Lab 3. Plant Module Lab 3 of 3, Fall biol 106

Biology 106: Organismal Biology Laboratory 3 The Influence of Nutrients on Plant Growth Plant Module lab 3 of 3 (This Document has been slightly modified from the original protocol written by Dr. Lisa Carloye and her Bio 106 teaching group at WSU) There are three parts to this lab 1. Final harvest of plants 2. Effect of fertilizer up the food chain: caterpillar feeding experiment 3. Conclusions PART 1: Final Measurements Our Brassica plants have been growing now for 3 weeks and received two fertilizer treatments (except for the control, of course). You have data for plant growth at two points so far. These measurements have been of aspects of plant vigor using measurements that did not harm the plant. These data are “non-destructive” parameters because you did not destroy plant tissue. This week we will conduct the final height measurement and then harvest the plants in order to weigh the above-ground parts of the plant (destructive measurements). This allows us to quantify both height and mass as we consider where the plant’s mass comes from and the role of fertilizers in plant growth. Exercise 1: Non-Destructive Parameters - Height Because you can measure the height of the plants without disturbing them, these measurements are “non-destructive”. Once you have the heights, you can harvest the plants, shake off the soil and weight them (but then your plants will be destroyed, so weight measurements are “destructive”. Begin with non-destructive measurements. PROCEDURE :  Open the ‘Lab 3 Prelab blurb.pdf’ for background and context.  Open the document ‘ Lab 3 of 3 SLIDES Brassica v3’ posted on Canvas.  Open the Excel DATA Spreadsheet you saved from last week.  Enter the height data for this week into the Excel data column for 2-weeks growth (columns for, ‘Plant Module Lab 3’) . Q. What are the main results and trends that you see in your data. Summarize this in the space below: Overall increase in plant size, with bigger plants that received low fertilizer as well as medium fertilizer

Biology 106: Organismal Biology Exercise 2: Destructive parameters - Biomass Your plants are made up of organic molecules – complex molecules built on a framework of carbon. These molecules are also rich in nitrogen and phosphorous. Your plant also uses a lot of water to support physiological processes (e.g., photosynthesis, transpiration) and to provide structure (turgor). You can determine how much of your plant’s mass comes from organic molecules and how much of it is water by first weighing your plant material (fresh weight) and then drying the plants to remove the water (dry weight); the difference is, of course, the weight of the water. Now it’s time to prepare your plants for obtaining fresh and dry weights. Follow the below procedures for the destructive , above ground fresh weight measurements. A. Fresh Weight Procedure: Each plant was trimmed with scissors at the soil surface and all plants in the pot were weighed together.  Slides 6-9 show the fresh weights of the plants.  Record the data in your Excel File on the t-Test tab under Fresh Weight . Exercise 3: Data Interpretation How much of the B. napus plant’s weight comes from water and how much comes from the organic matter of its body? To answer this question, we need to subtract the weight of the water in the plant from the fresh weight (which you just measured) by drying down the plant tissue.  Unfortunately, our balances are not sensitive enough to measure the dry weight so instead of actually drying your plants, we will infer how much of their fresh weight is due to organic tissues and how much is due to water.  In Fall 2017, the fresh weights and dry weights of 10 plants from each treatment group were recorded.  We can use these fresh weight:dry weight ratios as conversion factors to infer how much of the fresh weight you measured is due to water and how much is due to organic matter. INSTRUCTIONS:  Calculate the inferred dry weight of each plant using the appropriate conversion factor (see Table below): CONTROL LOW FERTILIZER MEDIUM FERTILIZER HIGH FERTILIZER Conversion factor: (Fresh weight x conversion factor = Dry weight) 0.10 0.23 0.16 0.13  Use the information in the Fresh Weight-to-Dry Weight Conversion table above to fill in the dry weight table.

Biology 106: Organismal Biology Copy and paste your graphs from Excel into this document in the spaces provided at the bottom, just before the Exit Questions. D. Conclusions (statistical analysis) Now, we need to draw conclusions. Recall that science uses statistics to objectively analyze data to help tease out differences that are just due to random variation in the plants themselves versus identifying differences that are due to the treatment itself (in this case the addition of nutrients). There are several approaches you could take for analyzing the data. The appropriate first test is called an Analysis of Variance (ANOVA) which makes comparisons among multiple treatments when you have three or more. Let us suppose the ANOVA indicates a significant difference(s) is present somewhere among the treatments, so we need to do some additional statistical work to determine where these differences occur. We will have you do a step-wise comparison using the t-test statistic with which you are familiar to answer the question of whether each fertilizer amount is better, worse, or the same as the others. You will conduct t-tests for each of the comparisons in the grid below (this is just a list, you will conduct the analyses below). Here is the gist of what you are asking: HIGH vs LOW MEDIUM vs LOW HIGH vs MEDIUM MEDIUM vs CONTROL HIGH vs CONTROL LOW vs CONTROL Exercise 4. Conduct statistical analysis (t-tests) of the dry-weight measurements. RESEARCH QUESTION: What effects do differing amounts of nutrients have on plant growth? STATISTICAL TESTING: EXCEL INSTRUCTIONS Refresher We can perform a t-test to compare these two treatments in Excel by using the following function. Array 1 and array 2 are the two groups you are comparing. The formula looks like this: =TTEST (array1,array2,tails,type) Dry weight of Brassica Plants (mg) Hypothetical #’s Replicate Control Low Medium High 1 2 22 77 98 2 3 34 59 90 3 2 36 79 99 4 0 37 80 100 array1 array2 t-test: control vs low type in: =TTEST Analyzing Plant Dry Weights USING THE ACTUAL DATA from this week, follow the directions to complete the first t-test comparison:

Biology 106: Organismal Biology  Record your statistical results in Table 1 , below. PROCEDURE :  Place your cursor in the box type in “ =TTEST” and then highlight each array in turn, separated by a comma (notice that once you type in the function, it shows you each component you need to supply).  Add a comma after the second array and then type “ 2 ”. This indicates you are running a “two-tailed” test.  Add a comma after the 2 and then type “ 3 ”. This choice indicates that we assume that the variances of the two treatments may not be equal.  Your final function will look something like this although the letters/numbers in the arrays will be different : =TTEST(C2:C8,D2:D8,2,3)  Finally, press Return . The number that appears is the p-value . Since we already determined what our criteria for significance (  ) is (0.05), all we have to do is compare our p-value to that. Recall: p-value vs  Conclusion p >  (p greater than  ) Accept (fail to reject) the null hypothesis; conclude there is no significant difference and any differences you see are due to chance, not the treatment. p ≤  (p less than or equal to  ) Reject null hypothesis & accept the alternate hypothesis; groups are significantly different and it is the treatment that caused the differences. Summary of Statistical Analysis of Plant Dry-Weight Data: p-value Conclusion ( are the treatment plants statistically different or are the differences due to natural variation between plants coupled with random chance for which treatment they happened to end up in ?) Low v Control 3.9946E-05 Reject Null hypothesis Medium v Control .0054 Reject null hypothesis High v Control .0001 Reject null hypothesis Low v Medium .0389 Reject null hypothesis Low v High 1.0509E-05 Reject null hypothesis Medium v High .0613 Accept null hypothesis B. Analyzing Plant Height-week 3

Biology 106: Organismal Biology Plants are the gateway for energy and nutrients into an ecological community. Plants obtain nutrients from the environment converting ‘raw materials’ such as carbon, nitrogen, phosphorous, etc., into organic molecules. They use these organic molecules to accomplish a wide range of tasks and overcome challenges, from staying alive, to staying healthy, to reproducing, to making toxic chemicals or physical structures to defend themselves against predators. Q. Where do primary consumers (herbivores) get their nutrients? _from plants___________ Another question we must consider regarding the effect of fertilizer on yield, is the effect it has up the food chain (web). Consider the following questions:  Does the amount of nutrients in the soil have ripple effects through the food web? Why would an herbivore eat more or less of either treatment? Do the nutrients make the plant tastier? How would they detect differences? Or, are caterpillars averse to some plants because the plants have additional defensive chemicals because of fertilizers? Q. What do you think? Write in full sentences: Consider the ecological context for these results. [ “ + Nutrients ” = fertilizer added, “ – Nutrients” = control ] Q. Why might a caterpillar have a preference for a “ + Nutrients ” leaf versus a “ – Nutrients ” leaf? Q. Conversely, why might a caterpillar have a preference for a “ – Nutrients ” leaf versus a “ + Nutrients ” leaf? You will explore the question of whether the fertilizer in the plants affects the herbivory by caterpillars by seeing if herbivores, specifically cabbage looper caterpillars ( Trichoplusia ni ), have a preference when offered a choice among leaves grown under different nutrient treatments . A. Procedure. Fertilizer can have major affects on the food web, Herbivores may eat more of the plants with higher nutrients but they also may like the natural plants that they do not detect changes in. Caterpillars are adverse to some plants because of fertilizers and chemicals. More nutrients, bigger plants and leaves Detects unnatural changes, plants have defense chemicals, if they are accustomed to – nutrients this may be what they prefer

Biology 106: Organismal Biology Look at the set-up of a typical leaf disc choice test shown (see slide #10 in the ‘ Lab 3 of 3 SLIDES Brassica v3’ data file ).  The dark circle are discs cut from a leaf of each treatment.  The caterpillar is the long worm-like critter eating the Medium Treatment leaf disc. This classic “choice experiment” is set up by cutting discs from each leaf, and arranging them equidistance around the edge of the arena. A single caterpillar is then placed in the center and allowed to choose a leaf and eat for a set amount of time (30 minutes in this case). When time is up, the researcher records which leaf each caterpillar is eating at that moment. This experiment was done for Bio 106 using a total of 52 arenas (and, therefore, 52 caterpillars). B. Caterpillar choice experiment set up. What is the hypothesis (H 1 )? Write it below. What is the prediction? What is the independent variable? What is the dependent variable? Write the null hypothesis (H 0 ) that is being tested with this experiment: Control Low Medium High H 1 : Caterpillar will prefer plants with higher fertilizer contents The caterpillar will eat more of the nutrient dense plants Treatment types water H o : Caterpillar will not have preferences

Biology 106: Organismal Biology # Caterpillars EXPECTED to be eating each leaf based on Null Hypothesis: # Caterpillars OBSERVED to be eating each leaf at end of experiment: The statistic equation is shown below. You can clearly see that the logic of this test is to compare the observed results with what one would expect to see if the caterpillars are just choosing a leaf randomly. If the observed results are close to what is expected, then the Chi-square value will be low. The more widely the observed values differ from the expected, the larger the Chi-square value. The associated probability (p-value) of getting the observed results under random choice allows us to draw conclusions. The symbol, Σ, means the ‘sum of,’ and in this case is the sum of the values, (O – E) 2 /E, from each of the four treatment conditions. When we run the Chi-square test, we get a value of Χ 2 = 12.4 which is associated with a p-value of 0.005 Draw Conclusions: Q. Does adding fertilizer impact the amount of herbivory? Q. What are the implications of this result? That is, if you are adding nutrients to increase yield in your canola plants, what do these herbivory results suggest? PART 3: Thought Question (no written response needed): Your rapeseed plants are now harvested and it’s time to think about what you’ve learned so far that will guide the growers’ business decisions for the next growing season, the basic premise for conducting the study. Fertilizers can be expensive so using them wisely is an important consideration for farmers. What do your results from the three-week study suggest will maximize farmers’ harvests? What recommendations would you make to them? The next step is to disseminate (communicate) your results. You will do this as part of the Exit Questions. Experimental #caterpillars Theoretical #caterpillars if they have no preference for one leaf over the others Theoretical #caterpillars if they have no preference for one leaf over the others Yes Adding some nutrients is helpful however too much damages herbivory however in general adding nutrients increases herbivory in low and medium nutrients.

Biology 106: Organismal Biology Insert your growth/height graph (column/vertical bar) for your third set of growth measurements here: Insert your growth/height graph (line) for all-three weeks of growth measurements here:

Biology 106: Organismal Biology Lab Week 3 Exit Questions Plant module lab 3 of 3 Participation in and Completeness of lab: [5 pts] _________ Plant Data: Final Plant Measurements 1. Drawing Conclusions : In the space below, write a formal, concise conclusion , based on your statistical analysis , regarding the effect of fertilizer on plant yield. Your statement should be written to be shared with the Canadian Canola Growers Association (the audience for your paper). Use complete sentences and cite your t-test and p-values to support your claims: [3 pts] Nutrients Overview 2a. What factors contributes to the plant’s mass? Highlight one option for each bullet [ 2 pts]  Is part of its weight from water in the plant ? YES / NO  Is part of its weight from carbon in the plant? YES / NO  Is part of its weight from nitrogen in the plant ? YES / NO  Is part of its weight from phosphorous in the plant ? YES / NO 2b. Water is a part of every plant but describe in your own words what happens to the majority of water that a plant takes up. [1 pt] 2c. Proteins and DNA both are made up of many carbon, nitrogen, and phosphorous atoms. Where does a plant get the majority of its … [1 pt total] a. carbon: b. nitrogen & phosphorus : 2d. What is the role of nutrients in plant growth? (1 pt) Caterpillar Data: Fertilizers effect on herbivory 3. Did fertilizing the plants affect herbivory? Give your conclusion and support your claim with the X 2 statistic. Use complete sentences. [2 pts] Based on our data we recommend an implementation of fertilizer into crops. Based on P values we found the medium vs control had a dry weight p value of .0054 indicated a rejection of the null hypothesis, and showing that the use of fertilizer does affect the height of the plants. Based on this I recommend implementation of medium fertilizer treatments. I do not recommend high amounts of fertilizer because plant size actually lessened, there was a p-value plant height of .3252 indicating accepting the null hypothesis and there was not a increase in plant height The majority of water is lost in transpiration, where water vapor is being released into the air from the leaves. From the atmosphere soil Nutrients feed the plants and allow the plant to grow Fertilizing plants affected herbivory, more treatment allowed for the caterpillar to eat more of it however with too much fertilizer there was a decrease in herbivory.