Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Z. A. M. Baka and S. Rabei Department of Botany, Faculty of Science, Damietta University, New Damietta, 34517, Egypt. E-Mail of corresponding author: zakariabaka@yahoo.com Abstract The aim of this work is an attempt to add a new tool for the classification of rust fungi particularly of the genus Puccinia by studying their uredinia and urediniospores. In this study, uredinia and urediniospores of seven Puccinia species attacking Poaceae were collected from different locations in Damietta Governorate at the Northern part of Egypt and morphologically compared using both light and scanning electron microscopy. The studied species were P. fragosoana, P. graminis, P. imperatae, P. magnusiana, P. polypogonis, P. recondita and P. striiformis. Many differences between uredinia (such as shape, colour, dimensions and presence or absence of paraphyses) and urediniospores (such as shape, colour, wall colour, dimensions, wall thickness, number of germ pores, spine density, spine length and distance between spines) of studied species were investigated. These differences may not be related to host plant but due to the species of Puccinia. Observations by scanning electron microscopy led to more information in distinguishing between these species particularly the density and length of spines over urediniospore surfaces. A key was built using computer generating key which included in DELTA packages. Phenetic analysis was conducted using UPGMA algorithm implemented in DELTA. The results showed that the seven Puccinia species were grouped into two distinct phenetic groups. Introduction Rust fungi (Uredinales) are a relatively well-known group of fungi in most parts of the world. However, in the African continent, the studies on rust fungi were few particularly in Egypt. All members of Uredinales are parasitic on plants, often causing dramatic losses in various important crop plants (Alexopoulos et al., 1996). Rust fungi, which are obligate pathogens of vascular plants, are unique in possessing up to six morphological and functional spore states, 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. Rust fungi are one of the largest natural taxa within the kingdom Eumycota. More than 7000 species belonging to about 125 genera and 14 families are accepted currently (Aime, 2006). Although species infecting hosts belong to Poaceae are economically important, the largest genus, Puccinia Pers., contains ca. 4000 spp., 650 of which occur on Poaceae (Abbasi 1996). Poaceae (grasses) is one of the most flowering plant families with rich species in the world and includes many economically important crops (Osman et al., 2011) and includes 1000 species and 700 genera worldwide (Linder and Rudall 2005). Poaceae is considered as the largest family of flowering plants in Egypt and represented by 110 genera and 288 species (Boulos 2005). Very few studies are available about the occurrence of Puccinia species on Poaceae in Egypt such as Reichert (1921), ElHelaly et al. (1966) and Baka and Gjaerum (1996). 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 systematic, 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 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. 407 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Urediniospores are produced in uredinia (singular-uredinium) and defined as repeating vegetative spores produced usually on dikaryotic mycelium and are repeatedly produced on a host several times during the growing season of the host. Because of this, the uredinial state is the most destructive spore state of such rusts as wheat stem rust (Puccinia graminis), coffee leaf rust (Hemileia vastatrix) and poplar leaf rust (Melampsora medusae) (Hiratsuka and Sato, 1982). Kenny (1970) recognized 14 different morphological types of uredinia among rust fungi based on such morphological characteristics as the presence or absence of bounding structures, position of the hymenium in the host tissue, and growth pattern of the hymenium. The uredinia may vary greatly in morphology, having a well-developed peridium or may contain paraphyses but with no or obscure peridium, or be devoid of both features (Cummins and Hiratsuka 2003). Since the 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, l98l). Scanning electron microscopy, which permits more detailed examination of additional morphological features on surfaces of spores, has been used extensively 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, l98l). Germ pore numbers is also a good taxonomic character (Cummins and Hiratsuka, 2003) and surface ornamentation, such as spines, warts and reticulum on the urediniospores is 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 Egyptian rusts, particularly using high resolution microscopy, have been reported. However, the purpose of the present work is to elucidate the morphology of seven Puccinia species infecting members of Poaceae, by comparing their uredinia and urediniospores, using both light and scanning electron microscopes. Material and methods The leaves of different hosts bearing uredinia and urediniospores of various Puccinia species were collected from different localities in Damietta Governorate at the northern part of Egypt (Table 1) and brought to the laboratory. Puccinia species were identified according to Cummins (1971), Hiratsuka et al. (1992) and Cummins and Hiratsuka (2003). The host plants were identified according to Boulos (2005). For light microscope (LM) examinations, hand sections were cut from fresh material, mounted in lactophenol cotton blue, examined (at 500 x) 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 have been taken from five different infected plants. For each specimen, 50 uredinia and urediniospores were measured. For scanning electron microscope (SEM) examinations, small pieces of leaf bearing uredinia or uredinospores were removed with a sharp razor blade and fixed for 24 h at 4oC in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.2. These small pieces were then postfixed in l % OsO3 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 408 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 examined with a JOEL SEM. Spine density was measured as the number of spines on and within a 100 µm2 circle on the spore surface. The program DELTA (Dallwitz et al. 2000) was used to construct keys phonetic analysis for these species of Puccinia. Keys were based on the characters from uredinia and urediniospores. The qualitative characters were used to avoid any mistake in the measurements. The characters were coded to DELTA format. The data were converted using TOKEY and the KEY (Coleman et al., 2010; Rabei, 2011; Rabei and El-Gazzar, 2007) was used to generate three different keys. Tree forming methods of clustering, including the unweighted pair-group method using arithmetic averages (UPGMA) were constructed. Cluster analyses were performed using the distance program DIST of the DELTA software (Dallwitz et al., 2000, El-Gazzar and Rabei, 2008), where distances were calculated using a modification of the Gower coefficient (Gower, 1971). Sequential agglomerative, hierarchic nest clustering was done with UPGMA (Sneath and Sokal, 1973). Results and Discussion The morphological characteristics, observed under LM of uredinia and urediniospores of the seven Puccinia species studied here are recorded in Tables 2-6. Light and SEM micrographs are presented in Figures 1-7. 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. Uredinia were abaxial, adaxial or amphigenous. All Puccinia species recorded here are macrocyclic and heterocious. The uredinia of P. fragosoana were mainly observed on adaxial leaf surface of Imperata cylindrica, oblong, naked, dark brown, measuring 507 x 91 µm. The uredinia include capitate colourles paraphyses measuring 10 x 5 µm, with thin wall measuring 2.5 µm at apex. Urediniospores were obovoid, golden-brown, 36.4 x 22.9 µm, wall yellow, 2.0 µm thick, with 4-5 equatorial germ pores, echinulate with spine density 11.9 µm2, spine length 0.8 µm. The distance between spines was 1.07 µm (Tables 2-6 and Fig. 1). Puccinia fragosoana was previously recorded on Imperata cylindrica in Africa (Cummins, l97l; Baka and Gjaerum, 1996; Evans, 1987), Asia (Cummins, 1971; Abbasi, et al., 2002) and Europe (Cummins, 1971). The uredinia of P. graminis were commonly observed on leaf sheaths and stems of Cynodon dactylon, elongated, cinnamon-brown surrounded by a ruptured epidermis measuring 969 x 730 µm. Urediniospores were ellipsoid, orange, 29.2 x 19.2 µm, wall cinnamon-brown, l.8 µm thick, with 3-5 equatorial germ pores, echinulate, spine density was 15.6, spine length was 0.65 µm. The distance between spines was 1.7 µm (Tables 2-6 and Fig. 2). Puccinia graminis, the black rust, is circumglobal, occurring on many genera of Poaceae. Cynodon dactylon is a new host for this rust in Egypt. The uredinia of P. imperatae were mainly observed on abaxial leaf surface of Imperata cylindrica, elongated, surrounded by a ruptured epidermis and cinnamon-brown in colour. Single uredinium measured 475 x 85 µm. Urediniospores were globoid, light-brown, 21.5 x 20 µm, wall cinnamon-brown, 2.5 µm thick with 2-3 equatorial germ pores, echinulate with spine density 12.8 µm2. The spine length was 0.75 µm and the distance between spines was 1.6 µm (Tables 2-6 and Fig. 3). Puccinia imperatae is distributed mainly in Mediterranean region and South Africa (Cummins, l97l). The uredinia of P. magnusiana mainly observed as amphigenous on the leaves of Phragmites australis, oblong, naked, yellowish- brown, measuring 820 x 318 µm. The uredinia include capitate colourles paraphyses measuring 15 x 6.5 µm, with thin wall measuring 2.0 µm at apex. Urediniospores were ellipsoid, brown, 30.0 x 15.2 µm, wall yellowish-brown, 2.0 µm thick, with 7-10 scattered germ pores echinulate, spine density was 13.7 µm2, and spine length was 0.76 µm. The distance between spines was 1.65 µm (Tables 2-6 and Fig. 4). Puccinia magnusiana is a circumglobal rust (Cummins, l97l; Baka and Gjaerum, 1996) living on Phragmites and Arundo. This rust was previously reported on Phragmites australis (previously P. communis) in Africa (Baka and Gjaerum, 1996) and USA (Cummins, 1971). 409 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 The uredinia of P. polypogonis mainly observed on abaxial leaf surface of Polypogon monspliensis, oblong, cinnamon-brown surrounded by a ruptured epidermis measuring 720 x 180 µm. Urediniospores were obovoid, golden, 26.0 x 23.0 µm, wall yellow, 2.0 µm thick, with 5-7 scattered germ pores, echinulate, spine density was 12.7 µm2, spine length was 0.86 µm. The distance between spines was 1.55 µm (Tables 2-6 and Fig. 5). Puccinia polypogonis described from South America and South Africa (Cummins, 1971) and Egypt (Baka, 1992). The uredinia of P. recondita mainly observed on leaves and stems of Lolium perenne, obovoid, cinnamon-brown, surrounded by a ruptured epidermis. Single uredinium is measuring 282 x 175 µm. Urediniospores of P. recondita were obovoid, yellow, 29.2 x 22.4 µm, wall yellowishbrown, 1.5 µm thick, with 6-9 scattered germ pores, echinulate, spine density was 16.2 µm2, and spine length was 0.95 µm. The distance between spines was 1.4 µm (Tables 2-6 and Fig. 6). Ptrccinia 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, 197l). This rust was previously reported in Africa on L. perenne (Baka and Gjaerum 1996), and L. temulentum (Reichert, l92l). The uredinia of P. striiformis appeared in linear series in chlorotic streaks on the leaves of Lolium perenne, surrounded by a ruptured epidermis, orange. Single uredinium was elongated measuring 288 x 125 µm. The uredinium contains saccate paraphyses with colourless wall. Paraphysis heads were measuring I5.0 x 7.5 µm, the wall thickness at apex was 0.8 µm. The urediniospores were broadly ellipsoid, yellow, 26.2 x 20.6 µm, wall pale yellowish, 1.8 µm thick, with 9-13 scattered germ pores, echinulate, spine density was 11.5 µm2, spine length was 0.55 µm. The distance between spines was 1.1 µm (Tables 2-6 and Fig. 7). P. striiformis, the stripe rust, is circumglobal, especially in the northern hemisphere, where it lives on many species of Poaceae. It was reported before in Africa on Lolium perenne (Baka and Gjaerum, 1996). There were qualitative morphological differences between uredinia and urediniospors of the different Puccinia species when observed under both light and electron microscopes. As indicated in Table 2, the uredinia of the species examined differ in location on host leaves, shape, colour, and in the presence or absence of sterile paraphyses. Apart from P. graminis, in which they occur mainly on stem and leaf sheaths, the uredinia of the other species are generally found between the veins on leaves, often forming linear series which, in P. recondita, occur in chlorotic streaks on leaves. Uredinia of P. imperatae, and P. polypogonis are abaxial, while in P. fragosoana are adaxial and in P. magnusiana are amphigenous. Uredinia of P. imperatae, P. graminis and P. striiformis are elongated but those of the other species are mainly oblong, sometimes becoming confluent. Uredinia of P.imperatae, P. graminis, P. polypogonis and P. recondita are cinnamon-brown in colour, those of P. fragosoana are dark brown, and those of P. striiformis are orange. Paraphyses, long-stalked, colourless, sterile capitate structures, of differing size and wall thickness, were present in uredinia of three species: P. fragosoana where they were found at the periphery of the uredinium, in P. magnusiana and P. striiformis where they appeared saccate and spread within the uredinium. The paraphysis heads of P. fragosoana were smallest in length and breadth and those of P. striiformis exhibited much thinner wall than those of the other species (Table 3). This study is the first report to compare Egyptian rusts attacking members of Poaceae using both qualitative and quantitative morphology. These methods indicated that urediniospores of the seven Puccinia species can be distinguished. This investigation showed that the differences between urediniospores of these Puccinia species are not related to the species of host plant, since both P. fragosoana and P. imperatae are growing on one host, Imperata cylindrica and P. recondita and P. striiformis are growing also on one host, Lolium perenne. Puccinia striiformis had 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 P. magnusiana, P. recondita and P. striiformis and in other Puccinia species were equatorial. These variations in germ pore number and distribution 410 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 may play a role in the classification of graminicolous rust fungi. Cummins and Hiratsuka (2003) reported that germ pores number is a good taxonomic character. Scanning electron microscopy of the urediniospores of seven Puccinia species further resolved their spore ornamentation and supported observations made with the light microscope. 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, 1979). Surface morphology and ornamentation features of rust urediniospores have been studied extensively with SEM by many authors (Stanbridge and Gay, 1969; Corlett, 1971; Hiratsuka, 1971; Amerson and Van Dyke, 1978; Brown and Brotzman, 1979; Littlefield and Heath, 1979; Traquair and Kokko, 1983; Harder, 1984; Gardner and Hodges, 1985, 1989; Woods and Beckett, 1987; Jennings et al., 1990; Baka l992, 1996a, 1996b, 2004; Baka and Losel, 1992; Gupta et al.,1994, Johnson et al., 1999; Littlefield, 2000). The depressions and raised annuli around spines observed here are common in Puccinia and Uromyces species (Littlefield and Heath, 1979). The first uredinia to be produced in the pathogen’s life cycle develop on mycelium resulting from the germination of aeciospores. Uredinia are produced on a dikaryotic mycelium, and the urediniospores also produce dikaryotic mycelium when they germinate (Cummins and Hiratsuka, 2003). The mycelium resulting from the germination of urediniospores usually produces more uredinia and this character was used to obtain genetically identical urediniospores for the present study. The identification and classification of grass rust fungi is often difficult since most traditionally used morphological characters are quantitative and subjective. Besides, when using the host range as a taxonomic criterion, it is important to realize that a rust fungus may have jumped to a new host species and that host range may also be affected by the variability and age of the host plant, and inoculation condition .In future work, Germlings 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. Figure 8 showed that the seven Puccinia species were grouped into two distinct phenetic groups. 411 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Key 1. Urediniospores shape obovoid ........................................................................................ 2 Urediniospores shape ellipsoid ....................................................................................... 4 Urediniospores shape globoid........................................................... Puccinia imperatae Urediniospores shape broadly ellipsoid ............................................ Puccinia striiformis 2 (1). Uredinia margin naked; paraphyses present; urediniospores germ pores position equatorial ..................................................................................................... Puccinia fragosoana Uredinia margin ruptured epidermis; paraphyses absent; urediniospores germ pores position scattered............................................................................................................. 3 3 (2). Uredinia shape oblong ................................................................... Puccinia polypogonis Uredinia shape obovoid ..................................................................... Puccinia recondita 4 (1). Uredinia margin naked; paraphyses present; urediniospores germ pores position scattered; uredinia shape oblong ................................................................ Puccinia magnusiana Uredinia margin ruptured epidermis; paraphyses absent; urediniospores germ pores position equatorial; uredinia shape elongated ............................................... Puccinia graminis References Abbasi, M. (1996) Contribution to the knowledge of Puccinia species in Iran. Iran. J. Plant Pathol., 32, 244-267. Abbasi, M. Hedlaroude, Gh. A., Gjaerum, H.B., Scholler, M. (2002) Puccinia ariorum sp. nov. and other noteworthy graminicolous rust fungi (Uredinales) from Iran. Mycotaxon 8l, 435-444. Aime, M. C. (2006) Toward resolving family-level relationships in rust fungi (Uredinales). Mycoscience 47, 112-122. Alexopoulos, C. J., Mims, C. W. and Blackwell, M. (1996) Introductory mycology. 4th ed, Wiley, New York, p 869 Amerson, H. V. and Van Dyke, C. G. (1978) The ontogeny of echinulation in urediniospores of Puccinia sparganioides. Exp. Mycol. 2, 41-50. Baka, Z. A. M. (1992) Observations on the ultrastructure of the uredinial stage of Puccinia polypogonis on Polypogon monspeliensis. Mycopathol., 120,103-111. Baka, Z. A. M. (l996a) Comparative ultrastructure of aecial and telial infections of the autoecious rust fungus Puccinia tuyutensis. Mycopathol., 4, 143-150. Baka, Z. A. M. (1996b) Occurrence and morphology of Puccinia lagenophorae on Senecio glaucus in Egypt. Microbiol. Res., 151, 81-85. Baka, Z. A. M. (2004) Occurrence of Puccinia isiacae on Phragmites australis in Saudi Arabia and its possibility as a biological control agent. Microbiol. Res., 159, 175-179 Baka, Z. A. M. and Gjærum, H. B. (1996) Egyptian uredinales. I. Rusts on wild plants from the Nile Delta. Mycotaxon 30, 291-303. Baka, Z. A. M. and Lösel, D. M. (l992) Ultrastructure of the thistle rust, Puccinia punctiformis. Mycol. Res., 96, 81-88. Brown, M. F. and Brotzman, H. G. (1979) Phytopathogenic fungi; a scanning electron microscopic survey. Univ. Missouri, Columbia Extension Division, USA, pp. 287-335. Boulos, L. (2005) Flora of Egypt. monocotyledons, Alismataceae-Orchidaceae. Vol IV, Al-Hadara Publishing, Cairo, Egypt, p 617 Coleman, C.O.; Lowery, J. K. and Macfarlance, T. (2010) DELTA for beginners: an introduction into the taxonomy software package DELTA. ZooKeys 45, 1-75. Corlett, M. (1970) Surface structure of the urediniospores of Puccinia coronata f. sp. avenae. Can. J. Bot., 48, 2159-2161. 412 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Cummins, G. B. (1971) The rust fungi of cereals, grasses and bamboos. Springer Verlag, New York, p 570 Cummins, G. B. and Hiratsuka, Y. (2003) Illustrated genera of rust fungi. 3rd ed, APS Press, St. Paul, Minnesota, USA, p 240 Dallwitz, M. J. et al. (2000) User’s guide to the DELTA system: A general system for processing taxonomic descriptions. 4th ed. CSIRO Division of Entomology, Canberra, ACT, Australia El-Gazzar, A. and Rabei, S. (2008) Taxonomic assessment of five numerical methods and its implications on the classification of Hyptis s.l. (Labiatae). Int. J. Bot. 4, 85-92. El-Helaly, A.F. Ibrahim, I. A., Assawah, M. W., El-Arosi, H. M., Abo-El-Dahab, M. K., Michail, S. H., Abd-El-Rehim, M. A., Wasfy, E. H. and El-Gorani, M. A. (1966) General survey of plant diseases and pathogenic organisms in UAR (Egypt) until 1965. Alex. J. Agric. Res. Egypt, No 15. Evans, H. C. (1987) Fungal pathogens of some subtropical and tropical weeds and possibilities for biological control. Biocont. News Inform. 8, 7-30. Gardner, D. E. and Hodges Jr, C. S. (1985) Spore surface morphology of Hawaiian Acacia rust fungi. Mycologia 77, 575-586. Gardner, D. E. and Hodges Jr, C. S. (1989) The rust fungi (Uredinales) of Hawaii. Pacific Sc. 43, 41-55. Gower, J. C. (1971) Euclidean distance geometry," Mathematical Sciemis. 7, 1-14. Gupta, V.P.; Tewari, S. K. and Datta, R.K. (1994) Surface ultrastructure of the uredinial stage of Cerotelium fici and its infection process on mulberry. Mycopathol., 128, 99-104. Harder, D. E. (1984) Developmental ultrastructure of hyphae and spores. In: Bushnell WR, Roelfs AP (eds.) The cereal rusts, Vol 1, Academic Press, New York, pp 331-373. Hiratsuka, Y. (1971) Spore surface morphology of pine stem rusts of Canada as observed under a scanning electron microscope. Can. J. Bot. 49, 371-372. Hiratsuka, Y. (1988) Ontogeny and morphology of teliospores (probasidia) in Uredinales and their significance in taxonomy and phylogeny. Mycotaxon 31, 517-531 Hiratsuka, Y. and Sato, S. (1982) Morphology and taxonomy of rust fungi. In: Scot KJ, Chakravory AK (eds) The rust fungi. Academic Press, London, New York, Pp 1-36. Hiratsuka, N.; Sato, T.; Katsuya, K.; Kakishima, M.; Hiratsuka, Y.; Kaneko, S.; Ono, Y.; Sato, S.; Harada, Y.; Hiratsuka, T. and Nakayama, K. (1992) The rust flora of Japan. Tsukuba Shuppankai, Ibaraki, Japan, p 1205 Jennings, D. M.; Ford-Lloyd, B.V. and Butler, G. M. (1990) Morphological analysis of spores from different Allium rust populations. Mycol. Res. 94, 83-93 Johnson, D. A.; Ball, T. E.and Mess, W.M. (1999) Image analysis of urediniospores that infect Mentha. Mycologia 91, 1016-1020 Kenny. M. J. (1970) Comparative morphology of the uredia of rust fungi. Ph D Thesis, Purdue University, W Lafayette, Indiana, USA, p 135. Linder, H. P. and Rudall, P. J. (2005) The evolutionary history of Poales. Ann. Rev. Ecol. System 36, 107-124 Littlefield, L.J. (1981) Biology of the plant rusts: An introduction. Ames, Iowa, USA, Iowa State University Press, p103. Littlefield, L. J. (2000) Ultrastructure and ornamentation of Puccinia carduorum urediniospores and teliospores. Mycologia 92, 381-389. Littlefield, L.J. and Heath, M. C. (1979) Ultrastructure of rust fungi. Academic Press, New York, p 275. Mercer, E. H. and Birbeck, M. S. C. (1972) Electron microscopy. A handbook for biologists. 3rd ed, Blackwell Scientific Publications, Oxford. p 94. 413 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Osman, A.; Zaki, M.; Hamed, S. and Nagwa, H. (2011) Numerical taxonomic study of some Tribes of Gramineae from Egypt. Amer. J. Plant Sci., 2, 1-14. Peterson, R. H. (1974) The rust fungus life cycle. Bot. Rev. 40, 453-513. Rabei, S. (2011) A morphological, anatomical and taxonomic study of Umbelliferae in Egypt. LAP Lambert Academic Publishing, Köln. Rabei, S. and El-Gazzar, A. (2007) Computer-generated keys to species of Hyptis (Labiatae).Taeckholmia 27, 11-39. Reichert, I. (1921) Die pilzflora Agyptens. Engler’s Jahrb 56, 595-727. Savile, D.B.O. (1965) Some fungal parasites of Umbelliferae. Can. J. Bot. 43, 571-596. Shoemaker, R. A. (1981) Changes in the taxonomy and nomenclature of important genera of plant pathogens. Ann. Rev. Phytopathol., 19, 297-307. Sneath, P. H. A, and Sokal, R. R. (1973) Numerical taxonomy - the principles and practice of numerical classification. WH Freeman, San Francisco Standbridge, B. and Gay, J. L. (1969) An electron microscope examination of the surfaces of the uredospores of four races of Puccinia striiformis. Trans. Brit. Mycol. Soc., 53, 149-153. Traquair, J.A. and Kokko, E. G. (1983) Urediniospore morphology of Puccinia species attacking Cardueae. Can. J. Bot. 61, 2047-2051. Woods, A.M. and Beckett, A. (1987) Wall structure and ornamentation of the urediniospores of Uromyces viciae-fabae. Can. J. Bot. 65, 2007-2016. Table 1 Puccinia species under study and their host plants. Puccinia species Host Plants P. fragosoana Beltron Imperata cylindrica (L.) Racuschel P. graminis Pers. Cynodon dactylon (L.) Pers P. imperatae G. Poirault Imperata cylindrica (L.) Racuschel P. magnusiana Koern. Phragmites australis (Cav.) Trin. Ex Seud. P. polypogonis Speg. Polypogon monspliensis (L.) Desf. P. recondita Roberge ex Desm Lolium perenne L. P. striiformis Westend. Lolium perenne L. Table 2 Qualitative features of uredinia of Puccinia species studied Species Location on the host Shape Colour Margin Paraphyses P. fragosoana Leaves, mainly adaxial oblong DB N + P. graminis Stem and leaf sheath Elongated CB RE P. imperatae Leaves, mainly Elongated Y RE amphigenous P. magnusiana. Leaves, amphigenous oblong YB N + P. polypogonis Leaves, mainly abaxial oblong CB RE P. recondita Leaves as chloretic oblong CB RE streaks P. striiformis Leaves as linear stricks Elongated O RE + CB = cinnamon-brown; DB = Dark-brown; YB = Yellowish-brown; O = Orange; Y = Yellow; RE = Ruptured epidermis; N = Naked; + = Presence; - = Absence 414 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Table 3 Mean of dimensions (µm) of uredinia and paraphysis heads of Puccinia species studied. Species Uredinia Paraphysis heads Length Breadth Length Breadth Wall thickness P. fragosoana 507 ± 12 91 ± 02 10 ± 0.5 5.0 ± 0.2 2.5 ± 0.2 P. graminis 969 ± 50 730 ± 18 P. imperatae 475 ± 19 85 ± 05 P. magnusiana. 820 ± 32 318 ± 12 15 ± 0.7 6.5 ± 0.1 2.0 ± 0.2 P. polypogonis 720 ± 20 180 ± 10 P. recondita 282 ± 02 175 ± 10 P. striiformis 288 ± 02 125 ± 01 15.± 1.0 7.5 ± 0.5 0.8 ± 0.05 ± = Standard error of mean; - = Absent Table 4. Qualitative features of urediniospores of Puccinia species studied. Species Shape Color Wall color P. fragosoana Obovoid Golden-brown Yellow P. graminis Ellipsoid Orange Cinnamon-brown P. imperatae Globoid Light yellow Yellow P. magnusiana. Ellipsoid Brown Yellowish-brown P. polypogonis Obovoid Golden Yellow P. recondita Obovoid Yellow Yellowish-brown P. striiformis Ellipsoid Yellow Pale yellowish Table 5 Mean of dimensions (µm) of urediniospores and number of germ pores of Puccinia species studied Species Length Breadth Wall thickness Germ pores P. fragosoana 36.4 ± 0.9 22.9 ± 0.5 2.0 ± 0.08 4-5 equatorial P. graminis 29.2 ± 1.0 19.2 ± 0.6 1.8 ± 0.05 3-5 equatorial P. imperatae 21.5 ± 0.5 20.0 ± 0.6 2.5 ± 0.05 3-4 equatorial P. magnusiana. 30.0 ± 1.0 15.2 ± 0.5 2.0 ± 0.05 7-10 scattered P. polypogonis 26.0 ± 0.9 23.0 ± 0.7 2.0 ± 0.05 5-7 scattered P. recondita 29.2 ± 0.7 22.4 ± 0.5 1.5 ± 0.08 6-9 scattered P. striiformis 26.2 ± 0.6 20.6 ± 0.5 1.8 ± 0.05 9-13 scattered Table 6 Mean of the dimensions (µm) of urediniospore spines of Puccinia species studied Species Spine density/µm2 Length Distance between spines P. fragosoana 11.90 ± 1.7 0.80 ± 0.02 1.07 ± 0.02 P. graminis 15.60 ± 2.1 0.65 ± 0.01 1.70 ± 0.05 P. imperatae 12.80 ± 2.0 0.75 ± 0.02 1.60 ± 0.02 P. magnusiana. 13.70 ± 2.2 0.76 ± 0.01 1.65 ± 0.05 P. polypogonis 12.70 ± 2.2 0.86 ± 0.01 1.55 ± 0.05 P. recondita 16.20 ± 2.8 0.95 ± 0.03 1.40 ± 0.05 P. striiformis 11.50 ± 1.5 0.55 ± 0.01 1.10 ± 0.02 ± = Standard error of mean 415 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Legends to figures Figure 1. SEM micrographs of P. fragosoana. A. A naked uredinium. Scale Bar = 100 µm. B. A magnified part of an uredinum is showing a group of uredinispores. Note the paraphyses (P). Scale Bar = 10.0 µm. C. A magnified urediniospore. Note the reticulation (arrowheads) and echinulation on its surface. Scale Bar = 5.0 µm. Figure 2. SEM micrographs of P. graminis. A. Number of uredinia (short arrows) in linear manner. Scale Bar = 500 µm. B. A magnified part of an uredinium with ruptured epidermis (E). Scale Bar = l0 µm. C. A magnified urediniospore is showing the echinulation on its surface. Note germ pore (arrowhead). Scale Bar = 5.0 µm. Figure 3. SEM micrographs of P. imperatae. A. Number of uredinia (arrowheads) in linear manner on host leaf surface. Scale Bar = 1000 µm. B. Single uredinium is showing a ruptured epidermis (E). Scale Bar = 100 µm. C. A magnified part of an uredinum is showing a group of uredinispores inside it. Scale Bar = 10 µm. D. A group of urediniospores is showing spines on their surfaces. Note the germ pore (gp) and a germ tube (gt) emerging from one spore. Scale Bar = 10 µm. Figure 4. SEM micrographs of P. magnusiana. A. An uredinium showing many urediniospores. Scale Bar = 100 µm. B. Magnified part of the uredinum showing many urediniospores with spines on its surface. Note the paraphyses (P). Scale Bar = 10 µm. Figure 5. SEM micrographs of P. polypogonis. A. An uredinum contains numerous urediniospores. Note ruptured epidermis (E). Scale Bar = 50 µm. B. A magnified part of the uredinum is showing mature urediniospores. Note a pedicel (pd) without urediniospore. Scale Bar = 1 µm. C. Mature urediniospores with mature spines emerging from the spore wall. The spines are located in circular depressions encircled by an annulus (arrows). Scale Bar = 2 µm. Figure 6. SEM micrographs of P. recondita. A. Uredinia with ruptured epidermis (E). Scale Bar =100 µm. B. A part of an uredinium showing urediniospores. Scale Bar =10 µm. C. A magnified urediniospore is showing the echinulation on its surface. Scale Bar = 5 µm. Figure 7. SEM micrographs of P. striiformis. A. An uredinium with ruptured epidermis (E). Scale Bar =100. B. A part of an uredinium showing urediniospores intermixed with paraphyses (p). Scale Bar = 10 µm. C. A magnified urediniospore is showing the echinulation on its surface. Scale Bar = 5 µm. D. A magnified part of an urediniospore is showing spines (arrows) located in depressions (D) and enclosed by annuli (A). Scale Bar = 0.5 µm. Figure 8. UPGMA-dendrogram of the seven Puccinia species which are grouped into two distinct phenetic groups. 416 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Fig. 1. (P. fragosoana) Fig. 2. (P. graminis) 417 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 D Fig. 3. (P. imperatae) Fig. 4. (P. magnusiana) 418 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Fig. 5. (P. polypogonis) Fig. 6. (P. recondita) 419 Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421 Fig. 7. (P. striiformis) 420 ‫‪Baka, Z.A.M. and Rabei, S. (2013) Morphology of uredinia and urediniospores of seven Puccinia species‬‬ ‫‪attacking Poaceae in Egypt. Egyptian J. of Botany 3rd int. con. 17-18 April, Helwan Uni., Egypt. 407 - 421‬‬ ‫‪Fig. 8.‬‬ ‫لشكل ل ه ي ل‬ ‫لي ر ي يني‬ ‫ك ي‬ ‫قس لن‬ ‫–كي ل‬ ‫–ج‬ ‫ص ل بي‬ ‫ل‬ ‫لج ثي لي ري يني لس‬ ‫لنجي ي في‬ ‫ح‬ ‫بق‬ ‫يط–‬ ‫أن‬ ‫ن فط بكسيني ت ج ل ئ‬ ‫س ي حسين ربي‬ ‫ي ط لج ي – رق ب ي ‪- 71543‬‬ ‫أص ء خ ص ج س ب س‬ ‫ل إض ف أ ج ل ص ف فط‬ ‫ل س‬ ‫ل ف‬ ‫ل‬ ‫لش ل ل‬ ‫ل س‬ ‫‪.‬ف‬ ‫ل ث ل‬ ‫ل‬ ‫ط ق ل‬ ‫ل ئ‬ ‫ل‬ ‫ل‬ ‫فط لص أ ب س ب س‬ ‫لس أ‬ ‫ل ث ل‬ ‫ل‬ ‫ف ب ف‬ ‫أ‬ ‫ل ئ ل‬ ‫ل صب ل‬ ‫ل س ‪ .‬ج ل‬ ‫إل‬ ‫ل‬ ‫س‪،‬‬ ‫‪،‬ب س ج‬ ‫‪:‬ب س ف ج‬ ‫س‬ ‫ل ف‬ ‫بس ل‬ ‫أ‬ ‫بص ‪.‬‬ ‫س‪.‬‬ ‫بس س ف‬ ‫س ‪ ،‬ب س ب ل ج س‪ ،‬ب س‬ ‫‪،‬ب س‬ ‫بس إ‬ ‫أب‬ ‫ف‬ ‫ثش ل لث ل‬ ‫أ‬ ‫إخ اف ب‬ ‫ث‬ ‫ل س أ‬ ‫أ ض‬ ‫أب‬ ‫ل ل‬ ‫ل‬ ‫شل ل ث ل‬ ‫أ‬ ‫ب خ‬ ‫ل‬ ‫ج ل‬ ‫ج أ‬ ‫ل ث ل سف ب ‪.‬‬ ‫سط‬ ‫ط ل‬ ‫ث ف أش‬ ‫إ‬ ‫ث‬ ‫س ل‬ ‫أ‬ ‫ف ل‬ ‫ل ف‬ ‫ل ص ‪.‬‬ ‫لفط فس ل س ل‬ ‫إل‬ ‫ب‬ ‫إخ اف‬ ‫أ‬ ‫ل س أ‬ ‫أ‬ ‫أ ‪.‬‬ ‫س ل ش ب إخ اف ب‬ ‫أ‬ ‫ب ج ل ‪.‬‬ ‫بس‬ ‫شب‬ ‫فص إل‬ ‫‪421‬‬