Characterization of Pepper leafroll chlorosis virus, a New Polerovirus Causing Yellowing Disease of Bell Pepper in Saudi Arabia.

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.

Characterization of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Saudi Arabia.

During 2014 and 2015, 97 lettuce plants that showed big-vein-disease-like symptoms and seven weed plants were collected from the Riyadh region. DAS-ELISA revealed that 25% and 9% of the lettuce plants were singly infected with LBVaV and MiLBVV, respectively, whereas 63% had a mixed infection with both viruses. The results were confirmed by multiplex reverse transcription polymerase chain reaction using primers specific for LBVaV and MiLBVV. LBVaV and MiLBVV were also detected in Sonchus oleraceus and Eruca sativa, respectively. The nucleotide sequence of LBVaV and MiLBVV Saudi isolates ranged from 94.3-100%, and their similarities to isolates with sequences in the GenBank database ranged from 93.9 to 99.6% and 93.8 to 99.3%, respectively. Olpidium sp. was present in the roots of lettuce plants with big-vein disease and it was shown to facilitate transmission of both viruses.

First Report of Tomato spotted wilt virus in Lettuce Crops in Saudi Arabia.

A survey for viruses in open field lettuce crops was carried out in March 2014 in the Al-Uyaynah area, central region of Saudi Arabia. In one plot, more than 50% of the lettuce plants (Lactuca sativa; hybrid: Romaine), with the majority of the affected plants in the edges of the plot, were showing virus-like symptoms such as necrotic lesions, necrosis of the lamina of the younger leaves, and leaf curling, indicating a possible infection by a Tospovirus, possibly Tomato spotted wilt virus (TSWV). Most of them were dead when the field was visited again 3 weeks later. Samples from 10 symptomatic and two asymptomatic plants were collected. Five of the samples from symptomatic and two from asymptomatic plants were mechanically inoculated onto Nicotiana benthamiana and N. glutinosa (three indicator plants of each species were used for each sample) using 0.1 M phosphate buffer (pH 7) containing 0.01M Na2SO3 mM. All the symptomatic lettuce samples were also tested serologically using polyclonal antisera (3) against TSWV, CMV, and by using monoclonal antibodies against potyviruses. Moreover, total RNA was extracted (1) and detection of TSWV was also attempted with reverse transcription (RT)-PCR using species specific primers (4) for a 276-bp fragment of the L RNA segment. In both serological and molecular methods, positive and negative controls were included. All the mechanically inoculated plants with tissue from the symptomatic lettuce plants of N. benthamiana showed chlorotic local lesions followed by systemic top necrosis 2 to 3 weeks post inoculation. Similarly, all inoculated N. glutinosa plants showed necrotic local lesions followed by systemic chlorosis. However, all the indicator plants mechanically inoculated with tissue from asymptomatic lettuce plants gave no reaction. All the symptomatic lettuce samples reacted positively, while asymptomatic samples reacted negatively in ELISA tests with TSWV antiserum and the presence of the virus was further confirmed by RT-PCR by using specific primers (method A) (4). PCR products of two randomly selected positive samples were directly sequenced and BLAST analysis of the obtained sequences (Accession Nos. KJ701035 and KJ701036) revealed 99% nucleotide and 100% amino acid identity with the deposit sequence in NCBI from South Korea (KC261947). Regarding mechanical inoculation, 10 days post-inoculation, both indicator plants showed typical symptoms of TSWV infection, such as necrotic local lesions, systemic necrotic patterns, and leaf deformation. None of the symptomatic plants was found to be infected with either CMV or potyvirus. To our knowledge, this is the first report of TSWV naturally infecting lettuce in Saudi Arabia; therefore, insect vector and weed management are necessary measures to control the virus spread to other crops such as tomato and pepper (2). References: (1) E. Chatzinasiou et al. J. Virol. Meth. 169:305, 2010. (2) E. K. Chatzivassiliou. Plant Dis. 92:1012, 2008. (3) E. K. Chatzivassiliou et al. Phytoparasitica 28:257, 2000. (4) R. A. Mumford et al. J. Virol. Meth. 46:303, 1994.

First Report of Tomato chlorosis virus (ToCV) in Tomato Crops in Saudi Arabia.

During January 2014, open field and greenhouse tomato (Solanum lycopersicum L.) crops in the peripheral areas of Riyadh region (Al-Aflaj, Al-Kharj, Al-Waseel, and Al-Dalam), Saudi Arabia, were surveyed. In all surveyed tomato crops, yellowing symptoms were observed on the lower leaves, possibly infected by a whitefly transmitted crinivirus (family Closteroviridae) such as Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV). Dense population of whiteflies (Bemisia tabaci G.) were present in all affected plants. Incidence of the yellowing disease varied between four greenhouses and three open field tomato crops, but in the majority of the tomato crops surveyed, symptoms typical of Begomovirus infection such as severe stunting, degeneration, upward cupping, distortion and interveinal yellowing of upper leaves, and flower abortion were also observed. Tomato yellow leaf curl virus (TYLCV) is endemic in Saudi Arabia causing severe crop losses (1). Twenty-six leaf samples from 24 symptomatic and two asymptomatic plants from four fields (three greenhouses and one open field crop) were collected and were processed in the lab at King Saud University. Whitefly transmission on tomato indicator plants was carried out using B. tabaci to fulfill Koch's postulates. Two hundred virus-free B. tabaci adults were confined to one of the collected symptomatic tomato sample singly infected with ToCV for a 48-h acquisition access period, followed by a 48-h inoculation access period on five healthy tomato plants Hybrid Super Strain B, using 40 whiteflies per plant. Crinivirus detection following transmission was conducted by RT-PCR. Total RNA was extracted from 26 collected leaf samples using the Total RNA Purification Kit and analyzed by SCRIPT One-Step RT-PCR Kit (Jena Bioscience). First, the degenerate primers HS-11/HS12 were used for amplification of a 587-bp fragment of the HSP70 gene of ToCV and TICV (3). Second, the RT-PCR product was subjected to a nested PCR using specific primers TIC-3/TIC-4 and TOC-5/TOC-6, for the detection of both TICV and ToCV, respectively (2). Finally, degenerate primers (AV494/AC1048) were used for detection of begomoviruses (4). No fragment was amplified by TIC-3/TIC-4 primer whereas TOC-5/TOC-6 amplified a size of 463 bp in all 24 symptomatic tested samples, including one mixed infection with TYLCV detected by AV494/AC1048. Asymptomatic samples did not produce any amplicon regarding TICV, ToCV, and Begomovirus detection. The amplicons of four positive fragments, each from one field, were further sequenced in both directions and all obtained sequences (KJ433488, KJ433489, KJ433490, and KJ433491) analyzed with BLAST and revealed 99% identity with the most closely deposited sequences in NCBI from Japan (AB513442) and Brazil (JQ952601). In the transmission tests, ToCV was detected to all tomato indicator plants which revealed yellowing symptoms 6 weeks post inoculation, whereas no transmission was obtained when non-viruliferous whitefly adults fed on two asymptomatic tomato leaves. To our knowledge, this is the first report of ToCV infecting tomato crops in Saudi Arabia. Further studies are being carried out to study epidemiology and genetic diversity of this virus associated with yellowing diseases of tomato in different regions of Saudi Arabia. This finding is important for the tomato crops and possibly other virus hosts as may cause serious epidemics and crop losses. References: (1) A. M. Ajlan et al. Arab J. Biotech. 10:179, 2007. (3) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (4) S. D. Whyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Insights into the Incidence of Watermelon chlorotic stunt virus Causing Yellowing Disease of Watermelon in Western and Southwestern Regions of Saudi Arabia.

During the spring season of 2014, a total of 148 melon and watermelon leaf samples were collected from symptomatic and asymptomatic plants in the western and southwestern regions of Saudi Arabia and were tested for the presence of Watermelon chlorotic stunt virus (WmCSV) and other suspected cucurbit viruses by double antibody sandwich enzyme-linked immunosorbent assays. Ninety-eight samples were found to be positive for the presence of WmCSV, nine samples were positive for the presence of Cucurbit yellow stunting disorder virus (CYSDV), and 22 showed a mixed infection with both WmCSV and CYSDV. No other cucurbit viruses were detected in any of the samples. Host range experiments revealed that eight out of fourteen tested plant species were susceptible to WmCSV. PCR products of approximately 1.2 kb were obtained after amplification using primers specifically targeting the coat protein region of WmCSV. Positive PCR results were confirmed by dot blot hybridization. Coat protein gene sequences from eleven WmCSV isolates indicated that the highest identity was between the 104WMA-SA isolate from the Wadi Baish location and a previously reported isolate from the AL-Lith location in Saudi Arabia. The lowest identity was observed between the 42WMA-SA isolate and an isolate from Palestine.