AN ABSTRACT OF THE THESIS OF. Cheryl A. Parris for the degree of Master of Science in Horticulture presented on March 25, 2016.

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2 AN ABSTRACT OF THE THESIS OF Cheryl A. Parris for the degree of Master of Science in Horticulture presented on March 25, Title: Soil Water Tension Irrigation Criteria Affects Both Stevia Rebaudiana Leaf Yield and Leaf Steviol Glycoside Composition Abstract approved: Clinton C. Shock Stevia (Stevia rebaudiana) is a perennial herbaceous plant native to Paraguay and used traditionally by the native Guarani peoples for centuries to sweeten medicinal teas. Stevia has only been commercially cultivated over the past 50 years and cultural practice information suitable for implementation by growers is limited. Currently stevia s commercial value is found in its steviol glycoside content, especially rebaudioside A. Growers seek information to maximize productivity of rebaudioside A through improved cultural practices. Irrigation management is one of several important agronomic techniques growers can use to obtain maximum yield while also pursuing water use efficiency. The current trial investigated the effect that soil water tension as an irrigation onset criteria had on stevia dry leaf yields, steviol glycoside content and yield, and steviol glycoside ratios. The experiment was conducted in a field of Owyhee silt loam (coarse-silty, mixed, mesic Xerollic Camborthid) at the Oregon State University, Malheur

3 Experiment Station, Ontario, Oregon, N 43 58' '' W 117 1' ''. Two stevia varieties, SW109 and SW129 (S&W Seed Company Inc., Fresno, California), were subjected to soil water tension irrigation criteria of 10, 20, 40, 60, 80, kpa over a 57-day trial period. Harvested plant material was measured for dry leaf yield and several steviol glycosides. Analysis of variance showed significant variety differences in leaf yield, leaf stevioside and rebaudioside A content and yield, and steviol glycoside ratios (P=0.05). Examining the data by analysis of variance, dry leaf yield, stevioside%, rebaudioside C %, total steviol glycoside (TSG), rebaudioside C yield, and TSG % yield had significant higher results among the wetter compared to the drier soil water tension criteria treatments, yet a preferred treatment regime was not was not clearly evident. When comparing varieties by regression analysis, results showed a highly significant decrease in stevioside % content, and an increase in the ratio of rebaudioside A to stevioside from drier to wetter irrigation onset criteria, whereas, rebaudioside A % content did not show any significant trend at differing irrigation criteria. The results indicate that maximizing dry leaf productivity directly affects the total rebaudioside A yield, which in turn can provide increased crop value to the grower. Irrigating near 10kPa produced higher dry leaf yield and total rebaudioside A yield, than irrigation at drier criteria. Irrigating stevia at 10 kpa at full canopy, the crop coefficient, kc, was equivalent to 0.97 ETo, averaging 6.4 mm d -1 for the days measured.

4 Copyright by Cheryl A. Parris March 25, 2016 All Rights Reserved

5 Soil Water Tension Irrigation Criteria Affects Both Stevia Rebaudiana Leaf Yield and Leaf Steviol Glycoside Composition by Cheryl A. Parris A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented March 25, 2016 Commencement June 2016

6 Master of Science thesis of Cheryl A. Parris presented on March 25, 2016 APPROVED: Major Professor, representing Horticulture Head of the Department of Horticulture Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Cheryl A. Parris, Author

7 ACKNOWLEDGEMENTS The author expresses sincere appreciation To family, friends, and co-worker s who have helped me in so many ways, I couldn t have done this without their encouragement, understanding and support. To Dr. Clinton C. Shock, for his motivation, persistence, understanding and valuable insight throughout this process. To Dr. Alexandra C Stone, for her help navigating Oregon State University campus curriculum and the graduate school process. To all my committee members for taking time from their busy schedules to evaluate my work. Last but not least, to my late husband Steve, who prodded me to begin this experience and I only wish he could have been here to see it completed. I hope he would be proud.

8 TABLE OF CONTENTS Page 1 INTRODUCTION Tables LITERATURE REVIEW MATERIALS AND METHODS FIGURES RESULTS Stevia response to irrigation criteria Crop evapotranspiration Stevia response to the crop coefficient kc, ETc as a proportion of ETo TABLES FIGURES DISCUSSION Differences in variety performance (leaf yield, steviol glycoside content, proportion of rebaudioside A, steviol glycoside yield per hectare) Difference in irrigation treatments (leaf yield, steviol glycoside content, and proportion of rebaudioside A, steviol glycoside yield per hectare) Leaf yield Stevioside responses Rebaudioside A responses Ratio of rebaudioside A to stevioside Ratio of rebaudioside A to TSG Total productivity of steviol glycosides Comparison of responses to ETc Water Use Efficiency Optimum irrigation criteria in terms of SWT and ETc The role of steviol glycosides in osmotic regulation CONCLUSION REFERENCES... 66

9 LIST OF FIGURES Figure Page FIGURE1. PLOT MAP FOR STEVIA REBAUDIANA, SHOWING COMPLETE SPLIT-PLOT DESIGN, 5 TREATMENTS, 4 REPLICATES, ONTARIO, OREGON, FIGURE 2. SOIL WATER TENSION FOR 10, 20, 40, 60, 80 KPA OVER CALENDAR DAYS , JULY 8, 2015-SEPT 5, 2015, ONTARIO, OREGON FIGURE 3. ACCUMULATED WATER APPLIED ON STEVIA REBAUDIANA IN RESPONSE TO SOIL TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 4. GRAVIMETRIC SOIL WATER CONTENT COMPARED TO THE SOIL WATER TENSION OF THE OWYHEE SILT LOAM USED FOR THE STEVIA IRRIGATION TRIAL, ONTARIO, OREGON, FIGURE 5. LEAF YIELD OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 6. LEAF STEVIOSIDE CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 7. LEAF REBAUDIOSIDE A CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 8. LEAF REBAUDIOSIDE C CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 9. LEAF TOTAL STEVIOL GLYCOSIDE CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 10. RATIO OF LEAF REBAUDIOSIDE A TO STEVIOSIDE CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON,

10 LIST OF FIGURES (Continued) Figure Page FIGURE 11. RATIO OF LEAF REBAUDIOSIDE A TO TOTAL STEVIOL GLYCOSIDE CONTENT OF TWO VARIETIES OF STEVIA REBAUDIANA IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 12. YIELD OF STEVIOL GLYCOSIDES IN THE LEAVES OF STEVIA REBAUDIANA VARIETY SW107 IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 13. YIELD OF STEVIOL GLYCOSIDES IN THE LEAVES OF STEVIA REBAUDIANA VARIETY SW129 IN RESPONSE TO SOIL WATER TENSION IRRIGATION CRITERIA, ONTARIO, OREGON, FIGURE 14. LEAF YIELD OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 15. LEAF STEVIOSIDE CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 16. LEAF REBAUDIOSIDE A CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 17. LEAF REBAUDIOSIDE C CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 18. LEAF REBAUDIOSIDE TOTAL STEVIOL GLYCOSIDE CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 19. RATIO OF LEAF REBAUDIOSIDE A TO STEVIOSIDE CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON

11 LIST OF FIGURES (Continued) Figure Page FIGURE 20, RATIO OF LEAF REBAUDIOSIDE A TO TOTAL STEVIOL GLYCOSIDE(TSG) CONTENT OF TWO STEVIA REBAUDIANA VARIETIES IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON FIGURE 21. YIELD OF STEVIOL GLYCOSIDES IN THE LEAVES OF STEVIA REBAUDIANA VARIETY SW107 IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON, FIGURE 22. YIELD OF STEVIOL GLYCOSIDES IN THE LEAVES OF STEVIA REBAUDIANA VARIETY SW129 IN RESPONSE TO VARIABLE IRRIGATION, KC, STEVIA ETC AS A PROPORTION OF REFERENCE ETO, ONTARIO, OREGON,

12 LIST OF TABLES Table Page TABLE STEVIA REBAUDIANA S&W SEED COMPANY YIELD TRIALS PERFORMED ON SEVEN VARIETIES AT FOUR WESTERN UNITED STATES TABLE 2. SOIL ANALYSIS COMPARISON DURING PRE-PLANT PREPARATIONS AT FOUR LOCATIONS SELECTED FOR S&W SEED COMPANY STEVIA REBAUDIANA YIELD TRIALS TABLE 3. FERTILIZER APPLICATION RATES APPLIED TO SELECTED FIELD LOCATIONS FOR S&W SEED COMPANY STEVIA REBAUDIANA VARIETIES FOR 2014 YIELD TRIAL STUDIES TABLE 4. STEVIA REBAUDIANA LEAF YIELD AND LEAF STEVIOL GLYCOSIDE CONTENT FROM THE FIRST HARVEST AT FOUR LOCATIONS IN TABLE 5. STEVIA REBAUDIANA LEAF YIELD AND LEAF STEVIOL GLYCOSIDE CONTENT FROM THE SECOND HARVEST AT FOUR LOCATIONS IN TABLE 6. STEVIA REBAUDIANA LEAF YIELD AND LEAF STEVIOL GLYCOSIDE CONTENT FROM THE SINGLE HARVEST AT FOUR LOCATIONS IN TABLE 7. STEVIA REBAUDIANA LEAF YIELD IN RESPONSE TO HARVEST STRATEGY AT FOUR LOCATIONS IN TABLE 8. REFERENCE EVAPOTRANSPIRATION, PAN EVAPORATION, RAINFALL, AND IRRIGATION, MM FOR PRE-TRIAL PERIOD, DAY 132 TO 188 AND TRIAL PERIOD, DAY 189 TO 245 APPLIED TO STEVIA REBAUDIANA, ONTARIO, OREGON, TABLE 9. ANALYSIS OF VARIANCE FOR STEVIA REBAUDIANA TRAITS BY A) VARIETY (SW107, SW129), B) SOIL WATER TENSION IRRIGATION TREATMENTS (10, 20, 40, 60, 80 KPA), AND C) INTERACTION OF VARIETIES AND TREATMENT. NOTE REB IS THE ABBREVIATION OF REBAUDIOSIDE TABLE 10. REGRESSION ANALYSIS FOR THE RESPONSE OF STEVIA REBAUDIANA TRAITS (Y) AS A FUNCTION OF THE IRRIGATION CRITERIA IN UNITS OF SOIL WATER TENSION (X), KPA

13 LIST OF TABLES (Continued) Table Page TABLE 11. REFERENCE EVAPOTRANSPIRATION, PRECIPITATION, AND PAN EVAPORATION DURING THE STEVIA IRRIGATION TRIAL FROM 8 JULY 2015 TO 1 SEPTEMBER 2015, ONTARIO, OREGON TABLE 12. CALCULATION OF THE STEVIA CROP EVAPOTRANSPIRATION (ETC) AS A PROPORTION OF THE REFERENCE EVAPOTRANSPIRATION (ETO) FROM 8 JULY TO 1 SEPTEMBER, 2015, ONTARIO, OREGON TABLE 13. REGRESSION ANALYSIS FOR THE RESPONSE OF STEVIA REBAUDIANA TRAITS (Y) AS A FUNCTION OF STEVIA ETC AS A PROPORTION OF ETO OR KC (X),... 45

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15 1 1 INTRODUCTION Stevia (Stevia rebaudiana) is an herbaceous perennial, endemic to a semi-tropical region in the highlands of Paraguay where it adapted to small niche environments between wetter marshlands and drier vegetated areas (Shock 1982a, 1982b). The acidic sandy soil is relatively infertile and has an underlying shallow water table providing a continuous source of water without soil saturation. Paraguay s native Guarani peoples have used stevia leaves for centuries as a sweetening agent for normally bitter medicinal teas (Madan et al. 2010; Ramesh et al. 2006) Stevia is among many species of the Asteraceae or Compositae family that have economic importance including sunflower, lettuce, and herbal teas. Stevia usually grows as an herbaceous perennial but can be cultivated in northern latitudes or other areas such as China, as an annual. Stevia is an obligate short day plant that has a critical day length of less than 13 hours for flower initiation, although day length sensitives are subject to varietal differences (Metivier et al. 1979; Zaidan et al. 1980; Valio et al. 1977). The sweet flavor, reported at upwards to 300 times sweeter than sugar, comes from approximately eight steviol glycoside compounds found in the leaf (Brandle et al. 1998). The two most prominent glycoside compounds by content are stevioside and rebaudioside A. Trends in US of sugar consumption by retail sales projects a 10 % decline from $2.175 billion in 2013 to $1.95 billion by In 2014, 48% of the sweetener market consisted

16 2 of sugar and 17% consisted of all non-caloric sugar substitutes (Packaged Facts, 2014). Within the non-caloric sugar substitutes, steviol glycosides comprised 29% of the market compared to 45% for sucralose. Over the previous two years 33% of US adults increased their consumption of stevia, 55% maintained their stevia consumption, and 12% reduced their stevia consumption. Manufactures of diet soft drinks are using stevia as a sweetener, reducing sugar content and associated costs, while marketing a potentially healthier alternative to soda sweetened with sugar or high fructose corn syrup. Currently, United States Federal Drug Administration (FDA) regulations limits stevia use in food products as processed or any combination of steviol glycosides as a stevioside, rebaudioside A, rebaudioside D, rebaudioside M, enzyme-modified steviol glycosides and other steviol glycosides and must be purified 95% (US Food and Drug Administration, 2015). However, the FDA does allow leaf product as a dietary supplement but not as a dietary ingredient or as a sweetener or flavoring agent. The chemical extraction process is currently only performed overseas and consequently, shipping costs may limit US dry leaf production. Commercial cultivation of stevia was reported in Paraguay and Japan during the early 1960 s (Madan et al. 2008; Yadav et al. 2011). Today, stevia has been introduced for crop production in many countries around the world including China, Brazil, Mexico, Canada, and to a lesser extent in the United States.

17 3 Limited information is available to growers who are interested in stevia production as an alternative crop to tobacco or as a rotational crop that may be of financial benefit to their current operation. Major stevia production costs include labor for clonal propagation and field transplanting, weed control, irrigation, leaf harvest, and drying. US leaf production may be limited in part by the unavailability of facilities that process dry leaf material into purified steviol glycosides. Planting densities, fertility requirements, and pest management practices have been discussed by several authors (Brandle et al.1998), yet there are many unsolved problems in stevia production. (Shock 1982a,b) studied stevia dry leaf and stem yield for possible cultivation in California, United States. The field study was conducted at the University of California, Davis, California, reported a dry leaf yield of 3.7 Mg ha -1 for a single growing season with a density of 191,400 plants ha -1. The irrigation treatment was not specified but mentioned that frequent shallow irrigations were used. Over the past decade, several agronomic stevia irrigation research trials have examined irrigation effect on stevia dry leaf yields, and in some instances, steviol glycoside content and were reported from several regions of the world including Paraguay, Italy, Iran, and Columbia (Cooperativa Colonias Unidas 2010; Lavini et al. 2008; Behera et al. 2013; Aladakatti et al. 2012; Niño et al. 2013).

18 4 In the United States, S&W Seed Company had an interest in stevia leaf production in California and planted stevia from seed purchased from China and the variety PC1 (Alvarez, 2011) in 2010, 2011, and Comparisons of the steviol glycoside analysis of leaves collected from plant materials grown at Chowchilla, CA, in 2011 and 2012, had higher steviol glycoside contents compared to subsequent trials grown at other locations where the plants did not suffer as much water stress. The reasons for the differences could have been caused by water stress, differences in plant sampling, or differences in laboratory analytical procedures. Stevia leaf and steviol glycoside productivity needed to be better understood in the western United States. In 2014 stevia yield trials were conducted by Clinton C. Shock and Cheryl A. Parris, for S&W Seed Company in four locations at various latitudes in the western United States, including Indio, California, Yuma, Arizona, Hanford, California, and Ontario, Oregon. The sites ranged in soil type, and irrigation practices. (Table 1.) Three sites were university agricultural research facilities and one site was a private organic farm. All field locations were analyzed for pre-plant nutrient levels. (Table 2). Six improved S&W stevia varieties 1049, 1090, 1108, SW107, SW129, and SW201, were selected for their plant vigor, leaf taste, and steviol glycoside content and were compared to a seedling check variety for their leaf yield and steviol glycoside content. Stevia cuttings were rooted and increased during the 2013 fall and winter seasons at either the S&W greenhouse facility, Ontario, Oregon or the TS&L Seed Company

19 5 greenhouse facility, Brentwood, California. The control was bulk seed from Genesis Seed Company, Ashalin, Israel. Control seedlings were grown at both nursery locations All stevia varieties were planted at each location in a randomized complete block, with split-plot designs. The varieties were the main plots and two harvest strategies were the split plots. Each main plot was divided into two subplots and randomly designated A and B. Split-plot A was harvested twice, first harvest at mid-season and second harvest at the end of the season. Split-plot B was a single harvest performed once at the end of the season (Table 1). At Indio, Yuma, and Ontario, each main plot consisted of two beds, with two rows of stevia plants on each bed with one drip line buried at 10 cm. The harvested areas used for yield estimates were from the adjoining interior plant rows of each bed. Plots at Hanford plots consisted of four rows of plants on single bed with two surface drip lines. At Hanford, the two rows of stevia were planted on opposite sides of each drip line in an alternate fashion creating four rows per plot and the harvested area consisted of the middle two rows of plants on the 4-row bed. Yuma utilized furrow irrigation. Supervised by local management teams, irrigation soil water tension were to be averaged near 20 kpa using a Watermark Monitor data-logger (Irrometer Company, Inc., Riverside, California) and five Watermark soil moisture sensors (Irrometer Company, Inc.) installed at 20-cm depth.

20 6 Harvested plant material was collected from the two innermost rows of each four row plot. The outside rows of each plot and the first and last plants on the inside rows were designated border plants and were not used in to estimate leaf yield. The in-row transplanting distances varied by bed width at each site and were adjusted to 40,000 plants ac -1 (99,000 plants ha -1 ). At Yuma, Indio, and Hanford twenty four main plots were measured, staked and numbered to accommodate six varieties with four replicates. At Ontario 28 main plots accommodated seven varieties and four replicates. All seven varieties would have been planted at the four locations if there had been adequate plant material. Nutrient supplements consisted of 56 kg N ha -1 as solution 32 (urea ammonium nitrate) and kg Fe ha -1 as Sprint 138 6% iron chelate (Becker Underwood, Inc., Ames, Iowa) were used to fertilize stevia plots at two weeks from initial transplanting, and directly after harvest dates (Table 3). The Hanford site was maintained organically with a pre-plant blood, bone and feather meal (8-4-4) (Phyta-Grow Salinas Veggie-Mix California Organic Fertilizer, Inc., Fresno, California). During the growing season, fish emulsion (4-3-4) was applied monthly (Westbridge Agricultural Products Vista, California). Entrust, an OMRI approved BT was used once to control beet armyworm (Spodoptera exigua) at Indio, California location. Weeds were controlled by hand weeding, with

21 7 Hanford furrow cultivated prior to the first harvest, Indio and Yuma had semi-monthly furrow cultivation while no mechanical cultivation was performed at Ontario Oregon. Prior to each harvest, plant observations were recorded for all split-plots. Average plant height of each split-plot, percent flowering, and subjective observations were recorded. For steviol glycoside analysis, stevia branches from inside each split-plot border rows were cut at 20 cm and bagged in corresponding plot numbered paper sacks. Each sack labeled with harvest treatment, variety, and replicate were transported to the greenhouse for drying. After drying the leaves were separated from the stems and ground in a Wiley #4 Mill Thomas Scientific, Swedesboro, New Jersey using a 2 mm screen. The ground samples were placed in labeled manila coin envelopes and shipped to Dr. Michael Qian, Oregon State University, Food Science and Technology Department, Corvallis, Oregon for leaf steviol glycoside analysis. During each harvest, plants for yield production were cut to 20 cm height from the splitplot two interior rows. Several branches from each of six average sized plants were harvested and placed in corresponding split-plot paper sacks labeled for harvest treatment, variety, and replicate. The paper sacks were weighed wet and recorded. The remainder of each split-plot was harvested in the same manner at all sites and the wet weight was recorded. The paper sacks were later dried in the greenhouse, weighed, and recorded. Stems were then separated from the leaves and discarded. The leaves were weighed and recorded to calculate leaf yield in Mg ha -1.

22 8 Differences in variety performance included dry leaf yields, steviol glycoside concentrations and harvest strategy were evaluated over the four sites using analysis of variance using NCSS 2007 (Hintze, 2007, NCSS 6.0, Kaysville, Utah) (Tables 4, 5, 6, and 7). Leaf production for the first harvest had significant differences between locations (Table 4). Indio (2.15 Mg ha -1 ) and Ontario (2.26 Mg ha -1 ) had greater leaf yield than Yuma (1.72 Mg ha -1 ) and Hanford (1.62 Mg ha -1 ). For the second harvest and the single harvest strategy, yield production was not significantly affected by location. Averaged over harvest strategies and varieties, season-long leaf yields were higher at Ontario (5.88 Mg ha -1 ) than at the other three locations (Table 7). Averaged over locations varieties had significant differences in dry leaf yield for all harvests. For the first harvest, SW107 (2.29 Mg ha -1 ) and SW201 (2.24 Mg ha -1 ) had significantly higher in dry leaf yields than the control (1.75 Mg ha -1 ) and SW129 (1.47 Mg ha -1 ). For the second harvest SW107 (2.81 Mg ha -1 ) and 1049 (3.57 Mg ha -1 ) had significantly higher in dry leaf yields than all other varieties. The single harvest dry leaf yield for varieties 1049 (6.46 Mg ha -1 ), SW107 (5.01 Mg ha -1 ), SW201 (4.91Mg ha -1 ), and 1108 (4.65 Mg ha -1 ) were significantly higher than the dry leaf yields of 1090 (3.64 Mg ha -1 ), the control (3.53 Mg ha -1 ), and SW129 (3.28 Mg ha -1 ). The only significant interaction of variety by location on leaf yield occurred at the first harvest.

23 9 Leaf steviol glycoside contents had few significant differences when comparing locations (Tables 4, 5, and 6). For the first harvest, leaf stevioside content was significantly higher for Indio (5.49 %) than for Ontario (4.61 %) and Yuma (4.96 %) while not being significantly different than Hanford (5.02 %). For the second harvest there were not any significant differences in leaf steviol glycosides between locations. For the single harvest, leaf stevioside content was significantly higher at Ontario (4.11 %) and Indio (4.09 %) than at Hanford (3.33 %) or Yuma (2.8 %). There were no significant interactions between locations and varieties in leaf steviol glycoside content. Varieties had significant differences in leaf steviol glycoside content at all harvests (Tables 4, 5, and 6). SW129 had significantly lower stevioside content compared to the other varieties at each harvest. SW129 had significantly higher rebaudioside A content than the other varieties at the single harvest and among the highest rebaudioside A content at the first and second harvest. At the first harvest, the rebaudioside A content of SW129 (13.58 %) was statistically similar to SW201 (12.63 %) and significantly greater than that of 1090, SW107, the control, 1108, and 1049, at %, %, 6.85 %, 6.46 %, and 6.34 %, respectively. SW129 rebaudioside A content (8.52 %) was statistically similar to that of line 1090 (7.75 %) and SW107 (7.49 %), and significantly greater than that of SW201, 1049, 1108, and the control, at 7.43 %, 4.30 %, 4.01 %, and, 3.25 %, respectively When comparing harvest frequencies of two harvests versus a single harvest, there was not a significant difference between the harvest strategies, nor in the location by harvest

24 10 frequency, nor in the variety by harvest frequency, or even the location by variety by frequency (Table 7). Based in part on the performance of SW107 and SW129 in the yield trial above, these varieties were used in the current irrigation trial. SW107had previously been selected for vigor and SW129 had been selected for steviol glycoside content (Shock and Parris, 2014, 2015). When SW107 and SW129 were first selected in California (2012) and later when they were grown in Oregon in 2013, their steviol glycoside contents were initially higher than in the subsequent yield trial. There was little information available to understand whether the difference in steviol glycoside contents between samplings were due to seasonal patterns, leaf sampling procedures, leaf analysis procedures, differences in irrigation practices, or some other cultural practice. One hypothesis was that stevia could be using steviol glycosides for osmotic adjustment against the effects of water stress. Generally, plants use accumulated solutes such as K + or sugars to adjust to water stress. Karimi et al. (2015) showed that stevia uses glucose (not fructose and sucrose) for osmotic regulation when water stressed and also showed that stevia only had small increases in total leaf steviol glycoside content with the drier treatments (irrigated at 45 % field capacity) compared to the total leaf steviol glycoside content with minimal water stress (irrigated at the wetter 90 % field capacity). Yet the total steviol glycosides for Karimi et al. (2015) only reached 5.5% of leaf dry

25 11 weight with water stress, a relatively low content, which places in question the role of steviol glycosides in osmoregulation. How does the content of steviol glycoside compounds change under different soil moisture criteria, and to what extent can correlations be found with that of stevia dry leaf yield and water stress?

26 Tables Table Stevia Rebaudiana S&W Seed Company yield trials performed on seven varieties at four western United States. Site Location Indio, CA Yuma, AZ Hanford, CA Ontario, OR Soil Type Sandy Loam Clay Loam Silt Loam Silt Loam Agricultural Agricultural Field Type Research Research Private Organic Agricultural Research Herbaceous Growth Perennial Perennial Perennial Annual Irrigation Drip Furrow Drip Drip Varieties 1090, 1164, SW107, SW129, SW201, Control 1090, 1164, SW107, SW129, SW201, Control 1049, 1090, 1164, SW107, SW129, Control 1049, 1090, 1164, SW107, SW129, SW201, Control Planting Date 8-Apr-14 9-Apr Apr-14 5-May-14 Split-plot A Harvest Date Split-plot A Harvest Date Split-plot B Harvest Date 10-Jul Jul Jul Jul-14 4-Sep-14 8-Sep Sep-14 3-Oct-14 4-Sep-14 8-Sep Sep-14 3-Oct-14 Table 2. Soil analysis comparison during pre-plant preparations at four locations selected for S&W Seed Company Stevia rebaudiana yield trials Indio, Yuma, Hanford, Ontario, Soil Analysis California Arizona California Oregon ph CEC OM NA 1.6 NA 2 Nitrate, ppm Ammonium, ppm NA 6 NA 4 Phosphorus, ppm Potassium, ppm Calcium, ppm Magnesium, ppm Sodium, ppm Sulfur, ppm Zinc, ppm Iron, ppm Copper, ppm Manganese, ppm Boron, ppm

27 13 Table 3. Fertilizer application rates applied to selected field locations for S&W Seed Company Stevia rebaudiana varieties for 2014 yield trial studies. Location Date Nitrogen Kgˑha -1 Iron Kgˑha -1 Copper Kgˑha -1 Zinc Kgˑha -1 Indio, California May 13, July 16, Yuma, Arizona June 2, July 21, Hanford, California pre-plant March 3, April 7, May 5, June 2, July 7, August 4, Ontario, Oregon May 30, July 28,

28 14 Table 4. Stevia rebaudiana leaf yield and leaf steviol glycoside content from the first harvest at four locations in First harvest Location Variety Yield Stevioside Rebaudioside A Rebaudioside C TSG Indio, California Yuma, Arizona Hanford, California Ontario, Oregon Mg ha -1 % % % % SW SW SW Control Average SW SW SW Control Average SW SW Control Average SW SW SW Control Average Average, Variety Average, Variety Average, Variety Average, Variety SW Average, Variety SW Average, Variety SW Average, Control Overall Average LSD(0.05) Location NS NS NS LSD(0.05) Variety LSD(0.05) Location x Variety 0.67 NS NS NS NS

29 15 Table 5. Stevia rebaudiana leaf yield and leaf steviol glycoside content from the second harvest at four locations in Second harvest Location Indio, California Yuma, Arizona Hanford, California Ontario, Oregon Variety Yield Stevioside Rebaudioside A Rebaudioside C Mg ha -1 % % % % SW SW SW Control Average SW SW SW Control Average SW SW Control Average SW SW SW Control Average TSG Average, Variety Average, Variety Average, Variety Average, Variety SW Average, Variety SW Average, Variety SW Average, Control Overall Average LSD(0.05) Location NS NS NS NS NS LSD(0.05) Variety LSD(0.05) Location x Variety NS NS NS NS NS

30 16 Table 6. Stevia rebaudiana leaf yield and leaf steviol glycoside content from the single harvest at four locations in Single harvest Location Variety Yield Stevioside Rebaudioside A Rebaudioside C TSG Indio, California Yuma, Arizona Hanford, California Ontario, Oregon Mg ha -1 % % % % SW SW SW Control Average SW SW SW Control Average SW SW Control Average SW SW SW Control Average Average, Variety Average, Variety Average, Variety Average, Variety SW Average, Variety SW Average, Variety SW Average, Control Overall Average LSD(0.05) Location NS 0.48 NS NS NS LSD(0.05) Variety LSD(0.05) Location x Variety NS NS NS NS NS

31 17 Table 7. Stevia rebaudiana leaf yield in response to harvest strategy at four locations in Harvest strategy Location Indio, California Yuma, Arizona Hanford, California Ontario, Oregon Variety First Second First + second Single Overall harvest harvest harvest harvest harvest frequency Mg ha -1 Mg ha -1 Mg ha -1 Mg ha -1 Mg ha SW SW SW Control Average SW SW SW Control Average SW SW Control Average SW SW SW Control Average Yield average, variety Yield average, variety Yield average, variety Yield average, variety SW Yield average, variety SW Yield average, variety SW Yield average, control Yield average, overall LSD(0.05) Location 0.23 NS NS 0.90 LSD(0.05) Variety LSD(0.05) Location x variety 0.67 NS NS 1.26 LSD(0.05) Overall harvest frequency NS LSD(0.05) Location x harvest frequency NS LSD(0.05) Variety x harvest frequency NS LSD(0.05) Location x variety x frequency NS

32 18 2 LITERATURE REVIEW A study in Prado, Columbia, (Niño et al. 2013) subjected stevia Morita II variety to soil water tensions (SWT) irrigation criteria, 0-10, 10-20, 30-40, and kpa. Differences in dry leaf yield, glycoside concentrations, and water use efficiency (WUE) were evaluated. Stevia were planted in soil where rows were covered in plastic at a density of 54,000/ha. Three harvests were performed, 38, 72, and 98 days post-transplant in one season. Total dry leaf yield showed significant decreases with drier soil water tensions (SWT) treatments kpa and kpa compared to the two wettest soil water treatments, 0-5 kpa and kpa, at all harvest dates. Nino (2013) reported that the increases in both stevioside and rebaudioside A content at the wetter irrigation criteria were not significantly different. The ratio of dry leaf yield to irrigation volume, WUE, was least when the SWT was managed from 0-5 kpa and increased to 50 to 60 kpa. In Paraguay where stevia is endemic (Prieto et al. 2010), Cooperativa Colonias Unidas compared micro sprinkler and drip irrigation with or without patchouli mulch in areas of high annual rainfall (2,019 mm), low soil fertility, and high erosion. Drip irrigation was installed at a depth of 10 cm with a watering frequency of 3-5 mm per day. Stevia dry leaf yields were compared at various densities. The authors did not specify field design pertaining to adequate plot size or limiting border effects. Additionally, after the initial planting, any dead plantlets were replaced with live stevia plants to maintain densities. Dry leaf yield improved from Mg ha -1 yr-3 with no irrigation, to Mg ha -1 yr -3 for

33 19 micro-sprinkler irrigation and Mg ha -1 yr-3 with drip irrigation at planting density of 60cm x 15cm. With the addition of mulch and replanting, micro-sprinkler irrigation harvest yields increased to Mg ha -1 yr-3. No comparative research was performed using drip irrigation, replant, and mulching. The authors claimed that production cost to dry leaf sales break-even in 2010 was 3 Mg ha -1 yr-3. In 2000, (Fronza and Folegatti 2003) studied the water consumption of stevia in San Piero, Italy (43ºN, 11ºE; altitude 5m) where the climate is Mediterranean, with rain in the fall and spring and little or non-existent rainfall in the summer and little winter water consumption by stevia, (ETc), from irrigation and rainfall was measured using two constant water table microlysimeters. The microlysimeters were 0.5 m deep, 1.4 m long and 1.2 m wide and placed within a 400 m 2 stevia plot. Plants were planted at a density of 55,000/ha. Water tables were maintained at a depth of 0.35 m by buoy mechanism designed by the Irrigation National Laboratory, University of Pisa, Italy. Water consumption was replaced and recorded by a pulse counter. ETc results were compared to the estimated reference evapotranspiration (ETo) calculations using the Penman-Monteith-FAO method (Allen et al. 1989). Data collected in the first 25 days of trial indicated a higher ETc use than was calculated from ETo during the same period. The crop coefficient value (kc), calculated as a ratio of ETc to ETo of 1.45 was higher during the initial 0-25 day growing season compared to 1.14 and 1.16 from 26 to 50 days and from days respectively. Fronza and Folegatti (2003) compared their results to similar findings (Sousa 1992) of an initial higher ETc in cotton that was

34 20 explained by higher exposure of the soil to the sun and comparative higher ETc with initial planting of small leafed seedlings (Sousa 1992; Fronza and Folegatti 2003). Once full leaf canopy was observed, the microlysimeters ETc findings were comparable to ETo. Total stevia evapotranspiration for the 80 day cycle was 464 mm and during the day period, average evapotranspiration was 5.44 mm day -1. Stevia leaf yield was 4.37 Mg ha -1, stevioside content 6.49 %, and rebaudioside A and total steviol glycosides were not reported. In south-western Belavatagi, India (15 o 34 N, 75 o 21 E, 578 m above sea level), (Aladakatti et al. 2012) studied flood-irrigated stevia, replacing 80 (I1), 100 (I2), and 120 (I3) percent of the cumulative pan evaporation (CPE) in 60 mm increments. Irrigation timings were based on the soil water content measured in all plots; at the time of planting, prior to irrigations, three days after irrigations, and after crop harvest, and repeated for each harvest interval. Two month old seedlings were planted at densities of 74,000, 83,000, 111,000, and 167,000 plants ha -1 in a vertisol soil with 78% water holding capacity. Harvest was initiated after a 90 day interval from planting date and subsequent harvests were performed on 70 day intervals totaling five harvests per year for two years. Plant material was harvested at 10 cm above ground level and dried in the field for one day, then dried for one week in the shade. Growth parameters, fresh biomass, average dry leaf yield, economic gains, and consumptive use and WUE were compared to irrigation criteria. Average dry leaf yield at 120 and 100 percent CPE were

35 21 statistically similar at Mg ha -1 yr-1 and Mg ha -1 yr-1, respectively and significantly higher than 80 percent CPE at 9.36 Mg ha -1 yr-1. Average total consumed water use, based on irrigation plus effective rainfall, for (I1), (I2), and (I3) was 1903, 2038, and 2373 mm respectively. Water use efficiency of 5.15 kg ha -1 mm -1 was achieved at 100 percent CPE. The combination of irrigation at 100 percent CPE and plant density 111,111 plants/ ha -1 showed WUE of 5.58 kg ha -1 mm-1. Additionally, with comparative soil and climate conditions the authors suggests 9-10 day flood irrigation (60mm) interval during the spring, 5-6 days in summer, and 7-8 days in the fall. A 62-day greenhouse experiment was carried out at the Agricultural Biotechnology Research Institute of Iran, Karaj, Iran, where dry leaf yield and metabolite concentration changes were compared in stevia, subjected to water stress (Karimi et al. 2015). Four soil moisture levels 90, 75, 60 and 45 % of field capacity (FC) were utilized. The potting mixture had a field capacity of 28.8 % by volume and watering frequency at 3, 6, 9 and 12-day irrigation intervals, corresponding to 90, 75, 60 and 45% FC were determined in a preliminary study. Metabolites compared in this study included steviol glycosides, soluble sugars, and antioxidant capacity. Results indicate that leaf dry weight and the ratio of dry leaf weight and total dry weight (harvest index) were only significantly reduced at the 12-day irrigation interval treatment 45 % FC. Rebaudioside A concentration significantly increased over 90, 75, and 60 % FC, and then decreased at 45 % FC. Stevioside concentrations slightly increased as treatment FC decreased.

36 22 The ratio of rebaudioside A/stevioside increased from the treatment irrigated at 90 % to the treatment irrigated at 60 %, then declined at the driest treatment of 45% FC. Steviol glycoside yield (g/plant) increased from 90 to 75 %FC and then decreased at 60 and 45 % FC. Soluble sugars analysis was performed only on the 45 % FC treatment and compared with the control (100% FC). The soluble sugars were greater in the 45% FC treatment. Glucose was responsible for the increase while fructose and sucrose were unchanged. Total antioxidant capacity increased with drier treatments, where 60% FC had the highest level. The authors concluded that a soil moisture content of 60% FC was applicable for stevia growth and that stevia may adjust to water stress with an increase in glucose and antioxidant concentrations. In Bhubaneswar, Odisha, India, (Behera et al. 2013) studied varying drip irrigation and fertigation regimes. This study compared three drip irrigation treatments, 100%, 80%, and 60% of pan evaporation (PE) against a surface irrigation treatment. Additionally fertigation treatments of 100, 75, and 50 % of the recommended N-P-K dose, kg ha -1 were compared to soil applied fertilizer. Annual average CPE during the twoyear cropping period was 902 mm. Consumptive water applications included 407 mm rainfall for the same period. Planting density configuration 60 cm x 30 cm (55,600/ha -1 ) was utilized. Irrigation water was adjusted by soil cover crop coefficient (k) values of 0.70, 1.10, and 0.70 for vegetative, full growth and later growth stages, respectively. Results at 100 % PE irrigation showed a significant dry leaf yield of 2.74 Mg ha -1

37 23 compared to 80 % PE, 60 % PE and the surface irrigation at 2.37, 2.19, and 2.34 Mg ha -1 respectively. Lavini et al. (2008) studied the effects of irrigation, accumulated rainfall, and ETo on stevia water consumption, leaf yield, and steviol glycoside content in Mediterraneantype climate of southern Italy. Average annual precipitation of 736 mm occurred mainly during non-growing seasons. Irrigation treatments were based on weekly replacement of 100% field capacity (T100), and two other treatments irrigated with 66% (T66) and 33% (T33) as much water as T100. Stevia was transplanted at a distance of 5 plants m - ² (50,000 ha -1 ) and harvested twice per season. During the two year study, crop coefficients (kc) compared a ratio between the water consumption and the mean ETo for each treatment during two separate harvest periods. Applied water assumed to be equivalent to ETc was calculated to be irrigation plus rainfall. Water applied plus soil water differences from the beginning to the end of the season were assumed to be consumed. Water applied to the treatments T100, T66, and T33 averaged ETc of 0.99, 0.67, and 0.38 of ETo respectively. The results over both study years showed that during the first harvest period, no significant differences in leaf yield were found with reduced irrigations, T33 and T66 compared to T100 perhaps due to the initial high soil water content. Leaf yield was only significant between treatments during the second harvests of both years. Dry leaf yield reported as a yearly sum increased 4.6, 3.9, and 3.3 Mg ha -1 for T100, T66, and T33 respectively. Conversely, WUE declined with each increment of water use, where T33 was most efficient. Steviol glycoside concentrations

38 24 lacked significant differences between treatments, and the increase in glycoside yield was seen as a result of greater leaf yield at the wetter irrigation criteria. Several irrigation monitoring methods are available to growers when managing agronomic crop health and include pan evaporation (PE), reference evapotranspiration (ETo), or soil water measurements. Soil water measurements can be based on soil water content or soil water tension (SWT). Increasingly higher water costs push growers from traditional methods based on intuition or experience of their fathers, to more efficient water use that can objectively increase their financial benefit. Onion growers using drip irrigation in the Treasure Valley of southwestern Idaho and southeastern Oregon have adopted SWT as an irrigation monitoring strategy because SWT is closely related to onion yield and grade. The SWT measurements help the grower provide accurate and timely delivery of water to the plant, reducing stress and loss of productivity (Shock and Wang 2011; Shock et al. 2013). The current trial explored the effects of soil water tension irrigation criteria on leaf yield and leaf steviol glycoside content. With this information, it is hoped to develop a protocol for grower irrigation guidelines to maximize both yield and glycoside content.

39 25 3 MATERIALS AND METHODS In September of 2014, cuttings of SW107 and SW129 were harvested at two separate field locations; OSU Malheur Experiment Field Station, Ontario, OR and UC Cooperative Extension Coachella Field Station, Indio, CA. Approximately 100 cuttings were placed into separate, air filled, gallon zip-lock bags, to prevent desiccation, and placed in a cooler containing ice. Air filling the bags prevents damage from compaction while in transit. The bagged cuttings from Ontario were transported to a local S&W Seed Company greenhouse facility, and the cuttings from Indio were shipped overnight in coolers containing cool-packs to Westside Transplant, Huron, CA. After arrival to greenhouse facilities, all cuttings were treated in the same manner to be root propagated. Prior to sticking the stevia cuttings into individual plug tray cells, the proximal end was dipped in a 1:30 diluted solution of 1.03% indole butyric acid rooting hormone (Wood s rooting compound, Earth Science Products, Wilsonville, Oregon). Cuttings were rooted in plug trays containing 162 individual cells filled with sterile Sunshine #4 potting mix (McConkey Company, Sumner, WA). Plug trays were placed on 92 cm x 245 cm galvanized metal trays in a mist chamber that maintained the environment at close to 100% humidity. Lighting was maintained at 14 hr. day length. On three separate dates through February 2015, cuttings were rooted. As the initial rooted cuttings reached a height of 12cm, additional cuttings were harvested and then