M. Al Rwahnih

Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus.

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a vine showing decline symptoms supported a mixed infection that included seven different RNA genomes. Fragments identified as derived from viruses or viroids spanned a approximately ten thousand fold range in relative prevalence, from 48,278 fragments derived from Rupestris stem pitting-associated virus to 4 fragments from Australian grapevine viroid. 1527 fragments were identified as derived from an unknown marafivirus. Its complete genome was sequenced and characterized, and an RT-PCR test was developed to analyze its field distribution and to demonstrate its presence in leafhoppers (vector for marafiviruses) collected from diseased vines. Initial surveys detected a limited presence of the virus in grape-growing regions of California.

Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.

Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.

Low genetic variability in the coat and movement proteins of American plum line pattern virus isolates from different geographic origins

SummaryUntil very recently isolates of American plum line pattern virus (APLPV) had not been reported from outside North America. The nucleotide sequences corresponding to the movement (MP) and coat (CP) proteins of eight APLPV isolates from five Mediterranean countries were determined. Sequence analysis showed that both MP and CP genes are highly conserved irrespective of geographic origin. The study of the distribution of synonymous and nonsynonymous changes along both open reading frames revealed that these proteins are under the effect of negative purifying selection. The MP and CP of APLPV possess most of the functional motifs described for other members of the genus Ilarvirus.

Evaluation of the phytosanitary status of the Prunus species in the National Clonal Germplasm Repository in California: survey of viruses and viroids.

The USDA National Clonal Germplasm Repository (NGCR) at the University of California, Davis is recognized as one of the richest sources of Prunus species material in the US. The repository maintains more than 2450 trees representing 96 taxa collected from around the world. However, the phytosanitary status of the NCGR Prunus collection has not been thoroughly evaluated. In this study, a comprehensive screening of a selected subset of the collection for virus and virus-like diseases affecting Prunus trees has been completed. Two factors were considered for selecting the trees: the country of origin and the observation of suspicious disease symptoms. A total of 221 trees representing 185 different cultivars of cherry, almond, peach, apricot and plum were sampled. Conventional RT-PCR detection was used to test for 13 different viruses and two viroids. The pathogens included in the survey were Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Plum bark necrosis stem pitting-associated virus (PBNSPaV), American plum line pattern virus (APLPV), Cherry virus A (CVA), Cherry leafroll virus (CLRV), Cherry rasp leaf virus (CRLV), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Apple chlorotic leafspot virus (ACLSV) Tomato ringspot virus (ToRSV), Little cherry virus 1 (LChV-1), Little cherry virus 2 (LChV-2), Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). Though the majority of these trees were asymptomatic, all tested pathogens were detected except for ToRSV and CRLV. Two of the viruses detected (ACLSV and LChV-2) had never been reported from California. Incidence of PNRSV in tested trees was the highest (55 trees) followed by the two viroids (PLMVd and HSVd), with 33 and 32 infected trees, respectively. The incidence for the rest of the pathogens ranged between 0 to 19 trees. The infection rate of all tested samples ranged from 0.5% to 24.9%.

A Mixed Infection of Lettuce chlorosis virus, Papaya ringspot virus, and Tomato yellow leaf curl virus-IL Detected in a Texas Papaya Orchard Affected by a Virus-Like Disease Outbreak

Severe virus-like symptoms consisting of mosaic, distortion, yellowing, and brittleness were observed on papaya plants in a 20-ha orchard in South Texas during the 2014-15 growing season. Incidence of symptomatic plants increased from ∼40 to 100% within 6 months of the outbreak; the most severely affected plants were stunted, and fruit yield and quality were reduced compared with asymptomatic plants. The orchard papaya plant virome was explored using the Illumina NextSeq 500 platform and results were validated by Sanger DNA sequencing of complete viral genomes obtained by PCR amplification. The combined results revealed the presence of Papaya ringspot virus (PRSV; Potyvirus), Lettuce chlorosis virus (LCV; Crinivirus), and Tomato yellow leaf curl virus-IL (TYLCV-IL; Begomovirus). The RT-PCR analyses of leaves from 51 randomly sampled papaya plants indicated the presence of PRSV, LCV, and TYLCV-IL in 100, 39.2, and 15.7% of the samples, respectively. Plants infected with PRSV, in combination with LCV and/or TYLCV-IL, exhibited more severe symptoms compared with plants infected with PRSV alone. Furthermore, successful whitefly-mediated transmission of TYLCV-IL and LCV was accomplished by exposing virus-free papaya seedlings to viruliferous Bemisia tabaci (Genn.) under greenhouse conditions. The results of this study document a new host record for LCV and the first successful whitefly-mediated transmission of TYLCV-IL and LCV to papaya. As a perennial crop, infected papaya serving as an over-seasoning reservoir for TYLCV-IL and LCV, presents a new challenge to viral disease management in papaya orchards.

Improved detection of ilarviruses and nepoviruses affecting fruit trees using quantitative RT-qPCR.

Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern virus (APLPV), and the nepoviruses Tomato ringspot virus (ToRSV) and Cherry leafroll virus (CLRV). These viruses affect various stone fruits such as apricots, cherries, peaches, plums, and almonds. The goal of this work was to improve the RT-qPCR detection of PNRSV, PDV, and ApMV in addition to developing three new RT-qPCR assays for the detection of APLPV, ToRSV and CLRV. Primers for conventional RT-PCR as well as primers and probes for RT-qPCR assays were designed after aligning coat protein (CP) gene sequences of geographically diverse isolates with the corresponding CP gene sequences from GenBank, targeting regions with 100% sequence identity. The efficiency of each RT-qPCR assay, as well as the intra- and inter-assay variability, were determined. These conventional RT-PCR and RT-qPCR assays were validated using purified total RNAs from 221 trees from the USDA Clonal Germplasm Repository orchards. The data showed that more isolates were detected by RT-qPCR than by RT-PCR.

First Report of Grapevine yellow speckle viroid 1, Grapevine yellow speckle viroid 2, and Hop stunt viroid Infecting Grapevines (Vitis spp.) in Nigeria

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2; genus Apscaviroid), and Hop stunt viroid (HSVd; genus Hostuviroid) are three of six viroid species documented globally from grapevines (Di Serio et al. 2017). These small plant-pathogenic, non-protein coding, circular RNAs may or may not elicit disease symptoms in grapevines depending on vagaries of the environment and their presence in mixed infection with other grapevine-infecting viruses. During 2016, surveys were conducted in the Northern Guinea Savannah agroecological zone of Nigeria to document the occurrence of grapevine viruses and viroids. A total of 318 leaf tissue samples belonging to five cultivars were collected during the survey across 28 vineyards. The samples were dried under CaCl₂ at room temperature, and then shipped under USDA-APHIS-PPQ permit to Texas AM MF576417–428) shared 96 to 100% identity with several HSVd GenBank isolates. The complete GYSVd-1 RNA derived in this study (365 to 367 nt; MF576399–407) shared 96 to 99% identity with several GYSVd-1 GenBank isolates. The complete GYSVd-2 RNA derived in this study (361 nt; MF576408–416) shared 97 to 99% identity with several GYSVD-2 GenBank isolates. No discernible symptoms were associated with the presence of all three viroid species in their source grapevines. To our knowledge, this is the first report of viroids infecting grapevine in Nigeria. Studies are ongoing to further elucidate the prevalence of all three viroids in Nigerian vineyards and their occurrences in mixed infections with other graft-transmissible agents of grapevine.

Grapevineleafroll-associated virus 7

Grapevine leafroll-associated virus 7 (GLRaV-7) is an asymptomatic virus in the newly established genus, Velarivirus, in the Closteroviridae family. GLRaV-7 has been detected in grape-growing regions across the globe and may be quite widespread. The virus is only known to infect grapevine, is transmitted through infected propagation material, and, at this time, has no known insect vector. Detection of the virus is most reliable and accurate using RT-qPCR.