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Cytological Characteristics and Detection of Viruses of Lilium Spp

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Cytological Characteristics and Detection of Viruses of Lilium Spp CYTOLOGICAL CHARACTERISTICS AND DETECTION OF VIRUSES OF LILIUM 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 virus 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' tulip 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 potyvirus 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 enzyme-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 strain 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 potato 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 tulip breaking virus, 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' tulips 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 bulbs 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.
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