Comparison of Abundance of Vesicular Arbuscular Mycorrhizal Fungi on Sun and Shade grown Coffea arabica plants John A. Colantonio Undergrad Energy Program and Department of Environmental Studies, University of Colorado, Boulder, Colorado 80310, U.S.A __________________________ ABSTRACT Large scale coffee agriculture can have significant detrimental effects on the environment. However, organic coffee production lowers these impacts and helps to conserve tropical diversity by reducing need for soil in puts. Naturally healthy soils contain beneficial vesicular mycorrhizal (VAM) fungi. These fungi help cycle nutrients and support plant health and growth. Mycorrhizal mutualisms are abundant and effective in particular habitats, particularly those in whi ch the fungi do not exhaust carbon resources for the plant. This study compares the abundance of VAM fungi vesicles on the roots of organic shade grown coffee plants and those grown in the sun. Root samples were collected from an organic coffee farm and stained, and observed under high power magnification to determine quantity of fungi vesicles. Data show no statistical significance in the mean number of vesicles between the two sample populations. This result implies that sunlight is not the only facto r determining plant health and need for soil inputs. INTRODUCTION Coffee is one of the most economically important exports in the world. A vast number of people around the world drink coffee, and there is high demand for the superior quality coffee th at the tropics produce. Coffee generates roughly $35 billion annually (Kopp 2010), and represents a crucial resource for tropical communities. As a result, coffee plantations are widespread throughout tropical countries, such as Costa Rica. Huge areas o f tropical rainforests are clear cut to provide more area for agriculture (Wallace 2007). As with any mass produced crop, large plantations typically grow acres upon acres of monoculture crops, like coffee. This technique depletes soil nutrients thus req uiring significant amounts of synthetic fertilizer inputs, while the need to control weeds around the crops requires large amounts of pesticide and herbicide inputs. These plantations often contain little to no tree coverage, and due to heavy precipitatio n in the tropics, significant runoff and topsoil erosion occur (Coffee and Conservation 2006), severely impacting watersheds and biodiversity. In
efforts to reduce environmental impact and conserve rainforest and biodiversity resources, organic coffee pla ntations have arisen. Organic coffee plantations attempt to maintain a balance between producing high quality coffee while conserving biodiversity. Migratory birds are a representative group of organisms that benefit from the crop diversification and con served tree patches present on organic coffee farms. But these farms also help protect the environment from toxic chemical runoff, which in turn protects watersheds and ecosystems downstream. Partial canopies over organic coffee farms catalyze nutrient c ycling by dropping leaves to the soil. The soils are kept healthy by a diversity of crops such as fruit trees, sugarcane, cacao, and coffee, and a major component of these healthy soils are mycorrhizal fungi (Coffee and Conservation 2006). Crop producti on is frequently dependent on naturally occurring populations of mycorrhizal fungi (Smith 1997), which increase absorptive plant root area and nutrient exchange (Mills 2006). Phosphorus (P), a fundamental nutrient for plant growth, is scarce in the tropic s. Mycorrhizal hyphae are more capable of taking up phosphorus ions in low concentrations than root hairs are, and therefore mycorrhizae significantly aide in P uptake (Janos 1983). For this reason, Janos (1983) proposes that the tropical rainforest as a whole may owe its existence to vesicular arbuscular mycorrhizal (VAM) fungi. Mycorrhizae require host plants as a substrate to live in and as a source of carbon (in the form of glucose and fructose), but in low light conditions, allocating a large amou nt of carbon to the fungal symbiont can be detrimental to plant growth (Smith 1997). Responsiveness to mycorrhizal colonization is strongly affected by exposure to sunlight and nutrient availability of the soil, and a significant proportion of coffee plan ts on organic farms grow in low irradiance conditions (Smith 1997). VAM are severely limited by soil inputs and tillage, both of which are necessary for sun grown coffee (Ishii 2009). I will examine coffee plants on organic farms that are in high sunligh t exposure contain stronger or more abundant mycorrhizal mutualisms than shade grown coffee plants. In this study, I compare the quantity of VAM fungi on organic coffee plants in the sun to the quantity on those in the shade. Increased presence of VAM fu ngi in the sun exposed plants could possibly indicate higher productivity, which could indicate potential economic advantages in comparison to organic shade coffee. I predict that organic coffee plants in the shade will contain more VAM fungi vesicles tha n those grown in the shade. MATERIALS AND METHODS STUDY SITE The study was conducted on an organic coffee farm in San Luis, Costa Rica, at 1100 m. The San Luis valley is surrounded by mountains and receives abundant rainfall and sunlight. Prior to co nversion to agriculture, San Luis was a premontane moist forest. The farm has significant canopy coverage and hosts a diverse polyculture of banana, orange, mango, and hardwood trees, as well as sugarcane and coffee plants. There are many coffee plants t hat are shaded by the trees for the duration of the day, as well as many plants that are exposed to sunlight at all hours of sunlight; all plants are of the same species, Coffea arabica . I chose to sample 20 plants that were constantly shaded and 20 plant s that were constantly in sunlight (weather providing). To the best of my knowledge, each plant was the caturra variety of C. arabica . All plants were healthy in appearance and roughly 2 meters tall.
METHODS I gathered root samples from each plant by cl earing away leaf litter and other organic debris and finding one large root stemming from the base of the plant. Digging just below the surface with a knife, I located and cut small roots growing underneath the large main root. I collected three root clus ters from each plant and placed them in labeled Ziploc bags. In the lab, I followed the procedures illustrated by Bagyaraj and StÃ¼rmer (2008), and Kopp (2010). After shaking excess dirt off and rinsing each root sample with tap water three times, I place d each sample in a 2% KOH solution in vials for 48 hours. After 48 hours, I rinsed the roots another three times with tap water and put them in vials containing a solution of 3 ml 20% NH4OH, 30 ml 3% H2O2, and 567 ml of tap water for one hour. After rins ing three more times, I acidified each sample in 1% HCl for five minutes, and without rinsing, transferred them to test tubes containing a 4:1 staining solution of acidic glycerol solution (500 ml glycerol, 450 ml distilled H2O, 50 ml 1%HCl) and 0.05% Tryp an Blue. The test tubes were then incubated in a 90Â° Celsius water bath for one hour. After incubation the staining solution was poured off and the samples were immersed and stored in the acidic glycerol solution mentioned above (without Trypan Blue). To quantify the average number of vesicular arbuscular mycorrhizal (VAM) fungi, I cut and selected three 0.5 cm root segments per sample. These lengths were selected based on lightest shade of blue in order to use the most readily observable samples. Un der 100X magnification with a compound light microscope, I counted each fungal vesicle from one end of the segment to the other. After quantifying vesicles on each segment, I calculated the mean number of vesicles for the entire root sample. RESULTS The number of VAM fungi vesicles observed in plants grown in the shade and plants grown in the sun are not significantly different (t=0.293, DF=38, P=0.77, Fig 1). Sun plants had an average of 74 (SD=32.358), and shade plants had an average of 71 (SD=33.201) .
Figure 1. The average number of mycorrhizal fungi vesicles (y axis) in plants from each location, sun and shade, with SD bars. DISCUSSION I predicted that sun plants would have more VAM fungi than shade plants because coffee plants in the sun can p hotosynthesize more and therefore provide more carbohydrates to mycorrhizae, whereas plants in the shade would photosynthesize less (providing fewer carbohydrates to a mutualist), evapotranspire less, and require less water absorption from the mycorrhizae. I expected the shade plants to reject the mutualism with mycorrhizae because plants often do so to avoid the carbon cost of the fungi (Janos 1983). Therefore, I predicted that sun plants would have more VAM. However, my data resulted in no significant difference, which implies that Coffea arabica plants grown in the same soil with different levels of irradiance accept the same mutualism with mycorrhizal fungi. In the tropics, plants are water stressed during the dry season and receive variably sunlight , so these growth constraints favor a mycorrhizal mutualism. The energetic/carbon cost of the mutualism is outweighed by the benefits shade plants receive. There is significant evidence that mycorrhizal colonization increases resistance to pathogens (Is hii 2009) and herbivorous insects and increases tolerance of water deficit (Smith 1997). Also, in many pot studies of mycorrhizal plants, colonization of the roots is followed by stimulated growth and phosphorus (P) uptake, and in controlled conditions wit h low P soil, there is significantly reduced plant growth which can be directly caused by lack of mycorrhizae (Smith 1997). Despite a lack of sunlight and the subsequent carbon (C) expense of the fungi, shaded coffee plants gain an increased ability to fi x carbon dioxide (CO2) and to photosynthesize, and this benefit offsets the cost of the fungi. This implies that the coffee plant must allocate significant carbohydrates (in the form of sugars) to the fungi, which would explain the comparatively smaller y ield of shade grown coffee plants.
In nutrient poor soils, as in the Tropics, plants benefit from mycorrhizal nutrient transfer. Mycorrhizal mutualisms represent a careful balance between costs and benefits as many mycorrhizal plants can survive without the symbionts. The production of hyphae is less carbon costly than the production of roots, and so in low fertility soils or areas of low irradiance, shade grown plants can benefit from the presence of mycorrhizae (Smith 1997). I expected to observe a sig nificant difference in VAM presence, but since plants in the sun evapotranspire more quickly and provide more C to the fungi, while shaded plants benefit in photosynthesis productivity and water absorption, it follows logically that coffee plants in both s un and shade require significant abundance of mycorrhizae. The fact that the two sample groups are grown in close proximity and in the same soil suggests that VAM fungi can easily colonize both sun and shade plants. This study shows that despite sun exp osure or a lack of sunlight, mycorrhizal mutualisms help supply coffee plants with sufficient nutrients and water. On large plantations of sun grown coffee, however, there is an insufficiency of organic debris such as leaf litter that allows for good nutr ient exchange between mycorrhizae and plants (Coffee and conservation 2006). This calls for increased synthetic soil inputs and therefore increased production costs, but sun grown coffee plants are typically more productive than shade grown, which can off set the higher costs. Therefore, there is no advantage to sun grown coffee regarding soil nutrition and VAM presence. A combination of crop diversification and higher price for organic coffee, as well as fewer to no soil inputs required for production in dicates that organic coffee can compete with monoculture sun grown coffee, but only if the market demand for organic coffee is high enough. ACKNOWLEDGEMENTS I would like to thank my advisor Karen Masters for guiding me through my research and data ana lysis, and for being patient. I thank Alan Masters as well for helping me decide to study this topic. Thanks are also in order for my friend Oldemar for allowing me access to his farm and coffee plants, and for showing me how to distinguish coffee roots from roots of nearby vegetation. I also appreciate the lab assistance from my homestay brother Eduardo, and my friend Mackenzie Most for photo documenting my lab work and helping me clean the numerous vials, test tubes, and various other lab equipment. A nd of course thanks to the University of Georgia San Luis campus for allowing me access to their lab and to CIEE Monteverde and Moncho Calderon for supplying all the materials and equipment I needed. LITERATURE CITED BAGYARAJ, J. D. AND S. L. STÃœRMER. 20 08. Arbuscular Mycorrhizal Fungi (AMF). In F. M. S. Moreira, E. J. Huising, and D. E. Bignell (Eds .). A Handbook of Tropical Soil Biology Sampling & Characterization of Belowground B iodiversity, Earthscan, London: 135 136. JANOS, D.P. 1983. Vesicular A rbuscular Mycorrhizal Fungi. Costa Rican Natural History. The University of Chicago Press: 340 345. KOPP, J. 2010. Arbuscular Mycorrhizal Fungi Vesicles in Coffea arabica (Rubiaceae): Agroforestry and Conventional Coffee Farms. Counc il on International Ed ucational Exchange, Monteverde, Costa Rica Spring 2010: 18 22. MILLS, T.J. 2006. Presence of endomycorrhizae on roots o f epiphytic orchids. Council on International Educational Exchange, Monteverde, Costa Rica Summer 2006: 10 14.
SMITH, S.E. AND D.J. REA D. 1997. Mycorrhizal Symbiosis, Second Edition. Academic Press: 105 118, 453 457. WALLACE, S. 2007. Last of the Amazon. N ational Geographic, January : 48 70. ISHII, T. 2009. Vesicular Arbuscular (VA) Mycorrhizal Fungi. Lab of Pomology in Kyoto Prefectura l University: http://www.bio.kpu.ac.jp/pomlab/vaminf.html SMITHSONIAN NATIONAL ZOOLOGICAL PARK. 2009. Shade grown coffee plantations. Smithsonian Institute: http://nationalzoo.si.edu/scbi/migratorybirds/coffee/ 2006. Problems with Sun Coffee. Coffee and Conservation: http://www.coffeehabitat.com/2006/02/the_prob lems_wi.html
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Colantonio, John, A.
Comparacin de la abundancia de los hongos micorrizas-arbusculares en las plantas de caf arabica en sitios de sol y sombra
Comparison of abundance of vesicular-arbuscular mycorrhizal fungi on sun- and shade-grown coffea arabica plants
Large-scale coffee agriculture can have significantly detrimental effects on the environment. However, organic coffee production lowers these impacts and helps to conserve tropical diversity by reducing need for soil inputs. Natural, healthy soils contain beneficial vesicularmycorrhizal
(VAM) fungi. These fungi help cycle nutrients, and support plant health and growth. Mycorrhizal mutualisms are abundant and effective in particular habitats, particularly those in which the fungi do not exhaust carbon resources for the plant. This study compares the abundance of VAM fungi vesicles on the roots of organic shade-grown coffee plants and those grown in the sun. Root samples were collected from an organic coffee farm and stained, and observed under high-power magnification to determine quantity of fungi vesicles. Data show no
statistical significance in the mean number of vesicles between the two sample populations. This result implies that sunlight is not the only factor determining plant health and need for soil inputs.
La agricultura de caf a gran escala puede tener efectos significativamente negativos en el medio ambiente. Sin embargo, la produccin de caf orgnico reduce estos impactos y ayuda a conservar la diversidad tropical mediante la reduccin de necesidad de insumos del suelo. Los suelos naturales y saludables contienen hongos micorrizas-arbulsculares (VAM) beneficiosos. Estos hongos ayudan a reciclar los nutrientes y apoyar la salud y el crecimiento de la planta. Los mutualismos micorrizas son abundantes y eficaces en hbitats particulares, especialmente aquellos en los que los hongos no agotan los recursos de carbn para la planta. Este estudio compara la abundancia de las vesculas de los hongos (VAM) en las races de las plantas de caf orgnico de sombra y aquellos que se cultivan en el sol. Las muestras de raz se obtuvieron de una finca de caf orgnico y desteido y se observan con una lupa de gran potencia para determinar la cantidad de vesculas del hongo. Los datos no muestran ningn significado estadstico en el nmero medio de vesculas entre las dos poblaciones de la muestra. Este resultado implica que la luz solar no es el nico factor determinante de la salud de las plantas y necesidad de entradas del suelo.
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Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Summer 2010
Ecologa Tropical Verano 2010
t Monteverde Institute : Tropical Ecology