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thesis entitled

EVALUATION OF DRY BEAN GENOTYPES FOR
PERFORMANCE UNDER ORGANIC PRODUCTION
SYSTEMS; EVALUATION OF EARLY NITROGEN
FIXATION IN DRY BEAN

presented by

James A. Heilig

has been accepted towards fulfillment
of the requirements for the

MS. degree in Plant Breeding, Genetics, and
Biotechnology

 

 

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EVALUATION OF DRY BEAN GENOTYPES FOR PERFORMANCE UNDER
ORGANIC PRODUCTION SYSTEMS; EVALUATION OF EARLY NITROGEN
FIXATION 1N DRY BEAN
By

James A. Heilig

A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of .
MASTER OF SCIENCE

Plant Breeding, Genetics, and Biotechnology

2010

ABSTRACT

EVALUATION OF DRY BEAN GENOTYPES FOR PERFORMANCE UNDER
ORGANIC PRODUCTION SYSTEMS; EVALUATION OF EARLY NITROGEN
FIXATION IN DRY BEAN

by
James A. Heilig

The performance of 32 diverse dry bean (Phaseolus vulgaris L.) genotypes, including one
non nodulating check, was evaluated under organic and conventional management
systems, in side by side plots. Research was conducted at multiple locations: Kellogg
Biological Station, Gull Lake, MI in 2007-2009, and in Gratiot County, MI in 2007 and
2008 and in Tuscola County in 2009. The conventional treatment was managed using
recommended practices, while organic plots were managed with approved methods for
certiﬁed organic production. The best performing bean genotypes were generally ﬁom
the Middle American gene pool. The black bean genotype ‘Zorro’ performed well at all

locations and years, and across both management practices systems.

Recognizing nitrogen as a limiting factor in organic production, the same 32 diverse
genotypes plus a high N-ﬁxing check and a non nodulating check, were screened under
greenhouse conditions for their ability to ﬁx nitrogen. Seed was sterilized, inoculated
with Rhizobium etli UMR 1597, and fertilized with a nitrogen free solution. Plants were
harvested at initial ﬂowering, weighed for root mass and biomass measured for height
and root length, rated for nodule growth and nitrogen content of biomass. Genotypes of
Middle American origin were superior to those of Andean origin for nitrogen ﬁxation and
biomass yield at the early establishment growth stage. Black, red, and pink seed classes

ranked highest in terms of both biomass and nitrogen accumulated.

Dedication: To my Grandmother, Theresa Wik and my Mother, Patricia Stier, for their
love, support, and encouragement.

iii

TABLE OF CONTENTS

LIST OF TABLES .................................................................................... v
LIST OF FIGURES ................................................................................. ix
CHAPTER 1

Introduction ........................................................................................................................ 1
History and Background ................................................................................. 1
Current Trends in Organic Production4
Crop Systems and Nitrogen Fixation... 7
NitrogenFixationandtheCommonBean 10
Methods to Study Nitrogen Fixation 1n the COmmon Bean 11
The Role of Rhizobia .................................................................................. 22
Objectives .............................................................................................. 25
CHAPTER 2

ORGANIC AND CONVENTIONAL PRODUCITON SYSTEMS...........................29
Abstract ................................................................................................. 29
Introduction ............................................................................................. 30
Materials and Methods ................................................................................. 34
Results ................................................................................................... 37
Discussion .............................................................................................. 42
Tables and Figures ..................................................................................... 49
CHAPTER 3

EVAULATION OF DRY BEAN GENOTYPES FOR BIOLOGICAL NITROGEN
FIXATION ............................................................................................ 63
ABSTRACT ............................................................................................ 63
Introduction ........................................................................................... 64
Materials and Methods .................................................................................. 71
Results ................................................................................................. 74
Discussion ............................................................................................. 76
Conclusion ............................................................................................ 87
Tables and Figures ................................................................................... 89
SUMMARY ..................................................................................................................... 102
APPENDICES ................................................................................................................. 106
Appendix A ...................................................................................................................... 106
Appendix B ...................................................................................................................... 127
Appendix C .................................................................. ' .................................................... 128
Appendix D ...................................................................................................................... 129
LITERATURE CITED ............................................................................ 130

iv

List of Tables

Table 1-1 . U.S.Organic dry bean (Phaseolus vulgaris L.) hectares by state, 1997 through
2005........... ................................................................................................................... 28

Table 2-1. Seed class, seed size, gene pool, race, and growth habit for 32 dry bean
(Phaseolus vulgaris L.) genotypes evaluated in organic and conventional production
systems at Kellogg Biological Station and Gratiot County in 2007, 2008, and 2009. . ....49

Table 22. Soil analysis and soil type of Kellogg Biological Station, Gratiot County, and
Tuscola County research sites from 2007 to 2009 .............................................. 50

Table 2-3. Precipitation (mm) from June to September at study sites where 32 dry bean

(Phaseolus vulgaris L.) genotypes were evaluated for production in organic systems in
2007, 2008, and 200951

Table 2-4. Mean yield of 32 dry bean (Phaseolus vulgaris L.) genotypes grown in
organic and conventional systems at KBS from 2007 to 2009 ................................ 52

Table 25. Mean yield of 9 Andean dry bean (Phaseolus vulgaris L.) genotypes and non

nodulating R99 check grown in organic and conventional production systems at Kellogg
Biological Station, 2007-200953

Table 2-6. Mean yield of 22 Middle American dry bean (Phaseolus vulgaris L.)
genotypes grown in organic and conventional production systems at Kellogg Biological
Station, 2007-200954

Table 2-7. Mean yield of 32 dry bean (Phaseon vulgaris L.) by seed class grown in
organic and conventional production systems at Kellogg Biological Station, 2007 to
2009 .................................................................................................................................... 55

Table 2-8. Mean yield of 32 dry bean (Phaseolus vulgaris L.) genotypes Gratiot County
in organic and conventional production systems from 2007 to 2008 ........................ 56

Table 29 Yield of 32 dry bean (Phaseolus vulgaris L.) genotypes grown in Tuscola
County in an organic production system in 2009. ............................................. 57

Table 2-10. Mean yield by 32 dry bean (Phaseolus vulgaris L. ) genotypes grown in

organic and conventional product systems at Kellogg Biological Station from 2007 to

Table 2-11. Mean Nitrogen accumulated by 32 dry bean (Phaseolus vulgaris L.)
genotypes in organic and conventional production systems at Kellogg Biological Station
from 2007 to 2009 .............................................................................................................. 58

Table 2-12. Mean Nitrogen Yield of 32 dry bean (Phaseolus vulgaris L.) genotypes
grown in organic and conventional production systems at Kellogg Biological Station
from 2007 to 2009 ................................................................................... 59

Table 2-13. Yield by seed class of 32 dry bean (Phaseolus vulgaris L.) genotypes grown
in Tuscola County in an organic production system in 2009. ................................. 60

Table 2-14. Pearson correlations for maturity, plant stand, days to ﬂower, height at
ﬂowering, 100 seed weight, yield and nitrogen yield for Organic and Conventional Trials.
......................................................................................................... 61

Table 3-1 Modiﬁed Hoagland’s nutrient solution utilized in the greenhouse study of
nitrogen ﬁxation of 34 dry bean (Phaseolus vulgaris L.) genotypes .......................... 89

Table 3-2. Nitrogen Fixed plant'1 of the 34 dry bean (Phaseolus vulgaris L.) genotypes
evaluated for nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in
September and December 2008 and March 2009 ............................................................ 91

Table 3-3. Nodule rating for 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation under greenhouse conditions in East Lansing, MI in September and
December 2008 and March 2009 ................................................................................. 92

Table 3-4. Nodule rating for 10 dry bean (Phaseolus vulgaris L.) averaged by seed class
evaluated under greenhouse conditions for nitrogen ﬁxation ability in East Lansing, MI in
September and December 2008 and March 2009 . . 93

Table 3-5. Average root mass of 10 dry bean (Phaseolus vulgaris L.) market classes
evaluated at the ﬁrst ﬂower stage for nitrogen ﬁxation ability under greenhouse
conditions in East Lansing, MI in September and December 2008 and March 2009. .....94

Table 3-6. Root mass of 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation ability under greenhouse conditions In East Lansing, MI In September
and December 2008 and March 2009... . 95

Table 3- 7. Average biomass of 10 dry bean (Phaseolus vulgaris L.) seed classes evaluated for
nitrogen ﬁxation ability under greenhouse conditions In East Lansing, MI In September and
December 2008 and March 2009... 96

Table 3-8. Biomass of 34 dry bean (Phaseon vulgaris L.) genotypes evaluated for nitrogen
ﬁxation ability under greenhouse conditions in East Lansing, MI in September and December

Table 3-9. Shoot to root ratio of 34 dry bean (Phaseolus vulgaris L.) genotypes
evaluated for nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in
September and December 2008 and March 200998

vi

Table 3-10. Shoot to root ratio of 10 dry bean (Phaseolus vulgaris L.) seed classes
evaluated under greenhouse conditions for nitrogen ﬁxation ability in East Lansing, MI in
September and December 2008 and March 2009 .......................................................... .99

Table 3-11. Nitrogen ﬁxed per day of 10 dry bean (Phaseolus vulgaris L.) seed classes
evaluated for nitrogen ﬁxation under greenhouse conditions in East Lansing, MI in
September December 2008 and March 200999

Table 3-12. Percent nitrogen derived from the atmosphere (%Ndfa) of 34 dry bean
(Phaseolus vulgaris L.) genotypes evaluated for nitrogen ﬁxation ability under
greenhouse conditions in East Lansing, MI in September and December 2008 and march

2009 ...................................................................................................... 100

Table 3-13. Correlations among growth characteristics of 34 dry bean (Phaseolus
vulgaris L.) genotypes evaluated for nitrogen ﬁxation under greenhouse conditions in
East Lansing, Mi in September and December 2008 and March 2009 ........................... 101

Summary Table 1. Pearson Correlations of 32 dry bean genotypes grown under organic
production at Kellogg Biological Station, Gratiot County, and Tuscola County from 2007
to 2009 ............................................................................................................................. 104

Summary Table 2. Ranking of the ﬁve highest yielding over all sites and years, and
highest nitrogen ﬁxed, of 32 dry bean genotypes ............................................................ 105

Table A1. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
at Kellogg Biological Station in 2007 .............................................................................. 106

Table A2. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically at Kellogg Biological Station in 2007 ........................................................... 107

Table A3. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2007 ..................................................... 108

Table A4. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2007 ..................................................... 109

Table A5. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
in Gratiot County in 2007 ................................................................................................ 110

Table A6. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically Gratiot County in 2007 ................................................................................. 111

Table A7. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally in Gratiot County in 2007 ....................................................................... 112

Table A8. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
at Kellogg Biological Station in 2008 .............................................................................. 113

vii

Table A9. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically at Kellogg Biological Station in 2008 ........................................................... 114

Table A10. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2008 ..................................................... 115

Table A11. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2008 ..................................................... 116

Table A12. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
in Gratiot County in 2008 ................................................................................................ 117

Table A13. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically Gratiot County in 2008 ................................................................................. 118

Table A14. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally in Gratiot County in 2008 ....................................................................... 119

Table A15. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally in Gratiot County in 2008 ....................................................................... 120

Table A16. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
at Kellogg Biological Station in 2009 .............................................................................. 121

Table A17. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically at Kellogg Biological Station in 2009 ........................................................... 122

Table A18. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2009 ..................................................... 123

Table A19. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
conventionally at Kellogg Biological Station in 2009 ..................................................... 124

Table A20. Small seeded dry bean (Phaseolus vulgaris L.) genotypes grown organically
in Tuscola County in 2009 ............................................................................................... 125

Table A21. Medium and large seeded dry bean (Phaseolus vulgaris L.) genotypes grown
organically in Tuscola County in 2007 ............................................................................ 126

Table B1.Average day of harvest of 34 dry bean (Phaseolus vulgaris L.) genotypes
evaluated for nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in
2008 and 2009 .................................................................................................................. 127

Table C1.Average percent seed N of 32 dry bean (Phaseolus vulgaris L.) genotypes each
year grown under organic and conventional production systems at Kellogg Biological
Station in 2007 to 2009 .................................................................................................... 128

Table D1. Average percent seed protein of 32 dry bean (Phaseolus vulgaris L.) genotypes
each year grown under organic and conventional production systems at Kellogg
Biological Station in 2007 to 2009 .................................................................................. 129

viii

List of Figures

Figure 1-1. Simpliﬁed equation of the process of nitrogen ﬁxation ......................... 10
Figure 1-2. Hectares of certiﬁed organic cropland in Michigan, 1997 through 2005 ..... 26

Figure 1-3. Hectares Organic Dry beans (Phaseolus vulgaris L.) produced in Michigan
1997 through 2005 .......................................................................................................... .27

Figure 2-1. Scatter plot of yield of 32 dry bean (Phaseolus vulgaris L.) genotyps in
organic and conventional treatments at Kellogg Biological Station. Conventional yield is
on the Y axis and Organic yield on the X axis .................................................. 62

Figure 3-1. Nitrogen ﬁxed per plant of 10 seed classes evaluated for nitrogen ﬁxation
under greenhouse conditions in East Lansing, MI in 2008 and 200990

ix

CHAPTER 1

HISTORY AND BACKGROUND

The common bean, Phaseolus vulgaris L., is indigenous to the Americas. The center of
origin for this species is in the Andes region of South America (Kami et al., 1995), from
where it spread south into South America and north into Central America and Mexico
and eventually was carried into North America through trade (Gepts, 1998). The
common bean was domesticated in two major geographic regions; the Andean gene pool
and the Middle American gene pool (Gepts, 1988). Within these regions there is evidence
of multiple domestication events. The two major gene pools are divided further into
races which are based on their agro-ecological adaptation (Singh et al., 1991). The
Andean gene pool is comprised of three races, Nueva Granada, Peru, and Chile whereas
the Middle American gene pool comprises the races Mesoamerica, Durango, and Jalisco.
Based on seed morphology, plant architecture, and RAPD analysis Beebe et a1. (2000)
identiﬁed a fourth group of beans in the Middle American gene pool which was
designated as Race Guatemala due to its presence primarily in that country. Crossing
between both gene pools is made difﬁcult by the presence of post zygotic fertility barriers
which result in hybrid seedlings that are weak and may die due to the combination lethal
genes present in both gene pools (Beaver and Osomo, 2009). The common bean is
inbreeding with a low level of out crossing, which can increase in certain tropical regions
in its various places of origin (Graham and Ranalli, 1997). In California Ibarra-Perez et
a1. (1997) found a 6.9% rate of outcrossing between a black seeded genotype with purple

hypocotyl and a white seeded genotype with green hypocotyl.

According to Gepts (1998), common beans from Middle American Race Mesoamerica
migrated from Central America into the Caribbean and from there north into the
Southeastern region of North America; these types generally consisted of small black or
white seeded beans. Medium seed sized beans belonging to Race Durango comprise both
white seed coat, or Great Northern, and mottled seed coat, or Pinto bean. Race Durango
beans moved north through Central America into Mexico and Southwestern North
America and north into the Great Plains region as far north as modern day Saskatchewan
(Gepts, 1998). Seed of the common bean from the Andean gene pool was taken to the
Iberian Peninsula by Spanish and Portuguese sailors where it subsequently spread largely
to highland regions of East Africa and into Europe. European settlers brought common
bean cultivars to North America, introducing the Andean gene pool into Northeast North
America. The diversity of bean seed types currently grown in the US. is classiﬁed into
the same races based on local adaptation and market opportunities. Kidney (N ueva
Granada race), cranberry (Chile race), and yellow (Peru race) beans are market classes
commonly grown in North America and are members of the Andean gene pool. Race
Mesoamerica is represented by the small seeded navy and black beans. The medium-
sized pinto and great northern beans are both of race Durango, and small reds and pinks
belong to race Jalisco. Race Guatemala types are climbing beans from Southern Mexico

and Guatemala are not grown in North America.

Currently, the United States is the sixth leading producer of dry beans in the world, after
Brazil, India, Myanmar, and Mexico. One ﬁfth of the dry beans produced in the US. are
exported. Michigan and North Dakota together produce half of the beans grown in the

US. Michigan is the number one producer of black beans, small reds, and cranberry
2

beans while ranking second in production of navy beans and kidney beans. The largest
single seed class produced in the United States is pinto bean.

(http://www.ers.usda.gov/Briefmg/drybeans/, accessed December 2009)

The major production area in Michigan is the central Saginaw valley and thumb,
consisting of Huron, Sanilac, Tuscola, Saginaw and Bay counties where primarily small
sized seed classes, such as black and navy beans are grown. Further west, there is
signiﬁcant production in Gratiot, Isabella, and Montcalm counties. In the sandy soils of
Montcalm and Isabella County large-seeded types such as kidney and cranberry are the
primary commercial classes produced. The majority (96%) of beans in Michigan are
produced under rain fed conditions though irrigation is employed in areas with light and
sandy soils such as Montcalm County. The majority of Andean beans possess an upright
determinate type I-growth habit, which produces a uniform large seeded bean. Producers
of small-seeded beans prefer the upright type II indeterminate upright architecture for
direct harvest as opposed to type III prostrate vine growth habit that favors development
of diseases such as white mold (Sclerotinia sclerotiorum). Type IV climbing types, such
as those of Race Guatemala, are not grown commercially in Michigan. Many producers
direct harvest the bean crops which is facilitated by taller, upright plants with
concentrated pod set in the middle of the canopy and a thicker stem that resists lodging.
Growers continue to windrow and thrash large seeded bush types to ensure seed quality,
though production trends toward direct harvest especially in the smaller seed classes such
as black and navy beans. Factors reducing productivity include drought, disease, and
insect pests in combination with insufﬁcient nutrient levels and low or high pH soils

(Kelly et al., 1999). In Michigan, precipitation can be erratic leading to periods of water
3

stress that reduce crop productivity. Growers typically band a side dress of fertilizer at a
rate of 40 to 55 kg ha.1 at planting time whereas, soybean, (Glycine max), is not usually

fertilized since it is capable of ﬁxing sufﬁcient nitrogen to produce an acceptable yield.
CURRENT TRENDS IN ORGANIC PRODUCTION

Organic production is increasing in Michigan, which consistently ranks in the top ﬁve
states for organic production of dry beans (ERS, http://wwwersusdagov/data /organic/
accessed December 2009). In the period from 1997 to 2005, area planted to organic
beans has increased nearly threefold, from 334 ha to 968 ha in Michigan (table l-l). In
the same period production of organic soy increased ﬁom 2,470 ha to 5,850 ha. In 1997,
there were fewer than 6,070 ha of certiﬁed organic crop land in Michigan. By 2005 that
ﬁgure had increased to over 17,401 ha (ﬁg 1-2). Production of organic dry beans is being
consolidated into fewer states as the top ﬁve states producing organic dry beans are
increasing in production area; the number of states producing dry beans is decreasing
(ERS-NOP, Organic Brieﬁng Room, http://www.ers.usda.gov/bdeﬁngiorganic/ accessed
December 2009). Nationally organic crop production has experienced considerable
growth recently. From a national value of $3.6 billion in 1997, organic crop value has
grown to $21.1 billion in 2008 (http://www.ers.usda.gov/Briefmgzmbeansh accessed
December 2009). Though there is an increase in certiﬁed organic acreage, supply still
does not meet demand (Dimitri and Oberholtzer, 2009). In 2002 the National Organic
Program (N OP) began regulating organic production by setting standards and certifying
third parties who certify farms and production facilities. The Organic Materials Review

Institute (OMRI) reviews products and practices and approves/disapproves for use in

organic production. Artiﬁcially produced chemicals and fertilizers are generally not
approved for application in organic production. Insects and disease must be controlled
with approved, naturally occurring pesticides. Herbicides are not approved, necessitating
weed control by mechanical means such as cultivation and hand pulling. The use of a
ﬂame to kill young weeds and sterilize the surface of the soil to prevent future
germination has gained attention in recent years. Soil nutrient levels are managed
through crop rotations, often involving a cover crop which may be a legume or other
crop, and application of manures and compost. (U SDA-ERS, http://www.ers.u&ggv_/

accessed December 2009)

Changes in weather patterns and movement of dry bean production to more marginal
crop lands has resulted in a need for dry beans with tolerance to drought stress and to
soils of marginal fertility. Different production systems such as organically grown dry
beans present their own unique challenges. In searching for germplasm useful in
developing dry bean genotypes adapted to drought stress in Idaho, Singh et al. (2009)
evaluated genotypes grown in Southwest North America where beans were cultivated for
thousands of years under drought stress in marginal soils. Singh et al. (2009) conducted
ﬁeld evaluations to ﬁnd genetic variation in landraces adapted to the Southwest for use in
other areas of production. The common Red Mexican bean was identiﬁed as tolerant of
such limiting conditions as drought and weeds following evaluation under seven
production systems, including on farm and on station systems as well as organic and
conventional with high and low inputs. Production systems affected crop characteristics
such as days to maturity was increased in the stressed environments (Singh et al., 2009).

Stressful growing conditions were also correlated with a reduction in seed weight. There

5

were also large differences in seed yield among the landraces and genotypes evaluated
across different production systems. Greatest yields were observed under the
conventional system with higher levels of inputs on research stations and the lowest
yields were produced under on-farm low input organic systems. The increased inputs of
the on farm system yielded nearly three times the on farm low input organic system. This
suggests that some cultivars are better adapted to different production systems than other
genotypes. Singh et a1. (2009) concluded that testing dry bean genotypes in organic
systems would be useful in identifying genotypes best suited to that system and better
control of environmental conditions could be achieved by having certiﬁed organic ground

available for research on research stations.

Costs of supplying nutrients are increasing on a wide scale as the cost of energy
increases. Crops that are better able to acquire more nutrients from the soil, or ﬁx
nitrogen from the atmosphere will help to relieve costs to producers in both developed
and developing countries. A reduction in the application of nutrients to ﬁelds may also
have the added beneﬁt of reducing the amount of pollution due to run off and ground
water contamination. Another major concern is the impact of agriculture on global
warming. Nitrous gases and nitrous oxides are more effective at trapping heat in the
atmosphere than C02. N “oxide” emissions have been associated with the application of
nitrogen containing fertilizers to agriculture systems, speciﬁcally the over application of

N to the soil (Peoples et al., 2009).

Crop legumes are often used in crop rotations to help increase the nitrogen level available
in the soils. Alternating nitrogen ﬁxing crops with non ﬁxing cereal crops which require

addition of nitrogen to the soil has been a means to improve yields of the non ﬁxing
6

crops. Those crops able to ﬁx nitrogen may be split into two groups based on harvested
product. One group is forages which are grown for biomass either for high-N feed for
animals or for integration into the soil as green manure in order to improve soil quality.
The other group is grain legumes which are annuals cultivated for the high value protein
rich seed they produce as well as for the residual nitrogen ﬁxed by the crop (Buttery et

al., 1992).
CROP SYSTEMS AND NITROGEN FIXATION

Forages consist of both perennial and annual plants such as alfalfa (Medicago sativa) and
clover (T rifolium spp.) and are harvested for their biomass to feed animals or are
incorporated into the soil to contribute to nutrient levels, especially nitrogen (Peoples et
al., 1995). Breeding of forage legumes focuses on production of quality biomass. Forages

are often cited as being quite efﬁcient in ﬁxing large quantities of nitrogen per year.
Estimates range up to 500 kg N ha"1 per year ﬁxed for perennial forages such as alfalfa

(Lindemann and Glover, 2003). This large amount of nitrogen ﬁxed is attributable to the
length of time the perennial forages are established in the ground compared to annual
legume crops. According to Bliss (1993b) there is a relationship between maturity and
nitrogen ﬁxed with later maturing dry bean genotypes ﬁxing more nitrogen owing to the
fact that they are in the ground longer than shorter season cultivars providing them more

time to ﬁx nitrogen.

Grain legumes, such as soybean, dry bean, and cowpea (Vigna unguiculata) are selected
to partition resources into the seed. This seed is then harvested, with the residual straw
typically left in the ﬁeld. These residues may contribute to increased nitrogen levels in

7

the soil; however, the plant is selected to translocate most of the nitrogen into the seed (as
reviewed in Buttery et al., 1992). As a result, the crop residue from grain legumes often

contributes little nitrogen to the following crop. Beckie and Brandt (1997) calculated that
integration of ﬁeld pea (Pisum sativum) residue contributed 15 kg N ha"1 for each 1000
kg of grain yield. Soon and Arshad (2002) estimated that pea straw contributed up to
41kg N ha.1 with an additional 2-3 kg N ha'1 in the root system indicating that the roots

may contribute a small percentage to the total nitrogen left for the next crop.

Some grain legume crops are more efﬁcient at ﬁxing nitrogen. Soybean has the ability to
ﬁx enough nitrogen through biological nitrogen ﬁxation (BNF) to achieve acceptable

yields without the need for additional fertilizer. Piha and Murms, (1997a), reported

soybean ﬁxing up to 189 kg N ha]. This was approximately two-fold higher than their

ﬁndings for common beans, where early maturing genotypes ﬁxed 35 kg N ha'1 as

compared to the 109 kg N ha'1 ﬁxed by later maturing genotypes.

Nitrogen ﬁxing plants form a symbiotic relationship with various soil bacteria such as
Rhizboium and Bradyrhizobium. Formation of nodules is preceded by the plant exuding
ﬂavonoides into the root zone which stimulates the Rhizobium to release nodulation
factors, initiating the process of infection. A root hair is modiﬁed by the legume plant
into an infection thread, or hook, through which the bacteria enters the plant cell. The
bacteria then move into the cortex of the root and begin to multiply. Through signaling
between host and bacteria, the cortex cells divide and enlarge to form a nodule. The
bacteria differentiate into bacteroids which is the phase of life that ﬁxes nitrogen. This

nodule serves as the housing for the nitrogen ﬁxation process. While Rhizobia are
8

aerobic bacteria, nitrogen ﬁxation requires the absence of oxygen, thus oxygen is limited
in the center of the nodule through the action of leghemoglobin, which is similar to
hemoglobin in the blood of animals and binds to oxygen. Leghemoglobin controls the
levels and movement of oxygen in the plant cells of the nodule and provides the bacteria
with oxygen for respiration but not enough to reduce the function of nitrogenase. The
presence of leghemoglobin results in the red, pink or orange color of a functioning
nodule. Other colors, such as cream, green or gray indicate the nodule is not ﬁxing
nitrogen either due to age or the symbiosis of a non-ﬁxing, or poorly ﬁxing Rhizobium.
Plants likely sanction non performing nodules by restricting carbohydrate ﬂow to those

nodules. Once the nodule has formed however, no other bacteria can colonize that
infection site. Inside the nodule N2 is combined with H2 to form ammonia, through the

activity of nitrate reductase, which can then be utilized by the plant. This process requires
an investment from the plant in the form of protection, carbohydrates, and proteins for
sustenance of the bacteria and energy for ﬁxation while the bacteria provide nitrogen in a
form usable by the plant. Disease, abiotic stress and developing seed may all compete

with the nodules for resources and thus reduce ﬁxation. (Maiti, 1997;

http://enwilciﬂ'aorgiwiki/Rhizobia, accessed November 2009).

Figure 1-1. Simpliﬁed equation of the process of nitrogen ﬁxation.

 

Energy

l
N2+3 H2->2NH3‘

 

 

 

NITROGEN FIXATION AND THE COMMON BEAN

Like most members of Fabaceae, the common bean P. vulgaris, has the ability to ﬁx
nitrogen from the air through a symbiotic relationship with Rhizobia spp. Unlike many

crop legumes, however, the ability to ﬁx a sufﬁcient amount of nitrogen to affect
competitive yields through this symbiosis is lacking in dry bean. Estimates of 50 kg N ha'
1 ﬁxed are reported as common by Bliss (1993a). Since it is unlikely that 50% of the
nitrogen applied as fertilizer is assimilated by bean plants, Bliss (1993a) points out that
50 kg N ha'1 ﬁxed is equivalent to applying nitrogen at a rate of 100 kg ha']. Typical
nitrogen application rates are closer to 50-60 kg N ha'1 with recommendations up to 100
kg 11 ha'1 when high yields (2,000 kg ha'1 or greater) are desired, and with certain

determinate seed classes such as kidney and cranberry beans that respond to N

t1m://www.2_rg.ndsu.nodak.edu/plantsci/breedingzmbean, accessed December 2009).

Accordingly, beans would have to ﬁx between 50 and 100 kg N ha'1 in order to achieve
competitive yields. Dry bean breeding programs typically supplement soil nitrogen with

fertilizer during the process of selection and yield evaluation. As a result, genotypes are

10

not selected for their ability to ﬁx nitrogen as they are being primarily selected for

disease resistance, maturity, yield, and quality traits.
METHODS TO STUDY NITROGEN FIXATION IN COMMON BEAN

Within the primary gene pool of P. vulgaris, there is variation for improved nitrogen
ﬁxation (Graham, 1981; Park and Buttery, 1990; Rennie and Kemp, 1983; Bliss, 1993).

In reviewing studies conducted at CIAT, Graham (1990) reports ﬁxation rates of bean
genotypes ranging from 3 kg N ha'1 to 125 kg N ha'l. There is a considerable variation

suggesting the opportunity for potential improvement in BNF. Graham states that
nitrogen ﬁxation is a quantitative trait, suggesting that improvements can be made by
cyclic or recurrent breeding methods to further improve nitrogen ﬁxation. Utilizing three
common bean genotypes identiﬁed in previous studies, Elizondo-Barron et a1. (1999)
demonstrated that following two generations of selection gain for seed yield was 10.2%
while there was an increase of 8.1% for seed nitrogen. Signiﬁcant increases were seen
between the average values of the original parent lines (C0 in their study) and the
selected C2 plants. And increase in nitrogen from selection was seen with the original
parent lines was 5.0 g N ﬁxed per four plants whereas the C2 selected lines achieved 6.2
g N ﬁxed per four plants. Seed yield for every four plants was 137 g for the parental
lines and averaged 180 g per four plants in the selected C2 lines. Elizondo-Barron et al.,
(1999) selected solely on seed nitrogen and shoot biomass to achieve these increases. St.
Clair et a1. (1988) measured gains over the recurrent parent, Sanilac, in population 24

which utilized Puebla 152 as the donor parent for nitrogen ﬁxation. One line in

particular, 24-17, ﬁxed 1,110 mg N plant'l, while Puebla 152 ﬁxed an average of 1,053

11

mg N plant'1 and Sanilac ﬁxed 629 mg N plant’l. Both Blizondo-Barron et al. (1999) and

St. Clair et a1. (1988) found progeny with signiﬁcant increases in nitrogen ﬁxed over one

or both parents used in their respective studies.

Bliss (1993b) suggested that there is a need to combine this variation into a single
agronomic package-that is a single genotype which has the appropriate agronomic
characteristics, adaptation to day length, and disease resistance-to be usable in
commercial situations. One obstacle to selecting genotypes for their BNF ability is the
difﬁculty in observing and measuring root traits, especially of ﬁeld grown plants.
Methods to measure the amount of nitrogen ﬁxed such as acetylene reduction analysis,
15N dilution, and total nitrogen content have been developed. Each method utilized in
studying BNF characteristics has advantages, but also has limitations and as such no

single method is adequate.

A once popular method to measure nitrogen ﬁxation is acetylene reduction analysis
(ARA) which allows the indirect measure of nitrogen ﬁxation by measuring the evolution
of ethylene from the process in the presence of acetylene. The level of ethylene is then
measured through gas chromatography. The level of ethylene is then interpreted as a
measure of nitrogenase activity, and thus nitrogen ﬁxation (Hardarson and Danso, 1993;
Herridge and Danso, 1995). ARA offers measurements at a very speciﬁc point in time,
however does not account for the level of ﬁxation during the entire growing season.

Also, nodule activity is dependent on time of day as well as soil moisture and
temperature. In addition nodules or roots containing nodules are typically detached from

the plant. The limitations to ﬁxation are not known when these tissues are removed from

12

their source of carbohydrates. Hardarson and Danso (1993) also suggest that there is the
potential of the presence of the acetylene to reduce the activity of nitrate reductase which
is an enzyme critical to nitrogen ﬁxation. All these factors might result in invalid
estimates of BNF. None the less, St. Clair and Bliss (1991) utilized ARA to identify the
high ﬁxing genotype Puebla 152. They also found that differences in nitrogen ﬁxation as

determined by ARA were a moderately heritable quantitative trait.

Tropical legumes transport nitrogen ﬁom ﬁxation within the plant in the form of ureides,
allontoin, and allontoic acid. Cool season legumes transport nitrogen from ﬁxation as
amides. Nitrogen derived from soil or fertilizer is transported in the form of nitrates
(Hardarson and Danso, 1993). For crops such as soybean, dry bean, and cowpea, the
amount of ureide in the sap bleeding from a cut stem has been used as a means to
measure nitrogen ﬁxation (Hardarson and Danso, 1993; Thomas et al., 1984). Thomas et
al. (1984) utilized a subset of the Puebla 152 x Sanilac population developed by St. Clair
and Bliss (1991) and discovered that Puebla 152 transported a greater amount of N than
Sanilac. Also, during vegetative growth of both parents most nitrogen transported was in
the form of nitrate, though Puebla 152 began transporting more ureides during late
vegetative growth. This changed, however during blooming and pod ﬁll when ureide
levels increased in both parents. Thomas et a1. (1984) concluded that percent of nitrogen
in the form of ureides in both high ﬁxing genotypes and low ﬁxing genotypes of those
studied was not necessarily different, though the rate of translocation was higher in those
that ﬁxed larger amounts of nitrogen. Rate of nitrogen ﬁxation in this study was

determined by ARA.

l3

Nodule number or mass could also provide an estimate of the potential for BNF, but this
does not take into account the activity of the nodules themselves. Also, edaphic and
weather conditions interact to effect this characteristic resulting in a reduced ability of
researchers to notice and document differences based on observing nodules (Herridge and
Danso, 1995). Measuring nodule characteristics is destructive and is only useful in
breeding programs practicing selection at the genetic family level where some plants may
be sacriﬁced to measure nodule characteristics while others are left to produce seed

(W olyn et al., 1991). Hardarson and Danso (1993) indicate that nodule number or mass
is best when used together with other measurements of nitrogen ﬁxation in order to be
useful in interpretation of data. This would help to determine if nodules present were
actually ﬁxing nitrogen while still giving an idea of potential based on the number and

condition of nodules present.

Other indirect methods include shoot dry weight and the total N of the shoots. According
to Hardarson and Danso (1993) measuring the biomass of the crop is a simple and
relatively accurate method to estimate nitrogen ﬁxation when comparing different
genotypes. Shoot dry weight can only be used to compare genotypes in the same study or
planting, as no value is given to the actual nitrogen ﬁxed thus comparisons with the work
of other researchers is limited. The primary assumption in this strategy is that nitrogen is
the limiting factor; if other limitations exist the comparisons between genotypes may not
be related to nitrogen ﬁxation. Differences in nitrogen ﬁxation between genotypes may
only be apparent when plants are grown in low N or no N soil conditions (St. Clair and
Bliss, 1991; Bliss, 1993b; Graham and Vance, 2000; Rennie, 1983) Evaluation is

facilitated by the visual appearance of the plants (Pereira et al., 1989). Plant type may
14

confound the results, however, as some growth habits in and of themselves may limit
nitrogen uptake by reduced soil mining, susceptibility of roots to pathogens, or

availability of Rhizobium infection sites.

Nitrogen difference methods rely on the availability of a reference crop. This reference
crop must acquire soil nitrogen in a similar manner to the crop being grown, but is not
able to ﬁx nitrogen itself. The amount of nitrogen in the reference crop represents the
available N, from both the soil and any nitrogen fertilizer applied. The difference
between a nitrogen ﬁxing genotype and the amount of nitrogen in the non-ﬁxing

reference crop represents the nitrogen ﬁxed by the ﬁxing genotype. This method has been
shown to be a good predictor of N ﬁxation and produces similar results to the 15N

dilution method, discussed below (Patterson and La Rue, 1983; Talbott et al., 1982;

Witty, 1983).

The 15N Dilution method requires the addition of fertilizer enhanced in the level of 15N,

or alternatively having a reduction of 15‘N contained in the fertilizer. Plants are fertilized
with the modiﬁed fertilizer and the percent N in the plant derived from the atmosphere
(%Ndfa) is determined by comparing the level of 15N in the plant tissue to determine how
much nitrogen came from the fertilizer. The proportion which did not originate from the
fertilizer as determined by the percent of 15N is the nitrogen that was ﬁxed. St. Clair and
Bliss (1991) evaluated a population of inbred back cross lines (IB) using a 15\N depleted

fertilizer. Lines from an IB cross were identiﬁed as high potential ﬁxers using ARA.

These high ﬁxing lines were then planted in the ﬁeld and fertilized with a solution

15

containing 0.01 atom % 15N (compared to a natural abundance of 15N in the atmosphere
of 0.386%). The lines planted included the parental checks, Puebla 152, donor parent and
Sanilac, the low ﬁxing recurrent parent along with the IB lines and a non ﬁxing soybean
cultivar. They sampled plants at mid-pod ﬁll and at maturity. At each harvest, plants
were dried and ground and digested using the Kj eldahl method. The distillate was then

analyzed by converting to N2 by LiOBr oxidation (Ross and Martin, 1970). The ratio of

15N to 14N was then determined and used to determine the percent nitrogen derived from
the atmosphere (%Ndfa). The purpose of this study was to identify lines with the
agronomic traits of Sanilac with the enhanced BNF of Puebla 152. They found that
several of the F3 families studied were superior to Sanilac in BNF, and four lines ﬁxed
nitrogen at levels similar to Puebla 152. These plants had acceptable agronomic
characteristics which demonstrate that BNF ability is not necessarily linked to late

maturity or indeterminate growth.

In working with Lotus sp. , Medicago sativa, and T rifolium repens, Steele et al. (1983)

found that the concentration of 15N was dependent on the strain of Rhizobia colonizing
the root nodule. Differences in the accumulation of 15N during nitrogen ﬁxation would
be one disadvantage to estimating nitrogen ﬁxation with 15N. Another disadvantage of

the 15N dilution method is the cost of the fertilizer. There may be difﬁculties in applying

the fertilizer in an even and consistent manner to ensure that all treatments and plants
within each treatment have the same access to the enriched or depleted fertilizer. Choice
of a reference crop can also affect the outcomes of the study. Danzo et al. (1993) indicate

that choice of the reference crop is the greatest obstacle to accurate nitrogen ﬁxation
16

measurement. Both the non ﬁxing reference crop and the ﬁxing crop must assimilate
nitrogen from the soil in the same way. Graham (1990) also attributes error in measuring
nitrogen ﬁxation due to the fact that applying nitrogen fertilizer has been associated with

the reduction or even inhibition of nodulation and nodule activity.

Reference crops are often grass species which have different root systems than legumes.
Roots are typically more ﬁbrous and are less deeply rooted than grain legumes (Danso et
al., 1993; Henidge and Danso, 1995; Rennie, 1983). This can inﬂuence the layers of soil
that the reference crop assimilates nitrogen from, either from the upper layer where the
15N enriched/depleted fertilizer is most likely to be, or perhaps deeper where the applied
fertilizer is less likely to have reached. An alternative reference crop is a non-nodulating
grain legume. St. Clair and Bliss (1991) used the non nodulating soybean genotype
“Clay” as their non-ﬁxing reference crop. Utilizing soybean allays some of the concerns
regarding problems of using a different species for a reference crop having a similar
growth habit and belonging to the same family (F abaceae) as bean with similar growth
habits and root architecture, though with differences such as longer maturity and higher
yield than common bean. Henson (1993) utilized dwarf sorghum line BR005 and wheat

line BRIO as reference crops for a study of 17 dry bean genotypes to compare results of
nitrogen ﬁxation estimates with different reference crops. The 15N dilution method was

used. Warm temperatures during reproductive stage of the beans inhibited ﬁxation, and
resulted in senescence of the nodules. The wheat assimilated nitrogen in a similar
manner to beans at most sampling dates while the sorghum assimilated nitrogen at a
much higher rate than the dry beans. This experiment underscores the importance of 1)

multiple seasons might be necessary to satisfactorily evaluate nitrogen ﬁxation and 2)
l7

reference crop behavior inﬂuences estimates of nitrogen ﬁxation. Henson (1993)

concludes that the bean genotype with the lowest amount of 15\N in its tissue is the most

efﬁcient nitrogen ﬁxer, and the one with the greatest amount of 15N is the least efficient

ﬁxer. Using this reasoning a reference crop is not needed.

Common bean lines unable to ﬁx nitrogen have been developed through mutation
breeding. These mutated genotypes either do not produce nodules or produce nodules
that do not possess the ability to ﬁx nitrogen. Park and Buttery (1992) utilized chemical
ethyl methyl sulfonate (EMS) mutagen to generate non-ﬁxing lines from common bean
genotype OAC Rico, belonging to the navy market class. Shirtliffe et al. (1995) were
able to identify R69 and R99 as being unable to ﬁx nitrogen by evaluating the growth of
the plants in Rhizobium-inoculated, nitrogen limiting conditions. R69 produced small
nodules which were pale in color and did not appear to be ﬁxing nitrogen. R99 produced
no nodules except in the presence of one strain of Rhizobium. These nodules were
similar to those in R69, though less than one per plant developed. R99 is considered a
non-nodulating mutant. Including a non-ﬁxing reference crop that is of the same species
addresses some of the limitations such as different root growth characteristics or nitrogen
assimilation differences since the non-ﬁxing reference crop has the same growth
characteristics as the crop being evaluated. Since there are differences in biomass or seed
yield potential between genotypes, care must be taken when making comparisons

between the non ﬁxing genotype and normal N-ﬁxing genotypes.

St. Clair and Bliss (1991) studied the potential to increase BNF by crossing a widely

recognized high ﬁxing line, Puebla 152 (Bliss 1993b; Bliss et al., 1989; Pereira and Bliss,

18

1987; St. Clair and Bliss, 1991; Thomas et al., 1984; Wolyn et al., 1991) with a poor
ﬁxing line, Sanilac. The purpose of the project was to produce a genotype with high

BNF potential with the agronomic traits necessary in the Midwestern United States. An
inbred backcross population was developed and the 11N dilution method was utilized to

screen the resulting lines. They discovered that some lines ﬁxed a high percentage of
plant nitrogen; this nitrogen did not always result in increased seed yield or seed nitrogen
which demonstrates that partitioning and efﬁciency are still important agronomic traits
when considering enhanced BNF. They also noted that if the plants being studied were
of the same growth type, shoot N was a good measure of nitrogen ﬁxation (St Clair and

Bliss, 1991).

The importance of the environment and other factors cannot be ignored (Buttery et al.,
1992). The interaction of the host plant and the Rhizobium strain infecting the roots
affect the rate of nitrogen ﬁxation. In their review, Buttery et al. (1992) found that
nitrogen ﬁxation can be inhibited or reduced by drought, excess soil moisture, and
disease. In addition, an excessive level of nitrate in the soil, either from a rich soil or that
applied through fertilizer may inhibit nitrogen ﬁxation. Any factor that reduces the
plant’s ability to provide carbohydrates may reduce the nitrogen ﬁxation which is
dependent on that source of energy. Plants supplied with combined nitrogen ﬁom the
soil or fertilizer exhibit more stability in terms of yield than those relying on nitrogen
ﬁxation alone. In comparing ﬁxation of common bean to other legumes, Buttery et a1.
(1992) mention that the timing of peak N ﬁxation and the duration of N ﬁxation differs.
Dry beans do not begin substantial ﬁxation until early reproductive phase and nitrogen

ﬁxation peaks during pod ﬁll, dropping off rapidly as carbohydrates are preferentially
l9

translocated to the seed and no longer to the roots. It should be noted that as mentioned
elsewhere in this literature review that Puebla 152 begins to ﬁx nitrogen in late vegetative
phase and continues beyond stages when declines are seen in other genotypes that ﬁx less
nitrogen. Extending the period of nitrogen ﬁxation might be an effective means to
improving the total nitrogen ﬁxed by commercial dry bean genotypes. Pena-Cabriales et
al. (1993) discovered that genotypes did differ in the timing of peak nitrogen ﬁxation and
also in partitioning of that ﬁxed nitrogen to pods and seeds. Evaluating several
genotypes in the ﬁeld and greenhouse they discovered that most nitrogen ﬁxation did not
occur until reproductive growth began. Common bean genotypes ‘Flor De Mayo’ and
‘Kallmet’ did not assimilate nitrogen during maturity, however remobilized nitrogen
from elsewhere in the plant into the pods. Kallmet partitioned less than half of its total N
into the pods, with the remainder retained in the straw. Nodule number and mass
decreased during maturity, with evidence that Flor De Mayo experienced nodule
senescence. This may be misleading, however, in that it is suspected that nodules on
lateral roots are likely still ﬁmctioning at this stage and contributing to an increase in
ﬁxed nitrogen (Hardarson et al., 1989). These ﬁndings reﬂect what has been reported in
soybean where sirrrilar periods of ﬁxation were reported (Zapata, 1987). Park and
Buttery (1989) showed that variation existed in dry beans for tolerance to high soil
nitrates and nodule development. Some genotypes were completely inhibited in nodule
development under nitrogen levels of 10.5 mM nitrogen, their highest nitrogen treatment.
Nodule dry weight increased from the 0 N treatments to the 3.5 mM nitrogen treatment
suggesting that some nitrogen early in the development of the plant may help to increase

later nitrogen ﬁxation. Since earlier nodulation may be important to later total nitrogen

20

ﬁxed, genotypes capable of forming nodules under higher initial levels of nitrogen should
be selected. Application of urea to the foliage at ﬂowering resulted in an increase in seed
yield while not reducing nodule activity and consequently nitrogen ﬁxed (Da Silva et al.,
1993). Foliar application might not inhibit the function of nitrogenase in the nodules,
which is an integral component of nitrogen ﬁxation. Applying urea to the soil at the
same rate as that applied to the foliage did not result in the same increases in seed yield.

Treatments receiving no fertilizer ﬁxed the greatest amount of nitrogen, nearly 50 kg N

ha], compared to the treatment receiving 50 kg N ha'1 applied as urea to the soil which

ﬁxed just over 10 kg N ha'1 (Da Silva et al., 1993). Working with common bean

genotypes Puebla 152 and ‘Negro Argel,’ Muller et al. (1993) found that nitrogen level in
the soil did not have a signiﬁcant effect on total nitrogen ﬁxed. Both Puebla 152 and

Negro Argel were identiﬁed as having superior BNF in this greenhouse study.

While considerable focus has been on nitrogen levels, either applied as fertilizer or
already present in the soil, Tsai et al. (1993) looked at the levels of other major nutrients:
P, K, and S. These nutrients are evidently important to both the proper development and

subsequent function of nodules on common bean. Highest nitrogen ﬁxation for bean

genotype Carioca was observed at medium soil fertility levels of 50 mg P kg'1 1.63 mg K
kg], and 10 mg S kg], at four different nitrogen levels. Greatest levels of ﬁxation

occurred at the lower soil nitrogen levels; 5 mg N kg'l soil and 15 mg N kg'l. These

ﬁndings have implications as to where dependence on nitrogen ﬁxation might be most

applicable. In soils generally deﬁcient in these nutrients, maximum nitrogen ﬁxation

21

may not be achieved. In regions with adequate levels of soil fertility nitrogen ﬁxation

may be sufﬁcient to provide nitrogen needed to produce acceptable yields.

Moisture levels have also been implicated in reducing nitrogen ﬁxation. Pena-Cabriales
and Castellanos (1993) showed that percent nitrogen ﬁxed was not affected as much by
drought stress at either vegetative growth or reproductive stage as was the grain yield.
Plants subjected to reproductive water stress likely had already ﬁxed a large portion of
the nitrogen they would have ﬁxed. Nodulation and BNF must have the ability to recover

from such stresses when water becomes available again.

The above ﬁndings demonstrate that there is an opportunity for improvement in BNF of
common bean. Identifying genotypes able to nodulate earlier in their life cycle would
expand the time (duration) during which N ﬁxation is occurring. Similarly, genotypes
that continue to ﬁx N up to physiological maturity would extend the period of nitrogen
ﬁxation. Coupling earlier nodulation with a greater ability to nodulate at high soil nitrate
levels would improve genotype N ﬁxation capacity considerably. Enhancing nitrogen
uptake during the vegetative and early reproductive growth stages would enhance

nitrogen available for remobilization to support the developing seed.

THE ROLE OF RHIZOBI UM

The other partner in the symbiosis needs to be addressed in discussing improving
nitrogen ﬁxation in grain legumes. Various Rhizobium strains have been Shown to have
different abilities to ﬁx nitrogen as well as compete with other strains for infection sites
on the plant roots. Where beans have been grown historically, there is likely an existing

population of Rhizobium. The indigenous strains may not be the best adapted to ﬁx
22

nitrogen with the genotype planted but may out compete those inoculant strains which are
superior in nitrogen ﬁxation (Alvaro et al., 1989; Dubois and Burris, 1986; Deoliveira
and Graham, 1990; Perret and Broughton, 1998; Rosas et al., 1998; Vasquez-Arroyo et

al., 1998; Weiser et al., 1985).

Vasquez-Arroyo et al. (1998) studied the occupancy of the nodules of three ﬁeld grown
common bean genotypes. They discovered that there was considerable variability in the
ARA values of the different strains isolated and that they had different abilities to
compete for nodulation sites. In addition there was a strain: genotype interaction. For
example strain N4, as identiﬁed in the study had poor ARA values with common bean
genotype FM-M-3 8, while the same bean genotype had high ARA values with strain
Q21. In the same study 64% of nodules on the roots of common bean genotype Negro
Queretaro were inhabited by Rhizobium strain Q21 (V asquez-Arroyo et al., 1998). Rosas
et al. (1998) performed competition studies by mutating Rhizobium etli strain KIMSs,
creating a non-ﬁxing strain. Plants were planted in the greenhouse in a low nitrogen soil
mix containing indigenous Rhizobi um sp. Pots were inoculated with the mutated strain,
called KM6001. Plants were later evaluated visually for color of their foliage. Plants
that formed nodules with the non-ﬁxing mutant Rhizobium strain would be lighter green
Since they were ﬁxing less nitrogen, while those that were dark green were nodulated
With indigenous strains able to ﬁx nitrogen. Of the 820 genotypes screened, two did not
nodIllate normally, the navy bean Sanilac and a non-nodulating line NOD125 developed
at CIAT. Those common bean genotypes showing N deﬁciency yellow color
Preferentially selected in some manner the non-ﬁxing Rhizobium etli strain KM6001. By

eXteIlsion the researchers identiﬁed common bean genotypes that preferentially nodulated

23

with Rhizobium etli strain KIMSs, which is a strain superior at ﬁxing nitrogen.
Identifying common bean genotypes which may form associations with speciﬁc applied
inoculant strains would circumvent the problem of forming nodules with inefﬁcient

indigenous strains of Rhizobium.

Bliss (1993a) encourages the utilization of indirect methods to measure nitrogen ﬁxation.
The nitrogen difference method, seed nitrogen content, and shoot mass are credited with
being quick and accurate in predicting nitrogen content as well as cost effective when
considering the size of many breeding programs where hundreds of lines need to be
evaluated. Elizondo-Barron et al. (1999) crossed common bean genotypes Puebla 152,
RIZ 21, and BAT271 in all possible combinations, bulking the reciprocals. These three
genotypes were originally identiﬁed in a previous study as having superior nitrogen
ﬁxation, though possessing different nitrogen ﬁxation traits. Seventeen lines were

identiﬁed from F23 lines which were originally derived ﬁ'om Puebla 152 x BAT 271 and

Puebla 152 x RIZ 21. After two cycles of recurrent selection these lines were evaluated
in the ﬁeld where soil conditions were low in nitrogen and seed was inoculated with a

single strain, Rhizobium etli UMR1632. Field data collected included seed yield and total

seed nitrogen. Selection was based on family means and appeared to be an effective
means of achieving increased seed yield and seed nitrogen. While methods such as total
seed nitrogen or those measuring nitrogen in biomass late in the crop cycle may provide
an accurate measure of total nitrogen ﬁxed, they may not identify critical events in the
Plant’s life cycle where attention needs to be given to increase nitrogen ﬁxation. For
CXtmrple, earlier nodulation and ﬁxation or the extension of ﬁxation late into

Physiological maturity may have direct impact on total nitrogen ﬁxed. BNF is a complex
24

system and improvements made at deﬁned steps in the process may be the most efficient

means to achieve results in a timelier manner.

OBJECTIVES

The objective of this research was to evaluate dry bean genotypes under an organic
production system and compare with conventional production to identify genotypes best
suited for organic production. Identifying characteristics that enhance the suitability of a
genotype to perform in organic production systems was investigated. Nitrogen
availability in organic systems appears to be a limiting factor thus a second objective was
to identify cost effective and accurate means of evaluating the BNF potential of bean
genotypes both for elite line screening and investigation of the genetic characteristics
inﬂuencing BNF for use in future breeding programs. Having a simple and efﬁcient
protocol to evaluate BNF would facilitate routine screening of bean breeding lines during

the breeding process to increase the BNF potential of future variety releases.

25

Figure 1-2. Hectares of certiﬁed organic cropland in Michigan, 1997 through 2005.

Hectares

 

1997 2000 2001 2002 2003 2004 2005

Year

Data from USDA-ERS.

26

Figure 1-3. Hectares Organic Dry beans (Phaseolus vulgaris L.) produced in Michigan.

 

Hectares

 

1997 2000 2001 2003 2005

Year

Data from USDA-ERS.

27

Table 1-1. U.S. organic dry bean (Phaseolus vulgaris L.) hectares by state, 1997 through
2005.

 

 

 

 

 

State 1997 2000 2001 2003 2005
Hectares

California 449 373 259 263 186

Michigan 334 728 390 664 968

Colorado 225 1525 2213 1409 1442

North Dakota 177 501 1126 692 409

Idaho 176 233 184

Wisconsin 97 1 10

Kansas 96

Texas 77 432

Oklahoma 61

Minnesota 432 139 192

Nebraska 347 104

Missouri 256 402

Utah 255 226

Iowa 243 362

Illinois 236

Arizona 57

Washington 226

Other 199 2468 826 183 126

Total 1891 7118 6105 3983 3981

Data from USDA-ERS.

28

CHAPTER 2

COMPARISON OF DRY BEAN GENOTYPES UNDER ORGANIC AND

CONVENTIONAL PRODUCTION SYSTEMS

ABSTRACT

Thirty two diverse dry bean genotypes were evaluated side by side under organic and
conventional production systems. Trial sites were located in grower ﬁelds in Gratiot
County, MI, in 2007 and 2008 and in Tuscola County in 2009. Trials were also conducted
at Kellogg Biological Station in Kalamazoo County, MI, in all three years. The ’
conventional plots were treated following standard acceptable management practices
including application of granular fertilizer at planting and use of chemical seed treatments
and foliar sprays to control pests. For the organic treatments certiﬁed organic land was
used and only approved methods for organic production were followed. Rhizobium
inoculant was applied to seeds in the organic treatment prior to planting. Higher yields
were observed in conventional treatments than in the organic treatment. Seed classes that
yielded well in the organic system included pink, red, and black seeded genotypes. These
groups also had the highest accumulation of nitrogen of the seed classes under organic
production. The black bean genotype Zorro was among the ﬁve highest ﬁve yielding

genotypes at all sites under both organic and conventional treatments. The small red
germplasm line TARS-SROS accumulated the highest nitrogen yield (> 100 kg ha'l)
under low soil nitrogen conditions. Some genotypes appear better suited to organic
PI‘Oduction than others; however, those genotypes performing poorly under the organic

treatment also performed poorly under conventional treatment. Genotypes of Andean

29

origin did not perform as well as genotypes of Middle American origin in either organic
or conventional systems. Older cultivars, such as the heirloom navy bean ‘Michelite,
commonly believed to be better suited to organic production, did not perform as well as

modern commercial cultivars.

INTRODUCTION

The increased interest in organic production of dry beans has emphasized the need to
identify dry bean genotypes that will perform successfully in an organic system. Modern
breeding programs utilize conventional production systems during the breeding process
to develop commercial cultivars. Application of fertilizer and chemical pesticides are
normally utilized to minimize pests, disease, and nutrient deﬁciencies in order to
maximize yield, eliminate variability, and provide a more uniform environment for
selection. With the low cost of nitrogen fertilizers, breeders paid little attention to
biological nitrogen-ﬁxation (BNF) so in the absence of direct selection for N-ﬁxing

ability this valuable characteristic may have been lost in current bean cultivars.

The area planted to organic dry beans has seen a considerable increase in recent years. In
the period from 1997 to 2005 the number of acres planted in Michigan expanded ﬁom
334 to 968 ha (ERS-NOP, Organic Brieﬁng Room,
http://wwwersnsdagov/briefmgiorganic/ accessed December 2009). Challenges
encountered in conventional production also affect the production of dry beans in an
Organic system. While insects may be controlled by insecticides in both systems, only
approved natural pesticides may be applied in organic systems. Nutrient levels are also

addressed differently between the two systems. Nutrients are typically applied in the

30

form of fertilizers in conventional production systems. Organic production systems rely
on the application of manures and compost as well as crop rotation to maintain nutrient
levels in soils, and forage legumes are often included in many crop rotations as they

contribute to soil fertility by ﬁxing nitrogen.

Studies have been conducted to compare the performance of different dry bean genotypes
in contrasting production systems. Singh et al. (2009) discovered that there was
conSiderable interaction between production system and genotype. Commercial dry bean
genotypes and land races were compared under seven different production systems
involving organic and conventional practices, high input and low input, as well as on
farm and on station treatments. Genotypes such as pinto bean ‘Othello’ and great
northern ‘Matterhorn’ were more stable across production systems. Others, such as pinto
bean ‘Buster’ and pinto bean ‘Bill Z’ were more responsive to high inputs and may be
better suited to systems where fertilizer and supplemental irrigation are utilized to

maximize yield.

Comparing yield of conventional and organic production systems in developed and
developing countries of the world, Badgley et al. (2007) showed that on average, the ratio
of the yield between organically produced and conventionally produced pulse crops was
1.86 and reached 3.99 in some locations. For the developed world the ratio was 0.86.
The differences observed between the developed world and the developing world
between organic and conventional production systems likely rely on the availability of
inputs in the developing world. Fertilizers and pesticides may not be readily available in
developing countries nor the technologies to utilize these inputs effectively, bringing

Productivity in both conventional and organic systems into a similar yield potential range.
31

Anecdotal evidence suggests that there is an expectation for organic systems to yield less
than conventional systems due to the reduced inputs. There seems to be potential to
increase yields in organic systems to be on parity, at least, with conventional system in
the developed world. Breeding programs typically select for disease resistant cultivars
which would be beneﬁcial in both conventional and organic production systems. Also,
plant architecture and other agronomic and phenological characteristics such as maturity
would likely be advantageous under both organic and conventional systems. However, a
more vigorous vegetative growth that is somewhat more open may help to reduce weed
competition by shading the ground more quickly, perhaps reducing the need to cultivate
as the plant canopy closes to control weeds. A canopy that closes more quickly may also
make it impossible to cultivate without damage to the bean plants. Thus, dry bean canopy

would need to be dense enough to eliminate the need for cultivation after canopy closure.

One area, however, that is generally ignored in conventional breeding programs is plant
nutrition. Since conventional production systems rely on the addition of synthetic
fertilizers to compensate for soil lacking in proper nutrient levels little effort has been
made to select for genotypes able to resist nutrient deﬁciencies or are better able to utilize
nutrients, or even provide their own nutrients through BNF. As discussed elsewhere,
many researchers have discovered variability in dry beans for their ability to ﬁx nitrogen.
Bliss (1993b) and Graham (1981) noted considerable variability in BNF between dry
bean genotypes, and suggested that there was potential to breed dry beans capable of

producing sufﬁcient nitrogen through BNF to meet production goals.

Ability of dry bean genotypes to ﬁx nitrogen may result in increased yield under

conditions of environmental stress. In two years of trials in Staples, MN, De Jensen et al.
32

(2004) found that inoculation of seed with Rhizobium as well as a biocontrol agent,
Bacillus subtilis, increased yield of dry beans and soybeans which had been planted in
soils heavily infected with root rot causing pathogens F usarium solani, F. oxysporum,
and Rhizoctonia solani. In addition to the inoculant, tillage resulting in the break up of
compacted soil also increased yield. Superior nitrogen ﬁxing dry bean genotypes such as
TARS-SROS which have been developed to tolerate poor soil conditions such as low
fertility, compaction, and the negative effects of root rot organisms (Smith et al., 2007)
may alleviate the need for application of nitrogen and thus help to reduce the negative

effects of root rot pathogens.

Soils in Michigan are often adequate in levels of phosphorus, potassium, and other
nutrients considered limiting in BNF (hgpz/ﬁpmnewsmsuedg, accessed December
2009). While external factors such as erratic rainfall limit BNF, the lack of ability of
some genotypes to ﬁx sufﬁcient nitrogen to achieve a competitive yield without addition

of exogenous nitrogen is a major factor limiting BNF.

Since BNF ability has not been a selection criterion in modern breeding programs, the
potential of elite breeding lines and commercial bean cultivars to ﬁx nitrogen may be
limited. Graham (1981) and Bliss (1993a) have suggested that selection based on yield
indirectly selects for improved BNF. Yield is a major component of modern breeding
programs, and perhaps a primary factor affecting the success of a genotype in being
considered for commercial release. How will selection based on conventional production

methods translate to cultivars adapted to organic production systems?

33

The objectives of the present study were to compare response of diverse dry bean
genotypes to organic production systems. In addition, the identiﬁcation of key traits that
contribute to the success of dry bean genotype(s) in organic production systems was
studied. The study was also intended to understand if dry bean genotype(s) developed for

conventional production systems would be competitive in an organic production system.
MATERIALS AND METHODS

Thirty two diverse dry bean genotypes, including one non-nodulating check, R99, were
selected for side by side comparison of performance in organic and conventional
production systems. These genotypes (table 2-1) were chosen to represent the market
classes grown in Michigan as well as representing the greatest diversity available among
modern dry bean genotypes. Important commercial cultivars were chosen in order to be
able to offer recommendations to growers wishing to produce beans organically. Elite
breeding lines were also chosen to evaluate them in an organic production system prior to
potential release. Field tests were conducted on both experiment station land and farmers
ﬁelds where conventional land and organically certiﬁed land was located adjacent to each
other. Kellogg Biological Station (KBS) is located in northern Kalamazoo County, Ross
Township MI, and has both conventionally maintained experimental ﬁelds as well as
organically certiﬁed ﬁelds. Soil type at the KBS sites is Kalamazoo loam, see table 2-2
for soil characteristics. Trial plots in Gratiot County in 2007 were planted on a
Metamora-Capac Loam; in 2008 the soil was a Selﬁidge loam (see table 2-2). The ﬁelds
in Gratiot County in both 2007-near Ithaca, MI and 2008-in St. Louis, M1, were
cultivated by the organic grower. Tests in 2009 were located in Tuscola County and were

Planted on an alkaline Tappan Loam. Corn was the previous crop at all locations and all
34

years except at KBS in 2009 where pumpkins and squash (Cucurbita spp.) were planted
the previous season. Precipitation was variable from year to year and throughout each
ﬁeld season (Table 2-3). Below normal rainfall, 217 mm at KBS in 2007 resulted in

severe drought stress.

Experimental design was a side by side RCB design. Genotypes were separated
according to seed size to facilitate planting, with 16 small seeded lines planted in one half
of the ﬁeld with 16 large and medium seeded planted in adjacent plots. The same
planting design was used in all ﬁelds in each season. Each location had one conventional
ﬁeld and one organic ﬁeld side by side. The conventional plot was lost to ﬂooding in

Tuscola County in 2009. Four reps were planted in each treatment.

Each plot at KBS consisted of four-6m rows spaced 0.5m apart. Seeds were planted at a
density of 267,000 plants ha]. The two outer rows served as guard rows to limit border

affects on the research station. The center 4.6m of the two center rows was harvested to
estimate seed yield. Plots on farmer’s ﬁelds in Gratiot County and Tuscola County
consisted of two-6m rows relying on the variety in the adjacent plot to limit border

affects. Plants were harvested by pulling and then thrashing mechanically.

Seed to be planted in the conventional treatment was coated with seed treatment
containing an insecticide-Cruiser, fungicide-ApronMax, and bactericide-Streptomycin as
is typically used on seed beans in Michigan. Weeds were controlled through chemical
means prior to planting, applying the preemergent herbicides Sonalan, Eptam, and Dual
in a single application prior to planting. Plots were mechanically cultivated prior to
Canopy closure. Under conventional management, granular fertilizer (19-19-19) was

35

applied at planting beneath the seed at a rate to provide 45 kg N ha]. Urea was applied at

50% bloom at a rate of approximately 34 kg ha'lat KBS. Insects, speciﬁcally potato leaf

hopper, Empoascafabae, were controlled with Asana (Esfenvalerate (S)-cyano (3-

phenoxyphenyl methyl (S)-4-chloro-alpha-(1-methylethyl) Benzeneacetate) at a rate of
190 ml ha'l as needed according to current recommendations.

Seed for the organic treatment was coated with a commercial preparation of Rhizobium
legyminosarum bv Meg]; (N itrastik-D, BMD Crop Bioscience, Brookﬁeld, WI) by
swirling seed in the powdered peat preparation with a small amount of water. No
fertilizer was applied. Weed control consisted of hand pulling and cultivation, including
a rotary hoe used to control early weeds just after seedling emergence. Insects,

speciﬁcally potato leaf hopper, were controlled with an OMRI approved insecticide,

Pyganic BC 5.0, labeled for potato leaf hopper applied at a rate of 95 ml ha'l resulting in

90.25 ml active ingredient ha'l.

Days to 50% bloom, maturity, and stand counts were recorded. After harvest seed was
air dried and cleaned to remove ﬁeld debris. Samples were measured for moisture content
and weighed. A sub sample of 30g was taken from each plot and ground to 40 mesh in a
Christy-Turner Lab Mill (Ipswitch, Suffolk, UK) to pass through a 1 mm screen.

Samples were sent to A&L Great Lakes Lab, (Fort Wayne, IN) for Kjeldahl analysis to
determine total seed nitrogen content of three reps of organic and convention treatments

from the KBS.

36

RESULTS

Yields varied substantially between years on both organic and conventional treatments at

KBS (table 2-4). In 2007 yield in both organic and conventional was similar, with

conventional plots yielding slightly higher (1934 kg ha'l) than organic treatment, (1862
kg had). In 2008 the difference between organic and conventional treatments was greater
with conventional yielding 1967 kg ha"1 and organic yielding 1432 kg ha]. The highest
yields were recorded in 2009 with conventional yielding an average of 3627 kg ha'1 and
the organic treatment averaging 2877 kg ha]. The three year mean yield of the organic

treatment at KBS was 2058 kg ha'1 compared to three year mean yield of 2507 kg ha'1

for the conventional treatment.

In Gratiot County in 2007 the difference in average yield between organic and

conventional was not signiﬁcant, with conventional yielding 1840 kg ha'1 and the organic
treatment averaging 1797 kg ha"1 (table 2-8). The difference in average yield between
organic and conventional increased substantially in 2008 with average yield 2493 kg ha'1

in conventional while the organic treatment yielded 1575 kg ha]. In 2009 the on farm
trial was moved to Tuscola County. Organic yield was higher in Tuscola County than the
previous two years in Gratiot County, with an average of 2675 kg ha'1 (Table 2-9). The
conventional treatment in Tuscola County was lost due to heavy rain the week after
planting that reduced plant emergence. The same reduction in emergence was not seen in

the side by side organic plot which emerged and grew normally the remainder of the

season. Though the ﬁelds were adjacent, with the required buffer strip separating the
37

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certiﬁed organic and conventional ﬁelds, the soils in each treatment were signiﬁcantly
different in ﬁiability. The conventional soil was more compacted either due to
differences in management or the use of heavy machinery on the conventional plot when
the soil was wet. Nutrient stress was observed in the Tuscola County organic trial. The
pH of the soil was high, 7.7, which may have resulted in Zinc and Manganese
deﬁciencies. Not all genotypes had symptoms of nutrient deﬁciency and deﬁciency
ratings were made on all entries. Air pollution damage was also evident at the Tuscola
County plots in 2009, which are located in the Wisner Oil Fields with active oil wells
surrounding the vicinity. In particular, ‘Jaguar’, B05039, BO4431, and ‘Bunsi’ showed

typical bronzing on the upper leaves due to ozone air pollution.

At the Gratiot County site in 2007 halo blight (Pseudomonas phaseolicola) was a
problem on ‘CELRK’ genotype causing early plant death in both organic and
conventional treatments. Symptoms were generally limited to CELRK and showed only
minor infection on neighboring plots, but did not result in early death of those plants. In
2007 and 2008 white mold (Sclerotinia sclerotioru_m_) was not observed on either organic
or conventional treatments. Increased precipitation, cool nights, and the resulting heavy
vegetative grth lead to a low level of white mold infection in 2009 in Tuscola County

on the organic treatment.

Total seed nitrogen was analyzed for all three years at KBS for both conventional and

organic treatments. Signiﬁcant differences were seen between years and treatments in

total nitrogen content of the seed. In 2007 the organic treatment averaged 63 kg N ha'1

while the conventional treatment averaged 73 kg N ha]. Average nitrogen levels for

38

organic were 52 kg ha'1 and 93 kg ha'1 for conventional in 2008. Average nitrogen levels
increased signiﬁcantly in 2009 for both treatments to 98 kg ha'1 in organic and 139 kg ha"

1 in conventional (table 2-13).

Climatic as well as pest factors resulted in increased levels of stress at KBS in both 2007
and 2008. Precipitation in 2007 at KBS was less than normal, especially early in the
season and during vegetative growth (table 2-3) resulting in drought conditions which
reduced yields in both conventional and organic treatments. After 6 weeks with little rain
37.5 mm of supplemental overhead irrigation was applied, with an additional application
of 37.5 mm applied later in the summer. The drought stress delayed maturity causing re-
growth in many of the genotypes. Re- growth was more prevalent in the conventional
treatment than the organic treatment. Neither the organic nor the conventional treatments
were irrigated at KBS in 2008. Irrigation was applied in 2009 to supplement natural

precipitation at a rate of 12.5 mm per week for 5 weeks in July and August.

Weed pressure was minor in the organic and conventional treatments in 2007. Higher
weed pressure in the organic treatment in 2008 may have resulted in reduced yield and
reduced total nitrogen values. Grass weeds, including crabgrass, Digitaria spp. and
foxtail, Setaria spp., were the primary component of the weed pressure in the organic
plot. Early cultivation and hand hoeing was inadequate to reduce weed pressure. None of
the bean genotypes seemed able to effectively compete with the weeds resulting in small
plants and reduced yields. Weed growth was a general problem in the organic plots when

compared to the conventional plots; however at all years and sites other than KBS

39

organic in 2008, cultivation and hand weeding reduced weed pressure to minor levels

comparable to that observed in conventional treatments.

The pink and red seed classes were the highest yielding group over all years for both
organic and conventional treatments, 2562 kg ha'1 and 2829 kg ha'1 respectively (table 2-
7). Genotypes of Andean origin-kidney and cranberry, grouped due to their relatedness,

had the lowest yield in the organic treatment at 1618 kg ha'1 while the average for the

conventional treatment was slightly higher 1928 kg ha]. In both organic and

conventional treatments the kidney and cranberry market class were statistically similar
to the non nodulating check, R99. Pink, red, and black seed classes produced the highest
yields at KBS. However, the pink and red seed class ranked third in the conventional
system with the pinto seed class producing more than the pink and red class. Pink and
red bean genotypes produced the highest nitrogen yield in organic while the black seed
class ranked second in nitrogen accumulated. When nitrogen fertilizer was provided there

was little variation in the nitrogen content of the seed. Most seed classes at KBS in the

conventional system had a nitrogen yield of 100 kg ha'1 or greater (table 2-12). The
lowest nitrogen yield was observed in the kidney seed class at 89.5 kg N ha]. The navy
seed class also tended toward the low end on nitrogen yield with Michelite, Bunsi, and
Vista possessing less than 70 kg N ha]. The only kidney genotypes having nitrogen yield

greater than the mean was Montcalm and Chinook Select in the organic treatment at
KBS. Chinook Select was the only kidney bean in the conventional treatment at KBS to

have seed nitrogen content greater than the mean.

40

The genotype with the lowest N yield was R99 in the organic treatment (table 2-13).
Nutrient levels on organic ﬁelds at KBS are controlled through the use of cover crops and
crop rotation including wheat (T riticum spp), corn (Zea mays), and a grain legume such
as soybean with occasional inclusion of other crops such as pumpkin and squash,
Cucurbita spp which were planted in 2008 at KBS in the ﬁeld used in this study in 2009.
It is expected that nitrogen levels are relatively low in such a treatment as no manures are

applied. The genotype with the highest N yield was the black bean cultivar Zorro in the

conventional treatment, yielding 133 kg N ha]. Zorro also had a high nitrogen yield in

the organic treatment, producing 97 kg N ha].

At KBS three genotypes ‘Zorro’, Buster, and the navy breeding line N05324 were in the
top ﬁve yielding cultivars for both conventional and organic treatments. All genotypes
yielding higher than the mean in both conventional and organic treatments at KBS were
of the Middle American gene pool. Except for the kidney bean USDK-CBB-IS the same
trend was observed among genotypes yielding above the mean in both years in Gratiot
County. Andean genotypes were heavily represented in the group of genotypes producing

below average yields.

No genotype yielded signiﬁcantly better in organic production than in conventional
production, whereas some genotypes yielded better in both treatments. The black bean
genotype Zorro was in the top ﬁve yielding genotypes in both organic and conventional
systems and at both KBS and Gratiot County. Except for the R99, the non nodulating

check, the bottom ﬁve yielders at all sites were all Andean kidney beans. Plant stand

41

showed a slight negative correlation with both seed yield (r2=-0.27, p_<_0.0001) and N

yield (r2=-0.23, 1950.001), respectively (table 2-14a).

DISCUSSION

Dry bean genotypes generally yielded more in the conventional than the organic plots.
Middle American genotypes such as Zorro showed competitive yields in organic
production, and Zorro was consistently represented in the ﬁve highest yielding genotypes

under both treatments.

Soils were generally adequate in nutrients such as P and K. The 2009 Tuscola County
site had a high pH, 7.7, resulting in typical Zn and Mn deﬁciencies. This site would be
useful in screening genotypes for nutrient deﬁciency. Symptoms of Zn and Mn
deﬁciency were visible on some genotypes and navy bean genotypes were particularly
susceptible to micronutrient deﬁciency. Navy beans N05324, N05311, Bunsi and R99
and the black bean cultivar Condor showed severe symptoms of Zn and Mn deﬁciency.

Neither TARS-SROS nor Zorro seemed to be affected by nutrient deﬁciencies.

Despite soil conditions resulting in nutrient deﬁciencies such as those encountered in
Gratiot County in 2007 and 2008 and Tuscola County in 2009 resulting from elevated
pH, the black bean genotype Zorro was in the group of 5 highest yielding genotypes. The
limited number of elite cultivars as well as the large number of black seeded genotypes
likely increased the range in yields reducing the mean for the seed class. Overall pinks
and reds as a seed class have the highest yield. The small red bean ‘Merlot’ as well as the

pink bean ‘Sedona’ produced the highest yields in the organic treatments. These two

42

genotypes remain in the group of 5 highest yielding genotypes in the conventional
system. It is possible that these representatives ﬁ'om race J alisco are better adapted than
other genotypes to high pH soils. Singh et al. (2007) cites the origin of the Common Red
Mexican bean as the arid highlands of Mexico. It is likely that selection under the
historically drought stressed and alkaline conditions have resulted in red and pink market
classes being generally better adapted to growth under high pH and low soil moisture. As
a class the pinks and reds had the highest nitrogen yield in the organic plot at KBS
supporting the suggestion that pink and red beans have characteristics, such as increased
drought tolerance, lending them to production in diverse production systems and varying
levels of stress. Singh and Westerrnann (2002) studied the inheritance of zinc deﬁciency
and found that the black bean ‘T39’ was susceptible to zinc deﬁciency while the great
northern Matterhorn was resistant to zinc deﬁciency. This trait was controlled by a single

dominant gene.

White mold was not observed on either organic or conventional treatments in 2007 and
2008. Increased precipitation, cool nights, and the resulting heavy vegetative growth lead
to a low level of white mold pressure in 2009. Only Zorro appeared to be affected in the
organic treatment in Tuscola County, likely due to its increased plant vigor and
susceptibility to white mold. Both organic and conventional treatments had slight white
mold pressure at KBS in both large and small seeded genotypes. Genotypes with type 111
growth habit such as the navy bean ‘Michelite’ were more susceptible to white mold
infection while growth types I and II seemed to avoid infection. The occurrence of white

mold in Michelite likely contributed to its poor performance.

43

Stand counts were taken to investigate potential damage from the use of the rotary hoe
shortly after emergence to control weeds and break the crust formed after rain. Plant

stand was slightly correlated with ﬂowering time and had a slight signiﬁcant negative
correlation with both seed yield and N yield (r2=-0.27, p50.0001, and r2=-0.23, p50.001,

respectively, Table 2-14). This suggests that any reduction in stand due to the use of the
rotary hoe was minor. Further reductions in stand may be detrimental as the seeding rate
for dry beans is lower than other crops such as soybean. In addition dry bean seedlings
are not as resilient to the practice of rotary hoeing; especially larger seeded genotypes

that produce bigger seedlings which may experience more plant damage due to the
mechanical damage caused by the rotary hoe. The strong negative correlation, (r2=-0.7,

p50.0001) observed between 100 seed weight and maturity is explained by the fact that
the determinate large seeded kidneys and cranberry genotypes, are earlier maturing than
the navy and black seed classes. Days to ﬂower were also negatively correlated with 100
seed weight. Other slight, non-signiﬁcant correlations were observed between plant
height at ﬂowering and seed yield, nitrogen yield, and 100 seed weight. Increased plant
height may be regarded as an indirect measure of plant vigor, and perhaps root growth in

certain genotypes.

At KBS, 2007 and 2008 were stressful years due to lack of precipitation (table 2-3). The
soil, a Kalamazoo Loam, at KBS is light and sandy and drains quickly. The organic ﬁeld

at KBS in 2008 provided extreme weed pressure. Crabgrass, Drggtaria spp. and foxtail,

Setaria spp. were the dominant weed species and grew thickly. These grasses were

 

difficult to control with cultivation and hand weeding. Weeds were a concern in all

organic treatments, though were better controlled in other years and locations. Weed
44

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pressure in the conventional plots was non existent due to the use of preemergence
herbicides. Late season weeds in conventional plots likely did not affect yield since the

plants had reached physiological maturity by the time the weeds had begun to compete.

Bliss (1993b) indicated that a ﬁxation rate of 100 kg N ha'1 would be enough to achieve
competitive yields of approximately 2000 kg ha]. Zorro yielded nearly 100 kg N ha'1

and TARS-SR05 yielded considerably more than 100 kg N ha'1 in the organic treatment.
In the low nitrogen levels at KBS, these two genotypes ﬁxed sufﬁcient nitrogen (100 kg

N ha'l) to achieve competitive yields. The kidney seed class acquired the lowest nitrogen

yield, 89.5 kg N ha'1 in the conventional treatment. In the organic treatment the kidney
seed class yielded slightly better than the non nodulating check with the kidney seed class
having a nitrogen yield of 64.6 kg ha]. This suggests that the plants are not as efﬁcient at

extracting nitrogen from the soil, or that they are inefﬁcient at partitioning nitrogen from
the roots or stems into the seed. Nodule evaluations from the previous study on screening
for nitrogen ﬁxation in the greenhouse suggests that Andean genotypes have well

developed nodules apparently capable nitrogen ﬁxation based on their size and red or

orange color.

In a scatter diagram of seed yield in the organic system versus the conventional system
over 3-years at KBS only (Figure 2-1.), Middle American gene pool genotypes grouped
in the upper right hand corner. These genotypes yielded well under both conventional
and organic production systems indicating that they were responsive to the application of
fertilizer but also ﬁxed sufficient nitrogen to produce a competitive yield in the absence

0f applied fertilizer. Characteristics that this group may have include roots better able to
45

extract soil nitrogen as well as the ability to partition acquired nitrogen into the seed.
Genotypes in the bottom right, however, would be less responsive to fertilizer. They may
be less able to extract soil nitrogen than the genotypes in the upper right quadrant. The

TARS-SROS genotype ﬁxed more nitrogen than Zorro, almost 112 kg N ha], and

produced similar seed yields. TARS-SROS was developed by Smith et al. (2007) in an

effort to produce a small red bean with resistance to multiple root diseases and stress.
Selection was carried out on F4; 5 lines in compacted and waterlogged ﬁelds with high
pressure from Rhizoctonia solam‘, Xanthomonas axonopodis pv. phaseoli, and F usarium

solani. TARS-SROS had an average yield higher in the organic treatment, 2588 kg ha],

than in the conventional treatment, 2390 kg ha,'lat KBS. Higher performance under

organic treatments demonstrates that this genotype may be tolerant of stress and performs
better under such conditions. Selection under low fertility, compaction and pathogen
pressure helped to produce a line that is stress tolerant which rrright prove useful in

breeding genotypes designed for organic production systems.

The presence of the non nodulating R99 in this quadrant causes some concern, though, as
the only nitrogen it should be able to acquire is that in the soil. One explanation could be
that the amount of fertilizer applied at planting and the additional nitrogen applied at
ﬂowering are leached from the soil or utilized early leaving the plant deﬁcient in nitrogen
later. The nitrogen in the organic system, however, may be more sustainable, since it is
derived from the decomposition of organic matter from previous crops and has a longer

release period than the nitrogen applied in more soluble fertilizer. Moreover, the

46

nitrogen may still be at an acceptable level in the soil at pod ﬁll to produce a yield greater

than is expected from a non nodulating dry bean.

Genotypes falling in the upper left quadrant may be responsive to the application of
nitrogen but not able to ﬁx nitrogen efficiently. The non nodulating R99 would be
expected to be present in this quadrant. These genotypes would be best suited to soils
rich in nutrients and where application of nitrogen in the form of fertilizer is possible. In
the group of 32 genotypes studied, however, only one genotype, the black seeded

breeding line B05039 was placed in the upper left quadrant.

The lower left quadrant represents genotypes that do not respond to application of
nitrogen by increasing yield, nor are these genotypes the most efficient nitrogen ﬁxers of
this group of genotypes evaluated. The genotypes in the lower left quadrant may not be
as efﬁcient at partitioning nitrogen to the seed as those in the upper right quadrant. The
genotypes in the lower left quadrant are dominated by the low N-ﬁxers in the kidney-

cranberry and navy seed classes implying lower overall yield.

There is a preconception that cultivars developed prior to the advent of the extensive use
of synthetic pesticides and fertilizers are better adapted to organic production. Organic
production is similar to conventional production prior to the advent of modern
technologies as both rely on composts, manure, and legumes and other cover crops to
improve soil fertility. Also, prior to application of pesticides, cultivars resisting disease
would be selected over those that were susceptible. However, these older cultivars have
not undergone the rigorous selection in breeding programs that modern genotypes have

for traits such as disease resistance, plant architecture and yield. Carr, et al. (2006)

47

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IIK'

den

investigated this supposition in wheat genotypes by comparing yields in organic
production to conventional production. They found that modern genotypes were much
better adapted to modern organic production than the older genotypes pre-dating common
use of synthetic fertilizers and pesticides. In this study, the navy bean Michelite was
included since it was released in 1938 thus was developed prior to the advent of synthetic
fertilizers and pesticides. T39, a selection from the landrace Black Turtle Soup, was ,
pure-line selected in the 19705. Michelite and T39 did not produce high yields and were
surpassed in organic production yield by many modern genotypes which had been
selected for yield under conventional systems. Those genotypes with the highest yield in
both conventional and organic production systems were generally those genotypes more
recently developed through modern breeding under conventional production systems.
These genotypes seem to be better suited to organic production than those genotypes

developed prior to the use of chemical pesticides and fertilizers.

Variability exists in the 32 dry bean genotypes studied in yield potential in both organic
and conventional systems. In addition to yield, there are differences in the nitrogen
content of the seeds between organic and conventional production systems and also
between genotypes. Genotypes showing the greatest yield potential in organic systems
belong to the Middle American gene pool represented by Zorro, a consistently high
yielding genotype in multiple environments and different production systems. Small reds
and pinks such as Merlot and Sedona, respectively, yield competitively and appear to
tolerate high pH conditions where rnicronutrients may be limiting as well as ﬁxing

sufficient nitrogen when grown under nitrogen limited conditions in organic systems.

43

Table 2-1. Seed class, seed size, gene pool, race, and growth habit for 32 dry bean
(Phaseolus vulgaris L.) genotypes evaluated in organic and conventional production
systems at Kellogg Biological Station and Gratiot County in 2007, 2008, and 2009.

 

 

Gene Growth
Genotype Seed class Seed Sizel Pool2 Race3 Habit4
B05039 Black Small M.A. M 11
Vista Navy Small M.A. M II
B04431 Black Small M.A. M 11
T39 Black Small M.A. M II
Condor Black Small M.A. M 11
R99 Navy/No No.15 Small MA. M 111
N05324 Navy Small M.A. M II
Michelite Navy Small M.A. M 111
305055 Black Small M.A. M II
Bunsi Navy Small M.A. M III
1 15-1 1M Black Small M.A. M II
N0531 1 Navy Small M.A. M II
Jaguar Black Small M.A. M II
Seahawk Navy Small M.A. M II
Zorro Black Small M.A. M II
TARS SR05 Small Red Small M.A. Jalisco II
Sedona Pink Medium M.A. J alisco II
Chinook Select Kidney Large Andean N.G. I
Red Hawk Kidney Large Andean N.G. I
K05604 Kidney Large Andean N.G. I
Matterhorn Great Northern Medium M.A. Durango II
USDK-CBB-IS Kidney Large Andean N.G. I
Buster Pinto Medium MLA. Durango II
P0613] Pinto Medium M.A. Durango II
Santa Fe Pinto Medium M.A. Durango II
CELRK Kidney Large Andean N.G. I
Capri Cranberry Large Andean Chile I
Montcalm Kidney Large Andean N.G. I
K03240 Kidney Large Andean N.G. I
Merlot Small Red Medium M.A. J alisco II
Beluga Kidney Large Andean N.G. I
Othello Pinto Medium M.A. Durango III

 

1Small=l8-29 g 100 seeds]; medium=30-45 g 100 seeds'l; large=46-60 g 100 seeds'1

2Gene pool according to Gepts (1988) M.A.=Middle American; 3Race according to Singh
et a1. (1991) N.G.=Race Nueva Granada; M=Race Mesoamerica; 4Growth habit

according to Singh, (1982). 5No Nod=non nodulating.

49

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50

Table 2-3. Precipitation (mm) from June to September at study sites where 32 dry bean

(Phaseolus vulgaris L.) genotypes were evaluated for production in organic systems in
2007, 2008, and 2009.

 

 

Year Kellogg Gratiot Tuscola
and Biological County County
Month Station‘

grim ------------ mm ---------
June 37 41

July 24 67

August 88 99
September 68 49

Total 217 256

M

June 26 90

July 131 40

August 16 36
September 333 82

Total 506 248

.20—09.

June 96 1 10
July 5 69
August 174 64
September 32 32
Total 307 275

 

lPrecipitation data for KBS in 2007 was from records at Ceresco, M1, the closest

monitoring site with functioning equipment that year. Data from 2008 and 2009 were
collected at KBS.

51

Table 2-4. Mean yield of 32 dry bean (Phaseolus vulgaris L.) genotypes grown in
organic and conventional systems at Kellogg Biological Station 2007-2009.

 

 

 

Qrggnic Conventional

Yield Yield
Genotype kg ha'1 Genotype kg ha'1
Zorro 2598 a N05324 3385 a
TARS-SROS 2588 a Buster 3239 ab
304431 2553 a Zorro 3203 ab
Buster 2463 ab B04431 3020 abc
N05324 2439 ab Condor 2953 abc
N0531] 2435 ab Merlot 2844 abcd
Seahawk 2380 abc Sedona 2831 abcd
Jaguar 2333 abc 115-11M 2756 abcde
Merlot 2310 abc B05039 2752 abcde
115-11M 2284 abcd Jaguar 2676 abcde
Condor 2221 abcd N0531] 2660 abcde
R99 2213 abcd Seahawk 2564 abcde
Santa Fe 2191 abcd Bunsi 2493 abcde
B05055 2189 abcd Santa Fe 2479 abcde
P061 31 2173 abcd B05055 2424 abcde
Sedona 2162 abcd Matterhorn 2407 abcde
Bunsi 2068 abcd Beluga 2406 abcde
Chinook Select 1994 abcd K03240 2403 abcde
T39 1966 abcd Chinook Select 2402 abcde
B05039 1957 abcd TARS-SOS 2390 abcde
Vista 1927 abcd T39 2353 abcde
Othello 1909 abcd Montcalm 2302 abcde
Matterhorn 1901 abcd Vista 2269 abcde
Red Hawk 1795 abcd Othello 2260 abcde
Capri 1747 abcd P0613] 2253 abcde
USDK-CBB-IS 1728 abcd Michelite 2198 bcde
Montcalm 1703 abcd Red Hawk 2179 bcde
Beluga 1695 abcd USDK-CBB-IS 2140 bcde
K03240 1686 abcd Capri 2130 bcde
Michelite 1604 bed CELRK 1946 cde
CELRK 1455 cd K05604 1811 de
K05604 1377 d R99 1641 e
Mean 2064 Mean 2507
LSD 931.2 LSD 1 142

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD

p=0.05.

52

Table 2-5. Mean yield of 9 Andean dry bean (Phaseolus vulgaris L.) genotypes and non

nodulating R99 check grown in organic and conventional systems at Kellogg Biological
Station 2007-2009.

 

 

 

thgnk Conventional

Yield Yield
Genotype kg ha'1 Genotype kg ha.1
R99 2213 a Beluga 2406 a1
Chinook Select 1994 ab K03240 2403 a
Red Hawk 1795 be Chinook Select 2402 a
Capri 1747 bed Montcalm 2302 a
USDK-CBB-IS 1728 bed Red Hawk 2179 ab
Montcalm 1703 bed USDK-CBB-IS 2140 ab
Beluga 1695 bed Capri 2130 ab
K03240 1686 bed CELRK 1946 b
CELRK 1455 cd K05604 1811 b
K05604 1377 d R99 1641 b
Mean 1739 Mean 2136
LSD 398 LSD 537

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD
p=0.05.

53

Table 2-6. Mean yield of 22 Middle American dry bean (Phaseolus vulgaris L.)
genotypes grown in organic and conventional systems at Kellogg Biological Station

 

 

 

2007-2009.1
QrgaLnic Conventional

Yield Yield
Genotype kg ha'1 Genotype kg ha"1
Zorro 2598 a N05324 3385 a
TARS-SROS 2588 a Buster 3239 ab
304431 2553 a Zorro 3203 ab
Buster 2463 ab 304431 3020 abc
N05324 2439 abc Condor 2953 abcd
N0531] 2435 abc Merlot 2844 abcde
Seahawk 2380 abcd Sedona 2831 abcde
Jaguar 2333 abcd 115-11M 2756 bcde
Merlot 2310 abcd 305039 2752 bcdef
115-11M 2284 abcd Jaguar 2676 bcdef
Condor 2221 abcd N0531] 2660 bcdef
R99 2213 abcd Seahawk 2564 cdef
Santa Fe 2191 abcd Bunsi 2493 cdef
305055 2189 abcd Santa Fe 2479 cdef
P0613] 2173 abcd 305055 2424 cdef
Sedona 2162 abcd Matterhorn 2407 def
Bunsi 2068 bcde TARS-SOS 2390 def
T39 1966 bcde T39 23 53 def
305039 1957 bcde Vista 2269 cf
Vista 1927 cde Othello 2260 ef
Othello 1909 cde P0613] 2253 cf
Matterhorn 1901 de Michelite 2198 fg
Michelite 1604 e R99 1641 g
Mean 2212 Mean 26]]
LSD 530 LSD 612

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD

p=0.05.

54

Table 2-7. Mean yield of 32 dry bean (Phaseolus vulgaris L.) by seed class grown in
organic and conventional production systems at Kellogg Biological Station, 2007 to 2009

 

 

 

Qrggnic Conventionﬂ DifferenceI
Seed Class kg ha'1 kg ha'1 kg ha-1
Pink/Red 2353 2688 3335
Black 2262 2767 -505
Navy 2142 2595 -453
Pinto 2127 2528 -401
Kidney 1687 2191 -504
No Nod 2213 1641 572
Mean 2064 2507 -443
LSD 273.8 442.2

 

I I O O
D1fference=organ1c-conventlonal

55

Table 2-8. Mean yield of 32 dry bean (Phaseolus vulgaris L.) genotypes Gratiot County
in organic and conventional production systems, 2007-2008.

 

 

 

_O_rg_anic Conventional

Yield Yield
Genotype kg ha'1 Genotype kgrha'l
Merlot 2817 a Buster 3075 a
Sedona 2537 ab Zorro 3019 a
T39 2533 ab N05324 2949 a
N0531] 2504 ab Sedona 2938 a
Zorro 2469 ab Merlot 2901 ab
P0613] 2463 ab 304431 2830 abc
115-1 1M 2408 abc Condor 2721 abcd
Condor 2397 abc 115-11M 2697 abcd
Santa Fe 2354 abc P0613] 2628 abcde
TARS-SROS 2214 abcd N0531] 2545 abcde
Jaguar 2170 abcd TARS-SROS 2530 abcde
304431 2137 abcd 305039 2520 abcde
Buster 2135 abcd Jaguar 2513 abcdef
305055 2110 abcd Santa Fe 2499 abcdefg
Matterhorn 2098 abcd Seahawk 2444 abcdefg
N05324 2075 abcd Matterhorn 2444 abcdefg
USDK-CBB-IS 2057 abcd T 39 2375 abcdefg
Othello 2002 abcd 305055 2364 abcdefg
Seahawk 1983 abcd Othello 2361 abcdefg
305039 1975 abcd Vista 2319 abcdefg
Vista 1922 abcd Chinook Select 2132 bcdefg
Capri 1905 abcd Bunsi 2112 cdefg
Sanilac 1611 abcd Capri 2087 cdefg
Bunsi 1608 abcd Michelite 2051 cdefg
Red Hawk 1603 abcd Montcalm 2027 defg
Montcalm 1504 abcd R99 1997 defg
Chinook Select 1482 abcd K03240 1962 defg
K03240 1441 bed USDK-CBB-IS 1956 defg
Beluga 1286 bed Beluga 1935 defg
K05604 1241 bcd Red Hawk 1871 efg
CELRK 1105 cd K05604 1699 fg
R99 896 d CELRK 1687 g
Mean 1910 Mean 2234
LSD 1332 LSD 826

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD

p=0.05.

56

Table 2-9. Yield of 32 dry bean (Phaseolus vulgaris L.) genotypes grown in Tuscola
County in an organic production system in 2009.

 

 

Yield
Genotype (kg ha'l)
Zorro 3846 aI
TARS-SROS 3583 ab
Merlot 3402 abc
P0613] 3373 abc
Vista 3348 abc
N0531 1 3309 abc
305055 3256 abc
Seahawk 3157 abcd
115-11M 3118 abcde
Condor 3083 abcde
305039 2937 abcde
Jaguar 2892 abcde
Santa Fe 2868 abcde
T39 2817 abcde
Sedona 2791 abcde
Matterhorn 2743 abcde
N05324 2728 abcde
Montcalm 2603 abcde
304431 2568 abcde
Sanilac 2565 abcde
Bunsi 2532 abcde
Capri 2516 abcde
R99 2467 abcde
Buster 23 84 abcde
Othello 2327 abcde
Red Hawk 1990 bcde
USDK-CBB-15 1947 bcde
K03240 1937 bcde
Chinook Select 1810 cde
K05640 1796 cde
Beluga 1523 de
CELRK 1398 e
Mean 2675

_LSD 1724

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD
p=0.05.

57

Table 2-10. Mean yield by 32 dry bean (Phaseolus vulgaris L.) genotypes grown in
organic and conventional production systems at Kellogg Biological Station ﬁ'om 2007 to
2009.

 

 

 

$291119 W Differencel
Seed Class kg ha'1 kg ha"1 kg ha'1
Pink/Red 2324 2726 -402
Black 2263 2767 -504
Navy 2142 2595 -453
Pinto 2127 2528 -401
Kidney 1687 2191 -504
No Nod 221 3 21 70 43
Mean 2126 2505 -3 79
LSD 273 .8 442.2

 

1 . . .
D1fference=organrc-conventlonal

Table 2-11. Mean nitrogen accumulated by 32 dry bean (Phaseolus vulgaris L.)
genotypes at Kellogg Biological Station in organic and conventional production systems
from 2007 to 2009.

 

organic conventional DifferenceI

 

Year kg N ha" kg N ha" kg N ha-1
2007 63.0 73.2 -1o.2
2008 52.1 93.2 41.1
2009 98.4 139.3 40.9
Mean 71.2 101.9 -30.7
LSD 9.2 9.6

 

l I O I
d1fference=organrc-conventronal

58

Table 2-12. Mean Nitrogen Yield of 32 dry bean (Phaseolus vulgaris L.) genotypes
grown in organic and conventional production systems at Kellogg Biological Station,
years 2007-2009.

 

 

Organic Organic

N-Yield N-Yield
Genotype kg ha'1 Genotype kg ha'1
TARS-SROS 111.9a Zorro 133.0 a
Zorro 96.7 ab TARS-SROS 127.7 ab
N05311 89.4 ab R99 119.6 abc
305055 88.2 ab Jaguar 118.4 abc
Jaguar 87.] ab Condor 118.2 abc
305039 84.3 ab Matterhorn 115.8 abc
304431 80.5 ab P0613] 115.5 abc
Santa Fe 78.6 ab N05311 114.5 abc
Seahawk 77.1 ab Santa Fe 114.3 abc
Merlot 75.9 ab Buster 112.7 abc
Sedona 75.8 ab 30443] 110.6 abc
Condor 74.5 ab Seahawk 110.6 abc
Montcalm 73.9 ab 305039 108.2 abc
Chinook Select 73.4 ab 305055 106.6 abc
P0613] 72.7 ab N05324 104.6 abc
115-11M 71.9 ab Merlot 104.0 abc
N05324 70.7 ab Sedona 103.8 abc
Buster 70.5 ab Chinook Select 103.4 abc
Beluga 69.8 ab T39 101.5 abc
T29 69.] ab Beluga 99.4 abc
Vista 68.4 ab USDK-CBB-IS 95.8 abc
Capri 66.8 ab Othello 93.4 abc
Othello 66.7 ab Montcalm 93.1 abc
Red Hawk 65.0 ab Capri 91.9 abc
K03240 63.9 ab K03240 89.6 abc
Matterhorn 62.6 b 115-11M 89.3 abc
USDK-CBB-IS 61.4 b Bunsi 89.0 abc
Bunsi 57.9 b Red Hawk 85.5 abc
Michelite 52.8 b Michelite 83.5 abc
CELRK 52.7 b Vista 82.3 abc
R99 52.6 b CELRK 78.0 bc
K05604 52.4 b K05604 68.0 c
Mean 71.7 Mean 101.9

_LSD 49.1 LSD 53.9

 

lGenotypes followed by the same letter are not signiﬁcantly different, Tukey’s HSD p=0.05.

S9

Table 2-13. Yield by seed class of 32 dry bean (Phaseolus vulgaris L.) genotypes grown
in Tuscola County in an organic production system in 2009.

 

Seed Yield
Class ﬁg ha'l)
Red 3142al
Black 3065a
Navy 2940a

Pinto 2741ab
No Nod 2467ab
Kidney 2196b
Average 2759
LSD 1063

 

lSeed classes followed by the same letter are not signiﬁcantly different, Tukey’s HSD
p=0.05.

6O

Table 2-14. Pearson correlations for maturity, plant stand, days to ﬂower, height at
ﬂowering, 100 seed weight, yield and nitrogen yield for 32 dry bean (Phaseolus vulgaris

L.) genotypes grown in organic and conventional Trials .

Table 2-14a. All treatments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100
Plant Days to Height at seed
Maturity Stand Flower Flowering Weight Yield
Maturity 1.00
Plant stand 1.00
Days to Flower 055*" 027*" 1.00
Height at Flowering -O. 12 -0.08 -0.22*** 1.00
100 Seed Weight -0.7*** -0.42*** -0.60*** 023*" 1.00
Yield -0.01 -0.27*** 0.16“” 0.16*** -0.08* 1.00
N yield 0.11 -0.23** 0.11* 029*" -0.02 089*"
1* sig at 0.05, H at 0.01, m at 0.001 or less
Table 2-l4b. Organic treatment.
100
Plant Days to Height at Seed
Oﬂanic Maturity Stand Flower Flowering Weight Yield
Maturity 1.00
Plant stand 0.25”“ 1.00
Days to Flower 0.66*** 0.2" 1.00
Height at Flowering 0.18“ -0.28"‘** -0.01 1.00
100 Seed Weiglt -0.33*** -0.47*** -0.57*** -0.07 1.00
Yield 047*" -0.22*** 0.37*** 0.49*** -0.06 1.00
N yield 0.43* -0.]6* 036*" 044*" 0.01 075*"
Table 2-14c. Conventional treatment
100
Plant Days to Height at Seed
Conventional Maturity Stand Flower ﬂowering Weight Yield
Maturity 1.00
Plant stand 027* 1.00
Days to Flower 0.76*** O. 16* 1.00
Height at Flowering 029*" -0.7*** -0.02 1.00
100 Seed Weight -0.48*** -0.6*** -O.53*** 0.26“" 1.00
Yield 034*" -0.44*** 032"” 04*" 0.07 1.00
N yield 0.44*** -0.49"'** 025*" 051*" 0.05 074*"

 

 

61

 

 

Figure 2-1. Scatter plot of yield of 32 dry bean (Phaseolus vulgaris L.) genotypes grown
in organic and conventional treatments at Kellogg Biological Station. Conventional yield

is on the Y axis and Organic yield on the X axis.

 

 

 

 

 

 

 

 

 

 

 

 

 

3800
.B
3300
.14 o D
2- ,1.“ .1
Q) .....-—
5'.‘ 230° 03 o x
g 00
“E Y OX 00 OR Q ‘ M o r.
3 2300 Z. -“:I . N
8 DD 0 0A2 0
U CC”BB
o as
1800 4—4
FF
0 P
1300 I T ﬁr j— T T I
1300 1500 1700 1900 2100 2300 2500 2700
Organic Yield, kgha'l

1The horizontal line represents the average yield for the conventional treatment and the
vertical line represents the average yield for the organic treatment from 2007 to 2009.

 

 

 

 

A Zorro I Condor Q Bunsi Y K03240
B N05324 J Merlot R Chinook Z Montcalm
C Buster K llS-llM S 305039 A RedHawk
D 30443] L Sedona T T39 B Capri

E TARS M Santa Fe U Matterhorn C 10510]

F N05311 N 305055 V Othello D Michelite
G Seahawk O P0613] W Vista E CELRK
H Jaguar P R99 X Beluga F K05604

 

 

62

CHAPTER 3

EVALUATION OF DRY BEAN GENOTYPES FOR EARLY ESTABLISHMENT
OF BIOLOGICAL NITROGEN-FIXATION

ABSTRACT

Bean genotypes with higher potential for nitrogen ﬁxation need to be identiﬁed to
support vigorous growth, competitive yields, and reduced reliance on external nitrogen
fertilizer. Thirty-three diverse dry bean genotypes representing seven commercial market
classes were evaluated for N ﬁxation at the early ﬂowering growth stage in a greenhouse
pot study. One high nitrogen ﬁxing check and one non-nodulating, non ﬁxing check were
included for comparison, and to determine N ﬁxed by the difference method. Plants were
grown in an inert medium with nutrients supplied as a modiﬁed nutrient solution lacking
nitrogen. Seed was inoculated with Rhizobium etli strain UMRl 597 at planting and again
10 days later. Plants were harvested at ﬁrst bloom, rated for nodule growth, measured for
plant height, root length, and biomass and root mass. Nitrogen levels were determined in
plant biomass. Dry beans of Middle American origin with indeterminate growth habit
appeared to acquire greater nitrogen than those genotypes of Andean origin with
determinate growth habit. Little variation was seen among elite breeding lines and
commercial genotypes. The high nitrogen ﬁxer, Puebla 152 accumulated nearly 60%
more nitrogen than genotypes that ﬁxed the next highest amount of nitrogen, the pinto
bean ‘Santa Fe’ and the black seeded ‘Zorro’. Selection criteria based on biomass, total

nitrogen accumulated in the biomass, and root mass of nodulating dry bean genotypes

63

grown in low nitrogen conditions are suitable measurements to identify bean genotypes

with superior biological nitrogen ﬁxation.

INTRODUCTION

Finding a method to efﬁciently evaluate dry bean genotypes for their ability to ﬁx
nitrogen has led researchers to utilize several methods such as acetylene reduction

analysis (ARA; St. Clair and Bliss, 1991), total biomass or total seed nitrogen (W olyn et
al., 1989), 15N methods (Kipe-Nolt and Giller, 1993), nitrogen difference method

(Rennie, 1983) as well as nodule and root measurements (Wolyn et al., 1989). Each
method offers different advantages and costs but breeders need a methodology to screen a

large number of genotypes in the most efficient manner with the minimum of cost.

St. Clair and Bliss (1991) used the ARA method to identify high and low ﬁxing lines in a
population derived ﬁom cross of the high N-ﬁxing black bean ‘Puebla 152’ and the low
N-ﬁxing navy cultivar ‘Sanilac’. St. Clair and Bliss (1991) asserted that signiﬁcant
differences in BNF between genotypes could be detected in a single growing season and
that the ranking of those genotypes would likely remain constant though levels of
nitrogen ﬁxed could change with changing environmental parameters. St. Clair and Bliss
(1991) found that ARA was highly correlated with simpler, indirect measures of nitrogen
ﬁxation. If genotypes are grown in nitrogen limited conditions shoot biomass was
reported to be strongly tied to the amount of nitrogen ﬁxed. In the absence of ARA,
however, St.- Clair and Bliss (1991) would not have discovered that some dry bean

genotypes were superior nitrogen ﬁxers based on the activity of their nodules. Since

64

nitrogen in the biomass is not completely translocated to seed yield, nitrogen partitioning
is as important a characteristic as is nodule activity. The cost associated with ARA as
well as calibrating issues and the fact that ARA only provides a measurement for a
speciﬁc time point during the season are major limitations to its use for assessing N-
ﬁxation. Another disadvantage to the ARA system of measuring nitrogen ﬁxation is that
ARA may falsely identify high nitrogen ﬁxers i.e. those individuals with high nodule

activity which did not translate N into increased yield (St. Clair and Bliss, 1991).

15N methods have been widely used to measure nitrogen ﬁxation. This method relies on

the fact that the natural abundance of 15N in the atmosphere is about 0.37% of nitrogen in
the atmosphere, with the rest of the nitrogen in the atmosphere being 14N. Kipe-Nolt and

Giller (1993) applied an isotope labeled fertilizer containing 29.3% 15N to one plot prior

to planting and the equivalent amount of unlabelled ammonium sulfate to another plot.
Seed was planted after allowing the fertilizer to distribute equally in the soil for one
week. Plants were harvested at 56 days in both treatments with an effort made to recover
the entire root system and nodules, along with the biomass including the stems, leaves
and pods. Plants in the unlabelled plot were sampled beginning at 13 days and weekly
there after to 56 days in the same manner as the isotope labeled plot with enough plants
left to grow to maturity and sampled for yield (Kipe-Nolt and Giller, 1993). At pod ﬁll,
the pods contained over half of the total shoot nitrogen. In the genotypes studied the

roots accounted for a minor amount of the nitrogen in the plant. Analysis of the total

nitrogen content in the seed, or biomass, at sampling time will give a ratio of 15N to 14N

65

in the tissue. This information can then be utilized to determine which percent of the
nitrogen was acquired from the fertilizer, the rest from nitrogen ﬁxation. Kipe-Nolt and
Giller (1993) discovered that less than 11% of the fertilizer N applied was recovered by
the plant noting that the nitrogen sources were soluble and easily leached from the soil
before plants were able to access them. A reference crop was utilized as a check to
measure the availability of nitrogen from the soil. Sorghum was chosen as the non ﬁxing
reference crop in the study of Kipe-Nolt and Giller (1993) though the plant growth rate
was slower and resulted in different nitrogen uptake rates from that of the bean genotypes

studied.

Kipe-Nolt and Giller (1993) observed results similar to St. Clair and Bliss (1991) in the
variability in partitioning of nitrogen in dry bean genotypes. Some high ﬁxing genotypes
as determined by total biomass nitrogen did not translocate the nitrogen into the pods or
seed as compared to other high ﬁxing lines which more effectively mobilized nitrogen
into the seed resulting in improved yield. Kipe-Nolt and Giller (1993) also discovered
that some of the higher ﬁxing genotypes also began nodulation earlier than those
genotypes with lower nitrogen ﬁxing ability. They attributed the increase in nitrogen
ﬁxation of certain lines to the earlier nodulation instead of increased or extended ﬁxation
at or beyond pod ﬁll due to the senescence of nodules at the 56 day sampling date when
all genotypes were in the pod ﬁlling stage. Kipe-Nolt and Giller (1993) associated
increased biomass accumulation with improved BNF as well as enhanced development of

nodules. However, some lines that were better able to acquire nitrogen from the soil may

66

be vigorous due to nitrogen from the soil instead of nodules thus evaluation of nodules

and biomass together would be useful in evaluating bean genotypes.

One limitation of 15N methods suggested by Kipe-Nolt and Giller (1993) is the mobility

of nitrogen in the soil. Irrigation and precipitation gradually remove the fertilizer from

the soil proﬁle reducing its availability to the plants. Costs of purchasing fertilizer
enriched with 15‘N as well as difﬁculties incorporating the fertilizer into the soil in a
consistent manner are limitations of this method. Other indirect measures, such as
nodulation and biomass were correlated with results from 15N method (Kipe-Nolt and

Giller, 1993). In reviewing methods used to measure nitrogen ﬁxation, Wolyn et al.
(1989) point out that application of nitrogen in any form may decrease nodule formation
and subsequent activity since nitrogen availability in the soil may inhibit nitrogen

ﬁxation.

Instead of applying a fertilizer enriched in 15N, a fertilizer depleted in 15N could be used,
or an alternative is not to rely on fertilizer (Danso etal., 1993). Usually, soil has a
slightly higher level of 15N isotope than that found in the atmosphere. The 15N:14N ratio,
natural abundance, also known as R can be used to determine how much nitrogen is

ﬁxed. Fixation of nitrogen from the atmosphere results in dilution of the 15‘N in the plant,
as nitrogen derived from the atmosphere has a lower 15N: 14N ratio than the soil. If the

plant were deriving all nitrogen from the soil, the 15N in the plant would be greater than a

plant deriving any nitrogen from the atmosphere. This natural abundance method relies

67

1 . .
on use of a reference crop as does other 5N methods to calibrate mtrogen measurements.

Since the natural abundance method deals with such small differences in 15N levels there

could be considerable error in measurement, along with the need to have access to
equipment sensitive enough to measure these differences (Danso et al., 1993). Working

with other nitrogen ﬁxing legumes such as T rifolium repens and Lotus spp., Steele et al.

(1983) discovered that nodules were enhanced in 15N. There was also evidence that

different strains of Rhizobium had different preferences for ”N, thus the 15N: 14N ratio
could vary from nodule to nodule based on which Rhizobium strain inhabiting the nodule.
If there was an expectation that a plant was ﬁxing nitrogen, and the ratio of 15N: 14N was

higher than expected, researchers may inadvertently underestimate the level of nitrogen

ﬁxation occurring.

Danso et al. (1993) discuss the choice of a reference crop and cite one of the major
problems with reference crops is the differences in growth habit between the crop being
studied and the reference crop. Grasses have commonly been utilized as reference crops
(Hardarson and Danso, 1993; Danso et al., 1993). Different plants have different root
system architecture and may access different layers of the soil proﬁle resulting in
inconsistent acquisition of nitrogen between the reference crop and the nitrogen ﬁxing
crop. Park and Buttery (2006) developed two dry bean genotypes, R99 and R69, lacking
the ability to ﬁx nitrogen since they do not form nodules, or form poorly developed
nodules incapable of nitrogen ﬁxation. The availability of a reference crop in the same

species as the crop being studied helps to relieve some problems with differences in

68

growth characteristics. However, genetic differences in growth habit and yield even
between genotypes of the same species may contribute to inaccurate estimations of

nitrogen ﬁxation.

With each of the methods to measure nitrogen ﬁxation discussed above come challenges
to interpreting the data. Traits that have been highly correlated to ARA and 15N methods

such as shoot biomass, root mass, nodule rating, and seed yield, would be suitable for the
evaluation of the BNF ability of a dry bean genotype. These indirect methods offer
efﬁciency in quickly determining which genotypes are superior nitrogen ﬁxers. These
screening methods could easily be integrated into a breeding program to develop superior

nitrogen ﬁxing commercial genotypes without considerable increases in cost or time.

Nitrogen ﬁxation research is best conducted in soils with extremely low soil nitrogen
levels (Bliss, 1993b). Wolyn et al. (1989) studied progeny from the population developed
by Bliss et al. (1989) derived ﬁom Puebla 152 and Sanilac. Test plots were considered
low in soil nitrogen and fertilizer lacking nitrogen was applied at planting. Several
progeny had total biomass nitrogen levels approaching that of Puebla 152, the high ﬁxing
parent, and others were more similar to the low ﬁxing parent Sanilac based on total

nitrogen, leghemeglobin levels and nodule mass ratings. Wolyn et al. (1989) found a
signiﬁcant correlation (r2=0.71 p50.001) between nodule mass from the entire root

system and seed yield. Nodule mass was determined to be the best indirect measure of

BNF.

69

While developing their non—ﬁxing bean genotypes using the chemical mutagen EMS,
Park and Buttery, (1994, 2006), utilized a greenhouse screening method where the
purported mutants were grown in a mix of perlite and vermiculite to facilitate the
observation of the plants and subsequent replanting of thOse individuals that exhibited
the appropriate non-nodulating phenotype. One-part vermiculite to two-parts perlite mix
was used along with the application of a nitrogen free solution. Similarly Souza et a1.
(2000) and Tsai et al. (1993) utilized an inert growing media for greenhouse screening
and provided nutrient solutions with different levels of nitrogen. Tsai et al. (1993) were
able to efﬁciently identify the level of nodulation of the genotypes studied, between
Puebla 152 and CIAT-125, a non-nodulating mutant, which had no nodule development
in either fertilized or nitrogen free systems with the addition of Rhizobium inoculant.
Utilizing this method they were able to identify four QTLs contributing to an increase in

nodule number and four QTLs contributing to a decrease in nodule number.

Use of an inert material as a growing medium is a suitable choice to support the growth
of plants while providing a controlled level of plant nutrients to the system. Growing
plants in a greenhouse also offers the beneﬁt of providing a consistent environment for all
plants being studied and removes the variation in soil encountered in a research ﬁeld
where pathogens and different root conditions exist. Plants grown in the soil are also
difﬁcult to extract with the entire root system intact, oﬁen leaving behind ﬁner roots and
those roots distant from the main stem. In addition to the ability to recover an entire root

system and have greater control on nutrient levels the plants are receiving, another

70

advantage is being able to use a greenhouse to screen at times of the year when ﬁeld

work is at a minimum.

The objective of the current study was to establish an efﬁcient screening system to
determine BNF potential of numerous dry bean breeding lines. Requirements for the
protocol include that it be simple and efﬁcient as well as economical in terms of cost and

time involved in obtaining reliable and reproducible results.

MATERIALS AND METHODS

Advanced breeding lines as well as commercial cultivars were included in the 34 diverse
genotypes selected for evaluation of BNF potential. Genotypes selected included
representatives of commercially important market classes such as navy, black, cranberry,
kidney, pinto and small red and pink seed types. A non nodulating check, R99, was
included along with a known high-N ﬁxing check, Puebla 152. R99 is derived from the
navy bean Bunsi which was also included. Puebla 152 is a black seeded genotype noted

for its improved BNF abilities. (Table 3-2. List of genotypes)

Plastic nursery trade containers were ﬁlled with an autoclaved 3 :2, perlite: vermiculite
volume for volume, mix. Seeds were surface sterilized by soaking in 70% Ethanol for 2
minutes, then soaked in 5% bleach solution for an additional 2 minutes. Seeds were
rinsed four times in water for 2 m each rinse. While moist from the rinse, seeds were
swirled in Rhizobium etli strain UMR 1597 (supplied by RH. Graham, University of
Minnesota) prepared in powdered peat to completely coat the seed. Three seeds were

planted into each container and watered thoroughly with tap water. A total of ﬁve pots

71

were planted of each genotype. Plants were grown in a greenhouse with supplemental
l-IPS lighting to day length of 12 h. Seedlings were thinned to one seedling per pot when
seedlings were large enough to determine that they were healthy but not so late that the
removal of a seedling would damage the remaining seedling. Thinning occurred prior to
ten days after sowing with seedlings removed by carefully pulling plant from the growing
media. At 10 DAP an additional amount, approximately 0.5g of inoculant was spread
over the surface of the media and lightly watered in with tap water. Plants were watered
as needed with tap water adjusted to a pH of 6.5 throughout the experiment and plants
were given 200 ml modiﬁed Hoagland’s solution twice weekly without nitrogen (Table
3-1). The ﬁnal nutrient solution was also adjusted for a pH of 6.5. The experiment was

repeated three times.

Measurement for plant height and nodule rating were conducted at opening of the ﬁrst
ﬂower plants (See appendix 1). Photographs of the plants were taken with an Olympus
camera mounted on a tripod at a distance of approximately 60 cm. Shoots were harvested
at the media surface and dried, then weight determined. Roots were removed from the
growing media and rinsed with tap water. After gently laying roots out, maximum root
length was recorded with a meter stick. The roots were then photographed at an
approximate distance of 40 cm such that nodules would be clearly visible in the
photograph. After drying, roots and shoots were weighed. Shoot tissue was ground with a
Christy-Turner Lab Mill (Ipswitch, Suffolk, UK) to pass through a 1 mm screen. Total N
analysis was performed on the shoot biomass using the Kj eldahl Method (A&L Great

Lakes Laboratories, Inc., Ft. Wayne, IN, USA).

72

Root nodule development was visually rated based on nodule number, size and
distribution. A scale of 0 to 6 was utilized where the roots of the non-nodulating check
R99 was used at the “0” reference. Roots of Puebla 152, the high nitrogen ﬁxing
genotype, were used as the reference for a score of “6.” Nodule number and size was
taken into account as well as placement of those nodules within the root system. Roots
with sparse, small, pale nodules were rated lower than roots with large well colored

nodules with a higher density on the root system.

The non nodulating check was harvested when its parent line, Bunsi, was harvested since
there was risk of the plants not surviving to ﬁrst bloom due to the lack of nitrogen in the

media. In the ﬁeld, Bunsi exhibited the same agronomic characteristics as R99.

Based on the method of Rennie, (1983), the percent of nitrogen derived from the

atmosphere (%Ndfa) was calculated as follows:

%Ndfa=(Nyield(fs)-Nyield(nfs)/Nyield(fs) x 100.

Where Nyield(fs)=the total amount of nitrogen in the biomass of the ﬁxing dry bean

genotypes.

Nyield(nfs)=the amount of nitrogen in the biomass of the non-ﬁxing dry bean genotype,

R99 (Park and Buttery, 2006)

Relative Nitrogen Growth Rate (RCN)) was calculated according to Park and Buttery,

1989:

R(N)=(logN2-logNl)/t

73

N2=nitrogen content at harvest time, N1=nitrogen content of seed, t=days from seeding

to harvest.

A concurrent study was conducted comparing performance of dry bean genotypes in side
by side plots of conventional production and organic production systems. One site for
this study was at Kellogg Biological Station where yield trials were conducted in 2007,
2008 and 2009. Seed nitrogen was measured at this site to determine nitrogen ﬁxed by
the genotypes in a ﬁeld setting. Seed nitrogen utilized in the above equation was the
average value of three reps obtained ﬁ'om the organic yield trial at Kellogg Biological

Station in 2008 since that was the seed source for the current experiment.

Data were analyzed using SAS 9.2 utilizing PROC MD(ED.

RESULTS

Plant biomass differed signiﬁcantly by bean genotype. Puebla 152, had the greatest

amount of biomass at an average of 4.36 g plant'l. At the low end of biomass

accumulated was the non ﬁxing genotype, R99, at 0.32 g plant'l. All of the market

classes accumulated less biomass than the high ﬁxer, and more biomass than the non
ﬁxer. Pink, pinto, red, great northern, and black market classes had a signiﬁcant increase
in biomass compared to navy and cranberry market classes. Kidney bean biomass did not
differ ﬁ'om navy or cranberry market classes (Table 3-7). Only three kidney bean
genotypes yielded greater than the mean of biomass accumulated, ‘Montcalm’, K05640

and K03204. All other genotypes yielding greater than the mean were of Middle

74

American origin. Root mass of all Andean genotypes, except the kidney K05 604, were
lower than the average root mass of all genotypes. Puebla 152 and the black bean

‘Zorro’, both Middle American genotypes had the highest root mass.

Root mass was positively correlated with nitrogen ﬁxed per plant (r2=0.83 p50.001).

Nodule rating was positively correlated with nitrogen ﬁxed per plant and root mass,

although the relationship was moderately strong (r2=0.52, p50.001 and r2=0.44 p50.001).

Values for nitrogen ﬁxed per plant ranged ﬁ'om 109mg for Puebla 152 to 6.3mg for R99.
Other genotypes that were not signiﬁcantly different from the non ﬁxing check include
115-11M, ‘Michelite’, ‘Bunsi’, ‘Seahawk’, and ‘Sanilac’. Of these ﬁve low ﬁxing
genotypes, four belong to the navy market class; only 115-11M is a black bean breeding

line (Table 3-2)

Puebla 152 had a higher amount of nitrogen ﬁxed per plant than any other genotype
studied. While not signiﬁcantly different than the majority of the genotypes studied, the
recently released black bean Zorro and the recently released pinto bean Santa Fe were at
the top of the list for nitrogen ﬁxed plant'l. The majority of the genotypes studied were
not signiﬁcantly different from one another in nitrogen ﬁxed (Table 3-2). The trend
observed with biomass was the same for nitrogen ﬁxed per plant. Those genotypes in the
cranberry, kidney, and navy market classes had the lowest amount of nitrogen
accumulated in biomass. Red Hawk and the elite breeding line K05 604, both kidney
beans, were the only genotypes of Andean origin to have nitrogen yield better than the

average.

75

Using the Nitrogen Balance method, or Nitrogen Difference, there is little variability in
the genotypes studied for the percent of nitrogen derived from the atmosphere. The range
was 88.7% for the pinto bean ‘Santa Fe’, to 59.4% for the navy bean ‘Vista’. Nitrogen
balance or the Nitrogen difference analysis yielded little difference between market

classes. (Table 3-12)

Root mass accumulated was similar to biomass with the high ﬁxer, Puebla 152, having a
greater average root mass than all other genotypes and the non nodulating R99 having
signiﬁcantly less root mass than any other genotype. Again, black bean Zorro was near
the top in accumulation of root mass though was not signiﬁcantly different from many of

the other genotypes studied. (See Tables 3-5 and 3-6).

For nitrogen ﬁxed per plant, a similar pattern emerges as in biomass and root mass.
Puebla 152 has the greatest amount of nitrogen ﬁxed at over 109 mg N plant'l. The non
nodulating check, R99, is at the bottom of the list with a total of 6.32 mg N plant'l. Pink,

small red, pinto, and great northern seed classes ﬁxed a signiﬁcantly greater amount of

nitrogen than navy and cranberry market classes.
DISCUSSION

Despite considerable work in the past to understand and breed for improved nitrogen
ﬁxation in dry bean, little progress has been made in using the technologies to actually
identify a genotype with superior BNF ability in the ﬁeld. Only ﬁve germplasm lines

have been released with improved BNF ability. Work of Bliss et al. (1989) resulted in

76

the registration ﬁve genotypes considered high N ﬁxing after testing in both Wisconsin
and Brazil. These germplasm lines were derived from an inbred backcross population
using Puebla 152 as the donor parent and the type II upright black bean ICA Pijao as the
recurrent parent. Bliss et al. (1989) utilized indirect methods in the selection process
including total shoot nitrogen, visual nodule score, seed yield and nodule mass of plants

grown where nitrogen was limiting along with more direct measures such as acetylene
reduction activity and 15N isotope dilution. Total shoot nitrogen was found to be highly

correlated with other measures of nitrogen ﬁxed and with seed yield.

Integrating screening methods for improved BNF into a modern breeding program would
be advantageous in selecting genotypes with improved agronomic characteristics and also
superior BNF. Combining agronomic characteristics with improved nitrogen ﬁxation
into a single genotype could offer growers a cultivar with improved productivity while
reducing the need for nitrogen inputs. While elite cultivars such as Zorro are an
improvement over other commercial cultivars there is still opportunity to increase the

level of nitrogen ﬁxation above that of current commercial cultivars.

Using 15N methods Pereira et al. (1989) found that Puebla 152 was the highest ﬁxing line
among the 17 bean genotypes studied. Sanilac, a navy bean, was the lowest ﬁxer in the
study. They also found that Puebla 152 began ﬁxing nitrogen earlier than most of the
bean genotypes studied and that it also nodulated well in the crown area. In the present
study, with harvest beginning at bloom, Puebla 152 had likely established a greater

number and mass of nodules than the other bean genotypes. Early nitrogen ﬁxation was

77

measured in the present study, since plants were harvested for analysis prior to peak
nitrogen ﬁxation at pod ﬁlling. However, the early establishment of BNF is an integral
component to increasing the seasonal BNF of a dry bean genotype. Increasing the days a
dry bean genotype is ﬁxing nitrogen increases the total nitrogen ﬁxed. At maturity
ﬁxation stops as seed become the primary sink for photosynthates and not the nodules,
favoring early growth nodulation as the most effective way to increase season long

ﬁxation.

St. Clair et al. (1988) measured ﬁxation potential of 12 different bean genotypes at
different growth stages in the ﬁeld. The study included four lines derived from an inbred
back cross of Puebla 152 and Sanilac and parents which had been identiﬁed as superior
nitrogen ﬁxers in relation to Sanilac, the low ﬁxing parent with acceptable agronomic
qualities. Comparing the R3 stage to the R9 stage, St. Clair et al. (1988) found that the
later sampling time was more effective at predicting the season long nitrogen ﬁxation
potential. The R3 stage would coincide with the harvest of the genotypes in the present
study. Peak nitrogen ﬁxation occurs during development of the seeds, which are a major
nitrogen sink, thus sampling prior to that stage may only be effective at measuring those
genotypes with improved early N-ﬁxation (St. Clair et al., 1988, Pereira et al., 1989). As
mentioned above, earlier ﬁxation may be a desirable trait to improve in order to develop
season long superior nitrogen ﬁxing genotypes since ﬁxation ends at maturity since the
seeds become a greater sink for resources than nodules. An ideal dry bean genotype with
superior nitrogen ﬁxation ability would ﬁx nitrogen earlier in its life cycle and also

efﬁciently mobilize nitrogen from roots and other organs into the seeds.

78

Aside from the checks, Puebla 152 and R99, the genotypes selected for this study were
not chosen based on their BNF ability but for their importance in the breeding program or
their importance as commercial cultivars. The 32 genotypes selected likely represent the
typical commercial genotypes which have not been selected for improved BNF during
their development. In fact selection would have been practiced under condition of applied
N that represses N-ﬁxation. This lack of variability for N-ﬁxation observed in this study
may indicate a reduction in genetic variability due to the process of selection or lack of
variability for the trait among parents. Sources of genetic material with improved BNF,
such as Puebla 152, may be necessary to bring in the variation needed to increase BNF in
commercial genotypes. Those genotypes more recently developed and selected heavily
for yield, such as Zorro or Santa Fe, were near the top in nitrogen ﬁxed and biomass
(Table 3-2 and Table 3-8 respectively). There has likely been indirect selection for
nitrogen use efﬁciency and partitioning of assimilates to seed, while directly selecting for
yield potential as Zorro and Santa Fe were deve10ped through intense selection for yield,
architecture and acceptable maturity for growing conditions in Michigan. However, the
majority of the commercial cultivars tested did not exhibit an ability to ﬁx nitrogen. The
difference between the best commercial cultivars, such as Zorro, and Puebla 152
demonstrate that there is substantial room for improvement in the BNF ability of modern
commercial cultivars. Utilizing methods such as those investigated in the current study,
breeding germplasm can be screened prior to inclusion in breeding programs in order to
select only those lines that have enhanced BNF ability. Also, early generation screening

of resulting breeding lines will help reduce the number of genotypes with improved BNF

79

for further inclusion in advanced yield trials either as parental lines or for potential

release as commercial cultivars.

It appears that the navy and cranberry seed classes are low in their nitrogen ﬁxation
ability (Table 3-2). The navy beans in this study may have reduced BNF ability do to the
inclusion of Sanilac, a known low ﬁxer and Bunsi, the parent line of R99 resulting in
skewing the navy seed class towards lower BNF levels. Sanilac has superior seed
quality along with determinant growth habit and early maturity which inﬂuenced St. Clair
and Bliss (1991) to select this genotype to utilize as the recurrent parent with Puebla 152
to develop a commercial genotype with superior BNF ability. Combining the superior
agronomic traits from Sanilac with the enhanced BNF and yield of Puebla 152 is a
desired outcome in breeding dry bean genotypes with enhanced BNF. There are likely
other aspects of the variability of this class that are contributing to reduced biomass
accumulation and nitrogen accumulation such as different growth habits or different yield
potentials. The cranberry seed class is of Andean origin which, along with the kidney
genotypes appear to have reduced ability for nitrogen ﬁxation. Those genotypes of
Mesoamerican origin-pinto, pink, red, and great northern seem to be better ﬁxers than
those genotypes of Andean origin as well as navy genotypes which are also
Mesoamerican. This may be an interaction with the Rhizobium strain used in this study
as there is evidence that the different gene pools prefer different Rhizobium strains.
Kipenolt et a1. (1992) found that Rhizobium tropici strain CIAT 899 preferentially
nodulated genotypes of Andean origin. Middle American genotypes were preferentially

nodulated by Rhizobium Ieguminosarum bv phaseoli strain CIAT 632. Graham

80

(www.rhizobium.umn.edu, accessed December 2009, and personal communication
March 2008) indicated that Rhizobium etli UMR1597, the strain used in the current study,
was isolated ﬁom Brazil. It has been the best overall inoculant for dry bean genotypes
regardless of dry bean origin in his studies. Graham (personal communication) indicated,
though, that efﬁciency of this strain may be superior when used with Middle American
genotypes as compared to Andean genotypes. Vasquez-Arroyo et al. (1998) discovered
that even within the indigenous strains (those that were already present in the ﬁeld soil)
in Durango, Mexico, that not all Rhizobium strains were as effective at competing for
nodulation sites. In addition, Vasques-Arroyo et al. (1998) found that different Rhizobium
strains had different abilities to ﬁx nitrogen. Differences between the Andean and
Middle American gene pools other than BNF ability may contribute to reduced biomass
and nitrogen yield. Characteristics such as nitrogen partitioning as well as maturity or
growth habit may also reduce yield in Andean genotypes when compared to Middle

American genotypes.

Seed yield and biomass are also components of nitrogen ﬁxation as seeds and vegetative
organs of the plant are sinks for nitrogen. Plants that yield a greater amount of seed or
biomass may have a larger pool of nitrogen available to them stored in the stems and
leaves. Kidney and cranberry seed classes typically experience lower yields than smaller
seed sizes (Kelly et al., 1987). This difference in productivity may explain the poor
performance of the larger seed classes when compared to the smaller, Middle American
derived genotypes. Cichy et al. (2009) noticed that a reduction in seed yield was linked to

the ﬁn, gene for determinacy in Andean genotypes which is located on linkage group B1.

81

In researching phosphorus uptake Cichy et al. (2009) hypothesize that either ﬁn has
pleiotropic effects resulting in a reduction of yield or is linked to other gene(s)
responsible for the reduction. The difference in growth habit that was responsible for
reduced phosphorus uptake in Andean bean genotypes may be responsible for the

reduced levels of nitrogen acquired found in the current study.

The lack of variation observed for %Ndfa in the nitrogen balance (difference) method
could be explained by different growth characteristics of the genotypes studied. The
estimates of %Ndfa below 100% demonstrate that there was some level of nitrogen
available to the plants, which would likely be around 6 mg, the average nitrogen in R99
biomass. This nitrogen may come from different sources, possibly the seed, water, and
dust settling from the air. Larger seeded types such as kidney beans have a larger amount
of stored nutrients for the developing seedling as opposed to genotypes with smaller
seeds and thus less capacity to store nitrogen. Those genotypes that are better at
scavenging this nitrogen from the soil may have an augmented nitrogen ﬁxed estimate.
Accordingly those genotypes could have an advantage throughout the experiment since
they would have more nitrogen to devote to developing nodules, increasing nitrogen
ﬁxed in the long term. Development of nodules is aided by a small application of
nitrogen early in plant development. An application of nitrogen immediately after
germination and subsequently eliminating nitrogen from the nutrient solution might
provide a more uniform test environment, allowing those genotypes that are good ﬁxers,

but poor nitrogen scavengers, to nodulate and reach levels closer to their potential.

82

Nodule rating has been shown to accurately predict those lines with improved nitrogen

ﬁxation potential (Park and Buttery, 1989). In this study, there was a high correlation
between nodule rating and nitrogen ﬁxation measurements (r2=0.52, p50.0001), with
smaller correlations between nodule rating and evaluations such as nitrogen balance
(r2=0.36, p50.0001) and relative growth in nitrogen (r2=0.3, p30.0001) (Table 3-13).

Kidney beans average 3.6 on a scale of 0 to 6 for nodule appearance. This is
considerably higher than seed classes that accumulated a larger amount of nitrogen, such
as pinto or red and pink seed classes. While the nodules were well developed it is
possible that the genotypes nodules were not as efﬁcient as those with lower nodule
ratings on a nodule for nodule basis. Additionally, there may be differences in
partitioning and mobilization of the nitrogen available into parts of the plant other than
the roots. Since seed was sterilized and growing media was autoclaved prior to sowing
and a known efﬁcient Rhizobium strain, identiﬁed by Graham (www.rhizobium.umn.edg,
accessed December 2009) as one of the best inoculants, was applied, it could be expected
that the kidney beans nodulated with the same rhizobia as the other genotypes did. This
difference in efﬁciency could be explained by the strain/host interaction, kidney beans
may not ﬁx nitrogen optimally with Rhizobium etli strain UMR 1597. Meschini et al.
(2008) determined that Rhizobium strains preferentially inoculate dry bean genotypes
from similar regions of origin. Rhizobium etli is likely of Middle American origin which
might help to explain the improved performance of Middle American bean genotypes

when compared to Andean bean genotypes inoculated with R. etli strain UMR1597.

83

Characteristics that are correlated with nitrogen ﬁxed include root mass, plant height and
nodule rating. The volume of roots produced would seem to determine the number of
nodules able to be formed, with more potential infection sites there would be in increase
in the number of nodules formed. It is likely that plants with upright architecture possess
a better anchorage which results in a more expansive root system to provide the needed
support with the resulting root area also having an increased amount of ﬁne roots which

are involved in nodulation.

Root mass shows some relationship to nitrogen ﬁxed based on high correlation (r2=0.83,

p50.0001). Selection of lines based on above ground architecture is common while
selection for root architecture is rarely practiced. It has been noted that indeterminate
plants also have indeterminate roots, root mass would be larger in relation to those that
are determinate (Wolyn et al., 1991). Plants approaching or exceeding shoot to root ratio
of 3.0 seem to be among those that are also better at ﬁxing nitrogen. Those genotypes
with a signiﬁcantly increased shoot to root ratio over R99 and Sanilac (table 3-9), are the

same lines that exhibited increased levels of nitrogen in the biomass (table 3-12).

Nodule rating has been considered an acceptable means to select plants for improved
nitrogen ﬁxation and the signiﬁcant correlation (r2=0.52 p50.001) between nitrogen ﬁxed
and nodule rating offers support for selecting plants based on visual appearance of
nodules (rating scale from 0 to 6). In addition, root length had a signiﬁcant positive
correlation with nodule rating, which could imply a greater number of infection sites for

Rhizobium. A root system that spreads a greater distance is able to access a greater range

84

of the soil proﬁle, both depth and horizontally, increasing access not only to different
nutrients in the soil but also has an increase opportunity to contact immobile rhizobia.
Roots in this study were conﬁned to the volume of the container and the long roots
exceeded the dimensions of the container, wrapping around the perimeter of the container
one or more times. Those genotypes with shorter root systems also wrapped around the
perimeter of the pot thus suggesting that the longer roots could form more associations

than the shorter roots since they all accessed the same area in the container. Root mass is

strongly correlated with nitrogen ﬁxed (r2=0.83 p50.001), Relative growth in nitrogen
(RN) (r2=0.65 p_<_0.001), and N Balance (r2=0.43 p50.001) while root length is correlated

with nodule rating (r2=0.43 p50.001). (See table 313).

It would seem that root characteristics are important in the BNF process and attention to

root attributes in considering the ﬁxation ability of a genotype would be advantageous.

Plant height is difﬁcult to evaluate in a greenhouse due to the difference in growth habit
when compared to ﬁeld conditions. Plants are generally more etiolated owing to reduced
light levels in the greenhouse while those plants that are type II, which do not vine
excessively in the ﬁeld, do so in the greenhouse. Plant height was measured as an
indication of vigor; however, biomass is a more encompassing and useful trait. Plants
such as Sanilac produced a vine that was very thin and wiry due to overall lack of vigor.
Sanilac was tall, yet had a low biomass (tables 3-8 and 3-10). The plants with the
lowest amount of biomass tended to be in the navy seed class. A plant that is ﬁxing a

greater amount of nitrogen would likely be able to grow a more of foliage, which would

85

photosynthesize at a greater rate thus having a greater amount of carbohydrates to supply
the nodules for greater nitrogen ﬁxation. Selecting for increased biomass, or yield,

indirectly selects those genotypes with improved BNF.

The ratio of the shoot weight to the root weight offers some perspective on where the
nitrogen is being partitioned, and a level of the efﬁciency of the growth. Investment in
roots is necessary to support plant growth, though increase in yield, the top portion of the
plant is critical for increased yield. While there were differences between seed classes
and several genotypes the rank of those genotypes and seed classes was not the same as
in nitrogen ﬁxed, nitrogen balance, or %Ndfa. The cranberry bean ‘Capri’, a consistently
low nitrogen ﬁxer, ranked in the middle of the scale of shoot to root ratio, whereas
‘Matterhorn’, a great northern, had a low shoot to root ratio. The efﬁcient transport of
nitrogen from nodules and roots to the rest of the plant is an important aspect of nitrogen
ﬁxation. Those plants retaining nitrogen in the roots are not as likely to have high levels
of biomass accumulation. Nitrogen partitioning is a critical trait that cannot be ignored
when selecting a genotype for improved BNF. The ultimate goal is to move the nitrogen
into the seed which is the harvestable organ. Though, nitrogen remaining in the straw
and roots may provide nitrogen for the next season’s crop, it does not contribute to the

economic yield of a grain legume crop.

Puebla 152 had the highest nitrogen ﬁxed, nodule rating, biomass, root mass, and height.
These combined characteristics would be a useful screen for identifying genotypes with

superior nitrogen ﬁxation abilities. Both the non nodulating R99 navy and Sanilac were

86

low in these characteristics. Those genotypes ﬁxing increased amounts of nitrogen above
the low/non frxers were more similar to Puebla 152 in these traits. There is some
evidence that later maturity helps increase BNF. There was no signiﬁcant difference
among the seed classes (table 3-13) and the genotypes for nitrogen ﬁxed per day. This
would suggest that an increase in days to maturity could explain the increase in nitrogen

ﬁxation in longer maturity genotypes.
CONCLUSION

Growing plants in a system that limits nitrogen and using indirect measures to evaluate
the BNF ability of different bean genotypes can be a useful tool in selecting bean
genotypes for improved nitrogen ﬁxation. The high ﬁxing check, Puebla 152,

accumulated greater biomass, larger root mass, had a higher nodule rating, and the

greatest accumulation of nitrogen, 109 mg N plant'l compared to the next highest ﬁxers,

the pinto bean Santa Fe and the black bean Zorro 72 mg N plant'1 and 70 mg N plant'l,

respectively. The lowest ﬁxers, mostly in the navy seed class, had low biomass, low root
mass, and a lower nodule rating than those determined to be greater nitrogen ﬁxers.
Traits such as total biomass and root mass may be used along with total biomass nitrogen
accumulation and a visual nodule score to screen genotypes quickly and in an efﬁcient
manner. This greenhouse assay can be performed during times when ﬁeld work is at a
minimum and also decrease the time necessary to properly evaluate genotypes based on
BNF. Genotypes which possess similar root mass, biomass, nodule score, and biomass

nitrogen combined with upright, vigorous, architecture could be selected in a breeding

87

program utilizing the above protocols. Reaching a balance between adequate root growth
with improved biomass, and resulting yield, is an essential requirement for a successful

bean genotype.

In addition to the above mentioned characteristics important for genotypes having
improved nitrogen ﬁxation, this study demonstrated that screening genotypes in the
greenhouse in inert media and fertilized with nitrogen free solution can be an effective
method to identify dry bean genotypes with enhanced nitrogen ﬁxation ability.
Convenience of observing the roots in an enclosed system as well as ﬂexibility in timing
of the screen in relation to ﬁeld season are advantages to this system. While considerably
different than conditions in the ﬁeld, the greenhouse screening identiﬁed both the poor
yielding genotypes in the ﬁeld as well as the higher yielding genotypes in the ﬁeld. Zorro
performed well under organic and conventional systems as well as being identiﬁed as a
superior nitrogen ﬁxer in the greenhouse. Similarly, the navy bean genotypes yielded
poorly in the ﬁeld and also performed poorly in amount of nitrogen ﬁxed per plant. While
the non nodulating genotype R99 was inconsistent in its performance between organic

and conventional sites, it ﬁxed the lowest amount of nitrogen in the greenhouse study.

88

 

Table 3-1 Modiﬁed Hoagland’s nutrient solution utilized in the greenhouse study of
nitrogen ﬁxation of 34 dry bean (Phaseolus vulgaris L.) genotypes.

 

 

Amount in 1 L Amount per L stock solution
stock solution for ﬁnal dilution.

K2I-IP04 116 g 3 ml

CaClz 366g lml

MgSO.r 245g lml

KHSO4 218g lml

The following were prepared as a lml of the minor nutrient

single stock solution solution

CuSO4 .05 1 g

NaMoO4*2HzO 0.12g

MnC12*4HzO 1.81g

H3BOz 2.86g

ZnSOr 0.22 g

 

89

Figure 3-1. Nitrogen ﬁxed per dry bean plant (Phaseolus vulgaris L.) from 10 seed
classes evaluated for nitrogen ﬁxation under greenhouse conditions in East Lansing, MI
in September and December 2008 and March 2009'.

140 Lrhgh , .7 , . , r.
120 ; 7' ’7’ 77* <7 7 i,

  
      
 
 

100 , Pink’” Northern, W77, , i if,
Pinto
80 - Red Kidney m 7,7
' Cranberry
Navy ,, , .7

  

nitrogen ﬁxed per plant (mg)
A O\
O O

N
O
l

 

Seed Class

1 The high genotype was Puebla 152 and the no-nod was R99.

90

Table 3-2. Nitrogen Fixed plant.1 of 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in September and

December 2008 and March 2009. ‘

 

Nitrogen ﬁxed plant-1(mg)

 

Genotype Seed class

Puebla 152 Black, High Fixer 109.48 a2
Santa Fe Pinto 72.34 b
Zorro Black 70.71 bc
T39 Black 69.97 bc
Sedona Pink 69.30 bc
P06l31 Pinto 68.65 bc
Buster Pinto 67.16 bcd
TARS-SR05 Red 66.25 bcde
Matterhorn Great Northern 62.06 bcdef
K05604 Kidney 61.94 bcdef
Jaguar Black 58.40 bcdef
Merlot Red 56.25 bcdef
Red Hawk Kidney 54.91 bcdef
Othello Pinto 54.65 bcdef
Montcalm Kidney 50.14 bcdefg
K03240 Kidney 49.97 bcdefg
USDK-CBB-15 Kidney 49.85 bcdefg
N05324 Navy 48.22 bcdefg
Chinook Select Kidney 46.13 bcdefg
N05311 Navy 46.12 bcdefg
B05039 Black 46.02 bcdefg
304431 Black 44.80 bcdefg
Beluga Kidney 44.10 bcdefg
305055 Black 42.79 bcdefg
CELRK Kidney 41.84 bcdefg
Condor Black 41.05 bcdefg
115-11M Black 38.56 bcdefgh
Michelite Navy 38.56 cdefgh
Capri Cranberry 33.56 defgh
Vista Navy 33.34 efgh
Bunsi Navy 29.32 fgh
Seahawk Navy 28.95 fgh
Sanilac Navy 19.98 gh
R99 Navy, No Nod 6.32 h

Mean 50.64
Tukey’s LSD (p50.05) 17.3

 

1See appendix A for days to ﬁrst bloom 2Genotypes followed by the same letter are not

signiﬁcantly different.

91

Table 3-3. Nodule rating for 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation under greenhouse conditions in East Lansing, MI September and December

2008 and March 2009. ‘

 

 

Genotype Seed class Nodule rating2
Puebla-152 High Fixer 5.2
Othello Pinto 4.0
Chinook Select Kidney 3.9
Buster Pinto 3 .8
USDK-CBB-IS Kidney 3.7
Red Hawk Kidney 3.7
K03240 Kidney 3 .7
CELRK Kidney 3.6
Beluga Kidney 3.6
TARS-SROS Red 3.5
Sedona Pink 3 .5
Montcalm Kidney 3 .5
K05604 Kidney 3 .4
Merlot Red 3.4
Capri Cranberry 3.3
Santa Fe Pinto 3.2
Zorro Black 3.1
T39 Black 3.0
N0531 1 Navy 2.8
305055 Black 2.8
Jaguar Black 2.8
Seahawk Navy 2.7
P0613l Pinto 2.6
Michelite Navy 2.6
305039 Black 2.6
Bunsi Navy 2.6
Matterhorn Great Northern 2.5
N05324 Navy 2.5
115-11M Black 2.5
Vista Navy 2.4
Sanilac Navy 2.3
B0443 1 Black 2.3
Condor Black 2.2
R99 No Nod 0
Mean 3.0

 

1 See Appendix A for days to ﬁrst bloom. 2Nodule rating scale 0= no nodules 6= extensive nodulation

92

Table 3-4. Nodule rating for 10 dry bean (Phaseolus vulgaris L.) averaged by seed class
evaluated under greenhouse conditions for nitrogen ﬁxation ability in East Lansing, MI in

September and December 2008 and March 2009.1

 

 

 

Seed class Nodule Rating2
High,Puebla 152 5.2
Kidney 3.6
Pink 3.5
Red 3.4
Pinto 3-4
Navy 3.4
Cranberry 3.3
Black 2.7
Great Northern 2.5
Non Nodulating, R99 0
Mean 3.1

 

1 See Appendix A for days to ﬁrst bloom 2Nodule rating scale 0= no nodules 6= heavy nodulation

93

Table 3-5. Average root mass of 10 dry bean (Phaseolus vulgaris L.) market classes
evaluated at the ﬁrst ﬂower stage for nitrogen ﬁxation ability under greenhouse conditions

in East Lansing, MI in September and December 2008 and March 2009.1

 

 

 

Seed Class2 Root Mass (g)
High, Puebla 152 1.62 a”
Great Northern 1.01 b
Pink 0.96 b
Pinto 0.89 bc
Black 0.87 bcd
Small Red 0.86 bcd
Kidney 0.73 bcd
Cranberry 0.61 cd
Navy 0.58 cd
Non Nodulating, R99 0.28 e
Mean 0.84

Tukey’s LSD(p50.05) 0.29

 

1See Appendix A for days to ﬁrst bloom 2The high genotype was Puebla 152 and the no—nod
was R99 3Seed classes followed by the same letter are not signiﬁcantly different.

94

Table 3-6. Root mass of 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in September

and December 2008 and March 2009.1

 

 

Genotype Seed Class Root Mass (g)
Puebla 152 High Fixer 1.62 a2
P0613] Pinto 1.17 ab
Zorro Black 1.16 ab
Matterhorn Great Northern 1.01 bc
Jaguar Black 0.98 bcd
Sedona Pink 0.96 bcde
B04431 Black 0.95 bcde
K05604 Kidney 0.95 bcde
T39 Black 0.93 bcde
805039 Black 0.9] bcde
TARS-SROS Red 0.90 bcde
Santa Fe Pinto 0.86 bcdef
Merlot Red 0.83 bcdef
Buster Pinto 0.79 bcdef
N0531 1 Navy 0.78 bcdef
Red Hawk Kidney 0.77 bcdef
USDK-CBB-IS Kidney 0.76 bcdef
805055 Black 0.73 bcdefg
Othello Pinto 0.72 bcdefg
K03240 Kidney 0.72 bcdefg
N05324 Navy 0.72 bcdefg
CELRK Kidney 0.70 cdefg
Condor Black 0.66 cdefg
Beluga Kidney 0.65 cdefg
115-11M Black 0.64 cdefg
Chinook Select Kidney 0.64 cdefg
Michelite Navy 0.63 cdefg
Montcalm Kidney 0.62 cdefg
Capri Cranberry 0.61 cdefg
Vista Navy 0.54 defg
Seahawk Navy 0.52 efg
Bunsi Navy 0.43 fg
Sanilac Navy 0.28 g

R99 No Nod 0.28 g
Mean 0.78
Tukey’s LSD (p30.05) 0.46

 

1See Appendix A, days to ﬁrst bloom 2Genotypes followed by the same letter are not

signiﬁcant.

95

 

Table 3-7. Average biomass of 10 dry bean (Phaseolus vulgaris L.) seed classes evaluated
for nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in September

and December 2008 and March 2009.1

 

 

 

Seed Class2 Biomass (g)
High, Puebla 152 4.36 a2
Pink 2.23 b
Pinto 2.22 b
Small Red 2.13 b
Great Northern 1.92 b
Black 1.89 b
Kidney 1.63 bc
Navy 1.22 c
Cranberry 1.17 c
Non Nodulating, R99 0.32 (1
Mean 1.91

Tukey’s LSD (p50.05) 0.65

 

1See Appendix A for days to ﬁrst bloom 2Averages followed by the same letter are not
signiﬁcantly different.

96

Table 3-8. Biomass of 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for nitrogen
ﬁxation ability under greenhouse conditions in East Lansing, MI in September and December

2008 and March 2009 .

 

 

Genotype Seed Class Biomass (g)
Puebla 152 Black 4.36 a2
Zorro Black 2.83 b
P0613] Pinto 2.47 bc
TARS-SROS Red 2.47 bcd
Santa Fe Pinto 2.37 bcde
Jaguar Black 2.35 bcdef
Sedona Pink 2.24 bcdefg
T39 Black 2.2] bcdefg
Buster Pinto 2.2] bcdefg
K03240 Kidney 1.96 bcdefgh
Matterhorn Great Northern 1.92 bcdefghi
K05604 Kidney 1.86 bcdefghi
Othello Pinto 1.83 bcdefghi
Merlot Red 1.80 cdefghi
B05039 Black 1.79 cdefghi
Red Hawk Kidney 1.78 cdefghi
B04431 Black 1.77 cdefghi
USDK-CBB-15 Kidney 1.65 cdefghi
B05055 Black 1.58 cdefghij
Condor Black 1.55 cdefghij
N05324 Navy 1.54 cdefghij
Montcalm Kidney 1.51 cdefghij
Beluga Kidney 1.46 defghij
N0531 1 Navy 1.45 efghij
CELRK Kidney 1.44 efghij
115-11M Black 1.39 efghij
Chinook Select Kidney 1.34 fghij
Michelite Navy 1.27 ghijk
Capri Cranberry 1.17 hijk
Vista Navy 1.17 hijk
Seahawk Navy 0.95 hijk
Bunsi Navy 0.87 ijk
Sanilac Navy 0.58 jk

R99 Navy No Nod 0.32 k

Mean 1.75

LSD 1.01

 

 

1See Appendix A for days to ﬁrst bloom 2Genotypes followed by the same letter are not
signiﬁcantly different.

97

Table 3-9. Shoot to root ratio of 34 dry bean (Phaseolus vulgaris L.) genotypes evaluated for
nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in September and

December 2008 and March 2009‘.

 

 

Genotype Seed Class Shoot to Root Ratio
TARS-SROS Red 3.3 32
Buster Pinto 3.3 ab
Puebla 152 High Fixer 2.9 abc
K03240 Kidney 2.9 abc
Santa Fe Pinto 2.8 abc
Othello Pinto 2.6 abc
Montcalm Kidney 2.6 abc
Beluga Kidney 2.5 abc
Zorro Black 2.5 abc
Jaguar Black 2.5 abc
Condor Black 2.4 abc
T39 Black 2.4 abcd
Red Hawk Kidney 2.4 abcd
Capri Cranberry 2.3 abcd
B05055 Black 2.3 abcd
N05324 Navy 2.3 abcd
115-11M Black 2.3 abcd
B04431 Black 2.2 abcd
Merlot Red 2.2 abcd
CELRK Kidney 2.2 abcd
USDK-CBB-IS Kidney 2.2 abcd
Vista Navy 2.2 abcd
Sedona Pink 2.2 abcd
Sanilac Navy 2.2 abcd
P0613 1 Pinto 2.] bed
181010 Bunsi 2.0 do
305039 Black 2.0 do
K05604 Kidney 2.0 dc
Chinook Select Kidney 1.9 dc
Matterhorn Great Northern 1.9 dc
Seahawk Navy 1.8 do
Michelite Navy 1.8 do
N05311 Navy 1.7 do
R99 No Nod 1.2 (1
Mean 2.29
LSD 1.23

 

 

1See Appendix B for days to ﬁrst bloom.
2Genotypes followed by the same letter are not signiﬁcantly different.

98

Table 3-10. Shoot to root ratio of 10 dry bean (Phaseolus vulgaris L.)seed classes
evaluated under greenhouse conditions for nitrogen ﬁxation ability in East Lansing,

MI in September and December 2008 and March 20091.

 

 

Seed Class Shoot to root ratio
High, Puebla 152 2.91 a2
Red 2.76 ab
Pinto 2.68 abc
Cranberry 2.34 abc
Kidney 2.32 abc
Black 2.28 abc
Pink 2.17 abc
Navy 2.07 bc
Great Northern 1.92 d
Non Nodulating, R99 1.18 (1
Mean 2.26

Tukey’s LSD @5005) 0.78

1See Appendix B for days to ﬁrst bloom.
2Seed classes followed by the same letter are not signiﬁcantly different.

Table 3-1 1. Nitrogen ﬁxed per day of 10 dry bean (Phaseolus vulgaris L.) seed classes
evaluated for nitrogen ﬁxation under greenhouse conditions in East Lansing, MI in

September and December 2008 and March 20091.

 

 

Seed class N ﬁxed per day (mg)
High 1.23 a2
Pink 1.23 a
Pinto 1.09 a
Red 1.06 a
Great Northern 1.02 a
Kidney 0.90 a
Black 0.83 a
Cranberry 0.73 ab
Navy 0.70 ab
Non Nodulating 0.09 b
Mean 0.89

Tukey’s LSD (pgog) 0.7

1See Appendix B for days to ﬁrst bloom.
2Seed classes followed by the same letter are not signiﬁcantly different.

99

Table 3-12. Percent nitrogen derived from the atmosphere (%Ndfa) of 34 dry bean
(Phaseon vulgaris L.) genotypes evaluated for nitrogen ﬁxation ability under
greenhouse conditions in East Lansing, MI in September and December 2008 and March

2009‘.

 

 

 

Genotype Seed Class %Ndfa
Puebla 152 High Fixer 94.2 a2
Santa Fe Pinto 91.3 a
T39 Black 91.1 a
Sedona Pink 90.9 a
Buster Pinto 90.6 a
TARS-SROS Red 90.5 a
P0613] Pinto 90. 1 a
K05604 Kidney 89.8 a
Matterhorn Great Northern 89.8 a
Jaguar Black 89.2 a
Zorro Black ' 89.0 a
Merlot Red 88.8 a
Red Hawk Kidney 88.5 a
805039 Black 86.3 a
Montcalm Kidney 87.4 a
K03240 Kidney 87.4 a
Othello Pinto 87.4 a
USDK-CBB-IS Kidney 87.3 a
N05324 Navy 86.9 a
N05311 Navy 86.3 a
Chinook Select Kidney 86.3 a
80443] Black 85.9 a
Beluga Kidney 85.7 a
B05055 Black 85.2 a
CELRK Kidney 84.9 a
Condor Black 84.6 a
115-11M Black 83.6 a
Michelite Navy 83.6 a
Capri Cranberry 81.2 a
Vista Navy 81.0 a
Bunsi Navy 78.4 a
Seahawk Navy 78.2 a
Sanilac Navy 68.4 a
R99 Non Nodulating 0 b
Mean 87.5
Tukey’s LSD (p<0.05) 63.5

 

1See Appendix B for days to ﬁrst bloom. 2Genotypes followed by the same letter are not
signiﬁcantly different.

100

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101

SUMMARY

A comparison of results from the nitrogen ﬁxation screening in the greenhouse to yield
trials conducted in the different production systems in the ﬁeld reveals a similar trend: In
the ﬁeld Middle American genotypes produced higher yields and acquired greater
amounts of nitrogen than Andean genotypes. Middle American genotypes produced
greater biomass and ﬁxed greater amounts of nitrogen than those genotypes of Andean
origin in the greenhouse screening. Black, pink and red seed classes as a group
performed well under low nitrogen conditions. Kidney and cranberry market classes

yielded poorly under low nitrogen conditions.

The black bean genotype ‘Zorro’ performed consistently well under organic and
conventional production systems as well as ﬁxing a high amount of nitrogen per plant in
greenhouse trials. Zorro is a modern commercial cultivar which was recently released
(Kelly et al., 2009). The black bean genotype ‘Jaguar’ also performed well under both

organic and conventional production systems.

The germplasm small red line TARS-SROS performed well under both organic and
conventional production systems. TARS-SR05 was ranked 8th for nitrogen ﬁxed per
plant (Table 3-2). This genotype will likely provide a source for improving nitrogen
ﬁxation but also development of genotypes better suited to organic production systems

and having better root rot resistance, stress tolerance and overall root health.

The navy genotype ‘Michelite’ released prior to the common use of pesticides and

fertilizers did not perform well under organic or conventional production systems.

102

Similar to the other navy beans evaluated, Michelite was inferior for nitrogen ﬁxation
(Table 3-2). Those modern genotypes selected for disease resistance and superior yield
appear better suited to organic production as they have undergone rigorous selection for

yield.

Under the organic production system at all sites and years, a moderate correlation
(r2=0.15, p50.0001) was observed between the amounts of nitrogen ﬁxed in the
greenhouse with yield as obtained in ﬁeld trials. Biomass in the greenhouse had a
slightly stronger correlation (r2=0.18, p50.0001) with yield from ﬁeld trials. The amount
of nitrogen ﬁxed per acre in ﬁeld trials correlated with nitrogen ﬁxed in the greenhouse,
(r2=0.12, p50.05) as well as greenhouse biomass (r2=0.17, p50.001). The genotypes
selected for evaluation were selected for their importance in breeding programs and
commercial production. TARS SR05 was included based on its development under stress
conditions which may be better suited for organic production. It is likely that stronger
relationships would be observed when evaluating populations which have been created
by crossing genotypes of divergent nitrogen ﬁxation ability. Such a population could be
generated by crossing a genotype identiﬁed as strong nitrogen ﬁxers such as Puebla 152

and a commercial cultivar that ﬁxes less nitrogen.

Nitrogen ﬁxed in the greenhouse along with biomass in the greenhouse may be used to
select genotypes with superior early nitrogen ﬁxation ability as well as greater yield

under ﬁeld evaluation in early generation selection programs.

103

Summary Table 1. Pearson Correlations of 32 dry bean (Phaseolus vulgaris L.)
genotypes grown under organic production at KBS, Gratiot County, and Tuscola County

 

 

from 2007 to 2009.
Greenhouse Greenhouse Greenhouse Field-
N Biomass N Balance Yield
Greenhouse 1.00
N
Greenhouse 0,9411'11111'1 1.00
Biomass
Greenhouse 0.77*** 0.72*** 1.00
N Balance
Field-Yield 0.15*** 0.18*** 0.05 1.00
Field-N Yield 0.12* 0.17 ** 0.11 075*"

 

 

‘*, pgoos, **, p50.01, ***p_<_0.001

104

Summary Table 2. Ranking of the ﬁve highest yielding over all sites and years, and
highest nitrogen ﬁxed, of 32 dry bean (Phaseolus vulgaris L.) genotypes.

 

 

 

Rank Organic Yield Conventional Yield Greenhouse
Nitrogen Fixed
-1
plant
1 TARS-SROS Zorro Puebla 152‘
2 Zorro TARS-SROS Santa Fe
3 N0531 1 R99 Zorro
4 B05055 Jaguar T39
5 Jaguar Condor Sedona

 

lPuebla 152 was not included in ﬁeld studies do to its lack of adaptation to production in
Michigan.

105

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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126

Appendix B

Appendix B1. Average day to harvest of 34 dry bean (Phaseon vulgaris L.) genotypes
evaluated for nitrogen ﬁxation ability under greenhouse conditions in East Lansing, MI in 2008

 

 

and 2009.
Average days

Genotype to harvest
Red Hawk 37.8
Sanilac 39.0
Chinook Select 39.9
USDK-CBB-IS 40.3
K03240 40.3
CELRK 40.7
Montcalm 40.8
Beluga 40.8
Capri 40.9
K05604 42.3
Bunsi 42.7
R99 46.4
Seahawk 47.8
Othello 48.1
T-39 48.3
Sedona 48.9
Merlot 50.5
Matterhorn 50.7
Santa Fe 50.7
Buster 51.0
N05324 52.6
1 15-M 52.8
TARS-SROS 53.0
P0613] 53.1
Jaguar 53.3
Zorro 53.4
Michelite 53.7
N053 l 1 54.4
Condor 54.9
B04431 56.3
Puebla 152 56.4
Vista 56.6
B05055 58.6
B05039 58.7
Mean 48.7

 

127

Appendix C1. Average Percent seed N of 32 dry bean (Phaseon vulgaris L.) genotypes
each year grown under organic and conventional production systems at Kellogg
Biological Station in 2007 to 2009.

 

 

 

Organic Conventional
M L08. m2 2902 M. 29.09.
Merlot 2.91 2.9 2.8 3.5 3.2 3.6
Sedona 3.4 2.3 2.9 3.7 3.1 3.7
T39 3.6 3.8 3.8 3.8 3.9 3.9
N05311 3.3 3.9 3.8 4.2 3.6 4.2
Zorro 3.6 3.5 3.6 4.2 3.5 3.8
P0613] 3.2 2.4 3.3 3.9 3.4 4.1
115-11M 3.4 3.4 3.6 3.9 3.9 3.4
Condor 2.6 2.9 3.7 3.9 3.9 3.9
Santa Fe 2.9 2.8 3.3 4.2 3.3 3.9
TARS-SROS 3.8 3.9 3.9 3.9
Jaguar 3.2 3.5 3.6 4.2 3.8 3.9
BO4431 3.4 3.2 3.4 3.5 3.8 3.7
Buster 2.8 2.7 2.8 3.7 3.6 3.6
B05055 3.7 2.9 4.1 4.5 4.2 4.4
Matterhorn 3.3 3.5 3.1 3.8 3.8 3.9
N05324 2.2 3.2 3.8 3.9 4.1 3.9
USDK-CBB-l 5 3.6 2.9 3.3 4.6 4.4 3.9
Othello 3.1 3.2 2.7 3.6 3.5 3.4
Seahawk 3.5 3.4 3.6 3.5 3.6 3.8
805039 3.7 3.3 3.8 3.7 4.3 4.4
Vista 3.5 3.7 3.6 3.9 4.1 4.4
Capri 3.4 3.1 3.2 3.6 3.9 3.8
Michelite 3.6 3.6 3.5 3.9 3.9 3.9
Bunsi 3.7 3.3 3.7 4.3 4.2 3.9
Red Hawk 4.1 3.9 3.2 4.6 4.4 3.8
Montcalm 3.9 3.7 3.4 4.4 4.4 3.9
Chinook Select 3.6 3.5 3.1 3.9 3.2 3.8
K03240 3.9 3.4 3.6 4.5 3.3 3.7
Beluga 3.8 4.2 3.4 4.4 4.3 3.9
K05604 3.9 3.3 3.3 4.4 4.2 3.9
CELRK 3.8 3.4 3.2 4.2 3.6 3.8
R99 2.2 3.4 2.8 2.9 3.7 3.8
Mean 3.3 3.3 3.4 4.0 3.8 3.9

 

lNitrogen values as determined by the Kj eldahl method.

128

Appendix D]. Average Percent seed protein of 32 dry bean (Phaseolus vulgaris L.)
genotypes each year grown under organic and conventional production systems at
Kellogg Biological Station in 2007 to 2009.

 

 

Organic
2001 M 2099. Mean 2.907. m M Mean

Merlot 1331 18.1 17.5 18.1 21.9 20.0 22.5 21.5
Sedona 21.3 14.4 18.1 17.9 23.1 19.4 23.] 21.9
T39 22.5 23.8 23.8 23.3 23.8 25.0 25.0 24.6
N0531] 20.6 25.0 23.8 23.1 26.3 22.5 26.3 25.0
Zorro 22.5 21.9 22.5 22.3 26.3 21.9 23.8 24.0
P0613] 20.0 15.0 20.6 18.5 25 .0 21.3 25.6 24.0
115-11M 21.3 21.3 22.5 21.7 25.0 24.4 21.3 23.5
Condor 16.3 18.1 23.1 19.2 24.4 25.0 24.4 24.6
Santa Fe 18.8 17 .5 20.6 19.0 26.3 20.6 24.4 23.8
TARS-SROS 23.8 24.4 24.1 25.0 24.4 24.7
Jaguar 20.0 21.9 22.5 21.5 26.3 23.8 24.4 24.8
B04431 21.3 20.0 21.3 20.8 21.9 23.8 23.1 22.9
Buster 17.5 16.9 17.5 17 .3 23.1 22.5 22.5 22.7
305055 23.1 18.8 25.6 22.5 28.1 26.3 27.5 27.3
Matterhorn 20.6 21.9 19.4 20.6 23.8 23.8 25.0 24.2
N05324 13.8 20.0 23.8 19.2 24.4 25 .6 24.4 24.8
USDK-CBB-IS 22.5 18.1 20.6 20.4 28.8 27.5 24.4 26.9
Othello 19.4 20.0 16.9 18.8 22.5 21.9 21.3 21.9
Seahawk 21.9 21.3 22.5 21.9 21.9 22.5 23.8 22.7
B05039 23.1 20.6 23.8 22.5 23.1 26.9 27.5 25.8
Vista 21.9 23.1 22.5 22.5 24.4 25.6 27.5 25.8
Capri 21.3 19.4 20.0 20.2 22.5 24.4 23.8 23.5
Michelite 22.5 22.5 21.9 22.3 24.4 24.4 25.0 24.6
Bunsi 23.] 20.6 23.1 22.3 26.9 26.3 24.4 25.8
Red Hawk 25.6 24.4 20.0 23.3 28.8 27.5 23.8 26.7
Montcalm 24.4 23.1 21.3 22.9 27.5 27.5 24.4 26.5
Chinook Select 22.5 21.9 19.4 21.3 25.0 20.0 23.8 22.9
K03240 24.4 21.3 22.5 22.7 28.1 20.6 23.1 24.0
Beluga 23.8 26.3 21.3 23.8 27 .5 26.9 25.0 26.5
K05604 24.4 20.6 20.6 21.9 27.5 26.3 24.4 26.0
CELRK 23.8 21.3 20.0 21.7 26.3 22.5 23.8 24.2
R99 13.8 21.3 17.5 17.5 18.8 23.1 23.8 21.9
Mean 20.5 20.7 21.3 21.1 24.2 23.9 24.3 24.4

 

lPercent Protein=%N x 6.25

129

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