Ann. Bot. Fennici 45: 281–289 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 29 August 2008 © Finnish Zoological and Botanical Publishing Board 2008 Mycorrhizas of Cephalanthera longifolia and Dactylorhiza majalis, two terrestrial orchids Aleš Látr1,*, Martina Čuříková1, Milan Baláž2 & Jaroslav Jurčák1 1) Department of Botany, Faculty of Science, Palacky University, Slechtitelu 11, CZ-783 71 Olomouc-Holice, Czech Republic (*corresponding author’s e-mail: [email protected]) 2) Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlarska 2, CZ- 611 37 Brno, Czech Republic Received 2 July 2007, revised version received 14 Sep. 2007, accepted 18 Sep. 2007 Látr, A., Čuříková, M. , Baláž, M. & Jurčák, J. 2008: Mycorrhizas of Cephalanthera longifolia and Dactylorhiza majalis, two terrestrial orchids. — Ann. Bot. Fennici 45: 281–289. Dynamics of mycorrhizal colonization of two orchid species, rhizomatous Cephalan- thera longifolia and tuberous Dactylorhiza majalis, was studied during two growing seasons, with special emphasis on the occurrence of collapsed pelotons. No complete lysis of pelotons in digestion cells was found. Mycorrhizal colonization of subterranean organs of both species was observed to take place even before aerial shoots developed, indicating that colonization is not restricted to leafy season, and was constantly present throughout the growing seasons with no distinct coincidence with flowering or fruiting time. Mycorrhizal colonization of C. longifolia was patchy and of low degree (mean = 4.4%, SE = 0.3%). It occurred mostly in the distal parts of main roots, lateral roots and/or in isolated root parts. Intensity of mycorrhizal colonization of D. majalis was generally higher (mean = 12.1%, SE = 0.7%). Tubers lacked colonization except in the root-like extensions. Key words: Cephalanthera, colonization, Dactylorhiza, peloton lysis, orchid mycor- rhiza, symbiosis Introduction of an orchid seed into a protocorm and then into a young plantlet, because orchid seeds pos- Regular occurrence of mycorrhizal fungi within sess only very limited reserves (Peterson et al. orchid tissues has been well documented for 1998). The fungus obtains carbon compounds over a century (Bernard 1909, Burgeff 1909). either saprotrophically (Hadley & Perombelon However, until now we have insufficient infor- 1963, Hadley 1969, Midgley et al. 2006) or from mation about the roles that mycorrhizas play ectomycorrhizal symbiosis with trees, which in in compound exchange between the host and some cases it is also able to form (Zelmer & its symbiotic fungus. It is widely accepted that Currah 1995, McKendrick et al. 2000, Gebauer during early developmental stages of all orchids & Meyer 2003). This kind of plant nourish- the fungus supplies carbon compounds. This is ment, known as myco-heterotrophy, persists in a prerequisite for the successful development some orchid species lacking chlorophyll in the 282 Látr et al • ANN. BOT. FENNICI Vol. 45 adult stage, e.g. Cephalanthera austinae, which rence of collapsed pelotons. We tested the pos- receives nutrients required for growth and devel- sibility that different patterns of mycorrhizal opment from its mycobionts belonging to Thel- colonization occur during flowering and/or seed ephoraceae (Taylor & Bruns 1997). Most adult maturation and during the rest of the growing orchid species, however, are capable of pho- season. Root phenology of the species was also tosynthesis. There is some evidence that upon recorded. these conditions mycorrhizal fungi and green orchids are mutually independent as regards their carbon demand (Hadley & Purves 1974, Smith Material and methods 1967, Cameron et al. 2006) and that the main function of mycorrhizal symbiosis is to enhance This study was carried out on plants from two the mineral nutrition of the host plants (mainly localities near Vsetin, in the Zlin region of of nitrogen and phosphorus) (Alexander et al. the Czech Republic, from April 2004 to Sep- 1984, Smith & Read 1997, Cameron et al. 2007), tember 2005. The investigated population of a situation well known in other mycorrhizal Cephalanthera longifolia, with approximately types, e.g. arbuscular, ecto- or ericoid mycor- ten plants per m–2 occurs in a Fagus sylvat- rhizas. Despite this experimental evidence, some ica forest (49°21´N, 18°02´E; elevation 480 m authors have speculated that even in these cir- above sea level), where Carex pilosa is abundant cumstances the fungus might supply the orchid and two other orchid species, Neottia nidus-avis with carbohydrates, which can supplement the and Orchis mascula subsp. signifera are also photosynthetically fixed carbon. The possibil- relatively abundant. The population of Dacty- ity that the green orchid can supply associated lorhiza majalis with a usual abundance of two mycorrhizal fungus with its own carbon com- or three plants per m–2 grows in a damp meadow pounds was often discussed and in some cases (49°23´N, 18°01´E; elevation 530 m above sea also experimentally studied, for a long time level) together with other orchid species such as without success. Cameron et al. (2006) were Listera ovata, Platanthera bifolia and Gymnad- the first to show that an orchid could supply the enia conopsea subsp. conopsea. The meadow is mycorrhizal fungus with a measurable amount of regularly mowed and/or grazed by cattle in June photosynthetically fixed carbon. and July. Although a considerable amount of informa- Both C. longifolia and D. majalis are tion has already been published on orchid myco- summer-green species that produce green leafy rrhizas, there is little evidence for a possibility shoots and inflorescences in spring and overwin- that hyphal pelotons may completely disappear ter underground, either as a tuber (D. majalis) or during their disintegration within host cells. as a rhizome with perennial roots (C. longifolia). Rasmussen and Whigham (2002) first observed Both species flower in May and June. this phenomenon in Corallorhiza odontorhiza, Owing to the protected status of the two a chlorophyll-deficient, presumably fully myco- species, a limited number of plants were har- heterotrophic species, as well as in the summer- vested in accordance to a permit issued by the green orchid Galearis spectabilis, which at least Regional authority of the Zlin region (KUZL supplements its myco-heterotrophic nutrition 5898/2003 and KUZL 5947/2003). From two with photosynthetic CO2 assimilation. If true, to eight mature specimens of C. longifolia were a complete lysis of the pelotons is essential collected per sampling (May to September/Octo- for proper understanding of the functioning of ber). Of D. majalis, two specimens were sampled orchid mycorrhizal symbiosis, especially regard- once a week from the beginning of the growing ing the myco-heterotrophic nutrition of orchids. period until the aerial shoots were visible (May For this reason we studied in detail the dynamics to June–July). Due to the inaccessibility of the of the mycorrhizal colonization in two summer- localities, no specimens were harvested during green orchid species, Cephalanthera longifolia the winter. and Dactylorhiza majalis, during two growing Mycorrhizal colonization was evaluated on seasons, with special emphasis on the occur- sections of all roots (D. majalis, C. longifolia), ANN. BOT. FENNICI Vol. 45 • Mycorrhizas of Cephalanthera longifolia and Dactylorhiza majalis 283 tubers (D. majalis) and rhizomes (C. longifo- roots of C. longifolia, and colonization between lia). Underground organs of each plant were adventitious roots and root-like extensions of carefully removed from the soil, washed in tap D. majalis. Analyses were performed using a water, fixed in FAA for 48 hours and transferred NCSS software package. Counts of digestion to a mixture of glycerol and 90% ethanol (1:1, cells in separate root zones were analyzed using vol/vol). Transverse sections were made either hierarchical ANOVA, where the random effect using a manually operated microtome or by free plant (assigning roots to individual plants in hand. The sections were stained with an aqueous every harvest) was tested within the fixed factor safranin and phluoroglucinol–HCl solution. Part harvest. When the overall ANOVA table showed of our study was made on unstained transverse the effect harvest to be significant, then pairwise sections. Transverse sections of rhizomes and comparisons between means were made using roots except lateral ones were taken from three a Scheffé test. Calculations were done using a zones: apical (5–11 mm behind the root/rhizome Statistica 6 software package. Values are given tip, zone A), middle (in the middle of the root/ as means ± 1 SE. rhizome, zone B) and basal (zone C, 5–11 mm from the rhizome/tuber in the roots and from the rhizome end in the rhizome itself). In tubers Results proper and lateral roots of C. longifolia, sec- tions were made approximately in the middle Cephalanthera longifolia of the tuber/root length. One thin and com- plete section from each zone was then randomly Adventitious roots of C. longifolia showed picked for analysis. The sections were mounted patchy and very low overall degree of myco- in distilled water or glycerol and immediately rrhizal colonization: 4.5% ± 0.5% (n = 26) in observed using an Olympus BX-40 compound 2004 and 4.3% ± 0.3% (n = 26) in 2005 (Table microscope. 1), concentrated in the distal parts of the long According to Jurčák (2003), degree of myco- roots, lateral roots and/or in short isolated root rrhizal colonization was