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INFLUENCE OF GLYPHOSATE ON RHIZOCTONIA CROWN
AND ROOT ROT IN GLYPHOSATE-RESISTANT
SUGARBEET

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Kelly Anna Barnett

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INFLUENCE OF GLYPHOSATE ON RHIZOCTONIA CROWN AND ROOT
ROT IN GLYPHOSATE-RESISTANT SUGARBEET

By

Kelly Anna Barnett

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

Crop and Soil Sciences

2010

ABSTRACT
INFLUENCE OF GLYPHOSATE ON RHIZOCTONIA CROWN AND ROOT
ROT IN GLYPHOSATE—RESISTANT SUGARBEET '
By

Kelly Anna Barnett

Previous greenhouse studies on experimental lines of glyphosate-resistant
sugarbeet indicated that tolerance to Rhizoctonia crown and root rot (Rhizoctonia solani
Kuhn) could be compromised alter glyphosate was applied. In initial greenhouse
experiments, exposure to glyphosate increased, did not affect, and decreased disease
severity in three glyphosate-resistant sugarbeet varieties. A laboratory experiment
indicated that R solam' mycelial grth did not increase in the presence of glyphosate,
however, glyphosate applied at a 10X rate decreased growth when compared with the
control. Additional greenhouse and ﬁeld experiments on four commercial glyphosate-
resistant sugarbeet varieties inoculated with R. solani indicated that herbicide did not
affect disease severity, disease indices, or plant fresh weight, or the percent of sugarbeet
considered harvestable or healthy. However, variety played a major role in differences of
these parameters. An additional ﬁeld experiment examining the effect of ﬁmgicide
applications of azoxystrobin on R. solani and interactions with tank-mixtures of
glyphosate and azoxystrobin indicated that herbicide treatments did not influence R.
solani disease index or effectiveness of azoxystrobin. Foliar azoxystrobin application
provided the greatest disease suppression when compared with in-furrow treatments and
either fungicide treatment was better than no ﬁJngicide treatment. Choosing varieties
with tolerance to Rhizoctonia crown and root and applying a foliar application of

fungicide like azoxystrobin will be the key factors to help growers manage this disease.

ACKNOWLEDGMENTS

I would like to thank Dr. Christy Sprague for giving me the opportunity to pursue
my Masters at Michigan State University. I appreciate the guidance and support that I
have received ﬁ‘om Dr. Sprague during my time here. I know I will use what I have
learned from Dr. Sprague as I continue my education in weed science. I also appreciate
Dr. Linda Hanson, Dr. Chris DiFonzo, and Dr. Wesley Everman for serving on my
guidance committee and providing assistance in helping me complete this research. You
all played an important role on my committee, and I truly appreciate what I have learned
from each of you.

I would also like to thank Gary Powell and Erin Taylor for their assistance in the
ofﬁce, lab, greenhouse, and ﬁeld. They are an essential part of the weed science program
at Michigan State, and I would not have been able to do this without them. Paul Horny
and Dennis Fleischman at the Saginaw Valley Research and Extension Center helped
manage the off-campus farm trials. I would also like to thank Jim Stewart, Lee Hubbell,
and Michigan Sugar Company for providing labor and funding to complete this research.

I also have many weed science graduate and undergraduate students to thank. Joe
Armstrong, Molly Buckham, Ryan Holmes, Megan Ross, Michelle Cole, Marc Hasenick,
David Reif, Nicole Phillips, and Anna Timmerman have all played an important role in
helping me complete this research. More importantly though, you all have been great
friends and will be. deeply missed. And last but not least, I would like to thank my
parents, grandparents, siblings, and all my additional family and friends who have

supported and encouraged me through all of my endeavors.

iii

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................. vi
LITERATURE REVIEW
INTRODUCTION ........................................................................................... 1
GLYPHOSATE-RESISANT CROPS .............................................................. 2
GLYPHOSATE AND DISEASE INTERACTIONS ........................................ 5
RHIZOCTONIA CROWN AND ROOT ROT ................................................. 9
LITERATURE CITED .................................................................................... 13

INFLUENCE OF GLYPHOSATE ON RHIZOCTONIA CROWN AND ROOT
ROT IN GLYPHOSATE-RESISTANT SUGARBEET

ABSTRACT .................................................................................................... 19
INTRODUCTION ........................................................................................... 20
MATERIALS AND METHODS ..................................................................... 23
Response of three sugarbeet varieties in the greenhouse
(Experiment 1) ..................................................................................... 23
Rhizoctonia solani growth in vitro ........................................................ 25
Response of four sugarbeet varieties in the ﬁeld ................................... 26
Response of four sugarbeet varieties in the greenhouse
(Experiment 2) ..................................................................................... 29
Statistical Analysis ............................................................................... 29
RESULTS AND DISCUSSION ....................................................................... 30
Response of three sugarbeet varieties in the greenhouse
(Experiment 1) ..................................................................................... 3O
Rhizoctonia solani growth in vitro ........................................................ 32
Response of four sugarbeet varieties in the ﬁeld ................................... 33
Response of four sugarbeet varieties in the greenhouse
(Experiment 2) ..................................................................................... 36
SOURCES OF MATERIALS .......................................................................... 40
LITERATURE CITED .................................................................................... 50

INFLUENCE OF GLYPHOSATE AND FUNGICIDE TREATMENTS ON
RHIZOCTON IA CROWN AND ROOT ROT IN GLYPHOSATE-RESISTANT
SUGARBEET

ABSTRACT .................................................................................................... 53
INTRODUCTION ........................................................................................... 54
MATERIALS AND METHODS ..................................................................... 58
RESULTS AND DISCUSSION ....................................................................... 62
Herbicide injury ................................................................................... 62
Effect of variety, herbicide, and fungicide on Rhizoctonia crown and root
rot ........................................................................................................ 62
Harvestable sugarbeet .......................................................................... 67

iv

SOURCES OF MATERIALS .......................................................................... 69

LITERATURE CITED .................................................................................... 75
APPENDICES

APPENDIX A: ADDITIONAL PARAMETERS FOR GREENHOUSE
EXPERIMENT 1 .............................................................................................. 78
APPENDIX B: ADDITIONAL PARAMETERS FOR GREENHOUSE
EXPERIMENT 2 .............................................................................................. 81
APPENDIX C: RESPONSE OF GLYPHOSATE—RESISTANT SUGARBEET
TO K SOLANI AG-2—2-IV .............................................................................. 83

LIST OF TABLES

Table 1. Monthly precipitation and the 30-year average for experiments located
in the Saginaw Valley region of Michigan in 2008 and 2009 ............................ 42

Table 2. Response of three glyphosate-resistant sugarbeet varieties to Rhizoctonia
solani isolate AG-2-2-IIIB in the presence and absence of herbicides ............... 43

Table 3. Fresh weights of three glyphosate-resistant sugarbeet varieties exposed
to Rhizoctonia solani isolate AG-2-2-IIIB in the presence and absence of
herbicides. ....................................................................................................... 44

Table 4. Mycelial growth othizoctonia solani isolate AG-2-2-IIIB in vitro in
the presence of varying rates of glyphosate and a standard sugarbeet herbicide
mixture. Glyphosate data are combined over treatments with and without
ammonium sulfate since there was not a signiﬁcant difference in the rate of
mycelial growth for these treatments ................................................................ 45

Table 5. P-values for main effects and interactions of herbicide treatments and
four Rhizoctonia solani inoculated glyphosate-resistant sugarbeet varieties for
ﬁeld experiments conducted in 2008 and 2009. ................................................ 46

Table 6. Response of four glyphosate-resistant sugarbeet varieties to Rhizoctonia
solani isolate AG-2-2-IIIB in ﬁeld experiments conducted in 2008 and 2009.
Data are combined over herbicide treatments since there was not a signiﬁcant
variety by herbicide interaction. ....................................................................... 47

Table 7. P-values for main effects and interactions of herbicide treatments on
Rhizoctonia solani isolate AG-2-2-IIIB disease severity and plant fresh weight of
four glyphosate-resistant sugarbeet varieties for greenhouse Experiment 2. ...... 48

Table 8. Response of four glyphosate-resistant sugarbeet varieties to Rhizoctonia
solani isolate AG-2-2-IIIB in greenhouse Experiment 2. Data are combined over
herbicide treatments since there was not a signiﬁcant variety by herbicide

interaction ........................................................................................................ 49

Table 9. Monthly precipitation and the 30-year average for experiments located
in the Saginaw Valley region of Michigan in 2008 and 2009 ............................ 70

Table 10. P-values for main effects and interactions of herbicide and fungicide
treatments on Rhizoctonia solani AG-2-2-IIIB disease index and healthy and
harvestable sugarbeet of four glyphosate-resistant sugarbeet

varieties. Data are combined across years ....................................................... 71

vi

Table 11. Disease index ratings and percent healthy sugarbeet of four glyphosate-
resistant sugarbeet varieties inoculated with Rhizoctonia solani. Data are
combined across herbicide treatments, fungicide treatments, and years. ........... 72

Table 12. Disease index ratings and percent healthy sugarbeet for fungicide
treatments applied to glyphosate-resistant sugarbeet inoculated with Rhizoctonia
solani. Data are combined across varieties, herbicide treatments, and years. 73

Table 13. Percent harvestable sugarbeet for ﬁtngicide treatment applied to four

glyphosate-resistant sugarbeet varieties inoculated with Rhizoctonia solani. Data
are combined across herbicide treatments and years. ........................................ 74

vii

CHAPTER 1

LITERATURE REVIEW

Introduction

Sugarbeet (Beta vulgaris L.) is a biennial crop that is treated like an annual when
grown for sucrose production. In Michigan, sugarbeet is typically planted early in the
spring as seed and roots are harvested in the fall (Asadi 2006). At harvest, leaf biomass
is removed at the crown by specialized equipment that contains a series of blades (Smith
2001 ). Sugarbeet roots are then mechanically harvested ﬁ'om the soil and transported to
sugar factories to be processed. Sugarbeet is a major source of sucrose, supplying 50 to
55% of the sucrose used in the United States and about 35% of the sucrose used
worldwide (Harveson et a1. 2009; Wilson 2001). Commercial production of sugarbeet
began in the United States around 1870 in California, and followed only a few years later
in Michigan (Harveson et al. 2009). Michigan is ranked the fourth highest state for
sugarbeet production in the United States behind Minnesota, North Dakota, and Idaho
(Harveson et al. 2009; NASS 2009). On average, 537,000 ha of sugarbeet were planted
in the United States each year ﬁ'om 2000 to 2009, with approximately 66,000 ha per year
grown in Michigan (N ASS 2009). In 2008, the total production value of sugarbeet per
year was over one billion dollars in the United States, with approximately 170 million

dollars coming from Michigan alone (NASS 2009).

Glyphosate-resistant Crops

Glyphosate is the most widely used herbicide in the world because of its ability to
control a broad spectrum of annual and perennial broadleaf and grass weed species (Duke
and Powles 2008; Pline-Srnic 2005). With its introduction in the early 1970’s,
glyphosate quickly became a valuable tool (Baylis 2000). The use of glyphosate
continued to increase with the introduction of glyphosate-resistant crops in 1996
(Gianessi 2008). Glyphosate use in glyphosate-resistant crops changed weed
management tactics by making weed control easier and more effective with fewer
herbicide applications and increasing proﬁts (Baylis 2000; Green 2009).

Currently, there are six commercialized glyphosate-resistant crops: soybean
[Glycine max (L.) Merr], corn (Zea mays L.), cotton (Gossypium hirsutum L.), canola
(Brassica napus L.), alfalfa (Medicago sativa L.) and most recently (2008) glyphosate-
resistant sugarbeet (Beta vulgaris L.) (Green 2009). Glyphosate-resistant sugarbeet
varieties were quickly adopted by growers in Michigan. Approximately 98% of
Michigan’s sugarbeet hectares were planted with a glyphosate-resistant variety in 2009
(C. Guza, Agronomist, Michigan Sugar Company, Bay City, MI, personal
communication). Competition from weeds is problematic for most sugarbeet growers
and multiple conventional herbicide applications, in addition to cultivation and hand
weeding, are the typical methods used to control weeds (Gianessi 2005). Weed control
costs for conventional sugarbeet are estimated at approximately $336 per acre (Gianessi
2005) and nationwide net economic return for conventional sugarbeet was negative for 4
out of 6 years ﬁom 1995-2001 (Gianessi et a1. 2002). The economic return for other

glyphosate-resistant crops such as corn and soybean is similar or greater when compared

with conventional systems (Johnson et a1. 2000; Nolte and Young 2002a, 2002b; Reddy
and Whiting 2000).

The use of glyphosate in glyphosate-resistant sugarbeet provides growers the
Opportunity for excellent control of many weed species that can affect sugarbeet yield
and quality (Kniss et al. 2004). Glyphosate applied to glyphosate-resistant sugarbeet
provided similar or superior weed control when compared with a mixture of conventional
herbicides including metamitron, phenmedipham plus desmedipham, and ethoﬁamesate
(Madsen and Jensen 1995). Two sequential applications of glyphosate applied to 10-cm
weeds provided similar weed control when compared with a conventional herbicide
combination of desmedipham plus phenmedipham, triﬂusulfuron, and clopyralid (Wilson
et al. 2002). Additionally, two applications of glyphosate in glyphosate-resistant
sugarbeet at a rate of 0.84 kg ae/ha provided 95% or greater weed control when
applications were made starting at the 2-1eaf stage (Dexter and Luecke 1999; Guza et a1.
2002). Conventional postemergence (POST) herbicides do not effectively control weeds
with more than two leaves, so many herbicide applications are necessary and seldom
result in 100% control of weeds (Dale et a1. 2006; Dale and Renner 2005). Wilson et. a1.
(2002) found that sucrose yields with a glyphosate herbicide program were as high as
10,000 kg/ha and that sucrose yield was reduced by as much as 15% where three
sequential applications of phenmedipham plus desmedipham, triﬂusulfuron, and
clopyralid were applied. In addition, Kemp et al. (2009) determined that when compared
with conventional sugarbeet, fewer herbicide applications were required for improved

weed control and higher yields in glyphosate-resistant sugarbeet varieties.

The introduction ofglyphosate-resistant sugarbeet also provides growers the
opportunity to adjust production practices. Narrowing row widths may be possible with
reduced cultivation, to obtain higher yields, and as a result, greater economic return
despite the additional seed costs associated with using glyphosate-resistant sugarbeet
varieties (Armstrong 2009). Glyphosate is less expensive when compared with
conventional sugarbeet weed control programs and the potential for greater economic
returns is also possible with fewer herbicide applications resulting in improved weed
control and increased yields (Kniss et al. 2004).

Glyphosate has a unique mode of action because it is the only herbicide that
prevents production of the 5-enolypyruvylshikimate-3-phosphate synthase (EPSPS)
enzyme, resulting in inhibition of the shikimic acid pathway (Steinrucken and Amrhein
1980). Glyphosate competes with the substrate phosphoenolpyruvate (PEP), preventing
the production of the EPSPS enzyme which is responsible for converting shikimate to
chorismate (Amrhein et a1. 1980; Bentley 1990; Dill 2005; Pline-Srnic 2005; Siehl 1997).
This inhibition of EPSPS blocks the shikimic acid pathway, therefore preventing the
production of the aromatic amino acids: tryptophan, tyrosine, and phenylalanine (Hanson
and Gregory 2002; Siehl 1997). Glyphosate also reduces the production of secondary
compounds including proteins, auxins, phytoalexins, folic acid, precursors of lignins,
glavonoids, plastoquinone, and many more phenolic and alkaloid compounds (Bentley
1990). These secondary compounds are important for plant defense against pathogens,
plant growth, and plant tolerance under stress (Pline-Smic 2005). If these secondary
compounds are inhibited, applications of glyphosate could lead to increased susceptibility

to certain plant pathogens.

Glyphosate-resistant crops contain a CP4-EPSPS gene that was isolated from
A grobacterium sp. and glyphosate-resistant crops expressing this enzyme exhibit a high
level of resistance to glyphosate (Dill 2005; Pline-Srnic 2005). While glyphosate-
resistant crops have a form of EPSPS that is not affected by glyphosate, the resistant
EPSPS may not be as efﬁcient as native EPSPS when exposed to glyphosate (Pline-Srnic
2005). The reduced efﬁciency of this non-native EPSPS enzyme may result in the
decreased production of secondary compounds that help protect the plant from pathogens
(Pline-Srnic 2005). Despite the ability of glyphosate-resistant crops to exhibit resistance
to glyphosate, applications of glyphosate may still have an eﬁea on the synthesis of plant
defense compounds (Pline-Srnic 2005). This may be important especially for diseases
caused by so il-bome pathogens, such as Rhizoctonia solani Kiihn (Altman and Campbell
1977). Limited resistance is available in commercial cultivars, therefore increasing the
importance of using cultural control methods to reduce the impact of these diseases

(Johal and Huber 2009).

Glyphosate and Disease Interactions
Prior to the introduction of glyphosate-resistant crops, studies on glyphosate
disease interactions have indicated that glyphosate may inﬂuence disease severity and
susceptibility to certain pathogens in non-glyphosate-resistant crops. Keen et aL (1982)
determined that by inhibiting phytoallexin production, soybean were more susceptible to
root rot (caused by the pathogen Phytophthora megasperma Drechsler f. sp. glycines
Kuan & Erwin) after glyphosate applications. Johal and Rahe (1984) determined that dry

bean grown in autoclaved soil or vermiculite survived a 10-ug dose of glyphosate while

dry bean grown in an unsterile soil (with Pythium and F usarium spp. present) or
autoclaved soil infested with Pythium spp. did not survive. This indicated that glyphosate
applications in the presence of Pythium or F usarium spp. increase the efﬁcacy of
glyphosate. Additional studies in dry bean demonstrated that glyphosate applications
reduced the production of phytoallexins and these plants were more susceptible to
anthracnose [Colletotrz'chum lindemuthanium (Sacc. & Magn.) Briosi & Cavara] (Johal
and Rahe 1988; Johal and Rahe 1990). In a Fusarium-susceptible tomato (Solanum
lycopersicum L.) cultivar, glyphosate increased the growth ofFusarium oxysporum f. sp.
radicis-lycopersici Synder and Hans when compared with tomatoes of the same cultivar
that did not receive glyphosate applications (Brammal and Higgins 1988).

More recent studies in glyphosate-resistant crops, including glyphosate-resistant
sugarbeet, have indicated a potential for increased susceptibility to some soil-bome
pathogens after glyphosate was applied (Larson et a1. 2006; Sanogo et a1. 2000; Sanogo et
al. 2001). In the late 1990’s after the introduction of glyphosate-resistant soybean,
growers raised concern about increased disease prevalence of sudden death syndrome
(caused by the pathogen F usarium solani (Mart) Sacc. f sp. glycines) (Sanogo et al.
2000; Sanogo et aL 2001). Growth chamber and greenhouse experiments were conducted
to determine the effect of glyphosate on the development of sudden death syndrome in
glyphosate-resistant soybean (Sanogo et a1. 2000). In vitro studies indicated that conidial
germination, mycelial growth, and sporulation were reduced by glyphosate. However,
there was a signiﬁcant increase in sudden death syndrome disease severity and the
frequency of isolation of F. solani from soybean roots in plants treated with glyphosate

when compared with plants with no herbicide application. Field studies supported

 

 

ﬁndings in the greenhouse and demonstrated that glyphosate-resistant soybean was more
susceptible to sudden death syndrome after glyphosate was applied (Sanogo et al. 2001).
Larson et a1. (2006) determined that two experimental varieties of glyphosate-
resistant sugarbeet, B4RR and H16, were more susceptible to certain isolates of
Rhizoctonia solani Kiihn and Fusarium oxysporum Schlecht. f. sp. betae Snyd. & Hans.
after glyphosate was applied. The variety B4RR demonstrated excellent tolerance to R.
solani AG-2-2-IIIB when a surfactant control treatment was applied. However, B4RR
plants treated with glyphosate had a signiﬁcant increase in disease severity when
compared with a no herbicide control. This indicated that in a variety tolerant to R.
solani, resistance may be lost after glyphosate was applied. The second variety, H16,
was more susceptible to R. solani and thus had a signiﬁcantly higher disease severity
rating than the B4RR variety, when treated with a surfactant control. After glyphosate
applications, disease severity was not statistically different between varieties, ﬁthher
demonstrating the loss of resistance in B4RR. However, glyphosate had no signiﬁcant
effect on ﬁlngal growth of R. solani and the production of overwintering structures when
compared with the control. Additional studies were conducted to determine the effect of
glyphosate on the production of shikimic acid. It was determined that for both
glyphosate-resistant varieties and at all growth stages, the rate of shikimic acid
accumulation was greater after glyphosate was applied compared with the surfactant
control. Although no differences in fungal growth or production of overwintering
structures were detected, it appears that glyphosate applications can increase disease
severity and the production of shikimic acid in at least some varieties of glyphosate-

resistant sugarbeet.

However, other studies demonstrated that in glyphosate-resistant crops,
glyphosate applications had no effect or decreased the severity of diseases caused by soil-
bome pathogens (N jiti et al. 2003; Pankey et a1. 2005). Field studies conducted in
glyphosate-resistant soybean determined that there were no signiﬁcant effects of
glyphosate on sudden death syndrome (F. solani) disease severity or soybean yield, and
that selecting cultivars with tolerance to sudden death syndrome was the best way to
manage this disease (N jiti et a1. 2003). These results were in contrast to greenhouse and
ﬁeld results reported by Sanogo et al. (2000) and (2001). Varietal differences as well as
environmental factors such as planting date, genotype, and other soil factors, may explain
why glyphosate has no effect on F. solani disease severity in certain varieties, but
increases disease severity in others. In greenhouse studies on glyphosate-resistant cotton,
applications of glyphosate had no effect on susceptibility to Rhizoctonia solani Kiihn
AG-2-2-IV (Pankey et al. 2005). In fact, ﬁeld studies indicated that glyphosate
applications actually reduced disease severity when compared with other preemergence
herbicides and the non-treated control.

Field studies also have been conducted to determine if glyphosate inﬂuenced
severity of foliar diseases in glyphosate-resistant crops. In glyphosate-resistant soybean,
glyphosate applications had no effect on the disease severity of white mold (Sclerotinia
stem rot), caused by the ﬁmgus Sclerotinia sclerotiorum (Lib) de Bary (Lee et al. 2000;
Nelson et al. 2002). Nelson et aL (2002) determined that glyphosate applications to
glyphosate-resistant soybean did not affect soybean response, reproductive development,
canopy development, ﬂower number, S. sclerotiorum lesion size, or phytoalexin

production, and that disease severity and grain yield were impacted by cultivar selection

rather than herbicide treatment. Lee et al. (2000) further demonstrated that neither the
glyphosate-resistant trait in glyphosate-resistant soybean nor glyphosate application
inﬂuenced soybean yield, disease severity, or S. sclerotiorum growth, and did not
increase soybean susceptibility to white mold.

Studies with glyphosate-resistant wheat (T riticum aestivum L.) indicated that
glyphosate actually decreased disease severity of leaf rust (caused by the pathogen
Puccinia triticina Eriks) and stem rust fungus (cause by the pathogen Puccinia graminis
f. sp. tritici Eriks) when exposed to glyphosate 21 d to 35 d after inoculation (Anderson
and Kolmer 2005). Additional studies by Feng et al. (2005) determined that glyphosate
also reduced the disease severity of leaf rust (caused by the pathogen P. triticina) and
stripe rust (caused by the pathogen Puccinia striiformis f. sp. tritici Westend) in
glyphosate-resistant wheat. Baley et a1. (2008) found that glyphosate-resistant wheat
cultivars were not more susceptible than glyphosate-susceptible cultivars to the pathogens
Rhizoctonia solani, R. oryzae Ryker & Gooch, Gaeumannomyces graminis (Sacc.) v. Arx

& J. Olivier var. tritici J. Walker, and Pythium ultimum Trow.

Rhizoctonia Crown and Root Rot
Rhizoctonia crown and root rot, caused by the soil-borne pathogen Rhizoctonia
solani, is a problematic disease in many crops throughout Michigan, including sugarbeet
(Windels et al. 2009; Kirk et al. 2008). Rhizoctonia crown and root rot reduces economic
retm'ns for sugarbeet by as much as 24% in the United States and causes up to 50% yield
loss, depending on disease severity (Franc et al. 2001; Windels et al. 2009). Although

AG-2-2-IIIB is the most common and virulent subgroup causing Rhizoctonia crown and

root rot in sugarbeet, another subgroup, AG-2-2-IV, is also found in Michigan (Engelkes
and Windels 1996; Kirk et a1. 2008). The ﬁrst symptoms that are observed with
Rhizoctonia crown and root rot are foliar (Franc et al. 2001; Windels et al. 2009). Leaves
permanently wilt and dark lesions form at the base of the petiole or on the crown of the
beet. Leaves then become dry and collapse, but remain attached to the crown and form a
dry, dark rosette. Root symptoms include black lesions that begin anywhere on the root,
but may coalesce and cover the entire root surface as the disease progresses. Root tissue
is typically ﬁrm underneath these lesions. However, root tissue begins to soften
underneath these lesions and cracks may also develop in advanced stages of the disease.

Rhizoctonia solani has many host crops in addition to sugarbeet, which makes it
difﬁcult to control with crop rotation alone (Rush and Winter 1990; Schuster and Harris
1960). Soybean, dry bean (Phaseolus vulgaris L.), corn, and cucumber (Cucumia sativus
L.), as well as many weed species, can act as alternate hosts for R. solani (Sneh et al.
1998; Windels et al. 2009). Many of these crops are commonly used in a rotation with
sugarbeet in Michigan and many potential weed hosts are common species found in
sugarbeet ﬁelds, further increasing the buildup of disease inoculum (Windels et al. 2009).

Varieties bred for tolerance to Rhizoctonia crown and root rot provide additional
options for managing this disease, and varieties with varying levels of tolerance are
readily available to Michigan sugarbeet growers. Although these varieties do not
completely prohibit infection, they certainly limit fungal colonization and disease
severity (Ruppel 1973).

Additional methods for controlling Rhizoctonia crown and root rot in sugarbeet

include applications of strobilurin fungicides, such as azoxystrobin (Jacobsen et al. 1998;

10

Kirk et a1. 2008). Applications of azoxystrobin in-furrow at sugarbeet planting can
reduce infection early in the season, but may not prevent later infections (J acobsen et. al.
1998; Karaoglanidis and Karadimos 2006; Kiewnick et al. 2001; Windels and Brantner
2000). Single ﬁmgicide applications are typically made either in-furrow at planting or
postemergence (POST) to sugarbeet between the 4- to 8-leaf stage (Karaoglanidis and
Karadimos 2006; Whitney and Dufﬁis 1986). If glyphosate-resistant sugarbeet are more
susceptible to plant pathogens after glyphosate is applied, then ﬁmgicide applications
may be important in controlling sugarbeet diseases such as Rhizoctonia crown and root
rot.

Potential interactions between ﬁmgicide and glyphosate applications could
inﬂuence the efﬁcacy of fungicide treatments used to manage Rhizoctonia crown and
root rot. Kataria and Gisi (1990) found that DNOC, dicamba, ioxynil, and bromoxynil
when used in combination with the fungicide cyproconazole were synergistic in reducing
disease severity Othizoctonia ceralis Van der Hoeven and Pseudocercosporella
herpotrichoides (Fron) Deighton in wheat. However, Jacobsen et al. (1998) determined
that there was no effect on Rhizoctonia solani control efﬁcacy, when azoxystrobin was
applied in a tank-mix of desmedipham plus phenmedipham and clopyralid. Additional
ﬁeld studies using a tank-mix of these same herbicides with triﬂuSulfuron and
sethoxydirn again indicated no reduction in R. solani control or sugarbeet yield when
combined with azoxystrobin (Jacobsen et al. 1998). These results are similar to earlier
studies which showed that preemergence (PRE) applications of diclo fop methyl and
ethoﬁimesate followed by POST applications of desmedipham plus phenmedipham,

EPTC, triﬂuralin, and metolachlor did not increase disease severity of Rhizoctonia crown

ll

and root rot (Ruppel et al. 1982). However, other studies have reported antagonistic
effects of glyphosate when tank-mixed with ﬁmgicide applications. In vitro studies
conducted by Hill and Stratton (1991) determined that metribuzin when used in
combination with the ﬁmgicide chlorothalonil were antagonistic and reduced control of
Alternaria solani (E11. and Mart.) Jones and Grout. Ward (1984) also reported that in
soybean, tank-mixed applications of metalaxyl and glyphosate resulted in reduced control
ofPhytophthora megasperma Drechs f sp. glycinea (Hildeb.) Kuan and Erwin. In
sugarbeet, Sprague et al. (2005) reported an increase in sugarbeet injury when
azoxystrobin was applied within 3 days prior to or after micro-rate herbicide applications.
Therefore, potential interactions between glyphosate and applications of azoxystrobin
may have an effect on disease severity if R. solani is present. Additionally, if glyphosate-
resistant crops are more susceptible to soil-borne pathogens such as R. solani, ﬁingicide
applications may be more important in controlling Rhizoctonia crown and root rot in

glyphosate-resistant sugarbeet.

12

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16

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. . d
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17

Windels, C. E., B. J. Jacobsen, and R. M. Harveson. 2009. Rhizoctonia root and crown
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of beet diseases and pests. 2nd edition. St. Paul, MN: APS Press.

18

CHAPTER 2
INFLUENCE OF GLYPHOSATE ON RHIZOCTONIA CROWN AND ROOT

ROT IN GLYPHOSATE-RESISTANT SUGARBEET

Abstract: Greenhouse experiments were conducted in 2008 to determine if glyphosate
had an effect on disease severity when compared with a conventional standard-split
herbicide treatment or no herbicide treatment. Three potential commercially-available
varieties of glyphosate-resistant sugarbeet were used for this experiment. HilleshOg
9027RR exhibited the most tolerance to Rhizoctonia crown and root rot when no
herbicide was applied. However, after exposure to either a 0.84 or 1.68 kg ae/ha rate of
glyphosate, this variety exhibited an increase in disease severity. There were no
signiﬁcant differences between herbicide treatments in the Hilleshbg 9028RR variety,
and glyphosate decreased disease severity in Hilleshbg 9032RR when compared with the
no herbicide treatment. Experiments conducted to determine if glyphosate had an effect
on Rhizoctonia solani Kiihn growth in vitro, indicated that glyphosate did not increase the
rate of radial growth. A 10x rate of glyphosate plus ammonium sulfate (AMS) actually
decreased the rate of radial growth of R. solani. Field and additional greenhouse
experiments were conducted in 2008 and 2009 to determine if glyphosate inﬂuenced the
disease severity of R. solani in four commercial varieties of glyphosate-resistant
sugarbeet. Differences in disease severity and the percent of harvestable sugarbeet in the
ﬁeld were observed when comparing the four varieties, but glyphosate did not
signiﬁcantly inﬂuence the disease severity when compared with the standard-split

treatment or no herbicide treatment. Despite the ﬁrst greenhouse experiment that

19

indicated that glyphosate may increase disease severity in some varieties, results from
additional experiments indicate that herbicide treatment, including glyphosate
applications, did not affect disease severity. Choosing a variety with tolerance to
Rhizoctonia crown and root rot is the most important factor in reducing disease severity
in commercial varieties of glyphosate-resistant sugarbeet.

Nomenclature: Glyphosate; Standard-split; Rhizoctonia crown and root rot, Rhizoctonia
solani Kiihn; sugarbeet, Beta vulgaris L.

Key words: Glyphosate-resistant crops; disease severity; fresh weight; dry weight;

disease index; harvestable sugarbeet; healthy sugarbeet

Introduction

For decades, glyphosate has played an important role in weed management
because of its broad spectrum control of annual and perennial broadleaf and grass weed
species (Duke and Powles 2008; PIine-Smic 2005). Glyphosate continues to be a
valuable weed management tool for growers with the introduction of glyphosate-resistant
crops. Currently, there are six commercialized glyphosate-resistant crops: soybean
[Glycine max (L.) Merr], corn (Zea mays L.), cotton (Gossypium hirsutum L.), canola
(Brassica napus L.), alfalfa (Medicago sativa L.), and sugarbeet (Beta vulgaris L.)
(Green 2009). The newest commercialized glyphosate-resistant crop is sugarbeet,
introduced in 2008. Since commercialization, glyphosate-resistant sugarbeet have
quickly been adopted, with almost 98% Of Michigan’s sugarbeet acres planted to
glyphosate-resistant varieties in 2009 (C. Guza, Agronomist, Michigan Sugar Company,

Bay City, MI, personal communication).

20

Competition from weeds is problematic for most sugarbeet growers.
Traditionally, multiple herbicide applications, in addition to cultivation and hand
weeding, were necessary to manage weeds (Gianessi 2005). Also, conventional
postemergence (POST) herbicides do not effectively control weeds with more than two
leaves, so many herbicide applications are necessary and seldom result in 100% control
(Dale et al. 2006; Dale and Renner 2005). However, with the introduction of glyphosate-
resistant sugarbeet, growers can achieve excellent control of many weed species that
affect sugar quality and yield (Kemp et al. 2009; Kniss et al. 2004). When compared
with conventional herbicide treatments, glyphosate is less expensive and fewer
applications are needed to control weeds with greater economic returns (Dexter and
Luecke 1999; Guza et al. 2002; Kemp et al. 2009; Kniss et al. 2004).

However, concerns have been raised about potential increases in disease pressure
after glyphosate is applied, due to physiological effects of the herbicide on plants. In
plants, glyphosate inhibits the shikimic acid pathway, preventing the production of
aromatic amino acids, as well as secondary compounds, including phytoalexins (Bentley
1990; Hanson and Gregory 2002; Siehl 1997). Some of these secondary compounds are
important for plant defense against pathogens, plant growth, and plant tolerance under
stress (Pline-Srnic 2005). If these secondary compounds are inhibited, applications of
glyphosate could lead to increased susceptibility to certain plant pathogens. Glyphosate-
resistant crops are not injured by glyphosate applications because they contain a CP4-
EPSPS gene that exhibits a high level of resistance to glyphosate. However, this enzyme
may not be as efﬁcient as native EPSPS when exposed to glyphosate and may result in

reduced production of secondary compounds that help protect the plant from pathogens.

21

Previous studies in glyphosate-resistant crops, including glyphosate-resistant
sugarbeet, demonstrated an increased susceptibility to soil-borne pathogens after
glyphosate was applied (Larson et a1. 2006; Sanogo et a1. 2000; Sanogo et a1. 2001). In
greenhouse and ﬁeld experiments, glyphosate-resistant soybean were more susceptible to
sudden death syndrome, caused by the pathogen F usarium solani (Mart.) Sacc. f. sp.
glycines, after glyphosate was applied (Sanogo et al. 2000; Sanogo et al. 2001). In
addition, Larson et al. (2006) determined that two non-commercial varieties of
glyphosate-resistant sugarbeet were more susceptible to certain isolates of both
Rhizoctonia solani Kiihn and Fusarium oxysporum Schlecht. f. sp. betae Snyd. & Hans
after glyphosate was applied.

In contrast, other studies demonstrated that glyphosate applications had no effect
on, or even decreased the severity of, diseases caused by soil-borne pathogens (N jiti et a1.
2003; Pankey et al. 2005). In glyphosate-resistant soybean, Njiti et a1. (2003) determined
that glyphosate had no effect on soybean yield or disease severity of sudden death
syndrome. These results conﬂicted with greenhouse and ﬁeld results reported by Sano go
et al. (2000) and (2001). There were differences between these studies concerning
variety selection and varietal response to the disease. In addition, there were differences
in environmental factors such as planting date, genotype, and other soil factors. This may
explain why glyphosate has no effect on E solani disease severity in certain varieties, but
increases disease severity in others. In glyphosate-resistant cotton, greenhouse
experiments conducted by Pankey et al. (2005) showed that glyphosate had no effect on
damping Off or soreshin (caused by the pathogen Rhizoctonia solani). Furthermore, in

the ﬁeld, glyphosate actually reduced R. solani induced disease severity.

22

Rhizoctonia solani is a soil-borne pathogen that can induce root disease in many
crops throughout Michigan, including Rhizoctonia crown and root rot in sugarbeet (Kirk
et al. 2008; Windels et al. 2009). Depending on disease pressure, Rhizoctonia crown and
root rot reduces economic returns for sugarbeet by as much as 24% and results in up to
50% yield loss (Franc et al. 2001; Windels et al. 2009). The greenhouse study by Larson
et. al (2006), indicating that applications ofglyphosate to glyphosate-resistant sugarbeet
increased Rhizoctonia disease severity, raised sugarbeet grower concerns about this
potential interaction with the 2008 commercialization of glyphosate-resistant sugarbeet.
To address these concerns, the objectives of this research were to: I) investigate the
effect ofglyphosate on the disease severity of Rhizoctonia crown and root rot in
glyphosate-resistant sugarbeet varieties in the greenhouse and the ﬁeld, and 2) determine

if glyphosate has an effect on mycelial growth othizoctonia solani in vitro.

Materials and methods

Response of three sugarbeet varieties in the greenhouse (Experiment I). Glyphosate-

resistant sugarbeet varieties, HilleshOg 9027RR,1 HilleshOg 9028RR, and HilleshOg

9032RR, were planted 2.54 cm deep in a pasteurized sandy loam soil with a soil pH of
7.1. Plants were grown in the greenhouse where temperature was maintained at 25 i 5 C

with a 16-h photoperiod of natural sunlight and supplemental lighting was provided at

1,000 umol/mz/s photosynthetic photon ﬂux. Plants were watered daily to maintain

adequate soil moisture for plant growth. One week after planting, seedlings were thinned

to one plant per pot. At 14 d after planting, sugarbeet were fertilized weekly with 50 ml

of a solution containing 6.61 g/L of 20:20:20 (N :P205:K20).

23

The experiment was arranged in a three-factor completely randomized design
with ﬁve replications, and repeated in time. Factors included Rhizoctonia solani
inoculation (inoculated or non-inoculated), sugarbeet variety (Hilleshbg 9027RR,

HilleshOg 9028RR, and Hilleshbg 9032RR), and herbicide treatment. Herbicide

treatments consisted of two rates of glyphosate2 (0.84 and 1.68 kg ae/ha) plus ammonium

sulfate at 2% v/v, a standard conventional sugarbeet herbicide mixture (phenmedipham at

270 g/ha plus desmedipham3 at 270 g/ha, triﬂusulfuron4 at 9 gm, and clopyralid5 at 104

g/ha), and a no-herbicide control. Herbicide applications were made when sugarbeet

. . . . . 6
were at the 6- to 8-leaf growth stage usrng a srngle tlp track-sprayer With a Teejet 8001B

ﬂat—fan nozzle. The sprayer was calibrated to deliver 187 L/ha at a pressure of 234 kPa at
a speed ofl.6 km/h.

Within 24 hours after herbicide application, treatments that were slated to be
inoculated were inoculated with R. solani AG-2-2-IIIB, the most common and virulent R.
solani subgroup found in Michigan (Kirk et al. 2008). Rhizoctonia inoculum was
prepared by growing R. solani AG-2-2-IIB on moist autoclaved millet (Panicum
miliaceum L.). Autoclaved millet seeds were spread over a water agar plate on which a 7
mm plug of the pathogen (Rhizoctonia solani) had been placed at the approximate center.
The millet was colonized as the fungus grew, and after 7 to 10 d, the plate was
completely covered with visible ﬁmgal growth. The millet was removed ﬁ'om the plate,
air dried in a biological safety cabinet for 2 to 3 d, and stored in a sterile closed container
at 4 to 7 C until it was ready to be used. Pots were inoculated by burying one millet seed

approximately 1 cm deep adjacent to the sugarbeet crown. Sterile-autoclaved millet seed

24

was used in the non-Rhizoctonia inoculated control pots. After inoculation, inoculum
was watered in.

Sugarbeet were harvested approximately 21 d after treatment (DAT) by removing
the whole plant from the pot and washing roots to remove any excess soil. Each
sugarbeet root was rated for disease severity using the 0 to 7 Rhizoctonia crown and root
rot rating scale as follows: 0 = no visible signs of disease; 1 = inactive lesions; 2 = less
than 5 % active lesions; 3 = 6 to 25 % of the root rotted; 4 = 26 to 50% of the root rotted;
5 = 51 to 75 % of the root rotted; 6 = greater than 75 % of the root rotted, but still some
living tissue; 7 = roots completely rotted and dead (Ruppel et al. 1979). Sugarbeet fresh
weights were recorded. One replication of sugarbeet roots was sliced into approximately

1 cm sections, surface disinfected for 60 s in 0.5 % sodium hypochlorite, and plated on

potato dextrose agar7 (PDA) to conﬁrm the presence of R. solani. The remaining

samples were air dried for one week at 28 C and dry weights were recorded. Dry weight
results followed similar trends as ﬁesh weight results, therefore only plant fresh weight

data are presented.

Rhizoctonia solani growth in vitro. A laboratory experiment measured the ﬁingal
growth othizoctonia solani AG-2-2-IIIB in the presence of glyphosate. The methods
used in this experiment were described by Harikrishnan and Yang (2001) and Larson et

al. (2006). Petri plates (100 x 15 mm) were ﬁlled with 25 ml ofherbicide-amended water

agar8 (1.5 % weight to water ratio). Herbicide rates were calculated based on the area of

the plate (56.5 cmz). All herbicide and additive aqueous stock solutions were ﬁlter-

25

sterilized (0.2 pm) before being added to autoclaved PDA. Herbicide treatments
included the following: glyphosate alone at 0, 9.5, 19, 38, or 190 pg ae/m1(0, 0.5, 1, 2,
and 10X the recommended use rate); glyphosate at the same rates plus ammonium sulfate
at 0, 41, 82, 164, or 818 ug/ml; ammonium sulfate alone at 82 ug/ml; and the standard
conventional sugarbeet herbicide mixture of phenmedipham plus desmedipham,
triﬂusulfuron, and clopyralid at 6, 6, 0.2, and 2.4 ug/ml, respectively. Mycelial plugs (7
mm diameter) of R. solani AG-2-2-IIIB were removed ﬁom three wk old stock cultures
and transferred to the center of each plate. Plates were paraﬁlmed and incubated in the
dark at 27 i 2 C. Radial growth was measured daily for 5 (1 until mycelia reached the
edge of the plate. Each treatment was replicated ﬁve times and the experiment was

repeated in time.

Response of four sugarbeet varieties in the field. A ﬁeld experiment was conducted in
2008 and 2009 in the Saginaw Valley region of Michigan. The 2008 experiment was
located in St. Charles, Michigan on a Misteguay silty clay (ﬁne, mixed, semiactive,
calcareous, mesic Aeric Endoaquepts) with a soil pH of 7.8 and 3.0 % organic matter.
The 2009 experiment was located in Frankenmuth, Michigan on a Tappan-Londo
complex (ﬁne-loamy, mixed, active, calcareous, mesic Typic Endoaquolls) with a soil pH
of 7.7 and 2.4 % organic matter. Following dry bean (Phaseolus vulgaris L.) harvest,
ﬁelds were fall-chisel plowed and in the spring, ﬁelds were cultivated twice prior to
planting. Fertilizer applications were standard for sugarbeet production in Michigan.

The glyphosate-resistant sugarbeet varieties HilleshOg 9027RR, HilleshOg 9028RR,

Hilleshbg 9029RR, and Crystal RR8279 were planted 2.5-cm deep in 76-cm rows at a

26

population of 122,000 seeds/ha on April 25, 2008 and April 16, 2009. Hilleshbg 9032RR
was removed from these experiments, since this variety was not being commercially
grown in Michigan. Plots were six rows wide by 9.1 m in length. Each variety was
planted, one per row, in rows two through ﬁve. Rows one and six served as border rows.
Commercial sugarbeet varieties selected for this experiment were approved by Michigan
Sugar Company and were thought to have varying degrees of Rhizoctonia crown and root
rot tolerance.

The experimental design was a split-strip-plot with all treatments replicated four
times. Herbicide treatment was the main-plot factor, R. solani inoculation was the sub-
plot factor, and variety was the strip-plot factor. When sugarbeet were at the 6- to 8-leaf
stage, plots were inoculated with R. solani AG-2-2-IIIB. Rhizoctonia inoculum was
grown on a barley medium Pans of barley, saturated with water, were autoclaved and 9
(7 mm) plugs of R. solani grown on potato dextrose agar were placed into the pans.
Paraﬁlm-sealed pans were incubated at 25 C i 2 for 3 wk. Once the barley was

colonized, it was air dried and ground into a ﬁne ﬂour. Inoculum was applied directly

over each sugarbeet row at 2 g/m of row using a modiﬁed drop spreader. 10 The

inoculum rate was conﬁrmed by determining the amount of leftover inoculum and

calculating the kg applied per m of row. Plots that were non-inoculated served as a control.

All plots were cultivated following inoculation to put soil and inoculum in the crown for
increased disease severity (Ruppel et al. 1979).

Herbicide treatments includedl) a glyphosate herbicide program, 2) a standard-
split program (standard herbicide program used in conventional sugarbeet), and 3) a

hand-weeded control (no herbicide). The glyphosate program consisted of glyphosate at

27

0.84 kg ae/ha plus ammonium sulfate at 2% v/v, applied three times at 2- to 4-leaf, 4- to
6-1eaf, and 6- to 8-leaf sugarbeet. The standard-split program consisted of a combination
of desmedipham at 180 g ai/ha plus phenmedipham at 180 g ai/ha, triﬂusulfuron at 9 g
ai/ha, clopyralid at 104 g ai/ha, and non-ionic surfactant at 0.25% v/v, applied twice at
the cotyledon to 2-leaf and 2- to 4-leaf stage sugarbeet. The rates of desmedipham plus
phenmedipham were increased to 270 g ai/ha in the second standard-split application.
All plots were maintained weed-free by hand-weeding throughout the growing season.

Herbicide treatments were applied with a tractor-mounted compressed-air sprayer

calibrated to deliver 178 L/ha at 207 kPa through 10003 AirMix11 nozzles, spaced 51 cm

apart at approximately 56 cm above the canopy. Plots were rated for herbicide injury 14
d after the last herbicide application timing.
Sugarbeet stand counts were recorded for each variety four weeks after planting

and at harvest. Approximately 8 wk after inoculation, sugarbeet were lifted item the soil

. . . 2 . . .
usmg a modified lift harvester.1 lndrvrdual sugarbeet roots were evaluated for disease

severity using the 0 to 7 scale described previously (Ruppel et a1. 1979). Stand counts
were used to determine how many sugarbeet were missing from each plot due to
advanced disease severity. Values were adjusted by assigning each of the missing
sugarbeet a disease severity rating of 7. An average disease index was determined for
each variety in each plot. The disease index was calculated as a weighted average based
on the number of sugarbeet in each of the eight disease classes (Ruppel et al. 1979). The
percent of healthy sugarbeet were determined by calculating the percent of sugarbeet that
had a disease severity rating of 0 or 1. Harvestable sugarbeet were determined by

calculating the percent of sugarbeet with a disease severity rating 3 or less.

28

Precipitation data was recorded by weather stations Operated by the Michigan

Automated Weather Network13 (Table 1) which were located within 3 km of the

experimental locations.

Response of four sugarbeet varieties in the greenhouse (Experiment 2). This
greenhouse experiment evaluated the four commercial sugarbeet varieties that were used
in the ﬁeld experiments in 2008 and 2009: HilleshOg 9027RR, Hilleshbg 9028RR,
HilleshOg 9029RR, and Crystal RR827. Two of these varieties, Hilleshbg 9027RR and
HilleshOg 9028RR, were also evaluated in greenhouse Experiment 1. Methods for this

experiment were similar to Experiment 1, with certain exceptions. Sugarbeet were

planted in a professional potting mix14 with a soil pH of 5.9. At the 4-leaf stage,

. . . . . 15 . . .
sugarbeet were fertilized once With a micronutrient solution containing boron and other

micronutrients. Similar procedures were used for Rhizoctonia inoculation, except the
inoculum was produced on barley (Hordeum vulgare L. subsp. vulgare). After
colonization, barley was air-dried and ground into a ﬁne ﬂour. Pots were inoculated by
spreading 0.5 ml of the barley inoculum around the sugarbeet crown. The non-inoculated
pots received 0.5 ml of sterile-autoclaved barley ﬂour. The experiment was arranged in
a three-factor completely randomized design with four replications, and repeated in time.

All other procedures and measurements were similar to Experiment 1.

Statistical Analysis. All data were analyzed using the PROC MIXED procedure in SAS

16 . . . . . . .
9.1. An analysrs of variance was performed to test for Signiﬁcant interactions and main

29

effects. Data were combined over experiments and/or years and main effects when
appropriate interactions were not signiﬁcant. Interactions between main effects were
analyzed using the SLICE Option in the LSMEANS statement. Mean separation for
treatment differences was performed using Fisher’s Protected LSD at the p _<_ 0.05
signiﬁcance level. In the laboratory experiment, radial ﬁingal growth of the different

treatments was compared by determining the slope of each replication with TableCurve

2D 5.0117 and analyzing this data in SAS, as described previously.

Results and Discussion

Response of three sugarbeet varieties in the greenhouse (Experiment 1). Two
experimental replications of greenhouse Experiment 1 were conducted in early 2008,
prior to the ﬁill-commercial release of glyphosate-resistant sugarbeet. Experimental
replication was not signiﬁcant; therefore the data were combined for analysis.
Inoculation with R. solani AG-2-2-IIIB was signiﬁcant and the average disease severity
for plants that were inoculated was 4.2 (Table 2). Rhizoctonia crown and root rot was not
present on any of the sugarbeet that were non-inoculated, indicating that the pathogen
was not present in the soil used in the greenhouse experiments. Therefore, the non-
inoculated treatments were dropped from further analysis. However, the non-inoculated
plants were used to standardize sugarbeet ﬁ'esh weight among the varieties. Fresh weight
data is presented as a percent of the non-inoculated treatments.

None of the glyphosate-resistant sugarbeet varieties used in Experiment I showed
visible signs of damage ﬁom the herbicide treatments (data not shown). However, there

were differences in disease severity and ultimately plant fresh weight, with the different

30

herbicide treatment-variety combinations. The most Rhizoctonia-tolerant variety of the
three glyphosate-resistant varieties evaluated when no herbicide was applied was
HilleshOg 9027RR, with a disease severity rating of 2.8 (Table 2). The other glyphosate-
resistant varieties, HilleshOg 9028RR and Hilleshbg 9032RR, were more susceptible to R.
solani, with disease severity ratings of 4.8 and 4.9, respectively, in the no herbicide
controls (Table 2).

Applications of glyphosate at 0.84 and 1.68 kg/ha tO Hilleshiig 9027RR increased
the disease severity rating from 2.8 to 4.7 and 5.9, respectively (Table 2). Increased
disease severity was also reﬂected with reduced plant fresh weight (Table 3). There was
a 39 and 61% reduction in plant fresh weight when glyphosate was applied at 0.84 and
1.68 kg/ha, respectively, as compared with the no herbicide control (Table 3). This
response was similar to results observed by Larson et al. (2006), where an increase in
Rhizoctonia crown and root rot disease severity occurred when glyphosate was applied to
a Rhizoctonia-tolerant glyphosate-resistant sugarbeet variety.

Although the glyphosate-resistant sugarbeet varieties HilleshOg 9028RR and
Hilleshbg 9032RR had similar disease severity ratings for the no herbicide control, they
responded differently to the herbicide treatments. None of the herbicide treatments
signiﬁcantly changed the disease severity rating or plant fresh weight for HilleshOg
9028RR (Tables 2 and 3). However, there was a signiﬁcant reduction in disease severity
when Hilleshbg 9032RR was exposed to the standard herbicide program or glyphosate at
0.84 kg when compared with the no herbicide control (Table 2). Sugarbeet ﬁesh weight
also was higher with the standard herbicide program as compared with the no herbicide

control (Table 3). This may indicate that certain herbicides could decrease disease

31

severity in certain varieties. Sanogo (2000) and (2001) demonstrated that glyphosate
applications inﬂuenced sudden death syndrome disease severity in some varieties of
glyphosate-resistant soybean, but this response was variety dependent. Differing results
in our experiment could also vary based on environmental differences. Pankey et al.
(2005) showed that in glyphosate-resistant cotton, glyphosate applications reduced
Rhizoctonia disease severity in the ﬁeld, even though there was no effect in the

greenhouse.

Rhizoctonia solani growth in vitro. In our initial greenhouse experiment, we observed
contrasting results among the three varieties evaluated. An increase in disease severity
was observed when glyphosate was applied to HilleshOg 9027RR and a decrease in
disease severity was found when glyphosate at 0.84 kg/ha or the standard conventional
herbicide mixture was applied to HilleshOg 9032RR. A laboratory experiment was
conducted to determine if these differences were explained by the rate of mycelial growth
othizoctonia solani in the presence of glyphosate. The addition of ammonium sulfate
to glyphosate did not have a signiﬁcant effect on the rate of mycelial growth. Therefore
data are combined over the glyphosate alone and the glyphosate plus ammonium sulfate
treatments.

There were signiﬁcant differences in mycelial growth for the different rates of
glyphosate (Table 4). The highest rate of glyphosate (190 ug/ml), equivalent to 10X the
normal use rate of glyphosate, inhibited mycelial growth when compared with the
control. However, lower rates of glyphosate (0.5, l, or 2X) and the standard

conventional herbicide mixture of phenmedipham plus desmedipham, triﬂusulfuron and

32

clopyralid treatment did not signiﬁcantly inﬂuence the growth rate of R. solani. Thus an
increase in the rate of mycelial growth of R. solani cannot explain the increased disease
severity after glyphosate was applied in Hilleshég 9027RR. Larson et al. (2006) also
concluded that fungal grth at varying rates of glyphosate were not signiﬁcantly
different ﬁom the control, except at the highest glyphosate concentration (40 ug/ml).
The reduction in the rate of mycelial growth at the highest rate of glyphosate may be due
to the adjuvants in the glyphosate formulation. Lee et a1. (2000) found that Sclerotinia
sclerotiorum mycelia were inhibited by a formulation blank with proprietary adjuvants at
100 mM ae glyphosate. The formulated glyphosate without an adjuvant did not inhibit
mycelial growth on herbicide amended PDA. It also is possible that glyphosate may have
anti-ﬁrngal activity and inhibit growth of R. solani. Feng et al. (2005) determined that in
glyphosate-resistant wheat, glyphosate decreased the disease severity of P. triticina and

P. Striiformis.

Response of four sugarbeet varieties in the field. Field experiments were conducted
using four commercial varieties of glyphosate-resistant sugarbeet to conﬁrm earlier
greenhouse results. Interactions between years were not signiﬁcant. Therefore, all data
are presented as a combination of the 2008 and 2009 experiments. The two-way
interaction of variety x herbicide was not signiﬁcant (Table 5) for any of the parameters
evaluated. Therefore, data are discussed as the main effects of variety and herbicide for

all parameters.

33

Rhizoctonia inoculation. Inoculation of R. solani subgroup AG-2-2-IIIB was highly
effective. The combination of cultivation and precipitation (Table 1) following
Rhizoctonia inoculation resulted in an average disease index of 5.9 in the ﬁeld (Table 6).
This provided a good basis for treatment separation. The natural R. solani infestations in
the ﬁeld were low each year based on the disease indices, 2 or less (data not shown).

Therefore, the non-inoculated treatments were dropped from ﬁrrther analysis.

Herbicide injury. The glyphosate-resistant sugarbeet varieties did not show visible signs
of damage from glyphosate treatments. However, applications of the standard-split
herbicide program (two applications) uniformly caused 13% injury to each of the four
glyphosate-resistant sugarbeet varieties evaluated (data not shown). Injury symptoms
consisted of yellowing and stunting compared with the non-treated control and are
consistent with what others have observed with this program (Wilson 1994, 1995).

Approximately 2 wks alter this evaluation, sugarbeet recovered from this damage.

Variety. The main effect of variety was signiﬁcant for Rhizoctonia disease indices and
the percentage of harvestable sugarbeet (Table 5). Sugarbeet that are considered
harvestable have a disease severity rating of 3 or less. The percentage of healthy
sugarbeet was not signiﬁcant. Sugarbeet that are considered healthy have a disease
severity rating of 0 or I. Averaged across all herbicide treatments, HilleshOg 9027RR
and Hilleshbg 9029RR were the most tolerant to R. solani infection, with disease index
ratings of 5.5 and 5.7, respectively (Table 6). The disease index rating for Hilleshbg

9028RR was signiﬁcantly higher than HilleshOg 9027RR, but was not signiﬁcantly

34

different than HilleshOg 9029RR. Crystal RR827 was the most susceptible variety to R.
solani infection, with a disease severity index of 6.6. The percentage ofharvestable
sugarbeet followed the same trend as the disease index ratings (Table 6). However,
regardless of variety, 15% or fewer of the sugarbeet were considered harvestable. Fewer

than 3% of the sugarbeet were considered healthy (Table 6).

Herbicide. The main effect of herbicide was not signiﬁcant (Table 5). These results
indicate that glyphosate had no effect on the development of Rhizoctonia crown and root
rot when compared with the standard conventional herbicide treatments or no herbicide
controls. This is in contrast to our Experiment 1 results and to the Larson et al. (2006)
ﬁndings.

In Experiment 1, applications of glyphosate increased disease severity for
HilleshOg 9027RR. However, the ﬁeld experiment did not support these ﬁndings. One
potential explanation for the contrasting results is the difference in inoculation media. In
the ﬁrst set of experiments, the Rhizoctonia inoculum was grown on millet, however, the
ﬁeld experiment used a ground barley media. Overall disease severity could have been
affected by the different soil types used in each of these experiments. The presence of
additional soil pathogens, as well as additional environmental factors, could have resulted
in differences between these experiments. In addition, other studies have indicated that
time of herbicide application in relation to disease infection may inﬂuence the
susceptibility of plants to pathogens. In the greenhouse, sugarbeet were inoculated within
24 h of herbicide treatment. However, in the ﬁeld, sugarbeet were inoculated days after

the last herbicide application. Studies with glyphosate-resistant wheat (Triticum aestivum

35

L.) have indicated that glyphosate actually decreased disease severity of leaf rust (caused
by the pathogen Puccinia triticina) and stem rust fungus (cause by the pathogen Puccinia
graminis f. sp. tritici Eriks) when exposed to glyphosate 21 d to 35 d after inoculation

(Anderson and Kolmer 2005).

Response of four sugarbeet varieties in the greenhouse (Experiment 2). An additional
greenhouse experiment (Experiment 2) was conducted using the four commercial
varieties ofglyphosate-resistant sugarbeet used in the ﬁeld to conﬁrm earlier ﬁeld and
greenhouse results. Experimental replications for the greenhouse studies were not
signiﬁcant, so data were combined for analysis. The two-way interaction of variety x
herbicide was not signiﬁcant in the greenhouse (Table 7) for any of the parameters
evaluated. Therefore, data are discussed as the main effects of variety and herbicide for

disease severity and fresh plant weight.

Rhizoctonia inoculation. Inoculation Of R. solani subgroup AG-2-2-IIIB was highly
effective in the greenhouse. Adequate moisture in the greenhouse following Rhizoctonia
inoculation resulted in an average disease severity rating of 5.9 (Table 8). Rhizoctonia
crown and root rot was not present on any of the non-inoculated sugarbeet, indicating that
the pathogen was not present in the potting mix used in the experiment. Therefore, the
non—inoculated treatments were dropped ﬁ'om ﬁirther analysis. However, the non-
inoculated plants were used to standardize sugarbeet fresh weight among the varieties.

Fresh weight data is presented as a percent of the non-inoculated treatments.

36

Variety. The main effect variety was signiﬁcant for Rhizoctonia disease severity and
sugarbeet fresh weight (Table 7). The order of Rhizoctonia tolerance of the varieties was
different in the greenhouse compared to the ﬁeld. In the greenhouse, Hilleshbg 9028RR
had the lowest disease severity rating (4.8) (Table 8). Hilleshbg 9027RR and Hilleshdg
9029RR had similar disease severity ratings of 5.9 and 6.1, respectively. Again Crystal
RR827 was the most susceptible variety with a disease severity rating of 6.7; however
this was not signiﬁcantly different from HilleshOg 9029RR. The fresh weight of
Rhizoctonia-inoculated sugarbeet was reduced by 61% or more when compared with the
non—inoculated control (Table 8). The fresh weight of HilleshOg 9028RR was

signiﬁcantly higher than fresh weights of the other varieties.

Herbicide. The glyphosate-resistant sugarbeet varieties did not show visible signs of
damage from glyphosate treatments or the standard conventional herbicide mixture. In
addition, the main effect of herbicide was not signiﬁcant for disease severity or sugarbeet
fresh weight in the greenhouse (Table 7). These results indicate that glyphosate had no
effect on the development of Rhizoctonia crown and root rot when compared with the
standard conventional herbicide treatments or no herbicide controls. This is in contrast to
our Experiment 1 results and to the Larson et al. (2006) ﬁndings.

In Experiment 1, applications of glyphosate increased disease severity for
HilleshOg 9027RR. However, ﬁeld and additional greenhouse experiments did not
support these ﬁndings. One potential explanation for the contrasting results is the
difference in inoculation media. In Experiment 1, the Rhizoctonia inoculum was grown

on millet, however, the ﬁeld and additional greenhouse experiment (Experiment 2) used a

37

ground barley media. The overall Rhizoctonia disease severity was lower for the
inoculum grown on millet (average disease severity rating = 4.2) when compared with the
barley source (average disease severity rating = 5.9). Overall disease severity could have
been affected by the different soil types used in each of these experiments. Issues with
other soil pathogens, such as F usarium spp., resulted in the switch ﬁ'om a pasteurized
ﬁeld soil in Experiment 1 to a professional potting mix in Experiment 2. The presence of
additional soil pathogens in these soil media sources could have resulted in differences
between these experiments. In addition, sugarbeet in Experiment 2 were fertilized with a
micronutrient solution and this may explain why herbicide had an inﬂuence on disease
severity in Experiment I, but not in Experiment 2. Previous studies have demonstrated
that some glyphosate-resistant soybean varieties exhibit an increase in manganese (Mn)
deﬁciency symptomalogy than conventional varieties (Dodds et al. 2001, 2002; Loecker
et al. 2010). Although this appears to be variety speciﬁc and more problematic in severe
Mn-deﬁcient soils, possible interactions between micronutrient applications to
glyphosate-resistant in Experiment 2 could explain differences in the level of disease
severity and response to herbicides when compared with Experiment 1.

We also observed a difference in the ranking of Rhizoctonia tolerance among the
varietieswhen comparing the greenhouse and ﬁeld experiments. Although Hilleshbg
9027RR was the most Rhizoctonia tolerant variety in two of the three experiments, it
appears there may not be vast differences in the tolerance levels within the three
Hilleshdg varieties (9027RR, 9028RR, and 9029RR). However, Crystal RR827 was
always the most susceptible variety to Rhizoctonia crown and root rot. In addition, the

micronutrient solution may also have resulted in differing results among the varieties. A

38

micronutrient solution was added to sugarbeet in Experiment 2 because sugarbeet showed
boron deﬁciency. HilleshO g 9027RR appeared to demonstrate the most severe deﬁciency
symptoms of the four varieties and this may explain the difference in ranking of
Rhizoctonia tolerance for Experiment 2 when compared with Experiment I and the ﬁeld
experiment.

Our results indicate that glyphosate does not inﬂuence disease severity of
Rhizoctonia crown and root rot in four commercially-available varieties of glyphosate-
resistant sugarbeet. Growers can make several glyphosate applications to glyphosate-
resistant sugarbeet varieties without increasing susceptibility to Rhizoctonia crown and
root rot. Although greenhouse Experiment 1 indicated that glyphosate may increase
disease severity, glyphosate applications did not inﬂuence disease severity in additional
ﬁeld and greenhouse experiments. Variety selection is the most important factor in
reducing disease severity of Rhizoctonia crown and root rot in glyphosate-resistant
sugarbeet. To prevent yield and sugar quality loss, using a variety with excellent

tolerance to R. solani is recommended.

39

SOURCES OF MATERIALS

1 Syngenta Seeds Inc., 1020 Sugarmill Rd., Longmont, CO 80501.
2 Roundup WeatherMAX, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167.

3 Betamix, Bayer CropScience AG, Alﬁed-Nobel-Str. 50, D-40789 Monheim am Rhein,
Germany.

4 UpBeet, E.I. du Pont de Nemours and Co., Crop Protection, 1007 Market St.,
Wilmington, DE 19898.

5 Stinger, Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN 46268.

6 Spraying Systems Co., PO. Box 7900, Wheaton, IL 60187.

7 Becton & Dickinson, and Co., 7 Loveton Circle, Sparks, MD 21 152.

8 Sigma Chemical Co., 6050 Spruce St., St. Louis, MO 63103.

9 BetaSeed, Inc., 1788 Marschall Road, Shakopee, MN 55379.

10 Gandy Company, 528 Gandrud Road, Owatonna, MN 55060.

H AirMix 11003, Greenleaf Technologies, PO. Box 1767, Covington, LA 70434.
12 Tractor Supply Company, 200 Powell Place, Brentwood, TN 37027.

13 Michigan Automated Weather Network, Web site:

http://www.agwcathergco. msu.cdu/

 

14 Baccto Professional Potting Mix, Michigan Peat Company, PO. Box 980129,
Houston, TX 77098.

15 MicroMax, Grace-Sierra, 1001 Yosemite Dr., Milpitas, CA 95035.

40

16 The SAS System for Windows, Version 9.1, SAS Institute, Inc., 100 SAS Campus Dr.,
Cary NC 27513.

17 TableCurve 2D 5.01, Systat Software Inc., 501 Canal Blvd., Richmond, CA 94804-
2028.

41

Table 1. Monthly precipitation and the 30-year average for experiments located in the
Saginaw Valley region of Michigan in 2008 and 2009.
Precipitation (mm)

 

 

2008 2009 30 yr.
April 51 l 19 72
May 29 31 71
June 99 122 83
July 100 69 70
August 53 88 96
Total 332 429 392

 

a Precipitation data was collected from the Michigan Automated Weather Network
(http://www.agweather.geo.msu.edu/mawn/).

42

Table 2. Response of three glyphosate-resistant sugarbeet varieties to Rhizoctonia
solania isolate AG-2-2-IIIB in the presence and absence of herbicides.

 

 

Herbicide treatment H 9027RR H 9028RR H 9032RR
. . ' C
disease severity (0 - 7 scale)
No herbicide 2.8 abd 4.8 cde 4.9 de
Standard conventional programb 4.0 abcd 4.7 cde 2.5 a
Glyphosate (0.84 kg ae/ha) 4.7 cde 4.4 cde 3.0 abc
Glyphosate (1.68 kg ae/ha) 5.9 e 4.7 cde 4.0 abcd

 

a . . . . . . .
Rhizoctonia solani inoculum was prepared With a mullet medium

b The standard conventional herbicide program included phenmedipham at 270 g ai/ha
plus desmedipham at 270 g ai/ha, triﬂusulﬁiron at 9 g ai/ha, and clopyralid at 104 g
tha.

c Sugarbeet roots were rated for disease severity on a 0 to 7 scale (0 = no disease and 7 =
completely rotted).

Means followed by the same letter are not different according to Fisher’s Protected
LSD atp g 0.05.

43

Table 3. Fresh weights of three glyphosate-resistant sugarbeet varieties exposed to
Rhizoctonia solania isolate AG-2-2-IIIB in the presence and absence of herbicides.

 

 

 

 

Herbicide treatment H 9027RR H 9028RR H 9032RR
% of non-inoculatedc

No herbicide 83 abd 54 be 33 cd

Standard conventional programb 59 abc 41 Cd 91 a

Glyphosate (0.84 kg ae/ha) 44 cd 62 abc 55 be

Glyphosate (1.68 kg ae/ha) 22 d 46 cd 63 abc

 

a . . . . . . .
Rhizoctonia solani inoculum was prepared wrth a millet medium

b The standard conventional herbicide program included phenmedipham at 270 g ai/ha
plus desmedipham at 270 g tha, triﬂusulfuron at 9 g ai/ha, and clopyralid at 104 g
ai/ha.

c Fresh weights were determined by dividing the fresh weight of the Rhizoctonia-
inoculated plants by the ﬂesh weight of non-inoculated plants for each treatment.
Means followed by the same letter are not different according to Fisher’s Protected
LSD atp _<_ 0.05.

44

Table 4. Mycelial growth othizoctonia solania isolate AG-2-2-IIIB in vitro in the
presence of varying rates of glyphosate and a standard sugarbeet herbicide mixture.
Glyphosate data are combined over treatments with and without ammonium sulfate since
there was not a signiﬁcant difference in the rate Of mycelial growth for these treatments.

 

 

 

 

Herbicide treatment Rate Mycelial grth rateb
— ug/ml — cm/d
Control — 1.05 bc
Glyphosate (0.5X) 9.5 1.05 b
Glyphosate (1X) 19 1.03 b
Glyphosate (2X) 38 1.02 ab
Glyphosate (10X) 190 0.96 a
Phenmedipham + desmedipham 6 + 6 + 1.04 b
+ triﬂusulﬁiron + clopyralid 0.2 + 2.4

 

a . . . . . . .
Rhizoctonia solani inoculum was prepared With a millet medium

b Growth rate was determined by the slope for mycelial growth from days 1 to 4
(cm/day).

c Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 5 0.05.

45

Table 5. P-values for main effects and interactions of herbicide treatments and four
Rhizoctonia solani inoculated glyphosate-resistant sugarbeet varieties for ﬁeld
experiments conducted in 2008 and 2009.

 

 

 

 

Effectsa Disease indexb Harvestablec Healthyd
p-value

Herbicide 0.8762 0.9714 0.5835

Variety <0.0001 <0.0001 0.5152

Variety x herbicide 0.9904 0.9991 0.7081

 

a Inoculation was removed from ﬁirther analysis since it was highly signiﬁcant and non-

inoculated plants had a disease severity rating of less than 2.

Disease is rated based on a 0 to 7 scale (0 = no disease and 7 = completely rotted) and
the disease index is calculated by determining a weighted average based on the number

of sugarbeet in each of the eight disease classes.

c Harvestable sugarbeet is the percentage of sugarbeet in the plot with a disease severity

rating of 3 or less.

Healthy sugarbeet is the percentage of sugarbeet in the plot with a disease severity
rating of l or less.

46

Table 6. Response of four glyphosate-resistant sugarbeet varieties to Rhizoctonia solania
isolate AG-2-2-IIIB in ﬁeld experiments conducted in 2008 and 2009. Data are combined
over herbicide treatments since there was not a signiﬁcant variety by herbicide
interaction.

 

 

 

 

Variety Disease indexb Harvestablec Healthyd
_ 0 - 7 scale — % %
Hilleshog 9027RR 5,5 a" 15 a 2 a
Hilleshog 9028RR 5.9 b 9 b 1 a
Hilleshog 9029RR 5.7 ab 12 ab 1 a
Crystal RR827 6.6 c 2 c 0 a

 

a Inoculation was removed ﬁ'om further analysis since it was highly signiﬁcant and non-
inoculated plants had a disease severity rating of less than 2.

Disease is rated based on a 0 to 7 scale (0 = no disease and 7 = completely rotted) and
the disease index is calculated by determining a weighted average based on the number
of sugarbeet in each of the eight disease classes.

6 Harvestable sugarbeet is the percent of sugarbeet in the plot with a disease severity
rating of 3 or less.
Healthy sugarbeet is the percent of sugarbeet in the plot with a disease severity rating
Of 1 or less.

6 Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 5 0.05.

47

Table 7. P-values for main effects and interactions of herbicide treatments on

Rhizoctonia solania isolate AG-2-2-IIIB disease severity and plant fresh weight of four
glyphosate-resistant sugarbeet varieties for greenhouse Experiment 2.

 

 

 

 

Effects Disease severity Fresh weight
p-value

Herbicide 0.3672 0.2024

Variety < 0.0001 0.0012

Variety x herbicide 0.2330 0.1667

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

b Inoculation was removed ﬁom further analysis since it was highly signiﬁcant and non—
inoculated plants had a disease severity rating of less than 1.

48

Table 8. Response of four glyphosate-resistant sugarbeet varieties to Rhizoctonia solania
isolate AG-2-2-IIIB in greenhouse Experiment 2. Data are combined over herbicide
treatments since there was not a signiﬁcant variety by herbicide interaction.

 

 

 

Variety Disease severityb Fresh weightc
0 - 7 scale——— —% of non-inoculated—
Hilleshog 9027RR 5,9 bd 22 b
Hilleshog 9028RR 4.8 a 39 a
Hilleshog 9029RR 6.1 be 25 b
Crystal RR827 6.7 c 13 b

 

a Rhizoctonia solani inoculum was prepared with a barley medium.
b Sugarbeet roots were rated for disease severity on a 0 to 7 scale (0 = no disease and 7 =
completely rotted).

c Fresh whole weight is determined by weighing the whole plant and dividing that weight
by the weight of the same non-inoculated treatment.

Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 3 0.05.

49

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following application of the herbicide glyphosate. Plant Dis. 89:1136-1142.

Bentley, R. 1990. The shikimate pathway: a metabolic tree with many branches. Pages
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Dale, T. M. and K A. Renner. 2005. Timing ofpostemergence micro-rate applications
based on growing degree days in sugarbeet. J. Sugar Beet Res. 42287-101.

Dale, T. M., K A. Renner, and A. N. Kravchenko. 2006. Effect of herbicides on weed
control and sugarbeet (Beta vulgaris) yield and quality. Weed Technol. 20: 1 50-156.

Dexter, A. G. and J. L. Luecke. 1999. Conventional herbicides at micro-rates,
glyphosate and glufosinate for weed control in sugarbeet. Proc. N. Cent. Weed Sci.
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Dodds, D. M., M. V. Hickman, and D. M. Huber. 2001. Comparison of micronutrient
uptake by glyphosate-resistant and non-resistant soybeans. Proc. North Central Weed
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Dodds, D. M., D. M. Huber, and M. V. Hickman. 2002. Micronutrient levels in normal
and glyphosate-resistant soybean varieties. Proc. North Central Weed SCi. Soc.
57:107.

Duke, S. O. and S. B Powles. 2008. Glyphosate: a once-in-a-century herbicide. Pest
Manag. Sci. 64:319-325.

Feng, P. C., G. J. Baley, W. P. Clinton, G. J. Bunkers, M. F. Alibhai, T. C. Paulitz. 2005.
Glyphosate inhibits rust diseases in glyphosate-resistant wheat and soybean. PNAS.
48: 17290-17295.

Franc, G. D., R. M. Harveson, E. D. Kerr, and B. J. Jacobsen. 2001. Disease
Management. Pages 143-145 in R. G. Wilson, J. A. Smith, and S. D. Miller, eds.
Sugarbeet Production Guide. Lincoln, NE: University of Nebraska.

Gianessi, L. P. 2005. Economic and herbicide use impacts of glyphosate-resistant crops.
Pest Manag. Sci. 61:241-245.

Green, J. M. 2009. Evolution ofglyphosate-resistant crop technology. Weed Sci.
57:108-117.

Guza, C. G., C. V. Ransom, and C. Mallory-Smith. 2002. Weed control in glyphosate-
resistant sugarbeet (Beta vulgaris L.). J. Sugar Beet Res. 39:109-123.

50

Hanson A. D. and Gregory J. F. 2002. Synthesis and turnover of folates in plants. Curr.
Opin. Plant Biol 52244-249.

Harikrishnan, R. and X. B. Yang. 2001. Inﬂuence of herbicides on growth and sclerotia
production in Rhizoctonia solani. Weed Sci. 49:241-247.

Kemp, N. J., E. C. Taylor, and K A. Renner. 2009. Weed management in glyphosate-
and glufosinate-resistant sugar beet. Weed Technol. 232416-424.

Kirk, W. W., P. S. Wharton, R. L. Schafer, P. Rumbalam, S. Poindexter, C. Guza, R.
Fogg, T. Schlatter, J. Stewart, L. Hubbell, and D. Ruppal. 2008. Optimizing ﬁingicide
timing for the control of Rhizoctonia crown and root rot of sugar beet using soil
temperature and plant growth stages. Plant Dis. 92:1091-1098.

Kniss, A. R., R. G. Wilson, A. R. Martin, P. A. Burgener, and D. M Feuz. 2004.
Economic evaluation of glyphosate-resistant and conventional sugar beet. Weed
Technol. 182388-396.

Larson, R. L., A. L. Hill, A. Fenwick, A. R. Kniss, L. E. Hanson, and S. D. Miller. 2006.
Inﬂuence of glyphosate on Rhizoctonia and Fusarium root rot in sugar beet. Pest
Manag. Sci. 62: 1182-1192.

Lee, C.D., D. Penner, and R. Hammerschmidt. 2000. Inﬂuence of formulated glyphosate

and activator adjuvants on Sclerotinia sclerotiorum in glyphosate-resistant and -
susceptible Glycine max. Weed Sci. 48:710-715.

Loecker, J. L., N. 0. Nelson, W. B. Gordon, L. D. Maddux, K A. Janssen, and W. T.
Schapaugh. 2010. Manganese response in conventional and glyphosate-resistant
soybean. Agron. J. 102:606-611.

Njiti, V. N, O. Myers Jr., D. Schroeder, and D. A. Lightfoot. 2003. Roundup Ready
soybean: glyphosate effects on F usarium solani root colonization and sudden death
syndrome. Agron. J. 95:1140-1 145.

Pankey, J. H., J. L. Grifﬁn, P. D. Colyer, R. W. Schneider, and D. K. Miller. 2005.
Preemergence herbicide and glyphosate effects on seedling diseases in glyphosate-
resistant cotton. Weed Technol. 192312-318.

Pline-Srnic, W. 2005. Technical performance of some commercial glyphosate-resistant
crops. Pest Manag. Sci. 61:225-234.

Ruppel, E.G., C.L. Schneider, R.J. Hecker, and G.J. Hogaboam. 1979. Creating

epiphytotics of Rhizoctonia root rot and evaluating for resistance to Rhizoctonia solani
in sugarbeet ﬁeld plots. Plant Dis. Rep. 63:518-522.

51

Sanogo, S., X. B. Yang, and H. Scherm. 2000. Effects of herbicides on Fusarium solani
f. sp. glycines and development of sudden death syndrome in glyphosate-tolerant
soybean. Phytopathol. 90:57-66.

Sanogo, S., X. B. Yang, and P. Lundeen. 2001. Field response ofglyphosate-tolerant
soybean to herbicide and sudden death syndrome. Plant Dis. 85:773-779.

Siehl, D. L. 1997. Inhibitors of EPSP synthase, glytamine synthase and histidine
synthesis. Pages 37-67 in R. M. Roe, ed. Herbicide activity: toxicology,
biomchemistry and molecular biology. Amsterdam, Netherlands: IOS Press.

Wilson, R.G. 1994. New herbicides for postemergence application in sugarbeet (Beta
vulgaris). Weed Technol. 82307-311.

Wilson, R.G. 1995. Response of sugarbeet, common sunﬂower, and common cocklebur
to clopyralid or desmedipham plus phenmedipham. J. Sugar Beet Res. 32:89-97.

Windels, C. E., B. J. Jacobsen, and R. M. Harveson. 2009. Rhizoctonia root and crown

rot. Pages 33-36 in R. M. Harveson, L. E. Hanson, and G. L. Hein, eds. Compendium '
of Beet Diseases and Pests, Second edition. St. Paul, MN: APS Press.

52

CHAPTER 3
INFLUENCE OF GLYPHOSATE AND F UNGICIDE TREATMENTS ON
RHIZOCTONIA CROWN AND ROOT ROT IN GLYPHOSATE-RESISTANT

SUGARBEET

Abstract: A ﬁeld experiment was conducted in 2008 and 2009 in the Saginaw Valley
region of Michigan to determine if there were potential interactions between applications
of glyphosate and the fungicide azoxystrobin and to determine the effectiveness of foliar
and in-ﬁirrow azoxystrobin applications when Rhizoctonia solani is present. Signiﬁcant
differences in disease indices, percentage ofharvestable sugarbeet, and percentage of
healthy sugarbeet were evident among the different varieties and fungicide treatments of
azoxystrobin, but herbicide treatment did not signiﬁcantly affect these parameters.
Hilleshbg 9027RR and HilleshOg 9029RR had the lowest disease indices and highest
percentage of healthy sugarbeet when compared with Crystal RR827 and HilleshOg
9028RR. When compared with the iii-furrow application or no fungicide treatment, foliar
fungicide applications of azoxystrobin resulted in the lowest disease index (2.0) and
highest percentage of healthy sugarbeet (42 %). In-ﬁirrow ﬁingicide application of
azoxystrobin reduced the disease index when compared with no fungicide application.
Similar trends were observed for harvestable sugarbeet, except for Crystal RR827 where
there was not a signiﬁcant difference between the in-furrow azoxystrobin application and
no fungicide treatment. HilleshOg 9027RR and Hilleshbg 9029RR exhibited the most
tolerance to Rhizoctonia crown and root rot. Hilleshd g 9028RR appeared to be

moderately tolerant and Crystal RR827 was the most susceptible of the four glyphosate-

53

 

resistant sugarbeet varieties. Foliar fungicide applications of azoxystrobin resulted in the
lowest disease index and highest percentage of healthy and harvestable sugarbeet when
compared with the in-furrow application or no ﬁrngicide treatment. Glyphosate did not
affect the efficacy of fungicide treatments, but choosing a Rhizoctonia-tolerant variety
and applying foliar fungicide applications appear to the best methods for managing
Rhizoctonia crown and root in glyphosate-resistant sugarbeet.

Nomenclature: Glyphosate; standard-split; azoxystrobin; Rhizoctonia crown and root
rot, Rhizoctonia solani Kiihn; sugarbeet, Beta vulgaris L.

Key words: Glyphosate-resistant crops; disease index; healthy sugarbeet; harvestable

sugarbeet

Introduction

Glyphosate is the most widely used herbicide in the world due to its ability to
control a broad spectrum of annual and perennial broadleaf and grass weed species (Duke
and Powles 2008; Pline-Srnic 2005). The introduction of glyphosate-resistant crops in
1996 changed the way many growers approach weed management. Growers widely
adopted glyphosate-resistant crops because glyphosate made weed control easier and
more effective with fewer applications, reduced the need for tillage, did not restrict crop
rotations, and increased proﬁtability (Green 2009). Currently, there are six
commercialized glyphosate-resistant crops: soybean [Glycine max (L.) Merr], corn (Zea
mays L.), cotton (Gossypium hirsutum L.), canola (Brassica napus L.), alfalfa (Medicago
sativa L.) and, most recently in 2008, sugarbeet (Beta vulgaris L.). Glyphosate-resistant

sugarbeet varieties were quickly adopted by growers in Michigan. Approximately 98%

54

of Michigan’s sugarbeet hectares were planted with a glyphosate-resistant variety in 2009
(C. Guza, Agronomist, Michigan Sugar Company, Bay City, MI, personal
communication).

The use of glyphosate in glyphosate-resistant sugarbeet provides growers the
opportunity to achieve excellent control of many weed species that can affect sugarbeet
yield and quality (Kniss et al. 2004). Conventional postemergence (POST) herbicides do
not effectively control weeds with more than two leaves, so many herbicide applications
are necessary and seldom result in 100% control (Dale et al. 2006; Dale and Renner
2005). Additionally, the time between herbicide applications in glyphosate-resistant
sugarbeet may be longer when compared with conventional sugarbeet herbicide
pro grams, because weed height at the time of application is generally not as limiting with
glyphosate. Kemp et al. (2009) determined that fewer herbicide applications were
required to improve weed control and yields in glyphosate-resistant sugarbeet. Growers
can also adjust production practices, such as narrowing row width, to obtain higher yields
and therefore greater economic returns despite the additional seed costs associated with
using glyphosate-resistant sugarbeet varieties (Armstrong 2009). Glyphosate is less
expensive when compared with conventional sugarbeet weed control programs and the
potential for greater economic return is possible with fewer herbicide applications,
improved weed control, and increased yields (Kniss et a1. 2004).

However, one potential issue with glyphosate-resistant sugarbeet is the possible
increase in diseases caused by soil-borne pathogens. Glyphosate inhibits the 5-
enolpyruvylshikimate—3-phosphate synthase (EPSPS) enzyme, an important component

in the shikimate acid pathway. This pathway produces aromatic amino acids and

55

 

A" “I“:

secondary compounds important for plant growth and protection (Amrhein et a1. 1980;
Bentley 1990; Dill 2005; Siehl 1997). While glyphosate-resistant crops have a form of
the EPSPS enzyme that is not affected by glyphosate, this enzyme may not be as efﬁcient
as native EPSPS when exposed to glyphosate and therefore may result in reduced
production of secondary compounds (Pline-Srnic 2005).

Studies in glyphosate-resistant crops, including glyphosate-resistant sugarbeet,
have indicated a potential for increased susceptibility to some soil-borne pathogens after
glyphosate was applied (Larson et a1. 2006; Sanogo et al. 2000; Sanogo et al. 2001).
Greenhouse and ﬁeld studies with glyphosate-resistant soybean showed that these plants
were more susceptible to sudden death syndrome, caused by Fusarium solani (Mart.)
Sacc. f. sp. glycines, after glyphosate was applied (Sanogo et al. 2000; Sanogo et al.
2001). Larson et al. (2006) determined that experimental varieties of glyphosate-resistant
sugarbeet were more susceptible to certain isolates of both Rhizoctonia solani Kiihn and
Fusarium oxysporum Schlecht. f sp. betae Snyd. & Hans. after exposure to glyphosate.

In contrast, other studies demonstrated that glyphosate applications had no effect,
or reduced the severity of diseases caused by soil-borne pathogens (N jiti et al. 2003;
Pankey et al. 2005). In glyphosate-resistant soybean, Njiti et al. (2003) determined
glyphosate had no effect on soybean yield or disease severity of sudden death syndrome.
These results conﬂicted with greenhouse and ﬁeld results reported by Sanogo et al.
(2000) and (2001). There were differences between these studies concerning variety
selection and varietal response to the disease. In addition, there were differences in
environmental factors such as planting date, genotype, and other soil factors. This may

explain why glyphosate has no effect on F. solani disease severity in certain varieties, but

56

.ns.
\

 

 

increases disease severity in others. In glyphosate-resistant cotton, greenhouse

 

 

experiments conducted by Pankey et al. (2005) showed that glyphosate had no effect on
damping off or soreshin (caused by the pathogen R. solani). Furthermore, in the ﬁeld,
glyphosate actually reduced Rhizoctonia induced disease severity.

Rhizoctonia crown and root rot, caused by the soil-borne pathogen Rhizoctonia
solani, is a problematic disease in many crops in Michigan, including sugarbeet (Kirk et
al. 2008; Windels et a1. 2009). Rhizoctonia crown and root rot reduces economic returns
in sugarbeet by as much as 24% in the United States and up to 50% yield loss may result,
depending on disease severity (Franc et al. 2001; Windels et al. 2009). The ﬁrst
symptoms of Rhizoctonia crown and root rot are foliar, consisting of a permanent wilting
of leaves and dark lesions at the base of the petiole or on the crown of the beet.
Sugarbeet leaves become dry and collapse, but remain attached to the crown and form a
dry, dark rosette. Root symptoms include dark lesions that begin anywhere on the root,
but may grow together and cover the entire root surface as the disease progresses. Root
tissue is typically ﬁrm underneath these lesions. However, root tissue will begin to soften
underneath these lesions and cracks may also develop in advanced stages of the disease.

Rhizoctonia solani has many host crops in addition to sugarbeet, which makes it
difﬁcult to control with crop rotation alone (Rush and Winter 1990; Schuster and Harris
1960). Soybean, dry bean (Phaseolus vulgaris L.), corn, and many weed species are
alternate hosts for Rhizoctonia, further increasing the buildup of disease inoculum
(Windels et al. 2009). The availability of sugarbeet varieties tolerant to Rhizoctonia
crown and root rot provides an additional option to manage this disease, and varieties

with varying levels of tolerance are readily available to Michigan sugarbeet growers.

57

Although these varieties do not completely prevent infection, they certainly limit fungal

 

colonization and disease severity (Ruppel 1973).

Additional methods for controlling Rhizoctonia crown and root rot in sugarbeet
include applications of strobilurin ﬁingicides, such as azoxystrobin. Single ﬁrngicide
treatments are typically applied either in-furrow at planting or postemergence (POST) to
sugarbeet at the 4- to 8-leaf stage (Karaolglanidis and Karadimos 2006; Whitney and
Duffus 1986). In-furrow applications of azoxystrobin can reduce infection early in the
season, but may not prevent later infections (Karaoglanidis and Karadimos 2006;
Kiewnick et al. 2001; Jacobsen et. al. 1999; Windels and Brantner 2000). If glyphosate-
resistant sugarbeet are more susceptible to plant pathogens after glyphosate is applied,
then ﬁrngicide applications may be important in controlling sugarbeet diseases such as
Rhizoctonia crown and root rot. Therefore, the objectives of this research were to: I)
investigate potential interactions between glyphosate and fungicide applications of
azoxystrobin on management of Rhizoctonia crown and root rot in four glyphosate-
resistant sugarbeet varieties, and 2) determine the effectiveness of in-ﬁirrow and foliar

applications of azoxystrobin when Rhizoctonia solani is present.

Materials and Methods
A ﬁeld experiment was conducted in 2008 and 2009 in the Saginaw Valley region
of Michigan. The 2008 experiment was located in St. Charles, Michigan on a Misteguay
silty clay (ﬁne, mixed, semiactive, calcareous, mesic Aerie Endoaquepts) soil with a pH
of 7.8 and 3.0 % organic matter. The 2009 experiment was located in Frankenmuth,

Michigan and the soil type was a Tappan-Londo complex (ﬁne-loamy, mixed, active,

58

calcareous, mesic Typic Endoaquolls) with a pH of 7.7 and 2.4 % organic matter.
Experiments followed dry bean in both 2008 and 2009. Fields were fall-chisel plowed
followed by spring ﬁeld cultivation twice prior to planting. Fertilizer applications were

standard for sugarbeet production in Michigan. The glyphosate-resistant sugarbeet

varieties Crystal RR827], Hilleshég 902711122, Hilleshbg 9028RR, and Hilleshbg

9029RR were planted 2.5-cm deep in 76-cm rows at a population of 122,000 seeds/ha on
April 25, 2008 and April 16, 2009. Plots were six rows wide by 9.1 m in length. Each
variety was planted, one per row, in rows two through ﬁve. Rows one and six served as
border rows. Commercial sugarbeet varieties selected for this experiment were approved
by Michigan Sugar Company and were thought to have varying degrees of Rhizoctonia
crown and root rot tolerance.

The experimental design was a split-strip-plot with four replications. The main
plot was herbicide treatment, the sub-plot was fungicide treatment, and the strip-plot was
variety. Herbicide treatments consisted of a glyphosate program, a standard-split

program (used in conventional sugarbeet), and a hand-weeded control (no herbicide).

The glyphosate program consisted of glyphosate3 at 0.84 kg ae/ha plus ammonium

sulfate at 2% v/v, applied three times at 2- to 4-leaf, 4- to 6-1eaf, and 6- to 8-leaf
sugarbeet. The standard-split program consisted of a combination ofdesmedipham at
180 g/ha plus phenmedipham4 at 180 g ai/ha, triﬂusulﬁiron5 at 9 g ai/ha, clopyralid6 at
104 g ai/ha, and non-ionic surfactant at 0.25% v/v, applied twice when sugarbeet was at

the cotyledon to 2-leaf and 2- to 4-leaf stages. The rates of desmedipham plus

phenmedipham were each increased to 270 g ai/ha for the second application. All plots

59

were maintained weed-ﬂee by hand-weeding throughout the growing season. Plots were

rated for herbicide injury 7 days after the last herbicide application timing. Fungicide

treatments consisted of azoxystrobin7 applied in-ﬁirrow at planting at 140 mg ai/m of

row, foliar applications of azoxystrobin at 0.82 kg ai/ha to 4- to 6-leaf sugarbeet, and a
no-fungicide control. Foliar applications of azoxystrobin were tank-mixed and applied
with glyphosate for the glyphosate program POST herbicide and fungicide treatments

were applied with a tractor-mounted compressed-air sprayer calibrated to deliver 178

L/ha at 207 kPa through 10003 AirMix8 nozzles. Nozzles were spaced 51 cm apart and

were positioned approximately 56 cm above the sugarbeet canopy.

All plots were inoculated with R. solani AG-2-2-IIIB when sugarbeet was at the
6- to 8-leaf stage. Subgroup AG-2-2-IIIB is the most common and virulent R. solani
subgroup found in Michigan (Kirk et al. 2008). R. solani inoculum was produced in bulk
on a barley medium. Pans of barley, saturated with water, were autoclaved and 9 plugs
(7 mm) of R. solani grown on potato dextrose agar were placed into the pans. The pans
were sealed with Paraﬁlm and incubated at 25 C i 2 for 3 wk. Once the barley was

colonized, it was air dried and ground into a ﬁne ﬂour. Inoculum was applied directly

over each sugarbeet row at 2 g/m using a modiﬁed drop spreader.9 Rate was conﬁrmed

by determining the amount of leftover inoculum and calculating the kg applied per m of
row. All plots were cultivated following inoculation to put soil and inoculum in the
crown for increased disease severity (Ruppel et a1. 1979).

Sugarbeet stand counts were recorded for each variety at 4 wk after planting and

at harvest. Approximately 8 wk after inoculation, sugarbeet were lifted from the soil

60

 

. . . 10 . .
usrng a modiﬁed lift harvester. Each sugarbeet root was rated for disease severity

 

using the 0 to 7 Rhizoctonia crown and root rot rating scale as follows: 0 -— no visible
signs of disease; 1 = inactive lesions; 2 = less than 5 % active lesions; 3 = 6 to 25 % Of
the root rotted; 4 = 26 to 50% ofthe root rotted; 5 = 51 to 75 % ofthe root rotted; 6 =
greater than 75 % of the root rotted, but still some living tissue; 7 = roots completely
rotted and dead (Ruppel et al. 1979). Stand counts were used to determine how many
sugarbeet were missing from each plot due to advanced disease severity. Values were
adjusted by assigning each of the missing sugarbeet a disease severity rating of 7. An
average disease index was determined for each variety in each plot. The disease index
was calculated as a weighted average based on the number of sugarbeet in each of the
eight disease classes (Ruppel et al. 1979). Healthy sugarbeet were determined by
calculating the percent of sugarbeet that had a disease severity rating of 0 or 1.
Harvestable sugarbeet were determined by calculating the percent of sugarbeet with a
disease severity rating of 3 or less.

Precipitation data was recorded by weather stations operated by the Michigan
Automated Weather Network11 (Table 9). Weather stations were located within 3 km of
the experimental locations.

. . . 12
Data were analyzed usmg the PROC MIXED procedure in SAS 9.1. An

. analysis of variance was performed and treatment means for disease index, percent of
healthy sugarbeet, and percent of harvestable sugarbeet were compared using Fisher’s
Protected LSD at the p 5 0.05 signiﬁcance level. Interactions between main effects were

analyzed using the SLICE option in the LSMEANS statement. Data were combined

61

across year, variety, herbicide treatment, or fungicide treatment when interactions were

 

not signiﬁcant.

Results and Discussion
Herbicide Injury. The glyphosate-resistant sugarbeet varieties did not show visible
signs of damage from glyphosate treatments. However, applications of the standard-split
herbicide program uniformly caused 13% injury for each of the four glyphosate-resistant
sugarbeet varieties evaluated (data not shown). Injury symptoms consisted of yellowing
and stunting when compared with the non-treated control, which are consistent with what
others have observed with this combination (Wilson 1994, 1995). Approximately 2 wks
after this evaluation sugarbeet recovered from this damage. In-furrow or foliar
applications of azoxystrobin neither signiﬁcantly increased nor decreased herbicide
injury. An increase in herbicide injury was a potential concern with the glyphosate and
azoxystrobin tank-mixture, since previous research has indicated an increase in sugarbeet
injury ﬁom tank-mixtures of azoxystrobin and other sugarbeet herbicides (Sprague et al.

2005).

Effect 01' Variety, Herbicide, and Fungicide on Rhizoctonia Crown and Root Rot.
Rhizoctonia solani subgroup AG—2-2-IIIB inoculation was highly effective. The
combination of cultivation and precipitation (Table 9) following Rhizoctonia inoculation
resulted in an average disease index of 5.9 in the non-fungicide controls which provided a

good basis for treatment separation. Natural R. solani infestations were low each year

62

based on disease index (2 or less) evaluations taken in adjacent non-inoculated sugarbeet
plots.

Interactions between the years were not signiﬁcant, therefore all data are
presented as a combination of the 2008 and 2009 experiments. The three-way interaction
of variety x herbicide x fungicide was not signiﬁcant for any of the parameters evaluated
(Table 10). All two-way interactions were not signiﬁcant for any of the parameters
measured, except for the variety x fungicide interaction for the percentage of harvestable
sugarbeet. Therefore, data are discussed as the main effects of variety, herbicide, and
fungicide, except for the variety x ﬁingicide interaction for the percentage of harvestable

sugarbeet.

Variety. There was a difference in how the four glyphosate-resistant sugarbeet varieties
responded to inoculation of R. solani. Averaged across herbicide and fungicide
treatments, Hilleshdg 9027RR and Hilleshég 9029RR were the most tolerant varieties to
R. solani subgroup AG-2-2-IIIB with disease index evaluations of 3.6 and 3.7,
respectively (Table 11). Crystal RR827 was the most susceptible glyphosate-resistant
variety with a disease index of 4.7 and HilleshOg 9028RR showed moderate tolerance
with a disease index of 4.0. The percentage of healthy sugarbeet, based on disease
severity ratings of 1 or less, followed a similar trend. The percentage of healthy
sugarbeet was less than 25% for all four glyphosate-resistant sugarbeet varieties.
However, the percentage of healthy sugarbeet for the most susceptible variety, Crystal
RR827 (14%), was considerably lower than the more Rhizoctonia tolerant varieties,

HilleshOg 9027RR and Hilleshéig 9029RR (Table l 1). As observed with other studies,

63

 

varieties have varying levels of Rhizoctonia crown and root rot susceptibility and
tolerance (Ruppel 1973). Although HilleshOg 9027RR and Hilleshbg 9029RR do not
completely prevent R. solani infection, they exhibited more tolerance and are more

effective at managing Rhizoctonia crown and root rot when compared with Crystal

RR827.

Herbicide. One of the objectives was to determine if there were interactions between
glyphosate and fungicide applications on Rhizoctonia crown and root rot. There were no
signiﬁcant interactions with herbicide and the main effect of herbicide was not signiﬁcant
(Table 10). This indicated that glyphosate had no inﬂuence on the disease index, the
percentage of harvestable sugarbeet, or percentage of healthy sugarbeet when compared
with the standard-split or no herbicide treatments. This is in contrast to what Larson et al.
(2006) observed in greenhouse experiments with non-commercial glyphosate-resistant
sugarbeet varieties. Their results indicated that a glyphosate-resistant sugarbeet variety
with tolerance to Rhizoctonia crown and root rot demonstrated increased susceptibility to
the disease after glyphosate was applied. The increased disease severity did not appear to
be a fungal response because there was not a signiﬁcant difference in the growth rate of
Rhizoctonia solani or in the production of sclerotia after exposure to glyphosate. They
concluded that differences in disease severity were explained by a particular cultivar or
isolate pathogen response. Only one of the glyphosate-resistant varieties demonstrated a
signiﬁcant increase in disease severity with AG-2-2-IIIB (not AG-2-2-IV) after
glyphosate application. In addition, other studies suggest the timing of glyphosate

application in relation to disease infection is important. In our ﬁeld experiment,

64

 

sugarbeet were inoculated days after the last herbicide application. However, if sugarbeet
were inoculated prior to herbicide applications, it may have inﬂuenced disease severity
differently than what was observed in our study. Experiments with glyphosate-resistant
wheat (T riticum aestivum L.) have indicated that glyphosate actually decreased disease
severity of leaf rust (caused by the pathogen Puccinia triticina) and stem rust fungus
(cause by the pathogen Puccinia graminis f. sp. tritici Eriks) when exposed to glyphosate
21 d to 35 d after inoculation (Anderson and Kolmer 2005). This may explain why
differences between herbicide treatments were not observed in our ﬁeld experiment,
while greenhouse studies by Larson et al. (2006) indicated that glyphosate applications
increased disease severity.

Herbicides may synergize or antagonize fungicide activity against different
diseases in different crops. Kataria and Gisi (1990) found that when used alone in wheat,
the herbicides DNOC, dicamba, ioxynil, and bromoxynil had a low to moderate effect on
reducing the disease severity of Rhizoctonia cerealis Van der Hoeven and
Pseudocercosporella herpotrichoides (Fron) Deighton. However, herbicide
combinations with the fungicide cyproconazole were synergistic and effective in reducing
the disease severity. Hill and Stratton (1991) concluded from in vitro tests, that the
herbicide metribuzin, when used in combination with the ﬁmgicide chlorothalonil, was
antagonistic and reduced efficacy on Altemaria solani (Ell. And Martin) Sor. Unlike
these examples, the herbicide treatments in our ﬁeld trial did not synergize or antagonize

Rhizoctonia crown and root rot management with azoxystrobin.

65

 

Fungicide. The main effect of fungicide was signiﬁcant for Rhizoctonia disease indices

.4}

and the percentage of healthy sugarbeet (Table 10). Combined across all varieties and
herbicide treatments, foliar application of azoxystrobin to 4— to 6- leaf sugarbeet provided
the greatest suppression of Rhizoctonia crown and root rot (Table 12). Foliar
applications of azoxystrobin resulted in a disease index rating of 2.0 and 42% of the
sugarbeet were considered healthy (disease severity rating of one or less). This was in
contrast to the no ﬁingicide treatment where the disease index rating was 4.0 and only 1%
of the sugarbeet were considered healthy. In-furrow applications of azoxystrobin also
provided some protection against Rhizoctonia crown and root rot. However, in-furrow
applications were not as effective as foliar applied azoxystrobin (Table 12). Others have
reported that in-furrow applications of azoxystrobin were just as effective as foliar
applications to 4- to 6-leaf sugarbeet in reducing Rhizoctonia crown and root rot (Kirk et
al. 2008). Differences in the results of our experiment may be related to the timing of R.
solani inoculation, which occurred when sugarbeet was at the 6- to 8-leaf stage. In-
ﬁirrow azoxystrobin applications may be more effective against earlier infections of R.
solani and may not last long enough to prevent later infections. In addition, method of
fungicide application may also have inﬂuenced fungicide efﬁcacy. In-furrow
applications were banded onto the rows; therefore soil in between rows would not have
been treated. Foliar applications were broadcast applied to the sugarbeet in the rows, as
well as soil in between the rows, and this may have been more effective in reducing
disease severity of Rhizoctonia crown and root rot. Several studies indicate that
environmental factors inﬂuence ﬁrngicide efficacy, therefore variations in temperature

and moisture may explain differences between in-furrow and foliar applications.

66

Previous studies indicated that fungicide treatments applied between 18 and 21 C are
optimal for disease management, therfore later fungicide application timings are more
effective with cool, spring temperatures (Jacobsen et al. 2004; Poindexter 2010). In
addition, Stump et al. (2004) determined that ﬁmgicide treatments applied at the time of
inoculation resulted in the lowest disease severity and that treatments (in- furrow) applied
at planting were too early for optimal control of Rhizoctonia crown and root rot in

sugarbeet.

Harvestable Sugarbeet. There was a ﬁrngicide by variety interaction for the percentage
of harvestable sugarbeet. Sugarbeet that were considered harvestable have a disease
severity rating of 3 or less, which means that less than 25% of the sugarbeet is rotted and
there are no deep penetrating cracks. Regardless of variety, fewer than 20% of sugarbeet
were harvestable when a fungicide was not applied (Table 13). In-furrow and foliar
applications of azoxystrobin increased the number of harvestable sugarbeet for all
varieties, excluding the in-furrow azoxystrobin treatment on the most susceptible variety,
Crystal RR827. A foliar application of azoxystrobin was the only treatment that
improved the percentage of harvestable sugarbeet for this variety (73%). In contrast,
HilleshOg 9027RR, HilleshOg 9028RR, and Hilleshbg 9029RR beneﬁted from both in-
furrow and foliar applications of azoxystrobin for the percentage ofharvestable sugarbeet
(Table 13), although the foliar azoxystrobin application resulted in the greatest
percentage of harvestable sugarbeet (88% or greater).

In summary, the four glyphosate-resistant sugarbeet varieties that we investigated

had a range of responses to R. solani. HilleshOg 9027RR and Hilleshdg 9029RR were

67

 

 

most tolerant, HilleshOg 9028RR was moderately tolerant, and Crystal RR827 was the
most susceptible variety to Rhizoctonia crown and root rot. Herbicide treatment, whether
it was the glyphosate program or the standard conventional herbicide program, did not
affect Rhizoctonia crown and root rot development or management in the ﬁeld. This is in
contrast to a greenhouse study by Larson et al. (2006) where applications ofglyphosate
increased the disease severity of Rhizoctonia crown and root rot in a Rhizoctonia—tolerant
glyphosate-resistant sugarbeet variety. Across the four glyphosate-resistant sugarbeet
varieties, a foliar application of azoxystrobin provided the most protection against
Rhizoctonia crown and root rot. However, both foliar and in-furrow applications of
azoxystrobin reduced the disease index and resulted in more healthy and harvestable
sugarbeet than treatments lacking a ﬁrngicide application. The exception was Crystal
RR827, the most susceptible variety to R. solani, where harvestable sugarbeet did not
differ between the in-furrow fungicide treatment and no fungicide application. From this
ﬁeld research, there is no evidence that Michigan sugarbeet growers should be concerned
about the potential for an increase in Rhizoctonia crown and root rot in glyphosate-
resistant sugarbeet when glyphosate is applied. Choosing varieties that exhibit some
tolerance to Rhizoctonia crown and root rot and applying a fungicide like azoxystrobin

will be the key factors to help growers manage this disease.

68

Sources of Materials

1 BetaSeed, Inc., 1788 Marschall Road, Shakopee, MN 55379.
2 Syngenta Seeds Inc., 1020 Sugarmill Rd., Longmont, CO 80501.
3 Roundup WeatherMAX, Monsanto Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167.

4 Betamix, Bayer CropScience AG, Alfred-Nobel-Str. 50, D-40789 Monheim am Rhein,
Germany.

5 UpBeet, E.I. du Pont de Nemours and Co., Crop Protection, 1007 Market St.,
Wilmington, DE 19898.

6 Stinger, Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN 46268.

7 Quadris, Syngenta International AG, PO. Box CH — 4002, Basel, Switzerland
8 AirMix 1 1003, Greenleaf Techno logies, PO. Box 1767, Covington, LA 70434.
9 Candy Company, 528 Gandrud Road, Owatonna, MN 55060.

10 Tractor Supply Company, 200 Powell Place, Brentwood, TN 37027.

1 1 . . .
Michigan Automated Weather Network, Web Site:
http://www.agwcathergccmsu.edu/

12 The SAS System for Windows, Version 9.1, SAS Institute, Inc., 100 SAS Campus Dr.,
Cary NC 27513.

69

Table 9. Monthly precipitationa and the 30-year average for experiments located in the

Saginaw Valley region of Michigan in 2008 and 2009.
Precipitation (mm)

 

 

2008 2009 30 yr.
April 5 1 1 19 72
May 29 3 1 71
June 99 1 22 83
July 1 00 69 70
August 53 88 96
Total 332 429 392

 

a Precipitation data was collected from the Michigan Automated Weather Network
(http://www.agweather.geo.msu.edu/mawn/).

70

 

 

 

 

 

if

Table 10. P-values for main effects and interactions of herbicide and ﬁingicide
treatments on Rhizoctonia solania AG-2-2-IIIB disease index and healthy and harvestable \
sugarbeet of four glyphosate-resistant sugarbeet varieties. Data are combined across 1
years.

Harvestable Healthy

Disease index sugarbeet sugarbeet
p-value

Variety <0.0001 <0.0001 0.0248
Herbicide 0.6361 0.5194 0.9533
Fungicide 0.0003 0.0006 <0.0001
Variety x herbicide 0.9514 0.9729 0.9326
Variety x fungicide 0.4919 0.0045 0.4484
Herbicide x fungicide 0.7364 0.5717 0.5662
Variety x herbicide x ﬁingicide 0.9999 0.9971 0.9966

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

71

Table 11. Disease index ratings and percent healthy sugarbeet of four glyphosate-

. . . . . , . a .
resrstant sugarbeet varieties inoculated wrth Rhizoctonia solani. Data are combined
across herbicide treatments, ﬁrngicide treatments, and years.

 

 

 

 

 

Variety Disease indexb Healthy sugarbeetc
0 to 7 scale %

Hilleshog 9027RR 3.6ad 20a

Hilleshog 9028RR 4.0b l9ab

Hilleshog 9029RR 3.7a 22a

Cgstal RR827 4.7c 14b

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

Disease is rated based on a 0 to 7 scale (0 = no disease and 7 = completely rotted) and

the disease index is calculated by determining a weighted average based on the number
of sugarbeet in each of the eight disease classes.

c Healthy sugarbeet is determined by calculating the percent of sugarbeet that have a
disease severity rating of 0 or 1.

Means followed by the same letter are not signiﬁcantly different according to Fisher’s
Protected LSD at p _<_ 0.05. '

72

Table 12. Disease index ratings and percent healthy sugarbeet for fungicide treatments

applied to glyphosate-resistant sugarbeet inoculated with Rhizoctonia solania Data are
combined across varieties, herbicide treatments, and years.

 

 

 

 

 

Fungicide Rate Disease indexb Healthy sugarbeetc
—— 0 to7 scale %

Foliar azoxystrobin 0.8 kg/ha 2.0ad 42a

In-furrow azoxystrobin 140 gm row 4.0b 13b

No fungicide — 5.90 lo

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

Disease is rated based on a 0 to 7 scale (0 = no disease and 7 = completely rotted) and
the disease index is calculated by determining a weighted average based on the number
of sugarbeet in each of the eight disease classes.

0 Healthy sugarbeet is determined by calculating the percent of sugarbeet that have a
disease severity rating of 0 or 1.

d Means followed by the same letter are not signiﬁcantly different according to Fisher’s
Protected LSD at p 5 0.05.

73

Table 13. Percent harvestablea sugarbeet for fungicide treatment applied to four

glyphosate-resrstant sugarbeet varieties inoculated wrth Rhizoctonia solani. Data are
combined across herbicide treatments and years.

 

 

 

 

 

Fungicide
Variety Foliar azoxystrobin ln-ﬁirrow azoxystrobin No ﬁmgicide
%
Hilleshog 9027RR 95a° 62bcd 15f
Hilleshog 9028RR 88abc 46c 9f
Hilleshog 9029RR 92ab 57cd 12f
Crystal RR827 73de 25 fg 2g

 

a Harvestable sugarbeet is determined by calculating the percent of sugarbeet that have a
disease severity rating of 3 or less.

Rhizoctonia solani inoculum was prepared with a barley medium

c Means followed by the same letter are not signiﬁcantly different according to Fisher’s
Protected LSD at p _<_ 0.05.

74

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77

Appendix A: Additional Parameters for Greenhouse Experiment 1

Fresh root weight and dry weight were also determined for sugarbeet in
greenhouse Experiment 1. Fresh root weights were determined by dividing the ﬂesh root
weight of the Rhizoctonia-inoculated plants by the ﬂesh root weight ofnon-inoculated
plants for each treatment. Dry weights were determined by dividing the dry weight of the
Rhizoctonia-inoculated plants by the dry weight of non-inoculated plants for each
treatment. Fresh root weight and dry weight are presented as a percent of the non-
inoculated. Data were combined across experiments when interactions were not
signiﬁcant.

Fresh root weight and dry weight followed a similar trend to disease severity and
ﬂesh plant weight. Inoculation with R. solani AG-2-2-IIIB was signiﬁcant and the
average disease severity for plants that were inoculated was 4.2. Non-inoculated plants
were removed for further analysis. In Hilleshbg 9027RR, glyphosate at 1.68 kg ae/ha
reduced ﬂesh root weight when compared with the nO-herbicide control (Table 14).
However glyphosate did not affect ﬂesh root weight in Hilleshbg 9028RR. In Hilleshbg
9032RR glyphosate at 0.84 kg ae/ha and 1.68 kg ae/ha did not reduce ﬂesh root weight
when compared with the no-herbicide control, but did signiﬁcantly reduce ﬂesh root
weight when compared with the standard conventional program. In Hilleshdg 9027RR,
glyphosate at 1.68 kg ae/ha also reduced dry weight when compared with the no-
herbicide control (Table 15). Glyphosate did not affect dry weight in Hilleshbg 9028RR,
However, in Hilleshbg 9032RR glyphosate applied at 0.84 kg ae/ha increased dry weight

when compared with the no—herbicide control.

78

Table 14. Fresh root weights of three glyphosate-resistant sugarbeet varieties exposed to

Rhizoctonia solani“ isolate AG-2-2-IIIB in the presence and absence of herbicides
(Experiment 1).

 

 

 

 

Herbicide treatment H 9027RR H 9028RR H 9032RR
% of non-inoculatedc

No herbicide 78abd 44c 34c

Standard conventional programb 57bC 320 97a

Glyphosate (0.84 kg ae/ha) 52bc 56bc 52bc

Glyphosate (1.68 kg ae/ha) 33c 51bc 56bc

 

a Rhizoctonia solani inoculum was prepared with a millet medium

b The standard conventional herbicide program included phenmedipham at 270 g ai/ha
plus desmedipham at 270 g ai/ha, triﬂusulfuron at 9 g ai/ha, and clopyralid at 104 g
ai/ha.

c Fresh root weights were determined by dividing the ﬂesh root weight of the
Rhizoctonia-inoculated plants by the ﬂesh root weight of non-inoculated plants for each
treatment.

d Means followed by the same letter are not different according to Fisher’s Protected
LSD atp 5 0.05.

79

Table 15. Dry weights of three glyphosate-resistant sugarbeet varieties exposed to
Rhizoctonia solania isolate AG-2-2-IIIB in the presence and absence of herbicides

 

 

 

 

(Experiment 1).

Herbicide treatment H 9027RR H 9028RR H 9032RR
% ofnon-inoculatedC

No herbicide 85abd 59bcd 48d

Standard conventional programb 75abcd 52cd 91a

Glyphosate (0.84 kg ae/ha) 59bcd 70abcd 77abc

Glyphosate (1.68 kg ae/ha) 52cd 64abcd 65abcd

 

Rhizoctonia solani inoculum was prepared With a rmIIet medium.

b The standard conventional herbicide program included phenmedipham at 270 g ai/ha
plus desmedipham at 270 g ai/ha, triﬂusulfuron at 9 g tha, and clopyralid at 104 g
ai/ha.

c Dry weights were determined by dividing the dry weight of the Rhizoctonia-inoculated
plants by the dry weight of non-inoculated plants for each treatment.

Means followed by the same letter are not different according to Fisher’s Protected
LSD atp g 0.05.

80

Appendix B: Additional Parameters for Greenhouse Experiment 2

Fresh root weight and dry weight were also determined for sugarbeet in
greenhouse Experiment 2. Fresh root weights were determined by dividing the ﬂesh root
weight of the Rhizoctonia-inoculated plants by the ﬂesh root weight of non-inoculated
plants for each treatment. Dry weights were determined by dividing the dry weight of the
Rhizoctonia-inoculated plants by the dry weight ofnon-inoculated plants for each
treatment. Fresh root weight and dry weight are presented as a percent of the non-
inoculated. Data were combined across experiments and herbicide treatments when
interactions were not signiﬁcant.

Fresh root weight and dry weight followed a similar trend to disease severity and
ﬂesh plant weight. Adequate moisture in the greenhouse following Rhizoctonia
inoculation resulted in an average disease severity rating of 5.9. Non-inoculated plants
were removed for ﬁirther analysis. In this greenhouse experiment, herbicide treatment
did not inﬂuence ﬂesh root weight or dry weight. Therefore, data are combined across
herbicide treatment. Hilleshbg 9028RR had the highest ﬂesh root weight and dry weight
when compared with Hilleshdg 9027RR and Crystal RR827 (Table 16). HilleshOg
9029RR also had a higher ﬂesh root weight and dry weight than Crystal RR827, but was

not signiﬁcantly different ﬂom Hilleshdg 9027RR for these parameters.

81

Table 16. Response of four glyphosate-resistant sugarbeet varieties to Rhizoctonia

solani“ isolate AG-2-2-IIIB in greenhouse Experiment 2. Data are combined over
herbicide treatments since there was not a signiﬁcant variety by herbicide interaction.

 

 

Variety Fresh root weightb Dry weightc
'—% of non-inoculated“ —% ofnon-inoculated_
Hilleshog 9027RR 23bcd 4lbc
Hilleshog 9028RR 39a 55a
Hilleshog 9029RR 30ab 45ab
gystal RR827 11c 33c

 

a Rhizoctonia solani inoculum was prepared with a barley medium

b Fresh root weight is determined by weighing the root and dividing that weight by the
weight of the same un-inoculated treatment.

0 Dry weight is determined by weighing the whole plant and dividing that weight by the
dry weight of the same un-inoculated treatment

Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 5 0.05.

82

Appendix C: Response of glyphosate-resistant sugarbeet to R. solani AG—2-2-IV
An additional greenhouse experiment was conducted to determine the response of
ﬁve glyphosate-resistant sugarbeet varieties to R. solani AG-2—2-IV. Similar methods for

previous greenhouse studies were also used in this experiment. Factors included R.
solani inoculation (inoculated or n0n_;n0culated), sugarbeet variety (HilleshOg 9027RR,

Hilleshdg 9028RR, Hilleshbg 9029RR, Hilleshbg 9032RR, and Crystal RR827), and
herbicide treatment. Herbicide treatments consisted of two rates of glyphosate (0.84 and
1.68 kg ae/ha) plus ammonium sulfate at 2% v/v, a standard conventional sugarbeet
herbicide mixture (phenmedipham at 270 g/ha plus desmedipham at 270 g/ha,
triﬂusulfuron at 9 g/ha, and clopyralid at 104 g/ha), and a nO-herbicide control. Disease
severity ( 0 to 7 scale), ﬂesh plant weight, ﬂesh root weight, and dry weight were the
parameters tested. Data were combined across experiments and herbicide treatments
when interactions were not signiﬁcant.

Adequate moisture in the greenhouse following Rhizoctonia inoculation resulted
in an average disease severity rating of 2. l. Non-inoculated plants were removed for
further analysis. In this greenhouse experiment, herbicide treatment did not inﬂuence
ﬂesh root weight or dry weight. Therefore, data are combined across herbicide treatment.
HilleshOg 9032RR had the lowest disease severity when compared with all other varieties
(Table 17). Crystal RR827 was the most susceptible to Rhizoctonia crown and root rot
and the highest disease severity when compared with the four HilleshOg varieties.
However, there were no signiﬁcant differences for ﬂesh plant weight. For ﬂesh root
weight and dry weight, Crystal RR827 had the lowest weights when compared with the

HilleshOg 9027RR, HilleshOg 9028RR, and Hilleshbg 9032RR varieties (Table 18).

83

 

 

Table l 7. Response of ﬁve glyphosate-resistant sugarbeet varieties to Rhizoctonia

solania isolate AG-2-2-IV in the greenhouse. Data are combined over herbicide
treatments since there was not a signiﬁcant variety by herbicide interaction.

 

 

 

Variety Disease severity Fresh weightC
—disease severity (0-7 scale)— —% ofnon-inoculated—

Hilleshog 9027RR 2.1bd 47a

Hilleshog 9028RR 2.3b 48a

Hilleshog 9029RR 2.3b 483

Hilleshog 9032RR 1.7a 47a

Crystal RR827 2.1c 46a

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

b Sugarbeet roots were rated for disease severity on a 0 to 7 scale (0 = no disease and 7 =
completely rotted).

0 Fresh whole weight is determined by weighing the whole plant and dividing that weight
by the weight of the same un-inoculated treatment.

Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 5 0.05.

84

Table 18. Fresh root weight and dry weight of ﬁve glyphosate-resistant sugarbeet
varieties to Rhizoctonia solania isolate AG-2-2-IV in the greenhouse. Data are combined
over herbicide treatments since there was not a signiﬁcant variety by herbicide
interaction.

 

 

Variety Fresh root weightb Dry weightc
—% of non-inoculated_ —°/o ofnon-inoculated—
Hilleshog 9027RR 97ad 95a
Hilleshog 9028RR 96a 93a
Hilleshog 9029RR 92bc 91 ab
Hilleshog 9032RR 9Sab 95a
Crystal RR827 90c 86b

 

 

a Rhizoctonia solani inoculum was prepared with a barley medium.

b Fresh root weight is determined by weighing the root and dividing that weight by the
weight of the same un-inoculated treatment.

C Dry weight is determined by weighing the whole plant and dividing that weight by the
dry weight of the same un-inoculated treatment
Means within each column followed by the same letter are not different according to
Fisher’s Protected LSD at p 5 0.05.

85