FABA BEAN GROWTH RESPONSE TO SOIL TEMPERATURE

AND NITROGEN

By

JOLENE ELISE MWENGI

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

MASTER OF SCIENCE IN SOIL SCIENCE

WASHINGTON STATE UNIVERSITY Department of Crop and Soil Sciences

May 2011

ii

To the Faculty of Washington State University: The members of the Committee appointed to examine the thesis of JOLENE ELISE

MWENGI find it satisfactory and recommend that it be accepted.

___________________________________ William L. Pan, Ph.D., Chair ___________________________________

Clarice J. Coyne, Ph. D. ___________________________________ Jinguo Hu, Ph.D.

___________________________________ Lynne Carpenter-Boggs, Ph.D.

iii

ACKNOWLEDGMENTS

Funding for this project was provided by a USDA Horticulture Crops Evaluation award,

USDA ARS CRIS Project 5348-21 000-026-00D, Washington State University

Agricultural Research Center Project 0373, and Washington State University Department

of Crop and Soil Sciences. Assistance from Landon Charlo, Wayne Olson, Kurt Tetrick,

Anne Kennedy, and Ron Bolton were greatly appreciated.

iv

FABA BEAN GROWTH RESPONSE TO SOIL TEMPERATURE

AND NITROGEN

Abstract

by Jolene Elise Mwengi, M.S.

Washington State University

May 2011

Chair: William L. Pan

The Palouse is known for dryland wheat production. Limited rainfall combined

with a Mediterranean distribution pattern limit what can be grown successfully. Fall

planted faba bean is grown commercially in Mediterranean climates where cold

temperatures are not limiting as well as in colder regions of Northern Europe where

excess moisture is more limiting than drought. A variety trial for fall planted faba beans

including 43 genotypes from the National Plant Germplasm System and 12 cultivars and

breeding lines previously identified as winter-hardy in European trials identified 13

genotypes as winter-hardy with high yield potential

Frost resistance in leaf tissue was found to correlate significantly to both plant

survival and seed yield, but low R2 values indicated winter-hardiness required additional

stress tolerances. Comparative soil temperature minimums between site-years were found

to better correlate to plant survival than ambient temperature minimums frost resistance

in root tissue may be an important genetic factor contributing to winter-hardiness.

Faba beans are known to have a high capacity to fix nitrogen (N) over a wide

range of climates and growing conditions. Tolerance to available soil nitrate may play a

v

role in greater rates of N2 fixation. A growth chamber investigation of nodulation

response to nitrate-N revealed that while pea (Pisum sativum) showed a negative

response in nodule number to increasing soil nitrate-N up to 132ppm at planting, faba

bean (Vicia faba L.) did not. At low levels of soil nitrate-N, less than 15 ppm, nodules for

both pea and faba bean appeared greater in size and pink in color when compared to

nodules formed at soil nitrate-N above 45ppm. Further work comparing tolerance to

nitrate-N for overall N2 fixation should target nodule growth and active fixation subjected

to soil nitrate-N ranging from 10 to 50 ppm with growing conditions supporting long

term nodule growth. This research supports that nodulation in faba bean does show

superior tolerance to soil nitrate-N.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ............................................................................................... iii

ABSTRACT .................................................................................................................... iv-v

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ........................................................................................................... ix

CHAPTER 1: Fall planted faba bean, a potential rotation for cropping systems in the

Palouse

1. AGRICULTURAL SYSTEMS ON THE PALOUSE ..........................................1

2. LEGUMES IN CROP ROTATIONS ...................................................................3

3. FABA BEAN ........................................................................................................4

LITERATURE CITED ......................................................................................................10

CHAPTER 2: Winter-Hardiness and Yield Potential of Faba Bean (Vicia faba L.)

Subjected to Subfreezing Temperatures

1. INTRODUCTION ........................................................................................................ 14

2. MATERIALS AND METHODS .................................................................................. 16

3. RESULTS AND DISCUSSION ................................................................................... 18

4. CONCLUSIONS ........................................................................................................... 26

LITERATURE CITED......................................................................................................41

CHAPTER 3: Nodulation Response to Available Soil Nitrate in Faba bean and Pea

1. INTRODUCTION................................................................................................43

2. MATERIALS AND METHODS …...................................................................46

3. RESULTS AND DISCUSSION………...………………………………………48

4. CONCLUSIONS………………………………………………………………..51

vii

LITERATURE CITED…………………………………………..………………………55

CHAPTER 4: Summary and Conclusions………………………………………………58

viii

LIST OF TABLES

1. Genotypes Included in Field Trial .................................................................................28

2. Comparative Plant Survival Among Site-years .............................................................29

3. Comparative Exposure to Sub-Freezing Temperatures .................................................30

4. Winter-hardy, High Yielding Genotypes Identified ......................................................37

5. Frost Resistance - Plant Survival/Growth Characteristics Correlation ..........................38

6. Comparative Plant Survival/Growth Characteristics by Country of Origin ..................40

7. Specific Nodulation .......................................................................................................52

ix

LIST OF FIGURES

1. Comparative Plant Survival Between Site-years ...........................................................29

2. Minimum Daily Ambient Temperature .........................................................................31

3. Minimum Soil Temperature: Surface ............................................................................32

4. Minimum Soil Temperature: 15cm ................................................................................32

5. Average Soil Moisture: 15cm .......................................................................................33

6. Field Conditions 2-Feb CF 09-10 ..................................................................................33

7. Comparative Plant Survival by Genotype......................................................................34

8. Genotypes Identified with Superior Winter-hardiness ..................................................35

9. Genotypes Identified with High Yield Potential ...........................................................36

10. Comparative Frost Resistance between years ..............................................................39

11. Root Length: Manual vs. Computer Image Analysis...................................................52

12. Nodulation Response to Soil Nitrate............................................................................53

13. Nitrate Affect on Timing of Nodulation ......................................................................53

14. Comparative Nodule Size and Color ...........................................................................53

1

CHAPTER ONE

Fall planted faba bean, a potential rotation for cropping systems in the Palouse

Agricultural systems on the Palouse

The Palouse region of eastern Washington and northern Idaho is known for its cereal and

cool season legume production in a dryland environment. In the high rainfall areas, 46 to 60 cm

annual rainfall, crop rotations are dominated by 2 and 3 year rotations of winter wheat, spring

cereals, and grain legumes. Winter wheat is the main cash crop, planted in rotation with spring

crops of barley or wheat, and a spring legume such as pea, lentil, or chickpea (Schillinger, 2006).

The climate is Mediterranean, receiving 60%-70% of precipitation between November and April,

but with the high elevations, 20% of that is snowfall (Schillinger et al., 2006). Average

temperatures for December and January are about 0°C, but will get down to -10°C and lower and

soils can freeze to depths of 41 cm (Schillinger et al., 2006). The landscape is made up of rolling

loessial hills with cultivation occurring on slopes as steep as 30% (Schillinger et al., 2006).

Cropping systems changed drastically over the last 60 years, becoming less reliant on

diversified rotations and more reliant on chemical fertilizers, herbicides, and pesticides

(Granatstein, 1992). The release of shorter wheat varieties meant yield response to added N was

no longer inhibited by induced lodging. The yield potential for wheat doubled (Khush, 1999;

Jennings, 1990). Inexpensive, readily available N fertilizers eliminated the dependence on

biological dinitrogen fixation (BNF) via legume rotations. It became more profitable to increase

the percentage of land planted into wheat and apply heavy rates of synthetic N fertilizers. High

residual levels in the area indicate a tendency to over-fertilize basing rates on maximum rather

than average yields despite the inability to predict growing conditions (Granatstein, 1992).

2

High input, industrial farming has led to soil degradation, but advances in new crop

varieties and improved technologies have hidden the negative impacts of these practices on soil

(McCool et al., 2001). Declining soil organic matter, increasing soil acidity and a reduction in

both biological activity and diversity are some of the environmental costs that threaten long term

productivity of the land (McCool et al., 2001). Soil pH has declined in proportion the

accumulated N fertilizer (Granatstein, 1992).

Increasing our reliance on legumes in cropping systems to add fixed N to the soil system

and/or food via proteins in harvested grains will have a positive impact on the sustainability of

cropping systems of this region. Sustaining food production requires management practices that

will not deplete and ultimately destroy requisite resources needed for the task such as soil, water,

and nonrenewable energy (Bohlool et al., 1992). From an energy standpoint BNF may be a more

sustainable source of N than fertilizer N with energy coming from the sun via photosynthesis

rather than non-renewable fossil fuel energy sources used in industrial production of fertilizer

NH3 (Crews and Peoples, 2004). Adding complexity and flexibility in cropping systems can

reduce weed and disease pressure resulting in decreased dependence on pesticide inputs as well

as increase nutrient and water use efficiency (Hanson et al., 2007).

There is interest in finding alternative rotation crops that provide agronomic advantages

(e.g., weed and disease control) and economic returns, increased sustainability, and flexibility.

Schillinger et al. (2006) listed alternative rotation crops that could be used instead of spring

cereals as an advance for dryland farming while at the same time calling for more field testing of

crops to create a greater diversity. A winter hardy legume capable of fixing high amounts of N

would make a good rotation crop for the Palouse. High N2-fixing legumes such as faba bean are

gaining attention as N fertilizer costs rise.

3

Legumes in crop rotations

Crop rotations increase wheat yields. A recent review found yield increases averaged

around 14% for wheat following a rotation crop vs. continuous wheat (Kirkegaard et al., 2008).

Legumes in rotation provide a source of N and other ecosystem services that enhance production

of subsequent crops (Peoples et al., 2009). Legumes in rotation increase yields beyond what can

be accounted for with added N. Stevenson and van Kessel (1996) found 92% of the 43% yield

advantage of wheat following pea vs. wheat was from non-N rotation benefits. Legumes provide

many ecosystem services including improved soil structure, increased soil porosity, recycling of

nutrients, increased biodiversity, and breaks in disease, weed, and pest cycles. The taproot root

structure of legumes improves soil structure by breaking up hardpans. Rochester et al. (2001)

attributed lower soil strength following legume rotations to increased root health and

development in cotton which ultimately resulted in higher lint yields. Legumes also improve soil

structure by improving soil aggregation and encourage greater biological activity (Jenson and

Hauggaard-Nielson, 2003). Disease and pest cycles in cereal crops are often reduced (Jenson and

Hauggard-Nielson, 2003; Peoples et al., 2009). In addition legumes have been found to enhance

P availability in wheat crops (Nuruzzaman et al., 2005).

Although legumes are often included in crop rotations for additions biologically fixed N,

it cannot be assumed that fixed N will be greater than the N that is removed. This is especially

true with grain legumes where the plant N removed with harvested grain ranges from 40 to 75%

(van Kessel and Hartley). With so much N removed, a net gain in soil N requires a sizable

amount of N2 fixed. The percentage of plant N derived from air (%Ndfa) and total plant biomass

are the factors that determine total fixed N. Legume crops utilize N from a combination of

sources that can include mineralized soil N, N derived from the atmosphere, and fertilizer N

4

(Salvagiotti, 2008). The extent to which legumes rely on BNF, and therefore the percentage of

plant N derived from air (%Ndfa) varies depending on species, variety, climate, soil properties,

and management practices as does overall plant growth (Beck, 1991; Peoples et al., 2009, van

Kessel and Hartly, 2000). Out of 505 published data sets reporting N2 fixation in soybean, 316 of

which did not involve fertilizer application, %Ndfa ranged from 0 to 98% (Salvagiotti et al.,

2008). Peoples et al. (2009) reviewed published studies on N2 fixation for difference species

around the world and found averages for species across environments ranged from 39% Ndfa for

common bean (Phaseoulus vulgaris) to 75%Ndfa for faba beans (Vicia faba). Soils high in

mineral N have a negative impact on N2 fixation (Salvagiotti, 2008), the extent of which is

species dependent (van Kessel and Hartley, 2000). An analysis of 637 published data sets on

soybeans showed that low ratios of fixed-N:N-uptake occurred with high nitrate soils, high

acidity, ineffective strains of Bradyrhizobium, and water deficits (Salvagiotti et al., 2008).

Faba Bean

Faba bean is a cool season grain legume grown in many regions of the world. In colder

regions faba bean is more often planted as a spring crop while in warm-temperate and

subtropical climates it is planted into the winter (Duc, 1997). There are three botanical types that

can roughly be distinguished by seed size: V. faba major with 1000 seed weights greater than

1kg; V. faba minor with 1000 seed weights less that 500g, and V. faba equina with 1000 seed

weights (Duc, 1997). Small seeded varieties tend to be more winter-hardy than the larger types,

but breeding efforts have been successful in transferring hardiness to equina types (Bond,1999).

Faba bean has proven to be a superior rotational crop for dryland wheat production under

limited rainfall. The results of a long term study carried out in Spain showed faba bean had the

greatest rotation effect on wheat yields as compared to chickpea, sunflower, fallow, and

5

continuous wheat. As in the Palouse, steep slopes and a Mediterranean climate make water and

N the most limiting to wheat yield. Further study showed significantly higher soil water content

at planting for wheat crops preceded by faba bean and fallow than preceded by chickpea, wheat,

or sunflower (Lopez-Bellido, 2007a). For additions of N fertilizer in wheat up to 150 kg∙ha-1

, the

faba-wheat rotation had higher wheat yields than all other rotations and maximum wheat yield

was obtained with less fertilizer N for the faba-wheat rotation than all other rotations (Lopez-

Bellido, 2007b). Within the same study, a two year field experiment was conducted to find the

contribution to the N balance of the cropping system. The mean residual N following faba bean

was18.3 kg N ha-1

with values as high as 40 kg N ha-1

. These values did not include the N

contribution from plant roots (Lopez-Bellido et al., 2006).

Residual N derived from legume crops are often underestimated because below-ground

N (BGN) is not considered. Khan et al. (2003) found BGN constituted 24% of total plant N for

faba bean and 68% for chickpea. The allocation of nutrients as well as the root:shoot ratio is

dependent on soil properties and environmental conditions. Averages from published studies for

BGN of faba bean and chickpea were 34% and 56% respectively (Khan et al., 2003).

Faba bean has a high capacity for N fixation when compared to other grain legumes.

Compiling published experimental data and reviews that include a wide range of agronomic,

environmental, and experimental factors, Herridge and Peoples (2008), found average %Ndfa in

faba bean to be higher than other legumes: faba bean averaged 75% while chickpea, lentil and

pea averaged 68%. Compiling data from > 800 farmers‟ fields, faba bean averaged 68%Ndfa

while chickpea, lentil, and pea averaged 65%Ndfa (Herridge and Peoples, 2008). A review of the

role of legumes in supplying N to cropping systems around the world showed faba bean fixed

high amounts of N across geographic regions (Peoples, 2009).

6

Faba beans and chickpeas are often used in rotation with wheat in dryland cropping

regions of Australia which like the Palouse have a Mediterranean rainfall pattern. A survey

evaluating N contributions of commercially grown faba beans and chickpeas showed a wide

variation in both N fixation and net N contributions. The use of faba beans usually resulted in a

net gain to the plant-soil system while net N balance under chickpeas showed both positive and

negative with an average of zero (Schwenke et al., 1998). The N contribution in chickpea

appeared to be more susceptible to drought (Schwenke et al, 1998).

Typical pulse crops used in rotation with cereals in the Northern Great Plains region are

similar to those grown in the Palouse. They include field pea (Pisum sativum L.), lentil (Lens

culinaris Medik.), chickpea (Cicer arietinum L.), and faba bean (Vicia faba L.). Walley et al.

(2007) prepared a meta-analysis of published studies in the region to evaluate and compare net N

contributions from the different pulse species. Comparing N contributions within each species

showed variable results among the studies due in large part to high variation in %Ndfa.

Comparing between species, faba bean contributed more N than pea or lentil and chickpea

contributed little to no N (Walley et al., 2007).

Biological N fixation is the product of the symbiotic relationship between legume and

rhizobium species. Mineral N has a negative impact on the BNF over multiple stages of the

symbiosis including nodulation, nodule growth, and N2 fixation activity. All stages play in role

in determining total biologically fixed N. The relative sensitivities of each stage to concentration

and form of N change with increasing N (Streeter, 1998). At high enough concentrations, all

forms of mineral N will inhibit N fixation (Guo, 1992). At low concentrations, nitrate has a

greater effect on both nodulation and nodule growth than ammonium (Bollman and Vessey,

2006). Over a certain range of soil nitrate, nodulation is less sensitive than nodule growth

7

(Steeter, 1988). At a N concentration of 5 mM combined N, soybeans showed no difference in

nodule number, but the nodules were much smaller and lacking in visible leghaemoglobin

(Cassman, 1980).

It has been reported that faba bean is more tolerant to mineral N than other legume

species. Through extrapolation Schwenke et al. (1998) estimated that faba beans could maintain

a net zero N balance with soil nitrate levels as high as 280 kg/ha while chickpea could only

maintain a net zero N balance with soil nitrate levels less than 43 kg/ha. Turpin et al.( 2002)

found that for soil nitrate levels between 71 and 86 Kg·ha-1

faba bean maintained a %Ndfa of

45% while chickpea maintained only 12%. A nutrient solution containing 18 mM Nitrate- N was

required to completely inhibit nodulation in faba bean while it took a concentration of only 7mM

inhibit nodulation in French bean (Chalifour and Nelson, 1988).

Chalifour and Nelson (1987) showed superior tolerance to mineral N by faba bean over

pea in a controlled experiment comparing N fixation response of the legume-rhizobium

symbiosis to increasing rates of nitrate - N. While the %Ndfa decreased for both species, the

suppression of N2 fixation with increasing nitrate was much larger for pea. The highest nitrate

treatment, 15 mM, resulted in zero Ndfa for pea while faba bean maintained 15.4 %Ndfa. Further

investigation showed that nitrate present at planting had a much larger impact on N fixation in

pea than later applications. This was not the case for faba bean leading to the conclusion that the

tolerance of the faba bean –rhizobium symbiosis to soil nitrate must be due in large part to its

ability to form effective nodules in the presence of high combined N (Chalifour and Nelson,

1988).

Winter crops have potential yield advantages over their spring counterparts depending on

varietal adaptation to climate. A field trial conducted across 3 locations in Syria and Lebanon

8

resulted in winter yields 70% higher than spring yields for chickpea (Singh, 1997). Chen et al.

(2006) found significantly higher yields for winter pea and lentil over spring pea and lentil

grown in the Palouse region of the Pacific Northwest, but no yield advantage for winter pea over

spring pea in the Northern Great Plains. Reasons cited for the advantage of winter pea in the

Palouse was milder winter temperatures and the winter concentrated precipitation distribution

(Chen et al., 2006). Wet springs with soils high in clay can delay planting of spring crops in the

Palouse which in turn shortens the growing season.

Winter varieties of faba beans have shown to produce higher yields than their spring

counterparts. Two series of field trials conducted between 1981 and 1988 for three locations in

Germany found winter yields to be 14% higher than spring yields (Herzog and Geisler, 1991).

More recent trials in the UK found winter yields were 47% higher than spring yields when

averaged over 3 locations (Link et al., 2010). It is likely that autumn sown faba beans would

show a yield advantage in the Palouse. Autumn sown winter varieties tend to mature before

spring types, which makes them suitable for a Mediterranean environment where they can take

full advantage of spring moisture and better avoid drought conditions (Duc, 1997, Link et al.,

2010).

As an added economic advantage, fall seeded faba beans require a lower seeding rate due

to their ability to develop more than one stem (Herzog, 1989). Winter cultivars have 4-6 stems

per plant while spring cultivars have 1-2 (Duc, 1997). First and second tiller have an equivalent

yield potential (Herzog, 1989). Roughly et al. (1983) found that autumn sowing faba beans can

have a positive impact N fixation. Field trials revealed that faba beans will start fixing N before

temperatures get below freezing and will fix a substantial amount of N in early spring before

spring planting.

9

The results of a field trial involving 29 faba bean varieties planted in fall across 5

European locations showed that frost tolerance was a contributing factor to plant survival and

yield, but varietal adaptation to site-specific biotic and climate stresses played a role as well. One

experimental line, identified as F7-29, had the highest yield for a single site-year, but did not

yield well across environments. Bulldog/1 was identified as one of five varieties that showed

better plant survival across all environments, but did not have a high frost tolerance. Authors

credited winter-hardiness for Bulldog/1 to good adaptation (Arbaoui et al., 2008).

Fall planted faba bean has potential to fit cropping systems in the Palouse. Although

susceptible to drought, fall planted faba bean yields well in Mediterranean environments.

Commercial production of fall planted faba bean has been successful in colder regions such as

UK and France. Breeding efforts for increased winter-hardiness are ongoing to expand

overwintering of faba beans into colder regions. A high capacity for N2 fixation along with a

high tolerance for residual N could mean an advantage over legume crops currently used in

rotation. An added alternative crop to wheat rotations would allow greater diversity in cropping

systems.

10

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commercial, dryland chickpea and faba bean crops in north-west New South Wales maintain or

enhance soil nitrogen? Aust. J. Exp. Agric. 38(1): 61 – 70.

Singh, K. B., R. S. Malhotra, M. C. Saxena, and G. Bejiga. 1997. Superiority of Winter Sowing

over Traditional Spring Sowing of chickpea in the Mediterranean Region. Agron. J. 89: 112 –

118.

Smith, S.C., D.F. Bezdicek, R.F. Turco, and H.H. Cheng. 1987. Seasonal N2 fixation by cool-

season pulses based on several 15

N methods. Plant Soil. 97: 3 – 13.

Stevenson, F.C. and C. van Kessel. The nitrogen and non-nitrogen rotation benefits of pea to

succeeding corps. Can. J. Plant Sci. 76: 735 – 745.

Streeter, J. 1988. Inhibition of legume nodule formation and N2 fixation by nitrate. Crit. Rev.

Plant Sci. 7(1): 1 – 23.

13

Turpin, J.E., D.F. Herridge, and M.J. Robertson. 2002. Nitrogen fixation and soil nitrate

interactions in field-grown chickpea (Cicer arietinum) and fababean (Vicia faba). Aust. J. Agric.

Res. 53: 599 – 608.

Van Kessel, C. and C. Hartley. 2000. Agricultural management of grain legumes: has it led to an

increase in nitrogen fixation? Field Crops Res. 65: 165 – 181.

Walley, F. L., G. W. Clayton, P. R. Miller, P. M. Carr, and G. P. Lafond. 2007. Nitrogen

Economy of Pulse Crop Production in the Northern Great Plains. Agron. J. 99: 1710 – 1718.

14

CHAPTER TWO

Winter-Hardiness and Yield Potential of Faba Bean (Vicia faba L.) Subjected to Sub-

Freezing Temperatures

Introduction

The rolling hills of the Palouse are located in Eastern Washington and Idaho and are

known for cereal and cool season legume production in a dryland environment. The Cascade

mountain range induces a rain shadow effect resulting in limited precipitation ranging from

15cm to 61cm increasing from west to east ( Schillinger et al., 2006). The pattern of precipitation

is Mediterranean, receiving 60%-70% of precipitation between November and April, but with the

high elevations, 20% of that is snowfall (Schillinger et al., 2006). Winter temperatures are cool

with average temperatures for December and January near 0°C and occasionally reaching -10°C

or lower. Soils can freeze to depths of 41 cm (Schillinger et al., 2006).

Faba bean (Vicia faba L.) is a cool season legume grown for its high nutritional value and

agronomic benefits. Faba bean is a good source of protein for both humans and animals with

protein content ranging from 27 to 34% (Duc, 1997; Bond et al., 1985). In cropping systems,

faba beans are used for breaking pest, weed, and disease cycles (Jenson and Hauggard-Nielson,

2003; Peoples et al., 2009). The cost of producing high-protein foods and the need to increase

diversity in agricultural systems continue to draw interest in introducing and or increasing faba

beans into cropping systems (Bond, 1985).

Traditionally autumn sowing of faba beans in a Mediterranean environment precedes

mild winter conditions followed by low moisture conditions that can limit yield (Lopez-Bellido,

2005). Autumn sowing allows crops to gain a longer growing season by establishing a stand

using fall-available water and subsequently take advantage of early spring moisture. Fall planting

is advantageous when wet, cool spring soil conditions can delay spring planting. In addition,

15

winter faba beans mature earlier than spring types potentially allowing them to avoid drought

conditions that occur in late spring and summer months (Link et al., 2010). The timing for

available moisture can be critical to faba bean production as they are susceptible to drought,

especially at flowering (Duc, 1997). Temperatures do not usually drop below -6°C in the

Mediterranean region which means chilling and freezing stresses are not often critical (Link et al.

2010). Non-hardened faba beans including both winter and spring types can tolerate

temperatures as low as -5°C without an impact on plant survival or yield (Herzog, 1987).

Faba beans grown in colder regions are normally planted as a spring crop. There is a

higher yield potential for winter genotypes, but most lack the necessary winter-hardiness to be

planted in the fall and survive through the winter (Arbaoui and Link 2008, Annicchiarico 2007).

Winter-hardiness describes overall resistance to multiple stresses present during the winter as

measured by plant survival. Low temperatures and excessive moisture are cited as most limiting

to winter faba bean production in Northern Europe (Lopez-Bellido, 2005). Trials in Germany

carried out in 3 locations over 7 years showed an average yield increase of 14% for winter faba

beans (Herzog and Geisler, 1991). In the UK, a trial taking place in 3 locations within a single

year showed a 47% increase (Link et al., 2010). Breeding efforts are taking place in France,

Germany and the UK to improve winter-hardiness which in turn will improve yield stability and

expand the region in which autumn sown faba beans can be grown (Link et al., 2010).

Winter-hardiness can correlate to a variety of stress tolerances depending on regional

climate and site specific growing conditions. Low temperature, duration of freezing, soil

moisture, soil type, freeze-thaw cycles, disease, and pests can influence plant survival over the

winter (Badaruddin and Meyer, 2001). For faba bean, resistance to freezing temperatures has

been identified as a key component of winter hardiness (Arbaoui et al., 2007, Link et al., 2010).

16

Because leaves are directly exposed to freezing temperatures and leaf injury can hinder crop

growth, visual scoring of leaf injury in controlled frost tests has been used as a method to

measure frost tolerance (Arbaoui et al., 2008).Cultivars identified as showing high frost

resistance include Cote d‟ Or (-16C), and Hiverna (-15C) (Link et al., 2010 referencing Herzog

1987). In the field, Cote d‟Or had 60% plant survival at -25°C without snow cover and often

cited for superior frost-resistance and winter-hardiness (Duc, 1997; Link et al., 2010).

The objectives of this study were to identify winter-hardy germplasm for autumn-sown faba

beans in the Palouse and to identify plant characteristics associated with higher yield potential.

Materials and Methods

A replicated field trial was conducted at 2 locations (Central Ferry and Pullman, WA)

over 2 years. Central Ferry (46°43‟52”N 117°39‟52”W; soil series, a fine-silty, mixed, mesic

Natrixerall) was drip irrigated, while Pullman (46°43‟28”N 117°08‟07”W; soil series, a fine-

silty, mixed, mesic Pachic Ultic Haploxerall) was rainfed. Forty-three faba bean accessions from

the National Plan Germplasm System (NPGS) were used, 40 of which were common to all four

site-years (Table 1). These accessions were collected from Afghanistan, Bulgaria, China,

Finland, Hungary, and Poland, and maintained by the USDA-ARS Western Regional Plant

Introduction Station in Pullman, WA. Twelve additional cultivars and breeding lines that were

identified as winter hardy were obtained from Professor W. Link, Department of Crop Sciences,

Georg-August University, Germany. The cultivars originated from UK, France, and Germany.

They were included in three of the four site-years: Central Ferry 08-09, Central Ferry 09-10, and

Pullman 09-10.

For both site-years, seeds were inoculated with Rhizobium leguminosarum biovar viceae

(N-Dure, INTX Microbials, LLC, Kentland, IN) and treated with Mo, fungicides and insecticide

17

dye prior to planting. Pre-plant Treflan was used for weed control. Planting was done using a

Hedge drill (Wintersteiger AG, Niederlassung, Germany).

The 2008-2009 field trials were planted using a randomized complete block design with

two replications. Two winter-pea varieties of known tolerance to winter conditions for the

Palouse were used as controls: Melrose, most cold-hardy pea in Palouse and Whistler,

moderately cold-hardy pea in Palouse. Plots were single rows approximately 1.5m long with

1.5m between each plot. Faba bean was seeded at 20 seeds∙m-1

and peas were seeded at 40

seeds·m-1

with a planting depth of 5 cm. Planting dates for Pullman and Central Ferry were

September 29, 2008 and October 8, 2008 respectively. Plots were harvested at 80% maturity. A

representative sample of five plants was taken for each plot for analyses below. The remaining

plot was harvested for total seed weight.

The 2009-2010 field trials were planted using a randomized complete block design with

three replications. Two commercially available varieties of bell beans (small-seeded V. faba)

were used as controls: variety name (SC) and variety name (PV). Plots were single rows

approximately 4.6m long with 1.5m between each plot. Faba bean was seeded at 10 seeds∙m-1

.

Planting dates for Pullman and Central Ferry were September 15, 2009 and September 16, 2009

respectively. Plots were harvested at 95% maturity. Whole plot samples were taken for each plot

for analyses below.

Samples from field plots were collected upon harvest for each site-year and brought back

to the lab for processing. In the field plants were dug up whole, collecting as much root as

possible. Above and below ground biomass were separated and placed into bags. In the lab all

samples were rinsed and placed in a dryer at 80°C for 48 hours. After drying, seeds and pods

18

were removed from stems and counted. Dry weights were recorded for seed, above ground

biomass (AGB) less seed, and below ground biomass (BGB).

Visual scoring of frost resistance to over-winter leaf tissue injury was conducted on

March 12, 2009 at the Central Ferry location with “1” indicating little to no leaf damage, “2”

indicating slight leaf damage, “3” indicating intermediate leaf damage and “4 indicating severe

leaf damage. Replicated plot scores were averaged to find a frost tolerance score for each

genotype.

Data loggers (EM-50, Decagon Devices, Pullman, WA) were used in conjunction with

temperature-moisture probes (5TE) over the growing season for each site-year. The data loggers

were put in place in the months of September or October and removed on the last day of harvest.

The 5TE probes were placed just beneath the soil surface and at depths of 7.5 and 15 cm.

Measurements for temperature and moisture were taken hourly.

Between site-years data were normalized on a per planted meter basis. For the 08-09 site

years, plant characteristics measured for each sample were “scaled up” using the ratio of whole

plot seed yield to sample seed yield.

Results and Discussion

The four site-years of this study provided a range of growing conditions in which to

evaluate the potential for faba beans to over winter in the Palouse. Considerable differences in

average survival and seed yield amongst site-years indicated a range severity of growing

conditions from mild to harsh. Survival and yield could be summarized as CF-08-09 > Pullman-

08-09 > CF-09-10 > Pullman-09-10 (Table 2). However, a year location genotype interaction

indicated that each site-year presented a distinct plant response to growing environment.

19

Significant variation in plant survival within each site-year made it possible to distinguish

winter-hardy genotypes within each environment (Figure 1).

Some genotypes varied in their response to environmental conditions of each site-year

evidenced by large changes in rank for plant survival among site-years. For example, W6 12026

ranked only in the 41st percentile for CF-08-09 with 48% survival, but in the harsher conditions

of Pullman 08-09 ranked in the 98th

percentile with 83% survival. Accession W6 12026 showed

no survival for CF-09-10 (Figure 1). Genes can vary greatly in phenotypic expression depending

on stresses present (Stoddard et al., 2006). Environmental preferences indicate different stresses

playing critical roles in plant survival for the different site-years.

Five-plant samples for the 08-09 site-years proved to be insufficient to adequately

represent the diversity within genotypes. The number of plants harvested, which is used to report

plant survival, was calculated from a five-plant sample using the proportion of sample seed

weight to whole plot seed weight. CF-08-09 had 26 out of 104 plots with the number of plants

harvested greater than the number of seed planted resulting in plant survival greater than 100%.

Herzog (1989a) reported a similar problem when taking 5 plant samples to study freezing

resistance in faba bean populations. He found a representative sample should include at least 20

plants. The five plants selected for sampling were visually representative of each plot suggesting

that calculated values, although subject to error, are still useful for comparative purposes.

Resistance to freezing temperature is a recognized component of winter-hardiness.

Ambient temperatures for all site-years included harsh freezing conditions in terms of

temperature minimums and/or duration of freezing. Lab experiments have shown that maximum

frost resistance, the temperature at which exposure results in 50% tissue injury, occurs around

­15°C for faba bean (Herzog, 1989a). The cultivar Cote d‟Or is often cited as the best source of

20

frost resistance in faba bean breeding because it showed 60% survival at -25°C (Picard et al.,

1985). Link et al. (2010) attributed a less than 1% survival to a long duration of harsh frost, five

consecutive nights of temperatures below -10°C with daytime temperatures remaining below

freezing, rather than a -16°C temperature minimum. One hundred percent survival was observed

the following year with a lower minimum over a shorter duration, -17°C for a single night. All

site-years for this study had minimum temperatures that were at or below -14° C with at least 5

consecutive nights of harsh frost (Table 3). The number of consecutive days that temperatures

stayed below zero ranged from 7 to 14 (Table 3). The ambient temperature of Pullman 08-09

reached a temperature minimum of -22.2 with 11 consecutive nights of harsh frost (Table 3).

Ambient temperature, temperature at the soil surface, and temperature 15cm beneath the

soil surface show different patterns in terms of comparative daily low temperatures among site-

years over the growing season (Figures 2 – 4). Pullman 08-09 had the lowest ambient

temperature as well as the lowest soil surface temperature in magnitude and duration, yet there

was greater plant survival than both CF-09-10 and Pullman-09-10. At the 15cm depth, Pullman-

09-10 had the lowest temperatures followed by CF-09-10, Pullman-08-09 and CF-08-09. The

ranking of site-years by decreasing temperature lower in the soil profile correlates to the ranking

of site-years by decreasing plant survival while the ranking of site-years based on minimum

ambient and soil surface temperatures do not (Figures 2 – 4).

Tests for frost resistance are conducted on stem and leaf plant tissue measuring chemical

and/or physical responses to freezing temperatures. Arbaoui and Link (2008) exposed hardened

potted plants grown in a controlled environment to natural sub-zero temperatures. Herzog (1987)

exposed leaf samples taken from potted plants grown under controlled conditions to controlled

freezing temperatures. Herzog (1989) exposed leaf samples taken from the field to controlled

21

freezing temperatures. Arbaoui et al. (2008) cites freezing injury of leaves due to direct exposure

to freezing and low non-freezing temperatures and subsequent retardation in crop development

as rationale for using leaves to test plant frost resistance. Roots of overwintering plants can also

be subjected to below freezing temperatures and are much more sensitive to cold than above

ground leaf tissue (Chen et al., 1983). Chen et al. (1983) found that while frost resistance in

above ground tissue changed with hardening for winter rye and wheat, frost resistance in roots

stayed the same. The results of the present field trial suggest that cold and frost resistance in

roots and whole plant response to cold temperatures may offer additional insight into winter-

hardiness. There may also be implications for using potted plants in controlled experiments on

frost resistance as soil temperatures would not be buffered as they would a field situation.

Plant survival for Pullman 09-10 was 0% (Figure 1). Low soil temperatures were

present, but soil moisture may have also played a role in total plant loss for this site-year. Soil

moisture as measured by volumetric water content was much higher than the other three site-

years during the winter months (Figure 5). The coldest temperatures occur between Dec 1 and

Jan 15. Pictures taken on Feb 4, 2010, show limited plant survival in the form of new shoot

growth suggesting that the winter temperature regime for that site-year was not the root cause for

total plant loss (Figure 7). Soil moisture stayed high throughout the month of February and into

March. Field conditions at this moisture level show saturated field conditions with evidence of

field erosion (Figure 6). It is possible that plants survived cold temperatures in December, but

could not tolerate the long durations of water-logging in the months of January and February

(Figure 5). The problem of excessive moisture for overwintering faba bean is common to

Northern Europe (Lopez-Bellido, 2005).

22

Central Ferry is irrigated therefore both site-years planted at the Central Ferry location

avoided moisture related stresses. Pullman 08-09 had soil moistures much lower than Central

Ferry 09-10 most of the growing season but had a higher rate of plant survival indicating that

drought was not the most limiting factor for plant survival or yield. Drought conditions may have

contributed to increased freezing resistance. Herzog (1987) found low soil moisture can increase

freezing resistance in leaves. Badaruddin and Meyer, (2001) found that the relationship between

soil moisture and freezing resistance was dependant on soil type. For clay soils, plant survival

was higher at 1/3 field capacity while for loam soil plant survival higher at field capacity.

Including a variety of landscape positions in future trials could provide more insight as to how

soil moisture contributes to winter-hardiness. Due to steep terrain in the Palouse region, there are

differences in soil moisture across the landscapes that become relevant for management

decisions.

Winter-hardy genotypes were identified as those that survived Pullman 08-09 and/or CF

09-10 with survival quantified by the percentage of plants harvested out of the number of seeds

planted. Thirty-four out of the 43 genotypes obtained from the NPGS for this evaluation showed

some survival in one or both site-years with 23 showing survival in Pullman-08-09 alone (figure

7C), two showing survival in CF-09-10 alone (figure 7D) and nine showing survival in both

(Figure 7B). Genotypes obtained from W. Link and PI 430725 from the NPGS were not included

for Pullman 08-09 because seed was not available at the time of planting. With the exception of

one, Bulldog/1, all cultivars and breeding lines from W. Link showed survival for CF 09-10

many of which had significantly higher survival than those from the NPGS (figure 7A).

Winter-hardy genotypes could be distinguished further by those that showed plant

survival significantly greater than zero in either of the Pullman 08-09 and CF 09-10 site-years

23

(figure 8). Those originating from Bulgaria proved to be more winter-hardy than others obtained

from the NPGS. The genotype with the highest ranking plant survival for Pullman 08-09 was

Bulgarian W612024 with no statistical differences found between it and other Bulgarian

genotypes, W6 12023, W6 12025, W6 12026, W6 12027, and W6 12028. All genotypes except

W6 12026 had some survival for CF 09-10. Three of the six Bulgarian genotypes had survival

significantly greater than zero in both environments (Fig 8B).

Six out of the 12 genotypes obtained from W. Link showed plant survival

significantly higher than zero for CF 09-10 (figure 8a). Go-wibo-pop proved to be the most

winter-hardy with the percentage of plant survival significantly higher than all other

genotypes. Bulldog 1-4 and PI 430725 did not survive Central Ferry 09-10, but should be

included in future trials for Pullman to fully assess winter-hardiness. Overall CF 09-10 was

a harsher growing environment than Pullman 08-09, but plant performance for CF 09-10 is

not a good indicator for plant performance for Pullman 08-09. Bulgarian W612026 and

Chinese PI 577721also did not survive CF-09-10, but were among the most winter-hardy

for Pullman 08-09.

Genotypes identified with high yield potential were those that showed significant yield

for Pullman 08-09 and/or CF 09-10 (Figure 9). With the exception of one, all genotypes with

high yield potentials also showed superior winter-hardiness. The reverse was not true. Three out

of the 16 genotypes showing superior winter-hardiness were not identified with high yield

potential. Wibo/1 had a significant yield, but was not identified with superior winter-hardiness.

Cultivars and breeding lines from W. Link were not planted for Pullman 08-09 and

therefore could not be fully evaluated for yield potential. Some genotypes had yields

significantly higher than zero for CF 09-10 and therefore can be considered high yielding while

24

others did not. One breeding line, Bulldog/1 had no yield for CF 09-10 due to its lack for

survival. As with winter-hardiness, from our data we cannot predict how these cultivars and

breeding lines would have performed if planted for Pullman 08-09. Table 4 shows 14 genotypes

identified with superior winter-hardiness and potential to be high yielding along with genotypes

that need to undergo further evaluation for winter-hardiness and yield potential.

Nine out of the 14 genotypes that showed both superior winter-hardiness and high yield

potential were among the highest yielding for the milder environment of CF-08-09 with seed

yields not significantly different from the top yielding genotype. There are three genotypes

within the highest yielding for CF-08-09 were neither winter-hardy or high yielding.The

genotype showing the highest yield for CF 08-09 did not show winter-hardiness or seed yield

potential that was statistically different from zero for Pullman 08-09 and did not survive CF-09-

10.The seed yield is based on a single replicate, Rep1, due to the loss of whole plot plant yield

data. Sample seed weights for Rep1 and Rep2 were 79.86 and 41.3 respectively. It is probable

that the reported seed yield is exaggerated from a representative mean. The other two genotypes,

Bulldog/1-4 and Clipper, were not tested for the Pullman-08-09 site-year.

Freezing temperatures for CF-08-09 were suitable for differentiating frost resistance

among genotypes. Leaf-tissue damage ranged from little or none to severe. No significant

correlation was found between frost resistance and winter-hardiness for the CF-08-09 site-year;

however, a significant correlation between frost resistance and seed yield suggests that the

temperature regime was harsh enough play a role in growth set-backs that affect yield (Table 5).

Highly significant correlations to frost resistance were found for both plant survival and

seed yield for Pullman-08-09 and CF-09-10 (Table 5). Frost resistance was not assessed for the

Pullman-08-09 and CF-09-10 site-years. Herzog (1989b) found that frost resistance in faba bean

25

does vary between environments, but ranking by genotype remains roughly the same. Freezing

resistance appeared to differ between site years with greater leaf damage occurring for CF-09-10

(Tmin -14.3°C) than for CF-08-09 (Tmin -16.8°C) (figure 10). This is consistent with Herzog

(1988) who found maximum freezing resistance varied between -12°C and -15°C for different

years when testing field-grown faba beans in Germany. Further investigation showed that the

plant growth stage during hardening had an effect on maximum frost resistance that could be

attained. Leaves that developed during hardening showed greater maximum frost resistance with

whole-plant characteristics being more correlated to frost resistance in the first leaf (Herzog,

1988). Frost resistance increased with a shorter time for pre-hardening conditions (Herzog,

1989b). Planting in 08-09 for Pullman and CF occurred 2 and 3 weeks later than in 09-10. It is

possible the later planting date induced a greater frost tolerance for the 08-09 site-years.

The low R2 value for CF-09-10 indicates that winter-hardiness involves resistance to

multiple stresses beyond frost resistance. Go-wibo-pop had a seed yield significantly higher than

all other genotypes for CF-09-10 yet a moderate frost tolerance score of 2.5. Field trials

conducted in Gottingen, Germany identified Bulldog/1 as having superior winter-hardiness along

with genotype Hiverna (included in this study), but artificial and natural frost tests showed it had

low frost resistance (Arbaoui et al., 2008).

Yield components of genotypes averaged over site-years showed differences in stems per

plant and pods per stem for CF-09-10 (Table 7). These two yield components play a major role

in compensatory response to decreased plant density (Lopez-Bellido, 2005). The seeding rate for

CF-09-10 was half that of CF-08-09 and Pullman 08-09. Turk and Tawaha (2002) investigated

the response of autumn-sown faba bean variety „Minor‟ to seeding rate and found increases in

both the number of stems per plant and the number of pods per stem with decreased seeding rate,

26

but this was in an environment that did not include temperatures below zero. In environments

that include sub-freezing temperatures, plant density and tiller number are also influenced by

winter kill and tissue injury. Herzog and Geisler (1991) found with seeding rates averaging 30

plants m-2

, plant densities ranged from 8 to 28 plants m-2

largely due to winter kill.

The average number of seeds per pod was not different among site years, which is

consistent with past findings that the number of seeds per pod is dependent on genotype, but

relatively stable ranging from about two to eight (Knott, 1990). Herzog and Geisler (1991) found

the same to be true in harsh, sub-freezing climates.

Conclusion

The Pullman and Central Ferry locations proved useful in evaluating fall planted faba

beans over a range of environments. Sixteen genotypes were identified as showing superior

winter-hardiness and 13 of those also showed significant yield. Six of the thirteen winter-hardy,

higher yielding genotypes were those cultivars and breeding lines previously identified as winter-

hardy in European trials originating from UK, France and Germany. The other seven were from

the NPGS with all but one genotype originating from Bulgaria. Full field trials are needed to

fully evaluate yield potential.

Low ambient and soil temperatures appear to be limiting to plant survival and yield.

Ambient temperatures are often used to communicate winter severity and to evaluate cold/frost

resistance in above ground plant tissue. This research suggests that soil temperature may play a

role in plant survival that is independent of ambient temperature and should be monitored to

better understand environmental factors that impact winter-hardiness. Root tissue should be

included in evaluations for cold/frost resistance.

27

The next step in this research should be additional field trials that vary planting density,

seeding date, and landscape position to better understand these variables in faba bean production

in a cool Mediterranean environment with limited rainfall. Sampling and plot size should be

increased to account for variability within each genotype.

28

Table 1: Genotypes for this study were obtained from the National Plant Germplasm System (NPGS). Cultivars and

breeding lines identified as winter-hardy were sent from Professor W. Link, Department of Crop Sciences, Georg-

August University, Germany.

*was not planted CF-09-10

**was not planted Pullman-08-09

Genotypes obtained from NPGS Genotypes obtained from NPGS cont.

Genotype Country of Origin Genotype Country of Origin

PI221516 Afghanistan PI577744 China PI221517 Afghanistan PI577745 China PI222128 Afghanistan PI577746* China PI222129 Afghanistan PI577747 China PI222216 Afghanistan PI577748 China PI223302 Afghanistan PI577749 China PI223303 Afghanistan W612023 Bulgaria PI223304 Afghanistan W612024 Bulgaria PI254005 Afghanistan W612025 Bulgaria PI254006 Afghanistan W612026 Bulgaria PI268477 Afghanistan W612027 Bulgaria PI284344* Afghanistan W612028 Bulgaria PI306699 Poland PI317499 Afghanistan PI317500 Afghanistan PI343831 Afghanistan PI415056 Nepal

PI415057 Nepal Genotypes obtained from W. Link

PI415058 China Genotype Country of Origin

PI415059 China Bulldog/1-4 Germany PI415060 China Clipper UK PI415061 China Cote d'Or/2-3 France PI430725** Hungary Go-Wibo-Pop Germany PI430726 Hungary Hiverna Germany PI458603 Finland Hiverna/2-5EP1 Germany PI458604 Finland I3 Diva Germany PI499959 China I3 Karl/2-3 France PI577721 China Irena France PI577722 China Scout Germany PI577740 Nepal Striker UK PI577741 Nepal Wibo/1 Germany

29

Figure 1: Plant survival based on the fraction of plants harvested per number of seeds planted. Site years listed in order of decreasing survival.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Pla

nt

Su

rviv

al:

Ha

rve

ste

d/

Pla

nte

d

CF-08-09 Pullman-08-09 CF-09-10 Pullman-09-10

Comparative Plant Survival Between Site-years

CF-08-09 NPGS

CF-08-09 W. Link

Pullman-08-09

CF-09-10 NPGS

CF-09-10 W.Link

Pullman-09-10

Table 2: Plant survival based on percentage average plant survival, the number of plots harvested and the average seed

yield for each site-year

All Plots Plant Survival (% harvest/planted)

Plot Survival (% harvest/planted)

Seed Yield (g/planted meter)

CF 08-09 81 100 163.2 Pullman 08-09 22 65 36.5 CF 09-10 5 32 17.3 Pullman 09-10 0 0 0

Hiverna/2-5EP1: 5% W6 12026: 0%

W6 12024: 12%

Go-Wibo-Pop: 34%

W6 12024: 84%

W6 12026: 83%

Hiverna/2-5EP1: 138%

W6 12026: 72%

W6 12024: 48%

Go-Wibo-Pop: 73%

30

Table 3: Comparative exposure to sub-freezing temperatures between site-years in terms of duration and absolute

minimum.

CF 08-09 Pullman 08-09 CF 09-10 Pullman 09-10

Minimum Daily Temperature

Ambient air -16.8 -22.2 -14.3 -18.9 Soil 0cm -9.7 -14.6 -11.9 -14 Soil 15cm -0.02 -3.3 -3.7 -7.7

Exposure to harsh frost conditions (temp < -10°C)

Consecutive nights 5 11 5 5 Number of frost periods

3 5 1 5

Duration of exposure to freezing temperatures

Days below 0˚C 19 29 13 16 Consecutive days < 0˚C 10 14 7 11

31

Figure 2: Comparative minimum ambient temperatures for each site year by date. Site years placed in order of

decreasing minimum temperature does not correlate with site-years placed in order of decreasing plant survival.

-25

-20

-15

-10

-5

0

5

10

15

20

23

-No

v

7-D

ec

21

-Dec

4-Jan

18

-Jan

1-F

eb

15

-Feb

29

-Feb

14

-Mar

-25

-20

-15

-10

-5

0

5

10

15

20

24

-Oct

7-N

ov

21

-No

v

5-D

ec

19

-Dec

2-Jan

16

-Jan

30

-Jan

13

-Feb

27

-Feb

12

-Mar

26

-Mar

9-A

pr

23

-Ap

r

7-M

ay

21

-May

4-Ju

n

Te

mp

era

ture

°C

Date

Minimum Daily Temperature:Ambient - Air

CF-08-09 Pullman 08-09 CF 09-10 Pullman 09-10

Decreasing Plant Survival Decreasing Temperature CF 08-09 (81%) CF 08-09 (-9.7°C) Pullman 08-09 (22%) CF 09-10 (-11°C) CF 09-10 (5%) Pullman 09-10 (-14°C) Pullman 09-10 (0%) Pullman 08-09 (-14.6°C)

32

Figure 3: Soil temperatures for each site-year at 0 cm depth. Site years placed in order of decreasing minimum

temperature does not correlate with site-years placed in order of decreasing plant survival.

Figure 4: Soil temperatures for each site-year at 15 cm depth. Site years placed in order of decreasing minimum

temperature correlates with site-years placed in order of decreasing plant survival.

-15

-10

-5

0

5

10

15

8-N

ov

22

-No

v

6-D

ec

20

-Dec

3-Jan

17

-Jan

31

-Jan

14

-Feb

28

-Feb

14

-Mar

So

il T

em

pe

ratu

re °

C

Date (Day-Month)

Minimum Daily Temperature: Soil Surface

CF08-09 Pullman 08-09 Central Ferry 00-10 Pullman 09-10

-10

-5

0

5

10

158

-No

v

22

-No

v

6-D

ec

20

-Dec

3-Jan

17

-Jan

31

-Jan

14

-Feb

28

-Feb

14

-Mar

So

il T

em

pe

ratu

re °

C

Date (Day-Month)

Minimum DailyTemperature: Soil 15cm

CF08-09 Pullman 08-09 Central Ferry 00-10 Pullman 09-10

Decreasing Plant Survival Decreasing Temperature CF 08-09 (81%) CF 08-09 (-9.7°C) Pullman 08-09 (22%) CF 09-10 (-11°C) CF 09-10 (5%) Pullman 09-10 (-14°C) Pullman 09-10 (0%) Pullman 08-09 (-14.6°C)

Decreasing Plant Survival Decreasing Temperature CF 08-09 (81%) CF 08-09 (-0.02°C) Pullman 08-09 (22%) Pullman 08-09 (-3.3°C) CF 09-10 (5%) CF 09-10 (-3.7°C) Pullman 09-10 (0%) Pullman 09-10 (-7.7°C)

33

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

24

-Oct

7-N

ov

21

-No

v

5-D

ec

19

-Dec

2-Jan

16

-Jan

30

-Jan

13

-Feb

27

-Feb

13

-Mar

27

-Mar

10

-Ap

r

24

-Ap

r

8-M

ay

22

-May

5-Ju

nV

olu

me

tric

Wa

ter

Co

nte

nt

m3

/m

3

Date (Day-Month)_

Average Soil Moisture 15cm

Central Ferry 08-09 Pullman 08-09 Central Ferry 09-10 Pullman 09-10

Figure 5: Average soil moisture for each site-year.

Figure 6: Pictures taken 4-Feb 2010 for Pullman 09-10 show saturated field conditions and sever erosion through the

plots. Few plots showed survival with new shoots.

34

0 0.5 1 1.5

PI 458604

PI 317500

PI 222216

PI 284344

PI 577749

PI 415059

PI 317499

PI 223304

PI 306699

PI 223303

PI 577745

PI 343831

PI 268477

PI 577746

PI 577740

PI 577747

PI 415057

PI 415056

PI 415060

PI 577741

PI 577744

PI 415061

PI 415058

PI 577721

W6 12026

PI 577748

PI 577722

PI 430726

W6 12023

PI 254006

W6 12027

W6 12024

W6 12025

W6 12028

PI 430725

Bulldog/1-4

Clipper

Striker

Irena

Hiverna/2-5EP1

Wibo/1

Hiverna

Scout

I3 Karl/2-3

I3 Diva

Cote d'Or/2-3

Go-Wibo-Pop

Plant Survival (harvested/planted)

Ge

no

typ

es

Ide

nti

fie

d a

s W

inte

r-h

ard

y

Winter-hardy: Comparative Plant Survival

CF 08-09

CF 09-10

Pullman 08-09

Figure 7A: Genotypes not planted for Pullman 08-09 listed in order of decreasing survival for CF-09-10; Figure 7B:

Genotypes that survived all three site-years listed in order of decreasing survival for CF-09-10; Figure 7C: Genotypes

that survived Pullman 08-09 but did not survive CF 09-10 listed in order of decreasing survival for Pullman 08-09; Figure

7D: Genotypes that survived CF 09-10 but did not survive Pullman 08-09 listed in order of decreasing survival for CF 09-

10

Fig 7A

Fig 7B

Fig 7C

Fig 7D

LSD (harvested/planted)

CF 08-09: 0.12

Pullman 08-09: 0.072

CF 09-10: 0.01

35

Figure 8: Genotypes identified as showing superior winter-hardiness. Fig 8A lists those with plant survival significantly

greater than zero for CF 09-10. These genotypes were not evaluated for Pullman 008-09; Fig 8B lists those with plant

survival significantly greater than zero for both Pullman 08-09 and CF 09-10. Fig 8C lists those with plant survival

significantly greater than zero for Pullman 08-09 only.

BCDE

ABCD

ABC

AB

AB

A

A

A

ABC

A

H

H

H

H

H

H

H

FG

F

F

EF

DE

BCD

BC

BC

A

0 0.2 0.4 0.6 0.8 1 1.2 1.4

PI 430726

W6 12023

PI 415061

W6 12027

PI 415058

PI 577721

W6 12026

W6 12024

W6 12025

W6 12028

Hiverna

Scout

I3 Karl/2-3

I3 Diva

Cote d'Or/2-3

Go-Wibo-Pop

Plant Survival (harvested/planted)

Ge

no

typ

s

Winter-hardy Genotypes

CF-09-10

Pullman-08-09

CF-08-09

36

Figure 9: Genotypes identified as showing superior yield.

A

CD

A

BC

A

B

B

H

GH

GH

FGH

DEFG

CD

CD

DEFG

DEF

DEF

CD

BC

B

A

0 150 300 450

W6 12026

PI 430726

W6 12027

W6 12023

W6 12028

W6 12025

W6 12024

Wibo/1

Hiverna

Cote d'Or/2-3

I3 Diva

I3 Karl/2-3

Scout

Go-Wibo-Pop

Seed Yield (g /planted m)

Ge

no

typ

es

High Yielding Potential

CF 09-10

Pullman 08-09

CF 08-09

Fig 11A

Fig 11B

Fig 11C

LSD (g)

CF 08-09: 85.6

Pullman 08-09: 54.7

CF 09-10: 37.1

37

Table 4: Genotypes identified with superior winter-hardiness and high yield potential based on plant survival and yield

for Pullman 08-09 and/or CF 09-10. Genotypes showing superior winter-hardiness that did not show high yield potential

for CF 09-10 but were not evaluated For Pullman 08-09, are also listed.

* Wibo/1 was not identified with superior winter-hardiness, but did have significant yield for CF 09-10.

Genotypes identified with superior winter-hardiness and high yield potential

-----------------Seed Yield (g m-1)-----------------

Genotype CF 08-09 Pullman 08-09 CF 09-10

PI 430726 175.74 65.04 6.18 W6 12023 242.43 115.34 21.11 W6 12024 253.44 126.76 69.61 W6 12025 200.91 135.81 60.8 W6 12026 304.44 241.54 0 W6 12027 293.21 243.18 8.59 W6 12028 251.45 253.31 39.63 Cote d'Or/2-3 75.16 Not planted 53.05 Go-Wibo-Pop 282.18 Not planted 163.01 Hiverna 281.79 Not planted 52.8 I3 Diva 245.98 Not planted 66.92 I3 Karl/2-3 217.65 Not planted 93.58 Scout 208.34 Not planted 115.54

Genotypes with winter-hardiness and yield potential pending further evaluation

-----------------Seed Yield (g m-1)-----------------

Genotype CF 08-09 Pullman 08-09 CF 09-10

PI 430725 178.16 Not planted . Bulldog/1-4 268.2 Not planted . Clipper 253.23 Not planted 10.21 Hiverna/2-5EP1 207.34 Not planted 23.64 Irena 123.36 Not planted 12.71 Striker 143.49 Not planted 16.82 Webo/1* 247.19 Not planted 39.63

38

Table 5: Regression analysis between frost resistance scores and plant survival/growth characteristics.

Mean R2 P <

Hardiness (harvested/ planted)

CF 08-09 0.81 0.01 0.36

Pullman 08-09 0.22 0.42 0.0001

CF 09-10 0.05 0.21 0.0001

Yield (g·planted m-1)

CF 08-09 161.41 0.19 0.0001

Pullman 08-09 37.79 0.45 0.0001

CF 09-10 17.26 0.16 0.0001

AGB (g·planted m-1)

CF 08-09 309.34 0.29 0.0001

Pullman 08-09 68.66 0.46 0.0001

CF 09-10 15.17 0.15 0.0001

Stem/Plant

CF 08-09 1.96 0.05 0.0001

Pullman 08-09 1.48 0.15 0.0001

CF 09-10 3.02 0 0.8

Pod/Stem

CF 08-09 5.3 0.06 0.0001

Pullman 08-09 3.74 0 0.89

CF 09-10 9.47 0 0.95

39

Figure 10: Pictured left are genotypes Hiverna and W6 12024 pictured Feb-12, March-12, and March-26 in after experiencing a minimum temperature of -16.8°C for

CF-08-09. A hardiness score of 1 was assigned to Hiverna based in little to no damage on March-12. A Hardiness score of 2 as assigned to W6 12024 based on slight

leaf damae. The same cultivars are pictured right, each show greater leaf damage after a minimum temperature of -14.3°C CF-09-10

CF 08-09 Tmin = -16.8°C

CF 09-10 Tmin = -14.3°C

Hiverna

W6 12024

12-Feb 12-March 26-March

Hiverna

W6 12024

40

Table 6: Comparative plant survival/growth characteristics among site-years by country of origin

Plant No Yield AGB Stem/Plant Pod/Stem Seeds/Pod

Overall CF 08-09 14.44 164.26 316.94 1.93 5.85 2.22

Pullman 08-09 4.62 42.2 76.78 1.38 4.29 2.04

Cf 09-10 0.47 17.03 15.02 3.34 8.14 2.12

Bulgaria CF 08-09 16.623 258.03 494.17 2.3325 5.798 2.268

Pullman 08-09 14.156 185.99 328.75 1.7833 5.133 2.3058

Cf 09-10 0.7412 33.29 33.32 2.474 11.505 2.3908

Germany CF 08-09 20.625 248.72 478.48 1.8561 5.596 2.3563

Cf 09-10 1.6313 66.1 54.55 3.197 9.734 2.6791

UK CF 08-09 9.401 198.36 379.9 2.25 6.208 2.2473

Cf 09-10 0.3281 13.52 10.08 3.75 7.256 3.0281

France CF 08-09 15.838 138.72 284.56 1.7333 3.484 2.4652

Cf 09-10 1.8713 53.12 49.16 3.362 8.52 2.4423

41

References

Annicchiarico, P. and A. Iannucci. 2007. Winter survival of pea, faba bean and white

lupin cultivars in contrasting Italian locations and sowing times, and implications for

selection. J. Agric. Sci. 145: 611 – 622.

Arbaoui, M., C. Balko and W. Link. 2008. Study of faba bean (Vicia faba L.) winter-

hardiness and development of screening methods. Field Crops Res. 106: 60 – 67.

Badaruddin, M. and D.W. Meyer. 2001. Factors modifying frost tolerance of legume

species. Crop Sci. 41: 1911 – 1916.

Bond, D.A., D.A. Lawes, G.C. Hawtin, M.C. Saxena and J.H. Stephens. 1985. Faba bean

(Vicia faba L.) p. 199 – 265. In R.J. Summerfield and E.H. Roberts (ed.) Grain Legume

Crops. Collins Professional and Technical Books. London, England.

Chen, T.H., L.V. Gusta, and D.B. Fowler. 1983. Freezing injury and root development in

winter cereals. Plant Physiol. 73: 773 – 777.

Duc, G. 1997. Faba bean (Vicia faba L.). Field Crops Res. 53: 99 – 109.

Herzog, H. 1997. Freezing resistance and development of faba beans as affected by

ambient temperature, soil moisture and variety. J. Agron. Crop Sci. 159: 90 – 100.

Herzog, H. 1988. Winter hardiness in faba beans: varietal differences and interrelations

among selection criteria. Plant Breed. 101: 269 – 276.

Herzog, H. 1989a. Freezing resistance and performance of faba bean populations during

winter seasons in northern Germany. J. Agron. Crop Sci. 162: 225 – 235.

Herzog, H. 1989b. Influence on pre-hardening duration and dehardening temperatures on

vaietal freezing resistance in faba beans (Vicia faba L.). Agronomie 9: 55 – 61.

Herzog, H. and G. Geisler. 1991. Yield structure of winter faba bean grown in northern

Germany in dependence of different environments, seed rates, sowing rates and genotyps.

J. Agron. Crop Sci. 167: 145 – 154.

Jensen, E.S. and H. Hauggaard-Nielsen. 2003. How can increased use of biological N2

fixation in agriculture benefit the environment? Plant Soil 252: 177 – 186.

Knott, M.C. 1990. A key for stages of development of the faba bean (Vicia faba). Ann.

Appl. Biol. 116: 391 – 404.

Link, W., C. Balko, F.L. Stoddard. 2010. Winter hardiness in faba bean: Physiology and

breeding. Field Crops Res. 115: 287 – 296.

Lopez-Bellido, F.J., L. Lopez-Bellido, and R.J. Lopez-Bellido. 2005. Competition,

growth, and yield of faba bean (Vicia faba L.). Europ. J. Agron. 23: 359 – 378.

42

Peoples, M.B., J. Brockwell, D.F. Herridge, I.J. Rochester, B.J.R. Alves, S. Urquiaga, R.

M. Boddey, F.D. Dakora, S. Bhattarai, S.L. Maskey, C. Sampet, B. Rerkasem, D.F.

Khan, H. Hauggaard-Nielsen, and E.S. Jensen. 2009. The contributions of nitrogen-fixing

crop legumes to the productivity of agricultural systems. Symbiosis. 48: 1 – 17.

Picard, J., G. Duc, and R. Pelletier. 1985. “Cote d‟Or”, a highly frost resistant population

of Vicia faba. Fabis Newslett 13:11-12.

Stoddard, F.L., C. Balko, W. Erskine, H.R. Khan, W. Link and A. Sarker. 2006.

Screening techniques and sources of resistance to abiotic stresses in cool-season food

legumes. Euphytica 147: 167 – 186.

Turk, M.A. and Abdel-Rahman M. Tawaha. 2002. Impact of seeding rate, seeding date,

rate and method of phosphorus application in faba bean (Vicia faba L. Minor) in the

absence of moisture stress. Biotechnol. Agron. Soc. Environ. 6(3): 171 – 178.

43

CHAPTER THREE

Nodulation Response to Available Soil Nitrate in Faba Bean and Pea

Introduction

Legumes are often included as a rotation crop for a N benefit. Biological nitrogen

fixation (BNF), the process by which N from the air is converted to a plant usable form,

is the result of a symbiotic relationship between legumes and soil bacteria of the genus

Rhizobium. Nodules develop on the roots that house the fixation process and will appear

pink when active due to the presence of leghaemoglobin (Roughley et al., 1983). In

addition to N derived from the atmosphere (Ndfa), legumes utilize N from a combination

of sources that can also include residual soil N and added fertilizer N (Salvagiotti, 2008).

The extent to which they rely on Ndfa and therefore the extent of the N benefit is

dependent on species, climate, and management practices (Roughley et al., 1983). Soil N

has a negative impact on N2 fixation, i.e. fertilizer N or residual N can inhibit nodulation

and N2 fixation (Streeter, 1988).

Faba bean (Vicia Faba L.) is known to have a high capacity for N fixation when

compared to other grain legumes. The percentage of N derived from air (%Ndfa) in faba

bean is generally reported to be higher than other legumes. Herridge and Peoples (2008)

found that reported %Ndfa of fababean averaged 75% while chickpea (Cicer arietinum),

lentil (Lens culinaris) and pea (Pisum sativum) averaged 68%. Rennie and Dubetz (1986)

compared N fixation in cool season grain legumes and found faba bean had 85% Ndfa

while chickpea, lentil, and pea had 82, 67, and 79 %Ndfa respecitively.

It has been suggested that a contributing factor to higher N fixation by faba bean

is a greater tolerance to soil N by the faba-rhizobium symbiosis. In a field experiment,

44

Schwenke et al. (1998) found the negative effects of soil nitrate on N fixation were

greater for chickpea than faba bean. At soil nitrate levels of 65 kg∙ha-1

, faba bean

maintained 48% Ndfa while chickpea had only 24% Ndfa. Turpin et al. (2002) found

similar results whereby at a soil nitrate levels of 78 kgN/ha faba bean maintained 45%

Ndfa while chickpea maintain only 12% Ndfa.

Chalifour and Nelson (1987) used a controlled experiment to compare the N

fixation response of the legume-rhizobium symbiosis of faba bean and pea to increasing

rates of nitrate - N. While the %Ndfa decreased for both species, the suppression of N2

fixation with increasing nitrate was much larger for pea. The highest nitrate treatment, 15

mol·m-3

, resulted in zero Ndfa for pea while faba bean maintained 15.4% Ndfa. Further

investigation showed that nitrate present at planting had a much larger impact on N

fixation in pea than later applications. This was not the case for faba bean leading to the

conclusion that the tolerance of the faba bean –rhizobium symbiosis to soil nitrate must

be due in large part to its ability to form effective nodules in the presence of high

combined N (Chalifour and Nelson, 1988).

Inorganic soil N affects nodules over multiple stages of the legume-rhizobial

symbiosis including nodulation, nodule growth, and N2 fixation activity (Streeter, 1998).

The extent to which each stage is affected is dependent on both N form and

concentration. At lower levels, NH4 is less inhibitory to the legume-rhizobia symbiosis

than NO3 and can even have a stimulating effect on nodulation (Steeter, 1988; Gulden

and Vessey, 2006). When both forms of mineral N are present, the effects of nitrate are

dominant (Bollman and Vessey, 2006). Nodulation is less sensitive to available soil

nitrate than nodule growth or N fixation (Streeter, 1988). Cassman (1980) found that N

45

concentration of 5 mM combined N had no impact on nodule number, but the nodules

were much smaller and lacking in visible leghaemoglobin (Cassman, 1980).

Nodule growth and fixation activity depressed by the presence of excess mineral

N will recover with the decrease in available soil nitrate. Fujikake et al. (2002) exposed

soybean plants to a 5mM nitrate solution resulting in decreased nodule size and fixation

activity. Both nodule growth and fixation activity recovered when transferred to a N free

growing solution without observation of new nodulation. Conversely, infection of root

hairs resulting in nodulation occurs soon after they emerge. Older cells have shown to be

resistant to infection (Bhuvaneswari et al., 1980; Pueppke, 1986; Tricot et al., 1997).

Nitrogen concentrations high enough to inhibit nodulation could be critical to overall N

fixation due to the limited window in root development in which infections will occur.

Hydroponic systems are used for non-destructive observation of root and nodule

development, but studies have shown that growing medium can impact the legume-

rhizobium symbiosis. Pueppke (1986) found changes in number and distribution of

nodules with a greater percentage of nodules found on lateral roots when grown in soil as

opposed to hydroponics. To observe nodulation patterns in the soil growing medium,

plants were removed from growing containers, roots were carefully rinsed, and nodule

infections were located and counted. The same process was carried out for plants in the

hydroponic medium. The soil also influenced the rank order for legume genotype –

rhizobium strain parings with a 95% reduction in nodulation for one of the parings

(Pueppke, 1986). Van Heerden et al. (2007) found that while no difference was observed

in shoot growth using sand, fine vermiculite, course vermiculite, and potting soil mixture,

46

nodulation and N fixation differed. Nodule numbers were higher in sand and potting soil

while fixation activity was higher in vermiculite.

The objectives of this study were to compare the nodulation response of faba bean

(Vicia faba L.) with pea (Pisum sativum), a legume currently used in rotations on the

Palouse, to varying levels of available soil nitrate at planting and evaluate the use of a

high resolution scanner for non-destructive observation of nodulation and nodule growth

in a soil growing medium. A difference in tolerance to soil nitrate in terms of capacity to

fix N could offer farmers more precision and flexibility for rotation decisions made

between and even within farms in the Palouse region.

Materials and Methods

A randomized complete block design with four replicates was used to compare

the effect of nitrate on nodulation and nodule growth between faba bean and pea. Soil

was collected from the Lester and Pat Boyd Farm near Pullman, WA, managed by WSU.

The soil type is a Palouse silt loam (Fine-silty, mixed, superactive, mesic Pachic Ultic

Haploxerolls. The soil was passed through a 19 mm sieve and large organic matter was

removed by hand. Water was added until soil moisture reached approximately 23%. Wet

soil (13.8 kg dry soil equivalent) was weighed for each growing container. Nitrate

treatments were added to the soil and mixed thoroughly by hand followed by the addition

of inoculum, also mixed thoroughly by hand

A range of soil nitrate levels was achieved by adding were prepared by adding 0g,

2.91g, 5.82g, and 8.74g of Ca(NO3)24·H2O to 25ml of distilled water. This equates to

additions of 0, 25, 50, and 75 ppm soil nitrate-N. A peat based inoculum of Rhizobium

leguminosarum (N-dure, INTX Microbials, Kentland, IN) was used to inoculate the soil

47

using 110g for each growing container. With observation possible on only a portion of

the root, soil inoculation was preferred to ensure placement of inoculums didn‟t affect

nodulation patterns. Inoculating the seed results in the majority of nodules being close to

the root crown as rhizobia are limited in their movement through soil (Kyei-Boahen et al.,

2002; Madsen and Alexander, 1982).

Growing containers for in situ high resolution imaging of nodulation and root

growth were assembled according to Hammac et al. (2010). Two Canon® LiDE 600 F

flatbed scanners were placed on each side of a 36 34 23.5cm Rubbermaid ® plastic

containers at a slight angle. Each plastic container houses a single replicate for one nitrate

treatment with pea and faba planted along opposite scanners as the split plot. Treated soil

was added to the container and left overnight to equilibrate before planting.

Soil samples were taken for each container to determine available soil nitrate on

the day of planting. Four soil cores taken down the middle of the containers and

composited for testing. Holes created by removed soil were filled with perlite to aid in

watering. Two plants were seeded across each scanner face. For replications one and two,

two seeds were sown at each position and later thinned to one plant. In replications three

and four, one seed was sown in each position. The surface was covered with perlite to

decrease water loss via soil evaporation.

Plants were grown in a growth chamber under a 14-hour photoperiod with three

sodium vapor and two metal halides, each 400 watts. The temperature control was set at

21°C. Scans were taken daily at a resolution of 1200 dots per inch (dpi) for 35 days.

Above and below ground biomass were separated and weighed. Nodule senescence did

not allow for whole plant nodule weights and nodule counts.

48

Scans that had more than a quarter of the primary roots missing from view were

removed from the analysis. Images were analyzed using Adobe® Photoshop CS5

Extended for root length, nodule count, and timing of nodulation. For replications one

and two, root lengths were measured manually summing up straight length measurements

along each root using the Photoshop “measure” tool. Computerized image analysis was

used to quantify root length for replications three and four according to Pan and Bolton

(1991). The soil background in the scanned images made it necessary to adjust the

computer output. Transform equations were found for pea and faba separately by plotting

daily root lengths measured manually against daily measurements found through

computer image analysis using scans for medium treatment replication 1 (figure 11).

Absolute root lengths determined by image analysis were much greater than manual

lengths due to inclusion of root hair lengths and other non-linear growth patterns not

measured manually. All nodules visible by day 28 were identified and labeled for nodule

counts. The timing of nodulation was qualified as the day the nodule became visible.

Regression analysis was done using an ANOVA procedure of SAS for the

relationship between measured soil nitrate and nodulation.

Results and Discussion

Available soil nitrate levels from 12.5 ppm to 132 ppm N, were not enough to

completely inhibit nodulation in either pea or faba bean. However, there was a

significant negative relationship between available soil nitrate-N and nodulation (nodule

number) in pea (p < .01) (Figure 12). Nodulation decreased between nitrate levels of 55

and 84 ppm for pea. Further investigation within that range could help to identify critical

levels of available soil nitrate for nodulation in pea. Nodulation in faba bean showed no

49

response to increased available soil nitrate indicating a higher tolerance for available soil

nitrate than pea. This supports the conclusion of Chalifour and Nelson (1988) that early

stages of the rhizobium-legume symbiosis in pea are less tolerant to nitrate than faba

bean. The conclusion was based on observations that delaying exposure of nitrate-N

decreased inhibition of N2 fixation in pea, but not in faba bean with nitrate concentrations

in a hydroponic solution ranging from 0 to 15mM. Direct comparison between nitrate

concentrations of a hydroponic growing medium and a soil solution are not possible, but

with beginning soil nitrate ranging from 12.5 to 132.2 ppm and a soil moisture of 23%,

nitrate concentrations in the soil solution would range roughly from 3.87 mM to 41 mM.

There were no significant relationships between increasing available soil nitrate

and timing of first nodulation for pea or faba bean (Figure 13). These findings are

inconsistent with Guo et al. (1992), who reported that increasing N concentrations (0mM,

5mM, and 10mM) delayed nodule initiation in faba bean, white lupin, and medic.

However, nodule counts taken 15, 25, and 35 DAP were limited to nodules that were

identified as active by pink color. Nitrogen fixation is more sensitive to available nitrate

than nodulation (Steeter, 1988; Cassman, 1980). Without counts of non-active nodules

they were more likely observing a delay was in nodule development and/or fixation

activity. Delays in both due to increasing available nitrate have been observed for pea in

lab and field experiments (Bollman and Vessey, 2006; Voisin et al., 2002).

Nodules on both pea and faba bean showed a distinct pink color, possibly

indicative of active N2 fixation, when available soil nitrate levels were less than 15 ppm

at planting (Figure 14). For available soil nitrate levels higher than 45ppm, no pink color

was observed for faba bean or pea. Chalifour and Nelson (1987) observed greater % Ndfa

50

in faba bean over pea for NO3 treatments ranging in concentration from 5 to 15 mM.

Future trials that allow greater observation of nodule development in the range of 10 to

50ppm available soil nitrate may show a difference in tolerance to available nitrate for

fixation that could be measured by nodule color.

Plateaued nodule growth followed by nodule senescence disallowed measurement

of appreciable size differences to track nodule growth. While primary nodules on faba

bean roots can grow to be over a centimeter in diameter (Roughly et at., 1983), the

nodules observed in this experiment grew to only a tenth of that, 0.1cm, before

deteriorating. Nodules were no longer present to count and weigh at the experiment‟s

end, 35 DAP. This indicates that growing conditions were not sufficient to support long

term nodule growth as would be observed in the field.

Water stress may have been a contributing factor to the cessation of nodule

growth and dieback. Ramos et al. (1999) found that nodule dry-weight, nodule number

and nitrogenase activity were all reduced when soil moisture decreased to 50% field

capacity for common bean (Phaseolus vulgaris L.). With soil moisture dropping to 30%

field capacity, nitrogenase activity stopped altogether. For these same levels of drought

stress, there was no measurable difference in plant dry weight meaning the components

of N fixation are more sensitive to drought than plant growth (Ramos et al., 1999). The

average soil moisture for all growing containers 35 DAP was 11.5%.

The distribution of nodules differed between pea and faba bean. Pea had very few

nodules on the lateral roots. Averaging over all treatments, pea had only 10% of nodules

located on lateral roots while faba bean had 58%. The lack of nodules found on lateral

roots of pea was consistent with Tricot et al. (1997) who looked at nodule distribution on

51

pea roots using a hydroponic growing medium. Roughly et al. (1983) reported that a

single faba plant can develop upwards of 1000 nodules with a larger number of small

lateral nodules becoming as, if not more, significant to overall N fixation than nodules

found on the primary root. It is possible that nodule distribution could impact capacity for

N fixation in that lateral roots would be more susceptible to root hair infection both in

time and space.

Conclusion

The use of high resolution scanners offers a solution for non-destructive observation of

nodule distribution, growth, and development. Nodulation in faba bean was more tolerant

to available soil nitrate than for pea. This could have implications for adding flexibility in

rotation choice to growers with high residual N looking to achieve a N benefit from a

legume rotation. Soil nitrate did not impact timing of nodulation. Further study with

growing conditions that allow for long term plant growth are needed to investigate the

impact of available N on nodule growth. Color identification of N fixation activity will

require a correlation between quantified values for nodule color and active N fixation

activity.

52

Nitrate-N at Planting (mg N/kg Soil)

Nodules·cm-1 visible root

----Faba---- ----Pea----

12.5 0.05 0.09

13.1 0.12 0.10

48.1 0.03 .

54.6 0.08 0.11

83.7 . 0.02

91.8 0.04 0.02

99.7 0.04 0.02

111.3 0.11 .

111.4 0.26 0.03

114.7 0.05 0.03

122.1 0.03 0.04

132.3 0.10 0.04

0

20

40

60

80

100

120

140

160

180

200

0 250 500 750 1000

Ma

nu

al M

ea

sure

me

nt

(cm

)

Root LAW measurement (cm)

Root Length: Manual vs. Computerized Image Analysis

Faba bean Pea

Peay = 0.39x - 60.2R2 = 0.885

Fabay = 0.29 x- 112.2R2 = 0.984

Figure 11: Root length found using computerized image analysis plotted against measured root length using straight line

approximation to find a transform equation.

Table 7: Specific nodulation, nodules per visible root length, for faba and pea for varying levels of nitrate-N at planting

53

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00

No

du

les

pe

r v

isib

le r

oo

t (N

od

ule

s p

er

cm)

Nitrate ppm

Nodulation Response to Soil Nitrate

Faba

Pea

Figure 13: Linear regression of nodulation timing response to available soil nitrate showed no significant relationship

exists for faba bean or pea.

0

2

4

6

8

10

12

14

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00

Da

y o

f N

od

ula

tio

n

Soil Nitrate (ppm)

Nitrate effect on timing of nodulation

Faba

Pea

Figure 12: Linear Regression for nodulation response to increasing soil nitrate. There was a significant negative

relationship found for pea (R2 = 0.59, P < .01), while no relationship was found for faba bean.

54

Figure 14: Nodules observed with 13.07 ppm nitrate-N at planting (left) appear larger and pink in color compared to

nodules observed with 111.43 ppm nitrate-N at planting (right)

55

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58

CHAPTER FOUR

Conclusion

Cool season grain legumes such as pea (Pisum sativum), chickpea (Cicer

arietinum), and lentil (Lens culinaris), are commonly used rotation crops for dryland

wheat production in the Palouse region. Faba bean (Vicia faba L.) is a cool season grain

legume that is most commonly overwintered in Mediterranean regions, but is increasingly

overwintered in colder regions due to efforts in plant breeding to increase cold and frost

tolance. It is well known that faba bean has a high capacity for N fixation across a wide

range of environments. A rotation crop that can overwinter and fix large quantities of N

could be a good addition to current cropping systems.

A variety trial was conducted field testing 43 genotypes housed in the National

Plant Germplasm System (NPGS) along with 12 cultivars and breeding lines previously

identified as winter-hardy in European trials. All genotypes were fall planted and

harvested in June or July. Sixteen of the 55 genotypes tested were identified with superior

winter-hardiness and 13 of those showed potential to be high yielding. Interestingly, all

winter-hardy genotypes that showed high yield potential out of the NPGS were collected

from Bulgaria with the exception of one that originated in Hungary.

Significant correlations were found between frost resistance and both winter-

hardiness and yield potential. Low R2 values emphasized that winter-hardiness requires

stress tolerances in addition to cold and freezing temperatures. Frost resistance varied

between years at the Central Ferry location, but greater leaf damage in 09-10 didn‟t

reflect increased minimum temperature and shorter durations of below freezing

temperatures. Frost resistance will fluctuate depending pre-harding/hardening conditions

59

during plant development. Later planting in 08-09 may have contributed to increased

frost resistance.

Winter conditions for site-years could be ranked from mild to harsh based

variation in plant survival. Looking at comparative temperature minimums among site-

years for air and soil at depths of zero and 15 centimeters, the correlation between site-

years ranked by decreasing survival and decreasing temperature improved moving down

the soil profile. Reporting minimum ambient temperature is a common way to

communicate winter severity. Exposure of leaf tissue to sub-freezing temperatures is

often studied to better understand frost resistance, a characteristic linked to winter-

hardiness. Monitoring soil temperatures and including root tissue in investigations of

frost resistance may prove useful for furthering our understanding of winter-hardiness.

Nodulation in faba bean is more tolerant to soil nitrate than pea. Regression

analysis showed that for available soil nitrate-N up to 132ppm, there was no relationship

between N concentration and nodulation in faba bean. For pea there was a significant

negative relationship with a considerable drop in nodulation between 55 and 85 ppm soil

nitrate-N. Both species appeared to show responses to available soil nitrate in terms of

nodule growth and active fixation. Nodules for both pea and faba bean appeared larger

and pink in color for soil nitrate-N 13ppm than at soil nitrate-N levels greater than 48

ppm.

Nodulation, nodule growth, and fixation activity are all important components of

total N fixation. While differences in tolerance to soil nitrate in terms of nodulation have

been identified between faba bean and pea, further study at nitrate concentrations less

than 48 ppm soil nitrate-N are needed to evaluate differences in tolerance in terms of

60

nodule growth and active fixation. Quantifying nodule growth will require growing

conditions that support long term plant growth. Decreasing soil moisture appeared to

affect nodule growth and survival.

The use of high resolution scanners may be a solution for non-destructive

observation of root and nodule development in a soil growing medium. Finding a way to

maintain soil moisture without affecting the soil scanner interface proves to be the next

challenge for observing long term growth. Computer image analysis using Root LAW

was effective for finding root length measurements. Some manual measurement is

required to find transform equations to correct for overestimation for each species.

Further investigation is needed to find the cause for over-estimation of root length.

Genotypes of faba bean were identified that survive and yield in the Palouse. Faba

bean shows a high tolerance to residual soil nitrate which could contribute to a greater

capacity to fix N. The results of these studies support continuing investigation of faba

bean as a potential crop rotation for the Palouse.