1 Arbuscular Mycorrhizal Fungi Vesicles in Coffea arabica (Rubiaceae): Agroforestry and Conventional Coffee Farms Jason C. Kopp Environmental Science, Policy, and Management, University of Minnesota Twin Cities ABSTRACT Due to their nutrient poor soils, a pressing issue in tropical countries is the conservation of land through more sustainable agricultural practices in order to continue to feed their booming populations. Agroforestry is a possible sustainable solution, which reduces negative impacts on t he environment, including impacts on mycorrhizal fungi. Mycorrhizal fungal associations within plants are important designators of a healthy crop and a more sustainable management system. This study investigates differences in arbuscular mycorrhizal fung i (AMF) vesicle abundance between the center and edge of an agroforestry coffee farm and the center of a conventional coffee farm. Eight different root samples were stained from each of the three locations using Trypan Blue (24 total root samples). The n umber of vesicles were then counted in three root segments from each of the 24 samples, and averaged for each location. Results showed a marginally statistically significant difference between all three locations (ANOVA, F=3.31, p=0.05, df=23). In additi on, the center coffee plants in agroforestry had more AMF vesicles per root segment (28.04 Â± 9.38 std) than roots at the center of conventional coffee (12.96 Â± 11.23 std) and were statistically different; but neither were statistically different from coffe e at agroforestry edge (21.17 Â± 14.08 std; Tukey's Multiple Comparison, q=2.52, p<0.05). Although there was little difference in AMF vesicle abundance on a micro scale within the agroforestry farm, there were more vesicles than in conventional coffee prod uction. This could be attributed to the use of biological fungicide, greater moisture retention, or younger trees in the agroforestry farm. RESUMEN Debido a sus suelos pobres en nutrientes, una presiÃ³n en los paÃses tropicales es la conservaciÃ³n de suel os a travÃ©s de prÃ¡cticas agrÃcolas sostenibles para continuar alimentando la poblaciones venideras. La agroecologÃa es una soluciÃ³n posible, reduciendo impactos negativos en el ambiente, incluyendo impactos en hongos micorrÃzicos. Las asociaciones micorr izicas con plantas son importantes evidencias de cultivos saludables y un mejor sistema de manejo. Este estudio investiga las diferencias en la abundancia de vesÃculas de hongos micorrÃzicos arborÃcolas (HMA) entre el centro y el borde de una finca de caf Ã© agroforestal y el centro de una finca convencional. Ocho muestras diferentes de raÃces fueron teÃ±idas de cada una de los tres sitios usando Azul de Tripano (24 muestras en total). El nÃºmero de vesÃculas se contaron en tres segmentos de cada una de las 24 muestras, y promediadas para cada sitio. Los resultados muestran una diferencia estadÃstica marginal entre los tres sitios (ANOVA, F=3.31, p=0.05, df=23). AdemÃ¡s, el centro de la plantaciÃ³n agroforestal presenta mÃ¡s vesÃculas de HMA por segmento de las raÃces (28.04 Â± 9.38 de) que las raÃces en el centro de la finca convencional (12.96 Â± 11.23 de), pero ninguna es estadÃsticamente diferente al borde de la finca agroforestal (21.17 Â± 14.08 std; Tukey's Multiple Comparison, q=2.52, p<0.05). AdemÃ¡s hay un a pequeÃ±a diferencia en la abundancia de vesÃculas de HMA a una microescala dentro de la finca agroforestal, hay mÃ¡s vesÃculas que en la finca convencional. Esto se puede atribuir al uso de fungicida biolÃ³gico, mayor retenciÃ³n de humedad, o Ã¡rboles mÃ¡s jÃ³ venes en la finca agroforestal.
2 INTRODUCTION Propelled by dramatic growth in the developing world, world population is rapidly approaching seven billion people. This growth is most apparent in tropical countries, where the population has increased from 1.8 billion in 1950 to 4.9 billion people in 2000 and is expected to grow by another two billion before 2030. In addition, tropical economies are projected to grow just as rapidly (Wright, 2005). This kind of growth is causing the need for grain to more than double, especially as demand for meat increases in developing countries (Tilman, 1999), which will cause a push to grow more and more crops using monocultures fed with chemical inputs. This kind of food production is causing a dramatic simplificatio ecosystems, increased nutrient runoff into waterways, decreased available potable water, and decreased soil fertility (Tilman, 1999). Thus, more research on sustainable, high yield, low input agriculture (which has less negative effects on the environment) is a necessity as we move into the future. Agroforestry, which is slowly becoming the norm in many tropical countries, is farmland combined with patches of forest (Garcia et al. , 2009). This type of agriculture suppor ts high levels of biodiversity and has less negative impacts on the environment ( Schroth et al. , 2004) . While tropical countries look to implement more of these strategies, they should consider the importance of arbuscular mycorrhizal fungi (AMF) within t heir crops and the benefits they have towards conservation. Arbuscular mycorrhizal fungi (AMF; Zygomycotina: Order Glomales) have hyphae that penetrate cortical root cell walls and form branched structures called arbuscules. These arbuscules transfer min eral nutrients from the fungi to the plant and sugars from the plant to the fungi. AMF also form storage organs called vesicles within the plant cell walls (Brady and Weil, 1996). A plant root with mycorrhizae has a lot of thick, short, lateral mycorrhiza l roots having abundant branching and plenty of hyphae. Stark, 1968). In nutrient deficient soils, mycorrhizae are essential for transforming dead organic matter into nutrients and minerals that can be passed directly into the roots of plants (Went and Stark, 1968). AMF increases available nutrients like Nitrogen, Phosphorus, Copper, and Zinc, increases resistance to pathogens and insect herbivores, and increases water deficit tolerance (Smith and Read, 1997). In addition, AMF improves soil structure and aggregation through hyphal hairs that hold the soil together and better connect soil particles (Rillig and Mummey, 2006). Given all of these factors, AMF are a key com ponent of a sustainable agricultural management model. Sustainable agriculture conserves the productivity of the soil, minimizes energy and resource use, and recycles nutrients (Jeffries and Barea, 2001). Due to the low fertility of tropical soils (Tilm an, 1999) and the booming populations within tropical countries, sustainable agriculture and soil conservation is of the utmost importance, of which AMF is a key factor. One of the largest exports in the tropics is coffee and, generating an estimated $35 billion dollars annually, it is very economically and socially important for the populations of tropical countries (Siqueira et al. , 1998). AMF associations are important in coffee because they help to produce greater yields and maintain soil quality, esp ecially in early years of growth (Siqueira et al. , 1998). Additionally, Cardoso et al. (2003) showed that coffee produced in an agroforestry
3 system had more AMF spores in the deeper soil than in a monocultural system. Thus, the association between coffee and AMF are a small but very important symbiotic relationship that needs continued investigation, especially as it relates to agroforestry and monocultural systems. This study investigates the effects of farm location and management practices on the abund ance of AMF in the roots of Coffea arabica (Rubiaceae) on a micro and macro scale. AMF abundances will be determined in the center and edge of separate coffee plots in a single agroforestry farm (AGRO CENTER and AGRO EDGE) and the center of a monocultural farm (MONO). My working hypothesis is that I will see more AMF vesicles in the roots of coffee trees on AGRO EDGE than AGRO CENTER or MONO and more AMF vesicles in the roots of coffee trees in AGRO CENTER than in MONO. Fungi generally grow best under mo ist conditions so the more shaded and cooler AGRO should have a greater abundance of AMF than in MONO. MATERIALS AND METHODS Study Sites I used two study sites both located in the CaÃ±itas area of Monteverde, Puntarenas, Costa Rica. Site 1, called LIFE M onteverde, is a farm managed by Guillermo Vargas and will henceforth be referred to as AGRO. It is comprised of 16.4 hectares of land at an elevation between 1200 and 1300m. 5.7 hectares of the farm is under coffee production (35% of the total area) and 8 hectares is comprised of secondary forest (49% of the total area). LIFE Monteverde produces around 16,000lbs of coffee per year. Biological fungicide, chemical fertilizer, and chemical herbicide are applied to the crop usually throughout the rainy seaso n (August coffee plots (between 68.8m 2 and 6459.2m 2 ) all separated by natural forest or windbreaks of native TubÃº ( Montanoa guatemalensis Asteraceae) and exotic Cypress (Cupressus lusitanica Cupressaceae). I sampled from 8 of the 33 plots (Figure 1). At each plot I took two root samples (from a depth of 1 20cm), one from the center of the plot, which I call AGRO CENTER, and one from the edge, AGRO EDGE, giving me 16 total root samples from LIFE Monteverde. Site 2 is owned by Juvenal Rodriguez Castro and will henceforth be referred to as MONO as it was a monoculture with more chemical inputs. It is comprised of four hectares of land at an elevation of 1200m. This farm is much more conventional than LIFE Mont everde in that Juvenal sprays chemical fertilizer, chemical fungicide, and chemical herbicide multiple times a year, and all four hectares of land are under coffee production in full sun. Other coffee farms surround his land, with only windbreaks but no f orest nearby. I took one root sample from eight different coffee trees from the center of his farm.
4 FIGURE 1. A map of LIFE Monteverde (AGRO), an agroforestry project that includes coffee production. The total area of the farm is 5.7 hectares. Brown areas are in coffee production. Dark green areas are forest or windbreaks. Light green areas are pasture. Sample areas for mycorrhizae on coffee roots are in red. Quantifying Arbuscular Mycorrhizal Fungi (AMF) I obtained a total of 24 root samples, 8 from each location. To quantify the number of AMF in the roots, I used methods modified from Bagyaraj and StÃ¼rmer (2008). In the field, all root samples were placed into a plastic bag and gently shaken to remove excess soil. I then cut the roots into t wo centimeter pieces, placed them into 250ml Erlenmeyer flasks, and rinsed them with tap water three times. They were then transported to and stored in labeled test tubes containing 2% KOH for 48 hours. After clearing the roots for 48 hours, the roots wer e rinsed with tap water three times. I then soaked the roots in an alkaline H 2 O 2 solution (3mL 20% NH 4 OH, 30mL 3% H 2 0 2 , 567mL tap water) for one hour. After this, I again rinsed the roots with tap water three times. Next, I acidified the roots in 1% HCl for five minutes. The samples were then removed from the HCl but were not rinsed. I stained the roots using a 4:1 staining solution of acidic glycerol (500mL glycerol, 450mL H 2 O, 50mL 1%HCl) and 0.05% Trypan Blue. I poured the staining solution into te st tubes that sat in a water bath at around 90Â° C, where they incubated for one hour. Finally, I removed the staining solution and stored the roots in the previously mentioned acidic glycerol solution (without Trypan Blue). Three root segments were then cut from each sample with a razor blade. I observed each segment under 40x magnification on a compound light microscope and took a photograph of the microscope image. I then quantified the number of vesicles in each photo using Adobe Photoshop by boostin g the contrast to better differentiate between cell walls and vesicles. Finally, I averaged the three AMF vesicle counts for each location.
5 RESULTS I found that all three locations were marginally statistically different in mean AMF vesicle abundance (AN OVA, F=3.31, p=0.05, df=23). AGRO CENTER had a mean AMF abundance of 28.04 with a standard deviation of 9.38. AGRO EDGE had a mean AMF abundance of 21.17 with a standard deviation of 14.08. MONO had a mean AMF abundance of 12.96 with a standard deviatio n of 11.23 (Figure 2). All of the sites had large variation in AMF abundance; meaning even trees side by side differed greatly in AMF abundance. Still, location did have some effect. MONO had low AMF abundance, which was less than half of what it was fo r AGRO CENTER and about 60% of what it was for AGRO EDGE. In addition, AGRO CENTER was 33% greater than AGRO EDGE. Finally, AGRO CENTER and MONO were statistically different but neither was different from AGRO EDGE (Tukey's Multiple Comparison, q=2.52, p <0.05). FIGURE 2. Mean AMF abundance Â± STD for each location (AGRO CENTER, AGRO EDGE, and MONO). The black bars, labeled A and B, represent those pairs that are not statistically different. The two sites had obvious differences in their physical appea rance. As stated, forest and/or windbreaks surrounded all of the coffee plots in AGRO. This meant that the soil retained more moisture and seemed to be better aerated. MONO was bigger than any of the plots I sampled from in AGRO, which meant many more of the coffee trees received direct sunlight throughout the day so the soil was much dryer. The trees in AGRO appeared to be younger than the trees in MONO, as well, due to their smaller size and less tough leaves, on average. It also appeared that there w as more leaf litter on the ground of MONO than on the ground of at least some of the plots in AGRO. Figure 3 shows the visual differences between the two locations.
6 FIGURE 3: (a) AGRO, surrounded by forest and windbreaks. Banana trees are also locate d in the center of the plot. (b) The center of MONO with no forest or surrounding windbreaks. DISCUSSION AGRO CENTER and AGRO EDGE had greater AMF abundance than MONO. This suggests that the conditions present within MONO are harsher for the developme nt of AMF. High input agroecosystems have been proven to have a lower diversity of AMF (Johnson and Pfleger, 1992). MONO, for example, uses all chemical inputs, which kill many microorganisms including AMF (Bethlenfalvay, 1992). AGRO EDGE had less AMF t han AGRO CENTER but they were not statistically different. So the edge may not have much of an effect on AMF abundance or may actually have a negative effect. This could mean there is very little difference in AMF abundance on such a small scale, or it co uld mean that the conditions on the edge are harsher for the development of AMF. Some of the edges are separated from the forest by small roads or walkways, which may increase soil compaction and erosion near the edge trees. Soil erosion is another facto r that negatively effects AMF abundance (Habte, 1989). Nonetheless, the edge is important in the maintenance of an agroforestry farm in that the windbreaks and forest increase biodiversity and reduce the negative effects of the managed farm. In addition, AGRO EDGE and MONO are also not statistically different. This means the edge of AGRO is similar to that of MONO. The increased erosion that may be occurring at the edge may lead to reduced AMF abundance. In addition, the edge may receive less fertilizer than the center of AGRO, which has been shown, in some cases, to decrease AMF abundance (Johnson and Pfleger, 1992). However, MONO still had dramatically fewer AMF leading me to believe that chemical fungicide inputs have more of a negative effect on AMF abundance. AGRO CENTER had more AMF abundance than MONO by about 10%. This could be attributed to many things. First, AGRO uses a biological fungicide, which is less detrimental to the environment and may be better at maintaining AMF in the roots of the coffee trees than the chemical fungicide used in MONO (Johnson and Pfleger, 1992). Secondly, the deep roots throughout the property of AGRO in the form of windbreaks and forest may increase nutrient cycling and aeration throughout the soils of AGRO, leadi ng to better growth conditions on average (Cardoso et al. , 2003). Finally, the plants
7 in AGRO may be younger than MONO, which would mean they have less root structure aiding in the development of AMF (Cardoso et al. , 2003). There are a few limitations of t his study, which should be improved before future investigation is to continue. More samples should be taken at each site and more segments should be cut from each sample to get a more representative average of the AMF counts at each site. A bigger sampl e size will better capture the extreme variability of AMF vesicle abundance. In addition, only coffee plots of similar age should be sampled as age has been shown to effect AMF abundance (Cardoso et al. , 2003). With these changes, the variable of age woul d be better accounted for and the overall variability reduced. These results show that there is little variation between the AMF abundance on a micro scale between the edge and center of coffee plots on a single agroforestry farm, though the conditions pre sent on the edge may have a somewhat negative effect on fungal growth. However, there is a statistically significant difference between the center of an agroforestry farm and the center of a conventional farm. This shows that farm management (biological fungicide and agroforestry) may lead to greater AMF abundance. In order for MONO to achieve a similar AMF abundance, it may need to increase the use of fertilizer, which may be beneficial to AMF growth. But this increase dependence on inputs would only f urther separate the tropics from a sustainable future. Agroforestry may be an important alternative to monoculture food production, which increases AMF abundance, decreases our dependency on chemical inputs, and may make sustainable food production in the tropics a reality. ACKNOWLEDGEMENTS I would like to thank my advisor, Alan Masters, for all of his guidance and inspiration throughout the entirety of this process; Guillermo Vargas for his support and for allowing me access to his farm; Juvenal Rodrig uez Castro for allowing me access to his farm; the EstaciÃ³n BiolÃ³gica for allowing me to use their chemistry room and equipment; Yimen Araya for his statistical genius; JosÃ© Carlos CalderÃ³n for his reliable guidance when I was lost and for translating my a bstract into Spanish; Pablo Allen and Anjali LITERATURE CITED Bagyaraj, J. D. and S. L. StÃ¼rmer. 2008. 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 Biodiversity, pg. 135 136. Earthscan, London, UK. Bet hlenfalvay, G. J. 1992. Mycorrhizae in the Agricultural Plant Soil System. Symbiosis. 14: 413 425. Brady, N. C. and R. R. Weil. 1996. The Nature and Properties of Soils. Prentice Hall, Upper Saddle River, New Jersey. Cardoso, I. M., C. Boddington, B. H. Janssen, O. Oenema, and T. W. Kuyper. 2003. Distribution of mycorrhizal fungal spores in soils under agroforestry and monocultural coffee systems in Brazil. Agroforestry System. 58: 33 43. Garcia, C. A., S. A. Bhagwat , J. Ghazoul , C. D. Nath , K. M. Nanaya , C. G. Kushalappa , Y. Raghuramulu , R. Nasi , and P. Vaast. 2009. Biodiversity Conservation in Agricultural Landscapes: Challenges and Opportunities of Coffee Agroforests in the Western Ghats, India. Conservation Biology. 24: 479 488. Habte, M . 1989. Impact of simulated erosion on the abundance and activity of indigenous vesicular arbuscular mycorrhizal endophytes in an Oxisol. Biology and Fertility of Soils. 7:164 167.
8 Jeffries, P. and J. M. Barea. 2001. Arbuscular mycorrhiza a key compo nent of sustainable plant soil ecosystems. In B. Hock (Ed.). The Mycota, Vol. IX: Fungal Associations. pp. 95 113. Springer Verlag, Berlin, Germany. Johnson, N. C. and F.L. Pfleger. 1992. Vescular Arbuscular Mycorrhizae and Cultural Stresses. In G. Bethlenfalvay, et al. (Eds.). VA Mycorrhizae in Sustainable Agriculture, pg. 71 99. ASA/SSSA Special Publication, Madison, Wisconsin. Rillig, M. C. and D. L. Mummey. 2006. Mycorrhizas and Soil Structure. New Phytologist. 171: 41 53. Schroth , G ., G. A. B . d. Fonseca , C. Harvey , C. Gascon , H. L. Vasconcelos , A. M. N. Izac editors . 2004 . Agroforestry and biodiversity conservation in tropical landscapes . Island Press , Washington , D.C . Siqueira, J. O., O. J. Saggin JÃºnior, W. W. Flores Aylas, and P. T. G. GuimarÃ£es. 1998. Arbuscular mycorrhizal inoculation and superphosphate application influence plant development and yield of coffee in Brazil. Mycorrhiza. 7: 293 300. Smith, S. E. and D. J. Read. 1997. Mycorrhizal Symbiosis. Academic Press, San Di ego, California. Tilman, D. Global environmental impacts of agricultural expansion: The need for sustainable and efficient practices. 1999. Proceedings of the National Academy of Science. 96: 5995 6000. Went F.W. And N. Stark. 1968. Mycorrhiza. BioS cience. 18: 1035 1039. Wright, S. J. Tropical forests in a changing environment. 2005. Trends in Ecology and Evolution. 20:553 560 .
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Kopp, Jason, C.
Las vesculas de los hongos arborcolas en Coffea arabica (Rubiaceae): Las fincas agroforestales y convencionales de caf
Arbuscular mycorrhizal fungi vesicles in Coffea arabica (Rubiaceae): Agroforestry and conventional coffee farms
Due to their nutrient-poor soils, a pressing issue in tropical countries is the conservation of land through more sustainable agricultural practices in order to continue to feed their booming populations. Agroforestry is a possible sustainable solution, which reduces negative impacts on the environment, including impacts on mycorrhizal fungi. Mycorrhizal fungal associations within plants are important
designators of a healthy crop and a more sustainable management system. This study investigates differences in arbuscular mycorrhizal fungi (AMF) vesicle abundance between the center and edge of an agroforestry coffee farm and the center of a conventional coffee farm. Eight different root samples were stained from each of the three locations using Trypan Blue (24 total root samples). The number of vesicles were then counted in three root segments from each of the 24 samples, and averaged for each location. Results showed a marginally statistically significant difference between all three locations (ANOVA, F=3.31, p=0.05, df=23). In addition, the center coffee plants in agroforestry had more AMF vesicles per
root segment (28.04 9.38 std) than roots at the center of conventional coffee (12.96 11.23 std) and were statistically different; but neither were statistically different from coffee at agroforestry edge (21.17 14.08
std; Tukey's Multiple Comparison, q=2.52, p<0.05). Although there was little difference in AMF vesicle abundance on a micro-scale within the agroforestry farm, there were more vesicles than in conventional coffee production. This could be attributed to the use of biological fungicide, greater moisture retention, or younger trees in the agroforestry farm.
Debido a sus suelos pobres en nutrientes, una presin en los pases tropicales es la conservacin de los suelos a travs de las prcticas agrcolas sostenibles para continuar alimentando a las poblaciones venideras. La agroecologa es una solucin posible, reduciendo los impactos negativos en el ambiente, incluyendo los impactos en los hongos micorrzicos. Las asociaciones micorrizicas con las plantas son importantes evidencias de los cultivos saludables y un mejor sistema de manejo. Este estudio investiga las diferencias en la abundancia de las vesculas de los hongos micorrzicos arborcolas (HMA) entre el centro y el borde de una finca de caf agroforestal y el centro de una finca convencional. Ocho muestras diferentes de races fueron teidas de cada una de los tres sitios usando Azul de Tripano (24 muestras en total). El nmero de vesculas se contaron en los tres segmentos de cada una de las 24 muestras, y promediadas para cada sitio. Los resultados muestran una diferencia estadstica marginal entre los tres sitios (ANOVA, F=3.31, p=0.05, df=23). Adems, el centro de la plantacin agroforestal presenta ms vesculas de HMA por segmento de las races (28.04 9.38 de) que las races en el centro de la finca convencional (12.96 11.23 de), pero ninguna es estadsticamente diferente al borde de la finca agroforestal (21.17 14.08 std; Tukey's Multiple Comparison, q=2.52, p<0.05). Adems hay una pequea diferencia en la abundancia de vesculas de HMA a una micro escala dentro de la finca agroforestal, hay ms vesculas que en la finca convencional. Esto se puede atribuir al uso de un fungicida biolgico, una mayor retencin de humedad, o a los rboles ms jvenes en la finca agroforestal.
Text in English.
Tropical Ecology Spring 2010
Ecologa Tropical Primavera 2010
t Monteverde Institute : Tropical Ecology