycete Brefeldiopycnis Petrak & Cif. (Beilharz, Giles & Joannides, unpublished data). Further examples of concentric bodies occurring in cercosporoid fungi are presented in this chapter. In addition, their distribution within the fungal thallus is recorded, and discussed with reference to the observation that in several fungi concentric bodies occur mainly in cells involved, or soon to be involved, in the production of ascospores or conidia, and less commonly in vegetative hyphae or the propagules themselves (Beilharz, 1985; Bellemère, 1973; Granett, 1974; Philipson, 1989 Pons et al., 1984; Rushing & Latham, 1991). 7.2 MATERIALS AND METHODS A list of specimens examined by transmission microscopy and the method of their preparation are found in Appendices C and E, respectively. 7.3 RESULTS Concentric bodies were found in every specimen examined by TEM in the present study. They were typical of those described in the literature in consisting of an electron-lucent inner core surrounded by an 151 electron-opaque layer which was slightly less opaque towards the outside, and from which an array of fine lamella-like projections extended into a surrounding electron-lucent layer. The organelles were usually clustered in a pale, homogeneous matrix situated close to the nucleus. No more than one cluster was present in any cell. Several clusters contained flattened as well as normal concentric bodies (Fig. 5.16). Concentric bodies were common in stroma cells, particularly those closest to the upper fertile layer, in conidiophore mother cells and in the conidiophores themselves. They were seen only once in a vegetative hypha and once in a conidium which was not germinating (P. platylobii, VPRI 17432). It should be noted, however, that mature conidia were not often observed at the ultrastructural level, because no special precautions were taken to prevent them being washed away during tissue preparation. There was a striking association of concentric bodies with endohyphae. Endohyphae (see Chapter 5.3.5) occurred in stroma cells (Fig. 5,20), conidiophore mother cells and conidiophores (Figs 5.14-5.19) of a number of species dealt with in this study. Concentric bodies and other organelles within endohyphae were invariably in much better condition than those in the non-endohyphal cells of the same specimen. Concentric bodies occurred in an endohypha within a conidium of P. loranthi, but were not transected in any other cell of that conidium (Fig. 5.16). Although the endohypha had not emerged from the conidium, at least in the plane of section, evidence from germination studies (Chapter 8.2.1) suggests that it would function as a basal germ tube. In P. loranthi, concentric bodies were also seen in two conidiophores connected by a presumed anastomosis peg (Fig. 8 2). Concentric bodies were transected in each of the three cells involved. 7.4 DISCUSSION The presence of concentric bodies in the cercosporoid fungi examined by TEM in the present study is not surpris ing, as they have been demonstrated in C. beticola (Pons et al., 1984). These authors pointed out that at least one species of Cercospora sensu stricto (C. arachidicola Hori) has a Mycosphaerella teleomorph, and that it is likely that C. beticola, another Cercospora species sensu stricto also has, or had, a connection with a member of the Dothideales. Kendrick & Dicosmo (1979) did not record any connections between Mycosphaerella and Pseudocercospora, but most literature up to that time pertaining to Pseudocercospora would have referred to Cercospora. A number of Pseudocercospora species dealt with in this study appear to have Mycosphaerella teleomorphs although the connection is often only one of close association (Chapter 8.2.4). Similarly, a Mycosphaerella state was closely associated with Verrucisporota daviesiae, another cercosporoid fungus containing concentric bodies. The order Dothideales is one of only eight orders of ascomycetes in which the presence of concentric bodies has been established (Pons et al., 1984). As in the Sphaceloma anamorph of Elsinoë rosarum and Pseudopeziza trifolii (Beilharz, 1985), concentric bodies in the cercosporoid fungi commonly occurred in cells closely associated with spore production. The first recorded association of concentric bodies with endohyphae was probably that of Corlett et al. (1976) in the Spilocaea anamorph of Venturia inaequalis, although the authors did not recognise the new 152 inner walls laid down in the conidiogenous cells to be endohyphal. The particularly clear definition of concentric bodies and other organelles in endohyphae may simply reflect the fact that endohyphae are among the most recently formed cells, and as such probably the most actively growing. On the other hand, endohyphae usually develop in cells associated with sporulation, a connection which has been demonstrated in other non-lichenized fungi (Beilharz, 1985). The apparent connection between concentric bodies and endohyphae is strengthened by their association in a germinating P. loranthi conidium. 154 Chapter 8 Miscellaneous Items 8.1. CULTURAL STUDIES 8.1.1. Isolation of cercosporoid pathogens from infected leaves The direct transfer of conidia and/or conidiophores to an agar plate proved to be the quickest, easiest and most effective way to isolate cercosporoid fungi. Transfers were performed under a dissecting microscope with a fine needle which had, after flaming, been plunged into agar to cool and moisten the tip. Oatmeal agar (OMA, Appendix H) was the preferred medium for the isolation and maintenance of cultures, which were routinely kept stacked in open plastic bags in the dark at 21-24°C. They did not sporulate under these conditions. All cultures were also stored under water in 20 ml McCartney bottles, according to the method of Boesewinkel (1976). They remained viable for at least four years, but some deteriorated over time, producing colonies with less aerial mycelium and of greater diameter than when originally isolated. 8.1.2. Cultural characteristics 8.1.2.1. Methods Cultural characteristics were recorded of 79 isolates which represented the full range of cercosporoid species isolated in the course of this study. Four 1-2 mm2 blocks taken from the edge of each colony on OMA were placed on each of two OMA plates. One plate of each isolate was sealed with Whatman's laboratory film and the other left unsealed in case the film affected colony characteristics. Plates were incubated in piles of ten in open plastic bags for 14 days at 25°C in the dark. 8.1.2.2 Results Cultures which had deteriorated with age were disregarded in the collation of results. Colonies on unsealed plates often had denser aerial mycelium than their equivalents on sealed plates. Species were categorised according to their average growth rates recorded after two weeks. Cercospora beticola grew fastest, the colonies attaining a diameter of 50 mm, while cultures of Pseudocercospora platylobii, the Pseudocercospora from Solanum and Cercospora zebrina all developed colonies 14-20 mm in diameter. The C. zebrina isolate had been stored under water for 6 years, and its original colony characteristics are not known. Colonies of Pseudocercospora loranthi, P. correae, P. uluruense, P. hardenbergiae, the Pseudocercospora from Pandorea doratoxylon and several Pseudocercospora isolates from eucalypts, all ranged from 10-16 mm in diameter. Colony diameters of 5-10 mm. were seen in P. pultenaeae and P. kennediicola, while colonies reached a diameter of only 3-7 mm in several isolates of P. loranthi, the Stenella isolate from P. formosum, one P. hardenbergiae isolate and one eucalypt isolate. Cultures of Verrucisporota daviesiae barely grew on OMA. After 2 weeks, the only growth was on the inoculum, 155 and even after several months the colonies had attained a radius of only 1-2 mm. Meanwhile, a tough, narrow plug of mycelium had grown straight down through the agar to the bottom of the Petri dish. All colonies had black reverses. The generally olivaceous buff to grey colour of the mycelium varied almost as much within as between species. However, isolates of P. loranthi had a distinctive khaki tinge, and the aerial mycelium of P. platylobii and the Pseudocercospora on Solanum readily peeled off the agar. 8.1.2.3 Discussion With few exceptions, the characteristics of Pseudocercospora species in non-sporing culture are non- diagnostic. Growth rates tend to be slow and colonies dense.