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thesis entitled

FIELD DETECTION OF PEACH X—DISEASE MYCOPLASMALIKE
ORGANISM IN SPECKLED LEAFHOPPER, Paraphlepsius irro-ntus
(Say) (Homoptera: Cicadellidae) USING A DNA PROBE

presented by

Utami Rahardja

has been accepted towards fulfillment
of the requirements for

Lster— degree in mm

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M310! professor

Date—JulLZLflBSL—

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MSU Is An Affirmative Action/Equal Opportunity Inuitution

FIELD DETECTION OF PEACH X-DISEASE NYCOPLASHALIKE ORGANISM
IN SPECKLED LEAFHOPPER, Paraphlepsius irroratus (Say)
(Honoptera: cicadellidae) USING A DNA PROBE

BY

Utami Rahardja

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirenents
for the degree of

MASTER OF SCIENCE

Departnent of Entomology

1989

ABSTRACT

FIELD DETECTION OF PEACH X-DISEASE HYCOPLASNALIKE ORGANISM
IN SPECKLED LEAFHOPPER, Paraphlepsius irroratus (Say)
(Holoptera: Cicadellidae) USING A DNA PROBE

BY
Utami Rahardja

X-disease in stone fruit is caused by a mycoplasmalike
organism (MLO). C6c, a fragment of pWXl (an x-disease
specific probe derived from infected Colladonus montanus
(Van Duzee)), was used to hybridize with DNA from several
procaryotic and eucaryotic organisms that are associated
with leafhoppers are close relation to the X-disease agent.
C60 gave a positive reaction with only Eastern X-disease
infected leafhoppers.

x-diseased P. irroratus seasonallity was observed in
Fennville and East Lansing, MI during 1988. Infected adult
leafhopper were detected in the first emergence flight.
The percentage of diseased adult leafhoppers in the first
generation was higher than in the second generation.

The extensive occurrence of X-diseased leafhoppers was
also monitored at Fennville, East Lansing, Lawrence,
Ludington and Suttons Bay during 1988. X-diseased
P. irroratus were observed at Fennville, East Lansing and

Lawrence but not at Ludington and Suttons Bay.

DEDICATIONS

To my father and mother
who introduced me to the beautiful and interesting creature,

the insect

iii

The fear of the LORD
is the beginning of wisdom,
and knowledge of the Holy One

is understanding

Proverbs 9: 10

iv

ACKNOWLEDGMENTS

I would like to thank Dr. Mark E. Whalon, my major
advisor for his guidance, prayers, patience and support
throughout my master's program, and for giving me the
opportunity to learn about the basics of biotechnology.

I sincerely appreciate the suggestions, ideas and
comments from the members of my graduate committee,

Dr. James Asher of the Department of Zoology, Dr. Stuart
Gage and Dr. Edward Grafius of the Department of
Entomology.

I also benefited from the cooperation of the Michigan
stone fruit growers whose farm were used for this study.

I gratefully thank Yong Tang Yan for introducing me the
’dot blot’ technique and to Scott Douglas for his assistance
in the field.

Without the cooperation of my co-worker, Carlos Garcia-
Salazar, this research would not have been possible. His
support, courage and patience in spending many late nights
and early mornings working with 32F is gratefully
acknowledged.

Finally, the continual prayers, courage, interest and

understanding of my best friend is gratefully appreciated.

TABLE OF CONTENTS

LIST OF FIGURES. . . . . . . . . . .

LIST OF TABLES . . . . . . . . . . .

CHAPTER I : GENERAL LITERATURE REVIEW .

Introduction. . . . . . . . . .
Objectives. . . . . . . . . . .
Hypothesis. . . . . . . . . . .

Refernces cited . . . . . . . .

CHAPTER II : DEVELOPMENT AND EVALUATION

Introduction. . . . . . . . . .
Materials and Methods . . . . .
Probe source . . . . . . .
Leafhopper samples . . . .

Procaryotic and eucaryotic

OF A DNA
HYBRIDIZATION TECHNIQUE FOR DETECTION OF
X-DISEASE MYCOPLASMALIKE ORGANISM IN
Paraphlepsius irroratus (Say) IN MICHIGAN.

DNA samples

MLO extraction from leafhoppers.

Probe radiolabelling . . .

Detection technique: Dot Blot Hybridization.

Evaluation of C6c DNA probe. .

Characterization of C6c DNA probe with

Eastern X-disease MLO. . .

Results. . .. . . . . . . . . .

vi

0 O

Page

viii

ix

11

15
16
19
19
19
19
22
22
23

25

25

27

Evaluation of C6c DNA probe. . . . . . . . .
Estimation of the presence of X-disease MLO.

The detection limit of X-disease MLO in
P. irroratus . . . . . . . . . . . . . . . .

DiscuSSion O O O O O O O O O O O O O O O O O O O O
References sited. . . . . . . . . . . . . . . . .
CHAPTER III : SEASONAL DISTRIBUTION OF X-DISEASE

MYCOPLASMALIKE ORGANISM IN Paraphlepsius irroratus
(Say) FIELD SAMPLES USING

A DNA PROBE O O O O O O O O O O O O O O O O O O O
Introduction. . . . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . . . .

Leafhopper sample. . . . . . . . . . . . . .

Leafhopper assay for seasonal and crude
distribution study . . . . . . . . . . . . .

MLO extraction and blotting. . . . . . . . .

DNA sample hybridization . . . . . . . . . .

Results . . . . . . . . . . . . . . . . . . . . .

P. irroratus population trends . . . . . . .
x-diseased P. irroratus seasonallity . . . .
X-diseased P. irroratus distribution . . . .
Discussion. . . . . . . . . . . . . . . . . . . .
References sited. . . . . . . . . . . . . . . . .
GENERAL CONCLUSIONS. . . . . . . . . . . . . . . . . .

APPENDIX A. LEAFHOPPER VOUCHER SPECIMENS PLACED IN
THE MICHIGAN STATE UNIVERSITY ENTOMOLOGY MUSEUM .

APPENDIX B. FIELD MONITORING DATA. . . . . . . . . . .

APPENDIX C. X-DISEASE MYCOPLASMALIKE ORGANISM DETECTION
DATA O O O O O O O O O O O O O O O O O O O O O O O

vii

Table

II.1

II.2

III.1

III.2

List of Tables

Page

Experimentally inoculated orchard herbaceous
plants (A) and symptom for plants from orchard
tested positive for the x-disease pathogen (B) 8

MLO symptomatology Chart . . . . . . . . . . . 9
X-disease symptomatology Chart . . . . . . . . 10

Sources of various DNAs used for C60
evaluation . . . . . . . . . . . . . . . . . . 26

The number of X—disease MLO cells in
experimentally infected Paraphlepsius irroratus
(saY)O O O O O O O O O O O O O O O O O O O O O 33

Total number of adult Paraphlepsius irroratus
(Say) and diseased leafhoppers observed at
Fennville and East Lansing, Michigan 1988. . . 48

The seasonal occurrence of X-diseased
Paraphlepsius irroratus (Say) in several stone
fruit orchards in Southern and Northern

Michigan 1988. . . . . . . . . . . . . . . . . 52

Number of Paraphlepsius irroratus (Say) caught
at East Lansing and Fennville, Michigan
1987-1988O O O O O O O O O O O O O O O O O O O 64

Number of total
irroratus (Say)

and diseased of Paraphlepsius
at East Lansing 1988 . . . . . 68

Number of total
irroratus (Say)

and diseased of Paraphlepsius
at Fennville 1988. . . . . . . 70

Integrated optical densities of C6c and
diseased Paraphlepsius irroratus (Say) . . . . 71

Number of Paraphlepsius irroratus (Say)

caught by light traps at Lawrence, Ludington
and Suttons Bay 1988 . . . . . . . . . . . . . 73

viii

Figure

II.1.

II.2

II.3

II.4

III.1

III.2

III.3

List of Figures
Page

pWXl, a pUC8 plasmid with insertion of a DNA
fragment from Colladonus montanus (van Duzee).
Double complete digestion with Hind III and

EcoRI resulted in 6 fragments 1.9, 0.58, 0.54,
o.5,o.45 and 0.42kb. . . . . . . . . . . . . . 20

Autoradiogram of C60 with specific activity of

2.7 x 108 cpm/ul hybridization with several
procaryotic and eucaryotic DNA samples.

8.5: Bacillus subtilis: C.a: Candide albicans;
M.a: Mycoplasma alkalscens; M.c: Mycoplasma
californicum; P.a: Pseudomonas aeriginosa;

Sacc.: Saccharomyces cereviceae;

S.c: Spiroplasma citri; S.a: Staphylococcus

aureus . . . . . . . . . . . . . . . . . . . . 28

Detection limit of C6c with specific activity
of 5.1 x 108 cpm/ul of hybridization solution. 29

Autoradiogram of diseased Paraphlepsius
irroratus (Say) with specific activity of
5.1 x 108 cpm/ul of hybridization solution . . 31

Estimated lines of the integrated optical
density values of C6c and diseased
Paraphlepsius irroratus (Say). . . . . . . . . 32

Total number of light trap and yellow sticky
board trapped Paraphlepsius irroratus (Say)

per day at Fennville and East Lansing, Michigan
1987-1988. . . . . . . . . . . . . . . . . . . 47

Total number of diseased Paraphlepsius

irroratus (Say) caught and the number of
leafhoppers at Fennville and East Lansing,
Michigan 1988 . . . . . . . . . . . . . . . . 50

The relationship between diseased Paraphlepsius
irroratus (Say) and mean temperature at Fennville
and East Lansing, Michigan 1988.

(_____ diseased P. irroratus, ..... Mean
temperature) . . . . . . . . . . . . . . . . . 51

ix

CHAPTERI

GENERAL LITERATURE REVIEW

INTRODUCTION

X-disease, a serious disease of stone fruit caused by
mycoplasma like organism (MLO), has been causing extensive
tree loss in Michigan. Berrien County, the most intense
peach growing area in Michigan, had approximately 1.4%
infection of 238,602 trees inspected in 1987. This
infection rate was down from 1986 (2.10%) which was up over
the previous year (1.65%) (Dreves 1985, 1986, and 1987).

This disease was first reported and named X-disease in
1933 in Connecticut (Stoddard 1933). By 1938 it had become
a major peach disease in most of the northern United States
(Stoddard 1938, 1941). In Michigan, X-disease was first
reported in 1941 (Cation 1941, Stoddard 1947).

The causative organism of X-disease of stone fruit
(Prunus spp) in the Northeastern and Western areas of the
United States is believed to be a mycoplasma-like organism
(MLO). The causal agent was first thought to be a virus
until studies demonstrated that the agent had similar
ultrastructural characteristics to mycoplasma. The X-
disease agent was found in infectious leafhoppers and in the

phloem sieve tube elements of diseased trees, yet this MLO

has never been cultured (Nasu et a1. 1970, Granet and
Gilmer 1971, Jones et a1. 1974, Agrios 1978). Transmission
of MLOs occurs during feeding of infected leafhoppers on a
susceptible host. The leafhopper injects the MLO into the
plant phloem where it multiplies and spreads through the
host-plant vascular tissue (Agrios 1978, Rosenberger 1982).
Stoddard (1947) first hypothesized with support from McCoy
(1979) that the MLO overwinters in the root and is
translocated up the tree during spring time. Meanwhile,
Rosenberger and Jones (1977) concluded that the X-disease
pathogen overwinters both in buds and roots of diseased
plants even though the percentage of infected buds was low
and remained low until mid-June. During cold weather, the
MLO agent apparently remains in the aerial portion of the
tree and in the roots with high titer. Recent unpublished
work by Dr. Bruce Kirkpatrick at the University California
at Davis supports both views (personal communication).
Leafhoppers are the only known vectors of x-disease in
commercial orchards. From 70 leafhopper species collected
on yellow sticky board traps placed in and around several X-
diseased peach and sour cherry orchards in Southwest
Michigan, Paraphlepsius irroratus (Say) and Scaphytopius
acutus (Say) were the most abundant (Taboada et a1. 1977).
P. irroratus is considered the most important X-disease
vector in Michigan because of its abundance and high
transmission efficiency (Rosenberger and Jones 1977, Larsen

and Whalon 1987).

Nymphs of P. irroratus generally stay in the orchard
ground cover where they feed on herbaceous plants
(Rosenberger and Jones 1978). Many herbaceous and grass
host have now been identified as alternate host for X-
disease (Table I.1). Adults appear to feed on woody plants
during the evening after a crepuscular activity period
(Larsen and Whalon 1987). To be able to transmit the MLO
from plant to plant, the leafhoppers have to acquire the MLO
by feeding on infected plants. They begin to transmit the
pathogen after an incubation period of 10 to 45 days
depending on temperature. The shortest incubation occured
at about 30 °C and the longest at 10 oC (Agrios 1978). In
the vector, the incubation period involves the MLO’s
circulation through the hemolymph and infection of other
tissues including eventually the salivary glands before the
leafhopper is competent to transmit the MLO (Agrios 1978,
Chiykowski and Sinha 1988).

In the past, several researchers have independently
reported the identification of mycoplasmas from plants or
insects infected with X-disease (Granet and Gilmer 1971,
Nasu et a1. 1974, Chiykowski and Sinha 1980). A few
researchers, furthermore, have tried to diagnose the
occurrence of X-disease both in plants and its vectors
(Sinha and Chiykowski 1984, 1986, Kirkpatrick 1987).
Identification of X-disease in plants has been based on
symptomatology, immunology, host range, vector-pathogen

relationship and characteristic DNA.

5

Rosenberger and Jones (1978), Chiykowski and Sinha
(1980), Suslow and Purcell (1982), all used symptomatology
to identify the MLO in its vector and host. There are a
number of descriptive symptoms used for MLO diagnosis, e.g.
virescence, phyllody, stunt, proliferation (’witches’
broom), etc (Table 1.2 and 1.3). But symptomatology is an
unreliable and therefore often unsatisfactory means of
detecting the presence of MLO.

Immunosorbent electron microscopy (ISEM) techniques
have been introduced to detect and identify plant mycoplasma
(Derrick and Brlansky 1976). The recent advances in immuno-
gold labelling have demonstrated the specificity and
sensitivity of this technique in detecting the occurrence of
Spiroplasma citri (Mowry et a1. 1985). However, this
technique requires considerable effort, time and is too
expensive to be applied for routine field detection. It is
clear that a rapid, simple technique to detect the
occurrence of X-disease in vectors and plants is desirable.

More rapid and specific methods than electron
microscopy have been developed. Polyclonal and monoclonal
sera have been used against MLO antigens through enzyme
linked immunoassays (Sinha and Chiykowski 1984, 1986, Lin
and Chen 1985). Purified and partially purified
preparation from celery leaves infected with peach X-disease
did not give a specific reaction in ELISA with antiserum
against the MLO. Detection of MLO from a single infected

leafhopper by ELISA was unsuccessful even with high

6

concentration of antigen. Several limitations arose
associated with these methods. Polyclonal antibodies showed
cross reactions to other MLO preparations and apparently
share the same common antigen possessed by the aster yellow
(AY) MLO agent and thus lack specificity. Monoclonal
antibodies have been developed but are too specific and thus
less usefull in evaluating the general presence of X-
disease.

Recent advances in molecular biology may prove to be
useful in developing detection systems for MLO disease in
vectors and plants. The use of specific DNA probes has
proved highly effective for assaying mycoplasma. This
approach was developed to detect the occurrence of X-disease
MLO in both purified and partially purified vector and plant
samples (Kirkpatrick 1987).

The overall goal of this research was to evaluate
several DNA probes as diagnostic tool for detecting Michigan
X-disease MLOs in leafhopper vectors sampled throughout
Michigan from 1987-1988. A secondary goal was to attempt to
relate the results of this work with the epidemiology of X-
disease of stone fruit in Michigan. This research was done

based on the following objectives and hypotheses.

OBJECTIVES

1. To evaluate several DNA probes for detecting Michigan
X-disease.

2. To develop the necessary laboratory protocols for use of
the DNA probe(s).

3. To preliminary determine the seasonal and geographical
incidence of x-disease in field collected Paraphlepsius

irroratus (Say) samples .

HYPOTHESIS

H1. A DNA probe can be used as a rapid, specific detection
system of x-disease MLO in leafhopper vector samples.

H2. The seasonal incidence of x-disease in leafhoppers and
the geographical distribution of X-disease leafhopper
can be established from field frozen leafhopper

samples.

Table 1.1. Experimentally inoculated orchard herbaceus plants
(A) and symptom for plants from orchard tested positive
for X-disease pathogen (B).

 

Common name Scientific Name

 

A. Assay test : ELISA and indicator plant1

Blueweed Echium vulgare L.

Bladder campion Silene cucubalus Wibel

Spinach Spinacia oleracea L.
Matricaria chamomilla L.

Pineappleweed Matricaria matricariodes
(Less.) Porter

Dandelion Taracacum officinale Weber

Meadow goat's beard Tragopogan pratensis L.

Shepperd's purse Capsella bursa-pastoris (L.) Medic.

Stinkweed Thlaspi arvense L.

Pin-clover Erodium cicutarium (L.) L’Her

Barley cv. Vanier HOrdeum vulgare L.

Flat pea Lathyrus sylvestris L.
Lapinus leucophyllus Dougl.

Black medic Medicago lupulina L.

Yellow sweet clover Melilotus officinalis (L) Lam.

Alsike clover Trifolium hybridum L.

B. Assay test : ELISA and Dot hybridization2

Filaree

Plantago L Plantago lanceolata

Ansinkia

Burr Clover Trifolium sp

Convolvulus Convolvulus sp

Pigged3 Amaranthus sp

Mustard3 Brassica mustum

Plantago M3 Plantago sp

 

IEach test plant was inoculated using 8 Paraphlepsius irroratus
(Say) that had acquired X-disease MLO from infected celery.
MLO-free leafhoppers were caged for 14 days on plants showing
symptoms and were tested singly on celery seedlings (Chiykowski
and Sinha 1988). Assessment based upon classical transmission
study and immunological test.

2The orchard weeds were inoculated experimentally by means of the
leafhopper Colladonus montanus (Van Duzee). Leaf and root
tissue were used for assay (Kirkpatrick personal communication).
Assessment based on immunological test and cdna hybridization
technique.

3Only roots tested positive

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REFERENCES CITED

Agrios, G.N. 1978. Plant pathology. 2nd edition. 703 p.
Academic Press, New York.

Cation, D. 1941. "X" disease of peach and chocke cherry
found in Michigan. Plant Dis. Rep. 25: 406-407.

Chiykowski, L.N. and Sinha, R.C., 1988. Some factors
affecting the transmission of eastern peach x-
mycolasmalike organism by the leafhopper Paraphlepsius
irroratus. Can. J. of Plant Pathol. 10:85-92.

Derrick, K.S. and Brlansky, R.H. 1976. Assay for viruses
and mycoplasma using serologically specific electron
microscopy. Phytopathology 66:815-820.

Dreves, J.C. 1985. Peach virus survey. Michigan Department
of Agr. Comm.

, 1986. Peach virus survey. Michigan Department of
Agr. Comm.

, 1987. Peach virus survey. Michigan Department of
Agr. Comm.

Granett, A.L. and Glimer, R.M., 1971. Mycoplasma
associated with x-disease in various prunus species.

Phytopathology 61:1036-1037.

Jones, A.L., Hooper, G.R. and Rosenberger, D.A., 1974.
Association of mycoplasmalike bodies with little
peach and X-disease. Phytopathology 64:755-756.

11

12

Kirkpatrick, B.C., Stenger, D.C., Morris, T.J. and Purcel,
A.H., 1987. Cloning and detection of DNA from a
nonculturable plant pathogenic mycoplasmalike
organism. Science 238:197-200.

Larsen, J.K. and Whalon, M.E., 1987. Crepuscular movement
of Paraphlepsius irroratus (Say) (Homoptera:
Cicadelidae), between the gorundcover and cherry
trees. Environ. Entomol. 16: 1103-1106.

Lin, C.P. and Chen, T.A., 1985. Production of monoclonal
antibodies against Spiroplasma citri.
Phytopathology 75:848-851.

Markham, P.G. and Townsend, R., 1979. Experimental vectors
of Spiroplasma, pp 413-446. In Leafhopper vectors and
plant disease agents,pp.413-446, K. Maramorosch and
K.F. Harris. Academic Press Inc. (London) Ltd.

Markham, P.G. 1982. The yellows plant disease: plant hosts
and their interaction with the pathogens. In Plant
and Insect Mycoplasma Techniques, pp. 82-100, M.J.
Daniels and Markham. John Wiley & Sons. New York -
Toronto.

Mowry, T., Baker, K. and Whalon, M., 1985. Immunogold
labelling of Spiroplasma citri: Effects of fixation
on surface and overall antigenicity. Micron. and
Micros. Acta 16:211-212.

McCoy, R.E., 1979. Mycoplasma and yellows diseases. In The
Mycoplasma, Vol. III, pp. 229-59, R.F. Whitcomb and
J.G. Tully (eds). New York: Academic Press.

McGrew, J.R. and Posnette, A.F., 1970. Virus diseases of
small fruits and grapevines (A handbook). Univ. of
California. Division of Agriculture Science.
Berkeley, California.

Nasu, S., Jensen, D.D. and Richardson, J., 1970. Electron
microscopy of mycoplasma-like bodies associated with
insect and plant hosts of peach western X-disease.
Virology 41, 583-595.

13

, 1974. Extraction of western X mycoplasmalike
organism from leafhoppers and celery infected with
peach western X-disease. Appl.Ent. 2001. 9 (2):53-57.

Rosenberger, D.A and Jones, A.L, 1977. Spread of X-disease
in Michigan peach orchard. Plant Dis. Reprt. 61:830-
834.

1978. Leafhopper vectors of the peach X-disease
pathogen and its seasonal transmission from
chockecherry. Phytopathology 68:782-790.

————I

Rosenberger. D, 1982. Leafhopper vectors, epidemiology, and
control of peach X-disease. PhD desertation.
Michigan State University, East LAnsing, MI.

Sinha, R.C., and Chiykowski, L.N., 1980. Transmission and
morphological features of mycoplasma-like bodies
associated with peach x-disease. Can. J. Plant
Pathol. 6:111-124.

Sinha, R.C., 1988. Purification and properties of
mycoplasma-like organisms from diseased plants. In
Mycoplasma Diseases of CrOps. Basic and Applied
Aspect, pp. 29-50, Maramorosch, K. and Raychaudhuri,
S.P. Spinger-VerlagNew York Inc.

, 1984. Purification and serological detection of
mycoplasma like organism from plants affected by peach
eastern x-disease. Can. J. Plant Pathol. 6:200-205.

 

1986. Detection of mycoplasmalike organism in
leafhopper vectors of aster yellow and peach x-disease
by immunosorbent electron microscopy. Can. J. Plant
Pathol. 8:387-393.

—,

Stoddard, E.M. 1933. Progress report of investigations on a
new peach trouble. Conn. Pomol. Soc.Proc. 43:115-117.

1938. The X-disease of peach. Connecticut
Agriculture Experiment Station. Conn. Agric. Sta.
Circ. 122:53-60.

———-——I

14

, 1947. The X-disease peach and its chemotherapy.
Conn. Agric. Exp. Sta. Bull. 506.19 p.

 

Suslow, K.G. and Purcel, A.H., 1982. Seasonal transmission
of X-disease agent from cherry by leafhopper
Colladonus montanus. Plant Disease 66:28-30.

Taboada, 0., Rosenberger, D.A. and Jones, A.L., 1975.
Leafhopper fauna of x-diseased peach and cherry
orchards in Southwest Michigan. J. of Econ. Entomol.
68:255-257

CHAPTER II

Development and evaluation of a DNA hybridization technique

for detection of x-disease mycoplasmalike organism in

Paraphlepsius irroratus (Say) in Michigan

15

INTRODUCTION

x-disease has been a major problem for Michigan peach
growers. Ten Michigan peach orchards were surveyed annually
from 1973 to 1976. The percentage of X-diseased trees
observed ranged from 2 to 75% (Rosenberger and Jones 1977).
Observations conducted by the Michigan Department of
Agriculture (MDA) in Bainbridge Township showed that the
percentage of x-diseased peach trees fluctuated annually but
generally increased during a 6 year period from 1980 to 1986
(1.09 to 1.80%) and decreased the following year (1987) to
1.25%. This decrease was presumably due to removal of the
infected trees but environmental and vector population
density may also have contributed. In three years (1985-
1987), an average of 15.48% of inspected trees has been
removed annually (Dreves 1985, 1986 and 1987).

The causal agent of X-disease was first assumed to be a
virus until researchers demonstrated that the agent had
similar ultrastructural characteristics to mycoplasma and
named X-disease mycoplasmalike organism (MLO) because it has
yet to be classified (Nasu et a1. 1970, Granet and Gilmer
1971, Jones et al. 1974). MLO was observed in infectious
leafhoppers and in the phloem sieve tube elements of

diseased trees (Agrios 1978), yet this MLO has never been

16

17

cultured (Sinha 1988). Since MLOs are uncultivable, they
can not be detected or classified by conventional media
preparations as with bacteria: therefore, there is a need
for developing a detection system for X-disease MLO.
Traditionally, x-disease MLO diagnosis has been developed
based on host symptomatology, and plant host-vector-MLO
relationship but these approach are time consuming and
expensive. Three major techniques have been developed to
diagnose MLO in plants and insects: 1) immunosorbent assay
(ELISA) with polyclonal or monoclonal antibody,

2) immunosorbent electron microscopy (ISEM), and 3) nucleic
acid hybridization.

Experimentally, ELISA has been used for detecting X-
disease MLO (Chiykowski and Sinha 1984, 1986). But it
failed to give detectable specific reactions with
experimentally infected celery. The progress in the
production of monoclonal antibodies against x-disease is
underway (Chiykowski, personal communication) although
monoclonals produced to aster yellow were not usefull (Lin
and Chen 1986) because the antibodies were too epitopes
conferring only subspecies specificity.

Nucleic acid hybridization techniques have been widely
used for various purposes. With this technique, single
stranded DNA is fixed on a membrane substrate (filter) and
is hybridized with 32P labelled single stranded DNA probes.
Complementary base pairing of the homologous strands results

in a probe-sample ligation. This hybrid molecule remains on

18

the membrane while the unpaired labelled DNA is washed away
during the washing step. The hybrid molecule or positive
samples can be visualized using autoradiography
(Strickberger, 1985).

A number of DNA probes have been developed for the
detection of procaryotic organisms (Amikam 1985,
Kirkpatrick 1987). However, none of these probes have been
used in extensive field studies, primarily because of the
long, complicated DNA extraction procedures. For routine
diagnostic use, a probe should be specific with known
hybridization potential against other procaryotes,
contaminants and possible strains of the target pathogen.
The most promising X-disease probes (Kirkpatrick 1987) were
not evaluated with Michigan x-disease or with crude
preparations from field sampled leafhopper vectors. Nor
were these probes evaluated with symbiotic or contaminating
procaryotic DNA associated with Michigan vectors.
Exploration of possible false positive reaction are critical
since leafhopper vector might carry several symbiotic
procaryotic and simple eucaryotic organisms (e.g. yeast).
In this study, C6c DNA probe derived from the pWXl X-disease
probe (Kirkpatrick 1987) was evaluated against several
procaryotic and simple eucaryotic organisms using nucleic

acid hybridization techniques.

19

NATREIALS AND METHODS

Probe source

C6c DNA probe is a 1.9 kb EcoRI-Hind III fragment of
pWXl (Figure I.1). pWXl, provided by Dr. Bruce Kirkpatrick
(Davis, CA), is a Western X-disease-specific plasmids
consisting of a 4.4 kb DNA fragment from HindIII and EcoRI
partially digested DNA of X-diseased Colladonus montanus
(Van Duzee) (Kirkpatrick 1976, 1987). pWXl was cloned in

pUC8 using Escherichia coli strain JM 83.

Leafhopper samples

Healthy P. irroratus were maintained in a caged plastic
pots (10 cm diameter, 17.5 cm height). Four males and four
females were put on three week old clover. To provide food
for nymphs that feed on barley, barley seeds were planted in
the same pots and the same time when putting the adults.
The second or third leafhopper instar were transferred onto
seven days old barley planted in caged plastic pots and
maintain them until they become adults. The diseased
leafhoppers were provided by Dr. L.N. Chiykowski (Chiykowski

and Sinha 1984).

Procaryotic and eucaryotic DNA samples
C6c DNA probe was evaluated for hybridization against
various procaryotic and eucaryotic DNA that might be

extracted from leafhoppers in routine sampling. These DNA

20

lfindfll EcoRI

l 1

H80" \\\\\\\\\\ Hag"
Lac 2 rig; I ” 3‘ " Q‘ ”” ‘;‘; III 0 Amp
,,,,,,,,,,,,,
.............
”All“

 

 

 

Complete
digestion

l

 

Figure 1.1. pWXl, a pUC8 plasmid with insertion of a DNA
fragment from Colladonus montanus (van Duzee). Double
complete digestion with HindIII and EcoRI resulted 6
fragments 1.9, 0.58, 0.54, 0.5, 0.45 and 0.42 kb.

21

sources are various MLOs, bacteria, yeast and healthy and
infected leafhoppers (Table II.1).

Mycoplasma alkalescens and MYcoplasma californicum were
cultured in broth medium described by ATCC (American Type
Culture Collection, Rockville, MD, 1982) that contained
Pleuropneumonialike organism (PPLO) broth (Difco) with 20%
horse serum and 10% (v/v) of 25% (w/v) fresh baker yeast
extract (Difco). Although these were not mycoplasma that
were likely to be in insect guts, they represented easily
obtained, and cultivable organisms which are closely related
to X-disease MLO.

Frozen bacteria cell cultures obtained from ATCC were
grown in a rich broth media containing 10 g tryptone, 5 g
yeast extract and 10 g NaCl. Media LD8 containing a-
Ketoglutaric acid and pyruvic acid as described by Lee and
Davis (1984) was used to culture Spiroplasma citri.

A standard colony amplification protocol was used for
obtaining enough cells for DNA extraction. Briefly, freeze
dried cells obtained from ATCC were streaked onto the
appropriate agar medium plates. Single colonies were then
selected, transferred to 5 ml liquid media and grown up in 1
liter quantities before DNA extraction.

DNA extraction was carried out following Carle et al.
(1983), Maniatis (1982), Sherman et al. (1970) for S. citri
and mycoplasma, bacteria and yeast respectively. The basic
procedure was to separate cells using differential

centrifugation, lyse cells, separate the DNA through

22

phenol/isoamylalcohol extraction procedure and precipitate

the DNA.

MLO extraction from leafhoppers

MLO was extracted from leafhoppers according to
Kirkpatrick (1987) with some modifications. Twenty healthy
or diseased leafhoppers were ground in 8 ml of MLO enriched
buffer (0.1 M NaZHPO4, 10% sucrose, 50 mM ascorbic acid, and
1% polyvinylpyrollidone pH 7.6), centrifuged at 3.000xg in a
Sorvall SS-34 rotor at 4 0C for 20 min and the supernatant
was harvested and centrifuged once more at 12.000xg for 30
min at 4 °C. The resulting pellet was resuspended in TE
buffer (10mM Tris-HCl, 1mM EDTA pH 8.0) and stored at -70 °c

until later use.

Probe radiolabelling

Nick translation of C6c DNA probe with 32F was carried
out using kit from Amersham (Arlington Heights, IL).
Unincorporated nucleotides were separated by passing the
reaction mixture through a 1 m1 Sephadex G-75 fine column
which was previously saturated with TE 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 only 32F
incorporated probe. The specific activity of each nick
translation was assayed by removing two 1.0 ul samples
before and two 1.0 ul samples after 32F incorporation.

These samples were blotted onto a nylon membrane and air

23

dried. One from each sample were washed according to the
first step of washing protocol. The specific activity of
the samples were measured using a scintillation counter

(Shelby 1987).

Detection technique: Dot Blot Hybridization

The samples were blotted onto the nylon membrane,
hybridized and autoradiographed according to the instruction
provided by the company (Method III, GeneScreenTM, New
England Nuclear, Dupont, Boston, MA). Before being blotted
the nylon membrane was soaked in 2x SSC buffer (diluted
appropriately from a stock of 20X SSC: 3 M NaCl, 0.3 M
sodium citrate pH 7.0) and air dried. MLO DNA was alkaline
denatured with 0.5 M NaOH for 10 min and chilled on ice for
another 10 min. After denaturation, the solution was
diluted with 10x SSC to a final volume of 400 ul. This
dilution was blotted onto the membrane fixed into the dot-
blot apparatus (BioRad, Richmond, CA) and then washed
carefully with 2x SSC for 2 min. The nucleic acid was
fixed onto the membrane by laying the wet membrane face up
on a clean glass plate and irradiated for 5 min with a 254
nm shortwave UV light sources (UVG-ll, Ultraviolet Products,
Inc., San Gabriel, CA) at a distance of 15 cm.

The wet membrane was then prehybridized in a plastic
bag with 10 ml of prehybridization solution (50% deionized
formamide, 0.2% polyvinyl-pyrrolidone, 0.2% bovine serum

albumin, 0.2% ficoll, 0.05 M Tris-HCl (ph 7.5), 1.0 M NaCl,

24

0.1% sodium pyrophosphate, 1.0% sodium dedoxyl sulfate, 10%
dextran sulfate and denatured salmon sperm DNA (2100 ug/ml).
The plastic bag was sealed off and incubated for 6 hr at

42 °C.

Hybridization was accomplished by cutting one corner of
the plastic bag and adding 2.5 m1 of hybridization solution
(prepared as prehybridization solution but without 1 M NaCl)
containing the radioactive labelled probe. Prior to use,
the probe and salmon sperm DNA were denatured by being
boiled for 10 min followed by immediately chilling on ice
for 5 min. The plastic bag was resealed and incubate for 16
hr at 42 °C. Before autoradiography, the membrane was
washed twice for 5 min each with 100 ml of a washing
solution (0.3 M sodium chloride, 0.06 M Tris-HCl pH 8.0 and
0.002 M EDTA) at room temperature with gentle rocking. A
second wash procedure was done twice for 30 min each at
60 0C using 100 ml of the above washing solution containing
1% SDS. The final wash was performed by washing the
membrane twice for 30 min at room temperature with 100 m1
ten fold diluted washing solution.

Autoradiography was carried out by exposing X-ray film
(Kodak XAR) for 24-48 hr with an intensifying screen (Sigma)
at -70 °C. x-ray film was developed in a dark room. The
Integrated Optical Density (IOD) of the hybrid dots were
quantified using 2-D/1-D soft laser scanning densitometer

(Biomed Instruments, Inc., Fullerton, Ca).

25

Evaluation of C6c DNA probe

To evaluate C6c DNA probe against several nucleic
acids, a 200 ng probe per 10 m1 hybridization solution to
hybridize the various samples (see Table II.1), and the
reaction membrane was exposed for 24 hr. A serial dilution
of C6c was blotted and hybridized with C6c itself to
determine the detection limit of the probe. These
experiments were performed twice with hybridization solution

with specific activity at the range of of 2x108 and 2x109

cpm/ug.

Characterization of C6c DNA probe with Eastern x-disease MLO
This experiment attempted to estimate the number of x-
disease MLO cells in leafhopper vector. Three membranes
were prepared to establish a standard curve for estimating
the amount of x-disease MLO DNA in leafhopper vector by
blotting a dilution series of healthy and diseased
P. irroratus extraction. The serial dilution was as follow:
1, 1/2, 1/4, 1/8 and 1/16 leafhopper extract containing
DNA. Each of these dilutions was further diluted with 7,
ten fold dilutions (see Figure II.2A). A serial dilution of
C6c DNA was blotted on the same membrane as a positive
control. The serial dilution was as follow: 1.0, 0.5, 0.25,
and 0.125 ngs. Each of these dilutions were further diluted
with 7, ten fold dilutions (see Figure II.2A). The number
C6c DNA copies in each dilutions blotted on the membrane

were estimated based on the molecular weight of single copy.

26

One ng of purified C6c DNA was estimated contain 480,390,461

copies of DNA (Stryer, 1988).

The use of C6c DNA to

estimate the number of X-disease MLO cells is based on the

following assumptions: 1) C6c DNA from Western X-disease is

homologous to the MLO DNA causing Eastern X-disease carry by

P. irroratus, 2) assuming the MLO cells are diploid, copy of

specific dsDNA represents a single cell.

Table II.1.

Sources of various DNA used for C60 evaluation.

 

Species

Source/Origin/Maintenance

 

MYcoplasma californicum
MYcoplasma alkalescens
Spiroplasma citri

Paraphlepsius irroratus (Say)

Frozen infected P. irroratus

Bacillus subtilis
Pseudomonas aeriginosa
Staphylococcus aureus
Saccharomyces cereviceae
Candide albicans

ATCC; 33461

Ach* 29103

(HRSC)C.E. Eastman,
Illinois.

Natural History Survey,
University Illinois,
Champaign, Il.(1982).
Maintained in greenhouse
cultures at 20—8 oC and
16L:8D.

L.N. Chiykowski,
Agriculture, Ottawa,
Ontario, Canada (1984).

Ach* 6051-U
ATCC* 10145-U
ATCC* 12600-U
ATCC* 2601-U
ATCC* 14053-U

 

*ATCC: American Type Culture Collection, Rockville, Md, 1982

27

RESULTS

Evaluation of C6c DNA Probe

No hybridization reactions were observed between C6c
DNA and DNA from Spiroplasma citri, MYcoplasma californicum,
Mycoplasma alkalescens, Bacillus subtilis, Pseudomonas
aeriginosa, Staphylococcus aureus, Saccharomyces cereviceae,
Candide albicans and healthy Paraphlepsius irroratus (Say)
(Figure II.1). C6c DNA probe hybridized to DNA from
infected leafhoppers which carried Eastern X-disease (Figure
II.2).

The probe was able to detect up to 0.0005 pg dilution

of C6c or equal to 240 copies of C6c (Figure 11.3).

Estimation of the presence of x-disease MLO

The Integrated Optical Density (IOD) values resulted
from dots initially increased proportionally with the
corresponding dilutions of the C6c DNA or P. irroratus.
After reaching a certain dilution, the IOD values did not
consistently proportionally increase with the corresponding
dilutions. These values were from dots which were not
perfectly blotted on the membrane possibly due to the
prevention of proper DNA denaturation and fixation to the
filter by protein deposited in crude homogenate.

To develop the equation to estimate the number of X-

disease MLO in single P. irroratus, the reliable IOD (which

28

.c
.1
BS
1’:
.S:
Shr
CI:

8
o

S."

10-
10'

 

Dilutions

10-

10

Figure II.1. Autoradiogram of C6c with specific activity of
2.7 x 108 cpm/ul hybridization with several procaryotic
and eucaryotic DNA samples. B.s: Bacillus subtilis:

C.a: Candide albicans: M.a: Mycoplasma alkalscens:

M.c: Mycoplasma californicum; P.a: Pseudomonas aeriginosa:
Sacc.: Saccharomyces cereviceae; S.c: Spiroplasma citri:
S.a: Staphylococcus aureus.

29

C6c(pyM00£fl)

 

' 1
l ‘4
!

 

dflnfions

Figure II.2. Detection limit of C6c with specific activity
of 5.1 x 108 cpm/ul of hybridization solution.

30

decreased with the dilutions) from autoradiographs of C6c
DNA and P. irroratus samples hybridized against C6c DNA
probe were selected and plotted against the number of C60
and the leafhoppers homogenate dilutions respectivelly
(Figure 11.4).

The resulting equations which best fit the actual IOD
of C6c DNA from three different dot blots using probes with

three different specific activities were °

y'== 0.003 x at 5x107 cpm/ug, 24 h, r=.99, p<o.001,

y'-— 0.0016 x at 2.6x108 cpm/ug, 24 h, r=.91, P=0.004,

and

Y — 0.0003 x at 5x108 cpm/ug, 24 h, r=.92,P=0.003,
where ’y' was the OD at 'x’ dilutions of C60 DNA. Based on
these equations, the IOD of one C6c DNA copy were: 0.003,
0.0016, and 0.0003 (Table II.2). These values were used to
estimate number of MLO cells in experimentally infected
leaf hoppers .

The resulting equations which best fit the actual IOD
of P. irroratus from three different dot blots using probes

With three different specific activities were °

Y

2,152,030 x or 2x106 x,

at 5x107 cpm/ug, r=.99, P<.001,

Y = 9,487,810 x or 9x106 x

at 2.6x108 cpm/ug, r=.99, p<.001, and

Y = 19,893,400 x or 2x107 x

at 5x108 cpm/ug, r=.99, p<.001,

31

diseased)". i
dilutions/400 an 066 (Ila/400 11)

V“ “HM" #7
(“<3
_. ., a 3. a: '0 8.3

pt

1 “00.00000
W 000000- 0

10‘ .. .

 

 

.5! citri
Healthyl’. i

dilutions
5

10‘5

10

10'7

Figure 11.3. Autoradiogram of diseased Paraphlepsius
irroratus (Say) with specific activity of 5 x 10
cpm/ul of hybridization solution.

32

 

 

 

250

200

§_ .5.
F
5.

‘3 100
O

50

0

0.00 0.01 0.02 0.03 0.04 0.05
Dilutions of leafhopper
225 -

180
8

O. 135
..
.5
o

O 90

45

 

 

 

Cop!“ of 05:: (x 1,000,000)

Figure II.4. Estimated lines of the integrated optical
density values of diseased Paraphlepsius irroratus
(Say) and C6c.

33

where ’y’ was the IOD at ’x' dilutions of diseased

leafhopper. The resulting equations were used to estimate

the number of X-disease MLO cells in one leafhopper. The

estimated number of X-disease MLO cells was: 7x109; 6x109

and 6.6x109 (Mean : S.E = 6.6x1o9 + 2x108) (Table II.2).

The detection limit of x-disease MLO in P. irroratus

With the given conditions, the smallest dilution that

can be used to detect MLO DNA was between one to the 400th

to 166,666th dilutions of one diseased leafhopper or

approximately equal to 1 among 400 to 166,666 leafhoppers

(see Appendix 1).

Table II.2. The number of X-disease MLO cells on

experimentally infected Paraphlepsius irroratus (Say).

 

 

 

 

Experiment IOD of IOD of Number 8f MLO
No.a C6c one P.ic per P.i
1 0.0003 2,152,030 7,173,433,335
2 0.0016 9,487,810 5,929,881,250
3 0.0030 19,893,400 6,631,133,334
lean 6,578,149,306
a The specific activity were 5x107, 2.6x1087and 5x108 cpm/ug
respectively.
b IOD of one C6c DNA copy
3 IOD of one P. irroratus

The number of X-disease MLO cells in P. irroratus: (c/b)/2

34

DISCUSSION

The nucleic acid hybridization techniques make use of
the fact that a hybrid molecule can be made between the two
hydrogen bonded strands of homologous nucleic acids. A
previous report describing the use of nucleic acid
hybridization for the detection of mycoplasma (Taylor 1984)
suggested that this method would be more specific than any
other means. The high sensitivity, speed and simplicity of
nucleic acid hybridization techniques make these approaches
very useful not only for basic research but also for
clinical diagnosis. This conclusion is confirmed by the
observation that C6c DNA probe detects only x-disease MLO
and not the other procaryotic and eucaryotic flora and fauna
that might be internally or externally present in
leafhopper samples.

Since the C6c DNA probe derived from Western X-disease
MLO infected C. montanus strongly hybridized under high
stringency with DNA from Eastern x-disease MLO infected
P. irroratus, this indicates that both strains of X-disease
MLO have high degree of DNA homology. Further studies need
to be done, including sequence analysis of the C6c fragment
from Eastern and Western X-disease MLO, to determine the
degree of similarity of these organisms.

Laboratory detection with C6c DNA probe showed high
sensitivity in detecting the occurrence of MLO in

P. irroratus. However, the fixation of MLO nucleic acid in

35

a crude extract of leafhoppers in a single dot blot well by
UV cross-link was partially blocked by protein (Figure
I.2.A). The observation suggests that a serial dilution of
leafhopper samples is needed to determine the optimum
hybridization result. The quantification of hybridization
results indicates that the C60 DNA probe under the reported
conditions can detect X-disease MLO in dilutions of total
DNA extracted from one diseased leafhopper. Thus only a
portion of the DNA extracted from a leafhopper needs to be
utilized for evaluation. This approach allows for multiple
testing on a single leafhopper's DNA with many different
probes.

The detection limit of C60, with the given conditions,
was 240 copies of C6c. Laboratory detection limit of this
probe in detecting X-disease MLO in P. irroratus was
approximately 1 among 400 to 166,666 leafhoppers.

The sensitivity of the detection of X-disease MLO using
C6c DNA probe may be due to the specificity of the C6c DNA
probe fragment. The specificity allows the probe to

hybridized a unique site of the genomic DNA samples.

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Amikam, D., Razin,S., Glaser,G., 1982. Ribosomal RNA genes
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Archer, D.B, Townsend,R. and Markham, P.G., 1982. Detection
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Carle, P., Saillard,C., and Bove,J.M., 1983. DNA
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D.D. Jensen and Richardson, J., 1970. Electron microscopy
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Dreves, J.C. 1985. Peach virus survey. Michigan Department
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Granett, A.L. and Glimer, R.M., 1971. Mycoplasma associated
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37

Jones, A.L., Hooper,G.R. and Rosenberger, D.A., 1974.
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Lee, I.M. and Davis, R.E., 1984. New media for rapid growth
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Lin, C.P. and Chen, T.A., 1986. Comparison of monoclonal
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Markham, P.G., 1982. The yellows plant disease: plant hosts
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, 1988. Detection of mycoplasma and Spiroplasma in
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, 1984. Purification and serological detection of
mycoplasma like organism from plants affected by peach
eastern X-disease. Can. J. Plant Pathol. 6:200-205.

 

38

, 1986. Detection of mycoplasmalike organism in
leafhopper vectors of aster yellow and peach X-disease
Can. J. Plant

by immunosorbent electron microscopy.

Pathol. 8:387-393.
Strickberger, M.W., 1985. Genetics, pp 85-86, 338. Third
edition. Macmillan Publishing Company. New York.

1984. Isolation of plant DNA

Taylor, B. and Powell, A.,

and RNA. Focus 4:3.
In Plant

Waters, H., 1982. Light and electron microscopy.
and insect mycoplasma techniques, pp 101-125, Daniels,
M.J. and P.G. Markham. John Wiley and Sons, New York-

Toronto.

CHAPTER III

Seasonal distribution of x-diseased

Paraphlepsius irroratus (Say) field samples using DNA probe

39

INTRODUCTION

Peach X-disease is one of the most serious
mycoplasmalike organism (MLO) disease of stone fruit in
Michigan. Observation done by the Michigan Department of
Agriculture (MDA) showed that the percentage of X-diseased
trees increased during 6 years period (1980-1986) that
resulted with the decrease of the production (Dreves 1985,
1986, 1987). It has been estimated that Michigan peach
growers suffer between $1.5 and $3.9 million annually in
tree losses from this disease (M.E. Whalon personal
communication ).

The increased incidence of the peach X-diseases in
Michigan has attract more research interest in recent years.
Stoddard (1938) reported that infected peach trees grew
normally until about the middle of June when small,
yellowish areas appeared on the leaves. Rosenberger and
Jones (1978) using chockecherry as an indicator plant,
demonstrated that the symptoms of x-diseases were observed
from mid June to late July. Larsen and Whalon (1987)
observed the phenology of X-disease symptomatology in
chockecherry from southwest to Northwest Michigan. This

study demonstrated the same incidence of x-disease from

40

41

Southwest to Northwest Michigan, although symptom
expression was progressively later in the North.
Peach x-disease transmission patterns by Paraphlepsius
irroratus (Say) (Rosenberger and Jones 1978, Chiykowski and
Sinha 1988), indicated that leafhoppers have the

capability to transmit the disease over a period of several
weeks. These observations were made on peach, chockecherry
and celery. Transmission of X-disease from cherry to
indicator plants by Colladonus montanus (Van Duzee) has been
recorded by Suslow and Purcell (1982). However, these
symptomatology were done under laboratory conditions, thus
the environmental factors that might affect the transmission
patterns and development of the x-disease MLO in the field
were not considered.

Throughout these studies the occurrence of the MLO in
its vectors was determined solely by symptomatology. There
are a number of symptoms caused by MLOs such as virescence,
phyllody, stunt, proliferation (’witches' broom), etc. But
symptomatology alone is not sufficient to determine the
presence of MLOs since many of these symptoms are caused by
other pathogens, water stress and poor nutrition. Because
symptomatology is very an unreliable method of establishing
the presence of X-disease MLO in its vector, a better method
with high specificity and sensitivity is needed.

The previous study (Chapter II) demonstrated the
utility of a dot blot hybridization technique to determine

the presence of the X-disease MLO in its vector. The

42

sensitivity and efficiency of this technique allows study
of the occurrence of the X-disease MLO in its leafhopper
vectors under field conditions. This approach allows direct
study of X-disease epidemiology. This chapter reports the
use of the dot blot hybridization technique to preliminary
establish the seasonal distribution of peach X-disease in
field populations of P.irroratus, as well as a crude
classification of the geographical distribution of X-
diseased leafhoppers. This understanding is critical to the
development of an integrated pest management program for X-

disease and its vectors.
MATERIALS AND METHODS

Leafhopper sample

Paraphlepsius irroratus (Say) were collected by
sweepnet, yellow sticky boards and light traps along a
transect from Southwest (Benton Harbor) to Northwest
(Northport), Michigan during the 1987 and 1988 growing
seasons. In each of the sites, 50 sweep net samples were
taken from the orchard ground cover and 50 in the
surrounding vegetation. Six yellow boards coated with bird-
tanglefoot (The Tanglefoot Co, Grand Rapid, MI) were placed
in each orchard site. The yellow sticky traps were replaced
every week. Light trapping was carried out in each site by
locating the three 1 m2 light trap boxes (Larsen and Whalon

1987) to capture the adult leafhoppers during their

43

crepuscular flight period which started at sunset and ended
approximately 45-75 min later. All the leafhoppers sampled
were transported on ice to the laboratory where they were
sorted, identified, counted and stored at -70 0C in dated

vials.

Leafhopper assay for seasonal and crude distribution study
The occurrence of x-disease in the leafhopper was
determined weekly by submitting three groups of 10
leafhoppers per light trap in two sites (Trevor Nicols
Research Station, Fennville and Collins Rd. Entomology
Research Station at East Lansing, MI) to dot blot
hybridization analysis. For weekly samples with fewer than
30 leafhoppers, the total number of leafhoppers were a
equally in three groups and analyzed. All of the light
trapped leafhoppers from Sottun Bay, Ludington and Lawrence,

MI, were assayed by date.

MLO extraction and blotting

X-disease MLO from frozen leafhoppers was extracted
according to the method of Kirkpatrick (1987) with some
modifications. Leafhoppers were placed individually into
1.5 ml microtubes containing 400 ul of MLO enrichment buffer
(0.1M NaZHPO4, 10% sucrose, 1% polyvinylpyrolliodine and
50mM ascorbic acid added prior used, pH 7.6) and mascerated
with siliconized glass rod by rotating the rod 6 to 8 times.

The mixture was centrifuged at 5,000 g for 20 min. The

44

supernatant was decanted in to a second 1.5 ml microtube and
centrifuged at 16,000 g for 30 min. The resulting pellet
was resuspended with 100 ul of TE buffer pH 8.00 prior to
storage at -70°C or resuspended in 10X SSC (1.5M sodium
chloride, 1.5M sodium citrate) for immediately blotting.

The sample was blotted onto a nylon membrane, hybridized
and visualized according to the instruction provided by the
company (Method III, GeneScreenTM, New England Nuclear,
Dupont, Boston, MA). Before blotting the nylon membrane was
soaked in 2X SSC buffer (diluted from 10X SSC) and air
dried. DNA was alkaline denatured by treating the
extraction solution with 0.5M NaOH for 10 min and chilled;on
ice for another 10 min. After denaturation, the solution
was diluted with 10x SSC to the final volume of 400 al.

The 400 ul dilution was then blotted onto the membrane with
a dot-blot apparatus (BioRad, Richmond, CA). After
blotting, the membrane was washed carefully with 2X SSC for
2 min. The nucleic acids were fixed onto the membrane by
laying it face up on a clean glass plate for irradiation for
5 min with 254 nm shortwave UV light source (UVG-11,
Ultraviolet Products, Inc., San Gabriel, CA) at a distance

of 15 cm.

DNA sample hybridization
The prepared membrane with sample dots was
prehybridized in a plastic bag by treating with 10 ml of a

mixture of 50% deionized formamide, 0.2% polyvinyl-

45

pyrrolidone, 0.2% bovine serum albumin, 0.2% ficoll, 0.05M
Tris-HCl (ph 7.5), 1.0M NaCl, 0.1% sodium pyrophosphate,
1.0% sodium dedoxyl sulfate, 10% dextran sulfate and
denatured salmon sperm DNA (2100 ug/ml). The plastic bag
containing the prehybridization solution and the membrane
was sealed and incubated for 6 hr at 42 °C.

Hybridization was carried out by opening the bag,
adding 2.5 ml of prehybridization solution minus NaCl
containing the radioactive labelled probe, and resealing the
bag before incubation for 16 hr at 42 °C. Based on the
previous study, P. irroratus field samples were hybridized
by 200 ng/10 ml of C6c DNA probe at a specific activity of
0.5 to 2x108 cpm/ug. The autoradiographs were exposed for
24 hr. Before autoradiography, the membrane was washed
twice for 5 min each with 100 ml of a washing solution
containing 0.3M sodium chloride, 0.06M Tris-HCl (pH 8.0) and
0.002 EDTA at room temperature with gentle rocking. The
second washing was repeated 2X for 30 min each at 60 °C
using 100 ml fresh washing solution containing 1% SDS.

The last washing was also performed twice for 30 min at room
temperature with a 100 ml ten fold dilution of the washing
solution.

Autoradiography was carried out by exposing X-ray film
(Kodak XAR) for 24-48 hr with an intensifying screen (Sigma)
at -70 °C. X-ray film was developed in a dark room. The

hybridized dots were quantified using 2-D/1-D soft laser

46

scanning densitometer (Biomed Instruments, Inc., Fullerton,

Ca).

RESULTS

P. irroratus population trends

1987 and 1988 East Lansing and Fennville leafhopper
trapped (Figure III.1) confirmed already established
seasonal population trends and voltinism for P. irroratus
(Taboada et al. 1975, Rosenberger and Jones 1978, Mowry
1982, Larsen and Whalon 1987). At both sites, population
peaked in both generations at approximately SOC-1,000 DD
(Base 55 °F) and 2,000-2,200 DD respectively. Total number
of leafhopper trapped in Fennville both year was smaller
comparing with total number trapped in E. Lansing. The
smaller number in Fennville was may be due to the
insectiside applications during the seasons. The number of
leafhopper trapped by light traps was averagelly higher
comparing with the number of leafhopper trapped by yellow

sticky board per day.

x-diseased P. irroratus seasonallity

The percentage of diseased leafhopper ranged from 0 to
43.33% (Table III.1). In the first generation, early in
season the percentage of diseased adult leafhopper was
already high. The percentage of X-diseased leafhopper

peaked at the same time with the peak of total number that

47

 

 

l l T ' l

FENNVILLE

1987 -
.......... 1988

 

 

Y .

 

680 1060 1440 1820 2200

 

 

I l I 1

EAST LANSING

’1

1987
.......... 1988

 

 

 

   

 

1000
y. 800 b
I
'0
\
2
g 600 —
5
C
.2
8 400 1
8
.9
E
5
z 200 ~
0
300
1000
p 800 "
C
'0
\
3
§ 800 —
5
I
.2
8 400 1
3
a
a
5 200 F
o
800

080 1000 1440 1820 2200

Degree-days (Base 55‘ F)

Figure III.1. Total number of light trap and yellow sticky
board trapped Paraphlepsius irroratus (Say) per day at
Fennville and East Lansing, Michigan 1987-1988

48

Table III.1. Total number of adult Paraphlepsius irroratus (Say)
and diseased leafhoppers observed at Fennville and East
Lansing, Michigan 1988.

 

% diseasedb Total Total

Jdate +/testeda
(meaniSE) trappedc diseasedd

 

A. Fennville

175 11/30 36.67 i 8.98 231 85

189 4/30 13.33 i 8.89 120 16

196 1/30 3.33 i 3.33 34 1

204 2/30 6.67 i 3.39 91 6

210 0/30 0 55 o

216 2/30 6.67 i 6.67 48 3

224 2/28 7.14 i 3.89 28 2

243 0/30 0 94 0

257 0/30 0 51 o

271 1/30 3.33 i 3.33 175 6

287 2/14 14.28 i 6.79 14 2

295 0/0 0 o 0

B. East Lansing

165 9/30 30 : 15.27 43 13
172 10/30 33.3 1 14.53 60 20
179 6/30 20 i 5.77 229 46
185 13/30 43.33 1 12.02 205 89
195 8/30 26.67 i 8.82 107 28
201 0/30 0 i 0 41 0
206 0/30 0 i 0 119 0
213 4/30 13.33 i 6.67 77 10
216 0/30 0 i 0 134 0
221 4/27 14.81 i 14.81 27 4
235 5/30 16.67 i 8.82 36 6
244 3/30 10 i 10 32 3
254 6/30 20 i 10 44 9
259 3/13 23.33 i 1.67 13 3
265 6/30 20 i 5.77 30 6
270 7/30 23.33 i 8.82 33 8
274 1/30 3.33 i 3.33 141 5
295 0/0 0 0 0

 

a+/tested : total number leafhopper with positive signal/total
number of 3 groups of 10 P. irroratus tested. Samples with
leafhoppers less than 30 were all tested
ean percentage diseased leafhopper from 3 groups of

P. irroratus tested:

cTotal number of adult leafhopper trapped in three light traps.
Estimated from percentage of diseased leafhoppers observed.

49

occurred between the end of June and early July when the
accumulated degree day were 500-1,000 DD which was in
(Figure III.2). In the middle of season the percentage of
diseased leafhopper was almost zero and then rebound in the
late season with lower total percentage than the early
season. The average percentage of X-diseased leafhopper per
week observed in Fennville and E. Lansing were (x : S.E) 7.6
i 3% and 16.56 i 2.99% respectively.

There appeared to be a temperature effect on the
percentage of diseased leafhopper observed (Figure III.3)
As mean temperature increased beyond 75 °F (after 1,000 DD)
the percentage of diseased leafhoppers declined.
Conversely, at the end of the season, when the mean
temperature dropped below 75 °C, the percentage of diseased
leafhoppers increased slowly until a killing frost occurred.

At both sites, there was a correlation between the
number of leafhoppers trapped with the estimated number of
diseased leafhoppers (r = 0.71 and 0.7 with P = 0.0001 and

0.002 respectively) (Figure III.2, Table III.1).

x-diseased P. irroratus distribution

The X-disease MLO occurred in trapped leafhoppers in
all sites except Ludington and Suttons Bay (Table III.2).
This observation is consistent with X-disease orchard
surveys which indicates that the disease is much less
frequent the further North samples were taken. Only an

average of 10 leafhoppers per sample were captured in

50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

80 _
70 ‘ {“1 total Fennvil|81988
g ”‘ ZZcfiwawn
'Efm-. fl
8
O 50 ‘7
e
g 40 .. -
E 30-. l l r
B
S 20 4
Z
101
H HJ fl
0 __ L ., 21l- IL
June July August WI 00008:
80
£- 70- East Lansing 1988
E 60 ,-
G.
G.
g 50-
8 r
.2 40- F
o _.
3‘? 30—. ;
g i
z 203 1.4
10f i E F
0__ l J J 1 1 JL-, Hm M MLJL
June July August quember ()(mbe:
Juliandate

Figure III.2. Total number of diseased Paraphlepsius
irroratus (Say) caught and the number of leafhoppers at
Fennville and East Lansing, Michigan 1988.

7. of dleeeeed leefhopper/trep/dey

% ot dleeeeed leeflaopper/trep/dey

Figure III.3.

Paraphlepsius irroratus (Say) and mean temperature at

60

50

40

30

20

10

0

450

60

50

40

30

20

10

450

51

FENNVILLE 1988

 

 

1 z: ‘. ,
1: . u! ' r.
5 '2 5:" :3. ‘3
:: ' : ',:'.
'. l ‘0‘.’ 1:.-
2 3' E-
ls;
.-: E25:
- :5 5:3:
1! : 1:1! 1
::;:5::.; _
.- Sauna;
" ::'--:2-
5' :; 3'5
‘9 '0 9: .
335 2%
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z :
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g...

 

 

1 050

 

  

 

EAST LANSING

   

5
.

853$)!!! , , .

l

 

 

 

1 050

1 650

Degree-days (Beee 55°F)

The relationship between diseased

Fennville and East Lansing, Michigan 1988.

( diseased P. irroratus,

75

75

2250

Temperature ('F)

Tempereture (° F)

....... Mean temperature)

52

Table III.2. The seasonal occurrence of X-diseased
Paraphlepsius irroratus (Say) in several stone fruit
orchards Southern to Northern Michigan 19881.

 

Date Fennville E. Lansing Lawrence Ludington Suttons Bay

 

June

Week
Week
Week

but.)
+-++

July

Week
Week
Week
Week

#UNH
|+-+-+
+1 +-+

+

u

I

August

I
+

Week
Week
Week
Week

:5me
+-++-+
+
+
I

September

Week 1 -
Week 2 -
Week 3 +
Week 4 +

+-++-+

+ —

 

1+ : the presence of X-disease MLO was observed
- : the X-disease MLO was not observed

53

Northern Michigan while an average of 110 leafhoppers per
sample were captured in Central and Southern Michigan.
Diseased leafhoppers in Fennville did not occur in the
fourth week of July or the first and second weeks of
September. The X-diseased leafhoppers were not observed in
East Lansing during the third week of July or the first week
of August. The Lawrence site yielded diseased leafhopper
every sample date. Incidentally, this site has a long

history of X-diseased orchards.

DISCUSSION

The results from field sampling support earlier reports
(Taboada et al. 1975, Rosenberger & Jones 1978, Larsen and
Whalon 1987) that P. irroratus has two generations each
season. The adult leafhopper populations reached their
peaks during mid June and September at 500 to 1,000 and
2,000 to 2,200 DD base 55 °F respectively.

Two hypothesis explaining the mechanism of leafhopper
acquisition of X-disease MLO has been postulated
(Rosenberger and Jones 1978). The first states that adult
leafhoppers acquire the MLO while feeding on woody host.
Therefore, X-diseased competent leafhoppers will begin to
transmit after 20 to 30 day incubation period. With this
type of acquisition, we would expect that emerging adult
leafhoppers would initially be uninfected then, as time

passes, the percentage of diseased adults should increase.

54

The second hypothesis states that leafhoppers acquire
the X-disease MLO during their nymphal development while
feeding in the ground cover. Since nymphs feeds primary on
grasses (Rosenberger and Jones 1978, Chiykowski and Sinha
1988), the hypothesis suggests that grasses within orchard
ground cover may be an important x-disease alternate host at
least for the first generation. My data demonstrate that
first generation adults are infected at emergence with peak
infection early in the adult period followed by declining
percentage of diseased adults during the peak population.
The second hypothesis is further supported with the study by
Chiykowski and Sinha (1988) which showed that nymphs can
acquire X-disease MLO as efficiently as adults. These
authors also demonstrated the susceptibility of a grass
species, HOrdeum vulgare (Graminae), to X-disease. The
presence of this and other susceptible grasses (Chiykowski,
personal communication) in orchards or nearby ground cover
would provide a good source of inoculum for nymphs.

Further research is needed on the detection of x-disease in
ground cover plants, particularly grasses.

The percentage of diseased leafhoppers was surprisingly
high considering the low rate of X-disease transmission to
orchard trees (Purcell and Elkinton 1980, Purcell 1987,
Whalon, unpublished data). However, the percentage of
diseased peaches and cherry in the study orchard and
surround area, as judged by symptomatology, was high (> 60%

in nearby Fennville peach site and 22% in the East Lansing

55

cherry site). There are many factors that contribute to the
competence of leafhoppers to vector the X-disease MLO. For
example, the pathological effect of X-disease on its Western
leafhopper vector has been studied in Colladonus montanus
(Van Duzee) (Jensen 1958, 1959, 1972). These works
demonstrated that the X-disease MLO causes premature death
and that temperature effects the longevity of infected
leafhoppers. A similar study on P. irroratus has yielded
similar results (Garcia-Salazar et al. 1990). The average
longevity of X-diseased adult P. irroratus at 25 °C (mean
temperature) significantly shortened (x i S.E= 13 i 5.8
days) when compared to x-diseased leafhoppers reared at 15°C
(x i S.E= 35 i 21.3 days). Another study indicated that
temperature does effect the incubation period from
acquisition to the transmission (Chiykowski and Sinha
1988). The average incubation period of leafhoppers that
acquired x-disease MLO by feeding at 28°C was significantly
shorter (32 days) than at 21°C (53.9 days).

The occurrence of X-diseased leafhoppers in East
Lansing, Fennville and Lawrence were similar. In Ludington
and Suttons Bay, occurrence of X-diseased leafhoppers could
not be ruled out since there were too few leafhoppers
captured. The population of the leafhopper vectors was very
low and leafhoppers were not always trapped during the
sampling periods.

These results suggest several future research needs

including: 1) the relationship between X-disease, ground

56

cover, leafhopper population density and resulting diseased
trees, 2) a search for ground cover host for the
leafhopper vector and the X-disease MLO, 3) the
relationship between temperature, X-disease infection,

diseased leafhopper longevity and transmission competence.

REFERENCES CITED

Chiykowski, L.N. and Sinha, R.C., 1988. Some factors
affecting the transmission of eastern peach
X-mycolasmalike organism by the leafhopper
Paraphlepsius irroratus. Can. J. of Plant Pathol.
10:85-92.

Dreves, J.C., 1985. Peach virus survey. Michigan
Department of Agr. Comm.

, 1986. Peach virus survey. Michigan Department of
Agr. Comm.

, 1987. Peach virus survey. Michigan Department of
Agr. Comm.

Garcia-Salazar, C., Whalon, M.E., and Rahardja, U., 1990.
Temperature dependent pathogenicity of the X-disease
mycoplasmalike organism (MLO) to its vector,
Paraphlepsius irroratus (Say) (Homoptera:
Cicadellidae). In Press.

Jensen, D.D., 1958. Reduction in longevity of leafhoppers
carrying peach yellow leaf roll viruses.
Phytopathology 48:394. (Abstract).

, 1959. A virus lethal to its vector. Virology
8:164-175.

, 1972. Temperature and transmission of the Western
X_Disease agent by Colladonus montanus.
Phytopathology 62:452-456.

57

58

Kirkpatrick, B.C., Stenger, D.C., Morris, T.J. and Purcel,
A.H., 1987. Cloning and detection of DNA from a
nonculturable plant pathogenic mycoplasmalike
organism. Science 238:197-200.

Larsen, J.K. and Whalon, M.E., 1987. Crepuscular movement
of Paraphlepsius irroratus (Say) (Homoptera:
Cicadellidae), between the gorundcover and cherry
trees. Environ. Entomol. 16: 1103-1106.

Purcell, A.H., and Elkinton, J.S. 1980. A comparison of
sampling methods for leafhopper vectors of x-disease
in California cherry orchards. J. Econ. Entomol
73:854-860.

Purcell, A.H., 1987. Comparative epidemiology of X-disease
in North America. Proceedings of 2nd International
Workshop on Leafhoppers and Planthoppers of Economic
Importance, pp. 175-185, Wilson, M.R. and L.R. Nault.
CAB International Institute of Entomology, London, UK.

Rosenberger, D.A and Jones, A.L. 1978. Leafhopper vectors
of the peach x-disease pathogen and its seasonal
transmission from chockecherry. Phytopathology
68:782-790.

Sinha, R.C., and Chiykowski,L.N., 1980. Transmission and
morphological features of mycoplasma-like bodies
associated with peach X-disease. Can. J. Plant
Pathol. 6:111-124.

Suslow, K.G. and Purcell,A.H., 1982. Seasonal transmission
of X-disease agent from cherry by leafhopper
Colladonus montanus. Plant Disease 66:28-30.

Taboada, 0., Rosenberger,D.A. and Jones, A.L., 1975.
Leafhopper fauna of x-diseased peach and cherry
orchards in Southwest Michigan. J. of Econ Entomol.
68:255-257

Stoddard 1938. The x-disease of peach. Connecticut
Agriculture Experiment Station. Conn. Agric. Sta.
Circ. 122:53-60.

59

GENERAL CONCLUSIONS

This research has developed the protocols and controls
necessary to evaluate a DNA probe for x-disease detection in
P. irroratus field collected samples using a dot blot
hybridization techniques. This information is important in
developing an understanding of the epidemiology of
leafhopper vectored x-disease in order to develop the bestfi
strategy to manage the X-disease of stone fruit in Michigan.

The evaluation of the C6c DNA probe, a restriction
fragment of pWXl was a more specific probe for detecting the
occurence of X-disease MLO in leafhopper vectors than pWX1.
As reported for many cDNA probes, the sensitivity of this
probe and signal stripping techniques allows for multiple
testing of a single leafhopper, as well as the estimation of
the number of cells of X-disease MLO present in individual
leafhoppers. This approach opens the possibility of
studying X-disease etiology and epidemiology.

Further DNA hybridization and sequencing studies using
other Mollicutes that are closely related to the X-disease
MLO will give more precision in estimating the number of x-
disease MLO in its leafhopper vectors. Development of a
nonradioactive diagnosis system is needed to overcome the
laborious and laboratory limited work of radio-detection of
field samples.

Leafhopper monitoring confirmed that P. irrotratus has

60

two generations per season. The X-disease was observed in
leafhoppers from Fennville, East Lansing, and Lawrence, but
not from sites farther North. The seasonal occurence of the
X-disease MLO in its leafhopper vector, P. irrotratus
occurres throughout most of the season. It appears that the
leafhoppers acquire the X-disease MLO during the nymphal
stage when they live and feed in the ground cover.
Apparently, temperature also effects the presence of the MLO
in its vectors because the percentage of diseased
leafhoppers decreased astemperature increased.

Further research is needed to identify the ground cover
that nymphs acquire X-disease from. Perhaps the best
managment approach will involve the development of ground
cover plants that are either resistant to X-disease or

leafhoppers or both.

APPENDIX A

LEAFHOPPER VOUCHER SPECIMENS PLACED IN THE MICHIGAN STATE
UNIVERSITY ENTOMOLOGY MUSEUM

61

62
APPENDIX C

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.: 1990-01

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

Field detection of peach x-disease mycoplasmalike organism in
speckled leafhopper, Paraphlepsius irroratus (Say) (Homoptera:
Cicadellidae) using a DNA probe 1

Museum(s) where deposited and abbreviations for table on following sheets:
Entomology Museum, Michigan State university (MSU)

Other Museums:

Investigator's Name (8) (typed)

HI . E l i'

 

 

 

Date 1091] 2%

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

Deposit as follows:

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

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

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

63

APPENDIX C.l
voucher Specimen Data

Page 1 of 1 Pages

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APPENDIX B

FIELD MONITORING DATA

64

65

Table 1. Number of Paraphlepsius irroratus (Say) caught at
East Lansing and Fennville, Michigan 1987-1988.

 

Jdate 00551 Light2 Yellow3 Total

 

East Lansing 1987

190 998.9 244 244
197 1113.4 72 72
205 1276.4 125 125
210 1399.9 10 10
217 1549.9 3 3
225 1676.3 1 1
232 1802.2 103 103
238 1849.1 198 198
242 1876.6 104 104
246 1899.6 72 72
248 1919.5 380 380
254 2000.5 132 507 639
258 2032.1 142 142
264 2076.2 381 381
270 2107.5 40 39 79
276 2127.6 219 219
283 2132.6 0 2 2
304 2148.6 95 95
311 2148.6 0 0

Fennville 1987

190 961.1 1 1
212 1381.1 3 3
217 1468.8 2 2
225 1618.9 2 2
232 1742.1 73 73
238 1794.5 50 50
245 1842.1 88 88
254 1953.3 109 109
261 2013.3 127 127
269 2048.5 139 139
276 2058.6 188 188
286 2086.8 8 8
304 2105.3 179 179
305 2105.3 0 0

East Lansing 1988

165 461.5 43 43
172 597.2 60 60
179 720.2 229 229
182 739.9 54 54
185 772.7 205 205

190 895.7 240 240

66

Table 1. (Cont’)

 

 

Jdate DD551 Light2 Yellow3 Total
195

998.7 107 107

197 1047.2 84 84
201 1147.2 41 153 195
206 1225.7 119 119
211 1321.4 509 504
213 1366.9 77 77
216 1461.9 134 134
218 1517.9 178 178
221 1583.4 27 63 90
235 1864.2 36 36
239 1900.1 60 60
244 1944.2 32 205 237
254 2017.1 44 44
259 2054.1 13 451 464
265 2107.1 30 309 339
270 2132.7 33 33
274 2160.0 141 774 917
281 2176.7 0 524 524
288 2176.7 97 97
298 2185.9 100 100
304 2185.9 0 0

Fennville 1988

175
182
189
196
204
210
216
218
224
231
238
243
252
257
271
281
287
290
302
311

586.7

677.0

788.2

924.3
1076.3
1171.6
1322.6
1376.1
1506.1
1675.1
1760.6
1794.4
1874.8
1930.7
2036.2
2062.5
2066.8
2078.8
2080.5
2080.5

231

120
34
91
55
48

28

94

51
175

21
22
33
40
20
52

61
14

10
20
96
52

16
14

252
22
152
74
111
102
48
61
42

100
10
71

271
52
14
16
14

 

IDegree-days base 55 °F

2 Total number of three traps
3 Total number of six traps

APPENDIX C

X-DISEASE DETECTION DATA

67

68

Table 1. Number of total and diseased of Paraphlepsius
irroratus (Say) at East Lansing 1988

 

Jdate Rep. Number tested Number positive

 

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

9

9

9
10
10
10
10
10
10
10
10
10

5

4

4
10
10
10

165

172

179

185

195

201

206

213

216

221

235

244

254

259

265

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69

Table 1 (Con’t)

 

 

Jdate Rep. Number tested Number positive
270 1 10 4

2 10 2

3 10 1
274 1 10 1

2 10 0

3 10 0

 

70

Table 2. Number of total and diseased of Paraphlepsius
irroratus (Say) at Fennville 1988.

 

Jdate Rep. Number tested Number positive

 

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

8
10
10
10
10
10
10
10
10
10

5

5

4

175

189

196

204

210

216

224

243

257

271

287

CONHwNHLAMP“NHwNHle-‘le-‘(ANHQNHLJNHMNH
OHHOOHOOOOOOHHONOOOOOl-‘l-‘OOl-‘Ol-‘WO-hNUl

 

71

 

 

Table 3. Integrated optical densities of C6c and diseased
Paraphlepsius irroratus (Say).
No. Exposure Specific3 C6C IODD P.iC IODD
time (hr) activity (ng)
cpm/ug
1 24 5x108 0.05 3195 0.005 2871
0.10 21304 0.00625 4702
0.125 13822 0.01 23920
0.25 30480 0.0125 21634
0.5 82329 0.025 51998
1 4623 0.05 111222
0.0625 100844
0.1 107642
1 7475
2 24 2x109 0.0025 18516 0.00025 1160
0.01 1226 0.0005 1806
0.0125 62964 0.000625 2176
0.05 26554 0.001 5655
0.1 79296 0.00125 7439
0.125 93104 0.0025 18516
0.25 87815 0.005 35501
0.5 140879 0.00625 67382
1 202876 0.01 93107
0.0125 122867
0.025 162171
0.05 197812
0.0625 220887
0.1 178746
0.125 256120
0.25 168931
0.5 96107
1 40501
3 48 2x109 0.0025 35835 0.000006 3606
0.01 4837 0.00025 6141
0.0125 115799 0.0005 8818
0.05 54384 0.000625 13087
0.1 128662 0.001 17620
0.125 200837 0.00125 27898
0.25 183938 0.0025 53319
0.5 242223 0.005 95094
1 341086 0.00625 126130
0.01 161246
0.0125 225287
0.025 284168
0.05 343535
0.0625 385833

72

Table 3. (Cont’)

 

 

No. Exposure Specifica C6C IODD P.iC IODD
time (hr) activity (ng)
cpm/ug
0.1 321926
0.125 427715
0.25 327067
0.5 231257
1 124641

 

a) Specific activity of probe

b) Optical density were quantified using 2-D/1-D Soft Laser
Scanning Densitometer (Biomed Instruments, Inc.,
Fullerton, Ca).

c) The data represent the fractions or dilutions of
diseased leafhopper

73

Table 4. Number of Paraphlepsius irroratus (Say) caught by
light traps at Lawrence, Ludington and Suttons Bay

 

 

1988.

Jdate Number1 Number2 Signal3
caught tested

Lawrence
6/27 78 20 +
7/4 140 20 +
7/11 99 20 +
7/25 36 20 +
8/1 73 20 +
8/8 25 20 +
9/29 90 20 +
Ludington
7/1 0 0
7/5 46 20 -
7/16 67 20 -
8/1 0 0
8/23 0 0
9/30 0 0
10/21 0 0
Suttons Bay
7/1 0 0
7/8 1 1 -
7/19 3 3 -
7/28 8 8 -
8/8 5 5 -
9/30 7 7 -
10/21 0 0

 

I Number of P. irroratus caught by three light traps
Number of P. irroratus subjected to dot hybridization
+ signal indicates the presence of X-disease MLO
- signal indicates the absence of x-disease MLO

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