- 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 et^ al., 1979; Hatta and Francki, 1979); bean golden mosaic (BGMV, Galvez and Castano, 1975; Goodman, Bird and Thongmeerkom,

1977c; Harrison ej: al., 1977; Kim, Shock and Goodman, 1978; Shepherd,

1979); euphorbia mosaic (EuMV, Shepherd, 1979). Bock et al. (1977, 1978b), isolated CLV from several CMD infected cassava varieties in East Africa and designated "the cryptogram D/l, 0.8/*:S/S:S/*, geminivirus group for the virus. The purified virus was found to consist of geminate particles isometrically arranged in pairs of 20 x 30 nm dimensions. Single particles presumably derived from geminate ones were also found and were 20 nm in diameter. Goodman et al. (1977c) and Goodman (1978) similarly found that

BGMV consisted of geminate particles and the virus had a restricted host range and was transmitted by whiteflies (B. tabaci).

Few ultrastructural studies have been carried out on members of

this virus group in vivo. Those that have been investigated have illustrated the following changes in infected cells viz. nuclear abnormal- ities, a disintegration and disappearance of nuclear membrane and chloroplast disruption. Hypertrophy of nucleoli and a segregation of nucleolar components with the appearance of fibrillar chromatin rings and virus like particles are all characteristic of a geminivirus infection in phloem and phloem parenchyma cells (Esau and Magyarosy, 1979; Francki et al., 1979;

Goodman et al., 1977c; Kim et al., 1978; Russo, Cohen and Martelli, 1980).

Crystalline arrays of MSV and tomato leaf curl virus (TLCV) have also been observed in the nuclei of infected hosts (Bock et al., 1974; Esau and

Magyarosy, 1979; Lana, 1976). - 10 -

MeristGm tissue culture

The main principle behind meristem tissue culture is based on the fact that cells at the extremities of new shoots on plants (often meristematic) are virus free. However, the reasons for this phenomenon are not known (Matthews, 1970). These tips can be aseptically removed, sterilised and cultured on a range of agar solidified media to regenerate whole plants (Cervantes and Victoria, 1978; Dale, 1977; Earle and

Langhans, 1974; Fereol, 1978; Gamborg and Wetter, 1975; Gamborg and

Kartha, 1976; Hasegawa, Murashige and Takatori, 1973; Hollings, 1965;

Kaiser and Teemba, 1979; Kartha, Gamborg, Constabel and Shyluk, 1974a;

Kartha, Gamborg and Constabel, 1974b; Kartha, 1975c; Kartha and Gamborg,

1975a, 1975b"and 1978; Lane, 1979; Murashige, 1974; Nair, Kartha and

Gamborg, 1978; Quak, 1961 and 1972; Slack, 1980; Tilquin, 1978; Walkey and Cooper, 1975; Westcott, Grout and Henshaw, 1976). Once tested for virus absence, new disease free plants could be used as source of clean cassava stocks for both cultivation and germ plasm exchange within and from one country to another (Hollings, 1965).

A large number of plant viruses causing systemic infection of

N. benthamiana have been outlined by Christie and Crawford (1978).

Quacquarelli and Avgelis (1975) also reported several plant viruses which

elicited both local and systemic infection in this variety.N.hybrida has al

been developed as a host for plant viruses by Christie (1969),Lana (1978)ca

for "a rapid infectivity test as a tool to virus research and identificatio

in developing countries" and N. benthamiana offers this possibility for

CLV. Bock and Guthrie (1978a) reported that it was possible to transmit

CMD mechanically to healthy cassava plants.However, Kartha and Gamborg (19/

used a grafting technique as a method of transmitting CMD into healthy

cassava plants as a check for the presence or absence of CMD in regenerated

cassava plants obtained by meristem tip culture. Lister (1959) attempted -11-

mechanical transmission of cassava brown streak virus.

Heat treatment and meristem culture techniques either in isolation or both together have been used to produce virus free clones of several plants, e.g. asparagus (Hasegawa, Murashige and Takatori, 1973); potato

(Cervantes and Victoria, 1978); banana (Berg and Bustamante, 1974);

cassava (Chant, 1959; Kaiser and Teemba, 1979; Kartha and Gamborg,

1975a and 1978; Kartha et ad., 1974a; Nair et al., 1978; Quak, 1961 and

1972); chrysanthemum (Earle and Langhans, 1974); cucumber, tomato and

tobacco (Kassanis, 1954); pea (Kartha al_., 1974b); pear (Lane, 1979).

The aims of this study include :

(i) Identification, purification and characterisation of the causal

agent of CMD as present in cassava plants imported from Nigeria.

(ii) Determination of a suitable host range for CMD and its causal

agent.

(iii) Attempting to obtain CMD-free cassava using heat therapy and

meristem culture.

(iv) A study of ultrastructural changes in CLV-infected Nicotiana

(N. benthamiana) leaves or stems. 2. MATERIALS

'Analar' (AR) grade chemicals were used throughout and solutions were made with glass double distilled water.

(i) Virus purification, virus protein and DNA extraction

Buffers used included phosphate buffer, citrate buffer prepared

from citric acid adjusted to the required pH with 40% sodium hydroxide, while borate buffer was prepared from borax and boric acid and the pH adjusted with

40% sodium hydroxide. All buffers were made to the required strength and the pH adjusted as outlined by Dawson, Elliot, Elliot and Jones (1969).

Carborundum 500 mesh (The Carborundum Company Limited, Trafford

Park, Manchester) was used as an abrasive. The sucrose gradient mixer,

peristatic pump and mechanical shaker were supplied by the LKB Company.

Centrifuges routinely used included an MSE 21, a Beckman LI and an L2—65B.

Rotors used in the Beckman centrifuge included the 39, 50, SW41 and Ti. 45

types. The ISCO fractionator and freeze drier were supplied by the

Biochemistry Department. Spectrophotometers used were both of the recording

type, namely a '.Gilford' 240 and a Beckman model 35 type. The homogenizer used was either of a Waring blender or Omnimix type.

Chemicals used are as shown in Appendix II.

(ii) Electron microscopy

The following materials were used in the EM investigations :

Sodium phosphotungstate at 3 to 4% bovine serum albumin, 0.0005%, the

Aerograph Super 63 air brush, copper grids (100 - 200 mesh) coated vith

either carbon, 0.6% formvar or necoloidine, gluteraldehyde, sodium

cacodylate buffer, osmium tetroxide, ethyl alcohol, epon resin, epoxy

propane, uranyl acetate, lead citrate. - 13 -

The 'Reichert' automatic microtome and the Siemens 102, Philips

301 and Philips 6B electron microscopes were located in the EM Unit.

(iii) Plant materials

CMD infected cassava cuttings of the cultivars or varieties 631004

MPIBN, Isunik-IITAIBN, and the Lagos/Agege-LGjN, LG2N and LG^N

Seeds of the Nicotiana species of N.benthamiana and N.hybrida were supplied by Dr. R. Christie, Institute of Food and Agricultural Sciences,

University of Florida, Gainesville, U.S.A. Others including N. clevelandii,

N. glutinosa, N. debneyi, N. tabacum var. Xanthi nc and the various seeds shown in section 3:3(i) were collected from The Royal Botanic Gardens, Kew,

Surrey and Rothamsted Experimental Station, Harpenden, Herts, England.

Seeds were germinated and grown in the greenhouse as indicated in section

3:1 either in John Innes No. 2 or Levingtons all peat compost.

Six types of cassava seed including 30211 (1977), 30395 (1977), 3055:

(1977), 30572 (1977), Isunikankiyan (1975) and 60444 (1973), were all

supplied by The International Institute of Tropical Agriculture (IITA),

Ibadan, Nigeria. Some were derived by breeding (Hahn, Howland and Wilson,

1977; Hahn, 1978). Crosses were made in the following way "Cassava from

Latin America and India which are susceptible to CMD, other pathogens and

lodging but have other desirable agronomic traits F1 + F2 were crossed with local sources and selections subsequently made. In this manner, these

exotics have been substantially improved for resistance to CMD and to

lodging while maintaining the other desirable traits" (Hahn et al., 1977;

Jennings, 1972).

(iv) Meristem tissue culture

Murashige and Skoog (1962) medium (modified in this study) and

Hoagland's nutrient medium were prepared according to methods of Gamborg - 14 -

and Wetter (1975). Plant growth substances and chemicals used in this study for virus purification, characterisation and meristem culture are as shown in Appendices I and II.

Gilson micro pipettes were supplied by Alfa Laboratories Limited.

The sterile air-cabinet used in this study was supplied by The Pathfinder

Company. The binocular microscope was of an 'Orthoplan1 type, and culture tubes were made of pyrex glass (2.5 x 10.0 cm). Vermiculite, garden soil

(of high humus content), John Innes No. 2 soil and *e"M gravel were purchased locally. 3. EXPERIMENTAL METHODS AND RESULTS

3.1. The growth of cassava (Manihot esculenta Crantz) and cassava mosaic

disease (CMD)

(i) Cassava seed germination

The seed viability of the six cassava types was checked by the water soaking technique. Seeds from each trial variety were dropped in distilled water in a beaker. The seeds that floated were discarded but those which soaked were collected for pre-germination treatments or direct planting in pots. Cassava seeds were germinated by either scarification or direct planting in pots/trays before transplantation (Hahn, personal communication)

Laboratory scarification method

A few cassava seeds from the water soaking test were put inside a bijoux bottle and a 1 - 2% sodium hypochlorite solution poured over them up to two-thirds the bottle capacity. The bottles were capped tight and shaken

(120 strokes/minute on a shaker) for five minutes. The seeds were then rinsed with sterile distilled water five times and scarified with a metal file (J.K. 6", flat type by the Smooth Line Company Limited) at the micropyl until the cotyledon just became visible. Twenty-five percent of the scarified seeds germinated (Table 1). Four seeds each were planted in four inch square pots containing sterile vermiculite (steam autoclaved at 15 psi,

120°C for 30 minutes at least three times before use). The pots were kept in trays, covered with transparent plastic covers to maintain high humidity and left in a growth cabinet at 25 - 30°C, 70 - 80% RH and an 18 hour photoperiod provided by warm white fluorescent tubes. Seedlings were

transplanted individually into eight inch square plastic pots containing

sterile garden/John Innes No. 2 soil and left in the same growth cabinet

for a further one to two weeks. Thereafter, seedlings were transferred to a glasshouse and potted into one or two.gallon plastic buckets containing - 16 -

gravel and John Innes soil as in section 3:1a (Fig. 1). Such seedlings were used in experiments on mechanical inoculation.

Cassava seedlings emerged 30 - 40 days after sowing unscarified seeds in Jiffy pots filled with Fisons potting compost (peat based) using

Hahn's greenhouse germination method (personal communication). With the laboratory scarification method and seed treatment prior to planting, seedlings appeared within 18 to 25 days. All cassava plants derived from seeds were apparently free of CMD (Fig. 1) which is endemic in West Africa on stem propagated plants (Fig. 2(i)) , (Thurston, 1973). Ten of such seedlings from the seed scarification grew into adult plants (Fig. 1) and were used for CMD/CLV transmission tests.

Table 1 The effect of scarification on the germination and

survival of cassava seeds

Cassava seed types Total No. Total No. No. of plants scarified germinated surviving (seedlings)

30211 (1977) 40 10 2

30395 (1977) 30 7 3

30572 (1977) 20 4 1

30555 (1977) 15 5 1

60444 (1973) 25 6 2

Isunikakiyan (1975) 10 3 1

Total 140 35 10

% Success 25% 7.1% - 17 -

(ii) Cassava mosaic disease (CMD)

The types of cassava used in this study were collected from the coastal and inland areas (Lagos and Ibadan) of Nigeria. The CMD was demonstrated in several types of infected cassava cuttings imported under licence from Nigeria (631004 MPIBN, Isunik-IITAIBN, Lagos/Agege (LGN) varieties 1, 2 and 3). The stems were aseptically cut into about 4-8 inch

long pieces bearing two to four dormant buds. The cuttings were then plante

in two gallon plastic buckets containing gravel roughly four inches deep.

John Innes No. 2 compost was then put into the buckets to two-thirds the

bucket volume and the buckets covered in plastic sheeting to maintain a high humidity. New infected shoots emerged within nine to fourteen days of planting (Fig. 2(i)). Infected cassava plants were then maintained under

insect-proof glasshouse conditions, at a temperature of 25 - 28°C, 70 - 80%

RH and 10-20 Klux light intensity supplied by mercury vapour bulbs of

400 watts power with an 18 hour photoperiod.

The effects of CMD on cassava plants are recognized by a generalized

leaf chlorosis, mottling/distortion and a reduction in leaf size in all

types. The symptoms start on the youngest emergent shoots with vein banding

appearing from the base of the midribs and extending towards the tips of

the leaves (Figs. 2(i), 3b and c). This is accompanied by leaf distortion

and as the leaves age, generalized chlorosis becomes pronounced (Figs. 2(i),

3b and c compared to 2(ii) and 3a). The effect of CMD on infected cassava

plants is not apparent on the stems but the fresh and dry weights as well

as leaf area are lower in infected plants (Chant, personal communication).

CMD infected cassava plants show a reduction in tuber yield and leaf area

when comparisons were made between healthy and infected cassava plants

(Beck and Chant, 1958; Chant and Beck, 1959; Chant et al., 1971). CMD

was maintained in an insect-proof glasshouse by cutting the infected stems

back or when the tips of similar plants were removed for meristem culture. - 18 -

These procedures were routinely carried out every month.

3.2. Cassava Latent Virus

It has been assumed that CLV acts as an assistor virus for the infection of cassava by CMD whose aetiological agent is unknown (Anon,

1978; Bock jBt al., 1978b). CLV however was easily transmitted from cassava leaves to susceptible Nicotiana species by standard methods of mechanical inoculation (Table 2).

Table 2 Symptom development of Nicotiana spp. following inoculation

with crude leaf extracts of CLV-infected N. benthamiana or

CMD-infected cassava

Extraction media Nicotiana species Post-•inoculation days

Either one inter- 6-7 8-9 12-16 18-21 25-30 changeable

1. Distilled water N. benthamiana nvs s c, sr c,sr,d ac,sr,d

2. 0.01 - 0.06M N. clevelandii " nvs s c,sr c,sr,d sodium phosphate pH 7.0 - 7.7

3. 0.01 - 0.1M N. hybrida n it ii ti II sodium citrate N. glutinosa it tt it tt tt pH 7.0 - 7.5 N. debneyi it it nvs nvs nvs

4. 0.1M borate N. tabacum var. ii it tt it it buffer pH Xanthi nc. 6.5 - 6.8

Symptom codes ac - advance chlorosis c - chlorosis d - dwarfing nvs - no visible symptom s - systemic top leaves sr - systemic rugosity - 19 -

(i) Mechanical inoculation

Young CMD-infected cassava leaves were ground (de Uzcategui and

Lastra, 1978) in either distilled water, 0.1M to 0.06M sodium phosphate pH 7.5, 0.1M sodium citrate pH 7.1 or 0.1M sodium borate pH 6.5 to 6.8 at a 1:2 w/v ratio of tissue to buffer (Bock ej: al., 1978b). The extracted sap was clarified by centrifuging at 5,000 rpm (MSE 21 centrifuge) for ten minutes at 4°C. The crude virus supernatant was inoculated onto leaves of test seedlings (Table 1) dusted previously with 500 mesh carborundum using sterile cotton buds or cheese-cloth pads. Some of the test plants were predarkened overnight for a period of 16 - 18 hours before inoculation with the infected extracts. The inoculated leaves were then rinsed briefly with tap water and maintained under the same glasshouse conditions as the cassava plants. All mechanical inoculations were routinely carried out in this manner.

Some Nicotiana species, N. benthamiana, N. clevelandii, N. tabacum var. Xanthi nc, N. hybrida and N. glutinosa when inoculated with buffered extracts prepared from the youngest cassava leaves exhibited the characteristic symptoms of CMD. The inoculated leaves of some Nicotiana species had a scattered chlorosis 8 to 12 days post-inoculation (Fig. 4) which was also apparent in newly emergent tobacco leaves (Fig. 5). There- after, systemically infected leaves become crinkled and mottled and the whole plant showed a characteristic rugosity, dwarfing and severe chlorosis,

20 days after inoculation especially on N. benthamiana (Fig. 5(i)). The

severity of elicited symptoms on test plants differed with the Nicotiana

species. The responses of N. benthamiana and N. clevelandii to infection were most recognisable while N. glutinosa showed only mild or diffuse

symptoms (Table 2). Predarkened test plants subsequently inoculated with

infectious CLV sap did not react any differently to unpredarkened plants

and disease development was synchroneous in both particularly N. benthamiana - 20 -

Neither N. tabacum var. Xanthi nc nor N. debneyi gave any symptoms (Fig. 6 and Table 2). The latter varieties did not give any reaction to cassava leaf sap, CLV passaged through N. benthamiana leaves or CLV purified from two tobacco varieties (Fig. 6 and Table 2).

It should be noted that CLV was routinely extracted from CMD infected plants after transmission onto N. benthamiana plants.

3.3. Transmission of CMD/CLV to healthy cassava plants

Attempts were made to transmit CMD from infected cassava plants and

its associated virus CLV from N. benthamiana crude sap, and partially or highly purified preparations from the same source to healthy seed derived

cassava by several methods of mechanical inoculation. Also, different methods of grafting infected scions unto healthy root stocks of cassava plants were investigated (Smith, 1980, Table 3).

No attempts at insect transmission of CMD were made because the

department was not licensed to carry such transmission by insects.

CMD from infected cassava was successfully transmitted to

N. benthamiana by standard methods of mechanical inoculation using either

distilled water or extraction buffer as in Table 2. Symptoms on N.

benthamiana developed 10 - 12 days as scattered chlorosis on the inoculated

leaves and similar chlorosis appearing later on the systemically infected

leaves. Rugosity and dwarfing of the whole plant with severe chlorosis

were evident 20 days after inoculation (Figs. 5 and 6).

Transmission of CMD from infected cassava (LG^N) to healthy cassava

(30395) was achieved by bud-grafting with the typical symptoms of CMD appeari

between 15-20 days after union (Fig. 7 and Table 3-3). Vein banding of

the newly infected leaves started from the base of the mid rib of young

apical leaves and progressed unto the leaf laminae, accompanied by a general

mottling of the leaves. Characteristic leaf chlorosis, typical of CMD,

progressed on to the new and old leaves of infected cassava plants (Figs.l Table 3 The effects of various techniques and additives on the transmission of CMD/CLV to healthy cassava plants

Techniques and media Transmission buffer Period of Observations* Conclusions observation

Standard mechanical inoculation 1. (a) Infected cassava leaves (i) 0.06M Sodium 60 days No typical CMD CMD not symptoms on transmitted (LGjN, LG2N & LG3N) homogenized phosphate pH 7.7. healthy cassava in buffer (1:2 w/v) and sap clarified at 5,000 rpm. for 5 mins. at 4°C. (ii) 0.06M Sodium 60 days Healthy cassava leaves (30211, 1977) phosphate/0.01M dusted with 500 mesh carborundum Sodium citrate were inoculated with clarified sap mixture pH 7.0. using either sterile muslin or cotton. Plants kept in an insect proof growth (iii) 0.01M Sodium 60 days room at 25 - 28°C, 70 - 80% RH, 10 - citrate pH 7.7. 12 klux light intensity supplied by white fluorescent tubes and tungsten (iv) Sterile distilled 60 days bulbs and with an 18h. photo-period. water.

(b) Clarified sap from CLV infected 0.06M Sodium phosphate 60 days N. benthamiana tissues processed as pH 7.7. in la and inoculated on healthy cassava leaves (60444, 1973)

* 30 plants were inoculated as indicated in Table 3 - la and b. Techniques and media Transmission buffer Period of Observations* Conclusions observation

2. Grafting All procedures were carried out using aseptic techniques. (a) Bridge grafting About 2 - 4 inches from the top of both None 60 days No union CMD not infected (LG^N) and healthy cassava established transmitted plants (30572, 1977 & 30555, 1977) cuts, and no disease in inverted 'V'-shapes were made. symptom was CMD infected scions were then inserted apparent on healthy into the healthy root stocks of cassava, cassava plants. bandaged and sealed with black sellotape.

(b) Tongue grafting Infected scions (LG^N) were cut into None 60 days No union 11 tongue shapes. The root stocks (Isunikakiyan established and no 1975, & 30211, 1977) were also cut in shapes typical CMD symptom to accommodate scions. The two were bandaged development on healthy and sealed with black sellotape. cassava plants Techniques and media Transmission buffer Period of Observations* Conclusions observation

3. Bud-grafting Internodal buds from both infected None 60 days Mosaic symptoms (LGjN) and healthy (30395, 1977) cassava of CMD appeared on plants were removed with small stems the youngest leaves from their parents. The small stems of cassava root were then removed from the buds. stock 20 days of bud- Infected buds (LGjN) were put into healthy grafting. The sympsoms plants (30395, 1977) at the positions were typical of those where similar buds had been removed. The found on any CMD buds were then placed four nodes below infected cassava plant. the terminal bud. (Fig. 7 compared to Figs. 3 b & c) The infected buds were then sealed with CMD transmitted black sellotape at the top and bottom within 30 days thus exposing the grafted buds to the (Fig. 7) light. The apex of the healthy cassava plant was then removed leaving only two buds above the grafted infected bud. All exposed surfaces were then sealed with red wax or parafilm (Smith, 1980)

5 plants each were grafted using either of the techniques i nc! i cn ted i n • Table 3 - ? Techniques and media Transmission buffer Period of Observations* Conclusions observation

4. (a) Standard mechanical inoculations (i) 0.06M Sodium 60 days No CLV/CMD CLV not as in 1(a) using CLV infected phosphate pH 7.7. typical symptoms transmitted. N. benthamiana sap. (ii) 0.1M Sodium 60 days on healthy citrate pH 7.1. cassava plant.

(b) Clarified sap from 4(a) injected 0.01M Sodium 60 days No CLV or CMD M into healthy cassava (30572, 1977) phosphate pH 7.7. typical symptoms using syringe and microlitre on healthy hypodermic needle. Injections were cassava plant. made into the intercellular spaces of leaves and also via phloem tissue in healthy cassava plants.

(c) Purified CLV inoculated by stand- 0.03M Sodium 60 days No CLV or CMD ii ard mechanical inoculation techniques phosphate pH 7.7. typical symptoms as in 4(a) and also by injection of on healthy cassava leaves and stem as in 4(b). plants (30211, 1977 & 30395, 1977).

* 30 plants were inoculated as indicated in Table 3 - 4a, b, c while 6 plants were used for the experiment indicated in Table 3 - 4d Techniques and media Transmission buffer Period of Observations* Conclusions observation

(d) Budding and grafting of CLV None 60 days CLV not infected N. benthamiana tobacco transmitted. buds on healthy cassava plants (Isunikankiyan 1975 & 30555, 1977) as in 3. - 26 -

and 7). It is assumed that if union between CMD infected cassava scions and healthy cassava root stocks had been achieved by bridge or tongue grafting techniques similar CMD transmission would have occurred as seen in bud-grafting.

Cassava latent virus was not transmitted to healthy cassava and Koch's postulates were not demonstrated. The virus was, however reproducibly trans- mitted on to some Nicotiana species (Table 2) amongst which N. benthamiana wa used as a propagation and assay host. Passage of CLV through N. benthamiana for virus purification studies after mechanical inoculation reproducibly eli- cited strong symptoms using any of the extraction buffers described in Table

(i) Host range of cassava latent virus (CLV)

All mechanical inoculations were made using carborundum and 0.06M sodium phosphate pH 7.7 with the normal methods as outlined in section

3:2(i). Inoculated plants were maintained under the same growth conditions as those previously described in section 3:1(ii)•

The host range for CLV is apparently restricted to the Solanaceae and

Euphorbiaceae. No local lesion hosts for CLV have as yet been found. Table

4 shows the responses of Nicotiana species to inoculation with either crude sap from CLV infected tobacco leaves, CMD infected cassava or highly purified virus preparations from N. benthamiana as prepared in section 3:4b.

In this study N. benthamiana, N. clevelandii, N. glutinosa and

N. hybrida all showed systemic and chlorotic symptoms when inoculated with

CMD/CLV from any of the above sources. D. stramonium also showed systemic vein-banding with severe leaf curling but this was not reproducible. The following species were not susceptible : Lycopersicon esculentum, Capsicum frutescens, Physalis floridana, Amaranthus caudatus, A. caudatus var albiflor

A. lividus var. rubricaula, A. retro-flexus, A. viridis, Solanum melongena,

S. nigrum, S. luteum, Pisum sativum, Glycine max, Vicia faba, Vigna sinensis, - 27 -

Phascolus vulgaris, Leonorus sibricus, Euphorbia prunifolia, E. orienta-lis,

Beta vulgaris, Chenopodium giganteum, C. amaranticolor, C. quinoa,

C. foetidum, C. foliosum, C. album, C. hybridum, C. murale.

Table 4 Host range responses to CLV/CMD inoculations

Plant reaction days post-inoculation

Plant species 8-10 13 - 21 22 - 30 After 30 days

Solanaceae

N. benthamiana m c,m c,r,d ac,r,d

N. clevelandii -ve m c,m c,r

N. hybrida II TI m_ c ,m

N. glutinosa it it ti it

N. tabacum var tt -ve -ve -ve

Xanthi nc

N. debneyi it II TI II

Key :- ac = advanced chlorosis, c = chlorosis, d = dwarfing,

m = mottling, r = rugosity, -ve = no symptoms

3.4. (a) Cassava latent virus (CLV) purification

CLV infected N. benthamiana tissues (100 - 500 g) (Figs. 4 and 5) were either processed immediately or frozen at -20°C for later use. Several purification methods were investigated as detailed under 3.4b. The elements of the better purification methods were combined finally to produce a purification schedule to obtain CLV of sufficient purity to conduct preliminary characterization of the viral coat protein and nucleic acid.

In the preparation procedures, low speed centrifugation (LS) was carried out on a MSE 21 centrifuge at 10,000 rpm whilst high speed centrifugation - 28 -

(HS) was performed on a Beckman LI ultracentrifuge in either a type 50 rotor at 36,000 rpm or on a Ti 45 type rotor at 40,000 rpm. Purified preparations, either highly purified or analysed at different stages during a purification were assessed by negative staining in the electron microscope

(EH), scoring the number of geminate virus particles/field of view (section

3.4b, Milne, 1978), and by assay on N. benthamiana plants, where the characteristic symptoms of CLV infection (see section 3.2) where scored positive for virus containing samples, 30 days after inoculation.

3.4 (b) Purification methods and results

Purification Method Results

1. Frozen leaves (50 g) blended in a top- drive homogenizer with 100 ml of 0.1M sodium borate buffer pH 8.1 - 8.4 containing 0.001M

EDTA and 1% 2-mercaptoethanol. The homogenate was clarified with an equal volume of chloroform No virus particles were

(CHCl^) and n-butanol (but-OH) (1:1 v/v mixture) observed in the EM. and no and given 3XLS and a HS centrifugation. symptoms were detected on

Pellets suspended in 0.5 ml of 0.005M sodium N. benthamiana. borate buffer containing pH 8.1 - 8.4 Bock et al. (1978b).

2. Frozen leaves (100 g) blended in a homogenizer with 200 ml of 0.1M sodium phosphate buffer pH 7.6 containing lOmM EDTA and 0.5% thioglycollic acid. The homogenate was divided into four equal parts and given one of four different treatments as over page : (i) Homogenate clarified with 0.4M sodium chloride (NaCl) and 30% polyethylene- No virus particles observed in glycol 6000 (PEG) in 0.03M sodium phosphate the EM and no symptoms developed buffer pH 7.6. The mixture was subjected on N. benthamiana. to 2 x LS and 1 x HS centrifugation.

Pellets suspended in 0.5 ml of 0.03M sodium phosphate buffer pH 7.6 containing O.lmM EDTA.

(ii) The homogenate was clarified with an equal volume of n-but OH/CHCl^ (1:1 As in 2(i) v/v mixture) and given 3 x LS and 2 x HS spins. Pellets-suspended in 0.5 ml of

0.03M sodium phosphate buffer pH 7.6 containing O.lmM EDTA.

(iii) Homogenate clarified with One virus particle per 10 EM an equal volume of n-but OH/CHCl^ (1:1 v/v fields observed,but preparations mixture) and 0.4M NaCl and given 2 x LS did not give any symptoms on spins. Pellets suspended as in 2(i). N. benthamiana.

(iv) Homogenate not clarified but Four virus particles per EM fiel given 3 x LS and 2 x HS spins. Pellets observed,but preparations did no suspended as in 2(i) . give any symptoms on N.

benthamiana.

3. Frozen leaves (50 g) blended in homogenizer with 100 ml of 0.1M sodium phosphate pH 7.8 containing lOmM EDTA and

ImM cysteine. Homogenate clarified then

0.2M PEG added to 4% (w/v) of the supernatant.

Given 4 x LS and 1 x HS centrifugations. - 30 -

Pellets suspended in 0.5 ml of 0.1M sodium No virus particles was seen in the EM and preparations did no phosphate buffer pH 7.8 (Goodman,- 1977b). give any symptoms on JL benthamiana. 4. Frozen leaves (50 g) blended in homogenizer with 100 ml of 0.01M sodium phosphate pH 7.0 containing 0.01M sodium sulphite (Na^SO^) and 0.001M EDTA. Homogenate

clarified with n-but OH/CHCl3 (1:1 v/v As in 3 mixture) then 1% NaCl and 10% PEG (w/v) was added to the homogenate and the mixture given

5 x LS centrifugations. Pellets suspended in 0.5 ml of 0.001M sodium phosphate pH 7.0 containing 10% (w/v) PEG (Mumford, 1974).

5. Frozen tissues (150 g) crushed to a 2 to 5 virus particles per powder in liquid nitrogen then blended in EM field observed in the in homogenizer with 0.1M sodium citrate initial supernatant, 10 virus buffer pH 7.1 containing 1% (w/v) of an particles per EM field observe enzyme mixture containing in the solvent interphase.

Macerozyme R and Cellulase R^ and 0.1%

TGA. Homogenate divided into six equal parts and subjected to one of the following

treatments :

(i) Homogenate clarified with n-but

OH/CHCl^ (1:1 v/v mixture) as before, then Both phases were infectious fo

NaCl and PEG added to bring homogenate to N. benthamiana. No virus

0.4M and 8% concentrations respectively. particles were observed in the

.Clarified homogenate was then given 3 x LS and EM and no symptoms were

1 x HS spins. Pellets resuspended in 0.5 ml observed on test plants

of 0.1M sodium phosphate buffer pH 7.8 inoculated with the final

(Goodman, 1977b). purified preparation. - 31 -

(ii) Homogenate clarified as in 5(i) Supernatant had 5 virus without the NaCl and PEG stage and given particles per EM field, the

3 x LS and 2 x HS centrifugations. Pellets interphase (from 5(ii) con- suspended as in 5(i). tained 10-20 particles

respectively and produced

symptoms on N. benthamiana.

(iii) Homogenate clarified as in 5(ii) Supernatant had 5 virus and given 1 x L$ and 1 x HS centrifugations. particles per EM field. No

Pellets suspended in equal amounts of 1% pronase virus was present in the int<

(w/v) and given 1 x LS centrifugation. phase. Neither preparation

produced any symptoms on _N.

benthamiana plants.

(iv) Homogenate clarified with an Between 20 to 50 virus parti- equal volume of n-but OH/CHCl^ (1:1 v/v) and cles per EM field were given 4 x LS and 3 x HS spins. Pellets observed in the final super- resuspended in 0.5 ml of 0.01M sodium natant and solvent interphase phosphate pH 7.1. Both were infectious for

N. benthamiana plants.

(v) Homogenate clarified as in 5(iv) Between 100 to 200 virus but given 7 x LS and 3 x LS centrifugations. particles per EM field, were

Pellets resuspended as in 5(iv). detected in both phases as

above. Both phases were

infectious for N.benthamiana, - 32 -

(vi) Homogenate clarified as in 5(iv) Between 200 - 300 virus particles but given 9 x LS and 3 x HS centrifugations. per EM field were observed and

Pellets resuspended as in 5(iv) . were infectious for

benthamiana.

6. Purification of CLV from N. benthamiana tissue infected with

two isolates of the virus derived from (A) 631004 MPIBN, and (B)

LGjN infected (CMD) cassava plants for preliminary chemical

characterization and host range studies of the virus

Initially, leaf dips of the infected tobacco leaves were examined in the EM to confirm the presence of the geminate CLV particles at a high concentration before purification commenced. Both CLV strains were at a high concentration in leaves as seen in Fig. 14, representing the 631004 MPIBN, isolate. Characteristic geminate particles could be readily seen (Figs.

14 - 16).

Sample A

(1) Infected tobacco tissue (350 g) was blended three times repeatedly in a homogenizer with 700 ml of 0.1M sodium citrate buffer pH 7.0 (SCI) containing ImM EDTA and 1% TGA. The homogenates were pooled and clarified with an equal volume of n-but OH/CHCl^ (1:1 v/v mixture) at 4°C for ten minutes and centrifuged at 5,000 rpm. for 15 minutes at 4°C. The super- natant was retained whilst the interphase was re-extracted three times with homogenization carried out as above and in SCI with similar low speed centrifugation.

(2) The supernatants were pooled and centrifuged at 36,000 rpm (Beckman

L2-65B) for 2 hours at 4°C in a Ti 45 rotor. The pellets were pooled resus-r. pended and kept overnight at 4°C in small amounts(0.5ml) of 5mM sodium citrate - 33 -

pH 7.0 containing 1 mM EDTA (SC2) .

(3) The resuspended pellets were suspended in a further amount of the SC2 to one-seventh the starting tissues (v/w) ratio. The suspension was centrifuged at 5,000 rpm. for 15 minutes at 4°C as above. The resultant pellets were washed four times with 4 ml of SC2 and subjected to further centrifugation.

(4) All the supernatants from step 3 were pooled and centrifuged at

36,000 rpm. for 2 hours at 4°C. The supernatants were discarded whilst the pellets were resuspended overnight at 4°C in 1 ml of SC2 as before.

(5) The resuspended pellets were finally suspended in a final volume of

SC2 one-tenth the starting tissue (v/w), and centrifuged at 15,000 rpm for

20 minutes at 4°C. Step 4 was repeated to give the final purified virus suspension.

Sample B

Infected tissue (150 g) was processed essentially in the same fashion as for sample A.

3.5 (a) Infectivity of CLV purified preparations and purification

results

The infectivity of the purified virus preparation was higher than that of crude sap and lasted up to 40 days, at 4°C as compared to 10 days for crude sap. Typical symptoms on N. benthamiana developed after only

7-9 days following inoculation with purified virus suspensions. Symptom descriptions are as given in section 3.2. A minimum of ten virus particles/

EM field elicited infection on N. benthamiana. - 34 -

Methods 1 to 4 (section 3.4b) yielded very low amounts of virus particularly so when the first supernatant after clarification and /or emulsification with n-but OH/CHCl^ mixture was used directly for further purification. The enzyme-assisted approach initially used by Takanami and Kubo (1979) for purifying tobacco necrotic dwarf virus (TNDV) and potato leaf roll virus (PLRV),two phloem-limited viruses, was modified for purification of CLV infected tobacco and gave better results particularly with regard to digesting leaf pectin and other plant materials that often contaminated virus pellets. Method 3.4b using sodium citrate containing EDTA and TGA without enzyme also gave good CLV yields but preparations were often associated with considerable amount of leaf pectin (Figs. 14 and 15). In all of the purification methods, it was consistently observed that many virus particles were found in discarded fractions rather than in the final preparations. Hence the routine use of several alternate high and particularly low speed centrifugation steps in an effort to release virus from the pellets.

3.5 Electron Microscopy

(b) Negative staining of cassava latent virus (CLV)

Negative staining and leaf dip techniques were used to confirm the presence or absence of CLV in young infected (CMD) cassava leaves, systemically infected leaves of N. benthamiana and partially or highly purified CLV preparations. Cut surfaces of infected leaves were dipped into a 2% sodium-

PTA (0.005%), pH 6.6, BSA mixture (1:1 v/v), on glass slides for several seconds. Droplet surfaces were then touched on to formvar (0.6%) coated electron microscope copper grids of 200 mesh and dried on filter paper in petri dishes. Similarly, infected leaves were ground in mortar with distilled water. Virus preparations from infected N. benthamiana either as partially or highly purified preparations and cassava crude sap (healthy or - 35 -

infectcd) were mixed with an equnl volume of 2% sodium-PTA and sprayed with an

Aerograph Super 63 air brush on to 200 mesh carbon/necoloidine coated copper grids and examined under the electron microscope. Electron micro- graphs were taken with a Siemens 102 electron microscope at 40,000 or

60,000 magnification (Figs. 14 - 16). Routine electron microscope examinations of all purified preparations enabled a completion of the results in the section on virus purification (3.4).

(c) Virus particles

Some 200 (Fig. 14) and 50 virus particles (Fig. 15) per EM field were observed from Samples A and B respectively. Both preparations were infectious for N. benthamiana. Virus preparations from N. benthamiana, partially or highly purified fresh virus, were found to contain numerous geminate, isometrically shaped virus particles with occasional single particles when the 'Aerograph Super 63' air brush technique and Siemens

102 electron microscope as used at Rothamsted Experimental Station, Harpenden.

Similar geminate forms of the virus were observed on formvar/carbon coated grids examined in the Philips 301 electron microscope at Imperial College,

London. Paired particles were of 30 x 19 nm dimensions and distinctive in their 'twinned' geminate appearance. The dimensions quoted above were from the measurements of 200 - 500 virus particles (paired or single). Single particles were found to be 19 nm in diameter. Small thread-like material seen in Figs. 14 and 15 may be fibrous proteins material from tobacco leaves or pectin. Pectin was particularly troublesome during resuspension of virus pellets, but the enzyme assisted purification method, partially alleviated this problem. - 36 -

3.6 Preliminary chemical characterization of cassava latent virus

(a) Virus concentration and purity

Samples A and B purified as in section 3.4 were examined in a

Beckman recording spectrophotometer, prior to further characterization, to determine their purity. Sample A had an 260/280 nm absorption ratio of 1.23, whilst B had a ratio of 1.38. Concentration of the virus was calculated

assuming an of 7.7, (BGMV, Goodman, 1978) for CLV. The yields 260 calculated for example as in Fig. 17 were for CLV sample A = 0.35 mg/ml

and for sample B =0.132 mg/ml.

(b) Iscflation of virus coat protein and electrophoresis to

determine its molecular weight

Virus coat protein was prepared for electrophoresis by boiling for

2 minutes (lcxrc) with one part of purified virus to two parts of double

strength disruption buffer (0.02M NaPO^, pH 7.2) containing 2%, 2-mercapto-

ethanol, 2% sodium dodecyl sulphate (SDS) and 1M sucrose (Maizel, 1971).

After cooling, 50 yl of the virus protein sample was layered on 10%

polyacrylamide cylindrical gels (each 65 mm long) on to which 10 pi bromo-

phenol blue (0.12%) had previously been layered containing a mixture of

proteins of known molecular weight. Similar gels were loaded with either

the protein mixture alone or the virus coat protein alone. The gels were

electrophoresed £*t 4mA/gel for the first 30 minutes and 9mA/gel for a further

4 to 5 hours until the bromophenol blue marker due had just left the gels

into the lower buffer compartment in a Shandon tank. The running buffer

used contained half strength stock gel buffer (Nal^PO^.21^=8.82g, Na2HP0^=

19.4g. and SDS = 2g. all per litre in distilled water and at pH 7.2

adjusted with ortho-phosphoric acid). The proteins in the mixture of known

molecular weight were bovine serum albumin (67,000d)., y-globulin-heavy - 37 -

chain (55,000d), ovalbumin (47,000d), y-globulin-light chain (23,500d) and cytochrome fcf (12,384d). The gels were stained after electrophoresis in 0.25% Coomassie blue in a water : ethylalcohol : acetic acid (5:5:1) mixture for 24 hours. Gels were then destained for 2-3 days in methanol :

7% acetic acid (1:1 v/v) and stored in 7% acetic acid for densitometer scanning in a Gilford spectrophotometer 240. Protein bands were measured on the traces and molecular weights estimated by calculating the Rf of the standard proteins from the gels and plotting the Rf values against the logarithm of their molecular weights. The best line of fit was drawn to the points and from this graph, the Rf of the viral protein was traced back to calculate its molecular weight (Table 5).

Table 5 Average Rf values for various marker proteins run in denaturing

gels as compared to cytochrome 'c' and their molecular weights

Ratio Combination A/X B/X C/X D/X X/X

Rf ratio to cytochrome 0.24 0.32 0.42 0.76 1 .0 'c'

Mol. weight (d) 67,000 55,000 47,000 23,500 12,384

Log. mol. wt. 4.826 4.740 4.672 4.371 4.093

CLV-isolates Average Rf Log. mo 1. wt. Molecular wt. (d) ratio to cytochrome 'c'

Sample A 0.52 4.55 35,481

B 0.49 4.58 38,018

Rf values were calculated from densitometer traces as outlined in section 3.5b.

Abbreviations

A = Bovine serum albumin B = y-globulin, heavy chain C = Ovalbumin

D = y-globulin, light chain X = Cytochrome 'c1 - 38 -

(c) Molecular weight of protein coat of CLV

From the molecular weight calibration curve shown in Fig. 18, constructed from the results tabulated in Table 5, it is clear that insufficient replicates of the gels were made, since the calculated molecular weight of the coat protein of both CLV-A and CLV-B differ markedly from those previously determined by other workers (Harrison et^ al. 1977). The molecular weight of CLV-A was determined as approximately 35,500d, and 38,000d for CLV-B. Similar experiments carried out in an identical fashion with other purified virus preparations estimated the molecular weight of MSV and

CLV protein to be between 28,000d and 32,5000d, respectively (Bock et al. ,

1977), illustrating further that the results of the molecular weight estimations in this study must be re-tested. Individual Rf values for the different proteins are not shown, but these varied widely even between identical gels.

(d) Fractionation of CLV - DNA on alkaline sucrose gradients and

neutral alkaline agarose electrophoresis

One part of purified virus was added to one part of 0.6M sodium hydroxide and left at 20°C for 20 minutes, 0.2 ml of the mixture was layered on alkaline sucrose gradients (12 mis) consisting of a 10 - 40% sucrose gradient in 0.3M NaOH, 0.7M NaCl and ImM EDTA. The gradients were then centrifuged in an SW41 rotor for 16 hours at 35,000 rpm at 20°C and then fractionated on an ISCO fractionator at 254 nm connected to a pen recorder and a peristaltic pump. Fractions (6 ml) covering the central ultraviolet absorbing peaks (Fig. 19) were collected, pooled and dialysed extensively against distilled water at 4°C over a 48 hours period. The dialysis tube was dried and sprinkled with a small amount of Sephadex G200 to remove residual water until only approximately 2 ml of viral DNA remained. The solution was transferred into a 10 ml round bottom flask and lyophilized on - 39 -

a Edwards Modulyo freeze dryer (code 10-F023-05 000) Sussex, England).

When 20 ml of the gradient was fractionated, the gradient showed a

fairly broad peak of ultraviolet absorbing material (Fig. 19) which was

later shown to consist mainly of ssDNA had a molecular weight of approximately

0.77 x 10 d, and contained approximately 2,450 nucleotides. The DNA was

analysed on 2% agarose gels under neutral conditions, and compared to heat-

denatured single-stranded DNA fragments of cauliflower mosaic virus (CaMV)

for molecular weight.estimations after ethidium bromide staining (Hull and

Shepherd, 1976).

4.1(a) Ultrastructural studies of cassava latent virus in tobacco

(N. benthamiana) tissue

A comparison of both healthy and diseased leaves and stems of N. benth-

amiana plants after inoculation with CLV was made following harvest at differ-

ent times after inoculation (5,15 and 21 days). Both gross and specific alter-

ations in plant leaf and organelles as viewed in thin section were noted.

Tissues infected for 15 days yielded more information on the effects of CLV on

infected cell ultrastructure than either leaves or stems infected for 21 days.

In tissue, infected for 21 days cells were so structurally disorganized that

any specific ultrastructural alterations were often impossible to define

and the tissue itself was difficult to process for routine observation of

thin sections.

(a) Embedding, fixing and sectioning

Both leaf and stem tissue were cut into pieces under fixative and

fixed for 3-4 hours in 2.5% glutaraldehyde in 0.1M sodium cacodylate

buffer (pH 7.0). The tissue slices were then washed in the same buffer

(0.1M sodium cacodylate pH 7.0) and post-fixed for one hour in cold 1%

osmium.tetroxide in cacodylate. Fixed material was then dehydrated stepwise - AO -

through an ethanol series (10 - 100%). The tissues in small embedding plastic cups were then flat embedded stepwise in 100% ethanol : 100% epoxy propane (1:1 v/v); 100% epoxy propane : 100% epon (resin) (1:1 v/v), and finally in pure epoxy resin. Sections gold/silver in colour (60 - 90 ym) , from trimmed blocks were cut transversely with a diamond/glass knife on

Reichert Austria automatic microtome. Sections were then mounted on copper grids (100 mesh) coated with 0.6% formvar and double stained with 2% aqueous uranyl acetate and lead citrate for 5 and 15 minutes respectively. The

stains were then washed away with glass distilled water several times and the grids dried with filter paper at the edges before examination in a Philips

301 transmission electron microscope operating at 80 KV.

4.1(b) Ultrastructural changes in N. benthamiana parenchyma cells following

infection with cassava latent virus (CLV)

Unless otherwise stated, electron micrographs were produced for both infected and comparable healthy control tissue 15 days after inoculation, when the characteristic symptoms of CLV infection (Figs. 4 and 5) were

pronounced on inoculated leaves. In infected tissue, the major cytological modifications occurred in the cells of the vascular bundles of petioles and

main veins, particularly in the nuclei of phloem parenchyma cells. Routine

comparison to normal healthy tissue was carried out when any abnormalities

in infected tissue were recorded.

(c) Electron microscopy observations.: Control (healthy) N.

benthamiana leaf mesophyll and phloem parenchyma cells

Both cells types possessed a normal nucleus, and were bounded by

the usual double membrane envelope, containing pores (Fig. 21). The nucleolus

in parenchyma cells was composed of granular and fibrillar regions but the - 41 -

two regions were not sharply defined (Fig. 21) indeed the fibrillar regions were almost impossible to define. Nucleoli contained chromatin, variable in density (Fig. 21). Chloroplasts when present in healthy parenchymatous

tissue were often much smaller than those found in mature mesophyll cells

(Figs. 20 and 26) and often resembled proplastids in appearance.

CLV infected N. benthamiana phloem parenchyma cells

The most recognisable features of infected cells were the alterations

in the structure of nuclei, because of the unusual segregation of the chromatin

in the nucleoplasm (Figs. 22 - 24 and Figs. 26 - 28), increased numbers of

recognisable nucleoli and the appearance of occasional fibrillar rings of

amorphous or a finely fibrillated matrix material (Figs. 26 - 28). These

alterations in the nuclei of geminivirus-infected cells are now thought to

be a common feature of infected cells, and a diagnostic test (Kim et al.,

1978; Russo et. al., 1980). The appearance of fibrillar regions of segregated

nucleoli, (Figs. 22 - 24) and the separation of granular chromatin (Figs.

24 and 25) were common in infected cells. Fibrillar rings were also detect-

able in infected parenchyma cells in this study (Figs. 22, 26 - 28). These

rings were however quite rare and often differed in size and number (Figs.

22 and 26).

The fibrillar rings observed in N. benthamiana closely resembled

those observed by Kim et al. (1979) in EUMV infected Euphorbia heterophylla

The segregation and distribution of the chromatin in the cell shown

in Fig. 26, illustrates another characteristic phenomena observed in the

nuclei of infected cells, there, the chromatin has become peripherally

arranged around a still intact nuclear membrane, and lies also in an axial

fashion across the cell (Fig. 26). This cell also contained two fibrillar

rings (Fig. 26). The matrices of the fibrillar rings were often homogenously

electron-dense, and readily identifiable (Figs. 27 and 28), but in a larger - 42 -

example "(Fig. 22) a clear area was visible. Characteristic alterations to

the chloroplasts of mesophyll cells were routinely observed some 20 days

after infection (Figs. 20 and 29). The alterations were reminiscent of those

seen in systemically infected tobacco leaf cells after tobacco mosaic virus

infection (Matthews, 1970). Chloroplasts were generally swollen, suffering from membrane disorganisation, grana loss, and large starch grain deposition as

a direct result of restricted transport of foodstuffs throughout the plant

and hormonal alterations. Both effects are presumably caused by the replication

of the virus in phloem-associated parenchyma cells.

5.1 Freeing cassava of CMD by meristem tip culture and regeneration of

whole plants

Cassava mosaic disease (CMD), was successfully eliminated from

infected cassava plants by meristem tip culture with or without prior heat

treatment of the infected plants. Whole plant regeneration was achieved by

careful adjustment of the hormone levels in the growth medium (Table 7C),

or by dipping regenerated plants showing only shoot development plus

associated callus into a 'hormone rooting powder1 (Boots hormone rooting

powder, BHRP) which contained a-naphthylacetic acid, 4-(indol-3-yl) butyric

acid, thiram and an inert diluent at percentage concentrations of 0.02%,

0.2%, 6.0% and 93.78% respectively (Boots' Company Limited, Nottingham,

personal communication). The Murashige and Skoog (MS) culture medium

constituents were prepared as indicated(Gamborg and Wetter, 1975),and inorganic

stock solutions were also prepared in a similar manner (see Tables 6 and 7).

Before the addition of the Difco Bacto-Agar, the pH of the MS medium was

adjusted to 5.8 with 0.2N KOH or 0.2M HC1 (Table 6A). The agar was allowed

to dissolve in the medium on a hot plate stirrer. Using a Gilson automatic

micro-pipette, MS medium was distributed into a series of pyrex test tubes

(25 x 100 cm), maintained in a hot water bath and hormonal stock solutions - 43 -

were added (Table 6D) to give a final volume of 2.5 ml. The tubes containing culture medium were capped with non-absorbent cotton wool and aluminium foil and autoclaved at 15 psi for 20 minutes and at 120°C. Tubes were left to cool before transference to 4°C for storage. Tables 7A and B illustrate the range of hormones used and their final concentrations in the above solidified

MS media which were initially screened for whole plant regeneration of healthy meristerns. The hormones used in liquid MS media at the final concentrations shown were not chosen empirically but were selected from a range of hormones used in similar experiments on cassava meristem regeneration as indicated previously (Fereol, 1978; Gamborg and Wetter, 1975; Gamborg and Kartha,

1976; Kaiser and Teemba, 1979; Kartha, 1975c; Kartha et al., 1974a, b;

Kartha and Gamborg., 1978; Nair _et al., 1979; Quak, 1972; Tilquin, 1978).

Table 6 Media requirements for meristem tissue culture

(A) Murashige and Skoog basal medium (prepared fresh in double distilled

water)

AR grade chemicals mg/litre

NH4.N03 1650

KNO3 1 900

MgS04.7H20 370

Ferric EDTA

(i) Na2 EDTA 37.3

(ii) FeSO..7H_0 27.8 4 2 Sucrose 30 x 103

Difco Bacto agar 8 x 103

CaCl2.2H20 2.9 ml stock from B2 (over)

Micro-nutrients 1.0ml " N B ( " ) KI 1.0 ml " M B1 ( ") Vitamin supplement 1.0 ml " II C ( " )

pH adjusted to 5.8 with either 0.2N KOH or 0.2N HC1 before adding - 44 -

agar. Thereafter the agar was dissolved on a hot plate stirrer at 100 C, and held at 49°C.

(B) Micro nutrient stock (- 20 C storage) : (AR grade in double distilled

water)

mg/100 ml

H3BO3 620 .

MnSO,.4Ho0 2230 4 2

ZnSO..7Ho0 860 4 I Na_MoO,.2H.0 25 2 4 2 CuSO,.5H.0 2.5 4 2 2.5 CoCl2.6H^0

(Bl) KI 75

(B2) CaCl2.2H20 15 x 10'

(C) Vitamin supplement stock mixture (- 20 C storage)

mg/100 ml

Nicotinic acid 100

Thiamine HC1 1000

Pyridoxine HC1 100

Myo-inositol 10,000

(D) MS meristem tip culture media : (All volumes in yl)

Medium MS + Agar Liquid MS Hormonal stocks (from Table 4)

Index 0.8% (A) NAA GA3 BA Kinetin

El 2300 98 1 .0 1.0 100 11 E2 it 188 It - 10 11 E3 it 193 II - 5

E4 n J 97 n M - J

E5 11 98 H H 100 — - 45 -

(D) contd

E6 2300 188 .0 1.0 10 5 E7 193

E8 197 1

Abbreviations

NAA = Naphathalene acetic acid GA^ - Gibberellic acid

BA = 6-Benzyladenine

Table 7 Plant growth substances used for regeneration of apical meris tems

(A) Hormonal stock solutions

Hormonal stock codes Composition and concentration

1. NAA at 10.0 yM plus 0.1 yM GA,

2. 10.0 yM minus 0.1 yM GA,

3. 1.0 yM plus 0.1 yM GA,

4. 1.0 yM minus 0.1 yM GA,

5. 0.1 yM plus 0.1 yM GA,

6. 0.1 yM minus 0.1 yM GA,

7. BA at 10.0, 1.0, 0.5 and 0.1 yM

8. Kinetin at 10.0, 1.0, 0.5 and 0.1 yM

(B) Hormone variations tested for whole plant regeneration of cassava

meristem as in 6D

Micro molar concentrations (yM)

Media index NAA GA3 BA Kinetin

El 0.1 0.1 0 10.0

E2 0.1 0.1 0 1.0

E3 0.1 0.1 0 0.5

E4 0.1 0.1 0 0.1

E5 0.1 0.1 10.0 0 - 46 -

(B) contd

E6 0.1 0.1 1.0 0

E7 0.1 0.1 0.5 0

E8 0.1 0.1 0.1 0

(C) Modified MS media for root induction (All volumes in yl)

Media index MS + Agar Liquid MS NAA GA3 Ba Kinetin Ze 2iP 0.8%

N9 2300 69 10 1 5 5 10 100

N10 2300 85 10 1 5 5 10 84

N1 1 2300 185 10

Abbreviations

Ze = Zeatin;

2iP = 6( y- y-dimethylallyl-(amino) purine

5.2 Effects of different media on regeneration of meristems into whole

plants

All the E-media (Tables 6D, 7A and B) were suitable for cassava meristem tip culture and after the eight days of culture, meristems were healthy and green (Fig. 8). Twelve days after culture, basal callus formation was clear and thereafter increased with shoot and/or root formation

(Fig. 9) in some cases, twenty to twenty-eight days after culture (Fig. 10).

The regeneration of healthy meristems was optimal in the E5 to E8 cultures containing BA concentrations of between 0.1 and 10.0 yM and NAA plus GA^ both at 0.1 yM concentrations without kinetin (Tables 7B and 9). Media El and E2 regenerated meristems into small shoots with a large amount of callus and often the callus overgrew the meristems (Fig. 9).. Few plantlets were obtained with poor shoot development and little callus in the E3 and E4 - 47 -

culture media. Media E5 and E6 produced good shoots and large amount of callus but few developed roots. Media E7 and E8 produced better plantlets with stronger shoots and occasionally one single long root (Fig. lib). A few meristems in these later media produced shoots with a large amount of callus only. Roots were induced in these cultures by either dipping the basal callus into hormone rooting powder or by transferring the cultured meristems into fresh MS media containing 2iP and Ze in a 0.1 to 1.0 yM concentration range omitting GA^ and IAA (Table 7C and Figs. 11 c and d) .

Meristem tips between 0.5 and 2.0 mm in size with one or two chlorophyllous leaf primordia regenerated reproducibly.

Table 8 Effect of media on regeneration of cassava meristems at

different stages of growth

State of meristems Medium CMD Healthy Total percentage infected cassava success cassava

3 2 5 Regenerated plant E3 7.1% 53 17 70

4 1 5 with callus (PC) E4 7.77. 48 17 65

31 5 36 E5 50.0% 56 16 72

28 4 32 E6 44.4% 50 22 72

2 2 Regenerated plants E5 — 3.61 56 56

8 4 12 with roots (PR) E6 16.7% 50 22 72

12 13 25 (plantlets) E7 42.4% 37 22 59

13 8 22 E8 40.7% 36 18 54

Total No. regenerated s= percentage Total No. cultured success - 48 -

However, tips of the same size dissected to the meristeraatic dome did not regenerate at all and did not produce any callus either. They were graded as no growth in Table 9. Initially meristems from both healthy

(variety 30211) and infected clones : (varieties 631004, Isunik, LGjN, LG^N and LG^N) were tested on the E-media range in order to establish if any differential existed with regard to the cultural capacities and regeneration potential. Later on when other media were tested, particularly with regard to initiating roots from partially regenerated meristems with a large amount of basal callus, only the infected clone (LGjN) was investigated (Table 10).

These initial comparisons did however allow for a selection of media from the E media range which would give reproducible results later on in studies using cassava plants subj ected to heat therapy to eradicate CMD. The media for meristem tip regeneration were those of Murashige and Skoog (1962),

Murashige (1974) as modified by Gamborg and Wetter (1975), Kartha and

Gamborg (1975b) and Nair et_ al. (1978). They included macro and micro elements, vitamins and 3% sucrose as in Table 6.

All operations were carried out in a Pathfinder sterile air flow cabinet under aspetic conditions. Meristem tips (0.5 to 2.0 mm) were asceptically dissected from both CMD infected and healthy cassava plants.

Meristems with one or two chlorophyllous leaf primordia (Quak, 1980, personal communication) were normally selected for culture and inserted into culture media. The tubes were then sealed with fresh cotton wool and aluminium foil and kept in growth cabinet at 25 - 27°C temperature, 70 - 80% relative humidity and 18 hours photo-period of 4 klux illumination supplied by warm white fluorescent tubes.

With the individual media El to E8, the percentage success of regenerating plantlets was as shown in Table 9. - 49 -

Table 9 Percentage success of plantlet regeneration in media El to

E8 (healthy and CMD infected cassava)

(A)

Medium % success

Healthy cassava clones El 0

(30211 seed derived), E2 0 and heat-treated LG^N - E3 28.6 originally infected with CMD E4 14.3

E5 0

E6 44.4

E7 36.4

E8 44.4

(B)

Medium % success

Infected cassava clones El 2.6

(631004 MP IBN, Isunik-IITAIBN, E2 0

LGjN, LG2N and LG^N) E3 12.0

E4 18.2

E5 3.6

E6 16.0

E7 32.4

E8 36.1 - 50 -

Table 10 Comparison of regeneration capacities of meristems derived

from CMD infected plants subjected to heat-therapy and those

grown under normal conditions

(A) Meristems from heat-treated infected plants (clone LG^N) at 20 to 60

days of culture

Medium E5 E6 E7 E8 Total No. of State of meristems cultures

Regenerated plants with 5 4 11 7 27 callus (PC)

Regenerated plants with root(s)

- 4 4 4 12 only (PR)

Regenerated callus only (C) 3 1 - - 4

No growth (-ve) - - • - - -

Total No. cultured 8 9 15 11 43

(B)

Regeneration of plants from meristems derived from heat-treated infected

plants after transfer on to fresh rooting medium or after dipping in

rooting powder

Experiments were carried out with meristems developing callus (C) and

regenerated plantlets with callus (PC) from varieties LGjN and LG^N as in

Table 1 OA, plantlets were cultured for a further 30 days after 4 to 28 weeks

of original culture on E5 to E8 media. - 51 -

Table 10 (B) contd....

Medium N9 N10 N1 I Rooting Total No. powder State of meristems plus E cultured

PC 3 2 5 3 13

PR 6 3 5 15 29

C 4 - - 1 5

-Ve 2 - 1 - 3

TRP 6 3 5 15 29

Total No, cultured 15 5 11 19 50

(C) Meristems from infected plants (631004 MPIBN, Isunik--IITAIBN, and LGN clones

1, 2 and 3) 4 to 30 weeks of culture

Medium Total No...... TT ^ E6 E7 E8 State of meristems cultured

PC 31 28 12 9 80

PR 2 8 12 13 35

C 16 7 7 5 35

-Ve 7 7 6 9 29

TRP 2 8 12 13 35

Total cultured 56 50 37 36 179

KEY :

PC = regenerated plant with callus PR = regenerated plant with root(s) only

C = regenerated callus only -Ve = no growth

TRP = transplanted plantlets - 52 -

Experiments were carried out as in Table 10B with C and PC meristem materials taken from 631004 MPIBN, Isunik-IITAIBN, LGN clones 1, 2 and 3 as

in Table 10C. Plantlets were cultured for 30 days after 4 to 28 weeks of original culture in any of the E-media.

Table 10 (D) Regeneration of plants derived from infected plants after

transfer unto fresh rooting medium or dipping in rooting

compound

Medium N9 N10 Nil Rooting Total No. powder State of medium plus 'E1 cul tured

PC 2 1 3 2 8

PR 1 - - 1 1 12

C 2 - - 2 4

-Ve - 2 1 2 5

TRP 1 - - 11 12

Total cultured 5 3 4 17 29

5.3 (i) Heat-•therapy of infected cassava plants prior to meristem

culture

Infected cassava clone Lagos/Agege (LG^N) plants were placed in

a growth chamber at 26°C with an 18 hour photoperiod and a light intensity

of 4 to 5 klux at the outset of the experiment. Subsequently, the air

temperature was gradually raised to a range of between 35 to 38°C over a ten

day period and the plants were maintained at this temperature for a further

thirty days (Walkey and Cooper, 1975). Thereafter, meristem tips were

aseptically dissected out and cultued on modified MS media. At the same time

of meristem tip removal, young emergent shoots on the heat treated plant

were harvested and screened for presence of CLV in N. benthamiana as in - 53 -

section 3.3.

5.3 (ii) Transplantation of regenerated plants into soil and

"hardening off"

After regeneration, plantlets were transferred into sterile garden soil (with a high humus content) or John Innes No. 2 compost in four inch plastic pots and covered with beakers or polythene bags to maintain high humidity. Plantlets were then gradually acclimatised to the growth cabinet conditions by gradual removal of covering. Plantlets were kept in the growth cabinet for four to six weeks and watered weekly with Hoagland's solution (Gamborg and Wetter, 1975; Kartha and Gamborg, 1975b).

Table 7 Constituents of a balanced salt solution for watering regenerated

plantlets

Hoagland's solution (molarity)

(a) Macronutrients

3 Ca(N03)2.4H20 3.98 x 10~

3 MgS04.7H20 2.11 x 1C"

3 KN03 6.53 x l(f

4 NH,HoP0, 9.67 x 1 (f 4 2 4

3 Na2EDTA 0.10 x 10~

3 FeSO,.5Ho0 4.13 x 10" 4 2

(b) Micronutrients(stock solution)

1 H3B03 . 0.05 x 10"

1 MnSO,.Ho0 0.02 x lO" 4 2

3 CuSO, .5Ho0 5.38 x 1 o"" 4 2

3 ZnSO..7Ho0 7.66 x 10~ 4 2. -3 (NH4)6Mo704.4H20 8.09 x 10

H f^onr"i 5.in X ID"2 - 54 -

The micronutrient stock solution (0.1 ml) was mixed with one litre of the macronutrient and the pH adjusted to 6.7 with either 0.2N NaOU/

KOH or 0.2N 11C1.

The beakers or polythene bags were removed from establishing plantlets

(Fig. 12) which were then left in the growth cabinet for a further two weeks before transfer to the greenhouse (Fig. 13).

Whole plants regenerated from initially CMD infected cassava were indexed for CLV at different stages of growth. Regenerated plants were visually inspected for the typical symptoms of CMD and leaf extracts from the regenerated plants (Fig. 13) were also prepared and assayed on N, benthamiana plants. Leaf dips of fresh leaves from both regenerated and seed-derived cassava plants and inoculated N. benthamiana leaves were also examined under the electron microscope.

5.3 (iii) Success of whole plant regeneration and heat-therapy treatment

Establishing plantlets in soil was initially not too successful mainly because of microbial contamination which often resulted in the death of the transferred plantlets. Both the garden soil and John Innes No. 2 compost which contained large numbers of micro-organisms, were mesh sieved(me;

No. 6; 1.1002 inches and 2800 mics.) and sterilized at 15 psi for 20 minutes and at temperature of 120°C at least three times prior to use. More success at establishing the plantlets came with practice and care but survival was assured for plantlets treated with the rooting powder not only because of increased root development but also presumably because of the presence of antifungal thiram in the rooting powder mixture. Both meristems from healthy cassava and heat treated infected cassava plants regenerated plantlets within four to six weeks (Tables 9 and 10C). But meristems from untreated infected plants took eight to twenty-eight weeks to regenerate

(Table 10C). Some 28% of the total of the cultured meristems from heat - 55 -

treated infected cassava produced plantlets within six weeks as compared to

a 28 - 30% regeneration rate of plantlets from healthy cassava meristems.

Only 15% of the meristems derived from untreated infected cassava

regenerated into plantlets within eight to twenty-eight weeks of culture.

These percentages are totals using all of the media investigated. Individual

growth media, particularly the E5 to E8 range were more effective than the El

to E4 media in the regeneration of whole plants(Table 9). Plants with regen-

erated shoots and callus only when treated with rooting powder induced roots at

many root hairs within twenty days of application (Fig. lid). The rooting

powder was more effective at inducing roots than the modified MS media

containing 2iP and Zeatin (N - N.. in Tables 10B and D). Often more than y ii

one large root formed in tissue cultured in the media with BHRP than in

the media containing either 2iP or Zeatin (Figs. 11D and C).

5.3 (iv) Indexing regenerated plants from meristem tip culture for the

presence of CLV

All regenerated plantlets derived from meristems originally taken

from infected cassava plants were indexed for the presence of virus. No

symptoms of CLV infection appeared on any inoculated N. benthamiana plants

30 days after inoculation and no particles resembling CLV or any other virus

were detected under the electron microscope at several stages after

establishment of all regenerated meristems in soil (Fig. 13). All regenerated

plantlets from meristems (0.5 - 0.8 mm) were apparently virus free. Most of

the regenerated cassava plants 'freed' of virus initially by heat-treatment

were also indexed in the similar fashion and were found to be free of virus,

as homogenised extracts of leaves from regenerated plants did not elicit

.typical symptoms of CLV infection when inoculated to N. benthamiana plants

up to 30 days post-inoculation and no virus particles were detected in the

electron microscope. Using heat-treatment it was possible using the growth - 56 -

conditions described, to produce virus-free cassava plants from raeristems growing in the soil in a greenhouse within 90 days of starting the treatment of whole plants. - 57 -

DISCUSSION

When sap from CMD-infected cassava was mechanically inoculated to

N. benthamiana, the latter became infected. The agent causing the infection was purported to be a virus, CLV (Bock et_ al^., 1976 and 1978b; and Harrison et al., 1977). In the present study, attempts were made to reinfect healthy cassava with either crude infectious sap of N. benthamiana or

N. benthamiana on to healthy cassava. The results in each case were negative.

Although back inoculations and grafting were not attempted in order to recover CLV from the apparently healthy cassava, it was considered logical to assume that the. agents of CLV and CMD may be different. This supports earlier findings by Bock jet al. (1978b) but the pathological status of CLV remains obscure and other results on successful mechanical inoculation await confirmation (Bock and Guthrie, 1978a).

The East African isolates of CLV contained paired and single particles of 30 x 20 nm and 20 nm respectively (Bock et al., 1978b). The Nigerian isolates of CLV, however, had 30 x 19 nm paired and 19 nm single particles.

Additionally, the molecular weight of the East African CLV was 32,000d

(Harrison _et al., 1977) as compared with 35,500d and 38,000d respectively for the two Nigerian isolates, CLV-A and CLVTB. These differences although minor, suggest that CLV from the two areas may represent strains of the same virus.

The Nigerian isolates of CLV were collected from coastal as well as from hinterland areas. CLV was propagated in tobacco from both isolates.

Bock _et al. (1978b),however, failed to obtain CLV from the coastal areas of

East Africa. This lends additional support to the possibility that the

Nigerian CLV and East African CLV constitute different strains and, that

East Africa may lack the coastal strain. - 58 -

My study has shown that CMD is not seed-borne in cassava. If methods of germinating cassava seed could be improved or perfected to achieve a high percentage of germination and if the resultant seedlings could be readily established in the fields, the incidence of CMD and CLV might be considerably reduced. Although re-infection is almost inevitable especially by whitefly transmission of the disease, it would be preferable to breed for resistant cassava varieties to alleviate the problem. This aspect needs to be further investigated probably with resistant clones.

Most of the methods of purification gave low yields of CLV from extracts of infected N. benthamiana. This may have been due to loss of considerable amounts of virus as a result of the high pectin content of

N. benthamiana or.the purification methods causing virus aggregation. The methods in which cellulolytic and pectolytic enzymes (cellulase RJQ and macerozyme R^) were incorporated during purification gave high yields of the virus. Inclusion of these enzymes may have eliminated plant proteins and pectins contaminating virus preparations. The purification method needs further refinement particularly when one considers that large amounts of the virus are still present in solvent interphase layers during the early part of the purification procedure and are discarded in pellet fractions.

The nucleic acid of CLV was positively identified as single stranded

DNA (ssDNA). This, therefore, confirms work by Harrison jet al. (1977) and agrees with this common feature of geminivirus group, (Goodman, 1977a).

Attempts made to characterize the viral proteins are only preliminary but gave indications of the capsid molecular weight.

Ultrastructural studies of CLV-infected cassava, although not illustrated in this thesis were problematical particularly with regard to

fixation and embedding of material. More successful ultrastructural studies were carried out on infected tobacco plants. The characteristic alterations caused by geminivirus infection were, however, seen only in a few cells.

Here striking cytological alterations including the occurrence of fibrillar - 59 -

rings Chat occurred in phloem parenchyma cells were characteristic of changes seen previously with other geminiviruses. It remains to be seen whether the small number of cells actually seen infected are representative of the total number of cells infected in systemically infected plants and thus reflect the low recovery of purified virus.

However, there did not appear to be any association of the fibrillar rings with the virus particles in contrast to previous studies (Francki et al.,

1979; Goodman et al., 1977c; Kim and Flores, 1979; Kim et. al., 1978;

Russo e_t aK, 1980) and the chromatin in the nucleoplasm of tobacco phloem-parenchyma infected with CLV did not appear to be replaced by virus particles. However the findings of Kim and Flores (1979), that cells often contained fibrillar rings without any visible virus particles was confirmed in my studies (Figs. 27 and 28). The possibility cannot be overlooked that cells similar to those observed actually did occur but that

I failed to observe them or possibly the CLV in infected cells in my system was considerably lower than in other types of geminivirus-infected cells.

Changes in the nucleus including the depletion of chromatin and a granular appearance of the nucleoplasm in CLV infected N. benthamiana cells (Fig. 22) have also been observed in the nuclei of cells infected with beet curly top virus (Duffus and Gold, 1973; Esau and Magyarosy, 1979); bean golden mosaic virus (Kim _et jal., 1978); chloris striate mosaic virus

(Francki et al_., 1979); and tomato yellow leaf curl virus (Osaki and Inouye,

1978; Russo et al., 1980). All the above mentioned viruses are considered to be geminiviruses. The nucleoli of some cells often appeared to be hyper- trophic (Figs. 22 and 25) as compared to healthy nucleoli (Fig. 20), (Kim and Flores (1979). Intranuclear ring-shaped inclusions have been detected in association with infections by beet curly top virus in these hosts (Esau and Magyarosy, 1979); golden mosaic virus in bean (Kim et al.., 1978); tobacco leaf curl virus in Lonicera japonica Thunb (Osaki _e_t al., 1979); euphorbia - 60 -

mosaic virus in Euphorbia heterophylla (Kim and Flores, 1979); and tomato yellow leaf curl virus in tomato and tobacco (Osaki and Inouye, 1978; and

Russo £tal.., 1980).

In N. benthamiana infected with CLV I failed to observe the development of large numbers of geminate virus particles in the nucleoplasm, which apparently occurs in tomato plants infected with tomato leaf curl virus

(Russo et al., 1980) and Chloris gayana or Hordeum vulgare infected with chloris striate mosaic virus (Francki et al,, 1979).

However, my observations of nuclear abnormalities in systemically

CLV-infected N. benthamiana phloem parenchyma cells suggest that the virus is restricted to vascular tissue. This might also be the case in cassava plants. This may also create difficulties for whole plant chemotherapy as most potential biological agents are not phloem transported (Ramaswamy,

1978). The inability to mechanically transmit CLV back to healthy cassava plants may well be a physical problem of establishing the virus in the appropriate tissues. The virus was successfully purified from systemically

infected N. benthamiana leaves but yields were low and greater

refinements of these techniques are required. Nevertheless, it was possible

to identify geminate particles by electron microscopy (Milne, 1978) by

negative staining methods confirming that CLV appears to be a true gemini-

virus.

The nucleic acid of the virus has been positively identified as

single stranded DNA (ssDNA) with a molecular weight similar to that previously

described (Harrison e_t al., 1 977) .

Freeing several different crop plants of virus by meristem

tip culture is now a routine procedure and has been successfully used for *

eradication of CLV from cassava plants. Meristem tip culture and thermo-

therapy techniques have been used to free East African cassava cultivars

of cassava mosaic (Kaiser and Teemba, 1979). Chant (1959) inactivated

mosaic virus in cassava (M. utilisima Pohl.) by t\eat treatment only but - 61 -

a proportion of.the plants eventually showed CMD symptoms after a fev weeks. In this study, however, the same result of virus inactivation was observed but typical CMD symptoms reappeared 30 days after heat treatment of infected cuttings when transferred to normal greenhouse conditions. The use of the hormone rooting powder was found suitable for the induction of roots on meristems 20 - 28 days after meristem establishment in nutrient agar where kinetin was omitted. Also meristems, measuring 0.5 to 0.8 mm in size with one or two primordia leaves when cultured, after heat treatment regenerated virus free plants.

Heat treatment of CMD infected cassava cuttings coupled with meristem tip culture and the use of hormone rooting powder were found to produce virus-free cassava seedlings which were later established in soil.

It is hoped that the incorporation of antiviral compounds for chemo- therapy into culture media, e.g. as indicated by Shepherd (1977) and

Tomlinson (1978), may assist in virus eradication. Subsequent meristem tip culture may be used to reduce the incidence of viral disease in cassava and possibly also other virus infected plants throughout the world (Shepherd,

1977; Varma, Sharma and Subramanian, 1978).

My successful attempts to eradicate CMD from infected cassava have involved the use of a range of media, based on those developed by other workers, some of which incorporated suitable levels of hormones for culturing Nigerian clones of cassava. Unfortunately root development was retarded in regenerating plantlets derived from meristems. However, I found that this problem could be partially overcome by the introduction of a step during the culture which involved the dipping of regenerating plant- lets without roots into a rooting powder (BHRP). It was possible in later studies to also induce a few roots on recalcitrant cultures by growing them on more hormonally defined media, e.g. with 2iP and Zeatin included. Using

these cultures it was possible to eradicate CMD by selecting meristems

(0.5 to 1.5 mm) which possessed at least one or two chlorophyllous leaf - 62 -

promordia as well as the meristernatic dome. Efforts at a prior heat-

therapy before meristem culture were wholly successful when meristems for culture were selected as above from treated plants. It was possible to

check the "cleanliness" of the regenerated plants by a back inoculation onto

susceptible tobacco varieties.

Unfortunately the host range of CLV appears to be somewhat restricted.

Only selected members of the Solanaceae and Euphorbiaceae gave any systemic

symptoms. No hosts which reacted hypersensitively to virus infection were

found. At the moment this rules out the quantitative estimation of the virus

by local lesion assay except by scoring for systemic symptoms which may take

10 days or more to develop.

In view of the increasing importance of cassava as a food crop staple,

and possible energy source, any advances in the production of disease free

clones must clearly be advantageous. Whilst this study has been successful

in most respects, it has served to emphasize that the problems of studying

plant viruses from the third world countries should receive much attention

than has been given previously. - 63 -

ACKNOWLEDGEMENTS

My profound gratitude goes to the Food and Agriculture Organization of the United Nations who supported my studies at Imperial College, London.

I would also like to thank the authorities of Imperial College who gave some financial assistance during the last three months of my stay in London enabling me to complete my thesis.

I would like to express my gratitude to Dr.R.H.A. Coutts for supervising this research and for valuable suggestions, guidance and assistance in preparing this thesis. Thanks also go to Professor R.K.S. Wood

Head of Botany Department who acted as an academic adviser throughout my stay at Imperial College.

My sincere gratitude goes to all staff of Imperial College, London,

Federal Department of Agriculture, Nigeria, International Institute of

Tropical Agriculture, Ibadan, Nigeria and all the other people who contribute to the successful completion of my studies.

The above research was carried out under licence (Number NH/2034A/57) issued under the destructive pests and diseases of plants act by the Ministry of Agriculture, Fisheries and Food (MAFF), England and Wales. - 64 -

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Anon. (1978). Etiology of cassava mosaic disease. In Research Highlights, International Institute of Tropical Agriculture, Ibadan - Nigeria, pp. 25 - 28.

Beck, B.D.A. and Chant, S.R. (1958). A preliminary investigation on the effect of mosaic virus on Manihot utilissima Pohl. in Nigeria. Tropical Agriculture, Trin. (a quarterly journal of the Imperial College of Tropical Agriculture) 15. (O : 59 - 64.

Berg, L.A. & Bustamante (1974). Heat treatment and meristem culture for the production of virus free bananas. Phytopathology 64 : 320 - 322.

Bock, K.R., Guthrie, E.J. & Woods, R.D. (1974). Purification of maize streak virus and its relationship to virus associated with streak diseases of sugar cane and Panicum maximum. Annals of Applied Biology 77 : 289 - 296.

Bock, K.R. & Guthrie, E.J. (1976). Recent advances in research on cassava viruses in East Africa. In African Mosaic : Report of an inter- disciplinary Workshop held at Muguga, Kenya. 19-22 February, 1976. Barry L. Nestel (ed.). International Development Research Centre, Mognor. IDRC-071e : p. 11 - 16.

Bock, K.R., Guthrie, E.J. & Meredith, (1977). RNA and protein components of maize streak and cassava latent viruses. Annals of Applied Biology 85 : 305 - 308.

Bock, K.R. & Guthrie, E.J. (1978a). Transmission of African cassava mosaic by mechanical inoculation. Plant Disease Reporter 62 : 580 - 581.

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APPENDIX I

Chemicals and plant growth substances for meristem tissue culture

Chemicals Source of supply

Thiamine HC1 The Sigma Chemical Company Limited, London.

Myo-inositol 11

naphthalene acetic acid (NAA) "

6 benzyl amino purine (BA) "

6 (dimethylally-amino)purine (2iP) 11

6 (4-hydroxy-3 methyl but-2 enylamino) purine (Zeatin) 11

Kinetin The BDH Chemical Company Limited, Poole, Dorset Pyridoxine - HC1

Indol 3-ylacetic acid (IAA)

Gibberellic acid (GA^) II

Hormone rooting powder (BHRP) The Boots Chemical Company Limited 9 Nottingham, England.

Nicotinic acid Hopkins and Williams Company Limited

Difco Bacto-agar Difco Labs. Michigan, U.S.A. - 73 -

APPENDIX II

Chemicals for virus purification and characterization

Chemicals Source of supply

Thioglycollic acid (TGA) BDH Chemical Company Limited

2-mercaptoethanol Poole, Dorset

Ethylene diamine tetra-acetic acid - disodium salt (EDTA)

Chloroform n-butanol

Sodium chloride

Polyethylene glycol (PEG) MW 6,000

Sodium sulphite

Sucrose

Formaldehyde

Polyacrylamide

Bis-methylene acrylamide

Sodium lauryl disulphate

Cytochrome ' cT Sigma Chemical Company Limited, London

Bovine serum albumin

Coomassie blue

Pronase

Cellulase R^ The Kinki Yakult Manufacturing Company Limited, Japan

Macerozyme R 10 - 74 -

ABBREVIATIONS ON PLATES

CH = Chloroplast

G = Golgi apparatus

M = Mitochondria

N = Nucleus

Nu = Nucleolus

Nup : = Nucleoplasm

NuPo = Nuclear membrane pore

NuM = Nuclear membrane

V = Vacuole

C- = Chromatin

FC = Fibrillar chromatin

GC = Granular chromatin

FR Fibrillar ring

GR = Grana

SG = Starch granule

DM = Disorganized membrane Figure 1

Seed-derived cassava plants representative of

types 30211, 30395, 30572, 30555, 60444 and

Isunikakiyan

Figure 2

Potted cassava plants

CMD-infected (631004 MPIBN) as compared to a

healthy (30211 IITAIBN) type - 586 -

i Fig 2 ii Figure 3

(a) A leaf from healthy seed-derived cassava

plant (type 30211 IITAIBN)

(b) A leaf from a mature shoot of a cassava plant

with CMD (type 631004 MPIBN)

(c) A leaf from a cassava plant with CMD on a young

emergent shoot (type 631004 MPIBN)

Figure 4

A N. benthamiana plant 12 days after inoculation with

partially clarified leaf sap derived from CMD affected

cassava leaves (type 631004 MPIBN) - 588 -

CASSAVA LEAS/TA

Fig 3

Mitotiojia beottamiana /NFECTED Por, iNOCUL/HftN VMITH CASSAVA Mt^C tyStASt

i. .W Fig 4 Figure 5

N, benthamiana plants.

Infected and control 20 days after inoculation with

partially clarified leaf sap derived from CMD affected

cassava leaves (type 631004 MPIBN), infected or

healthy leaves (control)

Figure 6

Similar plants to those in Figure 5 showing CLV in

N. benthamiana (front row left), N. hybrida (middle) and

N. glutinosa (right), with the respective controls

behind each of the infected plants - 77

Fig 6 Figure 7

CMD transmission from infected cassava (LGjN)-scion

onto healthy seed-derived cassava variety 30395 (root

stock) by the bud-grafting technique (20 days after

grafting)

Figure 10

Cassava meristem (0.8 mm diameter) asceptically excised

and cultured on agar medium (E7) (8 days old) Fig 8 Figure 9

Cassava meristem development into a 'plantlet'

with callus (15 days old) in E2 medium

Figure 10

Apical meristem developing into a plantlet bearing

roots (28 days old) in E7 medium

Figure 11

Regenerated 'plants' of cassava showing the effects of

the different rooting hormones and rooting powder,

15 days after treatment. All cultures (a - d) were of

the same ages (15 days) before treatment

(a) Shows plantlet with no treatment and no root development in E5 medium

(b) Shows plantlet with one root developing in E7 medium

(c) Shows plantlet with one long root after treatment with 2iP containing medium (N9)

(d) Shows plantlet with many roots after treatment with rooting powder (BHRP)

Figure 12

Plantlet of cassava in sterile soil covered with a

beaker, 30 - 40 days after transplantation to soil. - 596 -

Fig 11

Fig 12 Figure 13

Regenerated plant (50 days old) from meristem

(0.8 mm diameter) originally taken from CMD-infected

cassava (clone LG N) without prior heat treatment - 598 -

Fig 13 Figures 14 and 15

Cassava latent virus (CLV-A) and (CLV-B) respectively

Electron micrograph of purified preparation (with no

enzyme treatment) of cassava latent virus (CLV-A) and

(CLV-B) stained with sodium phosphotungstate. Most

particles are paired but a few occur singly (arrows)

CLV-A was isolated from cassava mosaic diseased leaves

(type 631004 MPIBN) while CLV-B was^from CMD infected

cassava leaves (type LG^N). Extracts from the infected

cassava leaves were inoculated into N. benthamiana

from whose leaves after passage, the virus was later

purified and negatively stained - 82 - Figure 16

Electron micrograph of purified preparation (with

enzyme treatment) of cassava latent virus (CLV-A),

stained with sodium phosphotungstate. Most

particles are paired but a few occur singly (arrows) - 83 - Figure 17

Ultraviolet absorption spectrum of purified cassava

latent virus isolates from 631004 MPIBN (CLV-A) and

LG N (CLV-B) clones 84 -

Fig 17

240 260 280 iSo wavelength nm - 85 - rig in

Pit '!.: • ».f Hw 1 ogc.rl thro of the MOICCU] E.i: v/o:i.

Alkaline sucrose density-gradient profile of

cassava latent virus-DNA, which is found in

the peak limited by the arrowed lines - 86 -

Fig 19

Ftc-VJ'cionafcion of a 10 bo 40;' alkaline c-\i"rose • g radio-nt containing CLV-A DIIA through an I SCO fractionator at 260nm. Figure 20

Thin-section (60 - 90 ym) of a healthy leaf of

N. benthamiana showing mesophyll cells, e.g. nucleus (N),

chloroplast (CH), vacuole (V), Golgi material (G)

Figure 21

Thin-section (60 - 90 ym) of a healthy leaf of

N. benthamiana showing a typical cell and illustrating

a normal nucleus (N)

C = chromatin CH = chloroplast G = golgi apparatus GR = grana N = nucleus NuM = nuclear membrane Nup = nucleoplasm NuPo = nuclear membrane pore V = vacuole Fig 21 Figures 22 and 23

Thin-section (60 - 90 ym) of CLV-infected

N. benthamiana phloem parenchyma cell showing

nuclear abnormalities, e.g. a fibrillar ring (FR),

segregation of granular chromatin (GC) and fibrillar

chromatin (FC). Deformation of nuclear membranes

(NuM) is also apparent

FC = fibrillar chromatin

FR = fibrillar ring

GC = granular chromatin

Nu = nucleus

NuM = nuclear membrane - 611 -

Fig 23 Figures 24 and 25

Thin-section (60 - 90 ym) of CLV-infected

N. benthamiana phloem parenchyma cell showing nuclear

abnormalities in the form of segregation of

fibrillar chromatin (FC),granular chromatin (GC),

deformation of nuclear membrane (NuM), hypertrophy

of nuclei (Nu) and deposition of starch granules (SG)

FC fibrillar chromatin

GC granular chromatin

Nu nucleus

NuM nuclear membrane - 613 -

Fig 24

500nm

Fig 25 Figure 26

Thin-section (60 - 90 ym) of CLV-infected

N. benthamiana phloem parenchyma cell showing a

deformed nucleus with segregated granular and

fibrillar chromatin (GC and FC); fibrillar rings

(FR), a swollen chloroplast (CH), peripheral and

axial chromatin (C)

Figure 27

A thin-section (60 - 90 ytm) of a fibrillar ring (FR)

in a matrix and the nuclear membrane (NuM)

C = chromatin

CH = chloroplast

FR = fibrillar ring

GC = granular chromatin

NuM = nuclear membrane - 615 - Figure 28

Similar section to that in Figure 27 but with a

fibrillar ring (FR) and possible virus-like particles

aggregates at the edge of the ring (arrowed) as in

Figures 27 and 28

Figure 29

Thin-section (60 - 90 ym) of CLV-infected N. benthamiana

leaf mesophyll cell showing the gross effects

of virus infection on chloroplast structure. Loss of

grana, starch granule (SG) deposition, and disorganized

membranes (DM) into ring-shaped forms, 20 days after

inoculation .

DM = disorganized membrane

FR = fibrillar ring

GR = grana

NuM = nuclear membrane

SG = starch granule

VP = virus particle(s) - 91 -

Fig 29