RNA silencing of South African cassava mosaic virus in transgenic cassava expressing AC1/AC4 hp- RNA induces tolerance.

Cassava mosaic disease (CMD), caused by geminiviruses, is a major hurdle to cassava production. Due to the heterozygous nature of cassava, breeding for virus resistance is difficult, but cassava has been shown to be a good candidate for genetic engineering using RNA interference (RNAi). T This study reports on the ability of a transgene-derived RNA hairpin, homologous to an overlapping region of the SACMV replication associated protein and putative virus suppressor of silencing protein (AC1/AC4), to confer tolerance in the CMD-susceptible model cassava cultivar 60444. Three of the fourteen transgenic lines expressing SACMV AC1/AC4 hairpin-derived siRNAs showed decreased symptoms and viral loads compared to untransformed control plants. Expression of SACMV AC1/AC4 homologous siRNAs showed that this tolerance is most likely associated with post-transcriptional gene silencing of the virus. This is the first report of targeting the overlapping AC1 and AC4 genes of SACMV conferring CMD tolerance in cassava.

Sequences enhancing cassava mosaic disease symptoms occur in the cassava genome and are associated with South African cassava mosaic virus infection.

Cassava is an important food security crop in Sub-Saharan Africa. Two episomal begomovirus-associated sequences, named Sequences Enhancing Geminivirus Symptoms (SEGS1 and SEGS2), were identified in field cassava affected by the devastating cassava mosaic disease (CMD). The sequences reportedly exacerbated CMD symptoms in the tolerant cassava landrace TME3, and the model plants Arabidopsis thaliana and Nicotiana benthamiana, when biolistically co-inoculated with African cassava mosaic virus-Cameroon (ACMV-CM) or East African cassava mosaic virus-UG2 (EACMV-UG2). Following the identification of small SEGS fragments in the cassava EST database, the intention of this study was to confirm their presence in the genome, and investigate a possible role for these sequences in CMD. We report that multiple copies of varying lengths of both SEGS1 and SEGS2 are widely distributed in the sequenced cassava genome and are present in several other cassava accessions screened by PCR. The endogenous SEGS1 and SEGS2 are in close proximity or overlapping with cassava genes, suggesting a possible role in regulation of specific biological processes. We confirm the expression of SEGS in planta using EST data and RT-PCR. The sequence features of endogenous SEGS (iSEGS) are unique but resemble non-autonomous transposable elements (TEs) such as MITEs and helitrons. Furthermore, many SEGS-associated genes, some involved in virus-host interactions, are differentially expressed in susceptible (T200) and tolerant TME3) cassava landraces infected by South African cassava mosaic virus (SACMV) of susceptible (T200) and tolerant (TME3) cassava landraces. Abundant SEGS-derived small RNAs were also present in mock-inoculated and SACMV-infected T200 and TME3 leaves. Given the known role of TEs and associated genes in gene regulation and plant immune responses, our observations are consistent with a role of these DNA elements in the host's regulatory response to geminiviruses.

Empowering biotechnology in southern Africa: establishment of a robust transformation platform for the production of transgenic industry-preferred cassava.

Knowledge and technology transfer to African laboratories and farmers is an important objective for achieving food security and sustainable crop production on the sub-Saharan African continent. Cassava (Manihot esculenta Crantz) is a vital source of calories for more than a billion people in developing countries, and its potential industrial use for starch and bioethanol in the tropics is increasingly being recognized. However, cassava production remains constrained by the susceptibility of the crop to several biotic and abiotic stresses. For more than a decade, biotechnology has been considered an attractive tool to improve cassava as it substantially circumvents the limitations of traditional breeding, which is particularly time-consuming and tedious because of the high heterozygosity of the crop. A major constraint to the development of biotechnological approaches for cassava improvement has been the lack of an efficient and robust transformation and regeneration system. Despite some success achieved in genetic modification of the model cassava cultivar Tropical Manihot Series (TMS), TMS 60444, in some European and U.S. laboratories, the lack of a reproducible and robust protocol has not allowed the establishment of a routine transformation system in sub-Saharan Africa. In this study, we optimized a robust and efficient protocol developed at ETH Zurich to successfully establish transformation of a commercially cultivated South African landrace, T200, and compared this with the benchmark model cultivar TMS 60444. Results from our study demonstrated high transformation rates for both T200 (23 transgenic lines from 100 friable embryogenic callus (FEC) clusters) compared with TMS 60444 (32 transgenic lines from 100 FEC clusters). The success in transforming landraces or farmer-preferred cultivars has been limited, and the high transformation rate of an industry-preferred landrace in this study is encouraging for a feasible transformation program for cassava improvement in South Africa (SA), which can potentially be extended to other countries in southern Africa. The successful establishment of a robust cassava transformation and regeneration system in SA demonstrates the relevance of technology transfer to sub-Saharan Africa and highlights the importance of developing suitable and reliable techniques before their transfer to laboratories offering less optimal conditions.

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.

Genetic identification of two sweet-potato-infecting begomoviruses in South Africa.

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) that occurred either alone or in mixed infection in sweet potato (Ipomoea batatas) plants collected in Waterpoort, South Africa, are presented. One of the isolates corresponds to sweet potato mosaic-associated virus (SPMaV; SPMaV-[ZA:WP:2011]), with which it shared 98.5 % nucleotide identity, whereas the second isolate corresponds to a new variant of sweet potato leaf curl Sao Paulo virus (SPLCSPV; SPLCSPV-[ZA:WP:2011]), with which it shared 91.4 % nucleotide identity. The phylogenetic and recombination relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses were also investigated. SPLCSPV-[ZA:WP:2011] was found to be a natural recombinant of swepoviruses consisting of two distinct parental genomic sequences from SPLCSPV and sweet potato leaf curl Georgia virus (SPLCGV).

Molecular interaction between two cassava geminiviruses exhibiting cross-protection.

There are increasing reports of geminivirus mixed infections of field plant hosts. These mixed infections have been suggested to result in recombinations, emergence of new viruses and new disease epidemics. We previously reported the occurrence of mixed infection between African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) resulting in severe symptoms in cassava fields in Cameroon. Here, we show that reassortment of DNA-A and DNA-B components of ACMV and EACMCV does not form viable recombinants. However, in the presence of both components of either virus, the DNA-A component of the other virus replicated and spread in the absence of its DNA-B component. This result suggests that failure of ACMV and EACMCV to form viable recombinants is due to the inability of each DNA-A component to trans-replicate the heterologous DNA-B component. This study also shows that ACMV DNA-A induces a resistance to ACMV and EACMCV as indicated by absence or late symptom development. Moreover, this resistance enabled plants to recover from severe symptoms caused by EACMCV in Nicotiana benthamiana, suggesting that the resistance induced is not specific to ACMV and is consistent with the phenomenon of cross-protection between related viruses.

Widespread occurrence and diversity of Cassava brown streak virus (Potyviridae: Ipomovirus) in Tanzania.

Cassava brown streak disease (CBSD) has been a problem in Tanzania since 1936. Existing literature indicated limited distribution of the disease to low altitudes, usually <100 m above sea level, but the current geographical distribution of the disease was not known. Whether a single or many strains for the virus exist in Tanzania had not been reported to date. In this study, CBSD was recorded from sea level to ≈1,800 m above sea level. In total, 2,730 cassava plants were assessed for CBSD leaf symptoms in 91 fields and root symptoms were assessed at 81 sites. A sample was taken from each site for laboratory screening for Cassava brown streak virus (CBSV). CBSD mean foliar and root incidences were 38 and 36%, respectively. Reverse-transcription polymerase chain reaction of a partial 3'-terminal coat protein (CP) region of CBSV indicated the presence of CBSV in 67 of the 91 (73%) samples. Forty-three amplicons were sequenced, and phylogenetic comparisons with nucleotide sequences from GenBank (National Center for Biotechnology Information database) suggested that one major clade of CBSV primarily exists in Tanzania. However, there was nucleotide sequence divergence of up to 19% among the 42 isolates. In all, 42 of the 43 sequences had 80 to 100% nucleotide identity with 6 previously reported CP-CBSV sequences (from Mozambique and Tanzania). In total, 13 of 42 isolates had <80% nucleotide identities with three previously reported Ugandan CBSV sequences. One isolate, FJ687177, shared <78% sequence identity with the other Tanzanian sequences but was closely related (93%) to Ugandan isolates. It is likely that isolate FJ687177 may belong to a less widely distributed recently described species (clade 2) of CBSV, named Ugandan cassava brown streak virus (UCBSV).

First Report of Pepino mosaic virus Infecting Tomato in South Africa.

Pepino mosaic virus (PepMV) (genus Potexvirus) is a highly infectious virus that is responsible for significant losses in yield of tomato fruit (Solanum lycopersicum) across Europe, Asia, and the Americas in the last decade (1). During the winter growing season of 2008, uneven discoloration of tomato fruit from farms in Limpopo Province, South Africa, was detected at the Pretoria fresh produce market. Twenty fruit were randomly selected from five different suppliers in this region and the 100 samples were batched into subsamples of five fruit. Leaves with suspect mosaic and bubbling symptoms were also detected from farms in Limpopo and were thus sampled. Leaf and fruit samples were tested by double antibody-sandwich (DAS)-ELISA (2) using polyclonal antibodies against PepMV (Agdia, Elkhart, IN) combined with appropriate positive and negative controls. Fruit samples from two of the suppliers, and all leaf samples tested, reacted strongly with PepMV antibodies. Inoculum was prepared from pooled DAS-ELISA-positive leaf samples and inoculated onto 10, 4-week-old, susceptible S. lycopersicum cv. Rooikhaki seedlings. After 3 weeks, all inoculated plants had developed characteristic PepMV symptoms (2) including leaf bubbling, distortion, and curled leaves. Older leaves developed yellow spots and light/dark green leaf mosaic while apical regions were stunted and branches were distorted to form 'nettle-head' symptoms. Fruit surfaces were marbled or displayed flaming and uneven discoloration. Leaves from symptomatic plants were sampled for confirmation of PepMV infection by DAS-ELISA and all samples reacted positively with PepMV antibodies. Total RNA was extracted from 500-µg replicates of pooled leaf samples from infected plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and amplified by conventional two-step reverse-transcription-PCR using a PepMV-specific primer set: Ker 1 (2) and PepCP-R (4) for a 986-bp region, including the coat protein, of the PepMV genome. PCR products were cloned into pTZ57R/T vector (Fermentas, Vilnius, Lithuania [UAB]) and six clones were purified and sequenced using universal M13 primers (3). Phylogenetic analysis clustered the sequence with EU (European), LP (Peruvian), US1 (United States)/CH1 (Chilean) and US2/CH2 PepMV isolates. The PepMV isolate accessions for US2/CH2 (AY509927, FJ612601, EF408821, FJ212288, and DQ000985) were identified as the closest relatives based on 98 to 99% nucleotide similarity obtained using BLASTN. The coat protein sequence of the South African isolate was submitted to GenBank (Accession No. HQ872607). To our knowledge, this is the first confirmed report of PepMV in South Africa. Further studies are necessary to determine its incidence and spread in this country. The presence of PepMV signals the urgent need for adoption of appropriate phytosanitary measures to restrict the spread and impact of this virus. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) I. M. Hanssen et al. Plant Pathol. 58:450, 2009. (3) J. Messing. Method Enzymol. 101:20, 1983. (4) I. Pagán et al. Phytopathology 96:274, 2006.

Sequence diversity among badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and Nigeria.

We analysed the sequence diversity in the reverse transcriptase (RT)/ribonuclease H (RNaseH) coding region of 19 badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin, and Nigeria. Phylogenetic analysis of the deduced amino acid sequences revealed that the isolates are broadly divided into two distinct species, each clustering with Dioscorea alata bacilliform virus (DaBV) and Dioscorea sansibarensis bacilliform virus (DsBV). Fourteen isolates had 90-96% amino acid identity with DaBV, while four isolates had 83-84% amino acid identity with DsBV. One isolate from Benin, BN4Dr, was distinct and had 77 and 75% amino acid identity with DaBV and DsBV, respectively, and may be a member of a new badnavirus species infecting yam in West Africa. Viruses of the two main species were present in Ghana, Togo and Benin and were observed to infect both D. alata and D. rotundata indiscriminately. This is the first confirmed report of DsBV infection in yam in Ghana and Togo. The results of this study demonstrate that members of two distinct species of badnaviruses infect yam in the West African yam zone and suggest a putative new species, BN4Dr. We also conclude that these species are not confined to limited geographic regions or specific for yam host species. However, the three badnavirus species are serologically related. The sequence information obtained from this study can be used to develop PCR-based diagnostics to detect members of the various species and/or strains of badnaviruses infecting yam in West Africa.

First Report of Cucumber mosaic virus in Yams (Dioscorea spp.) in Ghana, Togo, and Republic of Benin in West Africa.

Yam (Dioscorea spp., family Dioscoreaceae) is one of the most important food crops cultivated in the West African yam zone comprising the forest and savannah areas of Nigeria, Ghana, Côte d'Ivoire, Republic of Benin, and Togo, which account for more than 90% of the 4.59 million ha of yam cultivation worldwide (1). A survey was conducted in 2005 to document viruses in yams in Ghana, Togo, and the Republic of Benin. Samples (1,405) from five species of yam showing mosaic, chlorosis, and stunting as well as asymptomatic plants were tested for Dioscorea bacilliform virus (DBV, genus Badnavirus), Yam mosaic virus (YMV, genus Potyvirus), and Yam mild mosaic virus (YMMV, genus Potyvirus), the three most common viruses infecting yams. In addition, samples were tested for Cucumber mosaic virus (CMV), since CMV was previously reported to infect yams in Côte d'Ivoire (2) and Nigeria (3). In protein-A sandwich-ELISA with polyclonal antibodies to a cowpea isolate of CMV, 23 of the 1,405 samples (6 of 218 samples from Togo, 13 of 628 samples from Ghana, and 4 of 559 samples from Republic of Benin) tested positive for CMV. The CMV-positive samples were from D. alata (N = 16) and D. rotundata (N = 7), whereas all samples from D. cayenensis, D. dumetorum, and D. bulbifera tested negative. CMV was detected as mixed infections with DBV, YMV, or YMMV in 21 of 23 samples. Some of these samples showed puckering, chlorosis, mottling, and crinkling, whereas some plants infected by two or more viruses were asymptomatic. Only two samples from D. rotundata had a single infection of CMV and they showed mild chlorotic symptoms in young leaves that were inconspicuous in mature leaves. In sap inoculations, the virus induced systemic mosaic in Nicotiana glutinosa. The presence of CMV in ELISA-positive yam samples was further confirmed by immunocapture-reverse transcription (IC-RT)-PCR using CMV antibodies as trapping antibody and oligonucleotide primers specific for a 485 nt corresponding to 3' end of the coat protein gene and C-terminal noncoding region of RNA-3 (4). To confirm the specificity of IC-RT-PCR, the 485-bp amplicons from an isolate from the Republic of Benin was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and three independent clones were sequenced from both orientations. Pairwise comparison of a consensus sequence (Accession No. EU274471) with corresponding sequences of other CMV isolates deposited in GenBank showed 99% identity at the nucleotide sequence level (Accession No. U22821) and revealed that the CMV isolate from yam belongs to sub-Group IA. To our knowledge, this is the first report of CMV infection in yams (D. alata and D. rotundata) in Ghana, Togo, and the Republic of Benin. Together with a previous documentation of CMV in D. alata and D. trifida in Côte d'Ivoire and Nigeria (2,3), this report adds to existing knowledge on distribution of CMV in yams with implications for yam production and germplasm distribution in the West Africa Region. References: (1) FAO. Online publication. FAOSTAT, 2007. (2) C. Fauquet and J. C. Thouvenel. Plant Viral Diseases in the Ivory Coast. ORSTROM: Documentation Techniques. Paris, 1987. (3) Jd'A. Hughes et al. Phytopathology 87:S45, 1997. (4) S. Wylie et al. Aus. J. Agric. Res. 44:41, 1993.

Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules.

The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses.

Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.

Characteristics of a Latent Potyvirus Seedborne in Guar and of Guar Green-Sterile Virus.

A symptomless, seedborne potyvirus was isolated from guar (Cyamopsis tetragonoloba) germ plasm in Griffin, Georgia. The host range and serology were similar to those reported for guar green-sterile virus (GGSV) and guar symptomless virus. Biological, serological, and molecular comparisons of the Georgia isolate and the South African GGSV indicate they are similar and are closely related to bean common mosaic potyvirus (BCMV). The Georgia isolate is seed-transmitted at a rate of up to 94% in guar line PI 340385. Sequence analysis of the capsid protein (CP) gene and the 3'-untranslated region (3'-UTR) showed that both isolates are 96% homologous. GenBank searches indicate that both are related to various strains of BCMV. The highest CP nucleotide sequence and 3'-UTR identities of 91 and 93%, respectively, were with those of BCMV-NL4. On this basis, both isolates from guar should be considered as strains of BCMV.