Mycopathologia 134: 143-150, 1996. 9 1996KluwerAcademicPublishers. Printedin theNetherlands. 143 Comparative ultrastructure of aecial and telial infections of the autoecious rust fungus Puccinia tuyutensis Zakaria A.M. Baka Department of Botany, Faculty of Science, University of Mansoura, Mansoura, Egypt Received 17 October 1995;acceptedin revisedform 10 April 1996 Abstract This study demonstrates morphological differences between aecial and telial stages of the autoecious rust Puceinia tuyutensis. The aeciospores possess 'vermcose' ornamentation while the teliospores have smooth surfaces. The aecial and telial haustoria of this rust produced in the mesophyll of Cressa cretica differ morphologically in the following respects:(1) the haustorial mother cell of telial haustorium is more differentiated than that of aecial haustorium and its wall at the penetration site is composed of 4 layers; (2) the aecial haustorium is filamentous in appearance and slightly constricted at the point of entry into the host cell, while the telial haustorium is clavate and possesses a narrow neck with a densely staining neckband and swollen body; (3) the neck of the telial haustorium is always associated with numerous vesicles while that of the aecial haustorium is not. Vascular tissue of host leaves is heavily invaded by aecial haustoria but not by telial haustoria. Key words: Aecial stage, Puccinia tuyutensis, rust fimgi, telial stage, ultrastmcmre Introduction Puccinia tuyutensis Speg. (syn. P cressae Lagerh.), an autoecious macrocyclie rust fungus infecting Cressa spp. (Convolvulacae) [1], is known to infect Cressa cretica L. in Egypt [2, 3]. Aecia, uredinia and telia have been observed by the present author on this host species in Egypt but not pycnia. World distribution of Puccinia tuyutensis includes Argentina, Chile and Spain [1, 2], Algeria, Italy and Cyprus [2], Greece [2, 4], and the United States [5]. The morphology of different spore stages of this fungus has been described by several authors [1, 2, 4] but so far, no ultrastructural studies have been reported. Numerous ultrastructural studies indicate that the haustoria produced by the spermogonial and aecial stages of rust fimgi are morphologically different from those of the uredinial and telial stages [6-9]. However, most ultrastrucmral comparisons of aecial and telial stages have focused on heteroecious rusts, relatively few on autoecious rusts, for example P. podophylli [9], P. lagenophorae [10] and P. punctiformis [10, 11]. The purpose of this study is to use scanning and transmission electron microscopy to compare the ultra- structure of the aecial stage of the autoecious rust P tuyutensis with that of its telial stage. Material and methods Infected host. Leaves of Cressa cretica LI having aecia with aeciospores or telia with teliospores were collected in April (for aecia) and August (for telia) from the Kalabshow area at the Northern part of the Nile Delta, Egypt. Scanning electron microscopy (SEM). Infected leaves bearing aecial or telial stages were cut into approximately 1-cm2 segments with sharp razor blade and processed for examination by SEM according to the method of Baka [10]. Leaf segments were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.0 for 24 h, then transferred into 1% OsO4 in the same buffer at 4~ for 1 h. After fixation, the segments were dehydrated in a graded series of acetone concentrations, dried, coated with gold, examined and photographed using a Jeol JSM-6400 SEM. 144 Transmission electron microscopy (TEM). Infected leaf segments were prepared for TEM by the procedure of Baka and L6sel [11]. Ultrathin sections cut on a Reichert ultramicrotome, were transferred to copper grids and stained with uranyl acetate and lead citrate. The sections were examined and photographed using a Jeol 100-S TEM. Results Aecia and aeciospores. The aeciospores developed within the aecium and were surrounded by a clearly defined one cell thick peridium (Figure 1). Examination by SEM showed 'verrucose' ornamentation on the aeciospore surface (Figure 2). TEM examination revealed that mature aeciospore has electrondense cytoplasm, containing numerous lipid drops and glycogen granules (Figure 3). The mature aeciospore was enclosed by internal (secondary) and external (primary) walls. The latter was disintegrated leaving verrucae and remnants. The verrucae were smooth, knob-like, electron-lucent and situated in small depressions (Figure 4). Germ pores appeared as locallythickened regions of the electron-opaque secondary wall of mature spores containing fine electron-lucent interspersions (Figure 4). It was noted that the verrucae were continuous over the germ pore area (Figure 4). Telia and teliospores. The telia contained a compact growth of teliospores, as observed by SEM (Figure 5). The bicelled teliospores were characterized by smooth surfaces. Some spores showed abscission scars indicating positions of attachment to their pedicels (Figure 6). Aecial haustoria. The aecial stage of P. tuyutensis produced unmodified irregularly shaped filamentous haustoria (M-haustoria) (Figure 7), each of which arose from a relatively undifferentiated haustorial mother cell (HMC) on an intercellular hyphae. This type of HMC contained well-developed cytoplasm with mitochondria, ribosomes, and vacuoles (Figure 8). A typical complement of cellular components was present within the aecial haustorium including numerous mitochondria, endoplasmic reticulum, vacuoles, moderately electron-dense extrahaustorial matrix, and extrahanstorial membrane. TEM examination of serial sections revealed that the haustorium contained one nucleus (Figure 9), as indicated by LM examina- Figure 1. An aeciumwithaeciospores(AC)enclosedby a peridium (PR). Noterupturedepidermis(E). Bar = 50 #m. Figures1, 2, 5, 6 are SEmicrographs.Figures3, 4, 7-16 are TE micrographs. Figure2. A groupof aeciosporeswithverlucoseornamentation.Bar = 10#m. tion, although the aeciospores, aeciosporophores and hyphae under the aecium contained two nuclei. The examination of serial sections from blocks of tissue from different infected leaves bearing aecia revealed that the vascular systems are more heavily invaded by the fungus than mesophyll cells. Haustoria were particularly frequent in bundle sheath cells, companion cells, and phloem parenehyma (Figures 10, 11). Telial haustoria. The intercellular hyphae produced during the telial stage of infection were characterized by the presence of two nuclei, lipid drops associated with glycogen granules, dense cytoplasm, mitochon- 145 Figure 5. T.S. of host stem showing compact telium with crowded teliospores (T). Bar = 50/~m. Figure 3. Mature aeciospore containing elecuon-dense cytoplasm, lipid drops (L) and glycogen granules (GL). Note verrucae (arrowheads) and secondary wall (SW). Bar = 1.0/zm. Figure 6. Bicelled teliospores with smooth surfaces. Note points of detachment (arrowheads) of teliospores from their pedicels. Bar = 20 #m. Figure 4. A part of mature aeciospore showing verrucae (arrowheads). Note the remnants (arrows) of primary wall. Note also germ pore (GP) located within the secondary wall Bar = 0.5 #m. dria and ribosomes (Figure 12). These hyphae were attached to the host cell wall by extramural substances (Figure 12) as defined by Harder et al. [12]. The telial haustorium (D-haustorium) consisted of a narrow tubular neck with an electron-dense neckband and a swollen body, the latter surrounded by an extrahaustorial matrix and extrahaustorial membrane (Figure 13). This haustorium was produced by a more differentiated I-IMC than that of the aecial haustoriurn. At the point of haustorium entry into the host cell, the HMC of the telial haustorium was distinguished from other intercellular hyphae by its thicker and more complex wall structure in which four layers could be recognized (Figure 14). The outermost layer (4th layer) was probably not a rigid structural part of the wall but a mucilaginous coating substance, which apparently served to a ~ x the HMC to the host cell wall [12]. One striking feature, frequently noticed in mesophyll cells invaded by telial haustoria was the accumulation of vesicles and endoplasmic reticulum around the haustorial neck (Figures 13-15). The telial haustorium usually contained two nuclei (Figure 16). Mature aecial and telial haustoria differed further in the extrahanstorial matrix, between the haustorium wall and the invaginated host plasma membrane. This region tended to increase in thickness with age. In the former, the fibrillar material which accumulated in the extrahaustorial matrix was of a greater electron-density, similar to the haustorial wall (Figure 9), whereas the material in the extrahaustorial matrix around telial haustoria was of similar appearance to the host cell wall and less electron-dense than the haustorial wall (Figures 13, 16). The different characters of aecial and telial haustoria are listed and compared in Table 1. 146 Figure9. T.S. in a young aecial haustorium inside host cell showing Figure 7. Filamentous aecial haustorium (AH) produced by haustorial mother cell (HM). Note host nucleus (N), vacuole (V) and cell wall (W). Bar = 2.0 #m. one nucleus (n), mitochondria (m), vacuole (v) and endoplasmic reticulum (ER), Note haustorial wall (FW), extrahaustorial matrix (arrow) and extrahaustorial membrane (arrowhead). Bar = 1.0/zm. Figure 10. Bundle sheath cell (BS) invaded by numerous aecial haustoria (AH).Note host nucleus (N) is surroundedby chloroplasts (C). Bar = 2.0/zm. Figure 8. A magnified part of Figure 7 showing constriction of the aecial haustorium (AH) at penetration site. Note the continuity (arrowheads) between haustorial mothercell (HM) wall and haustorial wall (arrow). Bar = i .0 #m. Discussion The morphology and structure ofaecia and aeciospores o f P. tuyutensis, as observed by SEM and TEM, is similar to that described for P. lagenophorae [10], P. graminis [13], P recondita [14], and P poarum [15] although refractive bodies which have been reported on the aeciospore surface ofP. lagenophorae [10] and P. poarum [15] were not found in the present investi- gation. Cummins and Hiratsuka [16] defined five morphological types of aecia in rusts. The typically aecidioid aecium ofP. tuyutensis is similar to that o f many other Puccinia species. The 'verrucose' ornamentation o f aeciospores o f P. tuyutensis is similar to that observed in many Puccinia species. As previously described by Henderson et al. [17] for the development o f P poarum, aeciospore verrucae were synthesized within the primary wall and consisted of relatively solid material. The primary wall material was reabsorbed during spore maturation, leaving the verrucae free-standing on the surface of the underlying secondary wall. 147 Figure13. A clavate telial haustorium (TH) with narrow tubular neck (I-IN),neckband (NB) and swollen body. Note haustorial wall (FW), extrahaustorial matrix (large arrow) and extrahaustorial membrane (arrowhead). Note also host vesicles (small arrows) near to haustorial neck. C = chloroplast. Bar = 1.0 #m. Figure 11. T.S. of a minor vein of infected leaf invaded by aecial haustoria (AH), found inside companion cells (CC) and phloem parenchyma (PP). Note intercellular hyphae (h) with two nuclei (n) and sieve elements (SE). Bar = 2.0 #m. Figure 12. Intercellular hypha (h) during telial stage of infection showing dense cytoplasm with two nuclei (n) and lipid drops (L). Note the septum (arrowhead) and extramural substance (arrow) between the hypha and host cell wall. Bar = 1.0 #m. Littlefield and Neath [6] pointed out that teliospores generally exhibited a greater variety o f ornamentation types than do aeciospores and urediniospores and reported a list o f teliospore o r n a m e n t a t i o n based o n the definition o f Murrill cited in [6]. Teliospore ornam e n t a t i o n is a n important character in the t a x o n o m y Figure 14. Penetration site of the telial haustorium. The wall of haustorial mother cell (HM) is formed of 4 layers (1-4). Note host endoplasmic reticulum (ER) and vesicles (arrows) are close to haustorial neck. Note also neckband (NB) and host cell wall (W) with granular appearance. Bar = 0.5/~m. o f rust fungi, although the wall surface o f mature teliospores o f m a n y rusts is smooth [18]. The smooth walled teliospore ofP. tuyutensis is similar to those o f P. lagenophorae [10] andP. alli [19]. 148 Table 1. Comparison of aecial and teliat haustoria ofP. tuyutensis AH = aecial haustorium. TH = telial haustorium. + = present. - = absent Character AH TH Differentiationof HMC wall Specializationof HMC Constriction ofhaustorial neck at point of entry Very narrow neck Collar Neckband Association of host cell vesicles to haustorial neck Invasion of host vascular tissue + + + - - + + + + - Figure 15. Penetration site of telial haustorium showing haustorial neck with neckband (NB). Host vesicles (arrowheads) and ER profiles are located around the haustorial neck. Note host plasma membrane (arrow). Note also haustorial mother cell (HM). Bar = 0.5 #m. Figure 16. A telial haustorium (TH) with two nuclei (n) inside host mesophyll cell. Note intercellularhypha (h), host plasma membrane (arrowheads), and extrahaustorialmembrane (arrow). Bar = 1.0 #m. This study clearly demonstrates that the aecial and telial haustoria o f P. tuyutensis produced on similar cell types o f the same host plant are morphologically different from one another. The aecial haustorium o f P. tuyutensis is basically similar to that o f spermogonial and aecial haustoria in other rusts [6, 8-10, 20-23] Likewise, the m o r p h o l o g y o f the telial haustorium o f this fungus is similar to that o f uredinial and telial haustoria o f other rusts [6, 8, 9, 11, 20-26]. The extramural substances between intercellular hyphae and host cell wall are interpreted as having protective, adhesive, or specificity functions and m a y be related to the establishment o f compatibility or incompatibility [27]. Harder et al. [12] concluded that these substances appear to be highly complex and variable according to the location o f the hyphae within the rust colony. Components o f this material o f fungal origin m a y well be involved in adhesive functions, in conjunction with material o f host origin and are thought to consist basically o f glycoproteins [ 12, 27] In this study, no collars were detected at penetration sites o f either aecial or telial haustoria. The formation o f collars has frequently been linked to the degree o f host-fungus compatibility or related to the age o f hanstorium [28]. The latter interpretation m a y well fit the present investigation, since the presence o f dense cytoplasm and few vacuoles suggests the haustoria examined were probably young. The vesicles seen around the neck o f the telial haustorium resembled those reported around D-haustoria o f P. punctiformis [11], and P. coronata avenae [22, 28]. Heath and Heath [29] suggested that these vesicles were attached to host plasma membrane in cowpea infected with Uromyces phaseoli vat. vignae and possibly supplied the extra membrane needed to cover the developing extrahaustorial matrix. The present investigation supports this idea since, the vesicles are seen to be attached to the host-plasma membrane and its invaginated region around the neck o f telial haustoria. The invasion o f the host vascular system b y spermogonial-aecial haustoria has been relatively 149 infrequently recorded [3 0-34]. The invasion of the vascular system, particularly the phloem, which occurs in the monokaryotic but, apparently rarely in the dikaryotic infections [33-35] may relate to the presence or absence of a neckband. The neckband, found in Dhaustoria, forms a seal between the host and fungal plasmalemma, preventing leakage, from the extrahaustorial matrix, of solutes undergoing transport from host to pathogen [31]. Thus the neckband is instrumental in coupling the host and haustorial cytoplasms into a functional unit [34]. However, the absence of neckband from monokaryotic haustoria may be associated with intense invasion of vascular systems [34]. The high concentration of nutrients within the vascular system would provide a large diffusion gradient between host and fungus which may compensate for the loss of solutes to the general apoplast and/or reentry of solutes into the host from the extrahaustorial matrix [33, 34] In closing, it should pointed out that P tuyutensis is one of the few autoecious rusts in which the hostpathogen relationship has been examined [9, 31, 32]. The structural features of P. tuyutensis lend support to the evidence that aecial and telial haustoria may function differently. Acknowledgements I would like to thank Dr. Dorothy M. L6sel, Department of Animal and Plant Sciences, University of Sheffield, England for revising the manuscript and useful comments and Dr. Knut Krzywinski, Botanical Institute, University of Bergen, Norway for providing the facilities for electron microscopy. The expert technical assistance of Mr. Egil Erichsen, Electron Microscope Unit, University of Bergen, Norway, is gratefully acknowledged. References 1. LindquistJC. RoyasDe La RepublicaArgentinaY ZonasLimitrofes. ColeccionCientificaTomoXX. InstitutoNacionalDe TecnologiaAgropecuaria,Argentina, 1982,pp. 375-376. 2. SydowP, Sydow H. MonographiaUredinarum.Vol. 1, 1904, pp. 320-321. 3. E1-HelalyAF, IbrahimIA, AssawahMW,ElarosiHM, Abo-E1Dahab MK, Michall SH, Abd-E1-RehimMA, WasfyEH, E1GooraniMA. Generalsurveyof plant diseases and pathogenic organisms in U.A.R. (Egypt) until 1965. Alexandria J Agric Res 1966. 4. GjaerumHB, HansenA. Additionsto the rust flora&the Greek islands. Ann Musei Goulandris1990; 8: 81-96. 5. FarrDF, BillsGF, ChamurisGP, RossmanAY. Fungion plants and plant products in the United States. The American Phytopathol. Soc. St. Paul,MN, USA, 1989. 6. LittlefieldLJ, Heath MC. Ultrastructure of rust fungi. New York/London:AcademicPress, 1979. 7. Harder DE. Developmentalstructure of hyphae and spores. In: BushnellWR, Roelfs AP (eds.). The cereal rusts, Vol. 1. Orlando, AcademicPress, 1984, pp. 333-373. 8. HarderDE, Chong J. Structure and physiologyof haustoria. In: BushnellWR, Roelfs AP (eds.). The cereal rusts, Vol. 1. Orlando, AcademicPress, 1984, pp. 431-467. 9. BorlandJ, Mims CW. An ultrastructural comparison of the aecial and telial haustoria of the autoecious rust, Puccinia podophylli. Mycologia 1980; 72: 767-774. 10. Baka ZAM. Responses of plant tissues to infection by rust fungi:finestructure, cytochemistryandautoradiography.Ph.D. Thesis, Universityof Sheffield, England, 1987. 11. BakaZAM, L6sel DM. Ultrastrucmreof the thistle rust, Pucciniapunctiformis. MycolRes 1992; 96: 8148. 12. Harder DE, Chong J, Rohringer R, Mendgen K, Schneider A, Welter K, KnaufG. Ultrastructure and cytochemistry of extramural substancesassociated with intercellularhyphae of several rust fungi.Can J Bot 1989; 67:2043-2051. 13. HolmeL, TibellL. Studieson the finestructure of aeciospores III. Aeciosporesontogenyin Puccinia graminis. SvenBot Tidskr 1974; 68: 136-152. 14. GoldRE, LittlefieldLJ, Stellar GD. Ultrastructureof the pycnial and aecial stages ofPuccinia recondita. Can J Bot 1979; 57: 74-86. 15. A1-KhesrajiTO. Comparativeanatomyand histochemistryof the association of Puccinia poarurn with its alternate hosts. Ph.D. Thesis, Universityof Sheffield, England, 198i. 16. CumminsGB, Hiratsuka Y. Illustrated genera of rust fungi. The AmerPhytopathol Soc St. Paul, MN, USA, 1983. 17. HendersonDM, Prentice HT, Eudall R. The morphology of fungal spores. III. The aecidiospores of Puccinia poarum. Pollenet Spores 1972; 14: 17-24. 18. MimsCW,ThurstonEL. Ultrastrucmreofteliosporeformation in rust fungusPucciniapodophylli. Can J Bot 1979; 57: 25382553. 19. Baka ZAM. Light and electron microscopic study of teliospores of Puccinia aIli on garlic. Egyp J Bot 1989; 32: 21%226. 20. A1-KhesrajiTO, L6sel DM. The fine structure of haustoria, intracellularhyphae and intercellularhyphae of Puccinia poarum. Physiol Plant Pathol 198i; 19: 301-311. 21. Gray DJ, Amerson HV, Van Dyke CG. An ultrastrucmral comparison of monokaryoticand dikaryotic haustoria formed by the fusiform rust fungus, Cronaritum quercuum f. sp. fusiforme. Can J Bot 1982; 60: 2914-2922. 22. ChongJ, Harder DE, RohringerR. Ontogenyofmono- and dikaryntic rust hanstoria: cytochemicaland ultrastructuralstudies. Phytopathol 1981; 71: 975-983. 23. HarderDE. Comparativeultrastructureof the hanstoriain uredinialand pycnialinfectionsofPuccinia coronata avenae. Can J Bnt 1978; 56: 214-224. 24. Ferreira JF, Rijkenberg FHJ. Ultrastructural morphology of Uromyces transversalis infectionof resistant and susceptible gladiolus hosts and a nonhost, Zea Mays. Phytopathol 1991; 81: 596~02. 25. BakaZAM.Observationson the ultrastrucmreof the uredinial stage of Puccinia polypogonis on Polypogon monspeliensis. Mycopathol 1992; 120:103-111. 150 26. 27. 28. 29. 30. 31. Spiers AG, Hopcroft DH. Comparative studies of the poplar rusts Melampsora medusae, M. larici-populina and their interspecific hybrid M. medusae-populina. Mycol Res 1994; 98: 889-903. Rohringer R, Martens JW. Infection-related proteins in intercelluar washing fluid from stem rust-affected wheat leaves: race-associated protein differences revealed by PAGE and ConA blotting. Physiol Mol Plant Pathol 1987; 31: 375-386. Chong J, Harder DE. Ultrastructure of haustorium development ofPuccinia coronata avenae: some host responses. Phytopathol 1982; 72: 1527-1533. Heath MC, Heath IB. Ultrastructure of an immune and a susceptible reaction of cowpea leaves to rust infection. Physiol Plant Pathol 1971; 1: 277-287. AI-Khesraji TO, L6sel DM, Gay JL. The infection of vascular tissue in leaves of Tussilagofarfara L. by pycnial-aecial stage ofPucciniapoarum Niel. Physiol Plant Pathol 1980; 17: 19831997. Baka ZAM, L6sel, DM. Infection of vascular tissue by the autoecious rusts Puccinia punctiformis and Puccinia 32. 33. 34. 35. lagenophorae: a cytological study. Physiol Mol Plant Pathol 1992; 40:411-421. Larous L, L6sel DM. Vascular infection by Puccinia menthae and other rust fungi. Mycol Res 1993; 97: 407-414. Larous L, L6sel DM. Strategies of pathogenicity in monokaryotic and dikaryotic phases of rest fungi, with special reference to vascular infection. Mycol Res 1993; 97: 415-420. Woods AM, Gay JL. The interface between haustoria of Puccinia poarum (monokaryon) and Tussilago farfara. Physiol Plant Pathol 1987; 30: 167-185. Andreev LN, Plotnikova YuM, Serezhkina GV. Hanstoria of Puccinia graminis Pets. f. sp. tritici Eriks. & Henn. in the vascular system of wheat. Mikologia i Fitopathologiya 1982; 16: 335-338. Address for correspondence." Z.A.M. Baka, Department of Botany, Faculty of Science, University of Mansoura, Mansottra, Egypt. Tel. (work) 050-342388; Tel. (home) 050-355082; Fax. 050-347900.