Variability of m ycorrhizae in Coffea arabica Sime 1 Variability of mycorrhizal colonization amongst varieties of Coffea arabica in Monteverde, Costa Rica McKenzie Sime Department of Ecology, Evolution, and Marine Biology University of California Santa Barbara EAP Tropical Biology and Conservation Program, Fall 2017 15 December 2017 ABSTRAC T Mycorrhizae are a kind of fungus that forms symbiotic relationships with plants. They help plants deal with environmental stressors such as drought and disease. Almost all plants have this symbiosis with mycorrhizae, coffee being included in this group. Coffea arabica a species of coffee, is grown in tropical regions and multiple varieties have been developed to increase the productivity of the plants. However, tropical regions, like Monteverde, Costa Rica where this study was conducted, are going to be some of the ha rdest areas hit by climate change. Plants are going to be depending more and more on their mycorrhizae as these environmental stressors become stronger as the climate changes. I looked at the variability of mycorrhizal colonization amongst different coffee varieties. I also tested the effects of vermicompost on mycorrhizal colonization in an effort to find a simple and possibly applicable method to increase colonization in plants. There was a significant difference in mycorrhizal abundance between varieties with the CatuaÂ’ variety of coffee showing almost twice as much colonization as the Venecia variety (6.67% as opposed to 3.33%). There was also a significant change in the colonization of plants that were vermicomposted while control plants that were give n no vermicompost did not see a significant change (13.06% as opposed to 7.78%). These results might indicate that vermicompost is a viable method of increasing mycorrhizal colonization in Coffea arabica Variabilidad en colonizaciÂ—n de micorrizas en dife rentes variedades de Coffea arabica en Monteverde, Costa Rica RESUMEN Las micorrizas son hongos simbiÂ—ticos de las plantas. Se ha demostrado que ayudan a las plantas a lidiar con factores estresantes ambientales como la sequÂ’a y las enfermedades. Casi todas las plantas tienen esta simbiosis con micorrizas, incluyendo el cafÂŽ Coffea arabica una especie de cafÂŽ, se cultiva en regiones tropicales, y se ha desarrollado mÂœltiples variedades para aumentar la productividad de las plantas. Sin embargo, las regiones tropicales, como Monteverde, Costa Rica, donde se realizÂ— este estu dio, serÂ‡n algunas de las Â‡reas mÂ‡s afectadas por el cambio climÂ‡tico. Las plantas dependerÂ‡n cada vez mÂ‡s de sus micorrizas a medida que estos factores ambientales estresantes se vuelven mÂ‡s fuertes a medida que cambia el clima. ObservÂŽ la variabilidad de la colonizaciÂ—n de micorrizas entre las diferentes variedades de cafÂŽ. TambiÂŽn estudiÂŽ los efectos del vermicompost en la colonizaciÂ—n de micorrizas en un esfuerzo por encontrar un mÂŽtodo simple y posiblemente aplicable para aumentar la colonizaciÂ—n en la s plantas. Hubo una diferencia significativa en la abundancia de micorrizas entre variedades. La variedad de cafÂŽ CatuaÂ’ muestra casi el doble de colonizaciÂ—n que la variedad Venecia (6.67% en comparaciÂ—n con 3.33%). TambiÂŽn hubo un cambio significativo en la colonizaciÂ—n de plantas despuÂŽs de aplicar el vermicompost, mientras que las plantas de control que no recibieron
Variability of m ycorrhizae in Coffea arabica Sime 2 vermicompost no vieron un cambio significativo (13.06% en comparaciÂ—n con 7.78%). Estos resultados pueden indicar que el vermicompost es u n mÂŽtodo viable para aumentar la colonizaciÂ—n de micorrizas en Coffea arabica Mycorrhizae is a fungus that has a symbiotic relationship with plants. Mycorrhizae a fungus that depends on its host plant to survive helps plants to obtain key nutrients, such as magnesium, phosphorous, sulfur, and nitrogen that would otherwise be hard to access for the price of sugars (Harrier, 2001). It is this symbiosis that allowed plants to leave the oceans and colonize land (Sim on et al., 1993). Now plants and the fungus are still closely linked, with over 80% of plants still retaining the ability to host the fungus in their roots (Harrier, 2001). Today, mycorrhizae helps plants to cope with harsh environments that would otherw ise be uninhabitable. With the help of mycorrhizae plants are able to absorb water more effectively This is helpful when plants must endure tough abiotic conditions, such as droughts (Makoi and Ndakademi, 2009; Subramanian et al., 1995). The mycorrhizae can also help plants with fighting diseases, fungi, and pests that could potentially harm the plant (Newsham et al., 1995, Arancon et al., 2005). In the future, mycorrhizae may be even more necessary. Climate change is begin ning to affect the environment t hat the plants have evolved in. It is projected that Central America will be one of the hardest areas hit by climate change in the world (Giorgi, 2006). Coffee production does not respond well to increases in temperature, decreasing productivity significan tly (Gay et al., 2006). On top of decreased productivity, instances of drought, disease, and pests will increase as the climate heats up (Rosenzweig et al., 2001). The future of the natural enemies of the pests that affect humans' crops is also uncertain and the outlook is not favorable (Thomson et al., 2010). One of coffee's primary enemies, the broca beetle, is among the pests aided by the warming of the climate and the effects of this are already being felt in Africa (Jaramillo et al. 2011). Coffee farmers are going to need the help of their plants' mycorrhizae colonies to survive the coming changes. Behavioral a daptation by humans is necessary to mitigate the effect of climate change on agriculture and prevent food insecurity in the future (Turral, 2008)). A relatively simple potential adaptation is the increased use of vermicompost. Vermicompost is a method of breaking down organic matter that utiliz es earthworms' natural talents of decomposition. This method allows for a quicker c onversion of coffee pulp waste into compost with a high nutrient content and affects the microbiome of the soil ( Orozco et al., 1994; Lemtiri et al., 2014). This healthy microbiome in the soil could play a part in strengthening the mycorrhizae of the plant s. Another possibility is that there is a variety of Coffea arabica that naturally has a higher abundance of mycorrhizae. L IFE Monteverde, a coffee farm located in the Puntarenas Province of Costa Rica, is interested in sustainable methods of farming to help lessen the effects of climate change. This farm and most farms in Costa Rica traditionally grow the CatuaÂ’ and Caturra varieties of coffee (J.L. Vargas, pers. comm.). Currently, they are experimenting with two new varieties of coffee, ObatÂ‡ and Veneci a. CatuaÂ’, Caturra, and ObatÂ‡ have all been bred to have a dwarf mutation which allows for more plants per given area, while Venecia has longer branches (J.L. Vargas, pers. comm.). One advantage of Venecia though is that it matures later in the season, in January and February rather than in November and December (J.L. Vargas, pers. comm.). This is helpful to the farm because they can harvest after the rainy season and may be a possible way for them to adapt to climate change. ObatÂ‡, their other experimenta l variety, also has an advantage ; it has shown an ability to fight red rust, a fungus that targets coffee plants and can cause great
Variability of m ycorrhizae in Coffea arabica Sime 3 economic damage to a harvest (Camargo, 2010). However it is not known if the abundance of mycorrhizae differs between these varieties. Filling in this gap could provide insight into which of these varieties is more capable of adapting to the new conditions in the future. It is also not known what the effects of vermicompost are on the colonization of roots by mycorrhizae While plants are often inoculated and composted to improve their health, the effect of the compost on the success of the inoculation itself is often not investigated and inoculation is also not a reasonable solution on a large, agricultural scale due to hi gh costs (SÂ‡inz, 1998, Cardoso, 2006). In this study, I address ed whether ObatÂ‡, CatuaÂ’, and Venecia have different abundances of mycorrhizae I also discern ed if vermicompost has an effect on the density of mycorrhizae in Coffea arabica MATERIALS AND METHODS Field work : The site of the experiment was L IFE Monteverde, a coffee farm located at 1 3 00 m in the TilarÂ‡n Mountains of Puntarenas, Costa Rica. I chose three varieties of coffee for this experiment : ObatÂ‡, CatuaÂ’, and Venecia. The plants were five months old when the experiment began. They had been planted in bags containing 20% coffee pulp compost, 15% coffee shell compost, and 65% soil removed from land near the forest on the property (S. Barrios, pers. comm.). Trichoderma sp. (a fungus present in soil that helps suppress Ojo de Gallo or Rooster's Eye, a common fungus that afflicts coffee) was also added to this mixture (G. Vargas, pers. comm.). I began by flagging 12 plants in each variety. I chos e the plants randomly from each section, trying to keep the conditions (light, rain, etc.) consistent amongst the subjects. I did not move subjects from the display to avoid stressing out or damaging the plant s I flagged the chosen plants and gave them a unique label (O for ObatÂ‡, C for CatuaÂ’, V for Venecia and a number 0 12) I took two root samples by digging down next to the plant and removing an approximately 1 cm long root from the plant. I kept the roots of each plant separate in their own bags. Once I completed this, I applied coffee pulp vermicompost to the even numbered plants The odd numbered plants were left as a control. The compost had already been dried/ processed to not negatively affect soil moisture (J. Santamaria, pers. comm.) Vermicompost is dried because it is capable of retaining a large amount of water and, in the tropics, this can cause an increased incidence of root rot or fungal disease due to the already high humidity o f the environment I spread the vermicompost to cover the surface of the soil (about 1cm deep) and left the area directly around the stem free of the compost This prevented the conditions from becoming too humid around the stem (G. Vargas, pers. comm.). Lab work: First I cleared the roots and dyed the mycorrhiza e I rinsed the root sample in tap water to remove soil and other debris. I placed the sample in a 100 mL beaker with 10 20 mL of 10% KOH solution. I then placed the beaker containing the sample a nd the KOH solution in a bath of boiling water bath and allowed it to boil for 6 minutes. This step clears the cells in the root, leaving behind only cell walls and the mycorrhizae After 6 minutes, I then removed the sample from the beaker and rinsed it i n tap water once more. Following this, I placed the root in a 100 mL beaker with 10 20 mL of 5% blue ink vinegar solution. I placed the beaker in a boiling water bath for 4 minutes. After 4 minutes I removed the root sample and rinsed it in a 1% vinegar
Variability of m ycorrhizae in Coffea arabica Sime 4 so lution (Vierheilig et al., 1998). I kept the roots from each plant separate throughout this process. I then cut the root samples to be 1cm long. I mounted them on a slide and viewed them under a 10x ocular piece and a 10x objective. I assessed each root based on the percentage of its area that was colonized. I then took the average for the variety's percent colonization as a whole, percent colonization of those that did not have the vermicompost treatment, and percent colonization of those that did have t he vermicompost treatment. This followed Biermann and Linderman's method olo gy ( 1980 ) This is how I quantified changes in mycorrhizal abundance. I took samples again 5 days later, and once again 5 days later. For these two following collections I only col lected the roots and and did not apply vermicompost again I repeated the same process of dyeing and assessing the mycorrhizae for both of these samples. I analyzed the results using a t test to test for significant differences in colonization among varieties and a paired t test to test for significant changes in colonization within varieties over time. RESULTS CatuaÂ’ has a significantly higher st arting percentage of colonization than Veneci a has, 6.67% as opposed to 3.33% (Fig. 1, t(46) = 2.045, p = 0.047). The final difference between the composted plants and the control plants in the CatuaÂ’ variety was not significant. The final difference was a lso not significant in the Venecia variety. In the ObatÂ‡ variety, the difference was close to significant (t(22) = 1.773, p = 0.090). The difference in this variety could have possibly been significant if there were longer trials or more plants. The total difference between the final colonization percentages of composted and control plants was significant across all varieties (figure 2, t(71)= 2.708, p=0.009). For both CatuaÂ’ and Venecia, the plants that were vermicomposted had a larger increase in mycor rhizal colonization on average than those that were not vermicomposted. T he vermicomposted CatuaÂ’ plants had an increase of 6.67% 13.03% while the control plants had a 0% 11.28% (Fig. 3). Venecia composted plants had an increase of 13.3 % 11.65 % while the control plants had an increase of 9.17% 7.78 % (Fig. 4). The ObatÂ‡ variety does not hold true to this, as both the control and the treatment lost mycorrhizae, losing 7.5% in the vermicomposted plants and 2.5% in the control plants (Fig. 5). The first trial of this variety is what causes this decrease as it wa s unusually high compared to the other varieties. Due to an error with the identification of mycorrhizae I excluded this trial from the total calculations. The average increase in colonizat ion among all varieties ( not including Oba t Â‡'s first trial) is 7.6 4 % in composted plants and 3.19% in control plants (Fig. 2). I also tested the significance of the changes in mycorrhizal colonization from the initial colonization percentage to the final colonization percentage In the CatuaÂ’ variety, neither the composted or control plants had a statistically significant change in their colonization. In the Venecia variety, both the composted and the control plants had a statistically significant ch ange in their colonization. When the results were accumulated for both varieties the composted plants had a statistically significant positive change in mycorrhizal colonization while the control plants did not see a statistically significant positive change in mycorrhizal colonization.
Variability of m ycorrhizae in Coffea arabica Sime 5 FIGURES Fig. 1: A comparison of initial mycorrhizal abundance in the CatuaÂ’ and Venecia varieties E rror bars represent standard deviation (t(46) = 2.045, p = 0.047). Fig. 2: Percentage of mycorrhizal colonization in all varieties (excluding first trial of the ObatÂ‡ variety) and how it changed over time when composted Error bars represent standard deviation. The change in the composted plants' colonization was signific ant ( t(23) = 3.12, p < 0.05 ). Fig. 3: Percentage of mycorrhizal colonization in the CatuaÂ’ variety and how it changed over time with or without the addition of vermicompost Error bars represent standard deviation. Neither change was significant.
Variability of m ycorrhizae in Coffea arabica Sime 6 Fig. 4: Percentage of mycorrhizal colonization in the Venecia variety and how it changed over time with each treatment. Error bars represent standard deviation. The increases in both the composted and control plants was significant ( t(11) = 3.46, p < 0.05 for both). Fig. 5: Percentage of mycorrhizal colonization in the ObatÂ‡ variety and how it changed over time with each treatment. The error bars represent standard deviation. Neither change was significant. DISCUSSION When comparing the initial mycorrhizal colonization of the varieties, the CatuaÂ’ variety has a significantly higher colonization than the Venecia variety. The ObatÂ‡'s initial data was not included in this comparison as there was an error with the identific ation of mycorrhizae in this trial The colonization of the vermicomposted plants for all varieties combined was significantly higher than the plants that were not vermicomposte d. While all of the varieties did have an increase in mycorrhizae whether verm icomposted or not, the plants that were vermicomposted had a significant positive change in colonization overall while the plants that were left as controls did not. This might indicate that vermicompost does have a positive effect on mycorrhizae 's ability to colonize Coffea arabica There is also a significant difference in mycorrhizae 's ability to colonize different Coffea arabica varieties. This difference in mycorrhizal colonization between varieties could be explained by the different characteristics that the varieties display. One of the most important differences between Catua Â’ and Venecia is Venecia's late harvest season (J. Vargas, pers. comm.). The variety's tendency to take longer to mature could be a visi ble indicator of its weaker mutualism with
Variability of m ycorrhizae in Coffea arabica Sime 7 mycorrhizae. If the plants have less mycorrhizae supplying them with the nutrients that are hard for them to access on their own then it is very possible that it would take them a longer time to develop a fruit to maturity. The increase in coloniza tion can be explained by how the worms ingestion and excretion of the soil affects its microorganisms. Uningested soils have been shown to have microbiomes with different biomass and activity than soil that has been processed by worms (SchÂšnholzer et al. 1999). The gut of an earthworm greatly affects the populations of certain microorganisms as it grazes: digesting some, having no effect on some, and activating others (Pederson et al., 1993; Drake et al., 2007). The casts (excrement) of the worms then act as an inocula for these microorganisms that are activated, altering the microbial composition of the soil by giving some organisms an advantage and others a disadvantage (Brown, 1995). This alteration is most likely what causes the change in mycorrhizal co lonization after composting Worms have been shown to be a distributor of mycorrhizal fungi (Gange, 1993). Here the application of vermicompost has shown to be an effective method of increasing the colonization of mycorrhizae which is consistent with this literature. Worms also affect the nutrient balances of the soil that they ingest. In coffee pulp (the kind of compost that was used in this experiment), worms increase the amount of phosphorous, calcium, and magnesium available in the soil (Orozco et al ., 1996). Both phosphorous and magnesium are nutrients that mycorrhizae help plants absorb (Harrier, 2001). Also, i t is calcium that allows the mycorrhizae to communicate with the plant and begin colonization (Navazio, 2008). This increase in the nutrients that mycorrhizae help a plant absorb and the element that allows the fungus and the plants to communicate with eac h other most likely has a positive effect on the amount of mycorrhizae in the roots of the plants. Once again, the results of this study aligns with the literature mentioned on this topic. A factor that may have affected the results of this study is the short time period. While a change was seen in colonization, two weeks is not long in the life of a coffee plant that can live 20 30 years. Further research would need to be conducted to discern the true value of vermicompost to mycorrhizae whether the ben efit is transient or if the plants continue to have a strengthened mycorrhizae community. However there is literature that supports an increase in mycorrhizal colonization with properly applied vermicompost in clovers so it is likely that the same trend wo uld be observed in coffee (SÂ‡inz, 1998). The error in the initial percentages of the ObatÂ‡ variety also affected my ability to properly evaluate whether or not different varieties of coffee tend to have different amounts of colonization. However, I was sti ll able to answer that there are indeed differences between varieties of coffee since a strong difference was found between the other two varieties. There is also literature confirming that different species of plants have different amounts of colonization so it would not be unreasonable for this to be true (Bever, 1996). Therefore, with further experimentation, it would not be hard to define the different vari eties' abilities to host mycorrhizae Choosing plants that can host more mycorrhizae could be im portant in the future. In the tropics, increasing mycorrhizal abundance in crops is a possible way to aid plants' ability to absorb nutrients from tropical soils (Cardoso, 2006). Mycorrhizae helps plants to absorb the same nutrients that synthetic fertilizers provide and that are hard for plants to obtain on their own (Harrier, 2001). If farmers are able to help crops gain higher levels of mycorrhizal colonization there would be a reduced need for these h a rmful fertilizers and sustainable agricultu re could be a more reachable goal This is going to be especially important for Central America which is heavily dependent on agriculture, is going to be hit harder by climate change, and is seeing a
Variability of m ycorrhizae in Coffea arabica Sime 8 higher human population growth rate than the world avera ge (Leonard, 1987; Giorgi, 2006; Cincotta, 2000). Mycorrhizae could provide an alternative method to dealing with the often nutrient poor soils of tropical regions without cutting productivity or harming the unusually biodiverse habitats that often surroun d farms in these areas. ACKNOWLEDGEMENTS I would first like to thank SofÂ’a Arce Flores my primary advisor, for making this project possible by providing knowledge, transportation, advice, translation, and support. I would also like to thank Frank Joyce my secondary advisor for providing useful revisions and advice. I would also like to thank Laura Aldrich Wolfe who provided expertise on the identification of mycorrhizae in the roots. This project would not have been feasible without Guillermo Vargas al lowing me to conduct research on his farm. I would like to thank Justin Daniel as well for reviewing my paper for me. I would also like to thank Sergio Barrios, Jose Vargas, Jerson Santamaria, and Daniel Vargas for providing invaluable guidance and informa tion throughout the project. F i nally, I would like to thank the Monteverde Institute for providing the resources necessary for the completion of this project. LITERATURE CITED Arancon, N.Q., Galvis, P.A., Edwards, C.A. (2005). Suppression of insect pest populations and damage to plants by vermicomposts. Elsevier, 96, 1137 1142. Bever, J.D., Morton, J.B., Antonovics, J., Schultz, P.A. (1996). Host Dependent Sporulation and Species Diversity of Arbuscular Mycorrhizal Fungi in a Mown Grassland. Journal o f Ecology, 84(1), 71 82. Biermann, B., Linderman, R.G. (1981). Quantifying vesicular arbuscular mycorrhizae: a proposed method towards standardization. New Phytologist, 87, 63 67. Brown G.G. (1995). How do earthworms affect microfloral and faunal comm unity diversity? Plant Soil, 170(1), 209 231. Camargo, M. B. P. De. (2010). The impact of climatic variability and climate change on arabic coffee crop in Brazil. Bragantia, 69, 239 247 Cardoso, I.M., Kuyper, T.W. (2006). Mycorrhizas and tropical soi l fertility. Elsevier, 116, 72 84. Cincotta, R.P., Wisnewski, J., Engelman, R. (2000). Human population in the biodiversity hotspots. Nature, 404, 990 992. Drake H.L. & Horn M.A. (2007). As the worm turns: the earthworm gut as a transient habitat for soil microbial biomes. Annu. Rev. Microbiol., 61, 169 189. Gange, A.C. (1993). Translocation of mycorrhizal fungi by earthworms during early succession. Soil Biology and Biochemistry, 25(8), 1021 1026. Gay, C., Estrada, F., Conde, C. et al. (2006). Poten tial Impacts of Climate Change on Agriculture: A Case of Study of Coffee Production in Veracruz, Mexico. Climatic Change, 79, 259 288. Giorgi, F. (2006). Climate change hot spots. Geophysical Research Letters, 33(8). Harrier, L.A. (2001). The arbuscula r mycorrhizal symbiosis: a molecular review of the fungal dimension. Journal of Experimental Botany, 52(1), 469 478. Jaramillo, J., Muchugu, E., Vega, F.E., Davis, A., Borgemeister, C., Chabi Olaye, A. (2011). Some Like It Hot: The Influence and Implicat ions of Climate Change on Coffee Berry Borer (Hypothenemus hampei) and Coffee Production in East Africa. PLoS ONE, 6(9).
Variability of m ycorrhizae in Coffea arabica Sime 9 Lemtiri, A., Colinet, G., Alabi, T., Cluzeau, D., Zirbes, L., Haubruge, Âƒ., Francis, F. (2014). Impacts of earthworms on soil compon ents and dynamics. A review. Biotechnology, Agronomy, Society, Environment, 18(1), 121 133. Leonard, H.J. (1987). Natural resource and economic development in Central America. London, United Kingdom:International Institute for Environment and Developmen t. Makoi, J.H.J.R., Ndakidemi, P.A. (2009). The agronomic potential of vesicular arbuscular mycorrhiza (VAM) in cereals legume mixtures in Africa. African Journal of Microbiology Research, 3(11), 664 675. Navazio, L., Mariana, P. (2008). Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi. Plant Signaling and Behavior, 3(4), 229 230. Newsham, K.K., Fitter, A.H., Watterson, A.R. (1995). Arbuscular mycorrhiza protect an annual grass from root pathogenic fungi in the field. Journ al of Ecology, 83, 991 1000. Orozco, F.H., Cegarra, J., Trujillo, L.M., Roig, A. (1994). Vermicomposting of coffee pulp using the earthworm Eisenia fetida: Effects on C and N contents and the availability of nutrients. Biology and Fertility of Soils, 2 2(1 2), 162 166. Pedersen J.C. & Hendriksen N.B. (1993). Effect of passage through the intestinal track of detritivore earthworms (Lumbricus spp.) on the number of selected Gram negative and total bacterial. Biol. Fertil. Soils, 16(3), 227 232. Remy, W ., Taylor, T.N., Hass, H., Kerp, H. (1994). Four hundred million year old vesicular arbuscular mycorrhizae. Proceedings of the National Academy of Sciences, 91, 11841 11843. Rosenzweig, C., Iglesias, A., Yang, X. et al. (2001). Climate Change and Extrem e Weather Events; Implications for Food Production, Plant Diseases, and Pests. Global Change & Human Health, 2, 90 104. SÂ‡inz, M.J., Taboada Castro, M.T., VilariÂ–o, A. (1998). Growth, mineral nutrition and mycorrhizal colonization of red clover and cuc umber plants grown in a soil amended with composted urban wastes. Plant and Soil, 205(1), 85 92. SchÂšnholzer F., Hahn D. & Zeyer J. (1999). Origins and fate of fungi and bacteria in the gut of Lumbricus terrestris L. studied by image analysis. FEMS Micr obiol. Ecol., 28(3), 235 248. Simon, L., Bousquet, J., Levesque, R.C., Lalonde, M. (1993). Origin and diversification of endomycorrhizal fungi and coincidence with vascular plants. Nature, 363,67 69. Subramanian, K.S., Charest, C., Dwyer, L.M., Hamilton R.I. (1995). Arbuscular mycorrhizas and water relations in maize under drought stress at Tasseling. New Phytologist, 129,643 650. Thomson, L.J., Macfadyen, S., Hoffman, A.A. (2010). Predicting the effects of climate change on natural enemies of agricultural pests. Elsevier, 52(3), 296 306. Turral, H., Burke, J., FaurÂŽs, J.M. (2008). Climate change, water, and food security. Rome, Italy: FAO Water Reports. Vierheilig, H., Coughlan, A.P., Wyss, U., PichÂŽ, Y. (1998). Ink and Vinegar, a Simple Staining Technique for Arbuscular Mycorrhizal Fungi. Applied and Environmental Microbiology, 64(12), 5004 5