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BIONOMICS AND CONTROL OF TWO HETERODERA SPP.

IN MICHIGAN

presented by

CASSANDRA LEE BATES

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BIONOMICS AND CONTROL OF TWO HETERODERA spp. IN MICHIGAN
By

Cassandra Lee Bates

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTERS OF SCIENCE
Department of Entomology

2006

Abstract

BIONOMICS AND CONTROL OF
TWO HETERODERA SPP. IN MICHIGAN

By

Cassandra Lee Bates

Heterodera glycines (SCN) and H. schachtii (SBCN) are common
pathogens of soybeans and sugar beets in Michigan, respectively. Pratylenchus
penetrans (RLN) is another phytopathogenic nematode that parasitizes both
soybeans and sugar beets. It co-habits with SCN in ca 50% of Michigan soybean
fields. SCN resistant cultivars are widely used; however, there are no SBCN
resistant sugar beet varieties available. Michigan sugar beet growers have
adopted oilseed radish (OSR) cvs Colonel or Adagio as trap crops for control of
SBCN. This study evaluated 26 potential trap crops for the control of SCN.
Berseem clover, Dackon oilseed radish and Oriental mustard have potential as
trap crops for SCN. Another analysis evaluated RLN on SCN resistant
soybeans. RLN has the ability to reproduce on all seven plant introduction (Pl)
SCN resistant soybean lines. The nematode also has the ability to break down
resistance of a PI 88788 cultivar. Lastly, field and laboratory evaluation of over
30 sugar beet lines for resistance to SBCN was also conducted. All three USDA
lines tested showed resistance. Two commercial lines (Beta 5534N and Beta
5374) showed the most stability for resistance to SBCN in both the field and

greenhouse studies.

Dedication

I would like to dedicate this thesis to Philip Bates,
my husband and my best friend. He supported me through this process
and bore all my craziness with love and tenderness.

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Acknowledgements

Wow, where to even start, so many have helped me during this process. It
is tough to decide who should be listed first. So I have decided to thank my furry
feline friend, Yoda, first. She was and is always there to support me during the
good and bad. Next I would like to thank John Davenport. His guidance and
friendship through my time here has been immeasurable. And thanks for never
leaving me in a soybean field. Fred Warner is also another who I would like to
thank. He is the ultimate nematode guru.

My next group would be my committee. Many thanks are needed for Dr.
George Bird. Without his leadership and teaching my love for hematology would
not be complete. Dr. Chris DiFonzo with her guidance and humor has kept me
laughing through this whole process. Dr. Mitch McGrath, many words of wisdom
came from him, and for that I thank him very much.

Angie Tenney has also helped me tremendously. Either with advice or just
an ear to listen. Becky Gore, where to even start. Her statistical magical
manipulation of numbers is awesome. She was also someone whom I could
share a great laugh with and just talk to during long days in the lab.

Tom Kendle and Ken Arting are two Michigan soybean growers that
deserve so much of my thanks.

Family always has to be on this page. So I thank them. Without their
prayers and support I probably would have never had made it. My husband is an

awesome, Godly man who has been my rock of support throughout this whole

iv

process. Friends have also made the whole transition to Michigan easy. They
have also been a listening ear for me.

Finally, I have to give thanks to all the nematodes that without them this
project could have never happen. Heterodera glycines and Heterodera schachtii

this is for you!

Table of Contents

List of Tables ....................................................................................... viii
List of Figures ....................................................................................... x
Introduction .......................................................................................... 1
Literature Review .................................................................................. 3
Genus Heterodera ........................................................................ 3
Heterodera glycines ..................................................................... 8
Heterodera schachtii ................................................................... 14
Objectives and Hypotheses ................................................................... 20
Materials and Methods ......................................................................... 23
Heterodera glycines trap crop development
Primary screening ............................................................. 23
Host Status ...................................................................... 24
Demonstration Plot ............................................................ 25
Crop Management ............................................................. 26
Yield Impact ..................................................................... 27

Heterodera glycines resistant variety in the presence of P. penetrans
H. glycines resistant Pl lines host status for P. penetrans ........... 29

Development of H. glycines on an SCN resistant soybean cultivar in
the presence of P. penetrans ............................................... 30

Heterodera schachtii resistant variety development

Breeding line germplasm characterization .............................. 32
Commercial germplasm characterization ................................ 33
Field Evaluation ................................................................ 35
Beta 5534N Characterization ............................................... 36
Deposition of Voucher Specimens ................................................. 37

vi

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Results and Discussion ........................................................................ 38
Heterodera glycines trap crop development

Primary screening ............................................................. 38
Host Status ...................................................................... 39
Demonstration Plot ............................................................ 40
Crop Management ............................................................. 41
Yield Impact ..................................................................... 42

Heterodera glycines resistant variety in the presence of P. penetrans
H. glycines resistant Pl lines host status for P. penetrans ........... 44

Impact of P. penetrans on H. glycines development on a H.
glycines resistant soybean variety .......................................... 45

Heterodera schachtii resistant variety development

Breedling line germplasm characterization .............................. 47

Commercial germplasm characterization ................................ 48

Field Evaluation ................................................................ 49

Beta 5534N Characterization ............................................... 50
New Management Practices for the Soybean-Sugar beet Management System in
the Presence of Three Phytopathogenic Nematodes ................................... 51
Appendix :

Appendix 1. Relationship between Heterodera glycines to Pratylenchus penetrans on a
H. glycines resistant soybean variety under field conditions at Kendle Farm, Cass

County, MI. .......................................................................................... 76
Appendix 2. 2002 Greenhouse Trial to determine the influence of cover crop varieties on
the reproduction of Heterodera schachtii. ...................................................... 77
Apgndix 3. Handout for the Monroe County Farmer Field Day ............................ 78
Appendix 4: Record of Deposition of Voucher Specimens .................................. 80
Appendix 4.2: Voucher Specimen Data ......................................................... 81
Literature Cited ................................................................................... 82

vii

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List of Tables

Table 1. Cultivars screened in preliminary trap crop evaluation greenhouse trial
for the control of Heterodera glycines ....................................................... 55

Table 2. Seeding rate of Heterodera glycines trap crops in the Monroe County
field trial/demonstration plot .................................................................... 56

Table 3. Seeding rate of treatments for Saginaw County trap crop yield
determination field trial .......................................................................... 56

Table 4. Mean number of cyst and females of Heterodera glycines obtained from
100 cm3 of soil in the preliminary trap crop greenhouse trial ........................... 57

Table 5. Mean early second-stage juveniles of Heterodera glycines in roots of
seven plants at weekly intervals during host status trial ................................ 58

Table 6. Mean late fourth-stage (sausage-stage) Heterodera glycines in roots of
seven plants at weekly intervals during host status trial ................................ 58

Table 7. Mean difference (final — initial) of Heterodera glycines [eggs + J23]
population densities in Monroe County Farmer Education field trial ................. 59

Table 8. Mean difference (final — initial) of Heterodera glycines [eggs + J23]
population densities in greenhouse trap crop validation trial. ........................ 59

Table 9. Soybean yield in bu IA from Saginaw County trap crop field trial, all
treatments were spray killed and planted to an SCN susceptible soybean and
then harvested in October of 2006 ........................................................... 60

Table 10. Summary means of Heterodera schachtii cyst and female counts at
day 42 and 56 on four sugar beet breeding line germplasm greenhouse trial....60

viii

Table 11. Percentage of eggs or juvenile Heterodera schachtii that successfully
mature into cysts/ females on 20 commercial sugar beet varieties at the end of
42 days in the greenhouse ..................................................................... 61

Table 12. Percentage of eggs or juvenile Heterodera schachtii that successfully
mature into cysts/ females on 20 commercial sugar beet varieties at the end of
56 days in the greenhouse ..................................................................... 62

List of Figures

Figure 1. Conceptual model of the current Michigan soybean-sugar beet
management system in the presence of three phytopathogenic nematodes
(Heterodera glycines, H. schachtii, and Pratylenchus penetrans) ................... 63

Figure 2. Heterodera glycines and H. schachtii current and proposed
management practices for Michigan. Management practices in bold are the
primary focus of this research ................................................................. 64

Figure 3. 2005 Monroe County grower education field plot ............................ 65

Figure 4. Heterodera glycines development in Berseem clover (A) and H.
glycines susceptible soybean (B) after 14 days. ............................................ 65

Figure 5. Mean soil (15.24 cm below ground) and ambient (15.24 cm above
ground) temperatures over one month for Saginaw county yield impact trap crop
trial ................................................................................................... 66

Figure 6. Mean [egg + J2] H. glycines in 100mcm3 soil from three sampling
periods during the Saginaw County trap crop yield impact trial ....................... 67

Figure 7. Mean reproductive potential of Pratylenchus penetrans upon H.
glycines resistant Pl Lines. .................................................................... 68

Figure 8. Influence of Pratylenchus penetrans on Heterodera glycines
reproduction on H. glycines resistant Glycine max (Pl 88788) ....................... 69

Figure 9. Relationship among initial (Pi) Pratylenchus penetrans and final (Pf)
population densities of Heterodera glycines and Pratylenchus penetrans on H.
glycines resistant (Pl 88788) soybean. ..................................................... 70

Figure 10. Heterodera schachtii female development on 20 commercial sugar
beet varieties after 42 days under greenhouse conditions ............................. 71

Figure 11. Heterodera schachtii female development on 20 commercial sugar
beet varieties after 56 days under greenhouse conditions ............................. 72

Figure 12. Heterodera schachtii cyst development on 22 sugar beet lines under
Michigan growing conditions in Bay County, Michigan (Pi = 17.5 cyst/ 100 cm3
soil) ................................................................................................... 73

Figure 13. Reproductive potential ([eggs+J2$]) of Heterodera schachtii on 22
sugar beet lines under Michigan growing conditions in Bay County, Michigan (Pi
= 1387.5 [eggs+J2]/100 cm3 soil) ............................................................. 74

Figure 14. Heterodera schachtii female and [egg + J2] population densities on H.
schachtii susceptible Crystal 963 and H. schachtii tolerant Beta 5534N ........... 75

xi

Introduction

Heterodera glycines (soybean cyst nematode, SCN) and H. schachtii
(sugar beet cyst nematode, SBCN) are considerable economic pests throughout
the world, causing significant damage to their respective hosts.

In the State of Michigan, H. glycines and H. schachtii are major pests of
soybeans and sugar beets, respectively. To date, there is no H. schachtii
resistant sugar beet in commercial production, but there are dozens of H.
glycines resistant soybean varieties available to Michigan soybean growers. The
genetic variability of this resistance, however, is limited (verbal correspondence
with Warner, 2006). Pratylenchus penetrans (root-lesion nematode, RLN) is
another phytopathogenic nematode that is known to parasitize both soybeans
and sugar beets. Economic loss due to P. penetrans on these crops is still
unknown. It is speculated that P. penetrans and H. glycines interact to cause a
decrease in soybean production, but alone, P. penetrans, has limited effect upon
soybean production (Melakeberhan, 1998; Lawn and Noel, 1986).

The impact of current Michigan soybean-sugar beet management systems
in the presence of these three phytopathogenic nematodes is illustrated in Figure
1. Soybean production in Michigan is significantly impacted by H. glycines which
also increases the risk to Sudden Death Syndrome of Soybean (SDS), which is
caused by the fungal pathogen, Fusan'um solani, (Xing and Westphal, 2006).
Nematicides are costly and therefore not a viable option for control of H. glycines.
For that reason, an integrated management approach is vital, primarily focusing

on trap crop systems. By the author’s definition, a trap crop for nematodes is a

crop that allows the nematode to penetrate and enter the root; however, the
nematode is not able to complete its life cycle and therefore no reproduction
takes place. This in turn potentially lowers the field population without the use of
other control procedures.

Michigan sugar beet yield is significantly impacted by H. schachtii. The
result is fewer profits for Michigan sugar beet growers due to a decrease in sugar
content and a reduction in the number of viable stands. Control for this nematode
usually includes crop rotation and some pre-plant, and in the past, post-plant
nematicides (Caswell et al., 1986). These methods are highly labor intensive,
thus the focus has turned to discovering resistant germplasm lines with the hope
of a commercial sugar beet seed available to Michigan growers in the near
future.

The goal of this research was to develop H. glycines and H. schachtii
control tactics designed to increase the nematode management options for

Michigan growers, as proposed in Figure 2.

 

Literature Review

Nematode Taxonomy
Heterodera glycines and H. schachtii are classified in the phylum

Nematoda and the genus Heterodera. Their current classification (according to

DeLay, 2002) is as follows:

Genus Heterodera
Taxonomy

Classification: Nematoda = Phylum
Chromadorea = Class
Chromadoria = Subclass
Tylenchida = Order
Tylenchomorpha = Infraorder
Tylenchina = Suborder
Tylenchoidea = Superfamily
Heteroderidae = Family
Heteroderinae = Subfamily

Heterodera = Genus

Heterodera Morphology - Sexual dimorphism is present in the genus
Heterodera. The males are vermiform while the females are swollen, lemon
shaped, and white to cream colored. Cysts are various shades of brown. The
metacorpus of the females is enlarged and fills the neck region. The vulva is
subterminal and the anus is terminal. Males have a rounded cephalic region,

curved spicules and no bursa. The second-stage juveniles have a heavily

 

sclerotized offset cephalic framework. An infective juvenile’s stylet is also very
prominent with anterior-directed knobs. The juvenile’s tail is very pointed and
reminiscent of a “rattle snake tail.” There is a ventro-lateral overlap of the
esophageal glands over the intestines in all vermiform stages (Ferris, 2006 and
Tylka, 1994).

Life Cycle — Following embryogenesis, a first-stage juvenile is enclosed in
each egg. It molts into a second stage-juvenile and then hatches inside the cyst.
With the right chemical signals from its environment (usually food and hormonal
cues) the second-stage juvenile (J2) emerges from either the head or vulval
regions of the cyst.

Once a plant root is found, the J2 penetrates into the root and then moves
intracellularly until it reaches the vascular cylinder. Cellulases may aide the
nematode in intercellular migration through the root cortex to the vascular
cylinder (Univin et al., 1997). Cellulases aid in the break down of cell wall
material, allowing the nematode to move freely throughout the cell walls.

Once the juvenile reaches its final feeding location and becomes
sedentary, it initiates specialized feeding sites called syncytia (Burrows, 1992). A
syncytium consists of a multinucleate mass of protoplasm resulting from the
fusion of cells. This may happen due to the nematode injecting material secreted
by the pharyngeal glands (Smant et al., 1998). Little is known, however, as to the
exact make up of the secretions. The purpose of this feeding site is to transfer
nutrients from the plant’s vascular tissue to the feeding nematode (Burrows,

1992). Developing syncytia often consist of dense granular cytoplasm, usually

surrounded by highly vacuolated, normal cells (Burrows, 1992). The syncytia
often appear thick and opaque due to a proliferation of mitochondria,
endoplasmic reticulum, and ribosomes (Burrows, 1992). In order for the increase
of subcellular organelles, the genes for their productions must be turned on, or
up-regulated. This is explained by the “apparent metabolic stimulation within the
syncytial feeding sites” (Burrows, 1992). In addition to an increase of subcellular
organelles the “cell wall adjoining the xylem increases its thickness by forming
finger-like wall invaginations lined with plasma membrane” (Gheysen et al.,
2002). This allows water to be transported from the xylem to the feeding sites
(Gheysen et al., 2002).

Cytokinins play a role in the feeding site formation, as activators in the cell
cycle (Smant et al., 1998). Cytokinins are responsible for inducing the expression
of cyclin D3 at the start of S-phase and the cyclin-dependent kinase 2 prior to the
S phase (Smant et al. 1998). Cytokinins also influence the Gz-to-M phase
(Smant et al., 1998). SBCN produces a total cytokinin amount of z1.8x10'18
mol/J2 per hour; whereas, the average root produces z 10'18 mol/nL (Smant et
al., 1998). “The lower levels produced by H. schachtii, combined with the
pharyngeal secretions, may be sufficient for initiation of the syncytia” (Smant et
al., 1998). It is estimated that juveniles “withdraw from the syncytia an amount
equivalent to fourfold the total syncytia volLIme” (Gheysen et al., 2002).

As the nematode pushes its stylet into the root, a feeding tube is formed.
The feeding tube is located within the plant cell cytoplasm (Kosack-Hammond et

al., 2000). Every time the nematode feeds an entirely new feeding tube is

produced; by the completion of nematode infection, hundreds of feeding tubes
are present in the syncytial cell (Kosack-Hammond et al., 2000). There is,
however, a size exclusion limit of 20 to 40 kDa to the feeding tube (Kosack-
Hammond et al., 2000). This exclusion allows only small soluble proteins,
sugars, or other organic compounds to pass through the nematode feeding tube.
Once the juvenile is sedentary it then molts into a third stage sausage - shape
which is non-feeding. If the juvenile is predetermined to be a female, its body
expands and pushes out of the root and molts a final time to the classic lemon -
shaped white female.

If the juvenile is predetermined to be a male, it molts a final time from the
sausage-shape into a vermiform. The male then leaves the root in search of a
female. Males and females mate and the fertilized females begin to die, turning a
brown color. Her body becomes hardened and houses up to 250 eggs. The cyst
is tolerant of freezing temperatures, so the developing nematodes over-winter
(endure freezing temperatures) within the protection of the cyst. It is also possible
that the eggs over-winter in the soil (Ferris, 2006 and Tylka, 1994).

Symptomology - The stylet of the infective juvenile (J2) is persistently
thrusted into different sites of the epidermal cells until the cell wall is weakened
enough to cause a hole. The J2 then enters the root through the hole, moves
intracellularly until it reaches the vascular cylinder. Once there the J2 begins a
more subtle exploration of cells to find the initial syncytial cell (lSC). Cytoplasmic

streaming is seen as the lSC’s nucleus increases in size. Damage also occurs

when the male exits the root in search of a female, as well as when the female

pushes her body out of the root leaving her neck still in the root.

Heterodera glycines

History
H. glycines is suspected to have originated in China along with the

soybean (Riggs, 2004). Book 26 of The Annuls of Lt? Buwei, (China, 239 3.0. as
reviewed by Riggs, 2004) mentions H. glycines as one of the “three robbers” of
crops and gives rules of tillage for the management of the “three robbers.” One of
the robbers “the land stealing the crops” is suspected to be soil-borne pathogens,
soybean cyst nematode being one of them (Riggs, 2004). A report from China in
1899 confirms damage done to soybean seedlings was caused by H. glycines;
however, soybean farmers called the disease “fire-burned seedlings” long before
the report was released (Riggs, 2004). In Japan,” Moon-night Disease” of
soybeans was described in 1881 and confirmed as caused by H. glycines in
1916 (Riggs, 2004). In 1951, soybean cyst nematode was classified as
Heterodera gottingiana. One year later was renamed as a new species by
lchinohe, Heterodera glycines (Riggs, 2004).

In 1954, H. glycines lchinohe was reported in the United States in North
Carolina. It is suspected to have been introduced through infected soil from
flower bulbs from Japan (Riggs, 2004). It is also speculated that H. glycines was
in the United States long before the 1954 detection. In 1893 W. P. Brooks in
Massachusetts demonstrated the benefit of dusting soybean roots with soil from
three soybeans originally from Japan. It was later proven that Bradymizobium, a
nitrogen-fixing bacterium, was the benefit (Riggs, 2004). In the late 1950’s and
early 1960’s, soybean cyst nematodes were found in Missouri, Tennessee,

Arkansas, Kentucky, Mississippi, and Virginia (Riggs, 2004). H. glycines was first

detected in Michigan in Gratiot County in the spring of 1987 (Warner and Bird,
2000)

Distribution

H. glycines is widely distributed. It can withstand a wide range of
temperatures and is found world wide in Japan, China, Korea, Indonesia, South
America, Soviet Union, Canada and the United States (Riggs, 2004). In the
United States it is present in 26 states including Michigan, North Carolina, Texas,
Oklahoma, and Florida. In South Carolina alone, approximately one-third of all
soybean fields are infested. It is found in all soybean-producing states of the
Midwest (Riggs, 2004).

Host Range
H. glycines has a broad host range. A majority of crop plants, however, do

not support reproduction of H. glycines. It parasitizes most leguminous plants
such as soybeans (Glycines max), adzuki bean (Vigna angulan's) and snapbean
(Phaseolus vulgan‘s) (Riggs, 1992). A greenhouse study conducted in 2005, H.
glycines reproduced on all dry edible beans grown in Michigan (Bird et al.
unpublished data, 2005). It has also been reported to reproduce on sugar beets
and some cultivars of tomatoes (Riggs, 2004 and Riggs, 1992).

Symptomology
In the field, plants may appear yellowed and stunted; this is often referred

to as “yellow dwarf” disease in soybeans. These symptoms are most apparent on
sandy soil where moisture is low. However, in soils that are heavy and moisture
is optimum, there may be no symptoms. Infested plants have a lack of nitrogen-

fixing nodules and the roots may appear stunted.

 

Ecology

H. glycines can be found wherever soybeans are produced. Due to its
ability to reproduce on leguminous plants, it may also be found in regions where
dry-beans are raised as well as clovers and alfalfas. It co-exists with many other
plant-parasitic nematodes in many fields. This co-habitation is speculated to
cause an increase in nematode damage on certain plants.

Disease Complexes

H. glycines commonly occur in sites colonized by Pratylenchus penetrans
(root - lesion nematode, RLN). In Michigan, P. penetrans co-exists with H.
glycines in about 50% of all soybean fields (personal communication with F.
Warner, 2006). In one field in Cass County, Michigan there is a small correlation
between the number of P. penetrans and H. glycines on an H. glycines-resistant
soybean cultivar (Appendix 1). With the increase of P. penetrans there was also
an increase in success of H. glycines upon a resistant soybean (Bird,
unpublished data, 2005). Melakeberhan (1998) found that P. penetrans had no
effect upon plant growth of H. glycines-resistant soybean variety ‘Bryan.’ In
1986, Lawn and Noel provided evidence that when polyphagous communities of
nematodes were present in the field they presented a problem for control. When
P. penetrans and Meloidogyne incognita (non-target nematodes) are present
along with H. glycines a problem arises with the use of species-specific resistant
cultivars to control H. glycines.

Fusan'um solani is the causal agent for Sudden Death Syndrome (SDS) of
soybeans (Rupe et al., 1997). Rupe et al. suggested that rotation to any non-

soybean crop greatly reduced H. glycines populations as well as lowered the F.

10

solani populations. Recently, Xing (2006) and colleagues found that as H.
glycines populations increased in the field there was a higher probability of SDS
infestation (Xing et al., 2006).

Economic Loss
Heterodera glycines may reduce soybean yields between 5 to 90 percent

(Riggs and Wrather, 1992). In the US. alone, over 1.1 billion dollars are lost
annually due to this nematode. In 2003 and 2005, H. glycines caused 102,705
and 105,981 tons loss to soybean yield, respectively (Wrather and Koenning,
2006). In Michigan, H. glycines is detected in 36 soybean growing counties
(personal communication with G.W. Bird, 2006). Michigan soybean growers
experience an average of 5% annual yield loss due to H. glycines (personal
communication with F. Warner, 2006).

Current Control Strategies for Heterodera glycines
Crop Rotation - Current Michigan H. glycines crop rotation strategies are

based upon a SCN risk index. This is determined by viable units (eggs and
second-stage juveniles) per 100 cm3 of soil (MSU Diagnostic Services, 2006).
The risk system is on a scale from 0-3, where 0 (no SCN detected) is no risk and
3 (>10,000 [eggs+J23]) is high risk. For example, if the SCN risk index for a field
is a 3 (high risk), the recommendation would be two years without growing
soybeans. The third year the grower could plant a H. glycines - resistant soybean
variety. During the years out of soybean, a non-host should be grown (i.e. corn,
sugar beets, or wheat). In Michigan, the usual rotation is corn-corn-soybean (or

corn-wheat-soybean). If the risk index was 0, no H. glycines were detected and

11

 

the grower could grow a H. glycines susceptible soybean variety (MSU
Diagnostics Services, 2006).

Genetic Resistance - There are over 100 Plant Introduction Lines (PI) of
Glycines max (soybeans) with known resistance to H. glycines. Only seven have
been used in commercial variety development (Niblack et al., 2002). In Michigan,
only three are available in commercial varieties (personal communication with
G.W. Bird, 2006). These include Pl 54840 (Peking), Pl 88788, and PI 437654. Pl
88788 is the current dominate source of resistance used in commercially
available soybean varieties.

H. glycines is a parasite that has evolved diversity among and within field
populations. This enables some populations to reproduce upon certain H.
glycines resistant soybean varieties. In 1970, a bioassay was developed to
detect and quantify this diversity of field populations of H. glycines (Niblack et al.,
2002). The assay allowed for a more detailed protocol for development of
resistant cultivars (races). It also provided growers with specific information about
which race of H. glycines they had in their field and which resistant Pl Iine(s) to
plant. The race concept, however, had several significant faults. In 2002, Niblack
et al. published an alternative, the HG Type Test. The HG Type Test determines
if the field population reproduces on any of the seven Pl lines used in
commercially available seed. The most common HG Type in Michigan is 2.5.7
which indicates that the average Michigan field population of H. glycines is able
to reproduce upon Pl 88788, Pl 209332 and PI 548316; the growers should

refrain from planting soybean varieties that contain these resistance lines.

12

Soybean Cropping System
In Michigan, soybeans are the second largest commodity grown with 2.13

million acres planted to soybeans every year (Andersen, 2005). Soybeans are
typically planted as early as the end of April and as late as early June. The most
common rotation is corn-soybean-corn. Some producers grow dry edible beans
or green beans as well. There over 10,000 acres of organic edible soybeans in
Michigan which are usually shipped to Asia for consumption (personal
communication with J. Davenport, 2006). Additional pests found in soybean fields
include grubs, spider mites, and occasionally soybean aphids (DiFonzo et al.,

2006).

13

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Heterodera schach tii
History
The sugar beet cyst nematode disease of sugar beets was first described

in Halle, Germany in 1859 by Schacht (Gray and Kerr, 1992). Schacht described
the symptoms as rubenmudigkeit (beet weariness). The causal agent was
described by Schmidt in 1871 and classified as Tylenchus schactii. It was not
until 1930 that T. schachtii was placed in the genus Heterodera. H. schachtii is
believed to have been detected in the United States between 1895 and 1918
(Gray and Kerr, 1992). By 1992, H. schachtii was present in 17 states in the US
(Gray and Kerr, 1992).

The first field survey for H. schachtii in Michigan was conducted in 1920
by Gerald Thorne. H. schachtii was not detected, however, until 1948 (Knobloch
and Bird, 1981; Bockstahler, 1950). In 1998, Miller conducted an industry-wide
survey in Michigan and found that 54% of the 214 sugar beet fields sampled
were positive for H. schachtii (Miller, 1999).

Distribution
H. schachtii is present in 40 sugar beet growing countries as well as 17

states in the United States. In Europe, it ranges from Spain to Bulgaria. H.
shachtii is also found in regions of Russia, Turkey, Israel, South Africa, and
Australia. In the United States it is found in both eastern and western states and
it has been detected in parts of Canada (Ferris, 1999).

Host Range
H. schachtii parasitizes at least 200 plant species, most in the families

Chenopodiaceae and Cruciferae (Gray and Kerr, 1992). H. schachtii cysts have

14

been recovered from cabbage, Brussels sprouts, tomatoes, cauliflower, broccoli,
kale, radishes, turnips, spinach, and table beets (Gray and Kerr, 1992). H.
schachtii also parasitizes weed species. For example, mustard, pigweed, lambs-
quarter, shepardspurse, and purslane are all hosts for the nematode. H.
schachtii is categorized as the major pest on sugar beets in Michigan (Knobloch
and Bird, 1981).

Symptomology
H. schachtii is responsible for approximately 90% of all nematode-related

sugar beet damage (Steele, 1984). Fields affected by H. schachtii often appear
wilted and underdeveloped. Leaves of affected plants may remain green but can
develop a distinct yellowing (Steele, 1984). Beneath the soil the plant roots have
excessive fibrous root formation and the storage roots often appear sprangled or
have severe branching (Steele, 1984). When the infective juvenile enters into the
tap root it destroys the zone of elongation causing excessive branching. This
produces the occasional forked beet. This in turn severely decreases sugar
content of the beet and ultimately reduced economic return.

Ecology
H. schachtii co-exists with multiple different phytopathogenic nematode

species in fields. One in particular that causes increase economic loss in
Michigan is H. glycines. To date, Michigan is the only state in which these two
nematodes co-inhabit the same field. These two nematodes are very closely
related taxonomically, in that they are sibling species and are in the same
grouping on a parsimonious tree. Nematodes collected from a particular field in

Michigan have been collected and successfully mated under laboratory

15

conditions. The field nematodes have been confirmed to have similar molecular
patterns as that of the laboratory mated hybrids, confirming a possible hybrid
between H. gycines and H. schachtii (personal communications with G.W. Bird,
2006)

Disease Complexes
H. schachtii occurs not only with other nematodes but with fungal

pathogens in the field. In 1970, Jorgenson reported that in greenhouse trials,
damage to sugar beet plants was far greater when both Fusarium oxysporum
(wilt disease agent) and H. schachtii were present than when only the nematode
was present (reviewed by Powell, 1971). Root-rot fungal pathogens, such as
Rhizoctonia solani, are also known to occur with H. schachtii. Once the
nematode penetrates the root, it facilitates ensuing penetration by the fungus,
(Powell, 1971). Polychronopoulos and his colleagues looked at this interaction
and found that the syncytia cells induced by the nematode are very suitable
substrates for the fungal growth (reviewed by Powell, 1971).
Economic Loss

Heterodera schactii is responsible for approximately 90% of all nematode
related sugar beet damage (Steele, 1984). In Michigan, H. schachtii is a key pest
that lowers sugar beet yield potential significantly. In surveys conducted in 1999,
H. schachtii was present in approximately 50% of Michigan sugar beet acreage
surveyed (Miller, 1999). In fields with high nematode populations and visible foliar
symptoms yield loss was as great as 10 tons per acre. In fields with relatively low
population of H. schachtii and no visible foliar symptoms, yield loss range from 2

to 4 tons per acre (Miller, 1999).

16

Current Control Strategies for Heterodera schachtii
Crop Rotation - Similar to the crop rotation used for H. glycines control, H.

schachtii crop rotation strategies are based upon SBCN risk indices. This is
determined by viable units ([egg+J2]) per 100 cm3 of soil (MSU Diagnostics
Services, 2006). The SBCN risk system is on a scale from 0-5, where 0 (no H.
schachtii detected) is no risk and 5 (>5,000 [eggs+J2s]) is high risk.
Unfortunately, there is still not a clear understanding of the level of nematode
pressure that causes yield or economic loss on a sugar beet plant (personal
communication with F. Warner, 2006). Current rotation recommendations are the
responsibility of the Michigan Sugar Company (a grower cooperative, formally
Michigan Sugar and Monitor Sugar). The company makes the final
recommendation as to how long a field will be out of sugar beet production.
Currently, fields are planted to sugar beets one year out of three. The two years
out of sugar beets are planted to a H. schachtii non-host, such as corn, wheat,
potato, soybean, or dry beans.

Sugar beet growers have adopted oilseed radish (OSR) cultivars Colonel
or Adagio as trap crops for control of H. schachtii. Field trials indicate that a
spring crop of OSR before sugar beets is a more reliable practice for lowering H.
schachtii population densities and increasing subsequent sugar beet yields than
a late summer planting of OSR following wheat (Bird et al. unpublished data). H.
schachtii populations have been estimated to decrease as much as 50% the first
year in oil seed radish (Bird et al. unpublished data). Greenhouse trials indicate

that trap crop efficacy is cultivar specific. While OSR cvs Colonel and Adagio are

17

appropriate trap crops for H. shachtii, other cultivars of OSR are not (see
Appendix B).

Genetic Resistance - Currently there are no H. schachtii-resistant sugar
beet varieties available to Michigan sugar beet growers. In 2005, a H. schachtii
resistant variety (Beta 5534N) was field tested in 11 grower trials sponsored by
Michigan Sugar Company. In fields where H. schachtii populations were low,
there was a 6.9 ton per acre increase in yield. In fields where H. schachtii
populations were high, the yield increase was as high has 10.4 tons per acre
(personal communication with G.W. Bird, 2006).

Extensive research has been done in laboratories to identify H. schachtii

pm“, is on

resistance genes in a variety of plant species. One such gene, Hsl
chromosome 1 of Beta. procumbens (Cal et al., 1997). Plants that carry this gene
display an incompatible reaction between host and pathogen (Cai et al., 1997).
The nematode invades the roots, but most die in the late J2 stage. In this
process the syncytial cells degrades and leaving the nematode without adequate
nutrition by then it is incapable of moving to a new location (Cai et al., 1997). In
some cases females develop, although abnormally. The females are transparent
due to lack of eggs. This process prevents the nematode from completing its
lifecycle (Cai et al., 1997).

Also there has been some work in modifying sugar beets as well as other
plants to express resistance to H. schachtii. Some transgenic plants have been

developed with the incorporation of proteinase inhibitors as nematode anti-

feedants (Univin et al., 1997). A modified rice cystatin protein Oc-IA086,

18

expressed as a transgene in Arabidopsis thaliana has a profound effect on the
size and fecundity of females (Urwin et al., 1997). Ingestion of this cystatin by the
nematode caused a loss of cysteine proteinase activity in the intestine and
ultimately decreased normal nematode growth (Un/vin et al., 1997).

Biological Control - A proportion of the fungi associated with the soil
surrounding the plant roots can be nematophagous. Trapping fungi like
Hirsute/la rhossiliensis, aid in the plant defense methods of the nematode (Jaffee
et al., 1998). Trapping can occur with initial binding of the nematode then a Iectin
binding, followed by a penetration peg and hyphal development inside the
nematode. Other fungi have the advantage of attacking eggs inside cysts
(Pyrowolakis et al., 1999).

Sugar beet cropping system
In Michigan 160,000 acres of sugar beets are grown each year (Andersen,

2005). Sugar beets are grown in a three year rotation on a heavier soil, usually
on clay-loam or dry lake beds. Sugar beets are usually planted as soon as the
ground thaws from winter. This could be as early as the end of March. The
majority of planting takes place during the first two weeks of April. A corn crop
generally precedes sugar beets to decrease the chance of disease the following
year in sugar beets. Harvest begins in early October and continues into
November. Most rotations and variety choices are determined by the sugar
company under which all Michigan sugar beet growers are contracted (personal
communication with J. Davenport, 2006). Additional pests in sugar beet fields
include aphids (foliar and root), springtails, white grubs, and Spinach leaf miner

(DiFonzo et al., 2006).

19

wag-1mm: :33 (Inn-qr, I ."tnmmrrp

Objectives and Hypotheses

The goals of this research were to develop Heterodera glycines and H.
schachtii control tactics designed to increase the nematode management options

for Michigan growers.

Heterodera glycines trap crop development

The objective of this component of the research was to evaluate a large
number of potential trap crops and develop a technique that Michigan soybean
growers could incorporate into their current crop system to lower H. glycines field
populations.

Primary screening
Hypothesis 1: All plant cultivars tested will allow for successful H.
glycines reproduction.

Host Status
Hypothesis 2: Plant cultivars tested will allow for successful H.

glycines development.

Demonstration Plot
Hypothesis 3: Michigan growers will not accept an additional

method for the control of H. glycines.

Crop Management
Hypothesis 4: The trap crop system is difficult to incorporate into

Michigan soybean grower’s current soybean planting regime.

Yield Impact
Hypothesis 5: The trap crop has no impact upon soybean yields.

20

Illiluh'l 1' WNIOD‘I ‘..u- Rm,"

‘~ Ill"

Heterodera glycines resistant varieties in the presence of P.
penenans

The objective of this component of the research was to evaluate P.
penetrans reproductive potential on all seven Pl Lines. Evaluate the development
of H. glycines in the presence of P. penetrans on a PI 88788 sourced H. glycines
resistant soybean.

H. glycines resistant Pl Lines host status for P. penetrans
Hypothesis 6: P. penetrans will not reproduce on any of the Pl
Lines.

Impact of P. penetrans on H. glycines development on a H. glycines
resistant soybean variety

Hypothesis 7: P. penetrans has no effect upon H. glycines
development on a H. glycines resistant soybean variety.

Heterodera schachtii resistant variety development

The objective of this component of this research was to evaluate USDA
sugar beet germplasm and commercially available sugar beet varieties for
resistance to H. schachtii.

Breeding Line germplasm characterization
Hypothesis 8: USDA sugar beet germplasm does not express

resistance to H. schachtii.
Commercial germplasm characterization

vaothesis 9: Commercial varieties of sugar beets do not express
resistance to H. schachtii.

21

Field Evaluation
I_-I_ypothesis 10: All varieties and germplasm tested under field

conditions do not express resistance to H. schachtii.

Beta 5534N Characterization
vaothesis 11: There is no significant difference between Beta

5534N and Crystal 963 in relation to H. schachtii development and

reproduction.

22

 

Materials and Methods

Heterodera glycines trap crop development

Primary screening
H. glycines extraction and development - Cysts were collected from soil

from Cass County, Michigan. Soil (100 cm3) was processed using a centrifugal-
floatation method (Jenkins, 1964). The cysts were then crushed over a 60 mesh
sieve and rinsed into a 15 x 85 ml test tube. The supernanent contained second-
stage juveniles (J23) and eggs only. The [eggs + J2] were counted using a
dissecting microscope and calibrated to obtain a ratio of 2000 [J23 + eggs]/ml.
Plant development - Thirty seeds of each variety tested (26 varieties
tested, Table 1) were planted in moist (90% sand) soil in an aluminum container
(24.13 cm x 29.21 cm x 5.08 cm). The plant containers were placed on a
greenhouse (East Lansing, Michigan) bench under a 16 hour light period in a
230 day and 21C night atmosphere for 14 days. The plants were watered once a
day and fertilized with Peters 20—20-20 fertilizer solution (25 grams/3.78 L).
Plant inoculation - 14 days after planting, (150 cm3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized soil (90% sand). A hole
was formed, using a pencil, in the soil and a single seedling was placed inside.
One ml of J2 and egg mixture (1 ml. at 2000 [egg +J2] per ml) and 1 ml of water
was placed directly onto each plant’s roots using a syringe. Soil was then placed
on top of the roots and each plant was watered. A total of seven seedlings were
planted in seven Conetainers for each variety and replicated seven times. The

Conetainers were placed in a rack and arranged randomly, then placed into the

23

 

greenhouse under 16 hour light period at a temperature of 230 day and 21C
night for 35 days.

Nematode extraction - At the end of 35 days the soil was processed using
a centrifugal-floatation technique described by Jenkins (1964). Each sample was
inspected for the presence or absence of cysts and females.

Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison Test
was conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.) program
using a P-value 0.05 as alpha. A non-parametric test was also conducted to rank

the potential trap crops.

Host Status
Microfile preparation - Soil infected with H. glycines from Michigan Cass

County, Michigan was used to fill 4 liter microtiles. Soil (100 cm3) was separated
and processed for nematodes according to the above protocol. Cass County field
soil (3.5 L) was used to fill each microtile. Four trenches were dug in a field plot
located in East Lansing, Michigan. Each microtile was placed into the trench and
field soil was used to fill around the microtile. The cultivars used in this
experiment were the following: H. glycines susceptible soybean, H. glycines
resistant soybean, corn, Berseem clover, Oriental mustard, Dackon common
oilseed radish, and Common lespedeza. Five seeds of the soybeans were
planted in the middle of a microtile and replicated 12 times in 12 different
microtiles. Four seeds of the corn, replicated 12 times, and 10 seeds of the
remaining cultivars were planted in the middle of a microtile and replicated 12

times.

24

Sampling of microtile - At 14 days after planting a single microtile was dug
from the ground and placed into a plastic bag. The microtile was then
destructively sampled. The soil was knocked out of the tile using the side of a
table. The plant inside was then saved and the roots were washed. The soil was
placed into a plastic bag and put into a cooler that was kept at 40C. This was
done to each treatment once a week for six weeks. The roots were subjected to
acid fusion staining to detect the stages of nematodes.

Root Staining - All procedures were followed as outlined by Byrd et al. in

1983.

Demonstration Plot

A 9.14 m by 9.14 m plot was cleared in a recently planted soybean field
(Monroe County, Michigan) using garden hoes. The treatments were H. glycines
susceptible soybean, corn, Berseem clover, H. glycines resistant soybean,
Common Lespedeza, Oriental mustard, and Dackon common oilseed radish.The
treatments were replicated seven times; each plot measured 0.91 m by 0.91 m
with a 0.61 m alley in between replicates (Figure 3). Plots were arranged in a
complete random block design. The treatments were hand planted (Table 2)
plots were sampled at planting (mid-June) and at the harvest of the soybean field
(early October). Seeding rates can be seen in Table 2. Approximately 1 liter of
soil was sampled from each treatment at planting as well as at harvest.

Nematode extraction- Soil (100 cm3) was processed using a centrifugal-

floatation technique (Jenkins, 1964). Each sample was inspected for the

25

presence or absence of cyst and white females. Cysts were crushed by hand
using a tissue homogenizer and [eggs + J2] per each cyst were counted.

Data Analysis - An ANOVA and Tukey-Kramer Multiple Comparison Test
was conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.) program

using a P-value 0.05 as alpha.

Crop Management

Greenhouse study
H. glycines susceptible soybean, Berseem clover, Oriental mustard,

 

Dackon common oilseed radish and a fallow were all used to test the practicality
of a trap crop. H. glycines infested field soil from Cass County, Michigan, (800
ml) was used to fill plastic square pots. A sample of soil (100 cm3) was set aside
to get an initial nematode count. Two seeds of clover, radish, mustard and
soybean were planted individually in the middle of a pot. The fallow pot was left
undisturbed. The plant containers were placed in a greenhouse (East Lansing,
Michigan) under a 16 hour light period in a 230 day and 21C night atmosphere
for two weeks. The plants were watered once a day and fertilized with Peters 20-
20-20 fertilizer solution (25 grams/3.78 L). The plants were allowed to grow for 35
days and then were spray killed using Roundup WeatherMax at 33 oz/A +AMS at
7.7 kg/378.54 L of H20 using a Allen Spray Machine (Laboratory spray chamber
that delivers 187 Uha (20 GPA) at 173 KPa (25 psi) with a TeeJet 8001E
nozzle). Fourteen days post spray, two H. glycines resistant soybeans were

planted in each pot. After 43 days, the soybeans were destructively sampled.

26

Nematode extraction - The soil and roots were scrubbed and then
processed using a centrifugal-floatation technique (Jenkins, 1964). Each sample
was inspected for the presence or absence of cysts and white females. Cysts
were crushed by hand using a tissue homogenizer and the number of [eggs + J2]
per cyst was counted.

Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison Test
was conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.) program

using a P-value of P = 0.05 as alpha. A Fisher’s LSD was also conducted.

Yield Impact
A 91.44 m x 15.24 m plot was used and each treatment was planted using

a Great Plains® Drill in Saginaw County, Michigan. Soil samples were taken
before each treatment was planted to asses the initial H. glycines population
density. Approximately 1 liter of soil was sampled and processed using a
centrifugation-floatation method (Jenkins, 1964). Each plot was 9.14 m wide with
four rows in a randomized block design with seven replicates. Seeding rates of
each treatment are shown in Table 3. WatchDog® data loggers were placed in
three of the replicates in different areas of the field. One sensor was placed
15.24 cm below the soil and another was placed 15.24 cm above the soil. After
five weeks, the trap crops were spray killed using 24 oz/A of Roundup
WeatherMax + AMS at 7.7 kg/378.54 L of H20 using a flat-fan herbicide sprayer.
Fourteen days post-spray of the trap crops, an H. glycines susceptible soybean
was planted with a White Seed Boss 5100 using the same population as the H.

glycines resistant soybean. Soil samples were again taken before the soybeans

27

were planted. A pre-emergence was also sprayed, Pursuit Plus at 2pt/A + 24 oz
Roundup Weathermax using a flat-fan herbicide sprayer. At the end of the trial,
mid-October, another soil sample was taken from each plot. Soil (100 cm?) was
processed for nematodes using the centrifugation-floatation method as described
by Jenkins in 1964.

Harvest - At the end of the growing season the center two rows were
harvested using a small-plot combine (mid-October). The beans were run
through a fanning mill to remove soil pellets as well as debris. Beans were then
weighed and a sub-sample was tested for seed moisture and test weight.
Percent moisture and a test weight were recorded. Yield was then calculated
from the above information using a soybean yield conversion equation.

Nematode extraction - Soil (100 cm3) was processed using a centrifugal-
floatation technique (Jenkins, 1964). Each sample was inspected for the
presence or absence of cysts and white females. Cysts were crushed by hand
using a tissue homogenizer and eggs and J23 per each cyst were counted.

Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison Test
was conducted using SAS® program (Version 9.1 SAS Institute INC. Cary, NC.)

using a P-value of 0.05 as alpha.

28

Heterodera glycines resistant variety in the presence of
P. penetrans

H. glycines resistant PI Lines Host Status for Pratylenchus penetrans

Pratylechus penetrans extraction and development - Pratylechus
penetrans was obtained from greenhouse cultures of P. penetrans (RLN)
infested corn plants. The roots were processed using Bird’s method (1971) with
the substitution of Ethyl Mercuric Chloride (EMC) solution for a 0.01% NaOCI
solution. P. penetrans were counted using a dissecting microscope and diluted to
obtain 2000 P. penetrans/ml

Plant development - Seeds of each indicator line tested were planted in
moist (90% sand) sandy-loam in an aluminum container (24.13 cm x 29.21 cm x
5.08 cm). The containers were placed upon a greenhouse bench under a 16 hour
light period in a 230 day and 21C night atmosphere for two weeks. The plants
were watered once a day and fertilized with Peters 20-20-20 fertilizer solution (25
grams/3.78 L).

Plant inoculation - At the end of two weeks (150 cm3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized loam (90% sand). A hole
was formed in the soil, using a pencil, and a single plant was placed inside. P.
penetrans (1 ml at 2000 P. penetrans /ml) at different stages and 1 ml of water
was placed directly onto each plant’s roots using a syringe. Soil was then placed
on top of the roots and each plant was watered. A total of seven seedlings were
planted for each variety and replicated seven times and the Conetainers were

placed in racks, arranged randomly, then placed in a greenhouse (East Lansing,

29

 

Michigan) under 16 hour light period at a temperature of 230 day and 21C night
for 35 days.

Nematode extraction - The roots were processed using Bird’s method
(1971) with the substitution of Ethyl mercuric chloride (EMC) solution for a 0.01 %
NaOCl solution. Total amount of P. penetrans was counted from a 1 ml aliquot.

Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison test
was conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.)

program using a P-value of 0.05 as alpha.

Development of Heterodera glycines on an SCN Resistant Soybean Cultivar
in the Presence of Pratylenchus penetrans

Pratylechus penetrans extraction and development - Pratylechus
penetrans was obtained from greenhouse cultures of P. penetrans (RLN)
infested corn plants. The roots were processed using Bird’s method (1971) with
the substitution of Ethyl Mercuric Chloride (EMC) solution for a 0.01% NaOCl
solution. P. penetrans were counted using a dissecting microscope

H. glycines (SCN) extraction and development - Cysts were collected from
soil from Cass County, Michigan. Soil (100 cm3) was processed using a
centrifugal-floatation method (Jenkins, 1964). The cysts were then crushed over
a 60 mesh sieve and rinsed into a 15 x 85 ml test tube. The supernanent
contained second-stage juveniles (J23) and eggs only. The [eggs + J2] were
counted using a dissecting microscope

Plant development - Seeds of a PI 88788 resistance source and an H.

glycines (SCN) susceptible soybean were planted in moist (90% sand) soil. The

30

plant containers were placed in a greenhouse (East Lansing, Michigan) under a
16 hour light period in a 23C day and 21C night atmosphere for 14 days. The
plants were watered once a day and fertilized with Peters 20-20-20 fertilizer
solution (25 grams/3.78 L).

Plant inoculation - At the end of two weeks (150 cm3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized sandy soil (90% sand). A
hole was formed, using a pencil, in the soil and a single plant was placed inside.
Either 2000 SCN [J23 + eggs]/ml; 2000 RLN/ml; 1600 SCN [J23+eggs]/ml: 400
RLN/ml; 1000 SCN [J23+eggs]/ml: 1000 RLN/ml or 400 SCN [J2s+eggs]/ml:
1600 RLN/ml was placed directly onto each plant’s roots using a syringe. Soil
was then placed on top of the roots and each plant was watered. A total of seven
plants were planted for each variety and replicated seven times and the
Conetainers were placed into a rack and arranged at random. The plants were
then placed in the greenhouse under 16 hour light period at a temperature of 23C
day and 21C night for 35 days.

Nematode extraction - At the end 35 days the soil was processed using a
centrifugal-floatation technique (Jenkins, 1964). The roots were processed using
Bird’s method (1971) with the substitution of EMC solution for a 0.01% NaOCI
solution. Total P. penetrans was counted from a 1 ml aliquot. All stages of H.
glycines and each cyst was crushed using a tissue homogenizer and each J2
and egg were counted per a 1 ml aliquot.

Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison Test

was conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.)

31

 

program using a P-value of 0.05 as alpha.

Heterodera schachtii resistant variety development
Breeding line germplasm characterization

H. schachtii extraction and development - Cysts were collected from soil
from Michigan’s Saginaw Valley. Soil (100 cm3) was processed using a
centrifugal-floatation technique (Jenkins, 1964). The cysts were then crushed
over a 60 mesh sieve and rinsed into a 15 x 85 ml test tube. The solution
contained J23 and eggs only. The solution was then counted using a dissecting
microscope to obtain a ratio of 2000 [J23 + eggs]/ml

Plant development - Seeds of four germplasm ( Hil-2, HM E-17, N 224, N
172) lines tested were planted in moist (fine) grain vermiculite in an aluminum
container (24.13 cm x 29.21 cm x 5.08 cm). The plant containers were placed on
temperature tanks set at 240 under a 16 hour light period for 14 days. The plants
were watered once a day and fertilized with Peters 20-20-20 fertilizer solution (25
grams/3.78 L).

Plant inoculation - At the end of 14 days, (150 cm3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized sandy soil (90% sand). A
hole was formed, using a pencil, in the soil and a single plant was placed inside.
J23 and eggs (1 ml at 2000 [eggs+J2] per ml) were placed directly onto each
plant’s roots using a syringe. Soil was then placed on top of the roots and each
plant was watered. A total of seven seedlings were planted for each line and
replicated seven times. Conetainers were then placed into a rack and arranged

at random. Another Conetainer rack was filled with the same number of

32

Conetainers with the four different lines. The plants were then placed in the
greenhouse under 16 hour light period at a temperature of 230 day and 21C
night for 42 and 56 days.

Nematode extraction - At the end of both 42 and 56 days the plants were
taken out of the green house and the nematodes were extracted. Soil was then
processed using a centrifugal-floatation technique (Jenkins, 1964). Each sample
was inspected for the presence or absence of cysts and white females to give a
percent mature female.

Data analysis - An ANOVA and a Tukey—Kramer Multiple Comparison test
were conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.) program
using a P-value of 0.05 as alpha. Mature females were determined by adding
cyst and white females together. This sum was divided by cysts counts to give

the percentage of [egg + J23] that matured to cysts over each given time frame.

Commercial germplasm characterization

H. schachtii extraction and development - Cysts were collected from soil
from Michigan’s Saginaw Valley. Soil (100 cm3) was processed using a
centrifugal-floatation technique (Jenkins, 1964). The cysts were then crushed
over a 60 mesh sieve and rinsed into a 15 x 85 ml test tube. The solution left
over contained J2’s and eggs only. The solution was then counted using a
dissecting microscope to obtain a ratio of 2000 [egg +J23] /ml

Plant development - Seeds of 20 commercial sugar beet lines (obtained
from Michigan Sugar Company) were planted in moist (fine) grain vermiculite in

an aluminum container (24.13 cm x 29.21 cm x 5.08 cm). The plant containers

33

were placed on temperature tanks set at 240 under a 16 hour light period for 14
days. The plants were watered once a day and fertilized with Peters 20-20-20
fertilizer solution (25 grams/3.78 L).

Plant inoculation - At the end of two weeks, (150 cm3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized sandy soil (90% sand). A
hole was formed in the soil and a single plant was placed inside. J23 and eggs 1
ml. at 2000 [egg+J23] per ml) were placed directly onto each plant’s roots using a
syringe. Soil was then placed on top of the roots and each plant was watered. A
total of seven plants were planted for each variety and the Conetainers were
placed into a rack and arranged at random. The plants were then placed into a
green house under 16 hour light period at a temperature of 230 day and 21C
night for 42 and 56 days.

Nematode extraction - At the end of both 42 and 56 days the plants were
taken out of the greenhouse and the nematodes were extracted. Soil was then
processed using a centrifugal-floatation technique (Jenkins, 1964). Each sample
was inspected for the presence or absence of cysts and white females to give a
percent mature female.

Data analysis - An ANOVA, Tukey-Kramer Multiple Comparison and
Fisher’s Least Significance Difference tests were conducted using SAS® (Version
9.1 SAS Institute INC. Cary, NC.) program using a P-value of 0.05 as alpha.
Mature females were determined by adding cyst and females together. The sum
was divided by cyst counts to give the percentage of [egg + J23] that matured to

cysts over each given time frame. On the 56 day trial a Fisher’s Least Square

34

Difference was performed to allow for greater separation. Classification of
resistance was determined by a higher susceptibility index the greater the

probability of susceptibility (the percentage of mature females upon the variety)

Field Evaluation
Pre-plant sampling — A field in Saginaw County, Michigan was divided into

four sections and each section was sampled four times using a cone-soil probe,
15.24 cm below the soil surface. Approximately 1 liter of total soil was taken
during sampling.

Planting - Each of the four sections that the field was divided into became
a replicate. A wooden stake was pushed 5.08 cm into the soil and a single sugar
beet seed was placed into the hole by hand. This was done 10 times for each of
the 22 varieties and replicated 4 times.

Harvest - All sugar beets were hand dug.

Harvest sampling - Once the sugar beets were harvested one soil sample
was taken from each variety/line tested.

Nematode extraction - Soil (100 cm?) was processed using a centrifugal-
floatation technique (Jenkins, 1964). Each sample was inspected for the
presence or absence of cysts and females. The stages of development were
recorded. Also each cyst was crushed by hand using a tissue homogenizer and
the number of eggs and juveniles were also enumerated.

Data analysis - Initial sample field population density mean of H. schachtii
was calculated. If treatment sample mean was less than five times the initial

sample mean there was a good probability of resistances. If the treatment mean

35

was greater than 5 times the initial sample mean then it was a good probability of
susceptibility. If the treatment mean equaled 5 times the initial sample mean then

susceptibility or resistance was indeterminate.

Beta 5534N characterization
H. schachtii extraction and development - Cysts were collected from soil

from Michigan’s Saginaw Valley. Soil (100 cm3) was processed using a
centrifugal-floatation technique (Jenkins, 1964). The cysts were then crushed
over a 60 mesh sieve and rinsed into a 15 x 85 ml test tube. The supernanent left
over contained J23 and eggs only. The supernanent was then counted using a
dissecting microscope to obtain a ratio of 2000 [J23 + eggs]/ml.

Plant development - Seeds of two sugar beet varieties tested were planted
in moist (fine) grain vermiculite in an aluminum container (24.13 cm x 29.21 cm x
5.08 cm). The plant containers were placed on temperature tanks set at 240
under a 16 hour light period for 14 days. The plants were watered once a day
and fertilized with Peters 20-20-20 fertilizer solution (25 grams/3.78 L).

Plant inoculation - At the end of two weeks, (1500m3, 20.95 cm x 4.12 cm)
Conetainers were filled half way with steam pasteurized sandy soil (90% sand). A
hole was formed, using a pencil, in the soil and a single plant was placed inside.
J23 and eggs (1 ml at 2000 [eggs+J23] per ml) were placed directly onto each
plant’s roots using a syringe. Soil was then placed on top of the roots and each
plant was watered. A total of seven plants were planted for each variety the

Conetainers were then placed in a rack and arranged at random. The plants

36

were then placed in the greenhouse under 16 hour light period at a temperature
of 23C day and 21C night for 35 days.

Nematode extraction - At the end of 35 days the plants were taken out of
the greenhouse and the nematodes were extracted. Soil was processed using a
centrifugal-floatation technique (Jenkins, 1964). Each sample was inspected for
the presence or absence of cysts and females and cysts were crushed by hand
using a tissue homogenizer and eggs and J23 were counted.
Data analysis - An ANOVA and Tukey-Kramer Multiple Comparison test were
conducted using SAS® (Version 9.1 SAS Institute INC. Cary, NC.) program using

a P-value of 0.05 as alpha.

Deposition of Voucher Specimens

Cysts of Heterodera glycines were collected from Cass, Monroe, and
Saginaw Counties, Michigan. Cysts of Heterodera schachtii were collected from
Bay County, Michigan. Cysts of an unknown Heterodera spp. were collected from
Bay County, Michigan. The cysts were placed in glass vials of alcohol, labeled
and were deposited in the A.J. Cooke Arthropod Collection at Michigan State
University Entomology Museum under voucher number 2006-06 (Appendix 4 and

4.2).

37

Results and Discussion

Heterodera glycines trap crop development

Primary Screening
Results

Eighteen of the twenty-six crops tested had no detectable H. glycines
cysts or females (Table 4). There was a highly significant, (P<0.0001) impact of
host crops upon nematode populations. When crops with no H. glycines
development were excluded from the analysis, the remaining crops significantly
supported development for H. glycines (P=0.0018). Resistant soybean and sugar
daddy peas had a significant positive impact upon the nematode population
(P=0.0007 and P<0.0001, respectively). Since the resistant soybean and the pea
are considered hosts of H. glycines, they were also then removed from analysis.
The remaining crops were: Berseem clover, Sudax, Oriental Mustard, Dackon
Oilseed Radish and Generic Oilseed Radish. There was no significant difference
among treatments, (P = 0.4610). A non-parametiric test was conducted and
Oriental mustard, Dackon oilseed radish, and Berseem clover were found to
have a strong trend of positive influence on development for H. glycines.
Discussion

Much work has been done to determine a number of plant species that are
poor hosts for H. glycines (Miller and Ahrenes, 1969; Riga et al., 2001; Rao—Arelli
et al., 1991). Those poor hosts were the foundation for the development of an
efficient trap crop system. Twenty-six different crops were initially screened to

determine their nematode-trapping ability (Table 1). Riggs determined many of

38

155mm 1'. mr; as my

those crops to be poor hosts for the H. glycines (reproductive potential was very
low). Taking that into account, only reproductive potential was measured in the
initial screening trial. This allowed for creating a new list of those where no cyst
or females were detected, which could mean the plant trapped the nematode and
it was not allowed to continue on in its life cycle. The evaluation identified three
plant cultivars with potential for use as a trap crop for the control of H. glycines:
Berseem clover, Dackon Oilseed Radish, and Oriental Mustard. This decision

was based upon the low developmental success of H. glycines.

Host Status
Results

Based on visual obsenration of stained roots, H. glycines were not
detected in the corn roots. Common Lespedeza was included in this study due to
its suspected susceptibility for H. glycines. The lowest number of vermiform
nematodes were seen at the first sampling (Table 5), but towards the end of
sampling Lespedeza had a greater number of vermiform and sausage
nematodes than the H. glycines susceptible soybean (Table 5 and 6). Only
vermiform nematodes were detected throughout the sampling period on Berseem
clover (Tables 5 and 6 and Figure 4). Nematode numbers declined on Berseem
clover, Dackon oilseed radish and Oriental mustard. The vermiform nematodes
increased as sampling continued on the H. glycines resistant soybean. The

sausage nematodes increased as the sampling continued of that treatment.

39

‘1; fi‘I-I‘t “9*“ INTI-‘7‘?

“1'- rmr "a!!!

a?

Discussion

As expected the H. glycines susceptible soybean supported all stages of
nematodes throughout the sampling period. There appeared to be a difference in
mechanism of trapping occurring in Berseem clover, Dackon oilseed radish, and
Oriental mustard. Only vermiform nematodes were found throughout the
sampling of the Berseem clover, however; both vermiform and sausage
nematodes were observed but the number drastically decreased as sampling
continued in the oilseed radish and mustard. This could possibly be explained by
Berseem clover classically trapping the nematodes (allowing for entrance into the
root but failing to develop a feeding site) while oilseed radish and mustard may
not allow the nematode to develop a sufficient feeding site, so development is

incomplete. Further studies, however, are required to test this hypothesis.

Demonstration Plot
Field Trial:

The difference between harvest and at planting egg: J2 ratios were
calculated and an ANOVA was run. The H. glycines susceptible soybean had the
most significant effect on increasing reproduction and development of H. glycines
over one growing season approximately 4 months, (P=0.0039). Using an alpha of
0.1 both Oriental Mustard and Berseem clover had the most significant effect on
lowering H. glycines reproduction and development over one growing season,
(P=0.068 and P = 0.086, respectively, Table 7)

Overall, based on this field trial, growing a susceptible soybean as a trap

crop increased the H. glycines population. Growing Oriental mustard lowered the

40

 

nematode population substantially. Much more extensive field trials are needed
before the assessment of the feasibility of the effectiveness of the trap crop
system.
Farmer Education:

In late August a farmer field day was held. It was sponsored by Michigan
State University Monroe County extension. There were approximately 80
soybean growers in attendance. A handout was created to give to those in
attendance (Appendix C). Growers were able to walk by the field trial and see

what each of the possible trap crops look like as well as ask questions. A short

Y’fimmyrnm‘t W—‘ar‘tm mrcvfl;

talk was given to explain the objectives of the study and to define what a trap
crop is in relation to nematodes, in particular H. glycines. Most growers
responded very positively to the idea of a trap crop for H. glycines. Others were
concerned with the possibility of the trap crop becoming a nuisance or a problem

“weed” in the coming growing season.

Crop Management

Results

A difference between initial [egg + J2] count and final [egg + J2] counts
were calculated by subtracting harvest counts from initial counts. All treatments
were significant (P < 0.0001) at impacting H. glycines population densities. When
a Fisher’s LSD was conducted, the H. glycines susceptible soybean had the
most significant positive effect on [egg+J2] counts (mean difference of 22624, P

< 0.0001). Oriental mustard was separated out as having the most negative

41

effect on H. glycines [egg+J2] counts (mean difference of —1628, Table 8), but
with no significant difference between treatments.

Discussion
Overall based on the greenhouse trial it could be concluded that growing a

susceptible soybean as a trap crop followed by a H. glycines resistant soybean,
increases the H. glycines population. However, Oriental mustard does appear to
have the most significant effect on the nematode population based on
comparison of the mean difference calculation to the other treatments. It could be
concluded that growing Oriental mustard or possible any of the other treatments
(there was no significant difference between the treatments) except H. glycines
susceptible soybean prior to planting a H. glycines resistant soybean, the overall

H. glycines population were reduced at the end of the growing season.

Yield Impact
Results

Berseem clover had the best overall stand compared to the other trap
crops (fallow treatment excluded) over the course of one month. The ambient air
temperature during the time the trap crops were in the field was on average
13.7C and the soil was on average 14.20. Temperature per day can be seen in
Figure 5 (temperature in degrees F). The four treatments had no effect on the
yield of a H. glycines susceptible soybean over one growing season (Table 9).
The fallow control had an average yield of 41 .74 mm. The Oriental mustard had
the highest yield compared to the other treatments. It also had 1.14bu/A more

than the fallow control (Table 9). Mean H. glycines development over the course

42

of the growing season can be seen in Figure 6. Trap crop effect on H. glycines
population densities at both samplings of planting of the trap crop and planting of
the H. glycines susceptible soybean were not significant. Whereas, the harvest
samples were highly significant for H. glycines cyst and [eggs + J23] (P = 0.0004
and P < 0.001, respectively). Berseem clover had the most positive impact upon
H. glycines population at the harvest of the soybeans. The other trap crops
(Fallow, Oriental mustard, H. glycines resistant soybean, and Dackon oilseed
radish) had no difference among themselves. The H. glycines resistant soybean
showed the most reduction of the H. glycines population densities over the three

sampling periods (Figure 6).

Discussion
H. glycines is physically active in the soil at temperatures as low as 10C

(verbal correspondence with Bird, 2006). The optimum temperature for the
nematode is 23C (Caswell et al., 1986). Based on that knowledge, H. glycines
would have been active during the period in which the trap crops were in the
field. The nematode would have been able to penetrate the plant roots and
potentially do damage. The trap crop treatments had no effect on the overall
soybean yield which could be accounted for by the low stands of eachtrap cr0p.
This could have led to unclear conclusions and less reduction in nematode
population densities. Berseem clover was the only treatment that has the ability
to be frost seeded and would succeed if planted one month before soybeans.
Also, keeping the field fallow prior to soybeans also has the potential to decrease

nematode densities. Oriental mustard was the only treatment that had a positive

43

impact on yield. The mustard’s impact on yield could either be that the plant is
trapping the nematode or that it has nematicidal properties. Additional field trials

are required to further understand the yield impact of the H. glycines trap crops.

Heterodera glycines resistant cultivars in the presence of P.
penetrans

H. glycines Resistant Pl Lines Host Status for Pratylenchus penetrans

Results
Pratylenchus penetrans reproduced on all seven H. glycines resistant Pl

lines and the susceptible variety, Lee 74 (Figure 7). Final population densities
were highest on Lee 74 (~900/g root) and lowest on Pl 89722 (~300Ig root)
(Figure 6). The ANOVA was highly significant (P= 0.0060). The highest amount
of variability was in PI 88788 and the susceptible control, Lee 74.

Discussion
There are currently seven sources of H. glycines resistance used in

commercial soybean varieties (Niblack et al. 2002). The HG type test is used to
determine the diversity among H. glycines populations and their ability to develop
on resistant soybean varieties (Niblack et al. 2002). It is believed that P.
penetrans co-exists in ca 50% of Michigan soybean fields with H. glycines.

Data shows that each of the seven PI Lines are successful as hosts for P.
penetrans. Lee 74, which is susceptible to H. glycines, is also susceptible to P.
penetrans (success of host ability). This could lead to the conclusion that if a
cultivar is susceptible to H. glycines it may very well likely be susceptible to P.

penetrans. If a cultivar is resistant to H. glycines it may not be resistant to P.

44

penetrans. This could lead to many other problems. Having a soybean cultivar
resistant to only one type of nematode it may leave the plant susceptible to other
nematodes that may infect in a different mode, like P. penetrans.

Impact of P. penetrans on H. glycines development on a H. glycines
resistant soybean variety

Results

Where H. glycines was alone (2000 [eggs+J23]) upon the susceptible
bean there was an average of 265 times more H. glycines than compared to the
resistant bean. When P. penetrans was present at a low number (400 RLN/ml)
there were only 37 times more H. glycines on the susceptible than on the
resistant bean. Whereas, when P. penetrans was present at an equal number
(1000 nematodes/ml) to H. glycines there were 23 times more H. glycines upon
the susceptible than on the resistant (Figure 8 and 9). An ANOVA of the H.
glycines developmental potential upon the H. glycines susceptible soybean
showed that the treatments were all significant with a P-value of 0.0002. Each
treatment when compared to one another showed no significance for H. glycines
development (P-value 0.1349). The PI 88788 bean it was shown that the ANOVA
was significant for H. glycines development with a P-value of 0.0010. When
looking at the treatments where the nematodes were present separate from the
treatments where H. glycines (SCN) and P. penetrans (RLN) were alone it was
seen that together they were not significant (P-value 0.2351). Tukey-Kramer test
reviled that the treatment where the nematode ratio was 1000 SCN: 1000 RLN it

more significant than the other treatments (P-value 0.0003).

45

Discussion

Only three of the seven sources of resistance to H. glycines that have
been commercialized are available for use in Michigan (PI 54840, Pl 88788, and
PI 437654). Pl 88788 is the dominate source of resistance used in many of the
commercially available soybean seeds. It was noted that when P. penetrans was
not present or in very small numbers, either due to experimental error or failure to
develop, the total H. glycines numbers were similar to that of the control counts.
However, when P. penetrans was present there was a significant increase in cyst
counts. The nematode numbers upon the susceptible were what were to be
expected. When large numbers of H. glycines were added, large numbers were
observed in the final count. The same was true when small quantities of H.
glycines were added; small quantities were observed in the final count. Based on
the above results it can be concluded that in the presence of P.penetrans, H.
glycines has a higher rate of successfully developing upon a PI 88788 H.
glycines resistant source soybean (Figure 6 and 7). Pl 88788 is the primary
source of resistance to H. glycines in production and that P. penetrans is shown
to possibly break down that PI 88788 resistance a recommendation would be to
determine at what rate is P. penetrans present in the soil and notjust determining
which resistance would be the best for the field. This would be due to that an
equal number of RLN: SCN ratio there was a higher developmental potential of
H. glycines upon the PI88788 line as opposed to just inoculating with H. glycines

only.

46

Heterodera schachtii resistant variety development

Breeding line germplasm characterization
Results
Clear differences were observed between individuals and germplasm lines

in response to H. schachtii infection (Table 10), particularly by 56 days after
inoculation under the conditions used here. One germplasm, Hil-2, had uniform
resistance to H. schachtii. Apparent segregation was evident for N224 and N172,
also. Susceptible line E17 was variable, but counts of cysts and females were
higher than with other germplasm. By 42 days, it was evident but not statistically
significant, as to which germplasm was going to be more or less susceptible,
suggesting that this is not long enough for the nematode to complete its lifecycle.

Discussion
This experiment was an initial test into the development of a H. schachtii

assay in the greenhouse whereby selections for further breeding could be
performed. A small number (perhaps as few as 16) of breeding lines appear to
be resistant to H. schachtii, but no commercial cultivars are marketed at this time,
although the positive control used here (a breeding line from Syngenta, HiI-2)
may be the most advanced germplasm available. Resistance in Hil-2 is derived
from Beta procumbens. Differences were clearer on average at week eight,
however another time interval would have to be done to determine if the white
females seem would develop into viable cysts. The cysts that were seen on Hil-2
were small and, when crushed, not may eggs or J23 were seen, leading to
believe that the plant variety may have slowed the nematode’s development.

More time intervals may be needed to get a clearer picture of resistance of the

47

varieties. At six weeks may not be long enough to develop significant
discrimination. The plant’s response to the nematode could be monitored using
the method described here to find when the resistance takes place and how it

affects the plant and the nematode.

Commercial germplasm greenhouse screening
Results
At the end of the 42 day time interval, each of the 20 varieties could be

separated into 5 categories: resistant, somewhat resistant, susceptible, and
highly susceptible, based on H. schachtii development. This separation can be
seen in table 11 and figure 11. Six of the twenty varieties were shown to be
mostly resistant at the end of the 42 day period. Those varieties were: Beta 5471,
Beta 5374, 5X Spartan, Holly 02HX272, Crystal 963, and HM E-38. All of those
varieties had a female percent maturity of fewer than 33%. At the end of the 56
day time period, only three varieties could be classified as most resistant. Those
varieties were: Beta BK 1383R, Beta 5374, and Holly 02HX272. These varieties
had less than 25% female maturity. The 56 day values can be seen in Table 12
and Figure 12.

Discussion

Two time periods were used in this experiment to determine if resistance
broke down as time increased. It was shown that in certain varieties that
resistance does indeed break down in some varieties. The variety 5X Spartan by
the end of the eight weeks had dropped from most resistant to highly susceptible.

However, four of the varieties showed improvement at the end of the eight week

48

period. Crystal 271 and Beta BK 1381 R both were classified as somewhat
resistant at the end of the eight weeks, moving up from susceptible. Beta 5374
was only variety that was classified as most resistant at both 42 and 56 days and
remained at the same position. This would lead to the conclusion that Beta 5374
has the most stable resistance to H. schachtii as compared to the other

commercial varieties.

Field Evaluation
Results
Out of the twenty two different sugar beet varieties that were planted only

two showed probability of resistance. The initial sampling means can be seen in
Table 11. It was seen that HIl-2 and 2927-4 were the only varieties that showed a
probability of resistance based on the analysis done. Hil-2 had a mean cyst count
of 13.3 with a [J23 + egg] mean count of 1120 (Figures 12 and 13). The 5*mean
initial count was 17.5 for cyst and 1137.5 for [J23 + egg] counts. Hil-2 treatment
means were less than the initial means. 2927-4 was the other variety that
showed a probability of resistance. The variety 2927-4 had a mean cyst count of
13 and a [J23 + egg] count of 880. Both varieties showed numbers less than
5*initial mean.

Discussion
In the field it was seen that only two varieties had the probability of being a

resistant to H. schachtii, Hil-2 and 2927-4. Unfortunately, no definite conclusions

can be drawn due to many of the varieties had poor yield, thus leading to a

49

37"em '-‘ll rlPfliftlJllmnll

flu
.I‘.

decrease of replications. As well as, the initial sampling only obtained an average

nematode count for the entire field, rather than for each treatment.

Beta 5534N Characterization
Results
When each variety of sugar beet was inoculated with the same numbers

of H. schachtii J2’s and eggs, (2000), there was 40% less females (cysts or
white) upon Beta 5534N than the commercial susceptible variety. Further, the
number of eggs and juveniles per cyst significantly declined for the Beta host (P-
value <0.001). When the cysts were crushed an average of 32.8 eggs and
juveniles were accounted for as opposed to the average 91 found in the
commercial variety cysts (Figure 14).

Discussion

Recently, a sugar beet variety, Beta 5534N, was developed by a
commercial sugar beet seed company that is potentially resistant to H. schachtii.
Field tests conducted by Michigan Sugar Company found that beet yield was
increased 6-10 tons/A (personal communication with G.W. Bird, 2006). A
greenhouse experiment was conducted to determine the tolerance of Beta
5534N to H. schachtii as compared to a popular commercial H. schachtii
susceptible variety.

Overall, Beta 5534N decreased overall H. schachtii population as well as
decreased the fecundity of females that were successful enough to complete
development under greenhouse conditions. From this study, Beta 5534N is a

sugar beet variety that shows promise to being resistant to H. schachtii.

50

New Management Practices for the Soybean-
Sugar beet Management System in the Presence
of Three Phytopathogenic Nematodes

Heterodera glycines trap crop development

Out of 26 cultivars tested, three were carried into further experiments.
Those were Berseem clover, Dackon oilseed radish, and Oriental mustard. The
recommended planting of a trap crop is one month prior to the planting of the
cash crop (soybeans). Economically, the three potential trap crops are currently
not feasible. The oilseed radish is priced at $1 .80/lbs and was planting it at
22lbsIA this equates to $39.80/A. The clover is priced at $1 .50/lbs and was
planted it at 12lbs/A this equates to $18.00/A. The mustard is priced at $1 .50/lbs
and was planted it at 10lbs/A this equates to $15.00IA. (Verbal correspondence
with Davenport, 2006) However, if a grower is able to afford one of the above
crops it is recommended that the field be in the southern part of the state
(warmer climate) and that the trap crop be planted one month prior to planting of
soybeans. Then again, further field studies are required to illuminate the impact
of the trap crop under Michigan growing conditions. Also, additional laboratory
analyses are needed to better understand the mechanism of the three cultivars in

their reduction of H. glycines population densities.

51

Impact of Pratylenchus penetrans on Heterodera glycines
resistant cultivars

P. penetrans is able to reproduce on all seven Pl Lines used in
commercial varieties of soybeans. Since P. penetrans and H. glycines co-exsist
in ca 50% of all Michigan soybean fields and only Pl 54840, Pl 88788, and PI
437654 are grown, most of which is Pl 88788, and it is likely that P. penetrans
has the ability to successfully parasitize those varieties. Having this ability to
reproduce on all seven H. glycines resistant lines could enable the nematode to
cause soybean yield losses. Having a majority of all H. glycines resistant
varieties carry Pl 88788 resistances and knowing that P. penetrans has the
ability to successfully reproduce on this line, seeing how P. penetrans affected
the variety’s resistance to H. glycines was the most logical step. It was found that
P. penetrans does in fact affect the Pl 88788 resistance source variety to H.
glycines. It was seen that P. penetrans enables H. glycines to successful
reproduce on a PI 88788 resistant source variety, which without P. pentrans; H.
glycines would not be able to reproduce as well. When H. glycines control
recommendations are made they are based upon the H. glycines population of a
given field. Based on the above research the field population of P. penetrans
should also be considered when developing a H. glycines control strategy for a
grower. Pl 88788 may in fact not be the best H. glycines resistant source for a

grower to plant if the P. penetrans field population is relatively high.

52

Heterodera schachtii resistant variety development

All experiments that were conducted were preliminary in the development
of a H. schachtii resistant sugar beet variety for Michigan. In both USDA
greenhouse and field trials Hil-2 showed to be the most promising for a resistant
sugar beet variety. Unfortunately, this variety was a private sector developed
variety and could not continue into future USDA screening trials. The variety
2927-4 was another variety that showed probability of being a resistant variety in
the field trial. This variety was developed in Salinas, CA. and has the potential of
entering future H. schachtii resistant screening trials. The current commercial
varieties available for Michigan sugar beet growers all proved to be susceptible
to H. schachtii. In spite of this, at the end of both 6 and 8 week greenhouse trials
Beta 5374 proved to have the most resistance characteristics as compared to all
the other commercially available varieties tested. Recently, a private sector H.
schachtii resistant variety has been developed and field trials were set up all
around the state of Michigan. The greenhouse trial reveled that compared to a
commercially available H. schachtii susceptible variety that there were 40% less
cysts overall on the resistant variety. There was also 36% less eggs+J23 as
compared to the susceptible variety. Overall, H. schachtii resistance
development is a challenging endeavor. Based on the aforementioned research
USDA available germplasm for resistance to H. schachtii is small. The
commercially varieties available for Michigan sugar beet growers that were
tested only one proved to have resistance characteristics, majority are very

susceptible. For Michigan sugar beet growers Beta 5534N is a H. schachtii

53

tolerant variety that will be available in the very near future that will aide in the

control of H. schachtii.

54

Table 1. Cultivars screened in preliminary trap crop evaluation greenhouse trial
for the control of Heterodera glycines.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Common Name Variety H. glycines host status
(from Riggs, 2004)
Alfalfa Vernal Poor Host
Alfalfa wL252HQ Poor Host
Clover Berseem Poor Host
Clover Crimson Poor Host
Clover Kura Poor Host
Clover White Poor Host
Clover White Blossom, Sweet Poor Host
Clover Yellow Blossom, Sweet Poor Host
Corn DynaGrow SSK27 RR Non-Host
Soybean Kenwood 94 Host, Control
Sgbean INA Resistant Host
Sorghum-Sudan Sudax Unknown Host
Grass Hybrid
Sugar beet Crystal 963 Unknown Host
Vetch Hairy Poor Host
Cowpea Red Ripper Unknown Host
Cabbage Early Jersey Wakefield Unknown Host
Rye Grass Annual Unknown Host
Oil seed Radish Adagio Unknown Host
Oil Seed Radish Colonal Unknown Host
Oil Seed Radish Rimbo Unknown Host
Oil Seed Radish Generic Unknown Host
Oil Seed Radish Dackon Unknown Host
Oil Seed Radish Arena Unknown Host
Mustard Oriental Unknown Host
Oat IDA Unknown Host
Pea Sgugardaddy Host

 

55

 

Table 2. Seeding rate of Heterodera glycines trap crops in the Monroe County field
trial/demonstration plot.

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Per Plot Per Acre
SCN Susceptible Soybean 33 Seeds 160,000 Seeds
Corn 7 Seeds 31,000 Seeds
Clover, Berseem 1.12 g 5.44 kg Seed
SCN Resistant Soybean 33 Seeds 160,000 Seeds
Lespedeza, Common 1.88 g 9.07 kgSeed
Mustard, Oriental 0.94 g 4.53 kg Seed
Oilseed Radish, Dackon 2.1 g 9.939 Seed

 

Table 3. Seeding rate of treatments for Saginaw County trap crop yield
determination field trial.

 

 

 

 

 

 

 

 

Treatment Per Acre
SCN Resistant Soybean 142,000 seeds
Clover, Berseem 5.44 kg
Mustard, Oriental 4.53 kg
Oilseed Radish, Dackon 8.1659

 

56

Table 4. Mean number of cyst and females of Heterodera glycines obtained from
100cm” soil in the preliminary trap crop greenhouse trial.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Females1 Cyst1
Vernal Alfalfa 0 b 0 b
wL252HQ Alfalfa 0 b 0 b
Berseem Clover 0.2 a 0 b
Crimson Clover 0 b 0 b
Kura Clover 0 b 0 D
White Clover 0 b 0 b
White Blossom, Sweet Clover 0 b 0 6
Yellow Blossom, Sweet Clover 0 b 0 b
DynaGrow Corn 0 b 0 b
Kenwood 94 Soybean 3.2 N/A2 334.71 N/A2
INA Soybean 0.3 N/A2 6.5 N/A2
Sudax 0.14 a 0 b
Crystal 963 Sugarbeet 0 b 0 b
Hairy Vetch 0 b 0 b

Red Ripper Cowpea 0 b 0 b
Early Jersey Wakefield, Cabbage 0 b 0 6
Annual Rye Grass 0 b 0 b
Adagio Oilseed Radish 0 b 0 b
Colonel Oilseed Radish 0 b 0 b
Rimbo Oilseed Radish 0 b 0 b
Generic Oilseed Radish 0 b 0.2 b
Oriental Mustard 0 b 0.4 a
IDA Oats 0 b 0 b
Dackon Oilseed Radish 0 b 0.42 a
Sugardaddy Pea 0.6 N/A2 8.5 N/A2
Arena Oilseed Radish 0 b 0 b

 

 

 

1 Column means followed by the same letter are not significantly

different (P =0.05) according Fisher’s LSD.(N=7)

2 Not included in statistical analysis

57

 

Table 5. Mean early second-stage juveniles of Heterodera glycines in roots of

seven plants at weekly intervals during host status trial.

 

 

 

 

 

 

 

 

 

Treatment1 20-June 27-June 4-July 11-July 18-July
SCN Susceptible Scybean 45 28 186 17 8

Corn 0 0 0 MS2 0
Clover, Berseem 32 47 0 5 MS2
SCN Resistant Soybean 82 23 192 15 20
Lespedeza, Common 0 4 8 46 54
Mustard, Oriental 9 24 0 N/S2 N/S2
Oilseed Radish, Dackon 38 57 12 9 12

 

 

 

 

 

 

‘ N=7

2 No plants were found to be up on that sampling day

Table 6. Mean late fourth-stage (sausage-stage) juveniles of Heterodera glycines
in roots of seven plants at weekly intervals during host status trial.

 

 

 

 

 

 

 

 

 

Treatment‘ 20-June 27-June 4-July 11-July 18-July
SCN Susceptible Soybean 30 59 46 28 8

Corn 0 0 0 N/S2 0
Clover, Berseem 0 0 0 0 MS2
SCN Resistant Soybean 45 34 18 5 2
Lespedeza, Common 35 4 9 35 59
Mustard, Oriental 13 5 0 MS2 N/S2
Oilseed Radish, Dackon 3 2 0 0 6

 

 

 

 

 

 

‘ N=7

2 No plants were found to be up on that sampling day

58

 

 

Table 7. Mean difference (final-initial) of Heterodera glycines [egg +J2] population
densities in Monroe County Farmer Education field trial.

 

 

 

 

 

 

 

 

Treatment Mean difference (initial-final)1
H. glycines susceptible soybean 3528.6 b

Corn - 135.9 a

Clover, Berseem - 46.3 a

H. glycines resistant soybean - 76.6 a

Lespedeza, Common - 290.9 a

Mustard, Oriental - 486.7 a

Oilseed Radish, Dackon - 169.7 a

 

 

 

 

1 Column means followed by the same letter are not significantly
different (P =0.05) according to Fisher's LSD. (N = 7)

Table 8. Mean difference (final — initial) of Heterodera glycines [egg + J2]
population densities in greenhouse trap crop validation trial.

 

 

 

 

 

 

 

Treatment Egg + J2 mean difference (final
—initial)‘

SCN Susceptible Soybean 22624 a

Clover, Berseem -1439 b

Fallow -1468 b

Oilseed Radish, Dackon -1605 b

Mustard, Oriental -1628 b

 

 

 

1 Column means followed by the same letter are not significantly
different (P =0.05) according to Fisher’s LSD. (N=7)

59

Table 9. Soybean yield in bu. IA from Saginaw County trap crop field trial, all trap
crops were spray killed and plots were planted to an SCN susceptible soybean
and then harvested in October, 2006.

 

 

 

 

 

 

 

 

 

 

 

Treatment Mean1 Standard Range
(bu/A) Deviation

Fallow 41.74 :I: 4.75 36.99 - 46.49

Mustard, Oriental 42.88 t 4.29 38.59 - 47.17

SCN Resistant Soybean 40.07 :I: 2.31 37.70 - 42.38

Oilseed radish, Dackon 41.52 t 2.45 39.07 - 43.97

Clover, Berseem 41.40 :t 2.37 39.03 - 43.77

 

1 No significant difference in treatments

Table 10. Summary means of Heterodera schachtii cyst and female counts at day
42 and 56 on four sugar beet breeding line germplasm lines greenhouse trial.

 

 

 

 

 

 

 

 

 

 

 

Treatment 42 Day 56 Day
Females‘ Cyst Females1 Cyst1
Hil-2 7.0 a 8.0 0.0 a 3.2 a
HM E-17 48.3 ab 8.3 110.0 b 46.3 b
N224 49.4 ab 13.1 41.7 a 18.9 a
N 172 59.4 b 15.4 20.9 a 26.0 ab

 

 

1 Column means followed by the same letter are not significantly
different (P =0.05) according Fisher’s LSD. (N = 7)

60

Table 11. Percentage of eggs or juveniles of Heterodera schachtii that were able to
successfully mature into cysts! females on 20 commercial sugar beet varieties at
the end of 42 days in the greenhouse.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Female-Cyst (%)1 Clasification of Resistance2
Beta 5471 1 a Resistant

Beta 5374 1.2 a Resistant

5X Spartan 1.7 a Resistant

Holly 02HX272 1.8 a Resistant

Crystal 1353 1.9 a Resistant

HM E-38 4.2 a Resistant

HM E-33 14.9 ab Somewhat Resistant
Beta BK 1383R 16.4 ab Somewhat Resistant
Crystal 271 18.7 abc Susceptible

Beta BK 1381 R 20.6 abc Susceptible

Crystal R353 21.0 abc Susceptible

HM 7172RZ 23.5 abc Susceptible

Beta 5736 26.0 abc Susceptible

HM RH-5 27.9 abc Susceptible

HM 2761 R2 28.6 abc Susceptible

HM E-17 36.9 bcd Highly Susceptible
5X Prompt 39.4 bcd Highly Susceptible
Crystal 913 44.3 bcd Highly Susceptible
Beta 5310 46.4 bcd Highly Susceptible
Crystal 963 65.9 bcd Highly Susceptible

 

1 Column means following by the same letter are not significantly

different (P =0.05) according to Fisher’s LSD. (N=7)
2 Classification of resistance was determined by if there was a higher susceptibility index, the

greater the susceptibility (the percentage of mature females upon the variety)

61

 

Table 12. Percentage of eggs of juveniles of Heterodera schachtii that were able to
successfully mature into cysts! females on 20 commercial sugar beet varieties at
the end of 56 days in the greenhouse.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Female-Cyst (% )1 Classification of Developmental
Resistance Dynamics2
Beta BK 1383R 18.9 a Resistant +
Beta 5374 21 .5ab Resistant 0
Holly 02HX272 24.1 abc Resistant 0
HM E-33 30.5 abc Somewhat Resistant —
HM RH-5 30.7 abc Somewhat Resistant +
Crystal 1353 32.4 abc Somewhat Resistant -
HM E-38 32.6 abc Somewhat Resistant —
Crystal 271 35.6 abc Somewhat Resistant +
Beta BK 1381 R 36.4 abc Somewhat Resistant +
Beta 5471 37.7 bc Somewhat Resistant -
HM E-17 41.6 cd Susceptible +
HM 2761RZ 41.7 cd Susceptible 0
Crystal R353 43.3 cd Susceptible 0
Beta 5736 43.4 cd Susceptible 0
5X Prompt 44.3 cd Susceptible 0
5X Spartan 47.4 cde Susceptible 0
HM 7172RZ 58.9 de Highly Susceptible -
Beta 5310 61.2 e Highly Susceptible +
Crystal 913 61.4 e Highly Susceptible +
Crystal 963 80.9 f Most Susceptible 0

 

 

 

 

1 Column means following by the same letter are not significantly
different (P =0.05) according to Fisher’s LSD. (N = 7)
2 Comparative changes (42 vs. 56 days) in female-cyst development as a percent of original

population

+ = Increase
- = Decrease
0 = No change

62

 

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Figure 4. Photographs of Heterodera glycines development in Berseem clover (A)
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glycines stages)

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Appendix 3. Handout for Monroe County Farmer Field Day

 

 

Monroe County Soybean Cyst Trap Crop Trial
Treatments and Objective for Planting

1: SCN Susceptible Soybean (33 seeds or 160,000 seeds/A)

2: Corn (7 seeds or 31,000 seeds/A) (SCN non-host)

3: Clover, Berseem (1 .12g or 12 lbs seed/A) (Possible “trap crop”

4: INA SCN resistant soybean variety PI 437654 + PI 88788 (33 seeds or 160,000
seeds/A)

5: Lespedeza, Common (1.88g or 20 lbs/A) (Possible “trap crop”)

6: Mustard, Oriental (0.94 g or 10 lbs/A) (Possible “trap crop”

7: Oil Seed Radish, Dackon Common (2.1g. or 22 lbs/A) (Possible “trap crop)

Note: Planted on 6/16/05
There is a 2’ alley between each replicate
Each treatment is 3’x3’

      

VII 2 3 4 5 6 7 1

VI 3 4 5 6 7 1 2

V 4 5 6 7 1 2 3

IV 5 6 7 1 2 3 4

III 6 7 1 2 3 4 5

II 7 1 2 3 4 5 6

I 1 2 3 4 5 6 7

Road
NT

Berseem clover roots stained for SCN SCN susceptible soybean roots
at 2 weeks post-plant (photo shows stained for SCN at 2 weeks post-
SCN juveniles) plant (photo shows multiple stages

of SCN)

 

78

 

(Page two of handout)

 

 

Introduction:

In Michigan, soybeans are a highly economical crop. Unfortunately for soybean grower’s
soybean cyst nematode (SCN) was first detected in Michigan in 1987 and causes yield
loss in 31 soybean growing counties (Warner and Bird 2000). SCN can greatly reduce
soybean yields ranging from 5 percent to more than 90 percent (Riggs and Wrather,
1992). Regrettably, nematicides are extremely costly and therefore not a viable option for
control of SCN. Therefore, an integrated management approach is vital, primarily
focusing on trap crop systems. By the author’s definition a trap crop for nematodes is a
crop that allows the nematode to penetrate and enter the root; however, the nematode is
not able to complete a life cycle and therefore no reproduction will take place. Sugar beet
growers here in Michigan have adopted the use of oilseed radish (OSR) varieties for
control of Heterodera schachtii, sugar beet cyst nematode (SBCN). Field studies,
unpublished by the Bird laboratory indicate that a spring crop of OSR before soybeans is
a more reliable practice for lowering H. schachtii population densities and increasing
subsequent sugar beet yields than a late summer planting of OSR following wheat. In
addition, Warner, unpublished, in 2003, found in a greenhouse study that the role of
cover crops in the control of nematode populations is cultivar specific. He and his
colleagues found that while OSR cvs Colonel and Adagio are appropriate trap crops for
H. schachtii, other cultivars of OSR are not. In addition, OSR cv Colonel was found to
be a good host for reproduction of both Pratylenchus penetrans (Root-lesion nematode)
and Melodogyne hapla (Root-knot nematode). Confounding the problem more is the fact
that many fields in Michigan have all four nematodes, (SCN, SBCN, Root-lesion, and
Root-knot nematode). Additionally to the discovery of a trap crop for soybean cyst
nematode, further understanding of the interaction and contribution to biotic potential of
these four nematodes. Also, their interactions and how they affect resistant cash crops
need to also be expounded upon.

Objectives:

The primary focus of this project will be to develop a trap crop system to control soybean
cyst nematode due to its economical importance to the soybean industry. This is
complicated by the fact that most soybean fields in Michigan do not only have soybean
cyst nematode but three other semi-economical important nematodes. These would be
Root-lesion nematode, Sugar beet cyst nematode, and Root-Knot nematode. These
nematodes are found in fields that grow both sugar beets and soybeans. Fortunately, there
are soybean cyst nematode resistant soybeans and there is a sugar beet cyst nematode
resistant sugar beet variety in development. However, the author found in an unpublished
greenhouse study that having equal populations of Root-Lesion nematode and soybean
cyst nematode upon a SCN resistant variety soybean, it therefore becomes susceptible to
soybean cyst nematode. Further experiments into these interactions upon resistant
varieties will be looked at using greenhouse studies. In addition to the greenhouse studies
of interaction, two field trials for trap crops will be set up around the state.

 

79

 

Appendix 4
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.: 4006-06

Title of thesis or dissertation (or other research projects):

Bionomics and Control of Two Heterodera spp. in Michigan

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

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigators Name(s) (typed)
Cassandra Lee Bates

 

 

Date November 284006
*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix 4 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 4 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator, Michigan State
University Entomology Museum.

80

 

Appendix 4.2

Voucher Specimen Data

1 Pages

 

 

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81

Literature Cited

Anand, S.C. 1992a. Registration of ‘Hartwig’ soybean. Crop Science 32:1069-
1070.

Anand, S.C. 1992b. “Registration of ‘Delsoy 4710’ soybean.” Crop Science
32:1294.

Andersen, J. 2005. Growing Season Weather Summary. In: Michigan
Department of Agriculture 2004 Annual Report. pps. 18—19.

Bird, G.W. 1990. Soybean Cyst Nematode. Cooperative Extension Services,
Michigan State University. Extension Bulletin E-2200.

Brim,C.A. and Ross, JP. 1996. Registration of Pickett soybeans. Crop Science
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