Molecular Surveillance, Prevalence, and Distribution of Cacao Infecting Badnavirus Species in Côte d'Ivoire and Ghana.

The cacao swollen shoot disease (CSSD) caused by a complex of badnavirus species presents a major challenge for cacao production in West Africa, especially Ghana and Côte d'Ivoire. In this study, CSSD species detection efficiency, diversity, and geographic distribution patterns in cacao plantations in Ghana and Côte d'Ivoire were investigated through field surveillance, PCR detection assays, sequencing of positive amplicons, and phylogeographic clustering. Cumulatively, the detection efficiency of the tested CSSD primer sets that were targeting the movement protein domain of the virus ranged from 0.15% (CSSD-3 primer) to 66.91% (CSSD-1 primer) on all the symptomatic cacao leaf samples assessed. The identified CSSD species differed phylogenetically and overlapped in distribution, with the cacao swollen shoot Togo B virus (CSSTBV) (n = 588 sequences) being the most prevalent and widely distributed compared to the other CSSD species that were encountered in both countries. Geographically, the cacao swollen shoot CE virus (CSSCEV) species (n = 124 sequences) that was identified was largely restricted to the bordering regions of Ghana and Côte d'Ivoire. These results provide updated knowledge of the geographic distribution of the key CSSD species and their diagnostic efficiency and, thus, provide guidance in identifying locations for structured testing of cacao germplasm and optimal diagnostics for the predominant CSSD species in Ghana and Côte d'Ivoire.

Identification of a New Badnavirus in the Chinaberry (Melia azedarach) Tree and Establishment of a LAMP-LFD Assay for Its Rapid and Visual Detection.

The Chinaberry tree, a member of the Meliaceae family, is cultivated in China for use in traditional medicines. In 2020, Chinaberry trees with leaf deformation symptoms were found in Hangzhou, Zhejiang province, China. In order to identify possible pathogenic viruses, a symptomatic sample was subjected to deep sequencing of small interfering RNAs. Assembly of the resulting sequences led to the identification of a novel badnavirus, provisionally designated Chinaberry tree badnavirus 1 (ChTBV1). With the recent development of China's seedling industry and increasing online shopping platforms, the risk of tree virus transmission has increased substantially. Therefore, it is important to detect the occurrence of ChTBV1 to ensure the safety of the Chinaberry tree seedling industry. Here, we describe the development and validation of a sensitive and robust method relying on a loop-mediated isothermal amplification (LAMP) assay, targeting a 197 nt region, to detect ChTBV1 from Chinaberry tree leaves. The LAMP assay was also adapted for rapid visualization of results by a lateral flow dipstick chromatographic detection method.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Identification and distribution of novel badnaviral sequences integrated in the genome of cacao (Theobroma cacao).

Theobroma cacao is one of the most economically important tropical trees, being the source of chocolate. As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot virus disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous T. cacao bacilliform virus, varied in type with each predominating in a specific genetic group. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of one specific insert, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities and may also aid novel methods to reduce the impact of such viruses in this crop.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Molecular Viral Diagnosis and Sanitation of Yam Genetic Resources: Implications for Safe Yam Germplasm Exchange.

Yam (Dioscorea spp.) is an important crop in tropical and subtropical regions. Many viruses have been recently identified in yam, hampering genetic conservation and safe international exchanges of yam germplasm. We report on the implementation of reliable and cost-effective PCR-based detection tools targeting eight different yam-infecting viruses. Viral indexing of the in vitro yam collection maintained by the Biological Resources Center for Tropical Plants (BRC-TP) in Guadeloupe (French West Indies) unveiled a high prevalence of potyviruses, badnaviruses, Dioscorea mosaic associated virus (DMaV) and yam asymptomatic virus 1 (YaV1) and a high level of coinfections. Infected yam accessions were subjected to a combination of thermotherapy and meristem culture. Sanitation levels were monitored using PCR-based and high-throughput sequencing-based diagnosis, confirming the efficacy and reliability of PCR-based detection tools. Sanitation rates were highly variable depending on viruses. Sixteen accessions were successfully sanitized, paving the way to safe yam germplasm exchanges and the implementation of clean seed production programs worldwide.

First identification and molecular characterization of a new badnavirus infecting camellia.

A new badnavirus was identified in an ornamental camellia tree with yellow mottle symptom. The complete circular double-stranded DNA genome of this virus was found to consist of 8,203 bp. Its genome organization is typical of badnaviruses, containing three open reading frames (ORFs). ORFs 1 and 2 encode putative proteins with unknown functions. ORF3 encodes a large polyprotein that contains almost all of the conserved domains of badnaviruses. The virus shares 55-62% nucleotide sequence identities with other badnaviruses in the RT+RNase H region. Phylogenetic analyses placed it in group I of the genus Badnavirus. Therefore, this virus, which is tentatively named "camellia Lemon Glow virus", should represent a new species of the genus Badnavirus. This virus was found to be present in approximately a quarter of camellia trees tested.

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Genomic characterisation of a newly identified badnavirus infecting ivy (Hedera helix).

High-throughput sequencing (HTS) was used to investigate ringspots on ivy (Hedera helix) leaves. De novo assembly of HTS data generated from a total RNA extract from these leaves yielded a contig with sequence similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 8,885 nucleotides and has three open reading frames (ORFs). Genome organisation and phylogenetic analysis identifies this newly identified virus as a new member of the genus Badnavirus for which we propose the name "ivy ringspot-associated virus" (IRSaV).

Molecular characterization of a new badnavirus infecting green Sichuan pepper (Zanthoxylum schinifolium).

A new virus with a circular double-stranded DNA genome was discovered in green Sichuan pepper with vein clearing symptoms. Its complete genome of 8,014 bp contains three open reading frames (ORF) on the plus strand, which is typical of members of the genus Badnavirus in the family Caulimoviridae. Sequence comparisons revealed that the new virus has the highest nucleotide sequence identity with grapevine vein-clearing virus (GVCV). In particular, the identity of the two viruses in the ORF3 RT-RNase H region is 71.9%, which is below the species demarcation cutoff of 80% for badnaviruses. Phylogenetic analysis also placed the new virus with GVCV in a cluster. The virus was tentatively named "green Sichuan pepper vein clearing-associated virus" (GSPVCaV). The geographical distribution and genetic diversity of GSPVCaV were studied. Another isolate was found to be highly divergent.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

Complete genome sequence of a tentative new member of the genus Badnavirus identified in Codonopsis lanceolata.

High-throughput sequencing revealed a tentative new badnavirus infecting Codonopsis lanceolata, provisionally named Codonopsis vein clearing virus (CoVCV). The complete 8,112-nt CoVCV genomic DNA sequence (GenBank accession: MK044821) comprises three open reading frames (ORFs) encoding conserved domains, with typical features of badnaviruses. Additionally, BLASTn searches indicated the CoVCV genome sequence is most similar to the grapevine vein clearing virus (GVCV) genome (72% identity and 46% query coverage). Moreover, the polyprotein encoded in CoVCV ORF3 is most similar to the corresponding protein of GVCV, with 60% amino acid sequence identity (89% query coverage). These results suggest that CoVCV is a new member of the genus Badnavirus in the family Caulimoviridae.

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.

Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics.

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.

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.

Molecular characterization of two badnavirus genomes associated with Canna yellow mottle disease.

Members of the genus Badnavirus have a single non-covalently closed circular double-stranded DNA genome of 7.2-9.2kb. The genome encodes three open reading frames (ORFs) on the positive DNA strand. Canna yellow mottle virus (CaYMV) is a badnavirus that has been described as the etiological cause of yellow mottle disease in canna, although only a 565bp fragment of the genome has been previously reported from cannas. In this report, concentrated virions were recovered from infected canna plants and nucleic acids were extracted. Two full-length sequences represent two badnavirus genomes were recovered and were determined to be 6966bp and 7385bp in length. These DNAs represent a virus strain belonging to Canna yellow mottle virus and a novel species tentatively termed Canna yellow mottle associated virus. Phylogenetic analysis indicates that these two viruses are closely related to sugarcane bacilliform GD virus, pineapple bacilliform comosus virus, banana streak MY virus, and cycad leaf necrosis virus. We also showed naturally grown canna plants to be frequently co-infected by these two badnaviruses along with a potyvirus, Canna yellow streak virus.