Agronomic Practices to Reduce Alkaloid Levels in Dark Fire-Cured Tobacco

University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange

Masters Theses Graduate School

12-2018

Michael Ryan Brown University of Tennessee

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Recommended Citation Brown, Michael Ryan, "Agronomic Practices to Reduce Alkaloid Levels in Dark Fire-Cured Tobacco. " Master's Thesis, University of Tennessee, 2018. https://trace.tennessee.edu/utk_gradthes/5352

This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council:

I am submitting herewith a thesis written by Michael Ryan Brown entitled "Agronomic Practices to Reduce Alkaloid Levels in Dark Fire-Cured Tobacco." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Plant Sciences.

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Agronomic Practices to Reduce Alkaloid Levels in Dark Fire-Cured Tobacco

A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville

Michael Ryan Brown December 2018

Acknowledgments I would like to extend my gratitude to Dr. Eric Walker, for giving me a chance to pursue my M.S. in his program. Thank you for your guidance, patience, encouragement, and friendship.

I want to thank Dr. Andy Bailey and Dr. Bob Miller for serving on my graduate committee, and for their guidance, assistance, and friendship.

To Mr. Joe Beeler, for his constant encouragement, guidance, and friendship.

To Dr. Thomas C. Mueller, for his assistance and friendship.

To Mr. Rob Ellis and the personnel at Highland Rim Research and Education Center, for their assistance and great memories throughout my years at Tennessee.

To the personnel at the University of Kentucky Research and Education Center at Princeton, for their assistance with field plots in 2017.

To Dr. Virginia Sykes and Dr. Liesel Schneider, for their guidance and statistical counseling.

To my fellow graduate students and undergraduate assistants, Madison Cartwright, Trey Clark, Kacey Cannon, Johnny Richwine, Luke Shoffner, Shawn Butler, Mitchell Richmond, Andrea Keeney, Ethan Parker, Alinna Umphres-Lopez, Ragan Sloan, and Peyton Hix, for their assistance, encouragement, and friendships during my time at Tennessee.

To my parents, Mike and Christy Brown, for their constant love, patience, encouragement, and support.

To my grandmother, Betty Carol West, for her love, support, and encouragement.

To my uncle, Donnie Cherry, for his encouragement to pursue my career in agriculture.

To my brother, Austin Brown, for his love and friendship.

To Sarah Elise Ramsey, for her encouragement, support, friendship, and love.

Abstract

Field experiments were conducted at two locations over two years to evaluate the effects of agronomic practices on physical and chemical properties of dark fire-cured tobacco.

Locations were at the Highland Rim Research and Education Center in Springfield, TN, and at the University of Kentucky Research and Education Center in Princeton, KY. One experiment was conducted in Springfield, TN, in 2016 and experiments were conducted at both locations in

2017. These experiments evaluated two dark tobacco varieties (KT D14 LC and KT D18 LI), two nitrogen rates (84 kg N/ha applied preplant and 196 kg N/ha - 84 kg applied preplant followed by 112 kg at layby), three topping stages (early button, late bloom, and immediately prior to harvest), and two sucker control methods (dropline application with fatty alcohol solution and hand-suckered). The 2017 experiments did not evaluate the sucker control treatments. Soil amendments, pest control, and all other production practices were carried out according to university recommendations. The effects of variety and nitrogen rate on yield were inconsistent. However, similar yield and quality was observed for both varieties when averaged across all years and locations. Leaf chemistry was inconsistent between both varieties.

Reduction of nitrogen reduced alkaloids, nicotine, TSNA, and NNN, while earlier topping increased alkaloids, nicotine, TSNA, and NNN. However, results could have been influenced by the amount of days after topping until harvest. While topping immediately prior to harvest significantly reduced alkaloids, nicotine, TSNA, and NNN, this practice also severely decreased yield and quality. Leaf from higher stalk positions produced higher levels of all chemical constituents, except nicotine to nornicotine conversion and NNK. Conversely, lower stalk positions resulted in higher levels of nicotine to nornicotine conversion and NNK. Based on the

iii results of this study, reduced nitrogen and later toppings decreased the amount of certain chemical constituents in the cured leaf.

Table of Contents

CHAPTER 1: Introduction...... 1

CHAPTER 2: Literature Review ...... 4

Health Concerns Associated with Tobacco ...... 5

Tobacco Alkaloids ...... 6

Factors Influencing Alkaloids and TSNAs...... 7

TSNA Reduction ...... 9

CHAPTER 3: Research Methods & Procedures...... 12

Justification ...... 13

Objective...... 13

Materials & Methods...... 13

Statistical Analysis & Procedures ...... 15

Results & Discussion 2016 ...... 16

Results & Discussion 2017 ...... 23

Conclusions & Future Research...... 27

LITERATURE CITED ...... 30

APPENDIX ...... 37

VITA ...... 80

List of Tables

Table 1. Treatment list for the 2016 study in Springfield, TN...... 41

Table 2. Treatment list for the 2017 studies in Springfield, TN and Princeton, KY...... 42

Table 3. Total cured leaf yield by variety in 2016...... 46

Table 4. Total cured leaf yield in response to nitrogen rate in 2016...... 46

Table 5. Total cured leaf yield by topping stage and sucker control interaction in 2016...... 46

Table 6. Lug crop throw by variety in 2016...... 47

Table 7. Lug crop throw by sucker control method in 2016...... 47

Table 8. Seconds crop throw response by variety and topping stage interaction in 2016...... 48

Table 9. Leaf crop throw by variety in 2016 ...... 49

Table 10. Leaf crop throw by topping stage in 2016...... 49

Table 11. Leaf crop throw by sucker control method in 2016...... 49

Table 12. Quality grade index response to topping time in 2016...... 50

Table 13. Total alkaloids in response to nitrogen rate in 2016...... 51

Table 14. Total alkaloids by stalk position and variety interaction in 2016...... 51

Table 15. Total alkaloids by stalk position and topping stage interaction in 2016...... 52

Table 16. Total alkaloids by variety and topping stage interaction in 2016...... 52

Table 17. Total alkaloids response to sucker control method in 2016...... 53

Table 18. Nicotine response to nitrogen rate in 2016...... 56

Table 19. Nicotine percentage response to sucker control method in 2016...... 56

Table 20. Nicotine percentage by stalk position and variety interaction in 2016...... 56

Table 21. Nicotine percentage by stalk position and topping stage interaction in 2016...... 57

Table 22. Nicotine percentage by variety and topping stage interaction in 2016...... 57

Table 23. Nornicotine percentage by stalk position and nitrogen rate interaction in 2016...... 58

Table 24. Nornicotine percentage by stalk position and variety interaction in 2016...... 58

Table 25. Nornicotine percentage by stalk position and topping stae interaction in 2016...... 59

Table 26. Nornicotine response to sucker control method in 2016...... 59

Table 27. Effect of stalk position by variety interaction on nicotine to nornicotine conversion in 2016...... 60

Table 28. Effect of stalk position by nitrogen rate interaction on nicotine to nornicotine conversion in 2016...... 60

Table 29. Effect of stalk position by topping stage interaction on nicotine to nornicotine conversion in 2016...... 61

Table 30. Effect of variety by nitrogen rate by topping stage interaction on nicotine to nornicotine conversion in 2016...... 62

Table 31. Effect of stalk position by variety interaction on total TSNA in 2016...... 63

Table 32. Effect of stalk position by nitrogen rate interaction on total TSNA in 2016...... 63

Table 33. Effect of stalk position by topping stage interaction on total TSNA in 2016...... 64

Table 34. Effect of variety by nitrogen rate interaction on total TSNA in 2016...... 65

Table 35. Effect of variety by sucker control interaction on total TSNA in 2016...... 65

Table 36. Effect of stalk position on NNK in 2016...... 66

Table 37. Effect of nitrogen rate by topping stage interaction on NNK in 2016...... 66

Table 38. Effect of variety by sucker control method on NNK in 2016...... 67

Table 39. Effect of stalk position by nitrogen rate interaction on NNN in 2016...... 68

Table 40. Effect of stalk position by topping stage interaction on NNN in 2016...... 68

Table 41. Effect of variety by nitrogen rate by topping stage interaction on NNN in 2016...... 69

Table 42. Effect of variety by sucker control interaction on NNN in 2016...... 69

Table 43. Effect of variety by nitrogen rate interaction on total yield in 2017...... 70

vii

Table 44. Effect of topping stage on total yield in 2017...... 70

Table 45. Effect of topping stage on lug crop throw in 2017...... 71

Table 46. Effect of topping stage on leaf crop throw in 2017...... 72

Table 47. Effect of topping stage on quality grad index in 2017...... 73

Table 48. Effect of topping stage on total alkaloids in 2017...... 74

Table 49. Effect of stalk position on total alkaloids in 2017...... 74

Table 50. Effect of topping stage on nicotin in 2017...... 75

Table 51. Effect of stalk position on nicotine in 2017...... 75

Table 52. Effect of stalk position on nornicotine in 2017...... 76

Table 53. Effect of variety on TSNA in 2017...... 77

Table 54. TSNA response to nitrogen rate in 2017...... 77

Table 55. TSNA response to topping stage in 2017...... 77

Table 56. Effect of nitrogen rate on NNK in 2017...... 78

Table 57. Effect of stalk position on NNK in 2017...... 78

Table 58. NNN response to nitrogen rate in 2017...... 79

Table 59. NNN response to topping stage in 2017...... 79

viii

List of Figures

Figure 1. Total acres of tobacco production in the United States from 1990-2016...... 38

Figure 2. Total dark fire-cured tobacco production in the United States from 1990-2016...... 39

Figure 3. Total dark air-cured tobacco production in the United States from 1990-2016...... 40

Figure 4. Weather conditions in 2016 for Springfield, TN...... 41

Figure 5. Weather conditions in 2017 for Springfield, TN...... 44

Figure 6. Weather conditions in 2017 for Princeton, KY...... 45

Figure 7. Alkaloid profile of D14 & D18 in Springfield, TN in 2016...... 54

Figure 8. Alkaloid profile of D14 & D18 in 2017...... 55

CHAPTER 1

Introduction

Tobacco has been produced commercially in North America for over 400 years (Tso,

1990). Today, there are multiple types of tobacco grown in the United States, and it is an economically important crop for its producers. Flue-cured tobacco accounts for the largest portion of the U.S. tobacco market, making up approximately 68% of total production. It is mainly produced in Virginia, Georgia, North Carolina, and South Carolina. Flue-cured tobacco is primarily used in cigarettes. Burley tobacco accounts for around 22% of the United States total tobacco production. Burley is grown across a wide range of states in the U.S. The majority of burley tobacco is used in cigarettes, but a small portion is used for chewing tobacco. Dark tobacco, as a whole, makes up one of the smaller segments of the U.S. tobacco market. Dark air- cured tobacco accounts for about 1.5% of the tobacco production in the United States and is used in chewing or plug tobacco and moist snuff (Tso, 1990). Dark fire-cured tobacco accounts for about 6% of the total market annually (USDA, 2017). Dark fire-cured tobacco is distinctive from other U.S. tobacco types due to its curing process. Once the tobacco is housed in the barn, the producer fires the tobacco by setting small fires consisting of hardwood slabs covered by sawdust on the barn floor under the tobacco. These fires smolder and produce an abundance of smoke that, combined with the heat, gives this tobacco its distinctive aroma, finish, and flavor.

Dark fire-cured tobacco is used mainly in moist snuff, cigars, and roll-your-own products

(Bailey, 2017). While production of other types of tobacco has been decreasing in recent years

(Figure 1), both dark fire-cured (Figure 2) and dark air-cured (Figure 3) production remain strong (USDA, 2017).

The tobacco industry has been under scrutiny as early as the 1960’s due to the health risks associated with tobacco usage, particularly cigarette smoking. Awareness of tobacco- related harm to human health culminated during the 1990’s. In 1999, state attorney generals

2 from 46 states filed a lawsuit against large tobacco companies for health costs related to illnesses linked to cigarette smoking. The tobacco companies were found liable in this suit, and the resulting Master Settlement Agreement was met in the form of a financial allocation from the tobacco companies to the states (Jayawardhana et al., 2014; Jones and Silvestri, 2010). After disputing the need for price supports for tobacco, President George W. Bush signed ‘The Fair and Equitable Tobacco Reform Act’ in 2004, more commonly known as the ‘tobacco buyout’.

This act ended price supports and production allotments that had been in place for sixty-six years, resulting in fewer tobacco leaf auctions, more direct contracting with tobacco companies and leaf dealers, and at times, lower tobacco prices. Since then, the demand for American-grown tobacco has diminished somewhat due to the increasing production and quality of foreign tobacco (Brown et al., 2007). The decrease in overall production of tobacco in the United States coincided with both of these events (Figure 1). Dark fire-cured tobacco also experienced a similar drop in acreage during this time. However, since 2006, dark fire-cured tobacco production has increased, as shown in Figure 2 (USDA, 2017).

According to Snell (2013), the outlook for dark fire-cured tobacco is still strong in the current marketplace. Increasing sales of smokeless tobacco products and minimal competition from foreign markets provides a relatively stable future for dark fire-cured tobacco producers

(Brown & Snell, 2014). Dark fire-cured tobacco is a primary ingredient of smokeless products, including moist snuff, dry snuff, chewing tobacco, plug tobacco, and twists (Savitz et al., 2006;

Bailey, 2017). However, ongoing concerns, legislation, and pressure to reduce harm associated with the usage of tobacco products necessitates that the industry continually seeks ways to offer products with reduced health risks.

CHAPTER 2

Literature Review

Health Concerns Associated with Tobacco

Tobacco has long been consumed throughout the world for uptake and enjoyment of nicotine, an alkaloid produced by the plant (Tso, 1990). Nicotine is typically the major alkaloid and is responsible for the physiological effects caused by tobacco use (Andersen et al., 1990;

Ejrup 1965). It is an addictive psychostimulant and affects the central nervous system. These effects are determined by the dosage of nicotine that is received by the user. At lower doses, nicotine increases the effects that dopamine can have on the brain (Clark et al., 2008; Pesta et al.,

2013). At higher doses, nicotine increases the effects of serotonin and opiate activity, which can give the user a calming or relaxed feeling (Silvette et al., 1962; Pesta et al., 2013).

According to the National Cancer Institute (2017), tobacco use – particularly smoking – is one of the leading causes of cancer and cancer deaths. Tobacco use is also strongly correlated with other serious health problems, including cardiovascular problems, such as heart attack, stroke, and hypertension, and respiratory diseases, such as emphysema and chronic obstructive pulmonary disease (U.S. Department of Health and Human Services, 2010; U.S. Department of

Health and Human Services, 2014). Nicotine can also cause increased blood pressure, heart rate, vasoconstriction, and contractility (Walker et al., 1999; Pesta et al., 2013). The sympathetic nervous system experiences similar effects caused by nicotine from smoking cigarettes, chewing tobacco, or snuff (Savitz et al., 2006). However, different methods of tobacco use can be more efficient at transferring nicotine to the bloodstream than others. It has been noted that it takes about seven seconds for nicotine to reach the brain from cigarette smoke (Russell and

Geyerabend, 1978; Tso, 1990). The Center for Disease Control and Prevention (2017) has reported that smoking cigarettes can cause damage to almost all parts of the body and lessens the overall well-being of a person.

Although there is evidence of significant harm reduction associated with smokeless tobacco use in comparison with smoking, smokeless tobacco use carries significant health risks.

These risks include cancer, stroke, heart disease, and hypertension (Wyss et al., 2016; Piano et al., 2010; World Health Organization, 2007; Colilla, 2010; Bolinder et al., 1994; Hergens et al.,

2007; Teo et al., 2006; Rodu and Cole, 1994; Henley et al., 2007; Hatsukami and Severson,

1999; Scientific Committee on Emerging and Newly Identified Health Risks, 2007). Other health risks associated with smokeless tobacco product use are the development of dental cavities, gingivitis, and leukoplakia, which are patches or lesions that can be precancerous

(Savitz et al., 2006). Lowering the alkaloid concentration in tobacco plants has come to be an important objective for the tobacco industry due to the addictive properties of tobacco alkaloids and the health risks caused by their derivatives, tobacco specific nitrosamines, or commonly known as TSNAs, (Julio et al., 2008).

Tobacco Alkaloids

Alkaloids are naturally-occurring substances found in both plants and animals. There are numerous alkaloids found in tobacco plants, but only four account for the majority of the alkaloid pool in tobacco: nicotine, nornicotine, anabasine, and anatabine (Dewey and Xie, 2013;

Cai et al., 2016). Of these, nicotine is the most significant, comprising nearly 90% of the plant’s alkaloid content and responsible for tobacco being a cultivated and economically viable crop

(Saitoh et al., 1985; Tso, 1990).

It has long been established that the roots of tobacco plants are the only part of the plant that is capable of producing nicotine (Dawson, 1942). It has been confirmed by many studies to this date that the concentration of alkaloids, such as nicotine, is greater in the leaves than either the roots or shoots. After examining leaf lamina, Schlotzhauer et al. (1989) found alkaloid

6 concentrations to be higher in the upper leaf positions. This suggests alkaloids are present in leaves by translocation and accumulation (Dawson, 1942). Once synthesized in the roots, nicotine is then transported through the xylem to the shoot and into the leaves where it is stored in cell vacuoles (Baldwin, 1999; Morita et al., 2009; Wang et al., 2015).

Research has concentrated primarily on nicotine and nornicotine in recent years, as they are the major concerns to health risks for humans due to the TSNAs produced by their nitrosation

(Julio, 2007). Under certain conditions, some nicotine is converted to nornicotine by demethylation (Cai et al., 2016). According to Cai et al. (2016), alkaloids in tobacco leaves go through a nitrosation process and the end result of these reactions is a TSNA corresponding to a certain alkaloid. The four major TSNAs resulting from the nitrosation process include: NNK [4-

(methylnitrosamino)-1-(3-pyridyl)-1-butanone]; NNN [N’-nitrosonornicotine]; NAB [N’- nitrosoanabasine]; and NAT [N’nitrosoanatabine]. Therefore, nicotine only produces NNK, nornicotine produces NNN, anabasine produces NAB, and anatabine produces NAT upon nitrosation (Cai et al., 2016). Prior to these findings, it was believed that NNN could come from either nicotine or nornicotine (Caldwell et al., 1991; Dewey and Xie, 2013). These nitroso- compounds are carcinogenic to humans (Djordjevic et al., 1989; Bush et al., 2001; Cai et al.,

2016).

Factors Influencing Alkaloids and TSNAs

Multiple factors are capable of having an impact on alkaloid concentration and in turn,

TSNA levels. Nicotine synthesis and accumulation is affected by genetics, environmental conditions, injury from insects or mechanical devices, and agronomic practices, such as nitrogen rate, sucker control, and topping time (Campbell et al., 1982; Baldwin, 1988; Wang et al., 2008;

Wang et al., 2015).

Alkaloids, as a whole, are a class of secondary metabolites that contain nitrogen, and one of the precursors for TSNAs is the nitrate anion (Dewey and Xie, 2013; Chamberlain et al.,

1984; Brunnemann et al., 1977). Numerous sources show that nicotine levels are positively correlated with increasing nitrogen rate and uptake (Weybrew and Woltz, 1975; Campbell, 1982;

Collins and Hawks, 1994; Hu, 2000; Wang, 2008). Chamberlain and Chortyk (1992) concluded that there was a positive correlation between nitrogen rates and nicotine levels and total alkaloid levels in both burley and flue-cured tobacco. Chamberlain et al. (1984) recorded an increase in

NNN levels as nitrogen rate was increased under flue-cured practices. Mackown et al. (1984) found a positive correlation between TSNA accumulation and the total amount of nicotine and nornicotine. However, they reported no correlation between nitrogen rates and TSNA production in burley tobacco despite a 10-fold difference in nitrate levels across treatments.

Bailey (2014) concluded that different nitrogen levels had no effect on nicotine concentrations in dark fire-cured tobacco, but too much nitrogen increased the conversion of nicotine to nornicotine and the total amount of TSNA in some instances. These studies confirm that there is a relationship between alkaloids, nitrate anions, and how they interact with each other to create

TSNAs.

Other studies have investigated topping of tobacco plants as the main cause of alkaloid production, concentration, and TSNA accumulation. Wang et al. (2008) found no correlation between nicotine concentration and nitrogen fertility rate, but suggested that the removal of the shoot apex could be causing the increase in nicotine synthesis in tobacco. Arsenault (1985) determined that later topping times and a higher topping height decreased the amount of alkaloids in plants. This is likely due to the fact that nicotine production in the roots is initiated, or markedly increased, by topping, and with a higher topping height, the nicotine produced and

8 translocated into the shoot is in effect diluted by a taller shoot with more leaves in comparison with a shorter shoot with less leaves.

The harvest interval is a critical aspect of minimizing TSNA accumulation. In general,

TSNA accumulation will increase as the plant matures in the field. This is due to the higher amount of alkaloids in the plant after topping (Jack et al., 2016). The optimum time to harvest is dependent on multiple factors. Weather conditions, disease pressure, soil conditions, grower and industry preferences, and labor availability can all affect tobacco harvest timing (Heggestad and

Bowman, 1953). Literature has shown a positive correlation between increased alkaloid levels in tobacco when there was an increase in the period of time between topping date and harvest date.

Data from Heggestad and Bowman (1953) suggested that average nicotine concentration increased when harvest was delayed. As the plants continued to mature in the field, nicotine levels increased by an average of 2.35%. Their results indicated this was true for all leaf grades.

Gupton (1982) observed similar results, where total alkaloids increased when harvest was postponed. As tobacco harvest was delayed, the percentage of total alkaloids increased for both years of the study.

TSNA Reduction

The formation of TSNAs mostly occurs during the curing phase. It has been noted that

TSNAs can also be formed in the aging, processing, and combustion of tobacco leaves

(Chamberlain et al., 1986; Hecht et al., 1978; Hoffman et al., 1981; Parsons et al., 1986).

There are multiple ways to lower TSNA in tobacco. The genetic background of a tobacco plant largely impacts the chemical properties of the cured leaf (Campbell et al., 1982;

Long 1983; Terrill et al., 1985). Recently, much progress in lowering TSNA has been realized by selective breeding. Breeders have worked to develop tobacco varieties with desirable

9 agronomic traits and lower nicotine to nornicotine conversion levels, which can result in a lower propensity to produce TSNAs. Researchers have paid close attention to NNK and NNN since both have been considered the most carcinogenic TSNAs to humans (Lewis et al., 2012). In the last several years, researchers accomplished this by screening plants for the lowest nicotine to nornicotine conversion level and culling out any plants that do not have the correct nornicotine level. This process has given rise to the varieties currently on the market, known as low- converter (LC) varieties. However, some plants will revert back to producing more conversions than the desired levels, which is a natural occurrence. This requires breeders to screen plants each year for alkaloid levels and TSNAs (Miller, 2017). Researchers are now further reducing

NNN levels through conventional breeding, genetic engineering, and introducing mutations into certain genes that are responsible for the synthesis of NNN (Lewis et al., 2012). Inhibition of the nicotine demethylase gene has proven to be an effective practice for lowering NNN levels in tobacco, as demonstrated by Lewis et al. (2008), which resulted in an exponential decrease in nornicotine levels and fewer total TSNAs.

As mentioned in the previous text, agronomic practices have been shown to reduce

TSNAs, and some practices have also been shown to reduce tobacco alkaloid levels. Using low converter (LC) or screened seed, reducing nitrogen rate, reducing the time between topping and harvest, minimizing field wilting time, preventing the housing of tobacco with free moisture on it, housing with plant and stick spacing being such that adequate air ventilation throughout the barn is ensured, fire-curing at lower temperatures, avoiding artificial casing during market preparation, and timely delivery of tobacco that has been prepared for market will minimize

TSNA production and accumulation in tobacco (Jack et al., 2016). Similarly, variety selection, nitrogen rate, topping timing, length of time between topping and tobacco harvest, and water

10 availability during the season can impact alkaloid production and accumulation in tobacco

(Campbell et al., 1982; Gupton, 1982; Maw et al. 1998; Vepraskas and Miner 1987).

CHAPTER 3

Research Methods & Procedures

Justification In 2017, the Food and Drug Administration (FDA) proposed rules to limit the amount of alkaloids in cigarettes, particularly nicotine, and reduce NNN levels in smokeless tobacco products. These proposed rules are a culmination of years of research findings and tobacco industry movement toward reduced risk tobacco products. Additionally, these newly proposed levels may not be consistently attainable using current production practices. There has been increased interest in the development and release of low-alkaloid tobacco varieties, such as

DFHD18, an experimental breeding line. Previous data suggests this experimental breeding line produces lower alkaloid levels than conventional varieties. However, most lower alkaloid varieties tend to have lower quality than conventional varieties. While more time is needed to develop the tools, methods, and production recommendations to consistently meet FDA targets, there is a current need to evaluate available varieties under research-based production methods to minimize tobacco alkaloid and TSNA production and accumulation.

Objective

The objective of this research was to evaluate the effects of agronomic practices on the yield, quality grade index, and chemical properties of a conventional alkaloid dark tobacco variety (KT D14LC) and a lower alkaloid dark tobacco variety (DFHD18). Based on the findings of previous literature, variety, higher nitrogen rates, and earlier topping times will all have an effect on the cured leaf yield, quality grade index, and chemical properties of dark fire- cured tobacco.

Materials & Methods

Field studies evaluating the effects of agronomic practices on dark fire-cured tobacco were conducted at the University of Tennessee Highland Rim Research and Education Center in

Springfield, Tennessee, (2016 & 2017), and at the University of Kentucky Research and

Education Center in Princeton, Kentucky (2017).

The 2016 study employed a randomized complete block design with a factorial treatment arrangement with four treatment factors: two dark tobacco varieties, two nitrogen rates, three topping stages, and two sucker control methods, all of which resulted in 24 total treatments and four replications. A plant population of 10,800 plants per ha-1 was used on a Staser silt loam.

The sucker control treatments were eliminated from the 2017 studies due to the practicality of carrying out hand-suckered treatments in a field study, as well as the negligible effect these treatments had on the study compared to other treatment factors. In 2017, the study was repeated in Springfield with an additional location being added in Princeton, KY. These studies were blocked based on location. The experimental design for both locations in 2017 was a randomized complete block with a factorial treatment arrangement with three treatment factors: two dark tobacco varieties, two nitrogen rates, and three topping stages which resulted in 12 total treatments and four replications. The 2017 locations included plant populations of 10,800 plants ha-1 on a Hamblen silt loam in Springfield and 12,100 plants ha-1 on a Crider silt loam in

Princeton.

The tobacco varieties used for this study were KT D14 LC (D14) and DFHD18 (D18).

Two nitrogen rates were used for this study: 84 kg ha-1 and 196 kg ha-1. The lower nitrogen rate of 84 kg ha-1, was applied pre-plant at all locations for 2016 and 2017. At Springfield, urea (46-

0-0) was used as the pre-planting nitrogen source for both years. Soil tests indicated additional phosphorus be added to the selected site at Princeton in 2017. Therefore, diammonium phosphate (18-46-0) was used to meet this phosphorus requirement. Ammonium nitrate (34-0-0) was also applied prior to planting to achieve the 84 kg ha-1 needed for the Princeton location.

Ammonium nitrate was applied at layby as the nitrogen source for treatments requiring 196 kg N ha-1 for 2016 and 2017 at all locations. The topping stages used in this study were early button, late bloom, and immediately prior to harvest. The two sucker control methods in the 2016 experiment consisted of a chemical treatment, where a fatty alcohol solution was used (oiling), and a hand-suckered treatment.

Experimental plots consisted of four rows with plot dimensions of 12 meters x 4.2 meters. Conventional tillage was used at all locations and all other pest control and production practices were conducted according to university recommendations. A full list of treatments for each year and location can be found in Table 1 and Table 2. Weather data from each year and location can be found in Figures 4-6.

Data collected included total cured leaf yield, quality grade index, total alkaloids, and

TSNAs. Cured leaves were separated according to the following leaf positions on the stalk: lugs, seconds, and leaf. Whole, cured leaf samples were pulled from each plot for each leaf position to obtain chemical analyses. These whole leaf samples were ground in a Wiley Mill to pass through a 1-mm sieve and sent to Altria Client Services for leaf chemistry analysis of alkaloids and TSNA.

Statistical Analysis & Procedures

All data were analyzed in SAS (Statistical Analysis Systems 9.4). A mixed model

ANOVA using the proc glimmix procedure was used to measure the response variables for each year of the study. The following response variables were measured in this study: total yield (kg per ha-1), lug crop throw, seconds crop throw, leaf crop throw, quality grade index, total alkaloids, percent nicotine, percent nornicotine, percent conversion of nicotine to nornicotine, total TSNA, NNN, and NNK. Data for the 2016 experiment was analyzed separately from the

2017 experiments. Since there were no location interactions in 2017, data were combined across both locations for statistical analysis. Leaf chemistry data was averaged, but was not a weighted average based on crop throw.

Results & Discussion 2016 Yield. Total yield was significantly influenced by all four treatment factors in 2016.

Overall, D14 showed significantly greater yields when compared to D18 (Table 3). Previous reports from Gupton (1982) and Terrill et al. (1985) also reported yield differences between different cultivars. A positive correlation was seen in yield when nitrogen rate was increased to

196 kg ha-1 (Table 4). Both Campbell et al. (1982) and Link and Terrill (1982) saw an increase in yield when nitrogen rate was increased. There was a significant topping stage by sucker control method interaction on total yield in 2016 (Table 5). The highest yields were observed when plants were topped at the early button stage and oiled for sucker control. Lower yields occurred when plants were topped at later flowering stages. This agrees with findings from Jack et al. (2010) who saw a large yield decrease as topping was delayed in burley tobacco. It is important to note that plants topped at the early button stage and hand-suckered had much lower yields than oiled plants.

Crop Throw. The crop throw refers to a percentage or portion of the total yield. For this study, these percentages are based on the stalk position of the cured leaves: lugs, seconds, and leaf. The lug crop throw was significantly affected by variety, where D14 produced a higher percentage of lugs than D18 in 2016 (Table 6). Sucker control method also affected lug crop throw where hand-suckered treatments showed a small increase (Table 7). Nitrogen rate and topping stage did not show any significant effects on lug crop throw.

Seconds crop throw was influenced by an interaction between variety and topping stage.

In 2016, topping D18 immediately prior to harvest showed the highest percentage of seconds compared to other treatment combinations (Table 8). Palmer (1999) noted similar results when later topping occurred and reported an increase in flyings, the lowest stalk position in burley tobacco. Nitrogen rate and sucker control methods had no effect on seconds crop throw.

The leaf crop throw is especially important due to the higher market value of this leaf position. In 2016, variety, topping stage, and sucker control method influenced leaf crop throw, while nitrogen rate did not affect leaf crop throw. Although both varieties showed a leaf crop throw over 65%, D14 was slightly higher than D18 (Table 9). In optimum growing conditions, this could translate to a small increase in profits from a more valuable stalk position if a grower chose to grow D14 instead of D18, although further evaluation of D18 is needed. Topping at the early button and late bloom stages produced the highest leaf crop throws at 67% and 66% of the total crop, respectively (Table 10). Topping immediately prior to harvest drastically reduced the leaf crop throw, likely due to the lack of time for leaf growth, maturation, and ripening. Oiling plants for sucker control resulted in a higher leaf crop throw than hand-suckering (Table 11).

Quality Grade Index. Topping stage was the only factor that affected quality grade index in 2016 (Table 12). Topping at the early button and late bloom stages resulted in higher quality when compared to topping immediately prior to harvest. This is likely due to the longer period of time the leaves had to ripen and mature in the field. Gupton (1982) also recorded lower grade indexes in burley tobacco when topping was delayed. Furthermore, delayed topping could contribute to more immature leaves at harvest and possible curing problems in the barn

(Gupton, 1982). There was a numerical difference in quality grade index between varieties, but

17 this was not statistically different. Nitrogen rate and sucker control method did not influence quality grade index.

Chemistry Analyses. Total Alkaloids. The higher nitrogen rate of 196 kg ha-1 resulted in an increase in total alkaloid concentration (Table 13). Chamberlain and Chortyk (1992) also reported steady increases in total alkaloids as nitrogen rate was increased in air-cured and flue- cured tobacco. A significant stalk position by variety interaction showed a higher percentage of alkaloids in the leaf compared to seconds and lugs for both varieties (Table 14). Willamson and

Chaplin (1981) also observed differences in alkaloid levels based on stalk positioning in burley tobacco. While some values were not statistically different from each other, it was interesting to note that D18 consistently had numerically higher alkaloids for all three stalk positions than D14.

Another interaction between stalk position and topping stage indicated the highest amount of alkaloids was observed in the leaf from plants topped at the early button stage (Table 15).

Alkaloid levels were highest in the upper parts of the plant and gradually decreased down the stalk. This was a common trend for each topping stage. In general, it is understood that nicotine levels increase as a tobacco plant matures. Particularly after topping, the concentration of nicotine is highest in the upper part of the plant and decreases in a successional manner down the stalk (Tso, 1990), due to the source-sink relationship with the upper leaves requiring the most metabolic needs. Data from Gupton (1982) showed higher total alkaloids when harvest was delayed. Delaying harvest can give plants more time to mature due to the longer harvest interval, meaning more time for alkaloids to accumulate in the upper portions of the plant. A significant variety by topping stage interaction showed the highest alkaloid concentrations in D18 plants that were topped at the early button and late bloom stages (Table 16). Due to different developmental rates between the two varieties in this study, toppings occurred at various times

18 throughout the growing season in 2016. D18 plants grew at a faster pace than D14 plants and flowered earlier in the growing season. Therefore, toppings took place first on plots where D18 plants were topped at the early button stage. This also resulted in a longer harvest interval after topping for these plots, thus allowing more time for alkaloids to accumulate in these particular plants. Gupton (1982) reported the same issue due to physiological diversity between certain varieties. Plants that were oiled for sucker control showed higher alkaloids than hand-suckered plants (Table 17).

Nicotine. As stated previously, nicotine is the most dominant alkaloid in tobacco plants.

Both Figure 7 and Figure 8 show that this trend remained true in both years of this study. Plants that received a higher nitrogen rate of 196 kg ha-1 produced higher nicotine levels (Table 18).

MacKown et al. (1984), Bailey (2014), and Link and Terrill (1982) all reported similar results when nitrogen rates were increased. Sucker control method also influenced nicotine levels.

Plants that were oiled for sucker control had higher concentrations of nicotine compared to hand- suckered plants (Table 19). A significant stalk position by variety interaction indicated the highest amount of nicotine was in the leaf for both varieties (Table 20). It is important to note how nicotine levels dropped with each drop in stalk position as well. Much like total alkaloids,

D18 consistently had higher nicotine in each stalk position than D14. Another interaction between stalk position and topping stage showed nicotine levels were highest in the leaf stalk position after topping at the early button stage (Table 21). This is likely due to the longer amount of time for nicotine translocation and accumulation to occur in the upper most part of the plant prior to harvest. Nicotine was consistently higher in the upper leaves of the plant regardless of the topping stage. The final interaction that affected nicotine occurred between

19 variety and topping stage. D18 plants topped at early button and late bloom stages showed the highest nicotine percentages (Table 22).

Nornicotine. A significant stalk position by nitrogen rate interaction impacted nornicotine percentage in 2016. The combination of a higher nitrogen rate and higher stalk positions resulted in greater nornicotine levels in the cured leaf (Table 23). Bailey (2014) saw a small effect on nornicotine levels in dark air-cured tobacco due to different nitrogen rates, but data was not analyzed by stalk position. However, this disagrees with MacKown et al. (1984) who saw no effects on nornicotine percentage when the nitrogen rate was increased from 140 kg ha-1 to 280 kg ha-1. An interaction between stalk position and variety also affected nornicotine

(Table 24). D18 consistently showed more nornicotine content than D14 and the higher stalk positions continued to express higher concentrations of nornicotine as well. A third interaction of stalk position by topping stage indicated higher stalk positions and earlier topping stages resulted in higher nornicotine levels. This is similar to results seen from total alkaloids and nicotine (Table 25). Nornicotine signicantly differed based on sucker control methods where oiled plants showed increased levels of nornicotine (Table 26). McEvoy and Hoffman (1959) saw little effect on nornicotine levels when chemical agents were used for sucker control in cigar tobacco. While these values were not significant, each leaf grade varied from one another in nornicotine percentage (McEvoy and Hoffman, 1959).

Nicotine to Nornicotine Conversion. A significant stalk position by variety interaction indicated the lugs had a higher percentage of nicotine to nornicotine conversion for both varieties, but D14 had higher conversion on average (Table 27). An interaction between stalk position and nitrogen rate suggested that lugs with 84 kg N ha-1 had the highest conversion percentage (Table 28). Another interaction of stalk position by topping stage indicated that lugs

20 consistently had higher conversion when compared to seconds and leaf (Table 29). A three-way interaction between variety, nitrogen rate, and topping stage significantly affected nicotine to nornicotine conversion. (Table 30). D18 had a higher amount of conversion than D14.

Conversion increased as nitrogen rate was decreased and as topping was delayed. This interaction showed how delayed topping, nitrogen rates, and varietal differences can impact this conversion. Typically, lower nicotine, lower nitrogen rate, and later toppings all have the capability to slightly increase the conversion percentage (Jack, 2018). The trait involved in the conversion process is also very unstable. Some plants randomly revert back to higher conversion and some seed lots may be higher than others, even after screening (Jack, 2018).

Total TSNAs. A significant stalk position by variety interaction affected total TSNAs

(Table 31). Higher stalk positions contained the greatest amounts of TSNAs in both varieties, but D18 had much higher levels than D14 overall. A stalk position by nitrogen rate interaction indicated a significant linear increase in TSNAs when the higher nitrogen rate was used and stalk position increased (Table 32). A stalk position by topping stage interaction also affected TSNAs.

The combination of earlier topping stages and higher stalk positions showed increased amounts of TSNAs (Table 33). A three-way interaction between variety, nitrogen rate, and topping stage significantly affected TSNAs (Table 34). Overall, D18 consistently showed more TSNAs compared to D14, regardless of nitrogen rate or topping stage. This could be due to physiological differences as discussed previously, but could also be attributed to the timing of topping dates throughout the growing season. D18 plants topped at earlier flowering stages with a higher nitrogen rate collectively showed higher TSNAs than other treatment combinations. A variety by sucker control interaction showed D18 plants had significantly higher TSNAs than

D14, regardless of sucker control method (Table 35).

NNK. NNK levels varied significantly based by stalk position, where lugs contained more NNK than any other position (Table 36). An interaction between nitrogen rate and topping stage also affected NNK levels (Table 37). There was not a definite pattern of how these factors affected NNK, but the highest levels of NNK were recorded when late bloom toppings occurred at the lower nitrogen rate. It is important to note that the only treatment combination that was significantly different from all other combinations was when plants were topped immediately prior to harvest at the lower nitrogen rate. Data from 2016 showed no conclusive evidence of how treatment factors affected NNK levels. The interaction of variety by sucker control method revealed more total NNK in D18 than D14 overall, but the effect of sucker control method varied based on the variety (Table 38).

NNN. A significant interaction of stalk position by nitrogen rate showed higher nitrogen rates combined with higher stalk positions resulted in greater NNN concentrations (Table 39).

However, lugs and leaf at the lower nitrogen rate were statistically comparable to seconds and lugs at the higher nitrogen rate. The significant stalk position by topping stage interaction showed how later toppings and higher stalk positions can increase the amount of NNN present in the cured leaf (Table 40). The highest NNN levels were seen in the leaf stalk position from plants that were topped at the early button stage. NNN levels decreased drastically when harvest toppings occurred. This practice would minimize NNN levels in the cured leaf, but it is not practical from a production standpoint due to a large decrease in yield and quality that was observed with topping immediately prior to harvest. A significant three-way interaction between variety, nitrogen rate, and topping stage significantly affected NNN (Table 41). Generally, nitrogen rate and topping stage had larger impacts on this interaction than variety. Once again, a higher nitrogen rate combined with higher stalk positions led to the greatest NNN levels in the

22 cured leaf in both varieties. D18 had slightly higher NNN levels numerically, but D14 levels were comparable. An interaction of variety by sucker control method indicated a noteworthy difference in NNN (Table 42). D18 plants that were oiled had higher NNN compared to any other treatment combination, even up to a 40% difference when compared to D14 plants that were hand suckered. Other combinations were similar across variety and sucker control method.

Results & Discussion 2017

No location interactions were observed in the 2017 experiments. Therefore, data were combined across both locations.

Yield. A significant variety by nitrogen rate interaction influenced total yield in 2017

(Table 43). D18 plants at the lower nitrogen rate obtained the highest yields. This disagrees with data from 2016, where higher nitrogen rates maximized yields. However, yields for both varieties were statistically comparable to the top yield when the higher nitrogen rate was utilized.

This could enable producers to maximize yields while decreasing fertilizer inputs, overall production costs, and minimize environmental impacts of using too much nitrogen. Topping stage also affected yield significantly. Similar to 2016, topping at the early button and late bloom stages increased yields drastically compared to topping at harvest (Table 44).

Crop Throw. Seconds crop throw was not affected by any treatment factors in 2017.

Topping stage significantly affected both lug crop throw and leaf crop throw (Table 45 & Table

46). Earlier topping stages resulted in a higher percentage of leaf versus topping at harvest.

Conversely, topping at harvest increased the lug crop throw. Both of these reactions are likely due to lack of maturity and ripening of the leaves when the tobacco was topped, then immediately harvested. Variety and nitrogen rate had no effects on crop throw.

Quality Grade Index. Topping stage was the only factor having a significant effect on quality of cured leaves. Similar to 2016 data, earlier toppings drastically increased quality

(Table 47). Topping at harvest showed a large decrease in the quality of the cured leaf.

Chemistry Analysis. Total Alkaloids. Earlier topping stages greatly increased total alkaloids in 2017 (Table 48). It is important to note that all plots were harvested on the same day, regardless of topping stage each plot received. Therefore, plots that were topped earlier had more time to produce and accumulate higher alkaloid concentrations compared to those that received late toppings. Stalk position also significantly affected total alkaloids (Table 49). The concentration of alkaloids was highest in the upper part of the plant and decreased down the stalk. Lugs, seconds, and leaf were all significantly different from each other, where alkaloid concentration doubled in leaf compared to lugs. This agrees with 2016 data of this study and with findings from Willamson and Chaplin (1981), where total alkaloid levels differed significantly due to stalk position when averaged across four burley tobacco varieties. Neither variety nor nitrogen rate influenced total alkaloids.

Nicotine. Similar to data from the 2016 experiment in this study, topping stage and stalk position significantly influenced nicotine production in 2017. Earlier topping stages resulted in higher nicotine levels than topping at harvest (Table 50). In contrast to 2016, the same amount of nicotine was found in both the early button and late bloom stage toppings in 2017. Nicotine levels were greatest in the leaf stalk position and decreased significantly with each drop in stalk position (Table 51). Unlike data from 2016 of this study and other previous findings, the data from 2017 showed no effects from variety or nitrogen rate on nicotine.

These findings further establish a direct relationship between the amount of total alkaloids and the amount of nicotine found in tobacco plants when subjected to certain

24 agronomic practices. Both years of data from this study indicate that total alkaloid concentrations are heavily influenced by the amount of nicotine produced. This supports a strong correlation between total alkaloids and nicotine.

Nornicotine. Only stalk positioning affected nornicotine levels in 2017 (Table 52).

Much like total alkaloids and nicotine, nornicotine was greater in the upper parts of the plant while lower levels were observed down the stalk. Variety, nitrogen rate, and topping stage had no significant effects on nornicotine.

Nicotine to Nornicotine Conversion. There were no significant effects regarding nicotine to nornicotine conversion in 2017, although there were slight numerical differences for some agronomic factors.

Total TSNAs. Variety had a significant effect on total TSNA concentration. D18 contained 3.4 ppm while D14 resulted in 2.9 ppm on average (Table 53). As previously stated, this could be due to differences in the length of time between topping and harvest for experimental plots. Increased nitrogen rate resulted in a significant increase in TSNAs (Table

54). Topping stage also affected total TSNAs. Topping at the early button stage resulted in the highest accumulation of TSNAs and gradually decreased as toppings occurred at later flowering stages (Table 55).

NNK. Nitrogen rate had a large impact on NNK levels in 2017. The higher nitrogen rate of 196 kg ha-1 nearly doubled the NNK compared to 84 kg ha-1 (Table 56). This contradicts results from 2016, where lower nitrogen rates typically resulted in more NNK. However, nitrogen rate interacted with topping stage in 2016 and may have had an effect on the NNK levels for that year. There were also significant differences in NNK based on stalk position.

This agrees with data from 2016 of this study. Lugs contained the most NNK compared to any other stalk position (Table 57), accumulating over twice as much NNK when compared to leaf.

While more investigation is needed, a distinctive pattern was observed how NNK levels are affected relative to the stalk position on the plant in dark fire-cured tobacco. Variety and topping stage had no effect on NNK in 2017.

NNN. NNN was significantly influenced by nitrogen rate. The higher nitrogen rate nearly doubled NNN in 2017 (Table 58). Topping stage also showed a large influence on NNN.

Earlier toppings produced higher levels of NNN versus later toppings (Table 59). While this suggests that topping closer to harvest would reduce NNN, it is not a feasible practice due to the large yield decreases seen when plants are topped closer to harvest. However, data from 2017 may suggest a compromise that could meet the desired levels of chemical constituents and desired yields by farmers, in that topping at the late bloom stage produced similar yield and

NNN levels as early button toppings, but NNN concentrations were about 35% lower when topping occurred at the late bloom stage.

Conclusions & Future Research

Prior to this study, chemical analysis of D18 showed inherently lower alkaloids and

TSNA compared to other commercial dark tobacco varieties. The data presented in this thesis does not correspond with previous data collected on chemical constituents of D18. More research is needed to further investigate the effects of agronomic practices on dark tobacco varieties, especially D18. The differences seen in this study could be attributed to variable harvest intervals of the experimental plots after different topping treatments. Generally, D18 plants had a more rapid growth rate than D14 plants. The physiological maturity of the plants resulted in the same topping treatments occurring at different times, depending on the variety. In most cases, plots with D18 plants were topped prior to plots containing D14 plants undergoing the same topping treatment. This caused different harvest intervals, even though the same topping treatment was employed. In 2017, heavy rainfall and other environmental conditions caused topping treatments to occur in a different manner compared to 2016. These differences in growing conditions could have affected physical and chemical properties of the plants within experimental plots.

Nitrogen rates have previously been shown to significantly impact yields and chemical constituents in tobacco. Similar results were observed in this study. Nitrogen rate significantly influenced certain chemical constituents of the plant. There also was an instance where lower nitrogen rates significantly increased the amount of conversion of nicotine to nornicotine in

2016. However, this effect was not seen in 2017, and more research is needed to fully evaluate the effect of nitrogen rate on the conversion of nicotine to nornicotine in tobacco.

Topping stage heavily affected total yield, quality, and chemical properties of the tobacco plant in both years of this study. Earlier toppings repeatedly resulted in maximum yields and

27 higher quality of the cured leaf. However, earlier toppings also consistently showed increased levels of many chemical constituents, which is cause for concern when attempting to reduce alkaloids and TSNAs. This study also demonstrated the harvest intervals influence of minimizing alkaloid and TSNA accumulation. The longer a plant remains in the field after topping, the higher alkaloids and TSNA accumulations.

Sucker control had minimal effects during the 2016 experiment of this study, although it did affect total yield and various chemical properties of the plant. Using chemical agents, such as a fatty alcohol, provide for more uniform and efficient sucker control and limited human errors caused by hand-suckering, which could cause yield loss. On the other hand, chemical sucker control resulted in small increases in total alkaloids and TSNAs. This might be caused by escaped suckers becoming primary metabolic sinks or a stress response to further injury to the plant from the fatty alcohol application. Further research is needed to fully quantify the effects of sucker control methods on chemical properties of tobacco.

Stalk position played a critical role in the chemistry of the cured leaf during this study. In general, higher stalk positions consistently showed increased levels of all chemical constituents, except for nicotine to nornicotine conversion and NNK. Conversely, lower stalk positions resulted in greater amounts of nicotine to nornicotine conversion and NNK.

Consistently obtaining lower chemistry levels will be challenging with current dark tobacco varieties and production practices. Although this study did not use weighted leaf chemistry values, which would have increased NNN concentration values, leaf and seconds

NNN concentrations exceeded the FDA’s proposed threshold of 1ppm NNN. These grades make up approximately 80% of the total crop throw. Therefore, other strategies such as new varieties

28 or alternate tobacco types should be examined in the manufacturing of smokeless tobacco products.

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Wyss AB, Hashibe M, Lee YCA, Chuang SC, Muscat J, Chen C, Schwartz SM, Smith E, Zhang ZF, Morgenstern H, Wei Q, Li G, Kelsey KT, McClean M, Winn DM, Schantz S, Yu GP, Gillison ML, Zevallos JP, Bofetta P, Olshan AF. 2016. Smokeless Tobacco Use and the Risk of Head and Neck Cancer: Pooled Analysis of US Studies in the INHANCE Consortium. Am J Epidemiol 184:703-716.

Appendix

All Tobacco Types - Acres Harvested 900,000 800,000 700,000 600,000 500,000 400,000 300,000 200,000 100,000 0

Figure 1. Total acres of tobacco production in the United States from 1990-2016. Data gathered from USDA National Agricultural Statistical Service. Accessed March 2017.

TOBACCO, FIRE-CURED (CLASS 2) - ACRES HARVESTED

20,000 18,000 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0

Figure 2. Total dark fire-cured tobacco production in the United States from 1990-2016. Data gathered from USDA National Agricultural Statistical Service. Accessed March 2017.

TOBACCO, DARK AIR-CURED (CLASS 3B) ACRES HARVESTED 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0

Figure 3. Total dark air-cured tobacco production in the United States from 1990-2016. Data gathered from USDA National Agricultural Statistical Service. Accessed March 2017.

Table 1. Treatment list for the 2016 study in Springfield, TN.

Treatment # Description 1 D14 bud-topped, oiled, 84 kg N ha-1 2 D14 late bloom topped, oiled, 84 kg N ha-1 3 D14 topped immediately prior to harvest, oiled, 84 kg N ha-1 4 D14 bud-topped, hand suckered, 84 kg N ha-1 5 D14 late bloom topped, hand-suckered, 84 kg N ha-1 6 D14 topped immediately prior to harvest, oiled, 84 kg N ha-1 7 D14 bud-topped, oiled, 196 kg N ha-1 8 D14 late bloom topped, oiled, 196 kg N ha-1 9 D14 topped immediately prior to harvest, oiled, 196 kg N ha-1 10 D14 bud-topped, hand suckered, 196 kg N ha-1 11 D14 late bloom topped, hand-suckered, 196 kg N ha-1 12 D14 topped immediately prior to harvest, oiled, 196 kg N ha-1 13 D24 bud-topped, oiled, 84 kg N ha-1 14 D24 late bloom topped, oiled, 84 kg N ha-1 15 D24 topped immediately prior to harvest, oiled, 84 kg N ha-1 16 D24 bud-topped, hand suckered, 84 kg N ha-1 17 D24 late bloom topped, hand-suckered, 84 kg N ha-1 18 D24 topped immediately prior to harvest, oiled, 84 kg N ha-1 19 D24 bud-topped, oiled, 196 kg N ha-1 20 D24 late bloom topped, oiled, 196 kg N ha-1 21 D24 topped immediately prior to harvest, oiled, 196 kg N ha-1 22 D24 bud-topped, hand suckered, 196 kg N ha-1 23 D24 late bloom topped, hand-suckered, 196 kg N ha-1 24 D24 topped immediately prior to harvest, oiled, 196 kg N ha-1

Table 2. Treatment list for the 2017 studies in Springfield, TN and Princeton, KY.

Treatment # Description

1 D14 bud-topped, oiled, 84 kg N ha-1 2 D14 late bloom topped, oiled, 84 kg N ha-1 3 D14 topped immediately prior to harvest, oiled, 84 kg N ha-1 4 D14 bud-topped, oiled, 196 kg N ha-1 5 D14 late bloom topped, oiled, 196 kg N ha-1 6 D14 topped immediately prior to harvest, oiled, 196 kg N ha-1 7 D24 bud-topped, oiled, 84 kg N ha-1 8 D24 late bloom topped, oiled, 84 kg N ha-1 9 D24 topped immediately prior to harvest, oiled, 84 kg N ha-1 10 D24 bud-topped, oiled, 196 kg N ha-1 11 D24 late bloom topped, oiled, 196 kg N ha-1 12 D24 topped immediately prior to harvest, oiled, 196 kg N ha-1

Springfield 2016 100 30 90 80 25 70 20 60 50 15 40 10 30 (in) Rainfall 20 5 Air Temperature (oF) Temperature Air 10 0 0

Rainfall Avg High Avg Low Ovr Avg

Figure 4. Weather conditions in 2016 for Springfield, TN (maximum, minimum air temperature, and precipitation).

Springfield 2017 100 30 90 25

F) 80 o 70 20 60 50 15 40 10 30 (in) Rainfall 20

Air Temperature ( Temperature Air 5 10 0 0

Rainfall Avg High Avg Low Ovr Avg

Figure 5. Weather conditions in 2017 for Springfield, TN (maximum, minimum air temperature, and precipitation).

Princeton 2017 100 30 90

25 F)

o 80 70 20 60 50 15 40 10 30 (in)Rainfall 20

Air Temperature ( Temperature Air 5 10 0 0

Rainfall Avg High Avg Low Ovr Avg

Figure 6. Weather conditions in 2017 for Princeton, KY (maximum, minimum air temperature, and precipitation).

Table 3. Total cured leaf yield by variety in 2016. Variety Yielda kg ha-1 D14 2563 a D18 2373 b p=0.0034 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 4. Total cured leaf yield in response to nitrogen rate in 2016. Nitrogen Yielda kg/ha-1 kg ha-1 84 2395 b 196 2541 a p=0.0230 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 5. Total cured leaf yield by topping stage and sucker control interaction in 2016. Topping Stage Sucker Control Yielda kg ha-1 Button Oiled 2894 a Hand 2382 b Late bloom Oiled 2568 b Hand 2407 b Harvest Oiled 2268 b Hand 2289 b p=0.0031 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 6. Lug crop throw by variety in 2016. Variety Lug Crop Throwa % D14 10.15 a D18 9.18 b p=0.0480 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 7. Lug crop throw by sucker control method in 2016. Sucker Control Lug Crop Throwa % Oiled 9.04 b Hand 10.29 a p=0.0119 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 8. Seconds crop throw response by variety and topping stage interaction in 2016. Variety Topping Stage Seconds Crop Throwa % D14 Button 22.54 b Late bloom 23.63 b Harvest 23.86 b D18 Button 24.34 b Late bloom 23.98 b Harvest 28.41 a p=0.0066 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 9. Leaf crop throw by variety in 2016. Variety Leaf Crop Throwa % D14 66.50 a D18 65.24 b p=0.0459 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 10. Leaf crop throw by topping stage in 2016. Topping Stage Leaf Crop Throwa % Button 67.65 a Late bloom 66.12 a Harvest 63.85 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 11. Leaf crop throw by sucker control method in 2016. Sucker Control Leaf Crop Throwa % Oiled 66.74 a Hand 65.00 b p=0.0065 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 12. Quality grade index response to topping time in 2016. Topping Stage Grade Indexa 0-100 Button 41 a Late bloom 40 a Harvest 32 b p=0.0062 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 13. Total alkaloids in response to nitrogen rate in 2016. Nitrogen Total Alkaloidsa kg ha-1 % 84 1.51 b 196 1.74 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 14. Total alkaloids by stalk position and variety interaction in 2016. Variety Stalk Position Total Alkaloidsa % D14 Leaf 2.20 b Seconds 1.27 d Lugs 0.75 e D18 Leaf 2.89 a Seconds 1.63 c Lugs 1.01 de p=0.0047 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 15. Total alkaloids by stalk position and topping stage interaction in 2016. Topping Stage Stalk Position Total Alkaloidsa % Button Leaf 3.26 a Seconds 1.81 c Lugs 1.09 d Late bloom Leaf 2.75 b Seconds 1.54 c Lugs 0.91 e Harvest Leaf 1.63 c Seconds 0.99 de Lugs 0.63 e p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 16. Total alkaloids by variety and topping stage interaction in 2016. Variety Topping Stage Total Alkaloidsa % D14 Button 1.89 b Late bloom 1.40 c Harvest 0.93 d D18 Button 2.22 a Late bloom 2.07 ab Harvest 1.24 c p=0.0154 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 17. Total alkaloids response to sucker control method in 2016. Sucker Control Total Alkaloidsa % Oiled 1.71 a Hand 1.54 b p=0.0033 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Alkaloid Profile of D14 - Springfield 2016

Anabasine Anatabine 1% 3%

Nornicotine Nicotine 1% Nornicotine Anabasine Anatabine

Nicotine 95%

Alkaloid Profile of D18 - Springfield 2016

Anabasine Anatabine 1% 3%

Nornicotine Nicotine 1% Nornicotine Anabasine Anatabine

Nicotine 95%

Figure 7. Alkaloid profile of D14 (top) & D18 (bottom) in Springfield, TN in 2016.

Alkaloid Profile of D14 in 2017

Anabasine Anatabine 0% 3%

Nornicotine Nicotine 1% Nornicotine Anabasine Anatabine

Nicotine 96%

Alkaloid Profile of D18 in 2017

Anabasine Anatabine 0% 3%

Nornicotine Nicotine 1% Nornicotine Anabasine Anatabine

Nicotine 96%

Figure 8. Alkaloid profile of D14 (top) & D18 (bottom) in 2017

Table 18. Nicotine response to nitrogen rate in 2016. Nitrogen Nicotinea kg ha-1 % 84 1.43 b 196 1.66 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 19. Nicotine percentage response to sucker control method in 2016. Sucker Control Nicotinea % Oiled 1.62 a Hand 1.47 b p=<0.0039 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 20. Nicotine percentage by stalk position and variety interaction in 2016. Variety Stalk Position Nicotinea % D14 Leaf 2.10 b Seconds 1.21 d Lugs 0.71 e D18 Leaf 2.75 a Seconds 1.54 c Lugs 0.96 de p=0.0053 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 21. Nicotine percentage by stalk position and topping stage interaction in 2016. Topping Stage Stalk Position Nicotinea % Button Leaf 3.11 a Seconds 1.72 c Lugs 1.04 d Late bloom Leaf 2.63 b Seconds 1.46 c Lugs 0.86 de Harvest Leaf 1.55 c Seconds 0.95 d Lugs 0.60 e p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 22. Nicotine percentage by variety and topping stage interaction in 2016. Variety Topping Stage Nicotinea % D14 Button 1.80 b Late bloom 1.34 c Harvest 0.88 d D18 Button 2.11 a Late bloom 1.96 ab Harvest 1.18 c p=0.0141 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 23. Nornicotine percentage by stalk position and nitrogen rate interaction in 2016. Nitrogen Rate Stalk Position Nornicotinea kg ha-1 % 84 Leaf 0.0214 b 196 0.0280 a 84 Seconds 0.0138 cd 196 0.0154 c 84 Lugs 0.0110 d 196 0.0114 cd p=0.0104 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 24. Nornicotine percentage by stalk position and variety interaction in 2016. Variety Stalk Position Nornicotinea % D14 Leaf 0.0213 b Seconds 0.0132 cd Lugs 0.0104 d D18 Leaf 0.0281 a Seconds 0.0161 c Lugs 0.0121 cd p=0.0515 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 25. Nornicotine percentage by stalk position and topping stage interaction in 2016. Topping Stage Stalk Position Nornicotinea % Button Leaf 0.0333 a Seconds 0.0172 c Lugs 0.0127 cde Late bloom Leaf 0.0251 b Seconds 0.0153 cd Lugs 0.0116 cde Harvest Leaf 0.0156 cd Seconds 0.0114 de Lugs 0.0094 e p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 26. Nornicotine response to sucker control method in 2016. Sucker Control Nornicotinea % Oiled 0.0183 a Hand 0.0153 b p=0.0008 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 27. Effect of stalk position by variety interaction on nicotine to nornicotine conversion in 2016. Variety Stalk Position Conversiona % D14 Leaf 0.988 c Seconds 1.142 c Lugs 1.519 a D18 Leaf 0.998 c Seconds 1.093 c Lugs 1.332 b p=0.0417 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 28. Effect of stalk position by nitrogen rate interaction on nicotine to nornicotine conversion in 2016. Nitrogen Stalk Position Conversiona kg ha-1 % 84 Leaf 0.920 d Seconds 1.161 bc Lugs 1.586 a 196 Leaf 1.066 cd Seconds 1.074 cd Lugs 1.266 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 29. Effect of stalk position by topping stage interaction on nicotine to nornicotine converstion in 2016. Topping Stage Stalk Position Conversiona % Button Leaf 1.052 cd Seconds 0.997 d Lugs 1.237 bc Late bloom Leaf 0.945 d Seconds 1.063 cd Lugs 1.410 b Harvest Leaf 0.981 d Seconds 1.293 b Lugs 1.630 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 30. Effect of variety by nitrogen rate by topping stage interaction on nicotine to nornicotine conversion in 2016. Variety Nitrogen Topping Stage Conversiona kg ha-1 % D14 84 Button 1.110 bcde Late bloom 1.281 abcd Harvest 1.348 ab 196 Button 1.037 de Late bloom 1.212 abcde Harvest 1.310 abc D18 84 Button 1.101 bcde Late bloom 1.055 cde Harvest 1.437 a 196 Button 1.132 bcde Late bloom 1.010 e Harvest 1.112 bcde p=0.0366 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 31. Effect of stalk position by variety interaction on total TSNA in 2016. Variety Stalk Position TSNAa ppm D14 Leaf 3.075 c Seconds 2.938 c Lugs 2.911 c D18 Leaf 5.535 a Seconds 4.582 b Lugs 4.203 b p=0.0242 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 32. Effect of stalk position by nitrogen rate interaction on total TSNA in 2016. Nitrogen Stalk Position TSNAa kg ha-1 ppm 84 Leaf 3.196 c Seconds 3.288 c Lugs 3.125 c 196 Leaf 5.413 a Seconds 4.232 b Lugs 3.988 bc p=0.0027 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 33. Effect of stalk position by topping stage interaction on total TSNA in 2016. Topping Stage Stalk Position TSNAa ppm Button Leaf 6.187 a Seconds 4.558 b Lugs 4.107 b Late bloom Leaf 4.372 b Seconds 4.154 b Lugs 3.869 bc Harvest Leaf 2.354 d Seconds 2.569 d Lugs 2.694 cd p=0.0002 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 34. Effect of variety by nitrogen rate by topping stage interaction on total TSNA in 2016. Variety Nitrogen Topping Stage TSNAa kg ha-1 ppm D14 84 Button 3.351 def Late bloom 2.393 efg Harvest 1.744 g 196 Button 4.351 bcd Late bloom 3.496 def Harvest 2.511 efg D18 84 Button 4.409 bcd Late bloom 5.125 bc Harvest 2.197 fg 196 Button 7.692 a Late bloom 5.513 b Harvest 3.703 cde p=0.0031 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 35. Effect of variety by sucker control interaction on total TSNA in 2016. Variety Sucker Control TSNAa ppm D14 Oiled 3.062 c Hand 2.887 c D18 Oiled 5.261 a Hand 4.286 b p=0.0256 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 36. Effect of stalk position on NNK in 2016. Stalk Position NNKa ppm Leaf 0.279 c Seconds 0.433 b Lugs 0.620 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 37. Effect of nitrogen rate by topping stage interaction on NNK in 2016. Nitrogen Topping Stage NNKa ppm 84 Button 0.418 ab Late bloom 0.541 a Harvest 0.351 b 196 Button 0.471 ab Late bloom 0.404 ab Harvest 0.481 ab p=0.0264 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 38. Effect of variety by sucker control method on NNK in 2016. Variety Sucker Control NNKa ppm D14 Oiled 0.359 b Hand 0.416 ab D18 Oiled 0.559 a Hand 0.443 ab p=0.0379 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 39. Effect of stalk position by nitrogen rate interaction on NNN in 2016. Nitrogen Stalk Position NNNa kg ha-1 ppm 84 Leaf 0.484 bc Seconds 0.418 c Lugs 0.439 bc 196 Leaf 1.106 a Seconds 0.669 b Lugs 0.615 bc p=0.0002 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 40. Effect of stalk position by topping stage interaction on NNN in 2016. Topping Stage Stalk Position NNNa ppm Button Leaf 1.231 a Seconds 0.712 bc Lugs 0.607 bcd Late bloom Leaf 0.760 b Seconds 0.553 bcd Lugs 0.5574 bcd Harvest Leaf 0.394 d Seconds 0.366 d Lugs 0.417 cd p=0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 41. Effect of variety by nitrogen rate by topping stage interaction on NNN in 2016 Variety Nitrogen Topping Stage NNNa kg ha-1 ppm D14 84 Button 0.494 bcd Late bloom 0.360 cd Harvest 0.268 d 196 Button 0.841 b Late bloom 0.651 bc Harvest 0.423 cd D18 84 Button 0.594 bcd Late bloom 0.668 bc Harvest 0.297 cd 196 Button 1.472 a Late bloom 0.814 b Harvest 0.581 bcd p=0.0121 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 42. Effect of variety by sucker control interaction on NNN in 2016. Variety Sucker Control NNNa ppm D14 Oiled 0.524 b Hand 0.488 b D18 Oiled 0.859 a Hand 0.616 b p=0.0262 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 43. Effect of variety by nitrogen rate interaction on total yield in 2017. Variety Nitrogen Yielda kg ha-1 kg ha-1 D14 84 2332 b 196 2632 ab D18 84 2680 a 196 2576 ab p=0.0173 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 44. Effect of topping stage on total yield in 2017. Topping Stage Yielda kg ha-1 Button 2966 a Late bloom 2941 a Harvest 1759 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 45. Effect of topping stage on lug crop throw in 2017. Topping Stage Lug Crop Throwa % Button 10.13 b Late bloom 9.96 b Harvest 16.04 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 46. Effect of topping stage on leaf crop throw in 2017. Topping Stage Leaf Crop Throwa % Button 67.61 a Late bloom 68.10 a Harvest 60.37 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 47. Effect of topping stage on quality grade index in 2017. Topping Stage Grade Indexa 0-100 Button 55 a Late bloom 51 a Harvest 39 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 48. Effect of topping stage on total alkaloids in 2017. Topping Stage Total Alkaloidsa % Button 2.80 a Late bloom 2.80 a Harvest 1.70 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 49. Effect of stalk position on total alkaloids in 2017. Stalk Position Total Alkaloidsa % Leaf 3.31 a Seconds 2.36 b Lugs 1.61 c p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 50. Effect of topping stage on nicotine in 2017. Topping Stage Nicotinea % Button 2.68 a Late bloom 2.68 a Harvest 1.61 b p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 51. Effect of stalk position on nicotine in 2017. Stalk Position Nicotinea % Leaf 3.17 a Seconds 2.27 b Lugs 1.54 c p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 52. Effect of stalk position on nornicotine in 2017. Stalk Position Nornicotinea % Leaf 0.038 a Seconds 0.020 ab Lugs 0.015 b p=0.0223 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 53. Effect of variety on TSNA in 2017. Variety TSNAa ppm D14 2.90 b D18 3.40 a p=0.0494 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 54. TSNA response to nitrogen rate in 2017. Nitrogen TSNAa kg ha-1 ppm 84 2.53 b 196 3.78 a p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 55. TSNA response to topping stage in 2017. Topping Stage TSNAa ppm Button 4.15 a Late bloom 3.12 b Harvest 2.17 c p=<0.0001 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 56. Effect of nitrogen rate on NNK in 2017. Nitrogen NNKa kg ha-1 ppm 84 0.179 b 196 0.331 a p=0.0031 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 57. Effect of stalk position on NNK in 2017. Stalk Position NNKa ppm Leaf 0.166 b Seconds 0.245 ab Lugs 0.343 a p=0.0236 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 58. NNN response to nitrogen rate in 2017. Nitrogen NNNa kg ha-1 ppm 84 0.487 b 196 0.921 a p=0.0075 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Table 59. NNN response topping stage in 2017. Topping Stage NNNa ppm Button 1.022 a Late bloom 0.653 ab Harvest 0.474 b p=0.0119 aMeans with the same letter are not significantly different according to Tukey-Kramer multiple comparison test at P=0.05.

Vita

Michael Ryan Brown was born in Hendersonville, Tennessee, on May 13th, 1993. He is the son of Mike and Christy Brown. He graduated from Hendersonville High School in May of

2012. He entered the University of Tennessee, Knoxville in the fall of 2012 and completed a

Bachelor of Science degree in Agricultural Communications in December of 2015. He continued his academic career at the University of Tennessee, Knoxville, in the fall of 2016, where he completed a Master of Science degree in Plant Science in the fall of 2018. He accepted a position with the University of Georgia Cooperative Extension Service as the Agriculture and

Natural Resource Extension Agent in Meriwether County. The author is a member of Gamma

Sigma Delta, the international honor society of agriculture.