Novel circular DNA viruses associated with Apiaceae and Poaceae from South Africa and New Zealand.

Advances in molecular techniques used in viral metagenomics coupled with high throughput sequencing is rapidly expanding our knowledge of plant-associated virus diversity. Applying such approaches, we have identified five novel circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses from Poaceae and Apiaceae plant from South Africa and New Zealand. These viruses have a simple genomic organization, including two open reading frames that likely encode a Rep and a capsid protein (CP), a conserved nonanucleotide motif on the apex of a putative stem loop structure, and conserved rolling-circle replication and helicase motifs within their likely Rep: all suggesting that they replicate through rolling-circle replication. The Reps and the CPs putatively encoded by these five novel viruses share low to moderate degrees of similarity (22.1 - 44.6%) with other CRESS DNA viruses.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza).

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing.

High throughput sequencing (HTS) is a very powerful tool for detecting and discovering novel viral-like sequences without prior knowledge of the sequence. Here we describe the complete genome of a new vitivirus-like sequence that was found in arracacha (Arracacia xanthorrhiza) plants using HTS technology. The complete genome sequence was validated by Sanger sequencing. The genomic organization of the new putative vitivirus resembles that of grapevine virus B (GVB) and grapevine virus D (GVD). The putative coat protein showed 41 to 49% identity with similar proteins of known vitiviruses, while the RNA-dependent RNA polymerase shared 52 to 55% identity with those encoded by grapevine vitiviruses. Based on the demarcation criteria for the genus Vitivirus, the virus described in this work, provisionally named as "Arracacha virus V", represents a novel species in this taxon.

Genetic Characterization of 'Candidatus Liberibacter solanacearum' Haplotypes Associated with Apiaceous Crops in France.

'Candidatus Liberibacter solanacearum' (Lso) is an emerging phytopathogenic bacterium that causes significant crop losses worldwide. This bacterium has been identified in association with diseases of several solanaceous crops in the United States and New Zealand, and with carrot and celery crops in several European countries. Five Lso haplotypes (LsoA, LsoB, LsoC, LsoD, and LsoE) have now been described worldwide. In France, symptoms of Lso were observed on plants of the Apiaceae family in several regions. One hundred and ninety-two samples of apiaceous plants were collected from 2012 to 2016 in different geographical regions and were tested for the occurrence of Lso by real-time PCR assay. In addition to carrot and celery, Lso was detected in four other apiaceous crops: chervil, fennel, parsley, and parsnip. These new findings suggest that Lso has a wider natural host range within the Apiaceae family than expected. To identify the Lso haplotypes present in France, we sequenced and analyzed the 16S rRNA gene and the 50S ribosomal protein rpIJ-rpIL gene region from a representative bacterial collection of 44 Lso-positive samples. Our SNP analysis revealed the occurrence of two distinct bacterial lineages that correspond to haplotypes D and E. Then, we assessed the phylogenetic relationships between strains isolated from France and a worldwide collection of Lso isolates by using the rpIJ-rpIL gene region sequences. The neighbor-joining tree constructed delineated five clusters corresponding to the five Lso haplotypes, with LsoD and LsoE being closely related phylogenetically. Altogether, the data presented here constitute a first step toward a better understanding of the genetic diversity among Lso haplotypes in France, and provide new insights into the host range of this emerging bacterial species.

Complete genome sequence of arracacha mottle virus.

Arracacha mottle virus (AMoV) is the only potyvirus reported to infect arracacha (Arracacia xanthorrhiza) in Brazil. Here, the complete genome sequence of an isolate of AMoV was determined to be 9,630 nucleotides in length, excluding the 3' poly-A tail, and encoding a polyprotein of 3,135 amino acids and a putative P3N-PIPO protein. Its genomic organization is typical of a member of the genus Potyvirus, containing all conserved motifs. Its full genome sequence shared 56.2 % nucleotide identity with sunflower chlorotic mottle virus and verbena virus Y, the most closely related viruses.

[Disease survey and pathogen identification on Notopterygium incisum in Gansu province].

Six diseases have been found after disease surveys on Notopterygium incisum in Gansu province during 2004 to 2007. They were brown spot (Ascochyta levistici), powdery mildew (Erysiphe heraclei), grey spot (Alternaria sp. ; Alternaria burnsii), leaf spot (Septoria dearnessii), angular leaf spot (Pleospora sp.), leaf streak (Phoma sp.), bacterial angular leaf spot and a virus disease. Bacterial angular leaf spot and powdery mildew are the urgent problems waiting to be solved effectively. All these diseases were reported for the first time in China.

Characterization of a member of a new Potyvirus species infecting arracacha in Brazil.

An arracacha (Arracacia xanthorrhiza) plant collected in Brazil was found to be infected by a new virus. This viral isolate (named C17) systemically infected Nicotiana benthamiana and Apium graveolens. A polyclonal antibody was raised, and analysis of our arracacha germplasm collection showed a high infection rate of C17-like viruses (93% infection). Sequencing of the ca. 1.7 kb 3'-terminal genomic region revealed a typical potyvirus genome organization. It shared less than 70% nucleotide identity with any other potyvirus sequence, which thus indicated that it is possibly a member of a new Potyvirus species tentatively named Arracacha mottle virus (AMoV).

Potyviruses, novel and known, in cultivated and wild species of the family Apiaceae in Australia.

Three potyviruses were identified by gene sequencing and found to be widespread in species of Apiaceae in Australia. Only celery mosaic virus was found in celery crops and in one of 180 specimens of feral carrot ( Daucus carota). Another related but distinct novel potyvirus, carrot virus Y, was the only virus found in carrot crops and all except one feral carrot. A more distantly related novel potyvirus, apium virus Y, was found in plants of sea celery ( Apium prostratum), cultivated parsley ( Petroselinum crispum) and the immigrant weed species poison hemlock ( Conium maculatum). These three potyviruses, together with celery yellow mosaic virus of South America and the closely related carrot thin leaf virus and carrot virus B of North America, form a distinct subgenus of the Potyviridae most closely related to turnip mosaic virus and two potyviruses of yam; yam mosaic virus from the Ivory Coast and Japanese yam mosaic virus. Celery mosaic and carrot virus Y are probably recent migrants to Australia, but apium virus Y may have been endemic longer. In ELISA tests using polyclonal antibodies against virions of celery mosaic virus, some isolates of carrot virus Y were indistinguishable from celery mosaic virus, whereas others gave smaller absorbancy values, and those of apium virus Y did not react. This study shows the value of virus identification based on gene sequencing for planning control measures.

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family.

A universal primer (Sprimer: 5'-GGX AAY AAY AGY GGX CAZ CC-3', X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3'-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).

RT-PCR using redundant primers to detect the three viruses associated with carrot motley dwarf disease.

A method was developed for detecting and distinguishing the viruses associated with carrot motley dwarf (CMD) disease, i.e. Carrot mottle umbravirus (CMoV), Carrot red leaf virus (CRLV) and the virus known as carrot red leaf virus-associated RNA (CRLVaRNA). Redundant primers were made that targeted the RNA-dependent RNA polymerase (RdRp) gene in all available sequences of umbraviruses and in a subset of polerovirus genomes, and specific and redundant primers were made to target the same gene in CRLVaRNA. By using these primers in RT-PCR reactions, cDNAs were amplified from total RNA isolated from Belgian parsley with CMD disease. The cDNAs were sequenced and an analysis of this data showed that the plants contained all three viruses. RT-PCR assays were optimized for the simultaneous detection of the three viruses in infected parsley and chervil plants and in individual viruliferous aphids. This is the first report of a natural infection of CRLVaRNA in a plant other than carrot and the first report of this virus outside the USA. The study also confirmed that the umbravirus in parsley with CMD disease is CMoV, and that this virus is distinct from carrot mottle mimic umbravirus (CMoMV), which is also associated with CMD, but apparently not in Europe.