Growth of Ustilago bullata in culture

[ 1°3 ] Trans. Br, mycol. Soc. 70 (1) 103-108 (1978)

Printed in Great Britain

GROWTH OF USTILAGO BULLATA IN CULTURE By R. E. FALLOON Agricultural Botany Department, University College of Wales, Aberystwyth, U.K. Teliospore germination, metabasidium extension, development and proliferation of sporidia, plasmogamy and growth of dikaryotic infection hyphae of Ustilago bullata Berk, growing in culture were studied. Growth of this organism at all stages except proliferation of sporidia was linear, although growth rates differed at each stage. The pattern of early growth of U. bullata differs from that described previously for saprophytic fungi. The kinetics of growth in culture of several fungal genera have been described previously (Trinci, 1969, 1971; Koch, 1975). The fungi considered to date, all follow a similar pattern of early growth in which colonization of the growth medium by a vegetative mycelium occurs after spore germination. In most cases studied germ-tubes initially grew exponentially in length (Trinci, 1969, 1971). Members of the Ustilaginales do not progress directly from germinating spore to vegetative mycelium. T eliospores of this group usually germinate to form metabasidia (promycelia) of determinate length. On completion of metabasidial extension, sporidia are produced upon the metabasidium, and these may divide more or less profusely. Finally plasmogamy occurs between compatible metabasidial cells and/or sporidia, followed by growth of dikaryotic 'infection' hyphae. It is these processes, from teliospore germination to growth of the dikaryon, occurring in close proximity to the germinating host embryo, that precede infection by smut fungi that infect at the seedling stage of host growth. Teliospore germination in the Ustilaginales is important from a taxonomic viewpoint (Duran, 1973), and has been illustrated and described for Ustilago bullata Berk. by Fischer (1937). This paper reports a study on the growth of U. bullata, the causal organism of head smut of several grass genera. The study was carried out in order to understand more fully the processes leading up to infection of Bromus catharticus Vahl by this pathogen. MATERIALS AND METHODS

Teliospores of U. bullata used in this study were collected from infected, field-grown B. catharticus cv. 'Grasslands Matua' plants in Palmerston North, New Zealand. In examining cultural growth of U. bullata observations were made on both teliospore popu-

lations and individual teliospores and dikaryotic hyphae. Teliospore populations Teliospores of U. bullata were spread onto the surface of Potato Dextrose Agar (PDA; Oxoid) in Petri plates. The plates were incubated at 25°, (± 0'2°). At 1 h intervals, from 0 to 24 h, a plate was removed and spores on the agar surface were photographed microscopically. Plates were discarded after photographing. Photographic negatives were viewed with a film strip projector and measurements were made of teliospore dimensions, metabasidium lengths and numbers of sporidia produced by each germinated teliospore. At each time interval measurements were made of a sample of 100 teliospores, Individual teliospores and dikaryotic hyphae A microscope slide growth chamber (Falloon, 1977) was used to follow early growth of U. bullata on PDA. Observations were made ina constant temperature room. RESULTS

Increase in teliospore size prior to metabasidium emergence Increases in dimensions for populations of teliospores are indicated in Fig. 1. Teliospore diameters, both longest and shortest, increased linearly with time, the overall increase being about 8 %. This is equivalent to a volume increase of about 28 % (volume calculations made assuming teliospores to be prolate spheroids). Metabasidium growth Metabasidia began emerging from teliospores after a lag of 3 to 4 h at 25 °. Emergence of metabasidia from

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Fig. 3. Mean metabasidiurn lengths for U, bullata grown on PDA at 23° (±O'5) in the microscope slide growth chamber. D value obtained from Qat 1 % in the 'Studentized' range. populations of teliospores occurred over a period of about 18 h at this temperature. Of the teliospore sample used 91 % germinated. Metabasidia increased in length linearly until cessation of growth (Fig. 2). Examination of individual metabasidia in the slide growth chamber revealed a short lag phase at meta basidium emergence, followed by a period of linear extension (Fig. 3). Mean metabasidium growth rate at 23° was 10'2 pm h- 1 (S.E. ±0·56). At 23° metabasidium extension was completed in 1'5 to 2 h, and mean length at cessation of growth was 16,8 pm (S.E. ± 0'55). A number of U. bullata teliospores produced more than one, usually two, metabasidia. Secondary metabasidia were produced by about 25 % of germinated teliospores in two phases (Fig. 2); the first occurring 9 to 11 h after inoculation onto the growth medium, and the second 4 to 8 h later. Observation of metabasidia developing in the microscope slide growth chamber using high power phase contrast microscopy revealed that septation in metabasidia did not occur until after extension growth had ceased. Development and growth of sporidia Mean time from the cessation of metabasidium extension growth to appearance of the first

sporidia at 23° was 3'5 h (S.E. ± 0'13). Increase in numbers of sporidia from both teliospore populations (Fig. 4) and individual teliospores (Fig. 5) followed an exponential pattern. Data from the slide growth chamber (Fig. 5) showed that sporidia were produced erratically for about 4 h after which they increased in number at an exponential rate. Mean sporidium doubling times on PDA were 2'4 h at 25° (from teliospore populations) and 2'5 h at 23° (from individual teliospores). These doubling times gave mean specific growth rates (Trinci, 1969) of 0'29 h ? at 25° and 0'27 h-1 at 23°. Other work (unpubl.) has shown that optimum temperature for sporidium production on PDA was between 25'0 and 27'5°. Sporidia were produced both terminally and laterally on metabasidia and appeared to undergo enteroblastic development (Fig. 2; Falloon, 1977). Increase in numbers of sporidia from a single meta basidium occurred both by multiple proliferation from terminal and lateral loci on the metabasidium and by multiple proliferation from a single locus on each sporidium. At 23° the mean time interval between development of sporidia from single loci was 2'2 h (S.E. ± 0'09). Growth of fusion and dikaryotic hyphae Fusions between compatible cells (usually sporidia,

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R. E. Falloon but occasionally metabasidium cells) were first observed 15 h after inoculation onto PDA at 25° in the slide growth chamber, and by 22 h fusions were widely observed. In some instances fusion hyphae were quite extensive and growth of these could be measured. Growth of 7 fusion hyphae was measured, and in all cases hyphae grew linearly although growth rates varied considerably. Measured growth rates at 25° varied between 2'2 and 5'4 pm h- 1 (mean 3'9 pm h-l ~ S.E. ± 0'49). Dikaryotic hyphae commenced growth in some cases within 0'5 h of plasmogamy, but more commonly 2 to 3 h after fusion of compatible cells. From the outset growth of these hyphae was linear and at 25° mean growth rate was 37'2 pm h ? (S,E. ± 2-67). Dikaryotic hyphae were invariably unbranched and septate, usually growing in a straight line across or just above the agar surface. In some cases growth across the surface halted abruptly and the dikaryon grew vertically down into the air space of the growth chamber. Accurate measurement of aerial growth was not possible. DISCUSSION

In culture, growth of U. bullata progresses through several stages . From the time teliospores are placed upon the growth medium, measurable growth occurs, including increase in teliospore size, metabasidium extension, development and proliferation of sporidia, and extension of fusion hyphae. These stages precede plasmogamy and are thus essential for development of dikaryotic hyphae. Progression through these stages in vivo leads to growth of dikaryons which subsequently penetrate and infect host coleoptiles. Increase in size of fungus spores prior to germtube emergence is a common phenomenon (Allen, 1965; Barnes & Parker, 1966), but detailed examination of spore swelling has been carried out for only a few species (Ekundayo & Carlile, 1964; Fletcher, 1969; Trinci, 1971; Gull & Trinci, 1971). In all cases considered spores grew linearly with time. Linear increase in the dimensions of U. bullata teliospores recorded in the present study was considerably less than that reported for other genera. This may be due to the comparatively thick inextensible spore wall that occurs in Ustilago spp . (Robb, 1972). Spore swelling has been shown to be accompanied by increases in metabolic activity (Allen, 1965; Ekundayo, 1966) and internal complexity (Hawker & Abbott, 1963; Hawker, 1966; Robb, 1972). Allen (1965) suggested that spore swelling represented ' an intrinsic part of the growth process.' Unlike germ-tube growth in most fungi (Trinci, 1969, 1971), growth of metabasidia of U. bullata

1°7

was linear. Trinci (1971) suggested that exponential growth he had observed for germ-tubes may have been supported, in part at least, by endogenous spore organic reserves. As the organism became more reliant upon organic substances in the growth medium, germ-tube growth rate decreased. It seems possible that exponential growth of individual germ-tubes may be an adaptation in saprophytic fungi to increase the rate at which media supporting growth are initially colonized. Metabasidia differ from germ-tubes in that they are not concerned with colonization but are involved in processes leading more or less directly to plasmogamy. It thus seems reasonable that metabasidia need not follow a pattern of growth similar to that of colonizing saprophytes. Hyphal growth in U. bullata, both of fusion hyphae and dikaryons, was also linear. Both types of hyphae invariably remained unbranched. Single hyphae in other fungi have been shown to extend at constant rates (Plomley, 1959; Trinci, 1970, 1974), although exponential growth in total hyphal length occurred when mycelia developed multiplicities of growing tips (Plomley, 1959; Trinci, 1969, 1974; Koch, 1975). Cultural growth of U. bullata was thus linear at all stages observed except that of increase in numbers of sporidia. Proliferation of sporidia from a single metabasidium was exponential, and followed a pattern similar to that for cells in a young colony of fission yeasts (Morris, 1958). In the case of yeasts exponential growth would again appear to enhance initial colonization of the growth medium. In U . bullata exponential growth onl y occurred at the stage of increase in numbers of cells capable of plasmogamy. Whereas saprophytic fungi grow exponentially to increase the rate of substrate colonization, exponential growth in U. bullata would seem to facilitate successful plasmogametic fusions. However, as succes sful plasmogamy leading to development of dikaryons can occur directly between metabasidial cells, it is possible that extensive proliferation of sporidia occurs only under optimum growth conditions, and is a response to relatively high nutrient levels. Work reviewed and reported by Fischer & Holton (1957), indicated that rich culture media were necessary for maximum production of sporidia. It thus seems that development of large numbers of sporidia may be a characteristic of growth in artificial culture. Further work is necessary to establish the importance of sporidial growth in vivo and to determine the significance of this phase of growth in the infection process.

Ustilago bullata in culture Zealand National FLETCHER, J. (1969).

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Assistance given by a New Research Advisory Council Postgraduate Fellowsh ip is gratefully acknowledged. I thank Dr Ellis Griffiths for his helpful discussion and criticism of the manuscript. The assistance of Mr E . W. Wintle with processing of photomicrographs was appreciated.

REFERENCES

ALLEN, P. J. (1965). Metabolic aspects ofspore germination in fungi. Annual Review of Phytopathology 3, 3 13-342. BARNES, M. & PARKER, M. S. (1966). The increase in size of mould spores during germination. Transactions of the British Mycological Society 49, 487-494. DuRAN, R. (1973) . Ustilaginales. In The Fungi : An Advanced Treatise, vol. IVB (eds. G. C . Ainsworth, F . K. Sparrow and A. S. Sussman), pp . 281-300. New York, London: Academic Press. EKUNDAYO, J. A. (1966). Further studies on germination of sporangiospores of Rhizopus arrhizus. Journal of General Microbiology 42, 283-291. EKUNDAYO, J. A. & CARLILE, M . J. (1964). The germination of sporangiospores of Rh izopus arrhizus; spore swelling and germ-tube emergence. Journal of General Microbiology 35, 261-269. FALLOON, R. E. (1977). Chamber for continuous microscopic observation of fungal growth. Transactions of the British My cological Society 66, 41-44. FISCHER, G. W. (1937). Observations on the comparative morphology and taxonomic relationships of certa in gra ss smuts in Western North America. Mycologia 29, 408-4.25. FISCHER, G. W. & HOLTON, C. S. (1957). Biology and Control of the Smut Fungi. The Ronald Press Company, New York, pp . 221-233.

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(A ccepted fOT publication

20 June

1977)