- Email: [email protected]
Development of basidia and basidiospores in Uromyces species on wild barley and liliaceae in Israel
[ 377 ] Trans. Br. mycol. Soc. 7S (3) 377-382 (1980)
Printed in Great Britain
DEVELOPMENT OF BASIDIA AND BASIDIOSPORES IN UROM YCES SPECIES ON WILD BARLEY AND LILIACEAE IN ISRAEL By Y. ANIKSTER,* J. G. MOSEMANt
*
t
AND
1. WAHL*
Division of Mycology and Plant Pathology, The Tel Aviv University, Tel Aviv, Israel Plant Genetics and Germplasm Institute, USDA, Beltsville Agricultural Research Center, Beltsville, Md 20705, U.S.A.
Development of basidia and basidiospores varies in Uromyces rusts which have uredinia and telia on wild species of Hordeum and alternate with Liliaceous species. The same is true of short-cycled Uromyces species living on several members of the Liliaceae. Conventional rust types were observed in the two heteroecious species Uromyces hordeastri f.sp. bulbosi-autumnalidis, and U. reichertii, as well as in the microcyclic U. rayssiae which is correlated with U. reichertii. Simplified basidia have two binucleate cells and a basal cell lacking nuclei. Such basidia yield two binucleate basidiospores and are characteristic of U. christensenii. Several other heteroecious rusts, and the investigated formae speciales of the microcyclic U. scillarum have a similar basidial development. Basidia consisting of a binucleate cell with binucleate basidiospores, and uninucleate cells with uninucleate basidiospores were seldom found and were confined to heteroecious species. In U. oiennotbourginii and U. christensenii inoculation with a single basidiospore induced development of aecia, while in U. scillarum f.sp. leopoldiae-maritimae inoculation with one basidiospore resulted in the formation of telia. These rust fungi are therefore self-fertile. Israel is located in one of the centres of origin and diversification of species in the Liliaceae and also of Hordeum spontaneum C. Koch, the likely progenitor of cultivated barley. The wild barley species, H. bulbosum L. (of the type 2n = 28) and H. murinum L. are ubiquitous, varying in morphology and growth habit. This is true to a lesser degree of H. marinum Huds. The concept of host-parasite coevolution implies that genetic differentiation of the host is matched by a similar variability in the parasite. Our previous studies have revealed considerable diversification in the morphology, life-history and pathogenicity in rusts inhabiting some of the mentioned barley species and indigenous Liliaceae (Anikster, 1975; Anikster & Wahl, 1966a, b, 1979; Anikster, Moseman & Wahl, 1972, 1976). Buller (1950), Jackson (1931, 1935) and others emphasized the great variation in the nuclear cycle of rust fungi, as shown by the initiation of the dikaryotic condition and cytological processes in the development of the basidium. They postulated that cytological research in the Uredinales, and particularly in species correlated phylogenetically, could provide a better insight into the evolution of the rusts. Unfortunately, little attention has been given to these problems, and especially to a study on the
nuclear history in the basidium (Jackson, 1931). Kemp (1974, 1976) stressed the significance of cytological aspects in studies on the evolution of Hymenomycetes, their adaptation and speciation. Some of the above considerations prompted our study of the development of basidia and basidiospores in Uromyces species living on wild barley and Liliaceous species in Israel. Preliminary results have already been reported (Anikster, Moseman & Wahl, 1972, 1973, 1977; Anikster & WaW, 1979). MATERIALS AND METHODS
The rust fungi The diagnostic descriptions and information on the biology of the rust fungi have been published (Anikster, 1975; Anikster & WaW, 1966a, b; Anikster et al., 1977). The life-cycles of the fungi will be summarized in this paper. Teliospore germination The best germination was usually obtained with the method designed by Anikster (1975). Teliospores were scraped from green leaves or straw and floated for 24-28 h in Petri dishes on distilled
0007-1536/80/2828-6700 $01.00 © 1980 The British Mycological Society
Development of basidia and basidiospores or tap water at 18-20 °C. Similar results were obtained by soaking plant segments harbouring telia for 48-72 h and distributing the spore suspension on water agar. In some tests 100 % teliospore germination was obtained. The method had to be modified for teliospores of Uromyces reichertii Anikst. & WaW and U. rayssiae Anikst. & Wahl which are surrounded by thick walls (Anikster & Wahl, 1966a). A low to moderate germination rate was obtained by suspending teliospores in water in a Petri dish at 8° for 14 days and then transferring them to a chamber at 15° for 30-40 days. The nuclei in the teliospores, basidia and basidiospores were stained with Heidenhain's or Delafield's haematoxylin (Gurr, 1965). RESULTS
Uromyces reichertii Pycnia and aecia on Scilla hyacinthoides L., uredinia and telia on Hordeum bulbosum. The teliospores are uninucleate. The basidium consists of a basal cell without nuclei and four uninucleate cells (Fig. 1). A single uninucleate basidiospore is produced by each of the four cells. The fungus is self-sterile. The microcyclic species U. rayssiae with telia on S. hyacinthoides is correlated with U. reichertii. The basidium consists of a basal cell devoid of nuclei and four uninucleate cells, each forming a uninucleate basidiospore. Uromyces christensenii Aecia on Mus cari paruiflorum Desf., uredinia and telia on Hordeum bulbosum, Teliospores are uninucleate. At the initial stage of teliospore germination the nucleus migrates into the basidium. Upon elongation of the latter the nucleus divides once. Two cross walls divide the basidium into three cells. At first the middle and the upper cells are uninucleate and the basal cell has no nuclei. Then the nuclei divide and the two cells become binucleate (Fig. 2). Each of the two cells develops a single basidiospore, and both nuclei pass into the corresponding basidiospore. Inoculation of the alternate host with a single binucleate basidiospore results in the development of aecia with aeciospores infectious on H. bulbosum, The organism is obviously self-fertile. Its nuclear cycle parallels that of the microcyclic Puccinia arenariae (Schum.) Wint. (Lindfors, 1924). Only occasionally does a basidium contain a basal cell without nuclei; and two uninucleate cells which form two uninucleate basidiospores, and one binucleate cell with a binucleate basidiospore.
Uromyces viennot-bourginii The species contains several formae speciales, all living on Hordeum spontaneum (Anikster et al., 1977). The basidial formation, basidiospore development and nuclear conditions are the same as in U. christensenii Anikst. & WaW (F ig. 3). Aecia result from the inoculation of the alternate hosts Bellevalia eigii Feinbr. and B. fiexuosa Boiss. with a single basidiospore. Pycnia are rare but functional. Their appearance may be attributed to infection caused by uninucleate basidiospores (Anikster et al., 1977). Uromyces ofiveirae Anikst. & WaW is correlated with U. uiennot-bourginii WaW & Anikst . This species develops uredinia and telia on B. eigii. The germinating teliospore forms a three-celled basidium with a basal cell devoid of nuclei and two binucleate cells (Fig. 4). Uromyces hordeastri This species is represented in Israel by three formae speciales (Anikster, 1975). Uromyces hordeastri Guyot f.sp. bulbosi-scillaeautumnalidis Anikst. Uredinia and telia on Hordeum bulbosum, pycnia and aecia on Scilla autumnalis L. The basidium, like that of U. reichertii, consists of a basal cell lacking nuclei, and four uninucleate cells. Each of the four cells yields a uninucleate basidiospore. The fungus is self-sterile. Uromyces hordeastri Guyot f.sp. marini Anikst. Uredinia and telia on Hordeum marinum, the alternate host is unknown. The basidium usually contains a basal cell without nuclei, and two binucleate cells, each of which produces one binucleate basid iospore. Occasionally the basidium consists of a basal cell devoid of nuclei, two uninucleate cells and one binucleate cell. The binucleate cell gives rise to a binucleate basidiospore, and each of the two uninucleate cells forms a single uninucleate basidiospore. Uromyces hordeastri Guyot f.sp. bulbosi-bellevaliae-flexuosae Anikst. Uredinia and telia on Hordeum bulbosum, pycnia (rare) and aecia on Beflevalia fiexuo sa Boiss. The development of basidia, basidiospores and their nuclei is the same as in U. hordeastri-marini. Uromyces scillarum The species is of wide geographic distribution on a wide range of hosts. It was identified on 45 species of the Liliaceae (Lehoczky, 1959). In Israel U. scillarum (Grev.) Wint. is common in most parts of the country on a number of Liliaceous species, and on Pancratium paroiflorum Dec. in the Amaryllidaceae (Anikster & Wahl, 1979). The
Y. Anikster,]. G. Moseman and I. Wahl
2
.
•
3
Fig.
1.
4
Basidium of Uromyces reichertii with a basal cell devoid of nuclei and tour uninucleate cells
(x 1300).
Fig. 2 Basidium of Uromyces christensenii having a basal cell without nuclei and two binucleate cells. Note a germinating binucleate basidiospore ( x 1070). Fig. 3. Rare type of basidium with a basal cell devoid of nuclei, two uninucleate cells and one binucleate cell in Uromyces viennot-bourginii f.sp. eigii ( x 700). Fig. 4. Basidium of Uromyces oliveirae consisting of a basal cell without nuclei and two binucleate cells (x 270).
379
Development of basidia and basidiospores rust is considered by some researchers as a hemiform (Gaumann, 1959), and by others as a microcyclic species (Jackson, 1931). Cytological investigations by Blackman & Fraser (1906) revealed a high frequency of binucleate basidiospores, and that the vegetative mycelium usually contained conjugate nuclei. Jackson (1931) listed U. scilla rum among microcyclic species having a predominantly binucleate mycelium. The species is complex and was divided by Schneider (1927) into sub species differing in teliospore dimensions and parasitic spec ialization. Gaumann (1959) preferred to consider these units as autonomous 'small species' (K leinarten). Lehoczky (1959) classified U . scilla rum found on various ho st species in Hungary into formae speciales morphologically indistinguishable but diverging parasitically and compatible only with the respective host species. Research in Israel corroborated Lehoczky's conclusions, and the investigated organisms were tentatively classified by us as formae speciales. The following nine formae speciales were identified and studied. Uromyces scillarum (Grev.) Wint. f.sp, bellevaliae-fiexuosae Anikst., with telia on Bellevalia flexuosa . Basidia three-celled with the basal cell devoid of nuclei, and two binucleate cells. Each binucleate cell develops one binucleate basidiospore. Uromyces scillarum (Grev.) Wint. f.sp, belleualiae-desertorum Anikst. A herni-rust with telia on Bellevalia desertorum Eig & Feinbr. Pustules occasionally conta in urediniospores. The development of basidia, basidiospores and their nuclei as in U. scillarum f.sp. belleoaliae-flexuosae. Uromyces scilla rum (G rev.) Wint. f.sp , ornithogali-eigii Anikst. A hemi-rust with telia on Ornithogalum eigii Feinbr. Sometimes th ey contain urediniospores. The development of basidia, basidiospores and their nuclei is like that in U. scilla rum f.sp, belleualiae-flexuosae. Uromyces scilla rum (Grev.) Wint. f.sp. ornithogali-trichophylli Anikst. Telia on Ornithogalum trichophyllum Boiss. & Heldr. The development of basidia, basidiospores and their nuclei is like that in U. scillarum f.sp. belleoaliae-flexuosae. Uromyces scilla rum (Grev.) Wint. f. sp.leopoldiaemaritimaeAnikst. A hemi-rust, with telia occasionally forming urediniospores. The fungus inhabits Leopoldia marit ima (D esf.) ParI. The development of basidia, basidiospores and their nuclei is the same as in U. scilla rum f.sp , bellevaliae-fiexu osae. Inoculation with a single basidiospore produces telia. The rust is therefore self-fertile. Uromyces scillarum (Grev.) Wint. f.sp, mus cariparoiflori Anikst., is correlated with U. christensenii.
The development of basidia, basidio spores and the ir nuclei is like U. scillarum f.sp. bellevaliaeflexuosa e. Uromy ces scillarum (Grev.) Wint. f.sp. muscariracemosi Lky . Telia on Muscari racemosum (L.)
Mill. The development of basidium, basidiospores and nuclei is like U. scilla rum f.sp , bellevaliaeflexuosa e. Uromy ces scillarum (G rev.) Wint. f.sp. scillaeautumnalidis Anikst. Telia on Scilla autumnalis.
The development of basidia, basidiospores and their nuclei is like U. scillarum f.sp . belleoaliaefiexu osae. Ur omyces scillarum (Grev.) Wint. f.sp. urgineaemaritimae Anikst. Telia on Urginea maritima (L.) Bak. The development of basidium, basidiospores
and nuclei is similar to that of U. scillarum f.sp, bellevaliae-fiexuosae.
In artificial inoculation tests, each of the described long-cycled and short-cycled species was compatible only with its source host species. However, U . vie nnot-bourgi nii was also infectious on cultivated barle y. Significantly, every one of the species was found to be infectious in artificial inoculation experiments on Leopoldia eburnea Eig & Feinbr. (Anikster & Wahl, 1979). The macrocyclic species alternated with L. eburnea, while the herni-rusts and microcyclic fungi developed on it the same spores as on the parental plants. DISCUSSION
Uromy ces rusts in Israel have undergone a prolonged coevolution with their Hordeum and L iliaceous hosts indigenous to the region (Anikster & Wahl, 1979). The history of the process can be traced by an examination of series of species showing a regressive development of the fungus, and a gradual adaptation to the semi-arid and arid climate of the country. The prevalence of short-cycled Uromyces species is an adjustment of the organism to short seasons, and indicates the antiquity of the heteroecious progenitors (Savile, 1955, 1976). Another evolutionary lineage leading to shortseason adaptation involves the development of basidia, basidiospores and their nuclei. It extends in the heteroecious rus ts from self-sterile U. reichertii with normal pycnia and conventional basidia and basidio spore s to U. oiennot-bourginii and U. christensenii that are self-fertile with pycnia rare or lacking. Both fungi produce basidia with two binucleate cells and two binucleate basidiospores . Single basidiospore infection causes the formation of aecia on the alternate host. The lineage has transitional forms with basidia consisting of both uninucleate and binucleate cells
Y. Anihster, J. G. Moseman and I. Wahl and yielding, respectively, mononucleate and binucleate basidiospores. Parallel evolutionary tendencies were revealed in short-cycled Uromyces on native Liliaceae. They vary from self-sterile species like U. rayssiae with ordinary basidia and basidiospores to self-fertile organisms with basidia containing two binucleate cells which form two binucleate basidiospores. Single basidiospore infection gives a dikaryotic mycelium. Self-fertility is an effective adaptation to a short-growing season. In the arctic and alpine rusts, self-fertility associated with lack of pycnia saves perhaps 2 weeks of an 8-week growing season (Savile, 1976). Buller (1950) also, considered that self-fertility shortens the generation time and reduces the risk of non-diploidization and sterility under suboptimal environmental conditions. In his opinion, this advantage was gained at the cost of reduced variability, and eventually leads to 'evolutionary stagnation'. Savile (1976), however, has shown that self-fertile rusts have other means of nuclear reassortment which ensure genetic diversity. In Israel, Uromyces species on Hordeum and Liliaceae with binucleate basidiospores are common in regions with an annual rainfall of less than 200 mm and at elevations of 1300 m or more above sea level (Anikster & Wahl, 1979). Since we have not studied chromosome segregation during meiosis in these Uromyces rusts the genetic relationship of the two nuclei in the basidiospore is unknown. The basidiospore can be homokaryotic or heterokaryotic. In the first instance the fungus is homothallic and in the latter - secondarily homothallic, i.e. heterothallic (Fincham & Day, 1963). In Raper's (1966) opinion, 'these distinctions, while helpful in an appreciation of the broader aspects of sexuality, are often of little practical value', since they involve a very considerable amount oflabour. Blackman & Fraser (1906) assumed that the two nuclei in the basidiospore of U. seil/arum are not conjugate but 'the result of a precocious nuclear division to be followed later by cell division'. Hence they are sister nuclei. Savile (1976) asserted that in selffertile organisms, single basidiospore infection yields a dikaryotic mycelium of sister nuclei. Jackson (1935) postulated that suppression of pycnia is associated with development of homothallism involving homokaryosis. Buller (1950) surmised that U. scillarum is homothallic, but his concept of homothallism also includes rusts with nuclei of opposite mating types, paired by cell fusion. Jackson (1931, 1935) stressed the remarkable uniformity of the nuclear development in macro-
cyclic rusts, as compared with the pronounced variability in the nuclear history in short-cycled organisms. Our studies have shown that deviations from the usual pattern of basidial and basidiospore development are common in both macrocyclic and microcyclic rusts. The hemi-forms are treated here as microcyclic organisms since their urediniospores are functionless (Anikster, 1975). The most significant departure from normal is probably in the production of basidia with two binucleate cells and two binucleate basidiospores in the heteroecious self-fertile Uromyces species. To our knowledge, a similar phenomenon in heteroecious rusts has been found outside Israel only in the homothallic Puccinia penniseti Zimm. (Dalela & Sinha, 1962; Kapooria, 1968). Jackson (1931) also maintained 'that it is the variations from the usual type of life history which furnish some of the most important evidence with respect to evolutionary tendencies' in the rusts. The Uromyces rusts on Hordeum and Liliaceae indigenous to Israel support this view. They show a broad range of patterned variability with intergrading components produced by protracted host-parasite coevolution. Several of the microcyclic rusts are phylogenetically correlated with some of the heteroecious ones and their kinship is shown in the basidium and basidiospore structure and function. A better understanding of the development of basidia, basidiospores and their cytology, can provide an insight into the phylogeny of rust fungi. The authors gratefully acknowledge Prof. G. Viennot-Bourgin's most valuable assistance. This study was supported by P.L. 480 Research Grant FG-Is-260; A10-CR-96, USDA, and by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.
REFERENCES
ANIKSTER, Y. (1975). Studies on the taxonomy,biology and evolution of the genus Uromyces on barley in Israel. Ph.D. Thesis submitted to the Tel Aviv University(Hebrew,with Englishsummary),134 pp. ANIKSTER, Y. & WAHL, I. (1966a). Quatre especes nouvelles d' Uromyces recoltees en Israel sur orges sauvages et sur Liliacees, Bulletin de la Societe Mycologique de France 82, 546-560. ANIKSTER, I. & WAHL, I. (1966b). Uromyces rusts on barley in Israel. Israel Journal of Botany is, 91105· ANIKSTER, Y. & WAHL, I. (1979). Coevolution of the rust fungi on Gramineae and Liliaceae and their hosts. Annual Review of Phytopathology 17, 367-4°3.
382
Development of basidia and basidiospores
ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1972). Life cycles and evolutionary trends of some Uromyces species inhabiting wild barleys and Liliaceae in Israel. Proceedings of the European and M editerranean Cereal Rusts Conference, Praha, %, 81-86. ANIKSTER, Y., MOSEMAN, J. C. & WAHL, I. (1973). Nuclear development in Uromyces sp. on wild barleys and Liliaceae in Israel. znd International Congress of Plant Pathology, Abstracts of papers, No. 0068. Minneapolis, Minnesota. ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1976). Parasite specialization of Puccinia hordei Otth and sources ofresistance in Hordeum spontaneum C. Koch. Barley Genetics III, Proceedings of the Third International Barley Genetics Symposium, Garching, 1975. pp, 468-469. ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1977). Uromyces viennot-bourginii, its life cycle, pathogenicity and cytology. Travaux dedies aGeorges ViennotBourgin. Societe Francoise de Phytopathologic 9-17. BLACKMAN, V. H. & FRASER, H. C. 1. (1906). Further studies on the sexuality of the Uredinae. Annals of Botany 20, 35-48. BULLER, A. H . R. (1950). Researches on Fungi, vol. 7. Publ. for Royal Society of Canada, by Univ. Toronto Press. DALELA, G . G. & SINHA, S. (1962). HomothaIIism in Puccinia penniseti Zimm., a long cycled rust and the function of pycnia. Indian Phytopathology 15, 156161, FINCHAM, J. R. S. & DAY, P. R. (1963). Fungal Genetics. Oxford: Blackwell Scientific Publications. GAUMANN, E. (1959). Die Rostpilze Mitteleuropas. Bern: Buchler & Co. GURR, E. (1965). The Rational Use of Dy es in Biology. London: Leonard Hill Publ. JACKSON, H. S. (1931). Present evolutionary tendencies and the origin of life cycles in the Uredinales, Memoirs of the Torrey Botanical Club 18, 5-108.
JACKSON, H . S. (1935). The nuclear cycle in Herpobasidium filicinum with a discussion of the significance of homothallism in Basidiomycetes. Mycologia %7, 553-572. KApOORIA, R. G. (1968). Cytological studies of the germinating tcliospores and basidiospores of Puccinia penniseti, Netherlands Journal of Plant Pathology 74, 2-7. KEMP, R. F. O. (1974). Bifactorial incompatibility in the two-spored basidiomycetes Coprinus sassii and C. balanatus. Transactions of the British Mycological Society 6%, 547-555. KEMP, R. F. O. (1976). Oidia I homing and the taxonomy and speciation of basidiomycetes with special reference to the genus Coprinus. In The Species Concept in Hymenomycetes (ed. H. Clemencon), pp. 259-274. Hirschberg: J. Cramer. LEHOCZKY, J. (1959). Biological specialization of the rust fungus Uromyces sci//arum (Grev.) Wint. Acta botanica Academiae Scientiarum Hungaricae 5, 391398. LINDFORS, T. (1924). Studien uber den Entwicklungsverlaufbei einigen Rostpilzen aus zytologischen und anatomischen Gesichtspunkten. Svensk Botanisk Tidskrift 18, 1-87. RAPER, J . R. (1966). Genetics of Sexuality in Higher Fungi. New York: Ronald Press Co. SAVILE, D . B. O. (1955). A phylogeny of the Basidiomycetes. Canadian Journal of Botany 33, 60-104. SAVILE, D. B. O. (1976). Evolution of the rust fungi (Uredinales) as reflected by their ecological problems. Evolutionary Biology 9, 137-207. SCHNEIDER, W. (1927). Zur Biologie einiger Liliaceen bewohnenden Uredineen. Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten Abteilung 72, II. 246-265.
(Received for publication 28 September 1979)
Printed in Great Britain
DEVELOPMENT OF BASIDIA AND BASIDIOSPORES IN UROM YCES SPECIES ON WILD BARLEY AND LILIACEAE IN ISRAEL By Y. ANIKSTER,* J. G. MOSEMANt
*
t
AND
1. WAHL*
Division of Mycology and Plant Pathology, The Tel Aviv University, Tel Aviv, Israel Plant Genetics and Germplasm Institute, USDA, Beltsville Agricultural Research Center, Beltsville, Md 20705, U.S.A.
Development of basidia and basidiospores varies in Uromyces rusts which have uredinia and telia on wild species of Hordeum and alternate with Liliaceous species. The same is true of short-cycled Uromyces species living on several members of the Liliaceae. Conventional rust types were observed in the two heteroecious species Uromyces hordeastri f.sp. bulbosi-autumnalidis, and U. reichertii, as well as in the microcyclic U. rayssiae which is correlated with U. reichertii. Simplified basidia have two binucleate cells and a basal cell lacking nuclei. Such basidia yield two binucleate basidiospores and are characteristic of U. christensenii. Several other heteroecious rusts, and the investigated formae speciales of the microcyclic U. scillarum have a similar basidial development. Basidia consisting of a binucleate cell with binucleate basidiospores, and uninucleate cells with uninucleate basidiospores were seldom found and were confined to heteroecious species. In U. oiennotbourginii and U. christensenii inoculation with a single basidiospore induced development of aecia, while in U. scillarum f.sp. leopoldiae-maritimae inoculation with one basidiospore resulted in the formation of telia. These rust fungi are therefore self-fertile. Israel is located in one of the centres of origin and diversification of species in the Liliaceae and also of Hordeum spontaneum C. Koch, the likely progenitor of cultivated barley. The wild barley species, H. bulbosum L. (of the type 2n = 28) and H. murinum L. are ubiquitous, varying in morphology and growth habit. This is true to a lesser degree of H. marinum Huds. The concept of host-parasite coevolution implies that genetic differentiation of the host is matched by a similar variability in the parasite. Our previous studies have revealed considerable diversification in the morphology, life-history and pathogenicity in rusts inhabiting some of the mentioned barley species and indigenous Liliaceae (Anikster, 1975; Anikster & Wahl, 1966a, b, 1979; Anikster, Moseman & Wahl, 1972, 1976). Buller (1950), Jackson (1931, 1935) and others emphasized the great variation in the nuclear cycle of rust fungi, as shown by the initiation of the dikaryotic condition and cytological processes in the development of the basidium. They postulated that cytological research in the Uredinales, and particularly in species correlated phylogenetically, could provide a better insight into the evolution of the rusts. Unfortunately, little attention has been given to these problems, and especially to a study on the
nuclear history in the basidium (Jackson, 1931). Kemp (1974, 1976) stressed the significance of cytological aspects in studies on the evolution of Hymenomycetes, their adaptation and speciation. Some of the above considerations prompted our study of the development of basidia and basidiospores in Uromyces species living on wild barley and Liliaceous species in Israel. Preliminary results have already been reported (Anikster, Moseman & Wahl, 1972, 1973, 1977; Anikster & WaW, 1979). MATERIALS AND METHODS
The rust fungi The diagnostic descriptions and information on the biology of the rust fungi have been published (Anikster, 1975; Anikster & WaW, 1966a, b; Anikster et al., 1977). The life-cycles of the fungi will be summarized in this paper. Teliospore germination The best germination was usually obtained with the method designed by Anikster (1975). Teliospores were scraped from green leaves or straw and floated for 24-28 h in Petri dishes on distilled
0007-1536/80/2828-6700 $01.00 © 1980 The British Mycological Society
Development of basidia and basidiospores or tap water at 18-20 °C. Similar results were obtained by soaking plant segments harbouring telia for 48-72 h and distributing the spore suspension on water agar. In some tests 100 % teliospore germination was obtained. The method had to be modified for teliospores of Uromyces reichertii Anikst. & WaW and U. rayssiae Anikst. & Wahl which are surrounded by thick walls (Anikster & Wahl, 1966a). A low to moderate germination rate was obtained by suspending teliospores in water in a Petri dish at 8° for 14 days and then transferring them to a chamber at 15° for 30-40 days. The nuclei in the teliospores, basidia and basidiospores were stained with Heidenhain's or Delafield's haematoxylin (Gurr, 1965). RESULTS
Uromyces reichertii Pycnia and aecia on Scilla hyacinthoides L., uredinia and telia on Hordeum bulbosum. The teliospores are uninucleate. The basidium consists of a basal cell without nuclei and four uninucleate cells (Fig. 1). A single uninucleate basidiospore is produced by each of the four cells. The fungus is self-sterile. The microcyclic species U. rayssiae with telia on S. hyacinthoides is correlated with U. reichertii. The basidium consists of a basal cell devoid of nuclei and four uninucleate cells, each forming a uninucleate basidiospore. Uromyces christensenii Aecia on Mus cari paruiflorum Desf., uredinia and telia on Hordeum bulbosum, Teliospores are uninucleate. At the initial stage of teliospore germination the nucleus migrates into the basidium. Upon elongation of the latter the nucleus divides once. Two cross walls divide the basidium into three cells. At first the middle and the upper cells are uninucleate and the basal cell has no nuclei. Then the nuclei divide and the two cells become binucleate (Fig. 2). Each of the two cells develops a single basidiospore, and both nuclei pass into the corresponding basidiospore. Inoculation of the alternate host with a single binucleate basidiospore results in the development of aecia with aeciospores infectious on H. bulbosum, The organism is obviously self-fertile. Its nuclear cycle parallels that of the microcyclic Puccinia arenariae (Schum.) Wint. (Lindfors, 1924). Only occasionally does a basidium contain a basal cell without nuclei; and two uninucleate cells which form two uninucleate basidiospores, and one binucleate cell with a binucleate basidiospore.
Uromyces viennot-bourginii The species contains several formae speciales, all living on Hordeum spontaneum (Anikster et al., 1977). The basidial formation, basidiospore development and nuclear conditions are the same as in U. christensenii Anikst. & WaW (F ig. 3). Aecia result from the inoculation of the alternate hosts Bellevalia eigii Feinbr. and B. fiexuosa Boiss. with a single basidiospore. Pycnia are rare but functional. Their appearance may be attributed to infection caused by uninucleate basidiospores (Anikster et al., 1977). Uromyces ofiveirae Anikst. & WaW is correlated with U. uiennot-bourginii WaW & Anikst . This species develops uredinia and telia on B. eigii. The germinating teliospore forms a three-celled basidium with a basal cell devoid of nuclei and two binucleate cells (Fig. 4). Uromyces hordeastri This species is represented in Israel by three formae speciales (Anikster, 1975). Uromyces hordeastri Guyot f.sp. bulbosi-scillaeautumnalidis Anikst. Uredinia and telia on Hordeum bulbosum, pycnia and aecia on Scilla autumnalis L. The basidium, like that of U. reichertii, consists of a basal cell lacking nuclei, and four uninucleate cells. Each of the four cells yields a uninucleate basidiospore. The fungus is self-sterile. Uromyces hordeastri Guyot f.sp. marini Anikst. Uredinia and telia on Hordeum marinum, the alternate host is unknown. The basidium usually contains a basal cell without nuclei, and two binucleate cells, each of which produces one binucleate basid iospore. Occasionally the basidium consists of a basal cell devoid of nuclei, two uninucleate cells and one binucleate cell. The binucleate cell gives rise to a binucleate basidiospore, and each of the two uninucleate cells forms a single uninucleate basidiospore. Uromyces hordeastri Guyot f.sp. bulbosi-bellevaliae-flexuosae Anikst. Uredinia and telia on Hordeum bulbosum, pycnia (rare) and aecia on Beflevalia fiexuo sa Boiss. The development of basidia, basidiospores and their nuclei is the same as in U. hordeastri-marini. Uromyces scillarum The species is of wide geographic distribution on a wide range of hosts. It was identified on 45 species of the Liliaceae (Lehoczky, 1959). In Israel U. scillarum (Grev.) Wint. is common in most parts of the country on a number of Liliaceous species, and on Pancratium paroiflorum Dec. in the Amaryllidaceae (Anikster & Wahl, 1979). The
Y. Anikster,]. G. Moseman and I. Wahl
2
.
•
3
Fig.
1.
4
Basidium of Uromyces reichertii with a basal cell devoid of nuclei and tour uninucleate cells
(x 1300).
Fig. 2 Basidium of Uromyces christensenii having a basal cell without nuclei and two binucleate cells. Note a germinating binucleate basidiospore ( x 1070). Fig. 3. Rare type of basidium with a basal cell devoid of nuclei, two uninucleate cells and one binucleate cell in Uromyces viennot-bourginii f.sp. eigii ( x 700). Fig. 4. Basidium of Uromyces oliveirae consisting of a basal cell without nuclei and two binucleate cells (x 270).
379
Development of basidia and basidiospores rust is considered by some researchers as a hemiform (Gaumann, 1959), and by others as a microcyclic species (Jackson, 1931). Cytological investigations by Blackman & Fraser (1906) revealed a high frequency of binucleate basidiospores, and that the vegetative mycelium usually contained conjugate nuclei. Jackson (1931) listed U. scilla rum among microcyclic species having a predominantly binucleate mycelium. The species is complex and was divided by Schneider (1927) into sub species differing in teliospore dimensions and parasitic spec ialization. Gaumann (1959) preferred to consider these units as autonomous 'small species' (K leinarten). Lehoczky (1959) classified U . scilla rum found on various ho st species in Hungary into formae speciales morphologically indistinguishable but diverging parasitically and compatible only with the respective host species. Research in Israel corroborated Lehoczky's conclusions, and the investigated organisms were tentatively classified by us as formae speciales. The following nine formae speciales were identified and studied. Uromyces scillarum (Grev.) Wint. f.sp, bellevaliae-fiexuosae Anikst., with telia on Bellevalia flexuosa . Basidia three-celled with the basal cell devoid of nuclei, and two binucleate cells. Each binucleate cell develops one binucleate basidiospore. Uromyces scillarum (Grev.) Wint. f.sp, belleualiae-desertorum Anikst. A herni-rust with telia on Bellevalia desertorum Eig & Feinbr. Pustules occasionally conta in urediniospores. The development of basidia, basidiospores and their nuclei as in U. scillarum f.sp. belleoaliae-flexuosae. Uromyces scilla rum (G rev.) Wint. f.sp , ornithogali-eigii Anikst. A hemi-rust with telia on Ornithogalum eigii Feinbr. Sometimes th ey contain urediniospores. The development of basidia, basidiospores and their nuclei is like that in U. scilla rum f.sp, belleualiae-flexuosae. Uromyces scilla rum (Grev.) Wint. f.sp. ornithogali-trichophylli Anikst. Telia on Ornithogalum trichophyllum Boiss. & Heldr. The development of basidia, basidiospores and their nuclei is like that in U. scillarum f.sp. belleoaliae-flexuosae. Uromyces scilla rum (Grev.) Wint. f. sp.leopoldiaemaritimaeAnikst. A hemi-rust, with telia occasionally forming urediniospores. The fungus inhabits Leopoldia marit ima (D esf.) ParI. The development of basidia, basidiospores and their nuclei is the same as in U. scilla rum f.sp , bellevaliae-fiexu osae. Inoculation with a single basidiospore produces telia. The rust is therefore self-fertile. Uromyces scillarum (Grev.) Wint. f.sp, mus cariparoiflori Anikst., is correlated with U. christensenii.
The development of basidia, basidio spores and the ir nuclei is like U. scillarum f.sp. bellevaliaeflexuosa e. Uromy ces scillarum (Grev.) Wint. f.sp. muscariracemosi Lky . Telia on Muscari racemosum (L.)
Mill. The development of basidium, basidiospores and nuclei is like U. scilla rum f.sp , bellevaliaeflexuosa e. Uromy ces scillarum (G rev.) Wint. f.sp. scillaeautumnalidis Anikst. Telia on Scilla autumnalis.
The development of basidia, basidiospores and their nuclei is like U. scillarum f.sp . belleoaliaefiexu osae. Ur omyces scillarum (Grev.) Wint. f.sp. urgineaemaritimae Anikst. Telia on Urginea maritima (L.) Bak. The development of basidium, basidiospores
and nuclei is similar to that of U. scillarum f.sp, bellevaliae-fiexuosae.
In artificial inoculation tests, each of the described long-cycled and short-cycled species was compatible only with its source host species. However, U . vie nnot-bourgi nii was also infectious on cultivated barle y. Significantly, every one of the species was found to be infectious in artificial inoculation experiments on Leopoldia eburnea Eig & Feinbr. (Anikster & Wahl, 1979). The macrocyclic species alternated with L. eburnea, while the herni-rusts and microcyclic fungi developed on it the same spores as on the parental plants. DISCUSSION
Uromy ces rusts in Israel have undergone a prolonged coevolution with their Hordeum and L iliaceous hosts indigenous to the region (Anikster & Wahl, 1979). The history of the process can be traced by an examination of series of species showing a regressive development of the fungus, and a gradual adaptation to the semi-arid and arid climate of the country. The prevalence of short-cycled Uromyces species is an adjustment of the organism to short seasons, and indicates the antiquity of the heteroecious progenitors (Savile, 1955, 1976). Another evolutionary lineage leading to shortseason adaptation involves the development of basidia, basidiospores and their nuclei. It extends in the heteroecious rus ts from self-sterile U. reichertii with normal pycnia and conventional basidia and basidio spore s to U. oiennot-bourginii and U. christensenii that are self-fertile with pycnia rare or lacking. Both fungi produce basidia with two binucleate cells and two binucleate basidiospores . Single basidiospore infection causes the formation of aecia on the alternate host. The lineage has transitional forms with basidia consisting of both uninucleate and binucleate cells
Y. Anihster, J. G. Moseman and I. Wahl and yielding, respectively, mononucleate and binucleate basidiospores. Parallel evolutionary tendencies were revealed in short-cycled Uromyces on native Liliaceae. They vary from self-sterile species like U. rayssiae with ordinary basidia and basidiospores to self-fertile organisms with basidia containing two binucleate cells which form two binucleate basidiospores. Single basidiospore infection gives a dikaryotic mycelium. Self-fertility is an effective adaptation to a short-growing season. In the arctic and alpine rusts, self-fertility associated with lack of pycnia saves perhaps 2 weeks of an 8-week growing season (Savile, 1976). Buller (1950) also, considered that self-fertility shortens the generation time and reduces the risk of non-diploidization and sterility under suboptimal environmental conditions. In his opinion, this advantage was gained at the cost of reduced variability, and eventually leads to 'evolutionary stagnation'. Savile (1976), however, has shown that self-fertile rusts have other means of nuclear reassortment which ensure genetic diversity. In Israel, Uromyces species on Hordeum and Liliaceae with binucleate basidiospores are common in regions with an annual rainfall of less than 200 mm and at elevations of 1300 m or more above sea level (Anikster & Wahl, 1979). Since we have not studied chromosome segregation during meiosis in these Uromyces rusts the genetic relationship of the two nuclei in the basidiospore is unknown. The basidiospore can be homokaryotic or heterokaryotic. In the first instance the fungus is homothallic and in the latter - secondarily homothallic, i.e. heterothallic (Fincham & Day, 1963). In Raper's (1966) opinion, 'these distinctions, while helpful in an appreciation of the broader aspects of sexuality, are often of little practical value', since they involve a very considerable amount oflabour. Blackman & Fraser (1906) assumed that the two nuclei in the basidiospore of U. seil/arum are not conjugate but 'the result of a precocious nuclear division to be followed later by cell division'. Hence they are sister nuclei. Savile (1976) asserted that in selffertile organisms, single basidiospore infection yields a dikaryotic mycelium of sister nuclei. Jackson (1935) postulated that suppression of pycnia is associated with development of homothallism involving homokaryosis. Buller (1950) surmised that U. scillarum is homothallic, but his concept of homothallism also includes rusts with nuclei of opposite mating types, paired by cell fusion. Jackson (1931, 1935) stressed the remarkable uniformity of the nuclear development in macro-
cyclic rusts, as compared with the pronounced variability in the nuclear history in short-cycled organisms. Our studies have shown that deviations from the usual pattern of basidial and basidiospore development are common in both macrocyclic and microcyclic rusts. The hemi-forms are treated here as microcyclic organisms since their urediniospores are functionless (Anikster, 1975). The most significant departure from normal is probably in the production of basidia with two binucleate cells and two binucleate basidiospores in the heteroecious self-fertile Uromyces species. To our knowledge, a similar phenomenon in heteroecious rusts has been found outside Israel only in the homothallic Puccinia penniseti Zimm. (Dalela & Sinha, 1962; Kapooria, 1968). Jackson (1931) also maintained 'that it is the variations from the usual type of life history which furnish some of the most important evidence with respect to evolutionary tendencies' in the rusts. The Uromyces rusts on Hordeum and Liliaceae indigenous to Israel support this view. They show a broad range of patterned variability with intergrading components produced by protracted host-parasite coevolution. Several of the microcyclic rusts are phylogenetically correlated with some of the heteroecious ones and their kinship is shown in the basidium and basidiospore structure and function. A better understanding of the development of basidia, basidiospores and their cytology, can provide an insight into the phylogeny of rust fungi. The authors gratefully acknowledge Prof. G. Viennot-Bourgin's most valuable assistance. This study was supported by P.L. 480 Research Grant FG-Is-260; A10-CR-96, USDA, and by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel.
REFERENCES
ANIKSTER, Y. (1975). Studies on the taxonomy,biology and evolution of the genus Uromyces on barley in Israel. Ph.D. Thesis submitted to the Tel Aviv University(Hebrew,with Englishsummary),134 pp. ANIKSTER, Y. & WAHL, I. (1966a). Quatre especes nouvelles d' Uromyces recoltees en Israel sur orges sauvages et sur Liliacees, Bulletin de la Societe Mycologique de France 82, 546-560. ANIKSTER, I. & WAHL, I. (1966b). Uromyces rusts on barley in Israel. Israel Journal of Botany is, 91105· ANIKSTER, Y. & WAHL, I. (1979). Coevolution of the rust fungi on Gramineae and Liliaceae and their hosts. Annual Review of Phytopathology 17, 367-4°3.
382
Development of basidia and basidiospores
ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1972). Life cycles and evolutionary trends of some Uromyces species inhabiting wild barleys and Liliaceae in Israel. Proceedings of the European and M editerranean Cereal Rusts Conference, Praha, %, 81-86. ANIKSTER, Y., MOSEMAN, J. C. & WAHL, I. (1973). Nuclear development in Uromyces sp. on wild barleys and Liliaceae in Israel. znd International Congress of Plant Pathology, Abstracts of papers, No. 0068. Minneapolis, Minnesota. ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1976). Parasite specialization of Puccinia hordei Otth and sources ofresistance in Hordeum spontaneum C. Koch. Barley Genetics III, Proceedings of the Third International Barley Genetics Symposium, Garching, 1975. pp, 468-469. ANIKSTER, Y., MOSEMAN, J. G. & WAHL, I. (1977). Uromyces viennot-bourginii, its life cycle, pathogenicity and cytology. Travaux dedies aGeorges ViennotBourgin. Societe Francoise de Phytopathologic 9-17. BLACKMAN, V. H. & FRASER, H. C. 1. (1906). Further studies on the sexuality of the Uredinae. Annals of Botany 20, 35-48. BULLER, A. H . R. (1950). Researches on Fungi, vol. 7. Publ. for Royal Society of Canada, by Univ. Toronto Press. DALELA, G . G. & SINHA, S. (1962). HomothaIIism in Puccinia penniseti Zimm., a long cycled rust and the function of pycnia. Indian Phytopathology 15, 156161, FINCHAM, J. R. S. & DAY, P. R. (1963). Fungal Genetics. Oxford: Blackwell Scientific Publications. GAUMANN, E. (1959). Die Rostpilze Mitteleuropas. Bern: Buchler & Co. GURR, E. (1965). The Rational Use of Dy es in Biology. London: Leonard Hill Publ. JACKSON, H. S. (1931). Present evolutionary tendencies and the origin of life cycles in the Uredinales, Memoirs of the Torrey Botanical Club 18, 5-108.
JACKSON, H . S. (1935). The nuclear cycle in Herpobasidium filicinum with a discussion of the significance of homothallism in Basidiomycetes. Mycologia %7, 553-572. KApOORIA, R. G. (1968). Cytological studies of the germinating tcliospores and basidiospores of Puccinia penniseti, Netherlands Journal of Plant Pathology 74, 2-7. KEMP, R. F. O. (1974). Bifactorial incompatibility in the two-spored basidiomycetes Coprinus sassii and C. balanatus. Transactions of the British Mycological Society 6%, 547-555. KEMP, R. F. O. (1976). Oidia I homing and the taxonomy and speciation of basidiomycetes with special reference to the genus Coprinus. In The Species Concept in Hymenomycetes (ed. H. Clemencon), pp. 259-274. Hirschberg: J. Cramer. LEHOCZKY, J. (1959). Biological specialization of the rust fungus Uromyces sci//arum (Grev.) Wint. Acta botanica Academiae Scientiarum Hungaricae 5, 391398. LINDFORS, T. (1924). Studien uber den Entwicklungsverlaufbei einigen Rostpilzen aus zytologischen und anatomischen Gesichtspunkten. Svensk Botanisk Tidskrift 18, 1-87. RAPER, J . R. (1966). Genetics of Sexuality in Higher Fungi. New York: Ronald Press Co. SAVILE, D . B. O. (1955). A phylogeny of the Basidiomycetes. Canadian Journal of Botany 33, 60-104. SAVILE, D. B. O. (1976). Evolution of the rust fungi (Uredinales) as reflected by their ecological problems. Evolutionary Biology 9, 137-207. SCHNEIDER, W. (1927). Zur Biologie einiger Liliaceen bewohnenden Uredineen. Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten Abteilung 72, II. 246-265.
(Received for publication 28 September 1979)