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Somatic fusion in Puccinia graminis f. sp. tritici
Notes and brief articles nants of the tuber walls are in the background. Groups of wart-like protuberances can be seen on each of the cysts and are clearly shown in Fig. 2. Ultrathin sections of resin-impregnated material confirm that the protuberances are, in fact, outgrowths of the cyst walls. High-magnification phase-contrast optical microscopy (oil-immersion lens) of cystosori, stained in lactophenol-cotton blue, did reveal some surface irregularity of the cysts but was not adequate to resolve the structures. Scanning electron microscopy is thus a valuable tool for determining surface features of propagules, but caution must be exercised in the choice of fixation and dehydration techniques. In these
293
studies freeze-drying and critical-point drying of material both resulted in gross distortion and collapse of the cysts and chemical fixation was a necessary prerequisite to avoid this. It is hoped that cystosori of other species of Spongospora will be examined as fresh material becomes available since it is not possible to cultivate these fungi on artificial media . REFERENCES
JONES, D. (1976). Infection of plant tissue by Sclero-
tinia sclerotiorum : a scanning electron microscope study. Micron 7, 275-279. KARLING, J. S. (1968). The Plasmodiophorales, New York, London: Hefner.
SOMATIC FUSION IN PUCCINIA GRAMINIS F. SP. TRITICI P. G. WILLIAMS*
Department of Agricultural Botany, University of Sydney, Sydney
A somatic diploid phase of a variable duration is common to several mechanisms proposed for the origin of new strains of cereal rust fungi by somatic hybridization (Watson, 1957; Ellingboe, 1961; Hartley & Williams, 1971). However, evidence of a somatic diploid phase in rust fungi was lacking until investigations showed that monokaryotic variants isolated from axenic cultures of Puccinia graminis f.sp, tritici are diploid (Williams & Hartley, 1971; Maclean, Tommerup & Scott, 1974). In addition, it is known from a cytological study (Williams, 1975) that a urediniospore produced by a rnonokaryotic diploid strain obtained from an axenic culture is the same size as one produced by a normal strain, but its single nucleus has twice the volume of a haploid nucleus. Nuclear size therefore appears to be a valid criterion for making a preliminary assessment of the occurrence of somatic diploidy during the normal development of the wheat stem rust fungus. Accordingly, nuclei were examined in urediniospores produced by a normal strain on adult wheat plants and this communication reports the formation of urediniospores with a single, large nucleus. Urediniospores were taken from infections of P. graminis f.sp. tritici, culture No. 334 (race 126-Anz 6,7) on stems of adult plants of susceptible wheat (cv. W2691) grown in a controlled environment chamber at 22-24°. Nuclei were stained according to the procedure previously described (Williams, 1975) but with one modification : good definition was obtained more consistently if, after * Permanent address: 6 Undercliff Street, Neutral Bay, N.S.W. 2089, Australia. Trans. Br, mycol, Soc. 70 (2) (1978).
2006
extraction in alkaline ethanol, the slides were left overnight in 70 % ethanol before washing and applying acetic acid-orcein. Eight samples from six rust pustules were examined using a 50 x oilimmersion objective . Teliospores were seen with the naked eye in three of the infections. Of a total of 8470 urediniospores which was scored, ten possessed one large nucleus instead of two smaller ones. Each urediniospore with a single nucleus had the same size and morphology as a normal, binucleate urediniospore. The diameter of one large nucleus was 5'8 pm and the mean of three haploid nuclei in the same preparation was 4.6 pm; thus, the large nucleus was 2'5 x greater in volume than a small nucleus. The uninucleate urediniospores formed by a normal strain therefore closely resembled those urediniospores formed by a monokaryotic diploid strain (Williams, 1975). Uninucleate urediniospores were found in three of the five samples taken from pustules in which teliospores were visible (5519 urediniospores were counted in the five samples). No uninucleate urediniospores were observed in samples from uredinia apparently free of teliospores. This agrees with a previous observation (Williams, 1975) that urediniospores not having two nuclei are very rare in infections on seedling leaves. An unusual structure combining features of a teliospore and a urediniospore was seen in one sample from a partly telial infection. The structure consisted of two cells and a non-sessile pedicel and had the general appearance of a teliospore. However, the walls of the cells bore spines and lacked the refractivity and pigment of a normal teliospore,
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294
Notes and brief articles
some of which were present in the same preparation. Each cell contained two nuclei like those in a urediniospore. These results emphasize the suitability of the formaldehyde-alkaline ethanol-acetic acid-orcein procedure for detecting urediniospores with diploid nuclei. An important advantage of the technique is that most of the storage lipid is extracted, usually making the nuclei clearly visible. The preparation of living urediniospores for bright field or Nomarski interference-contrast microscopy is technically simple but because of the mass of oil granules it is rarely possible to see the nuclei clearly. Little & Manners (1969) used embedding, sectioning and the staining of infected tissues in their search for diploid nuclei in urediniospores of P. striiformis. Their procedure is laborious and it is likely that the small objects (0'7!tm diam) stained by crystal violet in their sections of immature urediniospores were nucleoli (Goddard, 1976). The uninucleate urediniospores described here may have been formed (a) as the result of an error in the function of dikaryotic sporogenous cells, the error initiating the process of karyogamy which normally occurs only during the development of a teliospore, or (b) as products of an isolated sporogenous cell containing a diploid nucleus or of cells in a diploid sector of a uredinium. The apparent association of uninucleate urediniospores with uredinia which contained teliospores and the occurrence of structures with composite morphology favour the first suggestion although other methods of origin are not excluded. The first explanation is also consistent with the idea that urediniospores and teliospores of Puccinia are sympodioconidia (Hughes, 1970), i.e, propagules with different function generated by the same sporogenous (sympodial) cell. The observations therefore suggest that sporogenesis is liable to error in a uredinium which is begining to form teliospores, The effect of one type of rare accident is to trigger karyogamy in a cell having the morphology and perhaps also the function of a urediniospore. The foregoing proposal is important in the breeding biology of the wheat-stem rust fungus. The principal questions in need of an answer are whether the large nuclei are diploid and whether the diploid urediniospores can infect wheat. A simple and direct approach to these questions would be to inoculate seedlings of a susceptible wheat with urediniospores gathered from uredinia in which uninucleate urediniospores had been detected by staining. Monokaryotic diploid isolates have a distinctive uredinial topography and
Trans. Br. mycol. Soc. 70 (2) (1978).
an experienced observer can distinguish between a diploid and a normal haploid infection (G. J. Green, P. G. Williams & D. J. Maclean, unpubl.; 1. A. Watson & D. J. Maclean, unpubl.). Failure to find anomalous uredinia would leave the question about the nature of the large nuclei unanswered. But if an apparent diploid uredinium was found the ploidy of that infection could be investigated by measuring urediniospore and nuclear size (Williams, 1975) and content of nuclear DNA (Williams & Mendgen, 1975). It would be more satisfactory, of course, to bring such an atypical strain into axenic culture and make a genetical analysis as Ingram (1968) did to demonstrate the diploid nature of Verticillium dahliae var. longisporum, The work was supported by the Wheat Industry Research Council. REFERENCES ELLINGBOE, A. H. (1961). Somatic recombination in Pucciniagraminis var. tritici. Phytopathology 51,13-15. GODDARD, M. V. (1976). Cytological studies of Puccinia striiformis (yellow rust of wheat). Transactions of the British Mycological Society 66,433-437. HARTLEY, M. J. & WILLIAMS, P. G. (1971). Genotypic variation within a phenotype as a possible basis for somatic hybridization in rust fungi. Canadian Journal of Botany 49, 1085-1087. HUGHES, S. J. (1970). Ontogeny of spore forms in Uredinales. Canadian Journal of Botany 48, 21472157. INGRAM, R. (1968). Verticillium dahliae var, longisporum, a stable diploid. Transactions of the British Mycological Society 51, 339-341. LITTLE, R. & MANNERS, J. G. (1969). Somatic recombination in yellow rust of wheat (Puccinia striiformisi, II. Germ tube fusions, nuclear number and nuclear size. Transactions of the British Mycological Society 53,259-267. MACLEAN, D. J., TOMMERUP, I. C. & SCOTT, K.]. (1974). Genetic status of monokaryotic variants of the wheat stem rust fungus isolated from axenic culture. Journal of General Microbiology 84, 364-378. WATSON,!. A. (1957). Further studies on the production of new races of Puccinia graminis var. tritici on wheat seedlings. Phytopathology 47, 510-5 12 . WILLIAMS, P. G. (1975). Evidence for diploidy of a monokaryotic strain of Puccinia graminis f.sp. tritici. Transactions of the British Mycological Society 64, 15- 22. WILLIAMS, P. G. & HARTLEY, M. J. (1971). Occurrence of diploid lines of Puccinia graminis tritici in axenic culture. Nature, New Biology 229, 181-182. WILLIAMS, P. G. & MENDGEN, K. W. (1975). Cytofluorometry of DNA in uredospores of Puccinia graminis f.sp. tritici. Transactions of the British Mycological Society 64, 23- 28.
Printed in Great Britain
293
studies freeze-drying and critical-point drying of material both resulted in gross distortion and collapse of the cysts and chemical fixation was a necessary prerequisite to avoid this. It is hoped that cystosori of other species of Spongospora will be examined as fresh material becomes available since it is not possible to cultivate these fungi on artificial media . REFERENCES
JONES, D. (1976). Infection of plant tissue by Sclero-
tinia sclerotiorum : a scanning electron microscope study. Micron 7, 275-279. KARLING, J. S. (1968). The Plasmodiophorales, New York, London: Hefner.
SOMATIC FUSION IN PUCCINIA GRAMINIS F. SP. TRITICI P. G. WILLIAMS*
Department of Agricultural Botany, University of Sydney, Sydney
A somatic diploid phase of a variable duration is common to several mechanisms proposed for the origin of new strains of cereal rust fungi by somatic hybridization (Watson, 1957; Ellingboe, 1961; Hartley & Williams, 1971). However, evidence of a somatic diploid phase in rust fungi was lacking until investigations showed that monokaryotic variants isolated from axenic cultures of Puccinia graminis f.sp, tritici are diploid (Williams & Hartley, 1971; Maclean, Tommerup & Scott, 1974). In addition, it is known from a cytological study (Williams, 1975) that a urediniospore produced by a rnonokaryotic diploid strain obtained from an axenic culture is the same size as one produced by a normal strain, but its single nucleus has twice the volume of a haploid nucleus. Nuclear size therefore appears to be a valid criterion for making a preliminary assessment of the occurrence of somatic diploidy during the normal development of the wheat stem rust fungus. Accordingly, nuclei were examined in urediniospores produced by a normal strain on adult wheat plants and this communication reports the formation of urediniospores with a single, large nucleus. Urediniospores were taken from infections of P. graminis f.sp. tritici, culture No. 334 (race 126-Anz 6,7) on stems of adult plants of susceptible wheat (cv. W2691) grown in a controlled environment chamber at 22-24°. Nuclei were stained according to the procedure previously described (Williams, 1975) but with one modification : good definition was obtained more consistently if, after * Permanent address: 6 Undercliff Street, Neutral Bay, N.S.W. 2089, Australia. Trans. Br, mycol, Soc. 70 (2) (1978).
2006
extraction in alkaline ethanol, the slides were left overnight in 70 % ethanol before washing and applying acetic acid-orcein. Eight samples from six rust pustules were examined using a 50 x oilimmersion objective . Teliospores were seen with the naked eye in three of the infections. Of a total of 8470 urediniospores which was scored, ten possessed one large nucleus instead of two smaller ones. Each urediniospore with a single nucleus had the same size and morphology as a normal, binucleate urediniospore. The diameter of one large nucleus was 5'8 pm and the mean of three haploid nuclei in the same preparation was 4.6 pm; thus, the large nucleus was 2'5 x greater in volume than a small nucleus. The uninucleate urediniospores formed by a normal strain therefore closely resembled those urediniospores formed by a monokaryotic diploid strain (Williams, 1975). Uninucleate urediniospores were found in three of the five samples taken from pustules in which teliospores were visible (5519 urediniospores were counted in the five samples). No uninucleate urediniospores were observed in samples from uredinia apparently free of teliospores. This agrees with a previous observation (Williams, 1975) that urediniospores not having two nuclei are very rare in infections on seedling leaves. An unusual structure combining features of a teliospore and a urediniospore was seen in one sample from a partly telial infection. The structure consisted of two cells and a non-sessile pedicel and had the general appearance of a teliospore. However, the walls of the cells bore spines and lacked the refractivity and pigment of a normal teliospore,
Printed in Great Britain
294
Notes and brief articles
some of which were present in the same preparation. Each cell contained two nuclei like those in a urediniospore. These results emphasize the suitability of the formaldehyde-alkaline ethanol-acetic acid-orcein procedure for detecting urediniospores with diploid nuclei. An important advantage of the technique is that most of the storage lipid is extracted, usually making the nuclei clearly visible. The preparation of living urediniospores for bright field or Nomarski interference-contrast microscopy is technically simple but because of the mass of oil granules it is rarely possible to see the nuclei clearly. Little & Manners (1969) used embedding, sectioning and the staining of infected tissues in their search for diploid nuclei in urediniospores of P. striiformis. Their procedure is laborious and it is likely that the small objects (0'7!tm diam) stained by crystal violet in their sections of immature urediniospores were nucleoli (Goddard, 1976). The uninucleate urediniospores described here may have been formed (a) as the result of an error in the function of dikaryotic sporogenous cells, the error initiating the process of karyogamy which normally occurs only during the development of a teliospore, or (b) as products of an isolated sporogenous cell containing a diploid nucleus or of cells in a diploid sector of a uredinium. The apparent association of uninucleate urediniospores with uredinia which contained teliospores and the occurrence of structures with composite morphology favour the first suggestion although other methods of origin are not excluded. The first explanation is also consistent with the idea that urediniospores and teliospores of Puccinia are sympodioconidia (Hughes, 1970), i.e, propagules with different function generated by the same sporogenous (sympodial) cell. The observations therefore suggest that sporogenesis is liable to error in a uredinium which is begining to form teliospores, The effect of one type of rare accident is to trigger karyogamy in a cell having the morphology and perhaps also the function of a urediniospore. The foregoing proposal is important in the breeding biology of the wheat-stem rust fungus. The principal questions in need of an answer are whether the large nuclei are diploid and whether the diploid urediniospores can infect wheat. A simple and direct approach to these questions would be to inoculate seedlings of a susceptible wheat with urediniospores gathered from uredinia in which uninucleate urediniospores had been detected by staining. Monokaryotic diploid isolates have a distinctive uredinial topography and
Trans. Br. mycol. Soc. 70 (2) (1978).
an experienced observer can distinguish between a diploid and a normal haploid infection (G. J. Green, P. G. Williams & D. J. Maclean, unpubl.; 1. A. Watson & D. J. Maclean, unpubl.). Failure to find anomalous uredinia would leave the question about the nature of the large nuclei unanswered. But if an apparent diploid uredinium was found the ploidy of that infection could be investigated by measuring urediniospore and nuclear size (Williams, 1975) and content of nuclear DNA (Williams & Mendgen, 1975). It would be more satisfactory, of course, to bring such an atypical strain into axenic culture and make a genetical analysis as Ingram (1968) did to demonstrate the diploid nature of Verticillium dahliae var. longisporum, The work was supported by the Wheat Industry Research Council. REFERENCES ELLINGBOE, A. H. (1961). Somatic recombination in Pucciniagraminis var. tritici. Phytopathology 51,13-15. GODDARD, M. V. (1976). Cytological studies of Puccinia striiformis (yellow rust of wheat). Transactions of the British Mycological Society 66,433-437. HARTLEY, M. J. & WILLIAMS, P. G. (1971). Genotypic variation within a phenotype as a possible basis for somatic hybridization in rust fungi. Canadian Journal of Botany 49, 1085-1087. HUGHES, S. J. (1970). Ontogeny of spore forms in Uredinales. Canadian Journal of Botany 48, 21472157. INGRAM, R. (1968). Verticillium dahliae var, longisporum, a stable diploid. Transactions of the British Mycological Society 51, 339-341. LITTLE, R. & MANNERS, J. G. (1969). Somatic recombination in yellow rust of wheat (Puccinia striiformisi, II. Germ tube fusions, nuclear number and nuclear size. Transactions of the British Mycological Society 53,259-267. MACLEAN, D. J., TOMMERUP, I. C. & SCOTT, K.]. (1974). Genetic status of monokaryotic variants of the wheat stem rust fungus isolated from axenic culture. Journal of General Microbiology 84, 364-378. WATSON,!. A. (1957). Further studies on the production of new races of Puccinia graminis var. tritici on wheat seedlings. Phytopathology 47, 510-5 12 . WILLIAMS, P. G. (1975). Evidence for diploidy of a monokaryotic strain of Puccinia graminis f.sp. tritici. Transactions of the British Mycological Society 64, 15- 22. WILLIAMS, P. G. & HARTLEY, M. J. (1971). Occurrence of diploid lines of Puccinia graminis tritici in axenic culture. Nature, New Biology 229, 181-182. WILLIAMS, P. G. & MENDGEN, K. W. (1975). Cytofluorometry of DNA in uredospores of Puccinia graminis f.sp. tritici. Transactions of the British Mycological Society 64, 23- 28.
Printed in Great Britain