Climate Change and Uredinales (Rust Fungi) on Pleurothallid Orchids in Monteverde Lindsey Parsons Department of Biology, Berea College ABSTRACT The purpose of this experiment was to determine if the decrease in mist frequency observed in Monteverde, has an effect on Uredinales (rust fungi) infections in Pleurothallis aristata and Pleurothallis segoviensis orchids and to determine if the infection in P. segoviensis contributed to the rate of herbivory and number of flowers the orchid produced. In mist exp eriments infected samples of both species of orchids were placed in either a misted and control condition and observing the change in infection rates over a 20 day period. Under natural conditions, number of leaves, leaves with infection, leaves with herb ivory, and number of flowers were counted on samples of P. segoviensis to determine if there were any significant interactions among these factors. Results reveal that additional mist does have an effect on rust fungus infection rates; with a decrease in mist, rust fungus infection rates increased. Infection rates also cause higher rates of herbivory and lower rates of flower production. Thirty eight species of Pleurothallid orchids were found in the short twenty day study in the forest near the EstaciÃ³n BiolÃ³gica de Monteverde and in the JardÃn de OrquÃdeas. Monteverde, famous for Pleurothallid orchids is experiencing a decrease in mist frequency, which puts the orchids here in a dangerous situation when dealing with rusts. RESUMEN El propÃ³sito de est e experimento fue determinar si el cambio del clima, a saber, la disminuciÃ³n de la frecuencia de la niebla, tenÃa un efecto en las infecciones de Uredinales (Hongos de herrumbre) en las orquÃdeas de la familia Pleurothallid Pleurothallis aristata y Pleurot hallis segoviensis . TambiÃn se determinÃ³ si la infecciÃ³n en P. segoviensis contribuyÃ³ al Ãndice de herbivorÃa y al nÃºmero de flores que la orquÃdea produjo. Los mÃ©todos para las muestras incluyeron experimentos de la niebla en ambas especies de orquÃdeas que fueron colocadas en condiciones de niebla y de control y observaciones del cambios en Ãndices de infecciÃ³n. El nÃºmero de hojas, hojas infectadas, hojas con herbivorÃa, y el nÃºmero de flores fue contado en muestras de P. segoviensis. para determinar si habÃan interacciones significativas entre estos factores. Los resultados revelaron que la frecuencia de la niebla tieneun efecto en los Ãndices de infecciÃ³n del hongo de herrumbre cuaudo la frecuencia de al niebla disminuye, los Ãndices de infecciÃ³n de l hongo de herrumbre aumentan. Los Ãndices de infecciÃ³n tambiÃ©n causaron Ãndices mÃ¡s altos de herbivorÃa y Ãndices de producciÃ³n floral menores. Monteverde, famoso por las orquÃdeas de la familia Pleurothallid, estÃ¡ experimentando una disminuciÃ³n en la f recuencia de niebla que ponea las orquÃdeas esta zona en una situaciÃ³n peligrosa en referencia a la infecciÃ³n del hongo de herrumbre. INTRODUCTION Uredinales are one of the largest groups of plant parasitic fungi with an estimated 5,000 species (Alexopo ulous et al. 1996). They usually display high host specificity and use two unrelated host plants during their very complex life cycle (Heath 1997). Rust fungi appear to be obligate biotrophs, meaning they only live on living plants (Wingfield 2003). They gain entry to the host via either growing into stomatal openings or by direct penetration of host epidermal cells (Alexopoulous et. al. 1996). Heath (1997) points out that although these parasites usually do not kill their host plants they can cause damage . Intracellular invasion of the rust hyphae and haustoria has profound effects on the cellular structure and activity of the host plant (Heath 1997). I have observed rust fungus of the genus Trentopohlia on Pleurothallis aristata and Pleurothallis segov iensis at the EstaciÃ³n BiolÃ³gica de Monteverde. This relationship between Pleurothallid orchids and Uredinales is concerning in Monteverde and unfortunately virtually nothing is known about the interaction. This area is abundant with
2 epiphytic plants, in cluding Pleurothallids. The potential threat of rust fungus infections on these organisms could cause much damage to the plants. Along with this threat, Pleurothallid orchids in Monteverde are also experiencing climate changes that are compromising t heir health. The nighttime temperature is increasing and the amount of precipitation is decreasing, including mist (Pounds et al. 1999). This change has had a significant effect on the birds, anurans, and lizards in the region. Anoline lizard and anuran populations, both sensitive to rainfall variability, have declined with the decrease in mist frequency and increased nighttime temperatures (Pounds et al. 1999). It is very possible that this decline is also affecting the flora in Monteverde, specificall y Pleurothallids. Arboreal flora is exceptionally sensitive to climate; they are dependent on the atmosphere for moisture and required ions (Benzing 1998). These climate changes, particularly the decrease in mist frequency, may have an effect on Pleuroth with rust fungus. Additionally, these climate changes may affect the impact rust fungi are having on the overall health of the orchids, specifically the rate of herbivory and numb er of flowers produced. Finally, not knowing what orchids have what rust fungus infections puts scientist at a disadvantage when trying to take action to change the patterns of degradation. I hypothesize that Pleurothallid orchids experiencing decreas ing mist frequency climate changes will have a harder time dealing with rust fungi, as shown by pre verses post climate change rates of infection. I also hypothesize that the percentage of leaves with herbivory will increase with higher rust fungus infec tions. Likewise, the number of flowers produced will decrease with a higher rate of rust infection. METHODS The study consisted of three parts that will be referred to as the (1) mist experiment, (2) rust, herbivory and flower health observation a nd (3) Uredinales on Pleurothallids survey. Mist Experiment The study site for the mist experiment was a small orchid garden close to the EstaciÃ³n BiolÃ³gica de Monteverde. Forty samples of Pleurothallis aristata and 32 samples of Pleurothallis segoviensi s (nomenclature as in Hammel et al. 2003) were obtained and tied with nylon string to long branches for easy observation. All of P. aristata samples had leaves infected with an unidentified rust fungus and about half of P. segoviensis had leaves infected with what was thought to be the same fungus. Each P. aristata was cut back to 40 leaves with a varying number of infected leaves (3 20 with average of about 10). In the same way, each P. segoviensis was cut back to 5 leaves with a varying number of inf ected leaves (0 3). After the orchids were set up, pruned and counted, I spread the rust fungus spores with a paintbrush to the other leaves on the orchids. Twenty of P. aristata were in a misted group (approximately 6ml twice daily) and the other 20 wer e in a control group, receiving no added mist. Likewise, 17 P. segoviensis were treated with mist and 15 as a control. At five day intervals I counted the number of infected leaves and non infected leaves for each plant of each treatment. In order to tra ck the change of infection rate on each P. segoviensis leaf, each leaf was placed into one of five categories describing the amount of infection it was experiencing (1 : none, 2 : small spot, 3 : <50% infected, 4 : >50% infected and 5 : dead). A 2 Way AN OVA was conducted to determine if the number of infected leaves at the end of the experiment depended on the treatment and/or time. Several analyses of Co Variance (ANCOVA) were conducted as well to determine if the number or
3 proportion of infected leaves at the end of the experiment depended on the treatment and/or the number or proportion of infected leaves infected at the beginning of the experiment. For P. segoviensis frequency distributions were created to see if there was a change in infection rates on the individual leaves. Rust, Herbivory, and Flower Health Observation Observations of the relationships between rust, herbivory, and flower health were made for P. segoviensis in the forest and regenerating pasture around the EstaciÃ³n BiolÃ³gica de Monteverde and at a garden in CaÃ±itas. Samples of P. segoviensis were found and the number of leaves, leaves infected with rust fungi, leaves with herbivory, and flowers were recorded. A simple linear regression analysis was conducted to see if the p ercent of infected leaves had an effect on the percent of leaves with herbivory and the percent of flowers. Uredinales on Pleurothallids Survey Finally, the Uredinales on Pleurothallids survey was conducted in the forest and regenerating pastures around the EstaciÃ³n BiolÃ³gica de Monteverde and the JardÃn de OrquÃdeas. The orchid flora was inspected for rust fungi. Leaves that were infected with rust were collected for identification of the orchids. RESULTS Mist Experiment During the study perio d there were two days of precipitation and twice everyday the misted groups were misted. Results for the ANCOVA revealed that in both P. aristata and S, segoviensis the number of infected leaves at interval four significantly depended on the treatment ( P. aristata : p = 0.0049; P. segoviensis : p = 0.0010) and number of infected leaves at interval zero ( P. aristata and P. segoviensis p = <.0001). Similarly, the proportion of infected leaves at interval four significantly depended on the treatment ( P. aris tata : p = 0.0037; P. segoviensis : p = 0.0041) and proportion of infected leaves at interval zero ( P. aristata : p=<.0001; P. segoviensis : p = 0.0008). Finally, the increase of infected leaves from intervals zero to four significantly depended on the tr eatment ( P. aristata: p = 0.0049; P. segoviensis : p = 0.0010) but did not significantly depend on the number of leaves infected at interval zero ( P. aristata : p = 0.4421; P. segoviensis : p = 0.1324). The 2 Way ANOVA showed that the number of infected leaves for both species significantly depended on the treatment ( P. aristata and P. segoviensis p = <.0001) (Fig. 1) and time ( P. aristata: p = <.0001; P. segoviensis : p = 0.0039). The p values for the effect of time on number of infected leaves was typ ically only significant at a ten day interval (Table 1).
4 A B FIGURE 1. Rate of Uredinales (rust fungus) infection in (A) Pleurothallis aristata and (B) Pleurothallis segoviensis over five da y periods in two different treatments misted and control. Number of infected leaves significantly depends on treatment [(A) n = 40 (20 misted; 20 control), p = <.0001] [(B) n = 32 (17 misted; 15 control), p = <.0001] and time [(A) n = 40 (20 misted; 20 co ntrol), p = <.0001] [(B) n = 32 (17 misted; 15 control), p = 0.0039].
5 TABLE 1. with Uredinales (rust fungi) in Pleurothallid orchids (A) Pleurothallis aristata and (B) Pleurothallis seg oviensis. Intervals zero to one and all other intervals are five day periods. S notes significances. Notice that in most cases it takes ten or more days to see a significant effect of time on the infection rates. Week Mean Difference P Value 0 1 0.000 --0 2 .542 .0311 S 0 3 .542 .0311 S 0 4 .671 .0079 S 1 2 .542 .0311 S 1 3 .542 .0311 S 1 4 .671 .0079 S 2 3 0.000 --2 4 .129 .6082 3 4 .129 .6082 B The change in individual leaf rust infection for P. segoviensis was recorded by placing each leaf into a category of infection at intervals 2, 3, and 4. The frequency distributions showed that the frequency of leaves per category did not change over 15 days (Fig. 2). Rust, Herbivory, and Flower Health Observation The regression analysis for the 54 samples of P. segoviensis showed a significant positive relationship between the percent of leaves per plant infected with rust fungus and the percent of leaves per plant with herbivory (R 2 = 0.222, p = 0.0003) (Fig. 3). There was also a significant negative re lationship between the percent of infected leaves per plant and percent flowers produced per plant (R 2 = 0.221, p = 0.0003) (Fig. 3). Uredinales on Pleurothallids Survey The results for the Uredinales on Pleurothallids survey are summarized in Table 2. Week Mean Difference P Value 0 1 .62 5 .5014 0 2 3.875 <.0001 S 0 3 4.575 <.0001 S 0 4 6.175 <.0001 S 1 2 3.250 .0006 S 1 3 3.950 <.0001 S 1 4 5.550 <.0001 S 2 3 .700 .4515 2 4 2.300 .0141 S 3 4 1.600 .0863
6 A B C FIGURE 2. Frequency distribution of Uredinales (rust fungi) infection category in Pleurothallis segoviensis by number of leaves. Categories were (1) no infectio n, (2) small spot, (3) <50% infected, (4) >50% infected, and (5) dead. Graph A is 10 days into the experiment, B 15 days, and C 20 days. The frequency of leaves per category did not change over 15 day period.
7 A B FIGURE 3. Regression plots for P. segoviensis. (A) As percent of leaves infected with Uredinales (rust fungi) increases the percent of leaves with herbivory also increases (R 2 = 0.222, p = 0.0003). (B) As the percent of infected leav es increases the number of flowers produced decreases (R 2 = 0.221, p = 0.0003).
8 TABLE 2. Pleurothallid orchid species found in the wild around the EstaciÃ³n BiolÃ³gica de Monteverde and at JardÃn de Orqu Ãdeas with rust fungus on leaves Genus Specific Epithet Wild JardÃn Epidendrum spp. X Scaphyglottis spp. X Maxillaria spp X Dracula astute X Dracula carlueri X Dracula erthrochaete X Dracula pusilla X Lepanthes barbosae X Lepanthes confus a X Lepanthes gracillima X Lepanthes jimenezii X Lepanthes lindleyana X Lepanthes monteverdensis X Madevalia calura X Pleurothallis amparonana X Pleurothallis barbulata X Pleurothallis bothros X Pleurothallis calyptrostele X Pleurothall is cardiothallis X Pleurothallis cogniauziana X Pleurothallis colossus X Pleurothallis crescentilabia X Pleurothallis dolichopus X Pleurothallis immersa X Pleurothallis leucantha X Pleurothallis pantasmi X Pleurothallis pilostoma X Pleuro thallis pompalis X Pleurothallis quadrifida X Pleurothallis segoviensis X X Pleurothallis segregatifolia X Restrepia muscifera X X Restrepia trichoglossa X X Restrepiopsis tubulosa X Stelis microchila X X Trichosalpinx memor X X Zootrophion en dresianum X
DISCUSSION The hypotheses that I set forth were supported by my data. Decreased mist does have an effect on Uredinales infections on the Pleurothallid orchids P. aristata and P. segoviensis. During the study period there were only two day s of precipitation and the misted groups were misted twice everyday. With decreased rates of mist, the orchids are more vulnerable to rust fungus infections. Additionally, P. segoviensis with rust infections are more susceptible to herbivory and produce f ewer flowers. Over my 15 day experiment period for individual leaves in P. segoviensis , the frequency of leaves per category did not change. It would be interesting to run this experiment for a longer period to see how the leaves changes over time with rust fungus infections. The effect of time on infection rates was one of interest. The fact that it took approximately ten days for an increase of infection to be significant gives insight to the time frame that Pleurothallids are dealing with. This is a relatively short period of time, indicating that Uredinales have the capacity to spread quickly on their host. In conclusion, the health of Pleurothallid orchids in the Monteverde area is in jeopardy. This threat could also be extrapolated to other orchid species. The effects of climate change are having a negative effect on many organisms in the area (Pounds et al. 1999) and these orchids are not excluded. Pleurothallids, as part of the epiphyte flora, promote biodiversity and provide abundant and diverse resources for fauna (e.g., food, shelter, breeding sites, etc.) (Benzing 1998). They are essential for nutrient cycling in the Monteverde cloud forest and scientist do not know exactly what impact there absence would have on the ecosystem. ACKNOWLEDGMENTS Thanks to Karen Maters for her help with the idea for this project and support throughout the process. Thanks to Javer for helping me with my translation. Thanks to InBIO for identifying the genus of my rust fungus. LITERATURE CITED A LEXOPOULOUS , C.J, C.W. M IMS , AND M. B LACKWELL . 1996. Introductory mycology fourth edition. John Wiley & Sons, Inc, New York. B E NZING , D. H. 1998. Vulnerabilities of tropical forest to climate change: the significance of resident epiphytes. Climate Change 39: 519 540. H AMMEL , B. E., G RAYUM , M. H., H ERRERA , C. AND Z AMORA , N. editors. 2003. Manual de Plantas de Costa Rica, Volumen III: MonocotiledÃ³neas: Orchidaceae Zingerberaceae. Missouri Botanical Garden Press, St. Louis. H EATH , M. C. 1997. Signalling between pathogenic rust fungi and resistant or susceptible host plants. Annals of Botany 80: 713 720. P OUNDS , A. J., M. P. L. F OGDEN , J. H. C AMPBELL . 1999. Biological response to climate change on a tropical mountain. Nature 398, 611 615. W INGFIELD , B. D., L. E RICSON , T. S ZARO , J. J. B URDON . 2004. Phylogentic patterns in the Uredinales. Australasian Plant Patho logy 33, 327 335.
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El cambio climtico y Uredinales (hongos de herrumbre) en orquideas Pleurothallid en Monteverde
Climate change and Uredinales (rust fungi) on Pleurothallid orchids in Monteverde
The purpose of this experiment was to determine if the decrease in mist frequency observed in Monteverde, has an effect on Uredinales (rust fungi) infections in Pleurothallis aristata and Pleurothallis segoviensis orchids and to determine if the infection in P. segoviensis contributed to the rate of herbivory and number of flowers the orchid produced. In mist experiments infected samples of both species of orchids were placed in either a misted or control condition and the change in infection rates were observed over a 20-day period. Under natural conditions, number of leaves, leaves with infection, leaves with herbivory, and
number of flowers were counted on samples of P. segoviensis to determine if there were any significant interactions among these factors. Results reveal that additional mist does have an effect on rust fungus infection rates; with a decrease in mist, rust fungus infection rates increased. Infection rates also cause higher rates of herbivory and lower rates of flower production. Thirty-eight species of Pleurothallid orchids were found in the short twenty-day study in the forest near the Estacin Biolgica de Monteverde and in the Jardn de Orqudeas. Monteverde, famous for Pleurothallid orchids is experiencing a decrease in mist frequency, which puts the orchids here in a dangerous situation when dealing with rusts.
El propsito de este experimento fue determinar si la disminucin en la frecuencia de la niebla observado en Monteverde, tiene un efecto sobre las infecciones en Uredinales (Hongos de herrumbre) en las orqudeas de la familia Pleurothallid Pleurothallis aristata y Pleurothallis segoviensis. Tambin se determin si la infeccin en P. segoviensis contribuy al ndice de herbivora y al nmero de flores que la orqudea produjo. Los mtodos para las muestras incluyeron experimentos de niebla en ambas especies de orqudeas que fueron colocadas en condiciones de niebla y de control y observaciones de los cambios en ndices de infeccin. El nmero de hojas, hojas infectadas, hojas con herbivora, y el nmero de flores fue contado en muestras de P. segoviensis. para determinar si haban interacciones significativas entre estos factores. Los resultados revelaron que la frecuencia de la niebla tiene un efecto en los ndices de infeccin del hongo de herrumbre cuando la frecuencia de la niebla disminuye, los ndices de infeccin del hongo de herrumbre aumentan. Los ndices de infeccin tambin causaron ndices ms altos de herbivora e ndices de produccin floral menores. Monteverde, famoso por las orqudeas de la familia Pleurothallid, est experimentando una disminucin en la frecuencia de niebla que pone a las orqudeas de esta zona en una situacin peligrosa en referencia a la infeccin del hongo de herrumbre.
Text in English.
Monteverde Biological Station (Costa Rica)
Estacin Biolgica de Monteverde (Costa Rica)
Tropical Ecology Spring 2005
Ecologa Tropical Primavera 2005
t Monteverde Institute : Tropical Ecology