Effects of Trichoderma harzianum perimeter applications on preventing Mycena citricolor in Coffea arabica plots Nicholas J. Gallagher Department of Botany, University of Wisconsin at Madison ABSTRACT This study examine d the fungus Trichoderma harzia num , a proven effective biological control agent, for its potential of preventing the forest and windbreak instigated onset of the fungus Mycena citricolor in coffee plots. Fifteen plots on one farm in the Monteverde region of Puntarenas, Costa Rica were s urveyed for the presence of M. citricolor on coffee near forest edges and along transects away from the forest. T. harzianum was applied to the forest edge on half of the plots, and the coffee plants near the edge were resurveyed for new infestations of M. citricolor . N o significant difference between the treated and untreated plots was found . Other factors such as short length of time. A regress ion analysis of the transects confirmed that the presence of M. citricolor decrease with increased distance form the forest. This may also be attributed to the levels of light and humidity along the transects. Less time limited experiments may still prove edge applications of T. harzianum an effective method for inhibiting M. citricolor inception in coffee plots. RESUMEN Este estudio examina el hongo Trichoderma harzianum como un agente efectivo de control biolÃ³gico, por su potencial de prevenir el hongo Mycena citricolor en plantaciones de cafÃ©. Quince cuadrÃculas en una finca en la regiÃ³n de Monteverde en Puntarenas, Costa Rica se estudiaron para detectar la presencia de M. citricolor en plantas de cafÃ© cercanas al bosque y a lo largo de transectos ale jÃ¡ndose del mismo. T. harzianum se aplicÃ³ al borde de bosque y a la mitad de los cuadrantes, y las plantas de cafÃ© cercanas al borde de bosque se observaron para detectar nuevas infestaciones por M. citricolor. No se encontraron diferencias significativas entre los cuadrantes tratados y los no tratados. Otros factores como la humedad y exposiciÃ³n a la luz solar pueden influenciar el establecimiento y de esta manera anular el efecto del fungicida sobre un periodo corto de tiempo. Un anÃ¡lisis de regresiÃ³n de los transectos confirma que la presencia de M citricolor disminuye cuando aumenta la distancia al bosque. Esto se puede atribuir a los niveles de luz y humedad a lo largo de los transectos. Experimentos que una menor limitaciÃ³n de tiempo pueden provee r informaciÃ³n para determinar si la aplicaciÃ³n de T. harzianum como un mÃ©todo efectivo para inhibir el crecimiento de M. citricolor en plantaciones de cafÃ©. INTRODUCTION Coffee ( Coffea arabica ) was the first major export crop of Costa Rica (Boucher 1983) , and is U.S. Department of State 2009 ). Such economic status gives heed to the importance of preventing widespread infestation of the crop. Fungal diseases in particular pose a major threat to cof fee production in Costa Rica, especially Mycena citricolor , commonly known as American leaf spot or ojo de gallo (Boucher 1983). M. citricolor is a Basidiomycete, in the family Tricholomataceae, that has a wide variety of hosts in 45 families including Ru biaceae (Sequeira 1958). It causes brown spots on infected leaves and can lead to considerable defoliation of the coffee plant and reduced plant production (Waller et
al. 2007). M. citricolor can also infect the fruits causing decreased bean quality and l owered market value. Intercropping coffee plants with trees can greatly aid the crop production by reducing high temperature variations and increasing nitrogen availability through increased leaf litter (Beer 1998). Intercropping has also been shown to d ecrease variation in crop yield and thus increase long term yields (Beer 1987). Conversely, these favorable coffee conditions are ideal for the growth of M. citricolor . In Costa Rica, the fungus thrives at altitudes between 1,100 and 1,550 meters and in ar eas of high humidity (Avelino et al. 2007). Sunlight proves detrimental to its growth while shade provides favorable conditions for the fungus (Wellman 1950). Its spores are wind and contact dispersed, but generally only over short distances (Avelino et a l. 2007). Therefore coffee that is intercropped with small trees or adjacent to forested areas is more susceptible to the infection. Many practices have been employed in order to control M. citricolor . For many decades regular applications of lead arsenat e were used to manage the pest (Boucher 1983), but this was proven to have carcinogenic effects on humans (Abernathy et al . 1999). Less harmful fungicides replaced lead arsenate, such as triazole, calcium hydroxide, and cyproconozole (Waller et al. 2007). However, such treatments have negative environmental effects and are not accepted for organic and shade grown certification (Staver et al. 2001). Biological control agents, such as fungicides, present a potentially effective and environmentally friendly me ans of controlling crop infestation (Butt 2000). Trichoderma harzianum is an Ascomycete in the family Hypocreaceae. Strains of this species are used to fight many crop infestations (Howell 2003). It is one of few biological control agents to reach notewort hy levels of commercial sales, totaling around $3 million world wide in 1999 (Harman 2000). T. harzianum is effective in controlling the M. citricolor infestation of coffee plants (Vargas 1984) and has the potential to increase crop yields by improving the soil conditions and crop health (Chang 1986). The aim of this study was to survey the presence of M. citricolor on coffee plants at the forest edge and along transects into the plots and to determine if applications of T. harzianum to the forest edge wou ld reduce the onset of M. citricolor in the neighboring plot. I hypothesize T. harzianum perimeter applications will decrease the onset M. citricolor on the adjacent coffee plants and that the presence of M. citricolor will decrease with increased distance from the forest. METHODS Study Site This study was conducted on one coffee farm in the Monteverde region of the Puntarenas Province of Costa Rica at an altitude of about 1325 m. It was carried out in October and November of 2009. Fourteen coffee plots were chosen for their prevalence of coffee plants over one meter tall that were adjacent to forest or windbreak . The windbreaks and forests were a mixture of small shrubs and trees all over three meters in depth. All the coffee plants were Coffea arabica .
Edge survey and treatments The row of 15 coffee plants most contiguous to the forest was surveyed for the presence of M. citricolor , alive and dead. M. citricolor was identified by the small brown grey spots that it creates on the coffee leaves. The averages of the number of spots on each plant were used to order the plots by level of infection and alternately assign them as treated or untreated. All leaves with living M. citricolor were removed from the plants before treating. The treatment consis ted of one liter of T. harzianum , purchased from the Santa Elena Coop., two liters of sugarcane juice, purchased from El Trapiche sugar farms, and thirteen liters of water. The sugarcane juice acted as an adhesive and a source of carbohydrates for the T. h arzianum. The control consisted of two liters of sugarcane juice and fourteen liters of water. Two liters of the treatment was applied to the forest edge adjacent to each treated plot. Each forest edge adjacent to the untreated plots was sprayed with two l iters of the control. The treatments were applied evenly from the ground to a height of two meters on the trees and shrubs along the total length of each studied perimeter. The applications were done with a hand pumped, backpack sprayer (bomba espalda). I t is important to note that the coffee plots did not directly receive treatment; only the bordering forest edges were treated. Management of the were resurv eyed for the presence of live M. citricolor spots identified by the small yellow fruiting bodies on the brown spots. These levels were then compared to the pretreatment levels to determine the effects of the treatment. Transects Transects, perpendicula r to the forest edge, were taken on ten of the coffee plots. Each plot had had four coffee plants: at 0m, 2m, 4m, and 8m from the forest edge. The plants were surveyed for the presence of M. citricolor spots , alive and dead. Analysis Two tailed parametric t tests were performed on the pre and post treatment edge surveys to determine differences between treated and untreated plots. Transects were averaged, and a regression was performed on these averages to determine variation of infestation from the forest edge. RESULTS The pretreatment surveys of M. citricolor ensured that there was no significant difference between the previous levels of infestation in th e treated and untreated plots, with mean infestation values of 44.0 and 54.0 spots per plant, respectively (Fig. 1; t = 0.62, df = 12, P = 0.55). The maximum infestation observed was a plant with 253 spots and every surveyed plant had at least a few dead s pots. The post treatment average occurrence of fruiting M. citricolor for treated and untreated plots was 2.7 and 3.2 spots per plant, respectively. The maximum amount of live M. citricolor observed on a plant after treatment was 21 spots for treated and 1 7 spots for untreated, while many plants from each had no living M. citricolor . The treated and untreated plots were not significantly different in their post treatment levels of live infestation (Fig. 1; t = 0.38, df = 12, P = 0.71). Transects showed a si gnificant decrease in M. citricolor infestation with increased distance from forest edge (Fig. 2; F = 21.72, df = 1,2, P = 0.04).
FIGURE 1. Mean ( SEM) number of M ycena citricolor spots on coffee plants before ( live and dead) and after ( live) treatments for untreated ( N = 7) and treated ( N = 7) plots. FIGURE 2. Mean ( SEM) number of M ycena citricolor spots on coffee plants at varying distance from forest edge ( N = 30 / distance) with best fit linear regression. B efore treatm ent A fter
DISSCUSION The very low occurrence of M ycena citricolor fruiting bodies on the coffee after the treatments indicted that there were limiting factors of its onset more momentous than the presence of Trichoderma harzianum on the forest edge. Aspects such as humidity and amount of sunlight could have affected the onset of the fungus enough such that large variations between plots make the treatment statistically negligible over such a short time frame. Furthermore, the T. harzianum may not have had sufficient time to establish it self in the forest. The coffee plots that were studied had already been treated with T. harzianum . This highly established T. harzianum in the coffee plots would have a great effect in deterring the pest but not prevent it completely. Additionally, much t o my surprise, the crops were sprayed with Atemi about ten days prior to the treatment. Atemi is a mixture of two pesticides: triazole and cyproconozole. This would effectively kill most of the M. citricolor (Avelino et al. 2007), and it could possibly pr event the onset of the fungus too. Many factors could have led to the reduction of M. citricolor along the transects. Most of these factors are probably associated with the effects of the forest edge, such as level of sunlight, humidity, and temperature ( Beer 1987). Moreover, the fungal infestation could originate from the forest. Regardless, it is evident that the fungus is more persistent near the trees. This suggests that fungicide applications should be more concentrated at the forest edge. The method that the Santa Elena Coop. uses to apply T. harzianum to coffee plots consists of three applications spaced 45 days apart. This ensures that it establishes itself on the plant and in the soil and leaf litter. This study could improve with three T. harzian um applications to the forest edge instead of one. Ideally, the forest applications would occur pesticides. Furthermore, crop surveys should continue for a mu ch longer span of time after treatment. This would increase the post treatment levels of T. harzianum and help reduce the influence of variation in other factors on the results. ACKNOWLEDGMENTS For the use of their farm I thank the Vargas family, particu larly Guillermo for his insight and personal knowledge of the farm. For materials, information, and support I thank Diego Calderon at the Santa Elena Coop. Thanks to El Trapiche for sugarcane juice on short notice. Special thanks to Pablo Allen for insight on my project, and its significance, and for having endless patience. LITERATURE CITED Abernathy , C.O., Y.P. Liu, D. Longfellow, H.V. Aposhian, B. Beck, B. Fowler, R. Goyer, R. Menzer, T. Rossman, C. Thompson, and M. Waalkes. 1999. Arsenic: health eff ects, mechanisms of actions, and research issues. Environmental Health Perspectives 107(7): 593 597. Avelino, J., S. Cabut, B. Barboza, M. Barquero, R. Alfaro, C. Esquivel, J.F. Durand, and C. Cilas. 2007. Topography and crop management are key factors for the development of American leaf spot epidemics on coffee in Costa Rica. Phytopathology 97: 1532 1542. Beer, J. 1987. Advantages, disadvantages and desirable characteristics of shade trees for coffee, cacao and tea. Agroforestry Systems 5: 3 13. Beer, J., R. Muschler, D. Kass, and E. Somarriba. 1998. Shade management in coffee and cacao
plantations Agroforestry Systems 38 : 139 16. Boucher, D.H. 1983. Agriculture: Coffee . In Janzen D.H., ed. 1983. Costa Rican Natural History . The University of Chicago Press , Chicago. Butt, T.M. 2000. Fungal Biological Control Agents. Pesticide Outlook, October 2000: 186 191. Chang, Y.C., Y.C. Chang, R. Baker, O. Kleifeld, and I. Chet. 1986. Increased growth of plants in the presence of the biological control agent Trichoder ma harzinum . Plant Disease 70: 145 148. Harman, G.E. 2000. Myths and Dogmas of Biocontrol: Changes in Perceptions Derived from Research on Trichoderma harzinum T 22 . Plant Disease 84 (4): 377 393. Howell, C.R. 2003. Mechanisms employed by Trichoderma speci es in the biological control of plant diseases: the history and evolution of current concepts. Plant Disease 87(1): 4 10. Staver, C., F. Guharay, D. Monterroso, and R. G. Muschler. 2001. Designing pest suppressive multistrata perennial crop systems: shade grown coffee in Central America. Agroforestry Systems 53: 151 170 Sequeira, L. 1958. The host range of the Mycena citricolor (Berk & Curt) Sacc. Turrialba 8(4): 136 147. U.S. Department of State. July 2009. Background Note: Costa Rica. Retrieved October 20 09. Retrieved from: http://www.state.gov/r/pa/ei/bgn/2019.htm Vargas, E. 1984. Interaction of biological and chemical treatment in the control of "ojo de gallo" disease (Mycena citricolor ) in coffee. Agronomia Costarricense 8(2): 91 97. Waller, J.M., M. Bigger, and R.A. Hillocks. 2007. Coffee Pests, Diseases and Their Management. CAB International, Oxfordshire, UK. Wellman, F.L. 1950. Dissemination of Omphalia leaf spot of coffee. Turriable 1(1): 12 27
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Gallagher, Nicholas, J.
Efectos de las aplicaciones perimetrales de Trichoderma harzianum para prevenir el Mycena citricolor en las plantaciones de Coffea arabica
Effects of Trichoderma harzianum perimeter applications on preventing Mycena citricolor in Coffea arabica plots
This study examined the fungus Trichoderma harzianum, a proven effective biological control agent, for its potential of preventing the forest and windbreak instigated onset of the fungus Mycena citricolor in coffee plots. Fifteen plots on one farm in the Monteverde region of Puntarenas, Costa Rica were surveyed for the presence of M. citricolor on coffee near forest edges and along transects away from the forest. T. harzianum was applied to the
forest edge on half of the plots, and the coffee plants near the edge were resurveyed for new infestations of M. citricolor. No significant difference between the treated and untreated plots was found. Other factors such as humidity and light exposure may have influenced the onset enough to nullify the fungicides effects over such a short length of time. A regression analysis of the transects confirmed that the presence of M. citricolor decrease with increased distance form the forest. This may also be attributed to the levels of light and humidity along the transects. Less time limited experiments may still prove edge applications of T. harzianum an effective method for inhibiting M. citricolor inception in coffee plots.
Este estudio examina el hongo Trichoderma harzianum como un agente efectivo del control biolgico, por su potencial de prevenir el hongo Mycena citricolor en plantaciones de caf. Quince cuadrculas en una finca en la regin de Monteverde en Puntarenas, Costa Rica se estudiaron para detectar la presencia de M. citricolor en las plantas de caf cercanas al bosque y a lo largo de los transectos alejndose del mismo. T. harzianum se aplic al borde del bosque y a la mitad de los cuadrantes, y las plantas de caf cercanas al borde del bosque se observaron para detectar nuevas infestaciones por M. citricolor. No se encontraron diferencias significativas entre los cuadrantes tratados y los no tratados. Otros factores como la humedad y la exposicin a la luz solar pueden influenciar el establecimiento y de esta manera anular el efecto del fungicida sobre un periodo corto de tiempo. Un anlisis de la regresin de los transectos confirma que la presencia de M. citricolor disminuye cuando aumenta la distancia al bosque. Esto se puede atribuir a los niveles de luz y humedad a lo largo de los transectos. Experimentos con una menor limitacin de tiempo pueden proveer informacin para determinar si la aplicacin de T. harzianum como un mtodo efectivo para inhibir el crecimiento de M. citricolor en plantaciones de caf.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
Tropical Ecology Fall 2009
Ecologa Tropical Otoo 2009
t Monteverde Institute : Tropical Ecology