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The effects of Phosphorus, Copper, and plant age on mycorrhizal abundance in orchid roots Nathan Jespersen Department of Chemistry, University of Cincinnati Abstract Mycorrhizae increase growth and nutrient acquisition in orchids. I study which factors affect mycorrhizal infestation I organized three experiments: the first used wild orchids assumed to already be colonized by mycorrhizae and tested how receiving a nutrient solution that emphasized phosphorus affected mycorrhizae abundance. The second tested this same hypothesis with the exception that plants were initially devoid of mycorrhiza e. It also tested how adept plants with and without mycorrhizae were at absorbing heavy metals such as Copper and Manganese from the soil The third experiment focused on how age differences of plants affected mycorrhizae abundance. Roots were stained with Trypan Blue and mycorrhizae were then coun ted under a microscope Wild orchids showed no difference in mycorrhizae with or without NPK spray ( NPK = 13.33 +/ 11.2 VAM, water = 12.9 +/ 13.5 VAM) Lab orchids initially devoid of mycorrhizae showed that there was a trend for NPK orchids to have fewer hyphae than those offered only water ( NPK = 10.7+/ 11.9 mycorrhizae water = 18.3+/ 29.2 mycorrhizae ), although it lacked statistical significance (p = .12). The second part of experiment two showed that plants with mycorrhiz ae did have the potential to reduce heavy metals in soil because the orchids with mycorrhizae had 2.1 ppm of Copper left in their growth substrate whereas the group devoid of mycorrhizae had 5.99 ppm The third experiment found that orchids have consistent numbers of mycorrhizae at different life stages ( young = 7 .5+/ 4.83 mycorrhizae middle aged = 5.8+/ 4.36 mycorrhizae old = 8.8+/ 6.76 mycorrhizae). Mycorrhizae abundance appear s to vary greatly between individuals and infestation is not decid ed by a single factor Resumen Las micorrizas aumentan el crecimiento y la adquisicin de nutrientes en orqudeas. Estudie que factores afectan la infeccin en orqudeas. Organic tres experimentos: el primero usando orqudeas que asum estaban ya colon izadas por micorrizas y prov como al recibir una solucin con nutrientes rica en fsforo afecta la abundancia de micorrizas. La segunda prov esta misma hiptesis con la excepcin de que las plantas fueron inicialmente desprovistas de las micorrizas. Ta mbin prov como las plantas con y sin micorrizas fueron absorbiendo metales pesados como Cobre y Manganeso del suelo para determinar habilidades de remedio por las micorrizas. El tercer experimento lo enfoqu en como se afecta la abundancia de micorriza s segn la edad de la planta. Las races se tieron con azul de tripano y las micorrizas se contaron usando un microscopio. Las orqudeas salvajes no mostraron diferencias en micorrizas con o sin la solucin aplicada ( NPK = 13.33 +/ 11.2 VAM, water = 12.9 +/ 13.5 VAM) Las orqudeas de laboratorio inicialmente desprovistas de micorrizas, muestran una tendencia a tener menor cantidad de hifas al aplicar la solucin NPK que al aplicar solamente agua (NPK = 10.7+/ 11.9 micorrizas, agua = 18.3+/ 29.2 mi corrizas), aunque carece de significancia estadstica (p= .12). La segunda parte del experimento muestra que las plantas con micorrizas tienen el potencial de reducir los metales pesados en el suelo debido a que las orqudeas con micorrizas tienen 2.1 pp m de Cobre en el sustrato de crecimiento mientras que las desprovistas de micorrizas tienen 5.99 ppm. El tercer experimento encontr que las orqudeas tienen un nmero consistente de micorrizas a diferentes edades (joven = 7.5+/ 4.83 micorrizas, mediana e dad = 5.8+/ 4.36 micorrizas, viejo = 8.8+/ 6.76 micorrizas). La abundancia de micorrizas parece variar ampliamente entre individuos y la infeccin no es decidida por un factor nico.
Introduction Vesticular Arbuscular Mycorrhizae (VAM) have been found t o be associated with 80% of all angiosperms and are viewed as a n important diffuse mutualistic organism (Janzen 1983). While t he abiotic and biotic factors tha t alter mycorrhizae abundance are not completely understood, plant age and soil richness are tho ught to play a large role (Smith and Read, 1997). P lants receive vital elements such as Phosphorus (P), Nitrogen (N), and Potassium (K) as well as some heavy metals (Copper, Manganese and Cadmium for example) while mycorrhizae receive photosynthates fr om the plant Since VAM have extensive root area, they are more able to obtain food produced by the plant (Smith and Read, 1997) While this mutualistic relationship is extremely strong in all angiosperms it is especially so in orchids, with every single orchid studied to date having been found to contain VAM at some stage of life (Janzen 1983). Some orchids, however, have been found to secondarily lose them (Janzen 1983). The need for VAM in germinatin g orchids is caused by orchids to be wind dispersed and have very small seeds. This means that they have little or no endosperm and must rely heavily on my corrhizae during germination and initial growth to obtain nutrients Later orchids may find VAM draw more than they deliver and, at this point, do away with them. Since mycorrhizae can take as much as 30% of the Glucose produced by the orchids, and older orchids may have enough root area to obtain a sufficient amount of nutrients without my corrhizae aid, it can be more beneficial to omit the VAM (Smith and Read, 1997). Plants have the ability to stop the mycorrhizae infestation by apoptosis or, intentionally killing their cells to stop the spread of the fungi. They can also produce secondar y metabolites to kill the mycorrhizae (Waller et al. 2007) It is not thoroughly understood, however, which orchids do this nor the precise environmental conditions that facilitate this removal. Smith and Read (1997) hypothesized that, since Phosphorus is the most limiting main nutrient in tropical ecosystems, the loss of mycorrhizae may be related to sufficient supplies of P. It has been seen that areas where P is not limiting (i.e. temperate areas) usually have fewer VAM relations. Beyond supplying P, m ycorrhizae increased uptake of Copper (Cu) by 62% ( Smith and Read 1997) Also, soils with abnormally high levels of heavy metals, which would usually have a negative effect on plants, had a diminished effect when VAM were present. Moreover, the amoun t of heavy metals in soil was significantly reduced (Smith and Read 1997). This an aspect that will continue to grow in importance as more agricultural chemicals are dumped into natural systems. These observations are what led me to the three questions this experiment answers. First, how strongly does fertilizer availability affect VAM abundance in roots? Secondly, how do mycorrhizae affect heavy metal levels in soil ? Finally, w hat role does o rchid age play in mycorrhizae abundance?
Methods Study Site This study takes place in San Luis, Cost a Rica at an elevation of 11 00 m above sea level from October 30 th to November 17 th This area is in Tropical Premontaine wet forest (Fogden 1993) and receives 2.5 m of rainfall annually ( Hartshorn 1983 ). Experiment 1 : Loss of mycorrhizae with different nutrient loads. The first experiment focuses on the variation in nutrients and how it affects the abundance of mycorrhizae. This was accomplished by going into the nearby forest and finding 44 Cryptocentrum calcaratum (Orchidaceae). These were then brought back to a wind break in San Luis where they wer e tied to four different sticks; 22 were sprayed daily with a solution containing the directed amount of NPK solution (1/4 th tablespoon for every 1000 mL) until thoroughly saturated from October 30 th to November 15 th The other 22 orchids were spra yed equally with tap water each day. On November 15 th the C. calcaratum were harvested and root samples were taken They were then stained with Trypan Blue using the procedure outlined by Smestad (2010). The stained roots were examined at a 100 X magnification using an Olympus CX21 microscope. The number of VAM visible were counted (FIGURE 1) and recorded. FIGURE 1: Example of what VAM in root stains looks like. Note: this is not a picture of my actual samples but an example representing what I would have seen under the microsc ope in the first and third experiments. Black lines are hyphae and ovular parts are spores.
Experiment 2 : Acquisition of mycorrhizae with different nutrient loads. Samples from this experiment were Trichocentrum cebolleta (Orchidaceae) obtained from the orchid suppliers VitroPlant S.A. They were grown i n a nutrient rich medium and were initially devoid of mycorrhizae. These orchids were removed from the ir growth medium rinsed thoroughly to clean roots, and placed in four separate 8 in. X 4.5 in. con tainers (25 in each container) The four containers were differentiated based on two areas of study: firstly, how nutrient levels changed initial colonization of mycorrhizae, and secondly what e ffect the infestation of mycorrhizae had on Copper and Mangan ese levels. Copper and Manganese were selected because of their increasing levels in the environment as a result of fertilizer and fungicide use (Jaques 1987). Containers were set up as follows: one container had only water and mycorrhizae, the second a 10:50:10 NPK solution (1/4 th tablespoon/1000 mL tap water) and mycorrhizae, the third had Cu solution (250 mL of a 1.125 g/100 0 mL tap water) and the 10:50:10 NPK solution, and the final containe r was given the Cu solution, NPK solution, and mycorrhizae. Chemical solutions were made from powdered forms bought from an agriculture store. The Cu solution was actually a fungicide made up of 11.1% Metallic Cu, 50% Manganese Ethylenebis, and 38.9% inert materials. F ungicide was used because it was the availab le mixture containing both Copper and Manganese. M ycorrhizae were ensured in containers one, two, and four by using a solution containing mycorrhizae called Mycorr h iza Mycorrhiza is a solution containing mycorrhizae spores that can be added to plants by either dipping the roots in the solution or by diluting the solution to 1: 4 Mycorrhiza:water. The containers were all filled approximately a fifth of the way with mos s (on November 2 nd ) in order to provide a substrate for the materials to become trapped in and to act as a sponge to supply water to the T. cebolleta Each orchid was dipped in a mycorrhiza solution before being put into their corresponding container with the exception of those destined for container three. In order to ensure no mycorrhizae colonized the roots of those orchids in container three, the moss was boiled in water at an excess of 100 0 C for 1 0 minutes (Mosse 1956) Each container was then filled with 125 mL of either water if it was container one, or NPK solution if it was container two, three, or four. Mycorrhiza was also diluted to a 1:4 Mycorrhiza: water solution and approximat ely 166 mL was added to containers one, two, and four on the 9 th of November to ensure opportunity for infection Containers three and four also received 250 mL each of the Cu solution over days two four of experimentatio n. Solutions of NPK or water were drained and replaced every third day to e nsure consistent nutrient availability. Containers were left in diffuse light and were also left open two days a week to allow for a more natural air environment. The orchids were brought to the lab on November 17 th and stained and examined using the same procedure as experiment one. One root from each orchid was selected based on apparent health In addition to counting the mycorrhizae on each slide, a number 1 5 was given pertaining to the number of young myc orrhizae found on each slide (FIGURE 2) Examples of what would rate what are as follows:
FIGURE 2: little to no young. B) a rating of 2, few young. C) a rating of 3, medium number of young. D) a rating of 4, many young. E) a rating of 5, young VAM visible in very lar ge quantities. Note that spots were usually smaller than these and ratings were much more accurate and consistent way to quantify them because of the errors involved in focusing the microscope. More revealed themselves as the focus was shifted slightly. A ) C) E) D) B)
Tests for soil Copper and Manganese (Mn) levels were done for containers three and four using a LaMotte SMART water quality test kit and a soil testing kit. The Procedure for Cu testing consisted of obtaining a liquid sample by finely cutting up nine mL of moss and adding it to a container. I then added ten mL of Universal Solvent Solution (USS) (3% Acetic Acid, 30% Sodium Acetate, and pure water) and shook the solution for five minutes. Next, I filtered out the liquid using filter paper. T en mL of th is solution was put into the machine used to quantify concentrations in the water test kit to obtain a blank scan Five drops of Cu (I) was added to the solution and shaken. The resulting solution was placed back in the machine and a number readout was g iven in p arts per million (ppm ). The procedure for Manganese testing was similar in that a liquid solution was again needed; however it only called for seven mL of moss and seven mL of USS. After shaking the solution, 15 drops from a pipette were added t o a container I then added .05 g of Mn buffer, followed by .05 g of Manganese Periodate Reagent. The solution changed to a pinkish color and the severity of this color determined the amount of Mn, with darker colors signifying a higher concentration. Concentration levels were quantified as on Experiment 3 : Orchid age and mycorrhizal infest at ion. The third experiment hinged upon how age affected mycorrhizae abundance in C. calcaratum. To study this, 54 orchids were c ollected from a forest in San Luis and separated based upon size. The orchids were all within an approximate 300 m 2 area close to the edge of the forest. They also all tended to be on the underside of slightly angled trees. Plants were considered young if they were less than three cm medium in age if they we re between 4.3 and 5.3 cm and old if they were greater than 6.2 c m. There were 14 young, 20 medium, and 20 old plants with these parameters. One root selected from each plant was removed, stained and examined using the same procedure as experiment one. Samples were collected on the 15 th of November and stained that same day. Results Experiment 1 : Loss of mycorrhizae with different nutrient loads. The C. calcaratum analyzed in the first experiment showed no significant difference between the samples that were sprayed with the solution containing N P K and those that were sprayed with just water ( df = 36, t stat = 0.107896 P = .91). The averages were extremely similar (NPK = 13.33 +/ 11.2 VAM, water = 12.9 +/ 13.5 VAM) and the number varied greatly from orchid to orchid, ranging from 0 to 39 VAM with N P K and 0 to 45 VAM with only water (FIGURE 3)
. FIGURE 3 : Comparison of the average VAM number of Cryptocentrum calcaratum orchids supplied an NPK solution and those given only water ( df = 36, t stat = 0.107896, P = .91, N = 36) Samples were taken in San Luis, Costa Rica. Error bars are a single standard deviation. Experiment 2 : Acquisition of mycorrhizae with different nutrient loads. T. cebolleta supplied water and VAM and those supplied NPK solution and VAM were not considered statistically different ( df = 29, t stat = 1.15, P = .26), though they did show a general trend in which those that were not given the N P K had on average almost twice as many mycorrhizae (NPK = 10.7 +/ 11.9 VAM, water = 18.3 +/ 29.2 VAM ) The orchids in the container with N P K also had a much smaller range going only from 0 41 VAM as opposed to the water groups 0 125 VAM. Not only that, but when I was studying the slides I noticed the presence of a number of dots, whi ch were in fact the startings of mycorrhizae. There was also a N P K although this was not found to be statistically s ignificant (DF = 41, t stat = 1.60, P = .12 N = 45 ; water = 3.09 +/ 1.16, NPK = 2.59 + / .91). 0 5 10 15 20 25 30 with NPK without NPK Average Mycorrhizae number
FIGURE 4 : Comparison of average number of m ycorrhizae in Trichocentrum cebolleta orchids supplied water and VAM or NPK and VAM ( df = 41, t stat = 1.60, p = .12, N = 45) Orchids were kept in diffuse light in San Luis, Costa Rica. Error bars represent a single standard deviation FIGURE 5 : Comparison of average number of mycorrhizae in Trichocentrum cebolleta orchids supplied with Cu and VAM and those supplied Cu without VAM ( df = 24, t stat = 2.24, P = .03, N = 44 ). Orchids were kept in diffuse li ght in San Luis, Costa Rica. Error bars represent a single standard deviation. 0 5 10 15 20 25 30 35 40 45 50 Water and Myco NPK and Myco Average VAM number Supplied Nutrients 0 0.5 1 1.5 2 2.5 3 3.5 Cu without Myco Cu and Myco Average VAM number Supplied Nutrients
The T. cebolleta in containers three and four were found to be statistically different ( df = 24, t stat = 2.24, P < .05). Even though container three was suppose d to be devoid of mycorrhizae, it did end up having a small number, approximately 1/10 th the average number of those in container four .44 VAM, Cu with V AM = 1.13 +/ 2.16 VAM) While the fungicide did apparently kill off the VAM in the other container, they had begun to grow back in between the 9 th of November when I added more VAM and the 16 th of November when I began staining the roots. Also, juvenile mycorrhizae numbers were twice as high in container four (rating for Cu with VAM=2.34 +/ 1.11, Cu without VAM = 1.14 +/ .36) (DF = 27, t stat = 4.92, P < .005, N = 44). When comparing the levels of Cu in the two containers that were supplied Cu solution the container four had nearly three times less Copper. Container three 5.99 ppm, while the other container had 2.1 ppm. Also, in the test comparing levels of Mn, the container nearly devoid of Experiment 3 : Orchid age and mycorrhizal infestation. A ge had no impact on the mycorrhizal infestation of C. calcaratum from the field ( ANOVA test df = 2, F = 1.50, P = .23, N = 54). The plants showed very little difference in number of mycorrhizae at different life stages (young = 7.5 +/ 4.83 VAM, middle aged = 5.8 +/ 4.36 VAM, old = 8.8 +/ 6.76 VAM) FIGURE 6 : Comparison of number of VAM in young middle aged and old Cryptocentrum calcaratum orchids ( df = 2, F = 1.50, P = .23, N = 54). Age was determined based upon size with small being < 3 mm, middle aged is between 4.3 and 5.3 mm, and old is > 6.2 mm. The stu dy took place in San Luis Costa Rica. Error bars represent a single standard deviation. Discussion Increasing nutrients did not significantly impact mycor r hizae on roots of C. calcaratum. However, there was a trend for higher VAM numbers with decreased nutrients in T. cebolleta This difference may be due to interspecies differences, or a result of the substrate they were 0 2 4 6 8 10 12 14 16 18 young med old Average Number of Mycorrhizae Age
grown on. Since experiment two used a moss substrate in a closed container, the nutrients were consistently available and more accessible (Bigelow et al. 2010). Therefore, orchids supplied nutrients in experiment two had less need for VAM Although not statistically significant, NPK samples for orchids originally de void of mycorrhizae contained nearly half the average number of VAM as those supplied water alone It appears that those orchids supplied nutrients necessary for growth chose to minimize the reduction of glucose availability due to VAM presence ( Smith and Read 1997). The fact that the upper limit of VAM on a single root for plants without NPK was more than three times as great as the highest number on those given NPK leads me to believe that these orchids do in fact have a point at which keeping the mycorrhizae around ceases to be of overall benefit Using the fungicide made a pparent that n ot only does adding this fungicide work to keep away the harmful fungi, but it also kill s the beneficial ones. However, the time constraints placed on the experiment show that a population of VAM can not only begin to recolonize the roots in only a week, but they also can significantly diminish the concentration of heavy metals. In this time it effectively cut the density of Cu in third and greatly reduced the amount of Mn in the soil. This may even be an underestimate of the potential for Cu reduction as the test had an upper limit of 6 ppm, which means that the 5.99 reading may have been maxing out the scale. As these two nutrients become more and more common with their use in ferti lizers, fungicides, etc., (Copper (Cu) 2010) the abilities of mycorrhizae for remediation may become increasingly important. Repeating this experiment over a greater time range and with a different starting solution could yield very enlightening and benef icial results. The results of the third experiment revealed that, at least for C. calcaratum, VAM numbers do not vary greatly depending on the age of the plant. This refutes my hypothesis and shows that in this plant the VAM are equally important mutua listic partners for the entir ety of their lives. This finding proves that some orchids do indeed need VAM throughout all stages; however, it opens the door for questions about what causes these inter and intraspecific variations since some orchids secondarily lose mycorrhizae (Janzen 1983). These findings all contribute to our understanding of this advantageous relationship. They also pave the way for more studies about VAM and orchids such as a test of varying nutrient levels with bigger sample s izes to determine the point at which orchids no longer need their partners. Also, a test of heavy metal absorption over a much longer period of time in a toxic soil might just show the true environmental potential for this relationship. Another study tha t might help us delve deeply into the inner workings of these plant mutualism would be to survey how quickly colonization occurs in differing conditions to determine whether or not it is a consistent time or varied based upon how nutrient rich the soil i s. It would also be very interesting to examine how great the growth tradeoff is between having VAM and not since they are believed to reduce glucose levels for some plants growth as much as 30% (Smith and Read 1997). Understanding these aspects may ena ble us to finally get to the heart of the mycorrhizae/plant mutualism.
Acknowledgements I would like to extend my thanks first to Alan Masters for his incredible support and we alth of knowledge that he lent me, Karen Masters for help in Orchid identification, La Trocha for keeping me in fantastic shape when I had to walk to and from the station almost every day, and the entire staff of CIEE Monteverde Costa Rica Tropical Ecology and Conservation for inspirin g interest in Biological proc esses and helping me the cornucopia of times I was in need
Literature Cited Bigelow, Cale A., Dan Bowman, and Keith Cassel. Sand Based Rootzone Modification with Inorganic Soil Amendments and Sphagnum Peat Moss USGA Green Section Record, July Aug. 2000. Web. 22 Nov. 2010.
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Los efectos del fsforo, el cobre, y la edad de la planta sobre la abundancia de micorrizas en las races de orqudeas
The effects of phosphorus, copper, and plant age on mycorrhizal abundance in orchid roots
Mycorrhizae increase growth and nutrient acquisition in orchids. I study which factors affect mycorrhizal infestation. I organized three experiments: the first used wild orchids assumed to already be colonized by mycorrhizae and tested how receiving a nutrient solution that emphasized phosphorus affected mycorrhizae abundance. The second tested this same hypothesis with the exception that plants were initially devoid of mycorrhizae. It also tested how adept plants with and without mycorrhizae were at absorbing heavy metals such as copper and manganese from the soil to determine mycorrhizaes remediation abilities. The third experiment focused on how age differences of plants affected mycorrhizae abundance. Roots were stained with Trypan Blue and mycorrhizae were then counted under a microscope. Wild orchids showed no difference in mycorrhizae with or without NPK spray (NPK = 13.33 +/- 11.2 VAM, water = 12.9 +/- 13.5 VAM). Lab orchids initially devoid of mycorrhizae, showed that there was a trend for NPK orchids to have fewer hyphae than those offered only water (NPK = 10.7+/-11.9 mycorrhizae, water = 18.3+/-29.2 mycorrhizae), although it lacked statistical significance (p = .12). The second part of experiment two showed that plants with mycorrhizae did have the potential to reduce heavy metals in soil because the orchids with mycorrhizae had 2.1 ppm of copper left in their growth substrate whereas the
group devoid of mycorrhizae had 5.99 ppm. The third experiment found that orchids have consistent numbers of mycorrhizae at different life stages (young = 7.5+/-4.83 mycorrhizae, middle aged = 5.8+/-4.36 mycorrhizae, old = 8.8+/-6.76 mycorrhizae). Mycorrhizae abundance appears to vary greatly between individuals and infestation is not decided by a single factor.
Las micorrizas aumentan el crecimiento y la adquisicin de nutrientes en las orqudeas. Estudie que los factores afectan la infeccin en las orqudeas. Organic tres experimentos: el primero fue usando las orqudeas que asum ya estaban colonizadas por las micorrizas y prob como al recibir una solucin con nutrientes rica en fsforo afecta la abundancia de las micorrizas. En la segunda prob esta misma hiptesis con la excepcin de que las plantas fueron inicialmente desprovistas de las micorrizas. Tambin prob como las plantas con y sin micorrizas fueron absorbiendo metales pesados como el Cobre y el Manganeso del suelo para determinar las habilidades de remedio por las micorrizas. El tercer experimento lo enfoqu en cmo se ve afectada la abundancia de micorrizas segn la edad de la planta. Las races se tieron con azul de tripano y las micorrizas se contaron usando un microscopio. Las orqudeas salvajes no mostraron diferencias en las micorrizas con o sin la solucin aplicada (NPK = 13.33 +/- 11.2 VAM, agua = 12.9 +/- 13.5 VAM). Las orqudeas del laboratorio inicialmente desprovistas de micorrizas, muestran una tendencia a tener menor cantidad de hifas al aplicar la solucin NPK que al aplicar solamente agua (NPK = 10.7+/-11.9 micorrizas, agua = 18.3+/-29.2 micorrizas), aunque carece de significado estadstico (p= .12). La segunda parte del experimento muestra que las plantas con micorrizas tienen el potencial de reducir los metales pesados en el suelo debido a que las orqudeas con micorrizas tienen 2.1 ppm de Cobre en el sustrato de crecimiento mientras que las desprovistas de micorrizas tienen 5.99 ppm. El tercer experimento encontr que las orqudeas tienen un nmero consistente de micorrizas a diferentes edades (joven = 7.5+/-4.83 micorrizas, mediana edad = 5.8+/-4.36 micorrizas, viejo = 8.8+/-6.76 micorrizas). La abundancia de micorrizas parece variar ampliamente entre individuos y la infestacin no se decide por solo un factor.
Text in English.
Costa Rica--Puntarenas--Monteverde Zone--San Luis
Costa Rica--Puntarenas--Zona de Monteverde--San Luis
Tropical Ecology Fall 2010
Ecologa Tropical Otoo 2010
t Monteverde Institute : Tropical Ecology