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BIOECOLOGY OF Paraphlepsius irroratus (Say) (HOMOPTERA:
CICADELLIDAE) : THE EFFECT OF THE X-DISEASE
MYCOPLASMALIKE ORGANISM ON PHYSIOLOGICAL DEVELOPMENT

presented by

Carlos Garcia Salazar

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BIOECOLOGY 0F Paraphlepsius irroratus (Say) (HOHOPTERA:
CICADELLIDAE): THE EFFECT OF THE X-DISEASE HYOOPLASHALIKE

ORGANISM OH PHYSIOLOGICAL DEVELOPMENT

BY

Carlos Garcia Salazar

A DISSERTATION

Sublitted to
Michigan State university
in partial fulfill-ant of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Departnent of Entomology

1991

[1

ABSTRACT

BIOECOLOGY OF Paraphlepsius irroratus (Say) (HOHOPTERA:
CICADELLIDAE): THE EFFECT OF THE X-DISEASE HYCOPLASHALIKE
ORGANISM OH PHYSIOLOGICAL DEVELOPMENT

BY

Carlos Garcia Salazar

The vector-pathogen relationship between Paraphlepsius
irroratus (Say) and the Eastern strain of the X-disease
mycoplasmalike organism was studied, and the effect of
temperature on physiological development of both, leafhopper
vector and x-disease determined. Assessment of X-disease
MLO acquisition and multiplication in MLO injected P.
irroratus was done utilizing a DNA probe that showed
specific hybridization with the Eastern strain of X-disease.
Growth and development of P. irroratus, as well as the
effect of the x-disease MLO on leafhopper longevity and
survival was studied at constant and fluctuating
temperatures regimens.

Research showed that.P. irroratus required 602 i 31 and
518 i 66 DD (degree days base 8 °C) to complete the nymphal
stage at constant and fluctuating temperature, respectively.

At constant temperature, the mean generation time required

939 i 31 DD. The lower developmental threshold for P.
irroratus was observed at 8 °C, and the upper threshold
temperature was around 28 °C.

Concerning the vector-pathogen association, there was a
temperature-dependent pathogenicity of the X-disease MLO to
its vector P. irroratus. Below 20 °C, the MLO accounted for
32% reduction in leafhopper longevity, compared with less
than 6% above 25 °C. The X-disease MLO also affected
leafhopper fecundity and a 48.8% fecundity reduction with
respect to healthy females was observed in X-diseased P.
irroratus. Compared with the X-disease MLO, the effect of
the injection solution on leafhopper longevity and survival
was negligible, and no statistical difference (P S 0.05) was
observed between buffer or heat inactivated MLO injected
leafhoppers and healthy non injected individuals.

There was a temperature effect on MLO growth and
multiplication in X-disease injected P. irroratus. The
optimum multiplication of the Eastern strain of x-disease
MLO in its leafhopper vector occurred in the 30-35 °C range.
No multiplication was observed at 15 °C, and only a small
titer was observed at 20 °C. Beyond 35 °C, the MLO titer

declined.

I dedicate this work to my parents Jose Cruz Garcia and
Micaela Salazar de Garcia. Their love and moral support
gave me the strength to succeed. To my wife, Maria Luisa
Kiwen whose love, understanding and support in the most
difficult moments of my career helped me not to yield to
failure. To my daughters, Rocio and Isis who have always

been the joy of my life.

iv

ACKNOWLEDGMENT

I like to thank Dr. Mark E. Whalon, my Graduate Advisor
and mentor, for the friendship and kindness always
demonstrated to myself and my family during all of my years
at Michigan State University. His advice and the trust he
deposited in me gave direction to my dreams and made it
possible for me to succeed where others met failure. I want
to thank the other members of my guidance committee Drs.
George S. Ayers, Stuart H. Gage, David R. Smitlly and Amy F.
Iezzoni for their inputs and insightful advice throughout
the planning, conduction and interpretation of my research.

I am grateful to my good friend Dr. Thomas M. Mowry for
his advice and encouragement in the early part of my
research. His moral values and standards were always an
example for me and the people who had the fortune to
interact with him.

Dr. L. N. Chiykowski deserves a special gratitude, the
advice and biological material be supplied were critical for
the success of my research. Likewise, special thanks to Dr.
B. C. Kirkpatrick for his DNA probe from which the x-disease

probe utilized in my research was derived.

I also, want to thank my fellow graduate student Miss.
Utami Rahardja and Mrs. Yang Tang Yong, our technician, for

their invaluable contribution to the success of my research.

Finally, I gratefully acknowledge the Mexican
Government, who through the National Council of Sciences and
Technology (CONACyT) and the National Institute of Forestry,
Agricultural and Livestock Researches (INIFAP) financed my
studies at Michigan State University's Department of

Entomology.

vi

TABLE OF CONTENTS

LIST OF TABLE ............................................ xii
LIST OF FIGURES .......................................... xiv
GENERAL INTRODUCTION..... ...... . ............ .... ........... l
The x-disease Importance............. ....... ...............1
X-disease Leafhopper Vectors ............................... 3
Biology of Paraphlepsius irroratus (Say) ................... 3

vector-Pathogen RelationShip O I O O O O O O O O O I O O O O O O O O O O O O O O O O O O O 5
Vector-Pathogen-Temperature Relationship...................7
ObjectiveSOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0000000000000000011

Bibliography ............ . ................................. 13

CHAPTER I. Temperature Related Development for the
Speckled Leafhopper (Paraphlepsius irroratus
(Say)) (Homoptera: Cicadellidae)............21

IntrOductionOOOOOOOOOOOO0....OOOOOOOOOOOOOOOOOOOO000......22

Materials and Methods.....................................24

1 O Leafhopper cu1ture O 0 ...... O O O O O 0 O O O O O O O O O O O O O O O O 0 O O O O O O 2 4
2. Treatments-0.0.0.000... ...... 0. ...... 00.... ......... 0.024
3. Egg Incubation Period...................... ....... .....25

4. Development Under Fluctuating Temperature..............26

vii

5. Statistical Analysis...................................28
6. Degree Days Estimate........................... ...... ..29
7. Test of Significance...................................30
Results...................................................31
1. Leafhopper Development at Constant Temperature.........31
2. Development at Fluctuating Temperature.................33
3. Development at Constant vs. Fluctuating Temperature....33
4. Lower Developmental Threshold..........................33
5. Upper Developmental Threshold..........................35
6. Physiological Time (DD) and Development................35
Discussion................................................38

Bibliography..................... ..... ....................43

CHAPTER II Temperature Dependent Pathogenicity of the X-
disease Mycoplasmalike Organism (MLO) to its
vector, Paraphlepsius irroratus (Say)
(Homoptera: Cicadellidae)...................46

Introduction..............................................47
Materials and Methods.....................................49
1. Leafhopper Culture.....................................49
2. Temperature and Injection Treatments...................50
3. Injection Protocol.....................................52
4. MLO Acquisition Determination..........................53
5. Dot Blot Hybridization.................................53
Results...................................................56
1. MLO Effect on Leafhopper Longevity.....................56

2.M110AcquiSitionOOOO0.0.000......DOOOOOOOOOOOOOIOOOOOOO.57

viii

3. Temperature Effect on Leafhopper Longevity ............. 57
4. Injection Solution Effect.............. ..... ...........59
Discussion... .......................................... ...61

Bibliography. ......... .... ........... .....................61

CHAPTER III Effect of the Eastern Strain of the X-disease
Mycoplasmalike Organism on the Fecundity of
its Vector, Paraphlepsius irroratus (Say)
(Homoptera: Cicadellidae)...................71

Introduction......... ...... .................. ........... ..72
Material and Methods......................................73
1. Leafhopper Culture.....................................73
2. Treatments.............................................74
3. Healthy Leafhoppers....................................74
4. x-disease Injected Leafhoppers.. ..... ....... ......... ..75
5. x-disease Acquisition Test.............................76
6. Dot Blot Hybridization....... .......................... 77
7. Data Analysis..........................................78
Results...... ............... . ............. .... .......... ..79
Discussion................................................80

Bibliography................................. ...... .......85

ix

CHAPTER IV Response of Paraphlepsius irroratus (Say)‘
(Homoptera: Cicadellidae) to the injection of
Different Non-Pathogenic Substances and the
Eastern X-disease Mycoplasmalike Organism...88

Introduction................ ..... .. ...... . ..... ...........89
Materials and Methods. .................................. ..90
1. Insect Culture.........................................90
2. Treatments.............................................90
3. Injection Protocol.....................................91
4. Parameters and Analysis ............................... .92
5. X-MLO Diagnosis..... ...... ......... ....... . .......... ..93
Results...................................................94
1. Survival Analysis.......................... ...... ......95
2. MLO’s Acquisition. ..................... . ........ .......98
Discussion..... ......................................... ..98

Bibliography... ....... ...................................106

CHAPTER V x-disease Myc0plasmalike Organism: In Vivo
Developmental Threshold Temperatures
EstimatBOOOOOOOOOOOOOOOOOOOOOOOOO ..... 0.00.110

Introduction.............................................111
Materials and Methods....................................112
1. Insect Culture. ................. . ....... . ............ .112
2. Treatments............................................113
3. Preparation of Inoculum...............................1l3

4. Leafhopper Injection..................................114

5. X-disease MLO Detection Method........ ..... ...........115
6. MLO Extraction...... ........... . ............ ..........115
7. Titer determination.. ............................... ..116
8. Blotting Procedure....................................116
9. DNA Hybridization Protocol.... ..... ...................117
10. Probe Radiolabelling................................118

11. Data Analysis........................................119
Results..................................................120
1. X-disease MLO Multiplication..........................120
2. X-disease MLO Titer................. ..... .............123
3. Time Course of MLO Multiplication........... ..... .....124
4. Temperature Dependent X-MLO Titer.....................128
5. Protein Development Over Time.........................128
Discussion... ............ ..... ........... ................129

Bibliography.............................................139

GENERAL CONCLUSIONS........OOOOO........O................142

APPENDIX A Record of Deposition of Voucher Specimens
APPENDIX B Survival Curves, Autoradiographs

APPENDIX C Autoradiographs of X-disease Multiplication and
Controls

APPENDIX D System Diagram of Paraphlepsius irroratus (Say)

xi

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Developmental time in days for immature
stages of P. irroratus reared at nine
constant temperatures................... ...... 32

Developmental time in days for immature
stages of.P. irroratus reared at four
fluctuating temperatures......................34

Development of P. irroratus from
emergence of first instar to adult at
constant and fluctuating temperatures.. ....... 36

Lower (TL) and upper (T ) threshold

temperature and degree gays (DD)

estimates for biological stages of P.

irroratus at constant temperature.............37

The effect of the X-disease MLO on the
longevity of P. irroratus, at different
fluctuating temperatures......................60

The effect of temperature on X-disease
MLO acquisition by.P. irroratus...............6o

The fecundity summary of healthy and X-
disease injected P. irroratus............ ..... 81

Comparison of longevity of P. irroratus

injected with different solutions and

incubated at 20 °C, fluctuating
temperature...................................96

Weibull statistics for survival curves

of P. irroratus injected with different
solutions and incubated at 20 °C

fluctuating temperature.......................99

X-disease acquisition in MLO injected P.

irroratus incubated at constant
temperature..................................121

xii

Table

Table

Table
Table

Table

Table

Standard curve of X-disease MLO inoculum
used to inject healthy P. irroratus..........121

Taylor’s parameters for the optical
density (OD) of X-disease inoculated P.

'irroratus incubated at constant

temperature.......................... ....... .125

Maximum X-disease titer in MLO
inoculated P. irroratus incubated at
constant temperature.........................126

Titer of X-disease in MLO injected P.
irroratus with different incubation
periods at constant temperatures.............127

Protein accumulation in X-diseased P.
irroratus incubated at constant
temperature...........................O......131

Taylor's parameters for protein
production in X-diseased P. irroratus
incubated constant temperature...............135

xiii

Figure

Figure

Figure

Figure

Figure

Figure

3.1

LIST OF FIGURES

Temperature cycles utilized to study the
development P. irroratus éthirty, 30 °C;

tfive, 25 °C; twenty, 20 ; and

fifteen, 15 oC, average temperature...........27

Temperature regimens utilized to study

the effect of the X-disease MLO on the

longevity of P. irroratus (thirty, 30 °C;

tfive, 25 °C; twenty, 20 0C: and fifteen,

15 °C, average temperature) ..................51

The DNA probe C6c: 1 and 2, C6c

(partially digested EcoRI and HindIII

pWXl): 3, Undigested pWXl: kb, 1 kb

marker and L, Hind III digested lambda

DNA marker....................................55

Deleterious effect of X-disease MLO and
temperature on the longevity of P.
irroratus. Healthy leafhoppers lived
for 62 i 16 days at 15 °C. MLO effect
(OvO-O); temperature effect (O-O-O)..........58

Oviposition patterns of healthy and X-

disease injected P. irroratus. X-

diseased females (0-0-0), healthy

females (O~O-O)...............................83

Survival curves of populations of P.

irroratus injected with different

solutions. Control= healthy non-

injected leafhoppers, Buffera saline

buffer (PBS), Tetracyc= Tetracycline
hydrochlorite, EX_MLO= extract of X-

diseased leafhoppers, Healthy= extract

of healthy leafhoppers, TET-MLO= X-
MLO+Tetracycline, Inac_MLO= heat

inactivated X-MLO extract.....................97

xiv

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

8.4

Standard curve to transform the observed
OD (optical density) values from X-
diseased P. irroratus to C6c equivalents

(ng).........................................122

Titer curve of Eastern X-disease MLO

incubated in P. irroratus at different

constant temperatures. The MLO titer is
expressed as C6c equivalents (ng)............130

Protein concentration (ng/lo ul) in

homogenates from X-diseased P. irroratus
incubated at different fluctuating
temperatures............................ ..... 133

The effect of X-disease on longevity and
survival of.P. irroratus at 15 °C,

average temperature. Control, healthy
non-injected leafhoppers; Inac_MLO,

individuals injected with inactivated X-
disease; X_MLO,.P. irroratus injected

with X-disease...............................147

The effect of X-disease on longevity and
survival of P. irroratus at 20 °C,

average temperature. Control, healthy
non-injected leafhoppers: Inac_MLO,

individuals injected with inactivated X-
disease; X_MLO, P. irroratus injected

with X-disease...............................148

The effect of X-disease on longevity and
survival of P. irroratus at 25 °C,

average temperature. Control, healthy
non-injected leafhoppers; Inac_MLO,

individuals injected with inactivated X-
disease: X_MLO, P. irroratus injected

with X-disease...............................149

The effect of X-disease on longevity and
survival of P. irroratus at 30 °C,

average temperature. Control, healthy
non-injected leafhoppers; Inac_MLO,

individuals injected with inactivated X-
disease: X_MLO, P. irroratus injected

with X-disease.......................... ..... 150

The effect of temperature on longevity

and survival of X-disease injected P.
irroratus. Thirty, 30 °C: Tfive, 25 °C;

Twenty, 25 °C: Fifteen, 15 °C, average
temperature............................ ...... 151

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

The effect of temperature on longevity

and survival of healthy non-injected P.
irroratus. Thirty, 30 °C; Tfive, 25 °C;

Twenty, 25 °C; Fifteen, 15°C, average
temperature..................................152

DNA hybridization reaction of

radiolabelled C6c with leafhoppers

injected with inactivated X-disease MLO

after 48 h of exposure. Sc, Spiroplas-a

citri; C, the probe C60; PI, X-diseased

.P. irroratus; PH, healthy P. irroratus:

TL, inactivated MLO injected
leafhoppers..................................153

DNA hybridization reaction of

radiolabelled C6c with leafhoppers

injected with X-disease MLO after 48 h

of exposure. Sc, Spiroplas-a citri: C,

the probe C6c: PH, healthy P. irroratus:

PI, X-diseased P. irroratus: : TL, X—

disease injected leafhoppers.................154

X-disease MLO multiplication in MLO
injected P. irroratus incubated at 15

oCoooooooooooooooooooooooooooooooooooo ...... .155

X-disease MLO multiplication in MLO
injected P. irroratus incubated at 30

C...........................................156

X-disease MLO multiplication in MLO
injected P. irroratus incubated at 35

Geoooooooooooooooooooooo00.000.000.00...0.0.0157

X-disease MLO multiplication in MLO
injected P. irroratus incubated at 38

°C...........................................158

Positive controls for X-disease MLO
hybridization. A, serial dilution of X-

disease inoculum; 8, serial dilution of

the probe C6c................................159

System diagram of Paraphlepsius
irroratus (Say)..............................160

xvi

GENERAL INTRODUCTION

The X-disease Importance.

Yellows diseases are a group of plant diseases
associated with mycoplasmalike organisms (MLO) affecting
monocotyledons and dicotyledons, particularly in temperate
regions of the world (Whitecomb & Black 1982). MLOs
responsible for "Yellows" diseases are extracellular
organims inhabiting the sieve tubes of the vascular system
of infected plants. Plant MLO infections cause a more or
less uniform yellowing or reddening of leaves, smaller
leaves, shortening of the internodes and plant stunting,
sterility of flowers and reduced yields as well as
"witches'" brooms, rapid dieback, decline and death of
infected plants (Agrios 1978). In fruit trees, pear
decline, coconut lethal yellowing, apple proliferation and
X-disease are the most important representatives of this
destructive group of diseases. The X-disease mycoplasmalike
organism is the causal agent of the ”yellows" diseases in
peach and cherry trees. Its geographical distribution
comprises the United States of America and eastern Canada
(Nemeth 1986). Reeves et a1. (1951) suggested the name of

Western X-disease for the strain of X-disease present in

western USA. Meanwhile, the strain attacking stone fruits
in central and eastern USA and Canada has been termed Easter
X-disease (Thornberry 1954, Chiykowski & Sinha 1982). The
X-disease agent, has been probably the most devasting
pathogen for peach and cherries and a limiting factor to the
stone fruit industry of the United States and Canada. In
the United States, peach production began around 1633 and
until 1791 peach trees grew with no symptoms or problems of
"yellows" diseases (Whitecomb & Black 1982). However, the
decline of the peach industry in the states of Maryland,
Delaware and Michigan during the 1800's was due to the peach
yellows disease. An example of the distribution of the
disease was documented in Berrien County, Mich., between
1874 and 1884, the acreage in peach dropped from 6000 to 500
and the number of trees from 654,000 to fewer than 55,000
(Wood 1953). As of 1987, Michigan had 1,050,000 trees
planted on 8,800 acres, and approximately 58% located in
Berrien and Van Buren Counties (Fedewa & Pscodna 1982,
1987). During the same year, 238,602 peach trees were
inspected in Berrien County, and 1.4% of inspected trees
showed X-diseased symptoms (Whalon 1988, unpublished
results). On the Pacific Coast, a survey conducted in 1979
in peach orchards of northern California showed that 2.87%
all inspected trees were infected by x-disease (Purcell et
a1. 1981). A similar X-disease survey conducted in Ontario,

Canada, showed that 50 % of all sampled orchards were

affected by X-disease and the percentage of affected trees

in these orchards was around 2% (Dhanvantari & Kappel 1978).
X-disease Leafhopper Vectors.

Phloem feeding leafhoppers are responsible for the
transmission and dissemination of the X-disease MLO in the
peach growing regions of North America. Known leafhopper
vectors of the Western strain of X-disease are Acinopterus
angulatus Lawso, Cblladonus gelinatus (VanDuzee),
Cblladbnus lontanus (VanDuzee), Cblladonus clitellarius
(Say), scaphytopius acutus (Say), Scaphytopius delongi
Young, Scaphytopius nitridus (DeLong), Fieberiella florii
(Stal), xeonolla confluens (Uhler), Osbornellus borealis
(DeLong and Mohr), and Enscelidius variegatus (Kirschbaum).
For the Eastern strain of X-disease the following leafhopper
vectors have been identified: Gyponana Janina (DeLong),
Paraphlepsius irroratus (Say), Ebrvellina selinuda (Say),
Orientus ishidae (Mat.), Fieberiella florii (Salt.),
scaphoideus melanotus Osb., scaphoideus titanus Bell, and
Scaphytopius acutus (Say) (Taboada et al. 1975, Chiykowski

1981, Nielson 1979).
Biology of Paraphlepsius irroratus (Say)

In Michigan peach and cherry orchards the Speckled

leafhopper, Paraphlepsius irroratus (Say), is the principal

vector of the Easter X-disease MLO due to its abundance and
efficiency as a vector (Taboada et al. 1975, Rosenberger &
Jones 1978, Larsen & Whalon 1988). The Speckled leafhopper
inhabits the peach and cherry orchard ground cover and
surrounding vegetation. The adult leafhoppers spend the day
in the ground cover and at twilight they move into fruit
tree foliage to feed. Only adults feed on the fruit tree,
while immatures remain in the orchard ground cover feeding
on grasses (Larsen & Whalon 1988, Chiykowski 1985). Until
recently, few studies on the biology and ecology of P.
irroratus were possible. Difficulties in rearing this
leafhopper under laboratory conditions prevented major
studies on its bioecology. Chiykowski (1982, 1985) was able
to maintain a P. irroratus laboratory culture long enough to
study some aspects of its biology, host plants and factors
affecting X-disease vector competence. These studies
demonstrated that at 23 °C constant temperature the nymphal
period passes through five instars and depending on host
plants, it requires 45.58 t 7.32 days (Mean 1 SD) for
complete development. For adult P. irroratus, host plants
suitable for food and oviposition are those belonging to
composite and Legule families. Immatures prefer species in
the Grass family (Graminae). In Michigan, there are two
generations of P. irroratus a year. Spring adults emerge
from late May to July and the second generation occurs from
August to October. .P. irroratus requires 1950 i 150 degree

days (Base 50 °F) (cumulated since the beginning of the

spring emergence) to complete two generations (Taboada et

a1. 1975, Larsen & Whalon 1988).

Vector-Pathogen Relationship.

Mollicute phytopathogens can survive and multiply in a
wide variety of environments including plant phloem, insect
haemolymph, and insect gut and salivary glands (Purcell,
1982). The X-disease mycoplasmalike organism (X-MLO) is a
circulative propagative disease that requires vectors for
its dissemination. The X-MLO is circulative or persistent
because its transmission is characterized by an incubation
period (IP) between acquisition and first transmission and
by a continuation of MLO transmsission for many days
following the removal of the vector from the MLO source. It
is propagative because the X-MLO multiplies in the vector
tissues (Tinsley 1973, Harris 1979). After an access
acquisition period (AAP) on a MLO infected host, the X-MLO
is acquired by the vector via the maxillary food canal
absorbed, translocated, and invade internal tissues
including midgut, haemocoel and salivary glands. Following
an IP, leafhoppers transmit the MLO to plants in the saliva
ejected from the maxillary canal during the feeding process.
According to Purcell (1982), a large number of prokaryote
species, including MLOs, are insect-associated but few of
them are pathogenic to their hosts. Mycoplasmalike

organism-vector relationships vary from incidental

commensalistic to pathogenic in the instance of some
leafhopper vectors (Hackett & Clark 1989). Whitcomb &
Williamson (1979) defined some criteria to characterize
pathogenicity of wall-less prokaryotes infecting arthropods
that included effect on longevity and fecundity as well as
histological and physiological effects. Leafhopper examples
where associated MLO maybe pathogenic are colladonus
lontanus (VanDuzee) and X-disease; Apia Jellifera L.and
Spinoplasna neliferul: Circulifer tenellus (Baker),
Macrosteles fascifrons (Stal.), D. elinatus (Bell),.D.
gelbus DeLong and Spiroplas-a citri Saglio et al.(Jensen et
al. 1967, Hackett & Clark 1989, Calavan 8 Bove 1989, Madden
et al. 1984). In all these cases, longevity and/or
fecundity is severely affected by MLO infection.
Furthermore, histological studies revealed extensive
disruption of organ tissues such as salivary glands,
alimentary tract, oviducts and cephalic structures of MLO-
diseased leafhoppers (Whitecomb et al. 1968, Nasu et al.
1970, Sinha & Chiykowski 1967). Observed non-pathogenic
relationships include Dulbulus laidis (DeLong & Walcott)
and Spiroplas-a kunkelii, maize bushy stunt mycoplasma
(MBSM) and D. elilatus and.D. gelbus, also M; fascifrons
and Aster Yellows MLO (Petrson 1973, Nault et al. 1984,
Madden & Nault 1983). These vector-pathogen relationships
in some cases resulted in increased longevity and survival
of vectors such as M. fascifrons and D. laidis grown on AY-

diseased plants (Peterson 1973, Purcell 1988). According to

Purcell (1982), MLO pathogenicity to its vector may reflect
a lack of adaptation because an increased virulence may
reduce parasitic fitness since vector and parasite mortality
may be linked. From the point of view of vector-pathogen
relationship co-evolution it would be expected low virulence
to a vector that has been an implicit part of the long-term
evolution of the pathogen it transmits. Thus, minimizing
pathogenicity to its vectors is an advantage to the MLO
since the number of transmissions per infected vector will
be maximized in the absence of a pathogenic interaction

(Whitecomb 1989).
vector-Pathogen-Telperature Relationship.

The vector-pathogen relationship does not occur in
isolation but in the physical environment surrounding insect
vectors, pathogens and host plants. Since insects are
poikilothermic organisms, they depend on the environmental
temperature for physiological development and activity. The
same is true for pathogens and host plants. Thus,
temperature might be regarded as a driving factor in the
transmission dynamics of any insect-borne pathogen. Purcell
(1985) outlined several epidemiological factors that can be
affected by temperature including; 1) pathogen N
multiplication and survival in both insects and plants; 2)
pathogenic interaction with other microbes: 3) disease

development in insects and plants, including recovery or

death of hosts; 4) vector survival, reproduction and
developmental rates; and 5) pathogen transmission rate.
Likewise, three basic steps of the pathogen transmission
process involving the insect vector are temperature related;
a) access acquisition period (AAP): b) incubation period
(IP) and median incubation period (IP50) and c) inoculation
access period (IAP). Research on the effect of temperature
on the AAP of some viruses and MLOs showed that at 10 °C
only 7% of a population of HephotetiX'virescens (Distant)
fed on tungro virus infected plants acquired the pathogen,
but 85% did so at 31 °C (Lingo 8 Tiongco 1979). x-disease
MLO acquisition in X-disease injected c; nontanus maintained
at 10 and 20 0C was undetectable and 85% respectively
(Jensen 1972). In the case of MLO transmission, insect
vectors can not transmit the pathogen immediately after
feeding on infected host plants. Depending on the
temperature, insect vectors may start transmitting after an
incubation period varying between 10 and 45 days. In most
MLOs, the shortest incubation period occurs around 30 °C and
the longest at about 10 °C (Agrios 1978). For example, the
IP50 in X-disease injected C. lontanus was 122 and 26 days
at 15 and 26 °C, respectively (Jensen 1972). The expression
of symptoms after MLO inoculation (incubation period in host
plants) vary from disease to disease depending on
temperature. For 3. citri the classic symptoms occur at 30-

32 °C and at 18-24 °C for clover phyllody. On the other

hand, very high temperatures (greater than 30 °C) may cure

plants of MLOs infection (Markham 1982).

Very little research has been done on the effect of
temperature on the IAP. The published literature reports
differences in MLO acquisition given different IAPs, but
always under a single temperature regime. MLO-infected
C.tenellus, for example, required a 2 h feeding period on
indicator plants to transmit the MLO at 30 i 3 °C (Liu et
al. 1983). In a separate experiment with the same vector-
pathogen-host plant system, Golino et al. (1987) reported
MLO transmission with an IAP as little as 30 min at 24:20 i

3 °C day:night temperature regime.

Pathogen survival and multiplication both in insect
vectors and host plants is critically affected by
temperature. Under field conditions, the low temperatures
of late fall, winter and early spring are below the minimum
required by most pathogens and usually the shortest time for
the completion of a disease cycle occurs when temperatures
are optimum for pathogen development (Agrios 1978). The
growth rate and survival of most pathogens raises slowly as
temperature increases from a minimum to the optimum, then
it falls sharply as the temperature increases from the
optimum to the maximum. Below the minimum or above the
maximum temperature deleterious effects occur to most

pathogens (Van Der Plank 1975). In the case of Mollicutes,

10

animal mycoplasmas have an optimum growth temperature around
37 °C and most are unable to grow at 22 °C. Meanwhile,
plant and insect Mollicutes such as s. citri have an optimum
growth in the range 27-35 °C (Archer 8 Daniels 1982). The
Aster Yellows MLO optimum growth temperature is around 25
°C, but, no development occurs below 10 °C or above 30 °C
(Agrios 1978, McCoy et al. 1989). Despite the reports, the
full extent of the effect of temperature on MLO survival
(threshold temperatures) and multiplication remains unknown.
Two problems have prevented researchers from elucidating
this matter. The first problem is the impossibility of in
vitro MLO culture. With the exception of the genus
Spiroplasma, all attempts to in vitro culture

mycoplasmalike organisms associated with plant yellows
disease to date have met with failure (Chen et al. 1989).
The second problem is inappropriate and cumbersome MLO
detection and identification techniques. Given the lack of
ability to culture mycoplasmalike organisms, detection and
identification of these Mollicutes has relied heavily on
symptomatology in indicator plants and on examination of
plant and insect tissues through electron microscopy.
However, many symptoms associated with MLO infections have
not been unique to disease caused by MLOs alone (eg.
chlorosis or yellowing, witch’s brooms, floral reversion and
virescens). Such symptoms can be associated with insect
feeding, viral or fungal diseases, nutritional deficiencies,

natural leaf senescence or the genetics of indicator plants

11

(McCoy et al. 1989). More recently, the use of
immunosorbent electron microscopy (ISEM), polyclonal and
monoclonal antibodies for enzyme linked immunoassay analysis
and the development of cDNA probes have allowed a more
precise detection and identification of MLO agents such as
the Spiroplasma kunkelii, AY-mycoplasmalike organism and X-
disease MLO (Madden 8 Nault 1983, Sinha 8 Chiykowski 1986,

Chiykowski 8 Sinha 1988, Kirkpartrick et al. 1987)

Objectives.

The importance of P. irroratus as a X-disease vector in
Michigan peach and cherry orchards has been discussed.
However, relatively little is known about its biology and
the vector-X-disease relationship. The effect of
temperature on both the primary Michigan leafhopper vector
and X-disease MLO remain unknown. Important management and
prediction components such as developmental thresholds and
optimum growth temperatures for both organisms are critical
to predict the leafhoppers’ phenology and epidemiology of X-
disease under field conditions. In the emerging Integrated
Pest Management (1PM) strategy to combat X-disease, a
phenology model of P. irroratus, for management purposes is
vital for the success of a leafhopper suppression strategy
using chemical pesticides. Another important feature
essential to understand X-disease transmission rates under

filed conditions is the nature of the P. irroratus-X-disease

12

MLO relationship (commensalistic, mutualistic, pathogenic,
etc.). So far, there are indications that some Spiroplasmas
and mycoplasmalike organisms are pathogenic to some of their
leafhopper vectors, but this relationship has not been
studied in P. irroratus. Yet, the X-disease MLO has been
shown to be pathogenic to C. montanus, and this has been
important information for management. The effect of
temperature on X-MLO survival and multiplication is for the
most part unknown. Since the X-MLO is uncultivable, so far
its thresholds and optimum growth temperatures have been
partially elucidated through symptom development in
indicator plants. Given the state of our current knowledge,

the objectives of this research were:

1) quantify development of P. irroratus under constant and
fluctuating temperatures, and to estimate its upper and
lower thresholds as well as optimum growth

temperatures.

2) to elucidate the effect of the X-disease MLO on P.
irroratus longevity and fecundity as well as to

understand the nature of their relationship.

3) characterize the effect of temperature on X-disease MLO

survival and multiplication.

13

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21

CHAPTERI

Temperature Related Development of the Speckled Leafhopper
(Paraphlepsius irroratus (Say)) (Homoptera: Cicadellidae), a

Vector of x-disease .

22

Introduction.

Phloem-feeding leafhoppers are among the most serious
pests of stone fruits in the peach and cherry growing
regions of Michigan. Damage results from leafhopper
transmission of a disease of mycoplasma-like organism
etiology. This disease was called ”X-disease" because the
causal agent was unknown when described (Reeves et al.
1961). This disease is one of the most serious limiting
factor of peach and cherry production in Michigan. The
pathogen is a mycoplasmalike organism (MLO) found in the
phloem sieve tube elements of diseased trees and affected
trees become commercially worthless in two to four years.
Young peach trees are rendered useless within two to three
years of inoculation (Agrios 1978).

The leafhopper Paraphlepsius irroratus (Say) is a key
vector of the X-disease MLO because of its abundance,
transmission efficiency, range association with alternate
host, and movement from alternate host to stone fruit trees
(Taboada et al. 1975, Rosenberger 8 Jones 1978, Larsen 8
Whalon 1988). In a survey conducted from Bainbridg Center
(42° 7' NL) to Walkerville, Michigan (43° 43' NL), 61% of
the total leafhoppers caught in yellow sticky board traps
and sweep nets were.P. irroratus (Larsen 8 Whalon 1988).

The leafhopper was most abundant at southwest and

23

west-central Michigan where peach and cherry orchards are
the hardest hit by the X-disease MLO. The number of
generations varied from two in southwest and west-central
Michigan to one in northwest Michigan and there was an
apparent relationship between degree days (DD) accumulated
and the number of generations (Taboada et al 1975, Larsen 8
Whalon 1988). The occurrence of X-diseased leafhoppers in
the field has been assessed since 1987. X-disease was
detected in the first eclosing adult leafhoppers trapped in
each generation, but the proportion of infected individuals
decreased through mid-summer and in late generation flights
(Rahardja 1990).

X-disease control has been based on intense
insecticidal suppression, alternate host eradication and
antibiotic treatment of symptomatic trees. Yet, there has
been no scientific demonstration of reduction of X-disease
transmission as a result of insecticide application. On the
other hand, the chokecherry eradication program has not
prevented the spread of the X-disease in areas where most of
the alternate hosts were already removed (Larsen 8 Whalon
1988). Michigan State University has been developing an
integrated X-disease management program comprising the above
mentioned practices as well as ground cover management, X-
disease monitoring (Rahardja et al. unpublished data), and
efficient timing of insecticide applications. As a first
step in the design of a degree day phenology timing system

for insecticide application, this researche reports the

24

effect of constant and fluctuating temperature on the growth

and development of P. irroratus.

Materials and Methods

Leafhopper Culture. Biological material for this study
was obtained from a leafhopper laboratory culture maintained
at the Michigan State University Pesticide Research Center
since 1987 (Garcia-Salazar et al. 1991). The culture was
initiated with field collected P. irroratus and augmented
during each seasonal generation by adding approximately 500
new field collected individuals to ensure genetic diversity.
To select treatment cohorts of uniform age, every 12 h newly
emerged nymphs were transferred from oviposition cages to
separate cages containing 2-wk old barley (Herdeum vulgaris

L.) seedlings.

Treatments. The developmental thresholds and duration
of developmental stages of P. irroratus. were determined at
6, 9, 12, 15, 20, 25, 30, 35, and 38 all with a variance of
11 °C. Each constant temperature regime was randomly
assigned to a different growth chamber while cohorts of 40-
60 less than 12-h old nymphs were randomly assigned to three
replicates. Treatment nymphs were placed individually on a
barley seedling into a small cage constructed of screened
PlexiglasTM tubing (10.0 cm height and 2.5 cm diameter)

attached with paraffin to the lid of a 1 oz. plastic cup

25

containing tap water. The lid had an opening in the center
allowing the seedling roots to be immerse in tap water,
while the stem was into the cage serving as a feeding
substrate for the nymphs. Insects were introduced into the
cage through the top which was closed with foam.
Developmental stages were monitored every 24 h and their
duration determined by counting and removing exuviae. The
number of insects that survived to the next stage was
recorded for each temperature regime, and all insects dying
due to accidental handling were eliminated from the
statistical analysis. Because of limited availability of
insects and growth chambers, temperature treatments
corresponding to 15 to 38 °C were run simultaneously, while
treatments corresponding to 6 to 12 °C. were conducted

later.

Egg Incubation Period. The incubation period was
estimated at 25 and 27 °C with a variance of :1 °C. As P.
irroratus eggs are laid inside the stems of clover plants,
it is difficult to count or remove them nondestructively.
Therefore, to estimate the incubation period cohorts of
adult.P. irroratus (four females and four males per cohort)
were allowed to oviposit on clover plants for 24 h and then
transferred to a new oviposition cage and the previous cage
held until nymphs emerged. The incubation period was
estimated as the time elapsed between the removal of females

from the oviposition cage and the time when the first

26

batches of nymphs emerged. Cohorts rather than individual
females were utilized to ensure oviposition, expecting that
at least one female per cohort would oviposit. The total
number of P. irroratus cohorts utilized was 42 and 15 for 25
and 27 °C, respectively. Leafhopper cohorts were randomly
allocated to three replicates. There were 14 and 5 cohorts
per replicate at 25 and 27 °C, respectively. Each cohort
was caged on a plastic pot (11.5 cm diameter, 14.0 cm deep)
containing a mixture of white clover and barley. The cages
were made from 2 liter soda bottles to which the neck was
removed to fit snugly on the top of the pot. To prevent
moisture condensation, each cage had six screened windows
(4.5 cm diameter) and an opening (3.5 cm diameter) on the
top for insect handling which was covered with foam. Clover
plants provided the substrate for oviposition while barley
was the preferred food for nymphs. Due to limited
availability of insects, tests for obtaining the incubation

period at 27 and 25 °C were staggered.

Development Under Fluctuating Temperature. Fluctuating
temperatures representing early spring, late summer and hot
mid-summer at 45° N latitude were chosen to study the
temperature related development of P. irroratus. The
temperatures fluctuated according to a 24 h sinuosoidal
programmed wave between a maximum and a minimum temperature

averaging 15, 20, 25, and 30 °C with a variance of :1 OC

27

 

 

40
35
3
9 0
.
5 25
2
E; 20
h' 15
D THIRTY
10 I THVE
C>11VENTY
5 _ J l l I l l . FIFTEEN
0 5 15 24 32 40 45

Figure 1.1. Temperature cycles utilized to study the
development P. irroratus (thirt , 30 °C: tfive, 25 0C:
twenty, 20 °C: and fifteen, 15 C, average temperatur).

28

(Figure 1.1). The temperature cycles were assigned randomly
to four growth chambers (Percival Manufacturing Co. Boone,
Iowa 50036). As in the study under contestant temperature,
cohorts of 42-45 first instars were randomly allocated to
three replicates and held individually in small seedling
cages. Temperature treatments were run simultaneously and
nymphal development monitored every 24 h. The number of
instars and their duration was estimated by counting and
removing exuviae from the seedling cages. At each
temperature regime, insects surviving to the next stage were
counted recorded and any insects dying due to accidental

injury excluded from the statistical analysis.

Statistical Analysis. The lower developmental
temperature threshold (TL) was estimated by regressing the
log of 1/development against temperature (Meter 8 Wasserman
1974). Only data from the linear portion of the development
curve was used and the point where the regression line
crossed the X axes was the lower temperature threshold
(Taylor 1979). Thus, lower developmental thresholds for all
biological stages (except eggs) were determined. The upper
developmental, Th, threshold was estimated according to

Taylor's (1981) equation:

Rm = R.*exp(-o.5('r-'r./rs)2)

29

where R(t), insect growth rate, increases with temperature
to a maximum = R . The temperature in which R. occurs has
been called the optimum temperature T-. Beyond T.,
development declines due to the lethal effect of high
temperatures. The spread of the developmental curve is
determined by T3, the standard deviation of temperature
while T is the observed temperature at time (t). Parameters
for the Taylor’s equation were estimated through an
iterative process utilizing the statistical packages SYSTAT

and SYGRAPH (SYSTAT INC. Evanston, Il 60201).

Degree Davastimate. The durations of different
biological stages of.P. irroratus reared at constant and
fluctuating temperatures were transformed to physiological
time (DD) according to Allen (1976). This method allows to
estimate DD on a half-day basis. Three algorithms were
utilized depending on the position of the upper and lower
temperature threshold. In the first case, when development
occurred above both the lower (TI) and upper (Tb) threshold

the corresponding degree days were estimated as follows:

DD = 1/2(TU-TL) where:
Tb = Upper developmental threshold, and T1 = Lower
developmental threshold. When development occurred within
the developmental thresholds the estimate of DD was done

using the equation:

30

DD = 1/2(Tm-Ti) where:
T. = Mean temperature, and ft = Lower developmental
threshold. In the third case, when the maximum temperature
exceeded the upper developmental threshold the algorithm

utilized was:

DD = 1/(2*pi)*[(Th-TL)*(b+pi/2)+(Tb-TL)*((pi/2)-b)-a*008(b)J
where T. = Mean temperature, TL = lower threshold, pi =
3.1416, a = (Tmax-Tmin1)/2 for the lst half-day, and (Tmax-
Tmin2)/2 for the 2nd half-day, and finally b = sin'1 [(TU-

Tm)/a].

Test of Significance. Pairs of populations subjected
to constant and fluctuating temperature (i.e. insects
developing at 20 0C constant versus individuals developing
at 20 °C average, etc.) were compared using the Student’s t
test. Statistical differences between the mean degree days
required to complete the immature stage under both
conditions were determine at P S 0.05 and P S 0.01, assuming
that both populations were normally distributed with equal
variance 02 and n1 different from n2. Comparison was done

according to :

t = (Yl-Y2)/sqr( 82(n1+n2/n1*n2))
where Y1 = mean degree days at constant temperature; Y2 =
mean degree days at fluctuating temperature: 82 = pooled

estimator of the common variance 02: n1 = number of

31

observations in the population at constant temperature; n2 =
number of observations in the population at fluctuating
temperature (Steel 8 Torrie 1980, Bhattacharyya 8 Johnson

1977).

Results

Leafhopper Development at Constant Temperature. At
constant temperatures, P. irroratus developed over the range
9 to 38 °C. At some temperatures, however, adults failed to
develop. In general, younger instars tolerated extreme
temperature conditions better than later instars. The first
and second instars developed at the extreme temperatures; 9
and 38 0C, while adults were produced only at 20 to 30 °C
(Table 1.1). The longest developmental time, 80.6 i 18.7
days (Mean 1 SD), was recorded for the first instar at 9 °C,
while at 38 °C, it lasted only 7.3 i 0.5 days. In general,
the completion of the first, second and third instar took,
on the average, 20, 15 and 15% of the total nymphal period.
Development of the fourth and fifth instars required 19 and

32% of the immature stage.

Regardless the temperature treatment, the fifth instar
required the longest time for completion. Immatures reached
adulthood only at 20, 25, and 30 °C. The hatch-adult period
required 50.1 i 3.3 days to develop at 20 °C, and only 33.4:

2.4, and 32 i 2.3 at 25 and 30 °C, respectively (Table 1.1).

32

Table 1.1. Developmental time in days for immature stages
of P. irroratus reared at nine constant temperatures

 

Temperature regime (°C)

 

 

Stage 6 9 12 15 20 25 30 35 38
E99
Mean ND* ND ND ND ND 20.5 ND ND ND
SD 1.4
N= 40
Instar I
Mean 0.0 80.6 38.1 14.3 9.0 6.1 6.4 7.8 7.3
SD 0.0 18.7 9.4 3.0 1.5 1.5 0.8 1.0 0.5
N: 41 40 40 37 48 41 48 60 0
II
Mean 0.0 0.0 0.0 13.3 8.3 4.9 4.7 4.3 7.5
SD 0.0 0.0 0.0 2.7 2.0 1.4 1.1 0.5 0.5
= 41 24 33 34 45 40 47 38 8
III
Mean 0.0 0.0 0.0 13.1 7.0 4.8 5.2 0.0 0.0
SD 0.0 0.0 0.0 4.6 1.5 1.2 1.8 0.0 0.0
= 41 24 33 21 43 39 46 6 4
IV
Mean 0.0 0.0 0.0 19.2 9.5 5.8 6.4 0.0 0.0
SD 0.0 0.0 0.0 4.7 1.5 1.1 1.2 0.0 0.0
= 41 24 33 14 41 38 45 6 4
V
Mean 0.0 0.0 0.0 0.0 16.8 11.5 8.9 0.0 0.0
SD 0.0 0.0 0.0 0.0 2.7 1.4 1.9 0.0 0.0
= 41 24 33 9 40 37 40 6 4
Hatch-Adult
Mean 0.0 0.0 0.0 0.0 50.1 33.4 32.2 0.0 0.0
SD 0.0 0.0 0.0 0.0 3.3 2.4 2.3 0.0 0.0
N= 41 24 33 9 35 37 30 6 4

 

(*) Not done.

33

Development at Fluctuating Temperature. In contrast to
constant temperature, P. irroratus completed development at
15 °C mean temperature (max 20 OC, min 10 oC). However, at
30 °C (max 38 °C, min 20 0C) leafhopper survival was
severely reduced (only 56% survived). As in the constant
temperature treatments, the fifth instar was the longest at
each temperature regime. Immatures required 30% of the
total nymphal stage to reach the fifth instar. At 15 °C,
the fifth instar lasted 18.4 i 2.9 and 9.5 t 1.8 days at 30
°C. The total nymphal period required 59.8 i 3.3 and 33.5 t

3.3 days at 15 and 30 °C , respectively (Table 1.2).

Development at Constant vs. Fluctuating Temperature.
The developmental period in days to complete the immature
stage under both constant and fluctuating temperatures was
similar, except at 15 00 (Table 1.3). At 20-30 °C, the
difference between the mean developmental time of P.
irroratus at constant and fluctuating temperature differed
only by 2 to 3 days (Table 1.3). At 20 °C, completion of
the nymphal period required on the average 50 and 47 days
under constant and fluctuating temperature, respectively.
Development at 30 °C, took on average 32-33 days at both

constant and fluctuating temperatures.

Lower Developmental Threshold. Immatures of P.
irroratus developed at relatively low temperatures (Table

1.1). First instars survived and molted to the next stage

Table 1.2.

34

Developmental time in days for immature stages

of P. irroratus reared at four fluctuating temperature

 

Temperature regime (°C)

 

 

Stage 15 20 25 30
Eggs ND* ND ND ND
Instar

I

Mean 11.3 9.7 8.1 5.98
SD 1.5 2.3 1.4 1.0
N= 40 38 38 39
II

Mean 10.2 7.0 4.4 4.4
SD 1.6 1.6 0.8 1.0
N= 38 37 35 33
III

Mean 8.8 7.5 6.0 5.4
SD 1.9 1.2 1.7 2.2
= 38 36 35 30
IV

Mean 11.1 7.8 6.6 7.8
SD 1.5 1.4 2.0 2.9
= 38 34 32 26
v

Mean 18.4 15.2 9.8 9.5
SD 2.9 2.1 2.0 1.8
N= 36 33 31 22

Hatch-Adult

Mean 59.8 47.0 35.1 33.5
SD 3.3 3.9 2.0 3.3
N= 36 33 31 22

 

a) Mean developmental time (days) of three replicates.

*) Not done.

35

at 9 0C but not at 6 °C. Even though at 9 and 12 0C first
instars moulted to the next instar, second instars failed to
complete development and died before reaching the third
instar. At 15 oC, nymphs developed up to the fourth instar
but no beyond. Leafhoppers reared at 20 °C successfully
completed the five instars and became adults. According to
regression analyses, the lower developmental threshold
varied for the different biological stages of P. irroratus
(Table 1.4). A threshold temperature of 8 °C was required
for the first instar to develop; 15 °C was the threshold for

adult development.

Upper Developmental Threshold. The upper threshold
varied from 27 to 31 °C, but an increment in the threshold
temperature was not associated with later biological stages
of P. irroratus as in the case of the lower developmental
threshold. The threshold temperatures for development of
both first instars and adults were 27.5 and 27.8 °C,

respectively (Table 1.4).

Physiological Time (DD) and Development. Degree days
for each biological stage were estimated using 8 °C and 28
°C as lower (TL) and upper (Tb) threshold temperatures
(Table 1.4). Under constant temperature, the mean
incubation period was approximately 337 DD, while the
nymphal period averaged 602 DD. The whole cycle averaged

939 DD.

36

Table 1.3. Development of P. irroratus from emergence of
first instar to adult at constant and fluctuating
temperatures

 

 

 

Regime
Temperature Constant Fluctuating
Days DD Days 001
15 00
Mean2 0 0 59.8 419.0
SD 3.3 23.7
20 00
Mean 50.1 601.7 47.0 564.3*
SD 3.3 40.5 3.9 47.6
25 00
Mean 33.4 568.8 35.1 533.4*
SD 2.4 41.5 2.1 31.8
27 00
Mean 31.2 593.9 ND ND
SD 3.5 72.9
3o 00
Mean 32.20 644.00 33.59 $87.84*
so 2.31 46.20 3.36 58.82

 

ND= Not done.

1)

2)
*)

Degree days estimated using 8 and 28 0C as lower and

upper developmental thresholds.

Mean of three replicates per temperature regime.

Mean is significantly different from mean at constant
temperature using Student’s t test (P S 0.01) (Steel and
Torrie 1980).

37

Table 1.4. Lower (TL) and upper (T ) threshold temperature
and degree days (DD) estimates for giological stages of P.
irroratus at constant temperature

 

Parameters (SD)

 

 

Stage TL TU DD (8 °C)
Eggs ND* ND 337 (17.3)a
Instar I 8.0 27.5 122 (27.9)
II 11.0 28.5 91 ( 6.5)
III 10.5 27.0 90 (10.0)
IV 11.5 27.0 114 (14.1)
v 14.5 31.0 192 (12.3)
Hatch-Adultb 15.0 27.8 602 (31.2)
Adult-Adult 939 (31.5)

 

a) Degree days estimated at 25 and 27 °C.

b) Degree days estimated averaging the duration of the
hatch-adult period at 20, 25 and 30 °C, in which adults

developed.

*) Not done.

38

At constant temperature, second and third instars
required the shortest time to develop: 91 i 6 and 90 i 10
DD, respectively (Table 1.4). The fifth instars required
the longest 192 i 12 DD for development. The rate of
development of P. irroratus at fluctuating temperature was
faster than at constant temperature. All instars required
less DD to complete development than those reared at
constant temperatures (Table 1.5). At fluctuating
temperature, the hatch-adult period averaged 526 i 74 DD: 76
DD less than at constant temperature. In a one to one
comparison, DD required to complete the nymphal period at
15, 20, 25, and 30 °C were significantly less (P S 0.01) at
fluctuating than at constant temperature regimes (Table
1.3). At 15 °C no insect completed development at constant
temperature, but 90% of them reached adulthood at the

fluctuating regime.

Discussion

Growth and development of some species of insects
proceeds faster at fluctuating than at constant temperatures
(Messenger 1959, 1964), but others develop equally well at
both constant and fluctuating temperature regimes (Martinal
8 Allen 1987, Tingle 8 Copland 1988). We found that P.
irroratus is well suited to develop in the temperate regions

of North America. A low temperature threshold of 8 oC

39

(46.54 0F) allows P. irroratus to begin development in early
spring when the mean ambient temperatures start increasing
beyond the 8 °C. On the other hand, the upper developmental
threshold, 28 °C (82.90 °F), limits the seasonal and
geographical distribution of P. irroratus to places with a
cold spring and moderate warm summer, as in Michigan. It
is important to point out that the degree days required to
develop from egg to adult, 939 i 31 permit the development
of two summer generations in south and central Michigan and
one and a partial second in the northwest. These findings
support earlier observations that the abundance of P.
irroratus through Michigan was degree day accumulation

dependent (Larsen 8 Whalon 1988).

Difference in insect development at constant and
fluctuating temperatures needs to be considered carefully.
Using the DD estimates obtained from the constant
temperature experiment may result in a lack of predictive
accuracy with respect to field data. The difference of 84
DD between estimates is a problem particularly important
early in the season when minimum and maximum temperatures
fluctuate between 10 (50.18 °F) and 20 00 (68.39 0F). Under
these circumstances, the prediction based on constant
temperature will forecast no insect development when in
reality it takes place. Under field conditions, the
difference between the two estimates would be equivalent to

10 days in early May, and 4 days in early June.

40

Table 1.5. Development of P. irroratus at constant and
fluctuating temperatures.

 

Temperature (DD 8 oC)

 

 

Constant Fluctuating
Stage DD (SD) DD (SD)
Eggs 337 (17.3)a ND*
Instar I 122 (27.9) 106 (19.7)
II 91 ( 6.5) 75 ( 7.1)
III 90 (10.0) 84 (15.1)
IV 114 (14.1) 102 (25.3)
v 192 (12.3) 157 (23.1)
Hatch-Adult 602 (31.2)b 526 (74.8)C

 

a) Degree day estimated at 25 and 27 °C.

b) Degree days estimated averaging the duration of the
birth-adult period at 20, 25, 27 and 30 °C, in which
adults developed.

c) Degree days estimated from leafhopper development at 15,
20, 25, and 30 °C mean temperature.

*) Not done

41

We have demonstrated the importance of estimating
insect growth and development at both fluctuating and
constant temperature regimes. In doing so, we make sure
that growth rates and degree day estimates obtained in
constant temperature do not vary significantly from those
that may occur in the field under fluctuating temperature.

In some insects such as the corn leaf aphid,
Rophalosiphum maidis (Fitch), constant and variable
temperatures have a negligible effect on its growth rate and
DD required for development (Elliott et al. 1988). The same
is true for the Spodoptera litura (F.) whose temperature
requirements under fluctuating temperature were similar to
those observed in the laboratory at constant (Ranga Rao et
al. 1989). On the other hand, Messenger 8 Flitters (1959)
reported that under variable temperature eggs of the
oriental fruit fly, Decus dbrsalis Handel, the melon fly,
Deans cucurbdtae Coq., and the Mediterranean fruit fly,
ceratitis capitata (Wied.), developed faster than at
constant temperature only when the temperature was below 65
0F (18.15 °C). Above 85 OF (29.15 °C) egg development was
slower than at constant temperature. The spotted alfalfa
aphid, Therioaphis maculata (Buckton), performed similarly
at low temperature. At 8 °C mean temperature development
proceeded twice as fast as at constant temperature
(Messenger 1964). Fielding 8 Ruesink (1988), reported that
the squash bug, Anasa tristis DeGeer, developed faster at

fluctuating than at constant temperature. At 21 oC, egg

42

development required 24 DD less at fluctuating than at
constant temperature. Likewise, at 26.7 °C, the completion
of the nymphal period required 100 DD more at constant than
at fluctuating temperature. The observed response of P.
irroratus is most like the response of the spotted alfalfa

aphid and the squash bug.

Given the different results obtained under variable and
constant temperature, more research is needed on the effect
of temperature on the growth rate and development of .P.
irroratus. It is important that any predictive model
developed on the basis of degree days to predict the
phenology of P. irroratus should be validated using
estimates of both, constant and variable temperature
regimes. Furthermore, more research on egg development is
required to have a better estimate of the incubation period

at temperature lower than 25-27 °C.

43

Bibliography

Bhattacharyya, G. K. 8 R. A. Johnson. 1977. Statistical
concepts and methods, John Wiley 8 Sons. New York. pp

639.

Elliott, N. C., R. W. Kieckhefer 8 D. D. Walgenbach. 1988.
Effects of constant and fluctuating temperatures on
developmental rates and demographic statistics for the
corn leaf aphid (Homoptera: Aphididae). J. Econ.

Entomol. 81(5):1383-1389.

Fielding, D. J. 8 William G. Ruesink. 1988. Prediction of
egg and nymphal developmental times of squash bug
(Homoptera: Coreidae) in the field. J. Econ. Entomol.

81(5):1377-1382.

Larsen, K. J. 8 Mark E. Whalon. 1988. Field monitoring of
X-disease leafhopper vectors (Homoptera: Cicadellidae)
and infected chockecherry in Michigan peach and cherry

orchards. Great Lakes Entomol. 21(2):61-67.

Martinat, P. J. 8 Douglas C. Allen. 1987. Laboratory
response of the saddled prominent (Lepidoptera:

Notodontidae) egg and larvae to temperature and

44

humidity: Development and survivorship. Ann. Entomol.

SOC. Am. 80:541-546.

Messenger, P. S. 8 N. E. Flitters. 1959. Effect of variable
temperature environments on egg development of three
species of fruit flies. Ann. Entomol. Soc. Am.

52:191-204.

Messenger, P. S. 1964. The influence of rhythmically
fluctuating temperatures on the development and
reproduction of the spotted alfalfa aphid. J. Econ.

Entomol. 57(1):71-76.

Neter, J. 8 W. Wasserman. 1974. Applied linear statistical

models. Richard D. Irwin, Homewood, Illinois. pp 842.

Rahardja, U. (1989). Field detection of peach X-disease
mycoplasmalike organism in speckled leafhopper,
Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)
using a DNA probe. MS thesis, Michigan State

University, East Lansing, MI pp 73.

Ranga-Roa, G. V., J. A. Wightman, 8 D. V. Ranga R08. 1988.
Threshold temperatures and thermal requirements for the
development of Spodoptera litura (Lepidoptera:

Noctuidae). Environ. Entomol. 18(4):548-551.

45

Rosenberge, D. A. 8 A. L. Jones. 1978. Leafhopper vector of
the peach X-disease pathogen and its seasonal
transmission from chockecherry. Phytopathology

68:782-790.

Steel, R. G. D. 8 J. H. Torrie. 1980. Principles and
procedures of statistics: a biometric approach. McGraw-

Hill, New York. pp 86-121.

Tabodad, O., D. A. Rosenberge 8 A. L. Jones. 1975.
Leafhopper fauna of X-diseased peach and cherry
orchards in southwest Michigan. J. Econ. Entomol.

68:255-257.

Taylor, F. 1979. Convergence to the stable age distribution

in populations of insects. Am. Nat. 113:511-530.

Taylor, F. 1981. Ecology and evolution of physiological time

in insects. Am. Nat. 117:1-23

Tingle, C. C. D. 8 M. J. W. Copland. 1988. Predicting
development of the mealybug parasitoid Anagyrus
pseudbcocci, LeptomastiX'dactilopii and Leptomastidae
abnormis under glasshouse conditions. Entomol. exp.

appl. 46:19-28

46

CHAPTER II

Temperature Dependent Pathogenicity of the X-disease
Mycoplasmalike Organism (MLO) to its vector, Paraphlepsius

irroratus (Say) (Homoptera: Cicadellidae).

47

Introduction.

X-disease, a stone fruit disease of mycoplasmalike
organism (MLO) etiology, is of major importance in the peach
orchards of North America. In Michigan, it is the most
important pest problem in peaches and is a limiting factor
in the economic success of peach production (Larsen 8 Walon
1988). The causal agent of this disease reproduces in the
phloem tissue of its hosts. The disease causes a reduction
in fruit production and usually kills infected peach trees
within two or three years unless they are treated with
antibiotics (Agrios 1978).

In the eastern part of North America, the MLO
responsible for X-disease is transmitted by a complex of
leafhopper species of the subfamily Deltocephalinae
including: Cblladbnus clitellarius (Say), Fieberiella
florii (Stal.), Gyponana lamina DeLong, Nbrvellina seminuda
(Say), scaphytopius acutus (Say), and Paraphlepsius
irroratus (Say) (Lacy et al. 1979). In Michigan, the
complex of leafhopper species includes all of the
forementioned species as well as Orientus ishidhe (Mat.),
scaphoideus melanotus (Osb)., and scaphoideus titanus Bell
(Rosenberger 8 Jones 1978, Taboada et al. 1975).

The Speckled leafhopper,.P. irroratus, is thought to be

the most important X-disease vector in Michigan because of

48

its abundance, association with stone fruit crops and
efficiency as a vector (Larsen 8 Whalon 1987, Mowry 8 Whalon
1985, Mowry 1982, Rosenberger 1977).

This vector leafhopper develops primarily in grasses
and herbaceous weeds. Herbaceous and perennial host plants
that comprise the ground cover provide food and breeding
sites for P. irroratus and associated species before they
move onto peach and cherry trees (McClure 1980a, 1980b,
1982). Ground cover might also provide a reservoir for the
X-disease MLO because X-disease MLO's have experimentally
been transmitted from plants that occur in orchard ground
cover including Deucus carota L., Matricaria maritime L,
Trifolium repens L. and Trifolium pretenses L. (Chiykowski 8
Sinha 1982, Chiykowski 1985).

The interrelationship between vector leafhoppers and
the X-disease MLO is of primary importance in planning
strategies to control X-disease transmission, but little
research has been done in this area. Research on a Western
X-disease vector, Cblladonus montanus (Van Duzee), has shown
that the X-disease MLO has a deleterious effect on the
reproduction and longevity of infected individuals (Jensen
et a1. 1967, Jensen 1971). In another leafhopper-MLO
transmission system, Delbulus spp: Spiroplasma kunkelii
(Corn Stant Spirplasma): Zea spp, association varies from
lethality to Dalbulus elimatus (Bell) to non effect on
longevity and fecundity of Dalbulus maidis (DeLong 8

Wolcott) (Nault 1985, Madden et al. 1984).

49

The MLO-leafhopper association has not been studied in
P. irroratus and X-disease. The objective of the this
research was to study the temperature mediated

interrelationship of P. irroratus and X-disease.

Materials and Methods

Leafhopper Culture. The P. irroratus culture was
initiated in 1987 with adults collected in a cherry orchard
located at the Collins Rd., Department of Entomology
Research Farm on the campus of Michigan State University at
East Lansing, Mich. Adult leafhoppers were collected using
light traps placed in the orchard and monitored for
leafhoppers during their daily flight period (Larsen 8
Whalon 1987). The leafhopper culture was maintained in a
walk-in growth chamber at an environmental regime of 25 i 1
°C, 70 i 10% rh, and a photoperiod of 16:8 (L:D).
Leafhoppers were caged in small cylindrical cages made from
screened two liter plastic bottles placed over plastic pots
containing a mixture of red clover (Trifolium.repens L.) and
barley (Herdeum vulgaris L.) planted as a substrata for
leafhopper oviposition and feeding. The top of each cage
had an opening of 5 cm in diameter to allow the placement

and removal of leafhoppers.

50

Temperature and Injection Treatments. The experiment
was arranged in a 3 x 4 randomized complete block design and
replicated in four fluctuating temperatures (Figure 2.1).
Temperature regimes were selected to simulate Michigan’s
temperature conditions during early and late summer as well
as during a hot mid-summer. The four temperature regimens
averaged 15 (10 °c min, 20 °c max), 20 (10 °c min, 30 °c
max), 25 (15 °c min, 35 °c max) and 30 (20 °c min, 38 °c
max). Temperature cycles were adjusted to increase or
decrease 0.5 to 2.0 degrees/hour until reaching the
established maximum or minimum temperature. Experimental
treatments were healthy non-injected (Control), MLO-
injected, and inactivated MLO-injected leafhoppers.
Leafhoppers injected with the inactivated MLO extract were
used as a second control to determine the effect of
injecting a foreign substances into healthy leafhoppers.
Needle injection of the X-disease MLO was preferred over
natural acquisition in order to standarize the amount of
inoculum per treated individual as well as the beginning of
the incubation period (IP). With the exception of the MLO-
injected treatment (20 nymphs), there were 10 leafhoppers/
replicate (40 /injection treatment) and 160/temperature
regime, for a total of 640 third instar P. irroratus in the
experiment. A replicate consisted of a pot containing 10
barley seedlings where treated nymphs were kept until

adulthood was reached.

51

 

 

40
as
30
é)
; 26
E
a 20
E
I- 16
a mm)!
10 I mva
o TWENTY
s , . J - . e 1 o FIF‘l'E-«l
o a 16 24 32 40 48

Figure 2.1. Temperature regimens utilized to study the

effect of the X-disease MLO on the longevity of P. irroratus
8thirty, 30 °C: tfive, 25 °c; twenty, 20 °c: and fifteen, 15
C, average temperature).

52

Injection Protocol. Ten naturally infected leafhoppers
raised on X-diseased celery were mascerated in an Ependorf
tube and suspended in 0.603 ml of a 0.01 M PBS (0.01 M
potasium phosphate, pH 7.0, 0.15M NaCl) solution. The
solution was centrifuged at 5,000 rpm at 4 0C for 15 min,
the supernantant removed, and filtered through a 0.45 pm
filter unit ( MILLEX-HA R. Millpore Corporation, Bedford,
MA 01730). The liquid phase (supernantant) containing the
X-disease MLO's was used for injection. An aliquot of this
inoculum was placed at room temperature ( 25 i 1 0C) for 6 h
to inactivate the MLO.

Leafhoppers were injected using a capillary needle
forged from 20 ul micropipets connected to a 100 pl Hamilton
syringe (Hamilton Company, Reno, Nevada) by 20 cm long
TeflonR tubing. Leafhoppers were immobilized prior to
injection by a stream of C02 and placed upside down onto an
inverted petri dish covered with a Parafilm R layer. A
second layer of Parafilm was stretched over them to hold
them in place during injection. The inoculum was injected
into the abdomen between the second and third sternite.

Each leafhopper received a 0.50 ul dose of X-disease MLO
preparation delivered by a microapplicator (ISCOR

Instrumentation Specialties Co.).

After injection, leafhopper nymphs were randomly
allocated to treatments and replicates and caged on barley

plants to complete development. Leafhoppers reaching

53

adulthood were removed daily from the barley pots and
deposited in clover pots for the longevity study. Both, the
date of adult emergence for each individual as well as the
death date was recorded. Dead leafhoppers were removed,
labelled and frozen at -70 °C in vials for later DNA

analysis.

MLO Acquisition Determination. MLO extraction from
treated leafhoppers was according to Kirkpatrick et
al.(1987). Frozen cadavers of injected leafhoppers were
individually ground in 400 pl MLO enrichment buffer (0.1 M
NazHPO4, 10% sucrose, 50 mM ascorbic acid, and 1%
polyvinylpyrollidone at pH 7.6) using a pellet pestle. The
homogenate was transferred to a 1.5 ml Ependorf tube and
centrifuged at 6,500 rpm in a Sorvall ss-34 rotor at 4 °C
for 15 min. The supernatant was harvested and centrifuged
once more at 11,500 rpm (4 0C for 30 min). The resulting
pellet was air dried, resuspended in TE (10 mM Tris-HCl, 1
mM EDTA, pH 8.0) buffer (pH 8.0) and stored at -70 0c until

used in the dot blot hybridization assay.

Dot Blot Hybridization. The leafhopper samples were blotted
onto a GeenScreenTM nylon membrane (New England Nuclear,
Dupont, Boston, MA) in a dot-blot apparatus (BioRad,
Richmond, CA). MLO DNA was alkaline denatured with 0.5 M
NaOH for 10 min and chilled on ice for another 10 min.

After denaturation, the samples were diluted with 10 X

54

standard saline citrate (SSC) to a final volume of 400 pl
and blotted. The membrane was removed from the dot blot
apparatus and washed carefully with 0.4 N NaOH for 30-60
seconds to enusre complete denaturation of the immobilized
DNA. Next, the membrane was neutralized by dipping it in
0.025 M sodium phosphate fro 30-60 seconds. The nucleic
acid was fixed onto the membrane with UV cross-linkage by
laying the wet membrane face up on a clean glass plate and
irradiating it for 5 min at a distance of 15 cm with a 254
nm shortwave UV light sources (UVG-ll, Ultraviolet Products,
Inc., San Gabriel, CA). Then, the membrane was prehybridized
and hybridized according to the Method III for GeneScreenT"
(New England Nuclear, Dupont, Boston, MA).

The cDNA probe C6c (Fig. 2.2) (Whalon et al, 1990), a
1.9 kb EcoRI-HindIII fragment isolated from X-diseased
Cblladbnus.montanus (Van Duzee) and cloned in pUC8 vector
(Kirkpatrick et al. 1987) was nicktranslated with 32F using
a kit from Amersham (Arlington Heights, IL). Unincorporated
nucleotides were removed by passing the reaction products
through a 1 ml Sephadex G-75 fine column previously
saturated with TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0).
The mean specific activity of the radiolabelled probe was 5
x 107 cpm, and the amount of probe utilized was 200 ng/ul.
After completion of the hybridization process, the membrane

was submitted to autoradiography by exposing it to a X-ray

film (Kodak XAR) for 24-48 h with an intensifying screen

 

55

 

Figure 2.2. The DNA probe C6c: l and 2, C60 (partially
digestesd EcoRI and HindIII pWXl); 3, Undigested pWXl: kb, 1
kb marker and L, Hind III digested lambda DNA marker.

56

(Sigma) at -70 °C. The film was developed and the number of
positive X-diseased samples determined.

Results from the Dot Blot test for each one of the
treatments (healthy, X-MLO-injected and inactivated X-MLO
injected leafhoppers) and temperature regimes were compared,
and only positive results were considered when analyzing
longevity and survivorship of X-diseased leafhoppers. All
data were submitted to analysis of variance and statistical
differences between treatments were established through

orthogonal comparison (Steel 8 Torrie 1980).

Results

MLO Effect on Leafhopper Longevity. The X-disease MLO
had a deleterious effect on the longevity of P. irroratus,
and the effect was temperature dependent (Figure 2.3). The
MLO effect followed a sigmoidal curve described by the
equation:

Y= 23.5/(1+exp (-14.556+0.69*T) ), where;
Y= reduction in longevity (%), and T= mean temperature (°C)
(r= 0.97). The greatest effect occurred at/or below 20 °C,
with minimal effect at/or above 25 °C. In comparison with
healthy non-injected, the X-disease MLO shortened the life
of MLO injected leafhoppers by about 31 i 5 (Mean 1 SD) days
at 15 and 20 °C. The mean longevity of MLO infected
leafhoppers at 15 °C was 35 i 21 days, while healthy non-

injected leafhoppers lived on average 62 i 16 days. At 20

57

°C the longevity of X-diseased leafhoppers was 23 i 9, but
healthy individuals lived 59 i 22 days (Table 2.1).
Discounting any injection effect, longevity reduction due to
the X-disease MLO was 32% on average. The MLO effect
decreased drastically between 20 and 25 oC, and at 30 0C the
effect was essentially gone. At 25 0C, the MLO effect
accounted for only a 6% reduction in leafhopper longevity

(Figure 2.2).

MLO Acquisition. X-disease acquisition among
treatments and replicates after MLO injection ranged from 20
to 48%. The mean acquisition among temperature treatments
was 32.4, 37.6, 36.9 and 35.7% for 15, 20, 25, and 30 0C,
respectively (Table 2.2). No statistical difference in MLO
acquisition between temperature treatments was observed (P S
0.05). All leafhoppers injected with the inactivated MLO
extract tested negative for the presence of the X-disease

MLO agent.

Temperature Effect on Leafhopper Longevity. The effect
of temperature on the longevity of adult P. irroratus was
significant (P S 0.05). There was an overall reduction in
longevity with an increase in the average temperature.
Decreasing longevity due to temperature effects followed a
sigmoidal curve with minimal effect at 15 and 20 °C with a
maximum effect at 25 and 30 °C. The equation estimating the

temperature effect was a logistic curve:

58

 

 

 

 

 

 

 

 

1‘“) I I I I
5‘
-; 3C1 -
0
1’
C
O
-J
.5 5" '
C
.2
3
. 4" P
C
'E
3
5 2C, '
a.
C)
10 15 20 25 30 35

Mean Temperature, 06

Figure 2.3. Deleterious effect of X-disease MLO and
temperature on the longevity of.P. irroratus. Healthy
leafhoppers lived for 62 i 16 days at 15 oC. MLO effect
(H-O-O): Temperature effect (O-O-O-O) .

59

Y: 55.5/(1+exp (15.461-0.671*T) ), where ;
Y= reduction in longevity (%), and T= mean temperature (°C)
(r= 0.98). In comparison with leafhoppers maintained at 15
°C, longevity at 30 °C decreased up to 80 % both in healthy
and diseased leafhoppers. The longest and shortest
longevity was observed at 15 oC, and 30 °C respectively. At
15 °C, and 30 0C the mean longevity of healthy and X-
diseased leafhopper was 62 i 16, 7 i 4 and 35 i 21 and 7 i 4
days, respectively (Table 1.1). Overall, the temperature
mediated reduction in longevity ranged 50-55 days in healthy
and 28-30 days in X-diseased individuals with a minimal

effect at 15 °C and maximum effect at 30 °C (Fig.2 3)

Injection Solution Effect. Even though inactivated
MLO-injected leafhoppers showed no detectable X-disease when
tested by Dot hybridization, the injection of a foreign
substance affected the longevity of treated individuals.
This effect was reflected in a shorter longevity of treated
leafhoppers. However, a statistically significant
difference (P s 0.05) between the control (non-injected
leafhoppers) and inactivated MLO-injected individuals was
only observed at 20 00 (Table 2.1). In comparison with
diseased leafhoppers, inactivated MLO-injected individuals
lived longer than X-diseased. However, as in the case of
non-injected, the difference in leafhopper longevity only
occurred at 15 and 20 °C, with no significant (P s 0.05)

difference at 25 and 30 °C (Table 2.1).

60

Table 2.1. The effect of the X-disease MLO on the longevity
of P. irroratus, at different fluctuating temperatures.

 

Mean Temperature (°C) 1

 

Treatment 15 20 25 30
(Mean 1 SE)

 

MLo injected 35:21.3 a1 23:09.8 32 13:5.8 a3 7:4.7 a4
Inactivated MLo2 58:19.9 b 39:20.7 b 14:4.8 a 7:4.8 8

Healthy (Control)3 62:16.7 b 59:22.5 C 15:9.3 a 7:3.5 a

 

1) Means within colums followed by the same latter are not
statistically different at P S 0.05, using Tukey’s test
and orthogonal separations (Steel and Torrie 1980).

2) Injected with inactivated MLO.

3) Healthy non-injected leafhoppers.

Table 2.2. The effect of temperature on X-disease MLO
acquisition by P. irroratus.

 

 

Temperature Individuals Percent
(°C) Reps tested acquisition1 SD
15 4 69 . 32.4 10.45
20 4 102 37.6 4.42
25 4 100 36.9 5.68
30 4 89 35.7 8.19

 

SD= Standard deviation of the mean.
1) Leafhopper cadavers were analyzed for X-disease MLO’s
with dot blot hybridization using the C60 cDNA probe.

61

Discussion

In this study, the use of fluctuating rather than
constant temperatures was preferred because fluctuating
temperatures are the normal condition that insects
experience in their environment, and a better prediction of
leafhopper behavior results. Yet, very little data on the
effect of fluctuating temperature on insects is available in
the literature.

Studies under constant and fluctuating temperature
regimes have demonstrated that mycoplasmas are sometimes
lethal to their vectors. Liu et al.(l983) showed that S.
citri reduced the longevity of inoculated Circulifer
temellus (Baker). Other mollicutes such as the corn stunt
spiroplasma (CSS) and the maize bushy stunt mycoplasma
(MBSM) have demonstrated deleterious effects to their
leafhopper vectors. From six species of Dulbulus inoculated
with CSS, only D. maidis was not affected in its survival.
In the same species, MBSM shortende their median survival
time and time to 25% survival but D. elimatus and D. gelbus
were unaffected (Madden 8 Nault 1983, Madden et al. 1984).
In Dalbulus longulus DeLong, the mean longevity for healthy,
CSS and MBSM-diseased leafhoppers was 13, 7, and 6 weeks
(Nault et al. 1984).

As early as 1959, Jensen reported that longevity of X-
diseased C.montanus averaged 24 days while healthy

leafhoppers lived on average 51 days. Our research on

62

P. irroratus confirms that the X-disease MLO is pathogenic
to its vector leafhoppers in a similar manner to other
mollicutes and their vectors. Temperature increased the
effect of the X-disease MLO on the leafhopper’s longevity at
the two lower fluctuating temperatures (15 and 20 °C).
However, at 25 °C or above it, no significat MLO effect was
observed. The temperature effect on the vector’s longevity
overshadowed the MLO effect.

The effect of temperature on MLO acquisition has been
documented by Jensen (1972). He reported that X-disease
acquisition by C. montanus in a fluctuating temperature
regime (30 °C maximum and 15 °C minimum) increased to 75%
from 24 and 4% at 15 and 30 °C constant temperature. Jensen
concluded that constant temperatures are less favorable for
X-disease MLO multiplication and transmission. Accordingly,
even those temperatures fluctuating between limits that
individually are not conducive for good MLO acquisition
resulted in better X-disease multiplication and
transmission. In a constant temperature study, Jensen (
1972) demonstrated that high temperatures inactivate the
MLO, and X-disease acquisition was reduced from an average
70% at 20 °C to only a 4% at 30 °C in C. montanus. In the
same study, temperatures lower than 20 °C decreased MLO
acquisition, and only 24% of injected leafhoppers became
infective when placed at 15 °C after MLO injection.
Chiykowski 8 Sinha (1988) reported that X-disease

acquisition by P. irroratus was maximized at 21 °C constant

63

temperature. Our transmission data confirms their results
of 7 transmission experiments which yielded on average 36%

MLO acquisition of injected leafhoppers.

Our acquisition data reflects the results of an
extremely sensitive Dot hybridization technique with a lower
detection limit between 3,000 to 12,000 X-disease MLO cells
(Rahardja 1989). Thus, percent MLO acquisition after
injection reflects multiplication of the MLO in the
leafhopper, but does not reflect the actual number that will
transmit X-disease. In the past, host plant symptomatology
of indicator plants has been the confirmation of
acquisition. The disadvantage of using indicator plants is
that symptomatology develops only when competent or
transmitting leafhoppers feed on the plant and the MLO
multiplies resulting in symptoms. However, misleading
results could occur if some leafhoppers acquire the MLO and
may not be competent to transmit, the plant may not be
susceptible due to intrinsic or extrinsic reasons and/or the
MLO may be altered and not cause symptoms as in the case of
S. citri (Liu et al. 1983a).

There are few specific reports on the effect of the
injection solution on leafhopper vectors. C. montanus
injected with a healthy insect extract lived longer than
individuals injected with a extract prepared from X-diseased
leafhoppers (Jensen et al. 1967). In another study, the

longevity of C. tenellus injected with sterilized S. citri

64

culture media was intermediate between healthy non-injected
and S. citri injected C. tenellus (Liu et al. 1983). Our
results coincide with those findings, longevity of MLO-
inactivated injected P. irroratus was longer than X-disease
injected but shorter than healthy non-injected. Even though
there is an appearent deleterious effect of the injection
solution on leafhopper survival, longevity is greater than
in pathogen injected individuals.

These are the first fluctuating temperature-longevity
and temperature-pathogenicity effects reported for P.
irroratus. Our research has demonstrated that leafhopper
longevity is significantly affected by temperatures above 20
°C. These findings are important in understanding the
epidemiology of X-disease in the field. The rapid decline
in leafhopper longevity with higher temperatures may not
allow the full expression of the MLO effect on the vector
leafhopper. Temperatures higher than 20 °C may also cause a
reduction in X-disease transmission potential. Thus, as
Figure 2.3 shows, X-disease transmission will occur mainly
during the early and late summer when the daily temperatures
average 20 °C or less. Even though at lower temperatures
the MLO is pathogenic to P. irroratus, the vector may live
long enough to efficiently transmit the X-disease agent.
Recent studies on the dynamics of X-diseased injected P.
irroratus conducted in central and southwest Michigan have
demonstrated a high percentage of diseased leafhoppers in

both early and late summer and a minimal number during

65

mid-summer (Rahardja 1989). This data seems to support our
findings.

Temperature developmental thresholds for the X-disease
MLO in the leafhopper remain unclear. Research is also
needed to elucidate the effect of the X-disease MLO on the
fecundity of P. irroratus. This research along with the
effect of the temperature-MLO interaction will provide a
better understanding of X-disease transmission rates and
population dynamics of P. irroratus in Michigan peach

growing regions.

66

Bibliography

Agrios, G. N. 1978. Plant diseases caused by mycoplasmalike
organisms. In: Plant Pathology. Academic Press, New

York, San Francisco, London, pp: 511-535.

Chiykowski, L. N., 8 R. C. Sinha. 1982. Herbaceous host
plants of peach Eastern X-disease agent. Can. J. Plant

Pathol. 4: 8-15.

Chiykowski, L. N. 1985. Biology and rearing of Paraphlepsius
irroratus (Homoptera: Cicadellidae). Can. Ent. 117:

717-726.

Chiykowski, L. N., 8 R. C. Sinha. 1988. Some factors
affecting the transmission of Eastern peach X-disease
mycoplasmalike organism by the leafhopper Paraphlepsius

irroratus. Can. J. Plant Pathol. 10: 85-92.

Jensen, D. D., R. F. Whitecomb, 8 J. Richardson. 1966.
Lethality of injected peach Western X-disease virus to

its leafhopper vector. Virology 31: 532-538.

Jensen, D. D. 1971. Vector fecundity reduced by Western X-

disease. J. Invertebr. Pathol. 17: 389-394.

67

Jensen, D. D. 1972. Temperature and transmission of the

Western X-disease agent by Cblladonus montanus.

Phytopathology 62: 452-456.

Kirkpatrick, B. C., D. C. Stenger, T. J. Morris, 8 A. H.
Purcell. 1987. Cloning and detection of DNA from a

nonculturable plant pathogenic mycoplasmalike organism.

Science 238: 197-200.

Lacy, G. H., Mark S. McClure, 8 T. G. Andreadis. 1979.

Reducing populations of the vector leafhoppers is a new

approach to X-disease control. Frontiers of Plant

Science, Fall 1979, 2-4.

Larsen, K. J. 8 M. E. Whalon. 1987. Crepuscular movement of

Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)

between the groundcover and cherry trees. Environ.
Entomol. 16(5): 1103-1106.

Larsen, K. J. 8 M. E. Whalon. 1988. Field monitoring of X-

disease leafhopper vectors (Homoptera: Cicadellidae)
and infected chokecherry in Michigan peach and cherry

orchards. Great Lakes Entomol. 21(2):61-67.

68

Liu, Hesing-Yeh., D. J. Gumpf, G. N. Olfield, 8 E. C.
Calavan. 1983a. Transmission of Spiroplasma citri by

Circulifer tenellus. Phytopathology 73: 582-585.

Liu, Hesing-Yeh., D. J. Gumpf, G. N. Olfield, 8 E. C.
Calavan. 1983. The relationship of Spiroplasma citri

and Circulifer tenellus. Phytopathology 73: 585-590.

McClure, M. S. 1980a. Role of wild host plants in the
feeding, oviposition, and dispersal of Scaphytopius
acutus (Say), a vector of Peach X-disease. Environ.

Entomol. 9: 265--274.

McClure, M. S. 1980b. Spatial and seasonal distribution of
leafhopper vectors of Peach X-disease in Connecticut.

Environ. Entomol. 9: 668-672.

Madden, L. V. 8 L. R. Nault. 1983. Differential
pathogenicity of corn stunt mollicutest to leafhopper
vecotrs in Dalbulus and Baldulus species.

Phytopathology 73:1608-1614.

Madden, L. V., L. R. Nault, S. E. Heady, 8 W. E. Styer.
1984. Effect of maize stunting mollicutes on survival
and fecundity of Dalbulus leafhopper vectors. Ann.

appl. Biol. 105: 431-441.

69

Mowry, T. M. 1982. Leafhopper sampling in Michigan peach
orchards and serological detection of spiroplasma
associated with X-disease in plant tissue. M.S.
Thesis, Michigan State University, East Lansing, MI.

134 pp.

Mowry, T. M. 8 Mark E. Whalon. 1984. Comparison of
leafhopper species complexes in the ground cover of
sprayed and unsprayed peach orchards in Michigan

(Homoptera: Cicadellidae). The Great Lakes Entomologist

17:205-209.

Nault, L. R. 1985. Evolutionary relationships between maize
leafhoppers and their host plants. In: The Leafhoppers
and Planthoppers. L.N. Nault and G. Rodriguez. John

Wiley 8 Sons. p 309-330.

Nault, L. N., L. V. Madden, W. E. Styer, B. W. Triplehorn,
G. F. Shambaugh, 8 S. E. Heady. 1984. Pathogenicity of
Corn Stunt Spiroplasma and Maize Bushy Stunt Mycoplasma
to their vector, Dalbulus Iongulus. Phytopathology 74:

977-979.

Rahardja, U. 1989. Field detection of peach X-disease MLO in
Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)
using a cDNA probe. M.S. Thesis, Michigan State

University, East Lansing, Mi.. 46 pp.

70

Rosenberger, D. A. 1977. Leafhopper vectors, epidemiology
and control of peach X-disease. Ph.D. Dissertation,

Michigan State University, East Lansing, Mi.

Rosenberger, D. A. 8 A. L. Jones. 1978. Leafhopper vectors
of the peach X-disease pathogen and its seasonal
transmission from chokecherry. Phytopathology 68: 782-

790.

Steel, R. G. D. 8 J. H. Torrie. 1980. Principles and
procedures of statistics: A biometrical approach.

McGraw Hill Book Company. 633 pp.

Taboada, O., D. A. Rosenberger 8 A. L. Jones. 1975.
Leafhopper fauna of X-diseased peach and cherry
orchards in Southwest Michigan. J. Econ. Entomol.

68(2): 255-257.

Whalon, M. E., U. Rahardja, Y. T. Yan 8 C. Garcia-Salazar.
1990. Evaluation of cloned sequences for detecting
Mycoplasmalike Organism in Michigan Paraphlepsius
irroratus (Say) (Homoptera: Cicadellidae) and plants.

Annals of the Entomol. Soc. (Submitted).

71

CHAPTER III

Effect of the Eastern Strain of the X-disease Mycoplasmalike
Organism on the Fecundity of its Vector, Paraphlepsius

irroratus (Say) (Homoptera: Cicadellidae).

72

Introduction

A temperature-dependent pathogenicity has been
demonstrated between the Eastern strain of the X-disease
mycoplasmalike organism (X-MLO) and its vector the speckled
leafhoppers, Paraphlepsius irroratus (Say) (Garcia-Salazar
et al. 1991). Similar results were reported by Jensen et
al.(1967) in Cblladonus montanus VanDuzee, a vector of the
Western strain of the X-disease MLO. In both cases,
longevity and survival of X-diseased leafhoppers were
severely reduced in comparison with healthy non infected
individuals. Apart from its effect on longevity and
survival, the X-disease MLO has shown negative effects on
the reproductive capacity of X-diseased C. mantanus (VanDuz)
(Jensen 1971). Reduced fecundity as a result of pathogenic
infections has also been reported in other leafhoppers and
planthoppers. Purcell et al. (1987) reported that fecundity
of Ehscelidhs variegatus (Kirschbaum) was reduced by about
80% when infected with a Gram-negative bacterium only known
as BEV. Likewise, studies on the effect of the rice dwarf
virus on its vector nephotettIX'cincticeps Uhler showed that
fecundity of virus infected individuals was reduced up to
70% in comparison with healthy planthoppers (Nakasuji 8
Kiritani 1970). However, not all MLO’s have shown

deleterious effect on their leafhopper vectors. The Aster

73

Yellows MLO and the corn stunt spiroplasma (CSS) do not
affect the longevity or fecundity of Macrosteles fascifrons
(Stal) (Purcell 1982) and Dalbulus maidis (DeLong 8 Wolcott)
(Madden et a1. 1984). The fact that X-disease infection
might reduce the fecundity of.P. irroratus presents an
important component of the population dynamics of this
vector and of the epidemiology of X-disease transmission
patterns under field conditions. This research reports the
effect of the Eastern strain of X-disease on the fecundity

of P. irroratus.

Materials and Methods

Leafhopper Culture. Leafhoppers utilized in this
research were obtained from a laboratory culture of P.
irroratus established in 1987 at the Michigan State
University Pesticide Research Center from field collected
individuals (Garcia-Salazar et al. 1991). This culture was
augmented every season with newly field collected
individuals to assure genetic diversity. Leafhoppers were
raised on a mixture of barley (Herdeum bulgaris L.) and
white clover (Trifolium pretenses L.) in cages made from
screened two liter plastic bottles. The leafhopper colony
was maintained in a walk-in growth chamber at 25 t 1 °C,

16:8 (L:D) photocycle and 70-80% rh.

74

Treatments. In this experiment fecundity of X-disease
injected P. irroratus and healthy non-injected individuals
was compared. Healthy non-injected P. irroratus were used
as a control. A second control that could have been
included buffer of destilled water injected individuals was
not included due to the limited number of available insects.
Likewise, syringe X-disease inoculation was preferred over
natural acquisition because it allowed both, the
standardization of the dosage per treated individual and the
initiation of the MLO incubation period. The treatments

were prepared and established as follows.

Healthy Leafhoppers. A cohort of three hundred 3rd
instar healthy P. irroratus was divided into 15 cages
containing 20 barley seedlings and monitored daily for adult
emergence. To assure uniformity, cohorts of newly emerged
adults were removed daily from cages and placed in another
cage containing barley seedlings and labelled with the date
of adult emergence and the number of individuals in it.
Adults remained in this cage for three days before being
assigned to an oviposition cage. The three day holding
period provided a standardized sexual maturity period for
all experimental individuals. After this, cohorts of four
males and four females were placed into oviposition cages.
Each cage contained a five week old clover plant that was
the substrate for leafhopper oviposition . Before placing

leafhoppers into the cage, six to ten barley seeds were

75

planted around the clover to provide a feeding substrate for

emerging P. irroratus nymphs.

X-disease Injected Leafhoppers. Three hundred 3rd
instar.P. irroratus were injected with a preparation of the
X-disease MLO according to Garcia-Salazar et al. 1991.
Leafhoppers were injected using a capillary needle forged
from 20 ul micropipets connected to a 100 pl Hamilton
syringe (Hamilton Company, Reno, Nevada) by 20 cm long
TeflonR tubing. Leafhoppers were immobilized prior to
injection by a stream of 002 and placed upside down onto an
inverted petri dish covered with a Parafilm R layer. A
second layer of Parafilm R was stretched over them to hold
them in place during injection. The inoculum was injected
into the abdomen between the second and third sternite.
Each leafhopper received a 0.50 ul dose of X-disease MLO
preparation delivered by a microapplicator (ISCOR

Instrumentation Specialties Co.).

As in the case of healthy individuals, X-disease
injected leafhoppers were placed on barley seedling growing
into 15 screened cages. The cages were made of two liter
inverted soda bottles with the tops excised at the neck so
they fitted snugly over 11.5 cm diameter x 13.5 cm height
plastic pots used to grow barley. Each cage had four 5 cm
diameter screened holes and one on the top to allow the

handling of leafhoppers. Cages containing X-disease

76

inoculated leafhoppers were placed into a growth chamber at
20 0C to complete development and a 20 day incubation period
(Garcia-Salazar et al. 1991). Temperature in the growth
chamber fluctuated between 10 and 30 °C in a 24 h cycle.
The photoperiod in the growth chamber was 16:8 (L:D) and 70-

80% rH.

The experiment was established in a walk-in growth
chamber at 25 i 1 °C (constant temperature), 16:8 (L:D) and
70-80% rh and treatments run simultaneously. The
experimental unit was a cohort of eight individuals, four
males and four females and there were sixteen healthy and
twenty MLO injected cohorts randomly assigned to three
replicates. Except for the temperature regime for MLO
incubation, criteria for setting up this treatment were the
same as those utilized to set up the control treatment
(healthy non-injected leafhoppers). Oviposition cages were
monitored every 24 h and all new emerging nymphs were
counted and immediately removed from the experiment with a

mouth aspirator.

X-disease Acquisition Test. MLO acquisition was
determined with a 32F radiolabelled DNA probe according to
Garcia-Salazar et al. (1991). The percentage of MLO
infected individuals was determined in a cohort of 10 adult
leafhoppers reandomly removed from the X-disease injection

treatment after a 30 day MLO incubation period. Leafhoppers

77

were individually MLO extracted and blotted onto a nylon
membrane for DNA hybridization with the radiolabelled probe
(Garcia-Salazar et al. 1991). A second cohort of 10
individuals removed from the control treatment was processed

in similar manner and used as a control.

Dot Blot Hybridization. The leafhopper samples were
blotted onto a GeenScreenTM nylon membrane (New England
Nuclear, Dupont, Boston, MA) in a dot-blot apparatus
(BioRad, Richmond, CA). The nucleic acid was fixed onto the
membrane with UV cross-linkage by laying the membrane face
up on a clean glass plate and irradiating it for 5 min at a
distance of 15 cm with a 254 nm shortwave UV light sources
(UVG-ll, Ultraviolet Products, Inc., San Gabriel, CA). The
membrane was then prehybridized and hybridized according to
the Method III for GeneScreenTM (New England Nuclear,

Dupont , Boston , MA) .

The cDNA probe C6c (Rahardja 1989), a 1.9 kb EcoRI-
HindIII fragment isolated from X-diseased Cblladonus
montanus (Van Duzee) (Kirkpatrick et al. 1987) was
nicktranslated with 32P using a kit from Amersham (Arlington
Heights, IL). The mean specific activity of the
radiolabelled probe was 5 x 107 cpm, and the amount of probe
utilized was 200 ng/ul.

After completion of the hybridization process, the
membrane was submitted to autoradiography by exposing it to

a X-ray film (Kodak XAR) for 24-48 h with an intensifying

78

screen (Sigma) at -70 °C. The film was developed and the

number of positive X-diseased samples determined.

Data Analysis. Becasue P. irroratus oviposition
behavior does not allow direct egg counting (females
oviposit into the stems of clover plants) without destroying
the eggs, fecundity was estimated as a function of the
number of nymphs hatching in each treatment (Chiykowsky

1985). Variables considered were:

1) Days to first emerged nymph. It was defined as the time
period between the day when leafhoppers were deposited
into the oviposition cage and the time when the first

nymphs were observed in the oviposition cage.

2) Days at 50% offspring production. It was the interval
between the time when the cohort was introduced into
the oviposition cage and the time when 50% of all

produced offspring was recorded.

3) Reproductive period. It was the observed time period
between the placement of leafhoppers into the
oviposition cage and the day when the last nymph
hatched.

79

4) Length of the oviposition period. It was the time
elapsed between the day of the first nymphal eclosion

and the time when the last nymph hatched.

5) Number of offspring produced per cohort. It was the

total number of nymphs observed per oviposition cage.

6) Offspring produced per day. It was calculated by
dividing the total number of nymphs observed per
oviposition cage between the length of the oviposition

period.

7) Number of sterile cohorts. It was the number of

oviposition cages were no nymphs were observed.

All these variables were recorded for both treatments
and compared through a two-way analysis of variance

utilizing the statistical package SYSTAT (Wilkinson 1988).

Results

From all variables considered in this research, only
the length of the reproductive and oviposition period of MLO
injected individuals, were not significantly different (P S
0.05) from the observed in healthy non-injected P.
irroratus. The oviposition period in the X-disease injected

treatment lasted 10.6 i 6.7 versus 14 O t 4.1 days in the

80

healthy leafhopper treatment. The length of the
reproductive period in the X-disease injected treatment was
37.5 i 4.3 versus 36.1 i 4.1 days in the healthy treatment.
The most notable differences between treatments were in the
number of offspring produced by both healthy and X-diseased
leafhoppers. Healthy leafhoppers produced 58.7 i 27.2
nymphs per cohort, while X-disease injected individuals
produced only 28.6 t 26.3. Another important difference was
observed in the number of cohorts that produced no
offspring. In the healthy leafhoppers treatment there was
only one sterile cohort while in the X-disease injected
treatment there were five (Table 3.1). Results
corresponding to the X-disease acquisition among inoculated
individuals showed that 60% of the treated leafhoppers
tested positive for the X-disease MLO (six out of ten
leafhoppers hybridized with the DNA probe). None of the
control leafhoppers showed hybridization with the DNA probe.
A summary of the statistical analysis comparing the healthy

and X-disease injected treatments is presented in Table 3.1.

Discussion

The effect of the X-disease MLO (X-MLO) on the
fecundity the speckled leafhopper, was not different from
the previously reported for the Western X-MLO in Cblladonus
montanus (VanDuzee) (Jensen 1971, Amin 8 Jensen 1971). The

Eastern strain of the X-disease MLO caused a 48.8%

81

Table 3.1. The fecundity summary of healthy and X-disease
injected P. irroratus.

M

 

 

Variable Healthy X-diseased F=0.05
Days to lst nymph 22.06 26.93 **
(1.7)a (4.6)
Days at 50 %
offspring production 28.60 32.00 **
(3.2) (3.9)
Reproductive period 36.13 37.53 NS
Oviposition period 14.06 10.60 NS
(4.2) (6.8)
Offspring/cohort/days 4.17 2.26 **
(1.8) (1.2)
Total offspring/cohort 58.73 28.66 **
(27.3) (26.3)
Sterile cohorts 1.0 5.0
(ND) (ND) NS

 

a ) Standard deviation of the mean.
**) Highly significative at P s 0.05
NS= No significant at P S 0.05.

ND= Not done.

82

reduction in the number of offsprings produced in X-diseased
P. irroratus, while in C. montanus the Western X-MLO caused
a 61.3% reduction in relation to healthy leafhoppers (Amin 8
Jensen 1971). It is important to point out that fecundity
reduction in the speckled leafhopper was not the result of a
shortening of the reproductive or oviposition period from X-
diseased females (Figure 3.1). In both treatments, the
oviposition period was not statistically different (P S
0.05) from each other and the same was true for the length
of the reproductive period. In the later case, the time
from the beginning of the experiment to the time when the
last nymph hatched was slightly longer in X-diseased
individuals but was not statistically different (P S 0.05)
from that observed in healthy leafhoppers (Table 3.1). A
possible explanation for the effect of the X-disease MLO
agent on the fecundity of its leafhopper vectors is the
cytological changes that the X-MLO caused in diseased
individuals. As early as 1968, Whitecomb et al. reported a
systemic pathological condition of cells in oviducts of
injected or naturally infected C. montanus and this was
interpreted as the onset of a moribund condition. Nasu et
al. (1970) recognized X-disease mycoplasmalike bodies
invading cells of diverse organs including brain,
subesophagal ganglion, thoracic ganglia and fat bodies among
others. In most insects, all these organs are related in
one way or another to egg production, maturation and

oviposition (Berry 1985). Lesions caused by the X-disease

83

 

 

 

 

100 f1 rT 1 Y T I I I T I U ' Ifi I TTT T r I I
.- -I
‘- .
80 - -
L .
h b ‘
a ' cl
'6 60 h 4
a. ’ ‘
O ' ‘
.E . .
h
3' 40 " ' a 'J
= P f. ‘ d
o P all
I: : a
P e = a “ d
20 "" u n " ‘
0 \\
P 0 "
u
I- u ..
‘\
I- : 1
0 _LJ 1 l l L l l l L Ll I J l L l l l l l J LJ
0 5 1O 15 20 25

Ovlpoaltlon Period (Days at 25 °C)

Figure 3.1. Oviposition patterns of healthy and X-disease
injected P. irroratus. X-diseased females (0-0-0), healthy
females (O-O-O)

84

MLO in these organs may reduce their ability to function
properly, therefore affecting the leafhopper reproductive
capacity and function.

Research results herein discussed are an important
contribution to the understanding of the ecology of the
Eastern strain of the X-disease MLO agent and its role in
the population dynamics of its leafhopper vectors. This
research has shown that the X-disease MLO not only affects
leafhopper survival but also fecundity. From the
perspective of pest population control, the X-disease MLO is
another natural factor regulating populations by limiting
the size of the leafhopper vector populations and the spread
of the X-disease MLO under field conditions. By reducing
longevity of X-diseased leafhoppers, the MLO limits the
extent and number of inoculation access periods and
therefore, the probability of X-disease transmission. On
the other hand, a fecundity reduction in diseased
individuals limits the size of the vector populations and
the subsequent number of potential X-disease vectors. This
would be very significant in years when the percentage of X-
diseased individuals is large. It is possible that in
Michigan the X-disease MLO is partially responsible for the
observed speckled leafhopper population fluctuation from
year to year and from one location to another, and that
these fluctuations are expressed in the dynamics of disease

transmission in important economic hosts.

1 I

85

Bibliography

Amin, P. W. 8 D. D. Jensen. 1971. effects of tetracycline on
the transmission and pathogenicity of the Western X-
disease agent in its insect and plant hosts.

Phytopathology 16:696-702.

Berry, S. J. 1985. Reproductive system. In: Fundamentals of
insect physiology (ed) M.S. Blum. John Wiley 8 Sons.

New York. Chicester. Toronto. Singapore.

Chiykowsky, L. N. 1985. Biology and rearing of
Paraphlepsius irroratus (Homoptera: Cicadellidae), a

vector of peach x-disease. Can. Ent. 117:717-726.

Garcia-Salazar,C., Mark E. Whalon, and U. Raharja. 1991.
Temperature dependent pathogenicity of the X-disease
mycoplasmalike organism to its vector Paraphlepsius

irroratus (Say). Environ. Entomol. 20(1):179-184.

Jensen,D.D. 1971. Vector fecundity reduced by western X-

disease. J. Invert. Pathol. 17:386-394.

86

Jensen, D.D., R.F. Whitecomb, and J. Richardson. 1967.
Lethality of injected peach Western X-disease virus to

its leafhopper vector. Virology 31:532-538.

Madden, L.V., L.R. Nault, S.E. Heady 8 W.E. Styer. 1984.
Effect of the maize stunting mollicutes on survival and
fecundity of Dalbulus leafhopper vectors. Ann. appl.

Biol. 105:431-441.

Nakasuji, F. 8 K. Kiritani. 1970. Ill-effect of rice dwarf
virus upon its vector Hephotettix cincticeps Uhler
(Homoptera: Deltocephalinidae) and its significance for
changes in relative abundance of infected individuals

among vector populations. App. Ent. Zool. 5(1):1-12.

Purcell, A. H. 1982. Evolution of the insect vector
relationship. In: Pathogenic Prokaryotes, Vol. 1.

Academic Press, Inc. 121-156 pp.

Rahardja, U. 1989. Field detection of peach X-disease
mycoplasmalike organism in speckled leafhopper,
Paraphlepsius irroratus (Say) (Homoptera: Cicadellidae)
using a DNA probe. M.S. Thesis, Michigan State

University. East lansing, MI. 73 pp.

Whitecomb, R.F., D.D. Jensen 8 J. Richardson. 1968. The

infection of leafhoppers by Western X-disease virus.

87

VI. Cytophatological interrelationships. J. Inverteb.

Pathol. 12:202-221.

Wilkinson, L. 1988. SYSTAT: The system for statistics.

Evanston,IL: SYSTAT, Inc.

88

CHAPTER IV

Response of Paraphlepsius irroratus (Say) (Homoptera:
Cicadellidae) to the Injection of Different Non-Pathogenic
Substances and the Eastern X-disease Mycoplasmalike

Organism.

89

Introduction.

Insect injection is a very useful technique in vector-
pathogen relationship studies because it provides the means
to inoculate individuals with different substances and
pathogens (Markham 8 Townsend 1979). Very often, needle
inoculation is preferred over natural pathogen acquisition
because it standardizes the pathogen dose, access
acquisition period (AAP) and incubation period (IP) in
treated individuals (Garcia-Salazar et al. 1991). However,
this method has disadvantages including the deleterious
effects from the physical injection on individuals,
mortality due to secondary infections and the behavioral and
physiological consequences of handling. Another less
studied effect relates to the effect of the injection
solution on individual survival. Buffers as well as
biological material injected into the insect haemocoel may
have a predictable impact on insect survival and longevity
and even confuse the results of vector-pathogen relationship
studies where injection has been the primary means of
acquisition. This research reports the effect of several
injected substances on the longevity and survival of P.
irroratus and compares these with the effect of the Eastern
X-disease mycoplasmalike organism (EX-MLO) on longevity and

survival .

90

Materials and Methods

Insect Culture. A laboratory culture was initiated in
1987 with field collected P. irroratus adults trapped in a
cherry orchard at East Lansing, MI. Insects were reared
into screened cages made from a 2 liter plastic inverted
soda bottles (the tops were excised at the neck to fit
snuggly onto plastic pots, 11.5 cm in diameter and 13.5 cm
in height) on a mixture of White clover (Trifolium repens
L.) and barley (Harden. bulgaris L.) grown in plastic pots.
Screened cages were fitted onto the containers and attached
with tape. Insects were introduced through a whole (5 cm
diameter) in the upper part of the cage. The barley
seedlings were a feeding substrate for immatures and the
clover plants were oviposition as well as feeding substrate

for adult leafhoppers (Chiykowsky 1985).

Treatments. Two hundred twenty one 3rd instar P.
irroratus were randomly assigned to seven treatments and
replicated three times. Each treatment corresponded to six
different injection solutions and a non injected treatment
used as a control. Treatments in this research included: 1)
Control, healthy non-injected leafhoppers. 2) PBS buffer,
standard saline buffer (0.01 M KH2P04, pH 7.0, 0.15 NaCl).
3) Tetracycline Hydrochloride, 1000 ppm of Tetracycline

“Sigma, St Louis, M0.) in PBS w/v. 4) X-disease (X-MLO), an

91

extract prepared from P. irroratus infected with Eastern X-
diseased (Garica-Salazar et al. 1991). 5) Healthy insect
extract, an extract of healthy.P. irroratus prepared in
similar manner as the X-MLO inoculum (Chiykowski 1983). 6)
X-MLO+Tetracycline, 1000 ppm of Tetracycline in an extract
of X-MLO infected P. irroratus w/v. 7) Inactivated X-MLO,
an extract of the X-MLO inactivated by placing it in a water
bath at 60 0C for 1 h. The X-MLO extract was prepared as
mentioned earlier. All injection solutions were prepared in
PBS buffer. Before treatment assignment, insects were
placed in 21 numbered cages (10 nymphs per cage) containing
10 barley seedlings. The cages were randomly assigned to
each treatment and replicate utilizing a random numbers
table. After injection, treatment leafhoppers remained in
these cages until adulthood was reached. Emerging adults
were then transferred to 21 new cages containing a 5 week
old clover plant each where the experiment was continued.
The experiment was estblished in a growth chamber at 20
°C fluctuating temperature and constant photoperiod.
Temperature was programmed to fluctuate in a 24 h cycle

between a maximum at 35 and a minimum at 15 °C.

Injection Protocol. Treatment insects were injected
with capillary needles forged from 20 ul micropipets
connected to a 100 pl Hamilton syringe (Hamilton Company,
Reno, Nv 89510) by a piece of 20 cm long TeflonR tubing.

Leafhoppers were immobilized prior to injection with a

92

stream of C02 and placed upside down onto an inverted petri
dish covered with a layer of ParafilmR. A second layer of
ParafilmR was stretched over them to hold them in place
during injection. Insects were injected into the abdomen
between the second and third sternite. Each leafhopper
received a 0.50 ul dose of the respective solutions
delivered by a microapplicator (ISCO Instrumentation
Specialities Co. Lincoln, Nb. 68504) and the needle

assambly.

Parameters and Analysis. Cages containing immatures
and adult leafhoppers were monitored every 24 h. Emerging
adults were removed and deposited into their respective
treatment cages. In each sample period, the number of new
adults entering the treatment cages and the number of dead
leafhoppers were recorded and daily survival and mortality
estimated per treatment. Dead insects removed from the
cages were stored at -70 °C for later Eastern X-MLO
diagnosis. The time to 50% survival (t50) was estimated and
statistical comparison between treatments performed through
a two-way analysis of variance. Separation of treatment
means was carried out according to Tukey's test (Honestly
Significant Difference) (Steel and Torrie 1980). The
survival data for each treatment were analyzed through the
two-parameter Weibull cumulative distribution function
(Pinder et al. 1978) and their c and b coefficients compared

at P 5 0.05 according to Billmann et al.(l972). The Weibull

93

cumulative distribution function is described by the

equation:
Y= 1-exp(- (t/b)°), where:

Y= mortality at time(t), t= time, b= scale parameter and c=
shape parameter. The coefficient 0 determines the type of
survival curve (Type I, II, etc) and the coefficient b
describes the rate of decline of the population (Madden 8
Nault 1983). The right term of the equation, exp(-(t/b)°),
corresponds to the survival term of the Weibull cumulative
distribution function. The shape (0) and scale (b)
parameter were estimated through the Least Square estimate

method according to White (1969) and Wagner et al. (1984).

X-MLO Diagnosis. X-MLO injected leafhoppers were
tested for X-disease acquisition through Dot Blot
hybridization with a DNA probe ,C6c, showing specific
hybridization with the Eastern strain of X-disease. DNA
hybridization was executed according to the protocol
discussed in Chapter II and Garcia-Salazar et al. (1991).
Percent MLO acquisition was estimated in both X-MLO injected
and X-MLO+Tetracycline injected populations. Likewise,
insects injected with heat inactivated X-MLO were tested for
the presence of MLO’s to assure that no active MLO’s were
injected in this treatment. Healthy non-injected P.

irroratus processed in the same manner than leafhoppers from

94

MLO injection teatments were extracted and blotted in the
hybridization membranes as a control. Together with the
leafhopper samples two other control were blotted. A 10-
fold serial dilution (200, 20, 2, 0.2 ul) of the X-MLO
extraction used as inoculum was blotted as a positive

control.

Results

In comparison with non-injected leafhoppers, all
injection treatments affected longevity and survival of P.
irroratus. Table 4.1 shows the mean longevity of all
treatments. On the average, non injected individuals lived
43.6 i 13.0 days (Mean 1 SD), while X-MLO injected
leafhoppers had the shortest longevity, 21.8 i 10.4 days.
The Tukey's test showed that longevity of X-diseased
leafhoppers was statistically (P s 0.05) different from all
other treatments. Tetracycline alone had a deleterious
effect on leafhopper longevity, and treated P. irroratus
lived 31.8 i 10.4 days. But longevity was statistically
different (P s 0.05) from x-MLO injected individuals. No
statistical difference (P S 0.05) between X-MLO+Tetracycline
injected individuals and Tetracycline alone was observed
(Table 4.1). The solution prepared from healthy P.
irroratus had a deleterious effects when injected into
healthy individuals. But, no statistic difference (P s

0.05) was found between healthy non-injected leafhoppers and

95

individuals injected with a extract of healthy P. irroratus.
Heat inactivated X-MLO as well as PBS injection shortened
leafhopper longevity but not statistic (P50.05) different
between these and the control group (healthy non-injected

individuals) was observed (Table 4.1).

Survival Analysis. Survival curves estimated by the
Weibull cumulative distribution function (Figure 4.1) showed
that P. irroratus responded to injection treatments in two
ways. In one instance, curves with parameter c lesser than
2.0 indicated an early and approximately constant mortality
rate along the period of study. On the other hand, curves
with parameter c greater than 2.0 indicated increased
mortality with an increase in time (Figure 4.1). Statistic
comparison of the coefficients c of survival curves showed
that survival of X-MLO injected leafhoppers was affected by
early mortality as much as PBS injected individuals.
However, the coefficients b of these curves were
statistically different (P S 0.05) indicating greater
survival in the buffer than in X-disease injected P.
irroratus. (Table 4.2). Coefficients c from the other group
of survival curves were not statistically different (P S
0.05) from each other. Therefore, their t50 values were the
effect of the injection treatment rather than a change in

mortality patterns (Figure 4.1 and Table 4.2).

96

Table 4.1. Comparison of longevity of P. irroratus injected
with different solutions and incubated at 20 °C, fluctuating
temperature.

 

Longevity (1:50)1

 

 

Treatment* Mean SD

Control (non-injected) 43.628 13.04
PBS buffer 39.14abc 17.30
X-MLO inactivated 36.65abc 10.98
Healthy Extract 34.80abc 13.80
Tetracycline 31.83bc 10.43
X-MLO+Tetracycline 31.77bc 10.57
X-MLO injected 21.81d 10.44

 

1) Time to 50% survival.

*) Treatments followed by the same letter are not different
at P s 0.05 according to Tukey’s HSD test (Steel 8 Torrie
1980).

97

Survlval/ 1 00

O INACJILO
I:I TE'LMLO

O HEALTHY

m EXJILO

I v TETRACYC
v BUFFER

O CONTROL

 

 

 

0 1 0 20 30 40 70

Age Since Adult Emergence (Days)

Figure 4.1. Survival curves of populations of P. irroratus
injected with different solutions. Control= healthy non-
injected leafhoppers, Buffers saline buffer (PBS), Tetracyc=
Tetracycline hydrochlorite, EX_MLO= extract of X-diseased
leafhoppers, Healthy= extract of healthy leafhoppers, TET-

lfl..o= X-MLO+Tetracycline, Inac_MLO= heat inactivated X-MLO
extract.

98

MLO’s Acquisition. DNA hybridization with C6c revealed
that acquisition among X-MLO injected leafhoppers was
70.37%. That is 19 X-diseased out of 27 leafhoppers tested.
In the treatment including X-disease and Tetracycline,
aquisition was 23.0% (five out of 26 insects treated with X-
MLO+Tetracycline hybridized with C6c). This percentage
indicates that in a 77.0% of the treated population
Tetracycline supressed the multiplication of the X-MLO. No
positive hybridization was observed between healthy non

injected P. irroratus and C6c.

Discussion

Depending on the nature of the injected substance
deleterious effect on treated individuals varies from no to
high mortality. Muller 8 Rochow (1961) reported high
survival (86.61%) in aphids injected with crude haemolymph
containing BYDV (Barley Yellow Dwarf Virus) from diseased
aphids or water. In contrast, survival in insects injected
with crude oat extracts was invariably low (35.50%). Jensen
(1967) reported that the injection of haemolymph of healthy
leafhoppers caused a reduction in longevity of Cblladonus
montanus VanDuzee. However, when injected with an extract
of Western X-disease infected leafhoppers, the tso (time to
50% survival) was 31.08% shorter than in C. montanus
injected with an extract of healthy individuals (51 versus

74 days). P. irroratus survival in the present

99

Table 4.2. Weibull statistics for survival curves of P.
irroratus injected with different solutions and incubated at
20 oC fluctuating temperature.

 

 

Treatment c b Residual R2

Control (non-injected) 3.049bc 47.80a 0.14 0.92
PBS buffer 1.629a 44.21ab 0.49 0.92
X-MLO inactivated 3.279c 39.33abc 0.04 0.97
Healthy extract 2.398b 37.97abc 0.11 0.96
Tetracycline 3.040bc 34.64bc 0.01 0.96
X-MLO+Tetracycline 3.096bc 33.52c 0.04 0.96
X-MLO injected 1.776ab 23.52d 0.50 0.94

 

Values of coefficient c and b followed by the same letter
are not different at P s 0.05 according to Billman et al.
1972.

100

research varied from 96.97 in the control group (non-
injected) to 76.67% in the healthy extract injected
leafhopper population. Likewise, the t50 of P. irroratus
injected with an extract from healthy leafhoppers was
shorter than healthy non-injected but greater than X-
diseased leafhoppers (Table 4.1). In the present research,
the tso of X-disease injected P. irroratus was 37.36%
shorter than that from insects injected with an extract of
healthy leafhoppers, and 40.50% shorter than the observed in
the heat inactivated X-disease injection treatment. Our
results are in close agreement with these reported by Jensen
(1967) in C. montanus and Garcia-Salazar et al. (1991) for

P. irroratus.

A non-pathogenic agent such as PBS, had a small
deleterious effect on leafhopper longevity. Compared with
non-injected individuals, PBS caused a 10.27% reduction in
the t50 of treated leafhoppers. This result is different
from the observed in Circulifer tenellus (Baker) injected
with sterilized liquid media utilized to culture S. citri.
In c. tenellus, the injection solution caused a 28.57%
longevity reduction comparison with healthy non-injected

leafhoppers (Liu et al. 1983).

There was an interesting result concerning Tetracycline
treatments. In relation to control leafhoppers (non-

injected), 1000 ppm of Tetracycline in PBS caused a 27%

101

longevity reduction in both, Tetracycline alone or in a
mixture with X-MLO. However, 50% survival time (t5o) for
Tetracycline treated leafhoppers was 31.35% greater than for
X-diseased individuals (Table 4.1). There are two possible
explanations for the deleterious effect of Tetracycline on
leafhopper longevity and survival. It has been documented
that the primary target for the action of Tetracyclines is
protein syntheis in both, eukaryotes and prokaryotes (Hlavka
8 Boothe 1985). In eukaryote organisms, cytoplasmic protein
syntheis is not seriously affected but, to some extent,
mitochondrial protein synthesis is inhibited (Chopra 1985).
In a small organism such as P.1rroratus, it is possible that
the harmful effect of Tetracycline on mitochondrial protein
syntheis migth become a limiting factor for adequate
functioning of most tissues and organs. This effect may
result in reduced longevity and survival of Tetracycaline
treated individuals. However, a more likely explanation
could be that Tetracycline destroys symbiote organisms that
provide leafhoppers with essential aminoacids. The
existence of symbiotic endocellular prokaryotes in
Homopteran insects has been extensively documented
(Schwemmler 1974, Mitsuhashi 8 Kono 1975, Louis 8 Nicolas
1976, Waku 8 Endo 1987). The role of symbionts has been
considered primarily as mediators between the host and the
environment enabling insects to adapt to changing
nutritional and environmental conditions (Schwemmler 1974).

In the pea aphid, Acrythosipbon pisum (Harris), symbionts

102

synthesize free fatty acids, mono- and diglycerides,
phosphatidyl choline, phosphatidyl ethanolamine, and
cholesterol. The last one, plays a essential structural and
physiological role in insect development (Houk 1974, Houk et
al. 1976). The destruction of symbionts in A. pisum fed on
a diet containing antibiotics greately reduced its longevity
and survival. For example, the tso of insects treated with
100 ppm of Neomycin and control individuals was 6 and 23

days respectively (Srivastava 8 Auclair 1976).

In spite of the observed deleterious effect of
Tetracycaline on P. irroratus, there are reports showing
increased longevity and survival in MLO infected leafhoppers
treated with antibiotics. Whitecomb 8 Davis (1970) reported
that survival of Aster Yellow (AY) infected Macrosteles
fascifrons (Stal) was improved when injected with Kanamycin
or Neomycin or a mixture of Penicillin and Streptomycin.

But only Chlortetracycline effectively blocked Aster Yellow
infection in leafhoppers injected with a mixture of
antibiotic and AY agent. In another related experiment the
tso for healthy M. fascifrons fed on Oxytetracycline and for
control insects fed on a buffer solution was 53 and 34 days,
respectively (Sinha et al. 1972). The effect of
Tetracycline on the longevity of C. montanus and on the
Western X-disease agent was assessed by Amin 8 Jensen
(1971). The t50 of C. montanus injected simultaneously with

500 ppm of Tetracycline and an extract of WX-diseased

103

leafhoppers was 64 days but only 50 in control individuals
injected only with WX-disease MLO. Our MLO acquisition test
showed that Tetracycline was able to stop the multiplication
of the X-MLO in most insects injected with a mixture of X-
disease and Tetracycline. Yet, MLO multiplication was not
completely suppressed and 23% of the treated population
developed the disease. In agreement with Amin 8 Jensen’s
(1971), longevity was higher in P. irroratus injected with a
mixture of Tetracycline and MLO than in leafhoppers treated
with X-MLO alone. These results support their asseverations
that Tetracycline either eliminates the MLO’s from the

vector’s system or substantially reduces their titer.

The use of the Weibull cumulative distribution function
to characterize survival under different injection
treatments allowed a better understanding of the survival
pattern in treated leafhoppers. It is important to mention
that equal c parameter (i.e. same Type of survival curve)
among curves does not mean equal parameter b (i.e. same rate
of population decline), nor equal t50 (time to 50%
survival). In this research, the mean survival time was
associated with the coefficient b. A regression of the
coefficient b against the t50 values of the corresponding
treatments gave a corrected r2 = 0.99 that supports the use
of the c and b parameters to directly quantify the effect of
Mollicutes on leafhopper survival (Madden 8 Nault 1983). In

our research, coefficients c of X-disease and PBS buffer

104

injected leafhoppers were statistically equal (P S 0.05) but
coefficients b were statistically different. In this case,
the difference in tso was the result of the effect of the
treatment rather than a change in the survival pattern.

That means a change in the survival curve type (e.g. Type I
c 2 2 or Type II c E 1). According to Pinder et al. (1978)
a Type I survivorship curve results when the age-specific
mortality rate increases with increasing t (time), and a
Type II when the age-specific mortality rate is constant
over time. Accordingly, survivorship of PBS and X-disease
injected populations was close to a Type II, while all other
treatments were intermediate between Type II and Type I

(Table 4.2).

Results herein discussed clearly signal the need for
appropriate controls when using needle inoculation as a
method for insect pathogen acquisition. So far, this
research has shown that even saline buffer (PBS) causes some
deleterious effect on leafhopper survival. Extracts from
healthy insects or extracts containing heat inactivated
pathogens also affected survival in treated populations.
However, their effect is not different from the observed in
buffer treated populations. In general, survival and
longevity of populations treated with non pathogenic
substances (except Tetracycline) were not significantly
affected in relation to non-injected control leafhoppers.

Thus, the usage of any of these substances in

105

vector-pathogen relationship studies does provide
appropriate controls for pathogen injected treatments.
This research has also confirmed that, independently from
the deleterious effect of the injection solution on
leafhopper survival, the Eastern strain of X-disease

drastically reduces the survival of X-diseased P. irroratus.

106

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Amin, P.W. 8 D.D. Jensen. 1971. Effect of tetracycline on
the transmission and pathogenicity of the Western X
disease agent in its insect and plant hosts.

Phytopathology 61:696-702.

Chiykowski, L. N. 1983. Frozen leafhoppers as a vehicle for
long-term storage of different isolates of the aster

yellos agent. Can. J. Plant Pathol. 5:101-106.

Chopra, I. 1985. Mode of action of the Tetracyclines and the
nature of bacterial resistance to them. In: The
Tetracyclines (eds) Hlavka, J. J. 8 J. H. Boothe.318-

392 pp.

Garcia-Salazar, C.,M.E. Whalon 8 U. Rahardja. 1991.
Temperature dependent pathogenicity of the X-disease
mycoplasmalike organism to its vector, Paraphlepsius
irroratus (Say) (Homoptera: Cicadellidae). Environ.

Entomol. 20(1):179-184.

Hlavka, J. J. 8 J. H. Boothe. 1985. Handbook of Experimental
Pharmacology Vol. 78. Springer-Verlag Berlin Heidelberg

N. York.

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Houk, E. J. 1974. Lipids of the primary intracellular
symbiote of the pea aphid, Acyrthosiphon pisum. J.

Insect Physiol. 20:471-478.

Houk, E. J., G. W. Griffiths 8 S. D. Beck. 1976. Lipid
metabolism in the symbiotes of the pea aphid,
Acyrthosiphon pisum. Comp. Biochem. Physiol. 548:427-

431.

Jensen, D.D., R.F. Whitecomb 8 J. Richardson. 1967.
Lethality of injected peach Western X-disease virus to

its leafhopper vector. Virology 31:532-538.

Liu, H.Y., D.J. Gumpf, G.N. Oldfield 8 E.C. Calavan. 1983.
The Relationship of Spiroplasma citri and Circulifer

tenellus. Phytopathology 73(4):585-589.

Louis, C. 8 G. Nicolas. 1976. Ultrastructure of the
endocellular procaryotes of arthropods as revealed by
freez-etching. I. A study of "a"-type endosymbionts of
the leafhopper Enscelis plebejus (Homoptera: Jassidae).

J. Microscop. Biol. Cell. 26(2/3):121-126.

Madden, L.V. 8 L.R. Nault. 1983. Differential pathogenicity

of corn stunting mollicutes to leafhopper vectors in

108

Dalbulus and Baldulus species. Phytopathology

73(12):l608-1614.

Markham, P.G. 8 R. Townsend. 1979. Experimental vectors of

spiroplasmas. In: Leafhopper vectors and plant disease
agents, (eds) K. Maramorosch 8 K.F. Harris. Academic

Press, New York, San Francisco, London. p 413-445.

Mitsuhashi, J. 8 Y. Kono. 1975. Intracellular microorganisms
in the green rice leathpper, NephotettIX'cincticeps

Uhler (Homoptera: Deltocephalidae). Appl. Ent. Zool.

10(1):1-9.

Schwemmler, W. 1974. zikadenendosymbiose: Ein modell fur die

evolution hoherer zellen?. Acta Biotheor. 23: 132-169.

Sinha, R.C. 8 E.A. Peterson. 1972. Uptake and persistence
of Oxytetracycline in aster plants and vector
leafhoppers in relation to inhibition of clover

Phyllody agent. Phytopathology 62:377-383.

Srivastava, P. N. 8 J. L. Auclair. 1976. Effects of
antibiotics on feeding and development of the pea
aphid, Acyrthosipbon pisum (Harris) (Homoptera:

Aphididae). Can. J. Zool. 54:1025-1029.

109

Waku, Y. 8 Y. Endo. 1987. Ultrastructure and life cycle of
symbionts in a Homopteran insect, Anomaneura mori

Schwartz (Psyllidae). App. Ent. Zool. 22(4):630-637.

Whitecomb, R.F. 8 R.E. Davis. 1970. Evidences of possible
mycoplasma etiology of aster Yellows disease: II.
Suppresion of aster Yellows in insect vectors. Infec..

Immun. 2(2):209-215.

White, J.S. 1969. The Moments of Log-Weibull Order

Statistics. Technometrics 2(2):373-386.

110

CHAPTERV

X-disease Mycoplasmalike Organism: In vivo Developmental

Threshold Temperatures Estimate.

111

Introduction.

The X-disease mycoplasmalike organism is a cell wall-
free prokaryote belonging to the Class Mollicutes. Because
of the impossibility to in vitro culture this organism, its
correct classification remains unconfirmed (Chen et al.
1989). The Eastern strain of the X-dieseas MLO (EX-MLO) is
transmitted to cherry and peach trees by a complex of phloem
feeding leafhoppers that includes Cblladenus montanus Van
Duzee, Gyponana lamina DeLong, Nbrvellina seminuda (Say),
Paraphlepsius irroratus (Say), and scaphytopius acutus (Say)
among others (Taboada et al. 1975, Larsen 8 Whalon 1988).
Environmental factors, including temperature, have a major
influence on the dynamics of disease infections and
transmission. Tipically, at low temperature multiplication
of disease agents in insects proceeds slowly and reaches a
maximum after a long incubation period. At higher
temperatures, multiplication may be totally blocked, or may
peak and subside rapidly (Whitecomb 8 Davis 1970). In
previous filed work, it was observed a correlation between
X-disease and transmission rates with temperature (Rahardja
1989). Understanding the temperature effect on the
development of the X-MLO in its vector could have major
implications for marshalling monitoring efforts and timing

management practices in our integrated pest management

112

project for X-MLO. Determination of the range of
temperatures for multiplication of the X-MLO presents an
additional challange given its in vitro uncultivability.
Other MLO works attempted to estimate the developmental
threshold temperatures by using live insects as a culture
media and monitoring the disease progress in leafhoppers
through transmission to indicator plants (Jensen 1972).
This methodology, however, has several shortcomings
including the lack of differentiation between failure to
acquire the MLO versus inability to transmit once the MLO
was acquired. Other factors such as the age of the
indicator plant and length of the inoculation access period
(IAP) also may affect the leafhopper transmission pattern
(Chiykowski 8 Sinha 1988). Yet, before the development of
molecular diagnostic for MLO’s in leafhoppers (Kirkpatrick
eta a1 1987, Davis et al. 1988, Sears et al. 1989, Rahardja
1989) improvements on this approach were exceedingly

’ difficult. This research reports the developmental

threshold temperatures for the EX-MLO in live P. irroratus.

Materials and Methods

Insect culture. AAP. irroratus culture was established
at the MSU Pesticides Research Center in 1987 with adult
leafhoppers collected at Collins Road Department of
Entomology Experiment Station at Michigan State University,

East Lansing, MI. Rearing was conducted in a walk-in growth

113

chamber at 25 i 1 °C, 70-80% rH, and 16:8 (L:D) photocycle.
Adult leafhoppers were caged on a mixture of barley (Herdeum
vulgaris L.) and white clover plants (Trifolium repens L.)
and allowed to oviposit. Clover plants were the substrate
for oviposition while barley was the favorite feeding
substrate for immatures. Oviposition cages were constructed
of screened two liter clear plastic soda bottles. Clover
and barley plants were grown in 150 ml plastic pots and the

cages were attached on the top of them.

Treatments. This experiment comprised 550 third
instar P. irroratus needle injected with an MLO extract
prepared from X-diseased leafhoppers. Treatment leafhoppers
were randomly allocated to six constant temperature
treatments 15, 20, 25, 30, 35, and 38 °C, respectively. A
treatment consisted of 90 individuals randomly assigned to
three replicates. Depending on the temperature treatment,
the X-MLO was monitored on six occasions as follows: 1) 15,
20, and 25 °C were sampled every 10, 6, and 4 days, and 2)
30, 35 and 38 °C were monitored every two days. In each
instance, 4-5 individuals were removed from the experiment
and immediately frozen at -70 °C for further MLO titer

determination.

Preparation of Inoculum. Ten X-disasede. irroratus
were ground in a Ependorf tube using a disposable pellet

pestle (Kontes, Scientific Glassware/instruments, Vineland,

114

NJ.) in 0.673 ml of phosphate buffer saline (0.01 M
Potassium Phosphate, pH 7.0, 0.15 M NaCl) and centrifuged at
5,000 rpm for 10 min. The supernatant was removed with a
tuberculin syringe and filtred through a 0.40 pm Millex-Ha
filter unit (Millipore Products Division, Bedford, Ma.).

The filtred product had a 1:10 concentration and was further
diluted to a 1:20 inoculum concentration. In calculating
the dilution of inoculum, it was assumed that a quantity of
leafhoppers weighting 1.0 g had a volume of 1.0 ml
(Chiykowski 1983). The inoculum was stored at -70 °C and
aliquots of this were used for leafhopper injections.

Insect utilized to prepare the inoculum were P. irroratus
raised on Eastern X-disease infected celery (Apimum

graveolens L.) (Garcia-Salazar et al. 1991).

Leafhopper Injection. A capillary glass needle was
forged from a 20 pl pipet and utilized for.P. irroratus.
injection. The needle was connected to a TeflonR tubing
coupled to a 100 p1 Hamilton syringe installed in a
microapplicator apparatus. The microapplicator was
activated by a peristaltic pump and calibrated to deliver
0.25-0.50 pl at a time. Prior to injection, insects were
anesthetized with a continuous stream of C02 and deposited
on an inverted petri dish covered with a layer of ParafilmR.
A second layer of ParafilmR was stretched onto the insects
to immobilize them during the injection process. The

injection was carried out through the second layer of

115

ParafilmR and 0.25-0.5 pl of inoculum were delivered into
the insect abdomen between the second and third sternite.
After injection, treated leafhoppers were randomly allocated

to replicates and temperature treatments.

X-disease MLO Detection Method. The presence and titer
of X-disease MLO’s in the insect host was assessed through
DNA hybridization utilizing C6c, a DNA probe derived from X-
diseased Cblladbnus montanus (Van Duzee) (Raharja 1989,

Garcia-Salazar et al. 1991, Kirkpatrick 1987).

MLO Extraction. x-disease injected leafhoppers were
extracted individually according to Kirkpatrick (1987) with
some modifications. Each leafhopper was placed in a 2 ml
Ependorf tube to which 400 p1 of ice cold MLO enrichment
buffer (0.1 M NaZHPO4, 10% sucrose, 50 mM ascorbic acid, and
1% polyvinylpyrollidone Mr 10,000, pH 7.6) was added before
the leafhopper was mascerated on ice with a disposable
pellet pestle (Kontes, Scientific Glassware/instruments,
Vineland, NJ). The homogenate was centrifuged at 3,000 g
for 5 min in a Sorvall SS-34 rotor at 4 °C. The supernatant
was removed and transferred to a new tube and centrifuged
again at 12,000 g for 30 min at 4 oC. The resulting pellet
was resuspended in 400 pl of TE buffer (10 mM Tris-MCI, 1 mM
EDTA pH 8.0) and stored at -70 °C until blotted for DNA
hybridization. The pellet contained partially purified
MLO's and insect organells including mitochondria. Each

leafhopper sample was divided into two aliquots, 200 pl

116

each. One aliquot was used for X-MLO quantification through
Dot Blot hybridization and the other for protein content

determination.

Titer Determination. Two standard curves were
calculated using serial dilutions of a known amount of C6c
and X-diseased leafhoppers. In the first case, 0.1 pg of C6c
was diluted in 10 x SSC to produce a solution concentration
of 100, 50, 25 ng/400 pl. Each one of these dilutions was
further diluted with 4, ten-fold dilutions. The curve
corresponding to X-disease MLO was prepared by grinding 10
X-diseased leafhoppers and processing them in the same
manner as the inoculum for leafhopper injection. The first
four dilutions were 2806, 1403, 701.5 and 300.52 ng of MLO
DNA plus protein in 400 pl of 10 x SSC. Each dilution was
further diluted with 4, ten fold dilutions. The Optical
Density (OD) of each X-MLO DNA dilution was estimated and
regressed against the concentration of C6c and X-MLO extract
(Rahardja 1989). The C6c standard curve was utilized to
estimate X-MLO titre in leafhopper samples and the X-disease
MLO extract curve was used to estimate the amount of X-
disease MLO injected per insect. Both standard curve
dilutions were blotted onto a nylon membrane together with

the insect samples.

Blotting Procedure. All samples were blotted onto a

GeneScreenR hybridization transfer membrane (New England

117

Nuclear, Boston, MA.) using a Bio-Dot microfiltration
apparatus (Bio-Rad Laboratories, Richmond, CA.). Prior to
blotting, the nylon membrane was equilibrated in 2 x SSC
(0.3 M NaCl, 30 mM Na3Citrate.2H20, pH 7.0) for 20 min and
placed into the dot blot apparatus and was ready to be
blotted. One aliquot (200 p1) of MLO enriched fraction was
added to 200 pl of 10 x SSC and boiled for 10 min to
denature the DNA. Immediately after, samples were placed on
ice for another 10 min before being placed into the dot blot
apparatus. The apparatus was connected to a vacuum pump and
through rapid suction the buffer removed and the DNA
impinged to the membrane. Leafhopper as well as MLO’s DNA
were fixed to the membrane by UV crosslinking according to
the supplier’s protocol (New England Nuclear Research Tips
E94729, Richmon, CA), and baked under vacuum at 80 °C for 3

h.

DNA Hybridization Protocol. Blotted membranes were
hybridized according to Method III described in the
GeneScreenR Instruction Manual (New England Nuclear 1985).
The procedure was as follows: membranes were prehybridized
in 10 ml of prehybridization buffer ( 50% deionized
formamide, 0.2% ployvinylpyrrolidone M.W. 40,000, 0.2%
bovine serum albumin, 0.2% ficoll M.W.400,000, 0.05 M Tris-
HCl pH 7.5, 1.0 M NaCl, 0.1% Sodium pyrophosphate, 1.0% SDS,
10% dextran sulfate M.W. 500,000, and denatured salmon sperm

DNA (2 100 pg/ml)). Membranes were prehybridized with

118

constant agitation for 12 h at 42 0C in a sealed plastic
bag. After prehybridization, the bag was opened and 2-3 ml
of hybridization buffer (50% deionized formamide, 0.2%
ployvinylpyrrolidone M.W. 40,000, 0.2% bovine serum albumin,
0.2% ficoll M.W.400,000, 0.05 M Tris-HCl pH 7.5, 0.1% Sodium
pyrophosphate, 1.0% SDS, denatured salmon sperm DNA (2 100
pg/ml)) plus the radiolabelled C6c probe was added. The bag
was resealed and hybridized with constant agitation for 16-

24 h at 42 °C.

After hybridization, membranes were washed twice with
constant agitation for 5 min at room temperature in 200 ml
of a solution containing 0.3 M NaCl, 0.06 M Tris-HCl (pH
8.0), 2 mM EDTA. Another wash in a solution containing 0.3
M NaCl, 0.06 M Tris-HCl (pH 8.0), 2 mM EDTA, and 1% SDS was
given at 60 °C for 30 min with constant agitation. Finally,
membranes were washed for another 30 min at room temperature
in a buffer containing 0.03 M NaCl, 6 mM Tris-HCl (pH 8.0),
0.2 mM EDTA before drying and exposure for autoradiography
at -70 °C for 72 h using a Kodak XAR X-rye film in an

intensifying screen (SIGMA).

Probe Radiolabelling. A 200 ng 32F radiolabelled C6c
was prepared through nick translation using an Amersham nick
translation kit (Amersham International Plc. Arlington
Heights, 11). Unincorporated nucleotides were separated by
passing the reaction mixture through a 1.0 ml Sephadex G-75

fine column which was previously saturated for 1 h with TE

119

buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0). Labelled probe
was harvested as a fraction of the first column peak to
ensure that only 32P incorporated C6c were used in the
hybridization mixture. The specific activity of each nick
translation was assayed by removing two 1.0 pl samples
before and after 32p incorporation, blotted onto a nylon
membrane and air dried. One sample from before and other
from after 32p incorporation were washed for 5 min in a
solution of 0.3 M NaCl, 0.06 M Tris-HCl (pH 8.0), 2 mM EDTA.
The specific activity of the washed and unwashed samples was
measured in a scintillation counter and the percent of
incorporated 32p estimated (Rahardja. 1989). The measured
specific activity of the nick translated probe was 3.7 x 108

cpm/pg having 74% incorporated 32P.

Data Analysis. The autoradiography of hybridization
intensity was assessed using a 2-D/1-D soft laser scanning
densitometer (Biomed Instruments, Inc., Fullerton, CA).
Each dot hybridizing with C6c was read and its Integrated
Optical Density (IOD) and Optical Density (OD) estimated.
All hybridization measurements were corrected for the amount
of protein in the original sample. The IOD's and OD’s were
non linearly regressed on the incubation period. This
analysis was carried out for each temperature regime and
Taylor’s (1981) equation was utilized to describe the X-MLO
development over time in individual leafhoppers. Taylor's

equation was sellected for this analysis because at

120

difference of other curves its parameters have ecological
meaning and describe the effect of constant temperature on
developmental rates with reasonable accuracy for many
insects (Lamb et al. 1984). Taylor’s algorthm is as as

follows:

R(t)= Rm*exp(-0.5*(T-Tm/TS)2) where:

R(t)= X-MLO growth rate, Rm: maximum MLO growth observed,
Tm: optimum temperature where Rm occured, TS= spread of the
MLO growth curve estimated by the standard deviation of T,
and T= observed temperature at time (t). The same algorithm
was utilized to describe the amount of extracted protein
with the length of the incubation period at each of the six
temperature treatments. The first approximation to T8 was
obtained directly from the temperature data points. The
estimated values for Rm and Tm were obtained through
nonlinear regression coefficients estimation using the

NONLIN program in SYSTAT (Wilkison 1988).

Results.

X-diseas MLO Multiplication. After 72 h of exposure,
autoradiographs showed that the X-MLO had a differential
development depending on the temperature regime (Table 5.1).
Hybridization indicating MLO multiplication was not observed

at any incubation period for the treatment 15 °C. At 20 and

121

Table 5.1. X-disease acquisition in MLO injected P.
irroratus incubated at constant temperature.

 

 

Range of
Temperature Vectors incubation Percent
(°C) Reps Number w/MLO (days) acquisition
15 3 64 0 4-45 0.00
20 3 65 2 4-27 3.07
25 3 68 5 4-23 7.35
30 3 72 70 4-14 97.22
35 3 61 59 4-12 96.72
38 3 57 51 4-12 89.47

 

Table 5.2. Standard curve of X-disease MLO inoculum used to
inject healthy P. irroratus.

 

 

Protein+MLO Dilution Optical density C6c Equivalents
(n9) (#1) (Pixels)a (09)
0.35 0.05 7.0 (2.8) 0.037 (0.02)1
0.70 0.10 12.7 (5.3) 0.068 (0.03)
1.40 0.20 38.0 (5.2) 0.201 (0.03)
2.80 0.40 120.0 (14.1) 0.636 (0.08)
3.50 0.50 98.0 (8.5) 0.519 (0.05)

 

1) Standard deviation of the mean.
a) Mean of three replicats.

122

 

250

200

150

100

Optlcal Denelty (Pixels)

50

 

 

 

o 4 l l l 4

0.00 0.17 0.34 0.51 0.08 0.85 1.02

Nanegrame of 06c

Figure 5.1. Standard curve to transform the observed OD
(optical density) values from X-diseased P. irroratus to C6c
equivalents (ng)

123

25 °C, only 3.0 and 7.3% of treated individuals demonstrated
positive hybridization or MLO development. Detectable MLO
multiplication occurred after a 23-27 day incubation period.
Maximum X-MLO multiplication occurred at 30 and 35 °C but
declined at 38 0C with a MLO acquisition among injected

leafhoppers of 97.2, 96.7 and 89.4 %, respectively.

X-disease MLO Titer. Using both, the C6c standard
curve (Figure 5.1) and the inoculum standard dilution (Table
5.2) as measure units, the amount (C6c equivalents) of X-MLO
inoculated per leafhopper was estimated. Given the
concentration of X-MLO protein in the inoculum solution
(4.007 ng/pl) and approximated injected volume (0.25
pl/leafhopper), the C6c equivalents injected per leafhopper
were 0.150 ng, approximately. Equations used in this

calculus were:

1) MLO OD= (B) * 34.877, where:
MLO OD= optical density of the hybridization reaction of the
amount of X-MLO injected per leafhopper with radiollabeled
C6c, B= concentration of X-MLO and protein (ng/pl) in the

inoculum, and 34.877= constant.

2) C6c (ng)= (MLO OD)*0.0043, where:
C6c (ng)= Amount of C6c equivalent to the amount of X-MLO
present in the inoculum or leafhopper samples, MLO OD=

optical density from the intensity of the hybridization

124

reaction with radiollabeled C6c, and 0.0043= constant.
According with hybridization results, the DNA probe C6c, was
able to detect up to 0.039 C6c equivalents (ng) that
coresponded to approximately 0.05 pl of the X-MLO inoculum

solution used for P. irroratus injection (Table 5.2).

Time Course of MLO Multiplication. Multiplication of
X-MLO after inoculation followed a curve described by the
Taylor’s equation (1981) (Table 5.3 and 5.4). The amount of
hybridization increased from a minimum at four days past
injection to a maximum and decline again in the longest
incubation periods. Table 5.5 presents a summary of the X-
MLO titer over time. At 30 °C, the MLO increased 4.5 fold
in relation to the amount of X-MLO inoculated per leafhopper
(0.150 C6c ng). Four days after injection the X-MLO titer
averaged 0.271 t 0.01 C6c ng per leafhopper sample but,
0.654 : 0.235 eight days later, remaining almost constant
until the end of the study. A three fold MLO titer
increment was observed at 35 °C four days after the
injection. But a 10 day incubation period yielded a four
fold (0.588 : 0.226 C6c ng) increment in relation to the
amount inoculated. Incubation at 38 °C yielded a three-fold
increment in MLO titer four days following injection, and a
12 day incubation period did not result in increased x-MLO

titer (Table 5.5).

125

Table 5.3. Taylor’s parameters for the optical density (OD)
of X-disease inoculated P. irroratus incubated at constant
temperature.

 

 

Temperature
(°C) N= Rm Tm Ts Residual R2
30 65 163.4 10.0 4.26 256388.7 0.82
(20.2)a (0.9)
35 53 151.2 8.2 4.26 108849.8 0.86
(16.7) (0.9)
38 45 86.3 7.7 4.26 94442.5 0.71

(16.7) (1.9)

 

a) Confidence limit P(a-0.05).

Rm: Maximum X-MLO titer (ng in 200 pl of insect homogenate),
T = Optimum incubation period (days), and Ts= Spread of
t e X-MLO titer curve.

126

Table 5.4. Maximum X-disease titer in MLO inoculated P.
irroratus incubated at constant temperature.

 

 

Temperature C6c equivalents Conf. Limits P(a-0.05)

(°C) N= (ng)a lower upper
15 0 0.000

20 2 0.173 (0.012)b NS NS

25 5 0.198 (0.216) NS NS

30 64 0.710 0.622 0.797

35 53 0.657 0.584 0.728

38 45 0.375 0.302 0.447

 

a) Tylor’s R values transformed to C6c equivalents, and
titer estlmated in 200 pl of insect homogenate

b) Standar deviation of the mean.

NS= No estimated.

127

Table 5.5. Titer of X-disease in MLO injected P. irroratus
with different incubation periods at constant temperatures.

 

X-MLO titer (C6c equivalents)a

 

 

Temperature Initial Maximum Final

30 00
Mean= 0.271 0.654 0.651
SD= 0.101 0.235 0.211
IP= 4 12 14

35 °c
Mean= 0.534 0.588 0.537
SD= 0.262 0.226 0.189
IP= 4 10 12

38 °c
Mean= 0.395 0.410 0.410
SD= 0.204 0.120 0.120
IP= 4 12 12

 

a) Titer estimated in 200 pl of insect homogenate.

SD= Standar deviation, IP=Incubation period.

128

Temperature Dependent X-MLO Titer. Comparison of the
temperature effect on MLO development was done using the RI
parameter (observed maximum X-MLO titer) of the Taylor's
algorithm (Table 5.3). The first comparison was done using
the estimated mean OD corresponding to the R. values and
there after transforming the OD values to C6c equivalents
(Table 5.4). Results showed that the optimum X-MLO growth
occurred around 30 °C, and below or above this temperature
there was a decline in MLO titer (Figure 2). At 38 oC,
there was a one-fold decrease in MLO titer in relation to
the optimum observed at 30 0C. However, a six-fold
difference was observed between titers at 20 and 25 °C and
the optimum at 30 0C (Table 5.4). As mentioned before, no
detectable MLO multiplication or titer was observed at 15
0C, while data corresponding to MLO titer at 20 and 25 °C
was the average of only two and five individuals out of 65

and 68, respectively (Table 5.1).

Protein Development Over Time. As well as the X-MLO
titer, protein increment over time followed the Taylor’s
distribution (Table 5.6). Protein concentration in
leafhopper samples increased from a minimum, four days after
the beginning of the study, to a maximum that varied
depending on the incubation period and temperature regime
(Table 5.6). In all instances, protein concentration in the
longest incubation period was higher than that observed four

days after the beginning of the study, but was lower then

129

the maximum observed in the intermediate incubation periods.
Based on the R. parameter of the Taylor’s distribution,
incubation at 15 and 20 °C resulted in similar protein
concentration at similar incubation period (Table 5.7). The
highest protein concentrations, 7.327 t 0.587 and 7.238
i.525 ng/insect sample, were detected in insects incubated
at 25 and 30 °C, respectively. However, at 25 °C the
incubation period was 12 i 1.21 days, and 9 i 0.54 day at 30
°C. At 38 °C, protein was one-fold lower than the optimum
observed in the 25-30 °C range. Temperatures lower than 25
°C caused a reduction in protein concentration as well
(Table 5.7). Figure 5.3 shows the maximum protein
concentration observed in the range of the study

temperatures.

Discussion

MLO Developmental threshold temperatures. Few studies have
been conducted to determine the temperature growth range of
Mollicute parasites of plants and insects. Lack of progress
in this area is due to difficulties for in vitro culturing
most of the plant and insect pathogens belonging to this
Class. Some advances have been reported in relationto
Mycoplasmas, Acholeplasmas and Spiroplasmas (Whitecomb 8
Tully 1989). However, data for the temperature growth range

for Mycoplamsalike organisms is scarce.

130

 

 

 

 

1.0 I |
'5
O
5
'a 0.8 L —
.2
3
U
.
8 0.8 - _
9.
3
C
‘5 0.4 L- _
_l
a
. 1
2 02 _
5 ' '
‘P
><
0.0 I '
10 20 30 40

Incubation Temperature 00

Figure 5.2. Titer curve of Eastern X-disease MLO incubated
in P. irroratus at different constant temperatures. The MLO
titer is expressed as C6c equivalenst (ng).

131

Table 5.6. Protein accumulation in X-diseased P. irroratus
incubated at constant temperature.

 

Protein (ng)a

 

 

Temperature
(°C) Initial Maximum Final
15 Mean= 3.76 5.73 4.09
SD= 1.84 1.35 1.68
IP= 4 34 44
20 Mean= 2.59 5.92 5.77
SD= 1.70 1.54 1.21
IP= 4 22 28
25 Mean= 4.69 6.51 5.66
SD= 2.15 2.38 1.40
IP= 4 12 20
30 Mean= 3.02 6.54 5.92
SD= 1.27 1.85 1.18
IP= 4 12 14
35 Mean= 4.54 5.11 3.88
SD= 1.99 1.52 1.85
IP= 4 10 12
38 Mean= 2.36 3.36 2.42
SD= 1.10 2.36 1.61
IP= 4 8 12

 

a) Protein in 10 pl of insect homogenate.
SD= Standar deviation of the mean, IP= Incubation period.

132

Studies on the effect of temperature on plant
associated mycoplasmas showed that several strains so far
isolated and cultured have a 23-37 °C temperature range with
optimum growth at about 30 °C (Tully 1989). Acholeplasma
species have been recovered from plant and insects as well.
Acholeplasma spp. isolated from coconut palms affected by
the lethal yellowing disease had an optimum growth at
temperatures ranging from 25 to 29 °C but inhibition
occurred at 37 °C. However, some of the isolates had a 23-
37 °C temperature range (Eden-Green 8 Tully 1979, Tully
1989). Acholeplasma florum isolated from insect guts showed
a temperature optimum between 25 and 30 °C, very similar to
that observed in Acholeplasmas recovered from plants (Tully
1989).

The temperature growth range for some Spiroplasma (the
closest pathogens related to MLO’s) isolates from citrus,
corn, flowers, and honey bee has been investigated
(Whitecomb 8 Tully 1989, Daniels 8 Markham 1982). In vitro
grown Spiroplasma citri showed a temperature range that
varied from 9 to 39 °C. However, in the range 9-21 and 36-
39 °C very few colonies developed as compared to those in
the range 27-33 °C. Optimum growth occurred at 30 °C (Fudl-
ALLAH et al. 1973). Another Spiroplasma, the corn stunt
spiroplasma (CSS), grows in temperatures ranging from 15 to
35 °C with optimum multiplication in the 30-32 °C range

(Liao 8 Chen 1978).

133

 

 

 

 

 

10 I I
8
g 6
.5
3
o
E 4
2 - _
o I l
10 20 30 40

lncubatlon Temperature 06

Figure 5.3. Protein concentration (ng/10 pl) in homogenates
from X-diseased P. irroratus incubated at different
fluctuating temperatures.

134

The flower spiroplasma (strain 23-6) has a 22-37 °C
range, while the optimum is in the 34-35 °C range (Davis
1978). In our present research, the Eastern strain of X-
disease MLO only multiplied in the 20-38 °C range but,
optimum multiplication occurred between 30 and 35 °C. These
results are similar to those described by Fudl-allah et al.
(1973) for S. citri and the corn stunt spiroplasma
researched by Liao 8 Chen 1978. It is important to mention
that the maximum X-MLO titer and acquisition among insect
samples occurs at 30 0C. This temperature is within the 28-
30 °C range where optimum growth occurs in P. irroratus (see
Chapter I). This characteristic is not unique for the X-
MLO. Sterol-requiring Mycoplasma species found in plants
have a broader temperature range (25-37 0C) than other
sterol-requiring mollicutes found in vertebrate (35-37 °C)
which allows them to exist in non vertebrate hosts (Tully
1989). For example, in citrus, shoot growth is optimum in
the 25-31 °C range, this is the range in which S. citri
growth is maximal. Likewise, optimal development in
Circulifer tenellus (Baker), 8 S. citri vector, occurs in
‘the 31-35 °C range that also falls in the optimum
temperature growth range of S. citri (Calavan 8 Bove 1989,

Fudl-allah et al. 1973).

X-disease MLO Temperature Growth Range. Through
indicator plants and needle MLO inoculated leafhoppers,

attempts have been made to determine the effect of

Table 5.7.
diseased P. irroratus incubated constant temperature.

135

Taylor’s parameters for protein production in X-

 

 

Temperature

(°C) N Rm Tm TS Residual R2

15 68 6.20 25.67 16.80 223.94 0.87
(0.58) (2.76)a

20 61 6.05 26.14 16.80 122.93 0.91
(0.64) (3.05)

25 64 7.33 12.71 8.22 231.32 0.91
(0.59) (1.22)

30 67 7.24 9.85 4.26 172.84 0.92
(0.52) (0.54)

35 53 5.43 7.57 4.26 200.32 0.84
(0.65) (1.02)

38 49 3.45 7.88 4.26 144.75 0.74
(0.59) (1.42)

 

a) Confidence limit P(a-0.05)
Rm: Maximum protein content (ng in 10 pl of insect

homogenate), Tm= Optimum incubation period, and Ts=
Spread of the protein production curve.

136

temperature on X-disease MLO acquisition and indirectly, its
development threshold temperatures (Jensen 1972). MLO
aquisition is used as a synonimus for MLO multiplication
after the insect has been inoculated. Early research on
Western X-disease MLO (WX-MLO) showed that MLO injected C.
montanus failed in acquiring the WX-MLO at 10 0C. At 30 and
15 0C, WX-MLO acquisition was 4 and 24% respectively but, at
20 °C, acquisition was 66.5% (Jensen 1972). Jensen's data
suggested that the temperature growth range for the WX-MLO
was between 15 and 30 °C, with optimum growth at 20 °C.
Jensen reported MLO multiplication in the vector as a
function of the number of insects transmitting WX-MLO to
indicator plants after incubation at different temperatures.
A more sophisticated detection techniques such as
immunosorbent electron microscopy (ISEM) showed that at
constant 25 °C, 76% of a population of EX-MLO injected P.
irroratus acquired the EX-MLO but, 29.7% of the infected
population was unable to cause any symptoms in indicator
plants (Sinha 8 Chiykowski 1986). Our results are different
from Jensen's or Sinha 8 Chiykowski's. Several reasons may
explain this discrepancy. First, Jensen researched the
Western strain of X-disease and a different leafhopper
vector, C. montanus. Second, even though we used the same
strain of Eastern X-disease that Sinha 8 Chiykowski used for
their research, it is possibile that the strain of P.
irroratus utilized in the present research is less competent
to acquire the MLO than the utilized by Sinha 8 Chiykowski

137

(Purcell 1982). Furthermore, environmental factors during
the MLO incubation process may also contribute to

divergences in results.

Protein accumulation. The protein study was only a
control to standardize the amount of MLO in leafhoppers
sample. It was assummed that the X-MLO was diluted at
different concentrations in MLO extracted samples. Thus, to
perform a fair comparison X-MLO hybridization reaction among
samples had to be done over the same amount of protein per
sample. However, the protein concentration and its dynamics
at different temperatures and incubation periods were also
good indicators of the insect growth process. Data herein
discussed showed that optimum protein concentration occurred
in the 25-30 °C temperature range. This range has been
observed as optimum for P. irroratus growth and development

(see Chapter I).

This research has demonstrated that the use of DNA
probes and live insects as MLO culture media is a good
strategy to study the temperature growth range of
uncultivable mycoplasmalike organisms transmitted by
leafhopper vectors. Likewise, the progress of the MLO
infection in arthropod vectors can be followed up and
quantifyed rather precisely through DNA hybridization
techniques. MLO acquisition and titer can be estimated

through a titer curve using a DNA probe standard curve and a

138

densitometer or simply by counting the number of insect
samples showing hybridization with the appropriate DNA
probe. Regarding the Eastern X-disease MLO, its optimum
range of temperature lays between 30 and 35 oC. Beyond this
range EX-MLO growth and multiplication declines. In the 20-
25 oC range a long incubation period (more than 25 days)

results in MLO multiplication but the MLO titer is low.

139

Bibliography

Calavan, E.C. 8 J.M. Bove. 1989. Ecology of Spiroplasma
citri. In: The Mycoplasmas V. (eds). R.F. Whitecomb 8

J.G. Tully. Academic Press, Inc.

Chen, T.A., J.D. Lei, 8 C.P. Lin.1989. Detection and
identification of plant and insect Mollicutes. In: The
Mycoplasmas V, (eds) R.F. Whitecomb 8 J.G. Tully.

Academic Press, Inc.

Chiykowski, N.L. 1983. Frozen leafhoppers as a vehicle for
long-term storage of different isolates of the aster

yellow agent. Can. J. Plant Pathol. 5:101-106.

Chiykowski, L.N., 8 R.C. Sinha. 1988. Some factors affecting
the transmission of Eastern peach X-mycoplamsalike
organism by the leafhopper Paraphlepsius irroratus.

Can. J. Plant Pathol. 10(2):85-192.

Daniels, M.J. 8 P.G. Markham. 1982. Plant and insect
mycoplasma techniques. John Wiley 8 Sons. Nwe York-

Toronto.

140

Davis, M. J., J. H. Tsai, R. L. Cos, L. L. McDaniel 8 N. A.
Harrison 1988. Cloning of chromosomal and
extrachromosomal DNA of the mycoplasmalike organism
that causes maize bushy stunt disease. Mol. Plant-

Microbe Interac. 1:295-302.

Eden-Green, S.J. 8 J.G. Tully . 1979. Isolation of
Acholeplasma spp. from coconut plams affected by the
lethal yellowing disease in Jamaica. Current

Microbiology 2:311-316.

Kirkpatrick, B.C., D.C. Stenger, T.J. Morris, 8 A.H.
Purcell. 1987. Cloning and detection of DNA from
nonculturable plant pathogenic mycoplasma-like

organism. Science 238:197-199.

Lamb, R. J., G. H. Gerber 8 G. F. Atkison. 1984. Comparison
of developmental rate curves applied to egg hatching
data of Ehtomoscelis americana Brown (Coleoptera:

Chrysomelidae). Environ. Entomol. 13: 868-872.

Larsen,K.J. 8 M.E. Whalon. 1988. Field monitoring of X-
disease leafhopper vectors (Homoptera: Cicadellidae)
and infected chokecherry in Michigan peach and cherry

orchards. Great Lakes Entomol. 21(2):61-67.

141

Neter, J., 8 W. Wasserman. 1974. Applied linear statistical

models. Richard D. Irwing, Inc. Homewood, Ill.

Purcell, A.H. 1982. Evolution of the insect vector
relationship. Phytopathogenic Prokaryotes, Vol. 1:121-

156. Academic Press, Inc.

Sears, B. B., P. O. Lim, N. Holland, B. C. Kirkpatrick, 8 K.
L. Klompares. 1989. Isolation and characterization of
DNA from a mycoplasmalike organism. Mol. Plant-Microbe

Interac. 2:175-180.

Taboada, O., D.A. Rosenberger, 8 A.L. Jones. 1975.
Leafhopper fauna of X-diseased peach and cherry
orchards in southwest Michigan. J. Econ. Entomol.

68(2):255-257.

Tully, J.G. 1989. Class Mollicutes: New perspectives from
plant and arthropod studies. In: The mycoplasmas V,

(eds) R.F. Whitecomb 8 J.G. Tully. Academic Press, Inc.

Whitecomb, R.F., 8 R.E. Davis. 1970. Mycoplasma and
phytarboviruses as plant pathogens persistently

transmitted by insects. Ann. Rev. Entomol. 15:405-464.

Wilkison, L. 1988. SYSTAT: The system for statistics.

Evanson, Il: Systat, Inc.

142

GENERAL CONCLUSIONS

I. Development of Parapnlepsius irroratus (Say) at constant

and fluctuating temperatute.

From the foregoing research on the effect of constant
and fluctuating temperature on the development of P.
irroratus ,it is concluded that at constant temperature
immatures survived in the range 9 to 38 0C, but adulthood
was reached only at 20, 25, 27 and 30 °C. Fluctuating
temperatures accelerated development and allowed leafhoppers
to develop at lower mean temperature (15 0C). The lower
temperature threshold (Ti) for P. irroratus was 8 0C, while
the upper threshold (Tb) was 28 °C. The mean constant
temperature generation time required 939 i 31 SD degree-
days (DD base 8 °C), while the nymphal period required 602
:31 SD DD. Under fluctuating temperature the nymphal period
required 518 i 66 SD DD and was significantly (P S 0.01)
lesser than at constant temperature. In all tested
temperature regimes, the fifth instar required 29-32% of the

nymphal stage for completion.

143

II. Characteristics of the leafhopper vector-mycoplasmalike
organism relationship between X-disease MLO and the

speckled leafhopper.

The effect of the Eastern strain of X-disease
Mycoplasmalike organism (MLO) on the longevity of
Paraphlepsius irroratus (Say) was investigated at four
fluctuating temperatures averaging 15, 20, 25, and 30 °C.
Conclusions of this research were: At the two lower
temperatures, the X-disease MLO is pathogenic to P.
irroratus. At 15 and 20 oC, healthy leafhoppers lived 62 i
16 and 52 i 22 (Mean 1 SE) days respectively, while X-
diseased leafhoppers lived 35 t 21 and 23 i 9 days,
respectively. In the 25-30 0C temperatures, MLO
pathogenicity was masked by the temperature effect on
leafhopper survival. At 25 and 30 °C, longevity of healthy
leafhoppers was 15 i 9 and 7 i 3 days, while X-diseased
leafhoppers lived 14 i 4 and 7 t 4 days, respectively.
Below 20 °C, the MLO accounted for a 32% reduction in
leafhopper longevity, compared with less than 6% above 25
°C. The MLO acquisition ranged from 32.4 to 36.9% with no

difference (PS 0.05) between temperature regimes.

II.1. Effect of Eastern strain of X-disease on P. irroratus

fecundity.

X-diseased P. irroratus fecundity was studied at 25 °C

constant temperature. Our conclusion was that the Eastern

144

strain of X-disease MLO adversely affected fecundity of X-
disease injected P. irroratus. Healthy individuals produced
58.7 i 27.2 nymphs per cohort (four females per cohort)
while X-diseased females produced only 28.6 : 26.3. Thus, a
48.8% fecundity reduction with respect to healthy P.
irroratus females was an apparent result of X-disease

infection.

III. In vivo estimate of the effect of temperature on X-
disease MLO survival, optimum growth and

multiplication.

The temperature growth range of the Eastern strain of
X-disease mycoplasmalike organism (EX-MLO) was investigated
utilizing X-disease injected P. irroratus as a culture
media. This research allows to conclude that maximal titer
and MLO acquisition, 97.22 and 96.72 % respectively occurred
in the 30-35 °C temperature range. No X-MLO development was
observed at 15 °C. In the range 20-25 °C, only 3.07 and 7.35
% of a population of X-MLO inoculated.P. irroratus developed
the disease. X-disease acquisition and titer declined at 38

°C.

APPENDIX A

RECORD OF DEPOSITION OF VOUCHER SPECIMENS

145

APPENDIX A

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.: 1991-06
Title of thesis or dissertation (or other research projects):

BIOEOOLOGY OF Paraphlepsius irroratus (Say) (HOMOPTERA:
CICADELLIDAE): THE EFFECT OF THE X-DISEASE MYCOPLASMALIKE
ORGANISM ON PHYSIOLOGICAL DEVELOPMENT

 

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

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator's Name (a) (CYPed)
Carlos Garcia Salazar

 

 

Date 612611991

*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 A in ribbon copy of thesis or
dissertation.

Copies: Included as Appendix Arin copies of thesis or dissertation.
Mueeum(s) files.
Research project files.

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

 

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146
APPENDIX A. 1

voucher Specimen Data
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APPENDIX B

SURVIVAL CURVES, AUTORADIOGRAPHS

147

 

 

 

 

11)
(LB
8
,. 0.6
:> .
I
P.
>
5 0.4 y
d!
(12
I CONTROL
3 o INACJILO
Ono l—L I 1 L . XJLO

 

0 120
Age Slnce Adult Emergence (Days at 15 °C)

Figure 3.1. The effect of X-disease on longevity and
survival of P. irroratus at 15 °C, average temperature.
Control, healthy non-injected leafhoppers: Inac_MLO,
individuals injected with incativated X-disease: X_MLO, P.
irroratus injected with X-disease.

148

 

 

 

 

 

1 .0
0.8
8 P
,_ 0.6
> .
a
.2
>
5 0.4
m ..
0.2
I CONTROL
_ O INACJILO
0'0 0 XJILO
O 20 4O 60 80 1 00

Age Slnce Adult Emergence (Days at 20 °C)

Figure 3.2. The effect of X-disease on longevity and
survival of P. irroratus at 20 oC, average temperature.
Control, healthy non-injected leafhoppers: Inac_MLO,

individuals injected with incativated X-disease: X_MLO, P.
irroratus injected with X-disease.

149

 

 

 

 

 

1.0
0.8
,8. 0.6
\ .
a
3 .
>
- 0.4
3 .
0.2
m CONTROL
0 INACJILO
0.0 e XJILO
o 40

Age Slnce Adult Emergence (Days at 25 °C)

Figure 8.3. The effect of X-disease on longevity and
survival of P. irroratus at 25 °C, average temperature.
Control, healthy non-injected leafhoppers: Inac_MLO,

individuals injected with incativated X-disease: X_MLO, P.
irroratus injected with X-disease.

150

 

(L8

(L0

(L4

Survlval/ 1 OO

 

 

 

 

 

0.2 ~
4 m CONTROL
_ j o INACJALO
0.0 - , ,_ J 1 e x_NLO
0 10 20 30 40

Age Slnce Adult Emergence (Days at 30 °C)

Figure 8.4. The effect of X-disease on longevity and
survival of P. irroratus at 30 °C, average temperature.
Control, healthy non-injected leafhoppers; Inac_MLO,
individuals injected with incativated X-disease: X_MLO, P.
irroratus injected with X-disease.

 

151

 

 

 

 

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0.8
§ 0..
3
e
.2
>
5 0.4
GI
0.2 D THIRTY
I TFIVE
O TWENTY
0.0 * 0 FIFT-I

0 15 30 45 80 75
Days Slnce Adult Emergence (X-Dlseased)

Figure 8.5. The effect of temperature on longevity and
survival Of X-disease injected .P. irroratus. Thirty, 30
°C: Tfive, 25 °C: Twenty, 25 °C: Fifteen, 15 °C, average
temperature.

152

 

 

 

 

11)
(L8
8
,. 0.0
b.
s
.2
>
5 0.4
In
0.2 D THIRTY
m TFIVE
O TWENTY
0.0 w 0 FIFTEEN

0 20 A 40 60 80 1 00
Days Slnce Adult Emergence (Healthy Control)

Figure 8.6. The effect of temperature on longevity and
survival of healthy non-injected P. irroratus. Thirty, 30
°C: Tfive, 25 °C: Twenty, 25 °C: Fifteen, 15 °C, average
temperature.

153

TL
80 C Pl PH

‘ 0

   

 

 

Dilution
01 a 00 IO

TL

Figure 8.7. DNA hybridization reaction of radiolabelled C6c
with leafhoppers injected with inactivated X-disease MLO
after 48 h of exposure. Sc, Spiroplasma citri; C, the probe
C6c: PI, X-diseased P. irroratus: PH, healthy P. irroratus;

TL, inactivated MLO injected leafhopper.

p .A v.~ea..fllh

154

TL

Sc C PH PI

Dilution
a co IO

TL

 

Figure 8.8. DNA hybridization reaction of radiolabelled C6c
w1th leafhoppers injected with X-disease MLO after 48 h
exposure. Sc, Spiroplasma citri; C, the probe C6c; PH,
healthy P. irroratus; PI, X-diseased P. irroratus; TL, X-

disease injected leafhoppers.

APPENDIX C

AUTORADIOGRAPHS OF X-DISEASE MULTIPLICATION AND CONTROLS

155

Figure C.1. X-disease MLO multiplication in MLO injected P.
irroratus incubated at 15 0C.

156

Figure C.2. X-disease MLO multiplication in MLO injected P.
irroratus incubated at 30 0C.

157

Figure C.3. X-disease MLO multiplication in MLO injected P.
irroratus incubated at 35 0C.

158

Figure C.4. X-disease MLO multiplication in MLO injected P.
irroratus incubated at 38 0C.

159

A B

DilufiondZBngolinoculum DilutionollOngolCGc

l0

    

In N LO 8

O. “3. 9! ". O. '4'? 9! ".

v- O O O T- C O C
1.0
.01

.

.001 . . . '
0

.0001]

Figure C.5. Positive controls for X-disease MLO
hybridization. A, serial dilution of X-disease inoculum; 8,
serial dilution of the probe C6c.

APPENDIX D

SYSTEM DIAGRAM OF Paraphlepsius irroratus (Say)

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