- I - NIGERIAN ISOLATES OF CASSAVA LATENT VIRUS By GABRIEL OLAYIWOLA ADEJARE B.Sc. (Hons.) A Thesis submitted in part fulfilment of the requirements for the degree of Master of Philosophy of the University of London Department of Botany and Plant Technology, Imperial College of Science and Technology, London SW7 2BB MARCH 1981 TO THE GLORY OF GOD What shall I render unto the Lord for all his benefits toward me ? I will take the cup of salvation, and call upon the name of the Lord Holy Bible, Psalms 116: 12 & 13. Thanks to God and all mankind in Great Britain and all over the World. G. 0. Adejare - 2 - Nigerian Isolates of Cassava Latent Virus Gabriel Olayiwola Adejare ABSTRACT Cassava latent virus (CLV) is a single stranded DNA (ssDNA) virus containing characteristic geminate particles in purified preparations. It is believed to be the causal agent of cassava mosaic disease (CMD) which threatens the production and yield of cassava in several growing areas of the world. Meristem tissue culture and thermotherapy techniques have been successfully used to free several Nigerian cassava varieties (Manihot esculenta Crantz) from CMD. Whole plants have been regenerated from excised meristem tips (0.2 to 1.0 mm in length) including one or two chlorophyllous leaf primordia. Regenerated 'virus free' plants have also been produced from rootless plantlets by dipping in a hormone rooting powder and further culture. In both cases, complete regeneration of CMD free plants has been achieved within 90 days of culture. Regeneration of meristems from CMD affected plants was consistently slower than from comparable healthy plants or from infected cassava plants heat treated prior to meristem excision. Tobacco plants (Nicotiana benthamiana) were used as indicator plants for CMD/CLV, and characteristic systemic symptoms appeared 9 to 10 days post-inoculation when inoculated with extracts prepared from infected plants, or purified virus. Identification of CLV was made by passage onto tobacco plants and electron microscopy by the investigation of CLV infected leaves in thin section or negative staining of homogenized leaf sap samples. Characteristic geminate particles of CLV have been seen in extracts prepared from tobacco leaves but similar particles have not been identified in the sap of CMD infected cassava tissue. Ultrathin sections of CLV infected tobacco leaves when examined in the electron microscope showed striking alterations in both nuclear and chloroplast structures as compared to healthy material. The changes include (i) hypertrophy of the nucleolus to occupy more than 50% of the nuclear volume, (ii) segregation of nucleolar components into discrete granular and fibrillar regions, (iii) the appearance of characteristic fibrillar rings in the nucleoplasm and (iv) disintegration of chloroplast membranes and the deposition of large starch grains in chloroplasts eventually obscuring the grana. CLV has also been successfully purified from infected N. benthamiana leaves and stems by differential centrifugation and a preliminary characterization of the viral DNA and protein made. - 3 - CONTENTS Page ABSTRACT 2 LIST OF ABBREVIATIONS 4 INTRODUCTION 7 MATERIALS 12 EXPERIMENTAL METHODS AND RESULTS 15 The growth of cassava and cassava mosaic disease 15 Cassava seed germination 15 Cassava mosaic disease (CMD) 17 Cassava latent virus (CLV) 18 Transmission of CMD/CLV 20 Host range of CLV 26 CLV purification methods 27 Electron microscopy 34 Chemical characterisation of CLV 36 Ultrastructural studies of CLV 39 Meristem tip culture 42 DISCUSSION 57 ACKNOWLEDGEMENTS 63 REFERENCES 54 APPENDIX I 72 APPENDIX II 73 PICTURES AND PLATES 74 - 4 - LIST OF ABBREVIATIONS CMD Cassava mosaic disease CLV Cassava latent virus ssDNA Single stranded deoxyribonucleic acid ..MPIBN Moor Plantation, Ibadan, Nigeria ..IITAIBN International Institute of Tropical Agriculture, Ibadan, Nigeria LGjN Lagos/Agege (variety 1) Nigeria mm millimetre ml millilitre M mole yM micromole yi microlitre nm nanometre gram 8 milligram mg volume v weight w molecular weight MW species Spp. *MS Murashige & Skoog (modified*) Y gamma a alpha % percentage Klux kilolux (1,000 lux = 1 Klux) RH relative humidity mA milliampere pH acid content - 5 - revolutions per minute low speed spin (5,000 - 15,000 rpm) high speed spin (36,000 - 40,000 rpm) Analar grade degree centigrade benzyl-amino purine gibberellic acid indol-3-ylacetic acid naphthalene acetic acid 6(dimethylallyl-amino) purine 6-(4-hydroxy-3-methylbut-2-enylamino) purine thiamine HC1 nicotinic acid pyridoxine HC1 Boots' hormone rooting powder nitrogen leaf sodium hydroxide sodium chloride sodium lauryl sulphate (Cj sodium sulphite sodium phosphotungstate bovine serum albumin hydrochloric acid polyethylene glycol (MW = 6,000) polyacrylamide gel electrophoresis potassium hydroxide - 6 - EDTA - ethylenediamine tetra acetic acid,disodium salt 2-ME - 2-mercaptoethanol ri-butOH - n-butanol EM - electron microscope Psi - Ib/sq. in (pressure) Pel't - pellet tiss - plant tissue (whole plants without roots) TGA - thioglycollic acid MAFF - Ministry of Agriculture Fisheries and Food CHC10 - chloroform - 7 - 1 . INTRODUCTION Cassava (Manihot esculerita Crantz) a native plant of Latin America was introduced from Brazil into Africa by the Portuguese approximately 400 years ago (llahn and llowland, 1972; Hahn, 1978; Kawano, 1975; Lozano and Van Schoonhven, 1975; Lozano, 1977; Peterson and Yang, 1976). Cassava is grown in Nigeria commercially on a large scale for human consumption, animal feed and for starch production (Hahn, 1978; Lozano, 1972; Oyenuga, 1955- Seif and Chogoo, 1976). Cassava mosaic disease (CMD) is present in almost all cassava grown in Nigeria and is more severe in the southern states than in the north (Beck and Chant, 1958; Chant, 1959; Hahn, 1978 and Hahn, 1980 (personal communication); Jones, 1959; Thurston, 1973). At least ten out of the nineteen states of Nigeria grow cassava for the above usage. Spread of the CMD within a country and from one country to another is believed to have been caused mainly by the vegetative propagation of infected cuttings (Jones, 1959; Lozano and Van Schoonven, 1975; Terry, 1978). The whitefly (Bemisia tabaci Genn.) is reported to be the only insect vector for CMD in some countries (Chant, 1958; Bock and Guthrie, 1978a; Hahn and Howland, 1972; Lozano, 1972; Lozano and Van Schoonven, 1975; Peterson and Yang, 1976; Storey and Nichols, 1938), but unintentional mechanical spread by farmers through using contaminated implements during tillage of plants is another possible means of transmission. CMD is an easily recognizable disease on infected cassava leaves but is often found in association with other pathogenic agents e.g. viruses, bacteria and fungi (Costa and Kitajima, 1972; Fereol, 1978; Hahn, 1978; Lozano, 1972 and 1978; Lozano and Booth, 1974). Symptoms on cassava include a generalised leaf chlorosis, mottling, distortion and a reduction in leaf lamina and leaf size (Bock and Guthrie, 1978a; Kaiser and Teemba, 1979; Lozano, 1972). The symptoms appear primarily on the top leaves of infected plants with occasional etiolation of the stems. The aetiology of - 8 - CMD is thought to be viral (Storey arid Nichols, 1938). It may be caused by cassava latent virus (CLV) a member of the emergent group of viruses known as geminiviruses (Anon, 1978; Bock, Guthrie and Meredith, 1978b; Goodman, 1977a ; Kaiser and Teemba, 1979). However this has not been conclusively proven because of the failure to demonstrate Koch's postulates (Anon, 1978; Bock al., 1978b). Considering the current and increasing importance of cassava as a staple food crop, efforts are now being made to eradicate CMD from cassava and to exchange and distribute clean cassava stocks and germ-plasm. CMD and CLV have generated a considerable amount of interest in recent years (Chant, 1958 and 1959; Chant and Beck, 1959; Chant Bateman and Bates, 1971; Beck and Chant, 1958; Bock and Guthrie, 1976; Bock, Guthrie and Meredith, 1977; Bock et al., 1978b; Gamborg and Kartha, 1976; Jennings, 1972; Khan, 1978; Kawano, 1975; Luisoni, Victoria, Molue, Lovisolo and Dellavelle, 1976; Terry, 1975; Verhoyen, 1978). Geminiviruses The principal characteristics of plant viruses belonging to the geminivirus group have been outlined (Bock, Guthrie and Woods, 1974; Goodman, 1977a; Harrison, Barker, Bock, Guthrie, Meredith and Atkinson, 1977). The main characteristics of this virus group include their regular occurrence as quasi-isometric particles with single stranded DNA (ssDNA) (Bock _et_al., 1977 and 1978b; Dubern, 1979; Francki, Hatta, Grylls and Grivell, 1979; Matyis, Silva, Olivera and Costa, 1975; Mumford, 1974) and their close association with the nuclei of phloem parenchyma cells (Shepherd, 1979). Some members of this group are wholly restricted to the phloem and cannot be mechanically transmitted, inoculation of susceptible plants then being performed by the insect vector (B. tabaci) (Chant, 1958; Goodman, 1977b; Hollings and Stone, 1976; Kaiser and Teemba, 1979; Mumford, 1974). Geminiviruses have divergent host ranges - 9 - (Bock £t £l., 1978b; Duberri, 1979; Lozano, 1972; Menon and Raychaudhuri, 1970; Shepherd, 1979). However, hosts giving a local lesion reaction have been difficult to find for members of this group. Some of the confirmed geminiviruses include : cassava latent (CLV, Bock £t_ al., 1978b); maize streak (MSV, Bock et al., 1974, 1977; Shepherd, 1979); beet curly top (BCTV, Esau and Magyarosy, 1979; Shepherd, 1979); chloris striate mosaic (CSMV, Francki