Sustainable Intercropping of Switchgrass and Hybrid

Home , Panicum virgatum

SUSTAINABLE INTERCROPPING OF SWITCHGRASS AND

HYBRID POPLAR FOR BIOENERGY PRODUCTION

EMI KIMURA

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Crop and Soil Sciences

DECEMBER 2014

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of EMI KIMURA find it satisfactory and recommend that it be accepted.

______Steven C. Fransen, Ph. D., Chair

______Harold P. Collins, Ph. D

______Stephen O. Guy, Ph.D.

______William J. Johnston, Ph.D.

ACKNOWLEDGEMENTS

I have had the privilege to work with esteemed committee members, research staff, and graduate students at the Department of Crop and Soil Sciences at Washington State University and the United

States Department of Agriculture Agricultural Research Service, Prosser WA. I owe my gratitude to all those people who made this dissertation possible.

My deepest gratitude goes to my advisor Dr. Steven Fransen. I will be forevery grateful forever for having an amazing advisor who instilled in me the knowledge of research, statistics, and passion for grasses. His patience and support helped me finish my dissertation. I am also greatly indebted to Dr.

Harold Collins, who has always been there to give me advice. His willingness and quick response in editing my writings has provided an exemplary example which I hope to follow. I hope to be as good advisors as an advisor to my future students as Drs. Fransen and Collins. I am extremely grateful to my committee members, Dr. Stephen Guy and Dr. William Johnston, who were there to discuss research design, seedling and germination studies, and my dissertation.

Outside my committee, many people helped me in the lab and in the fields. Jason Mieirs was my friend and co-worker whose smiles and talk encouraged me. I am greatly thankful to Griselda Godinez,

Sonia Rivera, and Erika Rivera for their continuous efforts on grinding and running NIRS. Without them,

I could not have completed my dissertation. Many thanks also go to Becky Cochran, Monica Silva, and

Kent Morris. Becky’s cookies were a big part of my success in preliminary exam. I would like to express my gratitude to my second parents, Drs. An Hang and Peter Tran, who made my home in Prosser.

Because of An and Peter, I was able to enjoy my Ph.D. process without stress.

Finally, I would like to thank my parents who raised me with unconditional love and supported me in all my pursuits. And most of all, I would like to express my heart-felt gratitude to my life partner,

Kyong Park. His inspiration and encouragement sustained me throughout this endeavor. Thank you.

iii

SUSTAINABLE INTERCROPPING OF SWITCHGRASS AND

HYBRID POPLAR FOR BIOENERGY PRODUCTION

Abstract

By Emi Kimura, Ph.D. Washington State University December 2014

Chair: Steven C. Fransen

Switchgrass (Panicum virgatum L.) is a perennial warm-season grass that has been selected as an important lignocellulosic feedstock to support bioenergy production. Intercropping of two perennial species, switchgrass and hybrid poplar (Populus spp), will maximize the benefits of perennial cropping system through improving land and water use efficiency, soil N cycles, and diversifying the ecosystem.

However, establishment of switchgrass is hindered by the dormant seeds that produce weak seedlings in the fields. Limited information is available on switchgrass biomass production and forage quality in west of the Rocky Mountains under irrigation. The objectives of our study were to; (1) determine the influence of freeze-thaw treatment on the seed dormancy; (2) quantify the effect of freezing storage at -20°C or at -

80°C for five storage lengths to enhance seedling vigor of seven seed lots of switchgrass; (3) determine the influence of hybrid poplar on switchgrass biomass, architecture, and land use efficiency of the two perennial species under irrigation; and (4) determine the influence of intercropping switchgrass with hybrid poplar on the forage quality of three switchgrass cultivars over multiple growing seasons. No freeze-thaw treatments were effective on breaking seed dormancy of all seed lots used in this study. The extreme temperatures may have increased seed damage possibly by damaging the seed coat. Seed storage at freezing temperatures enhanced seedling vigor through increasing germination percentage, seedling emergence speed, and total shoot and root DM regardless of storage length up to eight months of freezing

storage. Switchgrass yield decreased each year under both cropping systems due to the water stress from excessive irrigation water that was supplied to maximize hybrid poplar growth. Despite the yield loss, land use efficiency of intercropping was maintained at LER 1.7. Significant quality differences developed after second production year due to slow growth rate under intercropping compared to monoculture cropping. Forage quality by plant component revealed that the influence of intercropping was different on leaves and stems. This work provides an understanding of switchgrass seed dormancy and effects of intercropping on biomass production and forage quality in the Pacific Northwest under irrigation.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES...... ix

LIST OF FIGURES ...... xiii

LIST OF APPENDICES ...... xvi

LIST OF PHOTOS ...... xviii

CHAPTER

1. INTRODUCTION AND OVERALL OBJECTIVES ...... 1

2. REVIEW OF LITERATURES ...... 4

Breaking seed dormancy of switchgrass (Panicum virgatum L.) ...... 4

Intercropping switchgrass with poplar tree ...... 16

References ...... 25

3. BREAKING DORMANCY OF SWITCHGRASS SEEDS BY FREEZE-THAW

SCARIFICATION

Abstract ...... 44

Introduction ...... 45

Materials and methods...... 47

Results and discussion ...... 49

Conclusion...... 52

References ...... 53

Tables ...... 57

Figures ...... 59

Appendices ...... 60

4. SEEDLING VIGOR OF SWITCHGRASS INFLUENCED BY COLD STORAGE

TEMPERATURES

Abstract ...... 63

Introduction ...... 64

Materials and methods...... 66

Results ...... 68

Discussions ...... 74

Conclusion...... 76

References ...... 78

Tables ...... 82

Figures ...... 85

Photos ...... 88

5. SWITCHGRASS GROWTH REPONSE IN INTERCROPPING WITH HYBRID

POPLAR UNDER IRRIGATIONS

Abstract ...... 91

Introduction ...... 92

Materials and methods...... 94

Results and discussion ...... 96

Conclusion...... 102

References ...... 103

Tables ...... 110

vii

Figures ...... 113

Appendices ...... 117

6. FORAGE QUALITY OF SWITCHGRASS AT DIFFERENT GROWTH STAGES IN

INTERCROPPING WITH HYBRID POPLAR

Abstract ...... 121

Introduction ...... 123

Materials and methods...... 124

Results and discussion ...... 125

Conclusion...... 131

References ...... 132

Tables ...... 136

Figures ...... 140

7. BIOMASS PRODUCTION AND NUTRIENT REMOVAL BY SWITCHGRASS

(PANICUM VIRGATUM L.) UNDER IRRIGATION

Abstract ...... 142

Introduction ...... 143

Materials and methods...... 145

Results and discussion ...... 147

Conclusion...... 154

References ...... 156

Tables ...... 161

8. GENERAL CONCLUSIONS ...... 168

viii

LIST OF TABLES

CHAPTER 2.

1. Summary of major cultivars of switchgrass (Modified from Seepaul et al., 2011)

...... 42

2. Two seed lots with same germination percentage expressing different seed

characters (Adapted from Vogel, 2002) ...... 43

3. Summary of best yielding switchgrass cultivar at several locations in North

America ...... 43

CHAPTER 3.

1. Seed weight g-1 of seed lots for Kanlow, Blackwell, and Trailblazer ...... 57

2. Analysis of covariance and mean squares for adjusted germination percentage of

freeze-thaw treatment in response to seed lots [Kanlow (2008, 2009, and 2010),

Blackwell (2010 and 2011) and Trailblazer (2010 and 2011) of switchgrass],

treatment (-80ºC, -20ºC, Pre-chill, and 23 ºC), cycles (0-5 cycles), and two and

three ways interactions ...... 58

CHAPTER 4.

1. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new

tiller formation (# plant-1) of lowland cultivar of Kanlow switchgrass (seed lot:

2008, 2010, and 2011) influenced by storage temperatures at -80°C, -20°C, or room

temperature (23°C; RM) for storage lengths for one, two, three, six, and eight

months...... 82

2. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new

tiller formation (# plant-1) of upland cultivar of Blackwell switchgrass (seed lot:

2010, and 2011) influenced by storage temperatures at -80°C, -20°C, or room

temperature (23°C; RM) for storage lengths for one, two, three, six, and eight

months...... 83

3. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new

tiller formation (# plant-1) of upland cultivar of Trailblazer switchgrass (seed lot:

2010, and 2011) influenced by storage temperatures at -80°C, -20°C, or room

temperature (23°C; RM) for storage lengths for one, two, three, six, and eight

months...... 84

CHAPTER 5.

1. Analysis of variance and mean squares for yield of grass and tree, land equivalent

ratio (LER), tiller components during 2012-2014 growing seasons ...... 110

2. Aboveground biomass of Kanlow (KL), Blackwell (BW), and Trailblazer (TB)

switchgrass in monoculture plot or intercropped with hybrid poplar clones, OP367

(OP) or PC4 (PC) from 2012 to 2014 ...... 111

3. Tree yield and Land Equivalent Ratio (LER) of Kanlow (KL), Blackwell (BW), and

Trailblazer (TB) switchgrass in monoculture plot or intercropped with hybrid poplar

clones, OP367 (OP) or PC4 (PC) from 2012 to 2013...... 112

CHAPTER 6.

1. Analysis of variance and mean squares for forage quality attributes of whole plant by

growth stages for three cultivars of switchgrass grown alone or intercropped with

hybrid poplar during 2012 to 2014 study years ...... 136

2. Analysis of variance and mean squares for forage quality attributes for plant

component (stem and leaf) for three cultivars of switchgrass grown alone or

intercropped with hybrid poplar during 2012 to 2014 study years ...... 137

3. Crude Protein (CP), sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF),

and lignin of leaf and stem of switchgrass in grass monoculture (Mono) and

intercropped with hybrid poplar (IC) from June, 2012 to July, 2014 ...... 138

4. Tissue concentration of phosphorus (P), potassium (K), magnesium (Mg), and

calcium (Ca) of leaf and stem of switchgrass in grass monoculture (Mono) and

intercropped with hybrid poplar (IC) from June, 2012 to July, 2014 ...... 139

CHAPTER 7.

1. Physical and chemical properties of the Quincy sand (Xeric Torripsamments) soil under

switchgrass at the USDA-ARS Integrated Agricultural Research Field Station, Paterson, WA

...... 161

2. Monthly precipitation, air temperature, and total growing degree days from 2004 to 2009 ..

...... 162

3. Analysis of variance and mean squares for yield, macro- and micronutrients removal for three

cultivars of switchgrass during 2005-2009 study years ...... 163

4. Analysis of variance and mean squares for macro- and micronutrient concentration for three

cultivars of switchgrass during 2005-2009 study years ...... 164

5. Aboveground biomass DM (dry matter) under two N rates for three switchgrass cultivars

during the 2005-2009 study years ...... 165

6. Nutrient removal under two N rates for three switchgrass cultivars during 2005-2009 study

years ...... 166

7. Nutrient concentration under two N rates for three switchgrass cultivars during 2005-2009

study years...... 167

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LIST OF FIGURES

CHAPTER 3.

1. Germination percentage of Kanlow (seed lot 2008), Blackwell (seed lots 2010 and

2011), and Trailblazer (seed lots 2010 and 2011) influenced by freeze-thaw

scarification at -80°C, -20°C, Prechill treatment, and 23°C. Values followed by the

same letters are not significantly different at p < 0.05. Insignificant results were

obtained for Kanlow 2010 (avg. 79 %) and Kanlow 2011 (avg. 29 %)...... 59

CHAPTER 4.

1. Effect of storage temperatures (-80°C, -20°C, and room temperature (RM)) and

storage length (1, 2, 3, 6, and 8 months) on total shoot and root DM and shoot and

root DM plant-1 for Kanlow (seed lot 2008, 2010, and 2011), Blackwell (seed lot

2010 and 2011), and Trailblazer (seed lot 2010 and 2011). Values within a plant part

within a storage period by the same letter abc for total shoot and root DM and xyz for

DM plant-1 are not significantly different at p< 0.01...... 85

2. Effect of storage periods (1, 2, 3, 6, and 8 months) on leaf lengths and width of

Kanlow (seed lot: 2008, 2010, and 2011) and leaf lengths and width of Blackwell

(seed lot: 2010 and 2011) and Trailblazer (seed lot: 2010 and 2011). Values within a

leaf by the same letter are not significantly different at p< 0.05. * indicates sifnificant

differences of leaf length between seed lot for Blackwell and Trailblazer...... 86

3. Length of soil to the first node and internodes for Kanlow (seed lot: 2008, 2010, and

2011) and Trailblazer (seed lot: 2010 and 2011), and effect of storage lengths (1, 2, 3,

6, and 8 months) to node lengths. Values within a plant part by the same letter are not

significantly different at p< 0.01...... 87

xiii

CHAPTER 5.

1. Monthly temperature and precipitation during 2012 to 2014 growing seasons at

Boardman, OR...... 113

2. Layout of whole plot (left) and one replication of intercropping plot (right). There are

24 intercropped plot, 12 grass monoculture plots, and 8 tree monoculture plots,

resulting in 44 total plots...... 114

3. Leaf Area Index (LAI) taken under grass only plot or in intercropped with poplar

clones OP367 or PC4 during 2012 to 2014 growing seasons. Values within a day of

year followed by the same letter are not significantly different at p ≤ 0.05. The first

letter, second letter, and third letter correspond to LAI at grass only plot, OP367 plot,

and PC4 plot, respectively. LAI value of 0 was used to indicate the harvested stand

(DOY: 185)...... 115

4. Dry matter (g m-2) of tiller components (stem, leaf blade, and panicle) of switchgrass

cultivars Kanlow (KL), Blackwell (BW), and Trailblazer (TB) in monoculture and in

intercropped with hybrid poplar clones OP367 (OP) or PC4 (PC) in July and October

in 2012 and 2013, and July in 2014. Values within a cultivar and a plant component

followed by the same letter are not significantly different at p ≤ 0.05...... 116

CHAPTER 6.

1. Crude Protein (CP), sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF),

and lignin of switchgrass in grass monoculture ( ) and intercropped with

hybrid poplar ( ) from June, 2012 to July, 2014. * indicates that means between

cropping systems are significantly different at p = 0.05...... 140

xiv

2. Tissue concentration of phosphorus, potassium, magnesium, and calcium of

switchgrass in grass monoculture ( ) and intercropped with hybrid poplar

( ) from June, 2012 to July, 2014. * indicates that means between cropping

systems are significantly different at p = 0.05...... 141

LIST OF APPENDICES

CHAPTER 3

1. Germination percentage of three Kanlow seed lots by freeze-thaw scarification with

alternation of -80°C for one hour and 23°C for one hour (-80/23) or -20°C for one

hour and 23°C for one hour (-20/23) compared to seed germination stored at room

temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles...... 60

2. Germination percentage of two Blackwell seed lots by freeze-thaw scarification with

alternation of -80°C for one hour and 23°C for one hour (-80/23) or -20°C for one

hour and 23°C for one hour (-20/23) compared to seed germination stored at room

temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles ...... 61

3. Germination percentage of two Trailblazer seed lots by freeze-thaw scarification with

alternation of -80°C for one hour and 23°C for one hour (-80/23) or -20°C for one

hour and 23°C for one hour (-20/23) compared to seed germination stored at room

temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles ...... 62

CHAPTER 5.

1. Switchgrass coverage (%) across the plots. Row 1 and row 5 indicate west and east

side of plot, respectively...... 117

2. Percentage coverage of switchgrass cultivars Kanlow (KL), Blackwell (BW), and

Trailblazer (TB) in monoculture and in intercropped with hybrid poplar clones OP367

(OP) or PC4 (PC) and percentage of weed in May 2012 and September 2013. ... 118

3. Vigorous switchgrass observed in tree rows that received less water and less

compaction as compared to the middle of plots...... 119

xvi

4. Leaf temperature under monoculture (Mono) and intercropping (IC) from 10:10 am to

10:48am and from 10:50 am to 12:05pm. * and *** indicate that values are

significantly different at p < 0.05 and 0.0001, respectively...... 120

xvii

LIST OF PHOTOS

CHAPTER 4.

1. Seedling emergence at 15th day (top), shoot growth (middle) and root growth

(bottom) before harvesting for seeds stored at -80°C, -20°C, and room temperature

(RM) for Kanlow 2008 sotred for one month, Kanlow 2010 stored for eight months,

and Kanlow 2011 stored for one month ...... 88

2. Seedling emergence at 15th day (top), shoot growth (middle) and root growth

(bottom) before harvesting for seeds stored at -80°C, -20°C, and room temperature

(RM) for Blackwell 2010 and Blackwell 2011 seeds sotred for eight months...... 89

3. Seedling emergence at 15th day (top), shoot growth (middle) and root growth

(bottom) before harvesting for seeds stored at -80°C, -20°C, and room temperature

(RM) for Trailblazer 2010 seeds stored for six months and Trailblazer 2011 seeds

stored for eight months...... 90

CHAPTER 5.

1. Vigorous switchgrass was observed in tree rows, which received less water and less

compaction as compared to the middle of plots...... 111

xviii

Dedication

This dissertation is dedicated to the memory of my sister for providing inspiration and motivation.

xix

CHAPTER ONE

INTRODUCTION AND OBJECTIVES

Switchgrass (Panicum virgatum L.) is a perennial warm-season grass identified as a model species for bioenergy feedstock. The Oak Ridge National Laboratory administered the

Biofuels Feedstock Development Program (BFDP), and selected switchgrass through herbaceous crop screening trials conducted from 1985 to 1992 (Wright and Turhollow, 2010). This trial involved 34 herbaceous species and 31 sites over seven states in the USA: North Dakota, Iowa,

Indiana, Ohio, New York, Virginia, and Alabama (Wright and Turhollow, 2010). The

Renewable Fuel Standard (RFS) in the Energy Independence and Security Act of 2007 mandates that 16 billion gallons of cellulosic ethanol be produced by 2022. As political attention focused on the production of cellulosic ethanol, research highlighting switchgrass as a viable feedstock increased, with most studies concentrated in the eastern U.S. (McLaughlin and Kszos, 2005).

Among southeastern states (NC, TN, VA, WV), an average dry matter (DM) yield of

14.2 Mg ha-1 yr-1 was reported in seven year-old stands grown under dryland conditions (Fike et al., 2006a). Although switchgrass studies are concentrated east of the Rocky Mountains, switchgrass DM yields observed in western states are comparable or greater when grown under irrigation (Fransen et al., 2006; Fransen, 2008; Collins et al., 2010; Kimura et al., 2015). For example, a study established in 2004 in southeastern WA near the Colombia River showed the cultivar ‘Kanlow’ had a DM yield of 3.3, 21.0, and 22.6 Mg ha-1 during first, second, and third years, respectively (Collins et al., 2010) and maintained DM yield of 26.7 Mg ha-1 yr-1 for fourth to sixth year (Kimura et al., 2015). Switchgrass has high yield capacity throughout the USA; however, establishment is difficult due to the dormant seeds as well as slow seedling growth in the establishment year. The Association of Official Seed Analysts (AOSA) uses an effective

dormancy breaking technique called pre-chill treatment (AOSA, 2010). Pre-chill treatment increases germination of switchgrass seeds; however, it was shown that germination was decreased when seeds were dried after pre-chill treatment (Shen et al., 2001). These problems must be solved to enhance stand establishment of switchgrass to maximize yield per unit area.

Furthermore, a problem associated with switchgrass bioenergy production is limited land resources. Since there is a need to feed increasing population in the world, swichgrass production cannot displace food and fiber production. However, energy shortage in near future will be a critical problem; therefore, research efforts are necessary to improve land and water use efficiency of switchgrass production systems. In BFDP, woody species have gained attention as potential biofuel feedstock (Wright and Turhollow, 2010). It takes longer to grow woody species compared to herbaceous species; however, trees produce greater biomass than grasses.

Examples of such woody crops include black locust, silver maple, willow, and hybrid poplar

(Poplus spp.) Among the potential woody species, hybrid poplar became popular due to its fast growing habit (Newman et al., 1997).

GreenWood Resources’ Boardman Tree Farm (BTF) (10,850 ha), located in Boardman,

OR, is the largest hybrid poplar production in North America. While growing hybrid poplar trees at BTF, open ground may be utilized for herbaceous crop production. Incorporating an additional crop into tree production will improve crop diversity, weed control, SOC, wildlife habitat, and more importantly water and land use efficiency (Altieri, 1999). This resource- efficient intercropping system will eliminate the problem of using land for food and fiber production as well as increasing WUE by using micro-sprinkler irrigation set up for poplar trees.

A survey conducted in 2011 by National Agricultural Statistics Service (NASS) showed that

South Dakota had the highest state swichgrass proportions of 24.8%, followed by Texas (18.2%)

and Iowa (12.7%). In contrast, state switchgrass area in Washington and Oregon were less than

1% (Muelle, 2012). There is high potential to increase the switchgrass production area not only in the PNW but also in other areas of the USA if open ground under poplar trees can be used for production area.

The proposed project will intercrop switchgrass in alleys of hybrid poplar trees to create an alternative biofuel feedstock production system. This study will evaluate seed dormancy and seedling vigor of switchgrass to improve stand establishment, and determine the influence of inter-cropping of poplar tree and switchgrass biomass on both species. The specific objectives of the proposed research are to;

1) Investigate the effect of freeze-thaw scarification on germination and dormancy of

different cultivars and harvested years of switchgrass seeds;

2) Monitor the influence of storing periods and storing temperatures on seedling emergence,

seedling vigor, and growth rate of different cultivars and years of switchgrass;

3) Determine the influence of poplar cultivars of contrasting leaf area index and canopy

architecture on switchgrass biomass production within an intercropped production system

and;

4) Evaluate and compare the forage quality at vegetative, elongation, and reproductive

stages of switchgrass biomass in intercropped and monoculture production systems.

CHAPTER TWO

LITERATURE REVIEW

I. Breaking seed dormancy of switchgrass (Panicum virgatum L.)

Introduction

Swichgrass produces a diffuse panicle, from which 200 to 1000 kg ha -1 of seeds are produced, depending upon lodging level (Kassel et al., 1985; Brejda et al., 1994). Established switchgrass stands are very resilient to environmental fluctuation; however, seed dormancy and weak seedling vigor hinder stand establishment (Evers and Parson, 2003; Loch et al., 2004).

Seed dormancy is defined as failure of intact, viable seeds to germinate under favorable conditions (Loch et al., 2004), and represents an ecological adaptation of hardy plant species to survive under adverse environmental conditions. There are two known seed dormancy mechanisms: embryo-imposed and seed-coat-imposed dormancy (Adkins et al., 2002). Embryo imposed dormancy can be further categorized into morphological and physiological dormancy.

Morphological dormancy is caused when an embryo is immature or undeveloped, while physiological dormancy results from germination inhibitors present in the embryo (Adkins et al.,

2002). Examples of germination inhibitors include abscisic acid, coumarin, catechins, tannins, and phenols (Adkins and Bellairs, 1995).

Seed-coat-imposed dormancy prevents germination via chemical inhibitors or low permeability of the seed coat, which obstructs gas and water exchange, limiting embryo respiration (Adkins et al., 2002). A recent study using scanning electron microscopy revealed that seed-coat-imposed dormancy may be the major cause of switchgrass seed dormancy (Duclos et al., 2013), although embryo-imposed dormancy has also been reported (Sarath et al., 2006b).

Among all seed coat structures, the pericarp was the primary barrier to oxygen and water exchange, followed by the lemma and palea as secondary barriers. There was no evidence of germination inhibitors in these structures, and the endosperm had no influence upon dormancy

(Duclos et al., 2013). In the field, dormant seeds are exposed to variable temperature, humidity, soil moisture, and light regimes, through which seed dormancy may be reduced (Byers, 1973;

Emal and Conard, 1973; Hanson and Johnson, 2005).

Switchgrass produces large proportions of dormant seeds due to its short history as a cultivated plant species. Seed labels for native grasses often include viable dormant seeds in a total germination percentage. Despite the high total germination percentage on switchgrass labels (>90%), seedling emergence is very low in the field. The reseeding cost resulting from sporadic seedling emergence due to switchgrass seed dormancy was estimated at 36 % of the total cost of stand establishment, which cannot be paid off easily by increasing yield (Perrin et al.,

2008). Various treatments have been examined to break dormancy of switchgrass seeds because reduction of dormancy is the first critical step for successful stand establishment. Currently, the

Association of Official Seed Analysts (AOSA) recommends a moist pre-chill treatment at 5°C for 14 days for switchgrass seeds (AOSA, 2010). Pre-chill may mimic cold stratification during wintering of the seeds (Vogel, 2004; Sarath et al., 2007) and stimulate mobilization of seed carbohydrate (La Croix and Jaswal, 1967) and lipid (Ross, 1984) reserves. Although the moist pre-chill treatment breaks dormancy of switchgrass seeds, the effect is limited to situations in which seeds remain wet after the treatment (Shen et al., 2001). If pre-chilled seeds are desiccated for mechanical planting, they revert to dormancy (Shen et al., 2001). Based on this observation, Shen et al., (2001) explained that seed dormancy and germination are continuous processes, rather than on or off processes. To prevent reversion, primary dormancy must be

completely overcome by extending pre-chill periods to 42 days (Shen et al., 2001). Newly harvested seeds expressed more dormancy reversion than aged seeds (Grabowski et al., 2002;

Shaidaee et al., 1969; Shen et al., 2001). Moist pre-chill treatment, while useful for a small-scale seed germination improvement, is sometimes impractical due to the long duration of the treatment and often results in dormancy reversion (Shen et al., 2001).

Chemical treatments

Treatment time can be shortened with chemical scarification, which takes minutes to hours instead of weeks as required for the pre-chill method. A 15-minute treatment with 1.5 M chloroethanol solution increased germination of the lowland cultivar Alamo from 50 % in the untreated control to 87 % (Tischler et al., 1994). Despite the positive results, it was not recommended due to the toxicity. Sulfuric acid damages the margins of the lemma, allowing entry of water and gas into the seeds (Haynes et al., 1997). The acid concentration and duration of application are important criteria controlling efficacy (Haynes et al., 1997). Sulfuric acid at

16.8 M increased germination of freshly harvested Alamo (94 %), Kanlow (68 %), and Caddo

(68 %) switchgrass seeds compared to untreated seeds of these cultivars (52 %, 16 %, and 48 %, respectively) with 10 min of treatment time (Tischler et al., 1994). In contrast, diluted sulfuric acid (8 M) increased germination by 14 % with 5 min treatment from 46 % in the un-treated control (Haynes et al., 1997). The efficacy of chemical seed treatment depends upon many factors including cultivar, harvest method, seed production environment, and harvest year

(Tischler et al., 1994; Zarnstorff et al., 1994; Boe, 2009). For example, Sarath et al (2008) observed that several seed lots harvested in the same field had different responses to reactive nitrogen species and peroxide.

Physical treatments

Mechanical scarification breaks seed coat dormancy by cracking the pericarp and increasing gas exchange and imbibition (Jensen, 1985). Emery cloth (Sautter, 1962) and sand paper (Zhang and Maun, 1989) increased germination of switchgrass seeds (unknown cultivar) by 85%; however, rubbing seeds by hand is inefficient in a large-scale operation. The “Forsberg

Cylinder,” an emery-cloth-based scarifying device invented by Forsbergs, Inc., (Thief River

Falls, Minnesota) improved germination of Sunburst and North Dakota switchgrass seeds from

73 % and 30 % in controls to 82 % and 55 %, respectively (Jensen and Boe, 1991). Large-scale seed treatment may be possible with the scarifier. In the meantime, further research will reveal the effectiveness and applicability of mechanical scarification.

Germination of swtichgrass is sensitive to temperatures, and the sensitivity differs among cultivars. In the field switchgrass seeds germinate when soil temperatures are between 10.0°C

(Hsu et al., 1985a) and 15.5°C (Parrish and Fike, 2005), and germination and seedling growth are enhanced at 26.6°C (Hsu et al., 1985b; Seepaul et al., 2011) and with a 16/8 h day/night photoperiod (Norris and Decker, 1943). Optimum temperature for germination is cultivar- specific. ‘Summer’ tolerates higher temperature for germination (28.6ºC), while others (Cave in

Rock, Dacotah, Expresso, Forestburg, Kanlow, Sunburst, Trailblazer, and Tusca) prefers 24.0-

28.2 ºC for germinatnion (Seepaul et al., 2011). These cultivars did not germinate at temperatures above 45ºC (Seepaul et al., 2011). Germination of swichgrass was recorded as low as 8 ºC, and ‘Expresso’ switchgrass tolerates 3.7 ºC for germination (Seepaul et a., 2011). While extreme soil temperatures may inhibit switchgrass germination, temperature fluctuations enhance germination of switchgrass (Ahring et al., 1959). Germination of 6-month old switchgrass seeds at constant 20°C was 38 % lower than germination under 20/35 ºC day/night temperatures (88%)

(Ahring et al., 1959). Temperature alternation may mimic the surface soil temperature regime to which seeds are typically exposed in early spring, and may result in freeze-thaw scarification of seeds (Rinehart, 2006; Stout, 1990). Alternation between -80°C for two hours followed by thawing at 20°C (or room temperature) for two hours reduced dormancy of alfalfa seeds from 60 to 14 % (Stout, 1990). Similarly, seedling emergence of frozen switchgrass seeds was greater

(unknown number) than unfrozen seeds in three out of four years of study (Blake, 1935). Further research is needed to determine optimal treatment temperatures and duration of freeze-thaw scarification on grass seeds.

Plant hormones

Plant hormones are important growth regulators controlling initiation of germination

(Loch et al., 2004). Gibberellin promotes breakdown of starch stored in endosperm through enzymatic reactions, whereas abscisic acid depresses the action of gibberellin (Loch et al., 2004).

Water imbibition of seeds increased gibberellin concentration (Loch et al., 2004). Osmo- conditioning of Cave in Rock, Dakotah, and Jersey 50 switchgrass seeds in a solution of polyethylene glycol and 1 mM gibberellin increased germination by 19 % (Madkadze et al.,

2000). There is a strong correlation between nitrous oxide (NO) signaling pathways and dormancy mechanisms in warm season grasses (Sarath et al., 2006b). Exogenously applied nitric oxide (Sarath et al., 2006b) and hydrogen peroxide, which enhances NO production (Sarath et al.,

2007), increased germination of cold-stratified switchgrass, big bluestem, and indiangrass seeds.

Conversely, the presence of NO scavengers resulted in reduced germination (Sarath et al.,

2006b). Low concentrations of ethylene (25 µL L-1), gibberellin (0.25 mM), and kinetin (0.1 mM) did not increase germination of Balckwell and Cave in Rock (Zarnstorff et al., 1994).

Seed storage/after ripening periods

Thick seed coats that prevent radicle emergence are often weakened over time by temperature alternations, fungal attack, fire, after-ripening, or enzymes produced from the embryo both in natural and controlled environments (Adkins et al., 2002). However, when the storage periods become too long, seed viability decreases as a result of peroxidation of polyunsaturated fatty acids, and damage to cell membranes and DNA (Bewley and Black, 1994).

Seed aging was accelerated at above 55 g kg-1 humidity and at 60ºC (Shen et al., 1997; Shen et al., 1999), although optimal storage periods, temperature, and storage methods (e.g., seedbag, plastic containers, and paper bags) are cultivar- and seed lot-sepecific (Shaidaee et al., 1969;

Grabowski et al., 2002; Oliver, 2006; Boe, 2009). Two seed lots (1995 and 1996) of Alamo and accession 746 (upland ecotype) were stored at three different conditions: cold (7°C with 55 % humidity), room temperature (21°C with ambient humidity), and a warehouse condition (-1°C to

38°C with ambient humidity) for 1, 5, 7, 9, and 11 months (Grabowski et al., 2002). Initial germination percentages were 24 % and 76 % for Alamo and 1 % and less than 1 % for accession 746 for seed lots 1995 and 1996, respectively (Grabowski et al., 2002). For both seed lots, storage at room temperature was optimal for breaking dormancy. For the 1995 seed lot, germination was increased by 50 % after one month of storage for Alamo and after five months of storage for accession 746 at room temperature (Grabowski et al., 2002).

Seed storage at subfreezing temperatures increased germination of switchgrass.

Germination of switchgrass was improved from 10 to 76 % after storing at 4°C for 49 days

(Sautter, 1962), from 30 to 53 % at 4°C for 300 days (Oliver, 2006), from 78 to 90 % after storing at 23°C for 90 days (Zarnstorff et al., 1994), from 43 to 69 % after storing at 23°C for one year (Robocker et al., 1953), and from 27 to 88 % after storing at 25°C for one year (Byers,

1973). These results indicate that storage temperature between 4°C and ambient temperature may break switchgrass dormancy (Aho et al., 1989). Effect of storage temperatures below 4°C on dormancy breaking of switchgrass has been conflicting among studies with negative results

(Sautter, 1962; Byers, 1973; Zarnstorff et al., 1994; Burson et al., 2009) and positive results

(Blake, 1935). For instance, freezing switchgrass seeds for 14, 31, 49, and 54 days did not break seed dormancy (Sautter, 1962), and storing Blackwell and Cave in Rock seeds at -8°C for 90 days, 180 days, 2 years, and 4 years had little influence on germination (Zarnstorff et al., 1994).

Switchgrass seeds stored at -23°C for one year showed little germination improvement as compared to seeds stored at 25°C (Byers, 1973). Storage at -20°C had little influence upon dormancy of Alamo (Burson et al., 2009). Storage temperature at -20°C reduced germination of

TEM-LoDorm, Alamo-based cultivar with reduced post-harvest seed dormancy (Burson et al.,

2009). Although storage of switchgrass seeds at freezing temperatures have been found to have little influence on germination improvement, enhanced seedling emergence (Black, 1935) and vigor (personal communication with Dr. Fransen, 2011) was observed when seeds were stored at freezing temperatures for several weeks. Previous studies have mainly considered germination percentage in a petri dish followed by various storage temperatures and durations. It is important to examine how freezing seeds influence seedling vigor (e.g., shoot dry matter) of switchgrass when planted in pots or in the field.

Miscellaneous methods

Miscellaneous methods have been reported on the effort to break seed dormancy of switchgrass. Such methods include karrikinolide smoke (3-methyl-2H-furo [2, 3-c] pyran-2-one;

George, 2009), bacterial (Debebe, 2005) and fungal (Ghimire et al., 2009) inoculation, seed

priming (Debebe, 2005), electromagnetic radiation (Funk et al., 2010), and ultrasounds (Wang et al., 2012). Karrikinolide smoke was found to improve germination and seedling vigor in some plant species native to ecosystems with frequent fires (Brown and Staden, 1997; Daws et al.,

2007). In one study, two-month-old Alamo seeds were soaked in karrikinolide solution for 24 hours and planted in a pot with potting mix (George, 2009). The pots were placed in greenhouse under 12 hours day length at day/night temperatures of 32/20ºC. No difference was observed between the treated and untreated seeds; however, application of karrikinolide in different forms, such as gas applied with irrigation water, may improve germination of switchgrass seeds (George,

2009).

Germination of Kanlow strains (MAFF-305828, MAFF-305830, and MAFF-305842) was increased by 52 % upon inoculation with the mycorrhizal fungus Sebacina vermifera (Ghimire et al., 2009). This fungal inoculant also enhanced plant height and root length. Inoculated seeds produced 75, 113, and 18 % greater shoot biomass in the first, second, and third harvest, respectively, as compared to the shoot biomass from untreated seeds (Ghimire et al., 2009). The increased shoot biomass was attributed to improved nutrient acquisition through the symbiotic relationship with S. vermifera (Ghimire et al., 2009). Solid-matrix priming with post-priming heat treatment of Cave in Rock, Trailblazer, and Nebraska 28 seeds increased the average shoot dry matter by 56 %, the number of adventitious roots by 91 %, and the number of tillers by

138 % compared to untreated controls (Debebe, 2005).

Ultrasound treatments have been reported to promote breaking of seed dormancy in dicots (Kim et al., 2006; Shin et al., 2011) and monocots (Yaldagard et al., 2008). The ultrasound may disrupt plant cells surrounding the seeds, increasing gas and water entry (Wang et al., 2012). Wang et al. (2012) applied ultrasound treatments with various sonication times,

temperatures, and ultrasound output power levels to Alamo and Summer seeds. The optimum treatment was a sonication time of 22.5 min at 39.7ºC with an output power of 348 W (Wang et al., 2012). Funk et al. (2010) reported at the In Vitro Biology meeting in 2010 that electromagnetic radiation for 20 to 25 minutes increased germination of switchgrass and improved seedling vigor in terms of shoot and root length.

Effect of seed size upon seed dormancy

Seed properties vary greatly between the seed lots within and among cultivars (Boe,

2007). Two seed lots expressing the same germination percentage differ in their emergence percentage by 40 %, number of seeds g-1 by 200 seeds, and seeding rates by 5 kg ha-1 when aiming at 300 pure live seed m-2 (Table 2; Vogel, 2002). Larger seeds within a cultivar expressed a greater percentage of germination than smaller seeds (Kneebone and Cremer, 1955;

Green and Hansen, 1969; Aiken and Springer, 1995), although results between studies have been inconsistent (Aho et al., 1989). Blackwell seeds separated onto 1.27, 1.15, and 1.06 mm screens emerged at 82, 64, and 32 %, respectively (Kneebone and Cremer, 1955). In a study conducted by Aiken and Springer, (1995), seeds were separated by size using air valve settings of 40, 50, 60, and 80°. Seed weight between each valve setting was in the range of 8-46 mg/100 seeds depending upon cultivar. Germination of Alamo, Blackwell, Cave in Rock, Kanlow, and

Trailblazer was improved as seed size increased, while seed size had no influence on germination of Pathfinder. The time required for germination was longer in seeds less than 1.06 mm than larger seeds between 1.15 to 1.27 mm (Kneebone and Cremer, 1955). Seed weight is typically heavier in early-flowering than in late-flowering cultivars (Bortnem and Boe, 1998).

Upland cultivars flower earlier than lowland cultivars, while Sunburst flowers earlier than

Blackwell and Pathfinder. The 1000 seed weight of Sunburst was the heaviest (6.7 g) as compared to 1000 seed weight of Blackwell (5.2 g) and Pathfinder (5.3 g) (Bortnem and Boe,

1998). The advantage of larger and heavier seeds has been shown to last up to 10 weeks after emergence in greenhouse experiments (Zhang and Maun, 1991) and in the field (Smart and

Moser, 1999).

Effect of soil conditions upon seed dormancy

Seed dormancy of grasses is associated with soil conditions such as salinity (Ries and

Hofmann, 1983; Hanson and Johnson, 2005; Kim et al., 2012; Schmer et al., 2012), row spacing

(Newell, 1968a), and fertilizer rate (Smika and Newell, 1965; Traversa et al., 2013). Although established switchgrass stands are resilient to soil temperature and pH fluctuations, germinating seeds and emerging seedlings are sensitive to these soil parameters (Hanson and Johnson, 2005).

The optimum soil pH and temperature ranges for germination and emergence of Dacotah,

ND3743, Summer, Sunburst, Trailblazer, Shawnee, OK NU2, and Cave in Rock were pH 6-8 and 23-25ºC (Hanson and Johnson, 2005). Salt tolerance of switchgrass was examined with sodium sulfate (120 mmol Na+ L-1), magnesium sulfate (100 mmol Mg+2 L-1), and a mixture of these salts (66mmol Na+ L-1, 33 mmol Mg+2 L-1) (Ries and Hofmann, 1983). Switchgrass germination was decreased from 71 % in control to 57 % with the sodium sulfate treatment. A second report demonstrated that NaCl concentrations above 500 mM decreased germination of

Cave in Rock by 30% (Kim et al., 2012). The lowland cultivars Alamo and Kanlow expressed higher salt tolerance than the upland cultivars Dacotah, Forestburg, and PV-1777, with respect to germination (Schmer et al., 2012). Reduced germination under salt concentrations was shown to result from inhibition of adenosine monophosphate (AMP) for some cool-season grasses and

legumes (Underson, 1986). However, AMP had no observed effect on germination of switchgrass and western wheatgrass (Agropyron smithii Rydb).

Several studies have demonstrated that row-spacing influences seedling emergence.

Increased caryopsis weight was observed in side-oats grama (Bouteloua curtipendula Michx.) when seeds were planted in 101 cm-rows rather than in solid stands (Newell, 1968a). The relation between switchgrass dormancy (Blackwell, Cave in Rock, and Pathfinder) and agricultural practices, such as N rates and row spacing, was examined by Mullen et al. (1985) at

Ames, Iowa. Nitrogen rates and row spacing were 0, 90, and 180 kg ha-1; and 20, 60, and100 cm, respectively. The N rate at 180 kg ha-1 increased germination of pre-chilled Cave in Rock seeds but decreased germination of pre-chilled Blackwell and Pathfinder, and the results were highly variable between cultivars and years. In addition to N fertilizer, compost increased seedling emergence of switchgrass. Green compost, mixed compost, and coffee compost all increased switchgrass seedling emergence and vigor (Traversa et al., 2013). Coffee compost amendments between 50 and 200 mg L-1 increased germination of all tested cultivars except Alamo, and

Alamo germination was improved by green compost at 50 mg L-1 (Traversa et al., 2013).

Conclusion

Seed dormancy of switchgrass must be overcome for successful stand establishment.

This will be a first step to reduce establishment risk and costs associated with reseeding. Studies have been reported the effects of chemical, mechanical, thermal, and hormonal seed treatments upon switchgrass seed dormancy. Seed storage duration and conditions are associated upon switchgrass seed dormancy. Smaller seeds (e.g., lowland cultivars) typically have higher dormancy compared to larger seeds (e.g., upland cultivars); however, freshly harvested seeds

have a high level of dormancy regardless of seed size. Degree of dormancy cannot be predicted easily by cultivar since there are complex interactions between seed dormancy and environmental conditions such as soil parameters (e.g., salinity; Ries and Hofmann, 1983), micro- and macro-environments during seed production (Boe, 2009), and management practices

(e.g., row spacing; Newell, 1968a). Therefore, it is difficult to select one dormancy breaking treatment that reduces dormancy of all cultivars and seed lots of switchgrass seeds.

The effectiveness of dormancy-breaking treatments has been demonstrated on small amount of seeds in petri dishes and growth chambers. However, planting the treated seeds in pots or in the field more accurately reveals the true response of seeds following dormancy- breaking treatments because soil conditions significantly influence germination and seedling emergence of switchgrass.

II. Intercropping switchgrass with poplar trees

Introduction

Characteristics of switchgrass are associated with an origin of latitude (Gustafson et al.,

2003). Switchgrass cultivars originated from lower latitude (37° N latitude) expressed greater disease resistance (Gustafson et al., 2003), more phytomers tiller-1 (Boe and casler, 2005), and greater yield capacity (Casler and Boe, 2003; Boe and Casler, 2005) than cultivars from the northern Great Plains (Boe, 2007). Expression of morphological traits at a cultivar level was associated with biomass production because greater composition of reproductive tiller and the maximum number of phytomers tiller-1 were observed from the high yielding cultivars (Boe and

Casler, 2005). Latitude of the origin also determines timing of flowering because switchgrass is very sensitive to photoperiod (Moser and Vogel, 1995). Flowering of switchgrass is induced by a short day (Benedict, 1914 requested). Moser and Vogel (1995) reported that the adaptation of switchgrass is the most influenced by photoperiod, precipitation, and humidity. Due to the photosensitivity, vegetative stage of switchgrass can be extended if it is grown in the north of its originally evolved site (Newell, 1968a); however, it is recommended to grow switchgrass within one winter-hardiness zone from its originally adapted site because it may not tolerate winter freezing (Renz et al., 2009).

Harvesting switchgrass twice a year is a common practice in the PNW under irrigation

(Collins et al., 2010; Kimura et al., 2015), while harvest frequency varies depending on cultivars, locations, and use of the switchgrass biomass (Adler et al., 2006; Fike et al., 2006b).

Switchgrass becomes mature in four to five years after establishment and can maintain yield for

10 to 20 years under proper maintenance (Fike et al., 2006ab). It is critical to maintain 15 cm

stable heights for longevity of the stand and not to deplete carbohydrate reserves in below 15 cm of stems and roots. Nutrients removed during switchgrass harvest for bioenergy should be replaced to maintain biomass productivity and stands (Allison et al., 2012; Kering et al., 2012b;

Sadeghpout et al., 2014). Study conducted in the eastern U.S. showed that switchgrass stand produced 14 Mg DM ha-1 after 10 years (Fike et al., 2006ab). In southern WA in the Colombia

Basin, switchgrass stands have been grown since 2004 to 2009 under irrigation, where yield averaged 24.8 Mg DM ha-1 yr-1 (Kimura et al., 2015). Belowground biomass of switchgrass reached 5 Mg DM ha-1 in the top 15 cm of soil by the third year of production under irrigation

(Collins et al., 2010). Studies using PVC tubes (0.46-m diameter × 3.05-m tall) revealed that irrigated Alamo switchgrass root grew deeper (0.13 m below ground) than dryland switchgrass

(0.12 m below ground) at week 10, and irrigated switchgrass roots extended 0.5 m deeper than dryland switchgrass roots by week 30 (Mann et al., 2013). These observations indicate that irrigated switchgrass can use soil nutrients and moisture more efficiently than dryland switchgrass. Summary of aboveground productivity of switchgrass over several years and different locations within North America are presented in Table 3.

Intercropping between trees and switchgrass

In addition to the high above- and belowground biomass of switchgrass (24.8 Mg DM ha-

1 yr-1; Kimura et al., 2015), major research findings to date concludes that switchgrass has low greenhouse gas emissions (Schmer et al., 2008), produces greater net energy than oil-seeds

(Tilman et al., 2006), has high water use efficiency (Okwany et al, in progress; Wu et al., 2009), and sequesters a large amount of soil organic carbon (Collins et al., 2010). The benefits of growing switchgrass may be optimized when combining two perennial crops for bioenergy

production. The practice of ally cropping is gaining attention from forest landowners and commercial tree producers to utilize the land and resources for agricultural crops while trees are growing (Riffell et al., 2012; Susaeta et al., 2012). Intercropping of trees and crops improves N cycling (Allen et al., 2004), C sequestration (Fang et al., 2010), activity of soil microorganisms and wildlife habitat (Stainback and Alavalapati, 2004), reduction of ground water contamination

(Jose et al., 2004; Zamora et al., 2009; Bergeron et al., 2011) and early economic return (Zinkhan and Mercer, 1997; Gold et al. 2000). For the profitable intercropping between trees and switchgrass, the price of switchgrass needs to be higher than $30 Mg-1 when switchgrass is planted into ally of loblolly pine trees (Susaeta et al., 2012). Among the woody crops, hybrid poplar became a leading bioenergy woody species (Newman et al., 1997). It grows at 22 Mg ha-1 yr-1 in high yielding clone (Guo and Zhang, 2010) and is harvested after 9 to 10 years for traditional timber production (Carlson and Berger, 1998). Some studies reported positive N response of hybrid poplar by placing NPK tablets at 10 cm from tree at the depth of 10 cm belowground (Headlee et al, 2013), N fixed by alfalfa planted in ally (Sisi et al., 2012), and available soil nitrate (Fortieer et al., 2010). Increased biomass was observed as N application increased from 0 to 165 kg ha-1 yr-1 on silt loam and sandy loam soils, where amount of foliar N was proportional to increase in yield (Hansen et al., 1988). Intercropping between the fast growing poplar and switchgrass may mitigate issues with using lands for food and fiber production by maximizing land use efficiency.

Land equivalent ratio (LER) is utilized to measure the degree of land use efficiency in intercropping (Gliessman, 2007).

푌푖푒푙푑 표푓 푖푛푡푒푟푐푟표푝푝푒푑 푐푟표푝 푌푖푒푙푑 표푓 푖푛푡푒푟푐푟표푝푝푒푑 푡푟푒푒 LER = + 푌푖푒푙푑 표푓 푚표푛표푐푢푙푡푢푟푒 푐푟표푝 푌푖푒푙푑 표푓 푚표푛표푐푢푙푡푢푟푒 푡푟푒푒

The LER of greater than one indicates that total biomass produced under given area in intercropping is greater than the biomass produced in monoculture practice. The equation provides intuitive value to judge whether the crop can be utilized in the intercropping practice and yield better than monoculture cropping. Change in plant architecture between intercropping and solo cropping can be measured by a leaf area index (LAI) (Redfearn et al., 1997; Mitchell et al., 1998; Brye et al., 2002). The LAI is also related to light interception, forage productivity

(Perry et al., 2009), grazing accessibility (Mitchell et al., 1998), grassland stability (Brye et al.,

2002) and regrowth simulation (Jing et al., 2012). Burrows et al. (2002) reported that LAI and net primary productivity was positively correlated. In general, LAI in switchgrass was increased from June (1.6 m2 m-2) to August (2.7 m2 m-2), and declined in October (2.4 m2 m-2) (Albaugh et al., 2012). The reason for the declined LAI in October may be attributed to later season tiller senescence (Albaugh et al., 2012). Vertical distribution of LAI taken in switchgrass at every 20 cm up to 100 cm revealed that the top third of canopy contained more than 50 % of leaf area

(Madakadze et al., 1998). Measuring stomatal conductance also provides useful information about gas exchange as influenced by intercropping system. Switchgrass leaves are amphistomatic, meaning that the stomata are present at both side of leaf (Awada et al., 2002).

Stomatal conductance of switchgrass ranged from 0.05-0.187 mol m-2 s-1 depending on the environment, cultivars, and stresses (Awada et al., 2002; Hartman et al., 2012; O’Keefe et al.,

2013), where higher stomatal conductance is observed in summer than fall (O’Keefe et al., 2013).

Environmental stresses (e.g., drought, shading) reduces stomatal conductance (Awada et al.,

2003). For example, stomatal conductance of big bluestem (Andropogon gerardii Vitman.) and smooth bromegrass (Bromus inermis Leyss) declined when they were grown under 20 year-old green ash (Fraxinus pennsylvanica Marsh) (Awada et al., 2003). In contrast, conditions such as

flooding and N rate above 100 kg N ha-1 increase stomatal conductance (Barney et al., 2009;

Feng et al., 2012). Both lowland and upland cultivars of switchgrass increased stomatal conductance following flooding condition (0.13-0.14 mol m-2 s-1), while both cytotypes decreased stomatal conductance following drought condition (0.05-0.08 mol m-2 s-1) (Barney et al., 2009). The study conducted by Barnet et al. (2009) supported that increased water level increased stomatal conductance for switchgrass, foxtail millet (Setaria italica) and old world bluestem (Bothriochloa ischaemum) (Xu et al., 2006).

Weed management is complex in an intercropping than in a monoculture practice since there are limited registered herbicides for both trees and perennial herbaceous crops (Buhler et al., 1998). Seedling growth of switchgrass is very slow compared to annual weeds, and switchgrass seedlings are easily suppressed by vigorous weed invasion. It is critical to keep weed population low until a switchgrass stand become mature and resilient to disturbance; otherwise, establishment may fail. Integrated weed management is essential to control weeds in switchgrass grown in intercropping system (Buhler et al., 1998). In addition to the weed problem, shading may change plant architecture and decrease biomass yield (Suresh and Rao,

1999). Adverse effect of shading is higher in C4 grass species than C3 grass species due to a higher light saturation point and photosynthetic capacity of C4 species (Bjorkman, 1981). The yield reduction by the shading is attributed to the decreased photosynthetically active radiation

(PAR) necessary for normal plant growth (Suresh and Rao, 1999). In general, C4 species increase specific leaf area with response to increased shading (Murchie and Horton, 1997), while

C3 species increase chlorophyll content (Boardman, 1977). However, the increased leaf area does not compensate for decreased biomass yield of switchgrass grown under a tree canopy as compared to switchgrass grown in monoculture. Shading also changes forage quality (Buxton

and Fales, 1994). A densely shaded area has less light for plant growth, resulting in slow plant growth and maturity. Therefore, plants grown under shade may have higher forage quality than plants with saturated light sources because maturity of plant is inversely related to the forage quality (Kephart and Buxton, 1993). Lindgren and Sullivan (2013) determined a Crude Protein

(CP) value of 10.4 % in pinegrass (Calamagrostis rubescens Buckley) grown under less shade of

500 stems ha-1 of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) as compared to CP value of 12.5 % under more shade from 2000 stems ha-1 of lodgepole pine stand.

Benefits of intercropping between two perennial crops

Despite the negative influence on crop production intercropped with woody species, the intercropping between the two perennial crops (e.g., hybrid poplar and switchgrass) should be beneficial for many reasons. First, total biomass production per given area is higher in intercropping than monoculture practice; LER becomes greater than one. Herbaceous plants are able to maintain productivity for several years in tree intercropping until canopy closure. Big bluestem (C4) and smooth bromegrass (C3) planted under Scotch pine (Pinus sylvestris L.) maintained biomass productivity under partial shading (Perry et al., 2009). Switchgrass (Alamo) was planted in loblolly pine (Pinus taeda), spaced 6-m apart, in summer of 2009 to evaluate the intercropping of switchgrass and pine trees (Albaugh et al., 2012). There were no differences between switchgrass in monoculture and in intercropping plots, except that switchgrass height in the monoculture grew 16 cm taller on average than switchgrass in the intercropping plots. Yield between monoculture and intercropping averaged 2.65 Mg ha -1 in 2009 and 4.14 Mg ha-1 in 2010.

Shading had no effect on yield for the first two years of switchgrass production under pine trees planted in winter of 2008 (Albaugh et al., 2012).

High volume biomass production in the intercropping system will sequester large amount of C, reducing atmospheric CO2 (Peichl et al., 2006). Amount of C sequestered under tree and crop intercropping system ranged from 0.3 to 15.2 Mg C ha-1 yr-1 depending on tree, crop, management and environment (Nair et al., 2009). Intercropping between 13-year-old poplar and barley (Hordeum vulgare L. cv. OAC Kippen) at 111 trees ha-1 in Ontario, Canada proved that intercropping sequestered greater amount of C (96.5 Mg C ha-1) than barley solo cropping (68.5

Mg C ha-1) (Peichl et al., 2006). Estimated net C fluxes were positive value (+13.2 t C ha -1 y-1) for poplar intercropping and negative (-2.9 t C ha-1 yr-1) for barley solo cropping, indicating that the poplar intercropping successfully reduced atmospheric CO2 (Peichl et al., 2006). Amount of sequestered C varies by intercropping system and crops used in the system (Fang et al., 2010).

Three intercropping systems were compared using configurations A (250 trees ha-1), B (167 trees ha-1), and C (94 trees ha-1) with wheat-corn or wheat-soybean crop rotations (Fang et al., 2010).

The highest amount of C was sequestered in the configuration A with wheat-soybean system

(16.7 t C ha-1) and with wheat-corn system (18.9 t C ha-1) (Fang et al., 2010). The 200 trees ha-1 density of tree planting increased C sequestration presumably because of higher photosynthesis rate as compared to the 94 trees ha-1 of tree plantings. Amount of change in SOC was associated with the years of operation in intercropping (Bambrick et al., 2010). No differences were observed between SOC in four-years of intercropping system and SOC in conventional cropping system; however, the intercropping system increased SOC by 77 % after eight years compared to conventional cropping system at St. Remi, Quebec (Bambrik et al., 2010). Multispecies riparian buffers with poplar, switchgrass, or cool season grasses increased soil respiration and C sequestration as compared to buffers with annual species in Coland soil in Iowa (Tufekcioglu et al., 1999). Soil respiration rate (g C m-2 d-1) for poplar, switchgrass and cool season grass ranged

from 5 to 6 g C m-2 d-1 in August , which was higher than the soil respiration under corn and soybean (range: 3-4 g C m-2 d-1). Deeper root depth of poplar, switchgrass and a cool season grass compared to corn and soybean contributed to the higher C sequestration and soil respiration rate (Tufekcioglu et al., 1999). Poplar and switchgrass accumulated C at 2960 and 820 kg C ha -1 y-1 (Tufekcioglue et al., 2003).

Intercropping trees and crops has positive effects on soil parameters (Thevathasan and

Gordon, 2004). Trees shed leaves in fall, providing litterfall biomass near tree rows. Nitrogen accumulation caused by the litterfall was estimated to be 7 kg N ha-1 yr-1 (Thevathasan and

Gordon, 2004). The increased litterfall near poplar tree improved earthworm densities (Price and

Gordon, 1999) and microbial activity (Rivest et al., 2010). Soil microbial biomass (range: 0.23-

-1 0.92 g Cmin kg ) was 27 % higher in intercropping compared to harrowing at the end of growing season (Rivest et al., 2010). Perennial cropping system (e.g., switchgrass) enhanced the amount of microbial biomass than annual cropping system (e.g., corn) (Liang et al., 2012). Increased organic matter (OM) and soil worm densities indicate that the soil was healthy (Park et al., 1994).

Extractable-P, total N and mineralizable N were increased in intercropping plots of alternating hybrid poplar and green ash (Fraxinus pennsylvanica Marsh.) at 3-m interval and wheat. Wheat aboveground biomass was 2.0 g plant-1 in an intercropping treatment compared to 1.7 g plant-1 in a wheat monoculture plot (Rivest et al., 2013). Deep tree root help to mitigate the subsoil nitrate leaching (Bergeron et al., 2011). The 5-8 year-old poplar trees, hard wood species [black walnut

(Juglans nigra L.), and white ash (Fraxinus Americana L.)] reduced N leaching by 227 kg N ha -1 when planted in 6-m row spacing and 2-m within row spacing on clay loam soil in eastern

Canada (Bergeron et al., 2011).

Conclusion

Switchgrass is a unique and attractive long lived perennial herbaceous crop that demands cultivar- and site-specific management for a successful establishment. With proper fertility, weed, and harvesting management, switchgrass stands provide an average 14 Mg DM ha-1 yr-1 after ten years under dryland (Fike et al., 2006b) or 25 Mg DM ha-1 yr-1 after seven years under irrigation (Kimura et al., 2015). Intercropping switchgrass and woody species is a viable option for bioenergy production. In such cropping systems, LER provides an intuitive value to determine the intercropping system value (Gliessman, 2007). The LAI and stomatal conductance characterize the change in plant architecture and rate of gaseous exchange through stomata for switchgrass grown alone or under tree canopy (Barney et al., 2009; Albaugh et al., 2012). The unique intercropping systems may make management more complex than solo cropping system in terms of weed management, shading effects, and resource competition; however, the benefit obtained from the intercropping systems may compensate for the challenges. The innovative intercropping between woody species and switchgrass not only increase biomass production per given area (Albaugh et al., 2012), but also contribute to increased C sequestration (Fang et al.,

2010), enhanced soil microbial activity (Rivest et al., 2010) and reduced greenhouse gas (Peichl et al., 2006) and N leaching (Bergeron et al., 2011).

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Table 1. Summary of major cultivars of switchgrass (Modified from Seepaul et al., 2011)

Genotype Ploidy Cytotype Latitude Origin PHZ* Remarks References level Alamo T Lowland Southern TX 6 Selected for biomass Kanlow T Lowland N Wetumka, OK 5 Tusca Lowland Mississippi Selected for herbicide tolerance from Alamo Expresso Lowland Mississippi Selected for improved germination Cave in H Upland S Cave in Rock, IL 4b Riley and Vogel, 1982 Rock Shelter H Upland N St. Mary’s, WV 4 Wullschleger et al., 1996 Blackwell H Upland S Blackwell, OK 5a Riley and Vogel, 1982 Carthage O Upland Southern IL Dacotah T Upland North Dakota 4a Early maturity, winter hardy, high Barker et al., 1990 stand density, persistent Forestburg T Upland N Forestburg, SD 3b-4b Early maturity, excellent winter Barker et al., 1988 42 hardiness and persistence, good

seed potential Shawnee O Upland S Cave in Rock, IL High forage yield and quality from Vogel et al., 1996 Cave in Rock Summer T Upland Southern NE 4 Sunburst H Upland N South Dakota Winter hardy, leafy, heavy-seeded, Boe and Ross, 1998; superior seedling vigor Wullschleger et al., 1996 Trailblazer H Upland N Nebraska High forage quality, high IVDMD Vogel et al., 1991 Caddo Upland *PHZ: Plant hardiness zone

Table 2. Two seed lots with same germination percentage expressing different seed

characters (Adapted from Vogel, 2002)

Seed quality test Seed lot 2060 Seed lot 2061 Germination % 66 66 Total viable seed % 94 85 (Germinated and dormant seed %) Greenhouse pot test % 80 38 Seeds g-1 480 680 Desired seeding rate 300 PLS m-2 Greenhouse emerged seedlings g-1 seed 384 258 Bulk seeding rate kg ha-1 7.8 11.6

Table 3. Summary of best yielding switchgrass cultivar at several locations in North America

Establishment Locations Cultivar Harvest years Mg DM ha-1 Author(s) year Aboveground biomass West Paterson, WA Kanlow 2004 05/06/07/08/09 21.0/22.6/28.9/26.9/24.4 Kimura et al., 2015 (Irrigated) Middle Brookings, Shawnee 1998 98/99/00/01 4.9/6.0/4.7/4.9 Casler and Boe, 2003 SD Arlington, WI Shawnee 1998 98/99/00/01 10.2/12.0/17.3/16.4 Casler and Boe, 2003 George and Ames, IA Cave in Rock Unknown 94/95 9.3/7.8 Obermann, 1989

East Mean of three Rock Spring, cultivars 1999 01/02/03 6.7/7.0/7.0 Adler et al., 2006 PA (uplands) Knoxville, Mean of six 1992 93/94/95/96/97 15.6/22.3/16.8/23.2/15.4 Reynolds et al., 2000 TN cultivars Knoxville, West and Kincer, Alamo 2007 07/08/09/10 4.5/10.6/22.9/39.7 TN 2011 Princeton, KY Alamo 1992 99/00/01 15.2/17.0/15.0 Fike et al., 2006b Raleigh, NC Alamo 1992 99/00/01 16.0/17.0/10.0 Fike et al., 2006b

South Ardmore, OK Alamo 2008 08/09/10 6.6/15.7/16.6 Kering et al., 2012

CHAPTER THREE BREAKING DORMANCY OF SWITCHGRASS SEEDS BY FREEZE-THAW SCARIFICATION

ABSTRACT

Switchgrass (Panicum virgatum L.) has been selected as an important lignocellulosic feedstock to support bioenergy production. Established switchgrass stands can produce 25 Mg DM ha-1 of aboveground biomass under irrigation. Vigorous root systems contribute to C sequestration, reducing increasing CO2 due to the burning of fossil fuels. However, establishment of switchgrass is hindered by the high amount of dormant seeds that produce sporadic seedling emergence in the field. The objective of this study was to determine the influence of freeze-thaw and pre-chill treatments on the dormancy of two uplands and one lowland switchgrass cultivars harvested over multiple years. Seeds were subjected to freezing temperatures of -20°C or -80°C for one hour followed by one hour of thawing (one cycle of freeze-thaw) at room temperature for one to five cycles. No freeze-thaw treatments were effective on breaking seed dormancy of all seed lots used in this study. The extreme temperature alternations may have increased seed damage possibly by damaging the seed coat, oxidizing the endosperm and embryo. Wide variation of germination responses were observed among seed lots within a cultivar, harvested from the same field. The environmental differences during seed production may account for some of the variability in sample analyses. In addition, the environmental differences were

-1 reflected in the number of seeds gram . Seed lots with greater number of seeds gram-1 were susceptible to freeze-thaw treatment (Kanlow 2008 and Blackwell 2010), and seed lots with similar weights displayed similar response to seed treatments (Trailblazer 2010 and 2011). The smaller seeds may have thinner seed coats as well as undeveloped endosperm and embryos.

INTRODUCTION

Switchgrass (Panicum virgatum L.) is a perennial warm-season grass that is native to

North America (Vogel, 2004). It has been recommended by the Department of Energy (DOE) to use swichgrass as a viable lignocellulosic feedstock due to its high yield potential (Fike et al.,

2006a; Kimura et al., 2015) and vigorous root system for carbon sequestration (Collins et al,

2010). However, a high proportion of dormant seeds can hinder successful switchgrass establishment. Unlike intensively selected crop species such as alfalfa, neoteric switchgrass seeds contain high proportion of dormant seeds that reduce seedling emergence in the field.

Sporadic seedling emergence may result in weak competitive ability against weeds and failure of establishment, necessitating reseeding in the following year. The economic cost associated with reseeding has been estimated to be 36 % of the total establishment cost, which cannot be paid off by simply increasing the dry matter yield in the first year (Perrin et al., 2008). Breaking seed dormancy is the first critical step for successful stand establishment.

A myriad of studies have examined methods of breaking seed dormancy of switchgrass through chemical, mechanical, or using various extreme temperature treatments (Blake, 1935;

Jensen and Boe, 1991; Tischler et al., 1994). These approaches break seed coat dormancy and/or embryo dormancy. Sulfuric acid (16.8 mol L-1) effectively increased germination of freshly harvested Alamo (94 %), Kanlow (68 %), and Caddo (68%) switchgrass seeds compared to untreated seeds of these cultivars (52, 16, and 48%, respectively) (Tischler et al., 1994), while lower concentration (8 mol L-1) of sulfuric acid was ineffective in breaking dormancy of switchgrass (Haynes et al., 1997). The effect of sulfuric acid scarification was seed lot- and cultivar-specific (Tischler et al., 1994). Seed size differences may contribute to the specificity of germination response to the scarification. For instance, larger seeds within a cultivar express

greater germination than smaller seeds (Kneebone and Cremer, 1955; Aiken and Springer, 1995).

Sarath et al (2008) found that several seed lots harvested from the same field had different responses to reactive nitrogen species and peroxide. Germination was increased by 84 % and

85 % when switchgrass seeds were rubbed with emery cloth (Sautter, 1962) or sand paper

(Zhang and Maun, 1989). Thirty-second treatments with the scarifier ‘Forsberg Cylinder’

(Forsbergs, Inc. Thief River Falls, Minnesota, USA) improved germination of ‘Sunburst’ and

‘NDE’ switchgrass seeds by 9 % and 25 %, respectively (Jensen and Boe, 1991). Other efforts on breaking switchgrass seed dormancy include the use of karrikinolide smoke (3-methyl-2H- furo [2, 3-c] pyran-2-one; George, 2009), bacteria (Debebe, 2005) or fungal inoculation (Ghimire et al., 2009), seed priming (Debebe, 2005), electromagnetic radiation (Funk et al., 2010), or ultrasonic treatment (Wang et al., 2012). Although these scarification and induction methods have shown various degrees of successes in reducing switchgrass seed dormancy, uniformity of the treatment becomes very important when large amount of seeds need to be treated. Use of temperature regimes may be an effective alternative method to treat seeds uniformly. The

Association of Official Seed Analysts recommends a pre-chill treatment of switchgrass seeds at

5°C for 14 days to reduce dormancy (AOSA, 2010). It was suggested that pre-chill treatment stimulates seed starch conversion (Loch et al., 2004). However, pre-chill treatment is only effective under conditions where pre-chilled seeds remain wet (Shen et al., 2001).

Soil freezing and thawing in the early spring can be simulated by a freeze-thaw scarification (Rinehart, 2006). Effectiveness of freeze-thaw on breaking dormancy was reported for Fabaceae (Stout, 1990). The temperature alternation between -80°C for two hours upon thawing the seeds at 20°C at room temperature for two hours reduced dormancy of alfalfa from

60 to 14 % (Stout, 1990). This method may alter both the seed coat and embryo dormancy

because the extremes in temperature alteration from -80°C to 20°C may loosen the seed coat structure and the temperature difference may simulate soil condition in early spring. As information is scarce on freeze-thaw scarification for warm-season grasses, this investigation evaluate how freeze-thaw scarification affects seed dormancy of switchgrass seeds. The objective of this study was to induse freeze-thaw scarification on different seed lots and cultivars of switchgrass seeds and to determine if germination of switchgrass seeds improved.

MATERIALS AND METHODS

Switchgrass cultivars used in this study were Kanlow (Ernst Conservation Seed Inc.,

Meadville PA), Blackwell and Trailblazer (Sharp Bros. Seed Co. Greeley, CO). Kanlow seeds were harvested in 2008, 2010, and 2011 in Smithfield, North Carolina, while Blackwell and

Trailblazer were harvested in 2010 and 2011 in Kansas. Seed count g-1 for each seed lot and cultivar are summarized in Table 1. Kanlow seeds provided by Ernst Conservation Seed Inc. are typically harvested at 20 % seed moisture with a combine fitted with a stripper head to remove panicles in the field. Seeds are immediately dried to 10-15 % seed moisture at ambient temperature. Seeds are then cleaned and stored at 10°C at 50 % humidity (personal communication with Mr. Calvin Ernst at Ernst Conservation Seed Inc.). Blackwell and

Trailblazer seeds were stored at warehouse temperatures (personal communication with Mr.

Barnett at Sharp Bros. Seed Co.). All seeds were stored in the Washington State University Seed

Storage Room, Pullman WA (23°C) after receiving from the seed companies.

Freeze-thaw treatments consisted of two temperature treatments and five cycles. Seeds were put in a coin envelop (5.7 × 8.9 cm) and placed in freezers at -20 or -80°C for one hour followed by thawing for one hour at room temperature (23°C), resulting in one cycle of freeze- thaw treatment (one hour for freezing and one hour for thawing). There were five cycles in each

treatment temperature from zero, one, two, three, four, and five cycles. Seeds at zero cycle were stored at -20°C or -80°C for one day without temperature alteration. Each cycle of freeze-thaw treated seeds were compared with seeds stored at room temperature (23°C) and seeds subjected to pre-chill treatments (PC). Pre-chill procedure followed the AOSA (2010) pre-chill procedure, in which seeds were soaked in distilled water over night and kept at 5°C for two weeks. These two controls were used in germination tests at every cycle from zero to six for paired comparisons.

Germination tests were conducted in a petri dish (15 × 20 mm) with two layers of blotter paper (Manufac) moistened with 20 ml of distilled water. Blotter papers were moistened with additional 5 ml of distilled water when necessary. A growth chamber (Manufac) was set for alternating day/night temperatures of 30/15°C and light/dark periods of 8/16 hours (AOSA,

2010), which were consistent throughout all germination tests. Numbers of germinating seeds were counted on the 14th day. Seed moisture content was determined by weighing fresh and dry weight. Dry weight was obtained by drying seeds at 90°C in an oven for 4 hours until no change in weight was measured. The study was a completely randomized design with four replications.

One replicate consisted of 100 seeds. The study was repeated three times, described as Trial 1,

Trial 2, and Trial 3.

Analysis of variance was conducted for a complete randomized design with four replications using the GLM procedure in SAS 9.2 (SAS Institue, Cary, NC) to determine the interaction among the three trials. Due to the significant interaction observed among the trials, analysis of covariance was used to increase precision of the model influenced by after-ripening periods which increased germination over the three trials. Main effects (cycles and treatment temperatures) and seed lot interaction was determined, using the adjusted germination percentage.

Adjusted germination was reported as percentage basis. Orthogonal contrast was used to determine relationship between cultivars and seed lots. Mean separation was conducted among cycles and among treatment temperatures with Tukey’s analyses when F tests were statistically significant (P=0.05).

RESULTS AND DISCUSSION

Effects of freeze-thaw and pre-chill treatments

The germination response of switchgrass seed lots to freeze-thaw treatment widely varied among treatments temperatures and cycles shown by a significant seed lot × temperature and seed lot × cycle interactions for Kanlow and Blackwell, among which a strong temperature effect was observed for the three cultivars. Therefore, means were separated by seed lots and temperatures and pooled over cycles (Figure 1). Germination percentages of seed lot by temperature by cycle for each cultivar are summarized in Appendix A-C. Orthogonal contrast showed germination a significant response to freeze-thaw treatment and was different between upland and lowland cultivars. The difference was found between Kanlow and Blackwell (p <

0.0001) with no significant difference observed between Kanlow and Trailblazer (Table 1).

Kanlow and Trailblazer share similar traits in field, such as late maturing, vigorous, leafy, and are productive cultivars (Vogel et al., 1991). This could be the reason why there was no difference in response to freeze-thaw treatment between Kanlow and Trailblazer.

Freeze-thaw treatment at -80°C or -20°C among cultivars and seed lots did not improve germination. No differences in seed moisture were found among treatments and cultivars

(averaging 7.2 %). Germination of Kanlow 2008 and Blackwell 2010 decreased with the freeze- thaw treatment by 5 % and 1 % at -80°C and 7 % and 1 % at -20°C as compared to seeds stored at room temperature, respectively (Figure 1). On the other hand, all treatments were not different

compared to the control of Kanlow 2010 (79 %) and Kanlow 2011 (29%). Duclos et al. (2013) reported that the pericarp and testa were the primary structures for seed coat dormancy of switchgrass. Oxygen exchange, rather than water exchange, through the pericarp increases germination of switchgrass seeds (Duclos et al., 2013). The altermate freezing (one hour) and thawing (one hour) repeated for up to five cycles may have increased seed damage possibly by damaging the seed coat, oxidizing endosperm and embryo. The seed lots damaged by the freeze- thaw cycle may contain thinner pericarps than other seed lots. Seed lots from long term storage may be sensitive to alternation of freezing and thawing as Kanlow 2008 is the oldest seed lot among all seed lots used in this study.

Pre-chill treatment was reported to be on effective treatment to break seed dormancy of switchgrass when seeds were tested without drying (Shen et al., 2001); however, three upland seed lots (Blackwell 2010, Trailblazer 2010 and 2011) showed decreased germination by 3 %,

6 %, 7 % following the pre-chill treatment as compared to seeds stored at room temperature, respectively (Figure 1). In contrast, germination of two seed lots (Kanlow 2008 and Blackwell

2011) was improved with the pre-chill treatment (Figure 1). It is unclear why pre-chill treatment was ineffective in breaking seed dormancy of seed lots used in this study as positive effects of pre-chill treatment have been observed in other studies (Beckman et al., 1993; Zarnstroff et al.,

1994; Grabowski et al., 2002).

Variation within seed lots

Seed lots within each cultivar were harvested from the same field at different years; however, germination percentages of each seed lot stored at room temperature varied widely. As an example, germination of Kanlow was 34.6 %, 80.7 %, and 29 % for seed lot 2008, 2010, and

2011 when stored at room temperature, respectively (Table 2). The wide range of germination percentage was attributed to environmental differences during seed production (Sarath et al.,

2008; Germain and Filbert, 2014), seed age (Shaidaee et al., 1969), and storage conditions (Shen et al., 1997; Shen et al., 1999; Boe, 2009). This may explain the significant seed lot interaction observed for the three cultivars of switchgrass (p < 0.0001) (Table 1). As proposed by Sarath et al (2008), the genetic background may not be the only factor controlling dormancy, and more importantly micro-environment can contribute to the degree of dormancy, although some species, such as Italian rye-grass (Lolium multiflorum Lam.) and meadow brome (Bromus commutatus

Schrad.) receive less environmental effects of production in seed dormancy (Cook and Reverte,

2011). Germain and Gilbert (2014) reported that maternal environment causes significant changes in amount of dormant seed produced in later generations. Germination variations among seed lots observed in this study confirmed that maternal environment may have affected the level of dormancy produced in switchgrass seeds. The environmental variation during seed production may be related to seed size, which is an important factor for seed dormancy and germination (Green and Hansen, 1969; Aiken and Springer, 1995; Smart and Moser, 1999).

Also, the number of seeds gram-1 of switchgrass varied depending on the year the seed was produced and among cultivars (Table 2). In addition, seeds damaged by freeze-thaw scarification (Kanlow 2008 and Blackwell 2010) contained more seeds gram-1 as compared to other seed lots of the same cultivar. For example, the number of seeds gram-1 for Kanlow 2008 and Blackwell 2010 contained 600 and 230 seeds more seeds than the other seed lots of Kanlow and Blackwell, respectively, indicating that these seed lots may contain seeds with undeveloped embryo/endosperm or thinner seed coat structures. Such seeds may be more susceptible to extreme temperature treatments than seed lots with less seeds gram-1. As a result, little variation

in response to seed treatments was observed between the two seed lots of Trailblazer. It may be because that the two seed lots were similar in seed size and seed coat structures as number of seeds gram-1 was same (787 seeds g-1; Table 2).

CONCLUSION

The effect of freeze-thaw treatment varied widely over seed lots and cultivars used in this study. No freeze-thaw treatment was effective in breaking seed dormancy of seven seed lots used in this study. The freeze-thaw and pre-chill treatments decreased germination of five seed lots, except for Kanlow 2010 and Kanlow 2011, as compared to seeds stored at room temperature. The wide variation of germination responses to seed treatment among seed lots within the same cultivar may be attributed to environmental differences during seed production, seed age, and storage condition. In addition, the maternal environment during seed production may be reflected by the number of seeds gram-1 as observed in our study. Seed lots with greater number of seeds gram-1 were susceptible to freeze-thaw treatment, and seed lots with same weight (Trailblazer 2010 and 2011) displayed similar response to seed treatments. As breeding efforts proceed to reduce seed dormancy of switchgrass cultivars, the variation in germination among seed lots may also decrease.

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Table 1. Seed weight g-1 of seed lots for Kanlow, Blackwell, and Trailblazer

Cultivar Seed lot Seed count g-1 Kanlow 2008 1818 2010 1235 2011 1149

Blackwell 2010 917 2011 578

Trailblazer 2010 787 2011 787

Table 2. Analysis of covariance and mean squares for adjusted germination percentage of freeze-

thaw treatment in response to seed lots [Kanlow (2008, 2009, and 2010), Blackwell (2010 and

2011) and Trailblazer (2010 and 2011) of switchgrass], treatment (-80ºC, -20ºC, Pre-chill, and 23

ºC), cycles (0-5 cycles), and two and three ways interactions

Seed lot All seed lots Kanlow Blackwell Trailblazer Seed lot (S) 53534*** 63656*** 50450*** 1057* Treatment (T) 120* 1963*** 188* 388* Cycle (C) 242** 155* 62 259* S×T 236*** 1238*** 530** 18 S×C 82** 99** 61* 26 T×C 48 27 80*** 48 S×T×C 50*** 45 57** 44

Orthogonal contrasts Upland vs. lowland 7418*** Kanlow vs. Blackwell 18003*** Kanlow vs. Trailblazer 181 Blackwell vs. Trailblazer 12778*** ∗, ∗∗, ∗∗∗ Significant at P<0.1, P < 0.05, P < 0.001 and P < 0.0001, respectively

Figure 1. Germination percentage of Kanlow (seed lot 2008), Blackwell (seed lots 2010 and

2011), and Trailblazer (seed lots 2010 and 2011) influenced by freeze-thaw scarification at -80°C,

-20°C, Prechill treatment, and 23°C. Values followed by the same letters are not significantly different at p < 0.05. Insignificant results were obtained for Kanlow 2010 (avg. 79 %) and

Kanlow 2011 (avg. 29 %).

APPENDICES

Appendix A. Germination percentage of three Kanlow seed lots by freeze-thaw

scarification with alternation of -80°C for one hour and 23°C for one hour (-80/23) or -

20°C for one hour and 23°C for one hour (-20/23) compared to seed germination stored at

room temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles

Cycles -80/23°C -20/23°C PC 23°C LSD ------Germination %------2008 0 36.1 33.0 A‡ 36.4 45.2 A NS 1 31.4 34.0 A 38.6 32.6 BC NS 2 30.2 26.6 AB 38.6 36.2 AB NS 3 26.8 b† 24.0 Bb 36.5 a 24.0 Cb 8.5 4 24.9 24.7 B 34.7 30.7 BC NS 5 30.3 bc 23.4 Bc 33.8 ab 38.9 ABa 7.3 LSD NS 7.7 NS 10.1 Mean§ 29.9 b 27.6 b 36.4 a 34.6 a 3.9 Grand mean¶ 32.1 2010 0 78.8 82.8 75.8 75.5 NS 1 74.3 80.0 74.0 79.5 NS 2 80.0 78.3 79.0 85.5 NS 3 81.0 78.5 77.5 82.5 NS 4 78.5 71.0 78.8 78.3 NS 5 80.8 79.3 76.0 83.0 NS LSD NS NS NS NS Mean 78.9 78.3 76.8 80.7 NS Grand mean 78.7 2011 0 33.3 30.0 29.5 30.8 NS 1 29.3 24.7 29.8 25.0 NS 2 27.3 32.3 30.3 32.8 NS 3 26.8 27.2 30.3 26.3 NS 4 33.5 29.0 25.0 31.8 NS 5 30.8 27.3 32.5 27.5 NS LSD NS NS NS NS Mean 30.1 28.4 29.5 29.0 NS Grand mean 29.3 † Values within a row within cycle followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths. ¶ Values are averaged over all treatment for a seed lot.

Appendix B. Germination percentage of two Blackwell seed lots by freeze-thaw scarification with alternation of -80°C for one hour and 23°C for one hour (-80/23) or -20°C for one hour and

23°C for one hour (-20/23) compared to seed germination stored at room temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles

Cycles -80/23°C -20/23°C PC 23°C LSD Germ % 2010 0 98.4 96.8 94.7 97.4 NS 1 95.5 93.7 95.0 97.8 NS 2 93.5 94.2 94.8 96.8 NS 3 94.9 93.9 92.1 97.3 NS 4 96.7 a† 97.6 a 94.2 b 97.6 a 2.4 5 97.1 a 98.2 a 92.8 b 97.3a 3.7 LSD NS NS NS NS Mean§ 96.0 ab 95.7 b 93.9 c 97.4 a 1.6 Grand mean¶ 95.8 2011 0 69.5 AB‡a 54.5 Bb 75.0 a 56.9 Bb 9.2 1 60.9 Bb 59.3 BCb 74.6 a 58.9 BCb 6.9 2 61.8 ABb 62.7 ABb 71.3 a 47.4 Dc 8.5 3 64.8 ABb 62.0 ABCb 70.0 a 64.1 Ab 5.2 4 50.3 Cb 59.6 BCab 68.0 a 61.6 ABCa 10.3 5 70.8 A 67.8 A 65.8 62.5 AB NS LSD 9.9 8.1 NS 4.8 Mean 63.0 b 61.0bc 70.8 a 58.6 c 4.0 Grand mean 63.4 † Values within a row within cycle followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths. ¶ Values are averaged over all treatment for a seed lot.

Appendix C. Germination percentage of two Trailblazer seed lots by freeze-thaw scarification with alternation of -80°C for one hour and 23°C for one hour (-80/23) or -20°C for one hour and

23°C for one hour (-20/23) compared to seed germination stored at room temperature (23°C) or by pre-chill (PC) for 0, 1, 2, 3, 4, and 5 cycles

Cycles -80/23°C -20/23°C PC 23°C LSD Germ % 2010 0 54.9 D‡ 58.5 58.7 58.3 NS 1 63.8 AB 62.0 56.9 60.4 NS 2 68.1 A 60.5 56.9 65.8 NS 3 55.5 DC 57.3 55.3 59.8 NS 4 58.5 BCDa† 59.3 a 49.6 b 58.9 a NS 5 61.9 ABCab 59.1 ab 55.2 b 64.2 a NS LSD 6.5 NS NS NS Mean§ 60.4 a 59.5 a 55.4 b 61.2 a 3.2 Grand mean¶ 59.1 2011 0 60.5 67.0 59.0 70.9 NS 1 59.9 67.8 62.9 63.8 NS 2 74.1a 70.2 a 57.3 Bb 71.9 Aa 7.0 3 63.3 61.3 55.1 64.6 NS 4 59.0 58.8 57.5 61.1 NS 5 69.9 70.5 63.1 62.7 NS LSD NS NS NS NS Mean 64.4 a 65.9 a 59.1 b 65.8 a 4.3 Grand mean 63.8 † Values within a row within cycle followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths. ¶ Values are averaged over all treatment for a seed lot.

CHAPTER FOUR

SEEDLING VIGOR OF SWTICHGRASS INFLUENED BY COLD STORAGE TEMPERATURES AND DURATIONS

ABSTRACT

Switchgrass is a perennial warm season grass that produces high amount of above and below ground biomass for many years once the stand is established successfully. However, stand establishment can be difficult and may result in failure due to low seedling vigor. Switchgrass seedlings need to compete against vigorous annual and perennial weeds. Seed treatments that enhance seedling vigor of switchgrass are desired for successful stand establishment. The objective of this study was to quantify the effect of freezing storage at -20°C or at -80°C for five storage lengths to enhance seedling vigor of three cultivars and seven seed lots of switchgrass.

We found that storage temperature and lengths of storage influenced germination, and rate of seedling emergence. Storage temperature, regardless of the storage lengths, influenced shoot and root DM. Finally, storage length influenced seedling morphology, such as leaf length, width, and internode length. Storage temperature at -20°C enhanced seedling vigor of Kanlow seed lots used in this study with no seedlings damaged by freezing storage. Storage condition had effect on seedling vigor of lowland cultivar (Kanlow) than upland cultivars (Blackwell and Trailblazer), while the most recent seed lot (2011) received little influence on seedling vigor than older seed lots (2008 and 2010). We observed effect of freeze storage varied among cultivars and seed lots; however, storage temperature at -20°C increased germination percentage, seedling emergence speed, total shoot and root DM; therefore, increasing seedling vigor of switchgrass.

INTRODUCTION

Thick seed coats preventing radicle emergence are often weakened over time by temperature fluctuations, fungal attack, fire, after-ripening, or enzymes produced within the seed both in natural and controlled environments (Adkins et al., 2002). However, when the storage periods become too long, seed viability decreases as a result of peroxidation of polyunsaturated fatty acids, and damage to cell membranes and DNA (Bewley and Black, 1994). Such seed deterioration was shown to accelerate at high humidity (above 55 g kg-1) and high temperature

(60ºC) (Shen et al., 1997; Shen et al., 1999), while freezing temperature slows down seed damage druing long term storage (Rincker, 1983; Suszka et al., 2014). Germination percentage of wheat under low temperature (4ºC) and low moisture level (15 %) following 104 days storage decreased at a slower rate than at storage conditions at high temperature (40ºC) and high moisture level (24 %) (Al-Yahya, 2001). Kleingrass seeds (Panicum coloratum L.), a warm season perennial grass, stored at -20°C retained seed dormancy after six months storage (Tischler and Young, 1983). Sorghum seeds [Sorghum bicolor (L.) Moench.], warm season annual grass, maintained above 90 % germination percentage when seeds were stored for nine months at -20°C, while germination was reduced for the seeds stored at ambient temperature (25°C) (Owolade et al., 2011). Freezing temperatures are, therefore, used to preserve seeds for long term storage, such as for germplasm preservation at -10ºC to -20ºC (Sachs, 2009; Suszka et al., 2014).

A myriad of studies examined whether freezing storage could break seed dormancy of switchgrass (Panicum virgatum L.), a warm season perennial grass utilized for bioenergy production as well as C sequestration to reduce atmospheric carbon (McLaughlin and Kszos,

2005). However, results have been unsuccessful. Switchgrass seeds stored at 0°C, -8°C, -20°C, or -23°C showed no effect on germination of upland as well as lowland cultivars of switchgrass

(Sautter, 1962; Byers, 1973; Zarnstorff et al., 1994; Burson et al., 2009). On the other hand, storage temperature above 0°C increased germination of switchgrass. Studies reported that germination was increased when seeds were stored at 4°C (Sautter, 1962; Oliver, 2006) or 23-

25°C (Robocker et al., 1953; Zarnstorff et al., 1994; Grabowski et al., 2002; Oliver, 2006).

Previous studies have mainly considered germination percentage in a petri dish followed by various storage temperatures and durations; however, a few studies reported the relationship between freezing treatment and seedling emergence of switchgrass in soil. A 1935 study reported that planting frozen switchgrass seeds in the field increased seedling emergence for three successive years as compared to unfrozen seeds (Black, 1935). This observation was also made for other species of warm season perennials [big bluestem (Andropogon furcatus), blue grama (Bouteloua gracilis), Indiangrass (Sorghastrum nutans), little bluestem (Andropogon scoparius), and prairie cordgrass (Spartina michauxiana), and prairie threeawn (Aristida oligantha)]; and cool season perennials [Westeren wheatgrass (Agropyron smithii)], and forbs

[Western yarrow (Achillea occidentalis), leadplant (Amorpha canescens), lespedeza (Lespedeza capitata), and devil’s bite (Liatris scariosa)] (Blake, 1935). Although freezing storage may have little influence on germination undery laboratory conditions, temperature and length of freezing storage may be different for seedling vigor and seedling emergence in soil as reported by Black

(1935). No substantiating literature is available that describes the detailed relationship between freezing storage and seedling vigor of switchgrass. The objective of this study was to quantify the effect of freezing storage at -20°C or -80°C for five storage periods of one, two, three, six, and eight months on seedling emergence and vigor of three cultivars and seven seed lots of switchgrass under greenhouse conditions.

MATERIALS AND METHODS

Switchgrass cultivars used for this study were Kanlow (1526 seeds g-1) (Ernst

Conservation Seed Inc., Meadville PA), Blackwell (748 seeds g-1), and Trailblazer (787 seeds g-

1) (Sharp Bros. Seed Co. Greeley, CO). Kanlow was harvested in 2008, 2010, and 2011 in

Smithfield, North Carolina, named Kanlow 2008, Kanlow 2010, and Kanlow 2011. Blackwell and Trailblazer were harvested in 2010 and 2011 in, Kansas, named Blackwell 2010, Blackwell

2011. Trailblazer 2010, and Trailblazer 2011. Kanlow seeds provided by Ernst Conservation

Seed Inc. are typically harvested at 20 % seed moisture with a combine fitted with a stripper head to remove panicles in the field. Seeds are immediately dried down to 10-15 % seed moisture at ambient temperature. Seeds are then cleaned and stored at 10°C at 50 % humidity

(personal communication with Mr. Calvin Ernst at Ernst Conservation Seed Inc.). Blackwell and

Trailblazer seeds were stored under warehouse condition (personal communication with Mr.

Barnett at Sharp Bros. Seed Co.). After receipt, all seeds were stored in the Washington State

University Seed Storage Room, Pullman WA at 23°C.

Seeds from each cultivar and harvesting year were placed in -20°C, -80°C, or room temperature (23°C) starting in July, 2013 at Washington State University, Pullman WA. Seeds were stored for one, two, three, six, and eight months. Evaluation of seed moisture recommended by ISTA (1999) is to dry 5 g of samples at 130-133°C for one hour; however, seed moisture for this study was estimated by drying three replicates of 5 g of seed at 90°C for four hours. Twenty-five seeds from each treatment were planted 1cm deep in 14 cm × 15 cm pots filled with Professional Growing Mix (ingredients: 70-80% Canadian Sphagnum peat moss, horticultural grade perlite, and dolomite limestone, Sun Gro Hortiulture, Agawam, MA, USA. ).

Pots were placed in a greenhouse with a photoperiod of 16 hours, and day and night time

temperatures were 20-22°C and 14-17°C, respectively. The study was laid out as completely randomized design with four replications. Numbers of newly emerged seedlings were recorded for 14 days after the begning of seedling emergence. An emergence rate index (ERI) was calculated based on the newly emerged seedlings, using the following formula (Maguire, 1962).

Numbers of newly emerged seedling day 1 Numbers of newly emerged seedling day 10 ERI = + ···+ 1 day after seedling emerged 14 days after seedling emerged

High ERI scores indicate rapid seedling emergence. The seedlings were thinned to the largest 12 seedlings on day 15 after initiated seedling emergence with each quarter section of a pot having three seedlings to aid data collection. Measurements were made for each seedling (12 seedlings per pot and 48 seedlings pre replication) and included seedling height, length to the first node, internode lengths, leaf blade lengths and mid-leaf widths, and vigor score. Data were recorded every ten days until formation of four fully collared leaves. Seedling vigor was visually scored using a scale of one to nine; nine being the most vigorous and one being the least vigorous or weak seedling. Number of newly emerged tillers were counted for each seedling before harvesting. Seedlings in each pot quarter were clipped separately at the soil level to obtain shoot biomass, and roots were collected to obtain belowground biomass. Biomass was dried in an air-forced dryer at 55°C for at least 48 hours. Based on the dried sample, total shoot and root dry matter (DM) pot-1 and shoot and root DM plant-1 were estimated.

Statistical Analyses were conducted by analysis of variance using the procedures of SAS

(2009). Least significance differences (LSD) and paired t tests were used to separate means when

F tests were statistically significant (P = 0.01).

RESULTS

Kanlow

Stroing Kanlow 2008 seeds at -20°C increased seedling vigor (Photo 1). ERI was the highest for the seeds stored one month at -20°C (ERI 37) compared to seeds stored at -80°C (ERI

17) or room temperature (ERI 10) (Table 1). This indicates that seeds emerge faster from soil when seeds were stored at -20°C for one month than -80°C or room temperature storage. The -

20°C seed storage condition also increased germination percentage (51 %) (Table 1), total shoot

(2.6 g) and root (1.1 g) DM than seeds stored at -80°C (25 %, 1.4 g, and 0.5 g) or room temperature (23 %, 1.0 g, and 0.5 g), respectively (Figure 1). The effect of -20°C storage diminished as storage length increased from one to eight months for ERI (12), germination

(18 %), total shoot (1.0 g), and root DM (0.4 g) (Table 1 and Figure 1); however, -20°C storage averaged over storage lengths showed higher ERI and germination percentage than -80°C and room temperature. Seedling elongation rate showed no response to storage temperature and storage lengths for Kanlow 2008 (Table 1). Numbers of newly emerged tillers were in the range of 0.1-1.2 (Table 1). Storage temperature at -80°C stimulated the formation of new tiller among the three temperature treatments. Shoot DM plant-1 was 0.15 g higher from room temperature storage than freezing treatments (-20°C and -80°C), while root DM plant-1 was higher for freezing storage than room temperature in one month and eight months seed storage periods

(Figure 1). Leaf lengths and width showed little response to storage temperature for three seed lots of Kanlow; therefore, the data were pooled over three temperature treatments (Figure 2).

Leaf lengths and widths among the three seed lots averaged 7 cm and 0.2 mm, 11 cm and 0.5 mm, 20 cm and 0.6 mm, and 34 cm and 0.8 mm for leaf 1, leaf 2, leaf 3, and leaf 4, respectively

(Figure 2). Three months and six months storage enhanced leaf length. No treatment effects were observed for internode lengths; therefore, values were pooled over treatments (Figure 3).

Lengths from soil surface to the first node were 2.5 cm, 2.8 cm, and 2.7 cm for Kanlow 2008,

Kanlow 2010, and Kanlow 2011, respectively. Kanlow 2010 showed the longest first (3.6 cm) and second internodes (6.4 cm) compared to Kanlow 2008 (3.3 cm and 5.4 cm) and Kanlow

2011 (3.3 cm and 5.9 cm); however, the third internode was the longest for Kanlow 2011 (8.8 cm) than other two seed lots (Figure 3). Faster seedling emergence and higher germination percentage for seeds stored at -20°C accounted for vigorous seedling growth by the time seedlings produced four fully collared leaves for Kanlow 2008.

Seedling vigor of Kanlow 2010 was enhanced at room temperature storage or freezing storage for eight months (Photo 1). ERI was the highest at room temperature (ERI 43) than -

20°C (ERI 20) or -80°C (ERI 30), following one month of storage; however, ERI was increased as storage length increased to eight months at -20°C (ERI 50) and -80°C (ERI 51) (Table 1).

Seedling emergence rate showed little response to room temperature storage regardless of duration. One month storage at room temperature showed the highest germination (58 %) compared to one month storage at -20°C (27 %) or at -80°C (37 %) (Table 1). However, freezing treatment increased the germination percentage to 68 % at -20°C and 69 % at -80°C after eight months of storage. Elongation rate increased from two month storage (average: 1.6 cm d-1) to eight month storage (average: 2.0 cm d-1). New tiller emergence for Kanlow 2010 was in range of 0.2-0.6 plant-1 (Table 1). Total shoot and root DM were increased from one month

(average: 3.2 g and 1.2 g) to eight months storage (average: 4.4 g and 1.9 g) (Figure 1). No treatment effects were observed for shoot DM plant-1. Root DM plant-1 for seeds stored at room temperature increased at eight month storage, while root DM plant-1 for seeds stored at -20°C was the highest at one month storage. Seedling vigor of Kanlow 2010 seed was increased under

freezing condition as storage length increased or under room temperature storage regardless of the storage length.

Seed storage at -20°C showed increased seedling vigor for Kanlow 2011 (photo 1). No treatment effects were found for ERI (average: 11), new tiller (average: 0.6), shoot DM plant-1

(average:0.4 g DM plant-1), and root DM plant-1 (average:0.14 g DM plant-1) (Table 1 and Figure

1). Germination percentage was the highest at -20°C storage for one month (33 %) compared to room temperature (23 %) or -80°C storage (16 %) (Table 1). Aaverage germination percentage over storage lengths was the highest at -20°C. Total root DM of Kanlow 2011 increased at room temperature following three months of storage (1.5 g), after which total root DM decreased toward eight months storage (0.5 g) (Figure 1). No storage length effects were observed on total root DM for seeds stored at -20°C (average: 1.0 g) and -80°C (average: 0.7 g). Seedling elongation rate received no treatment effects (range: 1.6-2.0 cm d-1) (Table 1). Kanlow 2011 showed minor responses to the storage treatments compared to Kanlow 2008 and Kanlow 2010; however, storage temperature at -20°C increased germination percentage, which resulted in numerically higher values for total shoot (2.3 g) and root DM (1.0 g) than seeds stored at room temperature (2.0 g and 0.8 g) and -80°C storage (1.9 g and 0.7 g) (Figure 1).

Effects of storage temperatures and storage length varied widely among three seed lots of

Kanlow. One month storage at -20°C, longer freezing condition (e.g. eight months), and storage temperature at -20°C enhanced seedling vigor of Kanlow 2008, Kanlow 2010, and Kanlow 2011, respectively.

Blackwell

Seedling emergence of Blackwell 2010 seed was faster after room temperature storage

(ERI: 71) than both freezing storage (average: 62) treatments when the seeds were stored for two

months. After the two months storage, no treatment effect was observed for ERI (average: 66) until after eight months of storage (Table 2). Germination percentage of Blackwell 2010 seed was highest at room temperature storage at two months (92 %) than freezing storage for two months (79 %); however, germination percentage was increased following eight months of storage at -80°C (90 %) as compared to -20°C (71 %) or room temperature (86 %) at eight months storage (Table 2). Despite the reduction in germination at -20°C storage for eight months, total root (3.3 g) and total shoot DW (6.3 g) were higher at -20°C due to the higher individual shoot and root DM for -20°C storage as compared to -80°C (2.1 g and 5.1 g) or room temperature storage (2.9 g and 5.9 g) (Figure 1; Photo 2). Number of new tillers was in range of

0.5-0.9 plant-1. Elongation rate for the seeds stored at -20°C was declined from one month storage (2.0 cm d-1) to eight month storage (1.8 cm d-1). No treatment effects were observed for length and width of leaves for both seed lots of Blackwell; therefore, values were averaged over treatments (Figure 2). Leaf length and width among the two seed lots averaged 9 cm and 0.3 mm,

13 cm and 0.5 mm, 23 cm and 0.6 mm, and 35 cm and 0.7 mm for leaf 1, leaf 2, leaf 3, and leaf 4, respectively (Figure 2). No temperature effect was observed for internode lengths; therefore values were averaged over three temperature treatments. Internode lengths were longer when seeds were stored for 6 months for Blackwell 2010, while one, two, and three months storages showed longer internodes than six and eight months storage for Blackwell 2011(Figure 3).

Storage temperature at -20°C may enhance seedling vigor of Blackwell 2010 based on increased total root and shoot DM caused by increased individual shoot and root weights. Eight months seed storage at -20°C enhanced seedling vigor of Blackwell 2010 as compared to less than eight months storage.

Seedling emergence was the fastest for Blackwell 2011 seeds when stored at room temperature for two months (ERI: 54) compared to -20°C (ERI: 23) and -80°C (ERI: 34) (Table

2). After the two months, no treatment effect was observed for ERI. Germination percentage peaked at three months storage at -80°C (64 %), while no improvement in germination was observed over storage lengths at room temperature (average: 47 %) or at -20°C (45 %) (Table 2).

Root DM plant-1 was higher at -80°C (average: 0.23 g DM plant-1) compared to room temperature (average: 0.20 g DM plant-1) and -20°C (average: 0.18 g DM plant-1) (Figure 2).

Elongation rate was the highest at -80°C for one month storage (2.0 cm d-1) than room temperature (1.8 cm d-1) or -20°C (1.9 cm d-1) (Table 2). Elongation rate slowed down after two months storage to eight month storage (average: 1.6 cm d-1). Newly emerged tiller for Blackwell

2011 over treatments averaged 0.8 (Table 2). Storage temperature at -80°C enhanced seedling vigor based on increased germination percentage, root DM plant-1, and elongation rate (Photo 2).

Freezing seeds at -20°C increased seedling vigor of Blackwell 2010, while seedling vigor of Blackwell 2011 was enhanced when seeds were stored at -80°C. As observed in Kanlow 2011, younger seed lot (2011) showed little response to storage temperatures and storage lengths.

Trailblazer

Seedling emergence of Trailblazer 2010 seed was the fastest at -80°C for six months storage (ERI: 43) compared to room temperature (ERI: 33) or -20°C (ERI: 34) for six months storage (Table 3). Germination percentage for six months storage was higher for -80°C (57 %) than room temperature (49 %) or -20°C (46 %) (Table 3; Photo 3). Seedling elongation rate was higher at both freezing treatments (average: 2.0 cm d-1) compared to room temperature storage

(1.9 cm d-1) (Table 3). New tiller number was highest for seed in eight months storage at room temperature (1.3 tillers plant-1) and three months storage at -80°C (1.6 tillers plant-1) (Table 3).

New tiller formation over storage length at -20°C averaged 1.0 tiller plant-1. Total shoot and root

DM were high at six months storage for -80°C (7.4 g and 3.5 g), while eight months storage enhanced shoot (7.3 g) and root DM (4.1 g) for room temperature storage (Figure 1). Total shoot and root DM averaging over storage length were 6.8 g and 2.9 g for -20°C storage, respectively.

Leaf length and width among the two seed lots averaged 7 cm and 0.3 mm, 11 cm and 0.5 mm,

20 cm and 0.6 mm, and 33 cm and 0.7 mm for leaf 1, leaf 2, leaf 3, and leaf 4, respectively

(Figure 2). No treatment effects were observed for internode length; therefore, values were reported as averages over all treatments (Figure 3). For Trailblazer 2010, -80°C and room temperature storage increased seedling vigor through inceasing rate of seedling emergence, elongation rate, total shoot and root DM.

No treatment effect was observed for ERI for Trailblazer 2011 (average: 41) (Table 3).

Germination percentage was the highest at -20°C for eight months storage (74 %) compared to room temperature (42 %) and -80°C (53 %). Despite the higher germination percentage for the seeds stored at -20°C, no temperature effect was observed for total shoot DM (average: 6.5 g)

(Figure 1). It may be due to smaller shoot DM plant-1 for -20°C (0.53 g DM plant-1) than -80°C

(0.59 g DM plant-1) (Figure 1). Seedling elongation rate was the fastest at room temperature for eight months (2.0 cm d-1) than the freezing storage (1.7 cm d-1) (Table 2). Formation of new tillers averaged over treatments was 1.0 tiller plant-1 (Table 3). For Trailblazer 2011, it seemed less treatment effects were observed as compared to Trailblazer 2010. Increasing germination by

-20°C for eight months storage may help to increase seedling vigor for this seed lot (Photo 3).

Seedling vigor of Trailblazer 2010 was increased when seeds were stored at room temperature or -80°C for up to six months. Although less treatment effects were observed for

Trailblazer 2011, germination percentage was increased by storage condition at -20°C.

DISCUSSION

Seed storage duration and temperature are both important factors that affect the germination percentage and rate of seedling emergence, while storage temperature affects total plant DM and DM plant-1 rather than storage length for the three cultivars of switchgrass. The effect of storage condition on these parameters was higher for Kanlow than the two upland cultivars used in this study. Seed size differs between the two ecotypes of switchgrass. Kanlow seeds counts are twice the seeds gram-1 (1526 seeds g-1) of the upland cultivars (768 seeds g-1).

Small seeded switchgrass contain less energy for germination and reproduce late in the growing season (lowland ecotype), while larger seeded switchgrass contain more energy for germination and reproduce early in the growing season (upland ecotype) (Lit). Zhang amd Maun (1991) reported that the expression of diffrences from seed size continues up to 10 weeks after emergence in greenhouse experiments. In our study, the time of seedling emergence to seedling harvesting was within six weeks, indicating that effect of freezing storage also continued for 10 weeks and was similar to the findings of Zhang and Maun (1991). Environmental stresses may transmit to small Kanlow seeds easily through seed coat to endosperm, where starch is stored for germination. In addition to the seed size, origin of seeds may create ecotypic differences in response to freezing storage. Kanlow and Blackwell may be more sensitive to cold temperature than Trailblazer because former is originated from Oklahoma and latter is from Nebraska

(Seepaul et al., 2011).

Under favorable condition for seed germination, water imbibition stimulates breakdown of storage starch to soluble sugar through catalytic processes (Loch et al., 2004). The soluble sugars are translocated to the embryo to initiate germination (Loch et al., 2004). When the seeds are removed from freezer, frozen seeds are thawed before planting. Exposing the seeds to

freezing-thawing may stress the seed coat, endosperm, and/or embryo. For example, it may stimulate conversion processes in storage starch in the endosperm to produce soluble sugars so that germination process. Furthermore, seed responses to the freezing and thawing may be expressed when seeds were planted in soil. Some reports suggested that freezing storage for switchgrass has little effect on germination undern laboratory condition (Sautter, 1962; Byers,

1973; Zarnstorff et al., 1994; Burson et al., 2009). There are significant interactions between switchgrass seed germination and soil parameters, such as water potential (Demay et al., 2010), salinity (Ries and Hofmann, 1983; Hanson and Johnson, 2005; Kim et al., 2012; Schmer et al.,

2012), and fertilizer (Smika and Newell, 1965; Traversa et al., 2013). For instance, base water potential of Kanlow seed was -0.74 MPa, and germination was reduced at water potential of -

0.13 Mpa (Demay et al., 2010).

Storage length affected seedling morphologies, such as length and width of leaves, length from soil to the first node, and length of internodes. Long freezing may also affect seed coat and seed protein chemistry. The cuticle layer of seed consists of hydrophobic substances, such as cutin, suberin, and waxes (Ross et al., 2013). Hydrophobic interaction may weaken the memberane during freezing storage. It has been reported that water holding capacity of seed starch was reduced following freezing-thawing treatment for jackfruit seeds (Dutta et al., 2011).

Seed moisture is also critical in long term storage. Seed moisture averaged over three cultivars was 7.1 % of seed weight and no difference was observed among treatments (data not shown).

Nobukalu et al (2013) reported that seed moisture of sea oat (Uniola paniculata), a perennial warm season grass, remained unchanged (range: 10-16%) at -20°C storage regardless of the storage length up to 15 months; however, seed moisture was lower for the seeds stored at room temperature (range: 6-13%) than seeds stored at -20°C. By contrast, our seeds stored in room

temperature gained less moisture over time because seeds were stored at low humidity and did not absorb atmospheric moisture. In addition, seed moisture less than 13 % lowers biological activity (e.g., respiration) and which allows seed to be safely stored for long periods (Elias et al.,

2007). The relationship between seed viability and seed moisture was inversely proportional depending on storage period and species when storage temperature was above freezing point

(Harrington, 1963; Ellis and Robert, 1980). In addition, storage temperature above 10ºC was inversely proportional to germination percentage of sorghum seeds after 16 years of storage

(Bass and Stanwood, 1978). Small changes occur to seed coat structure and/or seed protein during seed freezing that could influence the morphology of switchgrass seedlings as we report, although specific mechanisms are unknown.

The seeds used in this study for each cultivar were from the same fields every year.

Variability among seed lots may result from differences due to seed ages and annual growing conditions. Life-history theory explains that dormancy evolved from environmental uncertainty.

Plants adjust their production of dormant seed each year depending on weather conditions (Rees,

1994). The more uncertain environments become, the greater number of dormancy seeds plants produce. Seed lots from 2011 received less effect compared to other seed lots for the three cultivars of switchgrass used in this study. The climatic differences, in addition to different seed ages, resulted in significant differences among seed lots in our study.

CONCLUSION

Freezing storage of switchgrass is a viable short-term and long-term storage method as freezing keeps seeds viable for long periods and may enhance seedling vigor. We found there were significant storage temperature × storage length interaction on germination percentage and

rate of seedling gemergence; shoot and root DM was affected by storage temperature; and seedling morphology (e.g. leaf length) was affected by storage lengths Storage condition had little effects on younger seed lots (e.g., 2011) and showed higher effect on seedling vigor for lowland cultivar than upland cultivars used in this study. Freezing storage caused no damage to seedlings and increased germination, rate of seedling emergence, and total shoot and root DM for switchgrass, especially in Kanlow. There are eigth-months periods between switchgrass fall harvesting to switchgrass planting in spring. Seed can be safely stored in freezing condition for the period to maintain seed viability as well as increase switchgrass seedling vigor.

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Table 1. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new tiller

formation (# plant-1) of lowland cultivar of Kanlow switchgrass (seed lot: 2008, 2010, and 2011)

influenced by storage temperatures at -80°C, -20°C, or room temperature (23°C; RM) for storage

lengths for one, two, three, six, and eight months

Storage Kanlow 2008 Kanlow 2010 Kanlow 2011 (mon) -80 °C -20 °C 23 °C LSD -80 °C -20 °C 23 °C LSD -80 °C -20 °C 23 °C LSD Emergence Rate Index 1 17 b† 37 A‡a 10 b 12.9 30 Bb 20 Cb 43 a 7 7 16 11 NS 2 10 14 B 11 NS 35 AB 30 BC 36 NS 8 12 11 NS 3 15 19 B 14 NS 29 Bb 28 Cb 43 a 10 12 15 16 NS 6 10 c 16 Bb 21 a 2.6 45 ABa 42 AB 44 NS 12 10 6 NS 8 18 a 12 Ba 10 a NS 51 A 50 A 43 NS 9 17 6 NS Mean§ 14 b 20 a 13 b NS 38 34 42 NS 10 b 14 a 10 b 3.1

Germination 1 25 Ab 51 Aa 14 BCb 15 37 Cb 27 Cb 58 a 17 16 Bb 33 a 23 b 7.6 2 13 B 22 B 12 C NS 48 BC 42 B 52 NS 16 B 26 24 NS 3 22 AB 23 B 22 AB NS 42 Cb 42 Bb 59 a 12 23 A 29 30 NS 6 13 Bb 26 B 25 A 6 58 AB 57 A 60 NS 25 A 21 15 NS 8 25 A 18 Bab 13 Cb 11 69 A 68 A 59 NS 13 Bb 33 a 13 b 11.2 Mean 20 b 28 a 17 b 5.7 51 47 58 NS 28 a 21 b NS

Elongation rate 1 1.6 A 1.7 1.9 NS 1.9 2.0 1.9 NS 1.9 2.0 1.9 NS 2 1.4 B 1.7 1.5 NS 1.6 1.7 1.6 NS 2.0 1.9 1.9 NS 3 1.9 Aa 1.7 ab 1.5 b 0.2 1.8 1.8 2.0 NS 2.0 1.6 2.0 NS 6 1.4 B 1.3 1.8 NS 2.0 2.0 2.0 NS 2.0 2.0 1.9 NS 8 2.0 A 1.6 1.6 NS 2.1 2.0 2.0 NS 2.3 1.5 2.3 NS Mean 1.7 1.6 1.7 NS 1.9 1.9 1.9 NS 2.0 a 1.8 b 2.0 a NS

Tiller 1 0.8 ab 0.5 b 1.0 Aa 0.4 0.5 0.7 0.3 NS 1.0 0.4 0.4 NS 2 0.5 b 0.7 ab 1.0 Aa 0.4 0.2 0.3 0.3 NS 0.7 0.4 0.7 NS 3 0.7 0.6 0.1 B NS 0.2 0.4 0.3 NS 0.3 0.3 0.4 NS 6 1.1 0.6 0.8 A NS 0.3 0.2 0.2 NS 0.4 0.8 0.7 NS 8 0.9 0.9 0.7 A NS 0.4 0.3 0.6 NS 1.1 0.5 0.4 NS Mean 0.8 0.7 0.7 NS 0.3 0.4 0.3 NS 0.7 0.5 0.5 NS † Values within a row within a seed lot rate followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths.

Table 2. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new tiller formation (# plant-1) of upland cultivar of Blackwell switchgrass (seed lot: 2010, and 2011) influenced by storage temperatures at -80°C, -20°C, or room temperature (23°C; RM) for storage lengths for one, two, three, six, and eight months

Storage Blackwell 2010 Blackwell 2011 (mon) -80 °C -20 °C 23 °C LSD -80 °C -20 °C 23 °C LSD Emergence Rate Index 1 65 68 72 NS 31 28 38 NS 2 63 ab 60 b 71 a 8 34 ab 23 b 54 a 24 3 67 65 63 NS 44 36 36 NS 6 64 69 65 NS 22 32 31 NS 8 64 63 67 NS 40 36 24 NS Mean§ 64 65 68 NS 34 31 37 NS

Germination 1 85 85 92 NS 49 B‡ 41 50 NS 2 80 b† 77 b 92 a 9 44 BC 33 57 NS 3 86 86 84 NS 64 Aa 52 ab 45 b 13 6 86 84 83 NS 33 C 45 45 NS 8 90 a 71 b 86 b 6 48 B 53 39 NS Mean 85 81 87 NS 48 45 47 NS

Elongation rate 1 2.0 a 2.0 ABa 1.9 BCa 0.1 2.0 AB a 1.9 Bab 1.8 ABb 0.1 2 2.0 a 2.1 Aa 1.9 Cb 0.1 2.3 A 2.1 A 2.0 A NS 3 2.0 a 1.9 BCb 1.9 BCab 0.2 1.7 B 1.9 B 1.7 B NS 6 2.0 2.0 ABC 2.1 A NS 1.7 B 2.0 AB 1.9 AB NS 8 2.0 ab 1.8 Cb 2.0 Ba 0.2 1.7 B 1.6 C 1.7 B NS Mean 2.0 1.9 2.0 NS 1.9 1.9 1.8 NS

Tiller 1 0.8 0.7 0.7 NS 0.5 B 0.7 0.8 NS 2 0.5 0.9 0.7 NS 1.1 A 0.9 0.7 NS 3 0.6 0.9 0.6 NS 0.6 B 0.5 0.8 NS 6 0.6 0.7 0.6 NS 1.0 Aa 0.6 b 1.0 a 0.3 8 0.6 0.8 0.8 NS 1.0 A 0.8 0.7 NS Mean 0.6 b 0.8 a 0.7 b 0.1 0.8 0.7 0.8 NS † Values within a row within a seed lot rate followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths.

Table 3. Emergence Rate Index (ERI), germination (%), elongation rate (cm d-1), and new tiller formation (# plant-1) of upland cultivar of Trailblazer switchgrass (seed lot: 2010, and 2011) influenced by storage temperatures at -80°C, -20°C, or room temperature (23°C; RM) for storage lengths for one, two, three, six, and eight months

Storage Trailblazer 2010 Trailblazer 2011 (mon) -80 °C -20 °C 23 °C LSD -80 °C -20 °C 23 °C LSD Emergence Rate Index 1 30 AB‡ 30 30 NS 45 42 47 A NS 2 38 AB 38 28 NS 44 37 47 A NS 3 27 B 36 41 NS 36 48 41 AB NS 6 43 Aa† 34 b 33 b 9 37 34 35 B NS 8 38 AB 27 45 NS 38 b 53 a 32 Bb 15 Mean§ 35 33 35 NS 40 43 40 NS

Germination 1 47 43 46 NS 63 57 59 A NS 2 52 52 40 NS 60 a 48 b 61 Aa 8 3 40 50 58 NS 55 66 58 A NS 6 57 46 49 NS 49 49 44 B NS 8 53 41 63 NS 53 b 74 a 42 Bb 11 Mean 50 46 51 NS 56 59 53 NS

Elongation rate 1 2.0 2.0 2.2 A NS 1.9 Aa 1.7 Bb 2.1 a 0.2 2 2.0 2.0 1.8 BC NS 1.7 B 2.1 A 2.1 NS 3 2.1 2.0 1.9 B NS 1.9 A 1.8 B 2.0 NS 6 2.0 a 2.0 a 1.7 Cb 0.2 1.9 A 1.8 B 1.9 0.2 8 2.0 2.2 1.9 B NS 1.7 B 1.7 B 2.0 0.2 Mean 2.0 2.0 1.9 NS 1.8 b 1.8 b 2.0 a 0.1

Tiller 1 1.2 AB 0.9 1.0 A NS 0.6 0.7 1.0 NS 2 0.9 B 1.0 0.7 AB NS 0.9 1.2 0.9 NS 3 1.6 Aa 0.9 b 0.5 Bb 0.6 1.0 ab 0.8 b 1.2 a 0.3 6 1.0 Ba 1.1 a 0.4 Bb 0.4 1.1 0.8 1.0 NS 8 0.7 Bb 1.1 ab 1.3 Aa 0.4 1.0 1.0 1.0 NS Mean 1.1 a 1.0 a 0.8 b 0.2 0.9 0.9 1.0 NS † Values within a row within a seed lot rate followed by the same lower case letter are not significantly different at p< 0.05. ‡ Values within a column within a temperature treatment within a seed lot followed by the same upper case letter are not significantly different at p< 0.05. § Values are average across storage lengths.

Figure 1. Effect of storage temperatures (-80°C, -20°C, and room temperature (RM)) and storage length (1, 2, 3, 6, and 8 months) on

total shoot and root DM and shoot and root DM plant-1 for Kanlow (seed lot 2008, 2010, and 2011), Blackwell (seed lot 2010 and

2011), and Trailblazer (seed lot 2010 and 2011). Values within a plant part within a storage period by the same letter abc for total

shoot and root DM and xyz for DM plant-1 are not significantly different at p< 0.01.

Figure 2. Effect of storage periods (1, 2, 3, 6, and 8 months) on leaf lengths and width of Kanlow (seed lot: 2008, 2010, and 2011) and

leaf lengths and width of Blackwell (seed lot: 2010 and 2011) and Trailblazer (seed lot: 2010 and 2011). Values within a leaf by the

same letter are not significantly different at p< 0.05. * indicates sifnificant differences of leaf length between seed lot for Blackwell

and Trailblazer.

Figure 3. Length from soil to the first node and internodes for Kanlow (seed lot: 2008, 2010, and

2011) and Trailblazer (seed lot: 2010 and 2011), and effect of storage lengths (1, 2, 3, 6, and 8 months) to node lengths. Values within a plant part by the same letter are not significantly different at p< 0.01.

Photo 1. Seedling emergence at 15th day (top), shoot growth (middle) and root growth (bottom) from seeds stored at -80°C, -20°C, and

room temperature (RM) of Kanlow 2008 sotred for one month, Kanlow 2010 stored for eight months, and Kanlow 2011 stored for one

month.

Photo 2. Seedling emergence at 15th day (top), shoot growth (middle) and root growth (bottom) before harvesting for seeds stored at -80°C, -20°C, and room temperature (RM) for Blackwell

2010 and Blackwell 2011 seeds sotred for eight months.

Photo 3. Seedling emergence at 15th day (top), shoot growth (middle) and root growth (bottom) before harvesting for seeds stored at -80°C, -20°C, and room temperature (RM) for Trailblazer

2010 seeds stored for six months and Trailblazer 2011 seeds stored for eight months.

CHAPTER FIVE

SWITCHGRASS GROWTH REPONSE IN INTERCROPPING WITH HYBRID POPLAR UNDER IRRIGATIONS

ABSTRACT

Biomass production for future energy has focused on perennial grass species over annual species due to high land and water use efficiency, high biomass production, and soil conservation. Land use efficiency can be maximized if ally is utilized to produce switchgrass biomass while growing hybrid poplar trees. The objective of this study was to quantify the influence of hybrid poplar on switchgrass biomass and architecture, and determine land use efficiency of the two perennial species under irrigation. Three cultivars of switchgrass (Kanlow, Blackwell, and Trailblazer) were planted in the ally of hybrid poplar trees (Clone: OP367 and PC4) at Boardman Tree Farm,

Boardman OR during 2011 to 2014. Mean grass yields for Kanlow, Blackwell, and Trailblazer under monoculture during 2012 and 2013 averaged 18.8, 14.6, and 15.0 Mg DM ha-1 yr-1, while grass yield under intercropping for the three cultivars were 9.1, 8.8, and 7.7 Mg DM ha-1 yr-1, respectively. Yield reduction in intercropping plots may be caused by compacted soil due to the excess water applied to the field, which was adjusted to the amount of water required for hybrid poplar. Reduction of PAR may have minimum influence on the yield reduction. Plant architecture and tiller demographic data displayed negative influence from flooded condition.

Declining trend of LAI was observed from 2012 to 2014 under intercropping, and tiller demographics displayed slow internode extension toward autumn. Land equivalent ratio (LER) with poplar-switchgrass intercropping maintained greater than 1.0 each year averaging 1.7; however, irrigation problems to satisfy both grass and tree crops need to be solved.

INTRODUCTION

Switchgrass is a model species for second generation biofuel feedstock due to its high yielding capacity (24.8 Mg DM ha-1 yr-1 under irrigation; Kimura et al., 2015), low greenhouse gas emissions (Schmer et al., 2008), greater net energy than oil-seeds (Tilman et al., 2006), high water use efficiency (Wu et al., 2009), and high C sequestration ability (Collins et al., 2010).

However, there are concerns of using limited land and water resources for biofuel feedstock that may reduce resource availability for food and fiber production. Ally cropping is the practice of intercropping herbaceous plants in open areas while trees are growing. This utilizes land and agricultural resources efficiently and provides early economic returns to forest landowners and commercial tree producers (Zinkhan and Mercer, 1997; Gold et al. 2000; Riffell et al., 2012;

Susaeta et al., 2012). In addition, intercropping of woody plants and herbaceous crops improves soil N cycling (Allen et al., 2004), C sequestration (Fang et al., 2010), activity of soil microorganisms and wildlife habitat (Stainback and Alavalapati, 2004), and reduction of ground water contamination (Jose et al., 2004; Bergeron et al., 2011). With use of the ally cropping practice, land and water use efficiency will substantially improve for biomass production.

Hybrid poplar is a fast growing leading bioenergy woody species for cogeneration of heat, electricity, and liquid fuel (Hansen et al., 1983; Newman et al., 1997). It grows at 22 Mg ha-1 yr-

1 in high yielding clone (Guo and Zhang, 2010) and is often harvested after 10 years for traditional timber production (Carlson and Berger, 1998). It yields up to 8600 Btu lb-1 of energy, equivalent to 11 barrels of oil acre-1 (Ranney et al., 1987). Among other woody species investigated for biofuel production by DOE (e.g., willow and eucalyptus), hybrid poplar composed of high cellulose (40 %) and low lignin (22 %), which makes the extraction process easier for liquid fuels (Advanced Hardwood Biofuels Northwest, 2013a). Currently, 36,400 ha

of hybrid poplar are planted in North America, which can potentially be utilized for intercropping practice with herbaceous biofuel crops, such as switchgrass (Kroll and Downing,

1995; Downing et al., 1996). Intercropping the two leading bioenergy species, switchgrass and hybrid poplar, will increase biomass production per unit land area with benefits of fixing atmospheric C and diversifying the ecosystem.

Although intercropping the two perennial crops is beneficial for many reasons, it also increases management difficulties. Weed management in intercropping is more complex than in single crop practice as there are a few registered products for two crops (Buhler et al., 1998).

Shading may change plant architecture and forage quality (Buxton and Fales, 1994), and decrease biomass yield (Suresh and Rao, 1999) especially in C4 species which has higher light saturation point than C3 grass (Bjorkman, 1981). Plant architecture and tiller demographics influenced by increased shading also affect forage yield and quality (Moor et al., 1991).

Quantified tiller information will generate important growth and developmental plant data base

(Mitchell et al., 1998; Boe and Beck, 2008; Boe, 2009). Forage quality was increased by shading due to the slower growth rate under reduced PAR (Buxton and Fales, 1994) because maturity of plant is inversely related to the forage quality (Kephart and Buxton, 1993). For example, the crude protein (CP) value of pinegrass (Calamagrostis rubescens Buckley) grown under 500 stems ha-1 of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) was 10.4 % as compared to 12.5 % CP under 2000 stems ha-1 of lodgepole pine (Lindgren and

Sullivan, 2013). Despite the ongoing challenges of ally cropping, limited information is available on establishment and management of the two leading biofuel crops, hybrid poplar and switchgrass, in an intercropping system. The objective of this study was to quantify the

influence of hybrid poplar canopy on switchgrass biomass, architecture, and tiller demographics, and determine land use efficiency of the two perennial species under irrigation.

MATERIALS AND METHODS

Field preparation

The poplar-switchgrass intercropping study was established in field at GreenWood

Resources’ BTF, Boardman, OR (45°44’54.21” N; elevation 192 m; Forst Free Days 162) in

2011. Weeds are controlled annually with glyphosate [N-(phosphonomethyl) glycine] at 13.0 g eq/ha in early March during switchgrass dormancy. Two hybrid poplar cultivars, P. ×generosa

‘PC4’ and P. ×canadensis ‘OP367’, were planted in March 2011, with two-year un-rooted boles.

Three switchgrass cultivars (Kanlow, Blackwell, and Trailblazer) of switchgrass were then seeded with seeding rate at 11.2 kg Pure Live Seed (PLS) ha-1 in early June 10 and 13, 2011.

The treatments consist of a randomized split-plot design with four replications (Figure 1). Two main treatments were cropping system (intercropping and monoculture) and two hybrid poplar cultivars (PC4 and OP367) that are split into four subplots of Kanlow, Blackwell, Trailbrazer, and tree only. Field plots comprise an area occupied by 30 poplar trees configured 5 rows × 6 trees within rows. Tree spacing was 6 m between rows and 3 m between trees within rows. There was a single grass/poplar buffer bordering each plot; so that the total plot dimension was 5 rows

× 12 trees (60 trees per plot). Each plot will occupied 792 m2. Each poplar varietal block containing four replicates was 6336 m2. The total area occupied by the four poplar blocks was

25,344 m2 or 2.5 ha. Grass monocultures were randomized and planted adjacent to the intercropped plots in a randomized complete-block design replicated four times.

Data collection

Leaf area index (LAI) was collected using PAR/LAI ceptometer (AccuPAR model LP-80,

Decagon Device, Pullman, WA, USA). Measurements were repeated six times across a plot.

Measurements were made every two weeks during growing seasons from May 2012 to July 2014.

Aboveground samples were clipped by hand before harvesting from each plot within randomly placed 0.25 m2 quadrate for tiller counting. Harvested tillers were separated into stems, leaf blades and panicles. Each plant part was dried separately at 55°C for at least 48 hours, and the dry weight was used to determine density of each tiller component.

Switchgrass biomass was harvested once in September 26, 2011 during the establishment year and twice (July1, 8, and 1, 2012, 2013, and 2014, respectively and October1, 1 and

September 30, 2012, 2013, and 2014, respectively) from 2012 to 2014. In this dissertation, data obtained from May, 2012 to July, 2014 are presented. Harvesting was conducted with a New

Holland 1496 forage swather (Sperry-NewHolland) equipped with 4-m-wide sickle bar and conditioner to a residual stubble height of 15 cm. Plot weight was estimated by weighing fresh weight collected within a 6-m by 4-m at each plot. Subsamples were taken to estimate DM yield

(forage quality will be discussed in Chapter 6). Harvested switchgrass was tedded for at least 5 times, baled with a New Holland Baler 8555 (New Holland Inc. New Holland, PA), and removed from the field. Tree yield was estimated with regression between tree weight obtained from tree samples of given age and clone by standing tree diameter and height (personal communication with Greenwood Resources Inc). Obtained tree yield was used to calculate Land Equivalent

Ratio (LER) with following formula;

푌푖푒푙푑 표푓 푖푛푡푒푟푐푟표푝푝푒푑 푔푟푎푠푠 푌푖푒푙푑 표푓 푖푛푡푒푟푐푟표푝푝푒푑 푡푟푒푒 LER = + 푌푖푒푙푑 표푓 푚표푛표푐푢푙푡푢푟푒 푔푟푎푠푠 푌푖푒푙푑 표푓 푚표푛표푐푢푙푡푢푟푒 푡푟푒푒

Analysis of variance using the GLM procedures of SAS (2008) were used to determine main effects and interactions of years, cultivars, and cropping systems (intercropping or monoculture) on grass and tree yields, LER, LAI, and tiller components. Least significance differences (LSD) and paired t tests were used to separate means when F tests were statistically significant (P = 0.05). Orthogonal contrast was used to compare between intercropping × monoculture and upland × lowland cultivars within each cropping system.

RESULTS AND DISCUSSIONS

Dry Matter Yield

Switchgrass cultivars were harvested twice a year during May, 2012 to July, 2014 growing seasons. Dry matter (DM) yield of three cultivars of switchgrass was summarized in

Table 2. Switchgrass annual DM yield was influenced by cropping system (p < 0.0001), cultivar

(p < 0.05), and year (p < 0.1) (Table 1); therefore, means were separated into cropping system, cultivar, and year (Table 2). Annual grass yields for Kanlow, Blackwell, and Trailblazer under monoculture during 2012 and 2013 averaged 18.8, 14.6, and 15.0 Mg DM ha-1 yr-1 respectively, while grass yield under intercropping for the three cultivars averaged 9.1, 8.8, and 7.7 Mg DM ha-1 yr-1, respectively (Table 2). Orthogonal contrast between monoculture and intercropping confirmed that intercropping had no influence on grass yield in the first year of biomass production (Table 2). Yield difference was attributed to switchgrass ecotype, where the lowland cultivar Kanlow out-yielded upland cultivar by 1.7 Mg DM ha-1 yr-1 in 2012 and 2.1 Mg DM ha-1 yr-1 in 2013. Higher yielding capacity of lowland cultivars (average: 18.4 Mg DM ha-1 yr-1; Fike et al., 2006b; West and Kincer, 2011; Kering et al., 2012; Kimura et al., 2015) as compared to upland cultivars (average: 8.8 Mg DM ha-1 yr-1; George and Obermann, 1989; Casler and Boe,

2003; Adler et al., 2006) are commonly observed in other studies (Cassida et al., 2005) due to the

extended vegetative growth periods of lowland cultivars (Esbroeck et al., 1997). Grass yield was reduced by 2.4 Mg DM ha-1 under intercropping from 2012 to 2013, while grass yield was increased by 2.8 Mg DM ha-1 under monoculture (Table 2). In the first harvest in 2014, yield was reduced by 13 % in monoculture and by 69 % in intercropping plots as compared to the first harvests in 2012 and 2013. Yield reduction in the study may be caused by excess water applied to the field rather than the reduced PAR from the poplar canopy. Field was irrigated at 195 cm ha-1 in 2012 and 236 cm ha-1 in 2013 during April to October (data obtained from BTF).

Switchgrass in our region requires 67 cm ha-1 of supplemental water during May to September to produce 23.0 Mg DM ha-1 yr-1 (Kimura et al., 2015). Having the irrigation water adjusted to the amount required for hybrid poplar trees, the field was saturated with water, which enhanced soil compaction by field equipment during harvesting and baling. Soil compaction reduces soil pores, which restricts gas and water fluxes and inhibits root growth (Dexter et al., 2008). Compacted soil reduces total root length of grass species (e.g., barley and triticale) by up to 79 % due to cell deformation in cortex and vascular cylinder (Lipiec et al., 2012). Growth of thick roots is inhibited more severely than the growth of fine roots because reduction of soil porosity is caused by reduction of large pores (> 30 µm) than small pores (< 6 µm) (Lipiec et al., 2012). The majority of switchgrass roots are distributed at top 30 cm of soils (Collins et al., 2010), which are comprised with 40 % of coarse roots (≥ 1 mm), 34 % of fine roots (< 1 mm), and 17 % of rhizomes or crown (Garten et al., 2010). Under the compacted soil, switchgrass root development is hindered at the surface soil. Switchgrass coverage was measured in November,

2013 and June 2014, by dividing each plot into five rows to estimate how growth depression was distributed within a plot along the equipment travel direction (Appendix A). Switchgrass coverage at each side of plot was 76 %, while three rows in the middle of plots had 40 % of

switchgrass coverage (Appendix A). It is speculated that the both sides of plot received less water as compared to the middle of the plots. Soil bulk density measured on July of 2014 at our study site revealed that the whole plots were heavily compacted with bulk density of 1.98 g/cm3 with no difference across the rows within a plot. Bulk density should be below 1.6 g/cm3 for proper root growth in sandy soil, while bulk density above 1.8 g/cm3 is considered as compacted soil (USDA NRCS, 2008). Consequently, in this study switchgrass root and shoot growth were inhibited, resulting in the annual yield loss of the stand. In addition, weed infestation increased from 27 % in May, 2012 to 41 % in September, 2013 due to the decreased switchgrass coverage in the field (Appendix A and B). Unlike annual grass species, accumulative root growth is critical in developing aboveground biomass in switchgrass. Grass yield in monoculture plots was twice greater than yield in intercropping plots although the amount of water applied was same for the two treatments. The reason for the greater yield reduction in intercropping plots than monoculture plots may be due to the decreased evaporation rate under tree canopy.

Evapotranspiration in the center of ally was 30 % less than plots without tree (Shapo and Adam,

2008), and 12 °C lower temperature was observed under tree canopy (Feldhake et al., 2001).

These cool and moist environment swill conserve soil moisture and improve plant growth under dry environment; however, it was detrimental to switchgrass plant growth in highly saturated condition observed in our study. At higher evaporation rate in monoculture plots, it is expected that switchgrass root growth was not as poor as root growth in intercropping plots. Finally, reduced PAR under poplar canopy may have minimum influence on yield reduction for the first three production years based on our field observation (Appendix C). There were volunteer switchgrass in the tree rows directly below the tree canopy, where the maximum shading may be formed. Despite the minimum PAR under the tree canopy, the volunteer switchgrass growth was

more vigorous than switchgrass inside the plots (Appendix C). The reason for the vigorous growth of the volunteer switchgrass in the tree rows may be due to receiving less water and compaction in the tree rows than the inside plots. The majority of water from overhead sprinkler goes to the middle of plots, while tree rows receives less water due to the sprinklers delivery pattern. Therefore, the volunteer switchgrass in tree rows are grown under less water and less compacted soils since no equipment is driven over the tree rows, providing favorable growing condition as compared to switchgrass in the plot. Other studies also reported that shading had little influence on biomass production for big bluestem (C4) and smooth bromegrass (C3) under

Scotch pine (Perry et al., 2009), and switchgrass (Alamo) under loblolly pine in the first year of tree and grass intercropping (Albaugh et al., 2012). These studies were conducted under dryland, thus had no disturbance by excessive water observed in our study.

Tree yield and LER were summarized in Table 3. Yield of tree averaged over the two clones were 3.5 Mg DM ha-1 in 2012 and 13.0 Mg DM ha-1 in 2013 in tree monoculture plots

(Table 3). Orthogonal contrast confirmed that no statistical difference was observed for tree yield between tree monoculture and intercropping plots in 2012, while 2.3 Mg DM ha -1 higher tree yield was observed in intercropping plots than in tree monoculture in 2013 (Table 3). The increased tree yield in intercropping may be attributed to fertilizer applied to the intercropping plots. Positive response of poplar yield to N application was reported elsewhere (Hansen et al.,

1988; Fortieer et al., 2010; Sisi et al., 2012; Headlee et al., 2013). The LER was influenced by cropping system (p < 0.05) (Table 1). The LER in intercropping plots averaging over the three cultivars was 1.7 in both 2012 and 2013, indicating that land use efficiency of intercropping was

70 % higher than monoculture plot. Despite the yield reduction of grass, LER was maintained above one because of increased tree yield in intercropping plots.

Switchgrass architecture and tiller demographics

Tiller was separated into stem, leaf, and panicles in July and October before harvesting and was presented as g m-2 in Figure 4 as well as tiller m-2 in text. Leaf biomass was 69 % of whole plant body in July, which was declined to 41 % in October in 2012 (Figure 4). Stem biomass and panicle were increased from 31 % and 0 % in July to 49 % and 8 % in October, respectively (Figure 4). The increased stem biomass contributes to the increased DM yield in the second harvest because stem is heavier than leaves. Such change in plant structures toward fall is commonly observed phenomenon in short-day plants (Griffin and Jung, 1983; Smart et al., 2004).

Leaf biomass of Blackwell switchgrass was twice more than stem biomass in June, while opposite was observed in August (Griffin and Jung, 1983). Decreased leafy biomass and increased stem biomass declined LAI values due to open area created by senesced leaves, resulting increased amount of sunbeam going through the plant (Figure 3). In 2013, tiller demographic patterns did not follow the pattern observed in 2012. Stem density in intercropping plots was decreased by 55 % from July to October. This indicates that extension of internodes was slowed down. The reason for the inhibited internodes extension probably is due to the root damage by the flooding condition and soil compaction. Having the slow growth, panicle formation was delayed and reduced from 31.5 g m-2 in 2012 to 0.73 g m-2 in 2013 (Figure 4).

High tiller density corresponds to high yielding cultivars in switchgrass (Boe and Casler, 2005), indicating that decreased tiller weight per unit area observed in our study is directly related to the decreased yield (Boe, 2007). Tiller density for lowland and upland cultivars averaged over the first two years (2012 and 2013) was 726 m-2 and 1387 m-2 in July and 581 m-2 and 850 m-2 in

October, respectively. The seasonal decrease in tiller density was also observed in the study conducted in Nebraska, where tiller density of Trailblazer was 2000 m-2 until DOY 168 and

100

declined to 1000 m-2 by DOY 224 (Mitchell et al., 1998). In July of 2014, tiller density was in range of 180-630 m-2, which is lower than tiller density observed in October in the first two years.

Tiller formation is controlled by the interaction between formation of new tiller and death of old tillers (Langer et al., 1964). The decreased tiller density is caused by the slow life cycle of tiller, from generation of new tiller to the death of old tiller. Again, plant development is deteriorated due probably to the excessive amount of water applied to field, which decreased soil pores and enhanced soil compaction.

Leaf Area Index (LAI) under grass monoculture and intercropping was measured using a leaf ceptometer. The increased LAI values indicate that the switchgrass forms dense canopy.

Means were pooled over cultivars and separated into years and cropping systems (Figure 3). The

LAI values increased from May (LAI: 1.5) to the end of June (LAI: 4.5) with no differences between grass monoculture and intercropping plots until July in 2012. Other study with one cutting system at NC also demonstrated that LAI was same between monoculture and tree intercropping in July (Albaugh et al., 2012). In the one cutting system, LAI was same between the cropping systems throughout the year. However, in two cutting system in our study, the LAI values for regrowth in grass monoculture was 4.1 as compared to LAI values of 3.0 in intercropping plots on August, indicating regrowth was faster under monoculture plots than intercropping plots. Under the tree canopy, switchgrass growth rate may be slowed down due to the flooded condition creating poor soil condition for proper root growth as preciously described under Dry Matter Yield section. The LAI peaked on the first week of September (LAI: 6.0 in monoculture and 4.4 in intercropping) and declined in both treatments (LAI: 4.3 in monoculture and 2.9 in intercropping) toward October before the second harvesting in 2012 (Figure 3). The similar seasonal change in LAI was observed in other studies (Madakadze et al., 1998; Albaugh

101

et al., 2012). The maximum LAI in monoculture observed in our study was similar to those obtained by Mitchell et al. (1998) (7.7) and Redfearn et al. (1997) (5.5). The declined LAI on

October before the second harvest may be attributed to leaf senescence toward fall (Smart et al.,

2004). Old tillers senesce leaves toward fall to translocate energy into reproductive parts (Smart et al., 2004). The LAI values well reflected the yield trend in intercropping plots. The LAI values in July before harvesting were decreased from 4.7, 4.6, to 2.7 from 2012, 2013, to 2014, respectively as yield decreased from 2012 to 2014 in intercropping plots (Figure 3). By contrast,

LAI values in monoculture plot in July was increased from 2013 (LAI 6.5) to 2014 (LAI 7.6) despite the yield reduction observed from 2013 (8.5 Mg DM ha-1) to 2014 (6.7 Mg DM ha-1). It indicates that switchgrass in monoculture plots developed dense crowns as compared to switchgrass in intercropping plots, resulting in the increased LAI. However, the young tillers produced from the crown had little contribution to yield due to the slow growth with poorly developed root system under the compacted soils.

CONCLUSION

Despite the water stress imposed during our study periods, biomass production per unit area was 4 Mg DM ha-1 greater in intercropping than monoculture cropping, indicating high productivity potential in tree-forage grass intercropping system. Incorporating the switchgrass production in tree allies will substantially increase land use efficiency for future bioenergy production. On the basis of these results, reduction of PAR by hybrid poplar had little influence on switchgrass growth especially if irrigation is controlled appropriately. Because the excess water applied for hybrid poplar production disturbed the growth of switchgrass in our study, further investigation of sufficient amount of water satisfying both crops is warranted.

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Table 1. Analysis of variance and mean squares for yield of grass and tree, land equivalent ratio

(LER), tiller components during 2012-2014 growing seasons

Grass Tree LER Variable Harvest 1 Harvest 2 Total Year (Y) 95.5*** 16.9* 7.3° 1787*** 0.016 Trt (T)† 2.3 32.2** 26.6* 0.3 0.044 Cropping system (C)‡ 120.1*** 231.1*** 469.1*** 14.9+ 3.71* Y*T 2.7 1.8 2.3 0.5 0.007 Y*C 22.5*** 8.5* 51.9* 0.5 0.019 T*C 0.96 4.2° 11.0° 4.4 0.069 Y*T*C 1.6° 2.0 4.4 1.4 0.036

Stem Blade Panicle Harvest 1 Harvest 2 Harvest 1 Harvest 2 Harvest 1 Harvest 2 Year (Y) 1064** 601.6° 1091** 2601.6* - 706.7* Trt (T) 340.7* 25.2 322.5* 9.8 - 65.5* Cropping system (C) 5.72 128.5 2.8 198.8 - 5.1 Y*T 1.06 114.3 0.83 47.8 - 11.7 Y*C 17.3 514.4* 20.7 627.4* - 24.4 T*C 30.3 75.3 32.4 149.6 - 15.6 Y*T*C 26.7 52.0 26.3 14.1 - 26.8

°, ∗, ∗∗, ∗∗∗ indicate significance at p < 0.1, p < 0.05, p < 0.001 and p < 0.0001, respectively. †Treatment represents switchgrass cultivars. ‡Cropping system represents monoculture planting and intercropping.

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Table 2. Aboveground biomass of Kanlow (KL), Blackwell (BW), and Trailblazer (TB) switchgrass in monoculture plot or

intercropped with hybrid poplar clones, OP367 (OP) or PC4 (PC) from 2012 to 2014

2012 2013 2014 Mean Yield Harvest 1 2 Total 1 2 Total 1 (2012-2013) ------Mg DM ha-1------

Kanlow 6.6 a *‡ 10.3 a 16.9 a 9.5 a ° 11.3 a 20.8 a 6.0 a 18.8 a KLOP 5.2 a† 6.3 b 11.5 b 5.9 b * 2.8 b 8.6 b 1.8 b 10.1 b KLPC 5.1 a 4.1 b 9.2 b 4.6 b * 2.5 b 7.1 b 1.4 b 8.1 b LSD NS 2.79 5.10 1.37 0.97 2.4 0.7 2.8

Blackwell 6.1 a 7.7 a 13.8 a 8.7 a 6.7 a 15.4 a 7.3 a 14.6 a BLOP 6.6 a * 4.0 b 10.6 b 6.6 a * 2.4 b 9.0 b 1.8 b 9.8 b BLPC 6.4 a * 3.0 b 9.4 b 4.7 b * 1.4 c 6.1 c 2.1 b 7.7 c LSD NS 1.65 2.41 1.70 0.75 2.24 1.8 2.0

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Trailblazer 6.8 a 6.8 a 13.5 a 8.8 a 7.7 a 16.4 a 6.9 a 15.0 a TBOP 5.7 a 3.3 b 9.0 b 5.3 b * 2.3 b 7.6 b 1.8 b 8.3 b TPC 6.4 a 2.5 b 8.8 b 3.9 b * 1.5 b 5.4 c 1.5 b 7.1 b LSD NS 2.01 2.15 1.65 1.02 1.44 1.4 1.2

Contrast§ Mono*IC NS NS NS NS *** * ° NS Mono Low*up NS NS NS NS *** NS *** + IC low*up NS NS NS * *** *** *** *** OP*PC KL*BW NS *** *** *** *** *** *** *** KL*TB NS * NS ** * *** NS * TB*BW NS *** *** *** *** *** *** *** † Values within a column within a harvest and cultivar followed by the same lower case letter are not significantly different at p< 0.05. ‡ Significance compares betwenn the first and second harvest at p< 0.05. § Mono, IC, low, and up indicate monocropping, intercropping, lowland cultivar, and upland cultivar, respectively.

Table 3. Tree yield and Land Equivalent Ratio (LER) of Kanlow (KL), Blackwell (BW), and

Trailblazer (TB) switchgrass in monoculture plot or intercropped with hybrid poplar clones,

OP367 (OP) or PC4 (PC) from 2012 to 2013

Tree LER 2012 2013 2012 2013 Mg DM ha-1 OP367 3.4 13.2 - - PC4 3.7 12.9 - - LSD NS† NS - -

KL - - 1.0 b 1.0 b KLOP 3.5 15.4*‡ 1.7 a 1.6 a KLPC 3.9 15.8 1.5 a 1.7 a LSD NS NS 0.3 0.3

BW - - 1.0 b 1.0 c BWOP 3.6 15.7* 1.9 a 1.9 a BWPC 4.1 15.5 1.7 a 1.6 b LSD NS NS 0.4 0.2

TB - - 1.0 1.0 b TBOP 3.1 13.7 1.6 1.5 a TPC 4.5 16.0 1.8 1.6 a LSD NS NS NS 0.4

Contrast§ Mono*IC NS * NS NS Low*Up NS NS * * IC OP*PC * NS NS NS † Values within a column within a cultivar and year followed by the same lower case letter are not significantly different at p< 0.05. ‡ Significance compares tree yield between intercropping and monoculture at p< 0.1. § Mono, IC, low, and up indicate monocropping, intercropping, lowland cultivar, and upland cultivar, respectively.

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Figure 1. Monthly temperature and precipitation during 2012 to 2014 growing seasons at

Boardman, OR.

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Figure 2. Layout of whole plot (left) and one replication of intercropping plot (right). There are

24 intercropped plot, 12 grass monoculture plots, and 8 tree monoculture plots, resulting in 44 total plots.

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Figure 3. Leaf Area Index (LAI) taken under grass only plot or in intercropped with poplar clones OP367 or PC4 during 2012 to 2014 growing seasons. Values within a day of year followed by the same letter are not significantly different at p ≤ 0.05. The first letter, second letter, and third letter correspond to LAI at grass only plot, OP367 plot, and PC4 plot, respectively. LAI value of 0 was used to indicate the harvested stand (DOY: 185).

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Figure 4. Dry matter (g m-2) of tiller components (stem, leaf blade, and panicle) of switchgrass cultivars Kanlow (KL), Blackwell (BW), and Trailblazer (TB) in monoculture and in intercropped with hybrid poplar clones OP367 (OP) or PC4 (PC) in July and October in 2012 and 2013, and July in 2014. Values within a cultivar and a plant component followed by the same letter are not significantly different at p ≤ 0.05.

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APPENDICES

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2 3 4 5

Row1 Row Row Row Row APPENDIX A. Switchgrass coverage (%) across the plots. Row 1 and row 5 indicate west and east side of plot, respectively.

APPENDIX B. Percentage coverage of switchgrass cultivars Kanlow (KL), Blackwell (BW), and Trailblazer (TB) in monoculture and in intercropped with hybrid poplar clones OP367 (OP) or PC4 (PC) and percentage of weed in May 2012 and September 2013.

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Appendix C. Vigorous switchgrass observed in tree rows that received less water and less compaction as compared to the middle of plots.

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Leaf temperature from 10:10am to 10:48am 32

Mono IC C)

° 30 28 26 Average leaf temperatures 24 Monoculture Intercrop

Temperature ( Temperature 22 28.9°C *** 26.3°C

IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC

Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono Mono

10:30am IC

10:40 am IC 10:48am IC

10:20Mono am 10:10amMono

Leaf temperature from 10:50am to 12:05pm 32

C) ° 28 26 24 Average leaf temperatures Monoculture Intercrop 22

Temperature ( Temperature 29.1°C * 28.1°C

IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC

Mono Mono Mono Mono Mono Mono Mono

11:30amIC

11:10am IC 11:20am IC 11:40am IC

12:00pmIC 12:05pmIC 10:50am Mono 11:00Mono am Appendix D. Leaf temperature under monoculture (Mono) and intercropping (IC) from 10:10 am to 10:48am and from 10:50 am to 12:05pm. * and *** indicate that values are significantly different at p < 0.05 and 0.0001, respectively.

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CHAPTER SIX FORAGE QUALITY OF SWITCHGRASS BY GROWTH STAGES AND PLANT COMPONENTS IN INTERCROPPING WITH HYBRID POPLAR UNDER IRRIGATION

ABSTRACT

Forage quality attributes are important factors when processing herbaceous biomass for energy.

Influence of seasons, fertility management, and cutting frequency on switchgrass quality are well documented under dryland systems; however, information is limited for switchgrass quality under irrigation. The objective of this study was to determine the influence of intercropping siwtchgrass with hybrid poplar on the forage quality of switchgrass over multiple growing seasons and by plant component (stem and leaf) under irrigation compared to monoculture production. Throughout the study years (2012-2014), switchgrass experienced water stress from excessive irrigation water that was supplied to maximize hybrid poplar growth. Forage quality was similar among switchgrass cultivars in the two cropping systems in the first production year, but subsequently significant quality differences developed. The crude protein (CP) concentration averaged over 2013 and 2014 was 2 mg kg-1 higher in intercropping at a July harvest, while concentrations of sugar and neutral detergent fiber (NDF) were 1 mg kg-1 lower in intercropped plot than monoculture plots. Slower growth caused higher CP and low fiber, and low photosynthetic rate produced lower sugars in intercropped plots with low intensity of photosynthetically active radiation (PAR). Switchgrass macronutrient concentrations under intercropping were higher than monoculture due to reduced growth under intercropping which inhibited mobilization of these nutrients to roots toward fall. Forage quality by plant component revealed that the influence of intercropping was different on leaves and stems. Leaf quality was higher in the intercropped than monoculture due to the slow growth observed in the intercropped plots. In contrast, the fiber concentrations (NDF, ADF, and lignin) in stems was higher in

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intercropped plots than monoculture plots, suggesting that switchgrass accumulated fiber in stem as well as stored N in leaf under excessive irrigation under intercropping.

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INTRODUCTION Lignocellulosic feedstocks are promising biofuel sources that have positive environmental impacts over fossil fuels in C-sequestration, mitigation of atmospheric CO2, ecosystem diversification, and nutrient cycling (Lemus and Lal, 2005). Intercropping between woody plants and herbaceous crops should maximize such benefits of lignocellulosic feedstocks as well as resource use efficiency (e.g., land, water, and fertilizer) (Xu et al., 2010; Susaeta et al.,

2012). Among herbaceous crops examined for biofuel by the DOE, switchgrass gained prominent attention because of high aboveground and belowground productivities (Fike et al.,

2006; Collins et al., 2010) and the potential as an animal forage feedstock, providing producers a flexible market for their production. Forage quality attributes of switchgrass are important factors for both animal forage (Sadeghpour et al., 2014) and biofuel biomass production under dryland and irrigation (Allison et al., 2012). Accumulation of belowground biomass is high in the first few years of switchgrass production (Collins et al., 2010), during which forage quality and mineral concentrations in tissue vary widely (Kimura et al., 2015), by plant component (e.g., stem and leaf) (Lemus et al., 2009), and by location within a stem (e.g., basal or apical) (Sarath et al., 2007). Furthermore, an intercropping system will add complexity to the forage biomass, morphology, and quality grown within an agroforestry setting tree as compared to the grass monoculture system (Albaugh et al., 2012; Kyriazopoulos et al., 2013; Mimenza et al., 2013;

Lindgren and Sullivan, 2014). When intercropping grass between tree alleys, higher CP value was observed due to the slow growth under low intensity of PAR and low temperature (Kephart and Buxton, 1993; Buxton and Fales, 1994; Lin et al., 2001; Lindgren and Sullivan, 2014).

Concentration of lignin and macronutrients in plant tissues are of great interest to chemical engineers in bioconversion of lignocellulose (Fu et al., 2011; Xu et al., 2011). Therefore, information of switchgrass quality grown within an agroforestry setting will be critical for the

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potential bioenergy intercropping production system of two important bioenergy crops, switchgrass and hybrid poplar. The objective of this study was to determine the influence of hybrid poplar on forage quality of switchgrass in an intercropped system over growing seasons and by plant component (stem and leaf) under irrigation.

MATERIALS AND METHODS Field preparation and weather data are described in Chapter Five. The plots were harvested twice annually in July and October. Aboveground biomass was clipped with a sickle by randomly placed 0.25 m2 quadrate from monoculture and intercropped plots at each growth stage of switchgrass; vegetative, elongation, and reproductive. Stored forage samples from fall

2012 were burned in a fire in 2012; therefore, those results are unable to be presented. Fresh aboveground biomass was collected before each harvesting, separated into stem, leaf blades, and panicles. All samples were dried in a forced-draft oven at 55 °C for at least 48 hours or until dried. Dried subsamples were ground by passing through a hammer mill and then a Wiley mill

(Thomas Scientific, Swedesboro, NJ) with 1 mm screen openings, and used for quality analysis by Near Infrared Spectroscopy (NIRS) at the Washington State University (WSU) Forage

Laboratory at the Irrigated Agriculture Research and Extension Center (IAREC) in Prosser, WA.

The NIRS standard curve was calibrated using wet chemistry results of representative samples. A bias adjustment was made based on wet chemistry results on the standard NIRS equations. These equations were then used to predict crude protein (CP), sugars, neutral detergent fiber (NDF), acid detergent fiber (ADF), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), and lignin. Statistical analyses were conducted by analysis of variance using the GLM procedures of

SAS Institute (2008) to determine main effects and interactions of years, growth stages, cropping systems, and cultivars on forage quality attributes. Years and replications were considered

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random effects. Least significance differences (LSD) and paired t tests were used to separate means when F tests were statistically significant (P = 0.05).

RESULTS AND DISCUSSION

Forage quality of whole plant

Forage quality of switchgrass in monoculture and intercropping with hybrid poplar was examined during 2012, 2013, and 2014 growing seasons. All main effects were significant, while cropping system × cultivar was insignificant (Table 1). Therefore, means were separated into years, cropping systems, and growth stages, and were pooled over three switchgrass cultivars (Figure 1 and 2).

Forage quality was similar between grass monoculture plots and intercropped plots in the first production year and displayed a difference in forage quality between the cropping systems in the second production year (Figure 1). Crude protein averaged over the cropping systems was

159 g kg-1 in July 2012. However, CP under intercropping was 8 g kg-1 and 41 g kg-1 greater than CP in monoculture in July 2013 and 2014, respectively (Figure 1). The higher CP values under intercropping were also observed in other months, where the differences were significant

(Figure 1). The tissue concentration of CP declined toward fall, and the magnitude of CP reduction was higher with swtichgrass in monoculture plots than intercropped plots. Rapid growth of switchgrass in monoculture plots utilized available N quickly as compared to the switchgrass in intercropped plots. By contrast, sugar levels of switchgrass in intercropped plots were 17 % lower on average than the switchgrass in monoculture plots. Photosynthetic rate may be slower in intercropped plots with low intensity of PAR than monoculture plots (Bidwell,

1974), resulting in the production of lower concentration of sugar in switchgrass in intercropped plots. After the summer solstice (June 21st) when the growth of short day plants accelerates,

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grass in monoculture displayed 3-5 % greater fiber levels than switchgrass in intercropped plots.

Generally, the nutritive values of warm-season grasses decreases from spring to summer as NDF and lignin concentrations increase (Jung and Vogel, 1992; MacAdam et al., 1996). Sugar and lignin tissue concentrations strongly depend on time of harvest (Waramit et al., 2011), plant maturity, (Grabber et al., 1991; Adler et al., 2006; Dien et al., 2006; Mann et al., 2009), plant component (e.g., stem and leaf), and location within a stem (e.g., basal or apical) (Sarath et al.,

2007). For example, lignin and NDF concentration in switchgrass increased over winter from

10 % to 33 % and 5 % to 14 %, while sugar concentration decreased due to the leaching of soluble sugars (Adler et al., 2006). The higher CP and lower fiber in intercropped plots indicate that forage quality was higher in intercropped plots than monoculture plots. The reason for higher forage quality in intercropped plots may be due to the slower growth of switchgrass due to shading by the hybrid poplar trees, delaying maturity (Kephart and Buxton, 1993; Buxton and

Fales, 1994; Lin et al., 2001; Lindgren and Sullivan, 2014) since forage quality is inversely related to the maturity (Kephart and Buxton, 1993). As discussed in the Chapter Five, the field was over-irrigated for optimal switchgrass production but supplied to meet the requirements of hybrid poplar. With excess irrigation, soil pores were filled with water, and the field was compacted to at bulk density of 1.98 g/cm3 by equipment conducting field operations. Although the situation was similar for monoculture plots, we propose that evaporation rates was higher in monoculture plots without the shading formed by tree canopy, which allowed switchgrass maturity in monoculture to occur faster than the maturity of switchgrass in intercropped plots.

Tissue concentrations of macronutrients were similar between cropping systems until

July 2013, after which macronutrient concentrations between cropping systems were different

(Figure 2). Under monoculture cropping, concentrations of P, K, Mg, and Ca declined as

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maturity proceeded from 3.3 g kg-1, 29.2 g kg-1, 2.8 g kg-1, and 5.2 g kg-1 in August, to 2.9 g kg-1,

24.9 g kg-1, 2.9 g kg-1, and 4.6 g kg-1 in September, and to 2.2 g kg-1, 19.1 g kg-1, 2.3 g kg-1, and

3.8 g kg-1 in October 2013, respectively (Figure 2). Similar seasonal changes in concentrations of macronutrients have been reported (Lemus et al., 2009; Kimura et al., 2015). Under intercropping, concentrations of K, Mg, and Ca ranged from 27.9 g kg-1, 30 g kg-1, and 50 g kg-1 in August, to 26.2 g kg-1, 3.4 g kg-1, and 5.2 g kg-1 in September, and to 26.5 g kg-1, 3.5 g kg-1, and 5.5 g kg-1 in October 2013, respectively. Although P concentration decreased toward fall, the magnitude of the reduction was small as compared to monoculture cropping. In intercropped plots, internode production was reduced by 55 % from July to October (Figure 4 in Chapter five).

With the slow growth of switchgrass under intercropping, switchgrass did not utilize the macronutrients effectively. As a consequence, mobilization of these nutrients toward the lower stem and roots were delayed. In addition, it is possible that damage to the root system caused by compacted soil did not transfer the nutrients downward to roots because mobilization of nutrients in plant requires transpiration streams (Lemus et al., 2009). Macronutrient concentrations of spring growth were similar in May and June of 2012 and 2013; however, the macronutrient concentrations were higher in intercropped than monoculture plots in the spring of 2014 (Figure

2). Spring growth of switchgrass under intercropping may be delayed compared to switchgrass in monoculture plots in 2014, resulting in a younger maturity stage as well as higher macronutrient concentrations in May of 2014 under intercropping. Fast growing spring tillers grow from apical meristems formed in the fall of previous year. In addition, the energy required to grow spring growth tillers is generated from C contained in lower stems and roots stored from previous year, as photosynthetic activity is low in early spring (Iqbal et al., 2012; Jing et al.,

2012). Sugar concentrations of switchgrass under intercropping is 34 g kg-1 compared to 62 g

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kg-1 in monoculture with 45 % lower sugar under intercropping (Figure 1). This explains the delayed spring growth of switchgrass under intercropping in 2014.

Forage quality by plant component

Forage quality attributes for each plant component (e.g., leaf blades and stems) are summarized in Table 3 and 4. Effect of year, plant component, plant component × cropping system, and plant component × cultivar were significant (Table 2). However, data were presented by year × plant component × cropping system, and means over cultivars were pooled to emphasize in effects of intercropping on forage quality by plant component.

The CP in leaf and ranged from 90 to 150 g kg-1 and 30 to 80 g kg-1, respectively, with

45 % higher CP observed in leaf than stem (Table 3). Structural carbohydrates, such as NDF,

ADF, and lignin, were 9 %, 10 %, and 35 % higher in stems than leaves (Table 3). This is commonly observed for fiber components in the stem and leaf (Lemus et al., 2009). The CP in stems decreased from 70 g kg-1 in spring to 40 g kg-1 in fall in 2013, while the opposite was observed in leaf CP concentrations that ranged from 100 g kg-1 in the spring to 120 g kg-1 in the fall (Table 3). Maturity of plants toward fall enhanced thickening and extension of internodes, increasing fiber concentration in stems and mobilization of N in leaf. Accumulations of NDF,

ADF, and lignin were found in stem with 720 g kg-1, 430 g kg-1, and 40 g kg-1 in the spring to

730 g kg-1, 470 g kg-1, and 50 g kg-1 in the fall 2013, while increases of fiber were smaller in the leaf tissue (Table 3). Griffin and Jung (1983) observed that concentrations of NDF and lignin in the stems increased with maturity and was greater than leaves. Sugar concentrations in leaf and stem tissues declined by 43 % and 40 % from July 2012 to July 2013, respectively, indicating that photosynthetic rate decreased from 2012 to 2013. Translocation of sugar from source to

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sink tissue was evidenced in the reduction of sugar concentrations in leaf from 36 g kg-1 in July to 27 g kg-1 in October, 2013. However, sugar concentration in stem under intercropping was twice that of stem under monoculture in October, 2013. According to Link (1959), the direction of movement of photosynthate varies by leaf age, where older leaves export photosynthate downwards to roots, while younger leaves export photosyntate toward the younger growing tip meristems. The slow growth rate under intercropping retained higher numbers of younger leaves than switchgrass in monoculture; therefore, sugar may remain in upper plant tissue rather than moving toward the lower stem and roots. The CP in leaf was 9 % and 20 % higher in intercropped plots than monoculture plots in July 2013 and 2014, respectively (Table 3). In contrast, NDF concentration of leaf tissue in intercropped plots was 3 % lower than monoculture plots in July 2014. The higher CP and lower fiber suggests that switchgrass leaves in intercropped plots are at younger stage than leaves in monoculture plots. However, CP and fiber of stems under intercropping followed the opposite trend from leaves. Stem CP concentration was 20 % higher in monoculture than intercropping in July 2013, while concentrations of NDF,

ADF, and lignin are 3 %, 2 %, and 12 % lower in monoculture than intercropping. Intercropping increased fiber content of stem tissue, possibly due to stresses from excessive water and microclimates formed under the hybrid poplar canopy. Lignification is directly or indirectly influenced by temperature, soil moisture, light, and soil fertility (Buxton and Casler, 1993;

Nelson and Moser, 1994). Warm temperature normally increases lignification due to the high activity of lignin synthetic enzymes (Buxton and Fales, 1994). In some cases, lignification is associated with increased plant partitioning (Cone and Engels 1990). In our case under intercropping, temperature may be cooler in intercropped plots with increased shading than monoculture plots; however, fiber components were increased under intercropping than

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monoculture plots. It is possible that small amounts of soil N was taken up by switchgrass roots due to the increased denitrification, enhanced N volatilization under anaerobic soil condition, and damaged root systems under excess water application. The majority of N may be mobilized to the leaves for storage. It was reported that stressed perennial plants mobilize N to storage organs

(Heckathorn and Delucia, 1994).

Macronutrient concentrations in leaf averaged over years were 2.6 and 2.7 g P kg-1, 22 and 24 g K kg-1, 2.6 and 3.0 g Mg kg-1, and 4.8 and 4.8 mg Ca kg-1 under monoculture and intercropping, respectively (Table 4). Switchgrass leaves under intercropping displayed higher concentrations of macronutrients than switchgrass in monoculture due probably to the long- lasting vegetative stage under intercropping as a result of slow growth. On the other hand, concentrations of P, K, and Ca in stem were similar except for the second harvesting in October,

2013, where the concentrations were 21 %, 31 %, and 36 % higher in intercropped than monoculture. Therefore, the reduction of macronutrients observed in the whole plant toward fall

(Figure 2) is mainly attributed to the decline in stem macronutrient concentrations rather than macronutrients in leaf tissues. Nutrient mobilization is governed by sink tissue, metabolic activity, and/or nutrient deficiency, and is dependent on the strength of the sink (Bidwell, 1974).

Root development of switchgrass in monoculture may have been more vigorous than intercropped plots, resulting in a greater concentration of macronutrients moved toward lower stem and roots.

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CONCLUSION Forage quality of switchgrass under intercropping with hybrid poplar was examined by whole plant and by plant component from 2012 to 2014 growing seasons under irrigation. The excess amount of irrigation water applied to the field inhibited switchgrass root growth, which resulted in slow growth of switchgrass, especially under the intercropping system. Switchgrass developed different quality in the second production year between the cropping systems. Higher

CP and macronutrient concentrations and lower fiber concentrations indicated that forage quality under intercropping was higher due to the younger maturity stage than monoculture. Indication of slower photosynthetic rate was observed from lower sugar concentration under intercropping.

Forage quality by plant component revealed that stresses such as low PAR along with soil compaction and low evaporation rates, accumulated fiber in stem tissues. Excess irrigation water applied to the field inhibited the root development of switchgrass. With the difficulties to satisfy water requirement of both herbaceous and tree crops, improving the irrigation system to a drip- irrigation system along the tree lines may reduce the standing water in the switchgrass plots.

This change in irrigation setting may improve soil quality by reducing soil compaction and anaerobic condition; therefore improving root development of switchgrass. Under the new proposed system, it is possible to examine the influence of reduced PAR by four years old hybrid poplar canopy to switchgrass forage quality.

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Table 1. Analysis of variance for forage quality attributes of whole plant by growth stages for three cultivars of switchgrass grown

alone or intercropped with hybrid poplar during 2012 to 2014 study years

CP Sugars NDF ADF P K Mg Ca Lignin Year (Y) ** *** *** *** *** ** ** *** *** Growth (G) *** *** *** *** *** *** * *** *** Cropping system (C) ** *** * NS *** ** ** * NS Cultivar (Cul) * NS * ** ** * * * NS Y*G NS * * * * ** * * ** Y*C * ** NS NS * * *** * NS Y*Cul NS NS NS NS * NS NS NS NS G*C ** * ** * *** ** ** *** NS G*Cul * NS NS NS NS * NS NS * C*Cul NS NS NS NS NS NS NS NS NS Y*G*C NS NS NS NS NS * NS NS *

136 Y*C*Cul NS NS NS NS NS NS NS NS NS

G*C*Cul NS NS NS NS NS NS NS NS NS Y*G*C*Cul NS * NS NS NS NS NS NS NS ∗, ∗∗, ∗∗∗ indicate significance at p < 0.05, p < 0.001 and p < 0.0001, respectively.

Table 2. Analysis of variance for forage quality attributes for plant component (stem and leaf) for three cultivars of switchgrass grown

alone or intercropped with hybrid poplar during 2012 to 2014 study years

CP Sugars NDF ADF P K Mg Ca Lignin Year (Y) ** *** *** *** ** ** *** ** *** Plant component (P) *** * *** *** *** ** * *** *** Cropping system (C) NS NS NS NS NS NS NS NS * Cultivar NS NS NS ** * * NS NS NS Y*P ** *** * *** *** ** *** *** *** Y*C NS * NS * NS NS * NS NS Y*Cul NS NS NS NS NS NS NS NS NS P*C * * NS * * * NS NS NS P*Cul * * * * * * NS NS NS C*Cul NS NS NS NS NS NS NS NS NS Y*P*C NS NS * * * ** NS ** **

137 Y*C*Cul NS NS NS NS NS NS NS NS NS

P*C*Cul NS NS * NS NS NS * NS NS Y*P*C*Cul * NS NS NS ** ** * NS * ∗, ∗∗, and ∗∗∗ indicate significance at p < 0.05, p < 0.001 and p < 0.0001, respectively.

Table 3. Crude Protein (CP), sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin of leaf and stem of

switchgrass in grass monoculture (Mono) and intercropped with hybrid poplar (IC) from June, 2012 to July, 2014

CP Sugar NDF ADF Lignin Leaf g kg -1 IC Mono IC Mono IC Mono IC Mono IC Mono 2012 July 90 c 88 d 62 a 63 a 682 a 673 a 388 b 387 ab 287 b 275 a 2013 July 104 bc ** 91 c 35 c 36 b 676 a 379 b 395 b 402 a 257 c 251 ab Oct. 113 b 119 b 19 d 27 c 676 a 346 b 427 a 393 ab 321 a 247 b 2014 July 153 a *** 120 a 43 b 42 b 612 b ** 633 a 350 c *** 378 b 246 c 236 b

Stem IC Mono IC Mono IC Mono IC Mono IC Mono 2012 July 56 c 56 b 62 a 66 a 750 a * 736 a 416 c 412 c 338 c 325 c 2013 July 64 b * 84 a 37 bc 39 c 730 b * 709 b 439 b * 429 b 410 b ** 362 b Oct. 49 c 31 c 33 c *** 64 a 735 ab 734 a 478 a * 453 a 561 a 529 a 2014 July 85 a 77 a 41 b ** 48 b 702 c 693 b 425 c * 412 c 397 c *** 335 c

138 ∗, ∗∗, and ∗∗∗ indicate that mean between the cropping systems are significantly different at p < 0.05, p < 0.001 and p < 0.0001,

respectively.

Table 4. Tissue concentration of phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca) of leaf and stem of switchgrass in

grass monoculture (Mono) and intercropped with hybrid poplar (IC) from June, 2012 to July, 2014

P K Mg Ca Leaf ------g kg-1------IC Mono IC Mono IC Mono IC Mono 2012 July 2.3 c 2.2 c 21.6 b 20.8 b 2.2 c 2.1 c 4.0 b 4.0 c 2013 July 2.8 b ** 2.5 b 23.1 b ** 21.5 ab 2.7 b 2.5 b 5.2 a 5.2 ab Oct. 2.8 b 2.7 a 25.4 a 22.8 a 3.4 a * 3.0 a 4.8 a 5.4 a 2014 July 3.0 a ** 2.7 a 24.7 a * 23.1 a 3.5 a * 2.9 a 5.0 a 4.8 b

Stem IC Mono IC Mono IC Mono IC Mono 2012 July 2.2 c 2.2 b 19.7 b 19.6 b 2.0 d 2.1 b 2.1 c 2.3 b 2013 July 2.5 b 2.7 a 21.7 a 22.3 a 2.6 c 2.9 a 3.2 a 3.7 a

139 Oct. 2.3 c *** 1.8 c 20.1 b *** 13.8 c 3.0 b *** 1.6 c 2.7 b * 1.7 c

2014 July 2.6 a 2.6 a 21.3 a 22.6 a 3.5 a * 2.8 a 3.5 a 3.3 a ∗, ∗∗, and ∗∗∗ indicate that mean between the cropping systems are significantly different at p < 0.05, p < 0.001 and p < 0.0001, respectively.

Figure 1. Crude Protein, soluble sugar, neutral detergent fiber, acid detergent fiber, and lignin of switchgrass in grass monoculture ( ) and intercropped with hybrid poplar ( ) from

June, 2012 to July, 2014. * indicates that means between cropping systems are significantly different at p = 0.05.

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Figure 2. Tissue concentration of phosphorus, potassium, magnesium, and calcium of switchgrass in grass monoculture ( ) and intercropped with hybrid poplar ( ) from

June, 2012 to July, 2014. * indicates that means between cropping systems are significantly different at p = 0.05.

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CHAPTER SEVEN

BIOMASS PRODUCTION AND NUTRIENT REMOVAL BY SWITCHGRASS

(PANICUM VIRGATUM) UNDER IRRIGATION

ABSTRACT

Switchgrass was identified to supply a major portion of U.S. energy needs when used as a fuel. Assessments of the export of essential plant nutrients are needed to determine impacts on soil fertility that will influence fertilizer recommendations since the nutrients contained in the above ground biomass will be removed from the field when processed as a bioenergy feedstock.

The objective of this study was to determine the influence of N fertilization rates (112 and 224 kg N ha-1 yr-1) on the biomass production, nutrient removal, and nutrient concentration of switchgrass grown under irrigation in the lower Columbia Basin of the Pacific Northwest.

Aboveground biomass yields averaged over five years of 23.0, 19.9, and 17.7 Mg DM ha-1 y-1 for

Kanlow, Shawnee, and Cave in Rock, respectively. Kanlow cv. increased biomass yield at the

224 kg ha-1 N rate compared to two upland cultivars. The annual removal of macronutrients from the field averaged 237 kg N ha-1, 37 kg P ha-1, 326 kg K ha-1, 15 kg S ha-1, 68 kg Ca ha-1, and 43 kg Mg ha-1 among cultivars. Switchgrass produced 93 kg of biomass per 1 kg of N, and S may play a key role in maintaining the yield under a high N regime. Micronutrients removed at harvest averaged less than 1 kg ha-1, while Fe removal was 3.4 kg ha-1. Increased yields and enhanced uptake of essential nutrients especially after reaching stand maturity in the third year resulted in high nutrient removal with the biomass harvest.

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INTRODUCTION

Perennial herbaceous plants can improve soil quality, enhance nutrient cycling, improve wildlife habitat, and sequester C (Lemus and Lal, 2005). Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that is native to the ecosystem east of the Rocky Mountains (Vogel,

2004). It is established with seeds and is categorized into two ecotypes (e.g., lowland and upland) that are separated by chloroplast DNA polymorphism (Hulquist et al., 1996).

Switchgrass has potential as a bioenergy crop due to its high above- and below-ground biomass, ability to grow on marginal lands and seemingly low demand for agricultural inputs

(McLaughlin and Kszos, 2005; Collins et al., 2010). Amount of C sequestered by switchgrass ranged from 2.4- 4.0 Mg C ha-1 yr-1 under rain-fed conditions at the 90-cm depth (Lee et al.,

2007), and 3.9 Mg C ha-1 in three-year old stand under irrigation (Collins et al., 2010).

Switchgrass enhances nitrogen mineralization and increases microbial biomass carbon in the top

15 cm of soil at a higher rate than annual crops (Chatterjee et al., 2013). Use of this perennial warm-season grass may address issues around energy security and mitigate increasing CO2 concentration in atmosphere.

Fertility management plays a key role in optimizing yield and cell wall composition for high quality energy biomass (Allison et al., 2012). Biomass production can be maximized for high energy yield per given land area with proper management. To maintain high yielding switchgrass stands nutrients removed by harvest need to be replaced. Fertility assessments on switchgrass have shown that N applications increased biomass yields in rain-fed environments

(Stroup et al., 2003; Lemus et al., 2008; Garten et al., 2011; Guretzky et al., 2011; Nikiema et al.,

2011, Allison et al., 2012; Kering et al., 2012b; Sadeghpour et al., 2014) and under irrigation

(Pedroso et al., 2013). Whereas, other research of N application rates have shown no influence

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on yield under rain-fed condition (Christian et al., 2002; Thomason et al., 2005; Jung and Lal,

2011; Kering et al., 2012a) depending on the cultivar planted, location, and harvest frequency.

Harvest frequency not only influences switchgrass biomass yield, but also determines nutrient removal and their concentration in plant tissues (Reynolds et al., 2000; Lemus et al., 2009;

Guretzky et al., 2011). Due to the higher leaf biomass during summer, higher nutrient removal has been observed in summer than fall harvests. Concentration of N, P, K, and Ca during midsummer and late fall averaged 10.8 and 6.4 g N kg-1, 1.8 and 1.1 g P kg-1, 12.7 and 5.1 g K kg-1 and 3.3 and 3.7 g Ca kg-1 at seven locations, in the upper Southeastern U.S., respectively, when a split application of 50 kg of N (34-0-0) was applied in May (Lemus et al., 2009).

Concentrations of P, K, S, and Ca in switchgrass straw from five locations in the Midwest and

South ranged 1.3 - 6.4 kg P Mg-1, 6.2 - 15.8 kg K Mg-1, 0.5 - 1.1 kg S Mg-1, and 2.3 - 5.3 kg Ca

Mg-1, respectively when the stand was harvested once in late summer with a spring application of

225 kg N ha-1 (El-Nashaar et al., 2009). Concentrations of macronutrients (S and Ca) and micronutrients (Cl, Al, and Si) varied widely by location and environment (El-Nashaar et al.,

2009). Despite many fertility studies on switchgrass, few studies have been conducted west of

Rocky Mountains and in the Pacific Northwest (PNW) under irrigation. Assessments of the extent to which essential plant nutrients (N, P, K, S, and micronutrients) would be exported from the soil system are needed. The objective of this study was to determine the biomass production, nutrient concentration, and response of switchgrass to N fertilization grown under irrigation.

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MATERIALS AND METHODS

This study was conducted at the USDA-ARS Integrated Cropping Systems Research

Field Station near Paterson, Benton County, WA (45o 56’ N, 119o 29’ W; 114 m above sea level) on a Quincy sand (Xeric Torripsamments) containing 4 g kg-1 organic C (11 g kg-1 organic matter) (Table 1). The study area was previously in a native shrub-steppe plant community that was converted to an irrigated agricultural field in 2002. The shrub-steppe was a portion of the semi-arid, shrub- and bunchgrass-dominated region in the western United States that stretches from British Columbia, Canada, to Mexico (Rogers and Rickard, 1988). The area is characterized by annual average precipitation of 178 mm, mostly occurring as rain/snow mix during winter months (Table 2). Selected physical and chemical characteristics of the soil are presented in Table 1. The surface soil (0-10 cm) has a bulk density of 1.33 kg m-3 and 917 and 56 g kg-1 of sand and silt, respectively. Total soil organic C and N were analyzed by dry combustion on a LECO, CNS-2000 Elemental Analyzer (St. Joseph, MI). Soil pH was determined using the

2:1 water method of Robertson et al. (1999).

Three switchgrass (Panicum virgatum, L) cultivars Kanlow (USDA-NRCS, 2011a),

Shawnee (Vogel et al., 1996), and Cave in Rock (USDA-NRCS, 2011b) were established in

2004 under solid set irrigation with an annual in-season (May-September) supplemental application of 670 mm irrigation water as 79 mm of rainfall from May to September is not sufficient to grow switchgrass (Table 2). The experimental design was a split-plot in randomized complete blocks with five replications (7.6 × 7.6 m plots). The main plot consisted of switchgrass cultivar, the sub-plot fertilization rate. In early April 2004, prior to planting each plot,

-1 -1 received a blended dry granular fertilizer containing 112 kg N ha , 64 kg P2O5 ha , 220 kg K2O ha-1, 22 kg S ha-1, and 1.3 kg B ha-1 applied with a tractor using a Valmor™ spreader. Each

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cultivar was planted at a seeding rate of 11 kg ha-1 of pure live seed on 01 June 2004 with a

Carter cone seeder (Brookston, IN). When grasses were four to five leaves, atrazine (2-chloro-4- ethylamino-6-isopropylamino-s-triazine) was applied at 10.2 g eq/ha post-emergence. As needed

2,4-D (dimethylamine salt of 2,4-Dichloro-phenoxyacetic acid) was applied at 8.1 g eq/ha for broad leaf weed control. In the establishment year, plots were also mowed when weeds, escaped herbicide treatment, and were greater than 15-cm tall. In subsequent years, to control winter annuals, glyphosate [N-(phosphonomethyl) glycine] was applied at 13.0 g eq/ha in late March, prior to grasses breaking winter dormancy. Fertilizer treatments consisted of two split N application rates, one ‘low’ of 56 kg N ha-1 (for a total annual rate of 112 kg N ha-1y-1) the second treatment ‘high’ at 112 kg N ha-1 per application (for a total annual rate of 224 kg N ha-1y-1). The first application occurred in May prior to breaking winter dormancy and the second in July following the first of two annual harvests. Nitrogen and S sources were urea (46-0-0) and ammonium sulfate (21-0-0-26) the P source was mono-ammonium phosphate (11-52-0) and the

K source was potassium chloride (0-0-62).

Aboveground biomass from the center of each plot (5.6 m2) was machine harvested twice each year with a John Deere F935 tractor (John Deere Co., Moline, IL) and 0.9 m flail harvester

(University of Wisconsin at Marshfield, WI) and cut to a stubble height of 15 cm. Harvest dates were 24 June and 14 Oct. in 2005, 13 July and 10 Oct. in 2006, 11 July and 12 Oct. in 2007, 14

July and Oct. 28 in 2008, and 09 July and 06 Oct. in 2009. Dry matter concentration was determined for each plot using 0.5- to 1.0-kg sub-samples dried at 50 oC and used to adjust plot yields to a dry matter basis.

All dried aboveground sub-samples were ground through a Wiley mill (Thomas

Scientific, Swedesboro, NJ) equipped with a 1-mm screen. Total N concentration of plant

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samples (0.2 g) were determined by dry combustion on a LECO, CNS-2000 Elemental Analyzer

(St. Joseph, MI). The macro- and micro-mineral composition (P, K, S, B, Ca, Cu, Fe, K, Mg, Mn, and Zn) of plant tissues after each harvest were determined by inductively coupled plasma atomic emission spectroscopy (Isaac and Johnson, 1998) after extraction by dry ashing (Miller,

1998). Elemental composition was expressed as g kg-1 or mg kg-1 dry matter. Statistical Analyses were conducted by analysis of variance using the MIXED procedures in SAS (2008) to determine main effects and interactions of years, cultivars, fertility rates on total biomass yield

(sum of two harvests), annual nutrient removal, and nutrient concentration at each harvest. Main plots were cultivars, and sub plots were fertility rates. Years and replications were considered random effects, while cultivars and fertility rates were considered fixed effects. Least significance differences (LSD) (P=0.05) were used to separate means when F tests were statistically significant. Paired t tests was used to compare two N rates (P=0.05).

RESULTS AND DISCUSSION

Dry Matter Yield, N Removal, and N Concentrations

Switchgrass cultivars were harvested twice a year during the 2005 to 2009 growing seasons. Table 5 provides dry matter production of the three switchgrass cultivars (Kanlow,

Shawnee, and Cave in Rock) for the five years of production after stand establishment in 2004.

Switchgrass yield was influenced by year (p < 0.0001) and cultivar (p < 0.0001), and cultivar × fertility rates (p < 0.001) (Table 3); therefore, means were separated into years, cultivars, and N rates (Table 5). Above ground biomass annual yields averaged 21, 20, and 17 Mg Dry Matter

(DM) ha-1 yr-1 at the 112 kg N ha-1 rate and 25, 20, and 18 Mg DM ha-1 yr-1 at the 224 kg N ha-1

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rate for the Kanlow, Shawnee, and Cave in Rock cultivars (range: 13 to 29 Mg DM ha-1) in five years, respectively (Table 5). No statistical difference was observed on the five year average yield under low N rate, while biomass yield of Kanlow was 5.8 Mg DM ha-1 yr-1 higher than upland cultivars under high N rate (p < 0.0005). Kanlow produced the highest yield with the maximum of 29 Mg ha–1 yr–1 under high N rate in 2007 (Table 5). Yield peaked in the third year of production and gradually declined at the low N rate for all cultivars, whereas Kanlow maintained yield above 24 Mg ha-1 yr-1 following peak production in 2007 with 224 kg N ha-1.

Yield of the two upland cultivars, Shawnee and Cave in Rock displayed a lower response to N applications than Kanlow, especially after 2007. This indicates that Kanlow, a lowland cultivar, is more responsive to N fertilization, leading to higher potential yield with N application.

Biomass yields were greater in the first harvest than second for all cultivars over the five years of production, except in 2005, when the first harvest occurred two weeks earlier than the typical first harvest date for this region. Dry matter yield in this study was comparable with an irrigated switchgrass study in CA (range: 13 to 27 Mg DM ha–1 yr–1; Pedroso et al., 2013) and was better than dryland production in southeastern U.S. (Fike et al., 2006a; average: 14 Mg ha-1 yr-1).

All main effects were significant on N removal (Table 3) and N concentration (Table 4).

Values for N removal (Table 6) and concentration (Table 7) were separated into cultivars and two N rates, and presented as mean over years. Nitrogen removal and N concentration were higher under the high N rate and during the first harvest in all cultivars over the five-year study

(Table 6 and 7). Total annual N removal averaged 224, 223, and 188 kg N ha–1 yr–1 at the low N rate and 311, 247, and 229 kg N ha–1 yr–1 at high N rate for Kanlow, Shawnee, and Cave in Rock, respectively (Table 6). Application of 224 kg N ha-1 enhanced N removal by Kanlow as compared to Shawnee and Cave in Rock. The N removal is enhanced in the summer harvest

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(Sadeghpour et al., 2014), under multiple harvest system (Vogel et al., 2004; Thomason et al.,

2004; Guretzky et al., 2011; Pedroso et al., 2013), and irrigation (Pederoso et al., 2013) but under dryland system (Kering et al., 2012). The reason for the enhanced N removal may be due to high

N concentration (Sadeghpour et al., 2014), increased yield with multiple harvests (Guretzky et al.,

2011), and with irrigation (Pedroso et al., 2013). Nitrogen removal averaged over three cultivars and years was 212 kg N ha-1 under low N rate (112 kg N ha-1), depleting 100 kg N ha-1 yr-1 from the soil (112 kg N ha-1 – 212 kg N ha-1 = -100 kg N ha-1). On the other hand, averaged N removal over cultivars and years was 262 kg N ha-1 under high N rate (224 kg N ha-1), depleting 38 kg N ha-1 yr-1 from the soil. Suggesting, switchgrass utilizes and depletes soil N reserves from seasonal

N mineralization of soil organic matter. Aboveground biomass N concentrations among cultivars averaged 1.4 and 0.7 g N kg-1 for low N rate, and 1.6 and 0.9 g N kg-1 for the high N rate in the first and second harvests, respectively (Table 7). Pedroso et al., (2013) reported N concentration increased as N rate increased from 0 to 300 kg N ha-1 with 75 kg N ha-1 increments in irrigated switchgrass study in CA. Similar seasonal N change was observed in dryland switchgrass production, where N concentration declined at the second harvest (Lemus et al., 2009; Kering et al., 2012a). This could be due to the decrease in leaf DM and extension of internodes as growth declines into fall dormancy. In general, multiple harvests and split N applications results in increased N and K plant removal (Thomason et al., 2004). Phosphorus uptake is influenced by harvest frequency, rather than timing of N application (Thomason et al., 2004). Multiple harvests with spring applied N at 448 kg N ha-1 maximized yield (Thomason et al., 2004). Total N concentration was higher with split N application at 224 kg ha-1 compared to the single N application in spring (Mulkey et al., 2006).

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Macro/Micro Nutrient Removal and Concentrations

Nutrient removal and concentrations of macro- and micronutrients were determined after harvest. Although year effect was significant (Table 3 and 4), means over years were presented and annual values were reported in text where important trends were observed (Table 7).

Removal of all macronutrients by Kanlow was enhanced under high N rate. High N rate enhanced nutrient removal of N, K, S, and Mg for Cave in Rock, while P and Ca removal was not enhanced under high N rate (Table 6). Nikiema et al. (2011) also reported that addition of N had no influence on P and Ca removal by Cave in Rock. By contrast, N rates had no significant influence on macronutrient removal by Shawnee, except for P (Table 6). The P removal was 4 kg

P ha-1 higher under low N rate instead of high N rate. The higher P removal by Shawnee is explained by 0.3 kg DM ha-1 higher yield of Shawnee under low N rate. Fertility rates had no significant influence on S removal by the three cultivars of switchgrass during the first two years of production (Table 6). However, all cultivars exhibited S deficiency symptoms starting in the second growth in 2006, and high N rate enhanced S removal after the third year. Sulfur concentration under high N and low N rates were 23 g S kg-1 and 19 g S kg-1, 18 g S kg-1,and 14 g S kg-1, and 16 g S kg-1, and 12 g S kg-1 in 2007, 2008, and 2009, respectively (Table 7). Thus,

S nutrition was important for the last three years of the study. A (NH4)2SO4 fertilizer was applied, which greatly reduced the deficiency symptoms. A similar observation was found for

Mg after year 2007, where high N rates enhanced Mg removal by switchgrass (Table 6). The highest P, S, Ca, and Mg removal in 2007 corresponds to the highest yielding year of the five year study, indicating that these macronutrients are necessary to support biomass production of switchgrass. Thomason et al. (2004) observed higher K, P, and S removal when yield increased for Kanlow, indicating that increasing yield enhanced nutrient uptake for K, P, and S. Proper

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application of macronutrients may enhance switchgrass yield in fall when yield declines compared to summer harvests as switchgrass responds to macronutrients (e.g., N, P, K, S, Ca, and Mg). Concentrations of macronutrients (P, K, S, and Mg) were higher in the first harvest than second harvest, except for concentration of Ca which varied depending on year (Table 7).

Concentration of P and K over years, cultivars, and fertility rates averaged 2.3 and 1.4 g P kg-1, and 19.2 and 11.5 g K kg-1 in the first and second harvest, respectively (Table 7). Tissue concentrations of P and K declined 40 % from the first to second harvest. Concentrations of S also declined by the second harvest (0.6 g S kg-1) compared to the first harvest (0.8 g S ka-1).

Similar seasonal changes in P and K concentration have been observed (Lemus et al., 2009). The decline in macronutrient concentration may be attributed to the reduction of leafy biomass produced into fall while increasing the biomass of stems and panicles (Smart et al., 2004).

Concentration of Ca in the second harvest was always highest in Cave in Rock, an earlier flowering cultivar among the three cultivars (Vogel et al., 1996). Average Ca concentration in our study was 3.5 g Ca kg-1, as compared to 2.3 g Ca kg-1 reported under dryland condition

(Sadeghpour et al., 2014). The 1.3 g Ca kg-1 less concentration in the study of Sadeghpour et al.

(2014) may be because the three harvest system reduced yield per each harvest (5.8 Mg DM ha-1 harvest-1) as compared to two harvest system in our study (10.1 Mg DM ha-1 harvest-1). The frequent cuts allows less time for Ca to deposit in the plant as Ca is an immobile nutrient and is mainly deposited by cumulative transpiration (Biddulph et al., 1958). High N rate enhanced macronutrient removal by Kanlow compared to Shawnee and Cave in Rock, especially after the third year. Suggesting S, Mg, and Ca fertilization is needed by Kanlow after three years of production.

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Removal of Fe, B, Cu, Mn, and Zn among cultivars and across years averaged 3.8, 1.2,

0.1, 1.6, and 0.3 kg ha-1 yr-1 under the high N rate, while removal of these micronutrients under low N rates were 3.0, 1.1, 0.1, 1.1, and 0.3 kg ha-1 yr-1, respectively (Table 6). Switchgrass removed 6.3 kg Fe ha-1 on average in 2005 when soil Fe concentrations were elevated in the first year of the study. The fields were first brought under cultivation from the native shrub-steppe two years prior to planting switchgrass. Soil samples analyzed in 2005 found average soil Fe across the site was 40 mg kg-1 soil that declined to 26 mg kg -1 soil by 2009. Soil Fe became low after 2006 as no Fe supplement was added throughout the study; therefore, the Fe concentration had decreased every year. Iron concentrations were higher in the first harvest than the second harvest, and under high N rates removed higher amount of Fe, especially in Kanlow and Cave in

Rock. First harvest tissue Fe concentrations among cultivars from 2005 to 2009 averaged 637 mg kg-1, 302 mg kg-1, 92 mg kg-1, 119 mg kg-1, and 103 mg kg-1 under high N rate, and 549 mg kg-1,

269 mg kg-1, 80 mg kg-1, 123 mg kg-1, and 85 mg kg-1 under low N rate, respectively. The interaction between high N rate and Fe removal of switchgrass was also reported in dryland study in Massachusetts (Sedeghpour et al., 2014). Sedeghpour et al. (2014) reported Fe content in Cave in Rock was higher (0.03 g Fe kg-1) for N rate at 134 kg N ha-1 as compared to 0.01 g Fe kg-1 for lower N rates at 0 and 67 kg N ha-1. Iron is important in production of leaves (Monti et al., 2008) and is essential in photosynthetic processes (Hänsch and Mendel, 2009). Tissue concentration of B in the first harvest decreased over the five years, regardless of the N rates

(Table 7). On the other hand, B concentration in the second harvest was steady from 2006 to

2009. This suggests that B may be actively taken up by switchgrass from spring to the first harvest. Copper concentrations among cultivars declined steeply at the first harvest from 2005

(12 mg kg-1) to 2006 (4 mg kg-1) and kept at that level through 2009. For the second harvest, the

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amount of Cu concentration peaked in 2006 (11 mg kg-1) and decreased every year. High N rate enhanced uptake of B and Cu for Kanlow compared to the upland cultivars (Table 6). It could be due to the higher yielding characteristic of Kanlow requiring higher levels of these micronutrients compared to uplands. No substantiating literature is available that describes the interactions among N rates, switchgrass uptake of B and Cu, and ecotypes. For long term switchgrass production under high N rates, B and Cu amendments may be necessary because B and Cu are essential for synthesis of cell walls and protein, respectively (Hänsch and Mendel,

2009). High N rate increased removal of Mn in all cultivars, and no seasonal effect was observed for Mn tissue concentration. Increased root system may have enhanced Mn uptake as above ground biomass increased under high N rate. Little seasonal effect was observed for Mn concentration in switchgrass in a dryland study conducted in Oklahoma (Makaju et al., 2013).

Manganese was the only micronutrient that had no reduction in tissue concentration over years.

Concentration of Zn decreased gradually from 2005 (17 mg kg-1) to 2006 (13 mg kg-1), after which the concentration was consistent through 2009 (Table 7). Seasonal effects were observed in Zn concentration, where the first harvest showed higher values than the second harvest. Zinc showed little response to N rates. We found switchgrass removed high amount of Fe, B and Cu, and small amount of Zn as early as the second year of production. Enhanced nutrient removal under high N rate as compared to low N rate could be explained by the increased yield under high N rate demanding all nutrients to support the additional growth, while enhanced nutrient concentration under high N rate may be caused by increased yield and seasonal effect.

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CONCLUSION

Switchgrass production under irrigation in the Columbia Basin of the PNW is a viable bioenergy feedstock with aboveground biomass yields averaging over five years, 21, 20, and 17

Mg DM ha-1 yr-1 at low N rate and 25, 20, and 18 Mg DM ha-1 yr-1 at high N rates for the

Kanlow, Shawnee, and Cave in Rock, respectively. High N rate increased yield of Kanlow (> 21

Mg DM ha-1 yr-1), while low or high N input had less influence on upland cultivars of Shawnee and Cave in Rock. Higher N input increased macro- and micronutrients uptake by Kanlow over the course of this study enhancing annual biomass production over either upland switchgrass cultivars. Under irrigated conditions in the PNW at 46° N latitude, the lowland cultivar Kanlow is the superior switchgrass cultivar when managed under a two harvest per season regime.

Limited land resource should be efficiently used to produce switchgrass biomass. Land use efficiency is increased with increased biomass production per unit area under irrigation as compared to dryland production. Having both irrigated and dryland switchgrass production system will be a significant contribution to future bioenergy production system.

The N removed exceeded the amount N applied; however, the high N rate depleted less N from soil than the low N rate. The amount of N depleted from soil after each year were 38 kg N ha-1 yr-1 (e.g., 224 kg N ha-1 added minus 262 kg N ha-1 removed) under the high N rate as compared to 100 kg N ha-1 yr-1 (e.g., 112 kg N ha-1 added minus 211 kg N ha-1 removed) under low N rate system. Although Nitrogen Use Efficiency (NUE) for low N rate was higher (169 kg

DM N kg-1) than high N rate (93 kg DM N kg-1), residual soil N will be depleted steeply under low N rate. Despite the limited influence of N on yield for upland cultivars, high N input is necessary to maintain soil N in subsequent production years. In addition, switchgrass depletes micronutrients, starting as early as second year of production, indicating further research of

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switchgrass yield response to micronutrients is warranted. To maintain long-term switchgrass productivity under irrigation and a two cut system, all cultivars must be adequately fertilized to supplement the high macro and micronutrient removal rates.

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Table 1. Physical and chemical properties of the Quincy sand (Xeric Torripsamments) soil under switchgrass at the USDA-ARS Integrated Agricultural Research Field Station,

Paterson, WA

Organic Depth BD† Sand Silt Clay pH C Total N cm Mg m-3 ------g kg-1------g kg-1------0-15 1.33 917 56 27 6.6 3.7 0.37 15-30 1.54 927 52 21 6.3 1.6 0.22 30-45 1.61 936 48 16 6.4 1.2 0.15 45-60 1.60 928 48 24 7.4 0.6 0.12 60-75 1.58 948 38 12 8.1 0.3 0.10 75-90 1.6 978 14 8 8.1 0.2 0.10 †BD- soil bulk density.

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Table 2. Monthly precipitation, air temperature, and total growing degree days from 2004 to 2009

Precipitation (mm) Air temperature (°C) Growing degree days at 10°C 2005 2006 2007 2008 2009 23 yr 2005 2006 2007 2008 2009 23 yr 2005 2006 2007 2008 2009 23 yr avg avg avg Apr 15 21 8 5 7 15 12 12 11 9 11 12 135 102 103 49 95 109 May 21 24 6 6 15 24 17 16 17 17 17 16 420 375 358 382 357 346 Jun 1 18 24 12 0 17 19 20 19 19 21 19 504 543 500 467 577 499 Jul 5 0 0 0 0 7 24 25 24 23 25 23 789 810 824 739 802 742 Aug 0 1 2 5 0 5 24 23 22 22 23 23 730 713 665 677 748 711 Sep 17 5 8 2 3 10 17 18 17 17 19 18 231 449 398 410 499 442 Oct 14 17 17 3 27 16 13 11 11 11 10 11 172 115 113 116 96 126 Nov 27 27 29 16 12 24 4 5 4 6 5 5 1 22 7 16 0 11 Weather data are obtained from Washington State University AgWeatherNet at http://weather.wsu.edu/awn.php.

23 years average data are obtained from 1991 to 2013..

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Table 3. Analysis of variance and F-values for yield, macro- and micronutrients removal for three cultivars of switchgrass

during 2005-2009 study years

Macronutrients removal Micronutrients removal Variable Yield N P K S Ca Mg Fe B Cu Mn Zn Year (Y) 58*** 24*** 81*** 125*** 86*** 35*** 36*** 122*** 89*** 37*** 35*** 8.3*** Cultivar (C) 64*** 28*** 23*** 111*** 16*** 17*** 37*** 6.8** 71*** 121*** 5.2* 51*** Fertility (F) 12** 62*** 0 29*** 37*** 1 42*** 4.8* 31*** 32*** 100*** 4.7* Y*C 1.3 0.7 1.4 2.3* 3.2* 3.9* 1.2 2.6* 7*** 4.7*** 1.3 0.5 Y*F 0.6 0.5 0.4 0.2 3.1* 7.8*** 0.9 0.5 0.7 0.6 6.4*** 2.1 C*F 7.2** 8.8** 8.7** 8.3** 7** 12*** 8.8** 0.7 7.9** 13*** 1.1 5.4* Y*F*C 0.9 0.8 1 0.4 1 6.3*** 0.3 3.3* 0.4 1.1 0.2 0.9 ∗, ∗∗, ∗∗∗ indicate significance at p < 0.05, p < 0.001 and p < 0.0001, respectively.

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Table 4. Analysis of variance and F-values for macro- and micronutrient concentration for three cultivars of switchgrass during 2005-2009 study years

Macronutrient concentrations Variables N P K S Ca Mg First harvest Year (Y) 163*** 121*** 114*** 84*** 37*** 88*** Cultivar (C) 2 11*** 57*** 35*** 35*** 4.1* Fertility (F) 51*** 5.5* 4.7* 18*** 5.5* 28*** Y*C 2.6* 4.1** 2.3* 4.4*** 3.5** 1.5 Y*F 0.7 0.7 0.6 5** 0.7 1.1 C*F 0.6 0.3 0.2 0.5 0.5 0.7 Y*F*C 1.1 0.4 0.6 1.4 1.4 0.7 Second harvest Y 20*** 35*** 84*** 9.7*** 55*** 50*** C 27*** 15*** 45*** 35*** 357*** 0.9 F 93*** 28*** 45*** 15** 57*** 46*** Y*C 3* 2.8* 2.6* 1.7 42*** 1.2 Y*F 4.5* 0.9 1.7 3.6* 41*** 2.5* C*F 0.6 1.7 2.9* 1.6 64*** 1.7 Y*F*C 1 0.3 1.7 1.1 39*** 1.5 Micronutrient concentrations Fe B Cu Mn Zn First harvest Year (Y) 193*** 505*** 429*** 22*** 41*** Cultivar (C) 2.3 37*** 56*** 12*** 34*** Fertility (F) 0.4 10** 7* 53*** 1.1 Y*C 1.7 10*** 7.4*** 3* 1.1 Y*F 0.5 1.6 1.1 5.7** 1.7 C*F 0.3 0.4 2.2 0.1 1.1 Y*F*C 0.8 2.3* 0.7 0.4 0.8 Second harvest Y 95*** 78*** 203*** 16*** 18*** C 0.8 1.3 60*** 12*** 18*** F 2.1 23 26*** 75*** 1.3 Y*C 5.6*** 3.3* 9.1*** 1.5 1.3 Y*F 0.7 0.5 0.7 5** 1.8 C*F 8.4** 1.9 7** 0.5 0.6 Y*F*C 6.5*** 1.7 2* 0.7 2.6* ∗, ∗∗, ∗∗∗ indicate significance at p < 0.05, p < 0.001 and p < 0.0001, respectively.

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Table 5. Aboveground biomass DM (dry matter) under two N rates for three switchgrass cultivars during the 2005-2009 study years

Cultivar Fertilizer Rate 2005 2006 2007 2008 2009 Average Low N Rate 112 kg N ha-1 ------Mg DM ha-1------Kanlow 17.0 a*† 20.9 a 27.3 a 21.5 a 19.8 a 21.3 Shawnee 15.8 b 18.0 b 24.7 a 22.1 a 19.6 a 20.0 Cave in Rock 12.7 b 14.2 c 20.9 b 18.3 b 18.9 a 17.0* High N Rate 224 kg N ha-1 Kanlow 21.0 a‡ 22.6 a 28.9 a 26.9 a 24.4 a 24.8 a Shawnee 15.1 b 18.4 ab 23.9 b 20.8 b 20.3 a 19.7 b Cave in Rock 13.9 b 16.7 b 21.4 b 19.6 b 20.0 a 18.3 b † Significance (*) compares an individual cultivar between low N rate and high N rate at p < 0.05. ‡ Values within a column within a fertilizer rate and year followed by the same letter are not significantly different at p < 0.05.

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Table 6. Nutrient removal under two N rates for three switchgrass cultivars during 2005-2009 study years

Cultivar Fertilizer Rate N P K S Ca Mg Fe B Cu Mn Zn Low N Rate 112 kg N ha-1 ------kg ha-1------Kanlow 224 a*† 39* 357 a* 14* 57 b* 43 a* 3.18 1.18 a* 0.12 a* 1.12* 0.32 a Shawnee 223 a 38* 316 a 13 66 ab 40 a 3.06 1.10 a 0.09 b 0.96* 0.22 b Cave in Rock 188 b* 35 248 b* 15* 78 a 35 b* 2.88 0.88 b* 0.07 c* 1.18* 0.22 b High N Rate 224 kg N ha-1 Kanlow 311 a‡ 44 a 434 a 20 69 57 a 4.88 1.42 a 0.17 a 1.70 0.32 a Shawnee 247 b 34 b 321 b 14 66 43 b 3.18 1.10 b 0.09 b 1.42 0.24 b Cave in Rock 229 b 34 b 282 b 18 70 40 b 3.32 0.98 b 0.09 b 1.60 0.20 b † Significance (*) compares an individual cultivar between low N rate and high N rate at p < 0.05. ‡ Values within a column within a fertilizer rate and year followed by the same letter are not significantly different at p < 0.05.

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Table 7. Nutrient concentration under two N rates for three switchgrass cultivars during 2005-2009 study years

Cultivar Fertilizer Rate N P K S Ca Mg Fe B Cu Mn Zn ------g kg-1------mg kg-1------

Low N Rate 112 kg N ha-1 First harvest † Kanlow 1.4* 2.2 21.0 a 0.8 ab* 2.9 b 2.4* 215 71* 7 57 b* 16 a Shawnee 1.5* 2.3* 18.5 b 0.7 b 3.2 ab 2.3 220 68 5 54 b* 13 b* Cave in Rock 1.4* 2.4 17.1 b* 0.9 a* 3.5 a* 2.2* 229 61 5* 71 a* 15 a Second harvest Kanlow 0.8 a* 1.5* 10.8 a* 0.5 b* 2.6 b* 1.7* 149 40* 6* 50 b* 13 a Shawnee 0.7 a* 1.4* 10.6 a 0.5 b 3.3 b 1.8* 126* 41* 5 46 b* 11 b

Cave in Rock 0.6 b* 1.4* 8.6 b* 0.6 a* 6.0 a* 1.8* 147 41 4* 63 a* 11 b Harvest dates‡ *** *** *** *** * *** NS *** NS * ***

-1 High N Rate 224 kg N ha First harvest Kanlow 1.6 2.2 21.4 a 0.9 ab 3.0 c 2.6 70 76 7 78 16 a

167 Shawnee 1.6 2.2 19.2 b 0.8 b 3.4 b 2.4 67 70 5 77 12 b

Cave in Rock 1.6 2.4 18.0 b 1.0 a 3.8 a 2.5 72 64 6 94 15 a Second harvest Kanlow 1.0 a§ 1.4 a 14.3 a 0.6 b 2.6 c 2.0 58 46 8 a 74 13 a Shawnee 0.9 a 1.2 b 12.9 ab 0.5 b 3.4 b 1.9 60 45 5 b 77 11 b Cave in Rock 0.7 b 1.2 b 11.9 b 0.7 a 4.2 a 2.0 68 43 4 b 93 12 ab Harvest dates *** *** *** *** NS *** *** *** NS NS ***

† Significance (*) compares an individual cultivar between low N rate and high N rate within the same harvest date at p < 0.05. ‡ Significance compares first harvest and second harvest over cultivars. ∗, ∗∗, and ∗∗∗ indicate significance at p < 0.05, p < 0.001 and p < 0.0001, respectively. § Values within a column within a fertilizer rate and harvest date followed by the same letter are not significantly different at p < 0.05.

CHAPTER EIGHT GENERAL CONCLUSION Switchgrass (Panicum virgatum L.) is a perennial warm-season grass that has been selected as an important lignocellulosic feedstock to support bioenergy production. However, the establishment is difficult due to the high amount of dormancy seeds that produce sporadic seedling emergence in the field. Freeze-thaw treatments used in this study, one hour of freezing followed by one hour of thawing for one cycle, were not effective in breaking seed dormancy of swtichgrass. The extreme temperature fluctuations may have damaged the seed coat. Longer treatment hours for freezing and thawing may have more accurately simulated the early spring soil conditions. Germination response to freeze-thaw treatments varied widely among seed lots within a cultivar. Environment during seed production may have created the large variation determined among seed lots. In addition, the maternal environment may be reflected to the number of seed g-1. Freezing storage of switchgrass seeds showed that there were significant storage temperature × storage length interactions on germination and rate of seedling emergence, while there were storage length influenced on shoot and root DM. Seeds harvested in the fall can be safely stored at freezing condition until following spring for planting, during which seedling vigor of switchgrass may be enhanced in terms of increased germination percentage, seedling emergence rate, and shoot and root DM. Accumulated yield of switchgrass from 2012 to 2013 showed that 36.1 Mg DM ha-1 under intercropping and 32.2 Mg DM ha-1 under grass monoculture with LER of 1.7. Forage quality of switchgrass was higher under intercropped plots due to the younger maturity stage of the switchgrass growth. Fiber accumulation was found from switchgrass stem under intercropping. We propose that the amount of PAR under five years hybrid poplar had no influence on switchgrass growth as the vigorous switchgrass growth along tree lines. The reduced switchgrass growth may be attributed to the water stress, field

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compaction, and reduced evaporation rates under the switchgrass canopy. Although switchgrass growth was stressed due to the excessive irrigation water adjusted for the maximum growth of poplar, the LER showed that intercropping of switchgrass and hybrid poplar increased land use efficiency as compared to switchgrass grown alone.

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