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thesis entitled

EFFECTS OF FERTILIZATION ON THE SOYBEAN APHID,
APHIS GL YCINES MATSUMURA AND THE EFFECTS OF
SOYBEAN APHIDS AND FERTILIZATION ON SOYBEAN
PLANTS

presented by

Abigail Jan Walter

has been accepted towards fulﬁllment
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EFFECTS OF FERTILIZATION ON THE SOYBEAN APHID, APHIS GL YCINES
MATSUMURA AND THE EFFECTS OF SOYBEAN APHIDS AND
FERTILIZATION ON SOYBEAN PLANTS
By

Abigail Jan Walter

A THESIS

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

MASTER OF SCIENCE
Department of Entomology

2005

ABSTRACT
EFFECTS OF FERTILIZATION ON THE SOYBEAN APHID, APHIS GL YCINES
MATSUMURA AND THE EFFECTS OF SOYBEAN APHIDS AND
FERTILIZATION ON SOYBEAN PLANTS
By
Abigail Jan Walter

Soybean aphid, Aphis glycines Matsumura, is an invasive agricultural pest ﬁrst reported
in North America in 2000. Soybean aphid populations appeared to be higher in areas
where plants exhibited symptoms of potassium deﬁciency. In order to determine whether
soil potassium deﬁciency affected aphid population levels, the effect of soybean aphids
and soil potassium level on growth and yield characteristics of soybeans, and a possible
mechanism for the soybean aphid-soil potassium interaction, ﬁeld surveys and controlled
cage studies were conducted. In both types of studies, soybean aphid pOpulations were
higher on plants with a lower level of potassium nutrition, and this was due to an increase
in individual aphid fecundity on deﬁcient plants. Phloem asparagine content was also
negatively correlated with soil potassium level, and corresponded to aphid populations.
Changes in asparagine content are suggested as the nutritional mechanism for the
soybean aphid-soil potassium interaction. Plants with high soybean aphid populations
had fewer leaves, pods, and nodes than uninfested plants; the effect of soil potassium on

plant characteristics was variable. When soybean aphid populations were low, they were

unaffected by foliar nutrient sprays.

To Vicki

iii

ACKNOWLEDGEMENTS

I would like to thank the soybean growers of southwest Michigan who allowed me access
to their ﬁelds for sampling, especially Jack Phillips, who allowed me to conduct cage
studies in his ﬁelds. I’d also like to thank the county extension agents that helped me
with ﬁnding sites and sampling, especially Bruce MacKellar and Mike Staton. I’d like to
thank Mike J ewett for help with ﬁeldwork and the people at the Macromolecular
Structure Facility for help with HPLC analysis. Mat Bet, Chris Dechelbur, Ben Kaeb,
Jessica Kolp, Becky Larson, Ben Nessia, and Dan Olds were a great help in ﬁeldwork.

Finally, I’d like to thank my advisor Chris DiFonzo for all of her guidance and support.

iv

TABLE OF CONTENTS

List of Tables ........................................................................................ vii
List of Figures ......................................................................................... x
CHAPTER 1
Introduction to the biology and nutritional ecology of the soybean aphid,
Aphis glycines, and the impact of potassium fertilization on soybean physiology .......... 1
CHAPTER 2
Soil Potassium Inﬂuences Soybean Aphid Population Size in Soybean ................... 19
Introduction ................................................................................. 19
Methods ..................................................................................... 22
Field Surveys ..................................................................... 22
Controlled Cage Studies ......................................................... 24
Clip Cages ......................................................................... 27
Results .............................................................................. 29
Field Surveys ....................................................................... 29
Controlled Cage Studies ....................................................... 3O
Clip Cages ......................................................................... 34
Discussion .......................................................................... 35
Conclusion ......................................................................... 38
CHAPTER 3
Improved nitrogen nutrition for the soybean aphid leads to higher aphid
populations on potassium deﬁcient soybean .................................................... 62
Introduction ................................................................................. 62
Methods ..................................................................................... 64
Field Surveys ...................................................................... 64
Controlled Cage Studies ......................................................... 65
Phloem Sampling and Analysis ................................................ 67
Statistical Analysis ............................................................... 69
Results .............................................................................. 70
Field Surveys ...................................................................... 70
Controlled Cage Studies ......................................................... 70
Discussion ........................................................................... 71
CHAPTER 4
The impacts of foliar fertilization on soybean aphids and soybeans ......................... 84
Introduction ................................................................................ 84
Methods ..................................................................................... 85
Aphid Counts ...................................................................... 86
Foliar Nutrient Sampling and Analysis ........................................ 86

Yield ............................................................................... 87

Statistical Analysis ............................................................... 87
Results ...................................................................................... 87
Aphid Counts ...................................................................... 87
F oliar Nutrient Sampling and Analysis ........................................ 88
Yield ............................................................................... 88
Discussion .................................................................................. 88
CHAPTER 5
Studies of the effects of sooty mold on the growth and yield of soybeans .................. 96
Introduction .................................................................................. 96
Methods ...................................................................................... 96
2002 Field Experiment ........................................................... 97
2002 Shade Experiment .......................................................... 98
2003 Cage Experiment ............................................................ 99
Results ..................................................................................... 100
2002 Field Experiment .......................................................... 100
2002 Shade Experiment ........................................................ 101
2003 Cage Experiment .......................................................... 102
Discussion ................................................................................. 103
APPENDICES ..................................................................................... 109
APPENDIX B—
Record of the Deposition of Voucher Specimens .................................... 110

vi

LIST OF TABLES

Table 1.1. Nutritional requirements of aphids ................................................. 14

Table 2.1: Table 2.1: Location of potassium deﬁcient commercial ﬁelds surveyed 14—15
August 2003 and 17-23 August 2004, Southwest Mi ................................. 45

Table 2.2. Initial soil test values for potassium deﬁcient ﬁelds used in controlled cage
studies, 2003 and 2004, Van Buren County, Michigan .............................. 46

Table 2.3. Comparisons of soil test levels, aphid number, and plant characteristics from
putative potassium deﬁcient (yellow) and less deﬁcient (green) pairs of sample
sites in eight commercial soybean ﬁelds, Southwest Michigan, 14-15 August
2003. p values based on a t-test to determine whether differences were
signiﬁcantly different from zero ......................................................... 47

Table 2.4. Comparisons of soil test levels, aphid number, plant characteristics, and
harvest characteristics (two ﬁelds) from putative potassium deﬁcient (yellow) and
less deﬁcient (green) pairs of sample sites in ﬁve commercial soybean ﬁelds,
Southwest Michigan, 2003. Surveys took place 17-23 August and harvest took
place in October. p values based on a t-test to determine whether differences
were signiﬁcantly different from zero .................................................. 48

Table 2.5. Soil potassium levels inside the cages in the 2003 controlled cage study ...... 49

Table 2.6. Soil potassium levels inside the cages in the 2004 controlled cage study after
treatment. Fertilization took place on 13 May and soil was sampled on 4 June
................................................................................................ 50

Table 2.7. Signiﬁcant differences in plant characteristics based on the presence of
soybean aphids in the 2003 controlled cage study. Cages were infested on 8 July
and sampling took place from 29 July through 28 August. t values and
probabilities are based on Fisher’s protected LSD .................................... 51

Table 2.8. Signiﬁcant differences in soybean yield parameters in the 2003 controlled
cage study based on the presence of absence of soybean aphids. Results are based
on an AN OVA with aphids as a ﬁxed factor and replicate as a random factor... 52

Table 2.9. Signiﬁcant differences in plant characteristics of uninfested soybean plants
based on potassium fertilization in the 2004 controlled cage study. The ﬁeld had
an initial soil potassium level of 67 ppm. Results are based on Fisher’s protected
LSD .......................................................................................... 53

vii

Table 2.10. Signiﬁcant differences in plant characteristics of soybean aphid-infested
soybean plants based on potassium fertilization in the 2004 controlled cage study.
The ﬁeld had an initial soil potassium level of 67 ppm. Results are based on
Fisher’s protected LSD ................................................................... 54

Table 2.11. Signiﬁcant differences in plant characteristics of soybean plants based on
soybean aphid infestation when the soil was not fertilized with potassium in the
2004 controlled cage study. Plants were infested on 28 May and the aphid
population was allowed to increase without control. The initial soil potassium
level of the ﬁeld was 67 ppm. Results are based on Fisher’s protected LSD... .. 55

Table 2.12. Signiﬁcant effects of soybean aphid infestation on soybean plant
characteristics of potassium fertilized plants in the 2004 controlled cage study.
Soybean aphids infestation took place on 28 May and the population was allowed
to increase without control. The ﬁeld had an initial soil test level of 67 ppm
potassium and was fertilized with 267 kg/ ha 0-0-62 fertilizer. Results are based
on Fisher’s protected LSD ................................................................ 56

Table 2.13. Signiﬁcant differences in soybean plant characteristics of caged plants based
on the presence of soybean aphids for characteristics without an aphid x
potassium interaction in the 2004 controlled cage study. Plants were infested on
28 May. Results are based on Fisher’s protected LSD ............................... 57

Table 2.14. Signiﬁcant differences in soybean plant characteristics based on potassium
amendment for those traits without a signiﬁcant aphid x potassium interaction in
the 2004 controlled cage study. Initial soil potassium level was 67 ppm and aphid
infestation took place on 28 May. Results are based on Fisher’s protected
LSD .......................................................................................... 58

Table 2.15. Signiﬁcant differences in soybean yield parameters based on soybean aphid
infestation in the 2004 controlled cage study. Infestation took place on 28 May,
and the soybean aphid population was allowed to increase without control. The
ﬁeld had an initial soil potassium level of 67 ppm and was fertilized with 267 kg
/ha of 0-0-62 fertilized. Results are based on Fisher’s protected LSD .......... 59

Table 2.16. Signiﬁcant differences in soybean yield parameters based on potassium
fertilization in the 2004 controlled cage study. Infestation took place on 28 May,
and the soybean aphid population was allowed to increase without control. The
ﬁeld had an initial soil potassium level of 67 ppm and was fertilized with 267 kg
/ha of 0-0-62 fertilized. Results are based on Fisher’s protected LSD ............ 59

Table 3.1. Slope, r-square value, and p value of the amino acids signiﬁcantly correlated
with soil potassium level ﬁve commercial soybean ﬁelds in the 2004 ﬁeld survey
on 17-23 August. Soybeans were setting seed and the soybean aphid populations
were extremely low ...................................................................... 79

viii

Table 3.2. Slope, r-square value, and p value of the amino acids signiﬁcantly correlated
with soil potassium level in the 2004 cage study 15 July sampling date. Soybean
were in full ﬂower. All phloem samples were taken from uninfested
plants ......................................................................................... 80

Table 4.1. Plot details and maintenance dates for the 4 sites in the study ................. 93

Table 5.1. Plant characteristics of potted soybeans receiving various levels of shading in
the 2002 shade experiment. Numbers in the same row followed by the same letter
are not statistically signiﬁcant at p<0.05 in a Fisher’s protected LSD ........... 105

Table 5.2. Effect of aphid infestation of plant characteristics throughout the season in the

2003 cage study t values and p values are the result of Fisher’s protected LSD
.............................................................................................. 106

Table 5.3. Effect of sugar application on plant characteristics throughout the season in
the 2003 cage study t values and p values are the result of Fisher’s protected LSD
.............................................................................................. 1 07

ix

LIST OF FIGURES

Figure 2.1. Schematic of ﬁeld cages used in the 2003 and 2004 controlled cage
experiments. Cages consisted of a PVC frame (dark bars) covered by a cage of
no-see-um mesh (gray shading) with a VelcroTM door (dotted
line) ................................ 41

Figure 2.2. Soybean aphids per plant during the 2004 controlled cage study on plants
with and without potassium fertilization in a ﬁeld with an initial potassium level
of 67 ppm. * Denotes sample dates where the treatments were different at
p<0.05. *** Denotes sample dates where the treatments were different at
p<0.0001..................... 42

Figure 2.3. Soybean aphid nymphs produced per mother per day of reproduction on
potassium fertilized and unfertilized plants in a ﬁeld with an initial soil potassium
level of 67 ppm in the earliest run of the clip cage experiment (June 10). The
results are signiﬁcantly different in an ANOVA. p =
0.0271 ............................................ 43

Figure 2.4. Response of soybean aphids to potassium soil amendment, expressed as A)
age of aphids at ﬁrst reproduction (days) and B) total number of young produced
per cage on potassium fertilized and unfertilized plants in a ﬁeld with an initial
soil potassium level of 67 ppm for the clip cage study begun on July 14, 2004.
Both are signiﬁcantly different in an ANOVA with
p<0.05 ............................................................. 44

Figure 3.1. Proportion of asparagine in the phloem amino acid proﬁle versus soil
potassium level in eight commercial soybean ﬁelds in the 2003 Survey (p = 0.03).
Samples were taken during a soybean aphid outbreak when aphid populations
were commonly over 10,000 per plant. Samples were taken August 13-14 2003,
just as the soybean were beginning to set seed ........................................ 75

Figure 3.2. Soybean aphids per leaf versus the proportion of asparagine in the phloem
free amino acids in three commercial soybean ﬁelds with active soybean aphid
populations in the 2003 ﬁeld survey (p = 0.0722) ..................................... 76

Figure 3.3. Proportion of asparagine in the phloem amino acid proﬁle versus soil
potassium level in ﬁve commercial soybean ﬁelds in the 2004 Survey (p = 0.055).
Samples were taken 17-23 August 2004 during soybean seed set. Aphid
populations were extremely low ......................................................... 77

Figure 3.4. Proportion of asparagine in the phloem amino acid proﬁle versus soil
potassium level in the 2004 Cage Study on the July 15 sampling date (p = 0.002).
Soybeans were in full ﬂower. All samples were taken from uninfested
plants ........................................................................................ 78

Figure 4.1. Aphid counts of fertilized and unfertilized plants at university farms sites
during the 2004 ﬁeld season. Counts are not signiﬁcantly different at either site

on any sampling date (p>0.05) .......................................................... 93

Figure 4.2. Yield of soybean at four experimental sites where foliar fertilizer was
applied. In an ANOVA, there were no differences at p<0.05 ........................ 94

xi

Chapter l—Literature Review
Introduction

Soybean aphid (Aphis glycines Matsumura) is an invasive agricultural pest that
ﬁrst appeared in the United States in 2000 (V enette and Ragsdale 2004). It is the most
important insect pest of soybean in Chine, where it originated (Wu et al. 2004), an it has
rapidly risen to become the most important insect pest of soybeans in North America.
There are currently no control strategies for soybean aphids in North America except for
the application of chemical insecticides. Other control strategies are sorely needed so
that soybean aphid control can be accomplished in the context of integrated pest
management.

In general, aphids are greatly affected by the nutritional quality of their host plant
(Honek 1991, Douglas 1993, Havlickova and Smetankova 1998, Ponder et a1. 2000,
Cisneros and Godfrey 2001, Nevo and C011 2001, J ansson and Ekbom 2002, Koyama et
a1. 2004). This can be manipulated through a variety of cultural practices, most notably

fertilization.

Distribution, Life History, and Phenology

The soybean aphid originated in China, where it cycles between species of
Rhamnus as the primary host and soybean, Glycine max, and wild relatives in the Glycine
genus such as Glycine soja. It is currently distributed in China, Japan, the Philippines,
South Korea, Indonesia, Malaysia, Thailand, Vietnam, Russia, Australia, the United

States, and Canada (Wu et a1. 2004).

Soybean aphid has a host-alternating life cycle typical of Aphis species. The
primary hosts are woody plants in the genus Rhamnus. In North America, the only
species known to be suitable for soybean aphid are Rhamnus cathartica L. and Rhamnus
alnifolia L’Hértier (Voegtlin et al. 2004). The eggs overwinter on the primary hosts,
hatching into fundatrices in the spring. The ﬁindatrices have several generations on
Rhamnus with each subsequent generation giving birth to proportionately more
fundatrigeniae that migrate to soybean. Many generations of alate and apterous soybean
aphids follow on soybean. Aphid populations on soybean can increase very rapidly,
doubling every two to three days in a predator free environment (Program 2004). In the
fall, soybean aphids on soybean give rise to gynoparae and winged males and then ﬂy to
the primary host. The gynoparae give birth to oviparae that mate with the males and

oviposit eggs.

Population Dynamics

Soybean aphid was ﬁrst discovered in North America in 2000. In 2000 and 2001,
soybean aphid infestations were severe, causing up to 40% yield reduction in different
parts of Michigan (DiFonzo 2002). In 2002, soybean aphid populations were low. There
was an aphid outbreak again in 2003 with yield loss up to 50% in unsprayed ﬁelds in
some parts of Michigan (DiFonzo, personnel communication). In 2004 aphid populations
were again very low. In China the soybean aphid is also a sporadic pest. Factors that
may induce a soybean aphid outbreak in China include higher temperatures, high
precipitation while soybean aphid is on buckthom, low precipitation when soybean aphid

is on soybean, low humidity, overwintering, planting date, soybean variety, the presence

of natural enemies especially coccinellids, and the synchronization of soybean and

soybean aphid phenology (Wu et al. 2004).

Agricultural Impact and Control

Soybeans are an important world crop, and especially important in the North
Central Region of North America, which has been severely impacted by the soybean
aphid. The North Central Region produces approximately 40% of the world’s soybean
crop (Anonymous 2005). The value of the 2003 US. soybeancrop exceeded $18.4
billion (Anonymous 2004b). In 2003 Michigan planted 809,000 ha of soybean,
producing 1.46 metric tones of beans with a value of US $387 million (Anonymous
2004b). In China severe infestations of soybean aphid can lead to 30% (Wang et al.
1996, Sun et al. 2000) to 70% (Wu et al. 2004) yield reduction, so the potential for yield
loss in North America is very great.

Since its initial discovery in North America in 2000, soybean aphid spread rapidly
in the United States and Canada and now affects much of the soybean-producing region
of North America. Predictive modeling suggests that it is likely to spread to all or nearly
all US. soybean-producing regions (Venette and Ragsdale 2004). In 2001 and 2003,
unsprayed ﬁelds in Michigan suffered yield losses of up to 40-50%. During 2003 an
estimated 770,000 acres or 36% of soybean acreage was sprayed with a chemical
insecticide in Michigan (Difonzo, personnel communication). Prior to the occurrence of
soybean aphid, soybeans were rarely treated with insecticide in Michigan, so these
numbers reﬂect a huge increase in the amount of insecticides being released into the

environment. Currently, there are no strategies for control of soybean aphid other than

chemical application. Organic production, an increasing market in Michigan, is also
severely impacted by the lack of non-chemical control options for soybean aphid.

The soybean aphid may damage the soybean beginning in the vegetative stages all
the way through seed set. In their review of the Chinese literature, Wu et al. reported that
soybean aphid may cause leaf distortion early defoliation, and stunting of the plant.
Soybeans with a severe aphid infestation have fewer branches, pods, seeds, and a lower
weight per seed than uninfested plants (Wu et al. 2004). Other studies have shown that
soybean aphid infestation reduces the number of pods per soybean plant and the total
weight of beans produced but does not affect individual bean weight (Wang et al. 1996).
In addition, sooty mold may grow on the honeydew excreted by soybean aphids. The
presence of honeydew on foliage may reduce the amount of light that penetrates the
foliage (Wood et al. 1988, Sparks and Yates 1991). In soybean, this shading effect has
been shown to reduce yield and seed quality (Wu et al. 2004).

Xibei et al. (year unknown) have published a soybean aphid threshold of 500
soybean aphids per 100 plants and a 35% colonization rate. However, the North
American experience shows that the threshold may be very different on this continent.
Extension specialists in the North-Central Region recommend chemical control of
soybean aphid when the population reaches 250 aphids per plant on 90% of the plants
examined, and when the population is actively increasing. Chemical control should take
place prior to the R6 (full seed) stage of soybeans (Anonymous 2004a). The preferred
method for chemical control is through foliar insecticide sprays, although seed treatments
may offer early season protection (Anonymous 2004a). Although there are efﬁcacy

differences among foliar insecticides, all of the products so far investigated offer the

same level of yield protection with the exception of dimethoate (DiFonzo 2002). All of
the conventional sprays would kill beneﬁcial insects as well as soybean aphids, so that
biological control later in the year may be impacted. Also, certain organic products
containing pyrethrum may fail to control soybean aphids or even cause a spike in soybean
aphid populations. This is probably due to the high activity of these products against

natural enemies.

Aphid Nutrition

Aphids feed by tapping into sieve elements and ingesting the phloem sap that is
being translocated through them (Srivastava 1987). Thus, they have access to only those
plant compounds that may be translocated in the phloem sap. Phloem is known to
contain water, sugars, alcohols, proteins, free amino acids and amides, plant hormones
(including steroids), ATP, auxins, gibberellins, cytokinins, certain herbicides (such as
2,4-D), vitamins (such as thiamine, niacin, pantothetic acid, Big-complex vitamins, myo-
inositol, and ascorbic acid), some viruses, and elemental potassium, phosphorus,
magnesium, iron, manganese, copper, molybdenum, cobalt, and titanium (Ziegler 1975).

The pH of phloem sap is 7.2-8.5 (Ziegler 1975).

Essential Nutrients

Aphids are prodigious feeders, ingesting their own weight in phloem sap every
day as adults and many times their own weight per day as nymphs (Dixon 1998). Yet
they are also remarkably efﬁcient feeders. Even on a nutritionally poor diet, they are able

to sustain not only their own lifelong embryogenesis but, because of their unique

telescoped generations, the beginning stages of embryogenesis in their progeny (Dixon
1998). In order to accomplish this, every aphid must be able to extract enough nutrients
from the phloem sap ingested each day to feed three generations.

Aphids have a respiratory quotient of 1.0, indicating that they utilize
carbohydrates for respiration. The main carbohydrate used by aphids is sucrose.
Individuals of the alate morph require more sucrose then their apterous counterparts
(Klingauf 1987). This is probably because the alatae must ﬁrst build and maintain wings
and the associated musculature, and must store enough food to embark on migrations that
may take several days and cover hundreds of kilometers. Thus, their energy requirements
are very different than those of apterae, which are assured of a constant energy rich food
source and do not develop or maintain wing muscles.

Although the exact nutritional requirements of soybean aphids are not known,
there are a number of nutrients commonly required by aphid species (Table 1) (Dadd et

al. 1967, Srivastava 1987, Nation 2001).

Amino Acids and N-limitation

The acquisition of nitrogenous compounds is the main challenge facing aphids
feeding on their natural host plants (Terra 1988). There is only one instance of a
proteinase (a cathepsin-I-like cysteine proteinase) occurring in the gut of an aphid,
Acyrthosiphon pisum (Harris). However, this enzyme probably plays a role in
detoxiﬁcation of ingested material rather than a role in the nutrition of the aphid
(Cristofoletti et al. 2003). In general, aphids are not thought to use proteinases as part of

their nutritional digestion. This is probably because the high levels of proteinase

inhibitors combined with the extremely low protein concentration typically encountered
in phloem sap make plant proteins a poor nitrogen source (Sandstrom and Moran 2001).
Furthermore, many of the proteins transported in the phloem are defensive proteins.
Often these proteins must be cleaved by herbivore proteinases in order to become active.
Thus, by not cleaving phloem proteins, aphids may circumvent this plant defense.
Aphids are thought to obtain all of their dietary nitrogen from amino acids being
translocated in the phloem sap. Rhopalosiphum padi (L.) have growth rates directly
correlated to concentration of amino acids in the phloem of their host plants (Weibull
1987)

There is evidence that the growth of aphid embryos, and hence the growth of an
aphid population, is limited by the availability of essential amino acids. Aphids have a
unique mode of reproduction in which aphid embryos receive nutrients from the mother’s
hemocoel until they are larviposited as ﬁrst instars. When the mother has a good quality
diet, nearly all of the essential amino acids phenylalanine, threonine, and lysine in her
hemolymph are taken up by her embryos. Larval development quickens and slows with
the maternal supply of these essential amino acids (Wilkinson and Ishikawa 1999).

Aphids are among the most versatile animals in respect to nitrogen nutrition
because they possess a primary intracellular symbiotic bacterium, Buchnera as well as
secondary gut symbionts, which are able to manufacture certain essential amino acids
from nonessential amino acids in the aphid diet (Moran et al. 2003). Buchnera may be
able to provide their hosts with synthesized essential amino acids at a higher rate than
aphids are able to bring these substances across the gut wall (Douglas 1998). For that

reason, not every aphid species requires all of the essential amino acids; the amino acids

that are required varies between aphid species and populations because of differences in
their symbionts (Srivastava 1987).

Among the amino acids, several are especially important to aphids. Dadd and
Kreiger (1968) showed that methionine has an important phagostimulatory effect that
increases host plant acceptance and reduces the tendency of aphids to leave a feeding site
(restlessness). They also found that without one of the non-essential amino acids
glutamine, glutamic acid, asparagine, aspartic acid, alanine, or serine almost no aphid
growth occurred (Dadd and Krieger 1968). Buchnera manufacture essential amino acids
from non-essential amino acids that occur more commonly in their hosts’ diets. Good
evidence exists that glutamate is the principal nitrogenous compound used by Buchnera
to synthesize essential amino acids (Douglas 1998, Wilkinson and Ishikawa 1999).
Indeed, glutamate and aspartate are known to be transported across the mycetome
membrane from aphid hemocoel to Buchnera cells (Moran et al. 2003). On oats and
barley, R. padi growth is greatest at the times in plant development when the most

asparagine is being transported in the phloem (Weibull 1987).

Nitrogen in the Soybean

In order to understand the nitrogen nutrition of soybean aphids, it is important to
understand the way nitrogen moves in a soybean plant. In nodulated soybeans, a
symbiosis occurs between the soybean plant and one of four genera of Rhizobiaceae,
usually Rhizobium or Bradyrhizobium (Schubert 1995). The bacterium supplies ﬁxed
nitrogen to the plant and receives ﬁxed carbon. Nitrogen ﬁxation by the bacteria can

supply up to ninety-ﬁve percent of the plant’s nitrogen demand (Unkovich and Pate

2000). The process of nitrogen ﬁxation is energetically very expensive; respiration by
the bacterial syrnbiont may account for 70% of total root respiration (Walsh et al. 1998).
For this reason, it is advantageous to the plant to slow or stop nitrogen ﬁxation under
environmental stress or when the plant reaches a growth stage where additional ﬁxed
nitrogen is no longer required. This is accomplished via signaling between the soybean
plant and the bacteria. The signaling compound is the amino acid asparagine
(Bacanamwo and Harper 1997).

Ammonium or ammonia travel from the syrnbiosome (bacteroid and surrounding
membrane) to the cytosol of the soybean plant (Whitehead et al. 2001). Inside the nodule
soybean cells, this nitrogen is converted to ureides, which are transported to the rest of
the plant via the xylem (Whitehead et al. 2001). Ureides are broken down into amino
acids, including asparagine, in the shoot and loaded into the phloem (Vadez et al. 2000)
or made into proteins. Asparagine that arrives at the nodules is broken down into
aspartate and glutamate which can then enter the bacteroid and inhibit nitrogen ﬁxation
(Bacanamwo and Harper 1997). When the plant requires a reduced rate of nitrogen
ﬁxation because of an environmental stress or the age, higher levels of asparagine are
loaded into the phloem. Higher levels of this signaling compound translate into reduced
nitrogen ﬁxation in the nodules, but can enhance the quality of phloem sap as a soybean
aphid food source.

Potassium status may affect the way that nitrogen is stored in a plants; an increase
in potassium levels leads to a decrease in soluble nitrogen, a correlate of phloem sap
amino acid concentration, in brussels sprouts (Van Emden 1966). Potassium is a cofactor

of the enzyme responsible for stringing amino acids into proteins (Mengel and Kirkby

2001). Thus, under potassium deﬁciency, amino acids may build up in the foliage. This
could trigger a soybean plant to slow down or shut down its nitrogen ﬁxation by

increasing phloem loading of the nodule signaling compound asparagine.

Role of Nutrition in Aphid Control

There are several examples of aphid populations that are affected by the
nutritional value of their food. Nitrogen fertilization has been shown to increase soybean
aphid populations (Wang and Ba 1998). High-input cotton cultivation practices common
in California have coincidentally elevated Aphis gossypii Glover from a secondary to a
primary pest. A. gossypii is more fecund, has a shorter development time, and occurs at
higher densities on cotton plants with high tissue nitrogen levels as a result of nitrogen
fertilization (Cisneros and Godfrey 2001, Nevo and C011 2001). The effects of nitrogen
fertilization appear to be stronger for nymphs than adults (N evo and C011 2001).

A. gossypii is not the only aphid to exhibit a response to nitrogen fertilization of
the host plant. Nitrogen fertilization of wheat and barley results in increased fecundity of
the cereal aphid, Metopolophium dirhodum (Walker), which feeds from sieve elements
located on the leaves of the plants (Awmack and Leather 2002). A positive correlation
has also been found between foliar nitrogen concentrations in corn and population levels
of the corn leaf aphid, Rhopalosiphon maidis (Fitch) (Morales et al. 2001). A related
species, R. padi, has a much reduced intrinsic growth rate when it is cultured on nitrogen
deﬁcient barley (Ponder et al. 2000). These studies, like many studies of the relationship
between plant fertilization and aphid populations measured foliar nitrogen concentration

rather than phloem exudate samples (Morales et al. 2001). However, foliar nitrogen

10

concentration is thought to follow the same general pattern as phloem sap amino acid
concentration, so a plant with high levels of foliar nitrogen is assumed to translocate high
concentrations of amino acids in its phloem sap (Douglas 1993). It is important to note
that nitrogen fertilization does not have predictable effects on the amino acid composition
of phloem sap (Prosser and Douglas 1992).

A few fertilization studies examined the effect of nitrogen fertilization on amino
acid concentration in the phloem sap, and these studies may shed some light onto the
mechanisms of this relationship. Nitrogen fertilization of oats and barley changes both
the total amount and proportions of amino acids present in the phloem sap. Most amino
acids remain at the same levels in the phloem sap following fertilization, but glutamic
acid and aspartic acid increase (Weibull 1987). Since these are the amino acids that
Buchnera most frequently use to manufacture essential amino acids and are the amino
acids most easily passed across the aphid gut wall (Douglas 1998), aphid nitrogen
nutrition probably improves dramatically following nitrogen fertilization of these crops.

Magnesium fertilization increases the fecundity of R. padi on barley. Magnesium
fertilization increases chlorophyll synthesis and presumably photosynthesis rates in plants
(Havlickova and Smetankova 1998). This may lead to an increased rate of assimilate
production, and thus increased assimilate loading in the phloem. This in turn would
increase total phloem ﬂux through osmotically generated pressure ﬂow and more total
phloem would be delivered to feeding aphids.

Potassium fertilization can affect aphids. Many sucking insects respond
negatively to potassium fertilization (Waring and Cobb 1989). Potassium fertilization

reduces lifespan and rate of reproduction of R. padi feeding on barley. In addition, R.

11

padi have lower landing rates on barley grown in potassium rich than barley grown in
potassium deﬁcient soil (Havlickova and Smetankova 1998). In low potassium soils,
fewer aphids were observed on potatoes receiving potassium fertilization in years when
aphid populations were high (Broadbent et a1. 1952). An interaction with potassium has
been observed with soybean aphid at high populations. In 2000 and 2001, ﬁelds or parts
of ﬁelds with potassium deﬁciency appeared to have higher soybean aphid populations
and greater soybean damage (DiFonzo 2002). However, potassium fertilization has also
been shown to contribute to higher soybean aphid populations, although the beginning
fertility of the ﬁeld was not reported (Wang and Ba 1998).

Lower concentrations of phloem amino acids may be a mechanism for plant
resistance to aphids. Peas that are resistant to Acyrthosiphon pisum (Harris) have a
decreased concentration of free amino acids in their phloem as compared to susceptible
cultivars (Weibull 1987). Wheat resistant to Diuraphis noxia Mordvilko decreases the
amount of translocated amino acids in response to infestation (Telang et al. 1999). Oat
and barley resistance to R. padi is not related to a change in total concentration of amino
acids, but resistant barley has a decreased concentration of one particularly important

amino acid, asparagine (Weibull 1988).

Conclusion

Aphids live in very intimate association with their host plants, and any factor that
causes internal changes to the host plant, such as fertilization, will also affect the aphids
living on that plant. Many studies show that these relationships do occur, and these

effects may exacerbate pest problems. It is critically important to understand not only the

12

gross effects of practices such as fertilization on soybean aphid populations, but also the
mechanisms. This information provides targets for plant breeders, facilitates the
movement of knowledge across systems, and allows agronomists to plan control
strategies in such a way as to minimize interference between the different strategies. This
knowledge may allow the design or alteration of cultural practices for control of soybean

aphid within an Integrated Pest Management system.

13

Table 1.1. Nutritional requirements of aphids (Dadd et al. 1967, Srivastava 1987, Nation

2001).

 

Nutrient Type Nutrient
Elemental Nutrients C

 

Ca

Vitamins Ascorbic Acid
Calcuim Pantothenate
Folic Acid
Meso-inositol
Pyridoxine
Nicotinic Acid
Thiamine
Niacin
Lipogenic Factors
Biotin
Choline
Riboﬂavin

Other Sterols

 

 

 

14

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Research Program.

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Bacanamwo, M., and J. E. Harper. 1997. The feedback mechanism of nitrate inhibition of
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Broadbent, L., P. H. Gregory, and T. W. Tinsley. 1952. The inﬂuence of planting date
and manuring on the incidence of virus diseases in potato crops. Applied Biology
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Cisneros, J ., and L. Godfrey. 2001. Midseason pest status of the cotton aphid
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Environmental Entomology 30: 501-5 10.

Cristofoletti, P. T., A. F. Ribeiro, C. Deraison, and Y. Rahbé. 2003. Midgut adaptation
and digestive enzyme distribution in a phloem feeding insect, the pea aphid
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Dadd, R., and D. Krieger. 1968. Dietary amino acid requirements of the aphid, Myzus
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Dixon, A. 1998. Aphid Ecology. Chapman and Hall, London.

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Douglas, A. 1998. Nutritional interactions in insect-microbial symbioses: Aphids and
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15

Havlickova, H., and M. Smetankova. 1998. Effect of potassium and magnesium
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Honek, A. 1991. Nitrogen fertilization and abundance of the cereal aphids
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Koyama, Y., I. Yao, and S. Akimoto. 2004. Aphid galls accumulate high concentrations
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Mengel, K., and E. A. Kirkby. 2001. Principles of Plant Nutrition. Kluwer Academic
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Moran, N. A., G. R. Plague, J. P. Sandstrom, and J. L. Wilcox. 2003. A genomic
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Nevo, E., and M. C011. 2001. Effect of nitrogen fertilization on Aphis gossypii
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Ponder, K., J. Pritchard, R. Harrington, and J. Bale. 2000. Difﬁculties in location and
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Prosser, W. A., and A. E. Douglas. 1992. A test of the hypotheses that nitrogen is
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Sandstrom, J. P., and N. A. Moran. 2001. Amino acid budgets in three aphid species
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Schubert, S. 1995. Nitrogen assimilation by legumes-processes and ecological
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Telang, A., J. Sandstrom, E. Dyreson, and N. Moran. 1999. Feeding damage by
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17

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New York.

18

Chapter 2—Soil Potassium Inﬂuences Soybean Aphid Population Size in Soybean

Introduction

Soybean aphid (Aphis glycines Matsumura) is an invasive agricultural pest that
was ﬁrst discovered in the United States in 2000 (Venette and Ragsdale 2004). It is the
most important insect pest of soybean in China (Wu et al. 2004) and has rapidly risen to
become the most important insect pest of soybeans in North America. The current
control strategy for soybean aphid in North America is the application of chemical
insecticides. Organic soybean production, an increasing market in Michigan, is severely
impacted by the lack of approved organic control Options for soybean aphid. Other
strategies, such as biological and cultural controls, are sorely needed so that soybean
aphid infestations can be managed in the context of integrated pest management.

A severe infestation of soybean aphids impacts the soybean plant in a variety of
ways. In a review of the Chinese literature on the soybean aphid, Wu et al. (2004)
reported that severely infested plants exhibited distorted foliage, early defoliation, and
stunting and had fewer branches, pods, and beans per plant as well as a lower individual
seed weight. In another study, a sever soybean aphid infestation reduced the number of
pods per plant and the weight of beans produced per plant but did not affect individual
bean weight (Wang et a1. 1996).

During the ﬁrst two seasons of soybean aphid infestation in Michigan (2000 and
2001), an apparent interaction between soybean aphid and soil potassium deﬁciency was

noted (DiFonzo 2002). Soybean aphid was found at higher populations in parts of ﬁelds

19

where soybeans exhibited an unusual top-down potassium deﬁciency symptom. It was
hypothesized that soybean aphid achieved greater populations on potassium deﬁcient
plants, but no mechanisms were suggested. Two non-exclusive mechanisms could
explain this effect: migrating soybean aphids could preferentially settle on the yellowed
plants or soybean aphid fecundity could be higher on potassium-deﬁcient soybeans,
probably because of improved nutrition.

Several studies support the ﬁrst hypothesis. The appropriate/inappropriate
landings hypothesis states that migrating insects will ﬁrst be attracted to an area by host
plant volatiles, then make a landing decision based on visual stimuli (Finch and Collier
2000). Aphids are known to be attracted to yellow pan traps over green pan traps
(Boiteau 1990). When studying a number of cereal aphids, De Barro (1991) showed that
yellow is always in the most preferred group of colors. Speciﬁcally, Rhopalosiphon
maidis and Sitobion nrfragariae prefer yellow and bright green to other colors, including
a specially designed green tile that mimicked the color of their host plant, wheat.
Rhopalosiphon padi and Metopolophium dirhodum prefer yellow to any other color (De
Barro 1991). Higher levels of migration to yellowing, deﬁcient plants could account for
higher aphid populations of those plants.

The plant stress hypothesis supports the idea that soybean aphid fecundity should
be higher on potassium-deﬁcient plants (Waring and Cobb 1989). It states that
herbivorous insects should perform better on host plants that are under some type of
environmental stress. However, experimental studies have yielded conﬂicting results. It
has been noted, however, that phloem feeders are particularly prone to exhibit a negative

response to potassium fertilization (Waring and Cobb 1989).

20

There are several examples of aphid populations being affected by fertilization of
their host plants. Nitrogen fertilization has been shown to contribute to higher
populations of the soybean aphid (Wang and Ba 1998). High-input cotton cultivation
practices, which include high nitrogen inputs, have become common in California and
have coincidentally elevated A. gossypii from a secondary to a primary pest. A. gossypii
is more fecund, has a shorter development time, and occurs at higher densities on cotton
plants with high tissue nitrogen levels as a result of nitrogen fertilization (Cisneros and
Godfrey 2001, Nevo and C011 2001). The effects appear to be stronger for nymphs than
adults (N evo and C011 2001). Nitrogen is not the only plant nutrient that affects aphids:
magnesium fertilization increases reproduction of the bird-cherry oat aphid,

Rhopalosiphon padi (L.) on barley (Havlickova and Smetankova 1998).

Aphid populations can also be affected by potassium. Potassium fertilization
reduces lifespan and rate of reproduction of R. padi feeding on barley. Barley grown in
potassium rich soil is less attractive to R. padi as measured by landing rates than barley
grown in potassium deﬁcient soil (Havlickova and Smetankova 1998). In low potassium
soils, potatoes fertilized with potassium had fewer aphids per plant in years when aphid
populations were high (Broadbent et al. 1952). An interaction was also observed in
Michigan soybean ﬁelds between soil potassium levels and soybean aphid at high
populations. In 2000 and 2001, ﬁelds with potassium deﬁciency appeared to have higher
soybean aphid populations and greater yield loss than non-deﬁcient ﬁelds (DiFonzo
2002). However, a Chinese study of a variety of cultural practices reported that
fertilization with potassium could actually beneﬁt soybean aphids (Wang and Ba 1998).

The initial potassium fertility of the ﬁeld used in this study was not reported.

21

The objectives of the study were to A) determine whether soybean aphid
population sizes differed on potassium deﬁcient and potassium sufﬁcient plants and B)
determine the type and magnitude of plant damage caused by soybean aphids and
potassium deﬁciency during the growing season and at harvest. This was done with a

combination of ﬁeld surveys and cage studies over two years.

Methods

Field Surveys

Field surveys were undertaken to examine the effect of soil potassium fertility on
soybean aphid populations and plant characteristics in commercial soybean ﬁelds.
Surveys took place on August 13-14 2003 and August 17-23 2004. The 2003 survey was
conducted in eight ﬁelds in Van Buren, Calhoun, and Kalamazoo Counties, Michigan;
the 2004 survey was conducted in ﬁve ﬁelds in Van Buren County and Calhoun County,
Michigan (Table 2.1). In each year, commercial soybean ﬁelds with patches of visual
symptoms of potassium deﬁciency were chosen. Three paired samples were selected in
each ﬁeld and soil, soybean aphid populations, and plant characteristics were sampled
(each year of the survey included one ﬁeld where only two paired samples were
collected). One site in each pair was in the center of an area of severe potassium
deﬁciency (stunted plants with chlorosis and necrosis around the outside of their leaves),
the other was a nearby topographically similar location without deﬁciency symptoms
(green foliage).

Soil samples were taken by extracting 25 cm soil cores from the center of each

site (1 core per sample in 2003, 3 cores per sample in 2004). The samples were

22

submitted to the Michigan State University Soil and Plant Nutrient Laboratory for
analysis.

For the aphid and plant characteristic counts, three plants were randomly chosen
and pulled from each site. These plants were then placed in coolers with ice packs that
maintained the temperature at approximately 4° C. Coolers were returned to the
laboratory and stored in a cold room at 4° C overnight. The next day, the following plant
characteristics were counted on each of the plants removed from the ﬁeld: primary
leaves (leaves on the mainstem), secondary leaves (leaves on branching stems), ﬂowers,
unﬁlled pods, ﬁlling pods (beans could be felt in the pod), full pods, and empty nodes.

In 2003, the number of soybean aphids per plant was extremely high, so plants
were placed in whirl-pack (N asco, Fort Atkinson, WI) bags ﬁlled with 70% ethanol and
stored in the cold room until the number of aphids could be counted under a dissecting
microscope. When the number of aphids was counted, the proportion of alates in the ﬁrst
hundred aphids counted was also recorded. In 2004, aphid numbers were much lower
and the aphids were counted the day following ﬁeld sampling. In 2004, no alates were
observed.

On 15 October 2004, GPS coordinates were used to return to ﬁve of the paired
sampling sites in two ﬁelds. Five plants per site were collected from a total of ten sites.
Nodes per plant, pods per plant, pods per node, beans per plant, beans per pod, total
weight of beans, and hundredweight of beans were measured.

To determine the differences between paired sites, the difference betWeen
averages of the green plants and deﬁcient yellow plants of each site were calculated.

These were analyzed via the probt option of PROC MEANS in SAS version 8.2 (Institute

23

1999). This option ran a T-test to determine the probability that the differences measured

were signiﬁcantly different from zero (p<0.05).

Controlled Cage Studies

In 2003 and 2004, cage trials were conducted to test the interaction between soil
potassium levels and soybean aphid population size in a controlled manner. The studies
were conducted in potassium-deﬁcient commercial soybean ﬁelds in Van Buren County,
Michigan. I

The 2003 study site (N 42 ° 7.55’ W 85 ° 79.95’) had a beginning soil potassium
level of 75 ppm as measured by a soil test prior to planting. Three replicated strips (5.1
m x 40.5 m) were established for each of three potassium treatments. On 9 May, Full
(recommended by MSU soil nutrient laboratory), Half, and no potassium amendments
were done with potash fertilizer (0-0-62) and were as follows: 196, 98, and 0 kg per ha.
The fertilizer was broadcast prior to planting and not incorporated. On 8 July 2003, four
ﬁeld cages in each strip were set up at approximately 10 m intervals in the plots.

Field cages were constructed of no-see-um mesh (Venture Textiles, Inc.,
Braintree, MA). The cages consisted of a 1 m2 frame of 1.880m (3/4 in) diameter PVC
tubing connected to 4 1.5m legs of the same material via PVC 3-way comer connectors
(PlumbingStore.com). The legs of the cage were buried 0.5 m in the soil. The frame was
covered with the mesh cage, which was buried about 0.25 m in the soil. On one side of
the cage, a strip of VelcroTM hook and loop fasteners was used as a door (Figure 2.1).
Cages were sampled by opening the door and leaning into the cage, then rescaling the

door when ﬁnished.

24

Two of the four cages (sacriﬁce cages) were sampled weekly by removing and
counting the number of soybean aphids and plant characteristics on ﬁve plants. The
remaining two cages (yield cages) were not disturbed until harvest. In each pair of
sacriﬁce or yield cages, one cage was infested with aphids while the other served as an
aphid-free control. This resulted in a factorial design with three levels of potassium
amendment and two levels of aphid infestation. On 9 July, counts of the soybean plants
in each cage were taken and the cages were assigned to a soybean aphid treatment.
Infested cages were inoculated by tapping an aphid-infested soybean plant above the
plants in the cage for approximately 30 5. Beginning on 29 July, ﬁve plants per week
were removed from the sacriﬁce cages, placed in a cooler at approximately 4 ° C, and
transported back to the laboratory. The coolers were stored overnight in a cold room at
the same temperature. The following day, the same plant characteristics measured in the
ﬁeld surveys were counted and the plants were preserved in whirl-paks as described
above. On 12 August, soil samples were taken from each cage as described above. One
core was taken from each cage.

In 2004, the study site (N 42 ° 7.50’ W 85 ° 80.00’) had an initial soil potassium
level of 67 ppm as measured by a soil test just after planting (Table 2.2). Two strips in
each of ﬁve replicates were established in the ﬁeld and randomly assigned to unfertilized
or fertilized treatments. Fertilization took place on 13 May by broadcasting 256.8 kg/ha
potash fertilizer (0-0-62) (Mason Elevator, Mason, MI). In 2004, fertilization took place
after planting. Two cages per strip were set up on 13 May 2004, when soybean
germination was almost complete. On 28 May 2004, the soybeans in the cages were

thinned to 13 plants per cage. One cage in each strip was infested with one soybean

25

aphid per plant on ten plants on 28 May 2004 and the second cage was maintained aphid-
free to serve as a comparison. This resulted in a 2x2 factorial design, with the presence
or absence of potash fertilizer and soybean aphids as factors. The source of the soybean
aphids was a laboratory colony established from a single ﬁeld-collected soybean aphid in
2003, and maintained at Michigan State University (27° C, 24 hour photoperiod). Soil
samples (three cores) from each cage were taken on 4 June 2004.

The number of soybean aphids per plant and plant characteristics were counted
two or three times a week from 28 May until 15 July. From 28 May till 30 June, all ten
plants in each cage were counted; ﬁve plants per cage were sampled on 2 July and three
plants per cage were sampled from 6 July till the end of the study.

At harvest in both years, the number of nodes per plant, pods per plant, pods per
node, beans per plant, beans per pod, weight of beans per cage, and hundredweight of
beans was measured. In 2003, yield data were collected on 30 September from plants in
the yield cages. In 2004, ten plants per cage were harvested on 12 October; these were
the same plants counted for aphid number and plant characteristics during the ﬁeld
season.

Because actual soil potassium levels did not correspond to the Full, Half, and
none categories, 2003 data were regressed to measure the effect of potassium and
contrasted by AN OVA for the effects of aphids. Because of this, exploring possible
interactions between aphids and potassium was impossible. The 2003 data were analyzed
via PROC REG in SAS version 8.2 (Institute 1999) for the effects of potassium, and
using PROC MIXED in SAS version 8.2 for the effects of aphids. In 2004, the data were

analyzed via PROC MIXED in SAS version 8.2 (Institute 1999) with potassium

26

treatment, date, and the potassium-date interaction as ﬁxed factors and replicate as a

random factor.

Clip Cages

Clip cage experiments were conducted to determine the effect of soil potassium
levels on individual aphids. Clip cages were constructed using the following method. A
1.88 cm diameter PVC pipe was cut into lengths of approximately 1cm, and the ends
were sanded until smooth. A piece of the no-see-um mesh (used to make the ﬁeld cages
described above) was glued over one end of the PVC piece. A hair roller clip (Discount
Beauty Supply, Mesquite, TX) was bent to enlarge the opening then the PVC piece was
glued to one side. The clip cages were suspended from wooden ﬁeld stakes using ﬁshing
line; when a cage was attached to a leaﬂet the ﬁshing line suspended the cage so it did
not weigh down the leaf.

Clip cage experiments took place in the same ﬁeld as the 2004 cage study. The
experiment was conducted two separate times (10 June and 14 July). In each case, four
clip cages per strip (4 cages/strip x 2 strips/replicate x 5 repetitions = 40 cages in each of
three experiments) were put out. Because birth order and maternal nutrition can have an
effect on aphid performance, the following procedure was used to control for maternal
effects. Adult aphids were removed from the colony maintained at Michigan State
University and placed on excised soybean leaves in Petri dishes. Every few hours, the
newly deposited nymphs were removed from these dishes. Groups of approximately 10
nymphs were placed on excised soybean leaves with the petiole inserted into a 1 mL

eppendorf tube ﬁlled with water and sealed with Paraﬁlm. Individual leaves were kept in

27

Petri dishes and maintained in a growth chamber at 4° C with a 24 h photoperiod. After
three to ﬁve days, the aphids were removed from these leaves and placed onto identically
treated fresh soybean leaves in groups of one to three aphids per leaf. The aphids were
checked daily. When the ﬁrst nymph appeared (usually after 7 days), the mother was
removed and the Petri dish was sealed with laboratory tape. Petri dishes containing
nymphs less than 24 hours old were placed into coolers at approximately 4° C and
transported to the ﬁeld. In the ﬁeld, individual nymphs were removed from the excised
leaves using a ﬁne camel hair paintbrush and placed onto the undersides of the second
fully-expanded leaf from the t0p of the plant. The clip cages were monitored two to three
times per week until live aphids were no longer present.

The ﬁrst set of forty clip cages was infested with soybean aphid nymphs on 10
June 2004. In these cages, the aphid ﬁrst placed in the cage was allowed to remain for
the duration of its lifetime. On each sample date, the number of nymphs she produced
was recorded and the nymphs were removed from the clip cage and killed.

Another set of forty clip cages was started on 14 July 2004. In these cages, the
aphid ﬁrst placed in the cage was allowed to remain until she had deposited ﬁve nymphs.
That aphid was then removed and her ﬁve offspring were evaluated for the remainder of
study. As in the ﬁrst trial, on each sample date, the number of nymphs produced by the
ﬁve sisters was counted and nymphs were removed and killed.

Clip cage data were analyzed using the PROC MIXED procedure of SAS version
8.2 (Institute 1999) with potassium treatment as the ﬁxed factor and replicate as the
random factor. Mean comparisons were conducted using Fisher’s protected LSD

(p<0.05).

28

Results

Field Surveys

Sample sites selected based on green (less deﬁcient) or yellow (more deﬁcient)
plants had signiﬁcantly different levels of soil potassium in both years (Tables 2.3 and
2.4); in 2004 soil magnesium was also different within pairs. In 2003, three of the
sample sites had already been invaded by entomopathogenic fungus, causing the aphid
population to crash. These ﬁelds were excluded from the analysis of aphid number and
proportions of alate aphids. In 2003, there was no difference in aphid number between
green healthy and yellow deﬁcient plants, but the yellow plants had signiﬁcantly fewer
leaves than plants from green areas. When expressed as the number of soybean aphids
per leaf, aphid populations were signiﬁcantly greater on the yellow plants (Table 2.3),
Showing that soybean aphids had a higher population density on potassium deﬁcient
plants than on nearby less deﬁcient plants. In 2004, no difference in total aphid number
or density was detected (Table 2.4). However, whereas 2003 was a soybean aphid
outbreak year (up to 17,000 aphids per plant), aphid numbers were very low in 2004
(usually less than 100 aphids per plant with a maximum of 500 aphids per plant).

In 2003, the proportion of soybean aphids with wings or wing pads in the ﬁrst
hundred aphids counted on each plant was also measured. There was no difference in the
proportion of aphids with wings (yellow plants = 1.3%, green plants = 1.8%, t = 1.11, p
= 0.30) or wing pads (yellow plants = 6.9%, green plants =5.4, t = -0.61, p = 0.55) . In

2004, aphid numbers were very low and no alates were observed.

29

In 2003, unﬁlled pods, ﬂowers, secondary leaves, total leaves, total nodes, and
total pods were signiﬁcantly higher in the green plants than the yellow plants (Table 2.3).
Green plants had about 50% more total leaves and total nodes and three times as many
pods as yellow plants.

In 2004, secondary leaves, ﬁlling pods, and total leaves were all signiﬁcantly
greater in the green plants, but there was no difference in yield based on a ﬁve-pair
sample (Table 4). Once again, green plants had 50% more leaves and nodes than yellow
plants. In 2004 green plants had only about 30% more pods, and the difference was

nonsigniﬁcant.

2003 Controlled Cage Studies

In both years, fertilizing strips with potash produced a range of soil potassium
levels in the study ﬁelds (Tables 2.5 and 2.6). Generally, the soil potassium levels in
2004 were lower than those in 2003.

Because the actual soil potassium levels in my cages did not always match the
treatment goal (i.e. some of the cages receiving the full rate of potassium had the lowest
actual potassium levels) (Table 2.5), 2003 plant characteristic and yield data Were
regressed against actual soil potassium levels. Cages with and without aphids were
regressed separately to avoid the confounding effects of soybean aphid damage.

Cages in the 2003 study were sampled on four dates: 29 July, 5 August, 19
August, and 28 August. In 2003, the cages were inoculated late with an inconsistent
number of aphids, and no differences were detected in the number of aphids per plant at

different soil potassium levels.

30

In both soybean aphid-infested and uninfested plants, differences in plant
characteristics based on soil potassium level did not appear until 28 August. In the aphid
cages, full pods (df = 39, F = 9.04, slope = 0.04, r2 = 0.0922, p = 0.0047), ﬁlling pods (df
= 39, F = 10.72, slope = 0.04, r2 = 0.2201, p = 0.0023), secondary leaves (df = 39, F =
4.90, slope = 0.02, r2 = 0.1038, p = 0.0426), total leaves (df= 39, F = 7.23, slope = 0.03,
r2 = 0.1598, p = 0.0106), total pods (df= 39, F = 11.98. slope = 0.09, r2 = 0.2397, p =
0.0013), and total nodes (df= 39, F = 4.40, slope = 0.03, r2 = 0.1038, p = 0.0426)
increased with soil potassium. In cages without aphids, only the number of unﬁlled pods
(df = 39, F = 4.45, slope = 0.04, r2 = 0.1049, p = 0.0415) increased with soil potassium.
At harvest, there was no relationship between yield and soil potassium levels.

Aphids affected a number of plant characteristics during the last two sampling
dates (Table 2.7). On 19 August, the presence of aphids signiﬁcantly decreased the
number of secondary leaves, unﬁlled pods, ﬁlling pods, total leaves, total pods, and total
nodes. On 28 August, the presence of aphids signiﬁcantly decreased the number of
primary leaves, secondary leaves, ﬁlling pods, ﬁlled pods, total leaves, total pods, and
total nodes.

At harvest, the effect of potassium was evaluated separately for aphid-infested
and uninfested plants because of the large differences between these plants. For both
types of plants, soil potassium level did not affect the total number of nodes, pods or
beans at harvest or the total weight or hundredweight of the beans in a cage (p>0.15).

The presence of soybean aphids affected a number of yield parameters (Table
2.8). Aphids decreased the total number of nodes, pods, and beans per plant as well as

the total weight of beans produced per cage.

31

2004 Controlled Cage Study

Aphid numbers were ﬁrst evaluated on 1 June then evaluated two to three times a
week until 15 July. Aphid and plant characteristic counts were also taken on 3 August
and 1 September. By 30 June, the average number of soybean aphids per plant in the
unfertilized cages was signiﬁcantly higher than in the fertilized cages (Figure 2.2). This
signiﬁcant difference lasted at least until 15 July, when the counting ended. The numbers
of aphids per plant had exceeded 22,000 in some cages and counting aphids was no
longer feasible.

Plant characteristics were evaluated on the same dates as the aphid counts.
Beginning on 9 June, plants in unfertilized, uninfested cages had more primary leaves
than those in fertilized, uninfested cages. This lasted until 1 September, when the trend
reversed. This same pattern was seen in infested cages, i.e. more primary leaves per plant
were found on unfertilized versus fertilized plants after 10 June. This pattern may be due
to compensatory growth. Beginning on 28 June, cages with no aphids had more primary
leaves than cages with aphids if both cages received potassium. This lasted through the
entire season. In the unfertilized cages, the uninfested plants had more primary leaves on
3 August through 1 September (Table 2.9, 2.10, 2.11, 2.12).

In secondary leaves, the date x potassium x soybean aphid interaction was non-
signiﬁcant. Thus, aphid treatments were pooled to compare potassium effects and
potassium treatments were pooled to compare aphid effects. Beginning on 8 July,
unfertilized cages had more secondary leaves than amended cages. On 1 September,

aphid-free plants had more secondary leaves than aphid cages (Table 2.13, 2.14).

32

On 8 July aphid-infested plants had more ﬂowers than uninfested plants
regardless of potassium treatment. However, after this date uninfested plants had more
ﬂowers than aphid-infested plants for the rest of the season. Also on 8 July, plants with
no potassium and aphids had more ﬂowers than plants with potassium and aphids. This
pattern continued for the rest of the season. The same pattern was seen for aphid-free
cages on 14 July and 3 August. By 1 September, unamended cages had more ﬂowers per
plant within soybean aphid treatments but fewer within the uninoculated cages (Table
2.9, 2.10, 2.11, 2.12).

In both potassium treatments, aphid-free plants had more unﬁlled pods per plant
than aphid plants on 8 July. On 14 July, aphid-infested plants had more unﬁlled pods
than aphid-free plants in the unamended treatment and there were no effects in the
amended treatment. On 3 August and 1 September, aphid-free plants had more unﬁlled
pods than aphid plants when both plants received the same fertilizer treatment. From 14
July onward, unfertilized had more unﬁlled pods than fertilized plants in the aphid
treatments. On 3 August and 1 September the same pattern appeared in the uninfested
plants (Table 2.9, 2.10, 2.11, 2.12).

The date x potassium x soybean aphid interaction was non-signiﬁcant for ﬁlling
pods. Thus, aphid treatments were pooled to compare potassium treatments and
potassium treatments were pooled to compare aphid treatments. Unamended treatments
had more ﬁlling pods on 3 August and 1 September. Aphid-free treatments had more

ﬁlling pods per plant on the same dates (Table 2.13, 2.14).

33

On 1 September, fertilized, aphid-infested plants had more full pods than
unfertilized, aphid-infested plants. On the same date, unfertilized, uninfested plants had
fewer full pods than unfertilized, aphid-infested plants (Table 2.10, 2.11).

At harvest, fertilized plants had signiﬁcantly fewer nodes per plant and more
beans per pod than unfertilized plants. Uninfested plants had more nodes per plant, pods
per plant, beans per plant, and beans per pod than infested plants. The total weight and
hundredweight of beans produced in uninfested cages was greater than that of infested
cages (Table 2.15, 2.16). Soybean aphids and soil potassium had only one interaction.
Plants in fertilized, aphid-infested cages produced more beans per cage than plants in
unfertilized, aphid-infested cages (fertilized, infested = 21.6 beans, unfertilized, infested

= 49.6 beans, t = -2.11, p = 0.0360).

Clip Cages

For the ﬁrst run of the clip cage experiment (10 June), the lifespan of the aphid
ﬁrst placed in the cage, age of the mother at ﬁrst reproduction, total number of nymphs
produced, and the number of nymphs produced per day during the reproductive period of
the aphid was measured and the data were analyzed by cage. The number of nymphs
produced per day during the mother’s reproductive lifespan was signiﬁcantly different
between treatments (Figure 2.3). Aphids caged on unfertilized plants produced on
average more nymphs per day then their counterparts on fertilized plants. However, this
was very early in the season.

In the later trial (July 14), the performance of the second generation of soybean

aphid in the clip cages was evaluated to further compensate for maternal effect. In these

34

cages, the lifespan of the second generation, total number of nymphs produced by the
adult aphids in the cage, nymphs produced per day, and the nymphs produced per mother
per day were evaluated. The mothers began to produce nymphs at an earlier age
(p=0.0282) and the total number of nymphs produced per cage was higher (p=0.0150) in

the clip cages placed in unfertilized strips (Figure 2.4).

Discussion

The results of the 2003 survey conﬁrmed the ﬁeld observations that were made in
2000 and 2001. In commercial soybean ﬁelds, areas of lower potassium fertility had
smaller plants with a higher density of soybean aphids than nearby areas with better
potassium nutrition. 2003 was a soybean aphid outbreak year; the aphids had escaped
environmental population regulations such as natural enemies, and host plant effects
probably had a large inﬂuence on the population. In 2004, aphid populations were much
lower, and there was no effect of soil potassium on aphid populations. This may be
because other controls (such as predation) suppressed populations enough that host plant
mediated effects were no longer distinguishable. It should be noted that all of the ﬁelds
included in this study had soil potassium levels far below the level of 150 ppm
recommended for Michigan (Vitosh et al. 1995).

There are two possible hypotheses to explain the results of the survey. Migrating
soybean aphids may be more attracted to yellowing plants than to healthier green plants.
In the 2003 survey, there was no difference in the proportion of soybean aphids with
wings or wing pads on the green and yellow plants. Thus, increased numbers of soybean

aphids on yellowed, potassium deﬁcient plants was not due to increased immigration

35

from migrating aphids. However, if the immigration occurred far before our sampling
the winged immigrants responsible for the increased populations may have already died
and would not be counted in this type of sampling. Also, if the immigrants had a large
number of babies, the proportions of immigrants on green and yellow plants might be
similar even though the total number of immigrants would be higher on the yellow plants.
More research in this area is needed to deﬁnitely determine whether soybean aphids
discriminate between yellowing and green soybean plants at the ﬁeld level.

The cage studies were designed to test the second hypothesis, that soybean aphid
populations increase at a higher rate on potassium deﬁcient soybeans. Although the 2003
cage study did not yield usable results because of the faulty aphid infestation, the 2004
study clearly showed that in the absence of human or environmental control, soybean
aphid populations were higher on soybeans growing in soils with lower potassium
fertility up until the aphid populations overwhelmed the host plant and crashed. The
results of the clip cage experiments show that this effect is probably due to higher
individual fecundity of soybean aphids on plants with a lower potassium status. In
different repetitions of the study, this was due to an earlier age of ﬁrst reproduction or a
faster rate of nymph production once reproduction had started.

In order to understand the relevance of the plant characteristic counts taken during
the season, it is necessary to look at them in the context of the ﬁnal yield. This is
impossible in the 2003 survey since no yield data were taken. In the 2004 survey, there
were no differences in any of the yield parameters measured. Thus, one can only assume
that, in years such as 2004 when soybean aphid populations are very low, there are no

differences between potassium-deﬁcient and healthy appearing plants at the levels

36

measured in terms of yield. It was assumed that the yellowed plants compensated for the
differences in the numbers of leaves and pods that were detected in mid-August.

In the 2003 cage study, only the presence or absence of soybean aphids affected
yield; aphid free cages had more nodes per plant, more pods per plant, more beans per
plant, greater weight of beans, and a greater hundredweight of beans. Although the mid-
season and yield data were taken from two separate sets of cages, it was assumed that
both would have experienced the same effects of soybean aphids and potassium.
Therefore, although the regression results appeared to show that pod set was lower or
delayed in the plants growing at lower potassium levels, especially when they were also
being attacked by aphids, it seems that the plants must have been able to compensate for
this effect between late August and harvest, possibly be reaching full maturity at a later
date. It was also observed that aphid-free plants had greater numbers of those traits that
contribute to yield, and that these effects appeared earlier or later in the season depending
on when the traits ﬁrst appear in the growing season.

In the 2004 cage study, both soybean aphids and potassium amendment affected
mid-season growth and ﬁnal yield. During the season, soybean aphids decreased the
number of leaves, ﬂowers, and pods. At harvest, plants in cages infested with soybean
aphids had fewer nodes, pods, beans, beans per pod, total weight of beans, and
hundredweight of beans than uninfested plants. The potassium amendment also affected
soybean plants. During the season, potassium amendment reduced soybean aphid
populations, leaves, ﬂowers, and pods. At harvest, the total number of beans, weight of
beans, and hundredweight of beans were the same in fertilized and unfertilized cages.

However, fertilized cages had more nodes per plant and beans per pod than unfertilized

37

cages. Since weight of beans probably correlates best with commercial soybean yield,
potassium fertilization did not reduce soybean yield. However, it also did not improve it
in this study. The reasons for these unexpected potassium results in our mid-season
counts are unknown, although they may be due to the small size of our cages and the
compounding effect of the soybean aphids. The beneﬁts of proper soil fertility are well
established (Mengel and Kirkby 2001), and this study does not show that potassium
amendment is without beneﬁt. It is more likely that the mid-season potassium effects are
some sort of experimental artifact, possibly due to compensatory growth.

Although potassium amendment did not improve soybean yield in either cage
study, it is important to note that the green, non-deﬁcient plants in both ﬁeld surveys had
higher mid-season characteristics than deﬁcient plants. This probably represents a more
realistic picture of what would happen in the ﬁeld. In 2004, the only year of the survey
when yield data was taken, these differences did not carry through to yield. However, the
deﬁcient plants lagged behind the other plants in mid-August. If the late summer had
poor conditions for soybean growth, these plants may have yielded less at harvest. In
2003, the soybean aphid out break year, only the non-deﬁcient plants had ﬁlling pods. In
that year, it did not appear that the yellow plants were likely to set seed at all, so in that
year, potassium level and it’s interaction with soybean aphid populations probably had a

large effect on ﬁnal yield.
Conclusion

In both ﬁeld surveys and controlled exclusion cages, soybean growing at lower

levels of potassium nutrition supported higher levels of soybean aphid populations.

38

Limited data did not support the hypothesis that migrating soybean aphids land
preferentially on yellowed soybean plants. However, studies in large exclusion cages and
clip cages supported the hypothesis that soybean aphids have a greater rate of increase on
potassium-deﬁcient soybean plants. Maintenance of proper potassium fertility levels will
result in a slower rate of increase and lower populations of the soybean aphid during
outbreak years. This has important management implications and is ideal for use in the
context of integrated pest management.

It is also interesting to note that the differences in plant characteristics between
green and yellow plants in 2003 were greater than in 2004. Speciﬁcally, green and
yellow plants had different numbers of pods, the trait that most directly translates to
yield. The low soybean aphid populations in 2004 probably did not affect on the soybean
plants. In 2003, both green and yellow plants were well above the aphid population level
required for plant damage. It would appear that in 2003, aphid-infested and deﬁcient
plants had more severe damage than aphid-infested and less-deﬁcient plants. Thus,
maintenance of proper soil potassium nutrition may help to alleviate part of the damage
caused by soybean aphids during outbreaks.

These results could have important implications for soybean aphid management.
Currently, soybean populations reach outbreak levels into the tens of thousands in some
years and in others the population remains at extremely low levels. Thus, there is the
potential for environmental control of this pest, but it is not achieved every year. Proper
potassium fertility will not by itself keep soybean aphid populations below damaging
levels. However, it will result in a slower increase of the population. This will allow

time for some other factor, such as predation, to provide soybean aphid control.

39

Furthermore, slower rates of population increase will be beneﬁcial when resistant or
tolerant cultivars are introduced because it will result in a lower level of pest pressure and
the resistance will be less likely to fail. Finally, a Slower rate of population increase is
beneﬁcial in the event that chemical control is employed. Slower increase of soybean
aphid populations will allow decision makers more time to determine whether a ﬁeld will
need to be sprayed and also will allow more time between the decision and the
application. In some areas, a lower level of aphid population rebound after chemical
control is applied may prevent the need for multiple applications.

As a cultural control, maintenance of proper fertility ﬁts very will into many
Integrated Pest Management schemes. It is compatible with most if not all other forms of
aphid population control including the use of resistant cultivars, biological control, or
chemical control. Thus, there will be no trade-offs between different control measures, as
in when an investment in biological control is lost when chemical control must be
undertaken. In addition, maintaining soil at the recommended potassium levels has a
number of other long-term and short-term beneﬁts. Even if soybean aphid control is not
needed in a given year, an investment in preventative control by fertilization need not be
lost. There are beneﬁts both for the crop being produced and also in long-term soil

maintenance.

40

 

 

\

Soil

\

 

 

\

Mesh Fabric

 

 

 

 

‘7 Door

Figure 2.1. Schematic of ﬁeld cages used in the 2003 and 2004 controlled

cage experiments. Cages consisted of a PVC frame (dark bars) covered by

a cage of no-see-um mesh (gray shading) with a VelcroTM door (dotted

line).

41

 

 

 

 

Soybean Aphids per Plant +l- SE

 

Figure 2.2. Soybean aphids per plant during the 2004 controlled cage study on plants
with and without potassium fertilization in a ﬁeld with an initial potassium level of 67
ppm. * Denotes sample dates where the treatments were different at p<0.05. ***

Denotes sample dates where the treatments were different at p<0.0001.

42

 

Nymphs produced per day of
Reproduction +I- SE

 

 

Unfertilized Fertilized

Figure 2.3. Soybean aphid nymphs produced per mother per day of reproduction on
potassium fertilized and unfertilized plants in a ﬁeld with an initial soil potassium level of
67 ppm in the earliest run of the clip cage experiment (June 10). The results are

signiﬁcantly different in an AN OVA. p = 0.0271

43

Age of Mothers at ﬁrst
reproduction (days) +I- SE

 

 

 

 

 

Total number of nymphs
produced per cage +I- SE

 

Figure 2.4. Response of soybean aphids to potassium soil amendment, expressed as A)
age of aphids at ﬁrst reproduction (days) and B) total number of young produced per cage
on potassium fertilized and unfertilized plants in a ﬁeld with an initial soil potassium
level of 67 ppm for the clip cage study begun on July 14, 2004. Both are signiﬁcantly

different in an ANOVA with p<0.05.

Table 2.1: Location of potassium deﬁcient commercial ﬁelds surveyed 14-15 August

2003 and 17-23 August 2004, Southwest Michigan.

 

Survey Year County Field Location

 

2003 Kalamazoo N 42°10'00" W 85°15'30"
Kalamazoo N 42°10'60" W 85°15'10"
Calhoun N 42°10'10" W 85°30'00"
Van Buren N 42°08'00" W 85°52'80"
Van Buren N 42°11'00" W 85°52'80"
Van Buren N 42°13'00" W 85°52'90"
Van Buren N 42°12'90" W 85°52'90"

Van Buren N 42°12'90" W 85°60'00"

 

2004 Calhoun N 41 °59.952' W 85°34.884'
Van Buren N 42 ° 7.50’ W 85 ° 8000’
Van Buren N 42°10.254' W 86°2.271'
Van Buren N 42°11.237' W 86°10.105'

Van Buren N 42°7.797' W 86°11.248'

 

45

Table 2.2. Initial soil test values for potassium deﬁcient ﬁelds used in controlled cage

studies, 2003 and 2004, Van Buren County, Michigan

 

 

2003 2004
pH 5.4 6.3
Limelndex 67 69
Phosphorus (ppm) 180 14
Potassium (ppm) 75 67
Calcium (ppm) 150 1173
Magnesium (ppm) 5 161
Cation Exchange Capacity (me/1009) 4.6 8.6

 

46

Table 2.3. Comparisons of soil test levels, aphid number, and plant characteristics from

putative potassium deﬁcient (yellow) and less deﬁcient (green) pairs of sample sites in

eight commercial soybean ﬁelds, Southwest Michigan, 14-15 August 2003. p values

based on a t-test to determine whether differences were signiﬁcantly different from zero.

 

 

Green Plant Mean Yellow Plant Mean t p
Total Soybean Aphids 1561 1760 -0.66 0.54
Soybean Aphids/Leaf 150 104 -2.57 0.03
Percentage of Winged
Aphids 1.83% 1.28% 1.11 0.3
Percentage of Aphids
with wing pads 6.86% 5.40% -0.61 0.55
Primary Leaves 8.46 7.39 2.1 0.05
Secondary Leaves 6.59 2.71 3.55 0.002
Empty Nodes 2.87 2.79 0.24 0.82
Flowers 33.97 25.33 2.27 0.04
Unﬁlled Pods 20.8 7.39 4.23 0.0005
Filling Pods 1.09 0 1.72 0.10
Full Pods 0 0 1.00 1.00
Total Leaves 15.05 10.10 3.34 0.003
Total Nodes 17.92 12.88 3.46 0.003
Total Pods at Survey 21.89 7.39 4.58 0.0002
Soil pH 6.13 6.05 0.44 0.67
Soil Phosphorus 35.23 27.43 0.34 0.74
Soil Potassium 42.33 29.05 3.4 0.004
Soil Magnesium 138.00 104.00 1.98 0.07
Soil Calcium 860.00 747.38 1.20 0.25

 

47

Table 2.4. Comparisons of soil test levels, aphid number, plant characteristics, and

harvest characteristics (two ﬁelds) from putative potassium deﬁcient (yellow) and less

deﬁcient (green) pairs of sample sites in ﬁve commercial soybean ﬁelds, Southwest

Michigan, 2003. Surveys took place 17-23 August and harvest took place in October. p

values based on a t-test to determine whether differences were signiﬁcantly different

 

 

fromzero.
Green Plant Mean Yellow Plant Mean t p

Total Soybean Aphids 42.3 18.8 1.57 0.1414
Soybean Aphids/Leaf 3.7 1.4 1.81 0.0935
Primary Leaves 8 6.7 1.78 0.0985
Secondary Leaves 6.9 3.5 2.27 0.0405
Empty Nodes 6 6.8 -1.11 0.289
Flowers 9.7 11 -0.81 0.4326
Unﬁlled Pods 24.1 18.1 1.73 0.1071
Filling Pods 24.1 9.5 3.83 0.0021
Full Pods 0 0 0 1
Total Leaves 14.9 10.2 2.41 0.0318
Total Nodes 48.2 27.6 3.01 0.01
Total Pods 21 17 2.12 0.0538
Nodes/Plantat Harvest 25.7 16.2 1.49 0.211
Pods/Plantat Harvest 35.7 9.7 2.01 0.114
Beans/Plant at Harvest 73 13.8 2.25 0.087
Weight of Beans 62.5 6.1 2.37 0.077
HundredweightofBeans 16.3 11.6 0.54 0.615
Soil pH 7.4 7.2 1.51 0.1552
Soil Phosphorus 136.1 178.2 -1.51 0.1554
Soil Potassium 53.3 33.5 5.48 0.0001
Soil Magnesium 115.2 74.1 2.81 0.0149
Soil Calcium 647.1 496.8 1.89 0.0806

 

48

 

Table 2.5. Soil potassium levels inside the cages in the 2003 controlled cage study.

 

Soil Potassium

 

Replicate Potassium Treatment Aphid Treatment Sacrifice (ppm)
1 Full Rate Yes Yes 56
1 Full Rate Yes No 108
1 Full Rate No Yes 100
1 Full Rate No No 62
1 Half Rate Yes Yes 131
1 Half Rate Yes No 134
1 Half Rate No Yes 155
1 Half Rate No No 103
1 No Ammendment Yes Yes 82
1 No Ammendment Yes No 137
1 No Ammendment No Yes 115
1 No Ammendment No No 165
2 Full Rate Yes Yes 218
2 Full Rate Yes No 100
2 Full Rate No Yes 114
2 Full Rate No No 116
2 Half Rate Yes Yes 1 14
2 Half Rate Yes No 53
2 Half Rate No Yes 54
2 Half Rate No No 82
2 No Ammendment Yes Yes 77
2 No Ammendment Yes No 62
2 No Ammendment No Yes 103
2 No Ammendment No No 108
3 Full Rate Yes Yes 104
3 Full Rate Yes No 131
3 Full Rate No Yes 196
3 Full Rate No No 100
3 No Ammendment Yes Yes 46
3 No Ammendment Yes No 110
3 No Ammendment No Yes 119
3 No Ammendment No No 94

 

49

Table 2.6. Soil potassium levels inside the cages in the 2004 controlled cage study after

treatment. Fertilization took place on 13 May and soil was sampled on 4 June.

 

Replicate Potassium TreatmentAphid Treatment Timing Soil Potassium (ppm)

 

1 Yes Yes Early 78
1 Yes No Early 1 15
1 No Yes Early 57
1 No No Early 59
2 Yes Yes Early 104
2 Yes No Early 52
2 No Yes Early 43
2 No No Early 31
3 Yes Yes Early 54
3 Yes No Early 46
3 No Yes Early 23
3 No No Early 27
4 Yes Yes Early 51
4 Yes No Early 71
4 No Yes Early 38
4 No No Early 45
5 Yes Yes Early 39
5 Yes No Early 52
5 No Yes Early 27
5 No No Early 23

 

50

Table 2.7. Signiﬁcant differences in plant characteristics based on the presence of
soybean aphids in the 2003 controlled cage study. Cages were infested on 8 July and
sampling took place from 29 July through 28 August. t values and probabilities are based

on Fisher’s protected LSD.

 

 

 

 

 

 

 

 

 

No Aphid Aphid Plant
Date t P
Plant Counts Counts

Primary 28 August 6.8 4.3 -4.73 <0.0001
Leaves

Secondary 19 August 7.4 3.3 -4. 10 <0.0001
Leaves

28 August 7.6 3.3 -4.33 <0.0001

Total Leaves 19 August 15.6 10.5 -4.82 <0.0001

28 August 14.4 7.6 -6.61 <0.0001

Total Nodes 19 August 19.2 15.2 -3.72 0.0002

28 August 19.1 14.0 -4.78 <0.0001

Unﬁlled 19 August 17.4 9.7 -5.52 <0.0001

Pods

Filling Pods 19 August 19.5 9.0 -8. 12 <0.0001

28 August 15.4 6.5 -7.07 <0.0001

Filled Pods 28 August 7.9 5.4 -3.81 0.0002

Total Pods 19 August 36.8 18.6 -727 <0.0001

28 August 30.0 15.9 -5.73 <0.0001

 

51

Table 2.8. Signiﬁcant differences in soybean yield parameters in the 2003 controlled
cage study based on the presence of absence of soybean aphids. Results are based on an

ANOVA with aphids as a ﬁxed factor and replicate as a random factor.

 

 

No Aphids Aphids F p
Nodes per Plant 13.8 7.4 33.90 <0.0001
Pods per Plant 24.1 9.2 48.61 <0.0001
Beans per Plant 52.2 18.7 44.35 <0.0001
Total Weight of Beans
226.5 72.0 24.47 0.0002
per Cage (g)

 

52

Table 2.9. Signiﬁcant differences in plant characteristics of uninfested soybean plants

based on potassium fertilization in the 2004 controlled cage study. The ﬁeld had an

initial soil potassium level of 67 ppm. Results are based on Fisher’s protected LSD.

 

 

 

 

 

Fertilized Uninfested Unfertilized
Date Plants Uninfested Plants t p value
Primary Leaves 9-Jun 2.1 2.4 2.02 0.0435
15-Jun 2.7 3.4 3.85 0.0001
18-Jun 3.2 3.8 3.40 0.0007
21-Jun 3.6 4.3 4.19 <0.0001
24-Jun 3.9 4.6 4.18 <0.0001
28—Jun 4.5 5.5 5.35 <0.0001
30-Jun 4.8 5.7 4.86 <0.0001
1-Jul 5.3 6.4 4.32 <0.0001
14-Jul 8.1 9.1 3.31 0.0010
3-Aug 12.3 13.7 4.34 <0.0001
1-Sep 12.4 11.6 -2.94 0.0033
Flowers 14-Jul 14.9 19.3 4.44 <0.0001
3-Aug 28.1 33.6 5.61 <0.0001
1-Sep 9.5 7.2 -2.78 0.0055
Unfilled Pods 3-Aug 13.7 19.1 6.55 <0.0001
1-Sep 13.1 21.8 12.34 <0.0001
Total Pods 3-Aug 27.0 42.4 7.12 <0.0001
1-Sep 82.6 101.3 9.94 <0.0001

 

53

Table 2.10. Signiﬁcant differences in plant characteristics of soybean aphid-infested

soybean plants based on potassium fertilization in the 2004 controlled cage study. The

ﬁeld had an initial soil potassium level of 67 ppm. Results are based on Fisher’s

 

 

 

 

 

 

protected LSD.
Fertilized Unfertilized
Date Infested Plants Infested Plants t p
Primary leaves 10-Jun 2.6 2.1 2.55 0.0107
15-Jun 3.2 2.7 2.71 0.0067
17-Jun 3.7 3.1 3.22 0.0013
18-Jun 3.8 3.1 3.78 0.0002
21-Jun 4.3 3.2 5.54 <0.0001
24-Jun 4.6 3.5 5.55 <0.0001
28-Jun 5.4 4.0 7.71 <0.0001
30-Jun 5.8 4.3 8.06 <0.0001
1-Jul 6.5 4.9 5.86 <0.0001
8-Jul 7.6 5.9 4.84 <0.0001
14-Jul 8.5 7.2 3.96 <0.0001
3-Aug 10.5 8.6 5.32 <0.0001
Flowers 8-Jul 12.3 7.5 4.64 <0.0001
14-Jul 13.5 8.7 4.72 <0.0001
3-Aug 12.4 8.6 3.56 0.0004
1-Sep 6.7 3.8 2.75 0.0060
Unﬁlled Pods 14-Jul 8.8‘ 6.1 3.25 0.0012
3-Aug 13.5 6.2 8.20 <0.0001
1-Sep 8.3 4.1 4.66 <0.0001
Full Pods 1-Sep 3.7 6.9 -10.95 <0.0001
Total Pods 3-Aug 19.0 10.9 3.39 0.0004

 

54

Table 2.11. Signiﬁcant differences in plant characteristics of soybean plants based on

soybean aphid infestation when the soil was not fertilized with potassium in the 2004

controlled cage study. Plants were infested on 28 May and the aphid population was

allowed to increase without control. The initial soil potassium level of the ﬁeld was 67

ppm. Results are based on Fisher’s protected LSD.

 

 

 

 

 

 

Unfertilized Unfertilized
Date Uninfested Cages Infested Cages t p value
Primary Leaves 3-Aug 13.7 10.5 9.26 <0.0001
1-Sep 11.6 8.8 8.05 <0.0001
Flowers 8-Jul 5.6 12.3 -6.45 <0.0001
14-Jul 19.3 13.5 5.68 <0.0001
3-Aug 33.6 12.4 19.96 <0.0001
Unﬁlled Pods 8-Jul 9.5 2.1 8.56 ~<0.0001
14-Jul 4.8 8.9 -4.83 <0.0001
3-Aug 19.1 13.5 6.25 <0.0001
Full Pods 1-Sep 0.3 3.7 -12.30 <0.0001
Total Pods 8-Jul 9.5 2.0 3.26 0.0008
3-Aug 42.4 19.0 9.94 <0.0001
1-Sep 101.3 36.6 28.18 <0.0001

 

Table 2.12. Signiﬁcant effects of soybean aphid infestation on soybean plant

characteristics of potassium fertilized plants in the 2004 controlled cage study. Soybean

aphids infestation took place on 28 May and the population was allowed to increase

without control. The ﬁeld had an initial soil test level of 67 ppm potassium and was

fertilized with 267 kg/ ha 0-0-62 fertilizer. Results are based on Fisher’s protected LSD.

 

 

 

 

 

Fertilized Fertilized
Date Uninfested Plants Infested Plants t p
Primary Leaves 28-Jun 4.5 4.0 3.05 0.0023
30-Jun 4.8 4.3 3.01 0.0027
8-Jul 6.8 5.9 2.69 0.0073
14-Jul 8.1 7.2 2.69 0.0073
3-Aug 12.3 8.6 11.41 <0.0001
1-Sep 12.4 8.3 14.30 <0.0001
Flowers 8-Jul 4.2 7.5 -3.35 0.0008
14-Jul 14.9 8.7 6.36 <0.0001
3-Aug 28.1 8.6 19.67 <0.0001
1-Sep 9.5 3.8 6.51 <0.0001
Unﬁlled Pods 8-Jul 8.3 2.7 6.88 <0.0001
3-Aug 13.7 6.2 9.14 <0.0001
1-Sep 13.1 4.1 12.23 <0.0001
Total Pods 8-Jul 8.3 2.7 2.59 0.0097
3-Aug 27.0 11.0 7.31 <0.0001
1-Sep 82.6 27.2 28.40 <0.0001

 

56

Table 2.13. Signiﬁcant differences in soybean plant characteristics of caged plants based
on the presence of soybean aphids for characteristics without an aphid x potassium
interaction in the 2004 controlled cage study. Plants were infested on 28 May. Results

are based on Fisher’s protected LSD.

 

Date Infested Plants Uninfested Plants t pvalue

 

 

 

 

Secondary Leaves 1-Sep 23.1 7.8 31.72 <0.0001
Filling Pods 3-Aug 18.3 5.1 10.31 <0.0001
1-Sep 74.0 20.4 44.85 <0.0001
Total Leaves 28-Jun 5.6 4.8 2.28 0.0201
30-Jun 6.0 5.3 1.99 0.0427
3-Aug 15.0 11.6 5.46 <0.0001
1-Sep 35.1 16.4 32.31 <0.0001
Total Nodes 3-Aug 17.0 13.7 4.75 <0.0001
1-Sep 41.4 27.0 22.65 <0.0001

 

57

Table 2.14. Signiﬁcant differences in soybean plant characteristics based on potassium

amendment for those traits without a signiﬁcant aphid x potassium interaction in the 2004

controlled cage study. Initial soil potassium level was 67 ppm and aphid infestation took

place on 28 May. Results are based on Fisher’s protected LSD.

 

 

 

 

 

Date Fertilized Plants Unfertilized plants t p
Secondary Leaves 8-Jul 2.0 3.3 2.48 0.0133
14-Jul 2.5 4.4 3.76 0.0002
3-Aug 1.5 2.9 2.56 0.0105
1-Sep 13.4 17.5 8.43 <0.0001
Filling Pods 3-Aug 9.0 14.4 4.23 <0.0001
1-Sep 42.6 51.9 7.80 <0.0001
Total Leaves 18-Jun 3.2 3.8 1.99 0.041
21-Jun 3.5 4.4 2.80 0.004
24-Jun 3.8 4.8 2.90 0.0029
28-Jun 4.7 5.8 3.18 0.0011
30-Jun 4.9 6.3 4.07 <0.0001
1-Jul 5.5 7.3 3.84 <0.0001
14-Jul 10.1 13.1 5.00 0.0038
3-Aug 12.0. 14.6 5.00 <0.0001
1-Sep 23.8 27.7 6.79 0.0036
Total Nodes 21-Jun 3.5 4.4 2.44 0.0134
24-Jun 3.9 4.8 2.41 0.0147
28-Jun 4.9 5.8 2.56 0.0095
30-Jun 5.1 6.4 3.55 0.0003
1-Jul 5.8 7.5 3.13 0.0015
8-Jul 8.7 11.0 3.44 0.0005
14-Jul 11.8 15.1 4.98 0.0023
3-Aug 14.1 16.6 3.69 0.0002
1-Sep 31.0 37.4 10.06 <0.0001

 

58

Table 2.15. Signiﬁcant differences in soybean yield parameters based on soybean aphid
infestation in the 2004 controlled cage study. Infestation took place on 28 May, and the
soybean aphid population was allowed to increase without control. The ﬁeld had an
initial soil potassium level of 67 ppm and was fertilized with 267 kg /ha of 0-0-62

fertilized. Results are based on Fisher’s protected LSD.

 

 

Uninfested Infested F p
Nodes per Plant 44.8 24.1 105.03 <0.0001
Pods per Plant 70.4 18.7 174.98 <0.0001
Beans per Plant 149.0 35.6 162.67 <0.0001
Beans per Pod 2.1 1.9 40.10 <0.0001
Weight of Beans per Cage (9) 205.5 37.3 30.21 0.0001
Hundredweight of Beans (g) 14.7 12.9 12.81 0.0027

 

Table 16. Signiﬁcant differences in soybean yield parameters based on potassium

fertilization in the 2004 controlled cage study. Infestation took place on 28 May, and
the soybean aphid population was allowed to increase without control. The field
had an initial soil potassium level of 67 ppm and was fertilized with 267 kg /ha of

0-0-62 fertilized. Results are based on Fisher’s protected LSD.

 

 

Fertilized Unfertilized F p
Nodes per Plant 31.0 37.9 9.82 0.0020
Beans per Pod 2.0 1.9 11.92 0.0007

 

59

Literature Cited

Boiteau, G. 1990. Effect of trap color and size on relative efﬁciency of water-pan traps
for sampling alate aphids (Homoptera: Aphididae) on potato. J. Econ. Entomol.
83: 937-942.

Broadbent, L., P. H. Gregory, and T. W. Tinsley. 1952. The inﬂuence of planting date
and manuring on the incidence of virus diseases in potato crops. Applied Biology
39: 509-524.

Cisneros, J ., and L. Godfrey. 2001. Midseason pest status of the cotton aphid
(Homoptera: Aphididae) in California cotton: Is nitrogen a key factor?
Environmental Entomology 30: 501 -5 10.

De Barro, P. 1991. Attractiveness of four colours of traps to cereal aphids (Hemiptera:
Aphididae) in south Australia. J. Aust. ent. Soc. 30: 263-264.

DiFonzo, C., Hines, R. 2002. Soybean aphid in Michigan Update from the 2001
season. Michigan State University, East Lansing, MI.

Finch, S., and R. H. Collier. 2000. Host-plant selection by insects--a theory based on
'appropriate/inappropriate landings' by pest insects of cruciferous plants.
Entomologia Experimentalis et Applicata 96: 91-102.

Havlickova, H., and M. Smetankova. 1998. Effect of potassium and magnesium
fertilization on barley preference by the bird cherry-oat aphid Rhopalosiphum
padi. Rostlinna Vyroba 44: 379-3 83.

Institute, S. 1999. SAS OnlineDoc ® computer program, version 8. By Institute, 8., Cary,
NC.

Mengel, K., and E. A. Kirkby. 2001. Principles of Plant Nutrition. Kluwer Academic
Publishers, Dordrecht, Netherlands.

Nevo, E., and M. C011. 2001. Effect of nitrogen fertilization on Aphis gossypii
(Homoptera: Aphididae): variation in size, color, and reproduction. Journal of
Economic Entomology 94: 27-32.

Venette, R., and D. W. Ragsdale. 2004. Assessing the invasion by soybean aphid
(Homoptera: Aphididae): Where will it end? Annals of the Entomological
Society of America 97: 219-226.

60

Vitosh, M. L., J. W. Johnson, and D. B. Mengel. 1995. Michigan State University
Extension Bulletin E-2567: Tri-state Fertilizer Recommendations for Corn,
Soybeans, Wheat, and Alfalfa.

Wang, S., X. Bao, T. Sun, R. Chen, and B. Zhai. 1996. Effects of soybean aphid, Aphis
glycines on soybean growth and yield. Soybean Science 15: 243-247.

Wang, Y., and F. Ba. 1998. Study on the optimum control of soybean aphid. Acta
Phytophylacica Sinica 25: 151-155.

Waring, G. L., and N. S. Cobb. 1989. The impact of plant stress on herbivore population
dynamics, pp. 167-226. In E. A. Bemays [ed.], Insect-Plant Interactions. CRC
Press, Boca Raton, Fl.

Wu, 2., D. Schenk-Hamlin, W. Zhan, D. W. Ragsdale, and G. Heimpel. 2004. The
soybean aphid in China: A historical review. Annals of the Entomological
Society of America 97: 209-218.

61

Chapter 3—Improved nitrogen nutrition for the soybean aphid leads to higher

aphid populations on potassium deﬁcient soybean

Introduction

The acquisition of nitrogenous compounds is the main challenge facing aphids
feeding on their natural diets (Terra 1988, Douglas 1993, Dixon 1998, Sandstrom 1999,
Sandstrom and Moran 2001, Douglas 2003). Aphids are thought to obtain all of their
dietary nitrogen from amino acids translocated in the phloem sap. Many species, such as
the bird cherry-oat aphid, Rhopalosiphum padi (L.), have growth rates directly correlated
to concentration of amino acids in the phloem of their host plants (Weibull 1987).
Proteinases have not been detected in the gut or saliva of any aphid species (Srivastava
1987, Foissac 2002), probably because the high levels of proteinase inhibitors, combined
with the extremely low protein concentration typically encountered in phloem sap, make
plant proteins a poor nitrogen source. In addition, many of the proteins transported in the
phloem are defensive chemicals that need to be cleaved in order to become toxic. Since
aphids do not cleave these proteins, they avoid the toxic effects.

Aphids are among the most versatile animals in respect to nitrogen nutrition
because they possess a primary intracellular symbiotic bacterium, Buchnera as well as
secondary gut symbionts, which are able to manufacture certain essential amino acids
from nonessential amino acids in the aphid diet (Moran et al. 2003). Buchnera may be
able to provide their aphid hosts with synthesized essential amino acids at a higher rate
than aphids are able to bring these substances across the gut wall (Douglas 1998). For

this reason, not all aphid species require all of the essential amino acids, and the amino

62

acids that are required vary between aphid species and populations (Srivastava 1987,
Wilkinson and Douglas 2003).

Among the amino acids, several stand out as having particular importance to
aphids. Dadd and Kreiger (1968) showed that methionine has an important
phagostimulatory effect that can inﬂuence host plant acceptance and restlessness in
aphids. Dadd and Kreiger (1968) also showed that, without one of the non-essential
amino acids glutamine, glutamic acid, asparagine, aspartic acid, alanine, or serine, almost
no aphid growth occurred. This is because Buchnera can manufacture essential amino
acids from non-essential amino acids that occur more commonly in aphid diets.
Glutamate may be the principle nitrogenous compound used by Buchnera to synthesize
essential amino acids (Douglas 1998, Wilkinson and Ishikawa 1999). Asparagine levels
increase the population size of R. padi on cat and barley (Weibull 1988).

Potassium fertilization is a good candidate for cultural control of soybean aphid,
Aphis glycines Matsumura. Potassium-deﬁcient soybeans support higher soybean aphid
populations in the ﬁeld, and cage studies showed a higher rate of soybean aphid increase
on soybean plants growing in soils with low potassium levels (Chapter 2). Potassium
fertilization reduces aphid lifespan and rate of reproduction, and this has been quantiﬁed
in the case of R. padi feeding on barley (Havlickova and Smetankova 1998). In addition,
R. padi land on barley growing in potassium-rich soil less often then barley growing in
potassium-poor soil (Havlickova and Smetankova 1998). Van Emden (1966) found that
an increase in potassium fertilization of Brussels sprout leads to a decrease in soluble
nitrogen, a correlate of phloem sap amino acid concentration. Thus, it is hypothesized

that the potassium nutrition of soybean affects the free amino acids being transported in

63

the phloem, and that potassium-deﬁcient soybean are of higher nutritional quality for the
soybean aphid.

The objectives of this study were A) to compare the free amino acid proﬁles of
the phloem sap of soybeans growing in potassium sufﬁcient and deﬁcient soils in
commercial ﬁelds and in artiﬁcially fertilized plots and B) to deﬁne the relationship

between phloem amino acid proﬁles with the soybean aphid populations on plants.

Methods

Surveys

Field surveys were undertaken to examine the effect of potassium fertility on
soybean aphid populations and soybean phloem amino acids in commercial ﬁelds.
Surveys took place on August 13-14 2003 and August 17-23 2004. The 2003 survey was
conducted in eight ﬁelds in Van Buren, Calhoun, and Kalamazoo Counties, Michigan;
the 2004 survey was conducted in ﬁve ﬁelds in Van Buren County and Calhoun County,
Michigan (Table 1). In each year, commercial soybean ﬁelds with patches of visual
symptoms of potassium deﬁciency were chosen. Three paired samples of soil, aphid
populations, and phloem sap were taken in each ﬁeld (each year of the survey included
one ﬁeld where only two pairs were sampled). One site in each pair was in the center of
an area of severe potassium deﬁciency (stunted plants with chlorosis and necrosis around
the outside of their leaves), the other was a nearby topographically similar location
without deﬁciency symptoms (green foliage).

Soil samples were taken by extracting 25 cm soil cores from the center of each

site (1 core per sample in 2003, 3 cores per sample in 2004). The samples were

64

submitted to the Michigan State University Soil and Plant Nutrient Laboratory for

analysis.

For the aphid counts, three plants were randomly chosen and pulled from each
site. These plants were then placed in coolers with ice packs that maintained the
temperature at approximately 4° C. Coolers were returned to the laboratory and stored in
a cold room at 4° C overnight. In 2003, the number of soybean aphids per plant was
extremely high, so plants were placed in whirl-pack (Nasco, Fort Atkinson, WI) bags
ﬁlled with 70% EtOH and stored in the cold room until the number of aphids could be
counted under a dissecting microscope. When the number of aphids was counted, the
proportion of alates in the ﬁrst hundred aphids counted was also recorded. In 2004, aphid
numbers were much lower and the aphids were counted the day following ﬁeld sampling.

In 2004, no alates were observed.

Controlled Cage Studies

In 2003 and 2004, we conducted cage trials to test the interaction between soil
potassium levels and soybean aphid population Size in a controlled manner. The studies
were conducted in potassium-deﬁcient commercial soybean ﬁelds in Van Buren County,
Michigan.

The 2003 study site (N 42 ° 7.55’ W 85 ° 79.95’) had a beginning soil potassium
level of 75 ppm as measured by a soil test prior to planting. Three replicated strips were
established for each of three potassium treatments. Full (recommended by MSU soil
nutrient laboratory), Half, and no potassium amendments were done with potash fertilizer

(0-0-62) and were as follows: 196, 98, and 0 kg per ha. On 8 July 2003, four ﬁeld cages

65

in each strip were set up. Two of the four cages (sacriﬁce cages) were sampled weekly
by removing ad counting the number of soybean aphids and plant characteristics on ﬁve
plants. The remaining two cages (yield cages) were not disturbed until harvest. In each
pair of sacriﬁce or yield cages, one cage was infested with aphids while the other served
as an aphid-free control. This resulted in a factorial design with three levels of potassium
amendment and two levels of aphid infestation. On 9 July, stand counts of the soybean
plants in each cage were taken and the cages were assigned to a soybean aphid treatment.
Infested cages were inoculated by tapping an aphid-infested soybean plant above the
plants in the cage for approximately 30 s.

Beginning on 29 July, ﬁve plants per week were removed from the sacriﬁce
cages, placed in a cooler at approximately 4 ° C, and transported back to the laboratory.
The coolers were stored overnight in a cold room at the same temperature. The following
day, the same plant characteristics measured in the ﬁeld surveys were counted and the
plants were preserved in whirl-paks as described above. On 12 August, soil samples
were taken from each cage as described above. One core was taken from each cage. In
addition, one phloem sample per cage was taken from the 2003 study. Sampling for
repetitions one and two took place on 5 September 2003 between 14:00 and 16:00.
Sampling for repetition three took place on 9 September 2003 between 12:00 and 14:00.
All phloem sampling took place while the plants were at the R6-R7 (full seed-beginning
maturity) growth stages.

In 2004, the study site (N 42 ° 7.50’ W 85 ° 80.00’) had an initial soil potassium
level of 67 ppm as measured by a soil test just after planting (Table 2). Two strips in

each of ﬁve replicates were established in the ﬁeld and either left unfertilized, or else

66

fertilized by broadcasting 256.8 kg/ha potash fertilizer (0-0-62) (Mason Elevator, Mason,
M1) on 13 May 2004. Two cages per strip were set up on 13 May 2004, when soybean
germination was almost complete. On 28 May 2004, the soybeans in the cages were
thinned to 13 plants per cage. One cage in each strip was infested with one soybean
aphid per plant on ten plants on 28 May 2004 and the second cage was maintained aphid-
free to serve as a comparison. This resulted in a 2x2 factorial design, with the presence
or absence of potash fertilizer and soybean aphids as factors. The source of the soybean
aphids was a laboratory colony established from a single ﬁeld-collected soybean aphid in
2003, and maintained at Michigan State University (27° C, 24 hour photoperiod). Soil
samples (three cores) from each cage were taken on 4 June 2004. The number of
soybean aphids per plant was counted two or three times a week from 28 May until 15
July.

In 2004, three phloem samples per cage were taken from three uninfested plants
in the cages receiving the aphid infestation (cages had 13 non-touching plants, only 10
were infested) and three plants in the uninfested cages on 18 June 2004 when the
soybeans were in the late vegetative or R1 (beginning ﬂower) growth stages. A second
set of 3 phloem samples per cage was taken from two remaining sets of uninfested cages
on 15 July 2004 when the soybeans were in the R2 (full ﬂower) growth stage. All 2004

phloem samples took place between 11:00 and 15:00.

Phloem sampling and analysis

Phloem samples were taken using a modiﬁcation of the phloem exudation method

of King and Zeevart (1974). A 10mM solution of ethylenediaminetetraacetate (EDTA)

67

was prepared and adjusted to pH 7.0 with 1 N NaOH. When a plant stem or petiole with
a cut sieve tube is immersed in this solution, the EDTA binds the calcium in the sieve
tube. Calcium acts as a signaling molecule that initiates callose formation. Thus,
immersing a cut sieve tube in EDTA solution prevents the formation of callose, and
phloem sap contained in both the cut sieve tube and connected phloem vessels exudes
into the solution. The entire solution is then analyzed as an indirect measure of phloem
contents. Five mL aliquots of the EDTA solution were placed in 2 dram vials that were
then sealed with Paraﬁhn. In the ﬁeld, a slit was cut in the Paraﬁlrn covering of each
vial. A soybean leaf (the second fully expanded leaf counting down from the top of the
plant) was cut off at the petiole in a dish of the EDTA solution. It was then immediately
transferred to one of the EDTA vials by passing the petiole through the slit in the
Paraﬁlm. Vials were placed in a cooler at approximately 4° C, returned to the lab, and
placed in a cool room at 4° C. Twenty-four hours after the samples were taken, leaves
were removed from the vials and the vials were sealed with a lid. The length and width
of the central leaﬂet of each soybean leaf was then recorded.

The concentration of potassium as well as the physiological amino acids was
determined for each phloem sample. The potassium concentration (ppm) of the exudate
in each vial was determined by measuring the potassium content of a 0.5 mL aliquot of
the exudate solution using a Cardy Potassium K+ Meter (Spectrum Technologies,
Plainﬁeld, IL). The samples were then stored at —80° C until free amino acid analysis
was performed. Free amino acid analysis was performed via high precision liquid
chromatography (HPLC) using a Waters system (Waters 2690 Separations Module,

Waters 474 Scanning Flourescence Detector, Waters Temperature Control Module,

68

Millennium Software Package, and the Acc-Q tag reagent kit, Waters Corportation,
Milford, MA). HPLC took place at the Michigan State University Macromolecular
Structure Facility. HPLC analysis included the following amino acids: alanine, arginine,
asparagine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,

lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine.

Statistical Analysis

Because phloem exudation is an indirect measure of phloem sap amino acid
contents, it is not possible to directly evaluate the concentration of amino acids in the
phloem. Instead, the proﬁle of amino acids, as deﬁned by the proportion of each amino
acid in all the free amino acids of the exudate, was compared among treatments. In the
surveys and the 2003 cage study, proportions of individual amino acids in the phloem
proﬁle and soybean aphid number per plant were regressed against soil nutrient levels
using PROC REG in SAS version 8.2 (The SAS Institute, 1999). For the 2004 cage
study, amino acid proportions and soil nutrient levels were regressed using Proc Reg;
because of a strong replicate effect soybean aphid numbers were analyzed via Proc
Mixed in SAS version 8.2 (The SAS Institute, 1999). For the ﬁeld surveys, any p value
less than 0.10 was considered signiﬁcant when regressing proportions of amino acids.
This is appropriate because of the large amount of variability encountered in the survey
results and because there were many factors that could not be measured or controled in
the ﬁeld surveys (variety, planting date, soil type, moisture levels, etc.). Amino acids
such as asparagine act as plant signaling molecules. Thus, they could be affected by a

wide range of environmental factors besides soil potassium. In 2003, phloem samples

69

were taken from plants in cages with and without aphids. Because some species of
aphids are able to inﬂuence the amino acid composition of their food source (Telang et al.
1999, Sandstrom 2000, Sandstrom and Moran 2001), samples taken from cages with and

without soybean aphids were analyzed separately.

Results

Surveys

In 2003, one amino acid, asparagine, had a signiﬁcant relationship with soil
potassium (Figure 3.1). The proportion of asparagine in the phloem amino acid proﬁle
was negatively correlated with the concentration of potassium in the soil where the plants
were growing. The average number of soybean aphids per leaf (20-200 aphids) was
positively correlated with the proportion of asparagine in the phloem (Figure 3.2.). In
2004, 11 amino acids were correlated with soil potassium (Table 3.1), including a
negative correlation between soil potassium concentrations and proportion of asparagine
in the phloem proﬁle (Figure 3.3). Other amino acids negatively correlated with soil
potassium level were histidine, threonine, valine, isoleucine and lysine. Aspartic acid,
glutamic acid, serine, glycine, and alanine were positively correlated with the soil
potassium level. There was no correlation between soybean aphid population and amino

acids, but soybean aphid populations were extremely low (fewer than 100 per plant).

Controlled Cage Studies

The amino acid proﬁles of infested and uninfested plants were analyzed

separately in the 2003 cage study. In plants from infested cages in the 2003 study,

70

 

glycine was the only amino acid signiﬁcantly correlated with soil potassium (slope =
0.0024, r-squared =0.35, p = 0.0131). In plants from uninfested cages, aspartic acid
(slope = 0.0035, r-squared = 0.46, p = 0.0078) and glutamic acid (slope = 0.0015, r-
squared = 0.33, p = 0.0333) were positively correlated with soil potassium levels.

In 2004, two sets of phloem samples were taken. All 2004 phloem samples were
taken from uninfested plants to avoid any effects of the aphids on phloem amino acids.
Early in the season (18 June), no amino acid had a signiﬁcant relationship with soil
potassium. At that time, soybean aphid populations were also not affected by soil
potassium levels. By 15 July, eleven amino acids were signiﬁcantly correlated with soil
potassium (Table 3.2). Once again asparagine was negatively correlated with soil
potassium (Figure 3.4). The amino acids positively correlated with soil potassium
included aspartic acid, glutamic acid, histidine, proline, tyrosine, valine, isoleucine,
leucine, lysine, and phenylalanine. Because all phloem samples were taken from
uninfested plants, it was not possible to directly assess the relationship between soybean
aphid population level and phloem amino acids. However, soybean aphid populations
were signiﬁcantly higher on plants growing in lower potassium soil from 30 June until

aphid populations crashed.

Discussion

In the 2003 survey, 2004 survey, and 2004 cage study, asparagine was negatively
correlated with soil potassium level. Also in the 2003 survey and 2004 cage study, aphid
population size was negatively correlated with soil potassium. The 2003 survey took

place during a soybean aphid outbreak (up to 17,000 aphids per plant) and the 2004 cage

71

study also represents outbreak conditions since no biological or chemical control was
present in the ﬁeld cages. The 2004 survey, however took place at extremely low aphid
populations (usually fewer than 100 aphids per plant). Therefore, the 2004 survey
probably represents a situation where the soybean aphid population was limited by
factors other than nutrition (i.e. predation) while during the 2003 survey and 2004 cage
study, the soybean aphid population was probably constrained by nutrition. Of those two
studies, soybean aphid population and asparagine may only be directly compared in the
2003 survey, and they are indeed positively correlated.

The 2003 cage study did not to follow this pattern. Neither soybean aphid
population nor phloem asparagine was correlated with soil potassium. However, this
should not be taken to contradict the patterns in the other studies. In 2003, cages were
infested by tapping an infested plant over the plants in the cage. Thus, aphid populations
were not standardized as they were in 2004. The aphid populations that were measured
probably reﬂect differences in initial infestation rather than a host-plant mediated
nutritional relationship. In addition, the phloem samples were taken at a late stage of
plant maturity after the soybean aphid population crash. Since asparagine acts as a
signaling molecule within the soybean plant (Bacanamwo and Harper 1997), it is possible
that it was no longer needed at that stage of maturity and thus did not vary with soil
potassium at the time that the plants were sampled. An interesting observation in the
2003 cage phloem samples is that different amino acids varied with potassium depending
on the presence of soybean aphids. Other species of aphids, such as Diuraphis noxia
Mordvilko are known to modify the amino acid proﬁle of their host plants (Telang et al.

1999), and it is possible that the soybean aphid does this as well. This is particularly

72

intriguing in view of the fact that aspartic acid and glutamic acid, which were positively
correlated with soil potassium in the uninfested plants, may be repellant to some aphids
(Abisgold et a1. 1994).

Asparagine may vary with soil potassium level because of its role as a signaling
molecule between the shoots and nodules of the soybean plant. In nodulated soybeans, a
symbiosis occurs between the soybean plant and one of four genera of Rhizobiaceae,
usually Rhizobium or Bradyrhizobium (Schubert 1995). The bacterium supplies ﬁxed
nitrogen to the plant and receives ﬁxed carbon. The process of nitrogen ﬁxation is
energetically expensive; respiration by the symbiont may account for 70% of total root
respiration (Walsh et al. 1998). Thus, it is advantageous for the plant to slow or stop
nitrogen ﬁxation under environmental stress or when the plant reaches a growth stage
where additional ﬁxed nitrogen is no longer required. The signal to slow nitrogen
ﬁxation is asparagine (Bacanamwo and Harper 1997).

The mechanism for increasing phloem asparagine under conditions of potassium
deﬁciency is as follows. Nitrogen is ﬁxed and converted to ureides in the nodule and
transported to the rest of the plant via the xylem (Whitehead et al. 2001). In the shoots,
ureides are broken down into amino acids and loaded into the phloem (V adez et al. 2000)
or made into proteins. Asparagine arriving at the nodules is broken down into aspartate
and glutamate which can enter the bacteroid and inhibit nitrogen ﬁxation (Bacanamwo
and Harper 1997). Potassium deﬁciency is likely to result in inhibition of nitrogen
ﬁxation because the enzyme responsible for converting amino acids into proteins requires
potassium as a cofactor(Menge1 and Kirkby 2001). When the plant is deﬁcient in

potassium, amino acids are especially likely to build up in the foliage (Mengel and

73

 

Kirkby 2001), causing increased levels of asparagine to be transported in the phloem in
order to slow nitrogen ﬁxation.

Through this mechanism, soil potassium deﬁciency is likely to increase levels of
phloem asparagine when nitrogen ﬁxation is occurring in soybean. Aphids are severely
nitrogen limited (Terra 1988), and asparagine is one of the most important nitrogen
sources in the aphid diet (Dadd and Krieger 1968) because it can be converted to
essential amino acids lacking in the aphid diet (Douglas 1998). In both ﬁeld surveys and
controlled cage studies, soybean aphid populations or density and phloem asparagine
levels are negatively correlated with soil potassium levels. The increased levels of
phloem asparagine encountered by soybean aphids feeding on potassium deﬁcient
soybean plants may alleviate part of their dietary nitrogen limitation, allowing for greater

soybean aphid populations.

74

 

0.3 -—-*i if i.,___ --__ _ 3E— f .—

 

 

 

 

 

. y=-0.0003x+0.0835 }
0.25 . — -—— —---#- —— m~— 2 v- m,
g 1 , R =0.0429 .
'- 0 O i
E 02 #;~r# ___ —- .r___ —— — —
N Q .
9': L 9. o
g 015 a A ——— —9—- - -----——.—— ,— — —
.2 0 ’ o
t , o
g 01 a—.—s— o ’ a
0 ~ ’ 0 00 ’0
l.—~-— seq-C" - we“
9 O
0 WW'. ’.“ . ___
0 20 4O 60 80 100 120 140
SoilK(ppm)

Figure 3.1. Proportion of asparagine in the phloem amino acid proﬁle versus soil
potassium level in eight commercial soybean ﬁelds in the 2003 Survey (p = 0.03).
Samples were taken during a soybean aphid outbreak when aphid populations were
commonly over 10,000 per plant. Samples were taken August 13-14 2003, just as the

soybean were beginning to set seed.

75

 

 

200 a - -- #~

180 '. . y=399X+82645 ............
l R2=0.2126 . . ’
160 ,, ,,,,,
I... O
I!
0
—| 1
. l
'0
E
Q.
<
C
0
a
>. ;
o .
U)

 

 

 

Percentage Asparagine

Figure 3.2. Soybean aphids per leaf versus the pr0portion of asparagine in the phloem
free amino acids in three commercial soybean ﬁelds with active soybean aphid

populations in the 2003 ﬁeld survey (p = 0.07 22).

76

 

 

 

 

 

 

 

 

 

 

 

 

 

 

09 T — W —* "W "' ‘Q #’ #r‘my: -0.0025X + 0.3113“ l
g 0.8 i— --—~— ——- ”—- -——- -~——w ~— R2=O.042 *i
g 0.7 e — 7.; — _ . He
goal 4 4.- 9—5—4 H-——— i —— e
< 1H *2- L ___ _7.- .___ﬁ ﬁ W
a 0.5 , ”—3— 4 l
.9 04 .- __- _ _____
t ' O l
O Q
8' 0'3 "# O , ' aﬁ—‘—'—*—‘ ,_______. l
a 0.2 . - o . 3 -.
0 1 m 4‘. ‘ ﬂ 9 i
' O 30 9,0 00 8 j
0 9. __.___._____Q_§___:__.____ _ ,_ O i
20 30 40 50 60 70 80 90
Soil K(ppm)

Figure 3.3. Proportion of asparagine in the phloem amino acid proﬁle versus soil
potassium level in ﬁve commercial soybean ﬁelds in the 2004 Survey (p = 0.055).
Samples were taken 17-23 August 2004 during soybean seed set. Aphid populations

were extremely low.

77

 

 

y = -0.0097x + 0.608 ——1
R2 = 0.2785 _;

 

 

 

 

 

 

 

 

 

Proportion Asparagine

 

 

 

 

Soil K (ppm)

 

60 7O 80

Figure 3.4. Proportion of asparagine in the phloem amino acid proﬁle versus soil

potassium level in the 2004 Cage Study on the July 15 sampling date (p = 0.002).

Soybeans were in ﬁrll ﬂower. All samples were taken from uninfested plants.

78

 

Table 3.1. Slope, r-square value, and p value of the amino acids signiﬁcantly correlated
with soil potassium level ﬁve commercial soybean ﬁelds in the 2004 ﬁeld survey on 17-
23 August. Soybeans were setting seed and the soybean aphid populations were

extremely low.

 

 

Amino Acid slope R2 p
Aspartic Acid 0.002 0.0949 0.0035
Glutamic Acid 0.0005 0.0394 0.0687
Serine 0.0016 0.0553 0.0275
Asparagine -0.0025 0.042 0.0553
Glycine 0.0012 0.043 0.0526
Histidine -0.0001 0.0815 0.007
Threonine -0.0002 0.0478 0.0406
Alanine 0.0005 0.0313 0.099
Valine -0.0003 0.0531 0.0437
lsoleucine -0.0003 0.0731 0.0109
Lysine -0.0009 0.159 0.0001

 

79

Table 3.2. Slope, r-square value, and p value of the amino acids signiﬁcantly correlated
with soil potassium level in the 2004 cage study 15 July sampling date. Soybean were in

full ﬂower. All phloem samples were taken from uninfested plants.

 

 

Amino Acid slope R2 p
Aspartic Acid 0.0045 0.1786 0.0179
Glutamic Acid 0.0021 0.1631 0.0269
Asparagine -0.0097 0.2785 0.0023
Histidine 0.0002 0.1928 0.0135
Proline 0.0009 0.3837 0.0002
Tyrosine 0.0001 0.1881 0.0148
Valine 0.0011 0.3571 0.0004
lsoleucine 0.0004 0.2741 0.0025
Leucine 0.0013 0.5623 <0.0001
Lysine 0.0004 0.2335 0.0059
Phenylalanine 0.0002 0.1439 0.0354

 

80

 

 

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Abisgold, J. D., S. J. Simpson, and A. E. Douglas. 1994. Nutrient regulation in the pea
aphid Acyrthosiphon pisum: application of a novel geometric framework to sugar
and amino acid consumption. Physiological Entomology 19: 95-102.

Bacanamwo, M., and J. E. Harper. 1997. The feedback mechanism of nitrate inhibition
of nitrogenase activity in soybean may involve asparagine and/or products of its
metabolism. Physiologia Plantarum 100: 371-377.

Dadd, R., and D. Krieger. 1968. Dietary amino acid requirements of the aphid, Myzus
persicae. Journal of Insect Physiology 14: 741-764.

Dixon, A. 1998. Aphid Ecology. Chapman and Hall, London.

Douglas, A. 1993. The nutritional quality of phloem sap utilized by natural aphid
populations. Ecological Entomology 18: 31-38.

Douglas, A. 1998. Nutritional interactions in insect-microbial symbioses: Aphids and
their symbiotic bacteria Buchnera. Annual Review of Entomology 43: 17-37.

Douglas, A. 2003. The Nutritional Physiology of Aphids. Advances in Insect Physiology
31: 73-140.

Foissac, X., Edwards, MG, Du, JP, Gatehouse, AMR, Gatehouse, JA. 2002. Putative
protein digestion in a sap-sucking homopteran plant pest (rice brown plant
hopper; Nilaparvata lugens: Delphacidae)-identiﬁcation of trypsin-like and
cathepsin B-like proteases. Insect Biochemistry and Molecular Biology 32: 967-
978.

Havlickova, H., and M. Smetankova. 1998. Effect of potassium and magnesium
fertilization on barley preference by the bird cherry-oat aphid Rhopalosiphum
padi. Rostlinna Vyroba 44: 379-383.

Institute, S. 1999. SAS OnlineDoc ® computer program, version 8. By Institute, S.,
Cary, NC.

King, R. W., and J. A. D. Zeevart. 1974. Enhancement of phloem exudation from cut
petioles by chelating agents. Plant Physiology 53: 96-103.

Mengel, K., and E. A. Kirkby. 2001. Principles of Plant Nutrition. Kluwer Academic
Publishers, Dordrecht, Netherlands.

Moran, N. A., G. R. Plague, J. P. Sandstrom, and J. L. Wilcox. 2003. A genomic
perspective on nutrient provisioning by bacterial symbionts of insects.
Proceedings of the National Academy of Sciences 100: 14543-14548:

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Sandstrom, J., Moran, N. 1999. How nutritionally imbalances is phloem sap for aphids?
Entomologia Experimentalis et Applicata 91: 203-210.

Sandstrom, J., Telang, A, Moran, NA. 2000. Nutritional enhancement of host plants by
aphids--a comparison of three aphid species on grasses. Journal of Insect
Physiology 46: 33-40.

Sandstrom, J. P., and N. A. Moran. 2001. Amino acid budgets in three aphid species
using the same host plant. Physiological Entomology 26: 202-211.

Schubert, S. 1995. Nitrogen assimilation by legumes-processes and ecological
limitations. Fertilizer Research 42: 99-107.

Srivastava, P. 1987. Nutritional Physiology, pp. 99-121. In A. Minks, Harrewijn, P [ed],
Aphids Their Biology, Natural Enemies and Control. Elsevier, Amsterdam.

Telang, A., J. Sandstrom, E. Dyreson, and N. Moran. 1999. Feeding damage by
Diuraphis noxia results in a nutritionally enhanced phloem diet. Entomologia
Experimentalis et Applicata 91: 403-412.

Terra, W. 1988. Physiology and biochemistry of insect digestion: an evolutionary
perspective. Brazilian Journal of Medical and Biological Research 21: 675-734.

Vadez, V., T. R. Sinclair, and R. Serraj. 2000. Asparagine and ureide accumulation in
nodules and shoots as feedback inhibitors of N2 ﬁxation in soybean. Physiologia
Plantarum 110: 215-223.

Van Emden, H. 1966. Studies on the relations of insect and host plant III. A comparison
of the reproduction of Brevicoryne brassicae and Myzus persicae: (Hemiptera:
Aphididae) on Brussels sprout plant suppied with different rates of nitrogen and
potassium. Entomologia Experimentalis et Applicata 9: 444-460.

Walsh, K. B., M. R. Thorpe, and P. E. H. Minchin. 1998. Photoassimilate partitioning
in nodulated soybean II. The effect of changes in photoassimilate availability
shows that nodule permeability to gases is not linked to the supply of solutes or
water. Journal of Experimental Botany 49: 1817-1825.

Weibull, J. 1987. Seasonal changes in the free amino acids of oat and barley phloem sap
in relation to plant grth stage and grth of Rhopalosiphum padi. Annals
of Applied Biology 111: 729-737.

Weibull, J. 1988. Free amino acids in the phloem sap from oats and barley resistant to
Rhopalosiphum padi. Phytochemistry 27: 2069-2072.

Whitehead, L. F., S. D. Tyerman, and D. A. Day. 2001. Polyamines as potential
regulators of nutrient exchange across the peribacteroid membrane in soybean
root nodules. Australian Journal of Plant Physiology 28: 675-681.

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Wilkinson, T., and H. Ishikawa. 1999. The assimilation and allocation of nutrients by
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Experimentalis et Applicata 91: 195-201.

Wilkinson, T. L., and A. E. Douglas. 2003. Phloem amino acids and the host plant
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Applicata 106: 103-113.

83

Chapter 4—The impact of foliar fertilization on soybean aphids and soybeans

Introduction

The purpose of foliar fertilization is to provide plants with nutrients at a critical
growth stage. In soybeans, foliar fertilizers are used at both early vegetative stages (Haq
and Mallarino 1998, 2000, Mallarino et al. 2001) and reproductive stages (Poole et al.
1983). Most of the foliar fertilizers used contain nitrogen, phosphorus, potassium, and
micronutrients. The impact of foliar fertilization on soybean yield is variable (Poole et a1.
1983, Haq and Mallarino 1998, 2000, Mallarino et al. 2001).

The soybean aphid was ﬁrst discovered in North America in 2000 (Venette and
Ragsdale 2004). It is the most severe insect pest of soybeans, and may reduce yield by
up to 40% (DiFonzo 2002). Currently, there are very few alternatives to chemical
insecticides for soybean aphid control. Some Michigan growers have been employing
foliar fertilization during the reproductive stages of soybean as a method of aphid control.
However, there are no controlled studies verifying this method.

Because foliar fertilizer contains a ninnber of nutrients, the anticipated effect on
aphids is unclear. Nitrogen fertilization of wheat increased the abundance of a leaf-
feeding aphid, probably by increasing the amount of nitrogen available in the phloem
(Honek 1991). However, nitrogen fertilization can depress nitrogen ﬁxation in legumes
(Bacanamwo and Harper 1997). If this resulted in a net decrease in the amount of
nitrogen translocated in the phloem, it could slow soybean aphid population increase.
The effects of the other nutrients on herbivorous insects are variable (Waring and Cobb

1989).

84

When used for aphid control, foliar fertilizers are applied to plants that are not
thought to be deﬁcient in any nutrient. However, it is possible that a hidden
micronutrient deﬁciency may be alleviated in some cases. The plant stress hypothesis
states that plants under stress are more susceptible to herbivores (Waring and Cobb
1989). Thus, if a foliar fertilizer reduced plant stress, it could allow the plant to better
combat soybean aphids, reducing aphid populations and/or aphid damage.

Finally, the foliar fertilizer may act directly on the aphids. The foliar spray could
coat the aphids. As it evaporates, the salty spray could draw water out of the bodies of
the insects, putting them under osmotic stress. This stress could slow aphid reproduction
or even kill the aphids, thus providing aphid control.

The objective of this study was to determine the effects of foliar fertilization of

soybeans on foliar nutrient level, soybean yield, and soybean aphid population.

Methods

The study took place during the 2004 growing season at four locations in
Michigan: on the Michigan State University Entomology Research Farm in Ingham
County, on the Michigan State University Bean and Beet Research Farm in Saginaw
County, and at two commercial soybean ﬁelds in Gratiot and Calhoun Counties.

At all locations, plots were established in existing soybean ﬁelds. The strips had
two treatments, with and without Alpine Fortiﬁed Liquid F oliar Fertilizer (Alpine) (10-
10-10 plus 0.1% Boron, 0.1% Iron, 0.05% Manganese, 0.05% Zinc, and 0.0006%
Molybdenum). The foliar fertilizer was applied according to label directions: 4.94 l per

ha of product at R3 (beginning seed) and again at R5 (full seed). See Table l for plot size

85

and treatment details. At one location (the Calhoun County site), an additional treatment

of a half rate of foliar fertilizer (2.5 l per ha) was included.

Aphid Counts

At the university farms sites, the number of aphids per plant was counted weekly
on ﬁve plants per plot. At the Ingham County site, counting began on 7 July 2004 and
lasted until 23 August 2004. At the Saginaw County site, counting began on 28 June
2004. The site was not evaluated again until 5 August 2004. It was then evaluated
weekly through 23 August 2004. In the commercial ﬁelds, cooperating extension agents

noted the presence of aphids, but did not conduct weekly counts.

F oliar Nutrient Sampling and Analysis

F oliar leaf samples were taken seven days after each foliar fertilizer application.
In each plot, the uppermost fully-unfurled leaf of 30 randomly selected plants was cut off
at the base (petioles were not removed). The leaves from each plot were combined and
placed a paper bag. Within 24 hours, the leaves were washed by placing them in a soapy
water bath (J oyTM phosphate-free dish soap) then rinsed thoroughly and patted dry. The
leaves were returned to the paper bags and dried in an oven at 32° C for three to four
days. The dried samples were stored until 21 October. The samples were sent to A&L
Labs (Fort Wayne, IN) and analyzed for their content of the following elements:
nitrogen, phosphorus, potassium, calcium, magnesium, sulphur, zinc, manganese, iron,

copper, boron, aluminum, and sodium.

86

Yield
All plots were mechanically harvested for yield. Plot area harvested depended on
the location and equipment (Table 1). All yield results were standardized to 13.5%

moisture prior to analysis.

Statistical Analysis

Nutrient content and yield data were analyzed using PROC GLM in SAS version
8.2 (The SAS Institute, 1999). The soybean aphid counts did have a normal distribution,
so they were analyzed with a Kruskall-Wallis test using PROC NPARlWAY in SAS

version 8.2 (The SAS Institute, 1999).

Results

Aphid Counts

Aphids were present at all locations throughout the season, but numbers were
extremely low. The highest populations observed were 120 aphids on a plant at the
Ingham County site (19 July). This single plant accounted for about half of the aphids
observed for the entire season at that site. At the Saginaw County site, the highest aphid
count was 73 aphids per plant (18 August). Aphid numbers in the fertilized and
unfertilized treatments did not signiﬁcantly differ at either site on any sampling date

(p>0.05) (Figure 4.1).

87

F oliar Nutrient Sampling and Analysis
There were no signiﬁcant differences in the concentrations of the selected foliar
nutrients between the fertilized and unfertilized plots at any location and sampling date

(p>0.05).

Yield
Although there were differences in yield between sites, there was no within-site

difference in yield between fertilized and unfertilized plots (p>0.05) (Figure 4.2).

Discussion

In 2004, foliar fertilization did not signiﬁcantly impact the soybean aphid
populations at either the Saginaw County or the Ingham County sites. However, the
soybean aphid populations at these sites were extremely low, and the results do not
deﬁnitely predict the effects of foliar fertilizer on outbreak populations of soybean aphid.
Although this does not appear to be a promising method for aphid control at this point,
additional replication under conditions of higher aphid pressure is needed to deﬁnitely
determine the effects of foliar fertilizer on soybean aphid populations.

We hypothesized that any effects on soybean aphids in this study would be due to
changes in the amount of nitrogen available to them. Aphids in general are very sensitive
to the amount of nitrogen in their diets (Dadd and Krieger 1968, Douglas 1993, Ponder et
al. 2000, Koyama et al. 2004), and if foliar fertilization treatments affected phloem
nitrogen, they could also affect aphid populations. There are several reports in the

literature of aphid populations increasing when their host plants are fertilized with

88

nitrogen (Honek 1991, Ponder et al. 2000, Cisneros and Godfrey 2001, Nevo and C011
2001, J ansson and Ekbom 2002). However, it is not known whether the small amount of
foliar fertilizer used in these treatments could affect the phloem nitrogen content of
soybean plants enough to affect the aphid population. Since soybeans are legumes, and
can slow or stop nitrogen ﬁxation if other nitrogen sources become available
(Bacanamwo and Harper 1997), it is also not clear that if foliar fertilization affected
phloem nitrogen that it would result in a net nitrogen increase. Thus changes in phloem
nitrogen content due to foliar fertilization could positively or negatively affect soybean
aphid populations.

The results of both the foliar nutrient analysis and yield comparison indicated that
the foliar fertilizer did not affect the plant in any way. All four of our sites were
nutritionally adequate ﬁelds because this study was designed to test the effects of
supplemental fertilizer as aphid control, not as a rescue treatment for deﬁcient plants.
Nonetheless, we had expected differences in the foliar nutrient analysis that would
indicate that the fertilizer had physiological effects on the plant; it would appear that it
does not.

The results of the foliar nutrient analysis indicate that if foliar fertilization had any
affect on soybean aphid populations, it is unlikely to be mediated by the host plant. It
appears that any possible effects of foliar fertilizer on soybean aphid populations is due to
the direct action of the fertilizer on the aphids, most likely by acting as a desiccating salt.
Although there is no evidence in the literature for foliar fertilization or any other
treatment to control aphid in this way, it is possible that it occurs. Spraying sugar

solutions on ﬁelds had also been suggested as an alternative to aphid control through

89

chemical insecticides. It is possible that both methods could work through the same
mechanism.

Foliar fertilization does not show great promise for soybean aphid control.
However, it has not been tested in a year when high populations of the pest were present.
Additional testing under higher aphid pressure is needed before the efﬁcacy of this

method can be deﬁnitely determined.

90

 

12 . ~-
‘ A. BayCountySite
10 t ...........................................

 

 

 

 

 

 

' 1+No Fertilizer
2 Ala—Fertilizer

 

Soybean Aphids per Plant +/- SE

 

 

6/22 7/12

 

 

 

 

 

 

 

 

 

   

 

 

 

 

a 7 ; - ——— + 5%
1' 6 l B. InghamCountySite . ,,,,,,,,,,,,,,,, 1
g a
E 5 l ---------------------
8 4 '1 iiiiiiiiiiiii
(I) ‘_. ._.________.
E 3 »« vi—o—No Fertilizer
2 hula—Fertilizer
C 2 i
g .
g 1 ’ ---- 1
o .
(D 0 . ,, ,__ _

7/10 7/1 5 7/20 7/25

Date

Figure 4.1. Aphid counts of fertilized and unfertilized plants at university farms sites
during the 2004 ﬁeld season. Counts are not signiﬁcantly different at either site on any

sampling date (p>0.05).

91

  

 

INo Foliar Fertilizer

     
  
 

UWith Foliar Fertilizer

  

IWith Half Rate Foliar ‘
Fertilizer h l

     
 

 

Yield (bu/acre) +I-SE

    

 

 

 

Ingham Gratiot Calhoun
Site

Figure 4.2. Yield of soybean at four experimental sites where foliar fertilizer was

applied. In an AN OVA, there were no differences at p<0.05.

92

Table 4.1. Plot details and maintenance dates for the 4 sites in the study.

 

 

County
Ingham Saginaw Calhoun Gratiot
Planting Date 5/19/2004 5/7/2004 6/8/2004 6/5/2004
148,000 125,000

Planting Rate 80-90 lbs/acre seeds/acre ** seeds/ acre
Planting Method Drilled 30 in rows 30 in rows 30 in rows

Randomized Randomized Randomized Alternating
Study Design Complete Block Complete Block Complete Block Strips
Plot Size 50 ft x 20 ft 4 rows x 50 ft 4 rows x 35 ft 6 rows x 70 ft
Replications 4 4 4 4
Foliar Fertilzer
Application--R3 8/16/2004 7/23/2004 7/29/2004 8/9/2004
Foliar Fertilzer
Application-—R5 9/2/2004 8/1 1/2004 8/24/2004 8/27/2004
Chemical
Application commercial
Method C02 sprayer C02 sprayer C02 sprayer applicator
Chemical 17.4 17.4
Application Rate gallons/acre gallons/acre 13 gallons/acre **
Chemical
Application
Pressure 30 psi 30 psi 24 psi **
Chemical
Application
Nozzle Type ﬂat fan ﬂat fan ** **
Harvest Date 10/26/2004 10/26/2004 1 1/8/2004 10/13/2004
Harvest Area 5 x 50 ft 2 rows x 50 ft 2 rows x 35 ft 4 rows x 70 ft

Combine +

Harvest Method Plot Combine Plot Combine Plot Combine Weigh Wagon

 

** = unknown.

93

Literature Cited

Bacanamwo, M., and J. E. Harper. 1997. The feedback mechanism of nitrate inhibition of
nitrogenase activity in soybean may involve asparagine and/or products of its
metabolism. Physiologia Plantarum 100: 371 -377.

C isneros, J., and L. Godfrey. 2001. Midseason pest status of the cotton aphid
(Homoptera: Aphididae) in California cotton: Is nitrogen a key factor?
Environmental Entomology 30: 501-510.

Dadd, R., and D. Krieger. 1968. Dietary amino acid requirements of the aphid, Myzus
persicae. Journal of Insect Physiology 14: 741-764.

DiFonzo, C., Hines, R. 2002. Soybean aphid in Michigan Update from the 2001
season. Michigan State University, East Lansing, MI.

Douglas, A. 1993. The nutritional quality of phloem sap utilized by natural aphid
populations. Ecological Entomology 18: 31-38.

Haq, M. U., and A. P. Mallarino. 1998. Foliar fertilization of soybean at early vegetative
stages. Agron. J. 90: 763-769.

Haq, M. U., and A. P. Mallarino. 2000. Soybean yield and nutrient composition as
affected by early season foliar fertilization. Agron. J. 92: 16-24.

Honek, A. 1991. Nitrogen fertilization and abundance of the cereal aphids
Metaopolophium dirhodum and Sitobion avenae (Homoptera, Aphididae). Journal
of Plant Diseases and Protection 98: 655-660.

Institute, S. 1999. SAS OnlineDoc ® computer program, version 8. By Institute, 8., Cary,
NC.

Jansson, J ., and B. Ekbom. 2002. The effect of different plant nutrient regimes on the
aphid Macrosiphum euphorbiae growing on petunia. Entomologia Experimentalis
et Applicata 104: 109-116.

94

Koyama, Y., I. Yao, and S. Akimoto. 2004. Aphid galls accumulate high concentrations
of amino acids: a support for the nutrition hypothesis for gall formation.
Entomologia Experimentalis et Applicata 113: 35-44.

Mallarino, A. P., M. U. Haq, D. Wittry, and M. Bermudez. 2001. Variation in soybean
response to early season foliar fertilization among and within ﬁelds. Agron. J. 93:
1220-1226.

Nevo, E., and M. C011. 2001. Effect of nitrogen fertilization on Aphis gossypii
(Homoptera: Aphididae): variation in size, color, and reproduction. Journal of
Economic Entomology 94: 27-32.

Ponder, K., J. Pritchard, R. Harrington, and J. Bale. 2000. Difﬁculties in location and
acceptance of phloem sap combined with reduced concentration of phloem amino
acids explain lowered performance of the aphid Rhopalosiphum padi of nitrogen
deﬁcient barley (Hordeum vulgare) seedlings. Entomologia Experimentalis et
Applicata 97: 203-210.

Poole, W. D., G. W. Randall, and G. E. Ham. 1983. Foliar fertilization of soybeans. I.
Effect of fertilizer sources, rates, and frequency of application. Agron. J. 75: 195-
200.

Venette, R., and D. W. Ragsdale. 2004. Assessing the invasion by soybean aphid
(Homoptera: Aphididae): Where will it end? Annals of the Entomological
Society of America 97: 219-226.

Waring, G. L., and N. S. Cobb. 1989. The impact of plant stress on herbivore population
dynamics, pp. 167-226. In E. A. Bemays [ed.], Insect-Plant Interactions. CRC
Press, Boca Raton, F l.

95

Chapter 5 — Experiments on the effects of sooty mold on soybeans.

Introduction

Sooty mold is a saprophytic fungus species or group of species that grow on the
foliage of plants where aphids have deposited honeydew. Sooty mold may block up to
98% of light penetration to the leaf surface of pecan, reducing net photosynthesis up to
70% (Wood et al. 1988). This reduction in assimilate production could have large
consequences on the production of energy-rich organs such as seeds and on yield.

During periods of high soybean aphid infestation, sooty mold was observed on
soybean leaves (DiFonzo 2002). Even after the aphids were no longer present because of
either chemical application, migration, or entomopathogenic fungus, the sooty mold
persisted for the rest of the season. Because of the potential of sooty mold to shade the
foliage, there was concern that sooty mold could be causing reductions in soybean yield
apart from the effects of the soybean aphids. Soybeans are especially sensitive to shading
in during pod set (R3-R5) (Board et al. 1990), which corresponds to the time period when
aphid-infested soybeans are likely to have high levels of sooty mold.

The objective of these studies was to determine whether sooty mold alone could
impact soybean yield and, if so, the relationship between mold coverage and yield

reduction.

Methods

Experiments took place over two years. In both years, the goal of the experiments

was to stimulate the growth of sooty mold on soybean foliage in the absence of aphids by

96

applying a honeydew-like substance to soybean plants. We hoped to achieve a range of
sooty mold coverage, which we could then use to determine the relationship between

sooty mold coverage and soybean yield reduction independent of aphids.

2002 Field Experiment

A large-scale ﬁeld experiment took place at the Michigan State University Bean
and Beet Research farm in Saginaw County, Michigan. On 25 June, 2002, 12.2 m x 30.5
m plots were cut out of a soybean ﬁeld. The ﬁeld was planted using a seed drill to assure
close plant spacing and full canopy coverage as early in the year as possible. On 11 July,
the following treatments were applied for the ﬁrst time: 1) molasses, 2) molasses and
Asana (esfenvalerate) insecticide, 3) molasses and Microsperse (elemental sulphur)
fungicide, 4) molasses, Asana, and Microsperse, and 5) an untreated control. Plots were
treated weekly with molasses and biweekly with the pesticides. Initially, plots were
treated with 4 gallons of molasses as a sugar source in 32 gallons of water, Asana at 5.4
oz/acre, and Microsperse at 12 oz/acre. On 8 August, molasses treatments were increased
to 6 gallons in 32 gallons of water and Asana treatments were increased to 9.6 oz/acre of
Asana. Microsperse treatments ceased on that day because of extremely low mold
infestations. The object of the treatments were to have natural soybean aphid
populations with natural sooty mold levels (untreated), natural soybean aphid populations
with enhanced sooty mold levels (molasses only), natural soybean aphid populations
without sooty mold (molasses and Microsperse), no soybean aphids with sooty mold
(molasses and Asana), and no soybean aphid populations with no sooty mold (molasses,

Asana, and Microsperse). Molasses was included in Microsperse treatments in order to

97

control for the effect of molasses application on the soybeans. This experiment was
replicated four times.

Soybean aphids and mold were sampled weekly. Soybean aphid populations were
sampled by counting the number of soybean aphids on one leaf from the top third of the
plant on 30 plants per plot. Mold populations were evaluated by visually estimating the
percent of mold coverage on one leaf from the middle third of the plant on ten plants per
plot.

Neither the mold coverage data nor the aphid data met assumptions of normality.
Therefore, the data were analyzed with the Kruskal-Wallis test in the NPARlWAY

procedure of SAS version 8.2 (The SAS Institute, 1999).

2002 Shade Experiments

The second experiment in 2002 measured the effects of artiﬁcial shading on
potted soybeans. On 16 July, tomato rings were placed over potted soybeans with one
true leaf (V2 stage) placed outdoors. Knitted shade cloth (Gothic Arch Greenhouses,
Mobile, AL) was then ﬁtted over the tomato ring to achieve 30, 50 70, or 90 percent
shading. An uncovered plant inside a tomato cage was also included as a control, and the
experiment was replicated four times. Plant characteristics (number of nodes, leaves,
ﬂowers, pods, and pod ﬁll stage) were monitored weekly until plant maturity. When
plants had senesced, the number of pods per node, beans per pod, and total weight of
beans per plant were measured.

Data were analyzed via Fisher’s protected LSD using the GLM procedure of SAS

version 8.2 (The SAS Institute, 1999).

98

2003 Cage Experiment

In 2003, a study was performed on the Michigan State University Entomology
Research Farm in Ingham County, Michigan. The ﬁeld was planted on 23 May using a
seed drill with Pioneer variety 92813 soybeans at a rate of 175,000 seeds/acre. On 18
June, 42-1m2 exclusion cages were placed in the ﬁeld. See chapter two for a description
of the cages. Cages were assigned to each of the six treatments: 1) negative control, 2)
aphids only, 3) sugar only, 4) ﬁmgicide only, 5) sugar and fungicide, and 6) aphids and
fungicide. The treatments were selected to provide planned contrasts measuring the
effect of soybean aphid in the absence of sooty mold, the effect of sooty mold in the
absence of soybean aphid, and the effect of both together. The experiment was replicated
seven times. Five plants per cage were removed each week for counting of plant
characteristics from the beginning of reproduction until late August. The fungicide used
was Penncozeb (ionic manganese 15.0%, ionic zinc 1.9%, ethylenebisdithiocarbamate
58.1%) at 1.1 kg/ha. Sugar treatments were 31.25 g or—D glucose and lmL liquid
fertilizer (27-0-0 plus 1% S) per cage applied in 300 mL per cage of water. Treatments
began on 1 July. Before treatment on the same day, soybean aphids were added to aphid
cages at the rate of one aphid per plant (unless the plant was already infested with aphids)
and the no aphid treatments were sprayed with Asana (esfenvalerate) at a rate of 19.2
oz/acre. Fungicide was applied weekly and sugar was applied twice a week. On 16 July,
the liquid fertilizer was cut to 0.25 mL per cage because the plants exhibited burning

symptoms. On 21 July, the sugar rate was adjusted to 93.75 g or—D glucose per cage to

99

encourage mold growth. Sugar treatments ceased on 1 August because of low mold
growth.

Between 30 June and 7 July, the number of aphids present per plant on ﬁve plants
per cage was counted four times in situ. At this time, the mold coverage on those same
ﬁve plants was visually estimated. At the beginning of soybean reproduction (R1 stage),
5 plants a week were removed from the sacriﬁce cages. Plant traits were counted and
sooty mold coverage was estimated. Although the plants and their aphids were
preserved, soybean aphid populations were not evaluated in view of the other

experimental results.

Results

2002 Field Experiment

Aphid numbers were extremely low in 2002, and very little sooty mold was
observed in plots with either natural aphid infestations or molasses sprays. When
molasses, insecticide, and fungicide (elemental sulphur) were evaluated for their effect on
aphid numbers, only insecticide had a signiﬁcant effect on aphids (season wide average
with insecticide = 2.37 aphids per leaf, untreated = 10.62 aphids per leaf, p<0.0001).
Molasses (with molasses = 0.4% mold coverage, untreated = 0.3% mold coverage,
p<0.0001) and fungicide (with fungicide = 0.42% mold coverage, untreated = 0.38%
mold coverage, p = 0.0061) signiﬁcantly increased mold coverage, while insecticide
decreased mold coverage (with insecticide = 0.3% mold coverage, untreated = 0.4% mold

coverage, p = 0.0018). Mold infestations were extremely low, and any spots leaf with

100

dark spots that may have been sooty mold was recorded as 1% coverage; the differences

in mold coverage could easily be due to artifacts of this practice.

2002 Shade Experiments

Shading had a signiﬁcant effect of soybean growth and reproduction, especially
when shading was greater than 50% (Table 5.1). The no shade treatment took less time
to ﬂower than the 70% and 90% treatments. The 30% and 50% treatments also took less
time than the 90% treatment. The 50% shade treatment set more ﬂowers and pods than
the 70% or 90% treatments; the no shade and 30% treatments set more ﬂowers and pods
than the 90% treatment (Table 5.1). The percent of ﬂowers aborted per plant did not vary
by shade treatment. The lower-shade treatments aborted a lower percentage of pods than
the 70% treatment and all of the treatments aborted a lower percentage of pods than the
90% treatment (Table 5.1). In the 90% treatment all pods aborted. The 90% shaded
treatment also had fewer nodes than the less-shaded plants (Table 5.1).

At harvest, plants shaded by 50% had more pods yielding beans than the 30%,
70% or 90% treatments (Table 5.1). The no shade treatment had more than the 70% or
90% treatments, and the 70% treatment had more than the 90% treatment. The no shade,
30%, and 50% treatments had a higher number of beans per plant and weight of beans
and thus a higher yield than the 70% and 90% treatments; the 70% treatment was
signiﬁcantly greater than the 90% treatment (Table 5.1). In ahrrost all measures of plant
growth, reproduction, and yield, there was a critical point for damage between 50% and

70% shading.

101

2003 Cage Experiment

In the early season aphid counts, neither date nor fungicide signiﬁcantly affected
aphid populations (date Chi-square = 5.19, df = 3 p=0.1587, fungicide Chi-square =
0.01, df = l, p = 0.9164). Although plants from the remainder of the experiment were
preserved, they were not counted in view of the other results from this study. Sooty mold
did not establish in the sugar treatments during the 2003 season.

The presence of aphids, sugar, and fungicide in the cages affected soybean
growth. Aphid-free plants had more primary leaves than infested plants from 30 July
through the last sample on 15 August (Table 5.2). Plants treated with sugar had a
reduced number of primary leaves from 7 August onward (Table 5.3). Aphid-infested
plants had fewer secondary leaves, ﬂowers, and unﬁlled pods from 30 July through the
end of the experiment (Table 5.2). On 15 August, aphid-infested plants had fewer ﬁlling
pods per plant (Table 5.2) and, within the soybean aphid treatment, infested, fungicide-
treated plants had fewer ﬁlling pods than infested untreated plants (15 August aphid
plants with fungicide = 4.3, 15 August aphid plants without fungicide = 10.9, t = 3.95,
p<0.0001). Aphid-infested plants had fewer leaves per plant from 30 July through the
ﬁnal sampling date, while plants receiving sugar had fewer leaves on 7 August and after
(Table 5.2, 5.3). Soybean aphid-infested plants had fewer nodes from 30 August through
the ﬁnal sampling date while sugar treated plants had fewer on 7 August only (Table 5.2,
5.3). The total number of pods per plant was lower on aphid infested plants from 30 July
onward; within the soybean aphid treatments, fungicide treated plants had fewer pods on
15 August (15 August aphid plants with fungicide = 5.15, 15 August aphid plants without

fungicide = 29.8, t = 3.06, p = 0.0023).

102

Discussion

The effects of sooty mold on soybean growth and yield were not determined in
the 2002 ﬁeld or 2003 cage experiment. With the two sugar sources and weekly or
biweekly applications, sooty mold was not successfully established in the absence of
aphids. A different type of sugar, more frequent applications of sugar, or a constant
supply of moisture may be needed to establish this fungus.

The 2002 shade experiment showed that if the primary effect of sooty mold is by
shading of the plants, at least 70% total shading is necessary for yield reduction to occur.
Soybean plants are partially shaded by their neighbors; thus less than 70% coverage by
sooty mold may reduce yield, but enough sooty mold would be necessary to bring the
amount of shading up to 70%.

Since these experiments began, an economic injury level of about 1,000 aphids
per plant through the R6 stage of soybean has been set by a committee of specialists from
the North Central Region (Anonymous 2004). This same committee recommends
chemical control at a level of 250 aphids per plant. At these population levels, sooty
mold is unlikely to occur as a result of aphid infestation. Therefore, if the soybean aphid
population is properly controlled, additional yield loss as a result of sooty mold growing
on aphid honeydew is not a concern.

The 2003 cage study showed the impacts that high soybean aphid populations can
have on soybean development. Although yield was not evaluated, soybean aphids

reduced the number of leaves, ﬂowers, pods, and nodes. Since all of these characteristics

103

may be affected, soybean aphids have the potential to injure soybeans from the vegetative
stages of soybean through at least seed ﬁll.

There were also some cases where the use of fungicide affected soybean plants.
More importantly, aphid-infested fungicide-treated plants had reduced ﬁlling pods and
total pods on 15 August. Soybean rust has recently been detected in North America, and
agronomists are anticipating the need for fungicide sprays in certain years throughout
much of the North American soybean producing region. Although the results of small
scale cages do not necessarily translate into effects that will be seen at the ﬁeld level, the

effects of ﬁmgicides on pod set and seed set should be monitored.

104

Table 5.1. Plant characteristics of potted soybeans receiving various levels of shading in

the 2002 shade experiment. Numbers in the same row followed by the same letter are not

statistically signiﬁcant at p<0.05 in a Fisher’s protected LSD.

 

 

Shading
0% 30% 50% 70% 90%
Days to Reach Flowering 24.3 a 26.5 ab 25.0 ab 31.0 bc 33.0 c
Flowers Set per Plant 87.5 ab 83.5 ab 95.3 a 70.3 b 10.5 c
Pods set per Plant 36 ab 37.5 ab 46.0 a 32.0 b 5.8 c
Percent of Pods Aborted 46.9 a 47.9 a 49.2 a 71.7 b 100.0 c
Nodes per Plant 42.3 a 32.5 b 36.8 ab 29.8 b 6.5 c
Pods Yielded per Plant 18.8 ab 19.0 bc 23.8 a 9.0 c 0.0 d
Beans per Plant 40.0 a 42.0 a 52.3 a 18.3 b 0.0 c
Weight ofBeans per Plant 4.8 a 5.6 a 6.5 a 2.3 b 0.0 c

 

105

Table 5.2. Effect of aphid infestation of plant characteristics throughout the season in the

2003 cage study t values and p values are the result of Fisher’s protected LSD.

 

 

 

 

 

 

 

 

 

Date No Aphids Aphids t p
Primary Leaves per Plant 30-Jul 9.1 7.3 5.85 <0.0001
5-Aug 9.0 7.3 7.17 <0.0001
15-Aug 10.3 7.6 8.74 <0.0001
Secondary Leaves per Plant 30-Jul 7.6 4.2 2.69 0.0074
5-Aug 8.4 2.5 2.93 0.0035
15-Aug 7.7 5.6 5.54 <0.0001
Flowers per Plant 30-Jul 38.4 29.9 2.60 0.0097
5-Aug 27.5 17.1 4.10 <0.0001
15-Aug 15.2 8.7 2.82 0.0050
Unﬁlled Pods per Plant 30-Ju1 18.2 8.1 3.99 <0.0001
5-Aug 38.7 26.2 6.48 <0.0001
15-Aug 24.7 14.9 4.14 <0.0001
Filling Pods per Plant 15-Aug 1.7 0.5 20.18 <0.0001
Total Leaves per Plant 30-Jul 16.8 1 1.5 3.80 0.0002
5-Aug 16.7 12.9 4.34 <0.0001
15-Aug 18.7 10.1 7.00 <0.0001
Total Nodes per Plant 30-Jul 16.8 11.5 3.68 0.0003
5-Aug 18.7 16.9 2.45 0.0147
15-Aug 23.0 16.8 4.70 <0.0001
Total Pods per Plant 30-Jul 16.8 11.5 3.27 0.0011
5-Aug 40.4 26.6 11.18 <0.0001
15-Aug 53.6 22.5 5.73 <0.0001

 

106

Table 5.3. Effect of sugar application on plant characteristics throughout the season in

the 2003 cage study t values and p values are the result of Fisher’s protected LSD.

 

 

 

 

Date No Sugar Sugar t p
Primary Leaves per Plant 7-Aug 8.7 7.9 5.59 <0.0001

15-Aug 9.3 9.6 3.58 0.0004
Total Leaves per Plant 7-Aug 16.2 13.8 3.77 0.0002

15-Aug 15.4 16.6 2.74 0.0063
Total Nodes per Plant 7-Aug 18.8 16.6 2.66 0.0082

 

107

Literature Cited

Anonymous. 2004. Soybean Aphid Research Update, pp. 16. North Central Soybean
Research Program.

Board, J. E., T. Qiang, and B. G. Harville. 1990. Effect of reduced light interception
during reproductive development on soybean yield, pp. 41-42. The Louisiana
Agricultural Experiment Station.

DiFonzo, C., Hines, R. 2002. Soybean aphid in Michigan Update from the 2001
season. Michigan State University, East Lansing, MI.

Institute, S. 1999. SAS OnlineDoc ® computer program, version 8. By Institute, 3.,
Cary, NC.

Wood, B. W., W. L. Tedders, and C. C. Reilly. 1988. Sooty mold fungus on pecan

foliage suppresses light penetration and net photosynthesis. HortScience 23: 851-
853.

108

Appendices

109

  

 

 

 

 

 

 

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Voucher Specimen Data

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110

Appendix 1
Record of Deposition of Voucher Specimens”
The specimens listed on the following sheet(s) have been deposited in the
named museum(s) as samples of those species or other taxa, which were used
in this research. Voucher recognition labels bearing the Voucher No. have been
attached or included in ﬂuid-preserved specimens.
Voucher No.: 2005-03
Title of thesis or dissertation (or other research projects):
EFFECTS OF FERTILIZATION ON THE SOYBEAN APHID, APHIS GL YCINES

MATSUMURA AND THE EFFECTS OF SOYBEAN APHIDS AND
FERTILIZATION ON SOYBEAN PLANTS

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

lll

 

 

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