Geminate structures of African cassava mosaic virus

Two types of geminate structures were purified from African cassava mosaic geminivirus (ACMV)-infected Nicotiana benthamiana plants and analyzed by electron cryomicroscopy and image reconstruction. After cesium sulfate density gradient centrifugation, they were separated into lighter top (T) and heavier bottom (B) components. T particles comigrated with host proteins, whereas B particles were concentrated in a cesium density typical for complete virions. Both particles were composed of two incomplete icosahedra of 11 capsomers each, but T particles were slightly larger (diameter, 22.5 nm) and less dense in the interior than B particles (diameter, 21.5 nm). T particles were frequently associated with small globules of approximately 14 nm diameter of unknown origin. The overall structure of ACMV, a begomovirus transmitted by whiteflies, was similar to that of Maize streak virus (MSV), a mastrevirus transmitted by leafhoppers, although the vertices of the icosahedra were less pronounced. Models of ACMV coat proteins based on Satellite tobacco necrosis virus support the exposure of parts of the molecule essential for transmission specificity by whiteflies and provide possible structural explanations for the smaller protrusion of the ACMV capsid relative to MSV. The differences of ACMV and MSV virion shapes are discussed with reference to their different animal vectors.     

Factors determining recovery and reversion in mosaic-affected African cassava mosaic virus resistant cassava

The severity and persistence of symptoms of mosaic virus disease were monitored during the first six months of two growing seasons in cassava of the African cassava mosaic virus (ACMV)-resistant cv. TMS 30572 either inoculated by grafting with a mild or severe strain or infected from the planted cutting. Symptomless shoots developed between January and March 1995 in two field trials differing in age by c. 6 months; this recovery occurred during particularly hot weather. Recovery was often only temporary in the plants inoculated with the severe strain and occurred later compared with those inoculated with the mild. In 1996, the weather was cooler and recovery that year was delayed until flowering, c. 7 months after planting, when recovered shoots were often produced from buds in the axils of symptomless leaves produced amongst diseased leaves. Most cuttings taken from the upper parts of diseased plants produced symptomless (reverted) progenies whereas most cuttings taken from the base of diseased plants produced diseased progenies. Reversion seemed to be associated with the recovery that had already occurred in the upper stems of the parent plants.     

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.     

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.     

Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV‐infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV‐infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus‐free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus‐free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus‐free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.     

The use of African cassava mosaic virus as a vector system for plants

This paper describes the development of a gene-displacement vector based on DNA1, one of two single stranded circular genomic components of a bipartite geminivirus, African cassava mosaic virus (ACMV). The DNA1 molecules of ACMV were cloned as dimers into a plant transformation vector and the constructs have been integrated into tobacco protoplasts by PEG-mediated DNA transfer. In transgenic plants extrachromosomal copies of DNA1 monomers could be detected. Deletion of the coat protein-encoding gene in chimeric constructs resulted in free DNA1 copies of reduced size, and extrachromosomal recombinant molecules were detected after displacement of the coat protein-encoding region by foreign DNA fragments of comparable size. Due to the absence of the second component of ACMV, DNA2, the transgenic plants are free from viral infection symptoms which allows the establishment of healthy transformants that carry a recombinant construct in an extrachromosomal form.     

Restriction of Virus Movement into Axillary Buds is an Important Aspect of Resistance in Cassava to African cassava mosaic virus

Axillary buds and bark samples of resistant, moderately resistant and susceptible (control) cassava genotypes either naturally infected under field conditions or experimentally inoculated by grafting were indexed for African cassava mosaic virus (ACMV). Virus detection was carried out using enzyme‐linked immunosorbent assay and polymerase chain reactions to determine the distribution of the virus within the plant and elucidate the genotypes response to virus movement. Significantly more bud and bark samples were positive for virus on the susceptible genotype TME 117 than resistant genotypes TMS 30001 and TMS 91/02319, or the moderately resistant genotype TMS 30572. Detectable virus concentration was significantly lower in the buds of moderately resistant and resistant genotypes than the susceptible control. Under field conditions, it was significant that more primary stem buds were infected than the buds of secondary and tertiary stems but such a gradient was not obvious with bark samples. Shoots that had asymptomic new leaves after the initial symptomatic leaves had no virus in their buds, but some of the bark samples from the same plants tested positive. A significant interaction was observed between year and stem type, and among year, genotype and stem type with respect to virus detection in bud and bark samples. Restriction of virus movement into axillary buds occurred in all the resistant and moderately resistant genotypes. This may explain ACMV‐infected stem cuttings of resistant genotypes producing healthy plants in subsequent generation.     

Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants inoculated with different strains of tobacco mosaic virus (TMV) inducing mosaic symptoms of widely varying severity were studied with in vivo chlorophyll fluorescence. This method was used to deduce photosynthetic electron transport efficiency in relation to symptom expression. The quantum yields of photosystem II (PS II) electron transport rate were significantly diminished in virus strains inducing loss of chlorophyll. The reduction in young mosaic-diseased leaves appeared to be due in part to a reduction in the fraction of open reaction centers, whereas in older leaves exhibiting less pronounced symptoms the reduction was mainly caused by a reduced efficiency of capture of excitation energy of open PS II reaction centers. Upon infection with any of the five virus strains PS II seemed to be irreversibly damaged in the inoculated leaves and the ones directly above, indicative of a possible increased susceptibility to photoinhibition in these leaves (Somersalo and Krause 1989) even when no symptoms were apparent. Symptom expression did not appear to be related to the influence of the virus on PS II activity, because the severest effects occurred in the inoculated leaves, which either remained symptomless or developed slight yellowing only. This study demonstrates the usefulness or modulated chlorophyll fluorescence measurements for the investigation of plant-virus interactions. It is particularly important when visual symptoms are absent.     

The effects of cassava mosaic virus disease on yield and compensation in mixed stands of healthy and infected cassava

The effects of cassava mosaic virus disease (CMD) on yield in fully and partly infected stands of cassava were investigated in field trials in Uganda in 1990-91 and 1991-92. Three cultivars (Ebwanateraka, Bao and Bukalasa 1 l), each at three levels of cutting infection (O%, 50% and 100%) and harvested 510 and 15 months after planting (MAP) were used in a randomised block design with split-split plots and four replicates. Moreover, yield and growth data for individual infected and uninfected plants were considered in relation to the health status of their nearest neighbours. In each experiment, fresh tuberous root yields of plants from 100% infected plots gave sigdicantly lower yields than those from 0% or 50% infected plots at each harvest date and the losses were greatest in cv. Bao. Yields of plants from 0% and 50% plots for each of the three cultivars were not significantly different, 10 and 15 MAP. The loss in yield differed between cultivars and harvest dates. Fresh stem, leaf and root yields and the number of tuberous roots were influenced by the health status of the plants harvested and that of their nearest neighbours. Uninfected plants surrounded by infected ones had more roots and heavier total fresh root, stem and leaf weights than those surrounded by uninfected ones. Overall, 26% and 42% compensation was recorded in 1990-91 and 1991-92, respectively. The effects of CMD on cassava production and of compensation in mixed stands of infected and uninfected plants are discussed, especially in relation to control strategies such as roguing.     

Field studies on the spread of African cassava mosaic

The spread of African cassava mosaic disease (ACMV) into healthy cassava fields was recorded at weekly intervals. In addition, 21 yellow water traps were placed in one field and the number of whiteflies caught was recorded twice a week. The number of Bemisia spp. feeding on cassava was also estimated. The results indicate that the pattern of disease spread is related to the pattern of infestation with Bemisia. Airborne whiteflies carried by the south-west prevailing wind alighted preferentially on cassava plants along the upwind edges (south and west borders) of the plantings. The pattern of incidence of mosaic disease resembled that of whiteflies. Along the SW-NE diagonal, there was a gradient of disease incidence with a maximum at the SW corner block. Similar gradients occurred in three different fields and they were maintained throughout the 6-month study, although gradually flattening with time. There were indications that the reservoirs both of the virus and of the vectors were located some distance upwind from the experimental fields.     

Characterisation of cauliflower mosaic virus DNA forms isolated from infected turnip leaves.

Several different forms of cauliflower mosaic virus (CaMV) DNA were detected in nucleic acid preparations from CaMV-infected turnip leaves. As well as supercoiled and open-circular molecules, various linear DNA structures were identified. The relative amounts of these DNA forms varied in plants infected with different CaMV isolates. Restriction enzyme mapping and one- and two-dimensional gel electrophoresis revealed the presence of linear molecules apparently formed by breaks in the second strand at each of the three discontinuities. Two major linear DNA forms are double-stranded over part of their length and appear to have single-stranded extensions of the -strand of variable length. Since these DNA forms are not produced during extraction and probably exist as unencapsidated or partially encapsidated molecules, they may represent intermediates either in DNA replication or in virion assembly.     

Dark and light green tissues of tobacco leaves systemically infected with tobacco mosaic virus

There are significant changes in the structure of the upper tobacco (Nicotiana tabacum L.) leaves systemically infected with tobacco mosaic virus (TMV) especially in the light green tissue (LGT). Dark green areas (DGI) had intermediate status between healthy tissue and LGT. DGI contained significantly less infectious TMV and viral antigen than the LGT. The DGI, LGT and healthy tissues did not differ in the permeability of cell membranes and in the set of acidic pathogenesis-related (PR) proteins but the total content of PR-proteins in the healthy plants was higher than in the infected ones with the DGI being intermediate between healthy tissue and LGT. The crude leaf extracts from DGI and LGT showed less total ribonuclease activity and ribonuclease isozymes in comparison with control.