T. asperellum growth on coffee waste DeVries & Van Tyne 1 Growing Trichoderma asperellum a natural fungicide, on coffee pulp and husks Amber DeVries University of California, Los Angeles Department of Microbiology, Immunology, and Molecular Genetics Georgia Van Tyne Department of Molecular, Cellular, and Developmental Biology University of California, Santa Barbara EAP Tropical Biology and Conservation, Fall 2017 13 December 2017 ABSTRACT Coffee farms often rely on chemical fungicides to control fungal crop diseases such as ojo de gallo ( Mycena citricolo r ) and red rust ( Hemileia vastatrix ). Trichoderma is a genus of fungus known to parasitize other fungi and it can be used to treat these crop diseases. Research has already proved that Trichoderma spp. can be cultivated on various organic substrates. Our goal was to help coffee farmers discover which coffee waste products could grow Trichoderma asperellum best. To do this, we collected three coffee by p, to create eight different coffee material test substrates. In addition to these test substrates, we chose goat waste and rice as test substrates The three test substrates that had the highest percent colonization of T. asperellum in order, were: (1) 50% old pulp and 50% coffee husk, (2) 75% old pulp and 25% coffee husk, and (3) 50% young pulp and 50% coffee husk. We saw conidial pigmentation indicating a m ore mature stage of Trichoderma growth in rice only. The pH had an optimal range of percent colonization of T. asperellum from about 5.5 7.5. Meanwhile, moisture had no observed correlation with percent colonization. Due to the colonization success of our T. asperellum we know it is possible for T. asperellum to grow on coffee byproducts in a non laboratory setting. Future research should study how long the T. asperellum needs to grow on the test substrates discussed in this experiment before it is concent rated enough for use as a fungicide and also how to make this procedure more effective for use on a larger scale. Cultivo de Trichoderma asperellum un fungicida natural, sobre pulpa y cscaras de caf RESUMEN Las fincas de caf dependen a menudo de fungicidas qumicos para controlar enfermedades fngicas de cultivos como ojo de gallo ( Mycena citricolor ) y roya ( Hemileia vastatrix ). Trichoderma es un gnero de hongo conocido por parasitar otros hongos y puede usarse para tratar estas enfermedades en l os cultivos. Estudios previos han demostrado que Trichoderma puede ser cultivada en diferentes sustratos orgnicos. Nuestro objetivo era ayudar a caficultores a descubrir
T. asperellum growth on coffee waste DeVries & Van Tyne 2 qu desechos de caf podran servir para cultivar Trichoderma asperellum Con este fi n, colec tamos diferentes para crear ocho sustratos. Adems de estos sustrato s, utilizamos boiga de cabra y arroz. Los tres sustratos que tuvieron la colonizacin ms alta de T. asperellum en orden, fueron: (1) 50% pulpa vieja y 50% cscara de caf, (2) 75% pulpa vieja y 25% cscara de caf, y (3) 50% pulpa joven y 50% cscara de caf. Observamos pigmentacin conidial en arroz solamente. La pigmentacin indicaba una etapa ms madura de crecimiento de Trichoderma El pH tena un rango ptimo del porcentaje de colonizacin de T. asperellum de aproximadamente 5.5 7.5. El porcentaje d e humedad no tuvo correlacin con el porcentaje de colonizacin. Debido al xito de colonizacin de nuestro T. asperellum sabemos que es posible que crezca en subproductos de caf sin tener que ser cultivado en laboratorio. Estudios futuros podran determ inar cunto tiempo necesita T. asperellum para crecer en los sustratos analizados en este experimento antes de que sea lo suficientemente concentrado para usarse como un fungicida y cmo hacer este procedimiento ms eficaz para un uso a gran escala. Coffee is the second most traded commodity in the world, only falling behind petroleum (Pushpa, 2012). Cultivation of this crop is not easy; coffee plants are susceptible to the fatal fungal diseases ojo de gallo ( Mycena citricolor ) and red rust ( Hemileia vastatrix ). These diseases wreak the most havoc in moist conditions. For countries that have intense wet periods and export significant amounts of coffee, such as Costa Rica, this poses a significant problem. In order to fight these fungal diseases and sav e their crops, many farms employ synthetic chemical fungicides that have the potential to harm the environment and human health (Martinez Toledo, 1998, option. Tr ichoderma is a genus of fungi that has evolved to parasitize other fungi (Harman, 2017). Many species in the genus Trichoderma possess fungicidal properties, especially Trichoderma asperellum which can parasitize 19 genera of other fungi (Harman, 2004 & W u, 2017). Coffee farms such as Caf de Monteverde, a sustainable coffee farm in Monteverde, Costa Rica, use Trichoderma spp. to eliminate risk of ojo de gallo and red rust, but many do not. According to staff at Caf de Monteverde, there are two limiting f actors preventing otherwise organic farms from replacing chemical fungicide use with this biocontrol method. These are its cost of $10 per kilogram, expensive for a coffee farm of many hectares, and its lack of availability (S. Barrios, pers. comm.). Trich oderma spp. sold for agricultural biocontrol use are usually grown in tubes and plates of potato dextrose agar (PDA) before they are transferred to rice, the substrate on which they are distributed (Dr. M. Obregn, pers. comm.). This method of growth is hi ghly specific to a laboratory setting and inaccessible to farms. However, Trichoderma spp. that are useful for biocontrol have been successfully cultured on various organic materials, such as maize meal, rice husk, sawdust, and wheat bran (Singh, 2007), although a method specific to utilizing coffee byproducts has yet to be simplified and made accessible to small farms. Fung i outside of the genus Trichoderma have been grown using coffee pulp (Rathinavelu, 2005 & Martnez Carrera, 2000), an organic waste that is readily available at Caf de Monteverde and other coffee farms. After growing the Trichoderma sp. cheaply on these
T. asperellum growth on coffee waste DeVries & Van Tyne 3 a Our goal is to provide a method for small scale organic farms to produce their own affordable supply of Trichoderma for use as a biocontrol agent. By working with sev eral easily accessible organic substrates we hope to answer the question: what mixes of coffee waste substrates work best to grow Trichoderma asperellum ? MATERIALS AND METHODS We collected three coffee byproducts from Caf de Monteverde: young coffee pulp, old coffee pulp, and coffee husks. The young coffee pulp was one week to two months old and the old coffee pulp was nine months to one year old. The young coffee pulp was more acidic and contained more liquid than the dry old coffee pulp. We collected fre sh goat waste from Caf de Monteverde. We chose goat waste as an additional test substrate because it is a nutrient rich waste material. Lastly, we chose rice as an additional test substrate because Trichoderma spp. are known to grow on cooked rice ( Cavalcante, 2008 ). We chose ten different mixes of substrates (Table 1, Figure 1). Table 1: Composition of the ten test substrates used in this experiment and their substrate codes. In this paper, test substrates are referred to by their substrate code. Al l percentages determined by volume. Substrate code Substrate composition YP young coffee pulp 75 YP 75% young coffee pulp + 25% coffee husk 50 YP 50% young coffee pulp + 50% coffee husk 25 YP 25% young coffee pulp + 75% coffee husk OP old coffee pulp 75 OP 75% old coffee pulp + 25% coffee husk 50 OP 50% old coffee pulp + 50% coffee husk 25 OP 25% old coffee pulp + 75% coffee husk Rice cooked rice Feces goat waste
T. asperellum growth on coffee waste DeVries & Van Tyne 4 Figure 1: Composition of ten test substrates and their corresponding substrate codes. We placed each substrate type into four separate bags with 750 mL of substrate each. The four bags were referred to as replicate 1 (R1), replicate 2 (R2), and replicate 3 (R3), and control. Controls were treated the same as the three replicates, but no T. asperellum was added. Pasteurization and inoculation of test substrates To pasteurize these materials before inoculating them with T. asperellum we floated e the pasteurization process ( van Loenen, 2003) We used this pasteurization to prevent other microorganisms already present in the substrate from outcompeting the Trichoderma asperellum for growth. After pasteurization, we labeled each bag with the type o f substrate and the replicate number and allowed them to cool at room temperature overnight. We sterilized PVC pipes 10 cm long and 2 cm in diameter. We did this by either (1) boiling for 10 minutes or (2) by washing with first 10% bleach, then 70% ethanol and finally rinsing with distilled water. We ordered one kilogram of T. asperellum from Laboratorios Dr. Obregn (San Francisco, Heredia Province, Costa Rica). The fungi came as dark green spores on rice. Once the bags had cooled after pasteurization, w e used this T. asperellum to inoculate the substrates, adding 10 grams of the rice with T. asperellum spores to each of the sterilized bags. We inserted the sterilized PVC pipe into the opening of each bag. We blocked the inside of the PVC pipe with cotton to allow airflow into the bag with less risk of contamination. Lastly, we used a r ubber band to tightly seal the plastic bag around the PVC pipe (Figure 2).
T. asperellum growth on coffee waste DeVries & Van Tyne 5 Figure 2: Experimental setup used in this experiment. Note that we pasteurized clear plastic bags and substrates and sterilized the PVC pipes before we added any T. asperellum to the mixes. Measurements On the first day of growth, we measured the pH and p ercent moisture of each bag. We took these measurements using a soil moisture meter and a pH meter with temperature capacities. After the first day, we only measured R1 of each test substrate every other day to decrease the risk of possible contamination f or most of the bags. After one week of growth at outside temperature rate. On the eleventh and final day of growth, we spread the contents of each individual bag ou t in a box and visually approximated the percentage of the substrate that had been colonized by T. asperellum We also took a picture of each spread. By measuring the approximate percent colonization per replicate of each substrate, we were able to qualita tively rank which substrates are best for T. asperellum proliferation. This method of estimating the amount of Trichoderma was used as an alternative to the Neubauer counting chamber, which did not work for this experiment. We would have used a Neubauer chamber to view a dilution of our substrate under the microscope and count the mycelium and spores in each sample. Because each grid on the Neubauer chamber is an exact volume, this mycelium and spore count would have allowed us to quantitatively determin e the exact amount of mycelium/mL of substrate. However, the complexity of the substrates and large, diverse amounts of microscopic material in each substrate made it difficult to distinguish the T. asperellum from other microscopic materials. Plat ing for Microscopic Confirmation of T. asperellum We sterilized plates and potato dextrose agar (PDA) in a pressure cooker used as an dishes with 25 mL PDA per plate. On day 9 of growth, we removed 1 mL of mycelium from replicate 1 of 75 YP and diluted it in 9 mL of water. We also diluted 1 mL of T. asperellum from Laboratorios Dr. Obreg n. in 9 mL of water. We plated 25 L of each solution on opposite ends
T. asperellum growth on coffee waste DeVries & Van Tyne 6 of the same plate. We left the plate in an incubator at 28 for two days. We then used a metal spatula to remove a small amount of both laboratory and experimental T. asperellum from the plate onto a glass slide. We looked at these slides with a compound microscope. S tatistical analysis of results We compared amount of growth of fungi (from percent colonization) by substrate using Kruskal Wallis to see if substrate growth differed significantly by substrate. We also used the Tukey Kramer test to group the substrates in to rank our various substrates by success of T. asperellum growth. This ranking is intended as a tool for coffee farms to use when deciding how to grow T. asperellum RESULTS Growth of Trichoderma by substrate T. asperellum grew visible mycelium on 50 OP, 75 OP, and 50 YP significantly better than on other substrates ( Figure 3, Tukey Kramer, p< 0.05,). We recorded t he growth of all substrates as measured by percent colonization with the initial pH a nd moisture of each test substrate (Table 2). The difference in the controls and experimental bags was clear after three days of growth. While on the second day of growth, we observed white mycelial growth on all replicates of 75 YP and 50 YP, there was no mycelial growth on any of the controls, even by the third day. We observed this qualitatively and did not open the bag to avoid introducing contamination. Because of this, although there were eventually small amounts of growth in some of the controls when opened (Table 2), the large amount of growth in the experimental replicates observed after three days and that grew thereafter was likely Trichoderma
T. asperellum growth on coffee waste DeVries & Van Tyne 7 Figure 3: Average percent colonization of each substrate type and respective controls. Error bars repr esent one standard deviation. Letters indicate significant groupings from Kruskal Wallis and Tukey Kramer analysis. For substrate codes, see table 1. Table 2: Substrates listed in order of decreasing average percent colonization. A control was not perform ed for goat feces because no colonization was observed on any of the replicates. For substrate codes, see table 1. Substrate Average percent colonization Percent colonization of control Initial pH Initial percent moisture 50 OP 88.3 2.9% 0% 5.44 3.13% 75 OP 78.3 16.1% 5% 6.80 9.53% 50 YP 48.3 31.8% 5% 5.56 >50% 25 YP 30.0 26.0% 0% 5.33 >50% 75 YP 30.0 35.0% 10% 6.50 >50% Rice 16.7 10.4% 0% 6.34 12.90% OP 13.3 2.9% 5% 8.47 1.27% 25 OP 6.7 7.6% 0% 4.98 22.93% YP 1.7 2.9% 0% 8.08 >50% Feces 0.0 0.0% NA 8.36 >50%
T. asperellum growth on coffee waste DeVries & Van Tyne 8 Substrate characteristics contributing to T. asperellum growth: pH and moisture The growth of T. asperellum as measured by percent colonization was the highest within the optimal pH range of 5.5 7.5 (Singh, 2014). The four substrates with the lowest average Trichoderma spp (Figu re 4). The growth of T. asperellum was not affected by percent moisture of the substrate (Figure 5). Figure 4: Average initial pH of each substrate affects the final average percent colonization. The three substrates with the highest percent colonization were all within about 1 pH unit of each other.
T. asperellum growth on coffee waste DeVries & Van Tyne 9 Figure 5: Initial percent moisture did not have a clear effect on average percent colonization. The moisture meter did not measure above 50%. Substrates measured as at least 50% moisture are graphed as 50% m oisture. Identification of experimental mycelium as Trichoderma We compared the laboratory T. asperellum to our experimental T. asperellum and found conid ial mycelium in both. Dr. Obreg n confirmed both samples in these photos are T. asperellum Figure 6A: Photo of laboratory T. asperellum conidial mycelium at 4x magnification. Figure 6B: Photo of experimental T. asperellum conidial mycelium at 10x magnification. Identification confirmed by Dr. M. Obregn (pers. comm.).
T. asperellum growth on coffee waste DeVries & Van Tyne 10 DISCUSSION T. asperellum grew best in 50 OP (50% old coffee pulp, 50% coffee husk), 75 OP (75% old coffee pulp, 25% coffee husk), and 50 YP (50% young coffee pulp, 50% coffee husk) (Figure 3). In this section, we consider the factors that made these three mixtures suitable test su bstrates as well as important points from this experiment for farms that intend to cultivate their own Trichoderma asperellum Trichoderma spp. grow from a wide range of pH, from pHs of 4 8 (Singh, 2014). Maximum growth occurs between pH 5.5 the more acidic end of the known maximum growth range. The four substrates with the least growth were more than 0.5 pH units away from known growth range (Figure 4). We did not find a single optimal pH value. This and the fact that we did not find successful substrates at the more basic end of the optimal pH range is likely due to other factors that affect Trichoderma growth such li ght, calcium, moisture, and oxygen (Kubicek, 1998 & Steyaert, 2010). Moisture and its effect on fungal growth The best three test substrates had a wide range of moisture (Figure 5). However, it seems as if excessively high or low moisture content can nega tively affect T. asperellum growth. The 25 OP showed minimum amounts of growth and had a 1.27% moisture content. Meanwhile, YP and feces also had minimal growth, but had moisture content above 50%. All three of these test substrates that grew the least amo unt of T. asperellum had extreme moisture contents in addition parameter: pH or moisture content. Relatively dry (moisture content of approximately 3 15%) substrate s could have each had a higher percent colonization due to the ease of mixing the sample. When we incorporated T. asperellum into the old pulp and coffee husk mixtures (the two driest coffee byproducts), it was much easier to evenly distribute the spores throughout the substrate. In the wetter young pulp mixtures, many clumps had very obvious T. asperellum growth, but it was not evenly distributed, leaving patches without any T. asperellum This patchy distribution may have contributed to the lower and more varied percent colonization of the best young pulp mixtures. Besides this, it was likely that moisture by itself could have affected growth of Trichoderma asperellum since moisture has been shown to affect spore germination times and rates of growth in other fungi (Ayerst, 1969). Conidial pigmentation and percent colonization in rice When measuring percent colonization on the last day of growth, we noticed that T. asperellum on rice had patches of dark green that no other test substrates showed. This coloration indicates that Trichoderma sp. has entered a more mature part of its life cycle and is about to sporulate (Kubicek, 1 998 & Steyaert, 2010). Nutritional factors, like the ratio of C:N in a substrate significantly affect conidiation of Trichoderma spp. (Steyaert, 2010) It is possible that the nutritional profile of rice promotes conidiation more than the nutritional profiles of coffee byproducts, which is why characteristic dark green was not seen on any of the coffee byproduct test substrates. The T. asperellum observed in the rice test substrate was more mature, as shown
T. asperellum growth on coffee waste DeVries & Van Tyne 11 by its green color ation, than the T. asperellum obs erved in other test substrates, such as 50 YP, that were more visibly colonized than rice. Although T. asperellum grown in rice may reach mature stages of growth faster, using coffee byproducts to grow the fungus is recomme nded because of the sheer amount of coffee waste that farms produce. For every two tons of coffee produced, one ton of coffee pulp and 0.36 tons of coffee husks are generated: waste that could be used to grow T. asperellum (Roussos, 1995 & Adams, 1981). Ou r visual method of quantifying growth introduced some bias. The greater visibility of green in rice is due to its color contrast. This may be why we did not see dark conidial pigmentation in our dark coffee byproduct substrates. By contrast, the mycelium w as white and thus difficult to see in the rice. Thus, the recorded T. asperellum growth in rice was only what we could see: the mature green sections. This may have accounted for the relatively small percent colonization measured in rice despite the maturi ty of the green T. asperellum Use of experiment for future farms We attempted to grow T. asperellum in a manner that would be possible for farms to imitate. However, temperature was a limit. T. asperellum mycelium growth is low at 20 and peaks around 27 (Domingues, 2016). Because of the constant 20 climate throughout the experiment, we had to move our test substrates from the outdoors into an incubator to promote more growth within a limited time period This method of using an incubator would be inaccessible to most farms. T he method we reported will work best during hot months or inside of an insulated space. Future research must also determine how long Trichoderma asperellum needs to grow before it i s concentrated enough for use as a fungicide. Farms would also benefit from further study on how to increase the efficiency of this procedure for use on a larger scale, making it more practical for farms to begin enough cultivation to replace chemical fung icides. Possible contamination Because a diverse and large community of fungal spores are present in large quantities in the air (Frhlich Nowoisky 2009 & Bauer, 2008), it is inevitable that the test substrate samples we had taken outdoors from Caf de Mo nteverde contained fungal spores. Some fungal spores can pasteurization process did not guarantee that all fungal competition was eliminated from our test substrates. However because of the aggressive nature of T. asperellum and its ability to parasitize other fungi ( Harman, 2004 & Wu, 2017 ), small amounts of contamination were likely to have been overtaken by the fungicidal fungus, and all mycelium we observed was T. asperel lum (Dr. M. Obregn, pers. comm.). Future research including cost analysis We have confirmed that it is possible to effectively grow T. asperellum on coffee byproducts. However, the effectiveness of the experimental T. asperellum as a fungicide has not been tested. One way the experimental T. asperellum differs from the lab grown T. asperellum is that the lab grown T. asperellum comes to farms in spores and the bulk of the experimental T. asperellum was still mycelium by the end of the growth period. Although the experimental Trichoderma would likely have sporulated with more time, after 10 days of growth it was still
T. asperellum growth on coffee waste DeVries & Van Tyne 12 mostly in the mycelial form. Thus, future research should also compare the effectiv eness of experimental mycelium to laboratory spores as a fungicide. This would give coffee farms more information to aid in deciding whether to purchase T. asperellum or to grow it themselves. A goal of our experiment was to make T. asperellum cultivation possible for farms as a cheaper alternative to buying Trichoderma from laboratories. In order to know if the methods in this experiment have succeeded in making this process effective and cheaper, the quantity of spores grown must exceed the 10g of T. asp erellum spores originally added, increasing in amount and concentration as the T. asperellum colonizes the substrates. Because of the limited timeframe of our experiment, we were not able to wait and see sporulation in all test substrates. If the amount of spores grown with these methods shows a significant increase in the amount of T. asperellum present, this method is excellent for farms who wish to use a biocontrol fungicide but have a limitation due to cost. ACKNOWLEDGMENTS Our biggest thank you goes to our primary advisor Sofa Arce Flores who encouraged us even when we thought we had failed. Without her, we would not have completed our goals and would have smiled significantly less (p < 0.05). Thank you to Federico Chinchil la for his help as our secondary advisor and his bad jokes. Thank you to the staff at Caf de Monteverde (especially Daniel and C sar ) for enthusiastically supporting our research by generously donating their coffee byproducts and answering our questions. This experiment would not have been possible without Dr. Obregn -he answered many of our questions and confirmed that we were indeed growing Trichoderma We owe a thank you to Cooperative Tarraz for their kind willingness to answer our questions. We woul d also like to thank the Monteverde Institute and Luisa Chinchilla for letting us use their lab space. Thank you to Frank Joyce, Emilia Triana, Andrs Camacho, and Flix Salazar for making this learning experience as enjoyable as possible. Also, thank you to Stephanie Li and Yibing Yu, who peer reviewed our paper for us. Lastly, thank you to all the other students in our program for being supportive friends and tolerating the smell that our experiment left in the Monteverde Institute lab. Thank you to empan adas for being a constant source of joy in our lives. LITERATURE CITED Adams, M. R. and Dougan, J. 1981. Biological management of coffee processing Trop. Sci 123 178 196. Ayerst G. 1969. The effects of moisture and temperature on growth and spore germination in some fungi. J. Stored Prod. Res 5 (2), 127 141. Baeur, H., Schueller, E., Weinke, G., Berger, A., Hitzenberger, R., Marr, I. L., and Puxbaum, H. 2008. Significant contrib utions of fungal spores to the organic carbon and to the aerosol mass balance of the urban atmospheric aerosol.. Atmos. Environ 42 (22), 5542 5549.
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