Mycologia, 106(2), 2014, pp. 216–223. DOI: 10.3852/106.2.216 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897 The interaction apparatus of Asteridiella callista (Meliolaceae, Ascomycota) De´lfida Rodrı´guez Justavino1 dark, thick-walled, branching hyphae with appressoria Universita¨t Tu¨ bingen, Institut fu¨ r Evolution und (capitate hyphopodia) and conidiogenous cells (mu- O¨ kologie, Evolutiona¨re O¨ kologie der Pflanzen, Auf der cronate hyphopodia); superficial, dark, perithecia Morgenstelle 1, 72076 Tu¨ bingen, Germany containing clavate asci with thin walls; and asci Universidad Auto´noma de Chiriquı´, El Cabrero, Chiriquı´, Panama´ typically containing 2–4 brown ascospores with four septa each (Hansford 1961, Kirk et al. 2008). Julieta Carranza Vela´squez Communication between fungi and plants was the Carlos O. Morales Sa´nchez focus of numerous papers (see e.g. Yi and Valent 2013 Universidad de Costa Rica, San Pedro Monte´s de Oca´, and references therein). However, in Meliolaceae San Jose´, Costa Rica studies have focused predominantly on morphologi- Rafael Rinco´n cal characteristics and consequently few studies have Universidad Auto´noma de Chiriquı´, El Cabrero, analyzed details of their parasitic interactions. For Chiriquı´, Panama´ example, Luttrell (1989) described microscopic struc- tures of the interaction between Meliola floridensis Franz Oberwinkler Hansf. and Persea borbonia (L.) Spreng. (Lauraceae) Robert Bauer and Mueller et al. (1991) reported on ultrastructural Universita¨t Tu¨ bingen, Institut fu¨ r Evolution und O¨ kologie, Evolutiona¨re O¨ kologie der Pflanzen, Auf der details of the interaction between Meliola sandwicen- Morgenstelle 1, 72076 Tu¨ bingen, Germany sis Ellis & Everhart and the host Kadua acuminata Cham. & Schltdl., syn. Hedyotis acuminata (Cham. & Schltdl.) Steud. (Rubiaceae). Abstract: We document here for the first time Theobjectiveofthepresentstudythereforewasto ultrastructural details of the cellular interaction of examine the ontogeny of structural characteristics in the Asteridiella callista and its host Stachytarpheta mutabilis cellular interaction between A. callista and its host. Our var. violacea from Costa Rica. A. callista attaches to the results provide evidence for the evolution of a unique host with appressoria, invades the epidermal cell wall interaction process in this biotrophic plant pathogen. and forms an apoplastic complex cisternal net, presumably for nutrient uptake from its host. This MATERIALS AND METHODS unique structure, called an interaction apparatus (Ia), consists of cisternae surrounded by a membrane Materials.—Colonies of Asteridiella callista (Rehm) Hansf. continuous with the fungal cytoplasmic membrane. were collected on leaves of Stachytarpheta mutabilis (Jacq.) Vahl var. violacea Moldenke (Verbenaceae) in Costa Rica, Subsequently the apoplastic trunk of the Ia extends Puntarenas, Canto´n Garabito, Distrito Ta´rcoles, National into the host epidermal cell wall and contacts the host Park Carara, 9u469N, 84u369W, 30–50 m, 5 Jul 2011, by Carlos cytoplasmic membrane. Electron-opaque material, O. Morales & De´lfida Rodrı´guez J.2324 (TUB, USJ). For probably of fungal origin appears at the host cytoplas- accuracy the material was compared with the type material of mic membrane. Finally these electron-opaque deposits Asteridiella callista (Rehm) Hansf. (BPI 693091). This species are encased by host material. Functional and systema- is recorded here for the first time for Costa Rica. It is known to tical aspects of this interaction scenario are discussed. infect members of Verbenaceae from Guyana, China, Key words: appressorium, cellular interaction, Ecuador, Grenada, Indonesia, Java, Philippines, Puerto Rico, cisternal tubules, concentric bodies, Costa Rica, Trinidad and Virgin Islands (Hansford 1961, Farr et al. 2013). Stachytarpheta mutabilis var. violacea, Verbenaceae Morphological analyses.—Hyphae with appressoria of A. callista were observed with a Zeiss Axioskop 2 Plus microscope. Small sections of the colonies were cut with INTRODUCTION aid of dissecting microscope, and preparations were Species of the Meliolaceae (Meliolales, Ascomycota), examined as slide mountings in water. also known as black or dark mildews, are parasitic Ultrastructural analyses.—The ultrastructure was studied fungi of mainly tropical distribution. They infect with a Zeiss EM 109 transmission electron microscope at foliage of members of numerous plant families. 80 kV with a transfaseroptic camera. Sections of leaves Species of this group are characterized by superficial, 2mm2 with colonies of A. callista were cut in the field and immediately placed in vials containing 2% of the fixative Submitted 7 May 2013; accepted for publication 25 Sep 2013. solution of Karnovsky (1965) and stored until further 1 Corresponding author. E-mail: [email protected] processing. In the laboratory samples were transferred six 216 JUSTAVINO ET AL.: ASTERIDIELLA CALLISTA 217 times in 0.1M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide in the same buffer for 1 h in the dark, washed in distilled water and stained in 1% aqueous uranyl acetate 1 h in the dark. After five washes in distilled water, samples were dehydrated in acetone, with 10 min changes at 25%,50%,70%,95%, and three times in 100% acetone, embedded in Spurr’s plastic (Spurr 1969) and sectioned with a diamond knife. Serial sections were mounted on formvar- coated, single-slot copper grids, stained with lead citrate at room temperature for 5 min and washed with distilled water. RESULTS Hyphae and ascomata of A. callista grow only on the upper epidermis of the leaves of S. mutabilis var. violacea (FIG. 1). Our morphological and ultrastruc- tural analyses of the interaction between the patho- gen and the host showed that initially appressoria attached to epidermal cell walls (FIG. 2A). Within the appressoria, a complex structure developed (FIG. 2B– F) which is designated here as an interaction apparatus (Ia). This structure, which resembles roughly the interaction apparatus described by Bauer et al. (1997) for the Exobasidiales and by Simon et al. (2004) for the ascomycete Cymadothea trifolii, consist- ed of elongate cisternae that fused to form a trunk (FIG. 2A, B, E, F). Opposite the epidermal cell wall the trunk was limited by an electron-opaque line (FIG. 2B, D, E). Mitochondria were numerous in the vicinity of the Ia (FIG. 2C–F), and sometimes concen- tric bodies of unknown origin and function could be observed (FIG. 2F). The tubular system of the Ia was continuous with the fungal cytoplasmic membrane (FIG. 2B, D). Thus, the intracisternal space represent- ed an apoplastic compartment. Within the apoplastic compartment of the Ia, electron-opaque granular particles were visible (FIG. 2D). At this stage of the infection, structural changes were observed at the contact zone of the appressorium and the epidermis cell wall of the host. The apoplastic trunk of the Ia, accompanied by partial desintegration FIG. 1. Colonies of Asteridiella callista showing hyphae of the basal appressorial wall, formed a penetration and appressoria. Bar 5 10 mm. pore (FIG. 2A, B, E) that extended through the cuticle and the thick cell wall of the host (FIG. 3A). Thus, the (FIG. 3B, C). Finally, the host cell added wall apoplastic trunk of the Ia contacted directly the host appositional material of different electron density cytoplasmic membrane (FIG. 3D). Within the resulting and unclear substructure to the site of interaction in apoplastic tubule, designated here as Ia-canal, mem- response to the fungal attack (FIG. 3D). This apposi- branes or components of the host cell wall could not be tional material led to the encasement of the original observed (FIG. 3A–D). electron-opaque deposition, which thereafter separat- An electron-opaque tubular structure (in three- ed from the cytoplasm and collapsed (FIG. 4A, B). dimensional reconstruction from serial sections) within the epidermal cell wall finally surrounded the DISCUSSION Ia-canal (FIG. 4A, B). Between the apex of the Ia canal and the host cytoplasmic membrane, electron-opaque Species of Meliolaceae have been considered fungi deposits appeared (FIG. 3B). As a consequence, the that inflict little damage on their hosts (Wellman host cytoplasmic membrane invaginated at this area 1972) and probably this is the reason that most 218 MYCOLOGIA FIG. 2. Sections showing ultrastructural details of the interaction apparatus of Asteridiella callista. A. Overview of an attached appressorium (App) of A. callista located on the leaf surface of Stachytarpheta mutabilis var. violacea with an initial stage of a penetration pore (Po), appressoria wall (Aw), host cuticle (HC) and host cell wall (CW). B. Detail from A showing the interaction apparatus. Note that the Ia consists of a cisterna net (Iac), which is continuous with the cytoplasmic membrane (Cm) of the fungus. Penetration pore (Po), appressoria wall (Aw) and the trunk of the Ia are visible. Note that in direction to the host the trunk is limited by an electron-opaque line (Am). C. Enlargement of an appressorium showing the cisternal net (Iac) of the interaction apparatus. Note that numerous mitochondria (Mi) are located in the vicinity of the Ia. The appressoria wall is visible at Aw. D. JUSTAVINO ET AL.: ASTERIDIELLA CALLISTA 219 FIG. 3. Penetration of the host epidermal cell wall. A. Attached appressorium (App) showing the complex cisterna net (Iac), the apoplastic trunk (Iat) and the penetration pore (Po) of the Ia. The electron-opaque