Effects of cattle feces on Fabaceae plants Appleseed 1 The effects of a possible soil nitrogen gradient on Mimosa pigra root nodule density and black bean plant growth Ronnie E Appleseed Department of Ecology and Evolutionary Biology University of California Santa Cruz EAP Tropical Biology and Conservation Program, Fall 2016 16 December 2016 A BSTRACT Cattle produce feces that contain organic nitrogen. The farm at the University of Georgia (UGA) station in San Luis, Monteverde is built on a sloping hill with nitrogen producing cattle present on the top and absent at the bottom. The legume Mimosa pigra has a mutualistic relationship with nitrogen fixing Rhizobia bacteria and is found growing as a weed on the UGA farm. I hypothesized that the possible decrease in soil [N] along the UGA farm hill would result in decreased Mimosa root nodule densities and that the greatest nodule densities would be found in plants growing closest to the cattle feces, the assumed N source, and at the bottom of the hill, an assumed N sink. After surveying the roots of ten Mimosa plants each at four collection sites going downhill and away from cattle feces, I found that increased root nodulation density does occur in M. pigr a growing in soil closest to the nitrogen producing cattle at the top of the UGA farm hill compared to further away. However, I did not find that plants grown in soil at the bottom of the hill also had increased root nodule density, suggesting that there m ay not be a nitrogen sink, as predicted. Additionally, black bean plants were grown for 17 days in soil collected from the same sites used to study Mimosa pigra along the UGA farm hill. I found no significant differences for leaf area or leaf dry matter de nsity, however I did find that bean plants growing in soil collected closest to the nitrogen producing cattle at the top of the hill did have increased plant height and stem dry matter density. Similar to the findings for Mimosa root nodule densities, pla nt height and stem dry matter density were actually lowest when grown in soil collected at the bottom of the hill, the assumed N sink. These results contradict my predictions and further support the idea that there may not be a nitrogen sink at the bottom of the UGA farm hill. The negative correlation between distance away from cattle feces at the top of the UGA farm hill and Mimosa root nodule density, as well as black bean plant height and stem dry matter indicates that there may be a decrease in soil nit rogen concentrations further away from cattle feces. However, because I was unable to directly test soil [N], future studies must be conducted to confirm this hypothesis.
Effects of cattle feces on Fabaceae plants Appleseed 2 Los efectos de una posible gradiente de nitr geno de suelo en la densidad de n dulos de raz de Mimosa pigra y en el crecimiento de frijoles negros. RESUMEN El ganado produce boiga que contiene nitrgeno orgnico. La finca en la estacin de la Universidad de Georgia (UGA) en San Luis, Monteverde se encuentra en una colina inclinada con ganado presente en la parte superior y ausente en la parte inferior. La le guminosa Mimosa pigra tiene una relacin mutualista con las bacterias fijadoras de nitrgeno y se encuentran creciendo como malezas en la finca de UGA. Predije que la posible disminucin de [N] del suelo a lo largo de la colina de la UGA resultara en una las races de di ez plantas de M.pigra en cuatro sitios de recoleccin que van cuesta abajo y lejos de la boiga, encontr la mayor densidad de ndulos en la M.pigra en el suelo ms cercano al ganado. Sin embargo, no encontr que las plantas en la parte inferior de la co lina tuvieran una alta densidad de ndulos, lo que sugiere que puede no haber un sumidero de nitrgeno. Adems, sembr plantas de frijol negro y despus de 17 das no encontr diferencias significativas en el rea foliar o en la densidad de materia seca fo liar, pero s encontr que las plantas de frijol que crecen en el suelo ms cercano al ganado eran ms altas y con mayor densidad de materia seca. Al igual que los hallazgos para las densidades de ndulos de la raz de M.pigra la altura de la planta y la densidad de la materia seca del tallo fueron ms bajas en el suelo al final de la colina. Estos resultados contradicen mis predicciones y apoyan la idea de que no hay un sumidero de nitrgeno al final de la colina de UGA. La correlacin negativa entre la distancia de la boiga de ganado en la parte superior de la colina y la densidad de ndulos, la altura de la planta y la materia seca del tallo indica que puede haber una disminucin en las concentraciones de nitrgeno del suelo ms lejos de la boiga. Si n embargo, debido a que no cuantifiqu directamente el [N] del suelo, deben realizarse estudios futuros para confirmar esta hiptesis. Besides water, plants rely on nitrogen more than any other chemical to support life (Lewis 1986). All agricultural crops are able to readily uptake nitrogen ions from the soil in forms such as nitrate and ammonium (NO NH ) (Peoples et al. 1995). Livestock manure is an important source of nitrogen ions for agricultural crops and can be produced directly on farms (An toun et al. 1998). On the other hand, atmospheric nitrogen gas (N ) comes from the air within the soil (Lewis 1986). Only plants that have mutualistic relationships with nitrogen fixing bacteria, such as Fabaceae legumes, are able to utilize N in the soil (De Faria et al. 1989). Nitrogen fixing bacteria, such as Rhizobia are free living organisms that grow directly in the soil (Gage 2004). Rhizobia infect the root tissue of Fabaceae plants from within the soil using infection thread tubules. Once the root is infected by Rhizobia a nodule structure is formed. In return for fixing N into a usable form, the Rhizobia receive carbohydrates from the plant and live within the nodules (Gag e 2004).
Effects of cattle feces on Fabaceae plants Appleseed 3 The farm at the University of Georgia (UGA) station in San Luis, Monteverde was developed on a sloped hillside with nitrogen ion producing cattle living at the top of the hill. As a result, gravity should cause a nutrient gradient to flow downhi ll from the livestock source to a presumed nitrogen sink at the bo ttom of the hill approximately 2 00 m from the top, with varying nitrogen levels in between. Previous studies have found that the addition of organic fertili zer, such as cattle manure, is p ositively correlated with increased legume root nodule abundance (Jannoura et al. 2014, Suryantini 2014 ). The cattle living at the top of the UGA farm slope provide organic fertilizer for the soil. This organic fertilizer should increase root nodule abunda nce for legumes growing on the farm. I observed Mimosa pigra legume plants growing as weeds throughout the sloped hill on the UGA farm. Because the cattle create organic fertilizer inputs at the top of the hill, I hypothesize that increased root nodule density will be found in Mimosa roots for plants at the top of the hill growing closer to cattle feces, the assumed nitrogen source, and at the bottom of the hill, the assumed nitrogen sink, compared to Mimosa growing in the soil found in the middle of the hill. Legume crops, such as black beans, should be able to ut ilize nitrogen along the entire UGA farm slope in the form of N According to studies by Jannoura et al. 2014, as well as Suryantini 2014, bean crops treated with manure have access to increased concentrations of ionic nitrogen and should produce more mut ualistic root nodules. However, neither Jannoura et al. 2014 nor Suryantini et al. 2014 discuss the mechanism causing increased root nodules in plants exposed to increased nitrogen. If increased root nodule density and nitrogen fixation occurs from an increased presence of cattle feces that provide ionic nitrogen inputs, black bean crops grow n in soil collected at the top and the bottom of the UGA farm hill, the nitrogen source and sink, should have increased plant growth. Based on the assumed nitrogen concentrations found throughout the UGA farm hill, I propose the question: What distance fro m the cattle feces at top of the UGA farm hill will have soil that result in the highest Mimosa root nodule density and the most black bean plant growth? METHODS and MATERIALS I conducted this study at the University of Georgia farm station in San Luis, Costa Rica from November 14 thru December 3, 2016. The necessary UGA farm research permit was obtained before collecting plants and soil for both field and greenhouse studies. I was unable to directly test soil nitrogen concentrations. Field Study Ten entire Mimosa pigra plants were collected from soil at four different sites totaling forty plants. The first collection site was located at the top of the hill and called Soil 1. Cattle feces were present at this site and absent from the remaining thre e sites. The next site was ~50 m downhill from Soil 1 and was called Soil 2. The next site was ~50 m downhill from Soil 2 and was called Soil 3. The final site was ~50 m downhill from Soil 3 and was called Soil 4. The roots were rinsed with water to remove soil and have a better view of the nodules. In the lab, I blocke d my vision to randomly select ten individual roots from each plant. The ten roots were then
Effects of cattle feces on Fabaceae plants Appleseed 4 measured for length and surveyed for nodule abundance using a dissecting scope. I calculated r oot nodule density by dividing the total number of nodules per root by the linear length of the root in cm. I then summed r oot nodule density for all ten roots for each plant. Mean nodule density was then calculated for each of the four soil sites. ANOVA tes t was used for all four sites and a Tukey HSD analysis was used to determine any significant differences to the 99% confidence interval between all sites. All statistics were run using R Studio v. 0.99.484. All data were graphed using box diagrams. The bot tom of the box represents the first quartile of the data the top of the box represent the third quartile, the horizontal line within the box represents the median value and the bars extending from the box represent the minimum and maximum data values. Any circles outside the box diagram represent outlier data points. Greenhouse Study I compared black bean plant growth by growing black bean plants from seeds planted in soil collected at the same four UGA farm hill sites used in the Mimosa field study: Soil 1, Soil 2, Soil 3 and Soil 4. To grow the black bean plants, I collected eight liters of soil from each site and took the collected soil from UGA to a privately owned greenhouse in Monteverde, Costa Rica. I partitioned the soil into 0.5 L plastic bags with 12 bags per soil site, 48 bags of soil in total. I then planted one bl ack bean seed per bag of soil for a total of 48 seeds approximately five cm deep. The seeds were watered every six days for 17 days. After 17 days, 32 bean seeds successfully germinated and grew. The 32 bean seeds germinated almost evenly across soil sites resulting in eight plants for Soil 1, nine plants for Soil 2, seven plants for Soil 3 and eight plants for Soil 4. I used a ruler to measure plant height (cm) for each plant that had successfully germinated and grown. After measuring plant height, I harvested each plant, rinsed the soil from the roots and aboveground plant matter and placed each plant into a labeled plastic bag. I then took the plants to the lab and used paper towels to absorb any excess water. I cut each plant into three sections: root matter, stem, and leaves. I used a dissecting microscope at 1.2x magnification to survey all roots for Rhizobia nodule s. I then measure d each stem ( cm ) with a ruler, placed each stem into an individually labeled envelope and placed the envelopes into a drying cabinet for two days. I selected the largest leaf from each plant, scanned each leaf and used ImageJ2 to measure leaf area (cm). I put each largest leaf that I had selected into a labeled envelope and placed all envelopes i nto the drying cabinet for two days as well. After two days of drying, I measured the mass of each individual stem and leaf. I then calculated specific stem length (SSL) a measure of dry matter density, by dividing stem length (cm) by stem dry mass (g). I also calculated specific leaf area (SLA) also a measure of dry matter density, by dividing leaf area (cm) by leaf dry mass (g). All statistics were also run usi ng R Studio v. 0.99.484 and box diagrams were used to represent all data. RESULTS Field Study Mimosa pigra mean root nodule density for plants grown in Soil 1 was 0.70 + 0.27 nodules/cm. Mean density for Soil 2 was 0.68 + 0.25 nodules/cm, Soil 3 was 0.52 + 0.32 nodules/cm, and
Effects of cattle feces on Fabaceae plants Appleseed 5 mean density for Soil 4 was 0.27 + 0.12 nodules/cm. Mimosa root nodules were two and a half times more dense for plants grown in soils close to cattle feces at the top of the UGA farm hill (Soil 1 an d Soil 2) versus plants grown in soil from the bottom of the hill in Soil 4 (Fig. 1). Mean nodule density from Soils 1 and 2 both differed significantly from mean nodule de nsity for plants grown in Soil 4 (F = 6.19, p<0.01). Mean Mimosa root nodule de nsity for plants grown in Soils 1, 2 and 3 were not significantly dif ferent from one another The two sites closest to the bottom of the hill, Soils 3 and 4, also had no significant difference when comparing mean root nodule density (Fig. 1). Figure 1. Mimosa pigra root nodule density for plants grown in soil along the UGA farm hill. Letters a and b above the data represent statistical significance. If different letters exist above the data boxes, the means are significantly different. Greenhouse Stu dy Root nodule survey After surveying the roots of all 32 black bean plants, no root nodules were found. Plant height Mean plant height for black bean plants grown in Soil 1 was 10.5 + 1.39 cm, Soil 2 was 10.7 + 2.45 cm, Soil 3 was 9.90 + 0.987 cm, and Soil 4 was 6.64 + 3.38 cm (Fig. 2). Black bean plant height for plants grown in Soils 1 and 2 were statistically different from plants grown in Soil 4
Effects of cattle feces on Fabaceae plants Appleseed 6 and were 1.6 times taller on average (F = 5.42, p < 0.01) (Fig. 2). I found o statisti cally significant difference for mean black bean plant height for plants grown in Soils 1, 2, and 3 (Fig. 2). Black bean plants grown in Soil 3 were one and a half times taller than plants grown in Soil 4 and were significantly different to a confidence in terval of 95% (F = 5.42, p = 0.05) (Fig. 2). Figure 2. Black bean plant height from plants grown in soil collected at sites with increasing distance, ~50 m apart, away from cattle feces. Letters a and b above the data represent statistical significance If different letters exist above the data boxes, the means are significantly different. Indicates significant difference only at the 95% CI. Specific stem length (SSL) Mean specific stem length for b lack bean plants grown in Soil 1 was 206.2 + 80.8 cm g Soil 2 was 282.1 + 65.4 cm g Soil 3 was 240.7 + 52.6 cm g and Soil 4 was 363.8 + 103.0 cm g (Fig. 3). No significant difference was found between SSL means for plants grown in Soils 1, 2, and 3, as well as between means for Soils 2 and 4. Mean black bean SSL was almost two times greater for plants grown in Soil 4 versus Soil 1 (F = 5.99, p < 0.01) (Fig. 3). Additionally, mean black bean SSL was almost two times greater for plants grown in Soil 4 versus Soil 3 and were statistically significant to a 95% confidence interval (Fig. 3).
Effects of cattle feces on Fabaceae plants Appleseed 7 Figure 3. Black bean specific stem length from plants grown in soil collected at sites with increasing distances, ~50 m apart, away from cattle feces. Letters a and b above the data represent statistical significance. If the same letter exist s above the data box, then the mean values are not significantly different between sites. If different letters exist above the data boxes, the means are significantly different. Indicates significant difference only at the 95% CI. Leaf area Mean leaf are a for black bean plants grown in Soil 1 was 34.6 + 10.3 cm, Soil 2 was 25.3 + 11.2 cm, Soil 3 was 30.9 + 7.8 cm, and Soil 4 was 21.3 + 4.31 cm (Fig. 4). Black bean mean leaf area had no statistically significant differences between plants grown in all four soil collection sites (F = 2.61 p=0.08) (Fig. 4).
Effects of cattle feces on Fabaceae plants Appleseed 8 Figure 4. Black bean leaf area from plants grown in soil collected at sites with increasing distances, ~50 m apart, away from cattle feces. The letter a above all data boxes represent no stat istically significant differences between mean values for plants grown in all soil sites. Specific leaf area (SLA) Black bean mean SLA for plants grown in Soil 1 was 579.8 + 71.1 cm g Soil 2 was 543.0 + 127.6 cm g Soil 3 was 550.5 + 27.1 cm g and Soil 4 was 576.8 + 95.2 cm g (Fig. 5). I found n o significant differences when comparing SLA from black bean plants grown in all four soil sites (F = 0.312, p = 0.82) (Fig. 5).
Effects of cattle feces on Fabaceae plants Appleseed 9 Figure 5. Black bean specific leaf area from plants grown in soil collected at sites with increasing distances, ~50 m apart, away from cattle feces. The letter a above all data boxes represent no statistically significant differences between mean values fo r plants grown in all soil sites. DISCUSSION Field Study Mimosa pigra root nodule density did significantly differ between collection sites at the top of the UGA gradient versus at the bottom. Though I predicted that the nodule density would differ b etween sites, I had predicted that the densities would be greatest near the cattle feces at the top of the UGA hill, the assumed nitrogen source, and from plants grown within the soil at the bottom of the hill, the assumed nitrogen sink. Contrary to my pre diction, root nodule density was highest for the two sites closest to the nitrogen source at the top of the hill and lowest for plants grown furthest away from the feces at the bottom of the hill in Soil 4. These results suggest that there may be no nitrog en sink at the bottom of the UGA farm hill slope and that nitrogen concentrations may actually be lowest at the bottom of the hill in Soil 4. Though there was no significant difference found between nodule density for plants grown in Soils 1, 2 and 3 and b etween Soils 3 and 4, a trend of decreasing mean nodule density does exist for soil sampled as the UGA farm hill slopes downward (Fig. 1). The decreasing trend and the significant difference
Effects of cattle feces on Fabaceae plants Appleseed 10 between nodule densities of Soils 1 and 4 does indicate that Mimo sa plants grown further away from cattle feces have decreased root nodule density. Further studies are needed to test the actual nitrogen concentrations in the soil along the UGA farm hill. If the soil nitrogen does decrease downhill from the cattle fece s, then these findings will support claims made by Jannoura et al. 2014 and Suryantini 2014 that increased organic nitrogen concentrations in soil are positively correlated with increased root nodule density. However, neither Jannoura et al. (2014) nor Suryantini (2014) discussed the mechanism driving the positive correlation between increased [N] and increased root nodules. I hypothesize that this positive correlation may be based on the costs and benefits associated with the plant bacteria mutualism. R hizobia fix nitrogen gas for Fabaceae plants, but they do not do it for free (Gage 2004). In return for their N fixing services, these bacteria require sugars supplied from the host plant (Gage 2004). A possible reason for this positive correlation may be that Fabaceae plants grown in soil with higher ionic nitrogen concentrations are larger, healthier and have increased carbon assimilation from photosynthesis. The increased carbon assimilation would result in more non structural carbohydrates available for supplying sugars to Rhizobia therefore, further resulting in increased bacterial infection and root nodule density. Future studies should be conducted to test this new hypothesis. Greenhouse Study Root nodule survey Because no root nodules were found o n any of the black bean plants, 17 days of growth were not sufficient for a mutualistic Rhizobia bacteria infection to occur. Previous studies have found that four weeks is often required before nodulation can occur on Fabaceae plants (Adjei et al. 1998). Therefore, young black bean plants must rely on nutrition within their seed endosperm and within the soil to grow (Adjei et al. 1998). Plant height Black bean plant height did significantly differ between plants grown in soil collected closer to cattle feces at the top two sites, Soils 1 and 2, on the UGA farm hill versus plants collected in Soil 4 at the bottom of the hill. Though I was unable to tes t soil [N], these differences in plant height may be due to differences in [N] caused by a gradation of nutrient runoff from the cattle feces. These results are contrary to my hypothesis that plant height would be highest at the top of the hill, the assume d nitrogen source, and at the bottom of the hill, the assumed nitrogen sink. The differences in plant height are similar to the findings of the field study where Mimosa root nodule density decreased to the lowest point at the bottom of the hill, also indic ating that there may not actually be a nitrogen sink, as predicted. Plants grown in Soil 3 did significantly differ from plants grown in Soil 4, but only by a 95% CI. Because plant height for black bean plants grown in Soil 3 did not differ between plants grown in Soil 2, these results are similar to the field study findings and allude to the area between Soils 3 and 4 as being the space where [N] begins to decrease to a less effective level. Though a 17 day growing period was not sufficient for root nodule s to form, ten days has been documented as sufficient time for plants to shift from relying on nutrition within the seed endosperm to the nutrition supplied within the soil (Fehr et al 1971). Previous studies have found that Fabaceae crops shift from rely ing on nutrition within the seed to nutrition within the soil after the first cotyledons appear, approximately ten days after sowing (Fehr et al. 1971). Shifting nutritional needs from seed to soil at such an early stage in the
Effects of cattle feces on Fabaceae plants Appleseed 11 lifecycle of bean plants may account for the differences in plant height measured after only 17 days of growth. However, because I was unable to test [N] directly, I cannot make an affirmative claim about the nitrogen soil levels effect on black bean plants. Future studies should be conducted to confirm if nitrogen inputs from cattle feces do cause a nutrient gradient that runs down the UGA farm hill. I will make the claim that black bean plants grown in soil collected ~200m downhill from cattle feces are unable to grow as tall as pla nts grown in soil collected closer to the cattle grazing site. Specific stem length Similar to the findings for Mimosa root nodule density and plant height, the most significant difference between mean values of SSL for black bean plants occurred between plants growing in soil collected at the top of the farm hill in Soil 1, closest to the cattle feces, versus plants grown in soil collected ~200 m away in Soil 4. These results also indicate that there may be a possible gradient in nitrogen availability as soil is collected further away from cattle feces. If there is a reduction in [N] for soils further away from cattle fece s at the top of the hill, then these results also support previous findings by Jannoura et al 2014 and Herath et al 1978 that increased dry matter is positively correlated with increased organic nitrogen inputs. As opposed to the results in Mimosa root nod ule density and black bean plant height, there was no significant difference between means for plants grown in Soil 2 versus Soil 4. This lack of difference is possibly due to the large variance in SSL data collected from plants in Soil 2 (Fig. 3). Leaf area and Specific Leaf Area Results for black bean leaf area and SLA failed to show a significant difference between mean values for plants grown in soil collected on the UGA farm hill gradient. These results contradict the findings by Herath et al 1978 wh o found that increased soil nitrogen levels do result in increased leaf area and dry matter density. These results may indicate that there actually are no differences in [N] throughout the soil on the UGA farm. Another possible reason for non significant r esults could be that the black bean leaves remain more reliant on the nutrients within their seed endosperm and not the nutrients within the soil at the beginning of development. Though studies have shown that Fabaceae crops become more reliant on nutritio n within the soil after ten days of development, the soil nutrition may have a greater effect on plant height as opposed to leaf organ area (Fehr et al 1971). The nutritional needs for leaf development in Fabaceae crops is another topic in need of further research. In conclusion, a trend of decreasing root nodule density, plant height and stem dry matter density was negatively correlated with being grown in soil collected further from cattle feces at the top of the UGA farm hill. Because leaf area and SL A had no significant differences between plants grown in the different soils, black bean leaf organs may have different nutritional needs for development than plant height The significant differences in plants growing on the UGA farm hill gradient for nod ule density, plant height and SSL may be due to decreasing [N] in soil collected further from the assumed nitrogen rich cattle feces. Future studies testing the actual soil [N] along the UGA farm need to be conducted to confirm my predictions.
Effects of cattle feces on Fabaceae plants Appleseed 12 ACKNOWLEDGMENTS I would like to acknowledge my primary advisors, Sof a Arce Flores and Justin Welch, along with my secondary advisor Federico Chinchilla and my mentor Frank Joyce for their guidance and time while assisting me with my study. I would like t o thank the local Solis and Mendez family from Cerro Plano, Costa Rica for their gracious hospitality when having me as a guest in their home. Additionally, I would also like to acknowledge and thank the Monteverde Research Institue, Biological Station of Monteverde, the University of Georgia field station and UPA the farmer supply collective for their support, resources and facilities. LITERATURE CITED Adjei, M. B., Quesenberry, K.H., Chambliss C.G. 1998. Nitrogen Fixation and Inoculation of Forage Legumes Plant and Soil 204: 57. Antoun, H., Beauchamp, C.J., Goussard, N., Chabot, R., Lalande, R. 1998. Potential of Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on non legumes: Effect on radishes (Raphanus sativus L.) Plant and Soil 204: 57. Fehr, W. R., C. E. Caviness, D. T. Burmood J. S. Pennington. 1971. Stage of Development Descriptions for Soybeans, Glycine Max (L.). Crop Science 11: 929 931. De Faria, S. M., Lewis, G. P., Sprent, J. I., Sutherland, J. M. 1989. Occurrence of nodulation in the Leguminosae. New Phytologist 111 : 607 619. Gage, DJ. 2004. Infection and invasion of roots by symbiotic, nitrogen fixing rhizobia during nodulation of temperate legumes. Microbiol Molecular Biology Review 68(2):280 300. Lewis, O.A.M. 1986. Plants and Nitrogen. Cambridge University Press Print. Jannoura, R., Joergensen R.G., Bruns C. 2 014. Organic fertilizer effects on growth, crop yield, and soil microbial biomass indices in sole and intercropped peas and oats under organ ic farming conditions. European Journal of Agronomy 52: 259 270. Peoples, M.B., Herridge, D.F., Ladha, J.K. 1995. Enhancing legume N2 fixation through plant and soil management. Plant Soil 174: 3. Suryantini, B. 2014. Effect of lime, organic and inorga nic fertilizer on nodulation and yield of soybean (Glycine max) varieties in ultisol soils. Journal of Experimental Biology and Agricultural Sciences 2(1) Thorup Kristensen, K. 2001. Are differences in root growth of nitrogen catch crops important for t heir ability to reduce soil nitrate N content, and how can this be measured? Plant and Soil 230: 185.