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Mycorrhizal Fungi in Aerial and Terrestrial Roots of an Epiphytic and Two Terrestrial Species of Orchidaceae Deren A. R. Eaton University of Minnesota, Twin Cities, Department of Plant Biology ABSTRACT: Very little is known about the mycorrhizal relatio nships of Costa Rican orchids, and even less is known about differences in the presence of mycorrhizae between aerial and substrate roots. All orchids require mycorrhizal symbionts during their germination stage, but few studies have examined mycorrhizal r elationships in adult orchids. Aerial and substrate roots from three closely related species of Orchidaceae (two terrestrial and one epiphytic) were collected and examined for the presence of mycorrhizal fungi. Mycorrhizal fungi were found in 45 of 51 individuals. The epiphytic species exhibited a much lower occurrence of mycorrhizae in its aerial roots than the two terrestrial species did. This may be attributed to the effects of nutrient limitations of the different habitats. The terrestrial speci es showed a much more variable occurrence of mycorrhizae in both their aerial and substrate roots. This may be a result of a changing frequency of infection of mycorrhizae at different growth stages of the orchid, which result from different nutrient avai lability and the costs imposed on the orchids by the mycorrhizae. RESUMEN: Se sabe muy poco acerca de las relaciones micorrizales de las orqudeas de Costa Rica, y se sabe todava menos acerca de las diferencias en la presencia de micorrizas entre las ra ces areas y terrestres. Todas las orqudeas requieren simbiontes micorrizales durante su etapa de germinacin, pero pocos estudios han examinado las relaciones micorrizales en orqudeas adultas. Se colectaron las races areas y terrestres de tres espe cies de orqudeas estrechamente relacionadas (Dos terrestres y una epiftica) y se determin la presencia del hongo micorrizal en 45 de 51 individuos. La especie epiftica mostr una ocurrencia de micorrizas ms baja en sus races areas que las dos especi es terrestres; esto puede ser atribuido al efecto del limitacin de nutrientes de los diferentes habitats. Las especies terrestres mostraron una ocurrencia de micorrizas mucho ms variada tanto en sus races areas como en sus races de sustrato. Esto pu ede ser el resultado de una frecuencia cambiante de la infeccin de micorrizas en los diferentes estados de crecimiento de la orqudea de vido a la diversa disponibilidad de alimento y los costos impuestos en la orqudea por las micorrizas.
INTR ODUCTION: vascular plants (Dressler 1990). The presence of mycorrhizal fungi with germinating Orchidaceae seedlings has been extensively studied and well documented. Ho wever, the occurrence of mycorrhizal fungi in adult orchids has been a subject of considerably less study (Smith 1997), and virtually no work has yet been reported on the mycorrhizal relationships of Costa Rican orchids, especially in aerial versus substra te roots (Walter 1983). All orchids require a mycorrhizal partner during germination because they are yet unable to photosynthesize and the seed alone is too small to provide sufficient nutrients, particularly phosphorus and carbohydrates (Dressler 1990 Rasmussen 2001). The symbionts most often are Rhizoctonia like fungi, and are always either vesicular arbuscular mycorrhiza (VAM) or another type of endo mycorrhiza termed orchid mycorrhiza (OM) (Otero 2002). Dependency of a plant on its mycorrhizal f ungi partner indisputably changes over the development of the plant; in most orchids a decrease occurs after the germination stage. Many species retain mycorrhizal relationships even after becoming photosynthetic, although the plants are not dependent on it (Walter 1983). The factors affecting whether an orchid will retain or reject mycorrhizal fungi require further study. Orchid mycorrhizae are different from other mycorrhizal associations in many respects. The exact nature of the relationship is not fully understood, but attempts to label it as a mutualism are often disputed. Orchids are capable of parasitizing fungi, and fungi are capable of parasitizing orchids in return (Smith & Read 1997). Orchids are often capable of rejecting a fungus if it i s no longer needed, or if it becomes harmful. Hadley (1970) described that the rejection of the fungus by the orchid is accompanied by thickening of the cell walls and producing phenolics. Therefore it is expected that orchids dispose of their mycorrhiza e if they are not necessary. Little is known about what controls the balance between the two organisms, but several studies have shown that nutrient availability plays a role (Rasmussen 2002). Several features of orchids affect their ability to attain nu trients. Orchids occur on a wide variety of substrates and can be epiphytic or terrestrial, although most are epiphytic (Otero 2002). Orchids are monocots and do not have taproots or primary roots like those of dicots. Their entire root system is made up of secondary roots which arise from the stem (Dressler 1990). Their roots contain a special epidermal layer made up of dead cells termed the velamen. This spongy sheath around the root acts to help absorb water and nutrients, especially useful in epiphy tes for use in absorbing the nutrient rich water from the canopy after a rainfall. Orchids have two types of roots which are recognizably different in appearance: ( a ) the first type are substrate roots, which usually extend from the base of the shoot to e ither enter the soil or attach to a substrate; and ( b ) aerial roots, which extend into the air (Walter 1983). Both types of roots function in nutrient and water collection. Aerial roots collect most nutrients from rain or mist water, and sometimes attach to surfaces for stability or climbing. Also, they usually have a more conspicuous velamen, and can be photosynthetic (Dressler 1990). VAM are particularly abundant in nutrient deficient environments. An epiphytic environment is a good example; very sma ll amounts of phosphorous can be absorbed in the canopy of the forest. Therefore it is hypothesized that mycorrhizal relationships
should be an important adaptation for epiphytic orchids (Lesica & Antibus 1990). However, water leached through the canopy can contain N, P, and K concentrations higher than in terrestrial soils (Benzing 1995). Other studies have shown that epiphytic orchids generally have lower intensity of mycorrhizal infection, compared to terrestrial species (Hadley & Williamson 1972). I n a study by Goh et al. (1992), they found that the aerial roots in epiphytic species generally have very little infection, where as roots in contact with a substrate have a high intensity of infection. The purpose of this study was to investigate the p resence of mycorrhizal fungi in the aerial and substrate roots of tropical epiphytic and terrestrial species of Orchidaceae. This study provided the first comparison of aerial and substrate roots for Costa Rican orchids. By focusing on related species th at occur in different habitats, the role of environmental factors was simultaneously investigated. MATERIALS AND METHODS: Orchid root samples were collected between Oct. 22 and Nov. 16, 2005 in Monteverde, Puntarenas, Costa Rica, from the two closely rel ated genera, Epidendrum and Oerstedella Two terrestrial species were studied: Epidendrum radicans and Oerstedella exasperata. Epidendrum radicans individuals were encountered along embankments on the road between Monteverde and San Luis, from 1200 1400 meters, where they often formed dense branching mats. Oerstedella exasperata individuals were collected along the towers road between 1600 1800 meters. Also collected was an epiphytic species, Oerstedella centropetala found commonly in Monteverde betwee n 1400 1580 meters. Many O. centropetala were collected on trees close to the main road in Monteverde near the Estacin Biologca de Monteverde. One hundred and two samples were collected. Each sample consists of two to three 3 cm long root segments fro m either aerial or substrate roots. Samples were collected in pairs from the same plant, consisting of an aerial and a substrate root sample. For terrestrial species, substrate roots were collected by both digging dirt out from around the root, and pulli ng a root out of the soil. Samples were collected from different sections of roots. In epiphytic samples, substrate root segments were removed from the host substrate, either soil or tree trunk. The aerial roots were sometimes difficult to locate, becau se many plants did not have them. Aerial roots were always less than three centimeters in length so the entire root was collected. Aerial roots that were attached to a substrate were not sampled. Once collected, the samples were placed in vials contai ning 10% KOH for approximately 24 hours, which acted to clear the roots. Root segments were then placed into test tubes containing H 2 O 2 for one hour to remove pigments, then transferred to test tubes containing 1% HCL for 20 minutes. Lastly the roots wer e transferred to test tubes containing a 4:2:1 solution of 50% glycerol: 1%HCL: 0.05% trypan blue dye for one hour to stain. After they were stained, the roots were viewed under a compound microscope at 40X and 100X, and the presence or absence of myc orrhizal fungi was noted. A contingency table was then constructed to find how many individuals of each species showed the following patterns: ( a )mycorrhizal fungi present in both its aerial and substrate roots (b) mycorrhizal fungi in its aerial roots bu t not in substrate roots ( c )
mycorrhizal fungi in its substrate roots but not aerial roots or ( d ) no presence of mycorrhizal fungi. RESULTS: Of the 51 orchid individuals examined, 45 showed the presence of mycorrhizae in either the aerial or substrate roots. In the epiphytic species O. centropetala, mycorrhizal fungi were found in 19 of 20 individuals; in the terrestrial species E radicans they were present in 15 of 20; and in O. exasperata ten of 11 had mycorrhizae. The frequency of mycorrhizal infe ction between the aerial and substrate roots differed among the three species (Table 1). The differences between the patterns in which the mycorrhizae were found in each species (e.g. present in aerial roots but not in substrate, or present in both) were tested in chi square tests for independence between two or more samples. The data for E. radicans were not statistically significant (X 2 = 0.303; P = 0.5820; N = 20), nor were they significant for O. centropetala or O. exasperata. For O. centropetala and O. exasperata this may be attributed to low sample sizes. To account for this, since the presence of mycorrhiza were almost ubiquitous in the substrate roots, the substrate condition was ignored, and a chi square test using a compressed table of results was used to examine the presence or absence in aerial roots. Oerstedella centropetala showed presence of mycorrhizal fungi in the roots of four individuals and no presence in 16, showing a significant statistical difference from expected values (X 2 = 6.1; P < 0.05, df = 1; N = 20). Oerstedella exasperata was also statistically significant (X 2 = 7.26; P < 0.05; df = 1; N = 11). DISCUSSION: The purpose of this study was to examine the difference in the presence or absence of mycorrhizae between the a erial and substrate roots of tropical epiphytic and terrestrial species of Orchidaceae, and also to make hypotheses as to what factors affect the frequency in distribution of the mycorrhizae between the different types of roots. The results differed for all three species examined. The general trend for O. exasperata seemed to point towards a common occurrence of mycorrhizal fungi in both the aerial and substrate roots. However, sufficient data were not collected to compare it to the other orchid species Mycorrhizal fungi were more common in the epiphytic species O. centropetala than in the terrestrial species. This was due to the very high presence of mycorrhizae in the substrate roots of O. centropetala since they showed very little presence of myco rrhizal fungi in their aerial roots. This leads to two questions: why did the epiphytic species have a lower presence of mycorrhizal fungi in their aerial roots; and why are mycorrhizal fungi distributed more evenly between the root types of the terrestri al species? There are several ways in which abiotic factors can affect orchids need for mycorrhizal symbionts in either their aerial or substrate roots. Nutrient limitation seems to be an important factor. Phosphorus is a limiting nutrient in tropi cal ecosystems. Soils contain very low levels of P and plants need to acquire many nutrients through rain or mist water. In Monteverde, the ion deposition of PO 4 3 P is 0.07 kg/ha/yr in bulk cloud water and precipitation, and 0.48 kg/ha/yr in through fall water, a much higher value, caused by leaching in the canopy (Nadkarni et al. 2000). One hypothesis to explain the
low frequency of mycorrhizae in the aerial roots of O. centropetala, is that differences in P between mist or rain water and water that is leached through the canopy can result in different levels of mycorrhizal infection in the aerial roots of orchids. The terrestrial species O. exasperata and E. radicans were both found and collected on roadsides in degraded, very clay rich soils with no c anopy cover. However, individuals of the epiphytic species O. centropetala were encountered on the base of trees usually under crown cover. A study using orchids found that with increased amounts of N and P levels, frequency of mycorrhizal fungi decrea se. In one study, after fertilizing an area with P, mycorrhizal abundance was found to decrease 32% (Treseder 2004). It has been hypothesized that this is a result of the orchid rejecting the fungus because it has a decreased need for it. The aerial roo ts in O. centropetala receive much higher levels of P than the terrestrial species; therefore, it is possible that they have less need for mycorrhizal fungi in their aerial roots. It is advantageous for them to dispose of fungi because they could they are not limited in P, and the fungus may impose a cost to the orchid. Conversely, the terrestrial species are much more limited in P and require mycorrhizal fungi in their aerial roots to collect as much as possible. The nutrient limitation hypothesis can e xplain why there is a higher frequency of mycorrhizal fungi in the aerial roots of terrestrial species than in epiphytic species, however, it does not explain why E. radicans was found to exhibit all four patterns of distribution relatively equally. To ex plain the high variability in occurrence of mycorrhizae between the aerial and terrestrial roots of E. radicans, a hypothesis was formed: E. radicans individuals can change frequency of infection often throughout their lives, and they have several stages a t which they attain, keep, or dispose of mycorrhizae. Epidendrum radicans differed from the other two species collected in that it forms dense branching mats, in which many shoots arise from one individual. It is hypothesized that one function of the a erial roots in E. radicans is for new shoots to reach non colonized areas and attach to the soil. When an aerial root enters the soil, it branches and forms terrestrial roots (Goh et al. 1983). This was observed. The four patterns of mycorrhizal distrib ution can each be matched to a pattern of growth and mycorrhizal infection in the life of an E. radicans individual (Figure 2). As stated earlier, in the germinating stage, mycorrhizal fungi are always present in the substrate roots. No samples were coll ected from this stage because aerial roots were not present. As the orchid becomes a photosynthetic juvenile, the fungal symbionts are no longer obligate, and the orchid can either retain or reject the fungus depending on nutrient availability and whether the OM are enforcing a cost on the orchid. Next the orchid becomes an adult and forms aerial roots; again, the orchid can either acquire or get rid of mycorrhizae in these roots. As the adult orchid creates new shoots, and the shoots grow long enough to reach the ground, the aerial roots will enter into the soil and become substrate roots. These roots can also accept a mycorrhizal symbiont or not. The nutrient availability of the habitat in combination with the costs imposed on the orchid by the rela tionship with the fungi, can both affect the frequency of distribution of the mycorrhizae between the two root types of E. radicans. The fact that E. radicans was found to show all patterns relatively equally, probably means that it experiences lots of v ariation in both of these parameters. This leads to high variation within and across individuals.
Differences were observed in the presence of mycorrhizal fungi between epiphytic and terrestrial species, and also between the aerial and substrate roots. This study provided new information into an area of little previous research. Future research can be done utilizing larger sample sizes, and more species of orchids to see if the trends found in this study are consistent across other genera and species o f orchids, or if they were specific to the species that were studied. ACKNOWLEDGMENTS: I thank Karen Masters for helping me to design methods, find research materials, and for endless advice. I also thank Alan Masters for added help, support, and hugs I would like to thank the family of Cristina Obando Leitn for putting up with me coming home late for dinner every night and never complaining, or at least not complaining slow enough for me to understand. I would also like to thank my own family, for love and support, and for whom without I would never have had a chance to study tropical orchids in Costa Rica. LITERATURE CITED: Benzing, D.H., 1995. Vascular Epiphytes In: Forest Canopies Lowman, M.D. and N.M. Nadkarni, ed. The Academic Pre ss, San Diego, CA, pp. 233. Dressler, R. L., 1990. The Orchids: Natural History and Classification : Harvard University Press. Goh, C. J., Sim, A. A. and Lim G., 1992. Mycorrhizal associations insome tropical orchids Lindleyana 7, 13 17. Hadley, G. and Williamson, B., 1972. Features of mycorrhizal infection in some Malayan orchids. New Phytologist 71: 1111 1118. Hadley, G. 1970. Non specificity of symbiotic infection in orchid mycorrhiza New Phytologist 69, 1015 1023. Lesica, P. and R. K. Ant ibus., 1990. The Occurrence of Mycorrhiza in Vascular Epiphytes of Two Costa Rican Rain Forests Biotropica 22(3): 250 258. Nadkarni, N. M., Lawton, R. O., Clark, K. L., Matelson, T. J., and D. Schaefer, 2000. Ecosystem Ecology and Forest Dynamics In: Monteverde: Ecology and Conservation of a Tropical Cloud Forest Nadkarni, N. M. and N. T. Wheelwright, ed. Oxford University Press, New York, NY, pp. 324 328. Otero, J. T., Ackerman, J. D. & Bayman, P., 2002. Diversity and host specificity of end ophytic Rhizoctonia like fungi from tropical orchids Am. J. Bot. 89 : 1852 1858. Rasmussen, H. N., 2002. Recent developments in the study of orchid mycorrhiza. Plant and Soil 244: 149 163, 2002. Row, A.R. and Pringle, A. 2005. Morphological and Molecu lar Evidence of Arbuscular Mycorrhizal Fungal Associations in Costa Rican Epiphytic Bromeliads Biotropica 27(2): 245 250 Smith, S.E. and Read D.J., 1997. Mycorrhizal Symbiosis San Diego, CA.: Academic Press Inc; 13:370 375. Treseder, K. K., 2004. A meta analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO 2 in field studies New Phytologist 164(2): 347. Walter, K.S. 1983. Orchidaceae. In: Costa Rican Natural History D.H. Janzen, ed. The University of Chicago Press, Chic ago, IL, pp. 282 285. Weller, S. Y., 2002. Abundance of Vesicular Arbuscular Mycorrhiza in Adult Epiphytic Orchidaceae Between Two Canopy Substrates. UCEAP Spring.
TABLES: TABLE 1: Contingency tables for three species of Orchidaceae, showing the number of individuals in each species that exhibited different patterns of infection by mycorrhizae on aerial and substrate roots. (a) Oerstedella centropetala, (b) Epidendrum radicans, (c) Oerstedella exasperata (a) O. centropetala Aerial roots present absent Substrate roots present 4 15 absent 0 1 (b) O. exasperata Aerial roots present absent Substrate roots present 9 1 absent 1 0 (c) E. radicans Aerial roots present absent Su bstrate roots present 6 6 absent 3 5
FIGURES: Figure 1: Presence or absence of mycorrhizae in the aerial roots of two orchids in the genus Oerstedella.
Figure 2: The four possible patterns of mycorrhizal distribution can each be matched to a pattern of growth and mycorrhizal infection in the life of an E. radicans individual. Epidendrum radicans individuals can change frequency of infection often throughout their lives, and they ha ve several stages at which they attain, keep, or dispose of mycorrhizae. (+) = mycorrhizae present; ( ) = mycorrhizae absent.
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Eaton, Derren A. R.
Los hongos micorrizas en las races de las epfitas areas y terrestres y dos especies terrestres de Orchidaceae
Mycorrhizal fungi in aerial and terrestrial roots of an epiphytic and two terrestrial species of Orchidaceae
Digitized by MVI
Very little is known about the mycorrhizal relationships of Costa Rican orchids, and even less is known about differences in the presence of mycorrhizae between aerial and substrate roots. All orchids require mycorrhizal symbionts during their germination stage, but few studies have examined mycorrhizal relationships in adult orchids. Aerial and substrate roots from three closely related species of Orchidaceae (two terrestrial and one epiphytic) were collected and examined for the presence of mycorrhizal fungi. Mycorrhizal fungi were found in 45 of 51 individuals. The epiphytic species exhibited a much lower occurrence of mycorrhizae in its aerial roots than the two terrestrial species did. This may be attributed to the effects of nutrient limitations of the different habitats. The terrestrial species showed a much more variable occurrence of mycorrhizae in both their aerial and substrate roots. This may be a result of a changing frequency of infection of mycorrhizae at different growth stages of the orchid, which result from different nutrient availability and the costs imposed on the orchids by the mycorrhizae.
Se sabe muy poco acerca de las relaciones micorrizales de las orqudeas de Costa Rica, y se sabe todava menos acerca de las diferencias en la presencia de micorrizas entre las races areas y terrestres. Todas las orqudeas requieren simbiontes micorrizales durante su etapa de germinacin, pero pocos estudios han examinado las relaciones micorrizales en orqudeas adultas. Se colectaron las races areas y terrestres de tres especies de orqudeas estrechamente relacionadas (Dos terrestres y una epfita) y se determin la presencia del hongo micorriza en 45 de los 51 individuos. La especie epfita mostr una ocurrencia de micorrizas ms baja en sus races areas que las dos especies terrestres; esto puede ser atribuido al efecto de la limitacin de nutrientes de los diferentes hbitats. Las especies terrestres mostraron una ocurrencia de micorrizas mucho ms variada tanto en sus races areas como en sus races de sustrato. Esto puede ser el resultado de una frecuencia cambiante de la infeccin de micorrizas en los diferentes estados de crecimiento de la orqudea de vida a la diversa disponibilidad de alimento y los costos impuestos en la orqudea por las micorrizas.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone--Monteverde
Monteverde Biological Station (Costa Rica)
Costa Rica--Puntarenas--Zona de Monteverde--Monteverde
Estacin Biolgica de Monteverde (Costa Rica)
Tropical Ecology Fall 2005
Ecologa Tropical Otoo 2005
t Monteverde Institute : Tropical Ecology