Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.

A total of 1368 symptomatic plant samples showing different virus-like symptoms such as mottling, chlorosis, mosaic, yellow mosaic, vein clearing and stunting were collected from alfalfa, weed and cultivated plant species growing in vicinity of alfalfa fields in five principal regions of alfalfa production in Saudi Arabia. DAS-ELISA test indicated occurrence of 11 different viruses in these samples, 10 of which were detected for the first time in Saudi Arabia. Eighty percent of the alfalfa samples and 97.5% of the weed and cultivated plants samples were found to be infected with one or more of these viruses. Nine weed plant species were found to harbor these viruses namely, spp., spp., ,, ,, , and. These viruses were also detected in seven cultivated crop plants growing adjacent to the alfalfa fields including ,, ,, ,, and . The newly reported viruses together with their respective percent of detection in alfalfa, and in both weeds and cultivated crop plant species together were as follows: Bean leaf roll virus (BLRV) {12.5 and 4.5%}, Lucerne transient streak virus (LTSV) {2.9 and 3.5%}, Bean yellow mosaic virus (BYMV) {1.4 and 4.5%}, Bean common mosaic virus (BCMV) {1.2 and 4.5%}, Red clover vein mosaic virus (RCVMV) {1.2 and 4%}, White clover mosaic virus (WCIMV) {1.0 and 5%}, Cucumber mosaic virus (CMV) {0.8 and 3%}, Pea streak virus (PeSV) {0.4 and 4.5%} and Tobacco streak virus (TSV) {0.3 and 2.5%}. Alfalfa mosaic virus (AMV), the previously reported virus in alfalfa, had the highest percentage of detection in alfalfa accounting for 58.4% and 62.8% in the weeds and cultivated plants. Peanut stunt virus (PSV) was also detected for the first time in Saudi Arabia with a 66.7% of infection in 90 alfalfa samples collected from the surveyed regions during the last visit that tested negative to all the previously detected viruses.

During the growing seasons of 2014 through 2016, a total of 336 leaf samples from bell pepper (showing leafroll and interveinal yellowing) and arable weeds were collected from Riyadh region, Saudi Arabia. The use of a polerovirus generic reverse transcription (RT)-PCR assay confirmed their presence in the bell pepper samples. Sequencing of the generic amplicon revealed high similarity (87.6 to 98.1% in nt) with four poleroviruses; Tobacco vein distorting virus, Pepper vein yellows virus, Pepper yellows virus, and Pepper yellow leaf curl virus. To further characterize one of these isolates (105D), a larger part of the genome (∼1,300 nt) spanning approximately from the 3′ end of ORF2 to the middle of ORF3, was amplified and sequenced. Blasting the resulting sequence revealed the low amino acid and nucleotide identity percentages in the coat protein and movement protein partial genes with viruses deposited in GenBank. Next-generation sequence was used to acquire a larger part of the genome, which resulted in the reconstruction of isolate 105D’s partial genome (5,496 nt). Sequence similarity analysis revealed the presence of a divergent polerovirus isolate belonging to a new species that was tentatively named Pepper leafroll chlorosis virus (PeLRCV). Using a specific RT-PCR assay for this isolate confirmed the presence of this new viral species in the symptomatic peppers. Aphid transmission experiments showed that PeLRCV is vectored by Aphis gossypii and that it can infect at least five out of the 15 different plants species tested. Based on our findings, PeLRCV is a new member of genus Polerovirus in the family Luteoviridae.

Agar double diffusion tests and, later, ELISAs were used to detect viruses associated with potato in 242 samples collected in 16 trips to Tabuk and Hail, northern regions of Saudi Arabia, in four consecutive growing seasons (autumn 1989, spring and autumn 1990 and spring 1991). Eleven different viruses were detected in Tabuk and 12 in Hail. The viruses detected in Tabuk were alfalfa mosaic (AMV), cucumber mosaic (CMV), tobacco mosaic (TMV), potato leaf roll (PLRV), tomato spotted wilt (TSWV), tobacco ringspot (TRSV) and potato A, M, S, X and Y. The same viruses, plus potato yellow dwarf (PYDV), were detected in Hail. AMV was most frequently and CMV least frequently detected in Tabuk, whereas in Hail the most and least common were PVA and PLRV respectively.

Mosaic symptoms were observed on bottlegourd in the College of Agriculture Research and Experimental Farm at Dirab. The following plant species: Lagenaria siceraria L., Cucumis sativus L., C. melo L., and Citrullus vulgaris L. showed mosaic symptoms when mechanically inoculated with extract from symptomatic bottlegourd. The rest of the inoculated plants did not show any symptoms. Properties of the infective plant extract were as follows: Dilution end point (DEP) was between 10−6–10−7, thermal inactivation point (TIP) was between 90–100 °C, and longevity in vitro was more than 32 days. Electron microscopic examination of negatively stained, partially purified preparations revealed rigid‐rod shaped virus particles measuring 300–325 nm. Positive serological reaction was observed between leaf extract from infected bottlegourd and CGMMV antiserum in immunodiffusion test in agar plates. This report is the first of the presence of CGMMV in this country. Zusammenfassung Ein Isolat des cucumber green mottle mosaic virus (CGMMV) aus dem Flaschenkürbis in Saudi‐Arabien Mosaiksymptome wurden an Flaschenkürbis bei dem College of Agriculture Research and Experimental Farm in Dirab, Saudi‐Arabien, beobachtet. Nach einer mechanischen Inokulation mit dem Extrakt aus befallenen Flaschenkürbispflanzen zeigten die folgenden Pflanzenarten Mosaiksymptome: Lagenaria siceraria L., Cucumis sativus L., C. melo L. und Citrullus vulgaris L. Andere Pflanzenarten, die gleichzeitig inokuliert worden waren, zeigten keine Symptome. Der infektiöse Pflanzenextrakt hatte folgende Eigenschaften: der Verdünnungsendpunkt (DEP) lag zwischen 10−6 und 10−7, der thermale Inaktivierungspunkt lag zwischen 90–100 °C, und die Langlebigkeit in vitro betrug mehr als 32 Tage. Elektronenmikroskopische Untersuchungen von negativ gefärbten, partiell gereinigten Präparaten offenbarten stabile, stabähnliche Viruspartikeln, die eine Größe von 300–325 nm hatten. Immunodiffusionstests in Agarplatten zeigten eine positive serologische Reaktion zwischen dem Blattextrakt von infizierten Flaschenkürbispflanzen und dem CGMMV‐Antiserum. Über das Vorhandensein vom CGMMV in Saudi‐Arabien wird hier zum ersten Mal berichtet.