J PRESENTED TO THE GRADUATE CiOUNCIL OFi
THE UNIVERSITY OF FLORIDA
JLFILLMENT OF THE REQUIREMENTS pJo TIM
U L J. O F T q ..... ib, iii h i ...................................
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The author wishes to acknowledge his sincere
appreciation to Dr. D.H. Habcck for his guidance and
patience while serving as chairman of his supervisory
lHe is also grateful to Dr. T.E. Summers,
Dr. H.L. Cromroyv, Dr. L.C. luitert,and Dr. E.S. Horner
for their critical review of this work.
He wishes to acknowledge Dr. S.C. Schankl and
Dr. J.L. Nation for serving on the supervisory committee.
Appreciation is also expressed to Ing. E. Morales
H. and Ing. C. Salas F. for their assistance in
conducting research in Costa Rica.
Special thanks are e::tended to Eliecer and Cccclia
Campos for their hospitalities extended to the author
and his wife during their stay in Costa Rica.
He vould especially like to thank his 'ijfe for her
patience and encouragement.
This investigation was supported by an assistantship
from the Center for Tropical Agriculture, for which the
author is sincerely appreciative.
TABLE OF CONTENTS
ACKNOWLEDGMENTS . . . . . . . . . i i
LIST OF TABLES . . . . . . . . . . .
ABSTRACT . . . . . . . . . ii
INTRODUCTION . . . . . . . . . . 1
LITERATURE REVIEl . . . . . . . . 8
Taxonomic Relationships of Several Corn Borers . 11
Bionomics of Several Borers of Maize . . . 12
Zeadiatraea lineolata . . . . . . 12
Diatraen saccharalis . . . . . . 14
Ostrinia nubilalis . . . . . . . 16
Maize Resistance to Ostrinia nubilalis . . . 17
Effect of plant height and maturity . . . 1
Effect of soil fertility . . . . . 19
Effect of insect behavior . . . . . . 20
Chemical basis of antibiosis . . . . 21
METHODS AND MATERIALS . . . . . . . . 25
Biological Materials . . . . . . . 25
Insect species ........... . 25
Collecting . . . . . . . . . 25
Mai e varieties, lines, and single crosses . 26
Insect-Pearing Techniques . . . . . . 30
Zeadiatraea lineolata . . . . . . 30
Diatraea saccharalis . . . . . . . 30
Resis;:ance in Maize to Diatraea saccharalis . 34
Ovipositional Studies . . . . . . . 35
Experiment 1 . . . . . . . . 35
Experiment 2 . . . . . . . . . 36
Table of Contents, Continued
Antibiosis Studies . .
Experiment 3 . .
Resistance in Maize to Zeadiatraea
Costa Rican field tests in 1967
Experiment 4 . .
Experiment 5 . . . .
Costa Rican field tests in 1968
Experiment 6 . . .
Experiment 7 . .
Experiment . . .
Analysis for 2,4-Dihhyd roxy-7-metho
3-one . . . . . . .
Synthesis of Benzo:,azolinones .
Technique e 1 . . .
Technique 2 .
Standard Curve for 6:1BOA and BOA
Recovery Standards for 6MBOA .
Analysis of Varieties from Experim
RESULTS . . . . . . .
Ovipositional Studies . . .
Diatraca saccharalis . .
Zeadiatraea lineolata . .
Antibiosis . . .
Diatraea saccharalis . . .
Zeadiatraea lineolata . . .
Effect of larvae on plants
Analysis of 6ilBOA . . . .
DIS USSIO . . . . . . .
SUHtlAfY . . . . . . . .
LITERATURE CITED . . . . .
BIOCRAPHIICAL Si'KETCH . . . . .
e n t s
. . 37
. . . 37
a . . 37
. . . 37
. . . 37
. . . 38
. . . 38
. . . 38
. . . 39
. . . 39
LIST OF TABLES
1. Pedigree and source of inbred lines,
varieties, and collections of maize tested
in Costa Rica and Florida . .. . . 27
2. Composition of Shorey and Hale's artificial
media and a modification of the diet . 32
3. Composition of artificial medium used by
Hensley and Hammond for rearing the
sugarcane borer larvae. . . . . . 33
4. Distributions of egg masses of D. saccharalis
per hill in Experiment 1 and their fit to
the expected Poisson and negative binomial
models . . . . . . . . . 50
5. Comparisons between means of ovipositional
sites of maize by 1). saccharalis in
Experiments 1 and 2 . . . . . . 51
6. Analysis for homogeneity of variance of
varieties . . . . . . . 55
7. Analysis of variance for eggs, larvae and
pupae, and tunnels for D. saccharalis and
Z. lineolata per maize variety. . . . 56
8. Comparisons between mean number of D. saccharalis
eggs per mass per maize variety in
Experiment 2 . . . . . . . . 57
9. Analysis for correlation of eggs, larvae and
pupae, and tunnels for D. saccharalis and Z.
lineolata. . . . . . . . . . 58
10. Mean number of D. sacchlaralis eggs per mass
and percent of masses laid on different
leaves in Experiment 1. . ... . . 59
11. Distributions of Z. lineojata egg masses per
hill in Experiment 5 and their fit to the
expected Poisson and negative binomial models 61
List of Tables, Continued
12 Distributions of Z. lineolata eggs per hill
in Experiment 5 and their fit to the
poisson and negative binomial models. . .62
13 Comparisons between mean number of Z.
lineolata eggs per maize variety in
Experiment 5 . . . . . . . .64
14 Correlation of Z. lineolata egg masses per
hill :ith eggs per hill and with eggs per
mass in E:xperimcents 4 & 5 . . . .65
15 Correlation of Z. lineolata eggs per hill
with tunnels in Experiment 4.... . . . 67
16 Correlation of Z. lineolata larvae pll.s
pupae with eggs per lill in Experiment 4.. 68
17 Comparisons between mean number of Z.
lineolata tunnels per maize variety in
Experiment 4 . . . . . . . 69
18 Comparisons between mean number of .
lineolata tunnels per maize variety
in Experiment 6.. . . . . . . 70
19 Comparisons between mean number of .
lineolata tunnels per maize variety in
Experiment 7 . . . . . . . 71
20 Comparisons between mean number of Z.
lineolata tunnels of maize varieties
in Experiment 5 . . . . . . 72
21 Correlation of Z. lineolata larvae and pupae
with tunnels per hill in Experiment 4 . 73
22 Comparisons between mean number of Z.
lineolata larvae and pupae per mai;:e
variety in Experiment 6. .. . . 7 4
23 Comparisons between mean number of Z.
lineolata larvae and pupae per maize
variety in Experiment 7 . . .75
List of Tables, Continued
24 Correlation of Z. lineolata tunnels in
plants of the same hill for
Experiment 5 . . . . . . . 76
25 Location of Z. lincolata tunnels in maize
stalks in Experiment !- . . . . 77
26 Recovery of 6MBOA from maize sample UF9
fortified and unforrified with 61BOA . 80
27 Results of analyses for 6MBOA in varieties
in Experiments 6 & 8S . . . . . 81
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of
Doctor of. Philosophy
RELATIONSHIP OF RESISTANCE IN MAIZE
(Z A MAYS I,.) TO TWO RELATED
SPECIES OF PYRALIDAE:
DIATRAEA SACCHARALIS (F.)
Z EA D IAT RAEA L I N E 0 LA T A (ULK.)
James Lynn Overman
Chairman: D.H. Habeck, Ph.D.
Magor Department: Entomology and Nematology
Tests for resistance in S4 varieties of maize to Diatrnea
saccharalis (F.) and 86 varieties of maize to Zeadiatraea
lineolata (11 ) :were conducted respectively in Gainesvil l ,
Florida and Alajuela, Costa Rica. Data were taken on thc
mean number of eggs, egg masses, tunnels, and larvae plus
pupae per variety for both insect species.
lean number of eggs per variety and mean number of eggs
per mass per variety were both good indicators of attractiveness
to oviposition to the two insect species. The ratio of eggs
per mass was independent of the density of egg masses per
plant. The physiological or morphological basis of maize
attractiveness to oviposition was not determined.
Different ovipositional sites on maize (upper leaf sur-
face, lower leaf surface, etc.), for D). saccharalis dif-
fered in nean number of eggs, egg masses and eggs per mass.
The upper leaf surface along the midrib was the preferred
Mean tunnel and larval counts per variety were correlated
for both species. Tunnel data were a more efficient index
than larval counts. Variety Poey T-66 consistently w'as
rated as most resistant to Z. lineolata. The physiochcmical
mechanism of this resistance was not determined.
Larval feeding of Z. lineolata resulted in an average of
21 cm of tunnel damage per plant. From 12 to 29% of the
plants had tunnels in their ear shanks.
Varieties differed little in their content of 6i1BOA and
all varieties were quite low in comparison to published
reports of varieties resistant to Ostrinia nubilalis (Hubn.).
Corn or maize (Zea mays L.) is an important crop,
both from an historical and current viewpoint, in the
Western Hemisphere. Unfortunately, consumption of this
crop exceeds production in several countries, e.g., Costa
Rica. This disparity has caused concern and prompted the
initiation of several programs to encourage greater pro-
duction through better cultural practices and allocation of
more land to this crop. The primary consideration for most
farmers involves the economics of the crop. Unless high
maize yields can be produced at a profit, such programs
stand little chance of success.
Edaphic, climatic, and biotic factors are all related
to the potential yield of maize for a given environment.
The objectives of the agricultural investigator are to
determine how these factors are related to eacl other and to
crop yield. his studies are directed toward finding v'ays
to manipulate these factors so higher yields can be obtained.
It is important, however, that changes ini the environment be
biologically :ournd both for the present and the future.
The biotic environment involves the interaLction of the
agronomic crop with other biological organisms. This environ-
ment is more complex in the tropics than in temperate zones,
and cultural methods as practiced in the temperate zones
have often proven unsatisfactory when applied indiscrimi-
nately in the tropics. Pesticides are widely used for the
control of many insects in the tropics, and their continual
use has led to the development of insecticidal resistance
in the pests while diminishing populations of beneficial
insects. Thus, many farmers have arrived at the untenable
position of lacking both effective insecticides and biolo-
gical controls to protect their crops. In addition,
chemical controls, when improperly used, can cause acute
effects in non--target organisms. Better methods of insect
control are essential to obtain higher crop yields and
reduction of the effects of ecologically unsound methods of
An integrated system, using a maximum of biotic
controls with a minimum of chemicals, is a more desirable
approach. One aspect of biotic control is breeding for
host resistance. The basic flaw in chemical control stens
fro. the use of a static entity, the insecticide, to
ccntrol a dynamic and mobile group of organisms. When not
all of a population is killed by pesticides, a selection
pressure is applied to the genetically variable insect
population. Selection pressure in this system favors
certain insect genotypes, often resulting in the develop-
mcnt of resistance. Biotic control, either by parasites,
predaors, or resistance in the host plant, differs from
chemical control in that selection operates against both
organisms. This interaction often reaches an evolutionary
equilibrium in which neither the host nor its parasite is
Breeding plants for resistance to insects involves
experimental evolution directed toward an increased
advantage for the host plant. Any plant breeding program
such as this necessitates a thorough understanding of the
two organisms; their physiology, behavior, morphology,
The adaptability of any plant species to any specific
environment is partly a function of the individual plant's
ability to develop and reproduce in spite of adverse
environmental factors such as insect attacks. The inter-
action of phytophagous insects with their host plants often
results in a selection pressure on the host plant for those
physicochemical and morphological factors that will permit
the plant to develop normally. The plant breeder may
accelerate the process of selection by intensifying selection
for those qualities he deems important for resistance.
Within a given randomly mating plant population, it
is expected that variance will exist between plants in
their response to insect attack. Plants may also change
in their expression of resistance as they mature. The
observed variance is composed of genetic and environmental
variance and an interaction of the genetic and cnviron-
mental components. In developing resistant varieties, we
are primarily interested in the inherited factors and the
stage or stages of the plant's growth at which selection
should be made.
Genetic advance for plant resistance to insects through
selection may follow the equation Gs=k cr 12 (Allard, 1960).
This refers to selection for a single trait when Gs repre-
sents gain from selection, k represents selection intensity
in standard units, m represents the phenotypic standard
deviation of the population, and h2 represents heritability.
Heritability, in the narrow sense, is the ratio of additive
genetic variance to total variance. Genetic progress
toward resistance is complicated by the great diversity of
insect species and their various developmental stages that
may interact with the host. In addition, there is the
possibility of races developing that are able to attack
resistant crops (Gallun, Deay, and Cartwright, 1961;
Resistance in plants to a single species of insect
may arise in any or all of three forms; antibiosis,
tolerance, or non-preference. For phytophagous Lepidoptera,
antibiosis and tolerance affect only the larval stage.
Antibiosis is the characteristic of the plant to adversely
affect the insect. Tolerance is a function of tlhe plant's
ability to survive despite insect damage. It does not
adversely affect the insect. Plants may avoid insect
damage by being unattractive for oviposition by the gravid
female moth. Non-preference may involve physicochemical
or morpological characteristics which cause the normal
sites for oviposition on the plant to be rejected or not
identified by the female insect. Thus, for a particular
species of insect, there are usually several factors
contributing to the total resistance of the plant. Each
factor nay be genetically independent, having its own
k, p, and h12 values.
The selection intensity v for any single factor follows
the formula v= 3C\ hen N equals the number of characters
being selected and V equals tle percent of the population
left after selection (Allard, 1960). Assuming equal
selection for two factors (A and B) with 5, of
the popular ion being saved, the selection intensity for
each trait would be equivalent to saving 22X of the
population. Therefor e, the intensity of selection for any
single genetic factor is reduced in proportion to the
number of independent resistance factors involved.
A more complex situation occurs when a plant popula-
tion is being selected for resistance to two or more insect
species. In this case, we may consider several possible
relationships between the resistance factors and the
parasites. Plant resistance to two or more species may be
positively correlated, negatively correlated, or not
correlated. Lack of correlation occurs when the resistance
is comprised of two distinct mechanisms, each of which
affects physiological systems that are peculiar to one of
the species. Selection for resistance in the host plant
to one of the species will not affect the host's relation-
ship to the other species. Positively correlated resis-
tance occurs when selection for resistance to one species
has a similar effect on the host's relationship to the other
species. Positive correlation may be partial or complete,
depending on the degree that the physiological systems in
the different species are affected the same by the
resistant mechanisms of the plant. Negative correlation
occurs wlhie resistance developed to the selecting species
renders the plant susceptible to other species.
Analysis for correlation of resistance to different
insect pests of agronomic crops needs to be investigated
for several reasons. Many closely related species are of
economic importance in the same crop gro.'n in widely
separated geographical areas. Despite quarantine measures,
new insect species are being introduced into agricultural
areas. Know-ledge of the type of resistance correlation in
agronomic hosts of the-se insects vould be of immense
importance in designing breeding programs for resistance.
The objectives of this investigation were to
dcternine some of the in erct iors between maize, Zen
na s L. and two related species of Pvra.lidae; the sugar-
ca o bore a Dia tr.ea s h ralis (i'.), in Florid a nd the.
n otropicnal cornstall: borer, Ze d i traea lin.?olata (l'1k.),
in Costa Rica, Central America. The study involved analysis
of resistance of the same lines of maize from Colombia,
South America, to both species. In addition, investiga-
tions in Florida were carried out on single crosses from
the Midwest which had previously been analyzed for
resistance to the European corn borer, Ostrinia nubilalis
(Hbn.). Zeadiatraea lineolata was also tested against
local varieties from Costa Rica and a collection of lines
from the Rockefeller Foundation in Mexico.
Cross resistance is the capacity of any single genetic
factor in a plant species to contribute to the plant's
resistance to more than one organism (i.e. different
species of insects, fungi, nematodes, etc.) in the biosphere.'
The importance of cross resistance becomes evident upon
consideration of the number and diversity of phytophagous
organisms associated with a plant species in a specific
locality. Among the more important phytophagous organisms
are the insects. Painter (1955) catalogued over 180
species of insects associated with maize in Guatemala,
Central America. Only about half of the species could be
identified by specialists of the different insect groups.
Although not all of these species were of significant
economic importance, the list did include large popula-
tions of several economic pests; Diatraea saccharalis
(Fabr.), Zeadiatraea lineolata (W1 k.), Euxesta major
V.ulp., S.pondio tera frugiperda (Smith), and over 15 species
of Diahroti.a and related genera. In addition, intra-
specific differences exist in many insect species in the
form of races. Each stage of an insect species may also
react differently to a resistant f-ctor ir. a plant species.
Bigger, Snelling. and Blanchard (1941), studying resistance
in nmize to the southern corn rooct:orm (Diahrotica
undecimpuncrata Fabra.), found no correlation between
resistance to larval feeding on roots and leaf injury
by adults. Painter (1955) stressed that resistance is
not developed to aphids, borers, or thrips as a group,
but to individual species.
It is expected that the maximum resistance developed
by a plant species would be toward the insect species most
closely associated with it. To the degree that other
insect species or biotypes differ in their behavior or
physiology, we can expect a lack of correlation of resis-
tance to these other species. Optimal plant resistance
may result from the combination of several factors; one,
a few, or all of which may be correlated to more than
one species. Only when the factors causing the resis-
tance are isolated can judgements be made concerning
cross resistance. Failure to isolate these factors has
led to contradictory results concerning cross resistance
in insects. Huber and Stringfield (1940) found a
correlation b<'.t.Leeu resistance in maize to the cor:n leaf
aphid and the European corn borer. Franklin (1964)
found that susceptibility to the corn leaf aphid and the
European corn borer is not consistent for all hybrids.
Whc.n an individual factor has been isolated, im re
meaningful data have been obtain.r concerning. cross
resistance. Resistance in maize or stall: rot, )ip]odia
zcae (Schw.), the European corn borer, dnd si':A-iirne may
all be correlated. Anderson (1964) found that five inbred
lines with known resistance to stalk rot, and the European
corn borer were much more resistant to the herbicides
simazine and atrazine than were two susceptible lines.
Resistance in the whorls of maize to the first instar
larvae of the European corn borer has been correlated with
a single factor; the concentration of 6-methoxybenzoxazo--
linone (6MBOA), a degradation product of 2, 4-dihydroxy-
7-methoxy-l, 4-benzoxazin-3-one (DIMBOA) (Klun and
Brindley, 1966; Klun and Robinson, 1969). EeMiller and
Pappelis (1965n,b) found correlation of concentrations of
glycosides, including DIMBOA, in maize, with stalk rot
resistance. The fungistatic effects of 6MBOA and its
analogues have been confirmed by several workers
(Wahlroos and Virtanen, 1958; Honkanen and Virtanen, 1960;
Loomis, Beck, and Stauffer, 1957). The aglucones, DIMBOA
and DIBOA (2, 4-dihydro:xy-l, 4-benzoxazin-3-one), and
their glucosides were isolated from maize sap and found
to deto:ify simazine (Roth and Knusli, 1961; Hamilton and
Moreland, 3962). Thus, a single factor, 6M1;OA, has been
shown to have a multifunctional effect, contributing to
the plant's resistance not only to a species of insect,
but also to a fungus and two herbicides.
Ta'i'onomic I~elationship of Several Corn Borers
The probability of a plant factor giving cross
resistance in insects is related to the similarity of
behavior and physiology of the insects involved. The
species of the closely related phytophagous genera,
Diatraea and Zeadiatraea (Lcpidoptera, Pyralidae,
Crambinae), are associated e::tensively with grasses
(Graminae) in the Ucstern Hemisphere. Their host plants
include all of the major food and forage grasses; maize,
Zea mays L.; sugarcane, Saccharum officinarum L.; rice,
Oryza sativa L.; what, Triticum aestivum L. ; sorghum,
Sorghun bicolor L. ; Johnson grass, Sorghum halepensis
Pers.; Grama grass, Tripsacum latifolium Hitch.;
Guatemala grass, Tripsacum laxum Nash.; bamboo, Bambusa
vulgarus Schrad.; and Digitaria horizontalis Wil-ld. (Box,
1935). Several species of Diatraea: P. saccharalis (F.)
and D. zeacolella Dvar; and Zeadiatraea: 7. lineolata
(013k.) and Z. grandiosella Dyar; are of economic importance
on maize (Kevan 1943, 1944; Painter, 1955; Holloway,
Haley, and Loftin, 1928; Metcalf, Flint, and lictcalf, 1962;
Henderson, Bennett, and MlcQueen, 1966). Box (1955)
Created the genus Zeadiatraea with D. lineolata U11k.
as the type specimen, but the two genera are closely
related with no reliable characters apparent by which
Zeadiatraea species can be separated as a group from those
of Diatraea. More distantly related and separated
morphologically is the European corn borer, Ostrinia
nubilalis (Hubn.) (Lepidoptera, Pyralidae, Pyraustinae).
Like species of Zeadiatraea and Diatraea, the European
corn borer is primarily a borer in graminaceous plants.
It has become well adapted to feeding on maize in the USA
Bionomics of Several Borers of Maize
Zeadiatraea lineolata:--Despite the economic
importance of Z. lineolata, little has been published
concerning its bionomics. The borer is almost completely
restricted to maize; however, it has been reared on wheat,
Guatemala grass, sorghum, and teosinte (Euchlaena
me:icana Schrad.) (l:ox, 1951; Painter, 1955). It has
occurred in sugarcane, but this infestation is thought to
be "accidental"; most published reports of its occurrence
in sugarcane are erroneous (Box, 1951). Kevan (1943)
found that larvae, when introduced on the plants, bored
into maize and teosinte but not adlay (Coi: sp.).
lMyers (1935) concluded that the borer is the only well-
studied borer of Diatraea or Zeadiatraea that is
restricted to a cultivated host plant. The borer is
unknown in the wild state as is its host plant maize.
Gravid females oviposit on the uppermost leaves or
on the husk:s of young ears (Kevan, 1943). Maize plants
are attractive for oviposition when they are between
knee- and shoulder-lieight (Painter, 1955). Adults
appear to be most attracted to maize shortly before
tasseling and scarcely at all after the ears are formed.
Females may lay eggs not only on maize, but also on the
sides of plastic containers (Painter, 1955). The number
of eggs deposired per moth in Trinidad ranged from 187
to 448, with an average of 377.5. The average number
of eggs per mass was 8.96 (Kevan, 1944). When two days
old, the eggs develop bright red transverse bands. The
average time from oviposition to eclosion is five days.
Prior to hatching, the black head capsule is apparent
through the chcrion.
Newly hatched larvae feed externally on the
epidermis of the leaves. Other leaf damage consists of
transverse rows of tiny holes caused by the boring of
the larvae in the uhori before the leaves unfold (Keva;,
1944). The larvae feed on the leaves up to the third
instar, at which time they bore into the stall. They
begin tunneling upward and may bore into the shanks of
ears. Burrows are usually continuous, but frequently the
larvae will leave their tunnels and re-enter the stalk
elsewhere (Kevan, 1944). Presence of tunnels is
indicated exteriorly by frass holes in the stalks.
There are from six to eight larval instars; the larval
stage in the laboratory lasts from 22 to 48 days :.'th an
average of 31.2 days (Kevan, 1944). In dry stalks the
larvae enter a resting stage and remain quiescent until
moisture becomes adequate. The duration of the pupal
stage is from 6 to 13 days; the pupal case may or may
not be left behind in the tunnel.
Adult females live from three to five days; males
live approximately three days (Kevan, 1944). Adults
are occasionally found at lights.
Damage to the host plant consists of disruption
of the vascular system by the tunneling in the stem,
loss of ears caused by tunneling in the pith of the
cob and the shank of the ear, breakage of the Lassel due
to a weakened condition of the stalk, and reduction in
Diatraea saccharalis:--Diatraea saccharalis is the
most ubiquitous species of the genera Diatraea and
Zeadiatraea. Its geographic distribution extends from
Louisiana, Texas, and Florida in the United States to
as far south as Buenos Aires Province in Argentina
(Box, 1935). The species has been found infesting over
56 different species of grasses (Box, 1935). It is of
economic importance on several major agronomic grasses;
sugarcane, maize, rice, and sorghum (Bo:, 1935;
Holloway et al., 1928; Painter, 1955; Kevan, 1943).
Oviposition begins at dusk and continues through-
out the night. The eggs may be deposited on either side
of the maize leaf, but usually are placed along the mid-
rib (Painter, 1955). Holloway et al. (1928) found that
the number of eggs per mass varied from 2 to 50 or more.
Kevan (1944) found the mean number of eggs per cluster
in the laboratory to be 10.0.
The eggs are flattened and oval, about 1.16 mm
long by 0.75 mm wide, and are deposited in clusters,
overlapping one another like fish scales. The egg stage
lasts from four to nine days, depending on temperature
(lolloway et al., 1923). One to two days before eclosion,
the black heads of the larvae are visible through the
chorion. The eggs lack transverse red bands but are
white in color when first laid and later take on an orange
cast (Hollouay et al 1928).
Following eclosion the first instar larvae congre-
gate in the whorl of the plant where they feed on patches
of the leaf epidermis. They may burrow through the leaves
of the whorl before the leaves unroll, leaving transverse
rows of holes in the leaves. After the first molt the
larvae may bore into the midrib of the leaves, feed in
the leaf sheath, or bore into the stalk. Larvae usually
do not bore into the stalk until the third instar. Upon
entering the stalk the larvae generally tunnel upward,
filling the tunnel behind them with frass. The shank of
the ears may also be tunneled (Painter, 1955). The number
of instars varies from 3 to 10, depending on the
temperature. Under favorable conditions the larval period
may be as short as 23 days, while for hibernating larvae
it can last up to 262 days (Holloway et al., 1928). In
the USA, the larvae overwinter in the stalks of sugarcane
and maize. The pupal period varies from 6 to 22 days when
subjected to an average temperature of 82.9F (Holloway
et al., 1928).
Ostrinia nubilalis:--The European corn borer had
its origin in Europe, being introduced in the United
States in 1917. Its original hosts in Europe were probably
graminaceous; but the insect is of economic importance on
hops, Humulus _1_i ulus L., and hemp, Cannabis sativa L.,
as veil as maize. Since its introduction into the United
States, the borer has become well adapted to maize and is
a major pest of the crop.
The gravid female begins oviposition within five
days after emergence, and the eggs are laid within two
weeks. The opaque white eggs are deposited in masses with
their edges overlapping. The number of eggs per mass
ranges from a few to over 100. The mo lh, in selecting
ovipositional sites, sho..s a preference for specific parts
of maize pla Lts of a certain height and stage of growth.
The eggs are usually deposited next to the midrib on the
underside of the leaves. Eggs nay also be placed on the
stem, on thel upper surfaces of leaves, on leaf sheaths,
and on ears (Everly, 1959).
The interval between oviposition and eclosion
varies fro:. 5 to 12 days and is related to temperature
(Hawkins and Devitt, 1953). Prior to eclosion the black
head of the developing embryo is visible through the
chorion of the egg.
The first instar larvae feed on the epidermis of
leaves and bore into the leaf sheaths and the leaf whorl.
Newly hatched larvae boring through the unfurled leaves
cause pin-hole damage in the leaves. Tassel buds and
stems are often bored by early instar larvae when the
plant is more mature. In the second and third instars,
the larvae tunnel in the stalks and may enter the base of
ear shanks. The larvae may pass through as many as seven
instars. They overwinter in stalks.
The pupal stage lasts approximately 14 days, and
the adult stage approximately 6.0 days for males and 8.3
days for females (Hawkins and Devitt, 1953).
Maize Resistance to Ostrinia nubilalis
The economic importance of the European corn borer
has engendered a vast amount of research. As early as
1925, a compilation of all known references pertaining to
this species amounted to approximately 900 titles (Wade,
1925). Since then many workers have investigated dif-
ferent aspects of the insect's relationship to maize. They
have shown that analy..is for resistance is a complex
process requiring the partitioning of the various factors;
edaphic, cliati. c, genetic, and biotic; and determination
of how these factors affect resistance. Many of the
concepts developed from these studies are applicable to
studies of other borers.
Effect of plant height and maturity:--Under natural
infestation, the uniformity of oviposition on the different
varieties determines the type of e:x:perimental design and
statistical analysis that can be used. Uniformity of egg
deposition is in part due to several inherent factors in
maize that affect the attractiveness of the plant to the
female moth. These factors are dependent on plant height
and stage of gro: .th (Everly, 1959).
In a study of hybrids, Patch, Holbert, and Everly
(1942) found that the half of the hybrids silking first
vere an average of 3.4 inches taller at the time of moth
flight than the other half. The early silhing varieties
received more eggs and therefore tended to have more
borers. In the early development of the plant, oviposition
is correlated with plant height. During tasseling, the
stage of the plant's development becomes the primary factor
influencing oviposition (Everly, 1959). Jackson and Peters
(1959), comparing brachytic and normal forms of the same
hybrid, found that the stage of the plant's grot.th was more
important than its height in determining infestation.
Everly (1959) concluded that plant height and maturity
are not in themselves the reason for attractiveness of the
plant, but are related to conditions in the plant that
affect attractiveness. The influence of height and stage
of growth on oviposition was found to be best expressed
by a second-degree parabola (Everly, 1959). The popula-
tion of borers in different strains has been predicted,
and based on the multiple regression of borer population
on strain height and silking date. The strains or
varieties that consistently received fewer eggs or had
fewer borers than predicted were classified as resistant
(Patch ct al., 1942).
Effect of soil fertility:--Various environmental
factors (soil fertility, tilth, rainfall, and temperature)
influence oviposition through their effect on factors
related to the plant's height and stage of development
(Everly, 1959). The effects of various plant nutrients
on borer survival in maize have been investigated by
several workers (Franklin, 1964; Taylor, Apple, and Berger,
1952 ; Cannon and Ortega, 1966). In field tests where
plants cerere manually infested, corn borer survival was
better on vigorous plants than on small, nutrient-
deficient plants of the same age (Taylor et al., 1952;
Frankly in, 1964). Borer survival and crop yield were both
higher under manure management than in the control plors
lacking manure. Survival of the first generation larvae
on the susceptible single-cross hybrid was 10-fold greater
at 200ppm of nitrogen than at lOppm, yet survival in the
resistant hybrid was low and not affected by nitrogen
level (Cannon and Ortega, 1966). Feu larvae survived on
plants of either hybrid when they received 2.5ppm or less
of phosphorous. Survival at 10ppm was triple that at 2.5ppm,
but did not improve at concentrations from 20 to SOppm.
Quantitative measurements of feeding damage and tunnels
were equally as effective in determining the effects of
nitrogen and phosphorous on survival. The factors causing
the differences in survival appeared to be operating whenn
the larvae were in the first and second instars.
Effect of insect behavior:--Spatial distribution of
the European corn borer population is due in part to factors
which are independent of the host plant. The behavior of
the insect in its different stages and the insects's
interaction with parasites, predators, and diseases
result in a heterogeneous distribution of the insecL.
Intraspecific interactions of a given stage of the insect
may be density dependent. These interactions also affect
the distribution of the borer (Beall, 1940; bliss, 1953;
McCuire, 1957; Cohen, 1960; Taylor, 1961; Katti and
Gurland, 1962; Waters, 1959; Fisher, 1953).
Field populations of the European corn borer
larvae are rarely, if ever, distributed at random, but
rather show aggregation or grouping of individuals in a
given spatial unit. Aggregation commonly results from
the behavioral characteristic of the moth laying eggs
in masses. This aggregation parameter can be computed
by either of two distribution formulae:k of the negative
binomial, or b of Taylor's power law (Harcourt, 1965;
Taylor, 1961). Based on the degree of aggregation and
the goodness of fit required, the data may be transformed
to make them amenable to analysis of variance.
Statistical analysis of biological data by analysis
of variance is based on the assumed additivity of the data
and the homogeneity of the variance. In addition, the
variance should be independent of the mean. Frequently
these assumptions are not fulfilled by the European corn
borer data, but in many cases the analysis of variance is
sufficiently robust to permit its use. When there is
doubt concerning the data, the k or b values can be used
in selecting the proper transformation technique for the
data (Southwood, 1966).
Chemical basis of antibiosis:--Antibiosis in maize
to the European corn borer is expressed by increased
larval mortality, the inhibition of larval growth, and
the reduction in larval feeding. Larval feeding by
first instar larvae on resistant maize lines causes smaller
and fewer leaf lesions than on susceptible lines (Becl,
Resistance to larval establishment is correlated with
the concentration of 6-metho:.:yben : o.:azolinone (6MBOA)
(leck, 1957; Klun and Brindley, 1966). Several techniques
were developed for quantitating 6MBOA in maize tissue (Beck
and Stauffer, 1957; Klun and Brindley, 1966; Beck, Kaske,
and Smissman, 1957; Bowman, Beroza, and Klun, 1968). The
6MEOA and related benzo:.azolinones have been isolated
from several graiinaceous plants and also synthesized by
several workers (Wahlroos and Virtanen, 1959; Honkanen
and Virtanen, 1960; Tipton, Klun, Husted, and Pierson,
1967; Gahagan and Mumma, 1967; Smissman, LaPidus, and
Beck, 1957 a,b; Hietala and Wahlroos, 1956).
Beck (1960) found that 6MBOA, when incorporated
into an artificial diet, caused inhibition of larval
growth and reduced feeding. Klun and Erindley (1966)
found resistance in 11 inbred lines to be correlated with
the concentration of 6MBOA. However, when 6MBOA was
placed in diet media containing a vitamin supplement, it
did not have a significant effect on borer development.
In addition, it is doubtful that 6MBOA occurs free in
plant tissue (Uahlroos and Virtanen, 1964). When the
plant tissue is crushed 6MBOA is rapidly released from
its precursors :2, 4-dihydroxy-7-methoxy-l, 4-benzo:xazin-
3-one (DIMBOA) and its glucoside. DIMBOA is found free
in appreciable concentrations in maize (Wahlroos and
The biosynthesis of DIMBOA was studied by applica-
tion of isotopically labeled metabolites to mazic seedlings
(Reimainn and Byerrum, 1964). The aromatic ring is
apparently derived from an intermediate in the shikimic acid
pathway. The 0-methyl group is formed from compounds
contributing to the one-carbon pool; such as methionine,
glycine, and glyceric acid. The tvo heterocvclic ring
carbons are derived from carbons 1 and 2 of ribose. The
compound DIMBOA exists as a monoglucoside in the seedlings
of maize and several other graminaceous plants, but the
aglucone is released en;:ymatically when the tissue is
crushed. Since there is a stoichiometric relation between
6MBOA and its precursors (Klun and Brindley, 1966), analysis
for 6MBOA is an indirect measure of its precursors in
Klun and Brindlcy (1966) suggested that the active
factor is DIMBOA and not 6MBOA. They subsequently found that
DIMBOA inhibited larval development and caused 25%
mortality when incorporated into a diet. The biological
activity was not attenuated by alteration of the vitamin
The concentration of DIMBOA is not static through-
out the development of the plant, nor is the concentration
the same in different parts of the plant (Klun and
Robinson, 1969). The levels of DIIBOA are initially very
high in both resistant and susceptible lines, but
decrease rapidly in susceptible lines and somewhat less
in resistant lines. The average level for five inbred
lines, 15-33 inches tall, was highest in the roots,
followed by the stem, whorl, and leaf tissues. Analysis
of the distribution in maize at pollen shedding
indicated that the concentration was highest in the
developing ear and next highest in the stalk. Concentra-
tions were lower in the leaf, sheath and collar, and
tassel portions. The inbred line B49, which is moderately
resistant to the first instar larvae of the second brood,
had higher concentrations of DIMBOA than the other lines.
The sheath and collar tissues are a primary site for the
first instar larvae on more mature maize.
The two other aglucones 2, 4-dihydroxy-l, 4-2H-
benzoxazin-3-one and 2-hydroxy-7mcthoxy-1, 4-2H-
benzoxazin-3-one, have been identified from maize (Tipton
et al., 1967; Gahagan and Humma, 1967). The relationship
of thesc two compounds and their glucosides to plant
resistance has not been investigated.
METHODS AND MATERIALS
Insect Species:--Determination of species of collected
male moths was made by examination of genitalia. The abdo-
mens were removed from the specimens and immersed in 10% KOH
overnight or in boiling 10% KOH for 20 minutes. The tissue
containing the genitalia was removed from the solution and
placed in 70, alcohol. The genitalia were cleaned of any
adhering tissue; the male genitalia were separated and
mounted in Hover's Modified Berlese Medium on a slide.
Diatraea snccharalis F. in Florida and Zeadiatraea lineolata
(11k.) in Costa Rica were identified by reference to
illustrations of Diatraca and Zeadiatraea sp. genitalia
(Box 1931; Dyar and Heinrich, 1927).
Co]lecting:--Pupae and late-instar larvae of D.
saccharalis collected from sugarcane fields east and south of
Lake Okeechobee, Florida, served as the parent generation for
a laboratory colony at Gainesville, Florida.
One hundred pupae of Z. lineolata were collected from
a maize field at the Finca La Pacifica in Guanacaste,
Costa Rica. Emerging adults were allowed to mate at
random and their egg masses collected for infesting maize
in field experiments in Alajuela, Costa Rica.
Maise varieties, lines, and single crosses:--Inbred
lines and varieties of maize were obtained from Dr. Dale
Harpstcad of the Rockefeller Foundation in Colombia and
Dr. E.J. Wellhausen of the Rockefeller Foundation in
Mexico. Several local Costa Rican varieties and a col-
lection of Chirripo varieties from the mountainous region
of Costa Rica were supplied by Ing. Carlos Salas F.,
of the University of Costa Rica. Mr. R.T. Everly of
Purdue University, .'est Lafayette, Indiana, supplied
several single-cross hybrids from the Midwest. The pedigree
and source of the maize samples that were tested are
shoi:n in Table 1, together with a reference number for
Table 1. Pedigree and source of inbred lines, varieties,
anc collections of maize tested in Costa Rica and Florida.
Pedigree Source Number
Variety Diacol V. 351
Variety Blanco Comun
Variety Diacol V. 153
Variety Am. Theobromina-lO
Variety Am. Monteria-9
Variety Cuba 362
Variety USA 342
Linea Ath. 13B-21 -4-1-4#-1-D
Variety Focy T-66
Variety Eto Blanco
Variety Eto Amarillo
Variety Rocamex V-520-C
Variety Tico H-1
Variety Tico H-2
Variety E1 Covol
Single Cross A x W23
Single Cross A x Os426
Single Cross 0s420 x A
Single Cross A x Oh02
Single Cross Tr x A
Single Cross L317 x A
Single Cross WF9 x A
Single Cross L317 :: Os426
Single Cross L317 x Oh02
Single Cross L337 x: 1123
Single Cross L317 x WJF9
Single Cross 1.317 x Tr
Single Cross L317 x Os420
Single Cross Oh02 x 0s426
Single Cross Oh02 x W23
Single Cross Oh02 x WF9
Single Cross Oh02 x Tr
Single Cross Oh02 x Os420
Mi d we s t
Mi dwes t
Mid wes t
- ~---- ------
Table 1, continued
Pedigree Source Number
S.L.P. Cpo. 1
S.L.P. Cpo. 1
Ver. Gpo. 6
Ver. Gpo. 7
Ver. Guo. 8
Oa Gpo. 5
Ver. r 43
0s420 x 0s426
0s420 x W23
0s420 x WF9
0s420 x Tr
0s426 x W23
0s426 x WF9
0s426 >: Tr
Tr x W23
Tr x WF9
W23 x WF9
Mid we s t
s iidwes t
Mex i co
Me i co
Mex i co
Mex i c o
Table 1, continued
Pedigree Source Number
Tuxp. F.P. (Peru Crist.)
Puerto Rico Cpo. 2
Granada Gpo. 2
Saint Croix Gpo. 2
A z t e c -Tu x p.
R. Dom. Gpo. 3
R. Dom. Gpo. 8
Pto. Rico Gpo. 6
Trinidad Gpo. 1 & 2
Sanvi b a
Antigua Gpo. 2
Cuba Gpo. 1
Cuba Gpo. 2
Cuba Cpo. 4
Cuba Gpo. 5
Haiti Cpo. 1
Pto. Rico Gpo. 1
Pto. Rico Gpo. 3
Pto. Rico Gpo. 6
Saint Croix Gpo. 3
San Vicente Gpo. 3
Sta. Lucin Cpo. 1
Tobago Gpo. 1
Guad. Gpo. 1A
Antigua Cpo. 1
Barbados Gpo. 1
Me >: c o
M e x i c o
Ml c> ico
1M e x i C o
e e x i c o
le :; i c o
M e > i c o
M1e >: i c o
i e i co
1 e x i c o
lHex i co
l e: i c 0
M e x i c o
e x i c o
Mie x: i c o
Se>x i C o
Mex i c o
Me x i co
lex i c o
Me:x i C o
-- -- --
Zeadietraea lineolata:--Emerging adults from field-
collected pupae were placed in gallon jars lined with
waxed paper. Crumpled waxed paper was placed in the jars
and the tops were covered with waxed paper. Moistened
cotton balls were placed in the jars to prevent dessica-
tion. The jars were kept at room temperature. Following
oviposition, the areas of paper containing egg masses were
cut out and kept in petri dishes lined with moist paper
toweling until a short time before hatching. The eggs
in each mass were counted and placed in the whorls of
maize plants that were to be tested for resistance.
Diatraea oaccharalis:--Ovipositional chambers were
prepared from gallon pasteboaid jie cream cartons. The
bottom of each container was covered with moist vermiculite
and the inside walls lined with waxed paper or green paper
toweling. Folded waxed paper or toweling was placed in
the chamber to provide additional surface for oviposition.
The container was covered with a single layer of cheese-
cloth secured with rubber bands.
Newly emerged adults were introduced into the
chamber and allowed to mate at random. The paper lining
and the folded paper were removed periodically and replaced
with fresh paper. Areas of paper containing egg masses
were cut out and placed in petri dishes that had been
lined with damp filter paper and their tops covered with
Alcoa Film (No. 5602). Pinholes were made in the covering
to provide aeration and reduce condensation. Various
sterilants were evaluated for surface sterilizing the egg
masses. Immersion for 30 seconds in 70% ethanol proved
the most effective.
Upon eclosion, one to five first instar larvae were
placed on artificial media in one-ounce clear plastic cups.
The cups were capped and the larvae left until pupation.
Pupae were then removed from the cups, separated according
to sex, and kept in half-pint ice cream containers until
they emerged. Cartons for the pupae were prepared by
covering the bottoms with moist vermiculite to prevent
dessication of the pupae.
Several artificial diets were evaluated for rearing
thb sugarcane borer. The composition of three of these
diets is given in Tables 2 and 3. The Shorcy and Hale
(1965) diet was used during the summer of 1967. Three
generations of borers were reared in the laboratory and
some of the progeny were used for field studies in
Gainesville, Florida. The Shorey and Hale diet was
modified by substituting soaked field corn for pinto
beans. Two generations of borers were reared on tils
During 1967, Hensley and Haimmond's diet (1968),
which had been used extensively in Louisiana, was used to
rear three generations.
Table 2. Composition of Shorey and Hale's artificial
media and a modification of the diet.
Scaked pinto beans
Soaked corn kernels
Original Diet Modified Diet
640 g ---
--- 640 g
100 g 100 g
10 g 10 g
6 g 6.g
3 g 3 g
6 ml 6 ml
40 g 40 g
1920 ml 1920 nil
*Nutritional Biochemicals Corp., Cleveland, Ohio.
Table 3. Composition of artificial medium used by Hensley
and Hammond for rearing the sugarcane borer larvae,
Water 3116 ml
Wesson's salt* 36 g
Casein* 108 g
Sucrose 180 g
Wheat germ* 108 g
Choline chloride'* 3.6 g
Vanderzant's vitamin mixture* 36 g
Ascorbic acid* 5 g
Formaldehyde (401) 5 ml
Methyl p-hydro:;ben zoate 5.4 g
Agn r* 70 g
*NuLtr tional Bic.chemical Corp., C]c veland, Ohio.
All diets were prepared in basically the same manner.
For the Shorey and Hale diet, either dry pinto beans or
corn kernels were soaked overnight and blended in a
Waring blender with half of the amount of water required
by the diet. Formaldehyde solution and other dry
ingredients, except agar, were added to the mi:x:ture while
blending. For Hensley and Hammond's diet the formaldehyde
and other dry ingredients, except the agar, were added
to one half of the total amount of water and blended.
In all cases, the rest of the water was brought
to a full boil and the agar added while stirring. The
agar solution was allowed to cool to about 700C before
blending with the other ingredients.
The Timedia were poured while still hot into one-
ounce clear plastic cups (Premium Plastic, Inc., Chicago,
Ill.). The cups were filled appro::inately half full,
allowed to cool, and capped with plastic-lined cardboard
lids (Smith-Lee Co., Inc., Oneida, N.Y.). The media
were then kept refrigerated until needed.
Resistance in Hlaize to Diatraea saccharalis
A cage 40 feet long, 16 feet wide, and 11 feet high, was
constructed of 20-mesh, 100" Saran insect screen
(Chicopce Hills Ind., New York, N.Y.) with aluminum
Diatraea saccharalis was used to study the relation-
ship of its host plant maize under a restricted biotic
environment. The bottom of the cage was covered with
crushed rock. The screen was found to appreciably
lower the wind velocity and reduce the light intensity
in the cage.
Experiment l:--Resistance in single-cross I idwestern
varieties of maize to the sugarcane borer was studied
under caged conditions in Gainesville. Seeds from source
numbers 18-45 (Table 1) were planted in green plastic
pots, 11 inches in diameter and 11 inches high. The pots
were filled with a mixture of 2/3 fumigated Arredondo
fine sand and 1/3 peat moss. The pots were placed ]-1/2
feet apart in the cage. A completely randomized design
was used with each variety represented twice in each
pot and each varietal pot replicated three times.
After the plants had reached an average height of
36 inches extended leaf measurement, 50 male and 50
virgin female adult sugarcane borers were placed in the
cage in ten half-pint ice cream containers and allowed to
emerge at dusk. The release stations were evenly dis-
tributed in the cage with the sexes in separate release
Data on the number of eggs, egg masses, and their
location on the plants were taken every other day for a
week following the release. Egg masses were circled
with India ink to prevent recounting the same egg masses.
Forty days after the initial infestation, the plants were
dissected and data taken on the number of tunnels and
larvae per plant. The length of each tunnel was also
Experiment 2:--Six Colombian varieties, references
nos. 1-3 and 5-7 (Table 1), were analyzed for resistance
to the sugarcane borer under caged conditions at
Cainesville. The same soil mixture and pots as in the
previous experiment tcre used. Two plants %were grown
per pot and each varietal pot was replicated six times.
The pots were situated in the cage in a Latin Square design.
Thirty virgin females vere placed in half-pint
containers which were used as release sites. Six
release sites containing five females per site were
evenly placed throughout the experimental plot. Thirty
males were placed in six separate release containers in the
Data were taken for the week on the number of eggs
and egg masses and their location on the plants. After
40 days, the plants were dissected and the number of
tunnels, their length, and the number of surviving larvae
for each plant recorded.
Experiment 3:--Seven Colombia varieties, reference
nos. 1-7 (Table 1) were planted in plastic pots. The
seven varieties,were grown with one plant per pot and
each variety replicated seven times. A completely
randomized design was used. When the plants averaged
approximately 36 inches in height, each plant was infested
with 10 newly hatched sugarcane borer larvae. After 30
days the plants were dissected and the number of tunnels
and surviving larvae recorded.
Resistance in Maize to Zeadiatraca lineolata
Costa Rican Field Tests in 1967:--Two initial experi-
ments were conducted in the summer, May through August,
1967, at the agronomy farm of the University of Costa Rica
at Alajuela, Costa Rica. The purpose of these experiments
was to survey maize sources for resistance to the
neotropical cornborer, Zeadiatraea lineolata. Maize
sources evaluated were reference nos. 1-16 (Table 1) in
Experiment 4 and nos. 61-115 (Table 1) in Experiment 5.
Borer infestation in the plots came from the natural
Experiment 4 :--The 16 entries were planted five seeds
per hill and later thinned to two plants per hill. The
experimental design consisted of 16 blocks, each having
16 varietal hills of two plants. The hills within each
block were one meter apart and the blocks two meters apart.
Cultiiral methods were under the supervision of Ing. Carlos
During the ovipositional period of the borer, the
number of eggs and egg masses on each plant was recorded.
The height of the tallest extended leaf was measured for
those plants receiving egg masses. At harvest, data
were taken on the number of tunnels, larvae, and pupae
Experiment 5:--The 56 entries were planted in six.
randomized blocks. Five seeds were planted per hill and
later thinned to two plants. The hills within blocks
were one meter apart and the blocks were two meters apart.
Measuremencs were recorded of the heights of plants
receiving eggs and the number of eggs and egg masses per
plant. At harvest, data were taken on the number of
tunnels and larvae per plant.
Costa Rican field tests in ]968:--Three experin ents
were conducted during June through August, 1968, at the
agronomy farm of the University of Costa Rica. The purpose
of these experiments was to continue screening of maize
varieties and lines from tests in 1967. In addition, a
collection of Chirripo varieties from Costa Pica was
surveyed for resistance under natural infestation.
Experiment 6:--Eight entries (reference nos. 1,3,6,
11, 15, and 61 in Table 1) were planted one hill. per
variety per block in 16 randomized blocks. Five seeds
were planted per hill and thinned to two plants per hill
when the average height of the plants was 36 inches.
Distances between hills and blocks were the same as in
Experiment 5. The plants were infested by placing 20 eggs
in the black-head stage in the whorl of each plant. At
maturity the plants were dissected and the number of
larvae and tunnels per plant recorded.
Experiment 7:--Twelve entries (references nos. 17,
61, 67, 71, 81, 82, 88, 90, 98, and 109 in Table l)were
planted one hill per variety per replicate in eight
randomized blocks. Distances between hills and blocks
were the same as in Experiment 5. The plants were thinned
to two per hill when their average height was 36 inches.
Twenty eggs in the black-head stage were placed in
the whorl of each plant. When the plants had matured,
they were dissected and the number of tunnels and larvae
Exoeriiaent 8:--Fifteen Chirripo varieties referencee
nos. 46-60 in Table 1) were planted in rows one meter
apart and hills within rows one meter apart. Hills were
thinned to two plants per hill. Infestation came from
the natural field population. Data were taken on the
first 10 plants in each varietal row. The number of
tunnels and larvae per plant were recorded.
Analysis for 2, 4-Dihydroxy-7-methoxy-l, 4-benzoxazin-3-one
Two cyclic hydroxamic acids; 2, 4-dihydroxy-7-
methoxy-l, 4-benzoxazin-3-one (DIMBOA) and 2, 4-dihydroxy-
1, 4-2H-benzoxazin-3-one (DIBOA), and their glucosides
have been isolated from maize. No technique has been
developed for directly quantitating these compounds, but
several methods have been developed for quantitating the
degradation products, 6-methoxybenzoxazolinone (6MBOA)
and benzoxazolinone (BOA). The concentrations of 6IIBOA
and BOA are stoichiometrically related to the concentra-
tions of their respective precursors in plant tissue,
thus, the two cyclic hydroxanic acids may be indirectly
Synthesis of Benzoxazolinones
Benzoxazolinone was commercially available from
Distillation Products Industries, Rochester 3, N.Y. The
6-methoxy-benzoxazolinone was not available commercially
and was synthesized by two different techniques.
Technique l:--To 6 & of 5-methoxy-2-nitrosophenol
(Distillation Products Ind.) in 150 ml water in a 250-mi
Erlenmeyer flask, 18 g Na2S204 (Fisher Sci. Co., Fiarlawn,
New Jersey) uere added slowly. The solution was then
heated to 60-65cC for 15 minutes. The solution was
subsequently cooled to room temperature and neutralized
with solid Na2CO3. The neutralized solution was then
extracted with three 50-ml aliquots of diethyl ether.
The ether fraction vas extracted with three 15-ml aliquots
of 4NHC1. The acid fractions, which turned a deep purple,
were combined and evaporated under reduced pressure. The
hydrochloride of 2-amino-5-metho::yphenol was added to an
Erlenmeyer flask along with approximately three grams of
urea. The flask was fitted with an air condenser, placed
in an oil bath, and heated at 1800C for two hours. Following
fusion, the residue was washed with dilute HC1 and the
acid fraction was extracted with diethvl ether. The ether
fraction was evaporated and the residue taken up in hot
water. Activated charcoal was added, the solution stirred,
and the ni::Lture filtered while still hot. Upon cooling,
white needle-like crystals appeared. The charcoal was
washed with diethyl ether, the ether evaporated, and the
residue taken up in hot water. This was filtered and
yielded crystals upon cooling. The two batches of needle-
like crystals were combined and stored in a dessicator
Technique 2:--To 10 g of 5-methoxy-2-nitrosophenol
in 200 ml water, 30 g Na2S204 were added slowly with
constant stirring. The solution was heated to 60-650C
for 15 minutes, cooled, neutralized with solid Na2C03,
and extracted with three 100-ml aliquots of diethyl ether.
The ether extracts were combined and evaporated to dryness
under reduced pressure. The crude residue (7.3 g) was
taken up in 200-ml anhydrous ether. Dichloroaicetyl
chloride (Distillation Products Ind.) was dissolved in
anhydrous ether (35-mi of a 10% solution by volume) and
added by drops with constant stirring.
The blue-purple precipitate of 2-amino-5-
methloxyphenol hydrochloride (2.15 g) was filtered. The
precipitate was added to an Erlenmeyer flask along which
0.75 g urea. The flask was fitted to an air condenser
and the mixture was heated at 180C'C for 2 hours. The
residue was washed with dilute HC1 and the remainder
taken up in anhydrous ether. The I1C1 wash was extracted
with diethyl ether and the ether fractions combined and
evaporated. The residue was taken up in a small quantity
of acetone, and hexane was added until the solution
became turbid. The red crystals which appeared were
placed in 50-ml boiling water, a small quantity of
activitated charcoal added, and the solution filtered.
Upon cooling, white needle-like crystals appeared.
Ultraviolet spectra were taken using a Beckman DI
spectrophotometer. Excitation and emission maxima for
fluorescence were determined with an Aminco-Bowman
spectrophotofluorometer (American Instrument Co., Silver
Spring, Maryland). Spectra compared favorably with
published spectra of 6MEOA isolated from maize seedings.
Stardard Curves for 6MBOA and BOA
The 95% ethanol was redistilled before using. The
synthesized 6:MiOA and the LOA were weighed and stock
solutions made up in 95% ethanol. The stock solution of
6MBOA was diluted with 95% ethanol to give concentrations
of 0.025, 0.05, 0.08, 0.1, 0.25, 0.50, 0.80, 1.0, 2.5, 5.0,
8.0, 10.0, 25.0, 50.0, 80,0, and 100.0 ig/ml. The stock
solution of BOA was diluted with 95% ethanol to give
concentrations of 0.1, 0.5, 1.0, 5.0, 3.0, 10.0, 50.0,
80.0, and 100 Jg/!ml.
Fluorescence was measured with an Aminco-Bowman
spectrophotofluorometer equipped with a xenon lamp and
1P21 detector tube; a 1-cm2 cell was used with a slit
program of 3-2-3-3-2-3-3 mm. The relative intensity
of 6MBOA was read with the excitation wavelength of 280 muL
and emission wavelength of 335 mL. The relative intensity
of BOA was read with excitation set at 264 mni and emission
at 327 mp The 95% redistilled ethanol w.as used as a
Recovery Standards for 6MBOA
The efficiency of the extraction procedure was
determined by fortifying plant samples with 6MBOA and
subjecting the samples to the complete extraction process
used for analysis of 6MBOA.
The Midwestern inbred line WF9 was grown in pots.
When the plants had reached an extended leaf height of
36 inches, the plant whorls were dried and ground in a
Wiley mill. One-gram amnples were placed in 250-ml
round-bottom flasks. The 6MBOA in 95% ethanol or only
the ethanol was added to the flasks to give plant samples
at four different levels of fortification (0, 75, 750, and
1500 ppm). Boiling chips and 150-ml distilled water vere
added to each flask and the flasks fitted with water-
cooled condensers. The flasks were placed on a hot plate
and refluxed for two hours. After extraction, the liquor
was cooled and filtered. The filtrate was brought up to
150-ml with distilled water and then kept refrigerated.
Twenty milliliters of the solution were placed in
a 60-ml separator, funnel and partitioned with three 10-ml
portions of diethyl ether. The ether fractions were
combined and filtered through a plug of 1a2S04 and the
ether was evaporated under reduced pressure.
Alumina columns were prepared for cleaning the
samples. A glass columnn (10 mm ID) was fitted with a
scintered glass disc and 5 g alumina (Fisher Scientific
Corp., no. A-540) were added. The column was washed
with 20-ml 95:' ethanol and the washings were discarded.
The residue from the etler extraction was taken
up in 10-ml 95% ethanol and added to the column. The
receptacle that had contained the residue was rinsed
with an additional 10-ml ethanol and the rinse added to
the column. The material on the column was eluted with
enough 952 ethanol to provide 100-ml effluent. The
effluent was collected in 100-ml volumetric fJ.asks.
The relative intensity of the ethanol solutions \as
determined by spectrophotofluorometry. The ppm of
6MBOA were determined by reference to the standard curve
for 611BOA. The calculated ppm of the unfortified
samples were subtracted from the readings for the fortified
samples to arrive at the level of added 611BOA that was
Analysis of Varieties from Experiments 6 and 7
Field plots 6 and 7 in Costa Rica in 1968 were
thinned to two plants per hill when the average height
of the plants vas 36 inches. The cut plants were placed
in bundles according to variety and dried at 30-500C for
3 days. The dried plants were individually ground in a
Wiley mill and the plant material derived from each was
weighed and placed in a plastic bag.
One-gram samples were placed in 250-ml round-
bottom flasks. Several boiling chips and 150-ml distilled
water were added. The flasks were fitted with water-
cooled condensers and heated on a hot plate for two hours
while the mixture reflu::ed. The contents were allowed
to cool to room temperature, filtered through Whatman
no. 1 filter paper on a Buchner funnel, and the filtrate
was brought up to 150-ml with distilled water.
Twenty milliliters of the filtrate were added to a
60-ml separatory funnel and shaken with three 10 -ml
portions of diethyl ether. The ether fraction was
filtered through a prewashed plug of Na2SO4 in a glass
column. Two boiling chips were added to the effluent,
which was then evaporated under reduced pressure.
The residue was taken up in 10-ml 95% ethanol
and added to an alumina column. The flask was rinsed
with another 10-ml ethanol, which was added to the column.
The column was eluted with ethanol to give 100-ml of
The 6NBOA content of the effluent was determined
by spectrophotof3 urometry, using the excitation and
emission values already mentioned. The relative intensity
(RI) was recorded and the amount of 6MBOA related to this
RI was read from the standard curve for 61IBOA. The
amount of 6HBOA in the original one-gram sample was
determined by the following formula:
ppm for one-gram sample=6MBOA ( g/ml) X dilution factor/ Ef.
Ef refers to the efficiency of the extracting procedure.
The expression of resistance in maize to Diatraea
saccharalis and Zeadiatraea lineolata results from the
interaction of plant factors with the insect's biology
to reduce the adaptation of the insects to their host
plants. Reduction in adaptation can result from antibiosis
or failure of the moth to oviposit on the plant. Conse-
quently, the analysis for resistance must be designed
to represent the effect of both factors on the insect,
unless from some prior knowledge it is known that one of
them is not significant.
Ovipositional Studies:--The distribution of insects
in an area determines the design and the efficiency of field
tests for resistance. Uneven distribution increases
variance within treatments and necessitates more repli-
cation. Unfortunately, for experimental purposes, insect
distributions are rarely, if ever, evenly distributed.
There are three basic distribution patterns;
regular, random, and aggregated. The regular pattern is
an overdispersed distribution in which the individuals
are not independent. Presence of an individual in a
unit area decreases the probability of finding other
individuals in that area. In the random pattern there is
no interaction of individuals. Each unit area has equal
of having other individuals in that area regardless of
how many are already there. The aggregated pattern is
commonly found among insects. There is a positive inter-
action among individuals. The presence of an individual
in a unit area increases the probability of finding other
individuals in that area.
The analysis of variance is the common test used
to determine if there are significant differences between
neans. This test is based on the assumption of normality,
homogeneity of variances for the treatments, additivity
of the data, and independence of the mean from the
variance. Insect counts seldom describe a normal curve.
Iany insect counts are approximated by the Poisson curve.
In this distribution, the mean is proportional to the
variance. Where the insects are aggregated, the binomial
distribution generally gives a better fit.
Diatraea saccharalis:--Replicated field experiments
with maize in south Florida gave non-significant results due
to the low infestations of the borer. To obtain a larger
insect population, further investigations were conducted in
the screen cage at Gainesville. Investigations under
caged conditions have the advantages of reducing the
effects of certain parasites and predators of D.
saccharalis and restricting the moths' choice of host
plants to those being tested. The screen reduced wind
velocity, evapotranspiration, and light intensity. These
factors probably affected plant growth and perhaps the
behavior of the insects. Whether the cage biased the
effects of only certain plant varieties is not known.
The distribution of egg masses per plant site
in Experiment 1 is given in Table 4. The fit of the
observed distribution to the expected Poisson w'as very
poor. The umderestimation of zero values indicated
that aggregation might be producing the poor fit. The
expected negative binomial distribution reduced the chi-
square value, but the fit was still above the rejection
There were several ovipositional sites on the
plant with the possibility of each site e:x:hibiting a
different distribution. A number of egg masses were
found at the bases of several plants in Experiment 1.
By considering only those egg masses placed on the leaves,
the fit of the distribution was improved to both the
Poisson and negative binomial distributions. By excluding
the egg masses found on the stalks, the category of 5 or
more masses per plant was significantly reduced.
The locations of the egg masses on the plants were
observed and compared (Table 5). Using the criterion of
eggs per mass as an indicator of preferred ovipositional
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sites, the leaf midrib was preferred over the leaf blade.
There was no difference between the upper leaf midrib and
the lower leaf rpidrib. In Experiment 2, the lower leaf
blade was preferred over the upper leaf blade.
Site preference may be exhibited as a difference
in egg masses between sites. In Experiment 1, the leaf
was preferred over the stem. The number of eggs per site
is determined by the number of eggs per mass and the
number of masses per site. Comparisons of total eggs per
site indicated that leaves were preferred over stalks,
upper leaf surfaces were preferred over lower, and the
midrib was preferred over the blade.
A comparison of the different varieties in
Experiment 2 was desired. Using the number of egg masses
per variety as an indicator of attractiveness, the
variance for each variety was determined. The variances
were found to be heterogeneous. Transformation of the
data (Table 6) removed the heterogeneity and permitted
analysis of variance (Table 7). No significant differences
were found between varieties.
Although no significant differences were found
between varieties in the number of egg masses, the
number of eggs per mass might have been different between
varieties. No correlation was found between the number
of eggs per mass and the number of egg masses per plant.
Analysis of variance for differences between varieties in
the number of eggs per mass indicated significant
differences between varieties (Table 7). Comparisons
between varieties by Duncan's multiple range test indicated
Diacol V-153 contained significantly more eggs per mass
than USA 342, Blanco Comun, and Cuba 362 (Table 8).
In Experiment 1, the number of egg masses per plant
was correlated with both the total number of eggs per
plant and the number of egg masses on plants in the same
pot (Table 9). The inajor it y of the eggs (68.6:2) were
placed on leaves 5-7 (Table 10). Several egg masses
were found on the pots.
Diatraea saccharalis in South Florida is adapted
to sugarcane, with other hosts being strictly secondary.
The ovipositional characteristics are adapted to the
dense plant population of sugarcane, a characteristic that
may not be adapted to the more open stands of maize.
Dispersion avay from the site of oviposition is thought
to occur for several reasons. No zero values were found
in the tunnel data from E::periments 1 and 2, as compared
to more than 15', zero values in the egg data. There was
no correlation between the numbers of eggs and tunnels
per plant. Distribution of tunnel data in Experiment 1
was found to approximate a binomial distribution; the chi.-
square value was 7.12 with 7 degrees of freedom. More
than half of the pots, each pot containing two plants,
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Table 7. Analysis of variance
and tunnels for D. saccharalis
for eggs, larvae and pupae,
and Z. lineolat3 per maize
Ex:per i- Da tt
ment No. Analyzed
df Square test
2 Egg masses per
va r iety
2 Eggs per mass
3 Tunnels per
va ri e t y
3 Larvae per
va r i e t y
4 Eggs per
v a r i e t v a
T Tunnels per
var i e y b
5 Eggs per
va r i e t 3
5 Tunnels per
va r i ety
6 Larvae + pu ae
6 Tunnels per
vari e ty
7 Tunnels per
7 Larvae + pupae
8 Tunnels per
va ri e t
S Larvae -I- pupae
Er r o r
Va r Io t Ic s
V a r i e t i e s
V a r i e t i e s
Va r I e t i e s
V a rieties
bData corrected for missing values.
cDifference significant of 5% level
eDifference significanL at 1% level
of probability .
2 2 d
_ __ ~I~_
Table 8. Comparisons between mean number of D. saccharalis
eggs per mass per maize variety in Experiment 2.
Mean No. of Eggs
Means of varieties having same letter are not significantly
different at 5% level.
24 .8 ab
Table 9. Analysis for correlation of eggs, larvae and
pupae, and tunnels for D. saccharalis and Z. lineolata.
Experiment No. Data Analyzed df Correlation
3 Larvae per plant with 41 .506a
tunnels per plant
1 Tunnels with larvae 81 .814a
1 Egg masses per plant 163 .497a
with total eggs
1 Egg masses on plants 81 .866a
in same pot
4 Tunnels per plant with 35 .846a
sum of tunnel lengths
4 Tunnels per plant with 35 .098
average of tunnel length
6 Larvae with tunnels 251 .764a
6 6MBOA per variety with 7 .2416
tunnels per variety
6 6MBOA per variety with 7 .512
larvae per variety
correlation at 1' level.
Table 10. Mean number of D. saccharalis eggs per mass
and percent of masses laid on different leaves in
Leaf No.a Mean Eggs Standard % of Total
Per Mass Deviation Leaf Masses
1 0 0 0
2 20.0 3.6 2.8
3 32.4 17.2 13.3
4 35.9 27.0 7.0
5 29.8 13.3 18.9
6 29.8 19.9 23.1
7 35.3 25.4 26.6
8 50.4 27.2 5.6
9 23.3 9.6 2.8
aLeaves verc numbered from base of plant to top.
received more than 50 eggs per pot, yet this apparently
had little effect on the number of tunnels.
The data concerning eggs and egg masses per plant
in Experiments 4 and 5 were recorded on two dates during
the peak of the ovipositional period. The distribution of
eggs and egg masses from Experiment 5 (Tables 11 and 12)
gave better fit to Lhe expected Poisson and negative
binomial distributions than did the D. saccharalis data
(Table 4). The number of egg masses per hill showed a
better fit to the negative binomial than the Poisson.
The Poisson distribution consistently underestimated the
number of zero values, thereby suggesting a degree of
aggregation. The negative binomial gave excellent fit
in all cases.
Varietal differences were examined in both experi-
ments. Variances associated with varieties were
sufficiently heterogeneous to require transformation
before analysis of variance could be performed (Table 6).
The transformations removed the heterogeneity so analysis
of variance (Table 7) and Duncan's multiple range test
(Table 13) were applied. Significant differences were
found between varieties in Experiment 5, but not in
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In both experiments the number of egg masses per hill
was not correlated with the number of eggs per mass (Table
14). In Experiment 5, the seven varieties rated as having
the most eggs were grouped together, as were the eight
varieties having the least number of eggs (Table 13). The
ratio of number of eggs per plant to masses per plant aver-
aged 1.56+.75 in the first group and 1.09+.27 in the
second group. Differences between means were significant
after analysis according to Snedecor (1956).
Assuming that no non-random errors were made in
counting eggs, the number of eggs received by each plant
should be correlated to the number of tunnels, larvae,
and pupae for each plant of a variety. Analysis for
correlation of eggs with tunnels and larvae plus pupae
was found to be nonsignificant (Tables 15 and 16). This
lack of correlation indicates that factors in addition to
oviposition are important in determining infestation per
Diatra:.a saccharalis :--The efficiency of testing for
antibiosis in maize is related to the evenness of insect
dispersal at the time of egg eclosion and the degree of
migration to surrounding plants. To insure that each
plant was exposed to the same number of insects, each plant
in Experiment 3 received tile same number of first instar
Table 13. Comparisons between mean number of Z.
lineolata eggs per maize variety in Experiment 5.
Mean No, Mean No.
Variety Transformed Variety Transformed
St. Croix Gpo.3
Ver. 4 3
V e r 14
Means of varieties having same letter are not significantly
different at 5. level.
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u a CJ
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larvae. Analysis of variance and Duncan's multiple range
test indicated no differences between larval and tunnel
data from the different varieties (Table 7). Larval
counts were significantly correlated with tunnel damage
Zeadiatraea lineolata:--Tunnel and larvae plus pupae
data from all experiments were tested for homogeneity of
variance to determine if transformation of the data was
required (Table 6). All variances were homogeneous.
Analysis of variance (Table 7) and Duncan's multiple range
test (Tables 17, 18, 19, and 20) were applied to tunnel
data. There were significant differences between varieties
in Experiments 4, 5, 6, and 7, but not in Experiment 8.
The number of tunnels per plant was correlated with the
larvae plus pupae data for most varieties at the time of
plant dissection (Tables 9 and 21).
Analysis of variance (Table 7) and Duncan's multiple
range test (Tables 22 and 23) were applied to the larvae
and pupae data. The number of tunnels per plant was
correlated for plants in the same hill (Table 24). The
number of tunnels per plant was correlated to the sum of
tunnel lengths per plant, but not to the mean length of
tunnels per plant (Table 9). Tunnels were located
throughout the upper portion of the stalk, with 85% of
the tunnels occurring in internodes 3 through 9 (Table 25).
Table 15. Correlation of Z. lineolata eggs per hill with
tunnels in Experiment 4.
Degrees of Correlation
Variety Freedom Coefficient
a = correlation coefficient not significantly different
from zero at 5% level.
Table 16. Correlation of Z. lineolata larvae plus pupae
with eggs per hill in Experiment 4.
Degrees of Correlation
Variety Freedom Coefficient
a = correlation coefficient
from zero at 5% level.
not significantly different
Table 17. Comparisons between mean number of Z. lineolata
tunnels per maize variety in Ex:periment 4.
Mean No. of Tunnels
Variety Per Hill*
Rocamex V-520-C 7.13 a
Amarillo Theobromina 6.94 a
Cuba 362 6.88 a
USA 342 6.75 a
Lto Amarillo 6.50 ab
Blanco Comun 6.31 ab
Tico H-2 5.94 abc
Tico 11-1 5.94 abc
Eto Blanco 5.69 abc
Diacol V-351 5.13 abc
Poev T-66 4.50 be
Diacol V-153 19 c
are not significantly
*leans of varieties having same letter
different at 5': level.
Table 18. Comparisons between mean number of Z.lincolata
tunnels per maize variety in Le:perimen t 6.
Variety MIean No. of Tunnels
lich 166 6.38 a
Cuba 362 5.47 ab
Eto Blanco 5.3 ab
Diacol V-153 5.22 abc
Tico 11-1 i .63 abc
Diacol V-351 i.09 abc
Etio Anarillo 3.91 bc
Pocy T-66 3.56 c
leans of varieties having same letter are not significantly
different at 5% level.
Table 19. Comparisons between mean number of Z.
lineolata tunnels per maize variety in Experiment 7.
Mean No. of Tunnels
Variety Per Plant
Collection 50 3.7 a
Collection 52 3.2 ab
Collection 46 2.9 abc
Collection 26 2.9 abc
Collection 15 2.7 abc
Collection 43 2.6 abc
Collection 62 2.2 abc
Collection 88 2.0 abc
Collection 13 2.0 abc
Collection 20 1.9 bc
Collection 151 1.5 bc
Collection 64 1.5 bc
Collection 83 1.3 c
Collection 65 1.3 c
Collection 11 1.3 c
Means of varieties having same letter are not significantly
different at 5% level.
Table 20. Comparisons between mean number of Z. lineolata
tunnels of maize varieties in Experiment 5.
Mean No. Mean No.
Variety of Tunnels Variety of Tunnels
per Plant per Plant
Trinidad Cpo. 1&2
Ver. Cpo. 8
Ver. Gpo. 6
Ver. Gpo. 7
Haita Cpo. 1
R. Dom. Cpo. 8
Pto Rico Gpo. 3
Cuba Cpo. 1
Sta.Lacin Gpo. 1
Tobago Cpo. 1
S.L.P. Gpo. 10
Pto.Rico Gpo. 6
Oax.. Cpo. 5
Cuba Cpo. 4
Saint Croix Cpo.3
Saint Vicente Cpo.2
Pto Rico Cpo.l
St. Croix GPO.1
Cuad. Cpo. 1A
Cuba Cpo. 5
Cuba Cpo. 2
Antigua Cpo. 1
Pto.Rico Cpo. 1
Means of varieties having same letter are not significantly
different at 5% level.
Table 21. Correlation of Z. lineolata larvae and pupae
with tunnels per hill in Experiment 4.
Degrees of Correlation
Variety Freedom Coefficient
Diacol V-153 15 0.727 h
Poey T-66 14 0.727 b
Diacol V-351 14 0.732 h
Eto Blanco 15 0.487 c
Tico 11-1 14 0.606 c
Tico 11-2 15 0.400 a
El.anco Conun 15 0.707 b
Eto Amarillo 13 0 434 a
lISA 342 13 0.457 a
Cuba 362 13 0.386 a
Amarillo Theobromina 15 0.600 c
Rocamc:;. V-520-C 15 0.566 c
a = not significant at 5' level.
b = significant at 1, level.
c = correlation significantly different from zero at 5. level.
Table 22. Comparisons bet)-eern mean number of Z
Sineolata larvae and pupae per maize variety in
E:: periment 6.
Mean No. of Larvae and
Pupae per Hill
V a rie L V
Eto Am arillo
Means of varieties having same letter are not significantly
different at 5,: level.
Table 23. Comparisons between mean number of Z. lineolata
larvae and pupae per maize variety in Ex;perinen t 7.
Variety HicMan No. of Larvae and
Pupae per Plant
Collection 26 1.8 a
Collection 50 1.5 ab
Collection 88 1.0 abc
Collection 20 0.9 abcd
Collection 62 0.9 abcd
Collection 15 0.9 abcd
Collection 13 0.8 bcd
Collection 52 0.7 bed
Collection 43 0.5 cd
Collection 83 0.4 cd
Collection 65 0. cd
Collection 46 0.4 cd
Collection 151 0.3 cd
Collection 11 0.1 cd
Collection 64 0.0 d
Means of varieties having same letter are not significanLly
different at 5% level.
Table 24. Correlation of Z. lineolata tunnels in plants
of the same hill for E.:periment 5.
Block Degrees of Correlation
A 53 0.333 a
E 52 0.766 b
C 50 0. 27 b
D 53 0.566 b
E 54 0.588 b
F 5 4 0.608 b
a = Significant correlation at 5 level of probability.
b = Significant correlation at 1 1 level of probability.
Table 25. Location of Z. lineolata tunnels in maize
stalks in Experiment 4.
Internodea No. of % of Tunnel
Tunnels Total Length in
1 1 0.4 10.0
2 6 2.3 2.0
3 21 8.1 4.8
4 41 15.8 6.0
5 40 15.4 6.6
6 43 16.6 -6.5
7 32 12.4 5.9
8 23 8.9 6.0
9 22 8.5 5.0
10 9 3.5 4.8
11 10 3.9 4.1
12+ 11 4.3 5.0
aThe higher the number, the higher the internode is on the
Effect of larvae on plants:--Larval damage to maize
consisted of leaf damage caused by first instar larvae,
rupture of the stems' vascular systems by tunneling larvae,
broken stems caused by tunnel-veakened stalks, and fallen
ears due to larvae tunneling in the shank of the ears.
The sum of all tunnel lengths per plant averaged
21 cm. A significant percentage of the ear slanks contained
tunnels. In Experiment 4, 9.9; of 303 ears contained
tunnels. Experiments 6, 7, and 8 had 19, 29, and 12',
respectively, of their plants with tunneled ears.
Analysis of 6MBCOA:--Plant samples from Ex:periments 6
and 8 were assayed for their content of 61IBOA. Readings
were compared with a standard curve (Fig. 1). A correction
factor of 80% was computed (Table 26) and used for
correcting plant sample readings. Results of the analysis
are reported in Table 27.
The 6MIBOA content was not correlated with either
tunnel or larval data in Experiment 6 (Table 9). The
analysis of (.IBOA was low for all varieties tested. Due
to technical problems, the dried tissue w.as not assayed
until a year after the plants were cut. The drying process
or storage might have resulted in a loss of activity in
the samples. Further testing is necessary to determine
the validity of the procedure used.
0 o --
*\ ~ d .
Table 26. Recovery of 61BOA from maize sample WF9
fortified and unfortified with 6MBOA
6MBOA 6MBOA Added % of added
Added Found 6MBOA 6MBOA
(ppm) (ppm) Recovered Recovered
Table 27. Results of analyses for 611BOA in varieties in
Experiments 6 and 8.
Variety No. Sanples Average ppm
Assayed of HILOA
IIich 166 8 430
Cuba 362 7 570
Tico H-1 7 380
Lto Blanco 7 379
Eto Amarillo 7 420
Diacol V153 7 510
Diacol V351 7 420
El Covol 7 330
J S. 7 600
Tuxp. San vibag 1 330
Ver. 8 1 520
Ver. 187 1 3 0
K. Dom. Gpo. 3 1 750
Ver. 39 1 600
Trinidad Gpo 1 & 2 1 1030
Ver. 41 1 520
Pto. Kico Gpo. 2 1 390
St. Croix Gpo. 3 1 490
The proper testing procedures and the determination
of indices of resistance are important considerations in
the analysis for resistance. The interaction of maize
with D. saccharalis and Z. lineolata may result in
ovipositional resistance and resistance to larval survival.
The mean number of eggs per variety and the mean
number of eggs per mass per variety were both good indices
for varietal differences caused by ovipositional attraction.
The eggs per mass ratio were not correlated with the number
of masses per plant. The efficiency of the test could
therefore be improved by considering each mass as a
replicate and using the number of eggs per mass as an
indicator of resistance. Dispersal of egg masses gave a
skewed distribution and resulted in data requiring
transformation prior to analysis of variance. By using
the ratio index, non-normality and heterogeneity of
variance associated with egg mass distribution were
removed. Using this index, Diacol V-153 was found to be
significantly more susceptible to oviposition by D.
saccharalis than USA 342, Blanco comun, and Cuba 362
The ratio index was tested in Costa Rica on varieties
that had shown differences in mean number of Z.lineolata
eggs per variety. The seven most susceptible varieties
composed Group 1, while the 8 most resistant composed
Group 2. The difference between ratios of the two groups
was significantly different.
The psysiological or morphological basis for
attraction resistance to oviposition by the two species
was not determined. Differences in oviposition sites for
D. saccharalis were shown to result in significant dif-
ferences between the mean number of eggs per site.
Varietal differences relating to differences in site
selection by the insect were not determined
The mean number of eggs per mass is a function of
the moths' behavior. The response of the insect to the
plant factors resulting in deposition of eggs probably
results from an interaction of the genotype with the
environment. The mean number of eggs per mass for Z.
lineolata averaged less than two and is comparable to
that of Z. grandiosella, which averages 3 eggs per mass
(Rolston, 1955). The average number of eggs per mass
found here is not consistent with the nine eggs per mass
reported for the same species by Kevan (1944) in Trinidad.
The heritability and environmental significance of this
difference night prove academically interesting and
Sites of oviposition of D.saccharalis were found
to be comparable to published data concerning sites of
oviposition of Z. grandiosella (Rolston, 1955). Both the
sugarcane borer and the southwestern corn borer showed
a preference for the upper surfaces of the leaves along
the midrib. Both species occasionally oviposit on the
stalk. In contrast, Ostrinia nubilalis oviposited more
than 80" of its eggs on the undersides of the leaves
with only about 5% on the upper surfaces. Less than 27
of the eggs were placed on the stem and leaf sheaths
(Everly, 1959). The importance of site differences in
resistance was difficult to evaluate without knowledge
of the mechanism or mechanisms of resistance to ovi-
Larval counts and tunnel damage in the stalks of
maize are common indices of resistance to any of the
stalk borers. Tunnel counts were found to be correlated
with the sum of tunnel lengths and larval counts per
stalk for both D. saccharalis and Z. lineclata. The
average length of each tunnel was not correlated with
the number of tunnels per stalk.
No significant difference was found between mean
number of tunnels per Colombian variety tested against
the sugarcane borer. In Costa Rica, these same varieties
were found to differ significantly between mean number
of tunnels and larvae per variety for Z. lineolata.
Based on larval survival and tunnel data from
e:-periments conducted under natural infestation of Z.
lineolata and natural infestations supplemented with egg
masses, variety Pocy T-66 consistently was found to have
the greatest degree of resistance. Significant differences
were also found between other varieties.
The location of tunnels caused by Z. lineolata
occurred principally in internodes 3 9. Appreciable
numbers of tunnels, more than 10 :cre found in ear
shanks. Unlike Z.grandiosella, Z.lineolata did not
girdle the stalks. Z. grandiosella occurs mainly in
the lower part of the stalk (Rolston, 1955). Ostrinia
nubi]alia is more frequently found in the upper portion
of the stalk and frequently caused breakage of tassels
(Haw-khins and Devitt, 1953). In cage studies, D.
saccharalis occurred throughout the stall, causing both
tassel damage and broken stalks.
Breeding programs for resistance to D. saccharalis
and Z. lineolata v'ill be more efficient when the
physiological or morphological basis for resistance is
recognized. Identification of the physico:hemical
resistant factors and their location in the plants, and
the development of laboratory techniques for their
idenLification and quantitation iill permit more efficient
selection procedures in breeding programs. Biological
variation and the non-uniform nature of D. saccharalis
and Z. lineolata distributions in the field experiments
reduce the efficiency of field testing.
Varieties from Cost Rica were analyzed for their
content of6HBOA to determine if maize resistance to Z.
lineolata was correlated with concentration of 6MBOA
or its precursors. Varieties differed little in their
content of 6NBOA and all were quite low in comparison with
published reports of varieties resistant to the European
corn borer. No correlation was found between field
resistance as measured by tunnel or larval counts and
average ppm of 6IBOA for 5 7 samples of each variety.
Further analysis of these varieties is needed to verify
the observations. The results obtained so far neither
prove nor disprove any relationship between 6EBOA and
resistance to D. saccharalis or Z. lineolata.
Ability to quantitate resistant factors in the
laboratory would be of great use in the study of cross
resistance of a plant variety to different insects in
S U MMA RY
Tests for resistance in 48 varieties of maize to
Diatraea saccharalis (F.) and 86 varieties of maize to
Zeadiatraea lineolata (W11k) were conducted respectively in
Cainesville, Florida, and Alajuela, Costa Rica. Data
were taken on the mean number of eggs, egg masses, tunnels,
and larvae plus pupae per variety for both insect species.
Mean number of eggs and mean number of eggs per mass
were both indicators of resistance in varieties to ovi-
position by the two insects. The number of eggs per
mass was independent of density of masses per plant.
Variety Diacol V-153 had significantly more D. saccharalis
eggs per mass than Cuba 362, USA 342, and Blanco comun.
The physiological or morphological basis of resistance
to oviposition was not known.
Different ovipositional sites on maize for D.
saccharalis differed in mean number of eggs, egg masses
and eggs per mass. The upper leaf along the midrib was
the preferred site.
Mean tunnel anrd larval counts per variety were
correlated for both species. Tunnel data \xre a more
efficient index in that dissection of the plant was not
required. Variety Poey T-66 consistently was rated as
most resistant to 7. lineolata. The physicochemical
mechanism of this resistance was not identified.
Larval feeding of Z. lineolata resulted in an average
of 21 cm of tunnel damage per plant. From 12 to 29%
of the plants had tunnels in their ear shanks.
Varieties differed little in their content of 6MBOA
and all varieties were quite low in comparison with
published reports of varieties resistant to Ostrinia
nubilalis (Hubn.). Further analysis of these varieties
is needed to verify the 6MBOA readings.
Ability to quantitate resistance in the laboratory
would be of great use in the study of resistance of a
plant variety to different species in different environ-
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