White yam (Dioscorea rotundata) plants exhibiting virus-like symptoms are co-infected with a new potyvirus and a new crinivirus in Ethiopia.

White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species.

Identification of yam mosaic virus as the main cause of yam mosaic diseases in Ethiopia.

Yam (Dioscorea spp.) is a staple food crop with cultural, nutritional and economic significance for millions of small-scale farmers in sub-Saharan Africa. While various virus-like symptoms such as mosaic and chlorosis are frequently observed in yam fields in Ethiopia, little information is available on the prevalence, distribution, and molecular characteristics of viruses causing these symptoms. The aim of this study was to investigate the incidence and distribution of yam viruses and determine the primary cause of yam mosaic diseases (YMD) in Ethiopia. Both symptomatic (n = 280) and asymptomatic (n = 110) yam leaf samples were collected and tested for potyviruses using ACP-ELISA. In addition, the symptomatic leaf samples were screened for yam mosaic virus (YMV), yam mild mosaic virus (YMMV), and cucumber mosaic virus (CMV) by DAS-ELISA. Subsequently, total RNA was extracted from 130 leaf samples comprising 94 symptomatic and 36 asymptomatic samples representing the different study areas. The representative RT-PCR amplicons (n = 6) were Sanger sequenced. The ACP-ELISA and DAS-ELISA results showed 9.2%, and 12.9% YMV infection, respectively, while the RT-PCR analysis showed 28.5% YMV positivity rate. Both CMV and YMMV were not detected in any of the samples tested. Thus, YMV is confirmed as the primary cause of YMD in Ethiopia. YMV isolates from Ethiopia shared 92-93% nucleotide identity among themselves and 85-99% with other YMV isolates from the GenBank. Phylogenetic analysis revealed that YMV isolates from Ethiopia, South America, and west-central Africa have the most recent common ancestor, while isolates from China and Japan are clustered as sister groups. This study enhances our understanding of YMV's genetic diversity and provides valuable information regarding the first report of YMV in Ethiopia.

Serological, biological and molecular characterization of viruses causing mosaic diseases on cabbage (Brassica sp L.) in Central Ethiopia.

The productivity of cabbage (Brassica oleracea var. capitata) in Ethiopia has been generally low due to several biotic and abiotic constraints among which are several viral diseases. There is a recent report indicating that this economically important vegetable is seriously affected in Ethiopia by cauliflower mosaic virus (CaMV) and turnip mosaic virus (TuMV). However, little information exists on the incidence and distribution of these viruses as the previous report is based on samples only from Addis Ababa. In this study, a total of 370 leaf samples were collected from 75 cabbage growing fields in Central Ethiopia in two rounds of survey. Two cabbage varieties locally known as "Habesha gomen" and "Tikur gomen" with virus-like symptoms were collected and tested with Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) using polyclonal antibodies specific to CaMV and TuMV. Results from serological diagnosis were confirmed with PCR and Sanger sequencing. The results indicated a high incidence and wide distribution of both viruses in Central Ethiopia with an average of 29.5% infection for CaMV and 40% for TuMV. Biological inoculation tests for CaMV or TuMV or both on healthy cabbage seedlings gave similar symptoms as those observed in the field. Symptom severity was higher with co-infection of CaMV and TuMV followed by TuMV single infection. BLAST analysis showed that TuMV and CaMV isolates from Ethiopia have nucleotide identity of 95-98% and 93-98%, respectively to previously reported isolates. Phylogenetic analysis revealed that CaMV isolates from Ethiopia are closely related to isolates from USA and Italy within Group II clade whereas TuMV isolates have close similarities with isolates from World B clade including isolates from Kenya, UK, Japan and the Netherlands. The identification of the causative agents of the mosaic disease observed on cabbage in Central Ethiopia may lay the foundation for future management studies.

Incidence of RNA viruses infecting taro and tannia in East Africa and molecular characterisation of dasheen mosaic virus isolates.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) plants growing in 25 districts across Ethiopia, Kenya, Tanzania and Uganda were surveyed for four RNA viruses. Leaf samples from 392 plants were tested for cucumber mosaic virus (CMV), dasheen mosaic virus (DsMV), taro vein chlorosis virus (TaVCV) and Colocasia bobone disease-associated virus (CBDaV) by RT-PCR. No samples tested positive for TaVCV or CBDaV, while CMV was only detected in three tannia samples with mosaic symptoms from Uganda. DsMV was detected in 40 samples, including 36 out of 171 from Ethiopia, one out of 94 from Uganda and three out of 41 from Tanzania, while none of the 86 samples from Kenya tested positive for any of the four viruses. The complete genomes of nine DsMV isolates from East Africa were cloned and sequenced. Phylogenetic analyses based on the amino acid sequence of the DsMV CP-coding region revealed two distinct clades. Isolates from Ethiopia were distributed in both clades, while samples from Uganda and Tanzania belong to different clades. Seven possible recombination events were identified from the analysis carried out on the available 15 full-length DsMV isolates. Nucleotide substitution ratio analysis revealed that all the DsMV genes are under strong negative selection pressure.

A novel nepovirus causing a chlorotic fleck disease on common bean (Phaseolus vulgaris L.).

Two common bean leaf samples from Ethiopia that had shown chlorotic fleck and veinal mosaic symptoms but tested ELISA-negative for known viruses were mechanically transmitted to herbaceous hosts to obtain virus isolates ET-773/4 and ET-779. Virus purification from Chenopodium quinoa systemically infected with ET-773/4 yielded icosahedral particles measuring ~ 30 nm in diameter and containing a single capsid protein of ~ 58 kDa, suggesting a nepovirus infection. Analysis of nucleotide sequences generated from RNA1 and RNA2 of the isolates indicated that they represent a distinct virus species in the genus Nepovirus. Surprisingly, the most closely related sequence in the GenBank database was that of Hobart nepovirus 3, an incompletely described metagenomic sequence obtained from honey bees in Tasmania. This new nepovirus from Ethiopia is provisionally named "bean chlorotic fleck virus".

A Review of Viruses Infecting Yam (Dioscorea spp.).

Yam is an important food staple for millions of people globally, particularly those in the developing countries of West Africa and the Pacific Islands. To sustain the growing population, yam production must be increased amidst the many biotic and abiotic stresses. Plant viruses are among the most detrimental of plant pathogens and have caused great losses of crop yield and quality, including those of yam. Knowledge and understanding of virus biology and ecology are important for the development of diagnostic tools and disease management strategies to combat the spread of yam-infecting viruses. This review aims to highlight current knowledge on key yam-infecting viruses by examining their characteristics, genetic diversity, disease symptoms, diagnostics, and elimination to provide a synopsis for consideration in developing diagnostic strategy and disease management for yam.

Molecular identification and characterization of badnaviruses infecting sugarcane in Ethiopia.

Sugarcane bacilliform virus (SCBV) is an economically important virus limiting sugarcane production worldwide. Although Ethiopia is a major sugarcane producer, and virus-like symptoms are frequently observed in sugarcane fields, there is a complete lack of information as to the occurrence, distribution and molecular properties of SCBV. This study was aimed to identify and characterize SCBV isolates in Ethiopia using molecular methods. Out of 292 leaf samples collected and tested by PCR, 76 samples (26% incidence level) were found SCBV-positive. Nucleotide sequence analysis results showed that three Ethiopian isolates (SCBV-EtS3, SCBV-EtS6 and SCBV-EtC10) shared high level of nucleotide identity (99.5-100%) among themselves and with SCBV isolates from China (accession numbers MH037614 and MH037915). Another isolate, SCBV-EtC2, shared maximum identity of 78% with the other three SCBV isolates from Ethiopia and 99.8% with SCBV isolates from China (KM214357 and KM214307). Based on phylogenetic analysis, isolates from Ethiopia were segregated into two different clusters. Isolates SCBV-EtS3, SCBV-EtS6 and SCBV-EtC10 clustered with SCBV-Q group and SCBV-EtC2 with SCBV-H group. This study provides information on the occurrence of SCBV for the first time in Ethiopia and also contributes to the understanding of the genetic diversity of SCBV. Keywords: Caulimoviridae; RNase H, Saccharum spp.; Sugarcane bacilliform virus.

Infectivity of an Infectious Clone of Banana Streak CA Virus in A-Genome Bananas (Musa acuminata ssp.).

We have characterized the complete genome sequence of an Australian isolate of banana streak CA virus (BSCAV). A greater-than-full-length, cloned copy of the virus genome was assembled and agroinoculated into five tissue-cultured plants of nine different Musa acuminata banana accessions. BSCAV was highly infectious in all nine accessions. All five inoculated plants from eight accessions developed symptoms by 28 weeks post-inoculation, while all five plants of M. acuminata AA subsp. zebrina remained symptomless. Symptoms were mild in six accessions but were severe in Khae Phrae (M. acuminata subsp. siamea) and the East African Highland banana accession Igisahira Gisanzwe. This is the first full-length BSCAV genome sequence reported from Australia and the first report of the infectivity of an infectious clone of banana streak virus.

Molecular characterisation of a putative new polerovirus infecting pumpkin (Cucurbita pepo) in Kenya.

Next-generation sequencing of RNA extracted from a pumpkin plant with mosaic symptoms in Kenya identified the presence of a polerovirus sequence closely related to pepo aphid-borne yellows virus (PABYV). The near-complete polerovirus sequence comprised 5,810 nucleotides and contained seven putative open reading frames (ORFs) with a genome organisation typical of poleroviruses. BLASTp analysis of the translated sequences of ORFs 0, 1 and 2 revealed that their amino acid sequences differed by more than 10% from the corresponding protein sequences of other poleroviruses. These results suggest that this virus is a putative novel member of the genus Polerovirus, which has been provisionally named "pumpkin polerovirus" (PuPV).

Assessment and optimization of rolling circle amplification protocols for the detection and characterization of badnaviruses.

The genus Badnavirus is characterized by members that are genetically and serologically heterogeneous which presents challenges for their detection and characterization. The presence of integrated badnavirus-like sequences in some host species further complicates detection using PCR-based protocols. To address these challenges, we have assessed and optimized various RCA protocols including random-primed RCA (RP-RCA), primer-spiked random-primed RCA (primer-spiked RP-RCA), directed RCA (D-RCA) and specific-primed RCA (SP-RCA). Using Dioscorea bacilliform AL virus (DBALV) as an example, we demonstrate that viral DNA amplified using the optimized D-RCA and SP-RCA protocols showed an 85-fold increase in badnavirus NGS reads compared with RP-RCA. The optimized RCA techniques described here were used to detect a range of badnaviruses infecting banana, sugar cane, taro and yam demonstrating the utility of RCA for detection of diverse badnaviruses infecting a variety of host plant species.

Characterization of a novel member of the family Caulimoviridae infecting Dioscorea nummularia in the Pacific, which may represent a new genus of dsDNA plant viruses.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

Characterization of an Australian isolate of taro bacilliform virus and development of an infectious clone.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

Characterization of badnaviruses infecting Dioscorea spp. in the Pacific reveals two putative novel species and the first report of dioscorea bacilliform RT virus 2.

The complete genome sequences of three new badnaviruses associated with yam (Dioscorea spp.) originating from Fiji, Papua New Guinea and Samoa were determined following rolling circle amplification of the virus genomes. The full-length genomes consisted of a single molecule of circular double-stranded DNA of 8106bp for isolate FJ14, 7871bp for isolate PNG10 and 7426bp for isolate SAM01. FJ14 and PNG10 contained three open reading frames while SAM01 had an additional open reading frame which partially overlapped the 3' end of ORF 3. Amino acid sequence analysis of ORF 3 from the three isolates confirmed the presence of conserved motifs typical of other badnaviruses. Phylogenetic analysis revealed the sequences to be closely related to other Dioscorea-infecting badnaviruses. FJ14 and PNG10 appear to be new species, which we have tentatively named dioscorea bacilliform ES virus (DBESV) and dioscorea bacilliform AL virus 2 (DBALV2), respectively, while SAM01 represents a Pacific isolate of the recently published dioscorea bacilliform RT virus 2 and is described as dioscorea bacilliform RT virus 2-[4RT] (DBRTV2-[4RT]).

Complete genome sequence of a novel zantedeschia mild mosaic virus isolate: the first report from Australia and from Alocasia sp.

The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMV-AU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.