Retrospective Theses and Dissertations
2008
Evaluation of Serbian commercial corn hybrid
tolerance to feeding by larval western corn
rootworm (Diabrotica virgifera virgifera LeConte)
using the novel difference approach
Stephanie Rose Kadličko
Iowa State University
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(Diabrotica virgifera virgifera LeConte) using the novel difference approach" (2008). Retrospective Theses and Dissertations. Paper
15421.
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Evaluation of Serbian commercial corn hybrid tolerance to feeding by larval western
corn rootworm (Diabrotica virgifera virgifera LeConte) using the novel difference
approach
by
Stephanie Rose Kadličko
A thesis submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Major: Entomology
Program of Study Committee:
Jon J. Tollefson, Major Professor
Linda M. Pollak
Kenneth H. Holscher
Iowa State University
Ames, Iowa
2008
Copyright © Stephanie Rose Kadličko, 2008. All rights reserved.
1457577
Copyright 2008 by
Kadlicko, Stephanie Rose
All rights reserved
2008
1457577
ii
TABLE OF CONTENTS
ABSTRACT
CHAPTER 1. LITERATURE REVIEW
iii
1
CHAPTER 2. EVALUATION OF SERBIAN COMMERCIAL CORN HYBRID
TOLERANCE TO FEEDING BY LARVAL WESTERN CORN ROOTWORM
(DIABROTICA VIRGIFERA VIRGIFERA LECONTE) USING THE NOVEL
DIFFERENCE APPROACH
Introduction
Materials and Methods
Results
Discussion
References Cited
23
23
25
28
30
35
REFERENCES CITED
39
ACKNOWLEDGMENTS
55
iii
ABSTRACT
Since the discovery of the pest in 1992, western corn rootworm (Diabrotica
virgifera virgifera LeConte (WCR)) populations in Serbia have successfully been kept low
with crop rotation. This has reduced the efficiency of screening for WCR resistance. A
cooperative project between Iowa State University and the Maize Research Institute,
Zemun Polje evaluated 13 Serbian commercial corn varieties in Ames, Iowa over a twoyear period. Corn hybrids were planted on trap crops where high WCR populations were
assumed. Hybrids were evaluated for WCR resistance using a randomized complete block
design with four replications. Treatments were paired rows arranged in split plots with one
row in each pair treated with insecticide and the other row left untreated. WCR injury was
evaluated using a rating of root size and root regrowth (1-6 scale), root injury
(0-3 Node Injury Scale), root mass, lodging, and yield. The results indicated significant
differences among the Serbian hybrids in the presence of moderate-to-high levels of
western corn rootworms. The relative benefit of insecticide treatments for maize lines was a
useful tool in evaluating resistant germplasm. However, conducting analyses on relative
differences between insecticide treated and untreated plots was not as effective at detecting
differences as comparing the plots independently.
CHAPTER 1. LITERATURE REVIEW
Corn
History of Corn in the United States. The word “maize” is derived from the Mayan
word for “grain of life,” signifying its importance to the subsistence of life in ancient
civilizations (Videnović and Drinić 2002). Corn, Zea mays Linnaeus, is a native of today’s
Mexico (Chiang 1973) and is now one of the most important cultivated crops in the world.
The domestication of corn is thought to have begun as long as 10,000 years ago, spreading
relatively slowly due to the equally slow migration of people across continents.
Introduction of corn into the present day United States (U.S.) is estimated to have occurred
circa 2,500 years ago, only reaching Europe in the 16th century after Columbus’s discovery
of America (Troyer 1999).
In the past, row spacing in the U.S. was 112 cm, a width that accommodated horsedrawn equipment (Olson and Sander 1988). With the mechanization of equipment, rowspacing began to decrease, resulting in increase in plant populations. Row-spacing in the
U.S. Corn Belt is now typically 76 cm (Cardwell 1982, Porter et al. 1997). In the future, it
is possible that equidistant spatial arrangements will be employed as they offer reduced
competition for sunlight between individual plants (Bullock 1988, Nielsen 1988, Olson and
Sander 1988, Porter et al. 1997). In equidistant spatial arrangement studies, plants have
shown up to an 8% increase in grain yield (Nielsen 1988, Polito and Voss 1991, Porter et al.
1997), likely as a result of higher energy absorption. The variation associated with the
increases in yield may be a result of hybrid adaptability or environmental conditions
(Fulton 1970, Olson and Sander 1988, Nielsen 1988, Porter et al. 1997).
2
However, equidistant spacing results in greater transpiration which may be
problematic in dry conditions, resulting in reduced grain yields (Fulton 1970, Porter et al.
1997).
History of Corn in Serbia. Corn has been planted on approximately 1.5 million
hectares in Serbia (Sivčev and Tomašev 2002) since the Second World War: 99.6% in
Serbia, and 0.4% (8,000 hectares) in Montenegro (Čamprag et al. 1995, Videnović and
Drinić 2002). As of 2002, most of this land was dedicated to continuous corn (Videnović
and Drinić 2002). In 1999, the Food and Agriculture Organization (FAO) ranked Serbia as
18th in land area devoted to corn and 14th in grain production (Videnović and Drinić 2002).
The total grain yield in 1947 was 2,153 million tons per year, which saw a consistent
increase to 8,413 million tons by 1986 (Videnović and Drinić 2002). By the millennium,
grain yield had tripled since WWII. In 1998, 11% of the corn land belonged to state farms
while the rest was privately owned (Republički Zavod za Statistiku 1998).
By the 1930s, the USA had begun production of commercial hybrid corn, heavily
influencing Serbia to follow their lead (Videnović and Drinić 2002). In 1953, Serbia sent a
group of corn scientists from the former Yugoslav Republic to visit the USA to investigate
advances in corn breeding (Videnović and Drinić 2002). The scientists were introduced to
inbreeding and crossing, and were able to observe successful American hybrids (Videnović
and Drinić 2002). The Serbians brought back to Yugoslavia both their training and the
American dent lines (Schnell 1992). U.S. dent lines crossed with European flint lines
created successful hybrids that were adapted to Europe and produced high yields
(Videnović and Drinić 2002, Schnell 1992). Between 1964 and 1999, the Federal
Commission for Variety Recognition recognized and approved 442 hybrids for
3
commercialization from national agricultural institutes. A main contributor was the Corn
Research Institute, Zemun Polje (Videnović and Drinić 2002).
The current importance of corn is based on its role as livestock feed (80% of total
corn usage) and human food and industrial uses (20%) (Videnović and Drinić 2002).
Serbia has exploited the chemical and biological composition of the corn plant and its grain
in fresh, stored, and dried forms. The country produces over 1,300 corn-derived products
(Berkić 1997). Much like in the U.S., the value of corn use in the production of fuel
continues to gain interest in Serbia (Videnović and Drinić 2002).
There is an increased demand for corn in the European and global markets, which
inevitably boosts prices (F. Bača, personal communication). In order for corn production to
meet high demands, larger areas of land need to be dedicated to corn grain production.
Unavoidably, demands have arisen in Serbia to grow monocultured corn in continuous
cropping (F. Bača, personal communication). This in turn brings with it the necessity of
finding host plant resistance traits, that is, tolerance to Western Corn Rootworm (WCR)
larval injury in corn (F. Bača, personal communication).
Additionally, the Serbian corn industry is faced with both political and economic
issues. The country was once governed by socialistic rule where growers had restrictions
limiting private land to 10 hectares (F. Bača, personal communication). Wages within the
country did not increase as they did in Western Europe and the economy of former
Yugoslavia, which is now Serbia, did not flourish (F. Bača, personal communication).
Instead, low incomes and war tore the country’s already struggling economy down
completely. When the land restrictions were lifted, growers who had adapted to the 10hectare limit showed no interest in expanding their crop land for several reasons: age,
4
equipment, and economic factors. Many farmers were close to or of retirement age, had
little to no mechanical equipment, or had very limited funds to spend on new seed,
fertilizers, or pesticides (F. Bača, personal communication). In combination, all these
factors have limited growers’ capabilities.
The younger generation of Serbian agriculturalists comprises the commercial
growers. The commercial growers have more crop land and are continually expanding their
land; their limitations are finances and available land for sale (F. Bača, personal
communication).
Corporations with established capital are privatizing former agricultural producers
by purchasing independently-owned land. The privatized individual farms, like commercial
farms, embark on intensive production with high use of pesticide, fertilizer and
mechanization at a robust, Western level. Such privatized farms collectively cover areas of
30 to 100 hectares at any given location (F. Bača, personal communication).
Western Corn Rootworm
Origin of the WCR. LeConte first documented the presence of galerucine
chrysomelids (Branson and Krysan 1981), Diabrotica virgifera virgifera, the western corn
rootworm (WCR), in 1868 in Kansas (Chiang 1973).The general consensus is that WCR
does not predate corn in the U.S. (Krysan and Smith 1987) and that the insect was likely
introduced into the U.S. along with corn from Central or South America (Branson and
Krysan 1981, Tollefson 2007). However, corn was not grown in Kansas at the time
(Goodman 1987, Weatherwax 1954). Moeser and Hibbard (2005) suggest that WCR may
have in fact jumped hosts from some unknown ancestral host to corn in the Kansas area
5
much later and spread as a result of corn production in that region. Corn has proven to be
the most suitable host for WCR, but scientists agree that it may not have been the original
host (Clark and Hibbard 2004, Oyediran et al. 2004, Branson and Ortman 1967).
WCR has not been documented to naturally complete its development on any host
other than corn, but a subspecies of Diabrotica virgifera, Diabrotica virgifera zeae Krysan
and Smith (the Mexican corn rootworm) has (Branson et al. 1982a). In 1986 Witkowski
reported damaging levels of MCR in areas over 250 km away from corn populations. This
was later confirmed by Krysan and Smith (1987).
Introduction and Spread of the WCR in the United States. The first report of
WCR as a pest was in 1909 (Gillette 1912), and the larval form of the beetle soon became
one of the most economically damaging insect pests in agriculture (Branson and Krysan
1981). The western corn rootworm spread across the North American corn growing areas at
the rate of 80 km/year, a rate that has been repeated in Central Europe (Tollefson 2007).
Introduction and Spread of the WCR in Serbia. WCR was first recorded in
Europe in July of 1992 (Bača 1994) when damaged corn was found, suggesting that
introduction must have occurred prior to the 1991 growing season (Čamprag et al. 1995).
The pest quickly spread across Serbia, into Hungary and Croatia, and then the rest of
Europe, making eradication unlikely and unfeasible (Kiss et al. 2005). The insect arrived in
Veneto, Italy in 1998, spreading to Pordenone in 2002 and Udine in 2003, Switzerland in
2000, and near Paris, France in 2002. In 2003 the insect arrived in Eastern France, Belgium,
UK, and the Netherlands (Kiss et al. 2005).
Miller et al. (2005) analyzed the genetic variation of adult WCR at several locations
in Europe, determining with high certainty that, while a few introductions were linked,
6
several were independent of each other. The population in Northeastern Italy originated in
Central Europe. The Eastern France population came from the Paris population, while the
Northwestern Italy and Paris populations came directly from North America. The group
also concluded that a minimum of three populations were introduced directly from North
America (Miller et al. 2005). The group attributed the sudden rise of introductions to:
adaptation of the insect, changes in control measures, and changes in transportation
procedures (Miller et al. 2005).
Serbian surveying for the insect pest began in 1993 with visual scouting in corn
fields. In 1996, use of sticky traps began; pheromone traps followed in 1997. In order to
quantify the influence of degree days (daily temperature high added to the daily
temperature low, divided by 2, subtracting the temperature threshold for WCR, 10, from the
overall quotient) and the hydrothermal coefficient (HTC) (total precipitation divided by
degree days) on WCR arrival, presence, and the total number of persistent adults, Bača et
al. (2006) trapped the insects from 1996 through 2005 using sticky traps (Multigard,
Csalomon, and Pherocon AM). They examined the influence of the degree days and the
HTC sums on the dynamics of the arrival, duration of injury, and the total number of caught
adults (Bača et al. 2006). During the eight-year study traps were put on corn plants in
continuous corn during the last 10 days of the month of June. Traps were replaced every 4
weeks and discontinued 2-3 weeks after the last adult was captured. The HTC was
calculated by adding the cumulative precipitation with the sum of the degree days (Bača et
al. 2006).
The arrival of the adults can be expected when the sum degree days between April
and June reaches 600˚C. The first 10.4% of the population was registered when the sum
7
degree days reached 704˚C. The dynamics of the arrival and the duration of the adults
depend on the magnitude of both the degree days and the HTC. Increases in numbers were
observed when the sum degree days was larger, and the duration of adult presence was
longer when the HTC was higher. These results were most easily observed in 2000, when
21.4% of the total population of adults was present as early as June, and in 1999, with a
recorded HTC of 0.31, adults were collected as late as October (Bača et al. 2006).
Uniform and dense populations of rootworms are necessary for proper evaluations
of germplasm. In Serbia and the rest of Europe, this may be hard to achieve due to two
primary factors: the clumped distribution of eggs by female adult WCR, which is also
observed in the U.S., and low pest densities in the region. While artificial infestation is a
solution to the non-uniform egg distribution, it requires special equipment and thousands of
eggs, which are expensive to purchase if they are not able to be produced. Thus, varietal
evaluations of corn may be performed on U.S. soil using European corn lines (Tollefson
2007).
Life-History of the WCR. WCR complete one generation per year. In the fall,
eggs are deposited in the top 20 cm of soil (Hein and Tollefson 1985), but may be found as
deep as 35 cm (Weiss 1983, Hein and Tollefson 1985). Eggs remain viable even through
very cold winters. While documentation has shown that egg populations have been
destroyed in laboratory freeze tests (Gustin 1981,1986), similar situations in nature have not
significantly reduced WCR populations. Eggs begin to hatch after 204 centigrade degree
days have accumulated (Chiang 1973). Carbon dioxide levels and gradients in soil help first
instars locate roots (Strand and Bergman 1987, Gustin and Schumacher 1989, Macdonald
8
and Ellis 1990, Bernlau and Bjostad 1998). However, extremely wet or dry soil may restrict
movement by the larvae and thus increase mortality (MacDonald and Ellis 1990).
Western Corn Rootworm adults are sexually dimorphic, with differences in elytra
coloration, antennal length, and size of the terminal abdominal segment (Sekulić and Kereši
1995). Both sexes can range from a yellow to greenish color with black stripes towards the
edges of each of the elytra; in females, the black stripes of the elytra are usually straight and
defined, while in the male the undefined stripes may bleed into one another (Sekulić and
Kereši 1995). The antennae of the male are longer than the length of the body, while in the
female the antennae are only about three-fourths as large (Sekulić and Kereši 1995). In
males, the second and the third segment of the antennae are of the same length, while in
females the third segment is noticeably longer than the second (Krysan and Smith 1987).
The length of the body can span from 4.2-6.8 mm long depending on the gender, with the
females the larger of the two sexes (Krysan and Smith 1987).
Immediately after larval emergence from the chorion they begin their host search.
They may travel as far as 40 cm horizontally in search of food (MacDonald and Ellis 1990),
but if host plants are not found within 12 – 24 hours, survival rates are dramatically reduced
(Branson 1989, Strand and Bergman 1987). Specific studies have shown that development
could be completed on several other plants including barley Hordeum vulgare Linnaeus and
wheat Triticum aestivum Linnaeus, among others (Branson and Ortman 1967; Clark and
Hibbard 2004). However, corn is the predominant dietary source.
First instar WCRs begin feeding on fine root hairs (Chiang 1973) and, in their
second and third instars, progress to the youngest roots where they begin to tunnel into the
cortex (Reidell and Kim 1990). Larval development almost always coincides with the
9
development of adventitious root tissue leading to detrimental effects if the larvae are in
abundance (Reidell and Evanson 1993). Through larval feeding, secondary infections can
be introduced via roots and lead to sickness or plant death (Levine and Oloumi-Sadeghi
1991). At ideal temperatures, larvae complete development in three weeks and progress to
the pupal stage in which they remain for approximately 10-12 days (Jackson and Elliot
1988).
Field collected adults are able to be maintained in the laboratory and usually survive
for 49-102 days (Ball 1957, Branson and Johnson 1973, Hill 1975, Boetel and Fuller 1997).
Field-caught beetles that emerged earliest had the longest laboratory longevity (Boetel and
Fuller 1997), but food quality also played an important role. Beetles fed on diets of young
plants lived the longest (Elliott et al. 1990).
Both male and female adults feed on pollen, silk, and foliage (Branson and Krysan
1981), usually not causing significant damage or economic loss. Through their feeding,
however, they may be vectors of corn stalk rot fungi (Levine and Oloumi-Sadeghi 1991).
Adults are active from July until the first frost (Levine and Oloumi-Sadeghi 1991,
Branson and Krysan 1981, Tollefson 2007). Males emerge first (Ruppel et al. 1978,
Branson 1987), beginning as early as late June. Emergence of both sexes continues for up
to 70 days (Ruppel et al. 1978). Soon after emergence, mating begins (Ball 1957). Males
mate an average of 8.2 times, while females mate only once (Branson et al. 1977) — but
retain the ability to lay multiple clutches of eggs if food is available. The female preovipositional period lasts 12-14 days (Branson and Johnson 1973, Hill 1975) and egg
production is induced by the digestion of protein in corn silks and pollen (Elliott et al.
1990). In laboratory environments the average number of egg clutches laid is 13, consisting
10
of approximately 1,000 eggs total, and are deposited over a period of 60-70 days (Branson
and Johnson 1973, Hill 1975). Oviposition occurs in the soil where the eggs remain until
the following spring (Levine and Oloumi-Sadeghi 1991), when they hatch (Branson and
Krysan 1981).
WCR Interactions with Corn. Plant damage caused by larval western corn
rootworm feeding is manifested in several ways. The affected root systems have limited
nutrient and water uptake, leaving plants without sufficient resources to complete proper
development (Levine and Oloumi-Sadeghi 1991). Root pruning by larvae also provides a
direct route for secondary infections and increases the likelihood of lodging.
An increase in a plants tendency to lodge, as a result of root pruning, may reduce
grain yield (Levine and Oloumi-Sadeghi 1991) up to 50% (Spike and Tollefson 1989).
Unfortunately, it remains difficult to predict what kind of yield loss will occur, as
environmental factors (moisture, soil type, nutrient availability, and weather), host plant
characteristics (root-system size, compensatory growth capabilities, and plant population),
and finally insect density all play a part in the ultimate damage to the plant (Levine and
Oloumi-Sadeghi 1991).
Host-Plant Resistance
Definition of Host-Plant Resistance. Many researchers in Europe are now
interested in the identification or development of corn lines that are resistant to WCR larval
feeding. Resistance is a heritable trait (Bigger 1941, Painter 1951) and leads to a reduction
in damage by the insect (Rausher 1992, Stowe et al. 2000). Painter (1951) described
resistance as the heritable qualities in a plant that limit the amount of injury or damage
11
caused on that plant by insect attack. The term can be further broken down into its
mechanisms: antixenosis; a plant’s ability to deter or decrease oviposition and colonization,
antibiosis; the ability to reduce or affect metabolic, developmental, or reproductive
pathways, and finally tolerance; the ability of a host plant to sustain injury, great enough to
damage a susceptible variety, without a reduction in yield (Painter 1951, Kogan and
Ortman 1978, Crawly 1983, Rausher 1992, Panda and Khush 1995, Stowe et al. 2000).
Resistance to WCR in Corn. As early as the 1920s, corn genotypes were shown to
react differently to rootworm feeding (Bigger et al. 1938). By the 1940s, host-plant
resistance research specific to WCR began in the Corn Belt states of the U.S. (Bigger et al.
1941).
Tolerance is not shown to be wide-spread, but it has been observed in some
cultivars (Chiang and French 1980). No hybrid has been documented to possess complete
tolerance (Rogers et al. 1975, Riedell and Evenson 1993, Gray and Steffey 1998).
However, since the 1960s, there have been constant improvements in cultivars (Riedell and
Evenson 1993, Gray and Steffey 1998). When present, the resistance mechanism manifests
itself in large root systems, which provide abundant compensatory growth after injury
(Chiang 1973, Branson et al. 1982a). Larger root-size and compensatory growth are often
correlated (Owens et al. 1974).
Compensatory growth is an important factor in sustaining elevated yields (Spike and
Tollefson 1988, Spike and Tollefson 1989) as it reduces the tendency to lodge (Levine and
Oloumi-Sadeghi 1991) and the uptake of nutrients and water. On the contrary, abundant
root tissue has been documented to negatively affect the yield by using any available
12
moisture for secondary root development in times of extreme drought (Gray and Steffey
1998).
Breeding for Resistance. Tremendous amounts of monetary resources are required
to conduct a corn breeding program. The labor is often a limiting factor (digging, handling,
and rating of roots) and results in small scale efforts by researchers. Advances in the
discovery or breeding of resistance lines have thus occurred in small steps over time.
Evaluations of resistance mechanisms have been conducted (Branson et al. 1982b, Kahler et
al. 1985a) and have lead to the release of resistant lines (Kahler et al 1985b, Russell et al.
1971).
In 1997 and 1998 Hibbard et al. (1999) documented crosses sustaining less
rootworm damage, but like other breeding programs they had limited success in developing
resistant germplasm (Branson and Rueda 1983; Branson et al. 1969; Branson 1971;
Branson et al. 1986; Riedell and Evan 1993; Owens et al. 1974; Wilson and Peters 1973;
Wilson et al. 1995; Rogers et al. 1975; Rogers et al. 1976a, 1976b; Rogers et al. 1977).
Tolerance has been documented as the typical resistance mechanism found in germplasm,
but other mechanisms (antibiosis and antixenosis) of resistance have been suggested
(Branson et al 1983, Kahler et al. 1985b, Assabgui et al. 1995).
There was limited success in developing or incorporating this germplasm into
commercial lines. This may be due to findings that showed a tendency to lose any tolerance
or resistance genes present in the germplasm when yield was targeted in selection trials
(Welter and Steggall 1993, Rosenthal and Dirzo 1997).
13
Western Corn Rootworm Control
Biological Control. Biological control experiments using ground beetles
(Coleoptera: Carabidae) suggested that the beetles were predators of WCR larvae (Tyler
and Ellis 1979). This research was challenged, however, by other scientists who suggested
that the ground beetles were mislabeled as predators and are not useful biological control
agents (Best and Beegle 1977, Kirk 1982). Stinner and House (1990) suggested that certain
non-insect arthropods such as mites in families Laelaptidae, Rhodacaridae, and
Amerosiidae could serve as control agents. These arthropods were found to be significant
predators of WCR larvae and eggs (Chiang 1970).
Susceptibility to a certain soil-dwelling nematode were found (Jackson and Brooks
1989, Steffey et al. 1987). However, inconsistencies were found in the results and were
suggested to be due to mechanical and edaphic factors (Munson and Helms 1970, Poinar et
al. 1983). No biological control methods have been shown to be sufficiently effective to be
adopted by corn producers as part of their integrated pest management tactics.
Rotation and the WCR Variant. Historically, WCR injury was avoided through
crop rotation with alfalfa, hay, clover, and small grains. In the 1940s, the utilization of corn
for a growing number of purposes caused a farming shift. Corn producers began growing
corn in fields where corn had been planted the previous season (continuous corn). This
production increase expanded the range of the WCR (Krysan and Branson 1983, Nowatski
2001), elevating the WCR to pest status. Since the recognition of WCR damage, many
growers have shifted back to crop rotation in combination with other control tactics in an
attempt to control the insect.
14
Regular, annual rotation of corn with another crop, primarily soybeans, resulted in
selection pressure against rotation and it become less effective. Finally, in the mid-1990s, a
behavioral change occurred and WCR females began ovipositing in soybean fields that had
been rotated annually with corn (Levine and Oloumi-Sadeghi 1996). In 1995 the first larval
WCR occurrences in corn after soybeans were reported along with adult collections in both
corn and soybean fields (Cook et al. 2005). This was just a few years after Levine and
Oloumi-Sadeghi (1991) reported that WCR did not show the extended diapause adaptation
to rotation that its close relative, the northern corn rootworm, (Diabrotica longicornis Say)
did (Krysan et al. 1986).
The adult females of the variant WCR strain lay eggs in soybeans, and in lesser
numbers in alfalfa, oat, and wheat, as well as corn (Cook et al. 2005). Eggs hatch the
following season when corn is planted again (Levine and Oloumi-Sadeghi 1988). Since the
mid-1990s, a dramatic increase in damage was observed in East-Central Illinois and
Northwest Indiana (Levine and Oloumi-Sadeghi 1996). In Iowa, WCR surviving on corn
planted after soybeans was recorded for the first time in 1999 (Rice and Tollefson 1999).
Since then, WCR has been reported numerous times in the major corn growing states in
cornfields planted after other crops (Levine et al. 2002). The presence of a WCR variant
presents major problems as larval control through crop rotation is overcome and thus calls
for stronger, alternative control measures.
Insecticide Use. The use of tolerant cultivars is not recommended as the sole
management tactic. Even the most tolerant root systems may be overwhelmed when planted
in areas with large populations of WCR (Levine and Oloumi-Sadeghi 1991). In lower insect
populations, Chiang and French (1980) found that larval survival was higher on tolerant
15
varieties. Experts and growers have integrated many management tactics into the control of
WCR including crop rotation, transgenic corn, and insecticide use. By 1986, insecticides
and crop losses from WCR were costing corn producers an estimated $1 billion (Metcalf
forward in Krysan et al. p260). Today, it is undoubtedly the most economically important
corn pest insect (Tollefson 2007).
Once applied, the presently used soil insecticides remain active for 6-10 weeks,
which coincides with primary larval feeding (Levine and Oloumi-Sadeghi 1991). The
materials are formulated on absorptive material, like clay or sand, and then incorporated
into the top layers of soil (Levine and Oloumi-Sadeghi 1991).
Cyclodienes were used in the late 1940s and were effective in their action against
larval WCR, but in just over 10 years, resistance was so prevalent that the insecticides were
abandoned (Meinke et al. 1998). By 1959, chlorinated hydrocarbon insecticides had been
widely accepted by corn growers for a decade, even though the WCR’s resistance to them
was documented (Ball and Weekman 1962) and was spreading 122 km per year (Metcalf
1983). As other classes of insecticides were developed, chemical control advanced beyond
chlorinated hydrocarbons. However, in 1998, WCR beetles in areas where adults were
controlled using the newest insecticides had already developed resistance (Meinke et al.
1998). Larval WCR have also shown elevated resistance levels to certain granular
insecticides (Wright et al. 2000) in those same areas.
Pesticide use persists in the US (Tollefson 1990, Gray et al. 1993). Unfortunately, in
some years, high numbers of emerging adults from treated areas have been reported (Gray
et al. 1992). Larvae may be developing in areas of insecticide application because they may
mature outside the treatment zone of insecticides, which are only applied in-furrow or in a
16
15 cm band over the row (Sutter et al. 1991, Gray et al. 1992). Due to the mechanical and
chemical nature of soil insecticides they are only active in a certain zone around the roots
(Felost and Steffey 1986). As a result, roots that grow outside this zone can be eaten by
WCR and insect development can be completed (Felost and Steffey 1986).
Over-reliance on soil insecticides is not wise. Soil insecticide efficacy is influenced
by numerous factors:
•
Environmental conditions like moisture, humidity, and wind
•
Chemical properties like soil composition and insecticidal stability (Felost and Lew
1989, Harris 1972)
•
Cultural techniques and equipment (Levine et al. 2002)
•
Biological factors like microbial insecticide degradation, extended diapause, and
insect resistance (Meinke et al. 1998)
•
Pesticide laws and regulations
All these issues make WCR management with soil insecticides challenging.
Host-Plant Resistance. Strauss and Agrawal (1999) further divided tolerance into
the resultant mechanisms that occur in response to herbivore plant feeding: an increase in
the photosynthetic rate after damage has occurred, elevated growth rates, increased
branching of roots and/or shoots, and the ability to redirect stored carbon from undamaged
to damaged plant parts. The mechanism(s) employed by a plant is dependent on the type
and location of the feeding damage and the species and variety of the plant (Strauss and
Agrawal 1999).
Researchers have found a negative association between nutrient availability and
tolerance, mainly when environmental nutrient levels were highest (Gertz and Bach 1995,
17
Irwin and Aarassen 1996). Olff et al. (1990) attribute this association to the reduction in the
root:shoot ratio by high nutrients as a reduction in the ratio reduces tolerance (Strauss and
Agrawal 1999). Endophytic fungal infections were also negatively associated with
tolerance (Strauss and Agrawal 1999), as infections were found to limit compensatory
growth in all grasses (Belesky and Fedders 1996). Conversely, a positive association was
found between water and light with tolerance (Maschinski and Whitham 1989).
Transgenic Corn. Transgenic corn is an alternative now offered to growers to
alleviate the concern regarding insecticide resistance. However, laws, regulations, and
technology fees accompany the use of transgenic seed. Additionally, WCR has already
shown resistance potential. A greenhouse WCR colony confirmed that resistance to Bt corn
can develop quickly in forced inbreeding that resulted in an 11.7 fold increase in tolerance
(B. E. Hibbard, personal communication).
The U.S. MON 863 corn, developed by Monsanto in the 90s offered the first
protection against WCR through expression of an insecticidal protein called Cry3Bb1
(Vaughn et al. 2005). Since the development of MON 863, Pioneer Hi-Bred International,
Inc. (Johnston, IA), Syngenta (Basel and Stein, Switzerland), and Dow AgroSciences
(Indianapolis, IN) have developed transgenic corn varieties that express insecticidal
proteins that are modified or different from Cry3Bb1 (Moellenbeck et al. 2001). There has
been varying control conferred by the different transgenic hybrids (Gray et al. 2007).
The European Union (EU) has not approved macro-scale use and production of
genetically modified organisms (GMO), including corn. EU countries have begun
experimentation on European soil, however, and are continuing the search for alternatives
18
to GMOs as no varieties are on the market or are approved for direct consumption (GMO
Compass).
Western Corn Rootworm Management
Sampling. While in some regions WCR continues to show resistance to chemical
control, insecticides are still in use. Turpin (1977) reported that 93% of soil-insecticide
applications in the state of Indiana were applied without sampling the WCR population.
Furthermore, although there were many areas of continuous corn production, approximately
50% of them did not have economically significant populations of WCR (Turpin et al.
1972, Gray et al. 1993). In 1986, Metcalf reported that 50-60% of land in the U.S. used for
corn production was treated with soil insecticides to control WCR. Today, this percentage
is presumably higher as states like Iowa, with its heavy corn production, concluded that soil
insecticides should be used at all times until more accurate sampling techniques that predict
damage for the following year are developed (Nowatski 2001).
Unnecessary applications of soil insecticides are avoided by sampling adult WCR
beetles at the end of a season to predict larval damage the following year (Steffey et al.
1982, Tollefson 1990). Initially, one adult beetle per plant in a field of continuous corn was
set as the standard to expect larval damage the following season (Pruess et al. 1974),
warranting insecticidal treatment. Godfrey and Turpin (1977) suggested that the economic
threshold would be attained when numbers reached 0.71 beetles per plant in first-year corn
fields. Researchers later re-confirmed that a one-beetle-per-plant assessment was 83%
accurate and that the associated economic damage was effectively estimated through the
19
use of adult sampling (Foster et al. 1986). Field sampling can be problematic because its
precision level is not consistently as high as desired as it is a very rough estimate.
Germplasm Management Programs. Root-injury ratings have been the preferred
method for evaluating corn tolerance (Tollefson 2007). Three root-injury scales have been
developed and commonly used. The 1-6 scale was introduced in 1971 by Hills and Peters.
The least amount of injury is represented by a rating of 1 and the greatest by 6; all other
ratings fall in between (Hills and Peters 1971). A second scale, developed about the same
time, also had categorical ranging from 1-9 (Musick and Suttle 1972) where the least
amount of injury was also represented by a rating of 1, but with this scale, the greatest
injury by 9; all other ratings again falling in between. The final and most intuitive scale is
the Node-Injury scale developed by Oleson et al. (2005), which provides a linear
representation of injury where 0 represents no injury and 1 represents a full node of roots
destroyed. Three is the highest injury rating representing three or more full nodes of injury.
Injury between full nodes is rated as a proportion of a node; for example, 1.25 represents
one and a quarter nodes destroyed.
In the evaluation of corn varieties, root sizes are also recorded. These ratings are
highly subjective due to variability in annual environments (Tollefson 2007). The Eiben
root-size scale classifies the sizes of roots on a scale of 1 (smallest) to 6 (largest) (Rogers et
al. 1975). A representative of the smallest and largest roots are chosen and assigned the
numbers 6 and 1, respectively; roots that best represent the sizes between the 1 and 6 are
then chosen and assigned ratings of 2-5. All other roots of that field are then rated against
the representative samples.
20
The root-compensatory-growth scale uses a similar rating as the Eiben root-size
scale, but assesses compensatory root growth (Rogers et al. 1975). A rating of 1 represents
the least amount of secondary growth and a rating of 6 the most. Hills and Peters (1971)
combined their own secondary root development rating (0-4 with 0 as least re-growth and 4
as most) with an injury rating (between 1 and 6) to calculate an index that predicts yield.
The re-growth rating was subtracted from the injury rating and the value was a categorical
index prediction of the yield.
Lodging, corn leaning or laying 30 or more degrees off the vertical, can be a clear
indicator of WCR presence (Tollefson 2007). Pruned roots are the primary cause of
lodging, leaving plants unsupported and leading to physiological and mechanical yield loss.
Plants that are significantly lodged experience decreased photosynthetic efficiency due to
the plant’s inability to receive adequate amounts of sunlight (Tollefson 2007). Mechanical
yield loss results when harvesting equipment is unable to pick up lodged corn (Tollefson
2007). Lodging is not directly useful in selecting resistant germplasm as it does not allow
for resistance mechanisms to be distinguished and, most importantly, it is also very
dependent on environmental conditions (Tollefson 2007).
American lines with documented resistance appear to derive their resistance from
large root systems and greater compensatory growth (Owens et al. 1974, Rogers et al.
1975). This finding indicates that the mechanism of resistance is most likely tolerance. Both
root size and compensatory growth are affected by environmental conditions including, but
not limited to, moisture and nutrients (Owens et al. 1974). Elevated levels of either
antibiosis or antixenosis to WCR have not been found in corn (Tollefson 2007). Branson
and Krysan (1981) suggested benefits in finding inbred and ancestral lines of corn that
21
exhibit antibiosis or antixenosis. However, the researchers predicted this as a difficult task
as they are evolutionarily unfavorable and require more energy from the plant (Branson
1971). If they exist, the traits would likely be found in relatives of corn that over long
periods of time were able to evolve.
Ultimately, growers are interested in the final grain yield (Tollefson 2007). Yield
tolerance measures the yield outcome between a single variety planted with paired rows
where one row is infested by an insect and the other is not. The difference between the two
grain yields is termed the yield tolerance, with the smallest difference representing the
greatest tolerance (Tollefson 2007). Yield tolerance might be used as an indicator of
varietal vigor and performance under rootworm pressure.
Rogers et al. (1975) were the first to conduct field evaluations for tolerance to yield
reduction as a result of WCR larval feeding. Their plots were planted on naturally infested
soil, resulting in significant differences between treated and untreated plots (Rogers et al.
1975), which provided a solid foundation on which to repeat such evaluations on more
naturally infested plots. The group found that feeding damage did not consistently result in
lodging, and that yield loss alone could serve as indicators of feeding (Rogers et al. 1975).
Corn on corn is still used today in both the USA (Branson and Krysan 1981, Krysan 1983)
and Serbia (Videnović and Drinić 2002).
If stresses are minimal growers may find that damage may not lead to injury and
thus see little or no yield loss (Levine and Oloumi-Sadeghi 1991). Control of adult
population is recommended the previous summer; the task involves direct action, but can
help the following season (Levine and Oloumi-Sadeghi 1991). Crop rotation and the
elimination of volunteer corn (Steffy and Gray 1989, Levine and Oloumi-Sadeghi 1991) in
22
combination with other management tactics provide a robust integrated pest management
strategy that confers results.
CHAPTER 2. EVALUATION OF SERBIAN COMMERCIAL CORN HYBRID
TOLERANCE TO FEEDING BY LARVAL WESTERN CORN ROOTWORM
(DIABROTICA VIRGIFERA VIRGIFERA LECONTE) USING THE NOVEL
DIFFERENCE APPROACH
Stephanie R. Kadličko
Introduction
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, was
first described in the United States in the late 19th century. As its population grew over time,
the insect became one of the most economically damaging agricultural insects in the
country and persists today. Nearly 200 years later, in 1992, the first European incidence of
corn damage by WCRs was reported in central Serbia close to the Belgrade airport in
Surčin (Bača et al. 1995). Within just three years the insect spread to neighboring Hungary
and Croatia; as a result of the rapid spread, eradication became unlikely and the insect is
now present across Europe (Kiss et al. 2005).
Until very recently US corn growers have used two primary tactics in WCR control:
crop rotation and insecticide application. While offering growers considerable control, the
tactics have applied selective pressures on the insect resulting in the evolution of resistance.
Genetically modified Bt-corn was later introduced to the North American market. It became
immediately popular among growers, offering an effective and time-efficient method of
protecting maize from WCR larvae. Europe, to date, has not approved of genetically
modified organisms (GMOs) and thus does not grow transgenic corn.
24
In Serbia crop rotation continues to be used as the primary WCR control strategy,
being found to be the most efficient and effective tactic (Bača et al. 1995). When rotation in
Serbia is employed, corn is preceded by crops including wheat, sugar beet, and field pea.
Unfortunately for subsistence growers, rotation is not always an option. Small land owners
who grow corn to feed livestock, primarily cattle and hogs, do not have the option of
rotating corn with other crops as the survival of their livestock and their farm depends on
corn. Likewise, the option of insecticide is not always available due to their high costs.
In Serbia, resistance breeding remains a viable option because WCR populations are
maintained at low to moderate levels with national, multi-crop rotation. Ideally a hybrid
utilizing a mechanism of resistance that withstands a moderate level of insect feeding
would provide the growers in the region with an option of producing corn in back-to-back
seasons.
The Maize Research Institute, Zemun Polje, Serbia has several vigorous hybrids of
various maturities that have been accepted and sold commercially for several years. Testing
the performance of these hybrids under rootworm infestations would allow growers in
Europe to make educated choices on seed that will offer protection from the devastating
pest. Due to Serbia’s low rootworm levels, evaluation on domestic soil was impractical
without employment of artificial infestation methods. To help determine whether resistance
to WCR is present in any of the hybrids grown in Serbia, Iowa State University in Ames,
IA and the United States Department of Agriculture - Agriculture Research Service in
Columbia, Missouri entered into a collaborative project with the Maize Research Institute
to evaluate Serbian germplasm. Specific objectives were to determine (1) how maize
performance varied among commercial Serbian hybrids in the presence of moderate-to-high
25
levels of western corn rootworms, and (2) whether analysis on the relative benefit of
insecticide treatments (performance differences between treated and untreated plots) for
maize hybrids would be a useful tool to guide selection for resistance.
Materials and Methods
Thirteen commercial hybrids were evaluated in Ames, IA over a two year period.
To ensure moderate-to-high levels of WCR, maize was planted onto land where corn had
been planted late the previous year to attract gravid females (trap crops). For each hybrid,
data on performance were collected in insecticide-treated and untreated plots. The
difference between treated and untreated plots for each hybrid essentially measures the
effect of insecticide use; smaller differences suggesting plant resistance or tolerance. If
successful, the ‘difference approach’ would permit assessment of WCR resistance among
the tested hybrids independent of differences not directly related to host-plant resistance.
Germplasm. Serbian commercial hybrids evaluated included ZP 341, ZP 360, ZP
434, ZP 42a, ZP 578, ZP 580 (2006 on), ZP 599, ZP 677, ZP 680, ZP 684, ZP 704, ZP 735
(2007 only), and ZP 737 (2006 only). Hybrid maturities ranged from 110 days in FAO
300s, to 130 days in FAO 700s. Hybrids were previously bred for various qualities (drought
tolerance, yield, etc.) and were chosen for WCR evaluation based on performance in
Serbian and European markets. Two public lines were used as control: CRW3 x LH51 as a
naturally resistant control and B37 x H84 as a susceptible control. No controls were used in
2006. In 2007, ZP 580 and ZP 737 were unavailable; ZP 737 was replaced with ZP 735 due
to genetic similarity, but the two hybrids were analyzed separately. ZP 580 was not
replaced in 2007.
26
Experimental Design and Field Use. The experiment was arranged in a
randomized complete block design with split plots and paired rows. One row in each pair
received a single, in-furrow application of a granular pyrethroid insecticide (0.1465 kg
(A.I.)/ha of Force ® 3G, Syngenta Crop Protection Inc., Greensboro, NC, USA) at the time
of planting. Four replications were planted with row spacing of 76.2 cm. In both seasons
hybrids were planted at a single location in Ames, Iowa: in 2006 at Johnson Farm, Iowa
State University and in 2007 at Bruner Farm, Iowa State University. Both locations had trap
crops grown on them the previous season and were chosen because high natural WCR
populations were assumed. Planting was conducted mechanically with a 4-row John Deere
Max-Emerge™ 7100 integral planter. Seed was deposited with 12.7 cm seed spacing. Plots
were subsequently thinned to 17.78 cm between plants when plants were approximately 30
cm tall. Each replication was divided into two sections that were separated by a narrow
alley. One section, 7.01 m in length, was designated for individual plant evaluations from
where representative plants were dug. The other section was left for yield measurements
and was 7.62 m in length.
Hybrid Evaluation. In 2006 the first evaluation was conducted on 13 July and in
2007 on 11 July. All ratings were completed within 2 days of the initial root digging, except
for root mass. Root mass was taken after roots had been cut from the plant, bagged, and
dried for 4 days at 100 degrees Celsius. At each evaluation 5 representative plants from
each row were chosen at random, dug, labeled, and cleaned with pressurized water. Plants
were rated for root size using the Eiben Root-Size Scale (Rogers et al. 1975) and root injury
using the Node-Injury Scale (Oleson et al. 2005). A root size rating of 1 indicated the
smallest root system, and a rating of 6 indicated the largest root mass. The Node-Injury
27
Scale is a linear scale from 0-3 where 0 indicates no injury, 1 indicates one full node of
pruning, and a partially pruned node is indicated as a percentage, i.e. a quarter node of
pruning is given a score of 0.25. The second evaluation was conducted on 2 August in 2006
and on 15 August in 2007; all of the same evaluations were conducted and completed
within 2 days of the root digging. Prior to harvest stand counts and lodging counts were
taken. Corn was considered lodged if it leaned more than 30 degrees off the vertical. Corn
was harvested on 30 October in 2006 and on 25 October in 2007.
Statistical Analysis. Preliminary tests were conducted using general linear models
(PROC GLM, SAS Institute Inc. 2004) to evaluate whether maturity class predicted
performance of varieties, and if study year interacted with other factors. To determine if the
effect of insecticide application (difference between insecticide-treated and untreated plots)
varied among commercial hybrids, models (PROC GLM) tested whether differences in
measures of maize performance were influenced by variety, year and replicate (within
year), with year and replicate treated as random effects. Separate analyses were conducted
for root injury (second evaluation), root mass (second evaluation), size, lodging and yield;
data from the first evaluations were excluded because it appeared that first evaluations of
root injury and mass took place before all WCR injury occurred in both seasons. When an
overall F-test indicated an effect of variety, t-tests were used to make pair-wise
comparisons among means.
Because several apparent differences among lines were shown not to be statistically
significant, two additional analyses were conducted. First, to determine whether the number
of replicates used was sufficient to detect variability among the hybrids using the
‘difference approach,’ a power analyses was conducted for each of the five tested measures
28
of maize performance using a routine designed for analysis of variance with a block effect
(NCSS 2002). Power (1 – β) is the probability that a false null hypothesis will be rejected,
thus avoiding a Type II error (β) (the chances of a β decreases with the increase of power).
Required inputs for the power analysis included differences between insecticidetreated and untreated plots for each hybrid and an estimate of random error (square root of
mean squared error). Data from 2007 were used in the power analyses because the
‘difference approach’ logically seems more likely to detect resistance when pest pressure is
highest. Second, a more conventional analysis of hybrid performance was conducted.
Separate models (PROC GLM) compare hybrids with or without insecticides. Each model
tested whether performance (e.g., injury, lodging, yield) was influenced by variety, year and
replicate (within year), with year and replicate treated as random effects. Dependant
variables expressed as percentages were modified using the arcsine-square root
transformation in order to make the data more normally distributed. Pair-wise comparisons
were made among maize varieties as indicated for the ‘difference approach,’ which is
equivalent to using Fisher’s Protected LSD.
Results
Maturity and Year Interactions. Variation among varieties in each maturity class
appeared greater than differences among maturity classes, and a significant year ×
insecticide interaction suggested the impact of Diabrotica on maize varieties increased
from 2006 to 2007. Difference analysis tested in models appeared to yield differences
among lines. However, most were not statistically significant.
29
Power Analysis. The power analyses showed that, for three of five parameters,
power could be increased significantly with the addition of replications. Root injury could
apparently have been elevated from a power of almost 0.7 with the addition of 4
replications (Fig. 1). Size and lodging were shown to require approximately double the
replication increase of injury, needing approximately 10 replications to achieve an
acceptable power. An increase in the number of replications for mass and yield parameter
measurements would apparently not gain any advantage, as an increase of 8 replications
would still result in a power of approximately only 0.7 and 0.5, respectively.
Lodging. Lodging results for insecticide untreated plots are presented in Table 1
and treated plots in Table 2. Averages for untreated plots ranged from a transformed log of
0.5 for ZP 599 to a transformed log of 0.09 for ZP 735 (Table 1). For treated plots, averages
ranged from a transformed log of 0.06 for ZP 580 to a transformed log of 0 for most of the
other hybrids (Table 2). All hybrids resisted lodging significantly more than the negative
control. Hybrids ZP 341, ZP 360, ZP 684, and ZP 735 resisted lodging significantly more
than ZP 42A, ZP 599, and ZP 704.
Yield. Average yields for untreated plots ranged from 1.48kg/ha for ZP 42A to
2.22kg/ha for ZP 684 (Table 1). For treated plots, averages ranged from 1.82kg/ha for ZP
580 to 2.63kg/ha for ZP 680 (Table 2). All hybrids yielded significantly higher than the
negative control. Hybrids ZP 578 and ZP 684 yielded significantly higher than 46% of the
hybrids: ZP 341, ZP 360, ZP 42A, ZP 434, ZP 599, and ZP 737. ZP 42A yielded
significantly less than 62% of the hybrids: ZP 341, ZP 434, ZP 578, ZP 580, ZP 677, ZP
680, ZP 684, and ZP 704.
30
Mass. Averages for untreated plots ranged from 9.48 g for ZP 599 to 16.05 g for
ZP 578 (Table 1). For treated plots, averages ranged from 8.37 g for ZP 580 to 17.70 for ZP
578 (Table 2). All hybrids but ZP 599, had a higher mass (dry weight) than the negative
control. ZP 578 had a higher mass than 46% of the hybrids, and ZP 677 had a higher mass
than 31% of the hybrids. ZP 599 did not have significantly more root mass than the
negative control or 46% of the hybrids.
Size. Averages for untreated plots ranged from 2.80 for ZP 360 to 3.64 for ZP 735
(Table 1). For treated plots, averages ranged from 2.90 for ZP 341 and 360 to 3.53 for ZP
580 (Table 2). All hybrids but ZP 42A were significantly larger than the negative control,
but were not significantly different among each other.
Injury. Averages for plots with no insecticide ranged from 1.75 for ZP 704 to 0.67
for ZP 735 (Table 1). For treated plots averages ranged from 0.35 for ZP 434 to 0.06 to 0.10
for ZP 737 (Table 2). ZP 735 suffered significantly less injury than the negative control as
well as ZP 434, ZP 578, ZP 580, ZP 677, ZP 684, and ZP 704. ZP 341 received
significantly less injury than the control and ZP 704.
Discussion
Insecticide Treatments as a Resistant Germplasm Evaluation Tool. Although a
preliminary analysis did not show significant differences among maturities, day length and
maturity at evaluation locations should always be considered. The lines that performed best
in this evaluation were of maturities that suited the region where they were tested.
The ‘difference approach’ is intuitively advantageous and theoretically would have
been ideal for line evaluation. However, its field performance indicated otherwise. When
31
margins of error were considered, large differences between treated and untreated
evaluation criteria for hybrids (the basis of the ‘difference approach’) were not statistically
significant. The power analysis indicated that an investment of twice as many resources
would be necessary to achieve high power and obtain significant results. Environmental
differences in soil composition and quality, nutrition and moisture, among other factors,
may account for the variability in yield and mass. Thus, the ‘difference approach’ was
rendered impractical. However, with relatively little resources the ‘comparison approach’
was shown to be an effective tool. Comparing separate models for insecticide treated and
untreated plots may be slightly more cumbersome, but shows distinct differences between
hybrids.
By analyzing hybrids according to each variable in independent models (insecticide
treated or untreated) significant differences become evident. Comparing hybrids that
appeared to show resistance in untreated plots to insecticide treated plots clearly indicated
the superior performance of certain hybrids. This method also allowed us to decipher
whether apparent strengths in hybrid resistance are truly manifestations of host-plant
resistance or just overall hybrid vigor, something that would not have been distinguishable
through use of the ‘difference approach.’ In these experiments, the ‘comparison approach’
was able to detect differences with eight replications. However, as with any other data,
statistical significance rises with an increase of replications over a wide range of
environments.
Hybrid Evaluation. Individual traits are relatively meaningless when not
combined with other traits for a specific hybrid. By considering multiple traits in the overall
32
evaluation certain lines were clearly exhibiting resistance, others demonstrated their
breeding potential, while some hybrids were weak overall.
ZP 42A had an extremely low yield even with a significantly large root system. The
yield was considerably larger when protected with insecticide, increasing 0.64 kg/ha,
indicating severely reduced vigor in the presence of WCR larvae. ZP 42A also had an
extremely low yield with a large root system, suggesting excess plant resources were
diverted from ear development to compensatory root development. ZP 434 performed
similarly to ZP 42A in terms of both root size as well as yield, gaining 0.64 kg/ha from
insecticide protection.
ZP 578 and ZP 684 performed very well among the other hybrids. ZP 578 had the
second largest yield of all the unprotected hybrids. Its yield did not increase by more than
0.16 kg/ha with insecticide protection, indicating tolerance to feeding by rootworms. Even
with high injury ZP 578 resisted lodging likely due to its large root system. The large mass
of ZP 578 in both the treated and untreated plots indicated that its large mass was not a
result of post-injury compensation. ZP 684, which was competitive with ZP 578, resisted
lodging and had the highest unprotected yield that did not increase with the application of
insecticide. It did not benefit from insecticide application, suggesting both plant vigor and
resistance to WCR damage. Its tolerance was likely conferred by its abundant root-mass.
ZP 580 did not have one of the highest yields, but did not receive additional
protection from insecticide application. The mass increased significantly between the
protected and unprotected plots, almost doubling. Resources devoted to growth apparently
did not take away from ear development as may have been occurring in other hybrids; ZP
580 yielded higher without insecticide protection than with insecticides applied.
33
ZP 735 did not have competitive yields with many of the other hybrids but showed
breeding potential. Its root size was the largest and among the heaviest. It also received the
least amount of injury in the untreated plots suggesting that ZP 735 may be exhibiting an
alternate resistance mechanism (antixenosis) than the other hybrids (tolerance). Even with
lower yields, the breeding potential of ZP 735 should be considered.
The negative control was a very weak hybrid. Its yield, even when protected with
insecticide, was 30 bushels lower than any other non-protected hybrid. The positive control
proved to have low vigor in comparison to some of the other hybrids. However, when
comparing insecticide treated and untreated plots it was evidently quite tolerant of
significant feeding injury. Size did not increase with feeding, nor did mass (in insecticide
untreated plots), and yield increased by only 0.08 kg/ha. This control demonstrated
perfectly that relative assumptions across lines can be misleading in predicting the true
tolerance of a given line.
One downfall to using this method of hybrid evaluation analysis is that it is less
direct and may be time-consuming if many lines are involved in an evaluation. In the case
of this study, the ‘comparison approach’ worked very well. The top hybrids (ZP 578, ZP
580, ZP 684, and ZP 735) were ranked high based on their ability to deliver unprotected
yields that were as high or higher than protected yields (ZP 578, ZP 580, and ZP 684), or
for their breeding potential (ZP 735). These hybrids appear to have natural host-plant
resistance and are able to tolerate WCR feeding. Because the primary resistance mechanism
observed was tolerance based, selection pressure by insects will likely be minimal and, as a
result, the resistance is more likely to be preserved over time.
34
Thus, the conclusion can be made that the Maize Research Institute, Zemun Polje
has several promising WCR resistant hybrids. These hybrids, in combination with crop
rotation, will perform well for Serbian farmers and corn breeders without the use of
insecticides for rootworm protection, suiting the current ecological WCR situation in the
region.
35
References Cited
Bača, F., D. Čamprag, T. Kereši, S. Krnjajić, B. Manojlović, R. Sekulić, and I. Sivčev.
1995. Western Corn Rootworm Diabrotica virgifera virgifera LeConte, pp. 98-112. In D.
Čamprag (ed.), Kukuruzna zlatica Diabrotica vifgifera virgifera LeConte. Društvo za
zaštitu bilja Srbije, Belgrade, Serbia.
Kiss, J., C. R. Edwards, H. K. Berger, P. Cate, M. Cean, S. Cheek, J. Derron, H.
Festic, L. Furlan, J. Igrc-Barčić, I. Ivanova, W. Lammers, V. Omelyuta, G.
Princzinger, P. Reynaud, I. Sivčev, I. Sivicek, G. Urek, and O. Varala. 2005.
Monitoring of western corn rootworm (Diabrotica virgifera virgifera LeConte) in Europe
1992–2003, pp. 29–39. In Vidal, S., U. Kuhlmann, and C. R. Edwards (eds.). Proceedings,
Western corn rootworm: ecology and management. CABI Publishing, Wallingford, United
Kingdom.
NCSS. 2002. PASS 2002: Power Analysis and Sample Size for Windows User’s Guide –
II. NCSS. Kaysville, UT.
Oleson, J. D., Y. Park, T. M. Nowatzki, and J. J. Tollefson. 2005. Node-injury scale to
evaluate root injury by corn rootworms (Coleoptera: Chrysomelidae). J. Econ. Entomol. 98:
1-8.
Rogers, R. R., J. C. Owens, J. J. Tollefson, and J. F. Witkowski. 1975. Evaluation of
commercial corn hybrids for tolerance to corn rootworms. Environ. Entomol. 4: 920-922.
SAS Institute Inc. 2004. SAS 9.1.3 Help and Documentation. SAS Institute Inc. Cary, NC.
Table 1. Adjusted means of evaluated traits combined over two years for insecticide untreated plots.
Root Size SEMa Injury
SEM Mass (g)
SEM Lodgingb SEM Yield (kg/ha)
SEM
3.10 ab
0.27 1.01 bc
0.21 11.74 cde
1.22 0.16 e
0.07 7382.02 bcd
462.79
2.80 ab
0.27 1.25 abc 0.21 12.13 cde
1.22 0.15 e
0.07 7054.37 bcde
462.79
2.65 bc
0.27 1.18 abc 0.21 14.21 abcd
1.22 0.39 bcd 0.07 5821.00 e
462.79
3.13 ab
0.27 1.42 ab
0.21 11.12 de
1.22 0.23 de 0.07 7314.23 bcd
462.79
2.98 ab
0.27 1.58 ab
0.21 16.05 a
1.22 0.18 de 0.07 8695.10 a
462.79
3.31 ab
0.38 1.63 ab
0.31 14.02 abcd
1.77 0.30 cde 0.10 7889.80 abcd
670.99
3.38 ab
0.27 1.27 abc 0.21 9.48e f
1.22 0.57 b
0.07 6287.36 de
462.79
3.20 ab
0.27 1.43 ab
0.21 15.71 ab
1.22 0.21 de 0.07 8018.47 ab
462.79
3.48 a
0.27 1.35 abc 0.21 12.51 bcde
1.22 0.30 cde 0.07 8014.08 ab
462.79
3.50 a
0.27 1.48 ab
0.21 14.16 abcd
1.22 0.16 e
0.07 8740.92 a
462.79
3.38 ab
0.27 1.75 a
0.21 11.71 cde
1.22 0.46 bc
0.07 7805.06 abcd
462.79
3.64 a
0.38 0.67 c
0.31 15.87 abc
1.77 0.09 e
0.10 7189.32 abcde 670.99
3.31 ab
0.38 1.44 abc 0.31 12.61 abcde 1.77 0.27 cde 0.10 6883.02 bcde
670.99
1.74 c
0.38 1.90 a
0.31 6.62 f
1.77 0.82 a
0.10 3123.28 f
670.99
3.14 ab
0.38 1.47 abc 0.31 11.97 abcde 1.77 0.11 e
0.10 6365.82 cde
670.99
a
SEM = standard error of the mean.
b
Log transformation of percentage of plants lodged.
Means in each column followed by the same letter are not significantly different (Least Significant Difference test; P ≤
0.05).
36
Hybrid
341
360
42A
434
578
580
599
677
680
684
704
735
737
neg
pos
Hybrid
341
360
42A
434
578
580
599
677
680
0.05).
37
684
704
735
737
neg
pos
Table 2. Adjusted means of evaluated traits combined over two years for insecticide treated plots.
Root Size SEMa Injury
SEM Mass (g)
SEM Lodgingb SEM Yield (kg/ha)
SEM
2.90 ab
0.23 0.18 abc 0.09 10.78 efgh 0.94 0.00 b
0.03 7918.67 def
423.29
2.90 ab
0.23 0.13 bc
0.09 12.76 cdef 0.94 0.00 b
0.03 8474.79 cd
454.64
3.15 ab
0.23 0.11 bc
0.09 10.50 fgh
0.94 0.00 b
0.03 8344.86 d
423.29
2.93 ab
0.23 0.34 ab 0.09 15.44 ab
0.94 0.00 b
0.03 9824.28 ab
423.29
2.92 ab
0.33 0.26 abc 0.12 17.70 a
1.36 0.00 b
0.04 9279.46 abcd 613.30
3.53 a
0.23 0.14 bc
0.09 8.37 h
0.94 0.06 b
0.03 7151.66 ef
423.29
3.25 ab
0.23 0.21 abc 0.09 14.35 bcd
0.94 0.00 b
0.03 8589.65 cd
423.29
3.18 ab
0.23 0.42 a
0.09 12.69 cdef 0.94 0.03 b
0.03 9642.25 abc
423.29
3.40 a
0.23 0.26 abc 0.09 12.81
0.94 0.00 b
0.03
bcdef
10345.24 a
423.29
3.20 ab
0.23 0.14 bc
0.09 13.16 bcde 0.94 0.00 b
0.03 8688.82 bcd
423.29
3.48 a
0.33 0.25 abc 0.12 14.68 abcd 1.36 0.00 b
0.04 7511.94 def
613.30
2.92 ab
0.33 0.18 abc 0.12 15.78 abc
1.36 0.00 b
0.04 8531.28 bcde 613.30
3.03 ab
0.23 0.10 c
0.09 12.18 defg 0.94 0.02 b
0.03 7859.67 def
423.29
2.53 b
0.33 0.35 abc 0.12 8.94 gh
1.36 0.69 a
0.04 3990.09 g
613.30
3.28 ab
0.33 0.06 bc
0.12 11.94
1.36 0.00 b
0.04
cdefg
6701.62 f
613.30
a
SEM = standard error of the mean.
b
Log transformation of percentage of plants lodged.
Means in each column followed by the same letter are not significantly different (Least Significant Difference test; P ≤
38
Size
Injury
Mass
Lodging
Yield
1.0
Power (1-ß)
0.8
0.6
0.4
0.2
0.0
4
5
6
7
8
9
10
11
12
Number of replications
Fig. 1. Results of five post-hoc power analyses, conducted for each of the tested measures
of maize performance, designed to determine whether the number of replications used was
sufficient to detect variability among the hybrids using the ‘difference approach’ with a
probability of 1-ß (where ß is the Type II error rate for testing a null hypothesis).
39
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ACKNOWLEDGMENTS
This project was made possible through the support and guidance of many important
people. Foremost, I would like to thank the three members of my program of study
committee Dr. Jon Tollefson, Dr. Linda Pollak, and Dr. Ken Holscher for their guidance
throughout my program. Their attention to all details improved not only the research I
conducted and my written work, but me as a scientist. I especially thankfully acknowledge
Dr. Tollefson for not only his guidance in the project, but as my academic advisor. It was
Dr. Tollefson who granted me the opportunity to pursue this research and my degree.
Through him my dreams became possible; his support, willingness to help, and kindness
provided me with the opportunity to complete an important chapter in my life and for this I
will be forever grateful. You and Mrs. Tollefson have touched my heart.
To Dr. Franja Bača and Mr. Goran Stanković, my mentors in Serbia, I send thanks
to overseas for the countless emails that provided academic and professional guidance. The
time you devoted to helping me is commendable and of course, without your work at the
MRIZP this work would not have existed. Dr. Bača, your benevolence is obvious and is
admirable. Thank you.
Thank you to Mr. Jim Oleson for his considerable donation of whole-hearted
support in my agricultural pursuit. His non-judgemental patience in educating and training
me in even the most rudimentary agricultural practices did not go unnoticed and was greatly
appreciated. Dr. Jarrad Prasifka receives a special thank you for his vigilance. When
frustration made itself at home in my mind, Dr. Prasifka came through with invaluable
56
input and aid in statistical analysis. Without him, the project would not have come together
as well and as timely as it did.
Thank you to Dr. Patti Prasifka, my in-office mentor and friend, to John Hinz for
offering his time and equipment for two seasons of plot-harvesting, and the seasonal
workers who helped me prepare and process samples. The enthusiasm of the summer
workers, even in the midst of the smoldering heat or blisters forming, made the days go by
quickly with the work completed well. The seasonal workers were Nick Kiley, Will Svec,
Rob Acker, Dado Stare.
An extra special thank you to Dado Stare. Dado not only worked the hours of the
day with me, but provided me with affectionate support, encouragement, and most
importantly a loving heart and face when the working hours were over. Your giving heart
deserves the highest regard.
To all of the faculty, staff, and students who I have crossed paths with during my
time at ISU, thank you. Each one of you, and there are many, have molded me into who I
am today. I have learned about science and about insects from you and I have had the
opportunity to grow among people of the highest caliber. But what I am happiest about is
that I can know that I have that many new friends; friends that I respect, friends that I look
up to, and friends I wish to keep for the rest of my life.
To my sister Lydia Rosanova and my brother George Kadlicko, thank you for your
patience, input and encouragement. Even when we are countries apart, knowing that we
would see each other ‘soon’ always gave me something to look forward to.
All my friends, especially Dr. and Mrs. Laurent and Linda Hodges, Jeff Maney,
Courtney Perry, Olivia Gismondi, Sonia Cosentino, and Jessica Belmonte, thank you. Your
57
affectionate gestures always came at the perfect time and in the perfect dose. Thank you for
your love and encouragement, you have showed me that distance means nothing and
friendship everything.
Finally, to my parents Ljuba and George Kadličko, the love that you provided, even
when it felt like we are a million miles apart meant that I knew when I had good news or
bad, that eager ears waited to listen and the biggest hearts were on my side. I remember and
treasure every word you have ever told me and everything you have ever taught me,
through the both of you I had the strength, inspiration, and willingness to do everything I
have done. To the two of you I owe everything; I hope that I can some day in some way
show you my gratitude. Thank you and I love you both with all my heart.