Recognition and differentiation of seven whitefly-transmitted geminiviruses from India, and their relationships to African cassava mosaic and Thailand mung bean yellow mosaic viruses

Particles resembling those of geminiviruses were found by immunosorbent electron microscopy in extracts of plants infected in India with bhendi yellow vein mosaic, croton yellow vein mosaic, dolichos yellow mosaic, horsegram yellow mosaic, Indian cassava mosaic and tomato leaf curl viruses. All these viruses were transmitted by Bemisia tabaci whiteflies, all reacted with at least one out of ten monoclonal antibodies to African cassava mosaic virus (ACMV), and all reacted with a probe for ACMV DNA-1, but scarcely or not at all with a full-length probe for ACMV DNA-2. Most of the viruses were distinguished by their host ranges when transmitted by whiteflies, and the rest could be distinguished by their pattern of reactions with the panel of monoclonal antibodies. Horsegram yellow mosaic virus was distinguished from Thailand mung bean yellow mosaic virus by its lack of sap transmissibility, ability to infect Arachis hypogaea, failure to react strongly with the probe for ACMV DNA-2 and its pattern of reactions with the monoclonal antibodies. Structures resembling a ‘string of pearls’, but not geminate particles, were found in leaf extracts containing malvastrum yellow vein mosaic virus. Such extracts reacted with two of the monoclonal antibodies, suggesting that this whitefly-transmitted virus too is a geminivirus. All seven viruses from India can therefore be considered whitefly-transmitted geminiviruses.     

Serological studies on cassava latent virus

Particles of cassava latent virus (CLV) were purified by a method that yielded up to 3 mg per 100 g of systemically infected Nicotiana benthamiana leaf. Specific antiserum was prepared and used for enzyme-linked immunosorbent assay (ELISA), which detected purified virus at 5 ng/ml. As estimated by ELISA, CLV antigen reached a greater concentration in leaves of N. benthamiana plants kept at 20–25 °C than in those at 15 °C or 30 °C. CLV was also detected in leaf extracts of naturally infected cassava plants kept at 25 C but its concentration was only 1–7% of that in comparable extracts from N. benthamiana. Staining sections of N. benthamiana leaves with fluorescent antibody indicated that CLV particle antigen accumulates in the nuclei of many phloem cells and of some cells in other tissues. In tests on mosaic-affected cassava plants of Angolan origin, three plants were found in which CLV could not be detected by either ELISA or immunosorbent electron microscopy, or by transmission to indicator plants. This suggests that the mosaic symptoms were caused by a pathogen other than CLV, but no such agent was detected by electron microscopy of leaf extracts. Three kinds of serological test indicated that CLV is related to bean golden mosaic virus. Evidence was also obtained of a distant relationship to beet curly top virus but none was detected to four other geminiviruses.     

Purification and Characterization of Indian Cassava Mosaic Virus

The Indian cassava mosaic virus (ICMV) was transmitted by the whitefly Bemisia tabaci and sap inoculation. ICMV was purified from cassava and from systemically infected Nicotiana benthamiana leaves. Geminate particles of 16–18 × 30 nm in size were observed by electron microscopy. The particles contained a single major protein of an estimated molecular weight of 34,000. Specific antiserum trapped geminate particles from the extracts of infected cassava and N. benthamiana plants in ISEM test. The virus was detected in crude extracts of infected cassava, ceara rubber, TV. benthamiana and N. tabacum cv. Jayasri plants by ELISA. ICMV appeared serologically related to the gemini viruses of Acalypha yellow mosaic, bhendi yellow vein mosaic, Croton yellow vein mosaic, Dolichos yellow mosaic, horsegram yellow mosaic, Malvastrum yellow vein mosaic and tobacco leaf curl.     

African Cassava Mosaic Virus (ACMV): Stability of Purified Virus and Improved Conditions for its Detection in Cassava Leaves by ELISA

African Cassava Mosaic Virus (ACMV) was purified by a method which allowed the separation of monomer from dimcr virus particles. Optimal conditions for storing purified virus to be used for immunization were determined by ELISA and inoculation on Nicotiana benthamiana. Purified virus could be stored without loss of infectivity and serological activity for more than 145 days at 4 °C or frozen at –20 °C, but not longer than 40 days in the presence of 50 % redistilled glycerol. Rabbit and chicken immunoglobulins were used to detect ACMV in cassava leaves by direct and indirect ELISA. To obtain the same absorbance values, it was necessary to use longer incubation times with the indirect method, but the virus detection end-point m sap from infected plants was the same for the two methods (1/512). Conditions for improving virus detection tn cassava samples were determined. The virus was better detected when leaves from diseased plants were ground in 100 mM Tris-HCl containing 1 % polyvinylpyrrolidone at pH 8.5 than in phosphate buffer. Plant inhibitors were the restricting factor in the detection of virus by ELISA, but this difficulty was avoided when leaves to be tested were harvested from the top of the cassava plants.     

Particle purification and properties of cassava Ivorian bacilliform virus

A virus found in cassava from the north-west of the Ivory Coast was transmitted by inoculation with sap extracts to herbaceous species in six plant families. Chenopodium quinoa was used as a propagation host and C. murale was used for local lesion assays. The virus particles are bacilliform, c. 18 nm in diameter, with predominant lengths of 42,49 and 76 nm and a structure apparently similar to that found in alfalfa mosaic virus. Purified preparations of virus particles had A₂₆₀/A₂₈₀ of 1.7 ±0.05, contained one protein of M_rc. 22 000, and yielded three species of RNA with M_r (× 10^-6) of c. 0.7, 0.8 and 1.2. Although the virus particles were poorly immunogenic, an antiserum was produced and the virus was detected by enzyme-linked immunosorbent assay (DAS-ELISA) in leaf extracts at concentrations down to c. 6 ng/ml. Four other field isolates were also detected, including a strain which caused only mild systemic symptoms in C. quinoa instead of necrosis. The naturally infected cassava source plants were also infected with African cassava mosaic virus (ACMV) but when the new virus was cultured in Nicotiana benthamiana, either separately or together with ACMV, its concentration was the same. The new virus did not react with antisera to several plant viruses with small bacilliform or quasi-bacilliform particles, and alfalfa mosaic virus reacted only weakly and inconsistently with antiserum to the cassava virus. The new virus, for which the name cassava Ivorian bacilliform virus is proposed, is tentatively classified as the second member of the alfalfa mosaic virus group.     

A strain of cassava latent virus occurring in coastal districts of Kenya

A strain of cassava latent geminivirus (CLV) was isolated from mosaic-affected cassava plants from coastal districts of Kenya. This virus (CLV-C) did not infect Nicotiana clevelandii, a diagnostic host of the type strain (CLV-T); experimental host range was very restricted and CLV-C only infected N. benthamiana and N. rustica out of several solanaceous hosts readily infected by CLV-T. CLV-C was also isolated from naturally infected Jatropha multifida (Euphorbiaceae) and Hewittia sublobata (Convolvulaceae). CLV-C was propagated in N. benthamiana with difficulty and only those isolates derived from cassava plants infected with severe mosaic symptoms were maintained more or less successfully; these sources usually contained a higher concentration of CLV than plants with mild symptoms. Symptom variants generally remained unchanged when grafted into a highly susceptible South American cassava variety. CLV-C and CLV-T seemed to occur respectively only in coastal and western districts but their ranges overlapped in central Kenya where they could have been introduced in infected material. CLV-C could be purified satisfactorily with the method used for CLV-T but only after modifying the procedure by substituting phosphate for borate in the extraction buffer, n-butanol for n-butanol/chloroform in clarification of extracts, and phosphate for borate buffer when resuspending concentrated virus. A virus serologically indistinguishable from CLV-T was isolated from mosaic- affected material obtained from Nigeria; East African and Nigerian isolates were essentially similar in host range and symptomatology. In gel-diffusion serology tests, pronounced precipitation spurs developed between CLV-T and CLV-C indicating that the isolates were related but not identical serologically. Symptoms typical of cassava mosaic disease appeared in only three of 105 plants in experiments on transmission of CLV-C and CLV-T by whiteflies, when attempted acquisition of either clarified CLV-infective sap or purified CLV was made through ‘Parafilm’ membranes. Because it is possible that the three infections resulted from contamination, they cannot constitute proof of transmission. The presence of CLV in relation to the etiology of cassava mosaic thus remains unresolved.     

Serological and biological variations of African cassava mosaic virus, in Nigeria

To determine the occurrence of variants of African cassava mosaic virus, 316 cassava leaf samples were collected from mosaic‐affected cassava plants in 254 farmers. fields in 1997 and 1998, covering the humid forest, coastal/derived, southern Guinea and northern Guinea savannas and arid and semi‐arid agroecologies of Nigeria. The samples were tested in triple antibody sandwich enzyme‐linked immunosorbent assay using a panel of 10 monoclonal antibodies (MAbs) against the virus in which 29 reaction patterns were observed. In cluster analysis, nine serotypes were obtained at 0.80 Jaccard similarity coefficient index in which at least 50% of isolates of each serotype reacted alike. The serotypes ranged between two extremes: serotype 1 with 90% isolates reacting with the 10 MAbs and serotype 8 in which 90% of its isolates failed to react with the antibodies. Isolates of serotypes 1, 2, 4 and 8 were widely distributed while those of the other serotypes were estricted to certain agroecologies. Four representative isolates 227 (serotype 1), 231 (serotype 2), 235 and 283 (serotype 8) elicited different responses in Nicotiana, benthamiana, with isolate 283 not able to infect this and other test plants used. The serological variations did not necessarily reflect the biological variations. In polymerase chain reaction tests, one out of the five pairs of ACMV primers tested distinguished only isolate 283. The humid forest, derived/coastal and southern Guinea savannas where most of the crop is grown in Nigeria had a high number of variants, which makes the agroecologies suitable for the selection of resistant cassava clones against ACMV.     

Virus content of leaves of cassava infected by African cassava mosaic virus

African cassava mosaic virus (ACMV) was detected in cassava leaves by ELISA. Some normal constituents of cassava leaves interfered with virus detection but leaf extracts of Nicotiana benthamiana did not. The symptom pattern was determined early in the growth of a leaf and subsequently changed little. ACMV was found only in the yellow or yellow green areas of the mosaic pattern. Virus content of the leaves increased with increasing symptom intensity, but decreased with leaf age and ACMV was not detected in mature leaves. Most whiteflies were found on young growing cassava leaves and the number decreased progressively with leaf age. This distribution will aid both the acquisition and inoculation of the virus.     

Some properties of a previously undescribed virus from cassava: Cassava American Latent Virus

A virus with spherical particles c. 28 nm in diameter was sap-transmitted from different cassava (Manihot esculenta) cultivars to a limited range of species in the families Chenopodiaceae and Solanaceae. Cassava seedlings infected by inoculation with sap or with purified virus preparations did not show any symptom, although the virus was readily detected by ELISA or by further inoculations. Leaf extracts from infected Nicotiana benthamiana were infective after dilution of 10^--³but not 10^--⁴, and after heating for 10 min at 70°C, but not at 72°C. The virus was purified from N. benthamiana, N. clevelandii or from cassava. On sucrose gradients, the virus particles sediment as three components all containing a protein of mol. wt c. 57000. The genome of the virus is composed of two RNAs of mol. wt c. 2.54 times 10⁶(RNA-1) and 1.44 times 10⁶(RNA-2). RNA-2 was detected in the middle and the bottom nucleoprotein components, and RNA-1 only in the bottom component. An antiserum prepared to purified virus particles was used to readily detect the virus in cassava and other host plants by ELISA and by ISEM. No serological relationship was shown between this virus and eight nepoviruses, including the recently described cassava green mottle nepovirus infecting cassava in the Solomon Islands (Lennon, Aiton & Harrison, 1987). The virus described here is the first nepovirus isolated from cassava in South America, and is named cassava American latent virus.     

Serological detection and antigenic variation of two whitefly-transmitted geminiviruses: tobacco leaf curl and croton yellow vein mosaic viruses

A panel of 25 monoclonal antibodies (MAbs) raised against particles of two heterologous whitefly-transmitted geminiviruses (begomoviruses) was used in triple antibody-sandwich ELISA (TAS-ELISA) to determine the detectability and epitope profiles of 26 Indian isolates of tobacco leaf curl virus (TLCV) and 13 of croton yellow vein mosaic virus (CYVMV). Stock cultures of the two viruses had indistinguishable epitope profiles although they differ in symptomatology and particle stability. Their epitope profiles also strongly resembled those of Indian isolates of bhendi (okra) yellow vein mosaic and Indian cassava mosaic (ICMV) viruses. TLCV isolates from Andhra Pradesh, Gujarat and Karnataka States differed slightly in epitope profile: they reacted with at least eight out of 10 MAbs raised to ICMV but only one to four out of 15 MAbs raised to African cassava mosaic virus (ACMV). Virus isolates serologically indistinguishable from TLCV were detected in symptom-bearing weeds (Acanthospermum hispidum, Ageratum conyzoides, Euphorbia geniculata, Parthenium hysterophorus) found in leaf curl-affected tobacco fields and shown previously to be experimental hosts of TLCV. Indian TLCV isolates had small, consistent differences in epitope profile from Pakistani isolates but large differences from isolates from Burkina Faso, Malawi or Uganda. Isolates from the three African countries reacted with four or five of the ACMV MAbs but only one or two of the ICMV MAbs, and there were small but consistent inter-country differences. CYVMV isolates from three Indian States showed less epitope variation than did Indian isolates of TLCV. TAS-ELISA with MAb SCR 18 was a more sensitive test for detecting Indian TLCV isolates than was double antibody-sandwich ELISA with polyclonal antibodies.     

Comparative epitope profiles of the particle proteins of whitefly-transmitted geminiviruses from nine crop legumes in India

Geminiviruses associated with yellow or golden mosaic diseases of legume crops in two regions of India were compared by testing their reactivity with 27 monoclonal antibodies (MAbs) prepared to the particles of African cassava mosaic (ACMV) or Indian cassava mosaic (ICMV) viruses. The viruses fell into two main groups. Group 1 comprised isolates of dolichos yellow mosaic virus; these reacted with three or four ACMV MAbs and four ICMV MAbs. Group 2 comprised isolates of horsegram yellow mosaic virus, together with isolates from blackgram, cowpea, French bean, pigeonpea, soybean, Indigofera hirsuta and probably also isolates from mungbean. These reacted with three or four ACMV MAbs but with few or no ICMV MAbs. Isolates within each group differed slightly in epitope profile, depending on the source species (Group 2) or geographical origin (Groups 1 and 2). Isolates from lima bean resembled those in Group 2 but had some antigenic differences, and their status is uncertain. The poor detectability of geminivirus isolates in mungbean may reflect a low virus concentration in this species.     

Occurrence of whitefly-transmitted geminiviruses in crops in Burkina Faso, and their serological detection and differentiation

Whitefly-transmitted geminiviruses were found to be associated with four diseases of crop plants in Burkina Faso: cassava mosaic, okra leaf curl, tobacco leaf curl and tomato yellow leaf curl. Tomato yellow leaf curl is an economically serious disease, reaching a high incidence in March, following a peak population of the vector whitefly, Bemisia tabaci, in December. Okra leaf curl is also a problem in the small area of okra grown in the dry season but is not important in the main period of okra production in the rainy season. The geminiviruses causing these four diseases, African cassava mosaic (ACMV), okra leaf curl (OLCV), tobacco leaf curl (TobLCV) and tomato yellow leaf curl (TYLCV) viruses, were each detected in field-collected samples by triple antibody sand-wich-ELISA with cross-reacting monoclonal antibodies (MAbs) to ACMV. Epitope profiles obtained by testing each virus isolate with panels of MAbs to ACMV, OLCV and Indian cassava mosaic virus enabled four viruses to be distinguished. ACMV and OLCV had similar but distinguishable profiles. The epitope profile of TobLCV was the same as that of one form of TYLCV (which may be the same virus) and was close to the profile of TYLCV from Sardinia. The other form of TYLCV reacted with several additional MAbs and had an epitope profile close to that of TYLCV from Senegal. Only minor variations within each of these four types of epitope profile were found among geminivirus isolates from Burkina Faso. Sida acuta is a wild host of OLCV.     

Purification and properties of cassava green mottle, a previously undescribed virus from the Solomon Islands

A sap-transmissible virus obtained from cassava with a green mottle disease occurring at Choiseul, Solomon Islands, was transmitted to 30 species in 12 plant families and was readily seed-borne in Nicotiana clevelandii. In cassava plants infected by inoculation with sap, the first leaves to be infected systemically developed a mottle with some necrosis whereas leaves produced subsequently were symptomless but contained the virus. Most other species developed chlorotic or necrotic local lesions and systemic mottle or necrosis. This was followed, in several species, by production of small symptomless virus-containing leaves. The virus was cultured in N. clevelandii; Chenopodium quinoa was used for local-lesion assays. Leaf extracts from infected N. clevelandii were infective after dilution to 10–⁵ but usually not at 10–⁶, after heating for 10 min at 60°C but not at 65°C, and after storage at 20°C for at least 12 days. The virus has isometric particles of 26 nm diameter which sediment as three components, all containing a protein of mol. wt c. 53000. The two fastest sedimenting components respectively contain single-stranded RNA of mol. wt, estimated after glyoxylation, c. 2.9 × 10⁶ and 2.3 × 10⁶. Both RNA species are needed for infection of plants. In tests with antiserum prepared to purified virus particles, the virus was detected in cassava and N. clevelandii by gel-diffusion precipitin tests, by immunosorbent electron microscopy and by ELISA. Despite its similarity to nepoviruses, the virus did not react with antisera to 18 members of the group. It was named cassava green mottle virus and is considered to be a previously undescribed nepovirus.     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.     

Role of a novel type of double infection in the geminivirus-induced epidemic of severe cassava mosaic in Uganda

To study the cause of the current epidemic of severe mosaic in Ugandan cassava, PCR analysis was used to detect and identify African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and the recently reported recombinant geminivirus (UgV), which is derived from ACMV and EACMV, in leaf extracts from cassava plants grown from cuttings in the glasshouse at Dundee. The cuttings were collected from plants showing symptoms of different severities and growing at different sites in Uganda inside, at the periphery of, and outside, the area affected by the epidemic. ACMV occurred throughout the nine districts sampled but UgV was detected only in the area affected by the epidemic. EACMV was not found in Uganda. Most plants containing ACMV alone expressed mild or moderate mosaic, whereas very severe mosaic developed in most plants containing UgV plus ACMV and a few of those containing UgV only. Very severe mosaic in cassava from southern Sudan was likewise associated with co-infection by UgV and ACMV. The very severe disease was reproduced by graft-inoculating geminivirus-free cassava with UgV plus ACMV; plants inoculated with either UgV or ACMV developed severe or moderate symptoms, respectively. Unlike ACMV, Malawian EACMV did not enhance the severity of symptoms induced by UgV. However, a very severely affected plant from Ukerewe Island, Tanzania, contained ACMV and EACMV but not UgV. UgV attained a much greater concentration in cassava than did ACMV but the opposite occurred in Nicotiana benthamiana. In neither host was total virus antigen concentration affected by co-infection. Factors affecting the genesis, selection and spread of UgV are discussed. The evidence indicates that UgV is probably of relatively recent origin, that such variants do not appear often, and that the current epidemic has resulted from the rapid spread of UgV to infect plants and to invade regions in which ACMV already occurred. The novel type of virus complex so produced, consisting of an interspecific recombinant virus (UgV) and one of its parents (ACMV), typically has even more severe effects than UgV alone.     

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue.Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized.Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum.Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.     

Genome affinities and epitope profiles of whitefly-transmitted geminiviruses from the Americas

The relationships among fifteen isolates of whitefly-transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple-antibody-sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA-A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV-T). Conversely, the probe for ACMV DNA-A did not detect any of the American viruses, and that for TYLCV-T DNA-A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA-B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA-B probe and both chino del tomate virus (CdTV)-DNA and SLCV-DNA. However, a probe for DNA-B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV-PR DNA, and a probe for DNA-B of CdTV from Mexico detected several American viruses. Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and-ACMV MAbs reacted with all, and one anti-ACMV MAb and two anti-ICMV MAbs reacted with nearly all the American viruses, one anti-ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV-CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA-A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA-B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA-B diverging to a greater extent than DNA-A and its particle-protein gene.     

The effect of cassava mosaic geminiviruses on symptom severity, growth and root yield of a cassava mosaic virus disease-susceptible cultivar in Uganda

A study was carried out to assess the effect of different cassava mosaic geminiviruses (CMGs) occurring in Uganda on the growth and yield of the susceptible local cultivar ‘Ebwanateraka’. Plants infected with African cassava mosaic virus (ACMV), ‘mild’ and ‘severe’ strains of East African cassava mosaic virus‐Uganda (EACMV‐UG2) and both ACMV and EACMV‐UG2 were grown in two experiments in Kabula, Lyantonde in western Uganda. The most severe disease developed in plants co‐infected with ACMV and EACMV‐UG2 and in those infected with the ‘severe’ form of EACMV‐UG2 alone; disease was least severe in plants infected with the ‘mild’ strain of EACMV‐UG2. ACMV‐infected plants and those infected with the ‘mild’ strain of EACMV‐UG2 were tallest in the 1999–2000 and 2000–2001 trials, respectively; plants dually infected with ACMV and EACMV‐UG2 were shortest in both trials. Plants infected with ‘mild’ EACMV‐UG2 yielded the largest number and the heaviest tuberous roots followed by ACMV and EACMV‐UG2 ‘severe’, respectively, whilst plants dually infected with ACMV and EACMV‐UG2 yielded the least considering the two trials together. Reduction in tuberous root weight was greatest in plants dually infected with ACMV and EACMV‐UG2, averaging 82%. Losses attributed to ACMV alone, EACMV‐UG2 ‘mild’ and EACMV‐UG2 ‘severe’ were 42%, 12% and 68%, respectively. Fifty percent and 48% of the plants infected with both ACMV and EACMV‐UG2 gave no root yield in 1999–2000 and 2000–2001, respectively. These results indicate that CMGs, whether in single or mixed infections, reduce root yield and numbers of tuberous roots produced and that losses are substantially increased following mixed infection.     

Particle purification, properties and epitope variability of Indian tomato leaf curl geminivirus

A whitefly-transmissible stock isolate of Indian tomato leaf curl geminivirus (ITmLCV) was cultured in graft-inoculated tomato plants and its particles purified from chloroform-clarified extracts in citrate buffer by precipitation with 70 g/litre polyethylene glycol, ultracentrifugation and sucrose density gradient centrifugation. Contaminating helical filaments were eliminated by banding in caesium sulphate gradients. ITmLCV particles had the shape typical for geminiviruses, measured c. 30 × 20 nm and contained a single major protein of estimated mol. wt c. 32 000. They reacted in immunosorbent electron microscopy with antisera to four other whitefly-transmitted geminiviruses. ITmLCV reacted with one out of 17 monoclonal antibodies specific for different epitopes in the particle protein of African cassava mosaic geminivirus and five or six out of 10 monoclonal antibodies to the particle protein of Indian cassava mosaic geminivirus. Virus isolates from tomato at nine locations in Karnataka State showed only slight differences in epitope profile, and isolates from four weed species in tomato fields were similar or identical to those from tomato.     

The cassava mosaic geminiviruses occurring in Uganda following the 1990s epidemic of severe cassava mosaic disease

The cassava mosaic geminiviruses (CMGs) isolated from cassava plants expressing mild and severe symptoms of cassava mosaic disease (CMD) in 2002 in Uganda were investigated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) molecular techniques and DNA sequencing. Two previously described cassava mosaic geminiviruses: African cassava mosaic virus (ACMV) said East African cassava mosaic virus - Uganda variant (EACMV-UG2) were detected in Uganda. The RFLP technique distinguished two polymorphic variants of ACMV (ACMV-UG1 and ACMV-UG2) and three of EACMV-UG2 (EACMV-UG2[1], EACMV-UG2[2] and EACMV-UG2[3]). ACMV-UG1 produced the fragments predicted for the published sequences of ACMV-[KE]/UGMld/ UGSvr, while ACMV-UG2, which produced the RFLP fragments predicted for the West African ACMV isolates ACMV-[NG], ACMV-[CM], ACMV-[CM/DO2] and ACMV-[CI], was shown to be ACMV-UGMld/UGSvr after DNA sequencing. EACMV-UG2[1] produced the RFLP fragments predicted for the published sequences of EACMV-UG2/UG2Mld/UG2Svr. However, both EACMV-UG2[2] and EACMV-UG2[3], which produced East African cassava mosaic vzras-[Tanzania]-like polymorphic fragments with RFLP analysis, were confirmed to be isolates of EACMV-UG2 after DNA sequencing. Thus, this study emphasises the importance of DNA sequence analysis for the identification of CMG isolates. EACMV-UG2 was the predominant virus and occurred in all the surveyed regions. It was detected in 73% of the severely and 53% of the mildly diseased plants, while ACMV was less widespread and occurred most frequently in the mildly diseased plants (in 27% of these plants). Mixed infections of ACMV and EACMV-UG2 were detected in only 18% of the field samples. Unlike previously reported results the mixed infection occurred almost equally in plants exhibiting mild or severe disease symptoms (21% and 16%, respectively). The increasing frequency of mild forms of EACMV-UG2 together with the continued occurrence of severe forms in the field warrants further studies of virus-virus and virus-host interactions.