Archives of Phytopathology and Plant Protection May, 2004, Vol. 37, pp. 89 – 101 COMPARATIVE MORPHOLOGY OF UREDINIA AND UREDINIOSPORES OF SIX PUCCINIA SPECIES PARASITIC ON POACEAE IN SAUDI ARABIA ZAKARIA A.M. BAKA*, HUSSIEN M. ALWADIE and YASSER S. MOSTAFA Department of Biology, College of Science, King Khalid University, Abha PO Box 9004, Saudi Arabia. (Received 20 November 2003) Uredinia and urediniospores of six Puccinia species growing on Poaceae in southwestern Saudi Arabia were morphologically compared by light and scanning electron microscopy (SEM). Puccinia cenchri, P. fragosoana and P. isiacae were recorded for the first time in Saudi Arabia. Many differences between uredinia and urediniospores of studied Puccinia species were recorded. These differences are not related to host plant but may be due to the species of Puccinia itself. Observations by SEM led to more information in distinguishing between these Puccinia species particularly the presence of paraphyses and density and length of spines. Keywords: Puccinia; Rust fungi; Saudi Arabia; SEM; Urediniospores INTRODUCTION Fungi belonging to the order Uredinales are commonly referred to as rust fungi. Rust fungi, which are obligate pathogens of vascular plants, are unique in possessing up to six morphological and functional spore state, which can exist in one species (Hiratsuka, 1988). Peterson (1974) pointed out that the life cycle of a rust fungus exhibits an extremely plastic and usually complex series of events. Puccinia is the major genus of the family Pucciniaceae and represents a significant proportion of all known rust fungi. It includes autoecious, heteroecious, macrocyclic and microcyclic species that occur on an extremely wide range of angiospermous hosts (Brown and Brotzman, 1979). Although various techniques of molecular biology now contribute to our understanding of fungal systematics, fungi are mostly classified on the basis of their morphology and a good knowledge of morphological features, life cycles, and biotrophic interactions with host plants remains necessary for the identification of rust fungi (Hiratsuka and Sato, 1982), and for their investigation as important plant *Corresponding author: Dr Zakaria A.M. Baka, Department of Biology, College of Science, King Khalid University, Abha PO Box 9004, Saudi Arabia. Tel: 00966 7 2280942. Fax: 00966 7 2290165. E-mail: zakariabaka@hotmail.com Permanent address for Dr Zakaria A.M. Baka: Department of Botany, Faculty of Science, University of Mansoura, Mansoura, Egypt ISSN 0323-5408 print; ISSN 1477-2906 online # 2004 Taylor & Francis Ltd DOI: 10.1080/0323540042000202685 90 ZAKARIA A.M. BAKA et al. pathogens or potential biological control agents. It is very important to have a good understanding of morphological features, life cycles, and host – rust relationships in the study of rust fungi as important plant pathogens or interesting biological agents. Uredinia, in which urediniospores, the abundant ‘repeating spore’ state of rust fungi, are produced on a dikaryotic mycelium, arise from infection of compatible host plants by dikaryotic urediniospores from the same host species or by aeciospores produced by dikaryotization of the haploid mycelium in the same, or the case of heteroecious rusts, the alternate host species. Kenny (1970) recognized 14 different types of uredinia among rust fungi, based on such characteristics as the presence or absence of bounding structures, position of the hymenium in the host tissue, and growth pattern of the hymenium. The uredinial sorus may vary greatly in morphology, having a well-developed peridium (e.g., Milesina, Pucciniastrum), or may contain paraphyses but with no or obscure peridium (e.g., Melampsora), or be devoid of both features (e.g., most species of Puccinia) (Cummins and Hiratsuka, 1983). The special taxonomical problems presented by host specialization within morphologically recognizable rust species have pointed out in relation to rusts on Allium (Jennings et al. 1990). Since such physiological characteristics and the occurrence of more than one rust species on a single host may not be reliable taxonomic criteria, the identification of rusts is based on morphological characters (Shoemaker, 1981). Scanning electron microscopy (SEM), which permits more detailed examination of additional morphological features on surfaces of spores, has been used extensively in for many taxa (Littlefield and Heath, 1979). Spore length and width have been shown to differ between pustules, leaves or individual host plants, and also with environmental conditions (Littlefield, 1981). Germ pore numbers is also a good taxonomic character (Cummins and Hiratsuka, 1983) and surface ornamentation, such as spines, warts, and reticulum on the urediniospores are sometimes of value in the identification of rust fungi (Corlett, 1970). Savile (1965) used differences in size, the height of spines and warts, and the average distances between their bases, to distinguish species, host limited varieties, and geographic populations. However, the height and diameter of such features are often less than one micron, close to the limits of resolution of the light microscope, and are difficult to measure accurately (Corlett, 1970). Although the diploid teliospores which usually form in the later development of the dikaryotic mycelium, are important in the classification of rust fungi (Littlefield and Heath, 1979), further information may be contributed by urediniospores produced earlier in the dikaryotic phase of the life cycle. So far, no detailed studies on Saudi rusts, particularly using high resolution microscopy, have been reported. The purpose of the present work is to elucidate the taxonomy of six graminincolous species of Puccinia, by comparing their uredinia and urediniospores, by both light microscopy (LM) and SEM. MATERIALS AND METHODS Rust material The leaves of hosts bearing uredinia and urediniospores of various Puccinia species were collected from different localities in southwestern Saudi Arabia (Table I) and brought into the laboratory. COMPARATIVE MORPHOLOGY OF PUCCINIA SPECIES FROM SAUDI ARABIA 91 TABLE I Puccinia species and their hosts under study Puccinia species Host Locality Puccinia cenchri Diet. & Holw P. fragosoana Beltron P. graminis Pers. P. isiacae Wint. P. recondita Roberge ex Desm. P. striiformis Westend. Cenchrus biflorus Roxb. Imperata cylindrica (L.) Raeuschel Cynodon dactylon (L.) Pers. Phragmites australis (Cav.) Trin. Lolium perenne L. Lolium perenne L. AsSuda Khamis Musheet AsSuda Abha Khamis Musheet Khamis Musheet Light microscopy For LM examinations, hand sections were cut from fresh material, mounted in lactophenol containing cotton blue, examined (at 500 6 ) and photographed. Dimensions of uredinia (length and width), urediniospores (length, width, wall thickness in optical section, at the centre of the spore), were measured, using a Leitz optical micrometer, and germ pore numbers recorded. Twenty-five leaf specimens has been taken from five different infected plants. For each specimen, 50 uredinia and urediniospores were measured. Scanning electron microscopy Small pieces of leaf were removed with a sharp razor blade and fixed for 24 h at 48C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2. These small pieces were then postfixed in 1% OsO4 in the same buffer and at the same pH, dehydrated through a graded series to 100% ethanol. At this point, the tissue was removed from the ethanol and cut into many smaller pieces (Mercer and Birbeck, 1972). The pieces were quickly returned to ethanol and then critical point dried with liquid CO2, mounted on aluminum stubs with silver paint, coated with gold-palladium and examined with a JOEL SEM. Spine density was measured as the number of spines on and within a 100 mm2 circle about the spore surface. RESULTS AND DISCUSSION The morphological characteristics, observed under LM, of uredinia and urediniospores of the six Puccinia species studied here are recorded in Tables II – VI. Light and SEM micrographs are presented in Figures 1 – 27. All Puccinia species studied here were characterized by uredinia arranged in elongated files on the leaf surface parallel to the main axis of the main blade. In each case uredinial postules were amphigenous. All Puccinia species recorded here are macrocyclic and heterocious. Puccinia cenchri Diet. & Holw Holway Bot. Gaz. 24: 28 (1897). Syn. Uredo cenchricola P. Henn., Mus. Congo Anal. 2: 223 (1908). On Cenchrus biflorus Roxb. 92 ZAKARIA A.M. BAKA et al. TABLE II Qualitative features of uredinia of Puccinia species studied Species Location Type P. cenchri P. fragosoana P. graminis P. isiacae P. recondita P. striiformis Leaves, mainly adaxial Leaves, mainly adaxial Stem and leaf-sheath Leaves, in large groups Leaves and stems Leaves as linear series Oblong Oblong Elongated Oblong Obovoid Elongated Colour Margin Paraphyses CB DB CB CB CB O RE N RE RE RE RE – + – – – + CB = cinnamon-brown; DB = Dark-brown; O = Orange; RE = Ruptured epidermis; N = Naked; + = Presence; 7 = Absence. TABLE III Mean of dimensions (mm) of uredinia and paraphysis heads of Puccinia species studied Uredinia Species P. cenchri P. fragosoana P. graminis P. isiacae P. recondita P. striiformis Paraphysis heads Length Breadth Length 750 + 36 507 + 16 977 + 60 1752 + 34 288 + 3.0 89 + 2.0 187 + 6.0 93 + 3.0 738 + 21 246 + 6.0 177 + 11 25 + 1.0 – 11.5 + 0.5 – – – 16.0 + 1.0 Breadth Wall thickness 8.2 + 0.20 2.5 + 0.2 7.5 + 0.5 0.8 + 0.05 + = Standard error of mean; 7 = Absent. TABLE IV Qualitative features of urediniospores of Puccinia species studied Species Type Colour Wall colour P. cenchri P. fragosoana P. graminis P. isiacae P. recondita P. striiformis Ellipsoid Obovoid Ellipsoid Ellipsoid Obovoid Broadly ellipsoid Brown Golden-brown Orange Golden Yellow Yellow Cinnamon-brown Yellow Cinnamon-brown Cinnamon-brown Yellowish-brown Pale yellowish The uredinia of P. cenchri are mainly on adaxial leaf surface, oblong, surrounded by a ruptured epidermis, cinnamon-brown. Single pustule is up to 750 6 187 mm. Urediniospores of P. cenchri were ellipsoid, brown, 32.8 6 25.3 mm; wall cinnamonbrown, 2.4 mm thick with 4 – 5 equatorial germ pores, echinulate, spine density was 11.52, spine length was 0.80 mm. The distance between spines was 1.53 mm. Puccinia cenchri was previously recorded on Cenchrus ciliaris and C. biflorus in Africa (Cummins, 1971; Baka and Gjaerum, 1996, respectively), on C. echinatus and C. brownii in Asia (Gardner and Hodges, 1989; Hiratsuka et al., 1992, respectively). Puccinia fragosoana Beltron Roy. Soc. Espan. Hist. Nat. 50: 249 (1921). COMPARATIVE MORPHOLOGY OF PUCCINIA SPECIES FROM SAUDI ARABIA 93 TABLE V Mean of dimensions (mm) of urediniospores and number of germ pores of Puccinia species studied Species P. cenchri P. fragosoana P. graminis P. isiacae P. recondita P. striiformis Length Breadth Wall thickness Germ pores 32.8 + 0.7 37.4 + 0.9 29.2 + 1.0 26.2 + 0.8 29.2 + 0.7 26.2 + 0.6 25.3 + 0.6 22.9 + 0.5 19.2 + 0.6 20.4 + 0.2 22.4 + 0.5 20.6 + 0.5 2.4 + 0.08 2.0 + 0.08 1.8 + 0.05 3.5 + 0.08 1.5 + 0.08 1.8 + 0.05 4 – 5 equatorial 4 – 5 equatorial 3 – 5 equatorial 3 equatorial 6 – 9 scattered 9 – 13 scattered + = Standard error of mean. TABLE VI Mean of the dimensions (mm) of urediniospore spines of Puccinia species studied Species Spine density/100 mm2 Length Distance between spines 11.52 + 1.8 11.92 + 1.9 15.60 + 2.1 12.80 + 1.6 16.20 + 2.8 11.50 + 1.5 0.80 + 0.01 0.80 + 0.02 0.65 + 0.01 0.55 + 0.01 0.94 + 0.03 0.55 + 0.01 1.53 + 0.05 1.07 + 0.02 1.75 + 0.05 2.72 + 0.04 1.39 + 0.05 1.10 + 0.02 P. cenchri P. fragosoana P. graminis P. isiacae P. recondita P. striiformis + = Standard error of mean. Syn. Uredo schizachyrii Doidge, Bothalia 2: 508 (1928). On Imperata cylindrica (L.) Raeuschel The uredinia of P. fragosoana are oblong, naked, dark brown measuring 507 – 593 mm. The uredinia includes capitate colourless paraphyses measuring 11.5 6 8.2 mm, with thin wall measuring 2.5 mm at apex. Urediniospores of P. fragosoana were obovoid, golden to brown, 37.4 6 22.9 mm, wall yellow, 2.0 mm thick, with 4 – 5 equatorial germ pores, echinulate, spine density was 11.92, spine length was 0.80 mm. The distance between spines was 1.07 mm. Puccinia fragosoana was previously recorded on Imperata cylindrica in Africa (Cummins, 1971; Baka and Gjaerum, 1996; Evans, 1987), Asia (Cummins, 1971; Abbasi et al., 2002) and Europe (Cummins, 1971). Puccinia graminis Pers. Syn. Meth. Fung. P. 228 (1801). Syn. P. albigensis Mayor, Rev. Mycol. 22: 279 (1957). On Cynodon dactylon. Uredinia are most commonly on sheaths and stems, cinnamon-brown, elongated, surrounded by a ruptured epidermis measuring 977 6 738 mm. Urediniospores of P. graminis were ellipsoid, orange, 29.2 6 19.2 mm, wall cinnamon-brown, 1.8 mm thick, with 3 – 5 equatorial germ pores, echinulate, spine density was 15.60, spine length was 0.65 mm. The distance between spines was 1.75 mm. Puccinia graminis, the black rust, is circumglobal, occurring on many genera of Poaceae. Cynodon dactylon is a new host for this rust in Saudi Arabia. 94 ZAKARIA A.M. BAKA et al. FIGURES 1 – 4 Puccinia cenchri. (1 – 3) are SEM micrographs. (1) An uredinium with ruptured epidermis (E). Bar = 100.0 mm. (2) A magnified part of an uredinium showing a group of urediniospores. Bar = 10.0 mm. (3) A magnified urediniospore showing the echinulation on its surface. Note the scar (arrowhead). Bar = 5.0 mm. (4) LM micrograph of a single urediniospore. Bar = 20.0 mm. Puccinia isiacae Wint. O. Kuntze Plantae orient-ross. P. 127 (1887). On Phragmites australis (Cav.) Trin. The uredinia scattered on leaves in large groups, oblong, surrounded by a ruptured epidermis measuring 1752 6 246 mm. Urediniospores of P. isiacae were ellipsoid, golden, 26.2 6 20.4 mm, wall cinnamon-brown, 3.5 mm thick, with three equatorial germ pores, echinulate, spine density was 12.80, spine length was 0.55 mm. The distance between spines was 2.72 mm. COMPARATIVE MORPHOLOGY OF PUCCINIA SPECIES FROM SAUDI ARABIA 95 FIGURES 5 – 8 Puccinia fragosoana. (5 – 7) are SEM micrographs. (5) A naked uredinium. Bar = 100.0 mm. (6) A magnified part of an uredinum showing a group of uredinispores. Note the paraphysis (P). Bar = 10.0 mm. (7) A magnified urediniospore. Note the reticulation (arrowheads) and echinulation on its surface. Bar = 5.0 mm. (8) LM micrograph of a an urediniospore. Note the equatorial germ pores (arrows). Bar = 20.0 mm. Puccinia isiacae was previously recorded on P. communis and P. maximus in Europe and Africa (Cummins, 1971), on P. australis in Africa (Baka and Gjaerum 1996). Puccinia recondita Roberge ex Desm. Bull. Soc. Bot. France, 4: 798 (1857). Syn. Puccinia dasypyri Guyot & Malen. Trav. Inst. Sci. Chefir Ser. Bot. 28: 62 (1963). On Lolium perenne L. 96 ZAKARIA A.M. BAKA et al. FIGUREs 9 – 12 Puccinia graminis. (9 – 11) are SEM micrographs. (9) A number of uredinia (short arrows) in linear manner. Bar = 500 mm. (10) A magnified part of an uredinium with ruptured epidermis (E). Bar = 10 mm. (11) A magnified urediniospore showing the echinulation on its surface. Note germ pore (arrowhead). Bar = 5.0 mm. (12) LM micrograph of urediniospores. Bar = 20.0 mm. The uredinia of P. recondita are obovoid, surrounded by a ruptured epidermis, cinnamon-brown. Single uredinium is measuring 288 6 177 mm. Urediniospores of P. recondita were broadly obovoid, yellowish, 29.2 6 22.4 mm, wall yellowish-brown, 1.5 mm thick, with 6 – 9 scattered germ pores, echinulate, spine density was 16.20, spine length was 0.94 mm. The distance between spines was 1.39 mm. Puccinia recondita, the brown leaf rust, is a circumglobal rust species, most common in temperate areas occurring on a large number of host genera of Poaceae (Cummins, 1971) and in Africa on L. perenne (Baka and Gjaerum, 1996), and L. temulentum (Reichert, 1921). Puccinia striiformis Westend Bull. Roy. Acad. Belg., Cl. Sci. 21: 235 (1854). COMPARATIVE MORPHOLOGY OF PUCCINIA SPECIES FROM SAUDI ARABIA 97 FIGURES 13 – 17 Puccinia isiacae. (13 – 15) are SEM micrographs. (13) An uredinium contains huge number of urediniospores. Note ruptured epidermis (E). Bar = 250.0 mm. (14) A magnified part of an uredinium showing a group of elongated urediniospores. Bar = 25.0 mm. (15) Magnified urediniospores showing the echinulation on their surfaces. Bar = 25.0 mm. (16 – 17) are LM micrographs. (16) A part of an uredinium showing a group of urediniospores. Bar = 20.0 mm. (17) Magnified urediniospores with equatorial germ pores (arrowheads). Bar = 20.0 mm. Syn. Uredo glumarum J. K. Schmidt, Allgem. Oekon-tech., Fl. 1: 27 (1827). On Lolium perenne The uredinia of P. striiformis appear in linear series in chlorotic streaks, surrounded by a ruptured epidermis, orange. Single uredinum was up to 89 6 25 mm. The uredinium contains saccate paraphyses with colourless wall. Paraphysis heads were measuring 16.0 6 7.5 mm, the wall thickness at apex was 0.8 mm. Urediniospores of P. striiformis were broadly ellipsoid, yellowish, 26.2 6 20.6 mm, wall pale yellowish, 1.8 mm thick, with 9 – 13 scattered germ pores, echinulate, spine density was 11.5, spine length was 0.55 mm. The distance between spines was 1.39 mm. Puccinia striiformis, the stripe rust, is circumglobal, specially in the northern hemisphere, where it lives on many species of Poaceae. It was reported before in Africa on L. perenne (Baka and Gjaerum, 1996). There were qualitative morphological differences between uredinia and urediniospores of the different Puccinia species when observed under both light and electron microscopes. As indicated in Table II, the uredinia of the species examined differ in 98 ZAKARIA A.M. BAKA et al. FIGURES 18 – 22 Puccinia recondita. (18 – 20) are SEM micrographs. (18) Uredinia with ruptured epidermis (E). Bar = 100.0 mm. (19) A part of an uredinium showing urediniospores. Bar = 10.0 mm. (20) A magnified urediniospore showing the echinulation on its surface. Bar = 5.0 mm. (21 – 22) LM micrographs. (21) An urediniospore showing germ pore (arrow). Bar = 20.0 mm. (22) Other view showing the wall (arrowhead) of urediniospores. Bar = 20.0 mm. location, form, colour, and in the presence of sterile paraphyses. Apart from P. graminis, in which they occur mainly on stem and leaf sheaths, the uredinia of these species are generally found between the veins on leaves, often forming linear series which, in P. recondita, occur in chlorotic streaks of leaf tissue, whereas those of P. isiaceae are scattered in large groups. Uredinia emerge on the adaxial surface of P. cenchri leaves but are abaxial in P. fragosoana. Uredinia of P. cenchri and P. graminis are elongated but those of the other species are mainly oblong, sometimes becoming confluent. Uredinia of P. cenchri, P. graminis, P. isiaceae and P. recondita are cinnamon-brown in colour, those of P. fragosoana dark brown, and those of P. striiformis orange. Paraphyses, long-stalked, colourless, sterile capitate structures, of differing size and wall thickness, were present in uredinia of two species: P. fragosoana where they were COMPARATIVE MORPHOLOGY OF PUCCINIA SPECIES FROM SAUDI ARABIA 99 FIGURES 23 – 27 Puccinia striiformis. (23 – 26) are SEM micrographs. (23) An uredinium with ruptured epidermis (E). (24) A part of an uredinium showing urediniospores intermixed with paraphyses (P). Bar = 10.0 mm. (25) A magnified urediniospore showing its echinulation. Bar = 5.0 mm. (26) A magnified part of an urediniospore showing spines (arrows) located in depressions (D) and enclosed by annuli (A). Bar = 0.5 mm. (27) LM micrograph showing urediniospores. Bar = 20.0 mm. found at the periphery of the uredinium, and in P. striiformis where the paraphyses appeared saccate (Table III). Those of P. fragosoana were smallest in length and breadth while the wall of P. striiformis paraphyses was much thinner than in the other species. This study is the first report to compare graminicolous Saudi rusts using both qualitative and quantitative morphology. These methods indicated that, urediniospore stages of the six Puccinia species can be distinguished. This investigation showed that the differences between urediniospores of these Puccinia species are not related to the type of host plant, since both P. recondita and P. striiformis are clearly recognized although they are growing on one host plant, Lolium prenne. Puccinia striiformis had 100 ZAKARIA A.M. BAKA et al. paraphyses and P. recondita had not. Moreover, the highest number of germ pores was recorded in case of P. striiformis. These germ pores were scattered in both P. striiformis and P. recondita and in other Puccinia species were equatorial. These variations in germ pore number and distribution may play a role in the classification of graminicolous rust fungi. Cummins and Hiratsuka (1983) reported that germ numbers is a good taxonomic character. Observation by SEM led to more information in distinguishing between the rust species studied here particularly urediniospore surface, spines density and spine length. Surface ornamentation, such as spines, warts, and reticulum on urediniospores are sometimes of value in the identification of rust fungi (Corlett, 1970). The spine morphology on the studied Puccinia species is in agreement with the descriptions for most other rust fungi (Littlefield and Heath, 1979) as being conical and sometimes curved at the tip, with the angle between the spines and the spore surface (Amerson and Van Dyke, 1978). Surface morphology and ornamentation features of rust urediniospores have been studied extensively with SEM (Stanbridge and Gay, 1969; Corlett, 1970; Amerson and Van Dyke, 1978; Brown and Brotzman, 1979; Littlefield and Heath, 1979; Harder, 1984; Gardner and Hodges, 1985; Woods and Beckett, 1987; Baka and Lösel, 1992; Baka, 1992, 1996a, 1996b, 2003). The depressions and raised annuli around spines observed here are common in Puccinia and Uromyces species (Littlefield and Heath, 1979). Germling morphology of urediniospores may clarify some of the difficulties of the identification and classification of grass rust fungi. In addition, nuclear DNA content or isozyme analysis may also add more information about rust fungi classification. Acknowledgement The authors would like to thank Dr Dorothy M. Lösel, Department of Animal and Plant Sciences, University of Sheffield, England for valuable comments. References Abbasi, M., Hedjaroude, Gh.A., Gjaerum, H.B. and Scholler, M. 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