1. Epidemiological and Molecular Characterisation of Cassava Mosaic Geminiviruses and their Effect on Yield of Cassava in Zambia
Mathias Tembo1,3, M. Mataa1, J. Legg2, P.C. Chikoti3 and P. Ntawuruhunga4
1The University of Zambia, School of Agricultural Sciences, P.O. Box 32379, Lusaka, Zambia, 2International Institute of Tropical Agriculture, P.O. Box 34441, Dar-es-Salaam, Tanzania, 3Zambia Agriculture Research Institute, P/Bag 7, Lusaka, Zambia, 4International Institute of Tropical Agriculture, 32 Poplar Road, Avondale, Lusaka, Zambia
SP02-08
Abstract
Results
An epidemiological and molecular characterization study was carried out in four cassava growing provinces of Zambia in The study involved cassava mosaic disease (CMD) survey, virus isolate characterization, and evaluation of landraces and improved cassava varieties to African cassava mosaic virus (ACMV) and East Africa cassava mosaic virus (EACMV). The objectives of the study were i) to determine the incidence and severity of Cassava mosaic disease (CMD); ii) to determine genetic diversity of Cassava mosaic geminiviruses (CMGs); iii) to evaluate the effect of cassava mosaic disease (CMD) on yield of cassava. The results of the study indicated that CMD incidence and severity was 61.5% and 2.7, respectively. The results also showed that most of the diseased cassava plants were due to cutting infection (57.7%) compared to whitefly infection (3.8%). Phylogenetic analysis categorised variable distinct groups of EACMV associated with CMD. Among the germplasm evaluated for CMD response, Manyopola was found to be susceptible (3.7) whilst Kampolombo was resistant (1). Mweru had the highest root yield (1.6 kg plant-1) while Kapumba had the lowest root yield of (0.2 kg plant-1).
Fig 2: CMD Incidences in surveyed provinces
Disease incidences were significantly different (P>0.05) between the provinces (Figure 2). Lusaka province had the highest CMD incidence of 87.5% while Eastern province had the lowest CMD incidence of 43%. The variations could be attributed to whitefly populations and the effect these have on CMD spread. Average CMD incidence recorded over the areas surveyed was 61.5% with 57.7% being due to cutting infection and 3.8% being whitefly infections, results consistent with studies done elsewhere in Africa (Legg and Raya, 1998). The primary source of infection of CMD in farmers fields is through infected cuttings as a result of farmers recycling and exchanging infected cuttings among themselves . CMD severity during this period averaged 2.7.
Fig 5: Phylogenetic tree (1000 boot strap replications) obtained from comparisons of the partial A (left) and B (right) component sequences from Zambia and available NCBI genebank cassava mosaic geminivirus DNA-A and DNA-B component sequences, respectively.
Introduction
A phylogenetic analysis of the CP of Zambian CMGs yielded a tree (Fig 5, left) that showed lower genetic divergence but with substantial homology with sequences of ACMV-UGMild Uganda (AF ), ACMV-UGSvr Uganda (AF ), ACMV-[MG:MG310A1] Madagascar, and ACMV-CM39 Cameroon (AY ) with sequence identities of 97 %, 97 %, 97 % and 98 %, respectively. Comparisons of the sequences of the Zambian EACMV isolates with those of the corresponding DNA-B genomic regions of other CMGs in GeneBank (Fig. 5, right), showed greater variability within the EACMV species with similarity to the Kenyan isolates (EACMV-KE), Malawian isolates (EACMMV) and Tanzanian isolates (EACMV-TZT) with sequence identities of 96 %, 90 % and 96 %, respectively. There was sequence identity to the isolates of EACMZV at 84 %, EACMCV at 80 % and SACMV at 89 %.
There has been a marked increase in cassava production in Zambia. However the yields are still low (5.8 t/ha) (FAO, 2010), compared to cassava yields of 25-40t/ha (Plucknett, 2000). This is only % of average cassava yields. The most important disease affecting cassava is cassava mosaic disease (CMD) caused by the Cassava mosaic geminiviruses (CMGs). The specific objectives of the study were to determine the incidence and severity of CMD, characterize the cassava mosaic geminiviruses infecting cassava and evaluate the response of improved and local popular cassava varieties and landraces to cassava mosaic geminiviruses at a “hot spot”.
774 bp
Materials and Methods
A total of 80 fields were surveyed in 2013 covering Central, Western and Eastern and Lusaka provinces using the method described by Sseruwagi et al. (2004). Data on incidence, severity, type of infection and whitefly population was collected from cassava fields on 3 – 6 months old plants. Geo-coordinates (longitude, latitude and altitude) were recorded for each field location using a GARMIN eTrex 20 Geographical Positioning System (GPS) handset. Plant genomic DNA was extracted from 186 fresh leaf samples collected from CMD symptomatic young shoots using the method described by Dellaporta et al. (1983). Polymerase Chain Reaction (PCR) was carried out to detect presence of Cassava mosaic geminiviruses (CMGs). The specific primers JSP 1/2 and EAB555-F/R were used to amplify the cassava mosaic geminivirus (CMG) isolates for African cassava mosaic virus (ACMV) (Fondong and Pita, 1998) and East African cassava mosaic virus (EACMV) DNA-B partial fragment with expected product sizes of 774bp and 556bp, respectively (Ndunguru et al., 2005). 89 amplified products were sequenced and the amplicons were analysed using CLC Main Workbench 7 (2014). Aligned nucleotide sequences were compared with corresponding fragment sequences of CMGs generated using the BLAST programme. A field experiment (Fig 1) was established in order to assess the response of cassava cultivars to CMD infection. Nine CMD symptomless cassava varieties were arranged in a randomized complete block design (RCBD) with four replications. Data on CMD incidence, CMD symptom severity, whitefly population and weight of root tubers was collected and analysed using GenStat, 16th Edition computer Package (VSN International, 2013).
556 bp
Fig 3: Gel electrophoresis of DNA fragments of representative Zambian isolates of (upper) African cassava mosaic virus (774bp) and (lower) East African cassava mosaic virus (556bp) using the specific primers JSP001/002 and EAB555F/R, respectively.
Preliminary identification of the CMG’s with differential primers distinguished EACMV and ACMV. The amplification products are as shown in figure % of the samples tested positive for either ACMV or EACMV. PCR results showed that 57.5% of the isolates analysed had dual infection, 32.8% of the sampled plants were infected with ACMV, and 4.3% with EACMV (Figure 4. The results show a significant increase in dual infections and corresponding significant reductions in both single infections of ACMV and EACMV when compared to previous studies (Chikoti et al., 2013; Muimba-Kankolongo et al., 1997). There were more dual infections in Western and Central provinces than in Eastern and Lusaka provinces. This may be due to whitefly population (especially in Western province) and recycling of infected materials by the farmers.
Field experimental results showed CMD incidences differed significantly (P≤0.05) among the cassava genotypes (Table 1). CMD severity differed significantly between the genotypes (P≤0.05) 6 months after planting (MAP) on a severity 1-5 scale. CMD-affected improved cassava varieties expressed mild symptoms. Manyopola was found to be susceptible (3.7) whilst Kampolombo was resistant (1). Despite a significantly high number of whiteflies (P≤0.05) observed on Chila (30.7) and Mweru (28.5), the genotypes had the highest significant root yields (P≤0.05) of 1.6 and 1.2 -kg plant-1 respectively, 12 MAP. However, results indicated a lack of association between whitefly populations and spread of CMD in improved varieties as found in studies by Legg et al. (2003) and Sserubombwe et al. (2001).
Conclusions
It was established that cassava mosaic disease is still widely prevalent and that infected cuttings remains the primary source of CMD. Phylogenetic analysis and Restriction Fragment Length Polymorphism (RFLP) showed less variability within the ACMV isolates from the surveyed fields compared to the EACMV isolates. Among the nine genotypes evaluated, Manyopola was susceptible and Kampolombo was resistant. Other cassava varieties, which are recommended and released for cultivation, are also moderately susceptible to the disease.
Acknowledgements
This work was supported by the USAID Feed the Future programme through the International Institute of Tropical Agriculture (IITA) who provided financial and logistical support.
Fig 1: Field experiment established at a “Hot Spot” area for CMD and whiteflies in Rufunsa, Zambia
Fig 4: Distribution of cassava viruses in surveyed areas, 2013