Anales de Biología 33: 127-134, 2011 ARTICLE Morphology, anatomy and mycorrhizae in subterranean parts of Zeuxine gracilis (Orchidaceae) Thangavelu Muthukumar1, Eswaranpillai Uma1, Arumugam Karthikeyan2, Kullaiyan Sathiyadash1, Sarah Jaison1, Perumalsamy Priyadharsini1, Ishworani Chongtham3 & Vellaisamy Muniappan1 1 Root and Soil Biology Laboratory, Department of Botany, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India. 2 Institute of Forest Genetics and Tree Breeding, R. S. Puram, Coimbatore 641 002, Tamil Nadu, India 3 Department of Life Sciences, Manipur University, Canchipur, Imphal 795 003, India Resumen Correspondence Morfología, anatomía y micorrizas en las partes subterráneas de T. Muthukumar Zeuxine gracilis (Orchidaceae) E-mail: [email protected] Zeuxine gracilis (Berda) Bl. es una orquídea terrestre endémica cuya Received: 27 July 2011 morfología, anatomía y micorrización es desconocida. A partir de plan- Accepted: 23 November 2011 tas colectadas en la región de los Ghats occidentales se investigó: (a) Published on-line: 14 December 2011 anatomía de la raíz y el rizoma; (b) características de los pelos radicu- lares y patrones de colonización de micorrizas. Los caracteres más re- levantes en raices fueron: ausencia de velamen y espirantosomas; exodermis simple y nueve protoxilemas arqueados. Rizoma con epi- dermis uniseriada, abundantes espirantosomas en células corticales internas, endodermis con bandas de Caspary y paquetes vasculares biseriados. Se descubrió la presencia de hongos micorrícicos tanto en las raíces como en los rizomas. Su entrada es principalmente a través de pelos radiculares y epidermis del rizoma. Los hongos forman pelo- tones y células monilioides en el cótex radicular. Ocasionalmente apa- recieron micorrízas arbusculares (AM), caracterizadas por hifas sifona- les, vesículas y esporas. La falta de arbúsculos en Z.gracilis indica que AM no son funcionales. Palabras clave: Hongos micorrícicos arbusculares, Células monilioides, Pelotones, Rizoma, Raíz. Abstract Zeuxine gracilis (Berda) Bl., is an endemic, terrestrial green orchid whose morphology, anatomy and mycorrhizal status is unknown. So we investigated: (a) root and rhizome anatomy; (b) root hair character- istics and mycorrhizal colonization patterns in Z. gracilis plants collec- ted from Western Ghats region of southern India. The prominent ana- tomical characters in the roots were: absence of velamen, spirantho- somes, and the presence of single layered exodermis and nine arched protoxylem. The rhizome had an uniseriate epidermis, abundant spir- anthosomes in the inner cortical cells, a distinct endodermis with cas- parian strips and biseriate vascular bundles. The presence of fungi both in the roots and rhizomes was revealed. The entry of fungi was chiefly through root hairs and through epidermis in the rhizome. Fungi formed pelotons and monilioid cells in the root cortex. Additionally, ar- buscular mycorrhizal (AM) fungi characterized by the presence of aseptate hyphae, vesicles and spores were present occasionally in roots. The lack of arbuscules in Z. gracilis indicated the AM to be non functional. Key words: Arbuscular mycorrhizal fungi, Monilioid cells, Pelotons, Rhizome, Root. 128 T. Muthukumar et al. Anales de Biología 33, 2011 Introduction ported AM-like colonization in the terrestrial orchid Corybas macranthus (Hook.f.) Reichb.f., Orchidaceae with around 25,000 to 30,000 species from New Zealand. Raman & Nagarajan (1999) is one of the largest families among flowering surveyed the occurrence of mycorrhizae in six plants. One of the peculiar features of this family epiphytic and five terrestrial orchids of Kodaik- includes the production of dust-like non endo- anal tropical forest of Western Ghats, South India. spermous seeds. Mode of seed germination and They found exclusive occurrence of AM associ- plant development in Orchidaceae is unique ations in all five terrestrial orchids examined. In- among flowering plants, which require association formation on mycorrhizal association in Zeuxine with the mycorrhizal fungi. Orchids have varied is limited. Burgeff (1932) reported mycorrhizal life-forms like epiphytes, lithophytes, or terrestri- association in Zeuxine clandestine Bl., Zeuxine al. Among these varied life-forms terrestrial orch- sp., and Zeuxine purpurascens Bl., and indicated a ids constitute less than one fourth number of the trend from the autotrophic condition to saprophyt- total orchid species. ism among these species which were strongly re- Zeuxine belonging to the subfamily Orch- lated to mycorrhizal dependence. Porter (1942) re- idoideae and tribe Cranichideae is one of the ported mycorrhizal association in Z. strateumat- largest genus among Orchidaceae. Taxa in Zeux- ica growing in Florida, USA. Apart from these ine are widely distributed in Asia with a few in there appear to be no information on mycorrhizal Africa and Australia. In India Zeuxine is represen- status of other species of Zeuxine. ted by 11 species of which Zeuxine gracilis The objective of the present study was to in- (Berda) Bl., and Zeuxine longilabris (Lindl.) vestigate the morphology and anatomy of roots Benth. ex Hook.f., are found in South India and rhizomes of Z. gracilis and also to record the (Hooker 1895). Zeuxine gracilis is a perennial mycorrhizal incidence and morphology. succulent herb found in shaded woodlands, with brown creeping rhizome, ascending to erect aerial Material and methods shoot with petiolate velvety green leaves (Abra- ham & Vatsala 1981). Zeuxine gracilis is endemic Fresh plant materials of Z. gracilis for our to South India. study were obtained from Top Slip, Indira Gandhi Published work on the anatomy of Zeuxine is Wildlife Sanctuary and National Park, Tamil limited. Stern et al. (1993b) studied the anatomy Nadu, India. This site lies in the Western Ghats of Zeuxine oblonga R. Rogers & C. White and south of the Palaghat Gap, known as Anaimalais Zeuxine strateumatica (L.) Schltr., while examin- with an area of 958 sq. km. The area lies between ing the vegetative anatomy and systematics of the 10.13’-10.33’N and 76.49’-77.21’E, 800 m a.s.l. subfamily Spiranthoideae in which the tribe Rainfall varies between 800-4500 mm. Cranichideae was included at that time. These au- Fresh plant materials of five individuals col- thors found the vascular bundles in Z. oblonga lected during May 2011, were preserved in FAA and Z. strateumatica arranged in one and two (9 parts 70% ethanol, 0.5 parts formalin and 0.5 series respectively and the absence of stored parts glacial acetic acid) for 24h and stored in starch in the former. In this study, Stern et al. 70% ethanol before sectioning. Transverse sec- (1993b) also reported ‘spiranthosomes’, a special- tions of roots and rhizomes were prepared by free ized type of amyloplast in many species of hand sections and stained with safranin or trypan Cranichideae including Z. strateumatica. blue. Observations were made using an Olympus Under natural conditions orchids are associ- BX-51 microscope and images were recorded us- ated with mycorrhizal fungi belonging to basidio- ing Proges 2 camera. mycota and ascomycota (Elena et al. 2010). The For mycorrhizal assessment the roots and fungal hyphae invade the cortical cells and form rhizomes were cut into 1-cm long bits, cleared in tightly interwoven coils called pelotons character- 2.5% aqueous potassium hydroxide at 90 ºC for istic of orchid mycorrhizae (Smith & Read 2008). 45 min., washed in running water, acidified with 5 Reports in literature also indicate the association N HCl and stained with 0.05% trypan blue in of arbuscular mycorrhizal (AM) fungi (Glomero- lactoglycerol. The extent of mycorrhizal coloniza- mycota) with terrestrial orchids. Hall (1976) re- Anales de Biología 33, 2011 Zeuxine gracilis: anatomy of mycorrhizal subterranean parts 129 tion was estimated according to magnified inter- chymatous, cells rounded with small triangular in- section method (McGonigle et al. 1990). Pelotons tercellular spaces. Cells of the inner cortical layer were considered intact if the constituting hyphae contain abundant spiranthosomes (Fig. 1E). Endo- were distinguishable and considered degenerating dermis uniseriate, cells tangentially flattened with if the fungal hyphae could not be clearly distin- casparian strips (Fig. 1F). Vascular cylinder 13 guished or found as an amorphous mass. Twenty arched with xylem elements arranged in two con- 1-cm long root bits were floated in water on a centric rings. Vascular tissue embedded in the par- glass slide to measure root thickness and to count enchyma. Pith cells: parenchymatous, thin walled, root hairs. Length and width of 50 root hairs, and polygonal, with small intercellular spaces. fungal variables (pelotons, monilioid cells, and in- tracellular hyphae) were measured using an ocular Mycorrhizal association micrometer. We measured only 10 to 12 AM Mycorrhizal colonization occurred uniformly fungal vesicles and spores due to their infrequent throughout the cortex in roots and rhizomes (Fig. occurrence. Measurements were presented as 2A). Fungal hyphae entered the roots by penetrat- (minimum value-) mean ±S.E. (-maximum value). ing root hairs or the rhizodermal cells in roots and rhizomes (Fig. 2B). Although single fungal hypha Results entered each root hair, occasionally more than one could be seen in a root hair (Fig. 2B). Fungal Root entry into roots or rhizomes was not characterized by the presence of vesicular or appresorial struc- Roots arise from the nodal region, off-white, ture.