MOLECULAR IDENTIFICATION AND COAT PROTEIN BASED GENOMIC CHARACTERISATION OF A VIRUS CAUSING YELLOW VEIN DISEASE ON CALENDULA X ABSTRACT •^ THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF Slnctnr of Piitlnsoplig m •W' /i BOTANY // l-r' / /' / /it •^' :>• ^'O''' BY AKILA. KHAN DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2005 ABSTRACT Calendula officinalis L. belongs to family Compositae/ Asteraceae which occupies the highest position among the angiosperms or at least among the dicots in the course of evolution. There are about 25 species of calendula but only two species viz. C. officinalis L. and C. arvense L. are found in India. Calendula is also known as pot marigold. A new viral disease was observed on calendula at Aligarh Muslim University, Aligarh campus and in the surrounding area with the symptoms of yellowing of vein, s\\in\mg of plants, shortening of the \eaf and petioles, reduction in the nMmbei of flowers. Mosaic symptoms also appeared on calyx and corolla in severely infected plants. The incidence of disease was about 10-15% in nature. However, to provide clean and healthy environment of calendula to the pharmaceutical industries, it was essential to identify the causal pathogen and to develop the diagnostic at molecular level. So, virus free material may be made available. The initial biological studies revealed that the causal pathogen of calendula was successfully transmitted from naturally infected calendula to the newly emerging seedlings of calendula through whitefly, which produces the symptoms alike to naturally infected plant. The calendula isolate was tested for host range studies. The yellow vein disease of calendula was successfully transmitted to Nicotiana tabacum var. White Burley, Lycopersicon esculentum, and Capsicum annuum, producing leaf curling, shortening of intemodes and petioles. While Datura stramonium did not show any type of symptoms. Based on symptomatology, host range studies and whitefly transmission, virus isolate causing yellow vein disease on calendula was suspected to be geminiviral in nature. Virus isolate causing yellow vein disease on calendula, was further checked by PCR amplification with begomovirus specific TLCV CP primers. The PCR results gave positive amplification at ~750 bp in the plant showing symptoms of the disease. The specificity of the PCR amplicon was checked by Southern hybridization using radiolabelled probe of a well-characterized begomoviral (ITLCV), which proved that PCR amplification was geminiviral in nature. The DNA band was LMP eluted and cloned in a suitable cloning vector (pGMT-T). The sequence data of clone of calendula isolate revealed that the gene was 519 bp in length and it encompassed the complete ORf with initiation codon (ATG) to termination codon (TAA), which encodes 173 amino acid residues. The nucleotides sequence of the coat protein gene, was deposited in gene bank under the accession number (AY887174). Amino acid alignment of calendula isolate revealed that four amino acid position "A2S", "K180R", "R180K" and "LKIILR-MRW218 to 227 YENHTENALM" were unique in the total amino acid sequence data. However, there were no unique sites from 61-120 and 241-253 amino acid. Pairwise nucleotides alignment of the virus isolate from calendula with the selected geminivirus revealed maximum similarities 96% with Tobacco curly shoot virus (ToCSV) and Tomato Geminivirus-China (TomGV), minimiun similarities 77% with TLCV-Banglore. While pairwise amino acid alignment of the virus, isolate from calendula revealed maximum 97% similarities with TomOV-China. Based on pairwise alignment of nucleotides and amino acids i. e. maximum similarities 96-97% with ToCSV and TomOV-China. Virus isolated from calendula seems to be similar to that of TomGV-China and ToCSV geminivirus. The dendrogram of nucleotides and amino acids showed close similarities of the virus isolate from calendula with ToCSV and TomGV-China. The association of P-DNA molecule, which is one of the characteristics of several begomoviruses, was also checked by PCR using P-DNA specific universal primers. PCR product showed -650 bp amplification, half of the genome (1.3 kbp). On the basis of results obtained by virus transmission, PCR amplification. Southern hybridization, sequence aligimient of nucleotides and amino acids and cluster analysis of CP gene, the virus has been identified as Calendula yellow vein virus (CYVV) which shows close similarity with Tobacco curly shoot virus and Tomato geminivirus-China. A P-DNA molecule has also been found to be associated with virus isolate from calendula. On the basis of above studies, the virus isolate from calendula comes under the subgroup of begomovirus, genus geminivirus and family geminiviridae. Yellow net disease of calendula associated with Cucumber mosaic virus has been previously reported. However, our report is the first record on natural infection of calendula by a begomovirus and association of (3-DNA with virus isolate. Moreover, the finding on molecular characterization of virus isolate causing yellow vein disease on calendula will open an understanding to the researchers on the genomic organization of a new begomovirus. Association of (3-DNA molecule with the virus isolate may have a definite role in symptoms severity or early development of symptoms as in case of Bhendi yellow mosaic disease. Tomato leaf curl disease, Cotton leaf curl disease. The sequence data generated on CP gene of the virus may be utilized in developing CP mediated resistance against the virus isolate in calendula as well as other economically important plant. The clones generated during the studies may be used to develop molecular diagnostic probes for sensitive reliable detection of geminivirus on calendula, so that a healthy virus free plants may be made available to the pharmaceutical industries. MOLECULAR IDENTIFICATION AND COAT PROTEIN BASED GENOMIC CHARACTERISATION OF A VIRUS CAUSING YELLOW VEIN DISEASE ON CALENDULA .^m'-"^' "'^4.*'S*rj ,..,=*»*^ ^ ^ •, \ THESIS \ SUBMITTED FOR THE AWARD OF THE DEGREE OF octor of ^l|iIojaopl|Q m BOTANY •^.. BY AKIL A. KHAN DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2005 '', ATSI* ;,>'>. T6739 2402016(0) T^'Vil^l2721763(R ) 'amaz c/fb"0.i. ^^aqtiL 9412593463 MSc . Mphil, PhD, FPSI Plant Virology Lab, Deptt of Botany M N A Sc , CIES Pans Aiigarh Muslim University, Aligarh (U.P) INDIA (Sitttxtxtvitt This is to certify that the thesis entitled "Molecular identification and coat protein based genomic characterization of a virus causing yellow vein disease on calendula {Calendula officinalis L.)" submitted by Mr. Akil A. Khan to the Aligarh Muslim University, Aligarh for the award of the degree of Doctor of Philosophy, is a faithful record of the bonafide research work carried out in my guidance and supervision. He is allowed to submit this thesis for consideration of the award of the degree of Doctor of Philosophy in Botany (Plant Virology). (Qam Resi. : 332, Greater Azad Enclave Wesi, Dhorra, Angarh-202002 ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Professor Qamar Abbas Naqvi, Department of Botany, Aligarh Muslim University, Aligarh, for suggesting the problem and his constant advice, guidance and encouragement during the course of investigation and in the writing up of the manuscript. I am also greatly indebted to Prof. Ishrat Hussain Khan, Ex-Chairman, Department of Botany, Aligarh Muslim University, Aligarh for giving him permission to complete his work in National Botanical Research Institute, Lucknow. The author is also thanhful to Prof. Ainul Hague Khan, the present Chairman, Department of Botany, Aligarh Muslim University, Aligarh for his encouragement and help during the course of my research work. The author is deeply indebted to Dr. P. Pushpangadon, Director, National Botanical Research Institute, Lucknow to carry out different experiments at the Mol. Virology Lab of his Institute. The author is also so much thankful to Dr. S.K., Raj (Scientist F) for allowing him to perform various experiments in his Mol. Virology Lab and providing necessary facilities, without his help experimental work could not be materialized. I am highly thankful to all lab members and friends Sajid Khan, Rachana Singh, Sushil, Dharmandra, Atul, Arti, Radha, Sunil and lab assistants Jamal A. Ansari and Yashwant Singh for their constant help and encouragement during my experimental work in National Botanical Research Institute. I am also thankful to my lab colleagues Nuzhat, Tabasum, Shahidfor providing me their help and support. I am also extremely thankful to my friends Shiv, Irfan, Romana, Abid, Rafiq, Naeem, Faisal, Kashifand Manzarfor their constant support during my research work. The financial assistance given by A.M.U Aligarh in the form of University Fellowship is also gratefully acknowledge. The author is grateful to his parents and all the family members for providing him indispensable support and continuous encouragements during the research work. (Akil A. Khan) DEDICATED TO MY BELOVED PARENTS CONTENTS Page Nos. 1. INTRODUCTION 1-4 2. REVIEW OF LITERATURE 5-34 3. MATERIALS AND METHODS 35 - 59 4. RESULTS 60 - 67 5. DISCUSSION 68 - 71 6. SUMMARY 72 - 73 7. REFERENCES 74 - 97 ABBREVIATIONS JO ACIDS Ala (A) Alanine Arg(R) Arginine Asn(N) Asparagine Asp(N) Aspartic acids Cys (C) Cysteine Gin (Q) Glutamic acid His (H) Histidine He (I) Isoleucine Lue (L) Luecine Lys (K) Lysine Met (M) Methinone Phe(F) Phenylalanine Pro (P) Proline Ser(S) Serine Thr (T) Threonine Trp(W) Tryptophan Tyr(Y)