gs - .. 3 ‘7‘ 7?
‘nC-"‘-“:

#1. :~
0 f". ,. .1.
Michigan ..-.. “e
I 0 O...
I -! . 531’- 19d"! ' '
LII-fl U will wag-i

 

 

 

This is to certify that the

thesis entitled

ONION FLY BEHAVIORAL RESPONSES TO BACTERIALLY
INFECTED ONION
presented by

Susan Mar 1e Hausmann

has been accepted towards fulfillment
of the requirements for

M. S . degree in W

   

Major professor

Date 3'5’86

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

MSU

LIBRARIES
M

.

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

 

 

 

ONION FLY BEHAVIORAL RESPONSES TO
BACTERIALLY INFECTED ONIONS

by

Susan Marie Hausmann

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Entomology

1986

42.3%

*¥D7

ABSTRACT
ONION FLY BEHAVIORAL RESPONSES TO MICROBIALLY INFECTED ONION

by

Susan Marie Hausmann

When cultured on sterilized onion tissue, Erwinia carotovora var.

 

carotovora (EC), a casual organism of soft rot, and Klebsiella

 

 

pneumoniae (KP), a predominate early colonizer of onions decomposing in

 

the field, produced strong attractants and ovipositional stimulants of

the onion fly Delia antiqua (Diptera: Anthomyiidae). EC cultured on

 

onion produced more potent attractants than axenic onion, KP-inoculated
onion, EC-inoculated potato, or chemical synthetic attractants. EC
cultured on potato (a nonhost of onion fly) produced attractants, but
did not stimulate oviposition. EC cultured on onion is a reproducible
source of attractants as well as ovipositional stimulants.

Q, antigua ovipositional response on mature onions with different
levels of physical and microbial damage was well correlated with larval
developmental performance on those treatments. The increased
suitability of those damaged plants most preferred by ovipositing
females may be due to the ability of first instar larvae to penetrate
the bulb. Since damaged bulbs can be an excellent food source for fall
generation onion flies, good sanitation practices in the fall may

decrease the number of overwintering pupae.

This work is dedicated to my sisters, Kathy Moss and Carol Ann.

ii

ACKNONLEDGEHENTS

My deepest respect and appreciation goes to my major professor,
Dr. James Miller, for his guidance, advice, and perhaps most of all for
his trust in me to develop my own lines of scientific inquiry. I also
thank the members of my guidance committee, Dr. Jack King, Dr. Karen
Klomparens, and Dr. Ed Grafius, for their considerable time and effort
devoted to editing manuscripts, offering unique insights, and sharing a
bottle of champaigne with me after the final oral exam. The other
members of the Miller lab, Marion, Joan, Paul, Jim, Martha, Dave, and
Debie, have become my friends, as well as my colleagues. I will miss

them very much. And thank you, Andrew, for everything.

iii

TABLE OF CONTENTS

LIST OF FIGURES ......................... vi
LIST OF TABLES ......................... viii
GENERAL INTRODUCTION ....................... 1

CHAPTER 1. Production of onion fly attractants and
ovipositional stimulants by bacterial isolates
cultured on onion . . . . . . . . . . . . . . . . . . . 11

Introduction . . . ......... . ........ 12
Materials and Methods ................ . 13
Insects O O O O O I O O O I O O O 000000 O O 13

Bacterial isolates . . . . . . . . . .
Preparation of axenic onion and potato . . . . . . 15
Experiment 1 - Attractancy of axenic onion . . . . 16
Experiment 2 - Attractancy of bacteria cultured

on onion over time . . . . . . . . . . . . . . 17
Experiment 3 - Relative attractancy of EC- and

KP- inoculated onion . . . . . . . . . . . . . 17
Experiment 4 - Attractancy of EC- inoculated

host- and nonhost- -plant. . . . . . . . .
Experiment 5 - Attractancy of EC- inoculated

onion vs. synthetics . . . . . . . . . 18
Experiment 6 - Oviposition on EC- inoculated

host- and nonhost- -plant . . . . . . . . . . . . 19

Results . . . . . . . . . . . . . . . . . . . . . . . 20

Identification of Erwinia carotovora var.

carotovora . . . . . . . . . . . . . . . . . . 20
Experiment 1 - Attractancy of axenic onion . . . . 20
Experiment 2 - Attractancy of bacteria cultured

on onion over time . . . . . . . . . . . . . . 22
Experiment 3 - Relative attractancy of EC- and

KP-inoculated onion . . . . . . . . . . . . . . 25
Experiment 4 — Attractancy of EC-inoculated

host- and nonhost-plant . . . . . . . . . . . . 25
Experiment 5 - Attractancy of EC-inoculated

onion vs. synthetics . . . . . . . . . . . 25
Experiment 6 - Oviposition on EC- inoculated

host- and nonhost- plant . . . . . . . . . . . . 3O

 

 

Discussion . . . . . . . . ....... . . ..... 30

CHAPTER 2. Onion fly ovipositional preference and larval
performance as influenced by physical and microbial

iv

damage to mature onion bulbs ............. 35

Introduction . . . .................. 36

Materials and Methods ............ . . . . . 37

Insects . . . . . . . ........... . . . 37

Host plant .................... 37

Ovipositional choice test ............ 38

Host plant suitability ............. 38

Results ........................ 39

Discussion ...................... 44

GENERAL DISCUSSION ........................ 47
LITERATURE CITED . . . . . ........... . . . . . . . . . 52
APPENDIX 1. Record of deposition of voucher specimens ..... . 59
APPENDIX 1.1 Voucher specimen data ................ 60

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

LIST OF FIGURES

The biosynthesis of the onion flavor precursor trans-
(+)-S-(propen-1-yl)-L-cysteine sulfoxide. L-cysteine
combines with 2-methylpropenoic acid, which is a
derivative of valine. The product is decarboxylated
to give propen- 1-yl-L- -cysteine, which is oxidized,
forming trans- (+)-—S-(propen-1-yl)-L-cysteine

sulfoxide . . . . . . . . . . . . . . . . . . . . . .

The degradation of trans-(+)-S-(propen-1-yl)-L-
cysteine sulfoxide when onion is cut or crushed. The
hydrolysis of the sulfoxide is catalyzed by

alliinases to give propen-l-ylsulfenic acid and the
by-products, pyruvate and ammonia. A transfer of
internal hydrogens in the sulfenic acid forms the
lacrimator, thiopropanal sulfoxide. After hydrolysis,
propionaldehyde, sulfuric acid, and hydrogen sulfide

are formed I O O O O O O C O I O O O C O O O O O O O 0

Onion fly catch as affected by onion bait aged in

the field after chopping . . . . . . . . . . . . . . .

Onion fly attractancy as influenced by length of time

sterilized onion tissue was held before assaying . . .

Influence of incubation time on onion fly attractancy
of EC-inoculated onion. Sterilized onion tissue
inoculated with Erwinia carotovora var. carotovora
(EC) and incubated 1, 2, or 3 days before the

 

 

bioassay O O O I O I O O O O I I O O O O O O C O O O 0

Influence of incubation time on onion fly attractancy
of KP-inoculated onion. Sterilized onion tissue
inoculated with Klebsiella pneumoniae (KP) and

 

incubated 1, 2, or 3 days before the bioassay . . . . .

Relative onion fly attractancy of EC and/or KP
inoculated on sterilized onion tissue and incubated 3

days before the bioassay . . . . . . . . . . . . . . .

Production of onion fly attractants by the
interaction of EC on onion fly host- and nonhost-
plant tissue. Sterilized onion tissue and sterilized
potato tissue inoculated with EC and incubated 3 days

before the bioassay . . . . . . . . . . . . . .....

vi

21

23

24

26

Figure 9.

Comparison of chemical synthetics and EC-inoculated
onion as onion fly attractants. Baits include
dipropyl disulfide (PrzS ) released from polyethylene
enclosures, an aqueous sglution of 2-phenylethanol
(2-PhEt) and n-pentanoic acid, and sterilized onion

inoculated with EC and incubated 3 days before the
bioassay ......

vii

28

Table 1.

Table 2.
Table 3.
Table 4.

Table 5.

LIST OF TABLES

Onion fly ovipositional responses to host- and
nonhost-plant tissue inoculated with EC. Treatments
presented in ovipositional dishes and as volatiles
pumped into ovipositional dishes . . . . . . . . . . .
Onion fly oviposition on undamaged and damaged onions .
Colonization rates of undamaged and damaged onions

Survival of first instar D. antigua larvae to pupal

. 29
. 40
. 41

stage on undamaged and damage on1ons . . . . . . . . . . 42

Heights of pupae and adults from D. antiqua larvae

reared on undamaged and damaged onions . . .......

viii

43

GENERAL INTRODUCTION

The onion fly, Delia antiqua (Meigen), is an economically important

 

insect pest of onion in the temperate Northern Hemisphere, including
many onion-growing regions of the United States, Canada, Europe, and
Asia (Ellis and Eckenrode, 1979). In the field, onion flies mate within
one to two weeks after eclosion (Loosjes, 1976). Female flies oviposit
on or near their host-plant, and the eggs hatch a few days later.
Larvae enter the base of the plant and feed on subterranean plant
tissues for two to four weeks before pupating in the soil. Most
populations overwinter as diapausing pupae. Diapause is not obligatory,
but is induced by lower temperatures and shorter photoperiods (Ramakers,
1973).

There are generally two to three generations a year, but exceptions
occur at the limits of its geographical range. For example, only one
generation occurs in northern Norway (Rygy, 1960). In southern Japan,
both diapause and aestivation occur (Kato, 1958). In Ingham County,
Michigan, there are three generations per year with temperatures and
photoperiods conducive to diapause beginning in August and September
(NCAA, 1984).

The primary host-plant of Q; antigua is onion, Allium cepa. Other

 

plants of the same genus are also attacked, including shallot (A;

ascalonicum), leek (A:_ ampeloprasum), Japanese bunching onion (5.

 

 

fistulosum) and chive (A; schoenoprasum) (Ellis and Eckenrode, 1979). A

 

characteristic common to alliums is the production of various volatile,

sulfur-containing organic molecules (Bernhard, 1970), many of which are
responsible for the familiar odors and tastes of this genus.

Because of some interesting and useful properties of Al_l_i_urr_1 spp.
extracts, scientists from fields as diverse as medicine and entomology
recognized the importance of identifying the secondary metabolites.
Allicin from garlic has antimicrobial (Cavallito and Bailey, 1944;
Tansey and Appleton, 1975) and insecticidal (Amonkar and Banerji, 1971)
properties. There is some evidence that consumption of onion and garlic
produces hypoglycemic effects (Brahmachari and Augusti, 1962) and may
reduce aortic atherosclerosis (Sainani et al., 1979). Many flavor
components in MI“. spp., especially onion and garlic, are also
important in the food and flavor industry.

Analytical chemists and biochemists have revealed much about the
molecular structures and biochemical pathways of onion flavor
components. The flavor precursors in onion are S-alk(en)yl-L-cysteine
sulfoxides, which are biosynthesized from L-cysteine through a series of
complex reactions (Block, 1985). The most abundant precursor is trans-
(+)-S-(propen-1-yl)-L-cysteine sulfoxide (Schwimmer and Friedman, 1972).
Its biosynthesis in an intact onion (Figure 1) starts with L-cysteine
(a) combining with Z-methylpropenoic acid (c), which is itself derived
from valine (b). The product, S-(2-carboxypropyl)-L-cysteine (d), is
decarboxylated to give propen-l-yl-L-cysteine (e), which is oxidised to
the corresponding sulfoxide (f) (Vickery and Vickery, 1981). In mature
onion, at least half of the ‘trans-(+)-S-(propen-1-yl)-L- cysteine
sulfoxide is bound as gamma-glutamyl peptide and is not accessible for
flavor production (Krull et al., 1970).

When onion is cut or crushed, the integrity of the cells is

destroyed. Alliinase enzymes (alliin allylsulphonate-lyases) are

2

HSCI-I CH NP CODE
2 ( 2)

L—cysteine
(a)

H C H C
3 3
CHCH (NHZ) COOH ————D C=COOH
H C H C
3 3
valine 2-methy1propenoic acid

(b) (C)

H3CCH=CHSCH2CH (NHZ) COOH ‘— H3CCI-l (COOH) CHZSCHZCH (NI-12) COOH
propen-l-yl-L—cysteine S-(2-carboxypropy1)-L-cysteine
(e) (d)

H3CCH=CHSOCH2CH (N82) COOH

trans-(+)-S-(propen-1-y1)-
L-cysteine sulfoxide
(f)

Figure 1. The biosynthesis of the onion flavor precursor .trans:(+)-S-
(propen-l-yl)-L-cysteine sulfoxide. L-cysteine (a) comb1nes w1th 2-
methylpropenoic acid (c), which is a derivative of valine (b). The
product (d) is decarboxylated to give propen-I-yl-L-cyste1ne (e), which
is oxidized, forming trans-(+)-S-(propen-1-yl)-L—cysteine sulfoxide (f)
(adapted from Schwimmer and Friedman, 1972; Block, 1985).

released to catalyze the degradation of the S-alk(en)yl-L-cysteine
sulfoxides. Sulfenic acids, RSDH, are formed with ammonia and pyruvate
as by-products (Schwimmer and Friedman, 1972). Because sulfenic acids
are highly reactive, they degrade and react spontaneously to form other
sulfur-containing volatiles giving onion its characteristic odor and
lacrimatory qualities (Block, 1985).

The formation of the lacrimator, thiopropanal sulfoxide provides a
good illustration of the biochemical fate of flavor precursors in onion
(Figure 2). When onion is cut or crushed, alliinases catalyze the
hydrolysis of transf(+)-S-propen-1-yl-L-cysteine sulfoxide (f) to
propen-I-ylsulfenic acid (9), pyruvate, and ammonia (Block, 1985). By
an internal transfer of hydrogens, this unstable sulfenic acid (9) forms
the lacrimator (h), which can undergo hydrolysis to form propionaldehyde
(i), sulfuric acid, and hydrogen sulfide (Block, 1985). Other
degradation products from the sulfenic acid are also possible. For
example, N-dipropyl disulfide is a predominate alkyl disulfide produced
by onion.

The exact composition of onion flavor components depends on the
specific methods and conditions of extraction (Block, 1985). For
example, n-dipropyl disulfide and propionaldehyde are the major products
obtained by steam distillation. A complex array of sulfur-containing
flavor components and associated by-products of onion has been
identified, including di- and tri-sulfides, thiosulphinates, and
mercaptans (Neigisch and Stahl, 1956; Carson and Wong, 1961; Saghir et
al., 1966; Bernhard, 197D; Boelens et al., 1971; Schwimmer and Friedman,
1972; Whitaker, 1976).

The role of non-pathogenic microorganisms in the mediation of onion

H
8+

. ./\/: \/\\..

trans-(+)-S-(propen-1-y1)-
L-cysteine sulfoxide

(f)
H C O
3\/\
S
.___..’ I + O + NH
0 11 c 3
// 3
H 0
propen-l-yl pyruvate ammonia
sulfenic acid
(9)
H H
3C
————§. S+ + S+
\_ \_
H C O H 0
3
(5 1n form) (anti form)
thiopropanal sulfoxide
(h)
H
---> O + H $04 + H28
H C
3
propionaldehyde sulfuric hydrogen
(1) acid sulfide

Figure 2. The degradation of trans- (+)-S-(propen-1-yl)-L-cysteine
sulfoxide when onion is cut or crushed. —The hydrolysis of the sulfoxide
(f) is catalyzed by alliinases to give propen- l-ylsulfenic acid (g), and
the by-products, pyruvate and ammonia. A transfer of internal hydrogens
in the sulfenic acid (9) forms the lacrimator, thiopropanal sulfoxide
(h). After hydrolysis, propionaldehyde (i), sulfuric acid, and hydrogen
igggide are formed (adapted from Schwimmer and Friedman, 1972; Block,
).

metabolite production has received some attention. Intact onions do not
release large amounts of sulfenic acids or their derivatives (Schwimmer
and Friedman, 1972). Yet, some sulfoxide precursors may be exuded by
the roots into the soil where common soil bacteria can convert them to
sulfides and related compounds (King and Coley-Smith, 1969). Following
the lead of Ellis et al. (1979), Hough et al. (1981) found that female
onion flies lay significantly more eggs on seedlings exposed to non-
pathogenic microorgansms than on axenic seedlings. Increased release

of sulfur-containing compounds from non-sterile, healthy seedlings
probably stimulated oviposition (Hough et al., 1981).

The earliest conclusive studies linking onion fly behavioral
responses to specific onion chemicals reported that n-dipropyl disulfide
stimulates oviposition of the onion fly (Matsumoto and Thorsteinson,
1968) and functions as an attractant in the field (Matsumoto, 1970). At
least 23 sulfur containing compounds, including mercaptans,
monosulfides, disulfides, and one trisulfide, have since been shown to
stimulate oviposition in the laboratory (Matsumoto and Thorsteinson,
1968; Vernon et al., 1978; Pierce et al., 1978; Ishikawa et al., 1978).
The attractancy of some of the compounds has been demonstrated in field
trials as well (Matsumoto, 1970; Loosjes, 1976; Vernon et al., 1981;
Vernon and Borden, 1983).

Several of these sulfur-containing onion fly
attractants/0vipositional stimulants have not been reported as
constituents of onion, e.g., dipropyl sulfide and propenylpropyl sulfide
catch significant numbers of onion flies when placed in field traps
(Vernon et al., 1981). Dipropyl sulfide, methylpropyl sulfide, and
dipropenyl sulfide stimulate oviposition in the laboratory (Vernon et

al., 1978; Ishikawa et al., 1978); yet, none of these compounds have

been found in onions.

Many host- and nonhost-produced ovipositional stimulants of onion
fly contain a thiopropyl moiety that may be responsible for olfactory
receptor stimulation resulting in an ovipositional response (Vernon et
al., 1978; Ishikawa et al, 1978). Active compounds generally have a
single sulfur bonded to a saturated hydrocarbon chain 3-5 carbons long
(Vernon et al., 1978).

Blending onion chemical synthetics to more closely resemble those
released by onion has led to some increase in attractancy (Vernon et
al., 1981; Dindonis and Miller, 1981a). Yet, to elicit normal insect
responses, release rates of chemical baits as well as chemical
composition are important. For example, super- or subnormal
concentrations of the pheromone components emitted by female oriental

fruit moth Grapholitha modesta significantly decreases field trap

 

catches (Baker et al., 1981). If too little (E)-8-dodecenyl acetate is
released relative to (Z)-8-dodecenyl acetate, the male moth loses
contact with the odor plume. If too much is released, the male moth is
arrested within the plume downwind of the source.

Controlled release rates have been achieved by putting volatile
chemicals in polyethylene enclosures (Kuhr et al., 1972). Optimizing
release rates of dipropyl disulfide in polyethylene enclosures
significantly increased onion fly attractancy in the field (Dindonis and
Miller, 1981b). Nevertheless, no synthetic chemical tested singly, in a
blend, or with an optimized release rate is consistently more attractive
than freshly cut or intact onion plants (Dindonis and Miller, 1980;
Dindonis and Miller, 1981a; Ishikawa et al., 1981; Vernon et al., 1981).

Decomposing onions at some stages of microbial and physical damage

produce more potent onion fly attractants and oviposition stimulants
than healthy or fresh cut onions (Tomioka, 1977; Dindonis and Miller,
1981a; Ishikawa et al., 1981; Miller et al., 1984). Vernon et al.
(1981) showed attractancy of decomposing onion in the field increased
with longer incubation periods. This changing attractancy was
correlated with qualitative and quantitative changes in the head space
profile as analyzed with gas-liquid chroatography. Miller et al.
(1984) baited traps with chopped onion exposed to microorganisms in the
field and also found that attractancy varied as decomposition
progressed, yet the attractancy of any decomposing onion proved
difficult to predict. In field trials conducted in May and June,
attractancy of onion tissue increased with incubation time (Figure 3),
which ranged from one to five days. In field trials conducted in late
August, trap catches increased with baits aged for one to three days,
but then dramatically declined with baits aged for four to five days
(Miller et al., 1984) (Figure 3). The authors hypothesized that warmer
temperatures increased the rate of decomposition during the August
trials. Yet, even during the same week, similiarly treated baits of
decomposing onion placed in different fields varied in attractancy (J.
R. Miller, personal communication).

The present research focused on the influence of bacterially
infected onion on onion fly behavior. The first objective was to
isolate and identify one or more bacteria associated with decomposition
that produced onion fly attractants and ovipositional stimulants when
cultured on onion (Chapter 1). The purpose was to clarify the role of
bacteria in the production of attractants/0vipositional stimulants, as
well as to define a reproducible source of these compounds that may be

used in future work aimed at identifying the potent chemical components.

8

 

 

 

 

 

(I
LU
(D 40 - '-
+I
.fii; Cl
46 30
o
820 _
.E: .
c
III 1!
o 10 H August 22-28
2 0—0 May 28-June 5

l l
1 2 3 4 5

Days chopped onion aged

Figure 3. Onion fly catch as affected by onion bait aged in the field
after chopping (adapted from Miller et al., 1984).

The second objective was to document how 9. antigua ovipositional
preference and subsequent larval performance was influenced by different
levels of physical and microbial damage to maturing onions (Chapter 2).
In the fall, female onion flies must oviposit on maturing bulbs. Their
offspring feed on these older plants and overwinter as diapausing pupae.
After emergence and mating the following spring, females oviposit on
onion seedlings. Onion maggot damage in the spring accounts for most of
the crop losses during the growing season. Any pest management scheme
aimed at reducing the number of overwintering Q. antigua pupae, must
begin with an understanding of ovipositional preference and larval
performance on the maturing bulbs in the fall. An important goal of
this study was to determine whether ovipositional preference may have

adaptive value for fall generation onion flies.

lO

CHAPTER 1

Production of onion fly attractants and ovipositional

stimulants by bacterial isolates cultured on onion

11

CHAPTER 1

Introduction

Decomposing onion produces more potent onion fly attractants and
ovipositional stimulants than does healthy or freshly cut onion
(Tomioka, 1977; Dindonis and Miller, 1981a; Ishikawa et al., 1981;
Miller et al., 1984), but only at certain successional stages of
microbial and physical damage (Ishikawa et al., 1981; Vernon et al.,
1981; Miller et al., 1984). Using gas-liquid chromatography (GLC),
Vernon et al. (1981) demonstrated that changes in onion fly attractancy
were associated with qualitative and quantitative changes in the
headspace chemical profile of onion as decomposition progressed.

Many factors influence volatile metabolite production in
decomposing onion. A complex interaction of environmental factors such
as temperature and relative humidity, the characteristics of the hosté
plant, and the presence of competing microorganisms and their
metabolites may influence which organisms and volatile metabolites
predominate.

A bacterial species on a nutritive medium produces a headspace
profile that is often unique. Indeed, using GLC for the qualitative and
quantitative determination of volatile compounds has become an accepted
technique in the identification of anaerobic bacteria (Zechman and
Labows, 1984). By analyzing the headspace profile of pure cultures,
anaerobic bacteria have been identified to the species level (Larsson et

al., 1980). The anaerobic bacteria infecting urine or other body fluids

12

have been detected and identified both by direct headspace analysis of
the clinical sample and by subculturing the bacteria onto an appropriate
medium before analysis (Taylor, 1980; Mardh et al., 1981). Some
aerobic bacteria are also known to produce distinctive volatile
metabolites (Bowman et al., 1983; Pittard et al., 1982; Freeman et al.,
1976; Lee et al., 1979).

The variable attractancy of decomposing onion has hindered work
aimed at identifying potent onion fly attractants and ovipositional
stimulants. A reproducible source of these compounds was obtained by
culturing bacterial isolates, which are associated with onion
decomposition, on sterilized onion tissue. The experiments described in
this chapter clarify the role of such bacteria in the production of

onion fly attractants.

Materials and Methods
Insects

Parental stock originated from commercial onion fields in Eaton
Rapids, MI. Insects used were 5-7 generations removed from the field.
Adults were fed water and a dry artificial diet (Ticheler, 1971) and
housed as described by Schneider et al. (1983), except sprigs of onion
seedling foliage was replaced by green surrogate stems (Harris and
Miller, 1983) in ovipositional dishes. Larvae were reared in sand-
filled boxes provisioned with an excess of cross-sectioned onion bulbs.

All behavioral bioassays were conducted at 35.: 5% RH, 23.: 10 C,
and in a 16:8 L:D light regime (alternating cool 55 N and warm 85 H
white flourescent bulbs 10 cm above the cages), corresponding to light
during the hours 0700-2300. Three days before assaying, flies from the

laboratory culture were placed in cylindrical, metal-screened cages (42

13

cm diameter x 46 cm height). Between assays, .flies were given food,
water, and access to ovipositional dishes of freshly cut onion covered
with moist sand. In the center of each dish was placed a surrogate
onion stem (4mm diameter x 9 cm length above substrate) constructed from
a hollow, sealed, glass cylinder, enclosing a strip of green paper.
Before each assay, the water dish was placed in the center of the cage,

but food and ovipositional dishes were removed.

Bacterial isolates

Erwinia carotovora var. carotovora (EC) is a casual organism of

 

 

soft rot on a wide range of vegetables. Onion maggots moving through
infested soil may spread the disease (Agrios, 1978). EC was isolated
from damaged Spartan Banner hybrid stored onions potted in moist muck
soil taken from a commercial onion field in East Lansing, MI. Damage
was inflicted by 20 g; antigua second instars taken from the laboratory
culture and added to each potted bulb. After five days of larval
feeding, 10 g of the decomposing plant tissue was homogenized in 25 ml
sterile buffer (0.2 M KH

P0 and 0.2 M K HPO

2 4 2 4’
mortar and pestle. Dilutions of the homogenate were made in buffer and

pH 7.0) with a sterilized

plated on CVP, a selective medium for the isolation of soft-rot bacteria
(Cuppels and Kelman, 1974). This medium, which contains crystal violet
and sodium polypectate, was modified by the addition of manganous
sulphate, an anti-foam agent (O'Neill and Logan, 1975). Plates were
incubated overnight at 25.: 20 C.

Colonies of Erwinia spp. formed deep, cuplike depressions in the
selective medium. They could be distinguished from pectolytic

Pseudomonas spp., which form shallow, wider depressions (Cuppels and

 

Kelman, 1974). A bacterial isolate of Erwinia sp. was obtained and

14

designated as strain EC6. Using current taxonomic keys and texts, the
isolate was identified (Kelman and Dickey, 1980; Lelliott, 1974; Dye,
1969).

Klebsiella pneumoniae was cultured from an isolate stored at -65° C

 

in 4% DMSO:nutrient broth (vzv). Nutrient broth consists of 3 g beef
extract and 5 g peptone (Difco) per liter of distilled water. The
original strain, JMI, was isolated and identified as a predominant
microorganism from decomposing onions that were five times as attractive
in the field than fresh cut onions (Miller et al., 1984).

For use in experiments, cultures of both species were grown on
nutrient agar (nutrient broth with 15 g/l of Bacto-agar) at 25.: 20 C.
Stock cultures were maintained on nutrient agar in the refrigerator at

4-60 c.

Preparation of axenic onion and potato

Axenic plant tissue was prepared from Russet Burbank potatoes and
an undetermined cultivar of yellow onions from commercial storage. The
outer, papery scales and upper and lower 2 cm of bulb were removed;
pototoes were peeled. Plant tissue was soaked in 75% ethanol for 20
minutes and flame sterilized 1-2 minutes. The flamed outer 0.5 cm of
potato and the flamed outer 2 onion scales were removed with sterilized
instruments and discarded.

Two sizes of potato and onion tissue were cut. Squares of tissue
measuring 3.5 x 3.5 x 0.5 cm and 2 x 2 x 0.5 cm were placed individually
in polystyrene petri dishes (diameters of 5.5 cm and 3.5 cm,
respectively). Onion squares were cut only from the 2-3 outer scales.
The scale thickness (ca. 0.5 cm) varied slightly. Potato squares were

cut from transverse tuber sections 0.5 cm thick. Enough sterilized,

15

distilled water was added to cover the dish bottom. Tissues were
incubated at 25 1.20 C. The concentration of sulfur- and nitrogen-
containing compounds in onion are known to differ in the outer and
inner scales (Freeman, 1975), so only tissue taken from the same onion

scale (or the same potato) was used within the same cage for bioassays.

Experiment 1 - Attractancy of axenic onion

Approximately 100-125 flies of both sexes and of mixed ages were
placed in each cylindrical cage. Between hrs 1100 and 2200, treatments
were assayed simultaneously by putting different test materials into
traps equally spaced along the periphery of cylindrical cages. After
the bioassay, flies in each trap were sexed and counted.

Traps are described in Heston and Miller (1985). They were
constructed from 400-ml glass beakers with three equally spaced holes (1
cm diam) around the circumference of the base. Glass tubing (0.8 cm 10
x 4 cm length) was inserted through the holes until ca. 1 cm projected
outside the beaker. Before addition of test materials, traps were
autoclaved. To minimize direct contact with captured flies, test
materials were placed in an autoclaved wire-screen basket suspended
from the top of the trap. A beaker lid made from a sterile, polystyrene
petri dish and the glass tubing were sealed onto the trap with
ParafilmTM.

Axenic onion was an important control in all the behavioral
bioassays, therefore, the influence of time axenic onion tissue was held
before use in the bioassays had to be established. In each of four
cages, traps were baited with an axenic onion squares (3.5 x 3.5 x 0.5

cm) cut immediately before assaying, cut axenic onion held for 1, 2,

and 3 days before assaying, and a petri dish filled with sterilized,

16

distilled water (water control). At the time of cutting, two parallel
grooves ca. 1 cm apart were cut into each square. Onion tissues were
assayed in the original water—filled polystyrene petri dishes with the

covers removed.

Experiment 2 - Attractancy of bacteria cultured on onion over time

To determine the minimal length of time necessary to incubate
bacteria with onion to obtain significant differences in attractancy,
traps were baited with axenic onion inoculated with one of the bacterial
isolates and incubated 1, 2, or 3 days before assaying. These treatment
were compared to freshly cut axenic onion, and a water control. To
inoculate onion with bacteria, two parallel grooves ca. 1 cm apart were
cut into each square. A loopful of 24 hr inoculum was placed in each
groove. Tissues were incubated at 25‘: 2° C. Experiments using EC and

using KP were replicated seven times as described in Experiment 1.

Experiment 3 - Relative attractancy of EC- and KP-inoculated onion

To determine the relative attractancy of the two isolates cultured
on onion, traps were baited with onion inoculated with EC, KP, or EC
plus KP and incubated for three days at 25 :_2° C before assaying and
with freshly cut axenic onion. Onion was inoculated as described in
Experiment 2 with EC or KP inoculum placed in both grooves of a square
or with EC inoculum in one groove and KP inoculum in the other groove.

Eight replications were conducted as described in Experiment 1.

Experiment 4 - Attractancy of EC-inoculated host- and nonhost-plant
The volatile onion fly attractants produced by EC-inoculated onion

may be due to EC, regardless of the nutritive medium, or to the

17

interaction of EC with onion, the primary host plant of the onion fly.
T0‘ determine the importance of the host plant in the production of
attractants, EC was cultured on axenic onion and axenic potato, a
nonhost plant of onion fly, as described in Experiment 2. The bioassay
was similiar to that described in Experiment 1 with traps baited with
EC-inoculated onion, EC-inoculated potato, axenic onion, axenic potato,
and a water control. All tissues were incubated three days before
assaying, except axenic onion, which was freshly cut. Experiments were

replicated seven times.

Experiment 5 - Attractancy of EC-inoculated onion vs. synthetics

In a bioassay similiar to that described in Experiment 1, the
attractancy of onion inoculated with EC and incubated 3 days before
assaying was compared to a water control and synthetic chemical
attractants.

One chemical attractant was a solution of 100 ul 2-phenylethanol
(2-PhEt) and 25 ul pentanoic acid was mixed in 50 ml distilled water.
These chemicals were identified by Ishikawa et al. (1984) as attractants
extracted from decomposing onions. This formulation was reported to
give an optimal release rate for onion fly attractancy in the field
(Ishikawa et al., 1984). A ten-ml glass beaker with 10 ml of this
aqueous solution was placed in traps immediately before testing.

A second chemical attractant was n-dipropyl disulfide (PrZSZ), a
predominate secondary metabolite obtained by steam distillation of fresh
onions (Block, 1985). For an optimal release rate (ca. 100 ug/hr), 100
ul of PrZSZ (purity 99%) was placed into size 3 BEEMTM (Ted Pella Co.,
Box 510, Tustin, CA, 92680) polyethylene enclosures (Dindonis and

Miller, 1981b). To attain a stable release rate, filled capsules were

18

held for six hours prior to assaying. One capsule per trap was used.

The experiment was replicated four times.

Experiment 6 - Oviposition on EC-inoculated host- and nonhost-plant

Six to eight ovipositing female onion flies were placed in each
cage. In the laboratory, peak oviposition occurs 9-11 hrs (1600-1800)
into scotophase (Miller, personal communication). Treatments were
assayed between hrs 1500 and 2100; the numbers of eggs were counted
by water flotation.

Ovipositional dishes (35 ml) were filled with sterilized sand
moistened with sterilized, distilled water. A surrogate onion stem was
placed in the center of each dish. By spacing ovipositional dishes
equally along the cage periphery, different test materials were assayed
simultaneously.

Two separate experimental set-ups were used. In the first
experiment, the small squares of plant tissue (2.0 x 2.0 x 0.5 cm)
along with associated water was placed ca. 0.5 cm beneath the sand
surface immediately before assaying. No plant material was placed in
the cup designated as a water control.

To determine whether test material volatiles alone are sufficient
to stimulate oviposition, a second experiment was conducted. Petri
dishes containing the larger squares of tissue (3.5 x 3.5 x 0.5 cm) and
water were placed in sterilized glass quart canning jars. A GLC septum
was inserted into each of two holes drilled into the jar lid. TeflonTM
tubing (1 mm ID) was inserted into each. septum. One tubing passed
through the side of the screened walls of a cage and was threaded
through a septum inserted into a hole in the side of an ovipositional

cup. The tubing opened ca. 5 cm from the surrogate stem and ca. 1.25 cm

19

below the sand surface. The second tube led from the jar lid to a small
plastic manifold and then to the filter of a compressed air tank. Air
flowed at a rate of 10mm/min through each tube leading from the tank to
a jar. Air carrying volatiles from the jar was then pumped into an
ovipositional cup.

In both experiments, ovipositional dishes or jars with tubes
leading to ovipositional dishes were baited with onion or potato
inoculated with EC and incubated for 3 days before assaying, axenic
potato held for 3 days before assaying, freshly cut axenic onion, and a
water control. Preparation of treatments followed the procedure
outlined in Experiments 1 and 2. Both experiments were replicated four

times.

Results

Identification of Erwinia carotovora var. carotovora

 

 

Bacterial cells of EC6 were motile, straight, short rods,
occurring singly and sometimes in pairs or short chains. This isolate
was gram-negative, facultatively anaerobic, grew at 360 C on nutrient
agar, did not produce reducing substances from sucrose, and utilized
Simmon's citrate. Based on these results and characteristic growth on

CVP medium, EC6 was identified as a strain of Erwinia carotovora var.

 

carotovora.

 

Experiment 1 - Attractancy of axenic onion

Freshly cut, sterilized onion tissue was a significantly more
attractive bait than the water control (P 5_ 0.05) (Figure 4).
Sterilized onion tissues incubated 1 day or more before being used as
bait lost activity (P 5 0.05). Indeed, the numbers of flies caught with

traps baited with sterilized onion tissues incubated 1, 2, and 3 days

20

 

 

 

LL]

0,13

"“11

.C

3 9 "
(U

U 7 _
O.

m 5- ..
8-

""' L-§water

C 3 -l
(U

G)

2 1 -

L 1 J J
0 1 2

3
Days sterile onion held

Figure 4. Onion fly attractancy as influenced by length of time
sterilized onion tissue was held before assaying. Orthogonal contrasts:
freshly cut onion vs. onion incubated 1, 2, and 3 days (P < 0.05);
freshly cut onion vs. water control (P < 0.05); and water control vs.
onion incubated 1, 2, and 3 days (P > 0.20).

21

before assaying or with the water control were not significantly
different (P > 0.20). In all remaining bioassays, sterilized onion

tissue controls were cut immediately before assaying.

Experiment 2 - Attractancy of bacteria cultured on onion over time

Attractancy of EC-inoculated onion increased with incubation times
from one to three days (Figure 5), however, the differences in mean
trap catch were significant only for EC-inoculated onion incubated 3
days vs. 1 day (P §_0.05). Onion inoculated and incubated 2 or 3 days
caught significantly more flies than either non-inoculated onion or the
water control (P §_0.05). Few flies were caught in traps baited with
non-inoculated onion, EC-inoculated onion incubated only 1 day, and the
water control. The mean differences between these three treatments were
not significant (P > 0.20).

A similiar pattern of increasing attractancy with incubation times
from one to three days was seen for KP-inoculated onion tissue (Figure
6). Traps baited with KP-inoculated onion incubated 3 days caught
significantly more flies than those baited with 1-day incubated tissue
(P 5_ 0.10). KP-inoculated onion that was incubated 2 days was
intermediate in attractancy between tissue incubated 1 and 3 days, but
these differences were not significant (P > 0.20). Regardless of
incubation time, KP-inoculated onion was more attractive than non-
inoculated onion (P.: 0.10) and the water control (P.: 0.05). Freshly
cut, sterile onion also caught significantly more flies than the water
control (P 5_0.05).

A three day incubation period was sufficient to obtain significant
differences in attractancy between bacterially-inoculated onion vs.

sterilized onion. In the remaining bioassays, EC- or KP-inoculated

22

 

7

N to
o 01
l l
I l
0,50

.4. .1.
0| 0 01
'

 

 

EAHP 3

water .

day 2 day 3 day

 

 

 

 

Mean trap catch 3: SE

-L

Figure 5. Influence of incubation time on onion fly attractancy of EC-
inoculated onion. Sterilized onion tissue ((3)) inoculated with Erwinia
carotovora var. carotovora (EC) and incubated 1, 2, or 3 days Before
theTbioassay. Treatments marked by different letters are significantly
different (P.: 0.05). ANOVA, Tukey‘s HSD all pair-wise comparisons.

23

 

N N

O 01

I 1
03.10

—L
01
l

 

—L

o
I
(9

-

-
water.

1 day 2 day 3 day

 

01

 

 

 

 

 

 

 

Mean trap catch :tSE

Figure 6. Influence of incubation time on onion fly attractancy of KP-
inoculated onion. Sterilized onion tissue ( C)) inoculated with .5;
pneumoniae (KP) and incubated 1, 2, or 3 days before the bioassay.
'Treatments marked by different letters are significantly different (P.g
0.10). ANOVA, Tukey's all pair-wise comparisons.

 

24

tissue was incubated three days before use in experiments.

Experiment 3 - Relative attractancy of EC- and KP-inoculated onion

Traps baited with onion inoculated with EC caught significantly
more flies than those baited with KP-inoculated onion (P_: 0.01) (Figure
7). Onion tissue inoculated with both bacteria did not catch
significantly more flies than onion inoculated with either bacterial
isolate alone (P > 0.20). Regardless of the bacterial isolate used, all
inoculated onion baits caught significantly more flies than traps
baited with non-inoculated onion (P §_0.01). The remaining experiments

focused on the bacterial isolate EC.

Experiment 4 - Attractancy of EC-inoculated host- and nonhost-plant

The effects of the two factors in this experiment, i.e., plant
tissue and the presence or absence of EC, interacted significantly (P.£
0.01) (Figure 8). The highest mean trap catch was attained with EC-
inoculated onion. Traps baited with EC-inoculated potato caught
significantly more flies than those baited with non-inoculated potato
(P 5_ 0.01), but significantly fewer flies than those baited with EC-
inoculated onion (P §_0.01). No significant differences in mean fly
catch were seen in traps baited with sterilized onion or potato (P >

0.20).

Experiment 5 - Attractancy of EC-inoculated onion vs. synthetics

Traps baited with EC-inoculated onion caught significantly more
flies than any other bait used (P §_0.01) (Figure 9). The mean fly
catches obtained with traps baited with either chemical attractant or
with non-inoculated onion were all low and did not differ significantly

from each other (P 3_0.20). All baits tested caught significantly

25

N
O

_

-Js
O

 

Mean trap catc

 

 

 

O KP

Figure 7. Relative onion fly attractancy of EC and/or KP inoculated
on sterilized onion tissue (0) and incubated 3 days

 

ab

  

bioassay. Treatments marked by different letters
different (P 5_0.01). ANOVA, Tukey's HSD all pair-wise comparisons.

 

 

before the
significantly

 

 

 

 

 

 

 

 

 

_ -9 ..
”'45 5‘0"
+I " ‘
:35~ "
U
14-: ._ -
co
025~ -
O.
E (- 1
q..-
c15- 4
«s
a) _. .-
2

5L!- !! i! , J

 

water D .

Figure 8. Production of onion fly attractants by the interaction of EC
on onion fly host- and nonhost-plant tissue. Sterilized onion tissue
(0) and sterilized potato tissue ([3) inoculated with EC and
incubated 3 days/Eefore the bioassay. Two-way ANOVA of data transformed
to (X + 0.5) . Significant interaction between plant tissue and
presence or absence of EC (P < 0.01). Orthogonal contrasts: potato vs.
EC-inoculated potato (P.§ 0(01); onion vs. EC-inoculated onion (P .3
0.01); EC-inoculated potato vs. EC-inoculated onion (P §_0.01); potato
vs. onion (P > 0.20).

27

 

N
01
a
1
+0
”:4

—L .3 N
01 O 01 O
l T

 

 

Mean trap catch i SE

water Pr2$2 2-PhET
&

1;}: i- =-
.@

pentanoic
acid

Figure 9. Comparison of chemical synthetics and EC-inoculated onion
as onion fly attractants. Baits include dipropyl disulfide (PrZS )
released from polyethylene enclosures, an aqueous solution of 2
phenylethanol (2-PhEt) and n-pentanoic acid, and sterilized onion ( C3)
inoculated with EC and incubated 3 days before the bioassay. Treatments
marked by different letters are significantly different (P 5_ 0.05).
ANOVA, Tukey's HSD all pair-wise comparison.

28

Table 1. Onion fly ovipositional responses to host- and
nonhost-plant tissue inoculated with EC. Treatments
presented in ovipositional dishes (Experiment A) and as
volatiles pumped into ovipositional dishes (Experiment 8).

 

1

 

 

 

Mean eggs laid
Treatment Experiment A Experiment 8
EC-inoculated onion 73.5; 3: 23.87 47.53: 10.54
non-inoculated onion 12'5c-i 2.96 1.0b i 0.56
EC-inoculated potato 1.0c i 1.00 1.0b.: 0.41
non-inoculated potato 0.0c 0.0b
water control 0.0 0.0
1

Means + SE followed by the same letter within a column
are not significantly diffgfient, one-way ANOVA of data
transformed to (X + 0.5) followed by Tukey's all-pair
wise comparisons (P §_0.05) (Gill, 1978).

29

more flies than the water control (P §_0.05), except PrZS2 (P > 0.20).

Experiment 6 - Oviposition on EC-inoculated host- and nonhost-plant

In the first ovipositional choice experiment, test materials were
placed directly in the ovipositional dishes (Table 1, Experiment A).
Dishes containing EC-inoculated onion received significantly more eggs
than any other treatment (P_§ 0.01). Freshly cut, sterilized onion
received significantly fewer eggs than EC-inoculated onion (P f. 0.01),
but significantly more eggs than all other treatments (P §_ 0.05). No
other significant differences were found (P > 0.20).

In the second ovipositional choice test, volatiles of these same
treatments were pumped into the ovipositional dishes (Table 1,
Experiment 8). Dishes with volatiles from EC-inoculated onion received
significantly more eggs than any other treatment group (P.: 0.01). No
other differences were found among the remaining treatments (P > 0.20),

all of which received very few eggs.

Discussion

It was not unexpected that freshly cut, sterilized onion tissue was
more attractive to onion flies than tissue incubated 1 day or more
before bioassaying. The unstable sulfenic acids released after cutting
onion tissue degrade to form various sulfur-containing volatiles that,
in part, give onion its characteristic odor and taste (Figure 2) (Block,
1985). Many of these volatiles, such as dipropyl disulfide, are known
to be attractive to and stimulate oviposition of onion fly (Matsumoto
and Thorsteinson, 1968; Vernon et al., 1978; Ishikawa et al., 1978).
Since the release of volatiles from damaged onion cells is exhausted

rather quickly, the attractancy of freshly cut sterilized onion would be

30

expected to decrease with time. Freshly cut sterilized onion was chosen
as a more appropriate control for the remaining assays testing the
influence of bacteria cultured on sterilized onion tissue.

Trap catches increased as the length of time EC- or KP-inoculated
onion was incubated before assaying. This increased activity may have
been due to changes in the volatiles produced over time. Quantitative
changes in volatiles may occur as the bacterial population increases,
perhaps producing larger amounts of pectolytic and cellulolytic enzymes
(Echandi et al., 1957). Qualitative changes are possible if the
initial bacterial and plant metabolites react chemically or are re-
metabolized by the bacteria, resulting in time-dependent changes in
headspace metabolites. In addition, as bacteria exhausted the supply of
available metabolites, one would expect changes in the composition of
volatiles produced.

The experiments demonstrating the effect of incubation time on
attractancy of bacteria cultured on onion illustrate the importance of
standardizing environmental factors. If conditions were not held
constant, the rate of successional changes in headspace metabolites may
have been altered. Factors such as temperature, size of plant tissue,
and length of incubation time all were controlled to produce a fairly
consistent headspace profiles from inoculated plant tissue.

Both KP and EC produced attractants when inoculated on onion. EC
was a better candidate under the given experimental conditions because
of its significantly higher attractancy than KP or KP plus EC when
cultured on onion. If the two bacteria on the same onion tissue had
produced more potent attractants than either one alone, it would have
been evidence that at least some of the attractant volatiles produced

were unique to one bacterium and capable of increasing the attractancy

31

of the other.
Human olfaction detects little differences between sterilized and
EC-inoculated potato tissue. Indeed, many vegetables infected with E;

carotovora var. carotovora alone do not release the unpleasant odors we

 

 

commonly associate with decomposing plants. Saprophytic bacteria,
which colonize plant tissue after the pathogen, generally are
responsible for the foul odor. Two notable exceptions are crucifers
and alliums, both of which release unpleasant sulforous odors when
inoculated with the soft rot pathogen (Agrios, 1978).

Peterson (1924) demonstrated that the addition of ethanol, 2-
propenol, and isopropanol to honey increased the attractancy of these
sweetened baits. EC-inoculated potato tissue may release some simple
fermentation products, such as short-chained alcohols that are not
particularly odoriferous but may still may function as mild onion fly
attractants. Since EC-inoculated potato was not nearly as attractive as
EC-inoculated onion (Figure 8), the potency of EC-inoculated onion was
due to the interaction of the bacterium with the primary host-plant of
&_ antiqua.

The aqueous solution of 2-PhEt and pentanoic acid was far less
attractive than EC-inoculated onion tissue (Figure 9), indicating that
one or more potent volatile attractants associated with decomposing
onions remain to be identified. Indeed, the low fly catches obtained
with 2-PhEt/pentanoic acid as well as with PrZS2 demonstrate that
currently known synthetic attractants do not closely simulate fresh or
maximally attractive decomposing onion.

In behavioral bioassays designed to test the efficacy of

attractants or deterrents, the use of appropriate controls is sometimes

32

neglected or infeasible. The relative effectiveness of a candidate
chemical synthetic must be compared not only with a negative control,
e.g., an unbaited or water-filled trap in attractancy assays, but also
with a positive control, e.g. the optimal source of the attractant.

Although aqueous solutions of 2-PhEt/pentanoic acid tend to catch
more flies than unbaited traps (Ishikawa et al., 1984; Ishikawa et al.,
1986), few data have been available on its efficacy relative to
decomposing onions of proven attractancy. The strength of 2-
PhEt/pentanoic acid as an onion fly attractant in laboratory and field
trials was reported by Ishikawa et al. (1984) to be similiar to that of
onion pulp exposed to undefined microorganisms and aged for 8 days at
250 C. Since it is not known whether such onion pulp was a reliable
source of onion fly attractants, traps with freshly cut onion should
have been included in these experiments as a standard to which the
attractancy of the decomposing pulp (the positive control) could be
compared.

EC-inoculated onion, whether presented as tissue or as volatiles,
stimulated the most oviposition in choice tests. Since female onion
flies will oviposit in response to freshly cut onion volatiles alone
(Matsumoto and Thorsteinson, 1968; Vernon et al., 1978; Ishikawa et al.,
1978), some oviposition on this treatment in Experiment 8 was expected.
Yet, because of the low response to any treatment except EC-inoculated
onion, the relative ovipositional preference among the remaining
treatments was not extant.

Even though attracted, female onion flies did not accept EC-
inoculated potato as an ovipositional site. Oviposition is a specific
behavioral response generally restricted to stimuli from the host-plant.

0n the other hand, attractancy responses include both host-finding and

33

food-finding. Thus, onion flies may be attracted to and ‘feed on
decomposing non-host plants (Miller et al., 1984), but may not accept

them for oviposition.

34

CHAPTER 2

Onion fly ovipositional preference and larval performance
as influenced by physical and microbial damage to

mature onion bulbs

35

CHAPTER 2

Introduction

Previously damaged and decomposing onions in the field have been
reported to be more susceptible to colonization by onion fly, .leia
antiqua (Meigen), than undamaged bulbs (Armstrong, 1924; Workman, 1958;
Crete and Tartier, 1972). Since newly hatched onion maggots are not able
to migrate very far from their initial feeding site (Kendall, 1932),
especially under dry environmental conditions, larval survival depends
strongly on the ability to utilize the plant on which oviposition
occurs. No study clearly demonstrates whether the observed
susceptibility of damaged, maturing, field onions is due to increased
oviposition, higher larval survival, or both.

Fall generation onion maggots feed on maturing bulbs then
overwinter as diapausing pupae. Upon emergence and mating the following
spring, the females oviposit on the new crop of onion seedlings. Onion
maggot damage in the spring accounts for most of the crop losses during
the growing season. Any pest management scheme aimed at reducing fly
populations in the fall, thus the subsequent number of overwintering
pupae, must begin with an understanding of onion fly ovipositional
behavior and the suitability of commericial onions available in the fall
for larval survival and development. The objective of this study was to
determine the influence of different levels of physical and microbial
damage to maturing bulbs on ovipositional preference and subsequent

larval performance.

36

Materials and Methods
Insects

Parental stock originated from commercial onion fields in Eaton
Rapids, MI. Insects used were 5-7 generations removed from the field.
Adults were fed water and a dry artificial diet (Ticheler 1971) and
housed as described by Schneider et al. (1983), except foliage was
replaced by green surrogate stems (Harris and Miller, 1983) in
ovipositional dishes. Eggs used in host plant suitability studies were
removed from laboratory culture ovipositional dishes by water flotation.
Larvae used to inflict damage to onions were taken directly from sand-
filled, rearing boxes provisioned with an excess of cross-sectioned

onion bulbs.

Host plant

Mature (5.5 - 6.5 cm diameter) Spartan Banner Hybrid onion bulbs
were transplanted from the field on September 29 - 30, 1984, into
plastic pots (15 cm diameter x 25 cm height) containing field-collected
muck soil. The senescing foliage was lodging, but the roots were
intact. Plants were kept at 22° c, 25-35% RH, and in a 16:8 L:D light
regime (cool 55 W and warm 85 W flourescent bulbs ca. 30 cm from
plants).

To obtain different levels of plant damage, groups of 20 late
second instar D; antigua larvae were added to some of the pots at five
day intervals. Individuals pupated ca. 5-8 days later. Fifteen days
after transplanting, four qualitative treatment groups had been
obtained: (1) undamaged onions with no added larvae, (2) onions damaged
by 5 days of active feeding with decomposition localized at the base of

the bulb, (3) onions damaged by 10 days of active feeding with bacterial

37

decomposition invading inner tissues, but bulbs firm to touch, and (4)
onions damaged by 15 days of active feeding with advanced decomposition
throughout the inner tissues and the majority of the outer tissues, and

bulbs soft to touch.

Ovipositional choice test

Behavioral bioassays were conducted in a randomized complete block
design. Two cages of flies were used during two different ovipositional
periods. Approximately 300 adult flies, one to two weeks old, were
placed in rectangular cages (3 x 1 x 1 m) with food and water. At ca.
200 C, maximal rates of oviposition begin ten to twelve days after pupal
eclosion.

At the start of the first experimental ovipositional period, five
plants from each treatment group were randomly placed equidistant from
each other in each cage. These were removed after 24 hours and replaced
with another set of plants for the second ovipositional period of 48
hours. Eggs were collected by water flotation. The total numbers of
eggs on all five plants from each treatment group and in each cage were

counted.

Host plant suitability

Using a white wheat flour and water mixture, groups of 20 eggs were
pasted gently on squares of black paper (1.5 x 1.5 cm) and subsequently
placed on top of the soil 1 cm from each plant. Eggs that did not hatch
after four days (less than 5%) were removed and replaced by newly
hatched larvae for a total of 20 larvae per plant. Ten replicates of
plants with 5-, 10-, and 15-day damage and twenty replicates of
undamaged plants were included. Soil was kept moist and was sifted

every five days to remove and discard the pupae that developed from

38

second instars added to cause the initial damage. Within each plant
treatment group, larvae added as newly hatched first instars pupated ca.
10 after the late second instars.

When possible, a single individual was weighed as both a fresh pupa
and an emerged adult or as a fresh and a freeze-dried pupa. When larvae
added as first instars were collected as pupae, they were placed
individually in 5 dram glass vials containing moist sand. Pupae were
kept in glass vials for five days before weighing to standardize pupal
moisture content. Unfortunately, many began to show evidence of
bacterial contamination before weighing. Perhaps the sand-filled vials
were too moist and temperatures too warm for healthy development of the
pupae to the adult stage. In these cases, only freeze-dried pupal
weights were taken. Pupae not eclosing after three weeks were freeze-
dried and weighed to avoid difficulties with possible bacterial
contamination. Some individuals emerged as adults before being removed
from the soil, so no fresh pupal weight was available. Adults were

weighed within twelve hours of emergence.

Results

Few eggs were laid on undamaged plants (Table 2). Females laid
significantly more eggs on plants with 5 or 10 days of active feeding
and microbial damage. Host plant acceptability decreased significantly
with increasing damage and decomposition beyond 10 days of active
feeding. Females showed no significant ovipositional preference for
15-day damaged vs. healthy plants.

A plant was considered to be successfully colonized if at least one
first instar pupated. Only 15% of the healthy plants were
successfully colonized (Table 3). Nearly all of the 5- and 10-day

39

Table 2. Onion fly oviposition on
undamaged and damaged onions

 

Treatment Mean number of eggs
per group of five plants

 

 

Undamaged 76.5
5-day damaged 584.5*
IO-day damaged 334.3*
15-day damaged 227.8ns
*

Means were significantly different
from oviposition on undamaged onions
(P < 0.05), randomized complete block
ANOVA of 9723 transformed to
(X + 0.5) followed by Dunnett's
test (Gill 1978).
"5 Mean was not significantly different
from oviposition on undamaged onion
(P > 0.20).

40

Table 3. Colonization rates
of undamaged and damaged

 

 

onions.

% Plants
Treatment colonized
Undamaged 15
5-day damaged 100
10-day damaged 90
15-day damaged 20

 

41

Table 4. Survival of first instar 0; anti ua
larvae to pupal stage on undamaged and amaged
onions.

 

 

 

Meanl larval survival per onion
(max = 20)
Treatment Colonized onions .All onions
Undamaged 16.0 i 1.6a 2.4:1.3b
5-day damaged 17.2;0.8a 17.2 1 0.8a
10-day damaged 6.3.12.0b 5.2 : 1.3b
15-day damaged 1.0 0.2

 

1Means + SE within a column followed by the same

letter—were not significantly different, one-
way ANOVA with Scheffe's all pair-wise
comparison for unbalanced data (P 5_0.05)
(Gill, 1978).

42

Table 5. Weights of pupae and adults from 01 antigua larvae reared
on undamaged and damaged onions.

 

 

 

 

Meanl weight, mg
Pupae
Treatment Fresh wt. (n)2 Dry wt. Emerged adults
Undamaged 16.4 1 0.4a (44) 5.5 1 0.2a (44) 14.3 1 0.93 (3)

5-day damaged 15.8 1 0.43 (66) 5.4 1 0.13 (98) 12.2 1 0.2ab (74)
10-day damaged 13.6 1 0.7b (33) 4.5 1 0.2b (39) 11.2 1 0.7b (13)
15-day damaged - - -

 

1Means + SE within a column followed by the same letter were not

signichantly different, one-way ANOVA with Scheffe's all pair-wise
comparison for unbalanced data (P 1_0.05) (Gill, 1978).

2n is the number of individuals weighed. The total numbers of
individuals recovered from undamaged, 5-, 10-, and 15-day damaged
onions were 47, 172, 52, and 2, respectively. Data for two weight
categories were available for some individuals.

43

damaged onions were successfully colonized, but the colonization rate
dropped from 90% to 20% between the 10-day and 15-day damaged plants,
respectively (Table 3).

0n the few healthy plants that were colonized, the number of larvae
surviving and developing to the pupal stage was not significantly
different from that on the 5-day damaged onions (Table 4). Yet, because
of very low colonization rates, overall larval survival on healthy
onions was significantly lower than that on 5-day damaged plants (Table
4). In spite of the high colonization rate on the 10-day damaged plants
(90%), the number of larvae surviving to the pupal stage was
significantly lower than the number surviving on 5-day damaged onions.
Overall larval survival on both 10-day damaged and healthy plants was
quite low and not significantly different (Table 4). In the 15-day
damage treatment group, only a single larva on each of two plants
survived to the pupal stage (Table 4).

Pupal and adult weights did not differ significantly between
larvae reared on undamaged and 5-day damaged onions, but weights
decreased significantly with more severe decomposition (Table 5). Since
only two pupae (fresh weights of 6.4 and 7.2 mg) were recovered from 15-

day damaged onions, their weights were not included in the analysis.

Discussion

The high colonization and overall larval survival rates in the 5-
day damaged onions indicate that these onions were more suitable host
plants for .01 antiqua development than healthy mature bulbs.
Apparently, if first instars penetrate and begin sustained feeding on
a healthy bulb, survival and weight gains will be similiar to those

attained on the more suitable onions that have some damage and microbial

44

infection. Since the soil was kept fairly moist throughout the
experiment, larval death due to dessication was unlikely. Increased
suitablity of 5-day damaged bulbs preferred by ovipositing females may
be due, in part, to increased ease of penetration by first instars.
Bulbs with more severe damage may be less suitable for larval survival
and development because of a decrease in food quality associated with
decomposition and in food quantity.

Microorganisms associated with decomposition also may contribute to
larval nutrition. Although larvae reared on pieces of axenic onion
tissue survive and develop nearly as well as those reared on onion
contaminated with naturally occurring microorganisms, inclusion of
microbes in the diet tends to accelerate developmental rates (Schneider
et al., 1983). Microorganisms may contain important nutrients or may
increase the availability of nutrients in onion (Eymann and Friend,
1985). Possible differences in the nutritive value of undamaged and the
more suitable 5-day damaged plants probably do not fully account for the
inability of first instars to successfully utilize a healthy, mature
bulb as food. Indeed, the few healthy plants that were colonized
resulted in larval survival rates and weight gains similiar to those on
5-day damaged plants.

Field traps baited with decomposing onions at certain stages of
microbial succession are attractive to adult onion flies (Ishikawa et
al., 1981; Vernon et al., 1981; Miller et al., 1984). Since the traps
prevent visual or physical contact with the bait before capture, the
flies must be responding to volatile compounds released from the bait.
Female onion flies ovipositing on decomposing onion bulbs may also be

responding to volatile attractants. In addition, bulbs at various

45

stages of physical and microbial damage may release metabolites that
influence ovipositional responses after females alight on or near the
plant. These metabolites may be nutritive or have other attractant or
deterrent properties that influence behavior.

Finch and Eckenrode (1985) showed that a high percentage of
overwintering pupae develop on damaged bulbs left in the field after
fall harvest. The results presented here support their conclusion that
damaged bulbs can serve as an excellent food source for fall generation
onion flies. In addition, this study demonstrates that the highest
densities of onion maggots are found on recently damaged fall onion
bulbs, both because of Q1 antiqua ovipositional preference and higher
offspring fitness. In these experiments, there appears to be a good
correlation between female ovipositional preference and subsequent

larval performance.

46

GENERAL DISCUSSION

. Volatile metabolites produced by Erwinia carotovora var. carotovora

 

 

(EC) and Klebsiella pneumoniae (KP) cultured on sterilized onion tissue

 

were more attractive to adult onion flies than volatiles of freshly cut,
axenic onion. During a three day incubation period, the attractancy of
onion inoculated with either bacterial isolate increased. Qualitative
or quantitative changes in the composition of volatiles during this time
were not determined. EC cultured on onion produced more potent
attractants than KP on onion. Furthermore, the attractancy of volatiles
produced by onion inoculated with both isolates was not significantly
different than those produced by onion inoculated by either isolate
alone.

Onion flies were attracted to headspace metabolites produced by EC
cultured on potato, a nonhost plant of onion fly. Yet, these
metabolites were significantly less potent than those produced by EC
cultured on onion, the primary host of .01. antigua; therefore, the
interaction of EC and onion, and not EC itself, was responsible for its
strong activity toward onion fly.

In laboratory bioassays, the efficacy of EC-inoculated onion as an
onion fly attractant was significantly better than an aqueous solution
of 2-phenylethanol and pentanoic acid, attractants reportedly extracted
from decomposing onion and formulated for an optimal release rate
(Ishikawa et al., 1984). One or more volatile components responsible

for the potent attractancy of some bacterially infected onion remain to

47

be identified.

Whether presented as plant tissue or as volatiles, EC-inoculated
onion stimulated significantly more onion fly oviposition than axenic
onion. Onion flies were attracted to EC cultured on potato, but females
did not accept it for oviposition, a specific behavioral response
generally restricted to stimuli from the host plant. Attractancy
comprises many behaviors, including host-finding by ovipositing females
food-finding responses. Therefore, female onion flies may be attracted
to and feed on bacterially infected, nonhost-plants without accepting
them for oviposition.

Female onion flies preferentially oviposited on those damaged, fall
onions most suitable for larval survival and development. Ovipositional
preference for decomposing bulbs significantly decreased as the physical
and microbial damage increased; the numbers of eggs laid on bulbs with
the most severe damage and on healthy bulbs were not significantly
different.

While colonizing 90-100% of the damaged bulbs (5-day and 10-day
damaged) preferred by ovipositing females, newly hatched larvae
successfully colonized only 15% of the healthy plants. Pupal and adult
weights were not significantly different for larvae reared on undamaged
or 5-day damaged bulbs, but did significantly decrease with further
plant damage. Overall larval survival was lowest for undamaged and
severely (IS-day) damaged bulbs, which were also the plant groups
stimulating the least oviposition.

These results support the hypothesis that damaged bulbs are an
excellent food source for fall generation onion flies. Healthy onions
may, in part, be resistant to onion maggot attack because of the

inability of first instars to penetrate undamaged bulbs. The high

48

densities of onion maggots found on recently damaged, fall onions was
due to both 91 antiqua ovipositional preference and higher offspring
fitness.

A logical step to the work reported in this thesis would be to
identify the biologically active, volatile metabolites from EC-
inoculated onion. Direct headspace analysis is possible and avoids some
of the complications introduced by extraction with solvents. It is
feasible to withdraw an aliquot of headspace volatiles with a syringe
and directly inject them into a gas-liquid chromatograph, but the
concentration of component chemicals may be too low for detection. An
automated headspace concentrator and high-resolution columns allow one
to obtain reproducible, detailed profiles of the headspace volatiles.

After identification, these chemicals may be purchased or
synthesized in pure form for further behavioral and neurophysiological
studies. Basic research needs to be directed toward the mechanisms by
which these attractants and ovipositional stimulants function. Their
reception by the peripheral nervous system and subsequent integration
in the central nervous system is an important area of research.

Attractants are currently used commercially as baits to monitor or
detect pest populations and for use in attracticides. For example,
traps baited with controlled-release attractants are used to detect
accidental importation of agricultural pests. To make informed pest
management decisions, growers and field scouts also may use baited
traps to monitor pest populations throughout a growing season. Using
traps baited with attractants to significantly decrease pest
populations, i.e., mass trapping, has met with little success in crops

and orchards, although this approach may be viable in confined areas

49

such as greenhouses or warehouses. Attracticides, which are attractants
laced with an insecticide, currently are used commercially. Since
attracticides often may be applied with conventional pesticide
equipment, they may be easily integrated into present pest management
practices. Although combining an attractant with an insecticide may lead
to more efficient use of smaller quantities of toxins, not all
attractants have high specificity. For example, yeast hydrolysate based
attracticides may attract and kill natural enemies of the pest along
with the target insect.

Applied uses for ovipositional stimulants have not been explored
in depth, but the study of ovipositional stimulants in the laboratory
and field has some advantages making them useful tools in the research
and development of behavior-modifying chemicals. Oviposition is a
specific, quantifiable, behavioral response. In many bioassays designed
to measure attractancy responses, it is not always possible to determine
whether insects are responding to food-, host-, or mate-finding stimuli.
An understanding of which insect behaviors are influenced by a
particular attractant is an important step in developing applications.
Of course, if only adult female insects at certain stages of development
respond, the use of ovipositional stimulants in the field may be
limited.

If new uses for behavior-modifying chemicals are to be developed
for agriculture, the ecology and economics of the crop system must be
considered. The influence of environmental factors, such as climate,
availability of food, presence of natural enemies, and the age structure
of the crop and insect pest must be studied in conjunction with
optimized, behavioral, laboratory studies. Furthermore, if the goal is

to develop a new product or technique, it must inexpensive enough to

50

warrent its use. More basic and applied research is needed to realize
the potential of behavior-modifying chemicals in management of

agricultural insect pests.

51

LITERATURE CITED

52

LITERATURE CITED

Amonkar, S. U. and A. Banerji. 1971. Isolation and characterization
of larvacidal principle of garlic. Science 174: 1342-1344.

Agrios, G. N. 1978. Bacterial soft rots. 13_Plant Pathology. Academic
Press, New York. pp. 477-483.

Armstrong, T. 1924. Onion maggot studies in the district of Montreal,
Quebec 1924. A. Rep. Ent. Soc. Ont. 54: 42-45.

Baker, T. C.. W. Mayer, and W. L. Roelofs, 1981. Sex pheromone dosage
and blend specificity of response by oriental fruit moth males. Ent.
exp. appl. 30: 269-279.

Bernhard, R. A. 1970. Chemotaxonomy: Distribution studies of sulfur
compounds in Allium. Phytochem. 9: 2019-2027.

Block. E. 1985. The chemistry of garlic and onion. Scientific
American 252: 114-119.

Boelens, M., P. DeValois, H. Wobben, and A. van der Gen. 1971.
Volatile flavor components from onion. J. Agric. Food Chem. 19: 984-
1000.

Bowman, E. V., L. R. Freeman, 0. W. Later, and M. L. Lee. 1983.
Comparison of volatiles produced by selected pseudomonads on chicken
skin. J. Food Sci. 48: 1358-1359.

Brachmachari, H.O. and K. T. Augusti. 1961. Hypoglycaemic agent from
onion. J. Pharm. Pharmocol. 13: 128.

Carson, J. F. and F. F. Wong. 1961. The volatile flavor components of
onions. J. Agric. Food Chem. 9: 140-143.

Cavallito, C. J. and J. H. Bailey. 1944. Allicin, the antibacterial
principle of Allium sativum. 1. Isolation, physical properties and
antibacterial action. J.‘Amer. Chem. Soc. 66: 1950-1954.

 

Crete, R. and L. Tartier. 1972. Chemical control of onion smut in
organic soils of southwestern Quebec in 1971. Can. Dept. Agric.
Pestic. Res. Rep. pp. 280-281.

Cuppels, D. and A. Kelman. 1974. Evaluation of selective media for
isolation of soft-rot bacteria from soil and plant tissue.
Phytopath. 64: 468-475.

Dindonis, L. L. and J. R. Miller. 1980. Host-finding responses of
53

onion and seedcorn flies to healthy and decomposing onions and
several synthetic constituents of onion. Environ. Entomol. 9: 467-
472.

Dindonis, L. L. and J. R. Miller. 1981a. Onion fly and little house
fly host finding selectively mediated by decomposing onion and
microbial volatiles. J. Chem. Ecol. 7: 419-426.

Dindonis, L. L. and J. R. Miller. 1981b. Onion fly trap catch as
affected by release rates of n-dipropyl disulfide from
polyethylene enclosures. J. Chem. Ecol. 7: 411-418.

Dye, D. W. 1969. A taxonomic study of the genus Erwinia. II. The
”carotovora" group. N. 2. JT Sci. 12: 81-97.

Echandi, E., S. D. Van Gundy, and J. C. Walker. 1957. Pectolytic enzymes
secreted by soft-rot bacteria. Phytopath. 47: 549-552.

Ellis, P. R. and C. J. Eckenrode. 1979. Factors influencing
resistance in Allium sp. to onion maggot. Bull. Entomol. Soc. Am. 25:
151-153.

Eymann, M. and W. G. Friend. 1985. Development of onion maggots
(Diptera: Anthomyiidae) on bacteria-free onion agar supplemented
with vitamins and amino acids. Ann. Entomol. Soc. Am. 78: 182-185.

Finch, S. and C. J. Eckenrode. 1985. Influence of unharvested, cull-
pile, and volunteer onions on onion maggot (intera:Anthomyiidae).
J. Econ. Entomol. 78: 542-546.

Freeman, G. G. 1975. Distribution of flavour components in onion
(Allium ce a L.), leek (Allium porrum) and garlic (Allium
sativum). . ci. Fd Agric. 26: 471-48 .

Freeman, L. R., G. J. Silverman, P. Angelini, C. Merritt, Jr., and W. B.
Essler. 1976. Volatiles produced by microorganisms isolated from
refrigerated chicken at spoilage. Appl. Environ. Microbiol. 32: 222-
231.

Gill, J. L. 1978. Design and Analysis of Experiments in the Animal
and Medical Sciences, Vol. 1. Iowa State Univ. Press, Ames, IA.

Hough, J. A., G. E. Harman, and C. J. Eckenrode. 1981. Microbial
stimulation of onion maggot oviposition. Environ. Entomol. 10:
206-210.

Harris, M. O. and J. R. Miller. 1983. Color stimuli and oviposition
behavior of the onion fly, Delia antigua (Meigen) (Diptera:
Anthomyiidae). Ann. Entomol. Soc. 76: 76 - .

Ishikawa, Y., T. Ikeshoji, and Y. Matsumoto. 1978. A propylthio moiety
essential to the oviposition attractant and stimulant of the onion
fly, Hylemyg antiqua Meigen. Appl. Ent. Zool. 13: 115-122.

 

Ishikawa, Y., T. Ikeshoji, and Y. Matsumoto. 1981. Field trapping of the
54

onion and seed-corn flies with baits of fresh and aged onion pulp.
Appl. Ent. Zool. 16: 490-493.

Ishikawa, Y., Y. Matsumoto, M. Tsutsumi, and Y. Mitsui. 1984. Mixture of
2-phenylethanol and n-valeric acid, a new attractant for the onion

and seed-corn flies, HElemyaw antigua and ___ platura
(Diptera:Anthomyiidae). Appl. 48- 455.

Ishikawa, Y., Y. Matsumoto, M. Tsutsumi, Y. Mitsui, K. Yamashita, M.
Yoshida, and E. Shirai. 1986. Controlled release formulation of the
attractant for the onion and seed- -corn flies, H lem a anti ua and H.
platura (Diptera: Anthomyiidae). Submitted to Appl. Ent. 20 01.

Kato, S. 1958. The turnip maggot and other species of Hylemya
(Diptera: Muscidae) of economic importance in Japan. Proc. 1nt.
Congr. Ent. 10: 445-447.

Kelman, A. and R. S. Dickey. 1980. Soft rot or 'carotovora' group.
In Laboratory Guide for Identification of Plant Pathogenic
'BEcteria (N. W. Schaad, ed.). Amer. Phytopath. Soc. St. Paul, MN.

Kendall, E. W. 1932. Notes on the onion maggot (Hylemya antiqua
Meig.). A. Rep. Ent. Soc. Ont. 62: 82-84.

 

King, J. E. and J. R. Coley-Smith. 1969. Production of volatile alkyl
sulphides by microbial degradation of synthetic alliin and alliin-
like compounds, in relation to germination of sclerotia of
Sclerotium cepivorum Berk. Ann. appl. Biol. 64: 303-314.

 

Kuhr, R. J., A. Comeau, W. L. Roelofs. 1972. Measuring release rates
of phermone analogues and synergists from polyethylene caps.
Environ. Entomol. 1: 625-627.

Krull, L. H., D. E. Gibbs, and M. Friedman. 1970. 2-Vinquuinoline, a
reagent to determine protein sulfhydryl groups spectrphotometrically.
Analyt. Biochem. 40: 80.

Larsson, L., E. Holst, C. G. Gemmell, and P.-A. Mardh. 1980.
Characterization of Clostridium difficle and its differentiation from
Clostridium 5 pro enes by automatic head-space gas chromatography.
Scan. J. Infect. Dis. Suppl. 22: 37-40.

 

 

Lee, M. L., D. L. Smith, and L. R. Freeman. 1979. High resolution gas
chromatographic profiles of volatile organic compounds produced by

microorganisms at refrigerated temperaturesnviron.
Microbiol. 37: 85-90.

Lelliott, R. A. 1974. Description of the species of the genus
Erwinia. In Bergey' 5 Manual of Determinative Bacteriology, 8th ed.
. . Buchanan and N. E. Gibbons, eds. ). The Williams and Wilkins
Co., Baltimore, MD.

Loosjes, M. 1976. Ecology and genetic control of the onion fly, Delia
anti ua (Meigen). Agric. Res. Rep. (Versl. landbouwk. Onde r2. )
udoc, Wageningen. 179 pp.

55

Mardh, P. -A., L. Larsson, and G. Odham. 1981. Head-space gas
chromatography as a tool in the identification of anaerobic bacteria
and diagnosis of anaerobic infections. Scand. J. Infect. Dis. Suppl.
26: 14-18.

Matsumoto, Y. and A. J. Thorsteinson. 1968. Effect of organic sulfur

compounds on oviposition in onion maggot, H lem a antiqua Meigen
(Diptera: Anthomyiidae). Appl. Ent. Zool. 3: 5-12.

Matsumoto, Y. 1970. Volatile organic sulfur compounds as insect
attractants with special reference to host selection. In Control
of Insect Behavior by Natural Products (0. L. W653, R. M.
Silverstein, and M. Nakajima, eds.). Academic Press, New York, pp.
133-160.

Miller, J. R., M. 0. Harris, and J. A. Breznak. 1984. Search for
potent attractants of onion flies. J. Chem. Ecol. 10: 1477-1488.

Niegisch, W. D. and W. H. Stahl. 1956. The onion: gaseous emanation
products. Fd. Res. 2: 657-665.

NOAA (National Oceanic and Atmospheric Administration). 1984. Local
climatological data. Annual summary with comparative data, Lansing,
MI. Temperature normals, means, and extremes based on the 1951-1980
record period.

O'Neill, R. and C. Logan. 1975. A comparison of various selective
isolation media for their efficiency in the diagnosis and
enumeration of soft rot coliform bacteria. J. appl. Bact. 39: 139-
146.

Peterson, A. 1924. Some chemicals attractive to adults of the onion
maggot and the seed corn maggot. J. econ. Ent. 17: 87-94.

Pierce, H. 0., Jr., R. S. Vernon, J. H. Borden, and A. C.
Oehlschlager. 1978. Host selection by Hylemya antiqua (Meigen),
identification of three new attraEtants andl oviposition
stimulants. J. Chem. Ecol. 4: 65-72.

 

Pittard, B. T., L. R. Freeman, 0. W. Later, and M. L. Lee. 1982.
I dentification of volatile organic compounds produced by flourescent
pseudomonads on chicken breast muscle. Appl. Environ. Microbiol. 43:
1504-1506. -

Ramakers, P. M. J. 1973. Oiapauze-induktie bij de uievlieg, Hylemya
nti ua (Meigen), onder invloed van fotoperiode en temperatuur.
Doctoraalverslag Afd. Entomologie, Landbouwhogeschool Wageningen, 37

a
PP-

Rygy, T. 1960. The onion fly (H lem a antiqua Meig.). Investigations
on its biology and control 1n orway. ( orwgian, Eng. summary)
Medd. St. Plantev. 18: 1-56.

Saghir, A. R., L. K. Mann, M. Ournbey, R. Y. Berg. 1966. Composition
56

of volatiles in relation to taxonomy of American Alliums. Am. J.
Bot. 53: 477-484.

Sainani, G. S., D. B. Desai, M. N. Natu, K. M. Katrodia, V. P. Valame,
and P. G. Sainani. 1979. Onion, garlic, and experimental
atherosclerosis. Jpn. Heart J. 20: 351-358.

Schneider, W. D., J. R. Miller, J. A. Breznak, and J. F. Fobes. 1983.
Onion maggot, Delia antiqua, survival and development on onions in

the presence and absence of’microorganisms. Ent. exp. 8 appl. 33:
50-56.

 

Schwimmer, S., M. Friedman. 1972. Genesis of volatile sulphur-
containing food flavours. Flavour Ind. 3: 137-145.

Tansey, M. R. and J. A. Appleton. 1975. Inhibition of fungal growth
by garlic extract. Mycologia. 67: 407-413.

Taylor, A. J. 1980. The application of gas chromatographic headspace
analysis to medical microbiology. In Applied Headspace Gas
Chromatography (B. Kolb, ed.). Heyden E—Son, Inc. Philadelphia, PN.
pp. 140-154.

Ticheler, J. 1971. Rearing of the onion fly, Hylemya antiqua
(Meigen), with view to release sterilized insects. Eh. Sterility
Principle for Insect Control or Eradication. (Proc. Symp., Athens,
1970). IAEA, Vienna pp. 341-346.

 

Tomioka, T. 1977. Plant Protection. (Shokubutsu Boeki) 31: 206-209.

Vernon, R. S., H. D. Pierce, Jr., J. H. Borden, and A. C. Oehlschlager.
1978. Host selection by Hylemyg antiqua: identification of
oviposition stimulants based on proposed active thioalkane
moieties. Environ. Entomol. 7: 728-731.

 

Vernon, R. S., G. J. R. Judd, J. H. Borden, H. D. Pierce, Jr., and A. C.
Oehlschlager. 1981. Attraction of H lem a anti ua (Meigen)
(Diptera: Anthomyiidae) in the field to as -pro uce oviposition
stimulants and their nonhost analogues. Can. J. Zool. 59: 872-881.

Vernon, R. S. and J. H. Borden. 1983. Dispersion of marked-released
Hylemya antigua (Meigen) (Diptera: Anthomyiidae) in an onion field.
nv1ron. n omol. 12: 646-649.

Vickery, M. L. and B. Vickery. 1981. Sulphur compounds derived from
cysteine. In Secondary Plant Metabolism. University Park Press,
Baltimore,'MD.

Weston, P. A. and J. R. Miller. 1985. Influence of cage design on
precision of tube-trap bioassay for attractants of the onion fly,
Delia antiqua. J. Chem. Ecol. 11: 435-440.

 

Whitaker, J. R. 1976. Development of flavor, odor, and pungency in
onion and garlic. Adv. Food Res. 22: 73-133.

57

Workman, R. B. 1958. The biology of the onion maggot (Hylemya antiqua
Meig.) under field and greenhouse conditions. Oregon State
College, Ph.D. Thesis, 82 pp.

 

Zechman, J. M. and J. N. Labows, Jr. 1984. Volatiles of Pseudomonas
aeruginosa and related species by automated headspace
concentration-gas chromatography. Can. J. Microbiol. 31: 232-237.

 

 

58

APPENDIX 1

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No.: 1986-2

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

Onion fly behavioral responses to microbially infected onion

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

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator's Name (s) (typed)

 

Susan Marie Hausmann

 

Date 3'2'86

 

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

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.
Research project files.

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

59

Pages

2

of

2

APPENDIX 1.1
Voucher Specimen Data

Page

 

 

sm-~-m ace;

was: scucpsu

 

 

.Esomzz >uo~o£cucm ccwEmzm: wwawx cmmsm
xudmpo>ac= macaw couaxowz use ea uwmcooc

Dem mcusfioucm woumv~ o>cow ecu vm>auuom
.oz poxoao> Acunxuv Amqumz m.u0unu«umw>c~

 

 

 

 

60

 

 

 

 

 

 

 

 

 

 

Ntmmmfi
Amumumouuc u“ mucosa fimcofiuwcvm wmav
33

am: as as o“ mm tosmaz.a .ou easesaz Seats ”a: cameo: wastage eases

Jubq voumuoacc car com: no cmuuw-cu ccxsu Ce:uc ac medcocm
m . s s s e mceefiumnm to» camp Huang
u e s r t t e .n a
Broadeallapns
s e p.e .n u u p. m .5
uhettddUYBoa
_M w.d.1 Au .5 .5 p. N_.L .L 1

"mo necesz