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EFFECT OF INOCULANT, FUNGICIDAL, AND INSECTICIDAL
SEED TREATMENTS ON SOYBEAN GROWTH AND YIELD IN

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Terry J. Schulz

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EFFECT OF INOCULANT, FUNGICIDAL, AND INSECTICIDAL SEED
TREATMENTS ON SOYBEAN GROWTH AND YIELD IN MICHIGAN

By

Terry J. Schulz

A THESIS

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
for the degree of

MASTERS OF SCIENCE

Department of Crop and Soil Sciences

2007

ABSTRACT
EFFECT OF INOCULANT, FUNGICIDAL, AND INSECTICIDAL SEED
TREATMENTS ON SOYBEAN GROWTH AND YIELD IN MICHIGAN
By

Terry J. Schulz

In recent years, several new varieties of soybean seed treatments have been introduced to
the marketplace, including inoculants and neonicotinoid insecticides. Researchers
throughout the Midwest have generated conﬂicting results regarding the beneﬁt of
inoculating soybean seed in successive plantings. One objective of this research was to
determine whether inoculation of soybean seed for successive plantings in Michigan.

Use of soybean seed inoculant increased soybean grain yields on 6 of 14 sites that had
been in a soybean rotation. Another objective was to determine whether interaction
between inoculant and Apron Maxx RTA (mefenoxam and ﬂudioxonil) fungicide could
be observed. Interaction between inoculant and fungicide was not observed on sites that
had been in soybean rotation. However, signiﬁcant interaction was observed at two sites
where soybeans had not been previously grown. The objectives of the neonicotinoid seed
treatment research were to 3) determine whether neonicotinoid seed treatments increase
soybean grain yield, b) determine the duration of soybean aphid control provided by
neonicotinoids, and c) determine whether neonicotinoids improve soybean plant health.
Yield increases from neonicotinoid seed treatments were only observed in 2005, a year of
signiﬁcant aphid pressure. Signiﬁcant neonicotinoid effects on soybean aphid
populations ended after R2. Neonicotinoid seed treatments did not contribute to soybean

plant health on a wide-spread basis

ACKNOWLEDGMENTS
I would like to sincerely thank the members of my graduate committee, Dr. Kurt Thelen,
Dr. Karen Renner, and Dr. Chris DiFonzo. Special thanks to Dr. Kurt Thelen who
provided me with the assistantship and served as my academic advisor. I got my start in
agricultural research thanks in large part to Dr. Karen Renner, who hired me as an
undergraduate, and has been a tremendous role model both as a researcher and an

instructor.

Special thanks to Bill Widdicombe, John Boyse, and Keith Dysinger, technicians who
help coordinate ﬁeld research. Mike Boring was instrumental in conducting these trials
in 2003 and 2004, and this thesis would not be possible without his work. Dr. Sasha
Kravchenko was extremely helpful with data analysis. Thanks to the Michigan Soybean
Promotion Committee for funding this research project. Thanks to the graduate and
undergraduate students who helped collect data for this study, including Steve Wagstaff,
Anatoily Kravchenko, Jim Williams, Deanne Sweeney, and Kelly Foote. My wife, Gina,
has also helped me along in many ways during this project, and I appreciate everything
she has done for me. Finally, I also thank my entire family for their patient support of my

educational pursuits over the year.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vi
LIST OF FIGURES .......................................................................................................... ix
LITERATURE REVIEW
Introduction ............................................................................................................ l
Taxonomy and Morphology of Soybean ................................................................ 3
History of Soybean Production .............................................................................. 4
Biological Nitrogen Fixation .................................................................................. 5
Lifespan of Soybean Nodules ................................................................................ 9
Environmental Factors ......................................................................................... I 1
Inoculant Rate Effect ........................................................................................... 12
Results in Past Trials ............................................................................................ l3
Advances in Soybean lnoculants .......................................................................... 15
Insecticidal Seed Treatments for Soybeans .......................................................... 17
Results in Past Trials ............................................................................................ 18
Insecticide-Rhizobium Interactions ...................................................................... 21
References ............................................................................................................ 23
CHAPTER I
SOYBEAN INOCU LANT AND FUNGICIDE EFFECTS
Abstract. ............................................................................................................... 32
Introduction .......................................................................................................... 33
Materials and Methods
Field Study ......................................................................................... 35
Greenhouse Study .............................................................................. 42
Results and Discussion
Climate ............................................................................................... 44
Inoculant Yield Effects ....................................................................... 47
Inoculant Brand Yield Effects ............................................................ 51
Fun gicide Yield Effects ...................................................................... 53
Grain Protein and Oil Effects ............................................................ 58
Greenhouse Results ............................................................................ 60
Conclusions ........................................................................................ 61
References .......................................................................................... 62
CHAPTER 2
NEONICOTINOID SEED TREATMENT EFFECTS
Abstract ................................................................................................................ 65
Introduction .......................................................................................................... 66
Materials and Methods
Field Study ......................................................................................... 68
Greenhouse Study .............................................................................. 74

iv

Results and Discussion

Climate ............................................................................................... 75
Insecticide Yield Effects .................................................................... 76
Insecticide-Inoculant Interaction ........................................................ 80
Soybean Aphid Population Effects ..................................................... 8]
Plant Stand Effects ............................................................................. 82
Plant Height Effects ............................................................ - ............... 84
Other Plant Growth Indicators ............................................................ 87
Greenhouse Trial ................................................................................ 89
Economic Return for Neonicotinoid Seed Treatment Use .................. 90
Conclusions ........................................................................................ 92
References .......................................................................................... 93

LIST OF TABLES

CHAPTER 1

Table 1.1:
Table 1.2:
Table 1.3:

Table 1.4:
Table 1.5:
Table 1.6:

Table 1.7:

Table 1.8:
Table 1.9:
Table 1.10:
Table 1.11:

Table 1.12:

Table 1.13:
Table 1.14:

Table 1.15:

Table 1.16:

Soybean inoculant ﬁeld trial site information ................................................ 38
Soil test results by site-year ............................................................................ 39
Inoculant and fungicide treatment rates .......................................................... 40

Precipitation by month(cm), ﬁeld Sites 2003-2005. Long term means
(1970-2000) included for comparsion. ........................................................... 45

Average temperature by month(°C), ﬁeld Sites 2003-2005. Long term means
(1970-2000) included for comparsion. ........................................................... 46

Summary of combined inoculants effects, ANOVA for grain yield by
site-year ......................................................................................................... 47

Inoculant grain yield effects, by site-year (kg ha- 1, treatments combined). 50

Inoculant treatment grain yield averages, by year (kg ha-l , sites combined,

excluding Montmorency ’04-‘05). ................................................................. 52

Inoculant treatment grain yield averages, Montmorency 2004-2005

(kg ha-l) ......................................................................................................... 52
Contrast of grain yield means for liquid and peat based lnoculants,
Montmorency 2004-2005 (kg ha-l, no fungicide plots only). ...................... 52
Summary of ANOVA for fungicide main eiTects on grain
yield by site-year. ......................................................................................... 54
Fungicide yield effects, by site-year (kg ha-I) .............................................. 54

Inoculant-fungicide interaction slicing effects at Montmorency,

2004-2005 ..................................................................................................... 56
Inoculant-fungicide interaction slicing effects at Montmorency, 2004-2005

(grain yield, kg ha—I). ................................................................................... 57
Fungicide plant stand effects by site (plants ha-l). ....................................... 58
Inoculant effects on soybean grain protein and oil content. .......................... 59

vi

Table 1.17: Inoculant-fungicide interaction effects on soybean grain protein and oil
content, Montmorency 2004. ........................................................................ 59

Table 1.18: Average nodule counts by inoculants treatment (50 DAP), greenhouse

study. ........................................................................................................... 60
CHAPTER 2
Table 2.1: Insecticide seed treatment trial site information. ............................................ 70
Table 2.2: Soil test results by site-year. ........................................................................... 71
Table 2.3: Pesticide and inoculant seed treatment rates. ................................................. 72

Table 2.4: Precipitation by month(cm), ﬁeld Sites 2003-2005. Long term means
(1970-2000) included for comparsion. ........................................................... 77

Table 2.5: Table 2.5: Average temperature by month(oC), ﬁeld Sites 2003-2005. Long
term means (1970-2000) included for comparsion. ........................................ 78

Table 2.6: Summary of combined insecticide effects, ANOVA for grain yield by

site-year. ......................................................................................................... 79

Table 2.7: Insecticide grain yield effects, 2004 by site (kg ha"). .................................... 79
Table 2.8: Insecticide grain yield effects, 2005 by site (kg ha!) .................................... 79
Table 2.9: Table 2.9: Insecticide grain yield effects, 2006 by site (kg ha'l). ................... 80
Table 2.10: Grain yield treatment means for soybean inoculated with Cell-Tech

inoculant, Montmorency 2004 and 2005 (kg ha"). ....................................... 81
Table 2.11: Effects of neonicotinoid seed treatment on soybean aphid pressure, Hillsdale

2005 (average adult aphids/plant) ................................................................. 82
Table 2.12: Effects of neonicotinoid seed treatment on soybean aphid pressure,

lngham 2005 (average aphids/plant) ............................................................. 82
Table 2.13: Summary of insecticide main effects on plant stand, by site-year. ............... 83
Table 2.14: Split plot contrasts of insecticide plant stand effects, sites with signiﬁcant

main effects (plants ha", insecticide treatments combined) .......................... 83
Table 2.15: Plant stand means of insecticide treated plots, sites combined for each year

(plants ha", insecticide treatments combined). ............................................. 84

vii

Table 2.16:

Table 2.17:

Table 2.18:

Table 2.19:

Table 2.20:

Table 2.21:

Table 2.22:

Summary of insecticide main effects on early plant height, by site (P-values

from ANOVA) .............................................................................................. 85
Contrasts of insecticide effects on early plant height (cm), site-years with

signiﬁcant positive insecticide effects. ......................................................... 85
Treatment means, plant height (cm), by year, sites combined. . .................... 86

Signiﬁcant contrasts of insecticide effects on harvest plant height (cm). ..... 87
Treatment means, pods per plant by year, sites combined. ........................... 88

Insecticide treatment contrasts, mean SPAD chlorophyll index,
Hillsdale 2006 ............................................................................................... 88

Economic return on use of neonicotinoid seed treatments ............................ 91

viii

LIST OF FIGURES
Figure 2.1: Plant height by insecticide treatment and week (cm, runs combined). . ........ 89

Figure 2.2: Figure 2.4: Greenhouse SPAD chlorOphyll indices by treatment and week
(runs combined). ........................................................................................... 90

ix

LITERATURE REVIEW

Introduction

Soybean (Glycine max (L.) Mcrr.) is the most economically important oilseed
crop grown in the United States, constituting 90% of US. annual oilseed production (Ash
et. al. 2006). Major expansion of soybean production in the United States has occurred
since World War II, when soybean was promoted as an alternative source of edible oils
for the war effort. Since 1923, US annual soybean production has increased from 5
million bushels to 2.97 billion bushels in 2005 (Hymowitz 1990, USDA). Soybean was
grown on 28.9 million ha and valued at $17.4 billion (Ash et. a1. 2006).

Soybean has become a pOpuIar crop in the United States for several reasons.
Export markets for US soybeans have been very strong, and soybean production is
economically competitive to other cash crops, as a result of relatively lower input costs.
Average variable cost per acre of soybean production was estimated to be half that of an
acre of corn in 2006 (Dobbins 2006). Differences in variable costs can be attributed to
ﬁJCI use in tillage, grain drying, and hauling, as well as pesticide costs. However, the
most signiﬁcant difference between corn and soybean cropping budgets is fertilizer
usage. For 2006, the fertilizer cost for corn on high productivity land was estimated at
$271 ha", compared to $94 ha“l for soybean (Dobbins and Miller 2006). This difference
is increasing due to the high cost of natural gas that is used to synthesize ammonia for
nitrogen fertilizer required for high yield corn production. Soybeans do not have a
fertilizer N requirement; therefore fertilizer costs in soybean are greatly reduced.

Soybean is a legume, and is therefore able to ﬁx its own nitrogen through an

association with symbiotic bacteria. This biological process is known as symbiotic

nitrogen ﬁxation. Symbiotic nitrogen ﬁxation is one of the most important plant
physiological functions in cropping systems worldwide, accounting for 60% of Earth’s
ﬁxed nitrogen (Zahran 1999). The ability for leguminous crops to ﬁx atmospheric N3
through symbioses with rhizobial bacteria allows crops such as soybean, cowpea (V.
unguiculata), and alfalfa (M. sativa) to be grown without added nitrogen fertilizer inputs
and on soils with marginal nitrogen reserves. Up to 280 kg ha'1 of N can be ﬁxed
through symbiotic nitrogen ﬁxation in soybean, accounting for about 70% of total plant
nitrogen requirements (Lindemann and Glover 2003, Tien et. a1. 2002). One of the
reasons soybean is capable of ﬁxing nitrogen is the efﬁciency of the symbiosis between
plant root and the symbiont, a rhizobial bacteria species speciﬁc to soybean. Other
legumes, such as common bean, do not have a high efﬁciency symbiont, and therefore
require supplemental nitrogen fertilization to obtain the highest yields (Berglund 1997).
The bacterial species that is the most effective symbiont to soybean cultivars in
the Americas is Bradyrhizobiumjaponicum. A second species, Rhizobiumﬁxedii will also
form symbioses with soybean (Dowdle and Bohlool 1985). However, this symbiont has
a wide range of host legumes, and will only nodulate speciﬁc, mostly Asian soybean
cultivars (Balatti and Pueppke, 1992; Heron and Pueppke, 1984; Keyser et al. 1982),
B. japoniwm is not ubiquitous; on lands where soybean was not produced in the past, it is
unlikely that a resident B. japonicum population is present. In cases where a new land
area is brought into soybean production, an inoculant is usually necessary to provide the
B. japonicum required for symbiotic nitrogen ﬁxation. Current inoculation techniques
usually involve a commercially produced, highly concentrated rhizobium product in a

packaged liquid suspension, a sterilized peat-based powder, or a granular concentrate.

These inoculants can be applied in-furrow near the seed, but are usually placed directly
on the seed as a seed treatment at or near planting time.

The yield beneﬁt from use of commercial inoculant on land where inoculation has
not occurred previously is substantial, and can reach 100% (Senviratne et. al. 2000,
Duong et. al. 1984). However, there are conﬂicting reports in the Midwest regarding the
effectiveness of successive inoculations of soybean plantings. Historically, successive
soybean inoculation was deemed unwarranted if healthy soybeans have been grown in a
ﬁeld in recent years (Vitosh 1997). Other recent studies provided evidence that
successive inoculation of soybean plantings is usually a proﬁtable practice and
encouraged farmers to inoculate their soybean plantings each year. Ohio State University
inoculant trials produced average yield increases of 175 kg ha'1 in 2004 and 121 kg ha’1
in 2005 on ground in soybean rotation (Beuerlein 2004, 2005). As a result,
recommendations regarding successive inoculation of soybean plantings differ between

states in the Midwest.

Taxonomy and Morphology of Soybean

Cultivated soybean (Glycine max (L.) Merr.) is in the soia subgenus of the
Glycine genus. The soia subgenus consists of the annual soybean species: the cultivated
form (Glycine max (L.) Men), and wild form (G. soia Sieb. & Zucc., Newell and
Hymowitz 1981). Cultivated soybean, an annual dicot with large cotyledons, produces
alternate trifoliate leaves after the V1 stage, where the ﬁrst pair of true leaves are
unifoliate. The ﬂower is a standard papilionoid with ﬁve united sepals forming the calyx,

ten diadelphous 9+1 stamens forming the androecium, and an apocarpous gynocium. The

 

ﬂowers are predominantly self pollinating, with less than 1% cross pollination, and pods
usually contain 2-4 seeds (Carlson and Lersten, 1987). The soybean plant grows
approximately 1 meter in height and can have a branched stem. Soybean varieties grown
in Northern latitudes of the United States such as Michigan are indeterminate. The root
system is characterized between taproot and diffuse types and is usually well nodulated

by rhizobacteria (Lersten and Carlson, 1987).

History of Soybean Production
Hymowitz (1990) suggests that soybean was ﬁrst domesticated in Northeastern

China about 3000 years ago, likely during the Shang dynasty. By the ﬁrst century AD,
soybean production spread throughout mainland China and parts of Korea. Soybean
cultivation spread toward other portions of Asia as new trade routes were developed and
populations emigrated from China to other lands such as Thailand. By the 1600’s,
soybean cultivation spread to most of Southeast Asia and India. Soy foods are important
sources of protein in this region, and were quickly adopted as a staple in Southeast Asia.
References to soy sauce and mm were mentioned in the diaries of European visitors since
the late 16th century. Soybean plantings probably did not occur in Europe until the early
1700’s, and North America until the late 1700’s. The use of soybean for forage was
studied throughout the 19th century in North America. USDA and agricultural
experiment stations continued to evaluate soybean cultivars and promoted new uses for
soybean grain in the United States. By the 1920’s soybean grain production had truly

become an industry in the United States (Hymowitz 1990).

Soybean grown commercially in the United States are generally planted in row
crop monocultures. Soybean in Michigan is usually planted in May, with row spacings
between 7.5 and 30 inches. Soybean seed can be saved from a previous crop and cleaned
by the farmer for use as seed the following year, as long as the variety is not proprietary.
Currently, most farmers in the US use some type of commercial proprietary seed source,
particularly glyphosate-resistant seed, which is illegal to save for use as seed the
following year. In 2005, glyphosate-resistant varieties comprised over 80% of all
soybean planted in the United States (Dill 2005). Soybean in Michigan is typically
harvested in late September or October using a mechanical combine and grain header. In
Michigan during 2004, 2 million acres of soybean was planted, producing 75.24 million
bushels, valued at $380 million. Soybean ranks second to corn among all Michigan crop
commodities in planted acres and production value (Kleweno and Matthews 2005).
Biological Nitrogen Fixation

Biological nitrogen ﬁxation is a symbiotic relationship requiring a system of
molecular signaling between plant roots and a bacterial organism. These few speciﬁc
species of prokaryotic bacteria are able to reduce atmospheric N2 into ammonia (NH 3), a
form that plants can assimilate into useful biomolecules, such as nucleic acids, amino
acids, and coenzymes (Gallon and Chaplin 1987). These speciﬁc bacteria are known as
diazotrophs. However, not all diazotrophs are symbiotic; some are free living and some
are associative. True symbiotic diazotrophs such as B. japonicum evolved physiological
and morphological adaptations with their symbiont plants, and these systems encourage

the continuation of the symbiosis (Gallon and Chaplin 1987).

A population of rhizobial bacteria live and reproduce as free-living organisms in
the soil, or on a seed in contact with soil in the case of inoculated soybean. Rhizobia
preferentially inhabit the rhizosphere, an environment surrounding the soybean root that
is rich in sugars and amino acids produced as root exudates by the plant (Uren 2000).
This makes the soybean root area favorable for growth and reproduction of all types of
soil bacteria. Soybean roots, like those of other legumes, have the ability to produce
chemotactant materials, which attract speciﬁc bacteria. In soybean root exudates, organic
acids such as succinate, malate, and malonate, and the amino acids glutamate and
aspartate elicit chemotactic responses from B. japonicum (Barbour et. al. 1991).

Evidence suggests that symbiotic bacteria attach to the legume root hairs via a
two step process. Initial attachment can involve root-produced lectins binding to
carbohydrate structures present on rhizobial bacteria (Dazzo et. al. 1984). Initial
attachment can also result from an adhesive bacterial protein, known as rhicadhesin (Smit
et. al. 1992). The second step involves permanent anchoring of the bacteria to the root. In
soybean, anchoring is promoted by the pili, ﬁbrous protein structures found on the
surface of gram negative bacteria used for bacterial attachment to other organisms (Ottow
1975). Strains of B. japonicum lacking pili have reduced attachment to soybean roots,
reducing nodule induction (Vesper and Bhuvaneswari 1988). B. japonicum preferentially
infects emerging root hairs because as root hairs mature, physical defenses are developed
that prevent such infection (Siu-Cheong Ho 1994).

Nod factors are signal compunds produced by rhizobia that induce a number of
physical changes that condition the root hair to accept the onset of infection and eventual

symbiosis. Their production depends on the expression of a set of nod genes by the

bacteria, which are induced by ﬂavenoids or isoﬂavenoids produced by the plant (Spaink
1995). These signal molecules are lipochitooligosaccharides, also known as the nod
factors. These nod factors elicit responses such as initiation of the nodule primordia in
the plant cortex, induction of plant nodulin genes expressed early in the nodulation
process, and curling of root hairs (Broughton et. al. 2000). These root responses occur in
the presence of nod factors at concentrations as low as picomolars (D’Haeze and Holsters
2002)

After the bacterium attaches itself to the root hair, nod factors elicit a response,
causing the hair to curl around the bacteria. This takes place as soon as within 12 hours
of bacterial attachment (Bauer and Turgeon 1982). This curling of the root hair traps the
bacteria within the curled root. Once the bacterium is encircled by the root hair, an area
of swelling develops near the site of infection. Then an area of existing cell wall is
hydrolyzed and a new tubular structure is synthesized, called an infection thread. This
infection thread allows the bacterium to penetrate the plant cell wall (Callaham and
Torrey, 1981). While the infection thread penetrates the root hair, a cell wall is formed
around the outside of the thread to contain the bacteria and prevent complete invasion and
death of the root hair (Gallon and Chaplin 1987). The infection thread then grows toward
the nodule primordium, an area of cells within the root cortex induced to resume active
division by nod factors. The nod factors also induce progressive differentiation of cells
within the nodule. As the infection thread reaches the nodule primordium, the bacteria
are released into the cytoplasm of the plant cell though structures called infection
droplets; this process is known as endocytosis (Brewin 1991). As the primordium

develops into a nodule, several plant nodulin genes are activated within all the cells

within the primordium, which differentiate their functions from typical root and shoot
meristems (Mylona et. al. 1995).

Nodule primordia become functional nodules after rhizobial bacteria move from
the infection thread into nodule primordial cells. As bacteria enter the cytoplasm of the
nodule primordial cells these cell are converted for use in nitrogen ﬁxation. These
converted cells are known as bacteroids. Bacterioids are surrounded by a plant derived
membrane, the peribacteroid membrane. This membrane protects the bacteroid from
plant defensive responses and serves as the interface where exchange of metabolites
between plant and bacterial cells occur (Mylona et. al. 1995). The ureides allantoin and
allantoic acid are the ﬁnal N metabolites; these are produced at the perioxisomes of
uninfected soybeans nodule cells and then exported through the peribacteroid membrane
and transported to the plant (Streeter 1972, McClure and Israel 1979, Hanks et al 1983).
Sucrose is the carbon metabolite transported from leaves through the vascular tissue to
the nodule. Sucrose is then hydrolyzed by sucrose synthase into the products fructose
and UDP—glucose. These products are used for starch and cellulose synthesis within the
nodule, or are further metabolized (Kavroulakis et. al. 2000, Day and Copeland 1991).

The conversion of N2 gas into NH 3 ammonium ions is catalyzed by the enzyme
nitrogenase. For this reaction to take place, a low oxygen content must be present within
the nodule, because a vital cofactor of the nitrogenase enzyme is deactivated in the
presence of oxygen (Shah and Brill 1977). To accomplish this, a nodule parenchyma
layer is formed around the nodule, eliminating oxygen difﬁrsion. Oxygen is then
delivered to bacteria by leghemoglobin canier proteins; this allows the bacteria to

continue aerobic respiration (Mylona et. al. 1995).

Life Span of Soybean Nodules

The time between ﬁrst soybean root exposure to B. japonicum and the onset of
symbiotic nitrogen ﬁxation is very dependent of the root zone temperature. Soybean
nodulation and nitrogen ﬁxation is optimal at 25 degrees centigrade, and is signiﬁcantly
reduced below 17°C, (Lynch and Smith 1993, Dart and Day 1971, Jones and Tisdale
1921). A laboratory study determined that at optimal temperature, soybeans can ﬁx
nitrogen approximately 20 days after initial exposure to B. japonicum. At 15°C, this
process takes between 39 and 45 days (Zhang and Smith, 1994).

Soybean nodules have a limited life span. Nodules ﬁrst form on soybean roots
around the V2 stage of development, and will continue to form until approximately seven
weeks after planting (Klukas 1974). Nodules of annual plants only ﬁx nitrogen at highest
capacity for about three to ﬁve weeks (Puppo et. al. 2005). An experiment observing
timing of nodule senescence of a group II variety soybean found that nitrogen ﬁxation
declined sharply between 58 and 65 days after planting, which was between two and
three weeks after ﬂowering in this study (Klucas 1974). As nitrogen ﬁxation slows, the
nodule will begin to senesce starting at the central tissue and working toward the outside
of the nodule over a few weeks. This makes the total life span of a nodule about 10-12
weeks in length (Puppo et. a1. 2005).

About half of the N used for seed ﬁll is remobilized from plant leaves, rather than
being ﬁxed and directly supplied by nodules or taken up as mineral N by roots (Imsande
and Touraine 1994). Over half of soybean leaf N content is moved to the developing
seed during podﬁll (Hanaway and Weber 1971). Lawn and Brun observed that as

soybean pod ﬁll begins, N ﬁxation by nodules peaks and then rapidly decreases. Plant

depodding allowed nodule activity to be maintained longer, while plant defoliation
caused N ﬁxation in nodules to decline earlier (Lawn and Brun 1974). Others have found
N ﬁxation to peak later than early pod ﬁll. (Thibodeau and Jaworski 1975, Abendroth and
Elmore 2006). One hypothesis to explain nodule death is that the plant-nodule
relationship may be a source-sink relationship, and that as photosynthate is supplied to
new seeds, it is diverted from nodules, particularly if resources are limited under stressﬁrl
conditions. While indirect evidence supporting this hypothesis has been observed (Lawn
and Brun 1974, Schulze 2003), this hypothesis has not been supported with direct
evidence relating carbon assimilate competition by nodules and nitrogen ﬁxation
(Schulze et. al. 2000, Puppo et. a1. 2005).

The signal to initiate nodule senescence can be triggered by shoot stresses.
However the exact signal that triggers the shutdown of symbiosis and senescence of
nodules has not been characterized. A possible trigger for nodule senescence may
involve the reduced levels of antioxidants within nodules as they age. Antioxidant
compounds glutathione and ascorbate have been shown to affect the nitrogenase activity
in nodules (Bashor and Dalton 1999, Ross et. al. 1999). These antioxidants may be
involved in signal transduction in root nodules as they are in plant leaves (Kiddle et. al.
2003, Arora et. al. 2002). Oxidative stress can occur in nodules in the absence of these
and other metabolites, which could damage the both the parenchyma layer that cordons
oxygen out of the nitrogen ﬁxing region of the nodule and infected nodule cells, which

could also adversely affect nodule ﬁJnction (Becana et. al 2000).

10

Environmental Factors

Many environmental factors are known to affect rhizobial populations and
biological nitrogen ﬁxation. Several studies concluded that acidic soils can reduce the
p0pulation of rhizobia and interfere with the symbiotic process even when a viable
population is present (Taylor et al. 1991, Graham 1992). A soil pH of 5.5 or less
decreases free living rhizobial populations (Daniels 1999). Initial contact and attachment
of rhizobium to root hairs can be affected by soil acidity, as low pH appears to destabilize
rhizobial binding to root hairs in alfalfa (Cactano-Anolles et. al. 1989).

Soil moisture is another environmental factor that affects biological nitrogen
ﬁxation. Soybean nodule function is much more susceptible to low water stress than
plant roots and shoots (Albrecht et. al. 1994). Deﬁcit water stress can lower rhizobial
population densities, reduce the formation of infection threads, and reduce the number
and size of nodules on soybean roots (Tate 1995, Williams and DeMallorca 1984,
Worrall and Roughly 1976). Soybean nodule functions are also more susceptible to
ﬂooding stress than root and shoot tissue, as inhibition of N ﬁxation and N accumulation
in plant tissue occurs earlier and more drastically than plant tissue biomass accumulation.
Supplementation of N fertilizer to ﬂood stressed plants reduces plant tissue biomass
responses, so soybean nodule functions appear to be more sensitive to ﬂooding than
soybean itself (Bacanamwo and Purcell 1999).

Agronomists consider conditions such as low soil pH, high sand content, and soil
ﬂooding in recommending where soybean seed should be inoculated (Pederson 2003,
Abendroth 2006). Rhizobia are heterotrophic, so they depend on soil organic matter for

nutrition when they are free living. Therefore, soil organic matter levels play a role in

II

maintaining B. japonieum in the soil. Other soil conditions that inﬂuence the
effectiveness of inoculation include use of no-till practices, use of seed applied fungicides
in previous soybean crops, planting date, and the frequency of water stresses on past

soybean crops (Abendroth and Elmore 2006).

Inoculant Rate Effects

Soybean grain yield generally responds positively to increased inoculant rates.
Depending on soybean size and the bacterial concentration of the inoculant, a typical
commercial inoculant rate provides between 8.5* 105 and 1.4*106 bacteria per seed.
Hume and Blair performed a study of inoculant rate effects and soybean grain yield on
virgin soybean ﬁelds by performing a set of logarithmic dilution applications of
commercial inoculant to soybean seed. Rates of 10", 102, 103, and 10‘4 of the
recommended rate showed signiﬁcant and regressing yield effects compared to the
standard inoculant rate, which was estimated to be approximately 106 bacteria per seed
(Hume and Blair 1992). Based on the regression curves relating inoculant rate and grain
yield produced in this study, use of a 10% standard inoculant rate would reduce grain
yield between 4 and 11%. Use of a 2X standard inoculant rate would increase grain yield
by 1.6 to 4.2% (Hume and Blair 1992).

Researchers have had difﬁculty determining the amount of inoculant required to
provide maximum yield on new soybean ground. More inoculant has typically provided
increased yield, yet in some studies, yield plateaus have been reached at varying
inoculant levels (Hume and Blair 1992, C atroux et. al. 2001). A greenhouse study

determined that while 105 rhizobium per seed did not provide maximum nodulation of

12

roots, it did provide maximum total N content in soybean plants (Weaver and Fredrick
1972). These studies have all been performed on soils that had no resident B. japonieum
population, and it is difﬁcult to extrapolate effects seen in these trials to expected effects
in soils that have been in soybean rotation. Also, very high inoculation rates are
constrained by economic cost to growers, seed adhesion difﬁculty, and issues with seed
metering and monitoring equipment.
Results in past trials

As nitrogen costs have risen dramatically in recent years, soybean seed inoculant
treatments have received increased attention in commercial production and in agronomic
research trials. Several ﬁeld trials investigating the efﬁcacy of soybean seed inoculant
treatments were conducted throughout the Midwest in the past decade. The common
recommendation of inoculating soybean seed only if a site has been out of soybean
production for three years or more has been challenged by some researchers in recent
years. Six ﬁeld trials performed in Nebraska between 2001 and 2004 on rotational
soybean ground show no yield differences between inoculated and non-inoculated plots
(Abendroth and Elmore 2006). Therefore, the University of Nebraska recommends
inoculation only when soybeans have not been grown in a ﬁeld for four years unless
environmental factors such as low soil pH, ﬂooding, drought, high soil sand content, etc.
are a factor. Soybean inoculant trials yielded similar results at Iowa State University in
2004 and 2005, and their recommendations are similar to the University of Nebraska
regarding consecutive years without soybean production in a ﬁeld (Pederson 2006).

Conley and Christmas compiled ten years of soybean inoculant trial data in

Indiana on soils in a com/soybean rotation (Conley and Christmas 2006). These trials

13

tested several inoculant products throughout the state over this period. Inoculated
soybeans yielded an average of 67 kg ha~l per acre higher than those that were not
treated. Some sites showed signiﬁcant yield increases from inoculation, while others did
not. They suggested that soybean seed inoculation may be an effective method of
increasing yields, and that farmers ought to create replicated strip trials on their own farm
to determine if inoculation enhances yield (Conley and Christmas 2006).

Beuerlein (2005) recommends inoculating soybean seed for every planting based
on trials conducted in Ohio. More consistent yield increases have been observed in these
trials than in other states. Results from the 2005 inoculant trial resulted in statistically
signiﬁcant yield increases for 17 out of 19 inoculant products and an average yield
increase of 121 kg ha’l above the untreated control over six sites (Beuerlein 2005). In
2004, a similar study resulted in a 175 kg ha'l yield increase with 18 products tested at six
sites. The average yield increase of the Ohio inoculation studies between 1995 and 2004
was 135 kg ha" (Beuerlein 2004).

Vitosh (1997) conducted several on-farm soybean inoculant trials around
Michigan between 1990 and 1995, as well as a set of six trials conducted at the Michigan
State University research farm between 1993 and 1996. No yield differences were
observed for any of these trials, individually or collectively. Another set of eight on-farm
replicated strip trials performed between 1990 and 1995 tested the yield effect of soybean
seed inoculant in ﬁelds that had a history of soybean production. Only one of these eight
trials showed a signiﬁcant yield increase for seed inoculant treated strips versus the
untreated checks. The average yield difference for these eight trials was only 6.7 kg

ha"(Vitosh 1997).

Clearly, conﬂicting results have been observed among university soybean
inoculant trials in recent years. It appears that some of these results may be associated
with geography, as inoculants have increased yields in eastern areas of the soybean belt,
including Ohio and Indiana. Studies in western states, including the Dakotas, Minnesota,
Iowa, and Nebraska have shown no effect from soybean seed inoculation (Abendroth and
Elmore 2006).

Advances in Soybean Inoculants

Soybean seed inoculant is available in three forms: peat-based, liquid, and
granular. Historically, peat based powdered inoculants have been the most popular
commercial form. Finely powdered peat provides adequate adhesion to soybean seed if a
sticking agent is included in the carrier. Most peat carriers in commercial products are
sterilized because sterilized peat provides advantages for sustaining rhizobial levels over
a longer period of time, thus increasing the product’s shelf life. However, peat sterilizing
treatments such as heat and radiation can create byproducts which can interfere with
rhizobial growth within the media. Liquid inoculants are either aqueous, oil, or polymer
based, and have become more popular in recent years because of their case of use (Xavier
et. a1. 2004).

In recent years, several new commercial soybean inoculant products of all three
forms have been introduced to the market. Newer products claim higher efﬁcacies than
past products for various reasons, such as the use of multiple strains of B. japonicum,
which are intended to be more competitive nodulators in different environments. Not all
strains of B. japonicum are equally effective in nodulating soybean roots. Indigenous B.

japonicum strains belonging to the serogroup USDA 123 are very competitive nodulators,

1.5

but much less effective at nitrogen ﬁxation than those used in soybean inoculants (Cregan
et. al. 1989). Soybeans grown where B. japonicum serogroup 123 is the dominant
symbiont yield signiﬁcantly less than soybeans grown where higher efﬁciency strains are
predominant (Ham 1980). Introduction of more efﬁcient strains of B. japonicum could
increase soybean yields if these introduced strains can effectively compete with
serogroup 123 strains for nodulation sites.

Other advancements in soybean inoculation include the increase in rhizobial
concentrations of commercial inoculant products, and the use of bacterial life extenders.
Concentrations of 2 billion or more bacteria per gram are now common in many
inoculant products, allowing for decreased application rates and increased efﬁciency of
use. The use of bacterial life extenders in liquid and granular inoculants has also been
recently introduced to the marketplace. These “extenders” provide a nutrient source for
the bacteria to allow them to live on the seed for a longer period of time. These nutrient
sources are a proprietary product; it is difﬁcult to know what nutrient concentrations are
in each of these products. Some of the newest encapsulated granular (ex. Excalibre,
Advanced Biological Marketing, VanWert, OH) and liquid products (ex. Optimize, EMD
Crop Bioscience, Brookﬁeld, WI) claim a 120-day application to planting window. This
time period allows for inoculant application by the seed dealer rather than the farmer in
some cases, which is of signiﬁcant convenience.

Genetic advances have also led to improvements in soybean inoculant technology.

One example is the development of B. japonicum strain Tal 1Nod+ as a result of
mutagenesis of the common H 10 strain (Kuykendall and Hunter 1991). Compared to

the [-1 10 strain, this mutated strain increased soybean root nodulation by 44%, and

16

increased yields by 135 to 202 kg ha". This mutant strain was patented in 1991, and is
still being produced commercially, more commonly known as "Nod+" or the USDA
strain (Suszkiw 1992).

Inoculant manufacturers have applied knowledge of the biochemistry involved in
symbiotic nitrogen ﬁxation by incorporating lipochitooligosaccarides produced by B.
japonicum into their product. The lipochitooligosaccharide class of molecules comprise
the nod factor signal molecules produced by B. japonicum that elicit responses such as
root hair curling, infection thread formation and nodule formation. Optimize (EMD Crop
BioScience lnc., Brookﬁeld, WI) inoculant is formulated with this molecular additive.
Nitragin Inc. (now EMD Crop BioScience Inc.) has also applied for a patent to use the
manufactured nod factor molecules in combination with insecticidal seed treatments such

as thiamethoxam and imidacloprid (Smith et. al. 2004).

Insecticidal Seed Treatments for Soybean

Neonicotinoids are the most recent class of insecticides introduced for crop
protection. Neonicotinoids are systemic insecticides, absorbed by plants from foliar or
soil application, or seed treatment. Generally, the neonicotinoids have lower logP
solubility values than organophosphates and other insecticides, meaning they exhibit
higher water solubility, an important pr0perty of systemic insecticides (Tomizawa and
Casida 2005, Elliot 1977). Neonicotinoids move to actively growing tissue, where they
are ingested by insect pests. The site of activity for neonicotinoids is the nervous system
of insects, where they act upon the acetylcholine receptor. As neonicotinoids bind to the
acetylcholine receptor, giant intemeurones are depolarized, leading to paralysis and death

of the insect (Matsuda et. al. 2001, Rice 2004).

I7

The neonicotinoids preferentially bind to insect acetylcholine receptors rather
than vertebrate acetylcholine receptors (Yamamoto and Casida 1999). This property
makes neonicotinoids less toxic to mammals than the organophosphates and
methylcarbamates, the two other common classes of soil-applied insecticides.
Neonicotinoids have the highest insect selectivity of any major class of systemic
insecticide, with an insect to mammal selectivity factor 14 and 28 times greater than the
organOphosphates and carbamates (Tomizawa and Casida 2005, Elliot 1977). This
favorable property has made the neonicotinoids popular options for insect control in
many crops.

Since 2004, two new neonicotinoid seed treatments for soybean were approved by
the EPA; Cruiser (thiamethoxam, Syngenta, Greensboro, NC), and Gaucho (imidacloprid,
Bayer Crop Science, Research Triangle Park, NC). These chemicals provide early season
control or suppression of insects such as soybean aphid, bean leaf beetle, leaﬂtoppers,
thrips, Mexican bean beetle, and wireworms. The products advertise crop beneﬁts such
as increased plant density, plant height, root, stem and leaf development, as well as

increased grain yields.

Results in past trials

Bean leaf beetle feeding in soybean causes signiﬁcant leaf defoliation and
transmits bean pod mottle virus, which can signiﬁcantly reduce soybean grain yield and
quality. Overwinterin g and ﬁrst generation bean leaf beetles feed on soybean only
through early June, and early feeding causes the highest risk for bean pod mottle virus

transmission (Ross 1969, Hopkins and Muller 1984, Krell et. al. 2004). Thiamethoxam or

18

imidacloprid seed treatment may be ideal to control early season bean leaf beetle
infestation and bean pod mottle virus transmission.

A pair of trials conducted at Janesville, WI in 2003 tested neonicotinoid control of
bean leaf beetle feeding and effects on soybean yield (Cullen et. al. 2004a). Bean leaf
beetle feeding was virtually absent in both thiamethoxam and imidacloprid treated plots
19 days after planting compared to over 60% of plants with feeding damage in untreated
plots. Although increases of 492 and 485 kg ha”1 were observed in one of two trials
where high rates of thiamethoxam (50 g a.i./ 100 kg) and imidacloprid (62.5 g a.i./ 100 kg)
were used, these yield increases were likely a result of soybean aphid suppression later in
the growing season, rather than the below threshold bean leaf beetle pressure observed
(Cullen et. al. 2004a). An Iowa trial tested the effects of thiamethoxam seed treatment on
soybean yield and bean leaf beetle control (Schmitt and VanDee 2005). Signiﬁcant
decreases in late August bean leaf beetle populations were observed, but this did not lead
to signiﬁcant yield differences.

Another trial conducted in Southern Minnesota tested effects of thiamethoxam
and imidacloprid on soybean stand, plant height, soybean aphid population, and bean leaf
beetle populations on two planting dates, May 2 and May 23 (Potter 2002). Soybean
stand was lower in untreated early-planted soybean, but plant populations later in the
growing season were not signiﬁcantly different. Early-planted soybean treated with high
rates of thiamethoxam and imidacloprid (50 g and 124 g/lOO kg seed) showed respective
plant height increases of 7.7 and 12.0 cm at R3. Bean leaf beetle defoliation was

signiﬁcantly reduced at V2 where soybean seed was treated with thiamethoxam or

19

imidacloprid. Soybean aphid population reduction was observed in early planted R3
soybean but not at R4, or in late planted soybean (Potter 2002).

Soybean aphid infestations occur later into the growing season relative to bean
leaf beetle. Soybean aphid pressure usually increases until late July or early August in
Michigan (DiFonzo 2003, 2005). This poses a challenge for the neonicotinoid seed
treatments, as the insecticide must persist longer and maintain potency in a much larger
soybean plant to provide any level of aphid control. Planting date may play an important
role as to whether seed treatment can control soybean aphid. Thiamethoxam has been
shown to provide systemic activity and signiﬁcant SBA mortality for 35 days after
planting and a reduction in nymph production up to 49 days after planting (McComack
and Ragsdale 2006). Therefore, insecticide treated soybeans planted in late May or early
June may maintain some systemic activity through July, while early planted soybeans
will likely lose systemic activity before the highest aphid pressure occurs.

Two Wisconsin trials in 2003 determined the effectiveness of neonicotinoid seed
treatments in controlling soybean aphid (Cullen et. al. 2004b). Soybean was planted in
these trials on June 9, and throughout the six aphid sample dates in both studies,
thiamethoxam and imidacloprid treated soybean had signiﬁcantly reduced aphid
populations. Untreated soybean averaged 1700 and 2600 aphids per plant on August 4,
while treated soybean averaged less than 700 aphids per plant, and less than 250 aphids
per plant for imidacloprid in one trial. Aphid pressure began to fall aﬁer August 4, while
aphid p0pulations in treated soybean continued to rise through August 13, indicating that

aphid control was weakening (Cullen et. al. 2004b).

20

A study conducted at Beresford, SD in 2003 determined the effectiveness of
neonicotinoid seed treatments in controlling soybean aphid (Catangui et. al. 2003). These
trials were planted on June 12, and aphids were counted on August 18, at the R5 stage.
High aphid pressure was observed at this site, with an average of 1,051 aphids per
untreated plant. No signiﬁcant difference between untreated and insecticide treated plots
in mean number of soybean aphids per plant was observed, nor was any resulting yield
advantage observed.

Trials in Iowa from 2002—2005 determined the effectiveness and duration of
soybean aphid control from neonicotinoid seed treatments (Pederson and Iang 2006). In
2003, soybean aphid pressure was high, with over 3,000 aphids per untreated plant by
July 27. The economic threshold was not reached in treated plots until July 20, compared
to June 30 for untreated soybean. In 2005, thiamethoxam and imidaCIOprid held soybean
aphid pressure under threshold through July 18, about one week longer than untreated
soybean. Pederson and Ian g concluded that seed treatments can control soybean aphid
approximately 60 days after planting, which may be useful in some late planting, replant,

or double-crop situations (Pederson and Iang 2006).

Insecticide-Rhizobium Interactions

Research is limited regarding potential interactions between neonicotinoids and
rhizobial bacteria. Thiamethoxam had little toxicity invitro to entomopathogenic bacteria
(Filho et. al. 2001). Irnidacloprid seed treatments had no antagonistic effect on soil

bacteria populations (Singh and Singh 2005).

21

Research has been conducted regarding rhizobial interactions with insecticides
from the carbamate and organophosphate groups. The soil applied insecticides
malathion, diazinon, acephate, carbaryl, and toxaphene at 5 and 10 times the
recommended rates had no effect on plant growth and total nodulation, although nitrogen
ﬁxation and nodulation sites were affected (Mallik and Tesfai 1985). Carbosulfan had
little effect on B. japonicum survival on soybean seed (Martyniuk et. al. 2002). Tandem
seed treatment of omethoate and inoculant reduced survival of rhizobium on subterranean
clover and alfalfa seeds, although little to no effect was seen when insecticide was
applied 16 h prior to inoculation (Evans et. al. 1991). Aldicarb inhibited in-vitro growth
of rhizobium bacteria at 5 and 10 ppm levels, and reduced nodulation and nitrogen
content of cowpea, although plant growth was enhanced (Sekar and Balasubramanian
1979). Lin et. al. conducted a study of nine different carbamate and organophosphate
insecticides on in-vitro rhizobium growth of four different species, including B.
japonicum (Lin et. al. 1972). B. japonicum was the least inhibited by the insecticide; only
2 of 9 chemicals tested at 2u concentration inhibited colony growth. Both of these

chemicals were organophosphates.

22

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31

CHAPTER 1
EFFECT OF SOYBEAN SEED INOCULANTS AND FUNGICIDAL SEED
TREATMENTS ON SOYBEAN YIELD IN MICHIGAN

Abstract
Several new bacterial inoculant products have been introduced for use as a soybean
(Glycine max (L.) Merr.) seed treatment. The advantages of these commercial soybean
inoculants over those used in the past may include higher B. japonicum concentrations,
bulk seed applications, additives that extend the life of B. japonicum on the seed, and
inclusion of nod factor molecules within the inoculant carrier. A three year study over 16
site-years determined whether soybean seed inoculation was a worthwhile practice on
ﬁelds that were in a soybean rotation, if yield or growth differences occurred between
soybean treated with commercial inoculant products, and whether use of Apron Maxx
RTA fungicide resulted in yield beneﬁts or interacted with commercial inoculants. Eight
commercial inoculants were tested both with and without fungicide treatment in a split-
plot randomized complete block conﬁguration. Use of soybean inoculant raised soybean
yield in 6 of 14 site-years that had been in soybean rotation. Differences between
inoculant products were observed at only at ﬁrst-time soybean sites. Apron Maxx RTA
seed treatment improved yield on 3 of 16 site-years, and only interacted with inoculant on

the two ﬁrst-time soybean sites.

Introduction

Soybean (Glycine max (L.) Merr.) is the most economically important oilseed
crop grown in the United States, constituting 90% of US. annual oilseed production (Ash
. et. al. 2006). Soybean, unlike some legumes, has the ability to derive most of its nitrogen
requirements from symbiotic nitrogen ﬁxation, and high yielding soybean depends on
adequate biological nitrogen ﬁxation (Duong et. al. 1984). Up to 280 kg ha’1 of N can be
ﬁxed through symbiotic nitrogen ﬁxation in soybean accounting for about 70% of total
plant nitrogen requirement (Lindemann and Glover 2003, Tien et. al. 2002). The reason
soybean is capable of ﬁxing a large source of nitrogen is the efﬁciency of the symbiosis
between plant root and Bradyrhizobium japonicum, the bacteria species speciﬁc to
soybean nitrogen ﬁxation in American cultivars (Balatti and Pueppke, 1992).

Inoculation of soybean seed or soil around the seed with B. japonicum is
commonly practiced to ensure biological nitrogen ﬁxation will take place. In ﬁelds
where soybean is crOpped for the ﬁrst time, inoculation can increase yields dramatically,
by 50% or more (Senviratne et. al. 2000, Duong et. al. 1984). Once a ﬁeld has been
cropped by soybean and inoculated with B. japonicum, a soil borne population of B.
japonicum will reside in that ﬁeld in following years, and future inoculations will not
likely have as great an effect on soybean yield as the initial inoculation.

Some farmers and researchers ﬁnd inoculation of soybean seed by B. japonicum
for each planting (successive inoculation) to be a worthwhile practice, as there is
evidence suggesting that successive soybean inoculation can increase soybean yield
(Beuerlein 2005, Conley and Christmas 2006). However, a number of trials saw no yield

increase from successive soybean inoculation (Abendroth 2006, Vitosh 1997, Pedersen

33

2003). Soil factors such as pH, soil water content, organic matter, and soil texture impact
B. japonicum populations (Albrect et. al. 1999, Graham 1992, Bacanamwo and Purcell
1999, Abendroth 2006). Successive inoculation of soybean seed may overcome these
environmentally mediated reductions of soil borne B. japonicum populations by
improving soybean root colonization.

Soybean seed inoculant is available in three forms, peat based, liquid, and
granular. Historically, peat based powdered inoculants are the most popular commercial
form. Liquid inoculants are either aqueous, oil, or polymer based, and have become more
popular in recent years because of their case of use (Xavier et. al. 2004). Soybean
inoculants are inexpensive and becoming more convenient, with new bulk seed
applications and bacterial life extenders allowing inoculant to live on stored seed for
much longer periods while maintaining effectiveness.

Newer products claim higher efﬁcacies than past products for various reasons,
such as the use of multiple strains of B. japonicum, which are intended to be more
competitive nodulators in different environments. Not all strains of B. japonicum are
equally effective in nodulating soybean roots. Indigenous B. japonicum strains belonging
to the serogroup USDA 123 are very competitive nodulators, but much less effective at
nitrogen ﬁxation than those found used in soybean inoculants such as USDA 110 (Cregan
et. al. 1989). Concentrations of 2 billion or more bacteria per gram are now common in
many liquid products, allowing for lower application rates and increased efﬁciency of
inoculant use. Genetic advances have also led to improvements in soybean inoculant
technology, such as the development Tal lNod+ from mutagenesis of the common [-110

B. japonicum strain (Kuykendall and Hunter 1991). Inoculant manufacturers have

34

applied knowledge of the biochemistry involved in symbiotic nitrogen ﬁxation by
incorporating lipochitooligosaccharide nod factors produced by B. japonicum into their
product. These signal molecules elicit responses such as root hair curling, infection
thread formation and nodule formation (Smith et. al. 2004).

This study was performed from 2003 to 2005 over 16 site-years. The objectives
of this study was to: 1) determine whether soybean seed inoculation was a worthwhile
practice on ﬁelds that were in a soybean rotation, 2) if yield or growth differences
occurred between soybean treated with commercial inoculant products, and 3) whether
use of Apron Maxx RTA fungicide resulted in yield beneﬁts or interacted with

commercial inoculants.

Materials and Methods
Field Study

In 2003 and 2004, six ﬁeld sites were chosen for the soybean inoculant trials,
conducted at sites adjacent to the Michigan Soybean Performance Trials throughout the
lower Peninsula of Michigan. In 2003, trials were performed in Montmorency, Grand
Traverse, Saginaw, In gham, Hillsdale, and Lenawee Counties. In 2004, trials were
performed in Montmorency, Grand Traverse, Sanilac, lngham, Hillsdale, and Lenawee
Counties. In 2005, four ﬁeld sites were used, including Montmorency, Sanilac, lngham,
and Hillsdale Counties. Sites at Hillsdale and Saginaw in 2003 and Ienawee in 2004 had
a previous crop of wheat. All other sites had a previous crop of com. All sites had a
recent history of soybean production except Montmorency County sites in 2004 and

2005.

35

Commercial inoculants selected for use in this trial included: Cell-Tech (2003-
2005, EMD Crop BioScience lnc.), Nod+ (2003-2005, Becker Underwood Inc.), SowFast
(2003-2005, Loveland Products Inc.), NitroFix Liquid (2003-2005, Trace Chemicals
LLC), PulseR HP (2003-2005, Agribiotics Inc.), HiStick2 (2003-2004, Becker
Underwood), HiStick N/T (2005 Becker Underwood Inc.) , Apex Extra (2004-2005,
Agribiotics Inc.) and Optimize (2005, EMD Crop BioScience Inc.). Hi-Stick, SowFast,
and PulseR HP are peat powder based inoculants. Cell-Tech, Nod+, NitroFix Liquid,
Apex Extra and Optimize are liquid products. Inoculant treatments were tested alone and
in combination with Apron Maxx RTA (mefenoxam and ﬂudioxonil) fungicide seed
treatment, to determine whether the fungicide treatment changed the efﬁcacy of the
inoculant. Soybean seed was treated with Apron Maxx RTA fungicide at the labeled rate
of 162 ml /50 kg seed in the week prior to soybean planting.

Each site was established as a randomized complete block design with split plots
acting as treatment and control. Each site had 6 complete blocks consisting of all
inoculant treatments used twice per block, once in a whole plot without fungicide and
once with Apron Maxx RTA. Each 12-row whole plot was divided into 2 subplots, 6
rows treated with one of the commercial inoculants, and six rows not treated. Rows were
38 cm in width. Using this design, split plots could be compared throughout the ﬁeld to
reduce the variance between treatments and controls and increase the reliability of
smaller observed yield differences. Each plot was 6.07 m length as planted. Alleys
measuring 1.82 m were cut in between plots during the growing season, making the plots

4.24 m in length for harvest.

36

Spring nitrogen applications were not made to sites except for at Grand Traverse
County in 2003 and 2004. A 190 kg ha'l application of 22-1 1-22 was broadcast on these
sites in the spring prior to planting (Table 1.1). This application contained 39 kg ha’1
total N. It is not uncommon for soybean farmers to use some type of low rate of early
season nitrogen fertilizer on sandy soils where low organic matter is present to provide
soybean plants an early nitrogen source until nodules are established (Lindemann and
Glover 2003). The Grand Traverse sites had both a high sand content and low organic
matter content (Table 1.2), which is common for farmers in northwest lower Michigan.

Many studies have shown that high levels of N fertilizer inputs inhibit soybean
nodulation and nitrogen ﬁxation. However, it is unlikely that the low pre-plant nitrogen
application made at the Grand Traverse sites signiﬁcantly impaired nodulation. A series
of experiments performed in Australia tested the effects of different fertilizer rates and
timings on soybean nodulation. An application of 34 kg ha’1 of N at planting had no
effect on the number of nodules per plant. Early nodule diameters were reduced where
fertilizer was applied, but by four weeks after ﬂowering, no difference in nodule size was
observed (Williamson and Diatloff 1975).

Inoculants were applied to seed using one gallon resealable plastic canisters, to

which 0.91 kg. of soybean seed and the labeled rate of inoculant was added. The

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Table 1.3: Inocul_ant and fungicide treatment rates.
Treatment Product Rate
1.. Cell-Tech I36 ml/50 kg seed
2. PulseR HP 118 g/50 kg seed
3. SowFast 124 g/50 kg seed
4. Nod+ 136 ml/SO kg seed
5. HiStick2/NT 87 g /50 kg seed
6. NitroFix Liquid 162 ml/50 kg seed
7. Apex Extra 136 ml/50 kg seed
8. Optimize 136 ml/50 kg seed
(110 ml inoculant + 26 ml extender)
Fungicide Apron Maxx RTA 162 ml/SO kg seed

 

 

canister for each treatment was then tumbled for several minutes on a mechanical
tumbling machine used for seed treatment, this ensured a uniform inoculant application.
After the inoculant was applied, the seed was packaged into small coin envelopes.
Approximately 670 seeds were packaged for planting in each plot, to obtain a seeding
rate of 445,000 seeds ha". Plots were planted using a 6 row, 2.28 m plot cone planter
(Almaco Inc., Nevada, IA). Two seed dump funnels fed the seed into the planting units.
This planter was split so that rows 1-3 on the planter were fed by a left seed dump funnel,
and rows 4-6 were fed by a right seed dump funnel. Each seed dump was fed with
separate enveIOpes, therefore, each subplot required two envelopes containing
approximately 335 seeds

The seed was packaged and planted in a manner as to avoid contamination of non-
inoculated plots with residual inoculum. To accomplish this, the planter’s fourth, ﬁfth,
and sixth row units were reserved for inoculant treatments, while the ﬁrst, second, and
third row units were reserved for control subplots that never received inoculated seed.

The planter would plant three rows of two subplots at a time. As the planter made an

40

away pass, the ﬁnal three rows of the control subplot from the previous whole plot, and
the ﬁrst three rows of the inoculated subplot from a new whole plot were planted. As the
planter made a return pass, the ﬁnal three rows of the inoculated subplot was planted
along with the ﬁrst three rows of the new control subplot. All sites were planted in a
conventionally prepared seedbed. Plot yields were taken using an Almaco plot combine
(Almaco Inc., Nevada, IA). This combine harvested the middle four rows of each six
row subplot in the ﬁeld. Total area harvested for each plot was 6.45 m2. An Almaco plot
weigh bucket controlled by a Harvestrnaster control on the combine allowed for seed
yield and moisture to be measured as plots were harvested (Almaco lnc., Nevada, IA and
Juniper Systems, Logan UT).

Yield data analysis was conducted separately for each location by year. Whole
plots were used as an error term to test the effect of fungicide, and split plots were used to
test the effect of inoculant. Mean square for residuals was used to test the effect of
interaction between fungicide and inoculant. The main model analysis was performed in
PROC MIXED (The SAS Institute, 2007). At sites where signiﬁcant inoculant effects
were observed, inoculant products were combined and contrasted against untreated plots
to determine if a signiﬁcant inoculant treatment effect occurred in each site-year. All
pairwise comparisons between the inoculant products were also performed with yield
data combined across sites for each year. At sites where signiﬁcant fungicide-inoculant
interactions were observed, the main inoculant effect was sliced between ﬁmgicide
treated plots and plots with no fungicide treatment.

Harvest samples (approx. 0.5 kg.) were taken from Lenawee in 2004, Hillsdale in

2005, and Montmorency in 2004 and 2005. These samples were analyzed for protein and

41

oil content using a Foss NIRSYSTEM 6500 near-infrared spectrosc0py instrument (Foss
North America, Eden Prairie, MN). Data analysis for protein and oil content proceeded
similarly to that of yield data, though analysis was only of inoculant treatments

combined.

Greenhouse Study

To observe growth effects of inoculant products in a controlled environment, a
greenhouse study was performed with several inoculant products. Soybean nodulation
was of particular interest in this study, as nodule data in ﬁeld studies is difficult to obtain
and typically highly variable. The experiment was a completely randomized design, with
10 pots of 7 different inoculant products: Hi-Stick N/T, Cell-Tech, Nod+, Sow Fast,
PulseR HP, Apex Extra, and Optimize, with an untreated control. Cell-Tech inoculant
was also tested with Apron Maxx RTA fungicide to determine whether there was a
detrimental effect by the fungicide on soybean nodulation.

Inoculants and fungicide treatments were applied to seed using one gallon
resealable plastic canisters, to which 0.91 kg of soybean seed and the labeled rate of
inoculant or fungicide was added. Pesticide seed treatments were applied the day prior to
inoculation and planting to allow the chemical to dry on the seed. The canister for each
treatment was then tumbled for several minutes on a mechanical tumbling machine used
for seed treatment; this ensured a uniform seed treatment application. Inoculants were
applied during the day of planting. Five treated soybean seeds (DF8251RR variety) were
planted approximately 2.5 cm deep into 15 cm diameter clay pots ﬁlled with BACTTO

high porosity planting mixture (Michigan Peat Company, Houston, TX). First emergence

42

occurred four days after planting. Untreated seed was planted ﬁrst, to avoid
contamination with inoculum, and rubber gloves were changed between seed treatments
to avoid contamination between treatments. Plants were grown using a 16/8 light/dark
day length cycle. The greenhouse was lit with S5] 400 watt high pressure sodium lamps
(Lithonia Lighting, Conyers, GA), and the average greenhouse temperature was 25°C.
Thrips were controlled weekly with an application of Conserve (spinosad, Dow
Agrosciences, Indianapolis, IN) at 0.46 ml/Iiter. At 14 DAP, pots were thinned to l bean
plant per pot. Pots were re-randomized every two weeks during the study.

SPAD meter readings were taken every 7 days between 14 and 35 DAP. Plant
heights were also taken every 7 days between 21 and 35 DAP. At 49 DAP, plant shoot
material was harvested. Leaf area meter readings were taken, as well as fresh biomass
and dry biomass. Finally, nodule counts were conducted at 50 DAP. Root masses were
removed from pots and gently washed in root washing sinks. Nodules were then counted
from the exposed root system.

This experiment was run twice. The ﬁrst run was planted on February 15, 2006.
Plants were harvested on April 7, 2006 and nodule counts were performed on April 7 and
8, 2006. The second run was planted on December 1, 2006. Plants were harvested on
January 19, 2007 and nodule counts were performed on January 19 and 20, 2007. Data
was analyzed as a single factor completely randomized design in PROC MIXED (The
SAS Institute, 2007). If the main effect was signiﬁcant, all paired comparisons were

performed between. treatments.

43

Results and Discussion
Climate

In 2003, precipitation was above average at all sites in May. However, rainfall
patterns through the state were quite variable later in the growing season. lngham
County (E. Lansing) had a rainfall deﬁcit in each month between June and September.
Montmorency County (Hawks) experienced a rainfall deﬁcit each month except July.
Lenawee County experienced rainfall surpluses in July, August and September, with
August nearly 10 cm above average. Other sites experienced normal precipitation in
2003. The 2004 growing season began with a remarkably wet spring, with widespread
record and near-record May rainfall throughout the state. July through August had nearer
to normal precipitation throughout the state, although Lenawee County continued to have
high precipitation through June. September was very dry throughout the state, with no
site receiving more than 3.12 cm of rainfall. The 2005 growing season began very dry,
with every site experiencing a rainfall deﬁcit for the month of May. June and July were
close to average overall, while lngham County became very dry in August and
September. September was again very dry throughout the state in 2005 (Table 1.4).

In 2003, lngham and Saginaw Counties saw temperatures below normal in May
and June, but above normal in August. Other sites had near normal temperatures
throughout the year, with the exception of August being above normal. In 2004,
Michigan experienced a cool July and August, as temperatures were over 1°C below
normal at every ﬁeld site for both months, with the exception of Lenawee County.
September temperatures were above normal however, and actually exceeded August

averages in Northern Michigan. In 2005, above average temperatures were observed at

44

every site in every month of the growing season except May. Average temperatures were

at least 23°C above normal at every site during the month of June (Table 1.5)

 

 

 

 

 

 

 

 

 

 

Table 1.4: Precipitation by month(cm), ﬁeld Sites 2003-2005. Long term means
(1970-2000) included for comparsion.
May June Julv August September
Grand Traverse 2003 7.59 5.41 10.31 11.43 7.31
(Kingsley) 2004 16.51 6.17 4.21 5.56 2.08
30 yr. 5.84 8.12 6.60 7.37 10.16
Hillsdale 2003 14.98 5.63 6.83 16.71 14.55
(Coldwater) 2004 15.16 11.45 22.55 5.61 3.20
2005 6.10 7.95 11.02 4.98 7.49
30yr. 9.62 9.44 9.65 10.01 9.42
lngham 2003 10.36 3.73 3.58 4.62 6.55
(E. Lansing) 2004 20.50 8.92 10.16 8.71 2.67
2005 3.33 10.87 8.71 1.63 7.67
30 yr. 6.90 8.07 7.56 8.74 9.22
Lenawee 2003 12.52 5.13 9.49 18.80 13.03
(Tecumseh) 2004 15.04 15.11 8.61 6.01 2.15
30 yr. 8.38 8.89 8.13 8.89 8.38
Montmorency 2003 5.92 5.13 8.18 3.99 5 .99
(Hawks) 2004 12.19 5.59 11.48 6.12 3.12
2005 1.91 8.20 6.30 13.44 4.50
30 yr. 7.36 6.85 7.62 9.65 8.89
Saginaw 2003 10.79 6.88 8.61 4.24 5.33
(Saginaw) 30 yr. 7.34 8.61 7.46 8.51 10.80
Sanilac 2004 16.08 8.51 12.67 2.97 1.96
(Sandusky) 2005 4.98 7.04 10.19 6.60 5.08
30 yr. 6.78 7.61 6.73 7.37 10.24

 

45

 

 

 

Table 1.5: Average temperature by month(°C), ﬁeld Sites 2003-2005. Long term
means Q970-2000) included for comparsion.

 

 

 

 

 

 

 

 

May June July August September
Grand Traverse 2003 11.7 17.3 20.0 21.1 16.3
(Kingsley) 2004 12.3 17.5 19.3 17.8 18.6
30 yr. 12.2 17.8 20.6 19.4 15.6
Hillsdale 2003 12.8 18.4 21.4 21.9 16.1
(Hillsdale) 2004 16.1 18.9 20.6 1. 8.9 18.9
2005 12.7 22.4 22.4 21.1 18.4
3Lvn 14.1 19.3 21.5 20.3 16.1
lngham 2003 12.6 18.1 21.0 21.1 15.7
(E. Lansing) 2004 14.6 18.1 20.1 18.4 17.6
2005 12.3 21.9 22.0 21.8 18.3
30 yr. 14.0 19.2 21.4 20.3 16.1
Lenawee 2003 14.3 19.1 22.3 22.4 16.6
(Tecumseh) 2004 16.4 19.9 21.6 19.6 18.9
30 yr. 14.4 19.4 21.7 20.6 16.7
Montmorency 2003 10.5 16.3 19.2 19.6 14.1
(Hawks) 2004 10.3 15.9 18.3 16.4 16.8
2005 10.6 19.8 20.7 18.9 16.6
30 W. 10.6 16.1 19.4 17.8 13.9
Presque Isle 2006 13.2 18.7 23.0 20.0 14.9
(Rogers City) 30 yr. 11.1 16.4 19.6 18.6 14.3
Saginaw 2003 13.1 18.3 21.5 22.3 17.1
(Saginaw) 30 yr. 15.4 20.4 22.8 21.5 17.3
Sanilac 2004 12.4 16.9 19.4 18.2 17.6
(Sandusky) 2005 11.8 21.2 21.6 20.9 18.1
30 yr. 13.5 18.5 21.2 20.1 15.8

 

46

 

Inoculant Yield Effects

Overall, for the 14 site-years on which inoculant trials were performed on soil
with a history of soybean production, the average yield difference between inoculated
and uninoculated plots was 85.58 kg ha". This result is consistent with results in Purdue
University trials, but somewhat less than results from Ohio State University trials
(Conley and Christmas 2006, Beuerlein 2005). Of these 14 site-years, 6 sites showed
signiﬁcant main inoculant effects (Table 1.6).

In 2003, inoculated plots at Hillsdale, Saginaw, and Montmorency County sites
showed signiﬁcant yield increases of 194 kg ha" (6.3%), 156 kg ha" (8.3%), and 185 kg
ha‘1 (5.5%) respectively. Sites in Lenawee, lngham, and Grand Traverse Counties
showed no signiﬁcant yield differences due to inoculation.

In 2004, the site at lngham County showed a signiﬁcant yield increase in
inoculated plots of 231 kg ha‘l (6.9%) Inoculated plots in Lenawee County showed a
signiﬁcant yield decrease of 281 kg ha"( 5.9%). However, this was the only site where a
signiﬁcant decrease in yield was observed in inoculated plots. Sites in Hillsdale, Grand

Traverse, and Sanilac Counties showed no signiﬁcant yield differences due to inoculant

 

 

 

(Table 1.7).
Table 1.6: Summary of combined inoculants effects, ANOVA for
ggain yield by site-year.
P-values from ANOVA

2003 2004 2005
Grand Traverse 0.0830 0.0905 ------
Hillsdale 0.0002 0.3790 <0.0001
lngham 0.6417 0.0015 0.5275
Lenawee 0.3355 <0.0001 ------
Montmorenc y 0.0001 <0.0001* <0.0001 *
Saginaw 0.0005 ----------------
Sanilac --------- 0.5027 0.0008
*virgin soybean ground

 

 

 

47

Researchers in other states have observed rare cases where soybean inoculation
led to decreases in yield. A trial at the Throckmorton-Purdue Agricultural Center in 2005
also showed a yield decrease as a result of soybean inoculation (Conley, unpublished
data). Both sites had very high trial yield averages. Untreated plots at the 2004 Lenawee
site averaged 4946 kg ha'l, and untreated plots at the 2005 Throckmorton site averaged
4804 kg ha".

Lenawee 2004 and lngham 2005, the two site years in the trial that had the most
numerically negative yield response to inoculant, had only 2.64 and 1.54 cm of rainfall in
the 30-60 day preharvest period when soybean would be in pod ﬁlling stages. At the site
of the 2005 Purdue study, only .01 inches of rain was accumulated during a 24 day period
between August 21 and September 13. This long period with lack of rainfall seems to ﬁt
a trend of negative inoculant results in years where a rainfall deﬁcit occurs during podﬁll
stages.

When rainfall in the 30-60 day preharvest period was plotted against combined
inoculant yield differences by site-year in this study, a moderate correlation existed when
a quadratic curve was ﬁtted to the data (R2=.403). The model used for this curve was y=
-2.33x2 + 1.2.51x — 10.08, with x equal to rainfall in the 30 to 60 day preharvest period
and y equal to the percent yield difference between inoculated and uninoculated plots by
site-year. Analysis of this model in PROC REG (The SAS Institute, 2007) resulted in a
Pr>F value of 0.0582. While the model was not signiﬁcant at the 95% conﬁdence level,

this may be an interesting trend worthy of more evaluation.

48

Lawn and Brun observed that as soybean pod ﬁll begins, N ﬁxation by nodules
peaks and then rapidly decreases. Plant depodding allowed nodule activity to be
maintained longer, while plant defoliation caused N ﬁxation in nodules to decline earlier
(lawn and Brun 1974). Others have found N ﬁxation to peak later than early pod ﬁll.
(Thibodeau and Jaworski 1975, Abendroth and Elmore 2006).

One hypothesis to explain nodule death may be that the plant-nodule relationship
is a source-sink relationship, and that as photosynthate is supplied to new seeds, it is
diverted from feeding nodules, particularly if resources are limited under stressful
conditions. If this source-sink relationship exists and photosynthate is competed over
between developing fruit and nodules particularly during a water-stressed period, this
may explain potential yield losses under extended dry periods during pod ﬁll. If
inoculated plots had superior nodulation, they may compete with deve10ping fruit for
photosynthate from leaves, at least during early pod ﬁll. While indirect evidence
supporting the source-sink hypothesis has been observed (Lawn and Brun 1974, Schulze
2003), this hypothesis has not been supported with direct evidence relating carbon
assimilate competition by nodules and nitrogen ﬁxation (Schulze et. al. 2000, Puppo et.
al. 2005). There has also been speculation that enhanced early season growth from
inoculants in high yielding beans could create a large plant vegetative sink, and if rainfall
is lacking late in the growing season, this could cause a negative yield response.

In 2005, Hillsdale and Sanilac County sites showed signiﬁcant yield increases.
Inoculated plots in Hillsdale County showed a yield increase of 358 kg ha", or 9.0%.
Inoculated plots in Sanilac County showed a yield increase of 273 kg ha'l, or 8.4%.

lngham County plots showed no signiﬁcant yield difference (Table 1.7).

49

Montmorency County plots in 2004 and 2005 were conducted on sites that did not
have a history of soybean production. As a result, yield increases were signiﬁcantly
higher than other locations. Inoculated plots in 2004 yielded 1433 kg ha'1 higher than
non-inoculated plots (82.2%). In 2005, inoculated plots yielded 551 kg ha'I higher than

non-inoculated plots (29.9%, Table 1.7).

 

 

 

 

Table 1.7: Inoculant grain yield effects, by site-year (kg ha",
treatments combined).
2003 2004 2005
Grand Traverse
Inoculated 2223 1 305
Untreated 2253 l 150
-30 155
Hillsdale
Inoculated 3262 3 1 I9 4348
Untreated 3067 3053 3990
195* 67 358*
lngham
Inoculated 2207 35 87 3149
Untreated 2151 3356 3287
56 231* -138
Lenawee
Inoculated 26 10 4666
Untreated 2639 4947
-29 -281***
Montmorency
Inoculated 3544 3 1 76 2412
Untreated 3359 1 743 1861
185* 1433* ** 551* **
Saginaw
Inoculated 2023
Untreated I867
156*
Sanilac
Inoculated 3942 3524
Untreated 3927 3250
15 274*
*signiﬁcant yield increase at P<0.05
**virgin soybean ground
***signiﬁcant yield decrease at P<0.05

 

50

 

Inoculant Brand Yield Effects

During certain site-years, treatment with particular inoculant brands produced
signiﬁcantly higher soybean grain yield than other brands, but averaged across site-years,
no signiﬁcant yield advantage was observed for any inoculant brand over another (Table
1.8). However, when the two virgin soybean sites, Montmorency 2004 and 2005, are
combined and analyzed, some interesting separation does occur. While six of the seven
brands are statistically similar, three of the four brands that also have a “b” grouping are
peat based powders: HiStick2/NT, PulseR, and SowFast (Table 1.9). When mean
inoculant treatment yield are combined and contrasted based on type of carrier, there is a
signiﬁcant yield advantage from liquid inoculants at these sites with no previous soybean
production (Table 1 . 10).

Most research regarding inoculant carrier materials found that in soybean, liquid
and peat based carriers provide similar yield effects. In the series of trials performed by
Ohio State, no signiﬁcant difference between peat based and liquid formulations were
observed, although their trials were performed exclusively on rotational soybean ground
(Beuerlein 2004, 2005). The differences observed between liquid and peat based
inoculants on ﬁrst-time soybean ﬁelds were interesting and unexpected. Trials on ﬁrst-
time soybean ﬁelds in Ontario found yields to be similar between peat based and liquid
inoculant treatments (Bohner 2002). There may be a coverage advantage using liquid
inoculants compared to peat-based inoculant. Whereas seed adhesion of liquid inoculants
is excellent, some percentage of peat inoculant will fail to adhere to seed or can fall off
during seed transport and seed movement within the planter and fall to the bottom of the

hopper (Smith 1992). A trial performed in Australia found that a water slurry application

51

 

 

 

 

 

 

 

Table 1.8: Inoculant treatment grain yield averages, by year
(kg ha") sites combined. excluding Montmorency ’04-‘05).
Inoculant 2003 2004 2005
Cell Tech 2685a 3281a 3543a
PulseR 2656a 3326a 3794a
SowFast 2616a 3294a 3541 a
Nod+ 2674a 3312a 3749a
HiStick2/NT 2656a 3346a 3630a
NitroFix Liquid 2616a 3372a 3729a
Apex Extra 3450a 3698a
Optimize 3656a
Eble 1.9: Inocul_ant treatment grain yield averages, Montrnorencv 2004-2005 (kg ha'1 ).
Inoculant yield carrier

NitroFix Liquid 3206a liquid

Cell Tech 3084a liquid

Nod+ 3056a liquid

Apex Extra 2922ab liquid

PulseR 2889ab peat

SowFast 27 96ab peat

HiStick2/NT 2442b peat

 

 

 

 

Table 1.10: Contrast of grain yield means for liquid and peat based
inoculants, Montmorency 2004-2005 (kg h_a". no ﬁmgicide plots only).

 

 

Yield Pr >t
Liquid Carrier Inoculants 3073
Peat Carrier Inoculants 2709
364 0.01 93

 

of inoculant did increase yields over a dry inoculant form (Brockwell et. al. 1988).
However, most peat based inoculants used today include sticking agents to minimize
these problems, and seed coverage was usually observed to be adequate during planting
of seed treated with the peat based products.

Overall results in these trials were similar to those performed in Ohio and Indiana,
in that certain site-years had obvious responses to soybean inoculation, while others
showed no response. In conclusion, it appears that in the long term, inoculation of

soybean seed is an economically viable practice, though not necessarily of a large

52

magnitude. It also appears that there are not signiﬁcant differences in the effectiveness of
any particular commercial brand of inoculant except when soybeans have not been grown
in the past, where it may be advantageous to use a liquid inoculant. There may also be a
trend for reduction in inoculant effectiveness or a potential for negative yield effects

when extremely dry conditions occur during pod ﬁll.

Fungicide Yield Effects

Signiﬁcant fungicide main effects were observed at ﬁve of sixteen site-years
(Table 1.11). In cases where an inoculant-fungicide interaction was observed in the
model or there was a signiﬁcant negative effect of fungicide within a contrast at a
particular site, the fungicide treatment was sliced between inoculated and uninoculated
plots to observe whether the inclusion of the fungicide treatment interacted with
inoculant. In cases where the main effect of the fungicide produced a yield increase,
interactions between fungicide and inoculant were not observed. Saginaw saw a yield
increase of 105 kg ha'1 in 2003. Hillsdale saw yield increases in both 2004 (260 kg ha")
and 2005 (102 kg ha"). However, signiﬁcant effects were observed at Montmorency in
2004 and 2005; and these effects produced yield decreases. These are the two sites which

had not previously had a soybean crop (Table 1.12).

53

 

 

Table 1.1 1: Summary of ANOVA for fungicide main effects on
grain yield by site-year.
P-values from ANOVA

2003 2004 2005
Grand Traverse 0.2600 0.5914 ------
Hillsdale 0.7541 0.0174 0.0205
lngham 0.7505 0.1225 0.4047
Lenawee 0.4176 0.2559 ------
Montmorency 0.0695 <0,()()0]* ** 00425:: **
Saginaw 0.0090 ............
Sanilac ------ 0.9321 0.6921

*new soybean ground
** signiﬁcant yield decrease, other signiﬁcant effects were
increases

 

 

Table 1.12: Fungicide yield effects, by site-year (kg ha'l).

 

 

2003 2004 2005
Grand Traverse
Apron Maxx 2257 1288
Untreated 2 l 96 1 258
61 -33
Hillsdale
Apron Maxx 3228 3247 4365
Untreated 3240 2974 4252
-12 273* 113*
lngham
Apron Maxx 2185 3518 3206
Untreated 2205 3601 3 148
-20 -83 58
Lenawee
Apron Maxx 2646 4729
Untreated 25 8 l 4676
65 53
Montmorency
Apron Maxx 3555 2803 2297
Untreated 3479 3 190 2412
76 -387* -1 15*
Saginaw
Apron Maxx 2053
Untreated 1948
105*
Sanilac
Apron Maxx 4063.7 3416
Untreated 4058.8 3445
*significant at P<0.05 4.9 -29

 

54

 

Slicing effects between inoculated and uninoculated subplots were highly
signiﬁcant at Montmorency in 2004. There was no signiﬁcant yield difference between
uninoculated subplots with or without Apron Maxx RTA. However, treatment of
inoculated subplots by Apron Maxx RTA resulted in a 504.4 kg ha“1 decrease in yield
compared to inoculated subplots with no fungicide. Slicing was also signiﬁcant at
Montmorency in 2005, where there was again no yield difference in uninoculated
soybean with or without Apron Maxx RTA, but a signiﬁcant yield decrease of 134 kg
ha“l when inoculants were used with Apron Maxx RTA treatment. Thus, the lack of main
fungicide effects at these two sites was masked by these inoculant-fungicide interactions
during these two site-years (Table 1.13).

Interestingly, when inoculant brands were compared based on carrier in the
presence of Apron Maxx RTA, the signiﬁcance of the difference between liquid and peat
products is lost. Also, when inoculant-fungicide interaction effects are sliced based on
the inoculant carrier used, the interaction remains signiﬁcant for liquid products, but is
not signiﬁcant when peat-based products are used. When the differences between
inoculant-fungicide interactions are contrasted, there is a signiﬁcant difference between
the interaction caused by Apron Maxx RTA to liquid inoculants and Apron Maxx RTA to
peat based inoculants. These results indicate that while liquid inoculants may be more
effective on virgin soil, these advantages may be mitigated when Apron Maxx RTA is

used as a seed treatment (Table 1.14).

55

Researchers have commented on the possibility that the particle matrix associated
with peat based inoculants provide some protection against environmental stresses,

particularly lack of moisture and resulting bacterial desiccation (Zdor and Pueppke, 1990,

 

Table 1.13: Inoculant-fungicide interaction slicing effects at Montmorency. 2004-2005.

 

 

 

Montmorency 2004
Yield Pr>F
Apron Maxx RTA 1799
Untreated 1687
l 1.2 0.2943
Apron Maxx RTA + Inoculant 3241
Inoculant Only 3746
-505 <0.0001
Montmorency 2005
Yield Pr>F
Apron Maxx RTA 1855
Untreated 1858
-3 0.9054
Apron Maxx RTA + Inoculant 2345
Inoculant Only 2479
-134 0.0377

 

 

Kyei-Boahen et al. 2002, Bashan 1998, Smith 1992). It is plausible that peat carriers
could provide a physical barrier against fungicide-bacteria interaction in the same
manner.

The Apron Maxx formulation used in this study (Apron Maxx RTA) is the most
common of two Apron Maxx formulations available for use on soybean today. The
second formulation is Apron Maxx RFC (Rhizobium Friendly Concentrate), which was
introduced in 2005 (Syngenta, Greensboro, NC) for use and co-application with liquid
inoculants. This formulation has a higher concentration of active ingredients

(mefenoxam and ﬂudioxonil), and is applied at a 48.6 ml/50 kg of seed rate, rather than

56

 

 

Table 1.14: Inoculant-fungicide interaction slicing effects at Montmorency, 2004-2005
(grain yield, kg ha").
Yield Pr>F
Peat Based Inoculants (No Fungicide) 2709
Peat Based Inoculants (With Apron Maxx RTA) 2558
151 0.1367
Liquid Inoculants (No Fungicide) 3074
Liquid Inoculants (With Apron Maxx RTA) 2677
397 0.0030
Liquid Inoculant/Apron Maxx Difference 397
Peat Based Inoculant/Apron Maxx Difference 151
246 0.0420
Peat Based Inoculants (With Apron Maxx RTA) 2558
Liquid Inoculants (With Apron Maxx RTA) 2677
-1 19 0.1874

 

 

 

162 mI/50 kg rate used with Apron Maxx RFC. Active ingredient rates are the same for
both products, but less total product is used in application of Apron Maxx RFC to seed.

While Apron Maxx RTA did not interact at the six rotational sites that were
responsive to inoculants, clear evidence of interaction between Apron Maxx RTA and
inoculants was observed at sites with no history of soybean production, where inoculant
effects were more pronounced. Use of Apron Maxx RFC may be an option for farmers
producing soybean at such sites.

Overall, Apron Maxx RTA did not provide widespread yield increases during this
study. Yields were increased at three of sixteen site-years throughout the span of the
trial. The largest increase was 272 kg ha'1 at Hillsdale in 2004. This yield increase could
be explained by climatic conditions experienced that year, as Hillsdale was planted on

April 30, which preceded the record setting rainfall pattern experience in May throughout

57

Central and Southern Michigan. This may have caused more early soil-borne disease
pressure. Apron Maxx RTA also increased yield at Hillsdale in 2005, which was the
earliest planted site and had temperatures that were well below normal throughout the
month of May. Early stands were somewhat reduced overall at Hillsdale in 2004, but no
fungicide treatment effects were observed on plant stands in 2004 or 2005 other than at

Lenawee (Table 1.15).

 

Table 1.15: Fungicide plant stand effects by site (plants ha‘l ).

2004 GT Hillsdale lngham Lenawee Mont. Sanilac Avg.

Apron Maxx 400695 356990 416052 380101 403422 410949 394717

No Fungicide 402921 368223 407727 359147 409521 395516 390479
-2226 -11223 8325 20954* -6099 15433 4238

2005

Apron Maxx 304036 357057 240460 247994 286801

No Fungicide 301096 362599 246971 247779 289361

2940 -5542 -6511 215 -2560

 

*significant at P<0.05

 

 

Grain Protein and Oil Effects

Inoculation had no effect on grain protein content at Lenawee in 2004 or Hillsdale
in 2005. Signiﬁcant differences were found at Montmorency in both 2004 and 2005, the
two ﬁrst-time soybean sites in this study. Grain protein content was 11.8% and 8.17%
higher in soybean harvested from inoculated plots at Montmorency in 2004 and 2005
(Table 1.16). An inoculant-fungicide interaction was observed at Montmorency in 2004,
as among inoculated subplots protein levels were reduced by 3% where fungicide seed
treatment was used in the whole plot (Table 1.17). This interaction was not observed at
Montmorency in 2005.

Similar results were observed for grain oil content analysis. Lenawee 2004 and

Hillsdale 2005 sites saw no signiﬁcant effect on grain oil content by inoculant. However,

58

both Montmorency sites saw signiﬁcant increases in oil content in soybean from
inoculated subplots. Grain oil content was 7.0% and 3.3% higher in soybean harvested
from inoculated plots at Montmorency in 2004 and 2005 (Table 1.16). Again, an
interaction between inoculant and fungicide was observed, as inoculated subplot grain oil
levels were increased by 3% where fungicide seed treatment was used in the whole plot
(Table 1.17). This interaction was not observed at Montmorency in 2005.

There is little question that yield differences observed at Montmorency in 2004
and 2005 correlated with the protein and oil content of each treatment. Yield was
positively correlated with grain protein content of subplots at both sites in 2004
(R220.53) and in 2005 (R22 0.63). Yield was negatively correlated with grain oil content

with an R2 value of 0.63 in both 2004 and 2005.

 

Table 1.16: Inoculant effects on soybean grain protein and oil content.

Protein (%) Oil (%)
Inoc. No Inoc. Pr<t Inoc. No Inoc. Pr<t
Lenawee 2004 42.29 41.86 20.72 20.76
Montmorency 2004 37.75 33.76 <0.0001 21.49 23.00 <0.0001
Hillsdale 2005 42.48 42.58 20.83 20.87

Montmorency2005 33.36 30.84 <0.0001 23.27 24.03 <0.0001

 

 

 

 

Table 1.17: Inoculant-fungicide interaction effects on soybean grain protein and oil
content, Montmorency 2004.

Protein (%) Pr<t Oil (%) Pr<t
Inoculated Only 38.30 21.27
Inoculant + Fungicide 37.20 0.0004 21.71 0.0017

 

 

 

Greenhouse Results

Inoculant brand had a signiﬁcant main effect on nodule count in both greenhouse
runs. In the ﬁrst run, average nodule counts ranged between 17 and 67 among inoculant
treatments, and only 6 for uninoculated plants. In the second run of the greenhouse trial,
average nodule counts ranged between 58 and 114 among inoculant treatments, and only
23 for uninoculated plants. Apex Extra and PulseR HP were in the “high nodule count”
group for means separation during both runs. Cell Tech was not signiﬁcantly different
from Cell Tech + Apron Maxx in the ﬁrst run, but averaged 47 nodules less per plant in
the second run of the experiment (Table 1.18).

Inoculant brand or carrier type was not a signiﬁcant factor for determining leaf
area index readings, whole plant fresh weight, or whole plant dry weight for either run.
Inoculant brand was also not a signiﬁcant factor in SPAD meter readings or plant height
at any measurement timing. Uninoculated soybean likely performed better in the
greenhouse than the ﬁeld due to the high organic matter peat potting mix used in the

greenhouse study, which may have provided more available N to compensate for the lack

 

 

of nodulation.

Table 1.18-Average nodule counts by inoculants treatment (50 DAP), greenhouse
study.

Run 1 Run 2
PulseR HP 67 a SowFast 114 a
Apex Extra 66 a Apex Extra 107 ab
Cell Tech + Apron Maxx 48 ab Cell Tech 106 ab
Cell Tech 39 b PulseR HP 102 ab
Nod+ 37 b Optimize 88 abc
SowFast 34 be HiStick N/T 83 abc
Optimize 30 be Nod + 69 be
HiStick N/T 17 cd Cell Tech + Apron Maxx 58 cd
Untreated 6 d Untreated 23 d

 

 

 

60

Conclusions

Soybean seed inoculants increased soybean grain yield on 6 of 14 site-years
which had been in a soybean rotation. The average yield increase as a result of soybean
seed inoculation at these 14 site-years was 85.58 kg ha". With inoculant costs of $7-$10
ha", soybean inoculation appears to be a proﬁtable practice in the long run, if not on an
annual basis. Soybean inoculant appeared to beneﬁt grain yield most often when normal
precipitation levels were observed during the pod ﬁll period.

No consistant advantage between inoculant products was observed on soils that
had been in soybean rotation, though on soil new to soybean production, liquid inoculants
provided larger yield increases. Inoculant brand effects on soybean nodulation were not
consistently observed in greenhouse studies.

Use of mefenoxam and ﬂudioxonil as a fungicidal seed treatment did not increase
soybean yields on a wide-spread basis. Fungicide-inoculant interactions were not
observed on soils in soybean rotation, but were observed on soils new to soybean
production. This interaction was observed in grain yields, protein content of grain, and

oil content of grain.

61

References

Albrecht, S. L., J. M. Bennett, and K. J. Boote. 1984. Relationship of nitrogenase activity
to plant water stress in ﬁeld grown soybeans. Field Crops Research 8:61-71.

Ash, M., J. Livesey, and E. Dohlman. 2006. Soybean Backgrounder. Electronic Outlook
Report from the Economic Research Service, USDA. Retrieved Nov 10, 2006 from
http://www.ers.usda.gov/pubIications/OCS/apr06/OC8200601/OCS200601.pdf

Bacanamwo, M. and L. Purcell. 1999. Soybean dry matter and N accumulation responses
to ﬂooding stress, N sources and hypoxia. Journal of Experimental Botany 50(334):689-
696.

Bashan, Y. 1998. Inoculants of plant growth-promoting bacteria for use in agriculture.
Biotechnology Advances 16(4):729-770.

Beuerlein, J. 2005. 2005 Ohio Inoculation Study. Ohio State University Agronomic
Crops Network. Retrieved Dec 5, 2006 from
http://agcrops.osu.edu/soybean/documents/ZOOSSoylnoculationReport.pdf

Beuerlein, J. 2004. 2004 Ohio Inoculation Study. Ohio State University Agronomic
Cr0ps Netwok. Retrieved Dec 5, 2006 from
httpzl/agcrops.osu.edu/researchl2004/04inoc%20reportl 1 .pdf

Bohner, H. 2002. Head-to-Head Comparison of Soybean Inoculants in 2002. Ontario
Ministry of Food, Agriculture, and Rural Affairs (Online). Retrieved February 18, 2007
from http://www.omafra.gov.on.ca/english/crops/ﬁeld/news/croptalk/2003/ct_0103a8.
Htrn

Brockwell J, D. F. Herridge, L. J. Morthorpe, and R. J. Roughley. 1988. Numerical
effects of Rhizobium p0pulation on legume symbiosis. In D. P. Beck and L. A. Materon
(eds.) Nitrogen Fixation by Legumes in Mediterranean Agriculture. 179—193. ICARDA.

Conley, S. and E. Christmas. 2005. Utilizing Inoculants in a Com-Soybean Rotation.
Purdue University Cooperative Extension Bulletin SPS-lOO—W.

Cregan, P. B., H. H. Keyser, and M. J. Sadowsky. 1989. Host plant effects on nodulation
and competitiveness of the Bradyrhizobiumjaponicum serotype strains constituting
serocluster 123. Applied Environmental Microbiology 55(10): 2532-2536.

Duong, T. P., C. N. Diep, N. T. Khiem, N. H. Hiep, N. V. Toi, N. V. Lich and T. K. L.

Nhan.'1984. Rhizobium inoculant for soybean [Glycine max (L.) Merrill] in Mekong
Delta. Plant and Soil 79(2):235-240.

62

Graham, P. H. 1992. Stress tolerance in Rhizobium and Bradyrhizobium, and
nodulation under adverse soil conditions. Canadian Journal of Microbiology 38: 475—
484.

Kuykendall, L. D. and W. J. Hunter. 1991. Enhancement of nitrogen ﬁxation with
Bradjirhizobiumjaponicum mutants. US patent 5,021,076.

Kyei-Boahen, S., A. E. Slinkard, and F. L. Walley. 2002. Evaluation of rhizobial
inoculation methods for chickpea. Agronomy Journal 94:851-859.

Lawn, R. J. and W. A. Brun. 1974. Symbiotic nitrogen ﬁxation in soybeans. I. Effect of
photosynthetic source-sink manipulation. Crop Science 14:11-16.

Lindemann, W. C. and C. R. Glover. 2003. Nitrogen ﬁxation by legumes. New Mexico
State University Cooperative Extension Service Guide A-129.

Pedersen, P. 2003. Soybean Seed Inoculation. Integrated Crop Management Newsletter,
Iowa State University Extension. Issue IC-490(5):12-l 3.

Puppo, A., K. Groten, F. Bastian, R. Carzaniga, M. Soussi, M. M. Lucas, M. Rosario de
Felipe, J. Harrison, H. Vanacker, and C. H. Foyer. 2005. Legume nodule senescence:
roles for redox and hormone signalling in the orchestration of the natural aging process
New Phytology 165(3): 683-701.

Schulze J., H. Beschow, E. Adgo, and W. Merbach. 2000. Efﬁciency of N2 ﬁxation
in Vicia faba L. in combination with different Rhizobium leguminosarum strains. Journal
of Plant Nutrition and Soil Science 1632367—373.

Schulze, J. 2003. Source-sink manipulations suggest an N-feedback mechanism for the
drop in N2 ﬁxation during pod-ﬁlling in pea and broad bean. Journal of Plant Physiology
160:531-537

Seneviratne, G., L. H. J. Van Holm and E. M. H. G. S. Ekanayake. 2000. Agronomic
beneﬁts of rhizobial inoculant use over nitrogen fertilizer application in tropical soybean.
Field Crop Research 68(3):]99-203.

Smith, R. S. 1992. Legume inoculant formulation and application. Canadian Journal of
Microbiology 38, 485-492.

Smith, S., R. M. Osborn, J. W. Kosanke. 2004. Methods and compositions providing
agronomically beneﬁcial effects in legumes and non-legumes. United States Patent

Application 200501 87 107.

Thibodeau, P. S. and E. G. Jaworski. 1975. Patterns of nitrogen utilization in the soybean.
Planta 127(2):133-147.

63

Tien, H. H., T. M. Hien, M. T. Son and D. Herridge. 2002. Rhizobial Inoculation and N2
Fixation of Soybean and Mungbean in the Eastern Region of South Vietnam. In D.
Herridge (ed.) Inoculants and Nitrogen Fixation of Legumes in Vietnam: ACIAR
Proceedings 10%.

Vitosh, M. L. 1997. Soybean Inoculation in Michigan. Soybean Facts Winter 1997.
Michigan State University Extention Facts Sheet 9708.

Xavier, I. J., G. Holloway, and M. Leggett 2004. Development of rhizobial inoculant
formulations. Online. Crop Management doi:10.1094/CM-2004-0301-06-RV.

Zdor, R. E. and S. G. Pueppke. 1990. Competition for nodulation of soybean by
Bradyrhizobiumjaponicum 123 and 138 in soil containing indigenous rhizobia. Soil
Biology and Biochemistry 22(5):607-613.

64

CHAPTER 2
EFFECT OF NEONICTINOID SEED TREATMENTS ON SOYBEAN GROWTH
AND YIELD IN MICHIGAN
Abstract
Two recently registered neonicotinoid seed treatments, thiamethoxam and

imidaCIOprid provide early season protection against several different insect pests,
including soybean aphid and bean leaf beetle. These seed treatments may also reduce
seedling mortality, increase plant height, and improve root, stem, and leaf development.
This research determined whether grain yield and plant health effects were observed as a
result of neonicotinoid seed treatments, as well the effectiveness and duration of soybean
aphid control. Positive neonicotinoid effects on grain yield were mainly limited to 2005,
a year in which signiﬁcant soybean aphid pressure was observed in Michigan. Both
thiamethoxam and imidac10prid provided signiﬁcant yield increases at two of four ﬁeld
sites in 2005. Signiﬁcant soybean aphid population suppression occurred through R2 in
2005, but was not signiﬁcant after R2. Improvement of plant health indicators such as
plant height, SPAD chlorophyll index, and stand counts from neonicotinoid seed

treatments was not observed on a widespread basis.

65

Introduction

Neonicotinoids are the most recent class of insecticides introduced for crop
protection. Neonicotinoids are systemic insecticides, absorbed by plants from foliar or
soil application, or seed treatment. Neonicotinoids have high water solubility, an
important property of systemic insecticides (Tomizawa and Casida 2005).
Neonicotinoids act upon the nervous system by depolarizing giant intemeurones at
nicotinic acetylcholine receptors, leading to paralysis and death of the insect (Matsuda et.
al. 2001, Rice 2004). Neonicotinoids preferentially bind to insect acetylcholine receptors
rather than vertebrate acetylcholine receptors, making them less toxic to humans than
other systemic insecticides (Yamamoto and Casida 1999, Tomizawa and Casida 2005,
Elliott 1977). This favorable property makes the neonicotinoids popular options for
insect control in many crOps.

Since 2004, two new neonicotinoid seed treatments for soybean have been
introduced. Thiamethoxam (Cruiser, Syngenta, Greensboro, NC), and imidacloprid
(Gaucho, Bayer Crop Science, Research Triangle Park, NC) have been approved by the
EPA for use as a soybean seed treatment. These chemicals provide early season control
or suppression of insects such as soybean aphid, bean leaf beetle, leafhoppers, thrips,
Mexican bean beetle, and wireworms. The product manufacturers advertise crop beneﬁts
such as increased plant density, plant height, root, stem and leaf development, as well as
increased yields.

Bean leaf beetles defoliate leaves and transmit bean pod mottle virus (BPMV),
which can signiﬁcantly reduce soybean yield and quality, particularly when damage

occurs early in the season (Krell et. al. 2004). Thiamethoxam or imidac10prid seed

66

treatment may be ideal to control early season bean leaf beetle infestation and bean pod
mottle virus transmission. Trials conducted in Wisconsin, Iowa, and Minnesota have
tested neonicotinoid control of bean leaf beetle feeding. These trials showed that
neonicotinoid seed treatments signiﬁcantly reduce bean leaf beetle feeding for ﬁrst
generation infestations (Cullen et. al. 2004a, Schmitt and VanDee 2005, Potter 2002).

Soybean aphid infestations usually occur later into the growing season than bean
leaf beetle. Soybean aphid pressure usually increases to its highest level in late July or
early August in Michigan (DiFonzo 2003, 2005a), which poses a challenge for
neonicotinoid seed treatments. The insecticide must persist longer and maintain potency
in a larger soybean plant to provide aphid control late in the growing season.
Thiamethoxam has been shown to provide systemic activity and signiﬁcant SBA
mortality for 35 days after planting and a reduction in nymph production up to 49 days
after planting (McComack and Ragsdale 2006). Wisconsin trials performed in 2003
determined the effectiveness ofneonicotinoid seed treatments in controlling soybean
aphid (Cullen et. al. 2004b). Soybean was planted in these trials on June 9, and
throughout the growing season, thiamethoxam and imidacloprid treated soybean had
signiﬁcantly reduced aphid populations, though by mid-August, aphid control appeared
to weaken.

A South Dakota study performed in 2003 produced no signiﬁcant difference
between untreated and insecticide treated plots in mean soybean aphids per plot at 67
days after planting. High aphid pressure was observed at this site, with an average of
1,051 aphids per untreated plant, and no resulting yield advantage was observed

(Catangui et. al. 2004). Trials performed in Iowa from 2002-2005 determined the

67

effectiveness and duration of soybean aphid control from neonicotinoid seed treatments
(Pederson and Lang 2006). In 2003, treated soybean did not reach economic threshold
aphid pressure until July 20, compared to June 30 for untreated soybean. In 2005,
neonicotinoid seed treatment held soybean aphid pressure under threshold through July
18, about one week longer than untreated soybean. Pederson concluded that seed
treatments can control soybean aphid approximately 60 days after planting, which may be
useful in some late planting or replant situations (Pederson and Iang 2006).

Research has been performed regarding rhizobial interactions with soil
insecticides from the carbamate and organophosphate groups. A few of these chemicals
reduced rhizobial growth in-vitro or rhizobial survival on seed, but were not detrimental
to nodulation or soybean growth (Mallik and Tesfai 1984, Martyniuk et. a1. 2002, Evans
et. al. 1991, Lin et. al. 1972). There is limited research regarding potential interactions
between neonicotinoids and rhizobial bacteria. Thiamethoxam had little toxicity in-vitro
to entomopathogenic bacteria (Filho et. al. 2001). Imidacloprid seed treatments had no

antagonistic effect on soil bacteria populations (Sin gh and Singh 2005).

Materials and Methods
Field Study

The experiment took place on farms throughout the Lower Peninsula of Michigan.
In 2004, trials were located at six sites: Montmorency, Grand Traverse, Saginaw,
lngham, Hillsdale, and [enawee Counties, at sites adjacent to the Michigan Soybean
Performance Trials. In 2005 and 2006, four ﬁeld sites were used, including

Montmorency County in 2005, Presque Isle County in 2006, and Sanilac, lngham, and

68

Hillsdale Counties both years. Lenawee in 2004 and Hillsdale in 2006 had previous
crops of wheat. All other sites had previous crops of corn (Table 2.1).

In 2004 and 2005, a randomized complete block design with split plots acting as
treatment and control was used. Both thiamethoxam and imidacloprid were used alone,
as well as in combination with Cell-Tech Inoculant. A Cell-Tech only subplot treatment
tested for insecticide-inoculant yield interaction; thus, a total of 5 different subplot
treatments were used. These treatments were used twice in each replicate, once in an
untreated whole plot, and once in a whole plot with mefenoxam + ﬂudioxonil treated
seed. Therefore, each replication had 10 whole plots. Each whole plot contained twelve
38.] cm rows, six rows treated with an insecticide and/or inoculant, the other six rows
acting as a control. Each plot was 6.1 m in length as planted. Alleys measuring 1.8 m
were cut in between ranges during the growing season, making the plots 4.3 m in length
for harvest.

In 2006, a standard randomized complete block design was used with whole
plots only. Three levels of insecticide treatment, (thiamethoxam, imidacloprid, and none)
and two levels of both fungicide (mefenoxam + ﬂudioxonil or none) and inoculant (Cell-
Tech and none) were tested in all possible combinations, for a total of twelve whole plots
per replication. Whole plots contained six 38.1 cm rows, and plot length was the same as
in 2004 and 2005.

Seed was treated with insecticides and fungicides inside a large drum, to which
4.53 kg of seed and the labeled rates of the chemicals were added. The drum was then

tumbled for several minutes on a mechanical tumbling machine used for seed treatment,

69

 

 

 

 
 

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7]

 

Table 2.3: Pesticide and inoculant seed treatment rates.

 

 

Product Rate

Insecticide

thiamethoxam (Cruiser S'FS) 83 ml/SO kg seed
imidacloprid (Gaucho 480) I30 ml/SO kg seed
Fungicide

mefenoxam/ 162 ml/SO kg seed
fludioxonil (Apron Maxx RTA)

Inoculant

Cell-Tech 136 ml/50 kg seed

 

ensuring uniform application. For inoculated treatments, inoculants were applied to seed
several days later using one gallon resealable plastic canisters, to which 0.91 kg of
soybean seed and the labeled rate of inoculant was added and then tumbled for several
minutes. Approximately 670 seeds were packaged for planting in each plot, to obtain a
seeding rate of 445,000 seeds ha". Plots were planted using a 6 row, 2.29 m plot cone
planter (Almaco lnc., Nevada, lA). Two seed dump funnels fed the seed into the planting
units. This planter was split so that rows l-3 on the planter were fed by a Ieﬁ seed dump
funnel, and rows 4-6 were fed by a right seed dump funnel. Each seed dump was fed
with separate enveIOpes, therefore, each subplot required two enveIOpes containing
approximately 335 seeds.

Plot yields were taken using an Almaco plot combine (Almaco lnc., Nevada, IA).
This combine harvested the middle four rows of each six row plot in the ﬁeld. Total area
harvested for each plot was 6.45 m2. An Almaco plot weight bucket controlled by a
Harvestmaster control on the combine allowed for seed yield and moisture to be

measured as plots were harvested (Almaco lnc., Nevada, [A and Juniper Systems, Logan

72

UT). Harvest samples (approx. 0.5 kg) were taken from each plot, and analyzed for
protein and oil content using a Foss NIRSYSTEM 6500 near-infrared spectroscopy
instrument (Foss North America, Eden Prairie, MN).

During the growing season, several observations were taken to observe plant
growth. Stand counts were performed approximately 1 month after planting at all sites.
Two 0.76 x 1.52 m quadrats were sampled from each plot to determine estimated plant
stand. Four plants in plot were measured for plant height. These measurements were
taken approximately 6 weeks after planting at each site. At Hillsdale and lngham sites in
2005, SPAD chlorophyll index readings (Spectrum Technologies, Inc., Plainﬁeld, IL)
were taken on each leaﬂet of a single newly expanded trifoliate leaf of ten random plants
in each plot. At harvest, four plants were removed from each plot at two sites in each
year, Montmorency and Lenawee in 2004, Montmorency and Hillsdale in 2005, and
Sanilac and Hillsdale in 2006. These plants were taken back to the laboratory where
plant heights were measured and pods per plant were counted.

The data analysis was conducted separately for each location by year. For yield
data in 2004 and 2005, whole plots were used as an error term for fungicide, and split
plots were initially used to test the effect of insecticide and inoculant. If the inoculant +
insecticide main effect was signiﬁcant, paired contrasts were performed to determine
whether an insecticide—inoculant interaction was present. After these contrasts were
performed, inoculated treatments were removed from the data set. Yield and all other
plant growth measurements were then analyzed with fungicide as the whole plot term and
insecticide as the split plot term. Reported P-values for main insecticide effects are from

the analysis of this model. In 2006, data was analyzed as a three-factor RCBD, and

73

 

reported P-values for main insecticide effects were reported from this model. Paired
comparisons among the means were conducted using contrasts when respective factor,
interaction or slicing effects were found to be statistically signiﬁcant at the 0.05 level.

The data analysis was conducted in PROC MIXED (The SAS Institute, 2007).

Greenhouse Study

To observe growth effects of neonicotinoid seed treatment products in a
controlled environment, a greenhouse study was performed. The experiment was a
completely randomized design, with 30 pots planted with soybeans treated with either
thiamethoxam or imidacloprid. An untreated check was included as well. All seed was
also treated with Cell-Tech inoculant.

Inoculants and pesticide treatments were applied to seed using one gallon
resealable plastic canisters, to which 0.91 kg of soybean seed and the labeled rate of
insecticide was added. Pesticide seed treatments were applied the day prior to
inoculation and planting to allow the chemical to dry on the seed. The canister for each
treatment was then tumbled for several minutes on a mechanical tumbling machine used
for seed treatment; this ensured a uniform application. Inoculants were applied during
the day of planting. Fivc treated soybean seeds (DF8251RR variety) were planted into 15
cm diameter clay pots ﬁlled with BACCTO potting mixture (Michigan Peat Company,
Houston, TX) at approximately 1 inch depth. Rubber gloves were changed between
chemical treatments to avoid contamination between treatments. First emergence
occurred four days after planting. Plants were grown using a 16/8 light/dark day length

cycle. The greenhouse was lit with $51 400 watt high pressure sodium lamps (Lithonia

74

Lighting, Conyers, GA), and the average greenhouse temperature was 25°C. Thrips were
controlled weekly with an application of Conserve (spinosad, Dow Agrosciences,
Indianapolis, IN) at 0.46 ml/liter. At 14 DAP, pots were thinned to l bean plant per pot.
Pots were re-randomized every two weeks during the study.

SPAD meter readings were taken every 7 days between 14 and 35 DAP, and
plant heights were taken every 7 days between 21 and 35 DAP. At 30, 40 and 50 DAP,
the plant shoot material of 10 randomly selected plants was harvested to analyse growth
habits over time. Leaf area meter readings were taken, as well as fresh biomass and dry
biomass at these 10 day intervals. Finally, nodule counts were conducted 50 DAP. Root
masses were removed from clay pots and gently washed in root washing sinks. Nodules
were then counted from the exposed root system.

This experiment was conducted two times. The ﬁrst run was planted on February
15, 2006. Plants were harvested on April 7, 2006 and nodule counts were performed on
April 8, 2006. The second run was planted on December 1, 2006. Plants were harvested

on January 19, 2007 and nodule counts were performed on January 20, 2007.

Results and Discussion
Climate

The 2004 growing season began with a remarkably wet spring, with widespread
record and near-record May rainfall throughout the state. July through August had near
normal precipitation throughout the state, though Lenawee County continued to see high
precipitation through June. September was very dry throughout the state, with no site

receiving more than 3.12 cm of rainfall. The 2005 growing season began very dry, with

75

every site experiencing a rainfall deﬁcit for the month of May. June and July were close
to average overall, while lngham County became very dry in August and September.
September was again very dry throughout the state in 2005. Aggregate precipitation in
2006 was near normal throughout Michigan. lngham and Hillsdale sites experienced
above normal rainfall in May, but near normal rainfall the rest of the season. Presque Isle
experienced below normal precipitation in June and July, and Sanilac experienced below
normal precipitation in August and September (Table 2.4).

In 2004, Michigan experienced an extremely cool July and August, as

temperatures were over 1°C below normal at every ﬁeld site for both months, with the

 

exception of Lenawee County. September temperatures were well above normal
however, and actually exceeded August averages in Northern Michigan.

2005 saw above average temperatures at every site in every month of the growing season
except May. Average temperatures were at least 23°C above normal at every site during
the month of June. In 2006, temperatures were near normal at most sites in May, June,
and August. Temperatures were 1°C or more above normal at every site in July. Presque
Isle experienced temperatures well above normal from May to August. September was

cooler than normal at all sites except Presque Isle (Table 2.5)

Insecticide Yield Effects

During the 14 site-years of these insecticide seed treatment trials, main insecticide
effects were seen only four times (Table 2.6). A yield increase of 338 kg ha'1 was
observed in thiamethoxam treated soybean at lngham in 2004. Signiﬁcant yield

decreases were observed from both insecticides at Lenawee in 2004. Four of six sites in

76

 

 

 

 

 

 

 

 

 

 

Table 2.4: Precipitation by month(cm), ﬁeld Sites 2003-2005. Long term means
(11970-2000) included for comparsion.
May June July August September
Grand Traverse 2004 16.51 6.17 4.21 5.56 2.08
(Kingslev) 30 yr. 5.84 8.12 6.60 7.37 10.16
Hillsdale 2004 15.16 11.45 22.55 5.61 3.20
(Coldwater) 2005 6.10 7.95 11.02 4.98 7.49
2006 14.75 8.12 13.08 7.06 10.41
30 yr. 9.62 9.44 9.65 10.01 9.42
lngham 2004 20.50 8.92 10.16 8.71 2.67
(E. Lansing) 2005 3.33 10.87 8.71 1.63 7.67
2006 11.07 7.08 8.03 9.25 7.47
30 yr. 6.90 8.07 7.56 8.74 9.22
Lenawee 2004 15.04 15.11 8.61 6.01 2.15
(Tecumseh) 30 yr. 8.38 8.89 8.13 8.89 8.38
Montmorency 2004 12.19 5.59 11.48 6.12 3.1.2
(Hawks) 2005 1.91 8.20 6.30 13.44 4.50
30 yr. 7.36 6.85 7.62 9.65 8.89
Presque Isle 2006 7.23 3.00 4.90 8.91 9.68
Eogers Citv) 30 yr. 6.47 6.78 7.46 9.34 7.51
Sanilac 2004 16.08 8.51 12.67 2.97 1.96
(Sandusky) 2005 4.98 7.04 10.19 6.60 5.08
2006 6.17 6.70 7.08 5.10 5.25
30 yr. 6.78 7.61 6.73 7.37 10.24

 

 

2004 had no signiﬁcant effect from insecticidal seed treatments (Table 2.7). Heavy
soybean aphid pressure was observed in Michigan during 2005, and main insecticide
effects were observed at three of four sites. When contrasts were performed, insecticidal
seed treatments had signiﬁcant effects at two of four sites during this year. Insecticide
treatments at Hillsdale in 2005 produced yield increases of 382 and 520 kg ha" for
thiamethoxam and imidacloprid, respectively. Thiamethoxam and imidacloprid

treatments at Sanilac at 2005 produced yield increases of 856 and 603 kg ha '1

77

respectively (Table 2.8). 2006 saw very little soybean aphid pressure, much like 2004.

As a result, no sites saw a signiﬁcant yield effect from insecticide seed treatment in 2006

 

 

 

 

 

 

 

 

 

 

(Table 2.9).
Table 2.5: Average temperature by month(°C), ﬁeld Sites 2003-2005. Long term
means (1970-2000) included for comparsion.
May June July August September
Grand Traverse 2003 11.7 17.3 20.0 21.1 16.3
(Kingsley) 2004 12.3 17.5 19.3 17.8 18.6
30 yr. 12.2 17.8 20.6 19.4 15.6
Hillsdale 2004 16.1 18.9 20.6 18.9 18.9
(Hillsdale) 2005 12.7 22.4 22.4 21.1 18.4
2006 14.2 19.2 22.8 20.9 15.3
30 yr. 14.1 19.3 21.5 20.3 16.1.
lngham 2004 14.6 18.1 20.1 18.4 1.7.6
(B. Iansing) 2005 12.3 21.9 22.0 21.8 18.3
2006 14.6 19.1 22.6 20.8 14.8
30 yr. 14.0 19.2 21.4 20.3 16.1
Lenawee 2004 16.4 19.9 21.6 19.6 18.9
(Tecumsem 30 yr. 14.4 19.4 21.7 20.6 16.7
Montmorency 2004 10.3 15.9 18.3 16.4 16.8
2005 10.6 19.8 20.7 18.9 16.6
30 yr. 10.6 16.1 19.4 17.8 13.9
Presque Isle 2006 13.2 18.7 23.0 20.0 14.9
(Rogers City) 30 yr. 11.1 16.4 19.6 18.6 14.3
Sanilac 2004 12.4 16.9 19.4 18.2 17.6
(Sandusky) 2005 11.8 21.2 21.6 20.9 18.1
2006 14.4 18.8 22.2 19.8 14.8
30 yr. 13.5 18.5 21.2 20.1 15.8

 

78

 

 

 

 

Table 2.6: Summary of combined insecticide effects, ANOVA for

grain yield by site-year.

 

P-values from ANOVA

2004 2005 2006
Grand Traverse 0.6807
Hillsdale 0.4064 0.0008 0.8199
lngham 0.0261 0.5069 0.5670
Lenawee 0.011 1*
Montmorency 0.4407 0.0478
Presque Isle 0.1430
Sanilac 0.2439 0.0003 0.1091

* signiﬁcant yield decrease, other signiﬁcant effects were increases

 

 

 

 

Table 2.7: Insecticide grain yield effects, 2004 by site (kg ha").

thiamethoxam 1 188
no insecticide 1074

(Corresponding Subplots) 114

1247
1241

imidacloprid
no insecticide

G.T. Hillsdale lngham Lenawee Mont.
3182 3727 4721 2137
3069 3389 4863 2109
113 338* -142* 28
3377 3750 4672 1886
311 1 3527 4848 1761
266 223 -174* 125

(Corresponding Subplots) 6

*signiﬁcant at P<0.05

Sanilac

4150

3991
159

3936
4108
-172

 

 

 

Table 2.8: Insecticide grain yield effects, 2005 by site (kg ha" ).

Hillsdale lngham Montmorency Sanilac

thiamethoxam 4298
no insecticide 3916
(Corresponding Subplots) 382*

imidacloprid 4265

no insecticide 3745
(Corresponding Subplots) 520*

*significant at P<0.05

3255 1858 3954
3034 1689 3098
221 169 856*
3104 2056 3818
3133 1933 3215
-29 123 603*

 

 

79

 

 

Table 2.9: Insecticide grain yield effects, 2006 by site (kg ha'1 ).

Hillsdale lngham Montmorency Sanilac

thiamethoxam 3386 3310 2278 3981
imidacloprid 3323 3233 2104 41 18
no insecticide 3356 3189 2227 4010

 

 

No significant insecticide effects at any site in 2006

 

In years when soybean aphid was not a pest problem in Michigan, only one site-
year showed a signiﬁcant positive response from insecticidal seed treatments.
Thiamethoxam and imidacloprid had highly signiﬁcant effects on yields at Hillsdale and
Sanilac sites in 2005, indicating that these products have efﬁcacy on soybean aphid early
in the season. Only the Sanilac site, which saw severe aphid pressure, was treated with a
foliar insecticide to control soybean aphid. By the time insecticide application occurred
at this site, signiﬁcant yield loss had already occurred. Yield contrasts showed no
signiﬁcant difference between thiamethoxam and imidacloprid at sites where insecticide

treatment was a signiﬁcant factor.

Insecticide-Inoculant Interaction

Contrasts were performed on yields at Montmorency sites in 2004 and 2005 for
Cell-Tech inoculated soybean between treatments which also had a neonicotinoid seed
treatment applied and treatments that did not. These were ﬁrst-time soybean production
sites where use of (Apron Maxx RTA) fungicide had signiﬁcant interaction effects in the
soybean inoculant study. The insecticides alone had no signiﬁcant impact on yield in

either year. The analysis showed that thiamethoxam and imidacloprid did not interact

80

 

Table 2.10: Grain yield treatment means for soybean inoculated with Cell-Tech
inoculanLMontmorency 2004 and 2005 (kg ha '1).

2004 2005

thiamethoxam 3405 thiamethoxam 2324
imidacloprid 35 5 1 imidacloprid 2417
no insecticide 3541 no insecticide 2353

No signiﬁcant dif erences

 

 

 

with soybean inoculant. Inoculated soybean yielded the same at these sites regardless of
insecticide seed treatment (Table 2.10).

Few studies found interactions between soil insecticides and bacteria. The limited
research on the neonicotinoid effects on bacteria has shown no ill effects on soil-home

bacteria (Singh and Singh 2005).

Soybean Aphid Population Effects

In 2005, neonicotinoid seed treatments signiﬁcantly effected on soybean aphid
pOpulations through R2. Soybean aphid p0pulation effects were signiﬁcant at both
lngham and Hillsdale sites, with aphid populations reduced by over 50% by both
treatments at both sites during the earliest count timings. However, aphid pressure was
very low at this time, averaging 6 and 10 adult aphids per plant at lngham and Hillsdale.
Insecticide effects on aphid p0pulation continued to be signiﬁcant through late R2, while
percent control fell for both treatments in each successive count timing. Late R2 was the
last growth stage in which insecticide treatments signiﬁcantly reduced aphid populations.
Aphid populations were reduced an average of 39% (15 vs. 9 adult aphids per plant) and
36% (42 vs. 26 adult aphids per plant) at Hillsdale and lngham sites at late R2. By the

R3 stage at Hillsdale (43 vs. 39 adult aphids per plant) and the R4 stage at lngham (450

81

vs. 348 total aphids per plant), there was no signiﬁcant difference in aphid number
between treated and untreated plots (Table 2.11 and 2.12).

These results corroborate observations made by Pedersen and Lang (2006); that
some aphid suppression can occur through about 60 days after planting (Pedersen and
Lang 2006). This study saw aphid suppression ending after late R2, which occurred at 57

and 69 days after planting at the In gham and Hillsdale sites, respectively.

 

 

 

 

 

Table 2.1 1: Effects of neonicotinoid seed treatment on soybean aphid pressure,

Hillsdale 2005 (average adult aphids/plant)

Count Timing Thiamethoxam Imidacloprid
Treated Control P-value Treated Control P-value

Early R2 (62 DAP) 6 11 0.0007 3 9 0.0004

Late R2 (67 DAP) 12 15 0.2194 5 14 0.0015

Early R3 (72 DAP) 37 45 0.2096 39 41 0.7530

 

 

 

Table 2.12: Effects of neonicotinoid seed treatment on soybean aphid pressure,
lngham 2005 (average aphids/plant)

 

 

Count Timing Thiamethoxam Imidacloyrid
Treated Control P-value Treated Control P-value
V7‘ll (43 DAP) 2 7 <0.0001 2 5 0.0005
Late R13 (49 DAP) 4 10 0.0022 8 16 <0.0001
Late R23 (57 DAP) 26 39 0.0343 28 45 0.0062
R41) (74 DAP) 388 546 0.2048 308 314 0.9624

a-Adult aphids counted
b-All aphids counted

 

 

 

Plant Stand Effects

As no pattern of interaction was observed between insecticide, ﬁJngicide, or
inoculant, mean contrasts were conducted across fungicide and inoculant classes.
Neonicotinoid seed treatments had signiﬁcant effects on plant stands at only 3 of 16 site-

years in this study (Table 2.13).

82

 

 

 

 

Table 2.13: Summary of insecticide main effects on plant stand, by site-
Lear.
P-values from ANOVA
2004 2005 2006

Grand Traverse 0.0962 ------------------
Hillsdale 0.0328 0.1722 0.1330
lngham 0.9276 0.0036 0.3382
Lenawee 0.2061 ------------------
Montmorency 0. 1914 0.9132 ---------
Presque Isle ----------------- 0.6615
Sanilac 0.0207 0.2796 0.1995

 

 

At Hillsdale and Sanilac in 2004, neonicotinoids protected soybean stand. At

lngham in 2005, negative effects on soybean stands were observed. (Table 2.14). With

sites combined for each year, no signiﬁcant difference in plant stand was observed

between insecticide treated and untreated soybean (Table 2.15).

No pattern of signiﬁcant stand improvement was observed. The greatest stand

improvement at any site—year was 10.4% at Hillsdale in 2004. While statistically

signiﬁcant, this level of stand loss at the plant populations observed would be unlikely to

cause signiﬁcant yield loss (Nafziger 2002). None of the sites that experienced a plant

stand difference also experienced a yield effect.

 

Table 2.14: Split plot contrasts of insecticide plant stand effects, sites with
signiﬁcant main effects (plants haLinsecticide treatments combined).

2004 Hillsdale 2004 Sanilac 2005 lngham

thiamethoxam 371,883 425 ,625 33 1,688
no insecticide 346,410 388,203 372,422
(Corresponding Subplots) 25,473 37,422* -40,734*
imidacloprid 385,752 404,792 359,903
no insecticide 349,280 401,957 367,325
(Corresponding Subplots) 36,472* 2,835 -7,422

 

*significant at P<0.05

 

 

83

 

Table 2.15: Plant stand means of insecticide treated plots, sites combined
for each year (plants ha", insecticide treatments combined).

 

 

2004 (Split Plots) 2005 (Split Plots)
thiamethoxam 396,734 thiamethoxam 288,01 7
no insecticide 392,004 no insecticide 289,766
imidacloprid 398,036 imidacloprid 285,239
no insecticide 389,944 no insecticide 282,438
2006 (Whole Plots)

 

thiamethoxam 358,878
imidacloprid 357,625
no insecticide 362,377

 

 

No signiﬁcant effects in any year

 

A Minnesota study conducted in 2002 found neonicotinoid seed treatments
improved soybean stand between 11.4 and 17.1% in early planted soybean (May 2), but
not in late planted soybean (May 23, Potter 2002). This study found the best overall
effects by the neonicotinoids on stand count was at Hillsdale in 2004. Planting at
Hillsdale in 2004 occurred on April 30th, the earliest of any site-year within this trial.
This planting was followed by record May rainfall. If these sites experienced delayed
emergence, some seedling loss due to early soil—borne insect pressure may have occurred.
However, early May plantings in other site-years did not experience increased plant

stands.

Plant Height Effects
Data from early season plant height measurements (5-6 weeks after planting)
showed little evidence to support the claim of increased plant height from neonicotinoid

insecticide treatments. Over 16 site-years, insecticide main effects were signiﬁcant ﬁve

84

times (Table 2.16). Contrasts performed at these 5 site-years showed only two sites
where signiﬁcant plant height increases resulted from insecticide seed treatments (Table
2.17). At Hillsdale in 2004, imidacloprid provided a 1.57 cm plant height increase over
untreated plants. However, thiamethoxam did not provide a signiﬁcant plant height
increase at this site. At In gham in 2005, both thiamethoxam and imidacloprid provided

increases in plant height, of 2.28 and 3.28 cm respectively.

 

 

 

 

 

 

 

Table 2.16: Summary of insecticide main effects on early plant height, by site (P-
values from ANOVA).
2004 2005 2006

Grand Traverse 0.4713 --------- ---------
Hillsdale 0.0271 0.4628 0.8202
lngham <0.0001 0.0145 0.0077
Lenawee 0.0720 --------- ---------
Montmorency 0.0523 0.2010 ---------
Presque Isle ----------------- 0.2926
Sanilac 0.0002 0.1040 0.5771
Table 2.17: Contrasts of insecticide effects on early plant height (cm), site-
years with signiﬁcant positive insecticide effects.
Hillsdale 2004 P-value P-value
thiamethoxam 30.49 imidacloprid 30.74
no insecticide 30.00 no insecticide 29.17

0.49 0.3428 1.57 0.0031
lngham 2005
thiamethoxam 24.79 imidacloprid 25.79
no insecticide 22.41 no insecticide 22.41

2.28 0.0303 3.28 0.0024

 

 

 

When sites are combined over year, not only were insecticide main effects not
signiﬁcant, but treatment means between insecticide treated and no insecticide plots were
nearly numerically identical in 2004 and 2005 (Table 2.18). The increased numerical

difference in 2006 is attributed to the signiﬁcant effects seen at the lngham site alone.

85

 

 

 

Table 2.18: Treatment means, plant height (cm), by year, sites combined.
2004 2005

thiamethoxam 2 1 .35 thiamethoxam 19.52
No Insecticide 2 1.45 no insecticide 19.53
imidacloprid 2 1 . l8 imidacloprid 19.40
no insecticide 21 . 15 no insecticide 18.92
2006

thiamethoxam 21 .26

imidacloprid 21 .63

no insecticide 20. 72

no signiﬁcant insecticide. elfects in any year

 

Overall, any positive effects of neonicotinoid seed treatments on early season plant height
were isolated.

Plant heights at harvest were taken for 6 site-years, at Montmorency and Lenawee
in 2004, Montmorency and Hillsdale in 2005, and Sanilac and Hillsdale in 2006. Results
for plant height at harvest were similar to those of the early plant heights. Of the six sites
that were sampled for plant height at harvest, two had signiﬁcant main insecticide effects:
Montmorency in 2005 and Hillsdale in 2006. At Montmorency in 2005, neither
thiamethoxam nor imidacloprid increased plant height compared with the insecticide
subplots, though the contrast between thiamethoxam and imidac10prid treatments gave
thiamethoxam a 3.38 cm height advantage. At Hillsdale in 2006, imidacloprid treated
plants were signiﬁcantly taller than both thiamethoxam treated plants and no insecticide
plants (Table 2.19).

No pattern of signiﬁcant plant height increase could be attributed to the use of

neonicotinoid insecticide seed treatments. Although in certain site-years some

86

differences could be observed, such as at lngham in 2006, height differences were

infrequent and small.

 

Table 2.19: Signiﬁcant contrasts of insecticide effects on harvest plant
height (cm).

Montmorency 2005 P-value
thiamethoxam 61 .60
imidacloprid 58.22

3.38 0.0246
Hillsdale 2006
imidacloprid 92.42 imidacloprid 92.42
thiamethoxam 88.30 no insecticide 87.95

4.12 0.0321 5.07 0.0209

 

 

 

Other Plant Growth Indicators

No site-years showed any signiﬁcant main effects of insecticide seed treatment on
average pods per plant. Annual pod count means were higher in 2006 than 2004 and
2005, consistent with overall grain yield for the speciﬁc site (Table 2.20). No signiﬁcant
differences in SPAD chlorophyll indexes were observed at three of four site years in
which these measurements were taken. The only site where insecticide main effects were
signiﬁcant was Hillsdale in 2006. Imidacloprid treated soybean plants at Hillsdale in
2006 had a higher mean SPAD index than thiamethoxam treated soybean plants (Table
2.21). This was the only signiﬁcant insecticide effect observed at any of the four site
years where SPAD readings were taken. Two other site-years, Hillsdale 2005 and
lngham 2006 saw both thiamethoxam and imidaCIOprid have numerically but not
signiﬁcantly higher SPAD chlorophyll indexes. So while a trend for increased SPAD
chlorOphyll index readings from neonicotinoid seed treatments may exist, signiﬁcant

differences again appear to be isolated.

87

 

 

 

 

Table 2.20: Treatment mean; pods per plant by year, sites combined.
2004 2005

thiamethoxam 27. 13 thiamethoxam 28.20
no insecticide 28.87 no insecticide 29.37
imidac10prid 29. 18 imidaCIOprid 27.06
no insecticide 30.12 no insecticide 27.96
2006

thiamethoxam 41 .44

imidacloprid 40.27

no insecticide 39.76

no signiﬁcant insecticide effects in any year

 

 

Table 2.21: Insecticide treatment contrasts, mean SPAD chlorophyll index,
Hillsdale 2006.
P-value P-value
imidacloprid 47.88
no insecticide 46.93
0.95 0.0524

thiamethoxam 46.73
no insecticide 46.96
0.23 0.6174

thiamethoxam 47.88
imidac10prid 46.73
1.15 0.0148

 

 

 

There has been much speculation about the potential for neonicotinoids to have
plant growth “vigor” effects even with negligible insect pressure. Claims have been
made and a United States patent has been granted regarding neonicotinoid plant vigor
effects (Senn et. a1. 2004). A recent press release by Syngenta (December 5, 2006) states
that the company has discovered the mechanism by which thiamethoxam produces a
vigor effect. This mechanism is purportedly a plant response elicited by the chemical
that results in an increase of plant produced proteins that convey stronger plant defenses
under stressful conditions. More published research to support these ﬁndings would be

beneﬁcial.

88

Greenhouse Trial

Five sets of measurements of plant growth were taken during the greenhouse
experiment, plant height, SPAD index readings, leaf area index, and plant dry weight.
There was no signiﬁcant difference in plant height between thiamethoxam-treated,
imidacloprid-treated, untreated plants at any measurement timing under greenhouse
conditions (Figure 2.1). SPAD chlorOphyll indices showed no signiﬁcant insecticide
treatment effect at any timing (Figure 2.2). Leaf area index, fresh plant weight, and dry
plant weight were statistically similar with runs 1 and 2 combined. The results of this
greenhouse trial corroborate the ﬁeld observations of negligible to no growth effects from

insecticidal seed treatments.

4 N
on O

.L
0)

Plant Hem (cm)

-—. _-....-—._......-. -Hw--.__~_ c.-.- .._....

14

12

10:

.-Wee_k 3,.

 

We?k4.._

,. _ v)-.. " *Untraaj
u.» thiamethom
—.- imidacloprid

Figure 2.1: Plant height by insecticide treatment and week (cm, runs combined).

89

39

37

35 ‘
X 1
o
‘g 33 *Untreated
0 .. .. . ‘
< I Crwser
31 ~k-Gaucho
31
29 »

 

Week 2 Week 3 Week 4 Week 5

Figure 2.2: Greenhouse SPAD chlorophyll indices by treatment and week (runs
combined).
Economic Return from Neonicotinoid Seed Treatment Use

Economic return on use of neonicotinoid seed treatment was calculated by taking
the numerical yield difference found in insecticide treated plots (Tables 2.6-2.8),
multiplying that difference by the average soybean price received by farmers in each
year, and subtracting the cost of the seed treatment. Substantial economic gain was seen
at Hillsdale and Sanilac in 2005. However, these gains are offset over the course of this

study, as most sites in 2004 and 2006 saw a net loss by using neonicotinoid seed

90

treatments (Table 2.21). Based on the cost of these products, a 130 to 170 kg ha'] yield

increase would typically be required to recuperate the cost of seed treatment.

 

Table 2.21: Economic return on use of neonicotinoid seed treatments

 

 

thiamethoxam imidac10prid
Yield Effect Net Return: Yield Effect Net Return.
Year Site (kg ha'l) ($ ha") (kg ha'l) ($ ha’1)
2004 Grand Traverse 114 -15.59 6 -34.32
2004 Hillsdale 11 -15.80 266 20.20
2004 lngham 338 31.37 233 13.28
2004 Lenawee -142 -65.35 -174 -72.06
2004 Montmorency 28 -33.62 125 -9.36
2004 Sanilac 159 -6.16 -172 -71.64
2005 Hillsdale 382 40.75 520 73.67
2005 lngham 221 6.93 -29 -41.67
2005 Montmorency 169 -3.99 123 -9.73
2005 Sanilac 859 140.97 603 91.11
2006 Hillsdale 30 -32.79 -33 -42.96
2006 lngham 121 -12.43 44 -25.73
2006 Montmorency 51 -28.09 123 -8.06
2006 Sanilac -29 -45.98 108 -11.42

*Economic Assumptions
2004 Average Price Paid in Michigan, $02097 kg" ($5.72/bu.)
2005 Average Price Paid in Michigan, $0.210] kg" ($5.73/bu.)
2006 Average Price Paid in Michigan, $02237 kg" ($6.10/bu.)
(USDA-NASS 2007, Prices do not include LDP and other government payments)

Cost of Cruiser Maxx treatment (thiamethoxam and mefenoxam + ﬂudioxonil),
$39.50/ha. (Cruiser only treatment no longer available)

Cost of Gaucho (imidacloprid) treatment, $35.58/ha
Determined through consultation with agribusiness

 

 

 

 

9I

Conclusions

Neonicotinoid seed treatments had a positive yield effect when soybean aphid
pressure was high. While aphid p0pulations were suppressed well into the growing
season, was lost 40 to 60 days after planting. Efﬁcacy of seed treatments on soybean
aphid control and soybean grain yield depends on the time at which soybean aphid begins
to colonize soybean in Michigan. In 2004 and 2006, only one site-year saw a positive
yield effect from use of neonicotinoid seed treatments. Based upon these results, it is
difﬁcult to recommend regular use of neonicotinoid seed treatments unless threshold soil
borne insect populations are present around planting time, or major pest infestations are
expected within the ﬁrst two months of the growing season. These experiments were
performed in a conventionally tilled system, and farmers may see a beneﬁt to using
neonicotinoids seed treatments in a no-till system, where soil-bome insect populations are
less disturbed in the spring and soil temperatures rise slowly.

Indicators of crop health such as stand count, plant height, SPAD meter readings,
leaf area indices both in the ﬁeld and in the greenhouse trial did not support claims of
improved crop health as an effect of neonicotinoid seed treatments. Only one site year,
lngham in 2006, showed a marked improvement in plant height by both thiamethoxam
and imidacloprid. Observed neonicotinoid impacts on soybean plant health appeared to

be very isolated in these trials, despite the stressful conditions observed at some sites.

92

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95

 

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ll

 

     

 

 

580

 

 

 

 

 

     

 

  

RSITY LIBRARIES

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