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I LIBRARY

Michigan State
Unlvoroity

 

 

 

This is to certify that the

thesis entitled

Allelic characterization and inheritance of
potyvirus resistance genes in cucumber

presented by
Eileen A Kabelka

has been accepted towards fulfillment
of the requirements for

Masters degree in Plant Breeding
& Genetics

 

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TO AVOID FINES return on or before dete due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU le An Affirmative Maud Opportunity IMRWOH
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ALLELIC CHARACTERIZATION AND INHERITAN CE OF POTYVIRUS
RESISTANCE GENES IN CUCUMBER

By

Eileen A. Kabelka

A THESIS

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

MASTER OF SCIENCE
Plant Breeding and Genetics Program
Department of Horticulture

1996

ABSTRACT

ALLELIC CHARACTERIZATION AND IN HERITAN CE OF POTYVIRUS
RESISTANCE GENES IN CUCUMBER

By

Eileen A. Kabelka

Several sources of potyvirus resistances have been identified within
the cucumber (Cucumis sativus L.) germplasm: ‘TMG- 1’ is resistant to
zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and
the watermelon strain of papaya ringspot virus (PRSV-W); ‘Dina’ is resistant
to ZYMV; and ‘Surinam’ is resistant to PRSV-W. These studies demonstrate
that TMG-l and Dina also possess resistance to the Moroccan watermelon
mosaic virus (MWMV). Determination of mode of inheritance, tests of
allelism among sources of resistance, and tests for cosegregation of the
resistances to the different potyviruses indicate that the resistances to
ZYMV, MWMV and PRSV-W in TMG-l, Dina, and Surinam are due to the
same, or tightly-linked gene(s). Although resistance to these potyviruses
may be conferred by a single gene, variation in dominance relationships,
resistance mechanisms, symptom expression, and which potyviruses are
protected against, support the possibility of a tightly-linked cluster of

potyvirus resistance genes.

ACKNOWLEDGMENTS

I would like to thank the members of my advisory committee
(Rebecca Grumet, Lowell Ewart, and Brian Diers) for their support
throughout this research project. I would especially like to thank Rebecca
Grumet for her time, patience, and encouragement. I consider it a privilege
to have worked with her. A special thanks to Sue Hammer for her assistance
and to all the undergraduate students who helped with greenhouse duties. I
would like to thank Lowell Ewart for sparking my interest in plant breeding,
John Biernbaum for excellent greenhouse training, Art Cameron for his
infectious enthusiasm, Rick Ward for his plant breeding expertise, Robert
Herner, Royal Heins, Joanne Whallon and Patricia Zandstra for their advise
and support. My learning experience, both as an undergraduate and

graduate student at Michigan State University, was excellent.

TABLE OF CONTENTS

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

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

CHAPTER 1

Introduction and Literature Review .......................................................... 1
1.1 Introduction ........................................................................... 1
1.2 General description and taxonomy of the potyviruses ........ 2

1.3 Occurrence, relationships among, and general
symptomatology of WMV, ZYMV, PRSV-W and MWMV.." 5
1.4 Sources of resistance in cucumber to WMV, ZYMV and

PRSV-W ................................................................................. 8
1.5 Cucumber genome and linkage analysis ............................. 10
1.6 Response to viral infection in plants - Definition of

terms ...................................................................................... l 1
1.7 Classical genetic analysis of plant resistance to viruses ..... 13
1.8 Purpose of thesis .................................................................... 20

References .............................................................................. 22

CHAPTER 2
Inheritance of resistance to the Moroccan watermelon mosaic virus in
the cucumber line TMG-l and cosegregation with zucchini yellow

mosaic virus resistance ............................................................................... 28
Abstract ............................................................................................ 28
Introduction ...................................................................................... 29
Materials and Methods .................................................................... 3 l

Cucumber genotypes ............................................................. 3 1

Virus inocula and inoculation procedures ............................ 32

Experimental designs and data analysis ............................. 33

Results .............................................................................................. 34

Response of TMG-l to inoculation with MWMV .................. 34

Inheritance of resistance to MWMV in TMG-l .................... 35
Genetic relationship between resistance to MWMV

and resistance to ZYMV in TMG-l ..................................... 37

Discussion ......................................................................................... 40

References ......................................................................................... 45

CHAPTER 3
Multiple alleles for potyvirus resistance at the zym locus in

cucumber ..................................................................................................... 48
Abstract ............................................................................................ 48
Introduction ...................................................................................... 49
Materials and Methods .................................................................... 51

Cucumber genotypes ............................................................. 5 1
Virus inocula, inoculation procedures, experimental
designs and data analysis ................................................... 51
Results .............................................................................................. 52
Relationship between the ZYMV resistance in
TMG-l and Dina .................................................................. 52
Differences in the performance of the resistance
alleles in TMG-l and Dina .................................................. 54
Response of Dina to inoculation with other
potyviruses ........................................................................... 62
Genetic relationship between resistance to ZYMV
and resistance to MWMV in Dina ...................................... 64
Discussion ......................................................................................... 66
References ......................................................................................... 73
CHAPTER 4

Characterization of genes controlling resistance to the watermelon
strain of papaya ringspot virus in cucumber: Allelism and

cosegregation with other potyviruses ........................................................ 75
Abstract ............................................................................................ 75
Introduction ...................................................................................... 76
Materials and Methods .................................................................... 77

Cucumber genotypes. ........................................................... 77
Virus inocula, inoculation procedures, experimental
designs and data analysis ................................................... 78
Results .............................................................................................. 79
Relationship between the PRSV-W resistance in
TMG-l and Surinam ........................................................... 79
Differences in the performance of the resistance
alleles in TMG-l and Surinam ........................................... 79
Genetic relationship between resistance to ZYMV
and resistance to PRSV-W in TMG-l ................................. 83
Discussion ......................................................................................... 86
References ........................................................................................ 95
SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS ................ 98

APPENDIX A:

Linkage relationship of potyvirus resistance to bitterfree

cotyledons .................................................................................................... 104
References ......................................................................................... 1 10

vi

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

LIST OF TABLES

Segregation data for response to cotyledon inoculation
with MWMV in populations derived from the inbred
cucumber lines TMG-l, WI-2 757 and Straight-8 ................. 36

Segregation data for response to true leaf inoculation
with MWMV in populations derived from the inbred
cucumber lines TMG-l and Straight-8 ................................. 38

Response to sequential inoculation with ZYMV and
MWMV in populations derived from the inbred
cucumber lines TMG-l and Straight-8 ................................. 39

MWMV and ZYMV inoculation of F3 families homozygous
recessive at the zym locus derived from the inbred

cucumber lines TMG-l and WI-2757. Families were
produced by self-pollinating F2 individuals selected

for resistance to ZYMV .......................................................... 41

Segregation for resistance to ZYMV in the progeny
derived from the cucumber lines TMG- 1 and Dina ............. 55

Response of the cucumber lines TMG-l, Dina and their
progeny 14 day post cotyledon inoculation with ZYMV ...... 56

Distribution of response in the cucumber lines TMG-l,
Dina, Straight 8, and their progeny to inoculation with
ZYMV ..................................................................................... 58

Response of the cucumber lines TMG-l, Dina and
Straight-8 to inoculation with several potyviruses .............. 6 1

Response of the cucumber lines Dina, Straight-8 and
their progeny to inoculation with MWMV ........................... 63

vii

Table 3.6

Table 3.7

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Table A. 1

Table A2

Response of the cucumber lines Dina, Straight-8 and

their progeny to cotyledon inoculation with ZYMV

followed by true leaf inoculation of resistant individuals

with MWMV ........................................................................... 65

Segregation for resistance to MWMV in the progeny
derived from the cucumber lines TMG-l and Dina ............. 67

Segregation for resistance to PRSV-W in the progeny
derived from the cucumber lines TMG-l and Surinam ....... 80

Response of the cucumber lines TMG-l, Surinam,
Straight-8, WI-2 7 57 and their progeny to inoculation
with PRSV-W ......................................................................... 82

Response of TMG-l, Straight-8, the F1 and backcross
progeny to sequential inoculation with ZYMV and
PRSV-W ................................................................................. 85

PRSV-W and ZYMV inoculation of F3 families

homozygous recessive at the zym locus derived from the
inbred cucumber lines TMG-l and WI-2 757. F3 families

were produced by self-pollinating F2 individuals selected

for resistance to ZYMV .......................................................... 87

Single trait goodness of fit tests for characters
analyzed in the F2 progeny of WI-27 57 x TMG-l
cucumber ................................................................................ 107

Tests for co-segregation of pairs of alleles.

Phenotypic classes and their frequencies in F2 progeny
derived from matings between the WI-27 57 x TMG-l
cucumber lines ....................................................................... 108

viii

Figure 3.1

Figure 3.2

LIST OF FIGURES

Phenotypic response of the cucumber lines Dina (left)

and TMG-l (right) 14 days post cotyledon inoculation

with ZYMV. Dina responds with a distinct veinal

chlorosis limited to the first and/or second true leaves

with subsequent leaves symptom-free. In TMG-l, this
distinct veinal chlorosis is not observed ............................... 53

Virus accumulation in the cucumber lines TMG-l, Dina

and Straight-8 two weeks post inoculation with ZYMV.

Each point is the mean +l- SE of five replicate plants.

Virus levels were measured by ELISA ................................ 60

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Cucumbers and other cucurbit crops are subject to severe losses due to
an array of potyviruses, including zucchini yellow mosaic virus (ZYMV),
watermelon mosaic virus (WMV) and the watermelon strain of papaya
ringspot virus (PRSV-W) (Lisa and Lecoq, 1984; Purcifull et al, 1984a,b).
Recently, an additional virus that infects cucurbits, the Moroccan
watermelon mosaic virus (MWMV) has been identified to be a distinct
member of the potyvirus group (McKern et a1, 1993). Sources of resistance to
several of these viruses have been identified within the cucumber germplasm
(Provvidenti, 1985). Inbred lines derived from the Chinese cucumber
cultivar, ‘Taichung Mou Gua’ (TMG) have been identified to be resistant to
ZYMV, WMV, PRSV-W (Provvidenti, 1985) and recently zucchini yellow fleck
virus (ZYFV) (Gilbert-Albertini, 1995). Inheritance of resistance in TMG to
these potyviruses have been characterized (Provvidenti, 1987 ; Wai and
Grumet, 1995a,b; Gilbert-Albertini, 1995). Two additional sources of

resistance include resistance to ZYMV in the Dutch hybrid ‘Dina’

(Abul Hayja and Al-Shahwan, 1991) and resistance to PRSV-W in the
cultivar ‘Surinam Local’ (Wang et a1, 1984).

The goals of this study are (1) determine if TMG possesses resistance
to MWMV and, if so, to investigate the relationship, if any, between
resistance to MWMV and the resistance to other cucurbit potyviruses,

(2) determine the relationships between resistance alleles in TMG and other
sources of resistance in cucumber to potyviruses and (3) determine the

relationship of different potyvirus resistances to each other.

1.2 General description and taxonomy of the potyviruses

The largest and most complex virus group in the Potyviridae is the
genus Potyvirus (Ward and Shukla, 1991; Shukla et a1, 1994). The potyvirus
group, named after its type member potato virus Y, has caused significant
loss in agricultural, horticultural and ornamental crops. The potyviral
genome is composed of a single-stranded, positive-sense RNA molecule of
approximately 10,000 bases. The 5’-end is covalently attached to a protein
VPg and the 3’-end contains a variable length polyadenylate tail. The
viruses belonging to this group are flexuous and rod-shaped, 680-900 nm
long and 11-15 nm in diameter. All members share the characteristic
pinwheel inclusion bodies in the cytoplasm of infected cells. Although a few
members can be transmitted by mites, whitefly and soil fungi, the

potyviruses are predominantly transmitted by aphids in a non-persistent

manner. Most members are easily transmitted mechanically while a few can

be transmitted through seed of infected plants.

An effective taxonomy for the potyviruses has been considered difficult
to establish (Shukla and Ward, 1989; Ward and Shukla, 1991; Shukla et a1,
1994). Assignment into the potyvirus group, in the past, has relied on
characteristics such as particle morphology, host range, symptomatology,
cross-protection, cytoplasmic inclusion morphology, serology and
transmission mechanisms. However, due to variation within the potyvirus
group and inadequacies of the traditional methods employed to identify
viruses and their strains, inconsistencies exist. In an effort to provide a
rational basis for potyvirus taxonomy, Ward and Shukla (1991) evaluate and
identify criteria, based on molecular and phenotypic characteristics, useful to

distinguish potyviral group membership and strain identification.

For membership into the potyvirus group, the most valuable molecular
characteristic is the complete nucleotide sequence which indicates the
number, nature and order of coding regions along with the mechanisms of
transcription, translation and replication (Shukla and Ward, 1989; Ward and
Shukla, 1991; Shukla et a1, 1994). Coat protein sequencing is considered the
next most valued characteristic as the coat protein virus product shares no
significant sequence identity with the coat protein virus products of other

virus groups. Considered the most valued phenotypic characteristic for

membership into the potyviruses is the appearance of pinwheel cylindrical
cytoplasmic inclusions (Ward and Shukla, 1991). This cylindrical shape
identifies only the potyviruses and no other virus group. The value of other
phenotypic characteristics such as particle morphology and transmission
mechanisms, for distinguishing potyviral membership, depends on how
accurately and uniquely they reflect the group. These phenotypic
characteristics, however, along with host range, symptomatology, cross-

protection, and serology, are considered useful tools for diagnostic purposes.

Molecular characteristics, as valuable criteria for potyviral group
membership, also provide valuable criteria to distinguish between viruses
and strains (Shukla and Ward, 1989; Ward and Shukla, 1991; Shukla et al,
1994). The sequence homology between distinct viruses and strains is
significantly different (distinct members range from 38-71% while strains
range from 90-99%). Because of this significant sequence homology
difference, gene sequencing, coat protein sequencing or simpler techniques,
such as nucleic acid hybridization, high-performance liquid chromatography
peptide profiling and N-terminal-targeted serology, can be useful in
distinguishing viruses and strains.

From an initial reliance based on morphological, biological, and
serological properties for assignment into the potyviruses, molecular

approaches, such as nucleic acid and coat protein sequencing, will ultimately

lead to a more accurate and detailed system of virus identification and

classification.

1.3 Occurrence, relationships among, and general symptomatology
of WMV, ZYMV, PRSV-W and MWMV

Watermelon mosaic virus (WMV) causes major loss in yield and
quality of cucurbit crops worldwide (Purcifufl et al, 1984a). With an
extensive range of natural hosts, WMV has been reported in Australia, Chile,
France, Hungary, Iran, Israel, Italy, Japan, Mexico, New Zealand, and
Venezuela. Reports of reduction in yield and quality in the United States
due, in part, to WMV dates back to the late 1940’s (Webb and Scott, 1965).

In 1965, Webb and Scott described WMV based on host range and
serological properties. They divided the isolates studied into two groups.
Isolates that did not infect non-cucurbitaceous plants were designated as
WMV-1 and isolates that infect species of Leguminosae, Chenopodiaceae and
Euphorbiaceae were designated WMV-2. WMV-1 and WMV-2 were first
considered to be strains of the same virus until biological, serological and
cDNA hybridization studies indicated WMV-1 was a strain of PRSV and
reclassified as the watermelon strain of papaya ringspot virus (PRSV-W)
(Gonsalves and Ishii, 1980; Purcifufl et al, 1984a,b). Later, this finding was
reinforced by coat protein sequence analysis (Quemada et al, 1990).

Symptoms of WMV generally consist of a systemic mosaic and/or

mottling pattern of the foliage with occasional leaf distortion. A reduction in

the production and quality of fruit following WMV infection is observed in
cucumber and other cucurbits (Purcifull et al, 1984a).

Zucchini yellow mosaic virus (ZYMV) was first observed in zucchini
crops of northern Italy in 197 3 (Lisa et al, 1981). ZYMV has since been
detected in Asia, Australia, central Europe, France, Germany, Israel, Japan,
Lebanon, Mauritius, Morocco, Spain and the United Kingdom (Lisa and
Lecoq, 1984; Davis, 1986; Sammons et al, 1989). In the United States,
Florida reported the occurrence of ZYMV in 1981, Connecticut, California
and Oregon in 1982, New York in 1983, New Jersey and Arkansas in 1985
(Sammons et al, 1989) and the Hawaiian Islands in 1988 and 1989 (Ullman
et al, 1991). The first report of ZYMV in Ontario occurred in 1989 (Stobbs
and Van Schagen, 1989).

ZYMV is serologically distantly related to WMV, PRSV-W, bean yellow
mosaic virus and to amaranthus leaf mottle virus (Lisa and Lecoq, 1984).
Strains of ZYMV differ in symptomatology but symptoms include mosaic,
yellowing, shoestringing, stunting and fruit distortion. Certain ZYMV
strains induce symptoms similar to those of other major cucurbit viruses such
as cucumber mosaic virus, PRSV-W and WMV and two or more of these
viruses will frequently occur together.

As with WMV and ZYMV, the watermelon strain of papaya ringspot
virus (PRSV-W) is considered a serious threat to cucurbit production and is

widely distributed geographically including Australia, the Caribbean,

France, Germany, India, Italy, the middle east, South America and the
United States (Purcifull et al, 1984b).

PRSV consists of three pathotypes - the papaya strain (PRSV-P), a
pathogen of papaya and cucurbits, the watermelon strain (PRSV-W), a
pathogen of cucurbits, but not papaya, and a strain isolated fiom squash
(PRSV-T) which also does not infect papaya (Baker et al, 1991).
Serologically, PRSV is closely related to WMV and ZYMV but distantly
related to bean yellow mosaic virus, potato virus Y, and tobacco etch virus
(Purcifull et al, 1984b). The type W strain of PRSV causes severe systemic
mottling and/or dark green blistering of leaves. Distortion of fruit prevails
following infection rendering them unmarketable.

The Moroccan watermelon mosaic virus (MWMV) was first reported
causing severe damage to cucurbits in Morocco in 197 4 (Fischer and
Lockhart, 197 4). Since then, isolates of this virus have been detected in
Cameron, the Canary Islands, Niger, South Africa, Spain, and Zimbabwe
(Van der Meer and Garnet, 1987; Quiot-Douine et al, 1990; McKern et al,
1993).

Based on biological properties, Fischer and Lockhart (1974) first
classified MWMV as a strain of WMV (formerly WMV-2). However,
subsequent comparative studies, based on biological and serological
properties, indicated this virus was related to but distinct from PRSV-W

(formerly WMV- 1) (Quiot-Douine et al, 1990). To clarify the taxonomic

relationship of this virus, McKern, et al (1993) compared a Moroccan isolate
to PRSV-W, WMV and soybean mosaic virus (a potyvirus related to WMV)
using high performance liquid chromatography of tryptic peptides and
limited amino acid sequencing of the coat protein. Their results concluded
that the isolate fiom Morocco was a distinct potyvirus. Similarly, sequence
analysis of cDN A clones of the coat protein gene show approximately 60%
homology with other cucurbit potyviruses confirming that MWMV is a
distinct potyvirus (Lanina and Grumet, unpublished).

Symptoms of MWMV consist of a foliar mosaic, green blistering, shoe
stringing and general stunting of the plant. Fruits show distortion, knobby-

like areas and color break (Fisher and Lockhart, 1974).

1.4 Sources of resistance in cucumber to WMV, ZYMV and PRSV-W
Naturally occurring resistances to viral disease have been identified in
existing cultivars, primitive cultivars or landraces, closely related wild or
cultivated species and genera of the same botanical family (Provvidenti,
1989). Provvidenti and Hampton (1992) report on sources of resistance to
56 viruses of the Potyviridae in 334 plant species. Of the Cucurbitaceae,
genes for resistance to WMV, ZYMV, PRSV-W and MWMV have been
identified in watermelon: Citrullus colocynthis, C. ecirrhosus, C. lanatus;

squash and pumpkins: Cucurbita ecuadorensis, C. ficifolia, C. foetidissima,

C. maxima, C. moschata, C. pedatifolia; and melon and cucumbers: Cucumis
melo, C. sativus, and C. metuliferus.

In cucumber (Cucumis sativus L.), Provvidenti (1985) identified two
accessions that possessed resistance to the most common potyviruses that
afflict cucurbits. Resistance to ZYMV, WMV, and PRSV-W was identified in
a single plant selection from the Chinese cucumber cultivar ‘Taichung Mou
Gua’ (TMG-1) and resistance to PRSV-W was identified in a cultivar from
South America, ‘Surinam Local’. The inheritance of resistance to ZYMV,
PRSV-W and WMV in TMG-1 has been characterized. In TMG-1, resistance
to ZYMV is conferred by a single recessive gene (Provvidenti, 1987) and
resistance to PRSV-W by a single dominant gene (W ai and Grumet, 1995a).
Inheritance of resistance to WMV in TMG-1 exhibits a tissue-specific
expression involving two independent types of resistances (W ai and Grumet,
1995b). In addition, their research suggests the ZYMV resistance gene
appears the same as, or tightly linked to, one of the WMV resistance genes.
The inheritance of resistance to PRSV-W in the cucumber cultivar ‘Surinam
Local’ was characterized by Wang, et al, in 1984. Their study revealed a
single recessive gene in Surinam confers resistance to PRSV-W. An
additional source of resistance to ZYMV was identified in a greenhouse
grown Dutch hybrid ‘Dina’ with characterization of this resistance

determined by Abul Hayja and Al-Shahwan (1991) as a single recessive gene.

10

1.5 Cucumber genome and linkage analysis

The Cucurbitaceae are among the more important plant families that
supply the human population with edible products (W ehner and Robinson,
1991). The three different genera of the cucurbit family are Cucumis
(cucumber and melons), Citrullus (watermelon) and Cucurbita (squash and
pumpkins). Cucumis sativus L, the cucumber, is grown fiom the extremes of
the temperate zones to the tropics. This species is believed to have originated
in Asia with ancestry similar to Cucumis sativus var. hardwickii.

The genetics of the cucumber (2n=14) has been extensively studied
with numerous morphological and disease resistance genes having been
identified since the 1930’s (Robinson et al, 1976). Since then, efforts have
been made to analyze linkage distances for the construction of genetic maps
through the use of RFLP, RAPD, isozyme, morphological and disease
resistance markers.

In 1987, Fanourakis and Simon investigated the linkage relationship
of 15 simply inherited traits to initiate construction of a genetic map of
cucumber. Linkage analysis arranged 13 of the 15 loci into three linkage
groups. In 1990, Pierce and Wehner, in their review of genetic linkages in
cucumber, assembled a morphological linkage map, identifying 40 map
positions on six linkage groups. Knerr and Staub in 1992, through joint

segregation analyses of 14 polymorphic isozyme loci, assigned 12 loci to four

11

linkage groups. Most recently, in 1994, Kennard, et al, constructed a
58 point genetic map assigned to ten linkage groups.

As characterization of genetically important traits in cucumber
continue to be identified and linkage distances analyzed, construction of a

detailed, saturated map of the cucumber will be attained.

1.6 Response to viral infection in plants — Definition of terms

Definitions of terms which describe different interactions and
responses of a host and a pathogen have evolved over the years and continue
to change. This is in part due to disagreement between mycologists,
virologists, and plant breeders as to the use of terms and in part due to not
fully understanding the mechanisms involved in host-pathogen interactions
(Fraser, 1986). In as much as this terminology is evolving, defining the
terms that have been used to describe the different host-pathogen
interactions and responses seem necessary. The following are definitions of
terms used to describe host-pathogen interactions and responses obtained
from several sources including Cooper and Jones, 1983; Fraser, 1986,1988.

Plants which allow virus multiplication and possible (but not always)
visible disease symptoms are susceptible and considered host to that
particular virus. Multiplication is defined as an increase of virus in time and
space. If an individual plant, or plant population, does not allow

multiplication of a particular virus and remains unaffected after repeated

12

inoculations, it is considered to possess nonhost immunity. Inhibition of
virus multiplication or inhibition of pathogenic effect on a host is termed
resistance. This resistance can operate at the cellular, plant or population
level. Individual plants or varieties that possess resistance to a particular
virus that normally affects that species of plant are considered to possess
constitutive resistance, or at time referred to as cultivar resistance. Tolerance
is a subjective description of disease severity and can present itself as a
complete absence, or only a mild degree, of symptoms causing little or no loss
in vigor or yield. Hypersensitivity is considered a type of pathological
response which takes the form of localized lesions or necrosis associated with
restricted virus invasion. Passive resistance has been used to describe
individuals that do not readily become infected, but once infected, replication
of the disease can occur. Plants can possess resistance to a virus but be
sensitive. Sensitivity is a subjective description of disease severity associated
with conspicuous symptoms that may diminish the rate or amount of plant
growth or yield.

A virus that multiplies and successfully infects a host is pathogenic to
that host. A virus that fails to establish infection is non-pathogenic to
nonhost plant species. Virulence is defined as the ability of a particular
strain of virus to multiply or cause disease within individuals of host species

that posses genes for resistance. Virus isolates not capable of multiplication

13

and disease within plant species containing susceptible individuals are

avirulent.

1.7 Classical genetic analysis of plant resistance to viruses

Classical genetic analysis of resistance to viruses in plants has been
accomplished by evaluating the progeny of crosses between true-breeding
resistant and susceptible lines to inoculation with a particular virus (Fraser,
1986). To evaluate the type of genetic control for resistance upon inoculation,
the observed genetic segregation ratios are compared to the expected genetic
segregation ratios. Scoring for response or reaction in plants to virus
inoculation is generally based on visual assessment of symptom severity on
individual plants, percentage of plants showing disease symptoms, back
inoculation to an indicator host, and quantitative measurement of virus
multiplication.

Resistance to viruses in most plant species is simply inherited
monogenetically (Fraser 1986; Provvidenti and Hampton, 1992). This single
locus resistance can manifest itself as either dominant or recessive and may
provide resistance to one particular virus, be pathotype specific, confer
resistance to two or more distinct viruses, or be tightly linked with other
resistance gene(s), in clusters, conferring resistance to multiple viruses or

strains.

14

Many simply inherited monogenetic disease resistances have been
identified in literature (Provvidenti and Hampton, 1992). For example,
resistance to blackeye cowpea mosaic virus in Vigna unguiculata is governed
by a single dominant gene (Ouattara and Chambliss, 1991) and resistance to
pea seed-home mosaic virus in Pisum sativum is conferred by a single
recessive gene (Hagedorn and Gritton, 1973). Examples of strain or
pathotype specific resistance includes a single dominant gene in Phaseolus
vulgaris that confers resistance to the PV2 strain of bean yellow mosaic virus
but susceptibility to other strains (Schroeder and Provvidenti, 1968) and a
single recessive gene in Pisum sativum that confers resistance to a third
pathotype of pea seed-home mosaic virus inherited independently but
possibly linked with two other single recessive genes conferring resistance to
other pathotypes of pea seed-home mosaic virus (Provvidenti and Alconero,
1988).

The existence of simply inherited genes, or clusters of separate tightly-
linked genes, that confer resistance to two or more distinct potyviruses have
been described previously in several species. For example, in Cucurbita
moschata, a single dominant gene confers resistance to both ZYMV and WMV
(Gilbert-Albertini et al, 1993), in Solanum stoloniferum, a single dominant
gene confers resistance to potato virus A and potato virus Y (Cockerham,
1970), and resistance to WMV and bean yellow mosaic virus is conferred by a

single recessive gene in Pisum sativum (Schroeder and Provvidenti, 1971).

15

In Phaseolus vulgaris, the possibility of a single gene, or cluster of tightly
linked genes cosegregating as a unit with the I gene, condition resistance
and/or lethal necrosis to a set of nine potyviruses including bean common
mosaic virus, WMV, blackeye cowpea mosaic virus, cowpea aphid-home
mosaic virus, azuki bean mosaic virus, Thailand Passiflora virus, soybean
mosaic virus, passionfruit woodiness virus-K and ZYMV. (Kyle and Dickson,
1988; Fisher and Kyle, 1994). In Pisum sativum, well defined clusters of
tightly-linked loci conferring resistance to a total of 1 1 potyviruses are
located on two chromosomes (Provvidenti and Hampton, 1993; Provvidenti
and Niblett, 1994). The first cluster, on chromosome 2, contains resistance to
WMV, bean yellow mosaic virus, pea mosaic virus, the NL-8 strain of bean
common mosaic virus, the lentil strain of pea seed-home mosaic virus, clover
yellow vein virus, and the Australian strain of passionfruit woodiness virus.
The second cluster, on chromosome 6, includes resistance to three pathotypes
of pea seed-home mosaic virus, clover yellow vein virus, and white lupin
mosaic virus.

Clusters of tightly linked genes have also been identified for disease
resistance other than viral diseases. Disease resistance genes in Lactuca L.
spp. have been arranged in three distinct clusters (Witsenboer et al, 1995).
The largest cluster contains eight genes (Dm 1, DmZ, Dm3, Dm6, Dm13,
Dm14, Dm15, Dm16') for resistance to downy mildew plus a gene (Ra) for

resistance to root-aphids (Crute and Dunn, 1980; Farrara et al, 1987). The

16

second largest cluster contains two downy mildew genes (Dm5 / 8, Dm10), a
dominant gene (Tu) conferring resistance to turnip mosaic virus and a
recessive gene (plr) conferring resistance to root downy mildew (V andemark
et al, 1992; Zink and Duffus, 197 3). Three additional downy mildew
resistance genes (Dm4, DM 7, Dm11) comprise the third cluster (Hulbert and
Michelmore, 1985).

Not all disease control systems in plants are simple. Single locus
resistance can also manifest itself as incomplete dominance or gene dosage-
dependent. Judgment as to whether a single locus resistance is dominant or
recessive is typically based on evaluating the F1 and segregating progeny of
the cross between pure-breeding resistant and susceptible parental lines for
symptoms of virus infection post inoculation with a particular virus. This
subjective observation of phenotypic symptom expression can be misleading.
An example of this is found in the resistance to tobacco mosaic virus in
tomato (Fraser and Loughlin, 1980). In the heterozygous state, the
resistance allele appears completely dominant with no viral symptoms
observed. However, measurement of virus multiplication of the heterozygote
revealed virus replication which is suggestive of a gene dosage-dependent
resistance. Resistance to soybean mosaic virus in common bean is reported
to be governed by an incompletely dominant gene (Kyle and Provvidenti,
1993). In the homozygous state, no detectable systemic symptoms were

observed, however, virus could be detected in inoculated leaves. In the

17

heterozygous state, under field or summer greenhouse conditions, individual
plants develop local chlorosis with systemic symptoms consisting of a mild to
moderate mottling. Plants, however, remain vigorous or only slightly
stunted. Interestingly, the incomplete dominant resistance allele of soybean
mosaic virus will appear completely dominant when plants are inoculated
and grown under winter greenhouse conditions. An additional example of a
single incomplete dominant system of control is reported in sugarbeet which
possesses resistance to beet mosaic virus (Lewellen, 197 3). Evaluation of the
parental and segregating populations, at an early stage of infection (10-14
days post virus inoculation) fell into discrete 3:1 [resistant(R):susceptible(S)]
classes for the F2 progeny and 1:0 (R2S) for the progeny backcrossed to the
resistant parent. However, at a later stage of infection (20-30 days post virus
inoculation) and depending on the time of year and greenhouse
temperatures, the F2 progeny segregated as a 1:2: 1 (R:intermediate:S) ratio
and the backcross to the resistant parent segregated as a 1: 1
(R:intermediate) ratio.

Effectiveness of resistance may depend on the genetic background of
the host cultivar. An example of this is found in barley which possesses
tolerance to barley yellow dwarf virus (Jones and Catherall, 1970). Reduced
effectiveness of tolerance, conferred by an incompletely dominant gene (Y d2)
was observed with slow growth rate considered either the result of host

genetic factors or environmental conditions. Interestingly, the effectiveness

18

of tolerance was recovered when introduced into rapidly growing genotypes.
In a follow-up study, Catherall, et al (1970), evaluated five gene donor
varieties that showed different levels of tolerance to barley yellow dwarf
virus. The one variety showing the greatest tolerance was the fastest
growing while the least tolerant was the slowest groWing variety. When the
tolerance factors from these varieties were introduced into similar genetic
backgrounds, the tolerance factor from the fastest growing variety
consistently provided the highest tolerance. Although the existence of
modifying genes efi'ecting the expression of the tolerance gene could not be
ruled out, these findings were suggestive of a series of alleles occurring at the
Yd2 gene locus differing in their effectiveness and their dominance
relationships.

The inheritance of resistance in plants to viral disease also can involve
more complex systems of control. This complexity can involve two or more
resistance factors, operating independently or additively. Kyle and
Provvidenti (198 7) report on two independent dominant genes that confer
resistance to WMV in Phaseolus vulgaris L. This pair of independent genes,
Wmv and st, are not pathotype-specific but can be distinguished
phenotypically at both low and high temperatures. The inheritance of
resistance to tobacco etch virus in Nicotiana tabacum cv. Havana 307 was
investigated by Rufty, et al (1988). In their investigation, parent and

progeny segregation ratios did not fit a simple Mendelian gene model but one

19

of resistance due to a few genes (possibly as few as two) with additive effects
based on a high frequency of resistant genotypes observed in the F2
generation, and the results of generation means analysis. Goodrick, et al, in
1991, reported on the inheritance of resistance to cowpea chlorotic mottle
virus in soybean. Their findings did not support a monogenetic control of
resistance but one of two recessive genes that conditioned resistance. A final
example of a more complex system of control is found in resistance to wheat
streak mosaic virus in maize (McMullen and Louie, 1991; McMullen et al,
1994). McMullen and Louie (1991) investigated the inheritance of resistance
to wheat streak mosaic virus in maize and identified a single dominant gene
for resistance (W31). However, in analyzing the segregating ratios of the
parental lines and progeny, additional genetic factors seemed involved as
different symptomatic responses were observed. To identify the additional
genetic factor(s) controlling resistance to wheat streak mosaic virus,
McMullen, et a1 (1994), through the use of bulked segregant analysis were
able to identify two additional genes. In addition, through the use of RFLP
analysis, they were able to determine the genotypes of the different
symptomatic responses to wheat streak mosaic virus infection. Plants that
exhibited a susceptible response of general mosaic were determined to be
wsml/wsm 1, wsm2/wsm2 and wsm3/wsm3 while the genotype of symptom-

free individuals were Wsm1/--, Wsm2/-- and Wsm3/--. Of interest were the

20

individual plants that exhibited dispersed chlorotic spots and rings. This

genotype was determined to be wsmI / wsm 1, wst/wst and Wsm3/--.

1.8 Purpose of thesis

Potyviruses, especially ZYMV, WMV, and PRSV-W, cause severe losses
in cucumber and other cucurbit crops worldwide (Lisa and Lecoq, 1984;
Purcifull et al, 1984a,b). Recently, an additional virus infecting cucurbits,
MWMV, has been identified to be a distinct member of the potyviruses
(McKern et al, 1993).

Potyvirus resistance can be governed by a single gene conferring
resistance to one specific pathotype, one particular virus, several distinct
viruses, or be tightly linked to other resistance gene(s) conferring resistance
to multiple viruses or strains (Fraser, 1986; Provvidenti and Hampton, 1992).
Disease resistance may also involve a more complex system such as
incomplete dominance (or gene dosage-dependence), effectiveness of
resistance may depend on the genetic background of the host, or may involve
two or more resistance factors, operating independently or additively.

A number of genes governing resistance to potyviruses in cucumber
have been identified and characterized. Inbred lines derived from ‘TMG’
govern resistance to ZYMV, WMV, PRSV-W (Provvidenti, 1985) and more

recently ZYFV (Gilbert-Albertini et al, 1995). Abul Hayja and Al-Shahwan

21

(1991) described resistance to ZYMV in the Dutch hybrid ‘Dina’ and Wang, et
al (1984), reported resistance to PRSV-W in the cultivar ‘Surinam Local’.

In TMG, resistance to ZYMV is conferred by a single recessive gene
(Provvidenti, 1987), resistance to PRSV-W by a single dominant gene (W ai
and Grumet, 1995a), and resistance to ZYFV by a single recessive gene
(Gilbert-Albertini et al, 1995). Resistance to WMV in TMG exhibits a tissue-
specific expression involving two independent genes (W ai and Grumet,
1995b). In addition, resistance to WMV and resistance to ZYMV in TMG
appear to be due to the same, or tightly linked genes (W ai and Grumet,
1995b). In Surinam Local, resistance to PRSV-W is conferred by a single
recessive gene (Wang et al, 1984) and in the Dutch hybrid Dina, resistance to
ZYMV is conferred by a single recessive gene (Abul Hayja and Al-Shahwan,
1991).

The purpose of this study is (1) determine if TMG possesses resistance
to MWMV and, if so, to investigate the genetic relationship, if any, between
resistance to MWMV and the resistance to other cucurbit potyviruses,

(2) determine the genetic relationship between resistance alleles in TMG to
other sources of resistance in cucumber to potyviruses and (3) determine the

relationship of different potyviruses to each other.

22

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CHAPTER 2
Inheritance of resistance to the Moroccan watermelon mosaic virus

in the cucumber line TMG-1 and cosegregation with zucchini yellow
mosaic virus resistance

ABSTRACT

Inbred lines derived from the Chinese cucumber cultivar, ‘Taichung
Mou Gua’ (TMG), have been identified to be resistant to several potyviruses
including: zucchini yellow mosaic virus (ZYMV), zucchini yellow fleck virus
(ZYFV), watermelon mosaic virus (WMV) and the watermelon strain of
papaya ringspot virus (PRSV-W). Recently, an additional virus that infects
cucurbits, the Moroccan watermelon mosaic virus (NIWMV), has been
identified to be a distinct member of the potyvirus group. This study
demonstrates that TMG-1 possesses resistance to MWMV. Rub or aphid
inoculated TMG-1 seedlings remain free of symptoms. Progeny analyses of
the F1, F2 and backcross generations show that resistance to MWMV is
conferred by a single recessive gene. Sequential inoculation of progeny
possessing resistance to ZYMV followed by MWMV (or MWMV followed by
ZYMV) and analysis of F3 families derived from F2 individuals selected for
resistance to ZYMV indicate that both resistances are conferred by the same

gene, or two tightly linked genes.

28

29

INTRODUCTION

Cucumbers (Cucumis sativus L.) and other cucurbit crops are subject
to severe losses due to an array of potyviruses including zucchini yellow
mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon
strain of papaya ringspot virus (PRSV-W) (Lisa and Lecoq, 1984; Purcifufl et
al, 1984a,b). An additional potyvirus, the Moroccan watermelon mosaic virus
(MWMV), was first reported causing severe damage to cucurbits in Morocco
in 1974 (Fischer and Lockhart, 1974). Since then, isolates of this virus have
been detected in Cameron, the Canary Islands, Niger, South Africa, Spain
and Zimbabwe (Van der Meer and Garnet, 1987 ; Quiot-Douine et al, 1990;
McKern et al, 1993). Although the relationship of MWMV to other
potyviruses had not been well defined, recent amino acid and nucleic acid
sequence analysis of the coat protein and its cDNA has verified that MWMV
is a distinct member of the potyvirus group (McKern et al, 1993; Lanina and
Grumet, unpublished). Predicted coat protein amino acid sequences indicate
that the core portion of the MWMV coat protein shares approximately 73%
identity with PRSV and approximately 67% sequence identity with WMV
and ZYMV, respectively (Lanina and Grumet, unpublished).

Sources of resistance to MWMV have been identified in Cucumis
metuliferus, Citrullus ecirrhosus, Coccinia sessifolia and Lu/fa aegyptica
(Provvidenti and Hampton, 1992) but not for Cucumis sativus. However, a

number of genes governing resistance to various other potyviruses in

30

cucumber have been identified. One particularly interesting genotype, the
Chinese cucumber cultivar ‘Taichung Mou Gau’ (TMG), is resistant to at
least four different potyviruses. Inbred lines derived from TMG have been
found to possess resistance to ZYMV, WMV, PRSV-W (Provvidenti, 1985),
and zucchini yellow fleck virus (ZYFV) (Gilbert-Albertini et al, 1995).

The potyvirus resistances in TMG show various genetic controls.
Resistance to ZYMV is conferred by a single recessive gene (Provvidenti
1987a) as is resistance to ZYFV (Gilbert-Albertini et al, 1995); resistance to
PRSV-W is due to a single dominant, or incompletely dominant, gene (W ai
and Grumet, 1995a). Resistance to WMV in TMG involves two types of
resistances (W ai and Grumet, 1995b). One is expressed in the cotyledons
and throughout the plant, the second is expressed only in the true leaves.
Interestingly, the gene conferring resistance to ZYMV appears to be the same
as, or tightly linked to, one of the WMV resistance genes.

Since the cucumber cultivar TMG possesses resistance to several
potyviruses, we sought to determine if it also possessed resistance to MWMV
and, if so, characterize the inheritance of resistance. In addition, as previous
studies with TMG indicated that the resistances to ZYMV and WMV may be
controlled by the same, or tightly linked, genes (W ai and Grumet, 1995b), we
also sought to determine if there is a genetic relationship between resistance

to ZYMV and MWMV.

31

MATERIALS AND METHODS
Cucumber genotypes

The inbred cucumber (Cucumis sativus L.) line ‘TMG-l’, resistant to
ZYMV, WMV, and PRSV-W (Provvidenti, 1985) was provided by Dr. Jack
Staub (USDA, University of Wisconsin at Madison). The two susceptible
parental genotypes used in this investigation were the inbred lines
‘Wisconsin 2757’ (WI-27 57 ; Peterson et al, 1982; provided by Dr. Jack Staub)
and ‘Straight-8’ (ST-8; W. Atlee Burpee & Company, Warminster, PA).

Production of the F1, F2 and backcross progeny of WI-27 57 and TMG-1
was described by Wai and Grumet (1995a). F3 families, used to analyze the
relationship between resistance to ZYMV and MWMV, were produced by first
screening 1,479 F2 (WI-27 57 x TMG-l) individuals for resistance to ZYMV in
the greenhouse. Three-hundred-seventy-three ZYMV resistant F2
individuals [3:1 (susceptiblezresistant); X2 = 0.08; non-significant] were then
transplanted to the field. As WI-2 7 57 is gynoecious (dominant trait; Peterson
et al, 1982), monoecious individuals were identified and self-pollinated by
hand under controlled conditions. Fifty-one F3 families were produced. The
progeny of the cross between ST-8 and TMG-1 were produced in the
greenhouse. The reciprocal F1 progeny were self-pollinated to produce the F2
generation. Backcrosses were made to both parents using the

F1 (ST-8 x TMG- 1) genotype.

32

Virus inocula and inoculation procedures

MWMV (provided by Dr. Purcifull, University of Florida, Gainesville,
FL) and ZYMV (Connecticut strain) were propagated in Cucurbita pepo L.
cvs. ‘Black Beauty’ (SeedWay Inc., Elizabethtown, PA) or ‘Midas’ (Willhite
Seed, Poolville, TX) and maintained in a growth chamber (16-h day, 24°C
constant temperature, ca 300 umol photons M'2 s1). Purity of the virus
source was verified by ELISA, as described by Wai and Grumet (1995a), and
by the use of the differential host, Phaseolus vulgaris cv. ‘Black Turtle 2’
(Provvidenti, 1983).

Young symptomatic leaves were harvested two to four weeks post
inoculation and macerated in ice-cold 20mM sodium phosphate buffer, pH
7.0, in a pre-chilled mortar and pestle. Cotyledons of 7-10 day old seedlings,
or the upper surface of the fully expanded second and half expanded third
true leaves, were lightly dusted with 320-grit Carborundum (Fisher
Scientific, Pittsburgh, PA) and rub inoculated with virus-infected sap
(approximately 124 dilution leaf materialzbufl'er) using sponge plugs. Mock-
inoculated control plants were included in each experiment. All non-
biological materials were sterilized prior to use.

TMG-l, WI-2757 and ST-8 were also inoculated with MWMV using
aphids. Virus-free aphids were harvested from Nicotiana tabacum cv.
‘Burley’ stock plants and allowed to feed on symptomatic virus-infected

leaves of Cucurbita pepo L. for one to three minutes. The aphids were

33

transferred to young cucumber seedlings (ten/plant) and allowed to feed for
two hours. All aphids were then removed by hand. Aphid inoculated
cucumber seedlings were transferred to a controlled growth chamber
environment, sprayed with the insecticide Orthene (Monsanto, St. Louis,

MO), and observed for symptom development.

Experimental designs and data analysis

Seed for all experiments were pregerminated in a 30°C incubator for
24 hours then sown in the greenhouse into 10.2 cm clay pots filled with
Baccto growing medium (Professional Planting Mix, Michigan Peat
Company, Houston, TX). Upon emergence of the cotyledons, a fertilization
regimen of 300 ppm N-P-K (Peters Professional All-Purpose Fertilizer,
20-20-20, Grace-Sierra Horticultural Products, Milpitas, CA) was applied
three times per week. Greenhouse temperatures ranged from 25°C to 35°C
throughout the year.

The F2 and backcross populations were inoculated at the cotyledon
stage. Sixteen rows of ten plants/row were interspersed with five internal
control rows consisting of inoculated, mock-inoculated and non-inoculated
parental and F1 progeny. Similarly, tests of F3 families included ten
individuals per family with five control rows evenly distributed along the
bench. Sequential inoculation experiments consisted of cotyledon inoculation
of parental genotypes and their progeny with one virus followed by true leaf

inoculation of the resistant individuals with the second virus. Sequential

34

inoculation experiments were performed in both directions: cotyledon
inoculation with MWMV followed by true leaf inoculation with ZYMV or
cotyledon inoculation with ZYMV followed by true leaf inoculation with
MWMV. Additional inoculated, mock-inoculated and non-inoculated control
plants were included for the true leaf inoculation portion of the experiment to
confirm successful virus inoculation. All experiments included border rows of
ST-8 (susceptible genotype) as a further check for any possible variation in
the inoculation technique and/or expression of viral symptoms.

In all experiments, plants were visually scored as either resistant
(symptom-free) or susceptible (evidence of virus) when symptom development
was optimal (approximately seven days post inoculation for ZYMV and
approximately 14 days post inoculation for MWMV). Segregation ratios were

analyzed by Chi square analysis.

RESULTS
Response of TMG-1 to inoculation with MWMV

Symptoms of MWMV generally developed 14 days post cotyledon or
true leaf inoculation. Following rub inoculation with MWMV, TMG-1
remained symptom-free. Symptom expression on the susceptible genotypes,
WI-27 57 and ST-8, showed a mild systemic rugosity and silvering of the true
leaves. Symptom intensity varied during the course of plant growth, the

rugosity and silvering of the true leaves would fade occasionally but then

35

resurge to full expression. Since potyviruses are normally transmitted by
aphids in nature, the response of TMG-1 to MWMV also was tested using
aphid inoculation. As for rub inoculation, TMG-1 remained symptom-free
while WI-2 7 57 and ST-8 developed mild systemic rugosity and silvering of

the true leaves (data not shown).

Inheritance of resistance to MWMV in TMG-1

The F1 progeny of the crosses between ST-8 and TMG-1 and WI-2 757
and TMG-1 reacted in a similar fashion and intensity as the susceptible
parents, showing systemic mild rugosity and silvering of the true leaves
(Table 2.1). The F2 progeny (ST-8 x TMG-1, TMG-1 x ST-8 or WI-2757 x
TMG- 1) segregated in a 1:3 [resistant (R):susceptible (8)] ratio while the
backcrosses to the resistant TMG-l parent segregated in a 1:1 (R28) ratio.
Progeny of the backcrosses to the susceptible parents were all susceptible.
These segregation ratios support a model in which resistance to MWMV in
TMG-1 is conferred by a single recessive gene (proposed gene designation
mwmw.

Previous studies by Wai and Grumet (1995b) indicated that resistance
to WMV in TMG-1 involves two types of resistances, one of which is
expressed only in the true leaves. To determine if TMG-1 had a similar true
leaf-specific system of resistance to MWMV, the parental genotypes of TMG-l

and ST-8 and their progeny were inoculated with MWMV at the true leaf

36

Table 2.1 Segregation data for response to cotyledon inoculation with MWMV
in populations derived from the inbred cucumber lines TMG-1,
Wl-2757, and Straight-8.

 

 

 

 

 

 

Parent or Number of plants Expected
progeny Resistant Susceptible ratio (R:S)a X2
TMG-1 (T) 36 0
Straight-8 (ST8) 0 36
Wl-2757 (2757) 0 12
F1 (ST8xT) 0 48 021
(T xST8)
F1 (2757xT) 0 12 021
F2 (ST8xT)” 78 241 1:3 0.037 ns
(T xST8)
F2 (2757xT) 39 121 1:3 0.008 ns
BC (ST8xT) xT° 111 129 1:1 1.204 ns
Tx (ST8xT)
BC (2757xT) xT 73 67 1:1 0.178 ns
BC (ST8xT) x ST8 0 160 0:1
ST8 x (ST8xT)
BC 2757 x (2757xT) 0 20 0:1

 

ns, non-SIgnIf cant X2 value

:expected ratios based on a single recessive gene model. R - resistant s= susceptible.
bdata pooled from two independent experiments. Each experiment fits the predicted ratios
X2 exp1- = 0.000, Xzexp2= 0.076; X2 homogeneity= 0. 053, df= 1.

°data pooled from two independent experiments. Each experiment fits the predicted ratios
X2 exp1- = 1.406, Xzexp2= 0.012; X homogeneity= 0.32, df= 1.

37

stage (Table 2.2). Symptom development following true leaf inoculation
occurred approm’mately 14 days post inoculation. Segregation ratios revealed
no difference between cotyledon and true leaf inoculation with MWMV.
Genetic relationship between resistance to MWMV and resistance to
ZYMV in TMG-1

To investigate whether a relationship exists between the resistance to
MWMV and resistance to ZYMV in TMG-1, two approaches were taken. The
first approach was sequential inoculation. This procedure consisted of
cotyledon inoculation of TMG-1, ST-8 and their progeny with MWMV
followed by true leaf inoculation of the resistant individuals with ZYMV
(Table 2.3A). In a separate experiment, this procedure was reversed
inoculating the cotyledons with ZYMV followed by true leaf inoculation of the
resistant individuals with MWMV (Table 2.3B). In all experiments,
additional control plants were included to verify successful inoculation at the
true leaf stage. Symptoms of MWMV in the susceptible genotypes, WI-27 57
and ST-8, were as described earlier. Symptoms of ZYMV, a systemic mosaic
pattern of the true leaves, generally developed on the susceptible genotypes
(WI-2 7 57 and ST-8) seven days post cotyledon or true leaf inoculation. The
resistant genotype, TMG-1, remained free of symptoms for both MWMV and
ZYMV rub inoculation. In all cases, those individuals resistant to cotyledon
inoculation with the first virus remained symptom-free upon true leaf

inoculation with the second virus.

 

38

 

 

Table 2.2 Segregation data for response to tme leaf inoculation with MWMV
in populations derived from the inbred cucumber lines TMG-1 and
Straight-8.
Parent or Number of plants Expected
progeny Resistant Susceptible ratio (R:S)a X2
TMG-1 (T) 16 0
Straight-8 (ST8) 0 16
F1 (ST8xT) 0 34 0:1
(T xST8)
F2 (ST8xT) 43 109 123 0.711 ns
(T xST8)
BC (ST8xT) x Th 76 83 1:1 0.226 ns
T x (ST8xT)
BC (ST8xT) x ST8 0 80 0:1
ST8 x (ST8xT)

 

ns, non-significant X2 value

aexpected ratios based on a single recessive gene model, R = resistant, S = susceptible.

bdata pooled from two independent experiments. Each experiment fits the predicted ratios
Xzexp1 = 0.81, Xzexp2 = 0.012; x2 homogeneity = 1.01, df = 1.

Table 2.3

39

Response to sequential inoculation with ZYMV and MWMV in populations
derived from the inbred cucumber lines TMG-1 and Straight-8.

 

A. Cotyledon inoculation with MWMV followed by true leaf inoculation of resistant individuals

 

 

 

 

 

with ZYMV.
Sequential inoculation of
Cotyledon inoculation MWMV resistant individuals
with MWMV with ZYMVfiat true leaf stage
Parent or Total Number of plants Number of plants
progeny plants Resistant Susceptible Resistant Susceptible
TMG-1 (T) 18 18 0 18 0
Straight-8 (ST8)" 9 0 9 - —
Straight-8 (ST8)" 9 — — 0 9
F1 (ST8xT)' 18 0 18 - -
F1(ST8xT)b 1e — — 0 13
F2 (ST8xT)c 319 73 243 73 0
ac (ST8xT)xT" 160 71 89 71 0

 

'control plants to verify successful inoculation at cotyledon stage with MWMV
”control plants to verify successful inoculation at true leaf stage with ZYMV
“data pooled from two independent experiments. Each experiment frts the expected and predicted segregation ratios based on

resistance to MWMV conferred by a single recessive gene. Xzexpt = 0.68, Xzexp2 = 0.08; X2 homogeneity = 0.16, df = 1
“data fits the emcted and predicted segregation ratio based on resistance to MWMV conferred by a single recessive gene.

x’=1.3

 

B. Cotyledon inoculation with ZYMV followed by true leaf inoculation of resistant individuals

 

 

 

 

with MWMV.
Sequential inoculation of
Cotyledon inoculation ZYMV resistant individuals
with ZYMV with MWMV at true leaf stage
Parent or Total Number of plgnts Number of plants
progeny plants Resistant Susceptible Resistant Susceptible
TMG-1 (T) 20 20 0 20 0
Straight-8 (ST8)' 10 0 10 - -
Straight-8 (ST8)° 10 - - 0 10
F1 (ST8xT)" 10 0 10 -- -
F1(ST8xT)" 10 — - 0 10
F2 (ST8xT)c 160 40 120 40 0
ac (ST8xT)xT‘ 80 36 44 36 0

 

'control plants to verify successful inoculation at cotybdon stage with ZYMV
l’control plants to verify successful inoculation at true leaf stage with MWMV

°data fits the expected and predicted segregation ratio based on resistance to ZYMV conferred by a single recessive gene.

x2 = 0.00

‘datafltstheexpectedand predicted segregahonmflobasedmresistancetoZYMVconfenedbyasingbrecesswegme.

x2 = 0.62

40

A second approach to evaluate whether a relationship exists between
the resistance to MWMV and resistance to ZYMV was to screen F3 families
that were produced by self-pollinations of F2 individuals selected for
resistance to ZYMV. Progeny from each ZYMV resistant F2 individual were
inoculated either with MWMV or ZYMV (Table 2.4). As expected, all families
were resistant to ZYMV. If the two resistances had been segregating
independently, 9/ 16th of the F3 families should be either susceptible or
segregating for susceptibility to MWMV. There was, however, no segregation

for susceptibility to MWMV either within or among the F3 families screened.

DISCUSSION

To our knowledge, this is the first report of resistance to MWMV in
Cucumis sativus. In this study, we have determined that the cucumber line
TMG-1, which possesses resistance to ZYMV, WMV, and PRSV-W, also
possesses resistance to MWMV. Rub and aphid inoculation with MWMV
revealed TMG-1 to remain symptom-free while the susceptible genotypes,
WI-27 57 and ST-8, developed a mild systemic rugosity and silvering of the
true leaves. Progeny analysis of the F1, F2, and backcross generations show
that resistance to MWMV is conferred by a single recessive gene (mwmv).
Sequential inoculation of progeny possessing resistance to ZYMV followed by
MWMV (or MWMV followed by ZYMV) and analysis of F3 families derived

from F2 individuals selected for resistance to ZYMV, indicate that both

41

Table 2.4 MWMV and ZYMV inoculation of F3 families
homozygous recessive at the zym locus derived from
the inbred cucumber lines TMG-1 and Wl-2757.
Families were produced by self-pollinating F2 individuals

 

selected for resistance to ZYMV.

No. F3 families“ No. F3 families susceptible
Potyyirus showing resistance or sggregating for resistance
MWMV 51 0
ZYMV 51 0

 

5’tests of F3 families included ten individuals/family.

42

resistances are conferred by the same gene, or two tightly linked genes (less
than 1 cM; product-ratio method). Earlier work with TMG-1 indicated that
the recessive gene for ZYMV resistance also conferred resistance to WMV
(W ai and Grumet, 1995b). Since our current investigation suggests that the
gene for ZYMV also confers resistance to MWMV, it appears a single
recessive gene, or cluster of recessive genes, in TMG-1 governs resistance to
at least three potyviruses, ZYMV, WMV and MWMV.

The existence of simply inherited genes, or clusters of separate tightly-
linked genes, that confer resistance to two or more distinct potyviruses have
been described previously in several species. For example, in Cucurbita
moschata, a single dominant gene confers resistance to both ZYMV and WMV
(Gilbert-Albertini et al, 1993), in Solanum stoloniferum, a single dominant
gene confers resistance to potato virus A and potato virus Y (Cockerham,
1970), and resistance to WMV and bean yellow mosaic virus is conferred by a
single recessive gene in Pisum sativum (Schroeder and Provvidenti, 1971).
In Phaseolus vulgaris, the possibility of a single gene, or cluster of tightly
linked genes cosegregating as a unit with the I gene, condition resistance
and/or lethal necrosis to a set of nine potyviruses including bean common
mosaic virus, WMV, blackeye cowpea mosaic virus, cowpea aphid-home
mosaic virus, azuki bean mosaic virus, Thailand Passiflora virus, soybean
mosaic virus, passionfruit woodiness virus-K and ZYMV. (Provvidenti et al,

1983; Kyle and Dickson, 1988; Fisher and Kyle, 1994). In Pisum sativum,

 

 

43

well defined clusters of tightly-linked loci conferring resistance to a total of

1 1 potyviruses are located on two chromosomes (Provvidenti and Hampton,
1993; Provvidenti and Niblett, 1994). The first cluster, on chromosome 2,
contains resistance to WMV, bean yellow mosaic virus, pea mosaic virus, the
NL-8 strain of bean common mosaic virus, the lentil strain of pea seed-home
mosaic virus, clover yellow vein virus, and the Australian strain of
passionfi'uit woodiness virus. The second cluster, on chromosome 6, includes
resistance to three pathotypes of pea seed-borne mosaic virus, clover yellow
vein virus, and white lupin mosaic virus.

It has been postulated that single genes, or clusters of related genes,
with conserved functions in plants could interrupt common pathogenic
processes shared by closely related viruses (Kyle and Provvidenti, 1993;
Fisher and Kyle, 1994). Sequence analyses have indicated that distinct
potyviruses share 38-71% sequence identity in their coat protein, whereas
strains within a given virus share 90-99% sequence identity (Shukla et al,
1994). It also has been suggested that viruses sharing intermediate levels of
homology (7 4-88% sequence identity) can be grouped together to form
subgroups within the potyviruses (Shukla et al, 1994). Of the viruses for
which the zym gene or gene cluster in cucumber confers resistance, ZYMV
and WMV share approximately 80% sequence and fall within the same
subgroup (Fang and Grumet, 1990; Shukla et al, 1994). MWMV, however,

does not appear to be a member of this subgroup as it shares only

 

 

44

approximately 60% sequence identity (Lanina and Grumet, unpublished). In
cucumber, it is therefore possible that one gene can confer resistance across
potyvirus subgroups or, alternatively, separate tightly-linked genes may be

responsible for the multiple potyvirus resistance.

 

 

45

REFERENCES

Cockerham, G. 1970. Genetical study on resistance to potato viruses X and
Y. Heredity 25:309-348.

Fischer, H.U., Lockhart, B.E.L. 1974. Serious losses in cucurbits caused by
watermelon mosaic virus in Morocco. Plant Disease Reporter 58:143-146.

Fisher, M.L., Kyle, M.M. 1994. Inheritance of resistance to potyviruses in
Phaseolus vulgaris L. III. Cosegregation of phenotypically similar dominant
responses to nine potyviruses. Theoretical and Applied Genetics 892818-823.

Gilbert-Albertini, F., Lecoq, H., Pitrat, M., Nicolet, J.L. 1993. Resistance of
Cucurbita moschata to watermelon mosaic virus type-2 and its genetic
relation to resistance to zucchini yellow mosaic virus. Euphytica 69:231-237.

Gilbert-Albertini, F., Pitrat, M., Lecoq, H. 1995. Inheritance of resistance to
zucchini yellow fleck virus in Cucumis sativus L. HortScience 302336-337.

Kyle, M.M., Dickson, M.H. 1988. Linkage of hypersensitivity to five viruses
with the B locus in Phaseolus vulgaris L. The Journal of Heredity 7 9:308-
3 l 1.

Kyle, M.M., Provvidenti, R. 1993. Genetics of broad spectrum resistance in
bean and pea. In: Kyle, M.M. (ed). Resistance to viral diseases of vegetables:
Genetics and breeding. Timber Press, Portland, OR pp. 153-166.

Lisa, V., Lecoq, H. 1984. Zucchini yellow mosaic virus. Descriptions of
Plant Viruses No. 282. Kew, Surrey, England: Commonwealth Agricultural
Bureax/Association of Applied Biologists.

McKern, N.M., Strike, P.M., Barnett, O.W., Ward, C.W., Shukla, DD. 1993.
Watermelon mosaic virus-Morocco is a distinct potyvirus. Archives of
Virology 131:467-473.

Peterson, C.E., Williams, P.H., Palmer, M., Louward, P. 1982.
Wisconsin-2757 Cucumber. HortScience 17:268.

Provvidenti, R. 1983. Two useful selections of the bean cultivar Black Turtle
Soup for viral identification. Bean Improvement Cooperative Annual Report
26:73-75.

 

46

Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of
Cucumis sativus. Cucurbit Genetics Cooperative Report 8212.

Provvidenti, R. 1987a. Inheritance of resistance to a strain of zucchini
yellow mosaic virus in cucumber. HortScience 22:102-103.

Provvidenti, R., Hampton, RD. 1992. Sources of resistance to viruses in the
Potyviridae. Archives of Virology Suppl 52189-211.

Provvidenti, R., Hampton, RD. 1993. Inheritance of resistance to white
lupin mosaic virus in common pea. HortScience 38:836-837.

Provvidenti, R., Niblett, CL. 1994. Inheritance of resistance to a strain of
passionfruit woodiness virus in pea (Pisum sativum L.). HortScience 29:901-
902.

Provvidenti, R., Gonsalves, D., Taiwo, MA. 1983. Inheritance of resistance
to blackeye cowpea mosaic virus in Pisum sativum. The Journal of Heredity
74260-6 1.

Purcifull, D., Hiebert, E., Edwardson, J. 1984a. Watermelon mosaic virus-2.
Descriptions of Plant Viruses No. 293 (N o. 63 revised). Kew, Surrey,
England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

Purcifull, D., Edwardson, J ., Hiebert. E., Gonsalves, D. 1984b. Papaya
ringspot virus. Descriptions of Plant Viruses N o. 292 (N o. 84 revised). Kew,
Surrey, England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

Quiot-Douine, L., Lecoq, H., Quiot, J .B., Pitrat, M., Labonne, G. 1990.
Serological and biological variability of virus isolates related to strains of
papaya ringspot virus. Phytopathology 80:256-263.

Schroeder, W.T., Provvidenti, R. 1971. A common gene for resistance to
bean yellow mosaic virus and watermelon mosaic virus-2 in Pisum sativum.
Phytopathology 61:846-848.

Shukla, D.D., Ward, C.W., Brunt, AA. 1994. Pages 322-392 In: The
Potyviridae. CAB International, Wallingford, UK.

Van der Meer, F.W., Garnett, HM. 1987 . Purification and identification of a
South African isolate of watermelon mosaic virus - Morocco. Journal of
Phytopathology 120:255-270.

47

Wai, T., Grumet, R. 1995a. Inheritance of resistance to the watermelon
strain of papaya ringspot virus in the cucumber line TMG-1. HortScience
302338-340.

Wai, T., Grumet, R. 1995b. Inheritance of resistance to watermelon mosaic
virus in the cucumber line TMG- 1: tissue-specific expression and relationship
to zucchini yellow mosaic virus resistance. Theoretical and Applied Genetics
91:699-706.

CHAPTER 3

Multiple alleles for potyvirus resistance at the zym locus in
cucumber

ABSTRACT

Sources of resistance to several potyviruses have been identified and
characterized within the cucumber (Cucumis sativus L.) germplasm.
Resistance to zucchini yellow mosaic virus (ZYMV) is found in the Dutch
hybrid ‘Dina’ and in an inbred line derived from the Chinese cultivar
‘Taichung Mou Gua’ (TMG-1). Tests of allelism indicate that the genes for
resistance to ZYMV in TMG-1 and Dina are at the same locus. However,
they exhibit different phenotypes in response to cotyledon inoculation with
ZYMV. Dina exhibits a distinct veinal chlorosis and accumulation of virus
limited to the first and/or second true leaves. TMG-1, on the other hand, is
symptom-free and lacks virus accumulation. In addition, the distinct veinal
chlorosis phenotype in Dina is dominant to the symptom-free phenotype in
TMG-1 and not due to a separate gene. These results indicate that a series of
alleles occurs at the zym locus and that the alleles differ in their effectiveness
and dominant relationships with Zym+ > zymDilrm > zymTMG'l. In addition to
ZYMV resistance, TMG-1 also possesses resistance to watermelon mosaic

virus (WMV), the watermelon strain of papaya ringspot virus (PRSV-W) and

48

49

the Moroccan watermelon mosaic virus (MWMV), all of which appear to be at
the same, or tightly linked loci. Our studies reveal that in addition to ZYMV
resistance, Dina also possesses resistance to PRSV-W and MWMV, and that
the gene for MWMV resistance is at the same locus as the ZYMV resistance.
In addition, tests of allelism indicate that the gene for resistance to MWMV
in TMG-1 and Dina are at the same locus. Interestingly, in contrast to
ZYMV inoculation, Dina does not exhibit the distinct veinal chlorosis
phenotype when inoculated with PRSV-W or MWMV. Collectively, these
observations suggest that the genes conferring resistance to ZYMV, WMV,
and MWMV may be part of a gene cluster for potyvirus resistance in

cucumber.

INTRODUCTION

Cucumbers (Cucumis sativus L.) are subject to severe losses due to an
array of potyviruses, including zucchini yellow mosaic virus (ZYMV),
watermelon mosaic virus (WMV), the watermelon strain of papaya ringspot
virus (PRSV-W), zucchini yellow fleck virus (ZYFV), and the Moroccan
watermelon mosaic virus (MWMV) (Lisa and Lecoq, 1984; Purcifull et al,
1984a,b; McKern et al, 1993; Gilbert-Albertini et al, 1995). Sources of
resistance to several of these viruses have been identified and characterized
within the cucumber germplasm. Resistance to ZYMV is found in the Dutch

hybrid ‘Dina’ (Abul Hayja and Al-Shahwan, 1991) and in an inbred line

50

derived from the Chinese cultivar ‘Taichung Mou Gua’ (TMG- 1) (Provvidenti,
1985). In addition, TMG also possesses resistance to WMV, ZYFV, PRSV-W
and MWMV (Provvidenti, 1985; Gilbert-Albertini et al, 1995; Kabelka and
Grumet, 1996).

In Dina, resistance to ZYMV is conferred by a single recessive gene
(Abul Hayja and Al-Shahwan, 1991). In TMG-1, resistances to ZYMV, ZYFV,
and MWMV (Provvidenti, 1987; Gilbert-Albertini et al, 1995; Kabelka and
Grumet, 1996, respectively) are each conferred by single recessive genes.
Resistance to PRSV-W is due to a single dominant, or incompletely dominant,
gene (W ai and Grumet, 1995a) while resistance to WMV involves two types of
resistances under separate genetic control (W ai and Grumet, 1995b). In
addition, the gene conferring resistance to ZYMV in TMG-1 appears to be the
same as, or tightly linked to, genes conferring resistance to WMV and
MWMV (W ai and Grumet, 1995b; Kabelka and Grumet, 1996).

Since there are two sources of resistance to ZYMV reported in the
literature, we sought to examine the relationship between the genes
conferring resistance to ZYMV in TMG-l and Dina, and to test for possible
differences in the performance of the resistance alleles. In addition, since a
genetic relationship exists between resistance to ZYMV and resistance to
other potyviruses in TMG-1, we sought to examine Dina for resistance to

other cucurbit potyviruses.

51

MATERIAL AND METHODS
Cucumber genotypes

The inbred cucumber (Cucumis sativus L.) line ‘TMG-l’, resistant to
ZYMV, WMV, PRSV-W, and MWMV (Provvidenti, 1985; Kabelka and
Grumet, 1996), was provided by Dr. Jack Staub (USDA, University of
Wisconsin at Madison). Progeny of the Dutch hybrid ‘Dina’, true-breeding for
resistance to ZYMV (Abul Hayja and Al-Shahwan, 1991), were initially
provided by Dr. Ken Owens (Peto Seed Company, Inc., Saticoy, CA) and then
increased by self- or sib-pollination in the greenhouse. The susceptible
parental genotype used in this investigation is the open-pollinated cucumber
cultivar ‘Straight-8’ (ST-8; W. Atlee Burpee & Company, Warminster, PA).
Progeny of the crosses between Dina and TMG-1, Dina and ST-8, and ST-8
and TMG-1 were produced in the greenhouse. The F1 progeny of each cross
were either self- or sib-pollinated to produce the F2 generations; backcrosses
were made to both parents.
Virus inocula, inoculation procedures, experimental designs and
data analysis

Propagation of virus inocula, methods of inoculation, experimental
designs, and data analysis were performed as described by Kabelka and
Grumet (1996). The relationship between symptom expression and virus
levels were examined by enzyme-linked immunosorbent assay (ELISA). The

ELISA procedure was performed as described by Wai and Grumet (1995a).

52

ELISA data was analyzed by analysis of variance of a randomized complete
block design with five replicates; individual comparisons were analyzed by
orthogonal contrasts. Various ZYMV isolates used to examine the allelic
relationship in TMG-1 and Dina include: Connecticut, Taiwan 1, Taiwan 2,
Florida, and California, (provided by Dr. R. Provvidenti, Cornell University,
Geneva, NY) and the Israeli non-aphid transmissible strain (NAT) (provided
by Dr. B. Raccah, The Volcani Institute, Bet Dagan, Israel). Two additional
potyviruses used in this investigation were MWMV, provided by Dr. D.
Purcifull (University of Florida, Gainesville, FL), and PRSV-W (PV-380;

American Type Culture Collection, Rockville, MD).

RESULTS
Relationship between the ZYMV resistance in TMG-1 and Dina

To determine if the resistance genes in TMG-1 and Dina are at the
same locus, the progeny of this cross were inoculated at the cotyledon stage
with ZYMV. Seven to ten days post inoculation, the susceptible genotype,
ST-8, developed a systemic mosaic pattern of the foliage while TMG-1, Dina
and their progeny initially appeared symptom-free. However, 14 days post
inoculation, the two resistant genotypes, TMG-1 and Dina, exhibited a
strikingly different phenotype (Figure 3.1). Dina responded with a distinct
veinal chlorosis limited to the first and/or second true leaves. Subsequent

leaves in Dina, however, remained symptom-free. In TMG-1, this distinct

Figure 3.1

53

 

Phenotypic response of the cucumber lines Dina (left) and
TMG-1 (right) 14 days post cotyledon inoculation with ZYMV.
Dina responds with a distinct veinal chlorosis limited to the
first and/or second true leaves with subsequent leaves
symptom-free. In TMG-1, this distinct veinal chlorosis
phenotype is not observed.

54

veinal chlorosis phenotype was not observed. TMG-1 remained symptom-free
or would occasionally exhibit sparse veinal chlorosis. For simplicity,
hereafter the response to ZYMV inoculation in Dina is referred to as a veinal
chlorosis phenotype and in TMG-1 as a symptom-free phenotype.

The distinct veinal chlorosis phenotype in Dina only occurs when
cotyledons are inoculated with ZYMV. When inoculated with ZYMV at the
second and third true leaf stage, no distinct veinal chlorosis on the inoculated
leaves or subsequent leaves was seen (data not shown).

Although Dina and TMG-1 have different phenotypes on the first
and/or second true leaves, evaluation of segregation for resistance and
susceptibility indicates that the two resistance alleles are at the same locus
(Table 3.1). From the third true leaf on, all F1, F2 and backcross progeny of
TMG-1 and Dina remained symptom-free, vigorous and healthy.
Difi’erences in the performance of the resistance alleles in TMG-1
and Dina

Although progeny from the cross between Dina and TMG-1 were all
resistant, there was segregation for the veinal chlorosis phenotype. The
veinal chlorosis appeared to be due to a single gene with the veinal chlorosis
phenotype dominant to the symptom-flee phenotype (Table 3.2). The
reciprocal F1 progeny exhibited the veinal chlorosis phenotype, the reciprocal
F2 progeny segregated in a 1:3 (symptom-free phenotypezveinal chlorosis

phenotype) ratio while the backcross to the TMG-1 parent segregated

Table 3.1 Segregation for resistance to ZYMV in the progeny derived from the

55

cucumber lines TMG-1 and Dina.

 

 

Expected ratio (R28)a

 

Parent or Num_ber of Plantsb If alleles at If alleles at

progeny Resistantc Susceptible same locus different loci

TMG-1 (resistant) 32 0

DINA (resistant) 31 0

Straight-8 (susceptible) 0 32

F1 (TMG x Dina) 64 0 1:0 0:1
(Dina x TMG)

F2(T MG x Dina) 320 0 1:0 7:9
(Dina x TMG)

BC (F1 x TMG) 160 0 1:0 1:1

aMchn
BC (F1 x Dina) 160 0 1:0 1:1

(Dina x F1)

 

llexpected ratios based on inheritance of resistance to ZYMV in TMG-1 as a single recessive

gene and in Dina as a single recessive gene.
bdata pooled from two independent experiments.

°in TMG-1, Dina, and their progeny, symptom expression ranged from no observable symptoms
(symptom-free) to a distinct veinal chlorosis confined to one or two leaves with subsequent

leaves vigorous and healthy.

56

Table 3.2 Response of the cucumber lines TMG-1, Dina and their progeny
14 days post cotyledon inoculation with ZYMV.

 

Number of plants

 

Parent or Symptom-a Distinct veinal Expectedc
progeny free chlorosis ratio X2
TMG-1 32 0 1:0
Dina 0 31 0:1
F1(Dina x TMG) 0 64 0:1
(TMG x Dina)
F2 (Dina x TMG)“ 90 230 1:3 1.51 ns
(T MG x Dina)
BC (F1 x TMG)° 72 88 121 1.40 ns
aMGan
ac (F1 x Dina)‘ 3 157 0:1 0.04 ns
(Dina x F1)

 

ns, non-srgnIf cant X2 value
:plants remain either symptom-free or exhibit an occasional sparse veinal chlorosis.
bsymptom expression of the distinct veinal chlorosis was confined to one or two tme
leaves with subsequent leaves symptom-free.
°expecled ratios based on the distinct veinal chlorosis phenotype dominant to the
dsymptom-free phenotype.
ddata pooled from two independent experiments. Each experiment fits the predicted
ratios. X2 exp1- = 1.41 ns; X2 exp2= 0. 21 ns; X2 homogeneity= 0. 32, df= 1
edata pooled from two independent experiments. Each experiment fits the predicted
ratios. X2 exp1- = 2. 82 ns; X2 exp2= 0. 00 ns; X2 homogeneity= 2.13, df= 1
data pooled from two independent experiments. Each experimentr fits the predicted
ratios. X2 exp1 = 0.08 ns; X2 exp2= 0. 00 ns; X2 homogeneity= 0.28 df= 1

57

1:1 symptom-free phenotypezveinal chlorosis phenotype. Backcrosses to the
parental genotype Dina, all exhibited the veinal chlorosis phenotype limited
to the first and/or second true leaves.

Possible explanations for the observed inheritance of the veinal
chlorosis phenotype are: (1) Dina and TMG-1 have different alleles for
resistance at the same locus, one of which causes the veinal chlorosis, or
(2) there is a separate locus responsible for the veinal chlorosis phenotype.
To distinguish between these possibilities, TMG-1 and Dina were each
crossed to a common susceptible genotype, ST-8, and their progeny tested for
response to cotyledon inoculation with ZYMV (Table 3.3). TMG-1 remained
symptom-free, Dina responded with a veinal chlorosis of the first and/or
second true leaves with subsequent leaves symptom-free and the susceptible
genotype, ST-8, exhibited a systemic mosaic pattern throughout. Consistent
with published results (Provvidenti, 1987 ; Abul Hayja and Al-Shahwan,
1991), segregation ratios of resistance vs. susceptibility indicate that
resistance to ZYMV in both Dina and TMG-1 is conferred by a single
recessive gene. Fourteen days post inoculation, individuals derived from the
Dina and ST-8 cross developed the veinal chlorosis phenotype while
individuals derived from the ST-8 and TMG-1 cross remained symptom-free.
If a separate gene was responsible for veinal chlorosis we would expect
segregating progeny within a cross to show all three phenotypes of mosaic,

veinal chlorosis and symptom-free. This was not the observed. Only one

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59

type of resistance phenotype, either veinal chlorosis or symptom-free was
observed among the progeny fiom a given cross. Thus segregation ratios for
the veinal chlorosis phenotype support a model in which the resistance
alleles in TMG-1 and Dina are performing differently; a separate locus is not
responsible for the veinal chlorosis phenotype.

The three parental genotypes were examined to determine whether the
different phenotypes reflected differences in virus accumulation (Figure 3.2).
Virus titers were measured from the first through the third true leaves post
inoculation with ZYMV. TMG-l remained symptom-flee and showed
essentially no significant virus accumulation (analysis of variance). Dina
exhibited the veinal chlorosis phenotype and significant virus levels in the
first and second true leaves. However, subsequent symptom-free leaves, in
Dina, revealed no detectable virus accumulation. As expected, ST—8 had high
levels of virus accumulation.

The alleles from Dina and TMG-l were also examined for differences
in response to various ZYMV isolates (Table 3.4). None of the isolates
(Taiwan 1, Taiwan 2, Florida, NAT and California) overcame the resistance
in either TMG-1 or Dina. TMG-l remained symptom-free in response to all
tested isolates; Dina exhibited the veinal chlorosis phenotype of the first
and/or second true leaves to all isolates except the California isolate where no

symptoms were observed.

60

 

1.4

1.2 .

Distinct veinal chlorosis

 

 

 

 

 

 

 

 

 

 

 

§ ox - observed on first true rear

é + \A

3 0.6 ‘ u

[Symptom-tree -
0.4 «
o . . f
1 2 3
Leaf Posltlon
[+me-1 ~—CJ-—Dina +374: +Cntrl|
Figure 3.2 Virus accumulation in the cucumber lines TMG-1, Dina and Straight-8

two weeks post cotyledon inoculation with ZYMV. Each point is the
mean +/- SE of five replicate plants. Virus levels were measured by
ELISA.

61

 

 

 

 

 

 

 

 

 

Table 3.4 Response of the cucumber lines TMG-1, Dina and Straight-8 to inoculation with
several potyviruses
Potyvirus TMG-1 Dina Straight-8
ZYMV - Connecticut Resistant Resistant Susceptible
- symptom-free - distinct veinal - systemic mosaic
chlorosis phenotype
- Taiwan 1 Resistant Resistant Susceptible
- symptom-free - distinct veinal - systemic mosaic
chlorosis phenotype
- Taiwan 2 Resistant Resistant Susceptible
- symptom-free - distinct veinal - systemic mosaic
chlorosis phenotype
- Florida Resistant Resistant Susceptible
- symptom-free - distinct veinal - systemic mosaic
chlorosis phenotype
- California Resistant Resistant Susceptible
- symptom-free - symptom-free - systemic mosaic
MWMV Resistant Resistant Susceptible
- symptom-free - symptom-free - systemic rugosity
and silverirg
PRSV-W Resistant Resistant Susceptible
- symptom-free - symptom-free - systemic mgosity

 

 

 

 

 

62

Response of Dina to inoculation with other potyviruses

Previous studies have identified TMG-1 to possess resistance to ZYMV,
WMV, PRSV-W and MWMV (Provvidenti, 1985; Kabelka and Grumet, 1996).
In addition, the resistance gene for ZYMV, in TMG-l, is the same as, or
tightly linked to, the genes for resistance to WMV and MWMV (W ai and
Grumet, 1995b; Kabelka and Grumet, 1996). Since Dina possesses resistance
to ZYMV, we sought to determine if it is also resistant to other potyviruses
and, if so, to examined whether a similar genetic relationship exists between
the resistance gene(s). We, therefore, tested Dina for response to PRSV-W
and MWMV. Following cotyledon inoculation with PRSV-W and MWMV,
Dina remained symptom-free and, interestingly, exhibited no veinal chlorosis
of the first and/or second true leaves as seen with ZYMV inoculation
(Table 3.4).

The resistance to MWMV in Dina was further examined by
characterizing the mode of inheritance and determining its relationship to
the ZYMV resistance gene. Following rub inoculation with MWMV, the
susceptible genotype, ST-8, showed a mild systemic rugosity and silvering of
the true leaves 14 days post inoculation (Table 3.5). Symptom intensity of
MWMV varied during the course of plant growth; the rugosity and silvering
of the true leaves would fade occasionally but then resurge to full expression.
Dina remained symptom-free. The F1 progeny of Dina and ST-8 were

susceptible, the F2 progeny segregated in a 1:3 [resistant(R):susceptn'ble(S)]

63

Table 3.5 Response of the cucumber lines Dina, Straight-8 and their
progeny to inoculation with MWMV

 

 

Parent or Number of plants Expected

progeny Resistant Susceptible ratio (R:S)a X2
Dina (D) 20 0

Straight-8 (ST8) 0 20

F1 (DxST8) 0 20 0:1

F2 (DxST8) 35 92 1:3 0.317 ns
BC (F1xD) 65 73 1:1 0.356 ns
BC (F1xST8) O 20 0:1

 

ns, non-significant X2 value
aexpected ratios based on a single recessive gene model, R = resistant,
S = susceptible.

64

ratio while the backcross to the resistant parent segregated in a 1:1 (R:S)
ratio. Backcrosses to the susceptible parent were all susceptible. These
segregation ratios support a model in which the inheritance of resistance to

MWMV in Dina is conferred by a single recessive gene.

Genetic relationship between resistance to ZYMV and resistance to
MWMV in Dina

By analogy to ZYMV resistance in TMG-l, one would predict that the
gene for MWMV resistance in Dina is the same as, or tightly linked to, the
gene for ZYMV resistance. This hypothesis was tested in two ways:

(1) sequential inoculation of Dina, ST-8, and their progeny with ZYMV and
MWMV and (2) tests for segregation of MWMV resistance in the progeny of
Dina and TMG— l.

The sequential inoculation procedure consisted of cotyledon
inoculation of Dina, ST-8, and their progeny with ZYMV followed by true leaf
inoculation of the resistant individuals with MWMV (Table 3.6). Additional
control plants were included to verify successful inoculation at the true leaf
stage with MWMV. Symptoms of ZYMV, a systemic mosaic pattern,
developed on the susceptible ST-8 plants approximately seven days post
cotyledon inoculation. Fourteen days post cotyledon inoculation with ZYMV,
all resistant individuals developed veinal chlorosis of the first and/or second
true leaves with subsequent leaves symptom-free. The resistant individuals

were then sequentially inoculated on the asymptomatic fourth and fifth true

65

Table 3.6 Response of the cucumber lines Dina, Straight-8 and their progeny to cotyledon
inoculation with ZYMV followed by true leaf inoculation of resistant individuals
with MWMV.

 

Sequential inoculation of

 

 

 

 

Cotyledon inoculation ZYMV resistant individuals
with ZYMV with MWMV at true leaf stage
Parent or Total Number of plants Number of leants
progeny plants Resistant Susceptible Resistant Susceptible
Dina (D) 20 20 0 20 0
Straight-8 (ST8): 10 0 10 -- --
Straight-8 (ST8)b 10 — — 0 10
F1 (DxST8): 10 0 10 -- —
F1 (DxST8)b 1O - — 0 10
F2 (DxST8)° 320 75 245 75 0
BC (DxST8)xD" so 38 42 38 o

 

:control plants to verify successful inoculation at cotyledon stage with ZYMV.
t’control plants to verify successful inoculation at true leaf stage with MWMV.
cdata fits the expected and predicted segregation ratios based on resistance to ZYMV conferred
by a single recessive gene. X2- - 0.33 (non-significant X2 value)
6data fits the expected and predicted segregation ratios based on resistance to ZYMV conferred by
a single recessive gene X2— - 0 12 (non-significant X2 value)

66

leaves with MWMV (18 days post cotyledon inoculation with ZYMV).
Symptoms of MWMV developed approximately 14 days post true leaf
inoculation on the susceptible genotype, ST-8, and consisted of mild rugosity
and silvering of the true leaves. In all cases, those individuals resistant to
cotyledon inoculation with ZYMV, although initially responding with a veinal
chlorosis limited to the first and/or second true leaves, remained free of
symptoms upon true leaf inoculation with MWMV.

The second approach tested for segregation of MWMV resistance
among the progeny of Dina and TMG-1 (Table 3.7). Fourteen days post
inoculation the susceptible genotype, ST-8, developed a mild rugosity and
silvering of the true leaves while the resistant genotypes TMG-1 and Dina
remained symptom-free throughout. Evaluation of segregation for resistance
and susceptibility in the progeny of TMG-l and Dina indicates that the two
MWMV resistance alleles are located at the same locus as all individuals of

the F1, F2 and backcross generations remained symptom-free.

DISCUSSION

Segregation ratios among the F1, F2 and backcross progeny of TMG-l
and Dina indicate that the ZYMV resistance alleles are at the same locus.
However, the two sources of ZYMV resistance seem to perform differently.
Dina responds to ZYMV cotyledon inoculation with a distinct veinal chlorosis

of the first and/or second true leaves with subsequent leaves symptom-flee.

67

Table 3.7 Segregation for resistance to MWMV in the progeny derived from the
cucumber lines TMG-1 and Dina.

 

 

Expected ratio (R:S)a

 

Parent or Number of Plants If alleles at If alleles at
progeny Resistant Susceptible same locus different loci
TMG-1 (resistant) 20 O
Dina (resistant) 20 O
Straight-8 (susceptible) 0 20
F1 (T MG x Dina) 20 0 1:0 0:1
(Dina x TMG)
F2(T MG x Dina) 140 0 1:0 7:9
(Dina x TMG)
BC (F1 x TMG) 80 0 1:0 1:1
(T MG x F1)
80 (F1 x Dina) 80 0 1:0 1:1

(Dina x F1)

 

a'expected ratios based on inheritance of resistance to MWMV in TMG-1 as a single recessive

gene and in Dina as a single recessive gene.

68

The progeny of TMG-l and Dina segregated for the observed distinct veinal
chlorosis phenotype which was dominant to the symptom-flee phenotype. An
alternate possibility, that an additional factor was responsible for the distinct
veinal chlorosis phenotype, was ruled out based on evaluation of the
segregating progeny of TMG-l and Dina crossed to a common susceptible
background.

The symptoms in Dina appear also to reflect virus replication and
movement. Consistent with previous studies (W ai and Grumet, 1995a;
Al-Shahwan et al, 1995), detectable virus, limited to the first and second true
leaves, was observed in Dina while no detection of virus accumulation was
present in TMG-l. The mechanism in Dina that limits virus to the first and
second true leaves is unknown. However, studies to examine the mechanism
of resistance to ZYMV in Dina are currently in progress.

The distinct veinal chlorosis phenotype in Dina only occurs when
cotyledons are inoculated with ZYMV. When inoculated with ZYMV at the
second and third true leaf stage, no distinct veinal chlorosis on the inoculated
leaves or subsequent leaves was seen. This suggests a tissue-specific
expression of the resistances (cotyledon vs. true leaf) to ZYMV in Dina.
Potyvirus resistance exhibiting a tissue-specific expression has been observed
previously in cucurbits. In TMG-1, resistance to WMV is conferred by two
independently segregating factors; one resistance is expressed in the

cotyledons and throughout the plant while the second resistance is expressed

69

only in true leave tissue (W ai and Grumet, 1995b). Tissue specificity was
also observed in muskmelon cultivars resistant to PRSV-W (Gibb et al, 1994).
One resistant cultivar ‘Cinco’ exhibited only occasional mild systemic
symptoms on cotyledon inoculated plants. The hybrid of Cinco crossed to a
susceptible cultivar ‘Planter’s Jumbo’ exhibited mild systemic symptoms
upon cotyledon inoculation but remained symptom-free upon true leaf
inoculation.

From the data obtained, it appears that a series of alleles occur at the
zym gene locus and that these alleles differ in their effectiveness and
dominance relationships. Both of the resistance alleles are recessive to the
wild type susceptible allele (Zym+). The veinal chlorosis phenotype
associated with the resistance allele from Dina (zymD‘M) is dominant to the
symptom-flee phenotype associated with the resistance allele from TMG-l
(zymTMG-l). Therefore, at the zym locus, Zym” > zymDim > zymTMG'l. Of the two
resistances examined, however, the allele from TMG-1 would represent the
most effective resistance as no distinct veinal chlorosis phenotype was seen
and no accumulation of virus was evident. Possibly, the relative
effectiveness of the different alleles at the zym gene locus is proportional to
the extent by which they reduce virus multiplication or prevent movement.

Series of alleles occurring at specific loci and differing in their
effectiveness have been described previously in literature. For example, in

Capsicum, response to infection by tobacco mosaic virus (TMV) was found to

70

be controlled by a series of alleles where L provides localization of TMV, Li
provides “imperfect” localization of TMV and L+ causes mottling (Holmes,
1937). In tomato, three resistance factors, Tm-I, Tm-2 and Tm-22 confer
resistance to TMV (Hall, 1980; Stobbs and MacNeill, 1980). Of the three,
Tm-2 and Tm-22 are considered allelic. Both Tm-2 and Tm-22 can induce a
hypersensitive response to TMV but Tm-22 is described as a more effective
allele as the hypersensitive response is more extreme. In addition, TMV
strains 1 and 2 overcome Tm-2 but not Tm-22.

As mentioned earlier, previous studies have identified TMG-l to
possess resistance to several potyviruses including ZYMV, WMV, PRSV-W
and MWMV (Provvidenti, 1985; Kabelka and Grumet, 1996). In addition, the
resistance gene for ZYMV, in TMG-l, appears to be the same as, or tightly
linked to, the genes for resistance to WMV and MWMV (W ai and Grumet,
1995b; Kabelka and Grumet, 1996). Our studies reveal that, like TMG-1,
Dina also possesses resistance to multiple potyviruses including PRSV-W
and MWMV. Interestingly, however, upon inoculation with PRSV-W or
MWMV, Dina remains symptom-free; not exhibiting the veinal chlorosis
phenotype as seen with several isolates of ZYMV except the California
isolate. Although MWMV, PRSV-W and ZYMV-California are distinct
potyviruses, one shared feature is the time interval preceding observable
symptoms in the susceptible genotype. While symptoms of most ZYMV

isolates developed approximately 7 -10 days post inoculation symptoms of

71

MWMV, PRSV-W and ZYMV-California did not become evident until
approximately 14-2 1 days post inoculation. Possibly this difference in time of
onset represents a slower rate of virus multiplication or movement resulting
in lack of veinal chlorosis in Dina. Possibly it represents a difference in
resistance mechanisms. As virus accumulation was observed in the first and
second true leaves of Dina post inoculation with ZYMV-Connecticut, it would
be interesting to determine if a lack of veinal chlorosis represents a lack of
virus accumulation. It is possible, however, to have virus accumulation in
the absence of symptoms. Previous studies with TMG-l and PRSV-W (in
contrast to ZYMV) have indicated that although TMG-l is symptom-flee post
inoculation with PRSV-W, there is PRSV-W accumulation (W ai and Grumet,
1995a).

The resistance to MWMV in Dina was further examined by
characterizing the mode of resistance and determining its relationship to the
ZYMV resistance gene. Resistance to MWMV in Dina, like resistance to
ZYMV, is conferred by a single recessive gene. Sequential inoculation of
progeny possessing resistance to ZYMV followed by MWMV suggests that
both resistances are conferred by the same gene, or two tightly linked genes.
In addition, tests for segregation of MWMV resistance in the progeny of Dina
and TMG-1 indicate the two MWMV resistance alleles are at the same locus.

Collectively, analysis of data suggests that one gene, or two tightly

linked genes, confer resistance to ZYMV and MWMV in both TMG-l and

72

Dina. Tests of allelism suggest the genes for resistance found in TMG-l and
Dina are at the same locus. However, as evidenced by Dina’s strikingly
different response to inoculation with ZYMV, both with regard to symptom
expression, virus accumulation and dominance relationships, the ZYMV
resistance alleles in TMG-l and Dina are likely to be different indicating that
a series of alleles exist at the zym locus where Zym+ > zymD‘M > zymTMG-l.
Since the differences in response were observed only for ZYMV and not
MWMV it is possible that rather than a single gene conferring resistance to
all three potyviruses, the MWMV, WMV and ZYMV genes may be part of a

gene cluster for potyvirus resistance in cucumber.

73

REFERENCES

Abul Hayja, Z., Al-Shahwan, I.M. 1991. Inheritance of resistance to zucchini
yellow mosaic virus in cucumber. Journal of Plant Diseases and Protection
98:301-304.

Al-Shahwan, I.M., Abdalla, O.A., Al-Saleh, MA. 1995. Response of
greenhouse-grown cucumber cultivars to an isolate of zucchini yellow mosaic
virus (ZYMV). Plant Disease 79:898-901.

Gibb, K.S., Padovan, A.C., Herrington, ME. 1994. Nature of resistance in
Cucumis melo cultivars to papaya ringspot virus type W. Australian Journal
of Agricultural Research 45:633-645.

Gilbert-Albertini, F., Pitrat, M., Lecoq, H. 1995. Inheritance of resistance to
zucchini yellow fleck virus in Cucumis sativus L. HortScience 30:336-337.

Hall, T.J. 1980. Resistance at the Tm-2 locus in the tomato to tomato mosaic
virus. Euphytica 29:189-197.

Holmes, FD. 1937. Inheritance of resistance to tobacco-mosaic disease in
the pepper. Phytopathology 27:637-642.

Kabelka, E., Grumet, R. 1996. Inheritance of resistance to the Moroccan
watermelon mosaic virus in the cucumber line TMG-1 and its relationship to
zucchini yellow mosaic virus resistance. Manuscript in preparation.

Lisa, V., Lecoq, H. 1984. Zucchini yellow mosaic virus. Descriptions of
Plant Viruses No. 282. Kew, Surrey, England: Commonwealth Agricultural
Bureax/Association of Applied Biologists.

McKern, N.M., Strike, P.M., Barnett, O.W., Ward, C.W., Shukla, DD. 1993.
Watermelon mosaic virus-Morocco is a distinct potyvirus. Archives of
Virology 131:467-473.

Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of
Cucumis sativus. Cucurbit Genetics Cooperative Report 8:12.

Provvidenti, R. 1987. Inheritance of resistance to a strain of zucchini yellow
mosaic virus in cucumber. HortScience 222102-103.

74

Purcifull, D., Hiebert, E., Edwardson, J. 1984a. Watermelon mosaic virus-2.
Descriptions of Plant Viruses No. 293 (No. 63 revised). Kew, Surrey,
England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

Purcifull, D., Edwardson, J ., Hiebert. E., Gonsalves, D. 1984b. Papaya
ringspot virus. Descriptions of Plant Viruses No. 292 (No. 84 revised). Kew,
Surrey, England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

Stobbs, L.W., MacNeill, EH. 1980. Response to tobacco mosaic virus of a
tomator cultivar homozygous for gene Tm-2. Canadian Journal of Plant
Pathology 225-11.

Wai, T., Grumet, R. 1995a. Inheritance of resistance to the watermelon
strain of papaya ringspot virus in the cucumber line TMG-1. HortScience
302338-340.

Wai, T., Grumet, R. 1995b. Inheritance of resistance to watermelon mosaic
virus in the cucumber line TMG-1: tissue-specific expression and relationship
to zucchini yellow mosaic virus resistance. Theoretical and Applied Genetics
91:699-706.

CHAPTER 4
Characterization of genes controlling resistance to the watermelon

strain of papaya ringspot virus in cucumber: allelism and
cosegregation with other potyviruses

ABSTRACT

The watermelon strain of papaya ringspot virus (PRSV-W) is one of
several potyviruses causing damage to cucurbit production worldwide.
Within the cucumber germplasm, sources of resistance to PRSV-W have been
identified in ‘Surinam’, a cultivar from South America, and ‘Taichung Mou
Gua’ (TMG-l), a single plant selection from a Chinese cultivar from Taiwan.
Each resistance is due to a single gene. Although resistance from Surinam is
recessive, and the source from TMG-l is incompletely dominant, segregation
ratios among the progeny of TMG-l and Surinam indicate that the
resistances to PRSV-W are at the same locus. The different dominance
relationships, which are exhibited even when crossed to a common
susceptible parent, may indicate different alleles or the influence of other
modifying factors. Unlike Surinam, TMG-1 also possesses resistances to
several other potyviruses all of which appear to be at the same locus, or
tightly-linked loci. This study indicated that resistance to PRSV-W in

TMG-1 is also at the same locus, or tightly-linked to ZYMV resistance.

75

76

INTRODUCTION

The watermelon strain of papaya ringspot virus (PRSV-W) is one of
several potyviruses causing damage to cucurbit production worldwide
(Purcifufl et al, 1984b). Other cucurbit potyviruses include zucchini yellow
mosaic virus (ZYMV), watermelon mosaic virus (WMV) and the Moroccan
watermelon mosaic virus (MWMV) (Lisa and Lecoq, 1984; Purcifull et al,
1984a; McKern et al, 1993). In cucumber (Cucumis sativus L.), Provvidenti
(1985) identified two accessions, ‘Surinam’, a cultivar from South America,
and ‘Taichung Mou Gua’ (TMG-1), a single plant selection from a Chinese
cultivar from Taiwan, as sources of resistance to PRSV-W. The inheritance
of resistance to PRSV-W in both Surinam and TMG-l have been
characterized. In the cultivar Surinam, resistance to PRSV-W is conferred by
a single recessive gene (Wang et al, 1984). In TMG-l, segregation ratios
indicated that resistance to PRSV-W is conditioned by a single dominant
gene. However, the effectiveness of this resistance varied at times suggesting
either incomplete dominance or the involvement of other genetic or
environmental factors (W ai and Grumet, 1995a).

Although Surinam only possesses resistance to PRSV-W, TMG, also is
resistant to ZYMV, WMV, MWMV and zucchini yellow fleck virus (ZYFV)
(Provvidenti, 1985; Gilbert-Albertini et al, 1995; Kabelka and Grumet,
1996a). Resistance to ZYMV is conferred by a single recessive gene

(Provvidenti, 1987), as is resistance to ZYFV and MWMV (Gilbert-Albertini

77

et al, 1995; Kabelka and Grumet, 1996a) while resistance to WMV involves
two types of resistances under separate genetic control (W ai and Grumet,
1995b). In addition, the gene conferring resistance to ZYMV appears to be
the same as, or tightly linked to, genes conferring resistance to WMV and
MWMV (W ai and Grumet, 1995b; Kabelka and Grumet, 1996a).

Since there are two sources of resistance to PRSV-W with reported
differences in dominance relationships, we sought to examine the allelic and
dominance relationships between the gene(s) conferring resistance to
PRSV-W in TMG-l and Surinam and to compare performance when crossed
to a common susceptible parent. In addition, since the gene conferring
resistance to ZYMV in TMG-1 appears to be the same as, or tightly linked to,
the genes conferring resistance to WMV and MWMV we sought to determine
if a similar relationship exists between resistance to ZYMV and resistance to

PRSV-W in TMG- 1.

MATERIAL AND METHODS
Cucumber genotypes

The inbred cucumber (Cucumis sativus L.) lines ‘TMG-l’, resistant to
ZYMV, WMV, PRSV-W and MWMV (Provvidenti, 1985; Kabelka and
Grumet, 1996a) and ‘Surinam Local’, resistant to PRSV-W (Wang et al,
1984), were provided by Dr. Jack Staub (USDA, University of Wisconsin at

Madison) and Dr. R. Provvidenti (Cornell University, Geneva, NY),

78

respectively. The two susceptible parental genotypes used in this
investigation were the inbred lines ‘Wisconsin-2 757’ (WI-27 57 ; Peterson et al,
1982; provided by Dr. Jack Staub) and ‘Straight-8’ (ST-8; W. Atlee Burpee
and Company, Warminster, PA). Progeny of the crosses between TMG-l and
Surinam, ST-8 and Surinam, and ST-8 and TMG-l, were produced in the
greenhouse. The F1 progeny of each cross were self-pollinated to produce the
F2 generation or backcrossed to both parents. Production of the F 1, F2 and
backcross progeny of WI-27 57 and TMG-l was described by Wai and Grumet
(1995a). F3 families, used to analyze the relationship between resistance to
ZYMV and PRSV-W, were produced by first screening 1,479 F2 (WI-2 757 x
TMG- 1) individuals for resistance to ZYMV in the greenhouse.
Three-hundred-seventy-three ZYMV resistant F2 individuals [3:1 (SzR);
X2 = 0.08, non-significant] were then transplanted to the field. As WI-2 7 57 is
gynoecious (dominant trait; Peterson et al, 1982), monoecious individuals
were identified and self-pollinated by hand under controlled conditions.
Fifty-one F3 families were produced.
Virus inocula, inoculation procedures, experimental designs and
data analysis

PRSV-W (PV-380; American Type Culture Collection, Rockville, MD)
and ZYMV (Connecticut Strain) were propagated in Cucurbita pepo L. cvs.
‘Black Beauty’ (SeedWay Inc., Elizabethtown, PA) or ‘Midas’ (Willhite Seed,

Poolville, TX). Propagation of virus inocula, methods of inoculation,

79

experimental designs, and data analysis were performed as described by

Kabelka and Grumet (1996a).

RESULTS

Relationship between the PRSV-W resistances in TMG-l and
Surinam

To determine if the resistance genes in TMG-1 and Surinam are at the
same locus, the progeny of this cross were inoculated with PRSV-W.
Approximately 14-2 1 days post inoculation, the susceptible genotype ST-8
developed a systemic rugosity of the foliage while the resistant genotypes,
TMG-1 and Surinam, remained symptom-flee (Table 4.1). Evaluation of
segregation for resistance and susceptibility in the progeny of TMG-1 and
Surinam indicated that the two resistance alleles are at the same locus as all
individuals of the F1, F2 and backcross generations remained free of
symptoms.

Difl'erences in the performance of the resistance alleles in TMG-l
and Surinam

Progeny analysis of TMG- l and Surinam indicated that the resistance
genes are at the same locus, however, one is reported to be recessive (Wang et
a1, 1984) and the other dominant, or incompletely dominant (W ai and
Grumet, 1995a). Since the reported inheritance studies for TMG-l and
Surinam were performed with different susceptible lines, a direct comparison

of their dominance relationships could not be made. Therefore, in this study,

80

Table 4.1 Segregation for resistance to PRSV-W in the
progeny derived from the cucumber lines
TMG-1 and Surinam.

 

 

 

 

Parent or No. Plants“
Progeny Resistant Susceptible
TMG-1 (resistant) 16 0
Surinam (resistant) 16 0
Straight-8 (susceptible) 0 16
F1 (T MGxSurinam) 16 0
F2 320 0
BC (F1xTMG) 80 0
(T MGxF 1)
BC (F1xSurinam) 200 0
(Suriname1)

 

“data pooled from two independent experiments.

81

TMG-l and Surinam were each crossed to a common susceptible genotype
(ST-8) and their progeny tested to further examine the dominance
relationships of each allele. In addition, since the inheritance of resistance to
PRSV-W was initially characterized utilizing WI-27 57 and TMG-l, their
progeny were included for comparative purposes.

Consistent with previous studies (Wang et al, 1984; Wai and Grumet,
1995a), segregation ratios indicated that the inheritance of resistance to
PRSV-W in Surinam is conferred by a single recessive gene and in TMG-l by
a single incompletely dominant gene (Table 4.2). Plants were scored visually
when symptoms were optimally expressed, approximately 14-21 days post
inoculation. Following rub inoculation with PRSV-W, the resistant
genotypes, TMG-l and Surinam, remained fiee of symptoms. Symptom
expression on the susceptible genotype ST-8 was a systemic rugosity while
the susceptible genotype WI-2 7 57 exhibited a systemic silver banding and/or
rugosity. The F1 progeny of ST-8 and Surinam were susceptible exhibiting
symptoms throughout the plant that were as severe as the susceptible
parent. The F2 progeny segregated in a 1:3 (R:S) ratio while the backcross to
the resistant parent segregated in a 1:1 (R:S) ratio. Progeny backcrossed to
the susceptible parent were all susceptible.

The F1 progeny of TMG-1 crossed with WI-27 57 or ST-8, however,
exhibited an intermediate phenotype where the young leaves and growing

points were symptom-free but the older leaves exhibited rugosity and/or

82

Table 4.2 Response of the cucumber lines TMG-1, Surinam, Straight-8, Wl-2757 and their progeny to inoculation with

 

 

 

 

 

 

 

PRSV-W
Number of Plants

Parent or Expected
progeny Rem mm mm _rat_ig_s_ >53
TMG-1 15 0 0
Surinam (S) 15 0 0
Straight-8 (ST8) 0 0 15
Wl-2757 (2757) 0 0 15
F1 (2757xT) 0 20 0
F1 (ST8xT) 0 20 0
F1 (ST8xS) 0 0 40
F2(2757xT) 14 29 15 1:2:1 0.04 ns
F2(ST8xT) 33 55 32 1:2:1 0.85 ns
F2(ST8xS) 33 0 86 1 :3 0.33 ns
BC (2757xT)xT 21 19 0 1 :1 0.04 ns
BC (ST8xT)xT 28 32 0 1 :1 0.46 ns
BC (ST8xS)xS 32 0 28 1 :1 0.04 ns
BC (ST8xT)xST8 0 29 31 1 :1 0.04 ns
BC 2757x(2757xT) 0 17 23 1 :1 0.62 ns
BC (ST8xS)xST8 0 0 60 1 :1 0.00 ns

 

ns, non-significant X2 value.

“plants symptom-free, vigorous and healthy.

:young leaves and growing points remain symptom-free but older leaves exhibit rugosity and/or silver banding.
:systemlc rugosity and/or silver banding throughout the plant similar to that exhibited by either susceptible genotype.
“expected ratios based on inheritance of resistance to PRSV-W in TMG-1 as an incompletely dominant gene
and in Surinam as a single recessive gene.

83

silver banding. The F2 progeny segregated in a 1:2:1
[resistant(R):intermediate(I):susceptible(S)] ratio. Progeny backcrossed to
the resistant parent segregated in a 1:1 (R:I) ratio while progeny backcrossed
to the susceptible parent segregated in a 1:1 (1:8) ratio. It should be noted,
however, that while the young leaves and growmg points of individuals
exhibiting the intermediate phenotype remained symptom-free, the rugosity
and/or silver banding of the older leaves varied from nearly symptom-free to
severe. Although symptom severity of the intermediate phenotype varied
with environment, it did not appear to be affected by the diflerent susceptible
parents. When the progeny of ST-8 x TlVIG-l and WI-2 7 57 x TMG-1 were
grown side-by-side, the symptom severity of the intermediate phenotype was

equivalent.

Genetic relationship between resistance to ZYMV and resistance to
PRSV-W in TMG-1

Earlier studies have identified sources of resistance in cucumber to
several potyviruses. As mentioned earlier, TMG possesses resistance to
PRSV-W, ZYMV, WMV, ZYFV, and MWMV (Provvidenti, 1985; Gilbert-
Albertini et al, 1995; Kabelka and Grumet, 1996a) while the Dutch hybrid
Dina is resistant to ZYMV, PRSV-W, and MWMV (Abul Hayja and
Al-Shahwan, 1991; Kabelka and Grumet, 1996b). In addition, the alleles for
ZYMV, WMV and MWMV resistance in TMG-l and the alleles for ZYMV

and MWMV resistance in Dina appear to be at the same locus, or tightly

84

linked loci (W ai and Grumet, 1995b; Kabelka and Grumet, 1996b). The
relationship of PRSV-W to the other potyviruses, however, is not known.
Interestingly, unlike TMG- l and Dina which are resistant to multiple
potyviruses including PRSV-W, Surinam is susceptible to ZYMV, WMV
(Wang et al, 1984), and MWMV (data not shown). As would be expected due
to the recessive inheritance of resistance to ZYMV (Provvidenti, 1987) the F1
progeny of TMG-1 and Surinam which are resistant to PRSV-W are
susceptible to ZYMV (data not shown).

To examine the relationship of PRSV-W resistance to the other
potyvirus resistances, we tested for association between ZYMV and PRSV-W.
To test this, two approaches were taken. The first approach was sequential
inoculation of backcross progeny of (ST-8 x TMG-l) x TMG- 1. This procedure
consisted of cotyledon inoculation with PRSV-W followed by true leaf
inoculation of the resistant individuals with ZYMV (Table 4.3A). In a
separate experiment, this procedure was reversed, the cotyledons were
inoculated with ZYMV and true leaves of the resistant individuals were
inoculated with PRSV-W (Table 4.3B). In all experiments, additional control
plants were included to verify successful inoculation at the true leaf stage.
Symptoms of PRSV-W in the susceptible genotype, ST-8, were as described
earlier. Symptoms of ZYMV, a systemic mosaic pattern of the true leaves,
developed on the susceptible genotype, ST-8, approximately seven days post

cotyledon or true leaf inoculation. The resistant genotype, TMG-1, remained

 

85

Table 4.3 Response of TMG-1, Straight-8, the F. and backcross progeny to sequential
inoculation with ZYMV and PRSV-W.

 

A. Cotyledon inoculation with PRSV-W followed by true leaf inoculation of resistant individuals
with ZYMV.
Sequential inoculation of

 

 

 

Cotyledon inoculation PRSV-W resistant individuals

with PRSV-W with ZYMV at true leaf stage
Parent or Total Number of plaints Number of plants
progeny plants Resistant intermediate“ Susceptible Resistant Susceptible
TMG-1 (T) 10 10 0 0 1 0 0
Straight-8 (sre)" 10 o o 10 -- .-
Straight-8 (ST8)° 5 - - - 0 5
F1(ST8xT)“ 10 o 10 o -- —
F1(ST8xT)c 5 - — - 0 S
ec (ST8xT)xT“ 160 77 83 o 77 o

 

“young leaves and growing points remain symptom-free but older leaves exhibit rugosity and/or silver
bbanding
bcontrol plants to verify successful inoculation at cotyledon stage with PRSV-W
:control plants to verify successful inoculation at true leaf stage with ZYMV
“data fits the expected and predicted2 segregation ratios based on resistance to PRSV-W conferred by
an incompletely dominant gene. X“ = 0.16 (non-SIgnIf cant X“ value)

 

B. Cotyledon inoculation with ZYMV followed by true leaf inoculation of resistant individuals
with PRSV-W.

Sequential inoculation of

 

 

 

 

Cotyledon inoculation ZYMV resistant individuals

with ZYMV with PRSV-W at true leaf stage
Parent or Total Number of giants Number of plants
progeny plants Resistant Susceptible Resistant Intermediate“ Susceptible
TMG-1 (T) 10 10 0 10 0 0
Straight-8 (ST8)b 10 o 10 - — —
Straight-8 (ST8)° 5 — — 0 0 5
F1(ST8xT)“ 10 o 10 — — -
F1(ST8xT)c 5 — — 0 5 0
3c (ST8xT)xT“ 160 78 82 78 o o

 

“young leaves and growing points remain symptom-free but older leaves exhibit rugosity and/or silver

banding

“control plants to verify successful inoculation at cotyledon stage with ZYMV

:control plants to verify successful inoculation at true leaf stage with PRSV-W

“data fits the expected and predicted segregation ratios based on resistance to ZYMV conferred by

a single recessive gene X“: 0.06 (non-SIgnIt' cant X“ value)

86

free of symptoms to both PRSV-W and ZYMV inoculation. In all cases, those
individuals resistant to cotyledon inoculation with the first virus remained
symptom-flee upon true leaf inoculation with the second virus.

The second approach to evaluate whether a relationship exists
between the resistance to ZYMV and resistance to PRSV-W was to screen F3
families that were produced by self-pollination of F2 individuals selected for
resistance to ZYMV. Progeny from each ZYMV-resistant F2 individual were
inoculated either with PRSV-W or ZYMV (Table 4.4). As expected, all
families were resistant to ZYMV. If the two resistances had been segregating
independently, 9/16th of the F3 families should be susceptible or segregating
for susceptibility to PRSV-W. There was, however, no segregation for
susceptibility to PRSV-W either within or among the F3 families screened.
These F3 families previously had been screened for response to MWMV and

all were resistant (Kabelka and Grumet, 1996a).

DISCUSSION

Segregation ratios among the progeny of TMG-l x Surinam,
ST-8 (susceptible genotype) x TMG-l, and ST-8 x Surinam, indicated that the
resistances to PRSV-W are at the same locus but may be due to difl'erent
alleles. The source of resistance from Surinam is recessive whereas the
source from TMG-l is incompletely dominant. While three distinct classes of

response (resistant, intermediate, and susceptible) were observed in the

 

87

Table 4.4 PRSV-W and ZYMV inoculation of F3 families
homozygous recessive at the zym locus derived from
the inbred cucumber lines TMG-1 and WI-2757.
F3 families were produced by self-pollinating F2 individuals

 

selected for resistance to ZYMV.

No. F3 families“ No. F3 families susceptible
Potflirus showing resistance or segregating for resistance
PRSV-W 51 0
ZYMV 51 0

 

“tests of F3 families included ten individuals/family

 

88

segregating population of TMG-l crossed with WI-2 7 57 or ST-8, the
intermediate phenotype varied; the young leaves and growing points
remained symptom-free but the older leaves exhibited various degrees of
rugosity and/or silver banding ranging fiom nearly symptom-free to severe.
Previous studies evaluating the inheritance of resistance to PRSV-W in
TMG-l (utilizing WI-2 7 57 as a susceptible genotype) describe resistance as a
single dominant gene (W ai and Grumet, 1996a). However, full expression of
resistance in the F1 progeny depended on optimal growing conditions. When
grown in cool weather or low light levels, occasional mild symptoms were
observed. Although the resistance to PRSV-W in TMG-l appeared
incompletely dominant in this study, possibly suggesting a series of alleles at
the Prsv-2 locus, the existence of modifying and/or environmental factors
influencing the effectiveness of the resistance gene in TMG-1 cannot be ruled
out. In fact, a combination of factors may be involved.

Judgment as to whether a single locus resistance is dominant or
incompletely dominant is typically based on phenotypic evaluation of the F1
and segregating progeny of the cross between pure-breeding resistant and
susceptible parental lines for symptoms of virus infection post inoculation
with a particular virus. This subjective observation of phenotypic symptom
expression can be misleading. An example of this is found in sugarbeet
which possesses resistance to beet mosaic virus (Lewellen, 197 3). Evaluation

of the parental and segregating populations, at an early stage of infection

89

(10-14 days post virus inoculation) fell into discrete 3:1 (R:S) classes for the
F2 progeny and 1:0 (R:S) for the progeny backcrossed to the resistant parent.
However, at a later stage of infection (20-30 days post virus inoculation) and
depending on the time of year and greenhouse temperatures, the F2 progeny
segregated as a 1:2:1 (R:IzS) ratio and the backcross to the resistant parent
segregated as a 1:1 (R:I) ratio. Another example is found in common bean
which possesses resistance to soybean mosaic virus governed by an
incompletely dominant gene (Kyle and Provvidenti, 1993). In the
homozygous state, no detectable systemic symptoms were observed but virus
could be detected in inoculated leaves. In the heterozygous state, under field
or summer greenhouse conditions, individual plants remained vigorous, or
only slightly stunted, and develop local chlorosis with systemic symptoms
consisting of a mild to moderate mottling. However, this incompletely
dominant gene will appear completely dominant when plants are inoculated
and grown under winter greenhouse conditions. A third example is
resistance to tobacco mosaic virus in tomato (Fraser and Loughlin, 1980). In
the heterozygous state, the resistance allele appears completely dominant
with no viral symptoms observed. However, measurement of virus levels of
the heterozygote revealed virus replication suggesting incomplete dominance
or gene dosage-dependence.

Effectiveness of resistance also may be influenced by the genetic

background of the host cultivar. An example of this is found in barley which

90

possesses tolerance to barley yellow dwarf virus (BYDV) (Jones and
Catherall, 1970). Reduced effectiveness of tolerance, conferred by an
incompletely dominant gene was observed with slow growth rate considered
either the result of host genetic factors or environmental conditions.
Interestingly, the effectiveness of tolerance was recovered when introduced
into rapidly growing genotypes. In a follow-up study, Catherall, et a1 (1970),
evaluated five gene donor varieties that showed different levels of tolerance
to BYDV. The one variety showing the greatest tolerance was the fastest
growing while the least tolerant was the slowest growing variety.
Interestingly, TMG-1 is a fast growing cultivar; this feature may be
influencing the apparent effectiveness of the resistance gene(s).

In addition to the growth rate component of BYDV resistance, there
may also be allelic differences. When the tolerance factors from the BYDV
resistant varieties were introduced into similar genetic backgrounds, the
tolerance factor from the fastest grong variety consistently provided the
highest tolerance suggesting a series of alleles occurring at the BYDV locus
differing in their effectiveness and dominance relationships.

Earlier studies have identified sources of resistance in cucumber to
several potyviruses. TMG possesses resistance to PRSV-W, ZYMV, WMV,
ZYFV, and MWMV (Provvidenti, 1985; Gilbert-Albertini et al, 1995; Kabelka
and Grumet, 1996a) and the Dutch hybrid Dina possesses resistance to

PRSV-W, ZYMV and MWMV (Abul Hayja and Al-Shahwan, 1991; Kabelka

91

and Grumet 1996a). In addition, the alleles for ZYMV, WMV and MWMV
resistance in TMG- 1 and the alleles for ZYMV and MWMV resistance in Dina
appear to be at the same locus, or tightly linked loci (W ai and Grumet,

1995b; Kabelka and Grumet, 1996a,b). In this study, an association between
resistance to PRSV-W and resistance to ZYMV has been identified in TMG-1.
Sequential inoculation of ZYMV-resistant backcross progeny with PRSV-W “’
(or PRSV-W-resistant backcross progeny with ZYMV), and analysis of F3 I

families derived from F2 individuals selected for resistance to ZYMV, indicate

 

that both resistances are conferred by the same gene, or tightly linked genes F
(less than 1 cM; product-ratio method). These results are also consistent
with the observation that both the PRSV-W and ZYMV resistances are linked
to the bi locus for bitterfree cotyledons (W ai et al, 1996).

The existence of simply inherited genes, or clusters of separate tightly-
linked genes, that confer resistance to two or more distinct potyviruses have
been described previously in several species. For example, resistance to
WMV and bean yellow mosaic virus is conferred by a single recessive gene in
Pisum sativum (Schroeder and Provvidenti, 1971), and in Cucurbita
moschata, a single dominant gene confers resistance to both ZYMV and WMV
(Gilbert-Albertini et al, 1993). In Phaseolus vulgaris, the possibility of a
single gene, or cluster of tightly linked genes cosegregating as a unit with the
I gene, conditions resistance and/or lethal necrosis to a set of nine

potyviruses including bean common mosaic virus, WMV, blackeye cowpea

92

mosaic virus, cowpea aphid-home mosaic virus, azuki bean mosaic virus,
Thailand Passiflora virus, soybean mosaic virus, passionfruit woodiness
virus-K and ZYMV. (Kyle and Dickson, 1988; Fisher and Kyle, 1994). In
Pisum sativum, well defined clusters of tightly-linked loci conferring
resistance to a total of l l potyviruses are located on two chromosomes
(Provvidenti and Hampton, 1993; Provvidenti and Niblett, 1994).
Chromosome 2 contains resistance to WMV, bean yellow mosaic virus, pea
mosaic virus, the NL-8 strain of bean common mosaic virus, the lentil strain
of pea seed-borne mosaic virus, clover yellow vein virus, and the Australian
strain of passionfruit woodiness virus while chromosome 6 includes
resistance to three pathotypes of pea seed-borne mosaic virus, clover yellow
vein virus, and white lupin mosaic virus.

Despite the inability to break the linkage among the resistances to
PRSV-W, ZYMV, WMV, and MWMV, in TMG-l, these resistances may not all
be due to a single gene. Lines of evidence suggesting that the resistances to
multiple potyviruses in cucumber may be conferred by a cluster of tightly
linked genes rather than a single gene include: (1) Resistance to PRSV-W in
TMG-1 appears incompletely dominant while resistance to ZYMV is
recessive. It is possible that these differences may be due to varying
effectiveness of one gene against different potyviruses, but it is also possible
that they are due to two different genes. (2) The mechanisms of resistance to

PRSV-W and ZYMV in TMG-l also appear to be different. Previous studies

93

indicated that when TMG-1 was inoculated with PRSV-W, high virus levels
were detected despite a lack of symptoms, however, little or no virus was
detected when TMG-1 was inoculated with ZYMV (W ai and Grumet, 1995a).
(3) Dina, which is resistant to ZYMV, PRSV-W and MWMV shows different
responses to ZYMV than to PRSV-W or MWMV inoculation. A distinct veinal
chlorosis phenotype limited to the first and second true leaves was observed
with ZYMV cotyledon inoculation whereas no symptoms were observed with
PRSV-W or MWMV inoculation (Kabelka and Grumet, 1996b). (4) Although
Surinam is resistant to PRSV-W, it is susceptible to ZYMV, WMV and
MWMV. Again this may be due to different responses of one gene to different
potyviruses or it may be that Surinam only possesses one member of a gene
cluster. As mentioned earlier, although resistance to PRSV-W in TMG-1
appeared incompletely dominant, the existence of modifying and/or
environmental factors influencing the effectiveness of the resistance gene in
TMG-l could not be ruled out. Possibly the effectiveness of PRSV-W
resistance in TMG-l is influenced other members of a cluster of resistance
genes.

In conclusion, although we have not been able to break the linkage
associations among the resistances to PRSV-W, ZYMV, WMV, and MWMV in
TMG-l, varying responses with regard to dominance relationships, resistance

mechanisms, symptom expression, and which viruses are protected against,

94

support the possibility that multiple potyvirus resistance in cucumber is

conferred by a tightly-linked cluster of resistance genes.

95

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McKern, N.M., Strike, P.M., Barnett, O.W., Ward, C.W., Shukla, DD. 1993.
Watermelon mosaic virus-Morocco is a distinct potyvirus. Archives of
Virology 131:467-473.

Peterson, C.E., Williams, P.H., Palmer, M., Louward, P. 1982.
Wisconsin-27 57 Cucumber. HortScience 17:268.

Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of
Cucumis sativus. Cucurbit Genetics Cooperative Report 8:12.

Provvidenti, R. 1987 . Inheritance of resistance to a strain of zucchini yellow
mosaic virus in cucumber. HortScience 222102-103.

Provvidenti, R., Hampton, RD. 1993. Inheritance of resistance to white
lupin mosaic virus in common pea. HortScience 282836-837.

Provvidenti, R., Niblett, CL. 1994. Inheritance of resistance to a strain of
passionfruit woodiness virus in pea (Pisum sativum L.) HortScience 29:901-
902.

Purcifull, D., Hiebert, E., Edwardson, J. 1984a. Watermelon mosaic virus-2.
Descriptions of Plant Viruses No. 293 (No. 63 revised). Kew, Surrey,
England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

Purcifull, D., Edwardson, J ., Hiebert. E., Gonsalves, D. 1984b. Papaya
ringspot virus. Descriptions of Plant Viruses No. 292 (No. 84 revised). Kew,
Surrey, England: Commonwealth Agricultural Bureax/Association of Applied
Biologists.

97

Schroeder, W.T., Provvidenti, R. 1971. A common gene for resistance to
bean yellow mosaic virus and watermelon mosaic virus-2 in Pisum sativum.
Phytopathology 612846-848.

Wai, T., Grumet, R. 1995a. Inheritance of resistance to the watermelon
strain of papaya ringspot virus in the cucumber line TMG-l. HortScience
30:338-340.

Wai, T., Grumet, R. 1995b. Inheritance of resistance to watermelon mosaic
virus in the cucumber line TMG-1: tissue-specific expression and relationship
to zucchini yellow mosaic virus resistance. Theoretical and Applied Genetics
91:699-706.

Wai, T., Staub, J ., Kabelka, E., Grumet, R. 1996. Linkage analysis of
potyvirus resistance alleles in cucumber. Journal of Heredity (in review).

Wang, Y.J., Provvidenti, R., Robinson, R.W. 1984. Inheritance of resistance
to watermelon mosaic virus 1 in cucumber. HortScience 19:587-588.

SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS

Cucumbers and other cucurbit crops are subject to severe losses due to
an array of potyviruses, including zucchini yellow mosaic virus (ZYMV),
watermelon mosaic virus (WMV), the watermelon strain of papaya ringspot
virus (PRSV-W) and the Moroccan watermelon mosaic virus (MWMV).
Sources of resistance to several of these viruses have been identified and
characterized within the cucumber (Cucumis sativus L.) germplasm. An
inbred line derived from the Chinese cucumber cultivar ‘Taichung Mou Gua’
(TMG-1) possesses resistance to ZYMV, WMV and PRSV-W. The Dutch
hybrid ‘Dina’ possesses resistance to ZYMV and the cultivar ‘Surinam Local’
is resistant to PRSV-W. This study demonstrated TMG- 1 also possesses
resistance to MWMV and that this resistance is conferred by a single
recessive gene. Sequential inoculation of progeny possessing resistance to
ZYMV followed by MWMV (or MWMV followed by ZYMV) and analysis of F3
families derived from F2 individuals selected for resistance to ZYMV indicate
that both resistances are conferred by the same, or two tightly linked genes.
Earlier work with TMG-l indicated that the recessive gene for ZYMV

resistance also conferred resistance to WMV. Therefore it appears that a

98

99

single recessive gene, or cluster of recessive genes, in TMG-1, governs
resistance to at least three potyviruses, ZYMV, WMV and MWMV.

Tests of allelism indicate that the genes for resistance to ZYMV in
TMG-l and Dina are at the same locus. However, they exhibit different
phenotypes in response to cotyledon inoculation with ZYMV. Dina exhibits a
distinct veinal chlorosis and accumulation of virus limited to the first and
second true leaves whereas TMG-l is symptom-free and lacks virus
accumulation. The distinct veinal chlorosis phenotype was dominant to the
symptom-free phenotype and shown not to be due to an additional separate
factor. Therefore, at the zym locus, a series of alleles occur differing in their
effectiveness and dominance relationships with Zym+ >zymD‘" >zymTMG°1.

Our studies also reveal that in addition to ZYMV resistance, Dina
possesses resistance to PRSV-W and MWMV and that the gene for MWMV
resistance is at the same locus as the ZYMV resistance. In addition, tests of
allelism indicate the gene for resistance to MWMV in TMG-1 and Dina are at
the same locus. In contrast to ZYMV inoculation, however, Dina does not
exhibit the distinct veinal chlorosis phenotype when inoculated with
PRSV-W or MWMV. Since the differences in response were observed only for
ZYMV and not MWMV, it is possible that rather than a single gene
conferring resistance to ZYMV, WMV and MWMV, the resistances may be

part of a gene cluster for potyvirus resistance in cucumber.

100

Segregation ratios among the progeny of TMG-1 and Surinam, and
TMG-1 and Surinam crossed with a common susceptible parent, indicate the
sources of resistance to PRSV-W are at the same locus but different
dominance relationships indicate either different alleles or the efi'ect of other
modifying factors as resistance to PRSV-W in Surinam is recessive while in
TMG-l it is incompletely dominant.

Although Surinam only possesses resistance to PRSV-W, TMG-l
possesses resistance to ZYMV, WMV, MWMV and PRSV-W. In addition, an
association between resistance to ZYMV and resistances to WMV and
MWMV was found in TMG-l. Our studies also indicated that in addition to
the tight linkage between resistances to ZYMV, WMV, and MWMV in
TMG-1, resistance to PRSV-W also cosegregated with resistance to ZYMV.
Sequential inoculation of ZYMV-resistant backcross progeny followed by
PRSV-W (or PRSV-W resistant backcross progeny followed by ZYMV) and
analysis of F3 families derived fiom F2 individual selected for resistance to
ZYMV, indicate that both resistances are conferred by the same gene, or
tightly-linked genes. Therefore, it appears that a single gene, or cluster of
tightly-linked genes, governs resistance to ZYMV, WMV, MWMV and
PRSV-W in TMG- 1.

Despite the inability to break the linkage among the resistances to
ZYMV, WMV, MWMV and PRSV-W in TMG-l, these resistances may not all

be due to a single gene. Several lines of evidence suggest that the resistances

101

to multiple potyviruses in cucumber may be conferred by a cluster of tightly-
linked genes: (1) the mode of inheritance of resistance to PRSV-W in TMG-1
appears incompletely dominant while to ZYMV it is recessive, (2) the
mechanisms of resistance with regards to viral accumulation also appears to
be different for PRSV-W vs. ZYMV in TMG-1, (3) Dina, which is resistant to
ZYMV, PRSV-W and MWMV shows different responses to inoculation with
these potyviruses, and (4) although Surinam is resistant to PRSV-W, it is
susceptible to ZYMV, WMV and MWMV. It is possible that these differences
may be due to varying effectiveness of one gene against different potyviruses,
but it is also possible that they are due to a cluster of potyvirus resistance
genes.

In conclusion, although we have not been able to break the linkage
associations among the resistances to PRSV-W, ZYMV, WMV, and MWMV in
TMG-1, varying responses with regard to dominance relationships, resistance
mechanisms, symptom expression, and which viruses are protected against,
support the possibility that multiple potyvirus resistance in cucumber is
conferred by a tightly-linked cluster of resistance genes.

Several additional experiments can be performed to further investigate
the relationship between the potyvirus resistance genes in cucumber.
Segregation ratios among the F1, F2 and backcross progeny of TMG-1 and
Dina indicated that the ZYMV resistance alleles are at the same locus,

however, the two sources of ZYMV resistance seem to perform differently.

102

Dina responds to ZYMV cotyledon inoculation with a distinct veinal chlorosis
phenotype and accumulation of virus limited to the first and second true
leaves whereas TMG-l remains symptom-free and lacks virus accumulation.
The distinct veinal chlorosis phenotype in Dina only occurs when cotyledons
are inoculated. When inoculated with ZYMV at the true leaf stage, no
distinct veinal chlorosis was seen. The mechanism in Dina that limits virus
to the first and second true leaves is unknown, however, studies to examine
the mechanism of resistance to ZYMV in Dina are currently in progress
utilizing a tissue-printing method.

Our studies also indicated that in addition to ZYMV resistance Dina
possesses resistance to PRSV-W and MWMV. However, upon inoculation
with PRSV-W or MWMV, Dina remains symptom-free not exhibiting the
distinct veinal chlorosis phenotype as seen with several isolates of ZYMV
except the California isolate. Although MWMV, PRSV-W and ZYMV-
California are distinct potyviruses, one shared feature is the time interval
preceding observable symptoms in the susceptible genotype. While
symptoms of most ZYMV isolates developed approximately 7-10 days post
inoculation symptoms of MWMV, PRSV-W and ZYMV-California did not
become evident until approximately 14-21 days post inoculation. Possibly
this difference in time of onset represents a slower rate of virus
multiplication or movement resulting in lack of veinal chlorosis in Dina.

Possibly it represents a difference in resistance mechanisms. It would be

103

interesting to determine if a lack of veinal chlorosis represents a lack of virus
accumulation. ELISA and/or tissue-printing can be used to address these
questions.

As mentioned, Dina also possesses resistance to PRSV-W. The
inheritance of resistance to PRSV-W in Dina was not characterized. In
addition, previous studies with TMG-l and PRSV-W (in contrast to ZYMV)
have indicated that although TMG-l is symptom-flee post inoculation with
PRSV-W, there is virus accumulation. Therefore, it would be interesting to

compare the resistances to PRSV-W found in TMG-l and Dina.

APPENDICES

APPENDIX A

APPENDIX A
Linkage relationship of potyvirus resistance to bitterfree cotyledons

To further understand the relationship of potyvirus resistances to each
other, linkage relationships were investigated. In Surinam and in TMG-1
the gene conferring resistance to PRSV-W is reported to be linked with
bitterfree cotyledons (Wang et al, 1987 ; Pierce and Wehner, 1990; Wai et al,
1996). Since the gene conferring resistance to PRSV-W appears to be the
same as, or tightly linked to, genes conferring resistance to ZYMV in TMG- 1,
we tested for cosegregation between resistance to ZYMV and the bitterfree
gene. For comparison we included tests for cosegregation between resistance
to PRSV-W and the bitterfree gene.

The inbred cucumber (Cucumis sativus L.) ‘TMG-l’, resistant to
ZYMV, WMV, PRSV-W, MWMV (Provvidenti, 1985; Kabelka and Grumet,
1996) and the susceptible parental genotype ‘Wisconsin 27 57 (WI-27 57 ;
Peterson et al, 1982) were provided by Dr. Jack Staub (USDA, University of
Wisconsin at Madison). Linkage tests between potyvirus resistance genes
and the bitterfree gene were performed using the F2 progeny of WI-27 57 and
TMG-l. Production of this progeny is as described by Wai and Grumet
(1995). Propagation of virus inocula, methods of inoculation, and

experimental designs were performed as described by Kabelka and Grumet

104

105

(1996). The presence of cucurbitacins (bitterness) was assayed by tasting a
small piece of the cotyledons (DeCosta and Jones, 1971). linkage
relationships and distances were analyzed using Chi square, the product-
ratio method, and Linkage 1 version 3.5 (Suiter et al, 1983).

WI-27 57 is bitterfree (bibr) while TMG-1 and the F1 progeny of this
cross are bitter (Bi--). Goodness-of-fit tests show that resistance to PRSV-W,
resistance to ZYMV, and bitterfree cotyledons fit expected ratios for single
gene traits (Fable A. 1). When traits were examined in pairwise combination
for linkage relationships there were variable results (Table A2). Resistance
to ZYMV appears to be linked to the bitterfree gene when evaluating a large
population (n=157; estimated map distance 21.3 cM) but independently
segregating when evaluating a smaller population (n=80). Pooled data
mapped to a distance of 24.8 cM. When considering resistance to PRSV-W as
a single incompletely dominant trait, linkage to the bitterfree gene, when
evaluating a large population (n=149), mapped to a distance of 37.2 cM but
segregated independently when evaluating a small population (n=79).
Pooled data (n=228) mapped to a distance of 46.4 cM. In previous reports,
the gene conferring resistance to PRSV-W was considered as a single
dominant trait and linked to the bitterfree gene at an approximate map
distance of 28 cM (W ai et al, 1996). Interestingly, evaluating the current
data as a single dominant trait brings the map distance between resistance

to PRSV-W and the bitterfree gene closer to the previous estimated map

106

distance in literature. When evaluating the large population (n=149),
linkage of PRSV-W resistance and the bitterfree gene mapped to a distance of
29.9 cM. However, the smaller population (n=79) still segregated
independently. Pooled data mapped to a distance of 35.5 cM.

It is possible that in order to accurately evaluate either the ZYMV or
the PRSV-W resistances to bitterfree cotyledons, a large population is
necessary. This conclusion is not unique and has been observed for other
groups of associated traits in cucumber such as andromonoecious sex
expression, protruding ovary, spine size and frequency, red mature fruit,
heavy netting of fruit, and black or brown spines (Pierce and Wehner, 1990).

Further experiments will be required to clarify these differing results.

 

107

 

 

 

 

 

 

 

 

Table A.1 Single trait goodness of 111 tests for characters analyzed in the F2 progeny
of Wl-2757 x TMG-1 cucumber.
Expected Observed
Allele Ratio“ Ratio Phenotype“ X2
Experiment 1 bi 3:1 115245 Bizbi 0.68 ns
zym 3:1 115242 S:R 0.17 ns
bi 3:1 103:46 Bizbi 2.44 ns
Prsv-2 1:2:1 32:76:41 S:l:R 1.15 ns
Experiment 2 bi 3:1 57:23 Bizbi 0.42 ns
zym 3:1 58:22 S:R 0.15 ns
bi 3:1 59:21 Bizbi 0.02 ns
Prsv-2 1:2:1 23:32:24 S:I:R 2.86 ns
Pooled data bi 3:1 172:68 Bizbi 1.25 ns
zym 3:1 173264 S:R 0.40 ns
bi 3:1 162:67 Bizbi 2.00 ns
Prsv-2 1:2:1 552108265 S:l:R 1.51 ns

 

ns, non-significant X“ value

“expected ratios based on bitterfree inherited as a single recessive trait in Wl-2757. ZYMV

 

resistance conferred by a single recessive gene in TMG-1, and resistance to PRSV-W
conferred by an incompletely dominant gene in TMG-1.
“Bi: bitter", bi: bitterfree; S: susceptible; R: resistant; I: intermediate phenotype

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REFERENCES

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susceptibility controlled by the bitter gene in Cucumis sativus L. Science
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Kabelka, E., Grumet, R. 1996. Inheritance of resistance to the Moroccan
watermelon mosaic virus in the cucumber line TMG-l and cosegregation with
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Peterson, C.E., Williams, P.H., Palmer, M., Louward, P. 1982.
Wisconsin-2757 Cucumber. HortScience 17:268.

Pierce, L.K., Wehner, T.C. 1990. Review of genes and linkage groups in
cucumber. HortScience 252605-615.

Provvidenti, R. 1985. Sources of resistance to viruses in two accessions of
Cucumis sativus. Cucurbit Genetics Cooperative Report 8:12.

Suiter, K.A, Wendel, J .F., Case, J .S. 1983. Linkage-l: A Pascal computer
program for the detection and analysis of genetic linkage. Journal of
Heredity 74:203-204.

Wai, T., Grumet, R. 1995. Inheritance of resistance to the watermelon strain
of papaya ringspot virus in the cucumber line TMG-l. HortScience 30:338-
340.

Wai, T., Staub, J .E., Kabelka, E., Grumet, R. 1996. Linkage analysis of
potyvirus resistance alleles in cucumber. Journal of Heredity (in review).

Wang, Y.J., Robinson, R.W., Provvidenti, R. 1987. Linkage relationships of
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