CYTOLOGICAL CHARACTERISTICS AND DETECTION OF OF SPP.

By

TSO-CHI YANG

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1997 ACKNOWLEDGMENTS

Sincerest appreciation is expressed to Dr. E W. Zettler for his

encouragement, guidance, patience, and support throughout my stay here.

Special thanks also are expressed to Drs. E. Hiebert, M. E. Kane, N.-J. Ko, J. E.

Polston, and D. E. Purcifull for their advice in their specialties, encouragement,

patience, and their critique of this study.

I would like to express my deepest appreciation to Mr. R. G. Christie for

training me in the study of plant inclusions and for providing me the great

opportunity to attend the workshop held in Gainesville in 1996. Sincere

appreciation also is extended to other members of the Department of Plant

Pathology, especially K. A. Beckham, W. E. Crawford, M. S. Elliott, M. A.

Petersen and E. E. Philman, for their technical assistance and support.

Special thanks are offered to Dr. C.-Y. Lin, Director of the Taiwan

Agricultural Research Institute, Taichung, Taiwan, for his strong vote of

confidence and constant encouragement for me to undertake and complete this

study. I am also thankful to the National Science Council of the Republic of

China and to the Manatee Fruit Company of Palmetto, Florida, and the American

Floral Endowment for providing me with financial support.

Finally, I wish to thank my parents, Chu-Wen Yang and Hsi-Guay Yeh, for their love, support, and encouragement. At the same time, I thank my lovely wife,

11 Hui-Ying Ma, and our daughter, Ying-Ju Yang, and son, Ying-Jen Yang, for their sacrifice on my behalf. To them, I owe my deepest gratitude.

iii TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

KEY TO ABBREVIATIONS vi

ABSTRACT xii CHAPTERS

1. INTRODUCTION 1

Commercial Importance of Lilies 1

Lily Viruses 2

2. CHARACTERIZATION OF LILY X POTEXVIRUS IN LILIES 16

Introduction 16 Materials and Methods 17 Results 24 Discussion 45

3. CYTOLOGICAL CHARACTERISTICS OF LILY VIRUSES AND THEIR POTENTIAL FOR DIAGNOSIS 49

Introduction 49 Materials and Methods 51 Results 54 Discussion 56

4. SEROLOGICAL METHODS FOR DETECTING AND DISTINGUISHING LILY VIRUSES 71

Introduction 71 Materials and Methods 74 Results 79 Discussion 83

5. INCIDENCE AND POTENTIAL COMMERCIAL IMPACT OF LILY VIRUSES 127

Introduction 127 Materials and Methods 130 Results 132 Discussion 145

IV APPENDIX 150

LITERATURE CITED 151 BIOGRAPHICAL SKETCH 168

V KEY TO ABBREVIATIONS

A 405 absorbance values at wave length of 405 nm

ADMIX the admixture of monoclonal antisera of PTY-1, -2, -4, -8, -10,

-21 -24, and -30 from R. L. Jordan in U.S.A. ,

ATCC the American Type Culture Collection (Rockville, MD)

ATCC PVAS732 ATCC accession number of a TBV-specific MCA produced by H. T. Hsu against a TBV isolate from Texas Flame'

ATCC PVAS733 ATCC accession number of a different clone of ATCC PVAS 732

ATCC PVAS675 ATCC accession number of a different clone of ATCC PVAS 732

BaMV bamboo mosaic potexvirus

BaMV-Lin polyclonal antiserum against BaMV isolated from bamboo

from N.-S. Lin in Taiwan

BCIP 5-bromo-4-chloro-3-indolyl phosphate

ByMV bean yellow mosaic

ByMV-A993 polyclonal antiserum against a ByMV isolate from red clover provided by F. W. Zettler

CMV cucumber mosaic cucumovirus

CMV-J12 a CMV isolate isolated in this study from 'Juanita' lily

CMV-K45 a CMV isolate from lily provided by T.-C. Deng

CsCMV cassava common mosaic potexvirus

CsCMV-A1091 polyclonal antiserum against CsCMV-chaya isolate in Florida provided by F. W. Zettler

VI CymMV cymbidium mosaic potexvirus

CymMV-A862 polyclonal antiserum against a CymMV isolate in Florida from F. W. Zettler dsRNA double-stranded ribonucleic acid

DMSO dimethyl sulfoxide

DTB direct tissue blotting

ELISA -linked immunosorbent assay l-ELISA indirect-ELISA

TAS-ELISA triple antibody sandwich-ELISA

IgG immunoglobulin G

ISEM immunosorbent electron microscopy kDa kilodalton kb kilobase

LiMV lily mottle potyvirus (synonymous with a Lilium longiflorum of TBV, or TBV-L)

LiMV-D a LiMV isolate which was recovered from L. longiflorum by A.

F. L. M. Derks in the Netherlands

LiMV-T a LiMV which was isolated from L. longiflorum by C.-A. Chang In Taiwan

LiMV-Chang polyclonal antiserum against LiMV-T isolate

LiMV-Derks polyclonal antiserum against LiMV-D isolate

UMV-WS342 a LiMV isolate isolated in this study from L. longiflorum cv.

'White Sheen' Easter lily in Florida

LSV lily symptomless carlavirus

vii LSV-CB a LSV isolate isolated in this study from 'Casa Blanca'

Oriental lily

LSV-Mon a LSV isolate isolated in this study from 'Montreaux' Asiatic

lily

LSV-NW a LSV isolate isolated in this study from 'Nellie White' Easter

lily

LSV-SG a LSV isolate isolated in this study from 'Stargazer' Oriental

lily

LSV-A1189 polyclonal antiserum against LSV isolated from Asiatic lily produced in this study

LVX lily X potexvirus

LVX-D a LVX isolate from A F. L. M. Derks in the Netherlands

LVX-NW a LVX isolate isolated in this study from 'Nellie White' Easter

lily

LVX-SS a LVX isolate in this study isolated from 'Strawberry

Shortcake' Oriental lily

LVX-SG a LVX isolate in this study isolated from 'Stargazer' Oriental

lily

LVX-WS a LVX isolate in this study isolated from 'White Sheen' Easter

lily

LVX-Derks polyclonal antiserum against LVX-D

MCA monoclonal antibody

Mr molecular weight

NanMV nandina mosaic potexvirus

NanMV-A934 polyclonal antiserum against NanMV isolated from nandina in Florida from F. W. Zettler

NBT nitro blue tetrazolium

viii PAGE polyacrylamide gel electrophoresis

PapMV papaya mosaic potexvirus

PapMV-A747 polyclonal antiserum against PapMV isolated from Florida, provided by D. E. Purcifull

PBS phosphate buffered saline, pH 7.4

PBST PBS containing 0.5% Tween-20, pH 7.4

PCA polyclonal antiserum

PTY-1 a potyvirus group cross-reactive MCA produced by R. L. Jordan, and marketed commercially by Agdia, Inc. (Elkhart,

In).

PVX X potexvirus

PVX-A782 PCA against a PVX isolate from Florida provided by D. E. Purcifull

PVP-40 polyvinylpyrrolidine-40, Mr 40,000

PVY potato Y potyvirus

PVY-A709 PCA against a PVY isolate provided by D. E. Purcifull

RTBV Rembrandt , an isolate of TBV from 'Jack Laan" tulip described by A. F. L. M. Derks and synonymous with TBV-JL and TBV-lily

SDS sodium dodecyl sulfate

SLRV strawberry latent ringspot nepovirus

SLRV-B PCA against SLRV from A. A. Brunt in the UK

TBST Tris buffered saline containing 0.5% Tween-20, pH 7.4

TBV tulip breaking potyvirus

TBV-Derks PCA against TBV, which originally isolated from 'Jack Laan' tulip by A. F. L. M. Derks in the Netherlands

ix TBV-FA a TBV isolate isolated from Asiatic lily provided by T.-C. Deng

TBV-JL an isolate of TBV synonymous with RTBV and TBV-lily recovered from 'Jack Laan' tulip by A. F. L. M. Derks

TBV-L Lilium longiflorum isolate of TBV synonymous with LiMV and

recovered from Easter lily by A. F. L. M. Derks

TBV-lily a lily strain of TBV synonymous with TBV-JL and RTBV and

isolated by A. F. L. M. Derks from 'Jack Laan' tulip

TBV-NW a TBV isolate isolated in this study from 'Nellie White' Easter

lily

TBV-PPVB a TBV isolate isolated in this study from 'Pink Perfection'

Aurelian lily

TBV-Sir a TBV isolate isolated in this study from 'Sirocco' Asiatic lily

TBV-TDG29 a TBV isolate isolated in this study from a Trumpet lily

TBV-tulip a tulip strain of TBV synonymous with TBV-TF isolated by A.

F. L. M. Derks from 'Texas Flame' tulip

TBV-TF an isolate of TBV recovered from 'Texas Flame' by A. L. F. M. Derks and synonymous with TBV-tulip

TBBV tulip band-breaking potyvirus, an isolate of TBV recovered from 'Lucky Strike' tulip by C. J. Asjes in the Netherlands

TCBV tulip chlorotic blotch potyvirus, a virus isolated from tulips, but closely related serologically to TuMV

Tris tris (hydroxymethyl) aminomethane

TTBV tulip top breaking potyvirus, a virus isolated from tulip, but closely related serologically to TuMV

TuMV turnip mosaic potyvirus

TuMV-A825 PCA against a TuMV isolated from dames violet and provided by E. Hiebert

\ TuMV-3374 a TuMV isolate which was isolated from turnip in Florida and provided by D. E. Purcifull

UA uranyl acetate

WaTV Washington tulip potyvirus, a virus isolated from tulip by J.

Hammond, which is closely related serologically to TuMV

WCIMV white clover mosaic potexvirus

WCIMV-A923 antiserum against a WCIMV isolated from Florida, provided by D. E. Purcifull

xi Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

CYTOLOGICAL CHARACTERISTICS AND DETECTION OF VIRUSES OF LILIUM SPP

By

Tso-Chi Yang

August 1997

Chairman: F. W. Zettler

Cochairman: J. E. Polston Major Department: Plant Pathology

Lily X potexvirus (LVX) was found for the first time in Asiatic, Easter, and

Oriental lily collected in the U.S.A. LVX is a potexvirus and was

distinguished from lily symptomless carlavirus (LSV) by serological tests such as

immunogold labeling tests.

Virus inclusions were found in lilies infected with cucumber mosaic virus

(CMV), LSV, LVX, and with tulip breaking (TBV) and lily mottle viruses (LiMV),

respectively. These results were confirmed by serological methods. A possible

new cryptic virus was found in lily seedlings presumed to be healthy by

serological tests.

TBV and LiMV were concluded to be closely related strains of one

another based on the similarity of their cytoplasmic cylindrical inclusions and on

results of serological tests. Monoclonal antibodies (MCAs) 732 and 733 from the

Ml American Type Culture Collection, and polyclonal antisera against LiMV and

TBV reacted with all isolates of LiMV and TBV, whereas potyvirus-group, PTY-1 and Admix (PTY-1, -2, -4, -4, -10, -21, -24, and -30) antibodies did not, confirming that neither TBV nor LiMV has the universal potyvirus epitope.

Direct tissue blotting (DTB) was more satisfactory than indirect-enzyme linked immunosorbent assay (l-ELISA) for routine virus indexing. The outer scales and roots were satisfactory tissues to sample for DTB tests. I-ELISA and

DTB methods were both unreliable, however, when indexing plants grown at

high temperatures (>30 C) and on plantlets growing in vitro.

A survey of 1180 Asiatic, Easter, and Oriental lilies showed LSV to be the

most prevalent virus in commercial stocks. LVX was also found in some samples, with incidences ranging from 7.6 to 13.0%.

Virus-free 'Nellie White' lilies were significantly taller than their virus-

infected counterparts, had higher bulb yields, and more and flowers.

However, anthesis was reached later in healthy plants than in ones doubly

infected with LSV and LVX or with LSV and TBV. Timing for anthesis, especially

in relation to the Easter market, can be synchronized with bulbs that are either

all healthy or infected, but in stocks that are mixed, considerable losses could

result when large numbers of flowers do not bloom in a timely fashion.

xiii CHAPTER 1 INTRODUCTION

Commercial Importance of Lilies

There are more than 90 species of lilies (Lilium spp.). They are indigenous to the Northern Hemisphere, and they occur throughout Europe,

Northern Asia, and North America. A few species are native to the tropics, but these only occur at higher elevations (Beattie and White, 1993). Lilies are among the most important ornamental flowering plants in the world. Some Lilium

species in China and Japan have been used for human consumption and as

medicinal plants (Nakamura era/., 1994).

Ornamental lilies are classified into several groups, the most important of

which are Easter lilies (L longiflorum), Asiatic lilies, and Oriental lilies. In the

plants U.S.A. and Canada, Easter lily are traditionally grown as potted

during the Easter holidays. Recently, Asiatic and Oriental hybrid lilies, grown

primarily for cut flower production, have become increasingly popular, especially

in Japan. As a result, countries such as the U.S.A. and Taiwan have become

interested in increasing commercial production of lily bulbs.

The three major countries now producing lilies for propagation are the

Netherlands, the U.S.A. and Japan. The lily acreage in the Netherlands in 1988

was 2,211 hectares (Asjes, 1990). About 560 million lily bulbs were exported

from the Netherlands in the 1991-1992 growing season. In Japan, about 400

l 2 hectares were grown for cut flower and bulb production (Derks, 1995). In the

U.S.A. in 1995, the lily acreage was about 760 hectares (Table 1-1). In addition, small acreages are also grown in many other countries such as Italy, France,

U.K., Spain, Germany, and Taiwan. In 1992, Taiwan imported about 80% more

lily bulbs from the Netherlands than it did in 1991 (Table 1-3).

Several decades ago, a modest commercial Easter lily industry existed in

Florida but it declined and eventually disappeared because of necrotic-fleck

disease and other factors. Today, only about 40 hectares of potted lilies are

being produced in Florida annually (Table 1-2).

Since commercial propagation is primarily by vegetative means from

scales, diseases caused by viruses and other pathogens are common and cause

serious economic losses. If good means of detecting and controlling viral

diseases of lily could be achieved, the prospects for a strong commercial lily

industry both in Florida and Taiwan would be enhanced.

Lily Viruses

At least 10 viruses have been reported to infect lilies, of which lily symptomless

carlavirus (LSV)(Allen and Lyons, 1969; Allen and Linderman, 1970; Asjes etal.,

1973; Beijersbergen and van der Hulst, 1980; Civerolo et a/., 1968; Derks and

Asjes, 1975; Simmonds and Cumming, 1979; van Schadewijk, 1986; van

Slogteren et a/., 1976; Yang et al., 1993), cucumber mosaic cucumovirus

(CMV)(Allen and Lyons, 1969; Benetti and Tomassoli, 1988; Brierley and Smith,

1944a), lily X potexvirus (LVX)(Kimura et a/., 1990; Memelink 3

Table 1-1. Commercial production of potted lilies in the U.S.A.

Year No. of Production area Yield Value

producers (hectares) (1 ,uuu pots) ( i,uuu uo aoiiars; 1) _ o, out? in ofifi 1980 1,291 479 7,105 19,766 1985 1,493 466 7,071 25,485 1990 1,013 603 10,068 38,293 1991 1,006 611 10,075 38,599 1992 1,294 655 10,367 39,882 1993 1,333 670 10,158 39,979 1994 1,326 713 10,181 42,861 1995 1,338 760 11,712 45,654 Source: Floriculture Crops Summary. Agricultural Statistics Board, National Agricultural Statistics Serial, USDA. 1) -= Data not available.

Table 1-2. Commercial production of potted lilies in Florida

Year No. of Production area Yield Value producers (hectares) (1,000 pots) (1,000 US dollars) 1975 13 60.5 259 487 1980 15 31.3 331 847 1985 25 30.7 324 1,023 1990 30 46.5 509 1,649 1991 23 40.3 498 1,674 1992 20 43.1 536 1,655 1993 19 45.7 430 1,758 1) 1994 11 43.6 392 1,470 1995 14 1) 24.2 321 1,204 Source: Floriculture Crops Summary. Agricultural Statistics Board, National Agricultural Statistics Serial, USDA. 1) Data of other potted lilies are not available. Table 1-3. Quantity of lily bulbs exported from the Netherlands in 1990-1992

Number of bulbs (X 1 million)

Importer June 1990-May 1991 June 1991 -May 1992 Percent Change

Italy 152.4 150.5 -1.25

Japan 82.3 94.8 +15.19

U.S.A. 56.3 84.6 +50.27

France 43.3 41 .6 -o.yo

U. K. 36.6 33.4 -8.74

Spain 21.1 19.3 -8.53

Germany 20.5 19.4 -5.37

Taiwan 11.1 19.8 +78.38

Others 80.7 96.0 +18.96

Total 504.3 559.4 +10.93

Source: International Bloembollen Centrum. Hillegom, The Netherlands. 5

etal., 1990; Stone, 1980), and tulip breaking potyvirus (TBV)(Alperef a/., 1982;

Asjes etal., 1973; Boonekamp era/., 1982; Brierley and Smith, 1944b; Derks,

1976) are the most frequently encountered.

Other viruses, such as arabis mosaic nepovirus (Asjes and Segers, 1 983;

Marani and Faccioli, 1974), mosaic potexvirus (Bellardi et al., 1988), tobacco rattle tobravirus (Derks, 1975; McWhorter and Allen, 1964), tobacco ringspot nepovirus (Travis and Brierley, 1957), citrus tatter closterovirus

(Inouye et al., 1979), strawberry latent ringspot nepovirus (Cohen ef al., 1995), broad bean wilt fabavirus (Chang and Chung, 1987) and a possible plant

reovirus (Varma et al., 1979), are represented by solitary reports and, thus, appear to be much less common than CMV, LSV, LVX, and TBV.

Cucumber Mosaic Virus

Probably the earliest description of a lily virus was CMV by Stewart

virus was (1895), who referred to the disease it caused as "lily mosaic." This probably introduced into Bermuda about 1893 where, in association with the so-

there called "yellow flat disease," it caused a rapid decline of the lily industry

(Price, 1937).

Early workers demonstrated that the so-called lily mosaic virus could be transmitted by and could be mechanically transmitted to Nicotiana tabacum (Guterman, Ogilvie 1928; Price, 1937). Based on these studies, 1928; , they identified this virus as CMV. Since then, CMV has been detected in many

other lily species and the related ornamental, Fritillaria (Guterman, 1930). 6

Since the 1970s, CMV infections of lilies were reported in Belgium

(Verhoyen and Horvat, 1972), Great Britain (Mowat and Stefanac, 1974), Italy

(Benette and Tomassoli; 1988, Tomassoli and Benette, 1988), Japan (Hagita,

1989; Hagita etal., 1989; Maeda et al., 1996), Korea (Chang and Chung, 1987;

Kim et al., 1995), Latvia (Zhola et a/., 1992), the Netherlands (Asjes, 1976),

Taiwan (Chang and Pang, 1991), and the U.S.A. (Brierley and Smith, 1944a;

Allen, 1975). Despite its past history, however, CMV is not among the three viruses (TBV, LSV, LVX) included in the Netherlands Bulb Inspection Service's

lily virus indexing program (Asjes, 1990).

Lily Symptomless Virus

LSV was described by Allen (1972). LSV is a carlavirus with particles 640-

cultivars remain 650 nm long, and it occurs wherever lilies are grown. Most symptomless when infected with LSV. However, occasionally "curly-stripe" symptoms have been observed about 30 to 60 days after inoculation in L. longiflorum plants maintained at 15.5 C (McWhorter and Allen, 1964). Plants

.infected with LSV had reduced growth, smaller flowers, lower bulb yields, and shorter vase lives (Asjes, 1990; Boontjes, 1976, 1978).

The host range of LSV is restricted to the . Besides Lilium spp., it can also infect tulip (Derks and Asjes, 1975) and Alstroemeria (Bellardi et al.

1994; Phillips and Brunt, 1986). LSV is transmitted in a non-persistent manner

by the aphids, Myzus persicae, euphorbiae, and

(Derks and Asjes, 1975; Mowat and Stefanc, 1974). This virus is not seed-borne,

however (Allen, 1972). 7

LSV is detected most reliably by serological methods using enzyme- linked immunosorbent assay (ELISA) and direct tissue blotting (DTB) (Derks and

Vink-van den Abeele, 1980; Hsu etal., 1995; Kim era/., 1995; 1996). In addition,

LSV is also detectable by hybridization assays with LSV-specific primers in PCR

(Memelink et al., 1990). The combination of a barley barrier crop and

applications of mineral oil is reported to be effective in controlling the field-

spread of TBV and LSV (Mowat and Woodford, 1976). Meristem culture has

been used to obtain virus-free lilies (Allen, 1988; Allen and Anderson, 1980;

Allen et al., 1980; Asjes, 1980; Asjes et al., 1974; Blom-Barnhoorn and van

Aartrijk, 1985; Linderman etal., 1976, Takashashi etal., 1992). The addition of

Virazole to the meristem-inducing medium was effective in obtaining LSV-free

plants, but not plants free of TBV, however (Blom-Barnhoorn and van Aartrijk,

1985).

Lilies infected by LSV were reported in Australia (Varma et al., 1979),

Belgium (Verhoyen and Horvet, 1972), Great Britain (Mowat and Stefanc, 1974),

Cuba (Jotoff and Lago, 1972), Italy (Benetti and Tomassoli, 1988; Beraccini and

Marani, 1982), Japan (Hagita, 1989; Hagita etal., 1989; Takamatsu etal., 1994),

Korea (Chung, 1987; Chang et al., 1987), Latvia (Zhola et al., 1992), the

Netherlands (Asjes et al. 1973), Taiwan (Yang et al., 1993), and the U.S.A.

(Brierley and Smith, 1944a; Allen, 1972; Ballentyne era/., 1979).

Lily Virus X

LVX was identified as a potential problem for lilies grown in the

in Netherlands for export. Accordingly, it is now one of the three viruses included 8 the federal virus-indexing certification program by the Bulb Inspection Service,

Lisse, the Netherlands (Asjes, 1990). In this program, LVX was detected by

DAS-ELISA in leaves and bulbs using the same protocols as for LSV (Derks,

1995; Kimuraefa/., 1990).

LVX was first described in England by Stone (1980), who reported that

LVX-infected lilies grew more slowly and were smaller than healthy ones. Derks

(1995) reported that this virus caused very faint chlorotic spots and occasionally brown necrotic lesions on the leaves (Derks, 1995). Like other potexviruses, LVX has an average particle length of about 550 nm. This virus has only been found

naturally in lilies, although Tetragonia expansa and Chenopodium murale seedlings inoculated mechanically developed chlorotic local lesions (Stone,

1980). LVX has no known vector and does not spread rapidly under field

conditions. Nevertheless, Asjes (1991) speculated that it was transmitted in a persistent or semi-persistent manner by some unidentified vector.

This virus is serologically related to commelina X potexvirus but not to other potexviruses (Stone, 1980). The LVX nucleotide sequence in the

consensus region is reportedly very similar to that of other potexviruses including potato X, clover yellow mosaic, narcissus mosaic, papaya mosaic, white clover mosaic, and strawberry mild yellow edge-associated virus

(Jelkmann et a/., 1992). Nevertheless, the nucleotide sequence of LVX appears

to be unique among potexviruses inasmuch as it apparently lacks the small open reading frame, 5' to the coat protein cistron, common to all other potexviruses

(Memelinkef a/., 1990). 9

Tulip Breaking Virus and Other Potvviruses in Lilies and Tulips

TBV is a serious problem in both lilies and tulips. Indeed, tulip flowers

with color-breaking symptoms were featured in oil paintings of seventeen-

century Dutch artists (Derks ef a/., 1994; Mowat, 1995).

Cayley (1928) first reported that color break in tulips was caused by an infectious agent which could be transmitted to healthy plants. This agent was

subsequently shown to be transmissible by aphids (McKay et al., 1929).

McWhorter (1932) transmitted TBV mechanically from L. speciosum to tulips, which then developed symptoms indistinguishable from those noticed before in TBV-infected tulips (McWhorter, 1937). Using L. formosanum seedlings as indicator plants for detecting TBV in commercial Easter lily plantings, Brierley (1940) determined that this virus was present nearly

everywhere lilies were grown commercially. The synonyms used to describe TBV and isolates thereof included "tulip mosaic" (Yamaguchi et al., 1963) and

McWhorter's "color-adding" and "color-removing" viruses, which were later

referred to by the same author as "Tulip Viruses I and II," respectively

(McWhorter, 1935; 1936; 1937). While McWhorter considered his two viruses to be distinct, Van Slogteren (1971) considered them both to be strains of TBV, which he referred to as "severe" and "mild," respectively.

Unlike lilies, which appear to be susceptible only to TBV and strains thereof, tulips harbor a second potyvirus, which was just discovered in 1985 and later determined to be serologically related to turnip mosaic potyvirus (TuMV) 10

(Hammond and Chastagner, 1988; 1989; Mowat, 1985). This second potyvirus, unfortunately, has caused confusion for those investigating TBV in lilies. Indeed, antiserum prepared before 1985 and derived from virions in infected tulips

(assumed to be singly infected with TBV) may in fact contain antibodies to both (Deng, 1995). Moreover, confusion has been compounded by the failure to realize until recently (Deng, 1995) that TBV, like other potyviruses such as hippeastrum mosaic virus (Derks, 1992) and peanut mottle virus (Li et a/.,

1991), lacks certain epitopes common to most other potyviruses. As a result, universal monoclonal antibodies (MCAs) such as PTY-1 did not react readily

with TBV (Deng, 1995). Still more confusion has been created by the large number of synonyms that have been applied by various workers to both TBV and tulip-infecting isolates or strains of TuMV.

Isolates of TBV. The International Committee for the of Viruses

all of (ICTV) (Murphy et a/., 1995) lists three potyviruses that infect lilies, three which are closely related serologically. These include TBV from Texas Flame'

Lily tulip, Rembrandt tulip breaking Virus (RTBV) from 'Jack Laan' tulip, and

mottle virus (LiMV) from Easter lily (Table 1-4). All three infect L. formosanum

and are considered to be strains of TBV (Alper et at. 1982; Brierley and Smith,

1944b; Dekkeref a/., 1993; Derks etal., 1982; Langeveld etal., 1991).

Polyclonal antiserum against isolates of TBV from 'Jack Laan' tulip (TBV-

JL) and Texas Flame' tulip (TBV-TF) reacted differently when tested against lily

isolates of TBV. Whereas the TBV-JL antiserum reacted strongly to lily isolates

of TBV, the TBV-TF antiserum did not (Beijersbergen and van der Hulst, 1982; 11

Derks et al., 1982). Based on these differences, Boonekamp et al. (1990) distinguished the -JL and -TF serotypes as "lily- " and "tulip-strains," respectively.

Yet another isolate of TBV (TBV-L), originally isolated from L longiflorum, was described later by Derks (1985). This strain proved similar to a previously described one from Israel, which could infect C. amaranticolor (Alper et al.,

1982) and cross-reacted against both lily and tulip serotypes of TBV (Dekker et

al., 1993). However, a monoclonal antibody against TBV-L reacted with neither

the -JL nor the -TF isolates of TBV (Boonekamp and Pomp, 1986).

Langeveld et al. (1991) referred to a virus they tested from 'Flevo' Easter

lily as "TBV-lily," apparently not knowing that Boonekamp ef al. (1982)

previously used that term exclusively for the TBV-JL isolate from tulip. "TBV-lily"

and "TBV-tulip" have also been used by other researchers without regard to the

specific isolates studied by Dutch workers. Ohira ef al. (1994), for example, who

described the nucleotide sequences of TBV isolates in Japan, used these terms

to describe their respective isolates from lilies and tulips.

In 1993, Dekker ef al. used the name "lily mottle" (LiMV) to denote the

following three TBV-lily isolates: the 'Flevo' Easter lily described by Langeveld ef

from al. (1991), Derks's (1985) TBV-L isolate, and Alper ef a/.'s (1982) isolate

Easter lily. Dekker ef al. (1993) also identified the -JL isolate of TBV studied by

Derks ef al. (1982) as being RTBV. Another TBV isolate, which was recovered

from 'Lucky Strike' tulip, but was not infectious to lily, was named TBBV. Based

upon differences in host range, nucleotide sequence, and coat protein 12 serological properties, Dekker et al. (1993) concluded that TBV-JL, TBV-TF,

LiMV, and TBBV should be considered as distinct viruses, despite their close serological relationships to one another.

Lily plants infected with LiMV develop light to yellowish green mottle or mosaic symptoms on leaves and sometimes a malformation or a color breaking pattern on the flowers (Derks, 1995). Some isolates are transmissible to lilies and to such indicator plants as Nicotiana benthamiana, Tetragonia expansa, and

C. amaranticolor (Alper ef al., 1982; Dekker et al., 1993). This virus is also transmitted in a non-persistent manner by the aphids, A. gossypii, M. persicae,

and Macrosiphum solanifolii. It is apparently not seed-transmissible, however

(Alper ef al., 1982; Brierley and Smith, 1944b).

LiMV can be detected in ELISA tests using polyclonal antiserum and in

hybridization assays with specific probes (Derks et al., 1982; Dekker ef al.,

1993). Based on ISEM results, LiMV has been shown to be serologically related

to TBV and RTBV (Dekker et al., 1993). However, Ohira ef al. (1994) provided evidence that the amino acid sequence of the coat protein of LiMV is only 64% similar to that of TBV. Therefore, they strongly suggested that these are two different potyviruses.

Lilies infected by LiMV and other potyviruses were reported in Great

Britain (Mowat and Stefanc, 1974), Israel (Alper ef al., 1982), Italy (Bertaccini

and Marani, 1982), Japan (Hagita et al., 1989; Hagita and Sasaki, 1994), Korea

(Chang ef al., 1987), the Netherlands (Asjes ef al., 1973), and the U.S.A.

(Brierley and Smith, 1944b; Allen, 1975). 13

Isolates of TuMV. In 1985, Mowat described TCBV in tulip, which was distinct from TBV. Later, Hammond and Chastagner (1988; 1989) described two similar tulip viruses, WaTV and TuMV-L2, which were serologically related to

TuMV. Later, Dekker et al. (1993) reported that yet another tulip virus, TTBV, was also closely related to TuMV. These four viruses were proved to be closely

serologically related to bean yellow mosaic virus (Hammond and Chastagner,

1989) and could infect certain herbaceous dicotyledons such as C. quinoa and

N. benthamiana. None of these viruses, however, is apparently able to infect

lilies (Dekker et al., 1993; Hammond and Chastagner, 1988).

Other Viruses Reported to Infect Lilies

Viruses other than LSV, LVX, and TBV have been occasionally reported to

infect lilies, but do not appear to be widespread in lilies, and are not taken into

account in the Netherlands Bulb Inspection Service's virus-indexing program.

These included three nematode-transmitted viruses. Arabis mosaic nepovirus

was reported in Italy and the Netherlands (Marani and Facciolo, 1974; Asjes and

Segers, 1983), tobacco rattle tobravirus has been found in the U.S.A. and the

Netherlands (McWhorter and Allen, 1964; Derks, 1975), and strawberry latent

ringspot nepovirus was reported in Israel (Cohen et al., 1995). Solitary reports of

broad bean wilt fabavirus in Korea (Chang and Chung, 1987), citrus tatter leaf

capillovirus in Japan (Inouye et al., 1979), narcissus mosaic potexvirus in Italy

(Bellardi ef al., 1988), and a possible reovirus in Japan (Varma et al., 1979) in

lilies have also been published. 14

Although lily planting stocks are field-inspected, and samples are tested

in ELISA by the Netherlands Bulb Inspection Service, many imported Dutch bulbs, while often with high quality, are nevertheless frequently infected with viruses such as LSV, CMV and TBV (Deng, 1995). Thus, commercial growers in the U.S.A. and elsewhere run the risk of planting contaminated stock and suffer

yield losses accordingly, especially if they replant the same materials in their

fields in subsequent years.

For the past 40 years, studies on lily viruses were conducted primarily by only three research centers, namely the USDA Florist and Nursery Crops Center

(Beltsville, MD), the Plant Pathology Department at the University of Oregon

(Corvallis, OR), and the Flower Bulb Research Center (Lisse, the Netherlands).

Most of the recent published papers have been contributed by scientists at the

latter institution. Information on lily viruses that applies to Florida has only

recently been obtained (Deng, 1995).

The objectives of this study were 1) to identify and characterize LVX,

which has never been reported in the U.S.A.; 2) to establish reliable and user-

friendly protocols for virus-indexing plants to be grown for certification; 3) to

determine the serological and cytological relationships between LiMV and TBV;

and 4) to evaluate the potential impacts of LSV, LVX, and TBV on commercial lily

production. 15

Table 1-4. Potyviruses reported to infect lilies and tulips

Mechanical transmission to Nicotiana

V/in ^' References V II uocoiqpq Hosts Lilv benthamiana 1991. LiMV lily + Langeveld eta'., TBV-TF tulip + Derks et al., 1982. TBV-JL (RTBV) tulip + Derks etal., 1982. TBBV tulip Asjes and Segers, 1985. TCBV tulip + Mowat, 1985. TTBV tulip + Asjes and Segers, 1982. WaTV tulip + Hammond and Chastagner, 1988

TuMV-T5-2-7 tulip + Hammond and Chastagner, 1988 1) = isolates of tulip LiMV = lily mottle virus, TBV-TF and -JL TF- and JL- breaking virus, RTBV = Rembrandt tulip breaking virus, TBBV = tulip band- breaking breaking virus, TCBV = tulip chlorotic blotch virus. TTBV = tulip top of turnip virus, WaTV = Washington tulip virus, TuMV-T5-2-7 = T5-2-7 isolates mosaic virus. 2) + = infection; - = no infection. CHAPTER 2 CHARACTERIZATION OF LILY X POTEXVIRUS IN LILItS

Introduction

Lily X potexvirus (LVX) was first found in lily in the England by Stone

(1980). Based on its particle shape and length, LVX was determined to be a potexvirus. Although LVX-infected plants were symptomless, they were smaller than healthy ones. This virus was sap-transmitted to Tetragonia tetragonioides

(Pall.) Kuntse, Chenopodium murale L, Gomphrena globosa L, and Nicotiana clevelandii Gray. LVX was determined to be serologically related to commelina virus X, but not to the other nine potexviruses tested (Stone, 1980).

LVX was subsequently discovered in Japan by Kimura et al. (1990).

Incidence of LVX there ranged from 2.06% in 'Stargazer' lily to 2.1% in 'Casa

Blanca' lily (Kimura et al., 1990). Four years later, LVX was also found in the

Netherlands, where infection rates ranged from 4.8 to 8.9% depending on the (Asjes and Blom-Barnhoom, 1994).

Up to now, LVX has never been completely characterized. The only other descriptive paper about this virus besides that by Stone (1980) was of Memelink et al. (1990), who described a nucleotide sequence of part of the genome at the

3' end. However, this sequence is atypical of other potexviruses inasmuch as it lacks the small open reading frame 5' to the coat protein cistron common to all other potexviruses.

16 17

Materials and Methods

Virus Isolates

The LL-, SS- and SG- isolates of LVX used in this study were isolated from 'Laura Lee,' 'Strawberry Shortcake,' and 'Stargazer' lily bulbs, respectively,

all of which were imported from the Netherlands. The LVX-NW and -WS isolates were isolated from 'Nellie White' and 'White Sheen' lily plants obtained from

commercial sources and are assumed to have been field-grown in the Western

U.S.A..

The J12 isolate of CMV was maintained in 'Juanita' lily, and the K45

isolate of CMV was maintained in N. benthamiana.

The CB-, SG-, Mon-, and NW- isolates of LSV were isolated from 'Casa

Blanca,' 'Stargazer,' 'Montreaux,' and 'Nellie White' lilies, respectively.

The Sir- FA- and NW- isolates of TBV were maintained in 'Sirocco' lily, L

of formosanum and 'Nellie White' lily, respectively. The T- and WS-342 isolates

LiMV were maintained in N. benthamiana. Healthy L formosanum seedlings

and/or indexed 'Nellie White,' 'Montana,' and 'Montreaux' lily plants were used

as healthy controls in all experiments.

Polyclonal Antisera

Polyclonal antisera of LVX-Derks, LiMV-Derks and TBV-Derks were

purchased from A. F. L. M. Derks (Derks et al., 1982). Antiserum to LiMV-T was

provided by C.-A. Chang (Taiwan Agricultural Research Institute, Taichung,

Taiwan), and antiserum to BaMV was provided by N.-S. Lin (Lin and Chen,

1991). Polyclonal antisera PapMV-A747 and PVX-A782 (Batchelor, 1974), PVY- 18

A709 (Purcifull et a/., 1975), TuMV-A825 (McDonald and Hiebert, 1975), and

WCIMV-A923 (Purcifull et a/., 1981) were provided by D. E. Purcifull. Polyclonal antisera to CMV-K45, LSV-Sir, and TBV-FA were provided by T.-C. Deng

(1995). Polyclonal antiserum LSV-A1189 was produced from LSV-infected

Asiatic lily (Yang et a/., 1993). In addition, polyclonal antisera ByMV-A993

(Zettler and Abo El-Nil, 1977), CsCMV-A1091 (Zettler and Elliott, 1986), CyMV-

A862 (Wisler et a/., 1982), and NanMV-A934 (Zettler et ai, 1980) were provided by F. W. Zettler.

Host Range Tests

Samples of infected leaves or scales were triturated in 0.1 M phosphate buffer containing 0.1% sodium sulphate, pH 7.2. The extracts were then rubbed onto leaves of test plants dusted with 600-mesh carborundum. Seedlings of

Chenopodium amaranticolor Coste and Reyn., C. quinoa Willd., Datura metel L,

Gomphrena globosa L, L formosanum Stapf., L. longiflorum Thunb. 'Arai' and

'Nellie White,' Nicotiana benthamiana Domin., N. clevelandii Gray, N. X

Comes, edwardsonii Christie & D. W. Hall , N. glutinosa L, N. sylvestris Speg. &

Tetragonia expansa (Pall.) Kuntse, and Zinnia elegans Jacq. were the test plants

used. All test plants were maintained in a greenhouse at about 20 to 24 C.

Viral Inclusions

These tests were conducted by modifying the procedures of Christie and

Edwardson (1977) and Hoefert et ai (1992). The leaf epidermal strips or scale

sections were floated on an Azure A and 0.2 M Na2 HP04 (9:1) solution and 19

heated in a microwave oven (Sharp Carousel II, Mahwah, NJ) for 20 seconds

2- (800 W power). The stained tissues were rinsed for 1 to 2 min in a methoxyethyl acetate and 95% ethanol mixture (7: 3) to remove excess stain.

The tissues were then placed in 100% 2-methoxyethyl acetate for 1 to 2 min and

mounted in either Euparal or LR White medium. Finally, these tissues were

examined by light microscopy. Epidermal strips and sections from leaves or

scales of non-infected lilies served as controls.

Serological Tests

cross- Cross absorption and purification of LVX IgG . To eliminate

reactivity with healthy lily sap when using antisera of LiMV-Derks, LiMV-Chang,

LSV-1189, LVX-Derks, TBV-Derks, and TBV-RE102, leaves of healthy L

formosanum were extracted (1:10) in phosphate buffered saline (PBS) and

subjected to dialysis against the same buffer at 4 C overnight. Antiserum was

mixed with the healthy plant sap (1:10) and stirred at 37 C for 4 hours and then

at 4 C overnight. After centrifugation (5,000 g, 10 min) and freezing overnight,

the antiserum and plant sap were subjected to immunoglobulin (IgG) purification

through a DEAE-Sephacel column as described by Clark and Adams (1977).

according to the Indirect ELISA . The purification of IgG was done

procedures of Clark and Adams (1977). Indirect ELISA with plate-bound antigen

protocols (Converse and Martin, 1990) were used, except that only 100 pi of

reagents were added to each well and that the incubation periods were reduced

to one hour at 37 C. Test samples of lily leaf or bulb scale tissues, extracted in

10 volumes of coating buffer (50 mM sodium carbonate, pH 9.6), were added to 20 wells of microtiter plates. The plates were incubated overnight at 4 C and then washed with PBST (PBS, pH 7.4, containing 0.5% Tween 20). Polyclonal antisera (PCAs) or monoclonal antisera (MCAs) diluted with conjugate buffer

(PBST containing 2% polyvinylpyrrolidone-40, and 0.2% bovine serum albumin) were then added and incubated. After washing with PBST, plates were loaded with alkaline phosphatase-labeled goat anti-rabbit (for PCA) or anti-mouse (for

MCA) immunoglobulin (1:25,000). Plates were then incubated and washed as

before. Enzyme substrate, p-nitrophenyl phosphate, was used at 1 mg/ml in diethanolamine buffer, pH 9.8, and incubated at room temperature for color development. Absorbance values (A405) were determined with a Bio-Tek automated microplate reader (Bio-Tek Instruments, Winooski, VT), at 15-min

intervals for 1 hr or longer.

Western blot tests. The Western blot procedure was conducted as

described by Towbin et al. (1979) using a Bio-Rad (Hercules, CA) Mini-Protein II

Electrophoresis cell and Bio-Rad Trans-Blot Electrophoretic Transfer cell.

Test samples were prepared by extracting 1 g of fresh leaf or 0.1 g of

dried tissue in 1 ml of distilled water. Plant extracts were concentrated by adding equal volumes of cold acetone (A. Abouzid, personal communication), and

the incubating them at -20 C for 5 min. After centrifugation for 5 min at 10,000 g ,

pellet was air-dried and resuspended in 200 (il of Laemmli dissociation buffer

(20% 1.25 M Tris-HCI pH at 6.8, 2.5% SDS, 4% 2-mercaptoethanol, and 10% sucrose) (Laemmli, 1970). During electrophoresis at a constant voltage of 150 V

for 1 hr or 100 V for 90 min, the proteins were separated in a 12% 21 polyacrylamide gel. The proteins were transferred to nitrocellulose membranes

(0.45 urn pore size) by electrophoretic transfer at a constant voltage of 100 V for

1 hr. Nitrocellulose membranes were rinsed three times in Tris buffered saline

(TBS) (20 mM Tris-HCI at pH 8.2, 15 mM NaCI) containing 0.1% Tween 20

(TBST). After blocking with Blotto solution (5% nonfat dry milk in TBST) at room temperature for 15 min, virus-specific antibodies were added and incubated at 4

C overnight or at room temperature for I hr. The dilutions for polyclonal antiserum to bamboo mosaic (BaMV), cassava common mosaic (CsCMV), cymbidium mosaic (CymMV), LVX, nandina mosaic (NanMV), papaya mosaic

(PapMV), potato X (PVX) and white clover mosaic (WCIMV) potexviruses were

1:1,000 or 1:2,000 in Blotto solution. After rinsing in TBST, membranes treated with PCAs were incubated with goat anti-rabbit IgG-conjugated alkaline phosphatase (Cat. No. A-3687, Sigma Chemical Company, St. Louis, MO), whereas membranes treated with MCAs were incubated with goat anti-mouse

IgG-conjugated alkaline phosphatase (Cat. No. A-3562, Sigma Chemical

Company, St. Louis, MO) for 1 hr at room temperature. Both conjugates were used at a dilution of 1:25,000. After rinsing as described above, and then rinsing

the again in substrate buffer (0.1M Tris-HCI, pH 9.5, 0.1 M NaCI, 5 mM MgCI2 ), nitrocellulose membrane was developed in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT)(Gibco-BRL, Gaithersburg,

MD) and observed for color reactions. 22

Immunogold labeling

modifying the Light microscopy . These tests were conducted by procedures of Ko (1988). Leaf epidermal strips were digested with a mixture of

10% pectinase and 4% cellulase (1:1; v/v) in PBST buffer for 30 min. These were then rinsed with PBST buffer three times for 5 min each. The tissues were incubated with respective antiserum diluted in PBST containing 10% dimethyl sulfoxide (DMSO) at room temperature for one hr and then rinsed as above. The tissues were incubated with goat anti-rabbit gold conjugates diluted in PBST containing 10% DMSO at room temperature for 90 min, rinsed as above, and mounted in glycerin diluted to 10% in PBS buffer. The tissues were then examined by light microscopy using a blue filter (Wratten Filter No. 80 C) for inclusions as described by Ko (1988).

in few drops of 0.1 M K- Electron microscopy . Leaf tissues were chopped

Na phosphate buffer (pH 7.2). The virus extracts were dropped onto the formvar-coated grids and incubated for one to several min. The plant extracts

20-30 then were absorbed by strips of filter paper, and the grids were rinsed with

drops of K-Na phosphate buffer followed by 20 to 30 drops of distilled water. The

grids were incubated with primary antiserum diluted in HST buffer (0.05 M Tris-

HCI, pH 7.4, containing 0.5 M NaCI and 0.1 % Tween) at room temperature for

30 to 60 min. The grids were rinsed with HST buffer and then incubated in goat

anti-rabbit gold conjugate diluted in HST buffer. The grids again were rinsed in

HST and distilled water. Finally, the grids were stained with 2% uranyl acetate 23 containing 100 ng/ml of bacitracin (filtered through 0.2 |im microfilter) for 2-3 seconds and examined by electron microscopy.

Direct tissue blotting (DTB) . The DTB tests were similar to those

described by Lin et al. (1990) and Garnsey et al. (1993) The root, stem, scale, or

leaf tissues of lily plants were cut transversally with a new razor blade, and the cut surfaces were applied gently to nitrocellulose membranes (Bio-Rad,

Hercules, CA). To reduce the nonspecific reactions, the membranes were blocked at room temperature for 30 min with a Blotto solution (5% nonfat dry milk in TBST buffer) in 1:50 or 1:100 dilutions of healthy lily leaf sap. The blotted membranes were washed for five min three times in TBST buffer and then incubated in a solution of antiserum and Blotto solution (1:1000) at room

temperature for 1 hr. The blotted membranes were subsequently washed for five min three times in TBST buffer and incubated in a solution of goat anti-rabbit (for

polyclonal antibody) or goat anti-mouse (for monoclonal antibody) IgG-alkaline

phosphatase conjugate (Cat. No. A-3687 or A-3562, Sigma Chemical Company,

St. Louis, MO) and Blotto solution (1:25,000) at room temperature for one hr.

Blotted membranes were then washed for five min two times in TBST buffer

followed by a five min wash in substrate buffer (0.1 M Tris-HCI, pH 9.5, 0.1 M

NaCI, 5 mM MgC^). Finally, the blotted membranes were incubated in a solution

of NBT and BCIP in a substrate buffer for 5 to 1 5 min in the dark to allow time for

the purple color reaction to develop. The reaction was stopped with distilled

water, and the blotted membranes were then dried between paper towels. 24

Results

Host Range Tests

The reactions of inoculated indicator plants to LVX are listed in Table 2-1.

Both isolates of LVX were similar in host range and induced local lesions in C.

amaranticolor, C. quinoa, and T. tetragonioides (Fig. 2-1), and systemic

symptomless infections in 'Arai' and 'Nellie White' lily. However, only the WS- isolate of LVX infected N. benthamiana.

Viral Inclusions

Red-violet vacuolate and non-banded spindle-shaped inclusions were

noted in the epidermal cells of LVX-infected lily and T. tetragonioides leaves

(Fig. 2-2). Similar inclusions were observed in the epidermal cells of PVX-,

PapMV- and CsCMV-infected tobacco leaves (Fig. 2-3). These inclusions were usually located adjacent to cell walls as described by Christie and Edwardson

(1977) and Ko (1985) for other potexviruses.

Serological Tests

Indirect ELISA . LVX IgG diluted to 1:1,000 in conjugate buffer (pH 7.4) reacted with homologous antigen but not with CMV, LiMV, LSV, and TBV antigens. Likewise, purified antisera to CMV, LSV, and TBV specifically reacted only with their respective homologous antigens (Table 2-2). Strong nonspecific reactions were noted when diluted crude polyclonal antisera to each of the

above five viruses were tested. Thus, antisera cross-absorbed with healthy lily saps were used in this study to eliminate the nonspecific reactions of antiserum

with healthy lily saps. 25

The reactivities of LVX against polyclonal antisera of other potexviruses are listed in Table 2-3. Weak heterologous reactions (A405 values lower than

0.55) with LVX antigen against antisera to BaMV, CsCMV, CymMV, NanMV,

PapMV, PVX, and WCIMV were noted. In contrast, strong homologous reactions

(A405 values all were higher than 1.00) were noted for all eight potexviruses.

Whereas LVX antiserum reacted strongly with LVX antigen (A405 value was 1.06), only weak reactions (A405 values were lower than 0.10) with antigens of BaMV,

CsCMV, CymMV, NanMV, PapMV, PVX, or WCIMV were noted.

of Western blot tests . All five cross-absorbed polyclonal antisera CMV,

LiMV, LSV, LVX, and TBV reacted with their respective homologous antigens but not with heterologous antigens. For instance, LVX polyclonal antiserum reacted only with LVX antigen but failed to react with CMV, LiMV, LSV, TBV, healthy lily, and N. benthamiana sap (Fig. 2-4, Table 2-4). Bands at 22, 26, and 32 kDa were noted for LVX, CMV, and LSV, respectively, whereas LiMV and TBV had bands at 31 and 34 kDa (Figs. 2-5, 2-6, and 2-7).

In Western blot tests, polyclonal antisera to LVX reacted with 5 isolates of

LVX, but not with healthy lily saps (Table 2-5). Polyclonal antisera to LVX and other potexviruses cross-reacted against each other (Table 2-6). Strong homologous and heterologous reactions with LVX protein (about 22 kDa) were obtained with antisera to CsCMV, CymMV, LVX, PVX, and WCIMV (Fig. 2-8).

Whereas polyclonal antisera of BaMV, CsCMV, CymMV, NanMV, PVX, and

WCIMV reacted heterologously with other potexviruses, polyclonal antisera of

LVX and PapMV did not (Fig. 2-9, Table 2-6). 26

Immunoqold labeling

Light microscopy . Light pink-colored inclusions were noted in LVX-

infected T. tetragonioides epidermal tissues immunostained sequentially with

homologous antiserum and goat anti-rabbit-gold conjugate complex (GAG). No

specific reactions were noted in LVX-infected tissues treated with the GAG

complex alone, or with tissues treated with LSV antiserum. Likewise, pink-

colored LSV inclusions were seen when the homologous antiserum and GAG were applied to these tissues (Fig. 2-10).

of Electron microscopy . Virions ca. 550 nm long, assumed to be those

LVX, were decorated with 10 nm-colloidal gold particles in indirect immunogold

labeling tests, whereas virions ca. 650 and 750 nm (assumed to be those of LSV

and TBV, respectively) were not (Fig. 2-11). LSV and TBV virions were

decorated with 10 nm-gold particles, however, when respective homologous

antisera were used.

reactions were noted on the Direct tissue blotting (DTB) . Purple color

nitrocellulose membranes blotted with LVX-infected tissues and then treated with

LVX antiserum and goat anti-rabbit alkaline phosphatase conjugate (Fig. 2-12).

No such reactions were noted, however, on membrane surfaces blotted with

healthy controls or with cut-tissues of plants singly infected with CMV, LSV, or

TBV. 27

Table 2-1 . Plants susceptible to two isolates of lily virus X

1) Virus isolate

Indicator plant LVX-SS LVX-WS

2) C. amaranticolor CL, NL CL, NL Chenopodium quinoa CL, NL CL, NL

Datura metel ni ni

Gomphrena globosa ni

Lilium formosanum S S

L. longiflorum cv. Arai S S

L longiflorum cv. Nellie White S S

Nicotiana benthamiana ni S

N. clevelandii ni

N. edwardsonii ni

N. glutinosa ni

N. sylvestris ni

Tetragonia tetragonioides CL CL

Zinnia elegans ni

Isolates from lily cultivars Strawberry Shortcake (SS) and White Sheen (WS).

CL = chlorotic local lesion, NL = necrotic local lesion, S = symptomless

infection, ni = no infection, - = not tested. All results were confirmed by DTB or

l-ELISA. 28

Fig. 2-1. Top: Leaf necrosis, and necrotic tepal tip caused by LVX and LSV infection on 'Strawberry Shortcake' Oriental lily (top). Bottom left: Local lesions on Tetragonia tetragonioides caused by LVX infection. Bottom right: Healthy. 29

Fig. 2-2. Top: Light micrographs of spindle-shaped inclusions (I) induced by LVX

in Lilium. Bottom: Tetragonia tetragonioides leaf cells stained with Azure A. N = Nucleus. Bar = 10 ^m. Fig. 2-3. Top left: Azure A-stained inclusions ( ) induced by LVX in lily; Top I right: inclusions induced by CsCMV; Bottom left: PapMV; and Bottom = right : PVX in Nicotiana benthamiana, respectively N Nucleus. Bar = 10 |am. 31

Table 2-2. Heterologous and homologous reactions of five lily viruses against purified, cross-absorbed antisera

2) Antigen (1:5) Antiserum 1) Healthy LSV LVX LiMV TBV CMV

3) LSV-sir1 0.06 0.41 0.06 0.07 0.06 0.06 LSV-1189 0.06 0.53 0.06 0.08 0.06 0.05

LVX-Derks 0.16 0.22 1.87 0.10 0.13 0.20 LiMV-Derks 0.05 0.06 0.07 0.99 1.77 0.24 LiMV-Chang 0.05 0.07 0.07 2.63 2.20 0.20

TBV-Derks 0.11 0.11 0.10 0.58 1.33 0.08 CMV-K45 0.10 0.15 0.10 0.16 0.18 1.15

' All antisera were diluted 1:1000. Antisera LSV-sir1 and CMV-K45 were provided by T.-C. Deng; antisera LVX-Derks, LiMV-Derks, and TBV-Derks were from A. F. L. M. Derks; antiserum of LiMV-Chang was from C.-A. Chang; LSV-

1 1 89 antiserum was prepared by the author. 2> The antigens are: Healthy = healthy lily sap; LSV = lily symptomless virus, LVX = = lily virus X, LiMV = lily mottle virus, TBV = tulip breaking virus, CMV cucumber mosaic virus in lily. 3) All A 405 values were the mean of two replications and read after 30 min reaction. 32

Table 2-3. Homologous and heterologous reactivities of LVX and other potexviruses to polyclonal antisera in l-ELISA tests

Antiserum (1:2,000)

1 Antigen/tissue ' LVX BaMV CymMV NanMV CsCMV WCIMV PVX PapMV

HCK/lily 0.07 3) 0.00 0.00 0.00 0.05 0.00 0.00 0.00

LVX/lily 1.06 0.32 0.55 0.15 0.25 0.28 0.39 0.35

LSV/lily 0.02 0.12 0.00 0.00 0.00 0.00 0.04 0.02

CMV/tobacco 0.04 0.00 0.00 0.00 0.09 0.02 0.00 0.00

TBV/lily 0.05 0.00 0.00 0.01 0.11 0.06 0.00 0.00

HCK/bamboo 0.00 0.11 0.02 0.02 0.06 0.01 0.22 0.00

BaMV/bamboo 0.04 1.07 0.29 0.43 0.48 0.66 0.65 0.81

HCK/chaya 0.01 0.00 0.00 0.00 0.02 0.00 0.00 0.00

CsCMV/chaya 0.04 0.05 0.00 0.10 1.03 1.05 0.00 0.11

HCK/cattleya 0.00 0.17 0.14 0.10 0.19 0.25 0.20 0.24

CymMV/cattleya 0.04 0.32 1.79 0.09 0.15 0.40 0.11 0.47

HCK/clover 0.02 0.25 0.17 0.05 0.12 0.03 0.11 0.25

WCIMV/clover 0.02 0.00 0.24 0.00 0.27 2.03 0.92 0.45

HCK/tobacco 0.06 0.15 0.06 0.03 0.20 0.01 0.05 0.07

NanMV/tobacco 0.02 0.00 0.08 2.26 1.48 1.45 0.00 0.52

PapMV/tobacco 0.06 0.00 0.65 0.00 1.12 1.69 0.58 1.70

PVX/tobacco 0.02 0.10 0.23 0.03 0.09 1.10 1.99 0.57

' The antigens were HCK = healthy tissue sap; LVX = lily virus X; LSV = lily symptomless virus; CMV = cucumber mosaic virus; TBV = tulip breaking virus; BaMV = bamboo mosaic virus; CsCMV = cassava common mosaic virus, CymMV = cymbidium mosaic virus, NanMV = nandina mosaic virus, PapMV = papaya mosaic virus, PVX = potato virus X, WCIMV = white clover mosaic virus. 2) Antisera PapMV, PVX, and WCIMV were provided by D. E. Purcifull; antisera

LVX and BaMV were provided by A. F. L. M. Derks and L.-S. Lin, respectively; antisera CsCMV, CymMV, and NanMV were from F. W. Zettler. 3) All A 405 values were the mean of two replications and read after I hr reaction. 33

Table 2-4. Homologous and heterologous reactivities of five lily viruses against six polyclonal antisera in Western blot tests

Antigen 1) CMV- LSV- LVX- TBV- LiMV- LiMV- K45 1189 Perks Perks Perks Chang CMV-K45 26 3) - LSV-Mon 32 LVX-SS 22 LiMV-P 34 34 34 LiMV-T 34 34 34

TBV-FA 31 31 31 HCK

' The antigens are: CMV-K45 = K45 isolate of cucumber mosaic virus provided = by T, -C. Peng, LSV-Mon = lily symptomless virus in 'Montreaux' lily, LVX-SS lily virus X in 'Strawberry Shortcake' lily, LiMV-P = lily mottle virus provided by

Perks, LiMV-T = lily mottle virus provided by C.-A. Chang, TBV-FA = FA- isolate of tulip breaking virus provided by T.-C. Peng, HCK = healthy lily sap. 2) All purified IgG were diluted 1:1000. Antisera LVX-Perks, LiMV-Perks, and

TBV-Perks were provided by A. F. L. M. Perks, Antiserum LiMV-Chang was from C.-A. Chang, Antiserum CMV-K45 was provided by T.-C. Peng, Antiserum LSV- 1189 was provided by the author. 3) = Numbers are estimated sizes (kPa) of proteins in nitrocellulose membrane; - no reactions. 34

Table 2-5. Reactivities of four LVX isolates against LVX polyclonal antiserum in Western blot tests

Antigen 1) LVX antiserum

Healthy Nicotiana benthamiana - 3)

Healthy Lilium formosanum

LVX-Derks in unspecified lily +

LVX-WS in 'White Sheen' lily +

LVX-SS in 'Strawberry Shortcake' lily +

LVX-WS in Nicotiana benthamiana +

LVX-SG in 'Stargazer' lily +

The antigen LVX = lily virus X, LVX-D = LVX provided by A. F. L. M. Derks,

LVX-WS = WS-isolate of LVX from "White Sheen' lily, LVX-SS = SS-isolate of

LVX from 'Strawberry Shortcake' lily, LVX-SG = SG-isolate of LVX from

'Stargazer' lily.

LVX antiserum was diluted to 1 :1 ,000 in Blotto solution. Reactivity determined as - = no reaction, + = positive reactions based on 22 kDa protein bands noted in nitrocellulose membranes. 35

ABCDEFGHIJ

97- 68- 43 -

29 -

Fig. 2-4. Reactivities of lily virus X polyclonal antiserum with 4 lily viruses in Western blot tests. The lanes are: A = healthy lily sap, B = lily symptomless virus, C = K-45 isolate of cucumber mosaic virus in N. benthamiana provided by T.-C. Deng, D = FA- isolate of tulip breaking virus in lily provided by T.-C. Deng, E = standard marker proteins with the numbers to the left indicating the estimated sizes in kilodaltons, F = D- isolate of lily virus X in lily provided by A.

F. L. M. Derks, G = WS- isolate of lily virus X in lily, H = SS- isolate of lily virus X = = leaf sap. in lily, I SG- isolate of lily virus X in lily, J healthy N. benthamiana 36

ABCDEFGHI JKMNO

97 - 68 *

43 *

29 *-

Fig. 2-5. Reactivities of cucumber mosaic virus polyclonal antiserum with 5 N. viruses in Western blot tests. The lanes are: A = healthy lily sap, B = healthy virus, benthamiana leaf sap, C = SS- isolate of lily virus X, D = lily symptomless E = FA- isolate of tulip breaking virus, F = T- isolate of lily mottle virus provided Derks, by C.-A. Chang, G = D- isolate of lily mottle virus provided by A. F. L. M. = = = standard marker proteins H peanut stunt virus, I aspermy virus, J with the numbers to the left indicating the estimated sizes in kilodaltons, K = lily symptomless virus, M = K-45 isolate of cucumber mosaic virus provided by T.-C.

Deng, N = D- isolate of lily virus X provided by Derks, O = healthy lily leaf sap. 37

ABCDE FGHI J

97- 68- 43 -

29-

with lily Fig. 2-6. Reactivities of lily mottle virus-T isolate polyclonal antiserum 5 = viruses and in Western blot tests. The lanes are: A WS provided by C.-A. isolate of lily mottle virus, B = T- isolate of lily mottle virus D = Chang, C = FA- isolate of tulip breaking virus provided by T.-C. Deng, virus provided by T.-C healthy lily sap, E = K-45 isolate of cucumber mosaic = Deng, F = 3374- isolate of turnip mosaic virus provided by D. E. Purcifull, G to the left lily symptomless virus, H = standard marker proteins with the numbers = = isolate of lily virus X, J indicating the estimated sizes in kilodaltons, I SS-

healthy lily sap. 38

ABCDEFG H I JK L M NO

29-

18-

Fig. 2-7. Reactivities of lily symptomless virus polyclonal antiserum with 5 lily viruses and turnip mosaic virus in Western blot tests. The lanes are: A = healthy

lily sap, B = D- isolate of lily mottle virus provided by A. F. L. M. Derks, C = T-

isolate of lily mottle virus provided by C.-A. Chang, D = FA- isolate of tulip breaking virus provided by T.-C Deng, E = WS- isolate of lily symptomless

virus, F = TDG- isolate of lily symptomless virus, G = standard marker proteins with the numbers to the left indicating the estimated sizes in kilodaltons, H = Sir- = isolate of tulip breaking virus, I = CB- isolate of lily symptomless virus, J NW-

isolate of lily symptomless virus, K = 3374- isolate of turnip mosaic virus provided by D. E. Purcifull, L = K-45 isolate of cucumber mosaic virus provided

by T.-C. Deng, M = SS- isolate of lily virus X, N = ST- isolate of lily symptomless virus, 0 = healthy N. benthamiana leaf sap. 39

Table 2-6. Homologous and heterologous reactivities of LVX and other potexviruses against 8 potexvirus polyclonal antisera in Western blot tests

Antiserum (1:1,000 or 2,000) Antigen Host LVX BaMV CymMV NaMV CsCMV WCIMV PVX PapMV 1) 2)

HCK Lily _3) _ _ _ - _

LVX Lily 22 — 22 - 22 22 22 -

HCK Bamboo - _ _ _ — - - -

BaMV Bamboo _ 30.5 _ 30.5 _ — _ —

HCK Cattleya _ _ _ _ _

CymMV Cattleya _ _ 27.5 _ _

HCK Tobacco ------

NanMV Tobacco 22 22 22 22 22 22

HCK Chaya

CsCMV Chaya 15

HCK Clover

WCIMV Clover 23 23 23 23 23

PVX Tobacco 28.5 28.5

PapMV Tobacco 23 23 23 23 23

The antigens are: HCK = healthy control, LVX = lily virus X, BaMV = bamboo mosaic virus, CsCMV = cassava common mosaic virus, CymMV = cymbidium mosaic virus, NanMV = nandina mosaic virus, PapMV = papaya mosaic virus, PVX = potato virus X, WCIMV = white clover mosaic virus. The antisera are the same abbreviations as the corresponding antigens. Reactivity determined as - = no reaction; Numbers are the estimated sizes (kDa) of proteins in nitrocellulose membrane; Values in bold font indicate homologous reaction. 40

ABCDEF g h ij klmno

97 68 43

29

Fig. 2-8. Reactivities of lily virus X polyclonal antiserum with 8 potexviruses in Western blot tests. The lanes are: A = healthy lily sap, B = SS- isolate of lily virus X, C = healthy bamboo leaf sap, D = bamboo mosaic virus, E = healthy = chaya leaf sap, F = chaya- isolate of cassava common mosaic virus, G standard marker proteins with the numbers to the left indicating the estimated = virus, J sizes in kilodaltons, H = healthy cattleya leaf sap, I cymbidium mosaic = healthy N. benthamiana leaf sap, K = nandina mosaic virus, L = papaya mosaic virus, M = potato virus X, N = white clover mosaic virus, 0 = healthy clover. 41

Fig. 2-9. Reactivities of bamboo mosaic virus polyclonal antiserum with 8 potexviruses in Western blot tests. The labels are A = healthy lily sap, B = SS- = virus, E isolate of lily virus X, C = healthy bamboo leaf sap, D bamboo mosaic = healthy chaya leaf sap, F = chaya isolate of cassava common mosaic virus, G = standard marker proteins with the numbers to the left indicating the estimated = mosaic virus, J sizes in kilodaltons, H = healthy cattleya leaf sap, I cymbidium = nandina mosaic virus, K = papaya mosaic virus, L = potato virus X, M = white clover mosaic virus, N = healthy clover, 0 = healthy N. benthamiana leaf sap. Fig. 2-10. Top: Light micrographs of lily virus X inclusion (I). Bottom: lily

symptomless virus inclusion (I) reacted with homologous antiserum and goat anti-rabbit-gold complex. Bar = 10 ^m. Fig. 2-11. Electron micrograph of virions treated with goat anti-rabbit gold conjugates. LVX particles labeled with LVX antiserum (top) and LSV particles labeled with LSV antiserum (bottom). Bar = 300 nm. 44

2-12. Reactions of LVX-infected Lilium scales with polyclonal LVX antiserum

in direct tissue blotting on nitrocellulose membrane. LVX antigens were detected on tissue blots incubated with homologous antiserum = and goat anti-rabbit antiserum-alkaline phosphatase conjugate. B basal plate, S = bulb scale, R = root, N = negative control, P = positive control. 45

Discussion

Based on host reactions, viral inclusions, and serological results, the LL-,

NW-, SS-, SG-, and WS- isolates of LVX encountered in this study are similar, if not identical, to the ones described by Stone (1980) and Kimura et al. (1990).

This is only the third published study of LVX, and the findings support the

the potexvirus premise that it should be considered as a distinct member of

potexvirus group. Memelink et al. (1990) reported that LVX is an unusual

next to the coat because it apparently lacks an open reading frame immediately

protein gene. Nevertheless, in other respects it appears to be a typical member

of this group.

in Lilium The limited host range of LVX and its apparent lack of symptoms

spp. and N. benthamiana precluded the use of these indicator plants for LVX

diagnosis. However, local lesions on T. tetragonioides and C. amaranticolor-

inoculated leaves were seen 21 to 28 days after mechanical inoculation under

<22 C growth conditions. Under higher temperatures, however, inoculated

leaves senesced before symptoms were noted. Nevertheless, the host reactions

the tests by of LVX were like those described by Stone, who conducted

maintaining inoculated plants at 18 C (1980).

Vacuolate and spindle-shaped cytoplasmic inclusions similar to those of

other potexviruses such as CsCMV, PVX, and PapMV were found in LVX-

infected leaf epidermal tissues. These inclusions were typical of those induced

by members of the potexvirus group as described by Christie and Edwardson

(1977). Banded inclusions of LVX, however, were not observed even in samples 46 fixed in 4% glutaraldehyade. According to Edwardson et a/.'s (1993) report, banded inclusions of potexviruses may be dissociated by mechanical injury when peeling epidermal strips or by alcohol, aqueous stains, water, or buffers used in the staining process. LVX can be distinguished from LSV by the conspicuous spindle-shaped inclusions located adjacent to the cell walls of LVX- infected plants. The vacuolate inclusions induced by LVX were difficult to distinguish from those induced by LSV, however. Light microscopic immunogold labeling with homologous LVX antiserum can be used to circumvent this problem and helped to confirm that these inclusions were indeed composed of virus aggregates.

In l-ELISA and Western blot tests, antisera against LVX provided by A. F.

L. M. Derks and used in this study reacted specifically with virus only after cross-

absorption with healthy lily saps. It appears that the existing commercial LVX antiserum is heavily contaminated with host proteins and therefore crude antiserum would have to be used cautiously when interpreting serological tests.

LVX antigen reacted weakly against antisera to the other potexviruses tested: BaMV, CsCMV, CymMV, NanMV, PapMV, PVX, and WCIMV. LVX antiserum reacted strongly only with homologous antigen, thereby helping to confirm that LVX is a distinct member of the potexvirus group, as proposed by

Stone (1980).

LVX can be distinguished from LSV in SDS-PAGE tests by the presence of coat protein bands at 22 kDa and 32 kDa, respectively. In Western blot tests, protein bands at 22 kDa were noted for LVX-infected Lilium and N. benthamiana 47 saps, but were not detected in saps of healthy Lilium, healthy N. benthamiana, or in saps of lilies infected with viruses other than LVX. This 22 kDa coat protein value corresponds to that calculated on the basis of the amino acid sequences

published by Memelink ef al. (1990). The 22 kDa protein reacted with antisera to

CsCMV, CyMV, PVX, and WCIMV in Western blot tests and, thus, these four

PCAs could conceivably be used to diagnose LVX in lilies by this method.

In immunogold labeling studies involving lily leaf extracts and LVX polyclonal antiserum, particles of about 550 nm long, but not longer ones, were gold-tagged. Thus, virions of the shorter particles appear to be those of LVX

(Stone, 1980; Kimura et al., 1990), whereas those of the latter are not (Allen,

1972). Based on these results, it appears that the commercially available LVX antiserum could be used for diagnosis even without cross-absorption with

healthy lily extracts by immunogold labeling, as has been done for viruses of

orchids and other crops (Ko et al., 1988; Lin, 1984; van Lent and Venduin,

1985).

Based on my results, LVX can be indexed by light microscopy, l-ELISA,

DTB, and electron microscopic immunogold labeling. DTB, which has several advantages, such as being time- and labor-saving and more sensitive, can be used for routine indexing even by commercial growers with adequate training.

Although the Netherlands Bulb Inspection Service indexes for LVX by

ELISA in their program, limited availability of antiserum and the lack of much published information about this virus may result in problems for others seeking

to propagate lily planting stock free of LVX. The only available antiserum must 48

be purchased from the Netherlands Bulb Research Center, and it is both relatively expensive (347.00 $US per ml) and may not be suitable for certain types of serological tests because of high levels of host proteins. Attempts to produce our own LVX antiserum failed because only a few local lesions formed

on the propagation host, T. tetragonioides, and lilies singly infected with LVX

were not encountered in this investigation. Of 1,180 lily samples indexed in this study, 125 plants were infected with LVX, but in no instance was there any plant singly infected with this virus. All LVX-infected samples also had LSV, but a few were triply with LVX, LSV and TBV or CMV The failure to encounter any plants singly infected with LVX can be attributed to the lack of movement protein gene

as described by Memelink et al. (1990) for this virus. Other viruses, especially

LSV, could serve as helper viruses for LVX in the systemic infection of lily plants.

Since antiserum to LVX is difficult to obtain and the only commercial antiserum reacts nonspecifically with components in healthy lily leaf extracts, cloning and expression of LVX coat protein in Escherichia coli or the production

of monoclonal antibodies is suggested as an alternative means to acquire a

large quantities of high quality LVX antisera for further work. Li (1995), working with dasheen mosaic potyvirus and encountering considerable difficulties in

obtaining purified virions for antiserum production, resorted to this method to

obtain antiserum to this virus, which was then distributed to commercial

companies and academic institutions.

Information about the incidence and potential impact of LVX as a

pathogen of commercial lilies is discussed in Chapter 5. CHAPTER 3 CYTOLOGICAL CHARACTERISTICS OF LILY VIRUSES AND THEIR POTENTIAL FOR DIAGNOSIS

Introduction

Light microscopy, which can be used to visualize characteristic virus inclusions, has been used as a diagnostic tool, especially when serological or electron microscope techniques are not available. Light microscopy has been

successfully used to identify virus diseases in several crops, including orchids

(Ko et al., 1985), maize (Overman et al. 1992), and peppers (Edwardson and }

Christie, 1979). Light microscopy was also used on a preliminary basis to identify LSV in lilies (Yang era/., 1993); however, inclusions induced by other lily viruses such as LVX and TBV have not yet been described or assessed from the standpoint of being useful for diagnosis.

Lily viruses (i.e. CMV, LiMV, LSV, LVX, and TBV) belong to groups that can be diagnosed by light microscopy based on the inclusions they induce.

Carla- and potexviruses, for example, induce banded and vacuolate inclusions.

Cytoplasmic cylindrical inclusions are induced by potyviruses, and hollow crystalloid inclusions are induced by cucumoviruses such as CMV (Christie and

Edwardson, 1977; Edwardson era/., 1993; Edwardson and Christie, 1996).

Likewise, characteristic inclusions can be detected by electron microscopy. In thin sections, aggregates of elongated LSV particles, ca. 640 nm

49 50

long and organized in parallel arrays were seen in necrotic fleck-diseased L

longiflorum and in 'Pink Perfection' lily leaf cells (Allen and Lyons, 1969;

Bertaccini and Marani, 1982; Lyons and Allen, 1969). Isometric particles, ca. 20-

31 nm in diameter and assumed to be virions of CMV, were found in phloem

sieve elements in necrotic fleck-diseased L. longiflorum leaves (Allen and Lyons,

1969; Lyons and Allen, 1969). In similar studies of thin sections, cytoplasmic inclusions such as bundles, scrolls, and tubes were found in the TBV-infected

Lilium spp. (Bertaccini and Marani, 1982; Chang et a/., 1987) and in tulip (van

Slogteren, 1971; Yamaguchi et al., 1963). Likewise, similar cytoplasmic

inclusions were observed in TBV-infected lilies and C. amaranticolor (Alper et

al., 1982). Based on these studies, TBV was designated as a subdivision I potyvirus (Edwardson, 1992; Edwardson and Christie, 1991).

The International Committee for the Taxonomy of Viruses (Murphy et al.,

1995) lists several potyviruses that infect lilies, i.e. TBV, LiMV, and Rembrandt

tulip breaking virus (RTBV). However, little is known about the relationships between these viruses to one another. Moreover, many of the serological studies

conducted with lily potyviruses used antiserum that was obtained by immunization of rabbits with virus purified from tulips, which are also hosts of

strains of TuMV (Deng, 1995), a subdivision III or IV potyviruses (Edwardson and Christie, 1991; Edwardson and Purcifull, 1970; Tomlinson, 1970).

Cytological investigations have been helpful in ascertaining relationships between potyviruses. For instance, the inclusion types that virus induced were 51

used to distinguish bean yellow mosaic and bean common mosaic virus in certain legumes (Edwardson etal., 1972).

Reddish-violet, irregularly-shaped cytoplasmic inclusions associated with nuclei, which presumably contain RNA, were detected by N.-J. Ko (personal communication) in presumptively virus-free L formosanum seedlings grown in

isolation from other lilies. The identity of these Azure A-staining inclusions, remains unresolved, and could indicate the presence of a previously

undescribed virus-like agent of lily.

Materials and Methods

Virus Isolates

The J12 isolate of CMV was maintained in 'Juanita' lily, and the K45 isolate of CMV was maintained in N. benthamiana. The CB-, SG-, Mon- and

NW- isolates of LSV were isolated from 'Casa Blanca,' 'Stargazer,' 'Montreaux,'

and 'Nellie White' lilies, respectively. The Sir-, FA-, and NW- isolates of TBV

were maintained in 'Sirocco', L. formosanum, and 'Nellie White' lily, respectively.

The T- and WS-342 isolates of LiMV were maintained in N. benthamiana.

Healthy L. formosanum seedlings and healthy 'Nellie White,' 'Montana,' and

'Montreaux' lily plants were used as controls.

Light Microscopy

The procedures used were as described in Chapter 2. They involved the same basic procedures as those used by Christie and Edwardson (1977). 52

Electron Microscopy

Thin sections . These procedures were modified from Ko (1985) and

Martelli and Russo (1984). Leaf tissues were fixed with 4% glutaraldehyde in 0.1

M K-Na phosphate buffer (pH 7.2) at 4 C for 6 hr or overnight. The tissues were then rinsed 3 to 5 times in 0.1 M phosphate buffer (pH7.2) at 4 C for 15 min each. Then, these tissues were postfixed with 1% osmium tetroxide at 4 C overnight or for 4 hr at room temperature and washed in 0.1 M phosphate buffer

(pH7.2) as noted above. Tissues were then dehydrated in EM-grade acetone

(Cat. No. 012921, Polyscience, Inc., Ft. Washington, PA) series (20, 40, 60, 80,

95, and 100%) at 4 C for 15 to 60 min each. Tissues were infiltrated with a series of EM-graded Epon Araldite resin/acetone mixtures (1:3, 1:2, 1:1, 2:1, 3:1) and

then transferred three times to fresh solution of 100% acetone for 1 hr to overnight each. Infiltrated samples were transferred to embedding molds filled with fresh 100% Epon Araldite resin and allowed to polymerize at 60 to 70 C for

12-24 hrs. Sections were made with a glass knife and stained with uranyl acetate and lead citrate, and then were examined by electron microscopy.

Leaf dips . These procedures were modified from Ko (1985) and Christie

ef al. (1 987). Leaf tissues of Lilium spp. were chopped in a few drops of 0. 1 M K-

Na phosphate buffer (pH 7.2). A drop of extract was transferred to a formvar-

coated grid and allowed to stand for 1 to 5 min. Excess fluid extract was then blotted from grids with filter paper and then washed with 20 drops of the same buffer and 20 drops of distilled water. Grids were stained with 2% uranyl acetate containing 100 ng/ml of bacitracin for 2 to 3 seconds. The stained grids were 53 then transferred to a grid box and allowed to air dry and then were observed by electron microscopy.

Western Blots

In an attempt to identify the unknown RNA-staining entity in light microscopy described by Ko (unpublished), Western blot tests were conducted by the methods described in Chapter 2. Crude polyclonal antiserum to the FA- isolate of TBV was provided by T.-C. Deng, and the same antiserum cross-

absorbed with healthy sap was used to detect the virus-like pathogen in lilies.

Double-stranded RNA Analysis

Double-stranded RNA analyses have been used for virus detection and

identification (Dodds et al., 1984; Dodds and Bar-Joseph, 1983; Morris and

Dodds, 1979; Valverde et al., 1986; Valverde et al., 1990). This technique could be applied to help resolve whether or not the RNA-containing inclusions in the

apparently virus-free lily plants seen by light microscopy were due to a cryptic virus.

Double-stranded RNAs were isolated using phenol extraction modified from the procedures described by Morris and Dodds (1979). Seven grams of tissue were used in each sample for each extraction. Tissue samples were ground to a fine powder in liquid nitrogen with a sterile mortar and pestle. The ground tissues were transferred to 50-ml sterile beakers and mixed with extraction buffer consisting of 14 ml 2X STE buffer (200 mM NaCI, 20 mM Tris-

HCI, 2 mM EDTA, pH 6.8), 20 ml Tris-saturated phenol, 2 ml 10% SDS, and 14

mg of bentonite. Samples were stirred for 1 hr at 4 C, transferred to phenol- 54

resistant centrifuge tubes, and centrifuged for 15 min at 10,000 g. The aqueous phase was collected, transferred into a sterile 50-ml beaker, and adjusted to 20 ml with 1X STE buffer. Ethanol (95%) was then added to obtain a final alcohol concentration of 15% (v/v).

Double-stranded RNA was purified from the supernatant by cellulose

chromatography. The solution was mixed with 1 g of Whatman CF-11 powder

and stirred for 1 hr at 4 C. The solution was transferred to a column and then was washed with 100 ml of 1X STE buffer containing 15% ethanol. Double- stranded RNAs were eluted from columns with 14 ml of 1X STE buffer, precipitated with 1/10 volume of 3 M sodium acetate (pH 6.0) and 2.5 volumes of

95% ethanol, and stored at -20 C overnight. After centrifugation at 10,000 gf for

30 min, the pellets were washed with 70% ethanol, air-dried, resuspended in 100

M-l of sterile distilled deionized water, and stored at -70 C. Fifteen [i\ aliquots of each sample were used for electrophoresis. Double-stranded RNA was analyzed

by electrophoresis on 1 % agarose gels for 90 min at a constant voltage of 85 V and stained with ethidium bromide. The dsRNAs were viewed with an UV-light transilluminator and photographed.

Results

Light Microscopy

Crystalline inclusions were observed in CMV-infected 'Juanita' lily and N. benthamiana leaves (Fig. 3-1). LSV-induced paracrystalline and vacuolate inclusions were also seen in the leaf and scale tissues of Lilium spp. (Fig. 3-2). 55

Cytoplasmic inclusions were noted in the TBV-infected lily tissues and LiMV-

infected lily and N. benthamiana leaves (Fig. 3-3). In addition, reddish-violet irregularly-shaped cytoplasmic inclusions like those described by Ko (personal

communication) were found in cells of lily plants determined by DTB, l-ELISA, and inoculation studies to be free of CMV, LSV, LVX, SLRV, and TBV (Fig. 3-4).

Electron Microscopy

Thin sections . Both the FA- isolate of TBV and LiMV induced cytoplasmic cylindrical inclusions including pinwheel arms, bundles and scrolls in infected leaves of Lilium formosanum, but no laminated aggregates (Fig. 3-5). Virus particles aggregated in parallel arrays were noted in LSV-infected 'Laura Lee'

lily. Neither these cylindrical inclusions, virus aggregates, nor tubular structures enclosing rows of virus particles like those reported for nepoviruses (Roberts

and Harrison, 1970; Cohen et a/., 1995), were found in healthy lily leaves determined by DTB and l-ELISA to be free of CMV, LSV, LVX, SLRV, and TBV.

Leaf dips . Filamentous particles, approximately 750 nm and 650 nm long,

were found in TBV-infected L. formosanum and in LSV-infected 'Montreaux' lily, respectively (Fig. 3-6). Low numbers of isometric particles of unknown etiology, approximately 28 nm in diameter, were noted in L. formosanum plants (Fig. 3-7) determined to be free of CMV, LSV, CMV, SLRV, and TBV by DTB, l-ELISA, and mechanical inoculation to indicator plants. 56

Western Blots

Whereas 43 kDa immunoreactive protein bands were noted in healthy

leaf extracts of Lilium spp. on the nitrocellulose membranes incubated with crude

antiserum to TBV-FA isolate, no such bands were seen on the nitrocellulose

membranes treated with cross-absorbed antiserum. Likewise, no protein bands

at 43 kDa were seen in LiMV or TuMV-infected N. benthamiana leaf extracts.

Variability in bands between isolates of TBV was also noted when antiserum

against TBV-FA isolate was used in Western blot test (Fig. 3-8).

Double-stranded RNA Analysis

Two bands, approximately 1.0 and 1.5 kb, were noted in the RNA

extracts from 'Nellie White' plants that were determined by DTB and l-ELISA to

be virus-free of CMV, LSV, CMV, SLRV, and TBV and LSV-infected by DTB in

the 1% agarose gel (Fig. 3-9). Similar bands were also found in presumably

virus-free L. formosanum seedlings in agarose electrophoresis.

Discussion

Conventional methods for detecting plant viruses, such as symptom

expression, host range, serology, and electron microscopy can be augmented by

light microscopy as described by Christie and Edwardson (1977) for staining

inclusions. Light microscopy has several advantages, i.e., it is rapid,

reliable, and relatively inexpensive. Light microscopy can be especially useful

when working with plant systems in which little is known about their viruses or when antisera to their viruses are not available or prohibitively expensive. Plant Fig. 3-1. Light micrograph of crystalline inclusions (C and I) induced by

cucumber mosaic virus in 'Juanita' lily cells stained with Azure A. N = nucleus. Bar =10 ^m. 58

Fig. 3-2. Light micrographs of paracrystalline inclusion (top) and vacuolate

inclusions induced by lily symptomless virus in 'Stargazer' lily cells

stained with Azure A (bottom left) and orange-green combination (bottom right). P = paracrystalline and V = vacuolate inclusions. Bar =10 urn. 59

Fig. 3-3. Light micrographs of cytoplasmic inclusions (CI) induced by tulip

breaking virus in L. formosanum cell (top) and lily mottle virus in N. benthamiana cell (bottom) stained with orange-green combination. N = nucleus. Bar =10 |im. Fig. 3-4. Light micrographs of irregular inclusions (I) induced by a possible cryptic virus in healthy L. formosanum (top) and 'Nellie White' lily cell (bottom) stained with Azure A. N =nucleus, Bar = 10 |^m. Fig. 3-5. Electron micrograph of cytoplasmic cylindrical inclusions induced by

tulip breaking virus (top) and lily mottle virus (bottom) in L. formosanum leaf cell. B = bundle, P = Pinwheel arms, S = scrolls. Bar = 300 nm. Fig. 3-6. Electron micrographs of tulip breaking virus (top) and lily symptomless virus particles (bottom) stained with 2 % uranyl acetate. 63

Fig. 3-7. Electron micrograph of possible cryptic virus particles (arrows) in healthy L. formosanum leaf sap stained with 2 % urany acetate. 64

abcdefghijklmno

97 68 43

29

Fig. 3-8. Comparative reactivities of a polyclonal antiserum of tulip breaking virus-FA isolate purified from L formosanum to two potyviruses and turnip the Mr in mosaic virus in Western blot tests. The number to the left indicates kilodaltons. A = healthy L formosanum; B, C, E = lily mottle virus isolates -D, -T, and -WS342, respectively; D = the FA- isolate of tulip breaking virus in L. collected in this study from formosanum] F, H, I, J = tulip breaking virus isolates respectively; = trumpet lily, 'Sirocco' lily, 'Stargazer' lily, and 'Nellie White' lily, G -F standard marker proteins; K, L, M = turnip mosaic virus isolates -3374, -T, and in N. benthamiana, N = healthy 'Nellie White' lily; 0 = healthy N. benthamiana. 65

Fig. 3-9. Agarose gel electrophoresis of dsRNA from lily plants doubly infected with lily symptomless virus and lily virus X (A), plants singly infected with lily symptomless virus (B), and healthy lily plants (C). Lane D indicates the dsDNA markers; Arrows show the size of dsDNA in base pairs. 66 virus inclusions have been widely used as a convenient and reliable tool for detecting and diagnosing virus diseases of orchids, peppers and several other crops (Christie and Edwardson, 1977; 1986; Edwardson and Christie, 1979;

1996; Hoefert era/., 1992; Koefa/., 1985; Overman era/., 1992).

Based on its merits, light microscopy was proposed as a tool for

establishing a preliminary lily virus indexing program in Florida (Ko, unpublished). However, these studies were hampered by the unexpected

presence of red-violet-staining, irregular-shaped cytoplasmic inclusions in lily

seedlings and in other lily plants that were presumed to be virus-free and did not react positively in serological tests with LSV, LVX, TBV, CMV, or SLRV.

In my study, inclusion bodies typical of cucumoviruses, carlaviruses,

potexviruses and potyviruses were found in lilies infected with CMV, LSV, LVX, and with LiMV and TBV, respectively. These results were confirmed by serological methods. Typical inclusions of CMV and LiMV were also found in N. benthamiana.

Microwave treatments to reduce staining times proved useful in this

investigation. It also improved the staining intensity of vacuolate inclusions

induced by LSV in lily tissues. However, this procedure did not improve prospects for detecting putative banded inclusions of LVX.

Spindle-shaped cytoplasmic and vacuolate inclusions induced by LVX in this study have not been described previously. The spindle-shaped inclusions closely resemble those reported for such potexviruses as nandina mosaic

(Zettler et a/., 1980) and cymbidium mosaic (Ko, 1985). The inclusions of LVX 67

were, however, not banded, which may be attributed to dissociation by

mechanical injury involved in producing epidermal strips, or by alcohol, aqueous

stains, water, or buffers used in staining them (Edwardson et a/., 1993). Since

LSV, like other carlaviruses, can induce similar spindle-shaped and vacuolate

inclusions, light microscopy would not be a useful procedure to distinguish it from LVX.

LiMV and TBV are considered by Dekker (1993) and Derks (1994) to be

distinct potyviruses. Cylindrical inclusions induced by LiMV have not been

described previously, however. Whereas strains of a few potyviruses such as

TuMV induced dissimilar cylindrical inclusions, most do not (Edwardson and

Christie, 1991). In my study, cytoplasmic cylindrical inclusions induced by LiMV

could not be distinguished from those of TBV by light microscopy, thereby

supporting serological results (Chapter 4) that show them to be very closely

related, if not identical. Based on examinations of ultrathin sections, TBV and

LiMV are subdivision I potyviruses because they both induce cytoplasmic

cylindrical inclusions with pinwheel arms, bundles and scrolls, but not laminated

aggregates. These inclusions are very similar to those described by Bertaccini

and Marani (1982) and Chang et al. (1987) for TBV in lilies. Moreover, they also

closely resembled those described by Alper et al. (1982) for an isolate of TBV

that infected both lilies and C. amaranticolor.

The distinction between TBV and LiMV is narrowly based upon whether or

not it can infect N. benthamiana or C. amaranticolor. Alper et al. (1982), for

example, described an isolate of TBV from Israel that could infect the latter 68

species. According to the terminology of Derks et al. (1994), this virus would be an isolate of LiMV. Cytological and serological evidence supports the contention

that TBV and LiMV are very closely related and, thus, there is little practical advantage for applying different vernacular names for each. Since the name

TBV predates LiMV, it should be used instead.

Irregular-shaped cytoplasmic inclusions in lily seedlings noted by Ko and

in my study could indicate the presence of a previously undescribed lily virus

inasmuch as the inclusions were often found in lily plants determined by other means to be virus-infected or healthy. These inclusions were located adjacent to the nuclei of infected cells and were more widely scattered in the tissues than

the other established viruses investigated in this study.

Based on electron microscopy, Western blots, and double-stranded RNA

analysis, it appears that the irregular-shaped cytoplasmic inclusions may be

induced by a heretofore undescribed cryptic virus in lily. Isometric particles, approximately 28 nm in diameter, similar to carnation (Lisa et al., 1980) and poinsettia cryptic viruses (Koenig and Lesemann, 1980), but smaller than other

Partiviridae (Ghabrial et al., 1995), were observed in L. formosanum seedlings.

CMV and nepoviruses such as SLRV have virions with similar morphologies;

however, these viruses were not detected by serology in the lily seedlings under

investigation. Unfortunately, the unavailability of positive controls for SLRV

made it impossible to rule out the presence of this virus in the l-ELISA and DTB

tests. The isometric particles seen in lily seedlings differed from ribosomes, which are 20 nm in diameter, and are not entirely spherical in outline (Bozzola et 69

al., 1992). In Western blot tests, 43 kDa protein bands noted in leaf extracts of lily seedlings were similar to Mr of coat protein of the Partiviridae (42 to 73 kDa) but differed from the 55 kDa of SLRV (Murant, 1974) or the 26 kDa of CMV in this study.

Two cryptic virus-like dsRNA segments, approximately 1.1 and 1.5 kb, were noted in extracts of L formosanum seedlings and in mature, apparently

healthy, 'Nellie White' lily. These values closely agreed with those of cryptic

viruses, which have two or more major dsRNA segments at 1 .25 to 2.5 kb (Abou-

Elnasr et al., 1985). Cryptic viruses vary in the number of bands and the

molecular weight of their dsRNA segments (Dodds et al., 1984). A virus similar

to other Reoviridae was found in 'Uchida' lily in Japan (Varma et al., 1979).

However, Reoviridae members are readily distinguishable from the plant cryptic viruses in that the former have 10-12 dsRNA segments and have virions 60-80

nm in diameter (Murphy ef a/., 1995).

If the dsRNAs are replicative form of viruses, their size would correspond to twice those of encapsidated ssRNAs such as carlaviruses (8.6 kb), cucumoviruses (1.0, 2.1, 2.8, and 3.2 kb), potyviruses (10.2 kb), and

potexviruses (7.8 kb) (Valverde et al., 1986; Dodds and Bar-Joseph, 1983).

Such dsRNAs were not detected in lily plants, however.

Because cryptic viruses occur in very low concentrations, induce no visible symptoms in plants, and are transmitted exclusively by seed or pollen, they can be easily overlooked (Dodds etai, 1984). There have been few studies on the yield losses these viruses could inflict and, thus, they are not considered 70

to be serious threats to any agricultural crops (Kenten et ai, 1981; Xie et a/.,

1994). Antiserum to a putative lily cryptic virus may exist as a contaminant in antisera produced against unrelated viruses. Such antisera could conceivably complicate the interpretation of some serological tests. Antiserum cross- absorbed with cryptic-virus-infected plant saps should solve these problems. In

general, tests for dsRNA and inclusion bodies in apparently healthy plants will be a worthwhile precaution for virological studies. CHAPER 4 LILY SEROLOGICAL METHODS FOR DETECTING AND DISTINGUISHING VIRUSES

Introduction

prerequisite for obtaining virus-free lily A reliable indexing method is a

the lilies cannot be detected on planting stocks. In most cases, virus-infected basis of symptoms alone.

diagnosed by inoculating indicator Lilies infected with some viruses can be

Chenopodium quinoa for CMV (Mowat plants, such as Nicotians Cleveland* and

benthamiana for LiMV (Dekker et and Stefanac, 1974), C. amaranticolor and N.

for lily Tetragonia tetragonioides and C. murale a/., 1993; Derks et al., 1994),

for TBV (van Slogteren, 1971). virus X (Stone, 1980), and L. formosanum

for LSV (Allen, 1972). However, no such indicator plants have been described

according to environmental Moreover, bioassays using indicator plants may vary

conditions where they are grown (Matthews, 1992).

viruses, such as LSV Electron microscopy has been used to detect lily

and strawberry latent ringspot (Mowat and Stefanac, 1974; Verma et a/., 1979)

immunosorbent electron microscopy virus (SLRV) (Cohen et al., 1995). Likewise,

and LVX (Cohen et al., (ISEM) has been used for detecting the lily viruses, SLRV

1995; Kimura era/., 1990).

the most practical and most Serological tests, however, have proven to be

Immunodiffusion procedures were widely used means for detecting lily viruses.

71 72

adapted the Netherlands Bulb used initially for this purpose, and in 1976 were

Slogteren, 1976; van Inspection Service (Simmonds and Cumming, 1979; van

however, immunodiffusion tests were Slogteren et al., 1976; 1980). After 1980,

(Beijersbergen and van der replaced by ELISA for indexing Dutch-grown bulbs

1986; van Slogteren et al., Hulst, 1980; Derks et al., 1982; van Schadewijk,

1980).

viruses drop Based on ELISA test results, titers of LSV and other lily

temperature (Deng, 1995). Thus, precipitously in actively growing plants at high

such titer changes, especially virus-infected stock may escape detection due to

LSV, and TBV may vary within plant in plants grown in vitro. Since titers of CMV,

indexing lilies for viruses by tissues, sampling must be taken into account when

Van Schadewijk (1986) ELISA or other means (Deng, 1995; Hagita, 1989).

bulb scales and bulbs if they observed that viruses could readily be detected in

Beijersbergen and Van der are stored for 3 weeks at about 2 C before indexing.

hemicellulase to improve Hulst (1980) suggested incubating bulb extracts with

in vitro plantlets is not the detection of LSV in some cultivars. Virus testing in

leaves of plants already reliable for either LSV or TBV, however. Thus, indexing

(van Zaayen et al., 1992). established in soil in the glasshouse is a necessity

recently been applied Dot-blot or direct tissue blotting assays (DTB) have

al., 1996). DTB, which requires to detect LSV in lilies (Hsu et al., 1995; Kim et

different buffers for antigen neither trituration of plant samples nor preparing

ELISA. DTB can preparation, has been shown to be more sensitive than

(Hsu et al., 1995). Because overcome the titer variability problem inherent in lilies 73

routine diagnosis of viruses of these advantages, DTB is already being used for

et al., 1993), bamboo (Lin et a/., in crops other than lilies, such as citrus (Garnsey

1990,) and garlic (Tsuneyoshi and Sumi, 1996).

more The polymerase chain reaction method (PCR) is reported to be a

ELISA. PCR was, for sensitive detection method than conventional ones such

tungro bacilliform example, 1,000-10,000 times more sensitive for detecting rice

ELISA (Yakahashi et al., virus DNA extracted from infected rice plants than

virus RNA in amounts 1993). Likewise, PCR could detect bean yellow mosaic

4 5 molecular hybridization approximately 10" to 10" lower than ELISA or

techniques (Vunsh etal., 1990).

potyviruses when Problems were encountered in trying to detect lily

from tulip monoclonal antibodies and polyclonal antisera against TBV purified

Deng, 1995; were used (Boonekamp and Pomp, 1986; Boonekamp et al., 1990;

tulips were Franssen and van der Hulst, 1986). Deng (1995) reported that

potyviruses, namely TBV and a infected with at least two serologically distinct

epitope common to most strain of TuMV, and the former did not have the

Before that was potyviruses which reacts against the universal PTY-1 MCA.

polyclonal antiserum reacted realized, Derks et al. (1982) reported that TBV-JL

with the TBV-TF isolate from strongly with lily isolates of TBV, but only weakly

reacted strongly against tulips. In contrast, polyclonal antiserum against TBV-TF

der Hulst (1982) and Boonekamp TBV in tulip. Similarly, Beijersbergen and van

existed, one in tulips and the et al. (1990) concluded that two serotypes of TBV

Israel (Alper et al., 1982) other in lilies. A TBV isolate similar to an isolate from 74

TBV-TF and TBV-JK likewise failed to react with two polyclonal antisera against

that against a (Derks, 1985). Boonekamp and Pomp (1986) reported MCAs

with either TBV-JL or TBV-TF. Dutch isolate of TBV from Easter lily failed to react

tulip were selected by When the MCAs against TBV derived from Texas Flame'

used to differentiate strains of Hsu et al. (1986; 1988), some clones could be

This study was, therefore, TBV, in lilies whereas others could not (Deng, 1995).

and to detect lily designed to assess the relationships between LiMV and TBV

viruses by serological tests.

Materials and Materials

Virus Isolates

from The SS- and SG- isolates of LVX used in this study were isolated

both of which 'Strawberry Shortcake,' and 'Stargazer' lily bulbs, respectively,

were were imported from the Netherlands. The LVX-NW and -WS isolates

plants obtained from isolated from 'Nellie White' and 'White Sheen' lily

Western USA. commercial sources and are assumed to have been field-grown in

lily, and the K45 The J12 isolate of CMV was maintained in 'Juanita'

isolate of CMV was maintained in N. benthamiana.

from 'Casa The CB-, SG-, Mon- and NW- isolates of LSV were isolated

respectively. Blanca,' 'Stargazer,' 'Montreaux,' and 'Nellie White' lilies,

maintained in 'Sirocco' lily, L The Sir-, FA-, and NW- isolates of TBV were

and WS-342 isolates of formosanum and 'Nellie White' lily, respectively. The T-

isolates used in this LiMV were maintained in N. benthamiana. Other TBV 75

1 994-1 996. The investigation were obtained during virus surveys conducted in

lily bulb TDG29- and PPVB- isolates were isolated, respectively, from a trumpet

a 'Pink purchased from a commercial bulb distributor in New Jersey and from

bulb distributor in New Perfection' Aurelian lily purchased from a commercial

York.

PV-388) were Isolates of TCBV (ATCC PV-425), and WaTV (ATCC

TBBV, LiMV, obtained from the ATCC. Dried reference antigens of RTBV, TTBV,

Research Center, Lisse, and LVX were provided by A. F. L. M. Derks at the Bulb

the Netherlands.

White,' Healthy L formosanum seedlings and/or indexed 'Nellie

healthy controls in all 'Montana,' and 'Montreaux' lily plants were used as

experiments.

Test Plants for Inoculation

inocula were The reconstitution of freeze-dried tissues and preparation of

Other samples done according to the ATCC's guidelines (McDaniel et al., 1993).

buffer, pH 7.2, of infected leaves or scales were triturated in 0.1 M phosphate

with 600-mesh and the extracts were rubbed onto leaves of test plants dusted

N. carborundum. Seedlings of Chenopodium amaranticolor, L. formosanum,

test benthamiana, and Tetragonia tetragonioides were the test plants used. All

plants were maintained in a greenhouse at <30 C.

Other Polyclonal Antisera and Monoclonal Antibodies

Polyclonal antiserum TBV-Derks (derived from TBV-JL), and LiMV-Derks

TBV-specific (derived from TBV-L) were provided by A. F. L. M. Derks (1982). 76

monoclonal antibodies (MCAs) of ATCC PVAS675, ATCC PVAS732, and ATCC

The PTY-1 MCA PVAS733 (Hsu et al., 1988) were purchased from the ATCC.

PTY-2, was purchased from Agdia, Inc. (Elkhart, IN). A MCA Admix of PTY-1,

provided by R. L. PTY-4, PTY-8, PTY-1 0, PTY-21, PTY-24, and PTY-30 was

Jordan (Jordan and Hammond, 1988; 1991). Antiserum of a turnip mosaic virus

and isolate (TuMV-F825) from Florida was the same one used by McDonald

Hiebert(1975).

Western Blot Tests

differentiating These tests were conducted as described in Chapter 2 for

potyviruses in lily and tulip.

ELISA Tests

Efficacy comparison of coating buffer and CE P buffer in l-ELISA. In I-

carbonate, 35 ELISA tests, samples were triturated in CEP buffer (15 mM sodium

mM sodium bicarbonate, 0.02% polyvinyl pyrrolidine, and 0.2% BSA) or coating

pH 9.6) in a buffer (15 mM sodium carbonate and 35 mM sodium bicarbonate,

II polystyrene microelisa plates ratio of 1 to 10, and then added to the Type

conducted as (Dynatech Labs, Inc., Chantilly, VA). Other procedures were

described in Chapter 2.

Polyclonal and monoclonal Monoclonal antisera to detect lily potyviruses .

method as antibodies were used to detect lily potyviruses using the l-ELISA

described in Chapter 2.

against Triple antiserum sandwich-ELISA tests for monoclonal antibodies

conducted according to the protocols of lily potyviruses . TAS-ELISA tests were 77

Van Regenmortel (1982). One hundred |il of polyclonal antiserum against TBV

diluted 1:1,000 in coating buffer were added to the wells of microtiter plates and

incubated for 1 hr at 37 C. The plates were rinsed with three 5-min washes in

PBST. Test samples of lily leaf or bulb scale tissue, triturated in 10 volumes of coating buffer, were then added and incubated overnight at 4 C. Subsequent procedures, such as additions of MCAs, anti-mouse IgG conjugated with alkaline phosphatase, and enzyme substrate, respectively, were conducted as described in Chapter 2.

and Antiserum mixture of four viruses to detect lily viruses . To save time materials, polyclonal antisera against CMV, LSV, LVX, and TBV were diluted

(1:1,000) and combined (1:1:1:1) when testing certain samples by l-ELISA.

Comparison of The Detection Efficiency of Lily Viruses by l-ELISA AND DTB

Bulbs from virus-infected lily plants were used to compare the relative

efficiencies of l-ELISA and direct tissue blotting techniques for virus detection.

Each scale peeled from individual bulbs was cut by a new razor blade and then

directly blotted onto the nitrocellulose membrane for direct tissue blotting tests.

The residual scale pieces after blotting were triturated in coating buffer (pH 9.6)

to in a ratio of 1 to 10 for indirect-ELISA tests. The extractions were added

microtiter plates for ELISA tests.

Virus Distributions in Lily Tissues

Roots, scales, stems, leaves, pedicels, tepals, and ovaries collected from

virus-infected lily plants were detected for viruses by DTB tests as described in

Chapter 2. 78

Virus distribution in relation to scale position . Different bulb scales were collected from individual bulbs and tested by l-ELISA and DTB to determine virus distribution patterns. Most of the scale samples were removed from individual bulbs after storage. Based on the A405 values in l-ELISA tests or the index of

reaction area in DTB of bulb scales, it was possible to establish a linear pattern that reflected overall trends and distribution in titer. The index of reaction areas for DTB were visually determined as: 0 = no purple color reaction evident in the

= tissue stained purple; II = more tissue; I more than 1 % but less than 25% of the than 26% but less than 50% of the tissue stained purple; III = more than 51% was stained.

were Virus distribution in relation to leaf position . Most of the leaf samples collected from plants at the flowering stage. The patterns of virus distribution in different leaves of a given plant were studied by plotting the A405 values of ELISA tests and the index of reaction areas of DTB as described above. Based on the

A405 values in l-ELISA tests and the index of reaction areas of DTB, it was

possible to plot linearized patterns that reflected overall trends and titer

distributions in individual plants.

Virus Detection in in vitro Plantlets

Scales from virus-infected and virus-free lily bulbs were propagated under

a 12-hr photoperiods of dark and light at room temperature (average 28 C) in the

Mirashige lily multiplication medium (34.57 g/L of Mirashige's minimal organics

medium, 0.3 mg/L of 1 -naphthaleneacetic acid, 80 mg/L of adenine sulfate-2H 20,

and 3 mg/L of 2-isopentenyl adenine; Gibco BRL, Gaithersburg, MD). After 16 79

l-ELISA and DTB, and weeks in culture, plantlets were indexed for viruses using

these plants transferred to soil-free medium in a growth chamber. After 6 months, were indexed again for viruses using l-ELISA and DTB.

Results

Host Reactions

Mon- The reactions of indicator plants to the K45- isolate of CMV, the

isolate of LSV, and the Sir-, SG-1, FA-, NW-, PPVB, TDG-29 and WS-342

LSV-Mon infected but failed isolates of TBV are listed in Table 4-1 . CMV-K45 and

isolates of to produce any discernible symptoms in L. formosanum, whereas all 7

isolate of TBV induced foliar mosaic symptoms in this species. Only the WS-342

LiMV and the K-45 isolate of CMV produced local lesions in Chenopodium

amaranticolor and Tetragonia tetragonioides, and systemic mosaic symptoms in

N. benthamiana.

Western Blot tests

the Admix of In Western blot tests, monoclonal antibodies, PTY-1 and

failed to PTY-1, PTY-2, PTY-4, PTY-8, PTY-1 0, PTY-21, PTY-24, and PTY-30,

react with 3 isolates of LiMV or 7 isolates of TBV, but did react strongly with an

-733, isolate of TuMV. Conversely, monoclonal antibodies ATCC-732 and

reacted with 3 isolates of LiMV and 7 isolates of TBV, but failed to react with

of LiMV, 7 TuMV (Fig. 4-1). MCA of ATCC-675 failed to react against 3 isolates

isolates of TBV, or 1 isolate of TuMV. 80

Polyclonal antisera of LiMV-Chang, LiMV-Derks, and TBV-Derks all reacted with 3 isolates of LiMV, the FA-isolate of TBV, the 3374-isolate of TuMV, and the other 4 potyviruses from tulip (Fig. 4-2). In addition, polyclonal antisera of

PVY and ByMV reacted weakly with two isolates of LiMV and the FA-isolate of

TBV. The ByMV antisera (Fig.4-3) and PVY antiserum (not shown) also reacted strongly with the 3374-isolate of TuMV and other potyviruses from tulip.

Interestingly, polyclonal antisera against the D- and T-isolates of TuMV reacted weakly with the WS-342 isolate of TBV (Fig. 4-4 and Table 4-2).

Immunoreactive protein bands for 6 TBV and 1 LiMV-WS isolates were noted at 31kDa, whereas those of 2 isolates of LiMV, TuMV and other potyviruses in tulip were noted at 34 kDa.

ELISA Tests

Efficacy comparison of coating and CEP buffer in l-ELISA tests . Similar

Aws values were noted for the coating and CEP buffers when three filamentous viruses in lilies were tested in l-ELISA tests (Tables 4-3 and 4-4). Coating buffer was subsequently used routinely as an extraction buffer in this investigation for I-

ELISA tests.

Monoclonal antisera to detect lilv potyviruses . In l-ELISA tests, PTY-1 monoclonal antibody did not detect the 2 isolates of LiMV and 6 isolates of TBV tested. MCA of the Admix of PTY-1, PTY-2, PTY-4, PTY-8, PTY-1 0, PTY-21,

PTY-24, and PTY-30 reacted weakly against the isolates of LiMV. In contrast,

MCAs of ATCC-732 and -733 reacted strongly with the isolates of LiMV and 2 isolates of TBV (Table 4-5). However, PTY-1 reacted weakly with three isolates of ,

81

TBV when the antigens were stored at -20 C for 1 week. In addition, the ATCC-

733 reacted strongly with the frozen leaf extracts of 'Nellie White' lily infected with

TBV than with fresh ones (Table 4-6).

MCAs of ATCC-675 and PTY-1 reacted weakly with 7 isolates of TBV and

1 isolate of LiMV in TAS-ELISA tests. However, MCA of the Admix of PTY-1

only PTY-2, PTY-4, PTY-8, PTY-1 0, PTY-21, PTY-24, and PTY-30 reacted

moderately with the NW-G isolate of TBV in these tests (Table 4-7).

In DTB tests, neither polyclonal antisera of TuMV-825 and SLRV nor

monoclonal antibodies of ATCC-675, PTY-1, and the Admix of PTY-1, PTY-2,

PTY-4, PTY-8, PTY-10, PTY-21, PTY-24, and PTY-30 reacted with the scale

tissues infected with any of the three isolates of TBV tested (Table 4-12).

four Antiserum mixture of four viruses to detect lily viruses . The mixture of

polyclonal antisera against CMV, LSV, LVX, and TBV, respectively, readily

detected lily plants that were either singly infected or infected with two or more

viruses. (Table 4-8).

Comparison of The Detection Efficiency of Lilv Viruses bv l-ELISA AND DTB

About 28.6, 11.6, and 6.0% more LSV-, LVX-, and TBV-infected scales,

respectively, were detected by DTB than l-ELISA tests (Table 4-9). Likewise,

viruses could be efficiently detected by both methods, however, TBV was

detected more readily in 'Nellie White' leaf samples by DTB than l-ELISA (Table

4-10). 82

Virus Distribution in Lily Tissues

CMV, LSV, LVX, and TBV were detected by DTB in roots, scales, stems,

leaves, pedicels, and tepals, but not ovaries, of greenhouse-grown lily plants

(Table 4-11). Roots and scales proved to be the best tissues for virus indexing by

DTB.

tests, Virus distribution in relation to scale position . In l-ELISA and DTB

4- the outer lily bulb scales generally had higher LVX titers than inner ones (Figs.

5, 4-6, 4.7, and 4-8). Like LVX, the outer bulb scales had higher LSV titers than

inner ones, regardless of whether lily plants were maintained at >30 C (22-35 C)

or at <30 C (18-30 C)(Figs. 4-9, 4-10, and 4-11). Uneven distribution and lower

titers of TBV and CMV in bulb scales were noted at higher temperature (>30 C);

4- however, outer bulb scales still had higher titers than inner ones (Figs. 4-12,

13, and 4-14).

In general, the linear trends in distribution and titer determined by DTB

4- were similar to those by l-ELISA (Figs. 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, and

12). DTB was subsequently used routinely as a detection method in this

investigation for virus surveys. In addition, the outer bulb scales provided better

samples for virus indexing in this investigation.

Virus distribution in relation to leaf position . At high (>30 C) growing

temperatures, LVX and CMV titers were higher in the newer leaves of plants than

in the lower ones (Figs. 4-15, and 4-25). Conversely, LSV titers were higher in

the lower leaves of plants than in the newer ones near the shoot apex.

Differences in LSV titers were also noted between lily cultivars (Figs. 4-16, 4-17, 83

and 4-18). As with LSV, lower leaves had higher titers of TBV than newer ones

(Figs. 4-19 and 4-20).

At lower temperatures (<30 C), TBV and CMV had different patterns of distribution than at higher temperatures (Figs. 4-21, 4-22, 4-23, and 4-24).

Virus Detection in in vitro Plantlets

The indexing results for lily plants derived from tissue cultures indicated that plants free of LSV can be obtained through adventitious shoot cultures.

Differences in the success rate in doing so varied, however, according to lily cultivars (Table 4-13). The low A^s values in l-ELISA tests in the in vitro plantlets derived from TBV-infected scales were increased to higher values when the in vivo transplants from the same clones were tested (Table 4-14). LSV was distributed unevenly in the leaves, scales, and roots of lily plantlets (Table 4-15).

Discussion

Based on its ability to infect N. benthamiana and its close serological

relationship with LiMV, the WS-342 lily potyvirus isolate could be considered as an isolate of LiMV, which can infect both monocotyledons and dicotyledons as

described by Alper etal. (1982) and Derks et al. (1994). On that basis, this would

be the first report of LiMV in Florida. My results, however, show WS-342 and

other isolates of LiMV to be closely related serologically to TBV and RTBV The

WS-342 isolate reacted with PCAs of LiMV, TBV, and RTBV as did the other TBV

and LiMV isolates. However, in Western blot tests using intact virus antisera, the

WS-342 isolate had an immunoreactive protein at 31 kDa like the TBV isolates, 84

but differed from the T- and D-isolates of LiMV which had a protein at 34 kDa in this and Derks et a/.'s (1994) studies. Coat protein subunits size variability among isolates of LiMV is like that reported for isolates of dasheen mosaic virus

(Li, 1995) and turnip mosaic virus (Ling et a/., 1995).

Table 4-1 . Indicator plant reactions to virus isolates from lilies

Virus Isolates 1)

Indicator plant cmv- lsv- tbv- tbv- tbv- tbv- tbv- tbv- umv

K45 Mon FA TDG29 PPVB Sir SG NW -WS C. amaranticolor L : ' ____L L formosanum S SMM MMMMM

N. benthamiana M M

T. expansa L L

1} CMV-K45 and TBV-FA = K45-isolate of cucumber mosaic virus and FA-isolate of tulip breaking virus provided by T.-C. Deng; LSV-Mon = Mon-isolate of lily symptomless virus isolated from 'Montreaux' lily; TBV-TDG29, -PPVB, -Sir, -SG, NW, and -WS = TDG29-, PPVB-, Sir-, SG-, and NW- isolates of tulip breaking virus isolated from Trumpet, 'Pink Perfection,' 'Sirocco,' 'Stargazer,' and 'Nellie

White,' lilies, respectively; LiMV-WS = WS- isolate of lily mottle virus isolated from 'White Sheen' lily. 2) L = local lesions, - = no infection, S = symptomless, M = mosaic; all results were confirmed by DTB or l-ELISA. 85

Table 4-2. Reactivities of two monoclonal antibodies and seven polyclonal antisera with virus extracts isolated from lilies and tulips in Western blot tests

Antiserum 2) " Antigen ATCC Agdia TBV- LiMV- TuMV- TuMV-l- RTBV PVY ByMV-

-732 PTY-1 Derks Chana 825 T G

LiMV-T + 3) + + +

LiMV-D 4. +

TBV-FA + + + +

LiMV-WS + + + + NT NT NT

TuMV + + + + + + + +

TTBV + + + + + + +

TCBV + + + + + NT NT

WaTV + + + + + + + +

RTBV + + + + + + HCK 1} LiMV-T - T- isolates of lily mottle virus provided by C.-A. Chang; LiMV-D = D- isolate of lily mottle virus provided by A. F. L. M. Derks; TBV-FA = FA-isolate of tulip breaking virus provided by T.-C. Deng; LiMV-WS = WS-isolate of lily mottle virus; TuMV = 3374-isolate of turnip mosaic virus provided by D. E. Purcifull; TTBV and RTBV = tulip top breaking and Rembrandt tulip-breaking virus, respectively, were provided by A. F L. M. Derks; TCBV and WaTV = tulip chlorotic blotch and Washington tulip virus were obtained from the American

Type Culture Collection (ATCC); HCK = healthy lily sap. 2 ' ATCC-732 = monoclonal antibodies against tulip breaking virus purchased from ATCC; PTY-1 = monoclonal antibody purchased from Agdia Inc.; TBV- Derks = polyclonal antiserum against 'Jack Laan' strain of tulip breaking virus provided by A. F. L. M. Derks; LiMV-Chang = polyclonal antiserum against lily mottle virus provided by C.-A. Chang; TuMV-825 and TuMV-l-T = polyclonal antisera against D- and T-isolates of turnip mosaic virus provided by E. Hiebert; RTBV = polyclonal antiserum against Rembrandt tulip-breaking virus; PVY = polyclonal antiserum against provided by D. E. Purcifull; ByMV-G = polyclonal antiserum against bean yellow mosaic virus provided by F. W Zettler (Nagel etal., 1983). 3) Reactivity determined as + when viral protein band was visible in nitrocellulose membrane, - = no reaction, ± = weak reaction, NT = not tested. 86 ABCDEFGHIJKLMNO

97 * 68 43

29

BCDEFGH 1 J KLM NO

i

npi f*

2 9 -

Fig. 4-1. Reactivities of PTY-1 (top) and ATCC-732 (bottom) monoclonal antibodies to 11 viruses from lilies and turnip in Western blot tests. The lanes are:

A and D = healthy lily sap; B = D- isolate of lily mottle virus provided by A. F. L.

M. Derks; C = T- isolate of lily mottle virus provided by C.-A. Chang; E = WS- isolate of lily mottle virus; F, H, I, J, K, L, and M = TDG29-, Sir-, CB65-, CB67-, NW204-1-, NW253-, and NW186- isolates of tulip breaking virus; G = standard = marker proteins with the numbers to the left indicating the size in kilodaltons; N 3374-isolate of turnip mosaic virus provided by D. E. Purcifull; 0 = healthy N. benthamiana leaf sap. S7

ABCDEFGHiJ

97^ 68

43

29

viruses from lilies, Fig 4-2. Reactivities of TBV-Derks polyclonal antiserum to = D-isolate of lily mottle tulips and turnip in Western blot tests. The lanes are: A = lily mottle virus provided by virus provided by A. F. L. M. Derks, B T- isolate of = 3374- isolate of turnip C.-A. Chang, C = FA- isolate of tulip breaking virus, D proteins with the mosaic virus provided by D. E. Purcifull, E = standard marker = tulip top-breaking virus, numbers to the left indicating the size in kilodaltons, F tulip- = tulip virus, I = Rembrandt G = tulip chlorotic blotch virus, H Washington breaking virus, J = healthy lily sap. Fig. 4-3. Reactivities of ByMV-G polyclonal antiserum to potyviruses from are: = D- gladiolus, lilies, tulips and turnip in Western blot tests. The lanes A = isolate of lily mottle isolate of lily mottle virus provided by A. F. L. M. Derks, B T- virus provided by C.-A. Chang, C = FA- isolate of tulip breaking virus, D = 3374- isolate of turnip mosaic virus provided by D. E. Purcifull, E = standard marker proteins with the numbers to the left indicating the size in kilodaltons, F = tulip = top-breaking virus, G = bean yellow mosaic virus provided by F. W. Zettler, H = tulip-breaking virus, J = healthy lily sap. Washington tulip virus, I Rembrandt 89

ABCDEFGHIJKLMNO

97 - 68 - 43 -

29 -

Fig. 4^1. Reactivities of TuMV-D (top) and TuMV-l-T (bottom) polyclonal antisera to 8 isolates of TBV in Western blot tests. The lanes are: A and N = healthy lily sap; B = D- isolate of lily mottle virus provided by A. F. L. M. Derks; C = T- isolate of lily mottle virus provided by C.-A. Chang; D = FA- isolate of tulip breaking virus provided by T.-C. Deng; E = WS-342 isolate of lily mottle virus; F, virus; H, I, and J =, TDG-29, Sir-, SG-, and NW-204-1 isolates of tulip breaking G = standard marker proteins with the numbers to the left indicating the size in kilodaltons; K = 3374-isolate of turnip mosaic virus provided by D. E. Purcifull; L = and M = T-8 and F- isolates of turnip mosaic virus provided by E. Hiebert; 0 healthy N. benthamiana leaf sap. 90

Table 4-3. Comparison of coating and CEP buffers for detecting LSV by l-ELISA

No. Infected samples (Range of A405 values)

1) Antigen No. samples Coating buffer 2) CEP buffer

LSV-SG 26 26 (0.246-0.655) 3) 26 (0.159-0.568)

LSV-Mon 19 19(0.153-0.343) 19(0.111-0.490)

HCK 4 0 (0.036-0.042) 0(0.037-0.041)

1} Antigens are: HCK = healthy lily sap, LSV-SG = SG - isolate of lily symptomless virus isolated from 'Stargazer' lily, LSV-Mon = Mon- isolate of lily symptomless virus isolated from 'Montreaux' lily. 2) Coating buffer = 0.05 M sodium carbonate buffer, pH 9.6; CEP buffer = 0.05 M sodium carbonate buffer containing 0.02% PVP and 0.2% BSA, pH 9.6. 3) Absorbances were recorded 30 min after addition of substrate and represent the averages of 2 duplicate wells. 91

Table 4-4. Comparison of coating and CEP buffers to detect three lily viruses by l-ELISA

Absorbance at 405 nm a) Antigen 1) Extraction buffer 2) LSV LVX TBV

4) HCK coating 0.01 0.02 0.02

CEP 0.02 0.02 0.03

TBV coating 0.02 0.02 0.40

CEP 0.02 0.01 0.47

LSV + TBV coating 1.15 0.05 0.44

CEP 1.14 0.03 0.42

LVX + LSV coating 0.39 1.65 0.10

CEP 0.23 1.58 0.12

1} = lily Antigens are: HCK = healthy lily sap, LSV = lily symptomless virus, LVX virus X, and TBV = tulip breaking virus. 2) Coating buffer = 0.05 M sodium carbonate buffer, pH 9.6; CEP buffer = 0.05 M sodium carbonate buffer containing 0.02% PVP and 0.2% BSA, pH 9.6. 3) Antisera are: LSV = polyclonal antiserum against lily symptomless virus provided by author, LVX and TBV = polyclonal antiserum against lily virus X and tulip breaking virus, respectively, provided by A. F. L. M. Derks. 4) the Absorbances were recorded 1 hr after addition of substrate and represent averages of 2 duplicate wells. 92

Table 4-5. Reactivities in l-ELISA tests of two polyclonal antisera and four

monoclonal antibodies to potyvirus isolated from lilies and tulips

Absorbance at 405 nm TBV- LiMV- ATCC- ATCC- PTY- Admix 3) 1) 2) Antigen Host Derks Chang 732 733 1 4 LiMV-WS342 L 2.45 > 1.02 1.91 0.92 0.03 0.22 UMV-WS380 L 2.21 1.35 2.26 1.35 0.04 0.39 TBV-FA L 2.16 1.05 1.69 0.95 0.04 0.07 TBV-Sir L 2.41 1.24 0.96 0.32 0.05 0.08 TBV-NW L 0.77 0.15 0.08 0.10 0.05 0.00 TBV-CB L 2.45 0.63 1.07 0.10 0.05 0.06 5 TBV-PPVB L 0.18 0.16 _ ) 0.25 0.04 TBV-TDG-29 L 0.53 0.14 0.16 0.04 TuMV-3374 T 0.44 0.11 0.03 0.09 0.87 1.37 TBBV T 0.83 0.45 2.23 0.33 TCBV T 0.10 0.09 0.12 0.59 TTBV T 0.24 0.09 0.08 0.76 WaTV T 0.20 0.08 0.12 0.59 HCK L 0.07 0.07 0.02 0.07 0.04 0.00 HCK T 0.07 0.08 0.08 0.10 0.03 0.00 1} = The antigens are : UMV-WS342 and LiMV-WS380 WS342- and WS380-

isolates of lily mottle virus; TBV-FA = FA- isolate of tulip breaking virus provided by T.-C. Deng; TBV-Sir, -NW, -CB, PPVB, -TDG29 = Sir-, NW-, CB-, PPVB-, and

TDG-29 isolates of tulip breaking virus were provided by the author ; TuMV-3374 = 3374- isolate of turnip mosaic virus provided by D. E. Purcifull; TBBV and TTBV = tulip band-breaking and tulip top breaking virus were provided by A. F. L. M. Derks; TCBV and WaTV = tulip chlorotic blotch and Washington tulip virus were obtained from the American Type Culture Collection (ATCC); HCK = healthy sap. 2) List of host plants from which the antigens were derived: L = lily, T = tobacco. 3) The antisera are: TBV-Derks =antiserum against 'Jack Laan' strain of tulip

breaking virus provided by A. F. L. M. Derks; LiMV-Chang = antiserum against lily mottle virus provided by C.-A. Chang; ATCC-732 and -733 = monoclonal antibodies against tulip breaking virus purchased from the ATCC; PTY-1 = monoclonal antibody purchased from Agdia Inc.; Admix = an admixture of monoclonal antibodies of PTY-1, -2, -4, -8, -10, -21, -24, and -30 provided by R. L. Jordan. 4) Absorbances were recorded 2 hrs after addition of substrate and represent the averages of 2 duplicate wells. 5) - = not tested. 93

Table 4-6. Reactivities in l-ELISA tests of one polyclonal antiserum and two monoclonal antibodies of tulip breaking virus to fresh and frozen leaf extracts of healthy and infected lilies

No. Positive Reaction Samples (A405 ranges) No. Antigen 1J Samples Treatment 2) TBV-Derks 3) ATCC-733 PTY-1 TBV-NW 38 Fresh 38 0 0 41 (0.20-1.19) (0.05-0.10) (0.02-0.06) Frozen 36 26 0 (0.23-1.61) (0.11-0.23) (0.09-0.21) LiMV-WS 2 Fresh 2 2 0 (1.45-2.81) (0.92-1 .35) (0.03-0.04) Frozen 2 2 0 (2.06-2.45) (1.56-1.79) (0.11-0.15) TBV-FA 3 Fresh 3 3 0 (1.09-2.16) (0.30-0.32) (0.03-0.04)

Frozen 3 3 1 (0.64-1 .89) (1.69-3.00) (0.13-0.39)

TBV-PPVB 1 Fresh 1 0 0 (1.19) (0.07) (0.03)

Frozen 1 1 1 (1.37) (1.27) (0.26) TBV-Sir 2 Fresh 2 2 0 (1.18-2.41) (0.30-0.32) (0.03-0.05)

Frozen 2 2 1 (0.92-1.55) (1.56-1.79) (0.24-0.36) TBV-CB 7 Fresh 7 7 0 (0.87-2.45) (0.16-0.69) (0.03-0.06) Frozen 7 7 0 (0.50-2.36) (0.12-1.65) (0.07-0.21)

HCK 1 Fresh 0 0 0 (0.03) (0.07) (0.04) Frozen 0 0 0 (0.06) (0.06) (0.13)

' The antigens are: TBV-FA = FA- isolate of tulip breaking virus provided by T.-C. Deng; TBV-Sir, -NW, -CB, and -PPVB = Sir-, NW-, CB-, and PPVB- isolates of tulip breaking virus, respectively, provided by the author; LiMV-WS = WS- isolate of lily mottle virus isolated from 'White Sheen' lily; HCK = healthy lily sap.

' Fresh = Antigens were added after extraction in coating buffer, Frozen = Same antigens were frozen at -20 C for 1 week. 3) The antisera are : TBV-Derks =antiserum against 'Jack Laan' strain of tulip breaking virus provided by A. F. L. M. Derks; ATCC-733 = monoclonal antibodies against tulip breaking virus purchased from the American Type Culture Collection; PTY-1 = monoclonal antibody purchased from Agdia Inc. 4) Absorbances were recorded 2 hrs after addition of substrate and represent the averages of 2 duplicate wells. 94

Table 4-7. Reactivities of monoclonal antibodies against potyviruses in triple antibody sandwich-ELISA (TAS-ELISA) tests

Absorbance at 405 nm J) Extraction ATCC-732 ATCC-675 PTY-1 Admix 1) Antigen Buffer 2) TBV-FA Coating 0.19 4) 0.00 0.00 0.00 CEP 0.34 0.01 0.00 0.02 Extraction 0.00 0.00 0.00 0.00 TBV-NW507 Coating 1.04 0.00 0.03 0.03 CEP 0.96 0.01 0.02 0.09 Extraction 0.07 0.00 0.00 0.02 TBV-Sir Coating 1.23 0.00 0.13 0.09 CEP 0.71 0.00 0.01 0.06 Extraction 0.00 0.00 0.00 0.00 TBV-NW-G Coating 0.87 0.02 0.10 0.23 CEP 0.72 0.02 0.15 0.36 Extraction 0.05 0.00 0.00 0.02 TBV-CB Coating 0.26 0.01 0.01 0.00 CEP 0.07 0.01 0.01 0.02 Extraction 0.02 0.00 0.00 0.00 TBV-SG Coating 0.01 0.00 0.00 0.00 CEP 0.02 0.00 0.00 0.04 Extraction 0.00 0.00 0.00 0.03 TBV-TDG29 Coating 0.39 0.00 0.00 0.00 CEP 0.33 0.00 0.01 0.03 Extraction 0.05 0.00 0.00 0.01 UMV-WS342 Coating 0.17 0.00 0.00 0.00 CEP 0.20 0.00 0.00 0.00 Extraction 0.02 0.00 0.00 0.01

HCK lily Coating 0.01 0.00 0.00 0.00 CEP 0.04 0.01 0.00 0.03 Extraction 0.00 0.00 0.00 0.00 1} The antigens are: TBV-FA = FA-isolate of tulip breaking virus provided by T.-C. Deng; TBV-NW507, -Sir, -NW-G, -CB, -SG, TDG29 = NW-507-, Sir-, NW-G-, CB-, SG- and TDG-29 isolates of tulip breaking virus were provided by the

author; UMV-WS342 = WS-342 isolate of lily mottle virus was provided by the

author; HCK lily = healthy lily sap. 2) Antigens were triturated 1:10 in coating buffer (pH 9.6), CEP buffer (pH 9.6), or extraction buffer (pH 7.4). 3) The antisera are: ATCC-732 and -675 = monoclonal antibodies against tulip breaking virus purchased from American Type Culture Collection; PTY-1 = monoclonal antibody purchased from Agdia Inc.; Admix = an admixture of monoclonal antibodies of PTY-1, -2, -4, -8, -10, -21, -24, and -30 provided by R. L. Jordan. 41 Absorbances were recorded 2 hrs after addition of substrate and represent the averages of 2 duplicate wells. 95

Table 4-8. Reactivities in l-ELISA tests of the mixtures of antisera against CMV,

LSV, LVX, and TBV to lily viruses

Antigen 1

CMV TBV TBV LSV LVX TBV LSV LSV Antiserum 21 HCK CMV LVX LVX LSV TBV LSV LVX LVX

3) CMV 0.02 0.35 0.79 0.01 0.01 0.01 0.01 0.01 0.01

LSV 0.01 0.04 0.28 0.02 0.16 0.02 1.15 0.35 0.19

LVX 0.02 0.03 0.14 3.00 3.00 0.02 0.05 0.15 0.34

TBV 0.02 0.04 0.10 0.05 0.07 0.40 0.44 0.21 0.52

CMV+LSV 0.02 0.37 1.00 0.02 0.14 0.03 1.19 0.31 0.17

LVX+TBV 0.03 0.06 0.22 3.00 3.00 0.45 0.54 0.37 0.79

LSV+LVX+TBV 0.02 0.08 0.59 3.00 3.00 0.47 1.34 0.53 0.85

CMV+LSV+LVX+TBV 0.04 0.37 0.73 3.00 3.00 0.54 1.38 0.57 0.86

} = Antigens triturated 1:10 in coating buffer are: HCK = healthy lily sap; CMV = cucumber mosaic virus; LSV = lily symptomless virus; LVX = lily virus X; TBV tulip breaking virus. 2) Antisera are: CMV = polyclonal antiserum against K-45 isolate of cucumber mosaic virus provided by T.-C. Deng; LSV = polyclonal antiserum against lily symptomless virus provided by author; LVX and TBV = polyclonal antiserum against lily virus X and tulip breaking virus, respectively, provided by A. F. L. M. Derks. 3) Absorbances were recorded 2 hrs after addition of substrate and represent the averages of 2 duplicate wells. 96

Table 4-9. Comparison of l-ELISA and direct tissue blotting (DTB) tests for

detecting lily viruses in bulb scales

No. Infected samples No.

" 2 > Viruses Lily Cultivar Samples DTB l-ELISA A405 ranges

LSV Strawberry Shortcake 32 32 32 3) 0.1 6-1 .23 3

27 27 27 0.23-1.39 30 30 30 0.23-1.13 26 26 26 0.60-1 .42

Nellie White 87 87 47 0.05-0.34

71 71 33 0.04-0.28 LVX Strawberry Shortcake 24 24 24 0.25-1.59 23 23 23 0.21-1.27

21 21 19 0.14-1.59

Nellie White 87 87 82 0.14-1.21

71 71 71 0.31-1.73 Horizon 30 30 30 0.30-1.21 29 29 13 0.15-0.90

TBV Pink Perfection 27 16 14 0.04-0.32 30 17 15 0.04-0.37 27 19 18 0.04-0.48 CMV Juanita 25 6 6 0.04-0.38 1} LSV = lily symptomless virus, LVX = lily virus X, TBV = tulip breaking virus, CMV = cucumber mosaic virus. 2) Positive reactions were determined by visual assessment of purple color reactions on nitrocellulose membranes. 3) Absorbances were recorded 1 to 2 hrs after addition of the substrate and represent the averages of 2 duplicate wells. Samples were judged to be positive for virus when A^s values were at least two times greater than those of negative controls. 97

Table 4-10. Comparison of l-ELISA and direct tissue blotting (DTB) tests for

detecting lily viruses in leaf samples

No. Infected leaves No.

\/iri 1 * ICOC 1 i l\/ iltiwor A/inc ranaes VII Ubco Lily OUIUVdi Leaves LI 1 D l-ELISA

3> 1 C\/ 41 0 45-2 37 LoV uasa bianca H 1 H I

41 41 1 04-2 65 H 1

1 \/v LVA otargazer ZO ZO zo 0W.z_C_J28-1I . 36\J\J 23 23 23 0.74-1.61 Enchantment 23 23 23 0.58-1 .66 TBV White Sheen 60 28 28 0.07-0.75 Sirocco 42 42 42 0.14-1.09

Nellie White 23 23 16 0.09-0.24 CMV Jolanda 29 25 25 0.07-0.46

Lolly Pop 77 75 75 0.07-2.21 1} virus, LSV = lily symptomless virus, LVX = lily virus X, TBV = tulip breaking CMV = cucumber mosaic virus. 2) Positive reactions were determined by visual assessment of purple color reactions on nitrocellulose membranes. 3) and Absorbances were recorded 1 to 2 hrs after addition of the substrate represent the averages of 2 duplicate wells. Samples were judged to be positive for virus when A405 values were at least two times greater than those of negative controls. 98

Table 4-1 1 . Virus distribution in lily tissues by direct tissue blotting

Tissues

1) Virus Root Scale Stem Leaf Pedicel Tepal Ovary

LSV + + + + +

LVX + + + + + +

TBV + + + + + +

CMV + + + + + +

' LSV = lily symptomless virus, LVX = lily virus X, TBV =tulip breaking virus, CMV= cucumber mosaic virus. 2) + = Positive reactions were determined by visual assessments of purple color reactions on the nitrocellulose membranes, - = no reactions. 99

Table 4-12. Reactivities in direct tissue blotting tests of TBV-infected scales to six polyclonal antisera and 5 monoclonal antibodies

Scales

Antisera 1) TBV-Sir 2) TBV-TDG29 TBV-PPVB HCK TBV-Derks + + - LiMV-Derks + + + - RTBV + + + WaTV ± ± ± TuMV-825 SLRV ATCC-675 ATCC-732 + + + ATCC-733 + + + PTY-1 Admix 1J TBV-Derks, LiMV-Derks, RTBV, and WTV = polyclonal antiserum against the 'Jack Laan' strain of tulip breaking virus, lily mottle virus, Rembrandt tulip- breaking virus, and Washington tulip virus, respectively, provided by A. F. L. M. Derks; TuMV-825 = polyclonal antiserum against a D- isolates of turnip mosaic virus provided by E. Hiebert; SLRV = polyclonal antiserum against strawberry = latent ringspot virus provided by A. A. Brunt; ATCC-675, -732, and -733 monoclonal antibodies against tulip breaking virus purchased from the American Type Culture Collection; PTY-1 = monoclonal antibody purchased from Agdia - Inc.; Admix = a monoclonal antibodies admixture of PTY-1, -2, -4, -8, -10, -21, 24, and -30 provided by R. L. Jordan. 2) TBV-Sir, -TDG29, and -PPVB = Sir-, TDG-, and PPVB- isolates of tulip breaking virus in 'Sirocco,' Trumpet, and 'Pink Perfection' lily, respectively. HCK

= healthy lily. 3) + = Positive reactions were determined by visual assessment of purple color reactions on the nitrocellulose membranes, ± = weak reactions; - = no reactions. 100

'Stargazer' at < 30C

HC K = 3.0 9, LV XCK = D.4 5

1 3 5 7 9 11 13 15 17 19 21

Innermost to outer scales -->

Fig. 4-5. Lily virus X titers in scales of infected 'Stargazer' lily plants grown at <30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of three bulbs. 101

1 'Stargazer at >30C

HCK = 0.10; LVXCK=2.15

3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

1 'Stargazer at >30C

HCK = 0; LVXCK = 3 2 |o ra 01 i 1

3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

Fig. 4-6. Lily virus X titers in scales of an infected 'Stargazer' lily grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 102

'Horizon' at > 30C

= HCK = ( 3; U/>;< c 3

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Innermost to outer scales ->

Fig. 4-7. Lily virus X titers in scales of infected 'Horizon' lily plants grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 2 bulbs. >>

103

White Sheen' at >30C

E c 8

O C ro o <

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71

Innermost to outer scales —

'White Sheen' at >30C

o ro

T3i C

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71

Innermost to outer scales —

Fig. 4-8. Lily virus X titers in scales of infected 'White Sheen ' lily plants grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 2 bulbs. >>

104

'Strawberry Shortcake' at <30C

1 .2

HCK = 0.05; LSVCK = 1 .28 i 1 i 0.8

ro 0.6 o c 0.4 CD -Q L_

o 0.2 -I < 0

1 3 5 7 9 1 1 13 15 17 19 21 23 25

Innerm ost to outer scales —

'Strawberry Shortcake' at < 30C

ea HCK = 0; LSV CK = 2 CO .92 -

react

°1 - X CD "DC

3 5 7 9 11 13 15 17 19 21 23 25

Innermost to outer scales —

Fig. 4-9. Lily symptomless virus titers in scales of infected 'Strawberry Shortcake' lily plants grown at <30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 2 bulbs. 105

'Horizon' at >30C

E 0.2 c m HCK = 0.09; LSVCK = 3.00 o 0.15 tj-

03 CD O 0.1 C 03 -Q L. o 0.05 v> <

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Innermost to outer scales->

'Horizon' at >30C

03 CD k_ 03 C HCK = 0;LSVCK = 3 •2o 2 o CO CD

o 1 x CD XI

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Innermost to outer scales->

Fig. 4-10. Lily symptomless virus titers in scales of infected 'Horizon' lily plants grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 3 bulbs. 106

'Nellie White' at >30C

0.3 i

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71

Innermost to outer scales ->

i

'Nellie White' at >30C

3

HCK = 0; LSVCK= 3

£ 0

1 5 9 131721 2529 33 374145 49 53 5761 65 69

Innermost to outer scales ->

Fig. 4-11. Lily symptomless virus titers in scales of infected 'Nellie White' lily plants grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 2 bulbs. 107

'Pink Perfection' at >30C

HCK = 0 04; TBVCK = 0.31

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

'Pink Perfection' at >30C

i i i i i i i

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

Fig. 4-12. Tulip breaking virus titers in scales of infected 'Pink Perfection' lilies grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. Data represented the averages of 2 bulbs. 108

'Sirocco' at >30C 0.4 0.35 0.3 HCK = 0.04; TBVCK = 0.55 0.25 0.2 0.15

0.1 0.05 nfinnflfinnflnnfl 0 1 a

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

'Sirocco' at >30C

HCK = 0; TBVCK = 3 ra 0)

.2 2 o TO

•K 1

-o c

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Innermost to outer scales->

Fig. 4-13. Tulip breaking virus titers in scales of an infected 'Sirocco' lily grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 109

'Juanita' at >30C

E c ino HCK = 0.04; CMVCK = 0.61

« 0.5 o c Sro o

1 3 5 7 9 11 13 15 17 19 21 23 25

Innermost to outer scales->

'Juanita' at >3X

° 1

a) -a

3 5 7 9 11 13 15 17 19 21 23 25

Innermost to outer scales->

Fig. 4-14. Cucumber mosaic virus titers in scales of an infected 'Juanita' lily grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 110

'Strawberry Shortcake' at >30C

1.6 HCK = 0.24; LVXCK = 2.00 | 1.4 « 1-2

! 1 B g 0.8 I 0.6 § 0.4 < 0.2 II

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to lowest leaves-->

1 'Stargazer at >30C

1.8

1.6 HCK = 0.24; LVXCK = 2.00 1.4

1.2

1 0.8 06 0.4 0.2 0 J

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to lowest leaves~>

'Enchantment' at >30C

2.5

E = = c HCK 0.24; LVXCK 2.00 2 1

15 1-5 O i 1

I 0.5 < 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to lowest leaves->

Fig. 4-15. Lily virus X titers in leaves of infected 'Strawberry Shortcake,' 'Stargazer,' and 'Enchantment' lilies grown at >30 C as determined by l-ELISA. Line represented trends in distribution and titers. Ill

Nellie White! at >30C

Fig. 4-16. Lily symptomless virus titers in leaves of an infected 'Nellie White' lily grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 112

Wite Sheen' at >30C

1 4 7 10 13 16 19 222528 31 343740434649525558

Uppermost to lowest leewes->

Wite Sheen' at >30C

Fig. 4-17. Lily symptomless virus titers in leaves of an infected 'White Sheen' lily grown at >30 C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 113

'Casa Hanca' at >30C

1 3 5 7 9 11 13 15 17 19 21 23 25 Z7 29 31 33 35 37 39 41

Uppermost to lowest leaves->

'Montana' at >30C

Uppermost to lowest leaves->

'Stargazer' at >30C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to lowest leaves->

Fig. 4-18. Lily symptomless virus titers in leaves of infected 'Casa Blanca,' 'Montana,' and 'Stargazer' lilies grown at >30 C as determined by l-ELISA. Line represented trends in distribution and titers. 114

Wtte Sheen' at >30C

1 5 9 13 17 2125293337 41 4549S35761656973

Uppenrost to lowest lea\es->

Wife Sheen' at >30C

HCK=0,TBvCK = 3

4 7 10 13 16 19 22 25 2B 31 34 37 43 43 4549 S2 55 58 61 64677073

Uppermost to lowest leaves ->

Fig. 4-19. Tulip breaking virus titers in leaves of an infected White Sheen' lily grown at >30 C as determined by l-ELISA (top) and TBV (bottom). Line represented trends in distribution and titers. 115

'Nellie White at >30C

'Nellie White at >30C

Fig. 4-20. Tulip breaking virus titers in leaves of an infected 'Nellie White' lily grown at >30 C as determined by l-ELISA (top) and TBV (bottom). Line represented trends in distribution and titers. 116

'Nellie Wife' at <30C

03

0.25 HCK= 0.07; TBV3K= 0.87

0.2

0.15 ]

0.1

0.05

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to lowest leares ->

'Nellie Write' at O0C

HCK=0;TBVDK=3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Uppermost to la/vest leaves->

lily Fig. 4-21 . Tulip breaking virus titers in leaves of an infected 'Nellie White' grown at <30 C as determined by l-ELISA (top) and TBV (bottom). Line represented trends in distribution and titers. 117

'Sirocco' at <30C

'Sirocco' at <30C

Fig. 4-22. Tulip breaking virus titers in leaves of an infected 'Sirocco' lily grown at <30 C as determined by l-ELISA (top) and TBV (bottom). Line represented trends in distribution and titers. 118

'Juanita' at <30C

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71

Uppermost to lowest leaves ->

'Juanita' at <30C

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71

Uppermost to lowest leaves->

Fig. 4-23. Cucumber mosaic virus titers in leaves of an infected 'Juanita' lily grown at <30C as determined by l-ELISA (top) and DTB (bottom). Line represented trends in distribution and titers. 119

'Jdandaf at <30C E c 0.5 ID 0.4 o HCK = 0.05; = 0.65

0.3 o c 02 CO JQ t— o 0.1 <

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Uppermost to lowest leaves->

'Jdanda' at<30C

TO CD HCK = 0;CM/CK = 2 2 2 o TO CD

"S 1

- 0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Uppermost to lowest leaves ->

Fig. 4-24. Cucumber mosaic virus titers in leaves of an infected 'Jolanda' lily Line grown at <30C as determined by l-ELISA (top) and DTB (bottom). represented trends in distribution and titers. >

120

CMV in 'Jolanda' leaves at >30 C

0.35 E c 0.3 HCK = 0.04; CMVCK = 0.29 m o 0.25

0.2 o c 0.15 cc ja k_ - o 0.1 if) < 0.05 0

1 4 7 10 13 16 19 22 25 28 31

Uppermost to lowest leaves->

CMV in 'Juanita' leaves at > 30 C

0.35 -i

1 6 11 16 21 26 31 36 41 46 51 56

Uppermost to lowest leaves —

Fig. 4-25. Cucumber mosaic virus titers in leaves of infected 'Jolanda' and 'Juanita' lilies grown at >30C as determined by l-ELISA. Line represented trends in distribution and titers. 121

Table 4-13. Detection of LSV in lily plantlets derived from LSV-infected donor plants in adventitious shoot culture

No Dlantlets infected No. of 1) Cultivar plantlets LSV

Montreaux 11 0 Grand Paradiso 27 3 2) Laura Lee 17 15 Strawberry Shortcake 6 2

Nellie White 14 4 Stargazer 3 3

Casa Blanca 1 1

Montana 1 1

Red Tiger 1 1

LSV = lily symptomless virus. Number of plantlets reacting positively with antiserum in l-ELISA had A405 values at least two times greater than those of negative controls. 122

Table 4-14. Virus indexing of stage II lily plantlets and stage IV transplants derived from TBV-infected donor plants in adventitious shoot culture

No. of positive reactions No. of No. of

Variety plantlets Stage II Stage IV mosaic transplants 1) L. formosanum 17 8 17 17

Nellie White 11 4 11 11

' Number of plantlets reacting positively with antiserum in l-ELISA and A405 values at least two times greater than those of negative controls.

Table 4-15. Virus indexing of various tissues of lily plantlets derived from LSV- infected donor plants in adventitious shoot culture

No. of positive reaction/ A405 values Cultivar No. of Scales Roots Leaves plantlets Strawberry 3 0/0.01-0.02 0/0.01-0.02 0 /0.01 Shortcake Laura Lee 4 4/0.10-1. 10 1) 4 /0.34-2.25 4/0.21-0.53

Nellie White 3 3/0.13-1.47 3 /2.00-2.04 2/0.04-0.15

Stargazer 2 1 /0.01-0.70 1 /0.03-0.13 2 /0. 36-0. 92

Casa Blanca 1 1 /no 1 /1.95 1 /0.98

Montana 1 1 10.2 1 /0.29 1 /0.09

' Number of plantlets reacting positively with antiserum in l-ELISA had A405 values at least two times greater than those of negative controls. 123

Interestingly, PCAs against LiMV from C.-A. Chang and A. F. L. M. Derks both reacted with TBV, LiMV, TuMV, TTBV, TCBV, and WW, as did PCA of TBV in my study. Based on Deng's findings (1995), there is a high possibility that

Derk's TBV antiserum contains antiserum to TuMV as well. TuMV does not

appear to infect lilies (Dekker et al., 1993) and it was not known to be a pathogen of tulips prior to 1985 (Mowat, 1985) and, thus, some of the TBV antiserum obtained before that time and derived from virus isolated from tulips could conceivably be contaminated with that virus. Since Chang's LiMV antiserum was

derived from N. benthamiana plants singly infected with this virus, it is unlikely

that lily was contaminated with TuMV. Thus, the strong cross-reactivity between

TBV and TuMV isolates noted in my work is most likely a result of the highly conserved core region of the coat protein subunits among many potyviruses or to the antisera having been derived from rabbits which received booster injections every 6-12 months over a span of 4-6 years (Derks et al., 1988). A highly conserved core of 215-227 amino acids was found in the coat protein subunits of potyviruses and an antiserum to the dissociated core region of the coat protein of

Johnson grass mosaic virus was found to react with 15 potyviruses tested

(Shuklaef a/., 1988; 1989; 1994).

LiMV and TTBV were considered to be strains of one another based on the similarities of the 92 amino acid conserved region of their capsid subunits

(Dekker et al., 1993). Differences between TBV, LiMV, TTBV, and RTBV in this region, however, caused Dekker et al. (1993) to conclude that these were distinct potyviruses. Moreover, Ohira et al. (1994) concluded that LiMV should be 124

considered as a distinct potyvirus because it was only 52-64% similar to TBV at the 3'-terminal 1586 nucleotides. Before drawing final conclusions, however, more data on the amino acid sequences of the coat protein and other genes of

LiMV and TBV should be obtained. Shukla et al. (1994) found that there are major differences in the sequence of the N-terminal region but considerable sequence similarities in the C-terminal portions of the coat protein subunits of potyviruses.

The MCAs, PTY-1 and the Admix of PTY-1, -2, -4, -8, -10, -21, 24, and -

30, and ATCC-675, failed to detect any of the TBV and LiMV isolates in this study. These results agreed with Deng's report (1995), but differed from the results of Jordan and Hammond (1988; 1991) and Hsu et al. (1986; 1988).

According to Jordan and Hammond (1988; 1991), TBV is one of the 55 potyviruses that reacts with PTY-1 monoclonal antibody. Yet, these MCAs could readily react with TuMV, TBBV, TCBV, TTBV, and WaTV, which apparently do not infect lilies. The inability of MCAs to detect TBV or LiMV could be accounted for by some of the MCAs being specific to certain epitopes of tulip infecting strains of

TuMV. Since PTY-1 failed to readily detect any of the lily potyviruses, it should not be used for indexing and identifying potyviruses in lilies. In contrast, two

MCAs produced by Hsu et al. (1988) from tulips, ATCC-732 and -733, did react with all isolates of TBV and LiMV, and, thus, these two MCAs can be used to detect TBV.

To save time and costs in a routine virus-indexing program of lilies, the mixture of four polyclonal antisera against CMV, LSV, LVX, and TBV can be 125

applied in l-ELISA and DTB. There appears to be no interference between any of

the polyclonal antisera used for detecting their respective homologous viruses.

Meanwhile, DTB proved to be more satisfactory than l-ELISA tests in detecting

viruses whether in scale or leaf tissues. DTB is more sensitive and practical than

l-ELISA and does not require trituration of samples, different extraction buffers for

antigen preparation, enzyme treatment for other serological tests, and expensive

facilities such as ELISA readers. Accordingly, DTB may be a better alternative

serological means for routine virus indexing program for commercial lily growers.

Bulb scales and roots were the best tissues to sample for DTB tests in my

study. Pedicels, on the other hand, were the most convenient tissues to use when indexing field-grown plants.

Uneven virus distribution in lily tissues has been noted as a major factor

affecting virus indexing (Deng, 1995; van Schadewijk, 1986). At high

temperatures, the titers of CMV, TBV and LSV in leaves and scales varied

considerably in certain cultivars. However, in this investigation, outer bulb scales

of all varieties provided consistently more reliable results when using DTB to test for CMV, LSV, LVX, and TBV. These results were similar to those reported by

Deng (1995) and Hagita (1989), who used outer scales for virus indexing by

ELISA tests. On the other hand, virus titers in various leaves depended on lily

varieties and the temperatures under which they were grown. Generally at lower temperatures (<30 C), leaves collected from newly emerging leaves produced the most reliable results for virus indexing. 126

l-ELISA or DTB tests involving plantlets grown in vitro were not reliable because of very low titers and uneven distribution of viruses. Hence, combinations of scales, leaves, and roots should be used when indexing such materials. An even better approach, of course, would be to index plants and select virus-free ones beforehand, as is routinely done commercially for orchids.

LSV could not be detected in bulblets maintained in vitro at 30C or higher (Cohen

et a/., 1985). Accordingly, in vitro cultures and mother stocks should be

maintained at lower temperatures if they are to be indexed. CHAPTER 5 INCIDENCE AND POTENTIAL COMMERCIAL IMPACT OF LILY VIRUSES

Introduction

Epidemiological information is an essential component for developing reliable and practical control strategies for plant viruses. While surveys have

been conducted for infections of CMV, LSV, and TBV in lilies (Hagita et al.,

1989; Kim et al., 1996), no such information exists for LVX. Viral diseases in

Lilium spp. have caused problems since the late 1800's, and it is in generally

assumed that LSV is widespread in commercial stocks (Brierley, 1940; Hagita et al., 1989; Deng, 1995; Yang era/., 1993). Allen and Fernald (1972), for example, found that more than 99% of the commercial Easter lilies surveyed in 1972 contained long, rod-shaped virus particles, and thus assumed that LSV was ubiquitous in the Western U.S.A., where these plants were grown under field

conditions. In contrast, Ballantyne et al. (1979) found no LSV in the native Lilium species from California and Oregon, even though the plants were collected in

close proximity of fields where commercial Easter lilies were grown for many

years. In Japan, a similar field survey was conducted by Hagita et al. (1989),

who found more CMV than LSV or TBV in the edible Maximowicz's lily. In Asiatic

lilies, however, LSV was the most frequently encountered virus. Yang et al.

127 128

(1993) assayed 17 commercial lily samples from the U.S. and 173 samples from

Taiwan, all of which were determined to be infected with LSV.

The lack of apparent symptoms induced by LSV and LVX hampers our

ability to learn how wide-spread and significant these two lily viruses are (Asjes,

1990). Moreover, lily viruses tend to express symptoms intermittently. Indeed, necrotic fleck symptoms tend to be masked at high temperatures (McWhorter

and Allen, 1967). These problems notwithstanding, lily viruses have been shown

to cause serious yield losses. The vase life of cut flowers, for example, is

reduced by virus infection (Asjes et al., 1973). Also, infections with TBV can

cause conspicuous symptoms in certain lily cultivars and can render then unfit

for market (Asjes et al., 1973).

Since lilies singly infected with LSV are usually symptomless, this virus cannot be controlled by a program of roguing based on symptoms. This problem

could account in part why LSV is so often found in commercial plantings on a world-wide basis (Allen, 1975). In contrast, CMV, which causes conspicuous

symptoms, almost eliminated Bermuda's Easter lily industry until a program of roguing was put into place there (McWhorter and Allen, 1967). Similarly, a small

commercial lily industry existed in central Florida in the 1920's but died out in large part due to necrotic-fleck, which was caused ostensibly by mixed infections of CMV and LSV (Roberts and Blanet, 1967).

Viruses have been eliminated from lilies through meristem-tip culture

(Allen and Anderson, 1980; Walkey and Webb, 1968). Mori and Hosokawa

(1977) found, however, only the upper 0.1 - 0.3 mm portions of the shoot apex of 129

CMV-infected plants to be virus-free. Thus, the use of shoot-tip culture to obtain

healthy plants may have its limitations. Chemotherapy followed by shoot-tip culture has been suggested as an improvement for obtaining a larger proportion of virus-free lily plants (Bloem-Barnhoorn and van Aartrijk, 1985; van Aartrijk et al., 1990). Thermotherapy apparently has not been widely used to obtain virus- free lily planting stock, however, even though titers of both LSV and TBV drop

considerably when plants are grown at high temperatures of >30 C (Cohen et al.,

1985).

A certain proportion of bulblets obtained from mature CMV-infected

Easter lily bulbs may be virus-free (Brierley, 1940; Brierley, 1962). Likewise, some virus-free planting units can be obtained from LSV-infected Asiatic lilies simply by removing bulblets derived from infected scale explants grown

axenically (Allen, 1974; Allen et al., 1980). Regardless, none of the aforementioned techniques were fail-safe and subsequent indexing was required to verify that the propagating units were indeed virus-free.

To date, there have been only a few reports describing the effects of viral

diseases on quality and yields of lilies. Virus-free 'Enchantment' lilies were 25 to

50% taller than plants from virus-infected bulbs (Allen, 1975). In 1976, Boontjes

(1976) reported that the stem length, number of flowers per stem, length of the longest leaf, stem weight, and size of buds and flowers were all greater on virus- free 'Enchantment,' 'Harmony,' and 'Fire King' plants than on virus-infected ones.

Later, Boontjes (1978) found that virus-free plants were 20 to 26% higher in

'Enchantment' and 42 to 85% higher in 'Destiny' lilies than in diseased 130 commercial stocks studied for comparison. Likewise, plants with symptoms of

TBV produced shorter stems, needed a longer forcing period, and suffered more

leaf dehiscence than plants without symptoms (Bakker et al., 1976). Similar results were noted by Asjes (1990), who described that bulb yields were reduced

by 10 to 35% if plants were doubly infected with TBV and LSV.

Materials and Methods

Source of Lilies

lily Bulbs imported from the Netherlands . Five cultivars of imported Dutch bulbs ('Gran Paradiso,' 'Jolanda,' 'Juanita,' 'Laura Lee,' and 'Sirocco') were

provided by J. F. Tammen in 1993. Two cultivars of imported Dutch lily bulbs

('Strawberry Showcake' and 'Casa Blanca') were purchased from the Park Seed

Co. (Greenwood, SC). In 1995-1996, another 12 cultivars of imported Dutch lily bulbs ('Acapulco,' 'Casa Blanca,' 'Lolly Pop,' 'Marco Polo,' 'Montana,'

'Montreaux,' 'Muscadet; 'Pesaro,' 'Pollyana,' Royal Dutch,' 'Stargazer,' and 'Red

Tiger' lily) were provided by W. L. Preston (Manatee Fruit Co., Palmetto, FL).

lily was Bulbs purchased from various retailers . A collection of bulbs obtained through various mail-order catalogues and local retail markets. These

included three Asiatic lilies ('Dandy,' 'Enchantment,' 'Horizon,' 'Gran Paradiso,'

and 'Lotus') and 'Nellie White' Easter lily. In addition, 'Pink Perfection' Aurelian

lily and 'Red Tiger' Tiger lily were included, as were several Trumpet lily hybrids not designated by cultivar. Two Easter lilies ('Nellie White' and 'White Sheen') were provided by Dewar Nurseries, Inc. (Apopka, Florida) after the 1 996 Easter 131 holidays. In addition, four Easter lilies ('Ace,' 'Arai,' 'Nellie White,' and 'White

Europe') were provided by Oglevee, Ltd. (Connellsville, PA). All Easter lily bulbs tested in this study, except those from Oglevee, Ltd., were field-grown in

Western USA or Canada.

Virus Incidence

Antisera against CMV, LSV, LVX and TBV were adopted for indirect

ELISA and DTB tests as described in Chapter 2. For routine indexing, either the upper leaves or the outer scales were selected for testing.

Impact of Virus Disease on Lily Yield and Quality

Trial I . Ten to fourteen cm-wide bulbs of 'Montana' lily were obtained from the Manatee Fruit Company and determined serologically to be either virus-free or singly infected with LSV. These bulbs were selected as controls in yield and quality studies. After bulbs were harvested and stored at 4 C for 2 months, 15 bulbs of each treatment were selected and planted (August-December, 1996) in a greenhouse at 20 to 30 C. At the end of a four-month growing season, leaf and outer scale samples representing each treatment in the experiment were tested by DTB for CMV, LSV, LVX, and TBV. Data were collected and analyzed using the Analysis of Variance (ANOVA) and the Duncan's multiple range tests.

lily Trial II . Ten to fourteen cm-wide bulbs of 'Strawberry Shortcake' imported from the Netherlands and confirmed serologically to be either LSV- infected or doubly infected with LSV and LVX were planted (February-July,

1996) in greenhouses at 20-30 C or at 22-35 C and compared as described above. 132

Trial III . Twelve to sixteen cm-wide bulbs of 'Nellie White' lily were provided from Dewar Nurseries and determined serologically to be either virus- free, LSV-infected, LSV- and LVX-infected, or LSV- and TBV-infected. Fifteen bulbs each were selected and planted (November-January, 1996-1997) at 5-28

C and compared as described above.

Results

Virus Incidences

Surveys conducted in 1994-1996 showed that LSV is the predominant

virus infecting commercial lilies. LVX and TBV were also found but not as often.

Very low incidences of CMV were noted. Of 1,180 lily samples tested, 1.2, 69.7,

10.6, and 11.2% were infected with CMV, LSV, LVX, and TBV, respectively

(Table 5-1).

The relative incidences of the four viruses varied with Lilium types. In

imported Asiatic lilies, virus incidence was very similar to that noted in domestic bulbs. For instance, of 165 bulbs imported from the Netherlands, 3.6, 44.2, 12.7, and 1 .2 % were infected with CMV, LSV, LVX, and TBV, respectively (Table 5-

2). Likewise, of 161 bulbs obtained from domestic retailers, 0.6, 43.5, 13.0, and

1.2 % were infected with CMV, LSV, LVX, and TBV, respectively (Table 5-4).

'Enchantment,' 'Gran Paradiso,' 'Horizon,' 'Juanita,' and 'Sirocco' lilies with mixed infections of LSV and other viruses were found. Whereas many plants doubly infected with CMV and LSV were found in 'Juanita,' 'Enchantment,' and 'Gran Paradiso' bulbs, 'Horizon' bulbs were often doubly infected with LVX 133 and LSV. However, the varieties, 'Dandy,' 'Jolanda,' 'Lolly Pop,' 'Lotus,'

'Montreaux,' and 'Royal Dutch' were either singly infected with LSV or virus-free.

About 48.8% of the imported and domestic lilies surveyed were virus free

(Tables 5-2 and 5-4).

Slightly higher percentages (60-100%) of healthy Oriental lily bulbs

('Acapulco,' 'Marco Polo,' 'Muscadet,' and 'Pesaro') imported from the

Netherlands were noted (Table 5-3). Virus incidences in Oriental lilies were

similar to those in Asiatic lilies, however, in that LSV was the most prevalent virus followed by LVX. Surprisingly, 49.5 % of 101 'Strawberry Shortcake' bulbs imported from the Netherlands were doubly infected with LSV and LVX (Table 5-

3).

Of 316 Easter lilies bulbs tested, 0.9, 62.3, 7.6, and 26.9 % were infected with CMV, LSV, LVX, and TBV, respectively (Table 5-5). In contrast to imported

Asiatic and Oriental varieties, field-grown Easter lilies from the Western USA and Canada had very high incidences of both TBV and LSV (Table 5-5). All

'Ace,' 'Arai,' 'Nellie White' and 'White Europe' plants from one vendor, who

maintains and grows his stock exclusively under greenhouse conditions, were free of these four viruses. Similarly, 70 and 4% of 'Nellie White' and 'White

Sheen', respectively, from a grower in Florida were virus-free. As in Easter lily varieties, a relatively high incidence of TBV was found in Aurelian and Trumpet

lilies, and the incidence of TBV exceeded that of LSV. However, no CMV or LVX

was found in these lilies. In addition, plants doubly infected with LSV and LVX or

with LSV and TBV were found in 'Red Tiger' lily. No infections by any of the four 134 viruses was detected in the L. formosanum seedlings grown from seeds collected from TBV-infected plants (Table 5-6).

Impact of viral Disease on Lilv Yields and Quality

Trial I . Despite the lack of obvious foliar symptoms, LSV significantly

reduced bulb yield and circumference in 'Montana' lily, but not stem length, leaf number, or flower number (Table 5-7). LSV-infected bulbs weighed only 80.5% as much as healthy ones and were only 88.8% as large.

Trial II. LSV and LVX did not induce obvious foliar symptoms in doubly

infected 'Strawberry Shortcake' lily plants. Plants singly infected with LSV did not differ significantly in four categories from those doubly infected with LSV and

LVX, regardless of greenhouse temperature. However, bulbs from doubly infected plants maintained at >30 C or <30 C weighed only 63.5 and 57.5%, respectively, as much as bulbs from singly infected plants (Table 5-8).

Trial III . 'Nellie White' lilies singly infected with LSV had neither visible foliar symptoms nor deformed leaves. Nevertheless, significant differences between healthy plants and plants singly infected with LSV were noted in relation to bulb yields, leaf number, and flower number. Plants doubly infected with LSV and LVX developed faint yellow spots in younger leaves. In contrast, plants doubly infected with LSV and TBV had foliar mosaic symptoms, petaloid stamens, and/or distorted corollas (Fig. 5-1). Virus-free 'Nellie White' lilies were generally more vigorous than doubly infected ones; for example, they were taller plants, had higher bulb yields, and had more leaves. Anthesis of healthy plants 135 compared to ones doubly infected with LSV and TBV differed significantly in that blossoming of the latter occurred more rapidly (Table 5-9).

Table 5-1. Virus incidence in commercial lilies surveyed in 1994-1996

No. infected bulbs '

1) Lily No. Healthy types Source bulbs CMV LSV LVX TBV (%) 5> Asiatic Neth. 165 6 73 21 2 40.6

57.1 USA 161 1 70 21 2

Oriental Neth. 496 4 451 58 22 32.9

Easter USA 316 3 197 24 85 37.3

Others USA 42 0 32 1 21 23.8

Totals 1180 14 823 125 132 38.1

1) in lily types 8, 5, 10, 5, and 5 varieties, respectively, were representing each

from the Netherlands and USA . 2) = lily virus CMV = cucumber mosaic virus, LSV = lily symptomless virus, LVX X, TBV = tulip breaking virus. 3) Number of bulbs reacting positively with antiserum in direct tissue blotting tests. 136

Table 5-2. Virus incidence in Asiatic lily cultivars imported from the Netherlands

and indexed by direct tissue blotting in 1994-1996

1) No. infected bulbs

Cultivar No. bulbs CMV LSV LVX TBV Healthy (%) 2 Enchantment 20 0 > 20 15 2 0

Gran Paradiso 54 1 25 6 0 44.4

Juanita 12 5 12 0 0 0

Jolanda 13 0 0 0 0 100

Lolly Pop 6 0 0 0 0 100

Montreaux 20 0 0 0 0 100

Pollyana 20 0 16 0 0 20

Royal Dutch 20 0 0 0 0 100

Totals 165 6 73 21 2 40.6

CMV = cucumber mosaic virus, LSV = lily symptomless virus, LVX = lily virus X, TBV = tulip breaking virus. Number of samples reacting positively with antiserum in direct tissue blots. 137

Table 5-3. Virus incidence in Oriental lily cultivars imported from the Netherlands and indexed by direct tissue blotting in 1994-1996

1) No. infected bulbs

No. Healthy

Cultivar bulbs CMV LSV LVX TBV (%)\ -J-

2) AcaDuIco 20 0 8 0 1 60 Casa Blanca 140 3 140 0 11 0 Laura Lee 4 0 4 3 0 0 Marco Polo 51 0 0 0 0 100 Montana 84 0 18 0 0 76.2 Muscadet 12 0 0 0 0 100 Pesaro 20 0 0 0 0 100 Stargazer 84 0 80 5 10 4.8

Strawberry 101 1 101 50 0 0 Shortcake

Totals 496 4 451 58 22 32.9

CMV = cucumber mosaic virus, LSV = lily symptomless virus, LVX = lily virus X, TBV = tulip breaking virus. Number of bulbs reacting positively with antiserum in direct tissue blots. 138

Table 5-4. Virus incidence in Asiatic lily cultivars obtained from U.S. and indexed by direct tissue blotting in 1994-1996

1 No. infected bulbs

Cultivar No. samples CMV LSV LVX TBV Healthy (%) Dandy 17 0 2) 0 0 0 100 Horizon 75 0 58 19 0 22.7

Lotus 45 0 1 0 0 97.8

Montreaux 12 1 1 0 0 91.7 Sirocco 12 0 10 2 2 0

Totals 161 1 70 21 2 57.1

CMV = cucumber mosaic virus, LSV = lily symptomless virus, LVX = lily virus X, TBV = tulip breaking virus. Number of bulbs reacting positively with antiserum in direct tissue blots. 139

Table 5-5. Virus incidence in Easter lily cultivars obtained from U.S. and indexed by direct tissue blotting in 1994-1996

No. infected bulbs

Huttix/ar No bulbs CMV LSV LVX TBV Healthy (%)

Ace 2) 10 0 e) 0 0 0 100

Arai 2) 10 0 0 0 0 100

2) Nellie White 13 0 0 0 0 100

3) Nellie White 29 0 29 8 9 0

4) Nellie White 44 0 44 8 16 0

5) Nellie White 100 0 29 0 0 71

White Europe 2) 10 0 0 0 0 100

White Sheen 5) 100 3 95 8 60 4

Total 316 3 197 24 85 37.3

1) = lily virus CMV = cucumber mosaic virus, LSV = lily symptomless virus, LVX X, TBV = tulip breaking virus. 2) Bulbs from one vendor, who maintains plants exclusively in greenhouses. 3) Bulbs purchased from a local retail outlet after the 1996 Easter holidays. 4) Bulbs purchased from another local retail outlet after the 1996 Easter Holidays. 5) Bulbs provided by one grower in Florida. 6) Number of bulbs reacting positively with antiserum in direct tissue blots. 140

direct tissue blotting Table 5-6. Virus incidence in miscellaneous lilies indexed by or l-ELISA in 1994-1996

1)

No. i nfected samples

No. Healthy

Lily type samples CMV LSV LVX TBV (%)

6) Aurelian 2) 12 Q 2 0 9 25

3) 0 Tiger 20 0 20 1 2 Trumpet 4) 10 0 10 0 10 0 5) 0^ 0 100 L. formosanum 573 0 0 93.6 Totals 615 0 32 1 21 1) virus, LVX = lily virus CMV = cucumber mosaic virus, LSV = lily symptomless X, TBV = tulip breaking virus. 2) Bulbs purchased from one retail outlet in New York. 3) Bulbs provided from one grower in Florida. 4) Bulbs purchased from one retail outlet in New Jersey. 5) Seedlings from TBV-infected parent plants. 6) Number of bulbs reacting positively with antiserum in direct tissue blots. 70 Number of plants reacting positively with antiserum in l-ELISA. 141

Table 5-7. Effects of lily symptomless virus (LSV) on yield and quality of

'Montana' lily

Treatment Stem Length No. of No. of Bulb yield Circumference of (cm) leaves flowers (g) main bulb (cm) »1) Virus-free 42.7 a 36.7 a 1.5a 52.7 a 14.3 a

LSV 43.8 a 32.9 a 1.6 a 42.5 b 12.7 b

} Data are the means of 15 plants. Means in the same column followed by the same letter are not significantly different (p=0.05) according to the Duncan's multiple range test. 142

Table 5-8. Comparison of yield and quality between 'Strawberry Shortcake' lily plants infected with lily symptomless virus (LSV) and plants doubly infected with

LSV and lily virus X (LVX)

Average Virus Stem length No. of No. of Bulb yield temperature leaves flowers (g) (cm) » <30 C 1) LSV 35.1 a* 36.0 a 3.9 a 29.6 a

LSV and LVX 35.7 a 40.9 a 4.1 a 18.8 a

>30C LSV 40.1 a 39.0 a 4.6 a 22.8 a

LSV and LVX 39.3 a 40.4 a 4.0 a 13.1 a

<30 C = 20-30 C; >30 C = 22-35 C. Data are the means of 10 plants. Means in the same row followed by the same letter are not significantly different (p=0.05) according to the Duncan's multiple range test. 143

Table 5-9. Yields of virus-free 'Nellie White' lilies compared with yields of plants

infected with lily symptomless virus (LSV), lily virus X (LVX) and tulip breaking virus (TBV)

Treatment Symp- Stem No. No. First anthesis Bulb Bulb toms length leaves Flowers anthesis period yield circumfer -ence (cm) (days) (days) (g) (cm)

1) " Healthy NS 58.2 a 65.1 a 4.3 a 65.5 ab 8.2 b 71.3 a 19.9 a

LSV NS 53.5 ab 59.6 b 3.1 be 66.3 a 7.9 b 61.3 b 19.3 a

LSV+LVX S 47.6 c 55.6 b 24c 63.8 be 8.9 a 59.0 be 18.9 a

LSV+TBV M 49.3 be 56.0 b 3.6 ab 62.3 c 7.2 c 50.1 c 18.4 a

NS = no symptom, S = faint leaf spot, M = foliar mosaic. 2) Data are the means of 15 plants. Means in the same row followed by the same letter are not significantly different (p=0.05) according to the Duncan's multiple range test. 144

Fig. 5-1. Two types of abnormal flower symptoms: a petaloid stamen (top left) and a distorted corolla (bottom left), caused by tulip breaking virus and

lily symptomless virus on 'Nellie White' lily. 145

Discussion

The survey results confirmed reports by others (Deng, 1995; Kim ef al.,

1996; Yang et al., 1993) that LSV (69.7%) is the most prevalent virus of the

commercial lilies in the world. Despite high incidences of LSV in Asiatic, Easter, and Oriental varieties, however, many plants proved free of CMV, LSV, LVX, and

TBV These results indicate that healthy bulbs are being produced through virus certification programs currently underway in the Netherlands. In the U.S.A.,

plants of Easter lily varieties ('Ace,' 'Arai,' 'Nellie White,' and 'White Europe') from one bulb grower were determined to be virus-free. According to this grower, these plants were obtained through meristem-tip culture, indexed for virus, maintained in a mother stock system, and maintained exclusively in greenhouses prior to sale.

An overall CMV incidence of only 1.2% was found in commercial lilies.

Deng (1995) also noted low incidence with this virus. High CMV incidences in

'Georgia' and 'Hinomoto' Easter varieties as well as in 'Connecticut King' and

'Enchantment' Asiatic varieties were noted in Korea, however (Kim ef al., 1996).

The low CMV incidences in the U.S.A. and the Netherlands can be attributed to

the severe necrotic fleck symptoms in lily plants doubly infected with CMV and

LSV. Such stock is unsaleable and, hence, diseased plants are self-eliminating.

Aggressive roguing programs (Asjes, 1974) and the relatively slow rate of CMV spread (Jacono, 1989) may account for the observed low incidences of CMV noted in this and Deng's study. Necrotic fleck symptoms were also observed in

young CMV- and LSV-infected 'Juanita' lily plants. The symptoms, while still 146 visible, become less evident at flowering. Therefore, prompt roguing early in the growing season could reduce the necrotic fleck disease.

The 10.6% rate of LVX incidence in imported and domestic lilies in this study was quite similar to the 6.7-8.9% noted in 'Enchantment' and 'Connecticut

King' Asiatic lily and the 4.8% in 'Stargazer' Oriental lily by Asjes and Blom-

Barnhoorn (1994) in the Netherlands. It was likewise similar to the 1.1-17.9% in

'Stargazer' and 'Casa Blanca' Oriental lily noted in Japan by Kimura et al.

(1990). In my study, LVX was found not only in Asiatic and Oriental varieties, but also in 'Nellie White' (8.6%) and 'White Sheen' (8%) Easter lily varieties. This is

the first report of LVX infecting Easter lily varieties.

Higher TBV incidences were found in the Aurelian (75%), Easter (26.9%), and Trumpet (100%) varieties than in the Asiatic (1.2%) and Oriental varieties

(4.4%). It is possible that varieties with funnel-shaped flowers, like L. formosanum, may be unusually susceptible to TBV. However, TBV also induced

flower breaking symptoms in 'Sirocco' lily and produced petaloid stamens and/or distorted flowers in 'Nellie White' plants. These abnormal flowers could greatly decrease the plants' market values and, thus, TBV has the potential of being a serious threat to the industry.

Commercial lilies grown for bulb production are de-headed in the field prior to anthesis. This practice results in increased bulb sizes and yields (Mey,

1978), but risks manual transmission of viruses, particularly the potexvirus, LVX.

Potexviruses have become problems in crops such as orchids for this reason

(Zettler et al., 1990). My discovery that LVX was more prevalent, especially in 147

' 'Nellie White,' may be old lily varieties such as ' Enchantment, 'Stargazer,' and attributed to manual transmission by deheading procedures. Therefore, spraying with surfactants such as polyoxyethylene alkylether, polyoxyethylene alkylphenol or alkylbenzene sulphonate to cease bud formation when the first flower bud became visible could reduce both virus spread and labor costs (Masaaki, 1974).

The use of virus-free lily stocks, of course, would be a useful preliminary means of thwarting epidemics of this and other viruses.

In these studies, virus-free 'Nellie White' lily plants produced significantly taller plants, had higher bulb yields, and had more leaves and flowers. Anthesis was reached later, however, than in plants doubly infected with LSV and LVX, or in plants doubly infected with LSV and TBV. These results are similar to the results of Allen (1975), who found that virus-free 'Enchantment' lily plants were

25 to 50% taller than plants grown from virus-infected bulbs. Similar results were also noted by Hanks and Menhenett (1981) and Menhenett and Hanks (1982),

who described that virus-free lily plants produced more and larger florets than infected plants, but reached anthesis by an average of 6.5 days later than virus- infected ones. According to the results of Allen (1975) and Deng (1995), LSV-

infected lilies reduced stem length, and bulb yields of 'Enchantment' and

'Sirocco' lily. These results were similar to my results for LSV-infected 'Nellie

White' plants. However, in LSV-infected 'Montana' plants, the only statistically significant difference noted was in reduced bulb yields. Based on these results,

therefore, LSV may vary in its effects on different lily types and varieties. The impact of anthesis date noted in this study and Menhenett and Hanks (1982) has 148 important economic implications, especially for lily stock that consists of a mixture of healthy and diseased plants. For Easter lilies, the flower timing is

especially critical for meeting the Easter market. Timing can be synchronized with bulbs that are healthy or infected, but in stocks that are mixed with roughly equivalent numbers of both healthy and diseased, considerable losses could

result because of the variability in flowering times.

in Kimura et al. (1990) reported that the bulb size and cut-flower quality

this Oriental lilies were not affected by LVX infection. My results agreed with

conclusion since there were no significant differences between 'Strawberry

Shortcake' lilies singly infected with LSV or doubly infected with LSV and LVX.

However, 'Nellie White' lilies doubly infected with LSV and LVX were more

damaged than ones singly infected with LSV or healthy ones. It would appear

that LVX and LSV together have a greater synergetic effect on 'Nellie White'

than on 'Strawberry Shortcake' lilies.

In my study, higher incidences of root deterioration, presumably caused

by fungi, were noted in 'Strawberry Shortcake' lilies doubly infected with LSV

with and LVX than in singly infected ones. Roots of L. longiflorum plants infected

necrotic fleck (i.e. LSV and CMV) were more severely damaged by Pythium

splendens than roots of plants without such symptoms (Raabe, 1975). Mixed

infections of virus could, therefore, influence roots.

Based on my results, both LSV and LVX can inhibit growth and bulb

development of certain lilies even in plants showing no overt symptoms. 149

Therefore, it appears that commercial growers should be encouraged to plant virus-free stock. APPENDEX

LILY CULTIVARS AND THEIR CORRESPONDING TYPES

Type Cultivar Asiatic Dandy Enchantment Gran Paradiso Horizon Juanita Jolanda Lolly Pop Lotus Montreaux Pollyana Royal Dutch Sirocco Aurelian Pink Perfection Easter (Lilium longiflorum) Ace Arai Nellie White White Europe White Sheen Oriental Acapulco Casa Blanca Laura Lee Marco Polo Montana Muscadet Pesaro Stargazer Strawberry Shortcake Tiger Red Tiger Trumpet Lilium formosanum

cultivars not identified.

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healthy Lativian-bred lily cultivars by means of flower-buds. Acta Hort. 325:529-535. BIOGRAPHICAL SKETCH

Tso-Chi Yang was bom in Tainan, Taiwan, the Republic of China, on

October 17, 1960. He graduated from National Chung-Hsing University in 1984 with a Bachelor of Science degree in plant pathology. He continued his studies

there and received a Master of Science degree in June 1986.

After two years of military service, he began working as an assistant

researcher in the field of plant pathology at the Taiwan Seed Propagation and

Improvement Station in Hsin-She, Taiwan, where he is currently employed. In

August of 1994, he was sponsored by the National Science Council of the

Republic of China to begin his doctoral program at the University of Florida under

the direction of F. W. Zettler.

Tso-Chi is currently a member of the American Phytopathological Society,

North American Lily Society, Taiwan Plant Pathology Society, Taiwan Plant

Protection Society, and Chinese Seed and Seedling Society.

168 I it certify that I have read this study and that in my qpiriion conforms to acceptable standards of scholarly presentation and is f)itj^adejquate, in scope and quality, as a dissertation for the degree of Doctop

F. W. Zettler, Chairman Professor of Plant Pathology

it I certify that I have read this study and that in my opinion conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Jf.fc. Polston, Cochairman Associate Professor of Plant Pathology

it conforms to I certify that I have read this study and that in my opinion acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

E. Hiebert Professor of Plant Pathology

it conforms to I certify that I have read this study and that in my opinion acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

M. E. Kane Associate Professor of Horticultural Science

opinion it conforms to I certify that I have read this study and that in my acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

N.-J. Ko President of OKKO Agrotech, Inc. I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

D. E. Purcifull Professor of Plant Pathology

This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy^

August, 1997 )tacA Dean.^College of Agrfeulture

Dean, Graduate School