Conidium ontogeny in the Chalara state of Ceratocystis adiposa

[ 259 ] Trans. Br. mycol, Soc. 68 (2) 259-265 (1977)

Printed in Great Britain

CONIDIUM ONTOGENY IN THE CHALARA STATE OF CERATOCYSTIS ADIPOSA I. LIGHT MICROSCOPY By C. R. HAWES AND A. BECKETT Department of Botany, The University of Bristol

The Chalara state of Ceratocystis adiposa (Butl.) C. Moreau forms two types of conidial chain, one of which is easily fragmented and composed of hyaline conidia, the other persistent and composed of hyaline or pigmented conidia. A sheath exists around the persistent chains. Both chain types are ontogenetically similar but conidia in persistent chains are variable in shape. The mode of conidium ontogeny is discussed in relation to this variability, and it is suggested that the conidiogenous locus is a meristem. Ceratocystis adiposa (Butl.) C. Moreau was first described by Buder (1906) as Sphaeronaema adiposum, the causal organism of black rot of sugar cane. Sartoris (1927) transferred this fungus to the genus Ceratostomella. Both authors describe the asexual state as being very variable, conidia ranging from elongated, hyaline endoconidia to large, deep brown, rounded conidia. Anyone conidiophore may produce all of these types. C. adiposa is placed in group 1 of Hunt's (1956) classification of the genus Ceratocystis as a species with an endoconidial imperfect stage, and on the basis of ascospore morphology, in the Fimbriata group of Olchowecki & Reid (1974). In a recent monograph Nag Raj & Kendrick (1975) suggested that the 'endoconidial' imperfect state of C. adiposa should be classified as a Chalara. They distinguished two types of conidia: (1) phialoconidia, hyaline to pale brown and variable in shape; (2) larger chlamydospores which are enteroblastic, phialidic, sometimes holoblastic, and brown to reddish brown. The Chalara state of C. adiposa fits into section IV of Hughes's (1953) classification of the hyphomycetes and IVA of Tubaki's (1958) scheme as a phialidic species. Hutchinson (1939) described conidia in Ophiostoma majus (van Beyma) Goidanich (Ceratocystis major (van Beyma) C. Moreau) as endogenously formed spores. The inner wall of the conidiophore took no direct part in their formation, but formed a delicate sheath around the conidial chain . Bhat (1972) also claimed that the conidia of C. adiposa were held together in a chain by a continuous outer wall layer. The work reported here together with that described by Hawes & Beckett (1977a) was undertaken to determine the relationships of the wall

layers involved in conidiogenesis in C. adiposa with respect to the recommendations of the Kananaskis meeting (Kendrick, 1971). MA TERIALS AND METHODS The culture of C. adiposa used in this study is maintained at the University of Bristol, Department of Botany (no. A 35). Conidiogenesis was studied in cultures grown on 3 % (w/v) malt agar plates at 25 DC and in thin culture chambers as described by Cole & Kendrick (1968) using 3 % malt agar. The conidiophores in Fig. 4 were fixed in acetic alcohol and hydrolysed with 1 M-HCl. Micrographs were taken using Ilford Pan F 35 mm film on a Zeiss Photomicroscope II (Figs. 1-4), and on a Wild M 20 microscope fitted with a Bolex-Wild control unit, Variotimer and an Alpa automatic, motorized camera (Figs. 5-19). For fluorescence microscopy material was mounted in a 0'05 % (w/v) solution of the optical brightener Calcoflour White M2R New (American Cyanamid Co.) in potassium phosphate buffer at pH 8. Photographs were taken using Ilford HP 4 35 mm film on a Zeiss Photomicroscope III with epifluorescence condenser III/RS using dark ground illumination and ultraviolet light . RESULTS Conidia are produced in basipetal chains at the tip of a subcylindrical to cylindrical conidiogenous cell (Figs. 4, 5). Two chain types occur: (1) chains that easily fragment and consist of smooth-walled hyaline, doliiform conidia (Fig. 1) and (2) persistent chains in which the conidia remain joined to each other and to the conidiogenous

260

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C. R. Hawes and A. Beckett cell (Figs. 1-4). These latter conidia are commonly surrounded by a membranous sheath (seen disrupted in Fig. 2) and show considerable variation in size, shape, degree of pigmentation and wall ornamentation. Conidia may be globose, pyriform, ovoid or cylindrical (Figs. 1-4) and range in size between 4'5 and 17'0 pm in diameter. The terminal conidium is always globose to ovoid, but a chain may contain a mixture of the above conidial forms (Figs. 2-4). The conidial wall may be verrucose to smooth (Figs. 1-4) and multilayered (Figs. 4, 19). Details of conidial morphology will be reported elsewhere. Globose and ovoid conidia are produced from the tip of the conidiogenous cell (Figs. 4, 5-19). Pyriform conidia are formed with their bases within the neck of the conidiogenous cell (Fig. 3) and cylindrical conidia are formed within the neck of the conidiogenous cell (Fig. 4). Clearing of the cells by hydrolysis in HCI renders the walls of the conidiogenous cell and of the conidia clearly visible (Fig. 4). In such preparations the flared wall at the apices of the conidiogenous cells can be seen. Figs. 5-13 show a time-lapse sequence of the formation of the first two conidia in a chain. The first conidium forms as a 'blowout' of the tip of the conidiogenous cell (Figs. 5-10) and septation between the conidium and conidiogenous cell is complete after 40 min (Fig. 10). A possible break in the wall at the tip of the conidiogenous cell can be seen in figure 10 (arrows). Formation of the second conidium is completed after a further 90 min and both conidia are of the hyaline, ovoid type. No change occurs in the length of the conidiophore or conidiogenous cell. Figs. 14-19 show the. development of a large pigmented conidium. The interval between initiation of the first and second conidium was over 16 h. The flared wall can be seen (Fig. 19 arrows) where the second conidium is 'blowing out' from the neck. of the conidiogenous cell. Treatment with Calcofluor results in a strong fluorescence of the walls in the neck region of the conidiogenous cells and of the walls ofthe youngest conidia (Figs. 20-23).

261 DISCUSSION

The terminology used in this and our other papers (Hawes & Beckett, 1977a, b) is that which resulted from the Kananaskis conference (Kendrick 1971). Conidial chains The Chalara state of C. adiposa forms two distinct chain types, one of which, the easily fragmented type, composed of hyaline, doliiform conidia has not been reported elsewhere. The persistent chains are surrounded by a sheath which Hutchinson (1939) described as the inner wall of the conidiophore. In contrast, Bhat (1972) considered that the conidia were held together in persistent chains by a continuous outer wall layer. If the sheath is disrupted (Fig. 2), the chain remains intact, suggesting that the sheath is not solely responsible for the integrity of the chain. Subramanian (1972) divided conidial chains into two types: 'true' chains in which there is a continuity of a wall layer along the conidia of a chain such as in Aspergillus niger van Tieghem, and ' false' chains which easily fragment and have no continuity of wall layers in the chain, such as in Thielaviopsis basicola (Berk. & Br.) Ferraris. He suggested that the nature of conidial chains is an important taxonomic criterion in the hyphomycetes. C. adiposa, however, is a fungus producing both types of conidial chain which differ only in the presence of a sheath around the permanent chains. Conidium formation is the same in both types (Hawes & Beckett, 1977 a). Doubt has been cast on the validity of a distinction between 'true' and 'false' chains (Campbell, 1975). His studies of conidiogenesis in Stachybotrys (Campbell, 1972) and Memnoniella (Campbell, 1975) show there is no difference in the way these fungi produce their conidia although one forms coherent chains and the other does not. Campbell suggested that the main factor in keeping chains intact is the time at which the septum between adjacent conidia splits. Our observations on C. adiposa also suggest that the strength of the septum is probably the major factor contributing towards the permanence of conidial chains and that to describe such chains as 'true' or 'false' is misleading and of no taxonomic value.

Fig. 1. Persistent and fragmented conidial chains. x 1400. Fig. 2. Chains of pigmented conidia with ruptured sheath (arrows). x 1200. Fig. 3. Globose, cylindrical and pyriform conidia. x 1700. Fig. 4: Cylindrical conidium in neck of conidiophore. Note flared wall at tip of conidiogenous cell. Material hydrolysed in HC!. x 1700.

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Figs. 14-19. Time-lapse sequence of formation of large globose conidiwn. Note flared wall of conidiogenous cell (Fig. 19 arrows). Time in minutes at top right-hand comers. x 1900.

Conidium ontogeny in Ceratocystis. I

21 Figs. 20-21. Fluorescence and bright -field micrographs of conidiogenous cell with the first conidium at the tip. x 1800. Figs. 22-23. Fluorescence and bright field micrographs of a conidial chain. x 1800.

Conidialsize and shape Variation in conidium shape in permanent chains is dependent on the depth at which the conidium develops within the conidiogenous cell. Thus a conidium formed completely within the neck of the conidiogenous cell such as in figure 4 (see also Hawes & Beckett, 1977a) has the shape of the conidiogenous cell neck, i.e. it is cylindrical. The wall of such a conidium must presumably rigidify whilst still in the neck of the conidiogenous cell. In contrast, globose and pyriform conidia expand as they develop. The term 'chlamydospore' has been adopted for the large pigmented, globose conidia of C. adiposa and other Chalara states (Nag Raj & Kendrick, 1975), on the assumption that they perform the 'resting' or 'survival' function of chlamydospores rather than the dispersal function of phialoconidia (Kendrick, pers. comm.), However, our own unpublished results show that after 6 weeks storage on filter paper, the percentage germination of hyaline and slightly pigmented conidia was as high as that of the large pigmented

conidia, and so functionally the term 'chlamydospore' does not seem appropriate. Hyaline and deeply pigmented conidia represent extremes in the range of wall pigmentation found in permanent chains, and since no ontogenetic differences between these types have been found, we suggest that, on these grounds also, the term 'chlamydospore' as applied to the pigmented conidia of C. adiposa is inappropriate.

Conidium ontogeny The first conidium is apparently 'blown out' at the tip of the conidiogenous cell and seen with the light microscope, appears to be holoblastic (Figs. 5-10, apparent layering of wall is due to focus changes during growth). Subsequently the outer layer of the conidiogenous cell breaks and all further conidia are enteroblastic (Figs. 3, 4). This pattern is confirmed by time-lapse studies (Figs. 5-13). More obvious is the fact that the time taken for the first conidium to form is approximately half that for each subsequent conidium. This may be related to a difference in the initial processes

C. R. Hawes and A. Beckett involved, the one being a sudden and rapid expansion by stretching, the other being a more gradual expansion as a result of wall growth by intussusception. Such an interpretation is supported by the observation that fluorescence with CaIcofluor is brightest in the region of the conidiogenous locus and around very young second or later conidia. In these areas wall growth would be expected as a result of intussusception of new wall material (e.g, Figs. 20, 22, arrows). In the primary conidium however, the new wall material, as a result of stretching, would be more diffuse and a corresponding lack of fluorescence is seen (Fig. 20). Conversely the very much greater time taken to complete the formation of the large, globose, pigmented conidia (Figs. 14-19) could be a consequence of the increase in deposition and elaboration of wall material. There is no change in length of the conidiogenous cell during conidiation and, excluding the first conidium, all conidial wall material must be newly synthesized from within the neck of the conidiogenous cell. This mode of ontogeny complies, at least in part, with that defined for a phialide (K endrick 1971). Variation in the position of the septum within the neck of the conidiogenous cell, and the fact that fluorescence occurs in a region equivalent to almost half the length of the conidiogenous cell suggests that the conidiogenous locus is either variable in position along this region, or, more probably, the whole of this zone is the conidiogenous locus, i.e. it is a meristem. Ultrastructural evidence for this concept, and further discussion of its implications are presented in another paper (Hawes & Beckett, 1977a).

BUTLER, E. J. (1906). Fungus diseases of sugar cane in Bengal. M emoirs of the Department of Agriculture in India 1, 32-53.

CAMPBELL, R. (1972). Ultrastructure of conidium ontogeny in the deuteromycete fungus Stachybotrys atra Corda. New Phy tologist 71, 1143-1149. CAMPBELL, R. (1975). The ultrastructure of the formation of chains of conidia in Memnoniella echinata . M ycologia 67, 760-769.

COLE, G. T. & KENDRICK, W. B. (1968). A thin culture chamber for time-lapse photomicrography of fungi at high magnifications. Mycologia 60, 340-344. HAWES, C. R. & BECKETT, A. (1977 a). Conidium ontogeny in the Chalara state of Cerato cystis adipose. II. Electron microscopy. Transactions of the British M ycological Society 68, 267-276.

HAWES, C. R. & BECKETT, A. (1977b). Conidium ontogeny in Thielaviopsis basicola. Transactions of the British M ycological Society 68, 304-307.

HUGHES, S. J. (1953). Conidiophores, conidia and classification. Canadian Journal of Botany 31, 577659·

HUNT, J. (1956). Taxonomy of the genus Ceratocystis. Lloydia 19, 1-58.

HUTCHINSON, S. A. (1939). Macroconidial formation in Ophiostoma majus (van Beyrna) Goidanich. Annals of Botany 3, 795-802. KENDRICK, W. B. (1971). Conclusions and recommendat ions. Ta xonomy of Fungi Imperfecti (ed. W. B. Kendrick), ch. 16, pp. 253-262. Toronto: University of Toronto Press. NAG RAJ, T. R. & KENDRICK, W. B. (1975). A monograph of Chalara and allied genera. Wilfrid Laurier University Press. OLCHOWECKI, A. & REID, J. (1974). Taxonomy of the genus Ceratocystis in Manitoba. Canadian Journal of Botany 52, 1675-1711.

SARTORIS, G. B. (1927). A cytological study of Ceratostomella adiposum (Butl.) comb.nov., the black-rot fungus of sugar cane. Journal of Agricultural Research 35, 577-5 85.

We are grateful to Drs R. Campbell and M. F. Madelin for helpful discussion during preparation of the manuscript and to the Science Research Council for a Research Studentship (to C.R.H.) and for a Research Grant (B jSRj90718 to A.B.).

SUBRAMANIAN, C. V. (1972). Conidial chains, their nature and significance in the taxonomy of Hyphomycetes, Current Science 4 1 , 43-49.

TUBAKI, K. (1958). Studies on the Japanese hyphomycetes. V. Journal of Hattori Botanical Laboratory 20, 142 - 244.

REFERENCES BHAT, V. R. (1972). Observations on Ceratocystis adiposa and the conidial ontogeny of its imperfect state. Sydowia Annales M ycologici 26, 26-28.

(Accepted for publication 29 September 1976)