Analyses of seven new whole genome sequences of cassava brown streak viruses in Mozambique reveals two distinct clades: evidence for new species.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) is a major constraint to cassava production in Mozambique. Full genome sequences of CBSD-associated virus isolates contribute to the understanding of genetic diversity and the development of new diagnostic primers that can be used for early detection of the viruses for sustainable disease management. This study determined seven new whole CBSV genomes from total RNA isolated from cassava leaves with CBSD symptoms collected from Nampula and Zambezia in Mozambique. Phylogenetic analyses of the new genomes with published CBSV and UCBSV sequences in GenBank grouped the CBSV isolates from Mozambique into two distinct clades together with CBSV isolates from Tanzania. Clade 1 and 2 isolates shared low nucleotide (79.1-80.4%) and amino acid (86.5-88.2%) sequence identity. Further, comparisons within the seven new CBSV isolates, and between them and the single published complete CBSV sequence (CBSV_MO_83_FN434436) from Mozambique, revealed nucleotide sequence identities of 79.3-100% and 79.3-98%, respectively, and amino acid identities of 86.7-100% and 86.7-98.8%. In addition, using RDP4, a recombination analysis comprising all CBSV and UCBSV genome sequences from GenBank detect 11 recombination events. Using several comprehensive evolutionary models and statistical programs, it was confirmed that CBSV and UCBSV are distinct virus species, with an additional probable new species (clade 2).

Cassava brown streak disease in Rwanda, the associated viruses and disease phenotypes.

Cassava brown streak disease (CBSD) was first observed on cassava (Manihot esculenta) in Rwanda in 2009. In 2014 eight major cassava-growing districts in the country were surveyed to determine the distribution and variability of symptom phenotypes associated with CBSD, and the genetic diversity of cassava brown streak viruses. Distribution of the CBSD symptom phenotypes and their combinations varied greatly between districts, cultivars and their associated viruses. The symptoms on leaf alone recorded the highest (32.2%) incidence, followed by roots (25.7%), leaf + stem (20.3%), leaf + root (10.4%), leaf + stem + root (5.2%), stem + root (3.7%), and stem (2.5%) symptoms. Analysis by RT-PCR showed that single infections of Ugandan cassava brown streak virus (UCBSV) were most common (74.2% of total infections) and associated with all the seven phenotypes studied. Single infections of Cassava brown streak virus (CBSV) were predominant (15.3% of total infections) in CBSD-affected plants showing symptoms on stems alone. Mixed infections (CBSV + UCBSV) comprised 10.5% of total infections and predominated in the combinations of leaf + stem + root phenotypes. Phylogenetic analysis and the estimates of evolutionary divergence, using partial sequences (210 nt) of the coat protein gene, revealed that in Rwanda there is one type of CBSV and an indication of diverse UCBSV. This study is the first to report the occurrence and distribution of both CBSV and UCBSV based on molecular techniques in Rwanda.

Cassava whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in East African farming landscapes: a review of the factors determining abundance.

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a pest species complex that causes widespread damage to cassava, a staple food crop for millions of households in East Africa. Species in the complex cause direct feeding damage to cassava and are the vectors of multiple plant viruses. Whilst significant work has gone into developing virus-resistant cassava cultivars, there has been little research effort aimed at understanding the ecology of these insect vectors. Here we assess critically the knowledge base relating to factors that may lead to high population densities of sub-Saharan African (SSA) B. tabaci species in cassava production landscapes of East Africa. We focus first on empirical studies that have examined biotic or abiotic factors that may lead to high populations. We then identify knowledge gaps that need to be filled to deliver sustainable management solutions. We found that whilst many hypotheses have been put forward to explain the increases in abundance witnessed since the early 1990s, there are little published data and these tend to have been collected in a piecemeal manner. The most critical knowledge gaps identified were: (i) understanding how cassava cultivars and alternative host plants impact population dynamics and natural enemies; (ii) the impact of natural enemies in terms of reducing the frequency of outbreaks and (iii) the use and management of insecticides to delay the development of resistance. In addition, there are several fundamental methodologies that need to be developed and deployed in East Africa to address some of the more challenging knowledge gaps.

Phylogenetic characterization of East African cassava mosaic begomovirus (Geminiviridae) isolated from Manihot carthaginensis subsp. glaziovii (Müll.Arg.) Allem., from a non-cassava growing region in Tanzania.

Manihot carthaginensis subsp. glaziovii (Müll.Arg.) Allem., a wild relative of cassava, native to Brazil, is one of the popular agroforestry trees used for hedges and/or boundary plants surrounding homesteads and farms and also harbours cassava mosaic begomoviruses (CMBs) and cassava brown streak ipomoviruses. Sequences of the DNA-A component of East African cassava mosaic virus (EACMV) isolates from M. carthaginensis subsp. glaziovii (Müll.Arg.) Allem., collected from non-cassava growing areas of Tanzania were characterized. Thirteen full length DNA-A sequences were analysed together with 15 already reported EACMV sequences and six CMB species reference genomes. The results show 96 to 100% nucleotide sequence identity with EACMV isolates from Kenya. Phylogenetic analysis revealed that EACMV isolates from M. carthaginensis subsp. glaziovii (Müll.Arg.) Allem, belong to a single cassava mosaic begomovirus species. The EACMV monophyletic clade is distinct from all other CMB species. The presence of Cassava infecting begomoviruses in wild cassava relative growing from traditionally non cassava growing region serve as inoculum sources for cassava-infecting begomoviruses and therefore their eradication is key in the sustainable management of CMBs, especially in the non-cassava growing areas.

Cassava brown streak disease and the sustainability of a clean seed system.

One method of reducing disease in crops is the dissemination of disease-free planting material from a multiplication site to growers. This study assesses the validity and sustainability of this method for cassava brown streak disease, a threat to cassava crops across East Africa. Using mathematical modelling, the effects of different environmental and control conditions on pathogen spread were determined in a single-field multiplication site. High disease pressure, through large vector populations and disease in the surrounding area, combined with poor roguing practice, resulted in unsuccessful disease suppression. However, fields may produce sufficiently clean material for replanting if these factors can be overcome. Assessing the sustainability of a low-pressure system over multiple harvests, well-managed fields were found to maintain low disease levels, although producing sufficient cuttings may prove challenging. Replanting fields from the previous harvest does not lead to degeneration of planting material, only cutting numbers, and the importation of new clean material is not necessarily required. It is recommended that multiplication sites are only established in areas of low disease pressure and vector population density, and the importance of training in field management is emphasized. Cultivars displaying strong foliar symptoms are to be encouraged, as these allow for effective roguing, resulting in negative selection against the disease and reducing its spread. Finally, efforts to increase plant multiplication rates, the number of cuttings that can be obtained from each plant, have a significant impact on the sustainability of sites, as this represents the primary limiting factor to success.

A single-tube duplex and multiplex PCR for simultaneous detection of four cassava mosaic begomovirus species in cassava plants.

A single-tube duplex and multiplex PCR was developed for the simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava mosaic Zanzibar virus (EACMZV), four cassava mosaic begomoviruses (CMBs) affecting cassava in sub-Saharan Africa. Co-occurrence of the CMBs in cassava synergistically enhances disease symptoms and complicates their detection and diagnostics. Four primer pairs were designed to target DNA-A component sequences of cassava begomoviruses in a single tube PCR amplification using DNA extracted from dry-stored cassava leaves. Duplex and multiplex PCR enabled the simultaneous detection and differentiation of the four CMBs, namely ACMV (940bp), EACMCV (435bp), EACMMV (504bp) and EACMZV (260bp) in single and mixed infections, and sequencing results confirmed virus identities according to the respective published sequences of begomovirus species. In addition, we report here a modified Dellapotra et al. (1983) protocol, which was used to extract DNA from dry and fresh cassava leaves with comparable results. Using the duplex and multiplex techniques, time was saved and amount of reagents used were reduced, which translated into reduced cost of the diagnostics. This tool can be used by cassava breeders screening for disease resistance; scientists doing virus diagnostic studies; phytosanitary officers checking movement of diseased planting materials, and seed certification and multipliers for virus indexing.

First Report of Iris yellow spot virus Infecting Onion in Kenya and Uganda.

Onion (Allium cepa L.) is one of the key vegetables produced by small-holder farmers for the domestic markets in Sub-Saharan Africa. Biotic factors, including infestation by thrips pests such as Thrips tabaci Lindeman, can inflict as much as 60% yield loss. Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) transmitted by T. tabaci is an economically important viral pathogen of bulb and seed onion crops in many onion-growing areas of the world (2,4). In Africa, IYSV has been reported in Reunion (1) and South Africa (3). In September 2009, symptoms suspected to be caused by IYSV were observed on onions and leeks cultivated in Nairobi, Kenya. Symptoms consisted of spindle-shaped, straw-colored, irregular chlorotic lesions with occasional green islands on the leaves. The presence of the virus was confirmed with IYSV-specific Agdia Flash kits (Agdia Inc., Elkart, IN). Subsequently, surveys were undertaken in small-holder farms in onion production areas of Makueni (January 2010) and Mwea (August 2010) in Kenya and Kasese (January 2010) and Rwimi (January 2010) in Uganda. The incidence of disease in these locations ranged between 27 and 72%. Onion leaves showing symptoms of IYSV infection collected from both locations tested positive for the virus by double-antibody sandwich-ELISA with IYSV-specific antiserum (Agdia Inc). IYSV infection was confirmed by reverse transcription-PCR with primers IYSV-465c: 5'-AGCAAAGTGAGAGGACCACC-3' and IYSV-239f: 5'-TGAGCCCCAATCAAGACG3' (3) as forward and reverse primers, respectively. Amplicons of approximately 240 bp were obtained from all symptomatic test samples but not from healthy and water controls. The amplicons were cloned and sequenced from each of the sampled regions. Consensus sequence for each isolate was derived from at least three clones. The IYSV-Kenya isolate (GenBank Accession No. HQ711616) had the highest nucleotide sequence identity of 97% with the corresponding region of IYSV isolates from Sri Lanka (GenBank Accession No. GU901211), followed by the isolates from India (GenBank Accession Nos. EU310287 and EU310290). The IYSV-Uganda isolate (GenBank Accession No. HQ711615) showed the highest nucleotide sequence identity of 95% with the corresponding region of IYSV isolates from Sri Lanka (GenBank Accession No. GU901211) and India (95% with GenBank Accession Nos. EU310274 and EU310297). To our knowledge, this is the first report of IYSV infecting onion in Kenya and Uganda. Further surveys and monitoring of IYSV incidence and distribution in the region, along with its impact on the yield, are under investigation. References: (1) L. J. du Toit et al. Plant Dis. 91:1203, 2007. (2) D. H. Gent et al. Plant Dis. 88:446, 2004. (3) H. R. Pappu et al. Plant Dis 92:588, 2008. (4) H. R. Pappu et al. Virus Res. 141:219, 2009.

Mitochondrial DNA variability and development of a PCR diagnostic test for populations of the whitefly Bemisia afer (Priesner and Hosny).

The whitefly, Bemisia afer (Hemiptera; Aleyrodidae), is emerging as a major agricultural pest. The current identification methods based on adult and pupal morphology are laborious and unreliable. A diagnostic polymerase chain reaction (PCR) protocol was developed for the first time in this study to discriminate B. afer from other whitefly species. Primers specific to mitochondrial cytochrome oxidase I gene (mtCOI) were designed to amplify a band of approx 650 bp. The PCR products were sequenced from B. afer samples collected from Malawi, Tanzania, Uganda, Zanzibar, and the United Kingdom. Phylogenetic analyses of mtCOI sequences and those of reference B. afer sequences clustered the African B. afer separately from the UK and Chinese populations and from other whitefly species. The African cluster was divided into two clades by parsimony and neighbor-joining methods. This indicates the existence of at least two genotypic clusters of B. afer, which are diverged by 0.8 to 3.2% nucleotide (nt) identities. Analysis of molecular variance indicated that these differences were the result of within population variation but were insufficient to identify discrete populations. Among the whitefly species used in the analysis, B. afer was equally dissimilar to Bemisia tabaci and Bemisia tuberculata (21.3-26.2% nt identities). As is the case for B. tabaci, these data show that mtCOI sequences are informative also for identifying B. afer variants, which lack distinguishing morphological features.

Methods of surveying the incidence and severity of cassava mosaic disease and whitefly vector populations on cassava in Africa: a review.

Field surveys in many cassava growing areas of Africa have assessed the incidence and severity of cassava mosaic disease (CMD), populations of the whitefly vector (Bemisia tabaci), and the distribution of cassava mosaic begomoviruses (CMBs). The methods employed differ greatly between countries and attempts at standardization were made in recent CMD surveys in East and Central Africa, notably in the systemwide Whitefly IPM Project, which provides a paradigm for future work on CMBs and whiteflies on cassava in Africa and also elsewhere. However, there is a need for greater standardization so as to assess the continued expansion of the current CMD pandemic in eastern Africa. Standardized methods will facilitate the collection of reliable data, which can be used to predict future disease spread, develop appropriate management strategies and compare disease development between seasons and locations. In this review, the methods used and the problems encountered during such surveys are discussed and recommendations made on future procedure.