Methods

This document is a student project created in connection with a course at The Colorado College. It has not been altered in any form from the original document submitted by the student and is here on the web for STUDENT USE ONLY. No parts of this document may be used or reproduced in any form without written permission of the instructor, Sharon Hall. To email Dr. Hall, click here

The Effects of Fertilization and Irrigation On Tree Growth and Chlorophyll Levels In Ponderosa Pine Trees

Taylor Williamson and Arow Parsons-Pineda

By 208: Ecology

December 18, 2001

Prof. Sharon Hall

Abstract

The purpose of the experiment is to quantitatively determine the effects of fertilization and irrigation on tree growth and chlorophyll levels in Ponderosa pine trees in Colorado Springs. This question will be answered by measuring tree growth over the last year, nitrogen levels in the soil, soil moisture, and chlorophyll concentrations in Ponderosa pines on the campus of The Colorado College (fertilized) and Ponderosa pines found in Palmer Park (unfertilized). The expected response to the nitrogen fertilizer would be higher growth rates, higher levels of chlorophyll in the needles, and higher levels of nitrogen compounds in the soil. If the hypothesis is proved correct, then linear curves will be created which represent these phenomenons. Multiple procedures were used to determine this type of data. These procedures involved use of the spectrophotometer to determine concentrations of ammonia, nitrate, and chlorophyll. A soil moisture test was also performed. This data was then related to tree growth data that was taken on site in an attempt to correlate each compound with growth rates. We found that nitrogen compounds have a negative correlation with tree growth; this is fairly counter-intuitive as nitrogen is necessary for tree development. Also, chlorophyll levels did not vary with tree growth over the last year. We did find a positive correlation between high levels of water and tree growth, leading us to believe that the main limiting factor on Ponderosa pine growth is soil moisture.

Introduction

The focus of our paper is to consider the differences in ponderosa pine in a domestic environment versus a natural, unregulated environment. To consider these differences we measured soil moisture, ammonia and nitrate absorbencies, and chlorophyll concentrations. We hypothesized that higher levels of all these substances would be present in the domestic environment, Colorado College campus, and these higher levels would correlate with higher growth rates.

Although we are trying to find the effect of intentional human activity, it may be helpful to consider some of the known data about Ponderosa Pines in general. Ponderosa Pines are an evergreen tree that can grow to 50 meters tall and 2 meters in diameter. (BC adventure.com, INT). Ponderosa Pines are the "widest distributed pine in the North America"(Richardson, 1998). Their habitat is labeled Temperate Montane and they are found throughout Western North America in all types of soils, from the semi-arid plains of Northern Mexico to the dense, rainy woodlands of Western Canada (WWPA, INT). In a dry, warm environment, they have been shown to decrease growth in foliage in favor of more sapwood biomass (Carey, Callaway, Evan, 1998). This is to reduce transpiration from the stomata in their needles; however, these needles also have higher concentrations of chlorophyll and a greater water use efficiency to offset this lack of water. Therefore, a correlation between water levels and chlorophyll density has been established for Ponderosa pines that live in the desert as compared to those in forests of the Sierra Nevada. (Carey, 1998) For our study, we chose a less invasive method of measuring tree growth than coring, which was to measure from the previous year’s growth node to the tip of the branch. Considering that we measured growth in the foliage, it would make sense, in light of the Carey study, that there would be higher growth rates in the areas with more moisture. Additionally, Ponderosa Pines are well adapted to fire and drought, which is advantageous in a habitat where both these factors are prevalent.

The effect of nutrient stress is also an important factor in tree growth. Although we did not find a strong liner correlation between levels of nitrogen and tree growth, we did find that on average tree growth was greater in areas with more availability of these nutrients. The fact that ammonia and nitrate fertilization increases growth has been established in previous studies. Increased levels of ammonia nitrate (in combination with abundant water) have been shown to increase tree shoot growth (Neilsen, Millard, Neilsen, Hogue, 1997). Nitrogen fertilization was used successfully to stimulate foliage growth in defoliated Scott pine trees. Both of theses studies are evidence that increased nutrients aid tree growth in the foliage. Nutrient stress has also been shown to affect chlorophyll absorbencies (Carter, Knapp, 2001); pines exposed to chronic nitrogen deficiency demonstrated a lower absorbency in the red spectra (700nm). Our data shows apparent differences on and off campus in different ranges of spectrum absorbencies.

Taken as a whole, previous studies show that nutrient (ammonia and nitrate) and water deficiencies may have an adverse effect on plant growth and chlorophyll absorbencies.

Materials

75 cuvettes

DI water

1 razor blade

60 plastic bags

30 erlenmeyer flasks

2 buchner funnels

2 filter flasks

2 rubber stoppers

2 plastic tubings

30 .22 micron filters

Soil core

Tape measure

Spectrophotometer

Coffee grinder

Refrigerator

Balance

35 paper bags

Oven

Timer

30 specimen cups

30 glass whatman filters

Vortex genie

30 Ammonia salicylate

Methods

Tree Growth

To obtain the needed data, we did soil cores, measured last year’s growth, and pulled needles off 15 on campus ponderosa pines and 15 off campus ponderosa pines. The off campus pines were sampled from Palmer Park. The soil cores we took were between 10 and 15 cm. In order to measure last year’s growth, we measured the distance, in centimeters, between the second to last node of pine needles and the end of the terminal bud. This was done 3 times for each tree, on branches that were no higher than 7 _ feet. The three samples were taken randomly from within this range. In order to standardize tree size, the median trees for both on and off campus were set between 150 and 160 cm in diameter. After this was accomplished, a random sample of needles was pulled from four separate locations on each tree. Sample number 14 (on campus) was discarded as it turned out to be a pinion pine.

Soil Moisture

This procedure is from a method written by Professor Sharon Hall. First, we sifted our soil core samples (which had been refrigerated to preserve moisture) through a 2 mm sieve, which separates the soil from gravel or other large particulates. The samples were weighed and labeled directly after being sieved, so as to retain moisture. The contents are then poured into a paper bag and weighed in grams. Five paper bags were also weighed and averaged which was then subtracted from the total weight (both wet and dry). The samples were then put in a 100 degree C oven for 22 hours, along with the five paper bags. After the 22 hours the samples and bags were weighed again, and these weights (dry) were then subtracted from the original weights (wet) and then these total are divided by the wet weight to calculate percents of moisture.

Nitrogen Concentration

Before we could test the absorbance NO₃^- and NH₄⁺, we had to extract them from the soils. We measured ten grams of the pre-sifted soil samples and 50 ml of DI water, which were both put in a specimen cup and shaken vigorously for one minute. The mixtures were then undisturbed for 24 hours. The next day, we filtered the samples through a glass Whatman filter paper and porcelain funnel into a beaker that was connected to a vacuum. The filtered liquid was put back into clean specimen cups and stored in the refrigerator.

Originally, we attempted to use an ion chromatograph to test for nitrate and ammonia residues. However, it was found that interference was plentiful in the specimen cups. This leads us to believe that the filtering process was inadequately designed for our needs. However, we were able to use another method to test for ammonia and nitrate deposits. To test for ammonia (NH₄⁺), 0.5 ml of the filtered specimen and 4.5 ml of milliq water were put into a clean test tube along with an ammonia salicylate powder pillow. Along with the samples we tested six standards 0.1, 0.5, 1, 5, 10 parts per million and a blank for a collaboration curve. Next the test tube was covered and vortexed for one minute. It was placed aside to react for twenty minutes. After the designated reaction time, the tubes were cleaned off and put into spectrophotometer using a wavelength of 655 and the absorption was displayed and recorded.

The nitrate (NO₃^-) was tested first using a Nitraver 5 pillow packet and 10 ml of the sample, but the results were unusually high and some of the murkier samples did not register absorbency. We hypothesized that the high absorbencies were due to interference in the 550 nm range, possibility because of inadequate filtering. Therefore, we decided to retest the nitrates using the Nitrover 3 and Nitrover 6 pillow packets and five ml of the sample water. We then ran these through the spectrophotometer at a wavelength on 500 nm. Unfortunately, we had a limited number of pillow packets and we were only able to test ten samples and four standards but the nitrogen concentrations were much more in line with known data.

Chlorophyll Concentrations

In order to measure chlorophyll levels in the needles a method was devised using the spectrophotometer. First, both ends of the needles were cut off of the needle. This left only the chlorophyll containing part of the needle. These pieces were cut to _ of an inch and stored over night in a refrigerator. Next, a coffee grinder was used for 30 seconds to grind the pine needles to fine threads. This posed a rather interesting problem as sap from the pine needles gathered on the interior of the coffee grinder. Ethanol, acetone, 1 M hydrochloric acid, and 18 M sulfuric acid were all used in an attempt to remove the pinesap. However, none of them succeeded in removing the sap. As the coffee grinder became unusable, it had to be replaced. Two and a half grams of these threads were then placed into a buchner funnel containing .22 micron filter paper. 100 mL of DI water was then poured into the funnel. The powder was allowed to soak for 1 minute until the suction was turned on to pull the liquid through the filter paper and into a filter flask. The suction tube was then turned on by a one eighth turn of the faucet, so as to standardize the amount of suction. It was turned off after the first drop of foam came out of the funnel. This foam was a result of air being sucked into the liquid by the suction of the buchner funnel. The liquid was then poured into a 125 mL erlenmeyer flask. This procedure was done to give the chlorophyll time to diffuse throughout the solvent. 5 mL of each liquid was then pipetted into separate cuvettes for readings by the spectrophotometer. A blank cuvette was inserted into the spectrophotometer to zero it at a wavelength of 420 nm. Next, all 29 cuvettes were run through at this wavelength. This was continued with the same procedure for 460 nm, 620, nm, and 680 nm. 420 nm and 680 nm are the wavelengths at which chlorophyll A absorbs, while chlorophyll B absorbs at 460 nm and 620 nm. The results are then graphed as % absorbance relative to the blank. We would like to have obtained a standard curve for chlorophyll, but we could not find a standard for chlorophyll in either Environmental Science or the Biology department.

Results

After collection and analysis of these samples we attempted to make sense of the data we obtained. The first thing we looked at is the variation in tree growth. Figure 1 shows the wide variation we observed. By this graph it is obvious that, on average, trees grow quicker on campus than off campus. However, the question is begged, why is tree growth more variable on campus than off-campus? Our thoughts were that as the trees compete for nitrogen, they create nitrogen deficient zones around them, out competing other nearby trees. This is not found off campus because of the distance between Ponderosa pines in a non-landscaped situation. Figure 2 shows the coefficient of variation (a measure of how much a data set varies from each other), this graph shows that growth was 3 times more variable on campus than off campus. Why are these trees growing faster on campus than off campus? Every October, the grounds crew at The Colorado College deposits 2.6 pounds of nitrogen fertilizer per 1000 square feet onto the entire campus. It is a slow release method used specifically for the grass, not the Ponderosa pines. However, the trees were studied were either in, or nearby grass lawns. Therefore, nitrogen fertilizer must have an effect on these trees. However, as evidenced by Figure 3, nitrogen fertilization had a negative correlation with tree growth. The reason for this, we believe, is the same reason that growth was highly variable on campus, the creation of nitrogen deficient areas in the soil. However, in our study of the nitrogen concentrations we discovered that it was the faster growing trees that were pulling the nitrogen out of the soil in a more rapid manner. This seemed to make intuitive sense, as these trees would be the ones that need the extra nitrogen provided by the College. Soil moisture was the variable found to control tree growth. This variation in soil moisture also seemed to be the largest difference between on and off campus samples. The low, non-variable tree growth in off campus Ponderosa pines correlated with lower levels of soil moisture, as shown by figure 4. The high, variable tree growth of pines found on campus correlated with higher levels of soil moisture. This trend should be very intuitive, but after finding the higher levels of nitrogen correlated with lower levels of tree growth, we were not willing to take anything for granted. Due to this trend, we reasoned that the main limiting factor on Ponderosa pine growth in the Pikes Peak region is the availability of water. As the Pikes Peak region is a semi-arid grassland receiving only 14.76 inches of precipitation a year, it is no surprise than plants have become limited in their growth by the amount of water found in the soil underneath them. (Selley, INT) Also, water intake aides the uptake of nutrients, such as nitrogen, into the roots. With this in mind we graphed soil moisture vs. ammonia (Figure 5) and found some rather interesting patterns. First, we found that ammonia and soil moisture are positively correlated, i.e. more water in the soil equaled more ammonia in the soil. As water is necessary to pull up this nitrogen from the soil, we expected this type of a graph. However, it became clear that there was a contradiction on the way. Why do higher soil moisture correlate positively with both higher tree growth and higher ammonia levels, but higher ammonia levels correlated negatively with tree growth rates? First of all, we should point out that these are only rough trends and it appeared as if the outliers flip-flopped with the trend data; however, this only accounts for a few data points, not all of them. Also, ammonia concentrations on campus were highly variable (0.03-0.25 μg NH₃/g dry soil) compared to the off campus data (0.01-0.13 μg NH₃/g dry soil), and this is right in line with our data concerning tree growth variability. Therefore, it seems apparent that the growth variability is correlated positively with the ammonia variability; however, ammonia concentrations are negatively correlated with tree growth. This is obviously an area for further study into the question of tree uptake of ammonia. Chlorophyll, which is high in nitrogen content, is the molecule in plants that is responsible for photosynthesis. First, we had to make sure that we were actually measuring chlorophyll levels and not just random plant material that had gotten through the filter. To do this, we graphed the average absorbencies of chlorophyll at the four wavelengths we measured (Figure 6). If we had chlorophyll we would have gotten a polynomial curve that bottomed out at 620 nm and rose slightly to 670 nm. As shown by figure 7 for on campus samples and the graph below for off campus samples, we were 99.6% confident that we were looking at chlorophyll on campus and 99.85% confident that we were looking at chlorophyll off campus. This is very important to note, as we found no correlation between chlorophyll levels and tree growth (Figure 8). However, there was a correlation between ammonia levels and chlorophyll levels. Higher ammonia levels in the soil led to lower chlorophyll levels in the pine needles. This trend is revealed by (Figure 9). This only supports our postulation that the nitrogen being used by these trees had been absorbed quickly to promote growth in the form of higher chlorophyll levels and in the form of more tree growth.

Discussion

Overall, our results demonstrated that moisture was our limiting factor, while soil ammonia, soil nitrate, and leaf chlorophyll were not. Irrigation (or higher moisture percentages) correlated to higher growth rates in the foliage of the previous year. It seems logical that lower soil moisture would lead to lower growth rates. This phenomenon has been documented in other studies. In a study of peach trees, reduced irrigation limited tree growth by up to seventy percent (Boland, Jerie, Mitchell, Goodwin, Connor, 2000). A study of ponderosa pine growth in different unregulated (not irrigated or fertilized) environments revealed that growth rates in foliage decreased while resources were allocated to sapwood growth in a drier and hotter location (Carey, 1998). This could potentially be critical for our study although we did not record temperatures, because of the fact that we did find a lower average of moisture off campus and we measured growth in the foliage.

Our findings for nitrogen levels in the soil did not correspond with our hypothesis or some previous studies on the effect of ammonia and nitrate on tree growth. Counter to our thinking we found no positive correlation between increased soil nitrogen (ammonia and nitrate) and increased growth rates. When considering the reasons for these results, we theorized that the higher nitrogen rates did not correlate to higher growth rates because they would not be allocated to growth in this season (winter). Meaning that the tree had already grown for this year or it was in a dormant stage. This seems to the case in a study of apple trees, which stated that nitrogen is ‘remobilized’ from storage in the xylem, and reallocated to growth of the foliage in the spring (Malaguti, Millard, Wendler, Hepburn,Tagliavini, 2001). Additional in the study the trees were fertilized with a nitrogen compound (15NH415NO3) in the fall, this is relatively the same time of year that the Colorado College campus was fertilized. The fact that the trees may not be growing foliage at this time, and that they applied fertilizer two months ago, are possible explanation for higher nitrogen rates not corresponding to higher growth rate. It would be interesting to test soil and leaf nitrogen level over the course of a year in comparison to growth rates.

Chlorophyll absorption levels are similar to nitrogen level in that higher absorption levels did not correlate to higher growth rates at the time of our study, but this too may have been affected by the season. Acclimation to environmental changes often result in actions like dormancy or storage (Ricklefs, 20001). Another factor we noted in our study was that tree growth overall was greater on campus but that growth in trees was also much more variable on campus. Less tree growth off campus can be explained by the fact that, tree growth in the Palmer Park area was correlated with less water. Environments with less water and higher temperatures have been linked to less growth in ponderosa pines (as mentioned above, Carey, 1998), which would support our finding of lower growth with less water on average. Still, this does not rationalize higher variability on campus. This may be due to increased nutrient patches on campus, due to higher leaching or run off due (because of proximity to sprinklers). Nitrogen discharge has been attributed to water input, with greater irrigation causing leaching, resulting in harmful nitrate in an aquifer (Diez, Roman, Caballero, Caballero, 1997). In our study, nitrogen discharge is a factor because of large amount of water in a small time period could be causing patches that would lead high variation in ponderosa pine growth.

Conclusions

What conclusions can we reach from this breadth of data? First, it seems as if nitrogen was not a major limiting factor in the growth of these tree. Second, water, in the form of soil moisture, tended to have the greatest effect on how well these trees were able to grow. Third, since it is winter, trees had brought in as many nutrients as possible to prepare for the winter. This made measuring chlorophyll samples very difficult. Fourth, the faster growing Ponderosa pines did not have as much nitrogen in the soil underneath them because they had already pulled a great deal of that nitrogen out of the soil.

Further Research

As has already been stated, this research really should be conducted in the summer or over a whole year, when the tree are growing, is using nitrogen at a much faster rate, transpiring at a faster rate, and has all of its chlorophyll out in its needles. We feel like many of the problems associated with this project were due to seasonal variation in tree physiology. Also, we would like to know what the nitrogen levels are in open areas, i.e. not underneath a tree. This data would be used to test our hypothesis about nitrogen depletion zone. Also, it became apparent that nitrogen levels in the needles would also have been interesting data to collect for this same reason. For example, a sample question would be, how do nitrogen levels in the needles correspond with nitrogen levels in the soil? If it were a negative correlation, our hypothesis would be correct, this would help to justify our conclusions in this report; however, time did not allow us to go back into the field to answer these questions.

Bibliography

Boland, A.M., Jerie, P.H., Mitchell, P.D., Goodwin, I., Connor, D. (2000) Long-term effects of restricted root volume and regulated difict irrigation on peach; growth and mineral nutrition. Journal of the American Society for Horticultural Science125 no.1

British Columbia Adventures. (2001). www.bcadventure.com/advernture/wilderness/forest/ponderosa.

Carey, Eileen V., Calleway, Ragan M., and Evan H. (1998). Increased photosynthesis offset costs of allocation to sapwood in an arid environment. (Biosis doc.). Ecology v. 79 no.7.

Carter, Gregory A., Knapp, Alan K., (2001). Leaf optical properties in higher plants: linking spectral characteristics to stress and chlorophyll concentration. American Journal of Botany, v.88 no.4.

Diez, J.A., Roman, R., Caballero, R., Caballero, A. (1997). Nitrate leaching from soils under a maize-wheat-maize sequence, two irrigation schedules and three types of fertilizer. Agriculture Ecosystems and Environment, 65 no.3

Malaguti, Donatella., Millard, Peter., Wendler, Renate., Hepburn, Alan., Tagliavini, Massimo. (2001) Translocation of amino acids in the xylem of apple trees in spring a consequence of both N remobilization and root uptake. Journal of Experimental Botany. 52, no. 361.

Neilsen, D., Millard, P., Neilsen, G.H., Hogue, E.J. (1997). Source of N for leaf growth in high density apple (Malus Domestica) orchard irrigated with ammonia nitrate solution. Tree Physiology, 17. Victoria, Canada Heron: Heron Publishing.