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.

Lanai: A small, fast growing tomato variety is an excellent model system for studying geminiviruses.

Geminiviruses are devastating single-stranded DNA viruses that infect a wide variety of crops in tropical and subtropical areas of the world. Tomato, which is a host for more than 100 geminiviruses, is one of the most affected crops. Developing plant models to study geminivirus-host interaction is important for the design of virus management strategies. In this study, "Florida Lanai" tomato was broadly characterized using three begomoviruses (Tomato yellow leaf curl virus, TYLCV; Tomato mottle virus, ToMoV; Tomato golden mosaic virus, TGMV) and a curtovirus (Beet curly top virus, BCTV). Infection rates of 100% were achieved by agroinoculation of TYLCV, ToMoV or BCTV. Mechanical inoculation of ToMoV or TGMV using a microsprayer as well as whitefly transmission of TYLCV or ToMoV also resulted in 100% infection frequencies. Symptoms appeared as early as four days post inoculation when agroinoculation or bombardment was used. Symptoms were distinct for each virus and a range of features, including plant height, flower number, fruit number, fruit weight and ploidy, was characterized. Due to its small size, rapid growth, ease of characterization and maintenance, and distinct responses to different geminiviruses, "Florida Lanai" is an excellent choice for comparing geminivirus infection in a common host.

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.

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.

The complete genome sequence of the Tanzanian strain of Cassava brown streak virus and comparison with the Ugandan strain sequence.

The complete genome sequence for an isolate of the Ugandan and Tanzanian strain types of Cassava brown streak virus have been determined using the novel approach of non-directed next generation sequencing. Comparison of the genome sequences revealed that CBSV is highly heterogeneous at the isolate level as well as the strain level. The isolate of the Ugandan strain was found to have a genome 9,070 nucleotides long coding for a polypeptide with 2,902 amino acid residues. The isolate of the Tanzanian strain was 9,008 nucleotides long and coded for a polypeptide with 2,916 amino acid residues. Nucleotide identity between the isolates across the genome was 76%, with protein encoding regions 57-77% and individual proteins had 65-91% amino acid similarity. In addition between the two strains four protein products (PIPO, CI, NIa-Vpg and coat protein) varied in size and an unusual HAM1-like protein, whilst of identical nucleotide length, was found to have the lowest homology. The implication of diversity of CBSV is discussed in the context of speciation, evolution, development of diagnostics, and breeding for resistance.

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses.

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.

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.

First Reports of Soilborne wheat mosaic virus and Wheat spindle streak mosaic virus in Africa.

During 1997 and 1998, virus symptoms similar to those of Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV) were observed on nine wheat cultivars (Triticum aestivum cvs. Deka, Gamtoos, Lorie II, MM2, Nata, Nkwazi, P7, Scepter, and Scan) in the Central, Copper-Belt, Lusaka, and Southern provinces of Zambia. Symptoms were observed between June and August on wheat, which in Zambia is an irrigated crop grown during the cooler months (May to August). In fields suspected to be infected with SBWMV, irregularly distributed, circular patches of severely stunted sparse plants were observed. Because of these symptoms, the syndrome is described in Zambia as the "crater disease." Infection was more common on light to medium sandy-loam clay soils, and affected patches were particularly common along the field edges and in poorly drained areas. Such waterlogged conditions are conducive to the multiplication and spread of Polymyxa graminis, the protist vector of SBWMV (1). Affected plants initially showed chlorotic streaks on all leaves, which became uniformly yellow and eventually necrotic. The roots of these plants were slightly swollen and enlarged and are likened to "Rastafarian pleats" locally. In fields suspected to be infected with WSSMV, symptomatic plants were observed in the border rows of affected fields. Chlorotic streaks and mosaics were observed on the leaves of affected plants, and the tips of these leaves were also frequently twisted. Using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), SBWMV and WSSMV were positively identified in symptomatic plants. In total, 81 plants from the four provinces were tested, and 72 and 37% were infected with SBWMV and WSSMV, respectively. Identification was confirmed by DAS-ELISA using antisera from W. Huth (BBA-Braunschweig, Germany) and C. Rubies-Autonell (Bologna University, Italy) for SBWMV and using antisera from W. Huth (BBA-Braunschweig, Germany) and G. Bergstrom (Cornell University, New York) for WSSMV. Further confirmation of the identity of the two viruses was provided by the reaction of 12 indicator species (Chenopodium amaranticolor, C. quinoa, C. hybridum, Digitaria milanjiana, Eleusine indica, Oryza sativa (cv. IITA 212), Panicum maximum, Rottboellia cochinchinensis, Setaria verticillata, Sorghum bicolor(cv. Sima), S. halepense, and Triticum aestivum (cvs. Lucal, Kwale, Lorie II, Nkanga, 128, and GV 4-12) in mechanical transmission studies using infected leaf sap. This is the first report of SBWMV and WSSMV not only in Zambia but also Africa. The area of wheat grown in Zambia has risen in the past several years to approximately 18,000 ha per annum. However, annual wheat yield (60,000 tons) has not risen to match this increase. The effect of SBWMV and WSSMV on yield in Zambia has not yet been measured, but both viruses cause serious losses in other countries (1-3) and the severity of the symptoms suggests that significant yield reductions are likely. Furthermore, no Zambian wheat cultivars are known to be resistant to either virus. Generally, wheat production fails to meet demand in the country and therefore further yield losses due to virus infection could be disastrous. References: (1) M. K. Brakke. CMI/AAB Desc. of Plant Viruses 77, 1971. (2) J. T. Slykhuis. Phytopathology 60:319, 1970. (3) V. Vallega and C. Rubies-Autonell. Plant Dis. 69:64, 1985.

Rare symptoms and conidial variation in Septoria lycopersici in Zambia.

Surveys during the rainy season of 1996 showed that Septoria lycopersici developed two different types of leaf spots on tomatoes grown in kitchen gardens at the University of Zambia Campus and in nearby gardens. The two types of spots could be easily distinguished on the basis of their external morphology. One type, designated as T1, began as dark brown spots of less than 1 mm diameter. Upon increase in size, the spots differentiated into a dark brown outer ring and a grey centre, reaching a maximum diameter of 5 mm. Spots of this type are common and have been described in reports on Septoria leaf spot disease. A second type of spot found in our survey was designated as T2. This Septoria spot was greyish brown with several concentric rings of shrunken leaf tissue. The type T2 spots were larger and did not differentiate into two parts as in T1. The T2 spot diameter was 4-12 mm. The conidia showed differences in curvature, and significantly length, between T1 and T2. The study has shown that S. lycopersici in Zambia is variable.