M. A. Al-Saleh

Response of greenhouse-grown cucumber cultivars to an isolate of Zucchini yellow mosaic virus (ZYMV)

Mechanical inoculation of 12 greenhouse-grown cucumber (Cucumis sativus) cultivars at the three- to four-leaf stage with an isolate of zucchini yellow mosaic virus (ZYMV) from Saudi Arabia resulted in various symptoms in 11 cultivars. Significant differences in plant height, fruit numbers, and fruit weight were observed between the inoculated and the control plants of the 11 affected cultivars. The cultivar Dina was asymptomatic, and no significant differences were observed between the inoculated and control plants for any of the parameters tested. Virus recovery test from cultivar Dina performed on the susceptible cultivar, Farol, together with lack of virus detection by enzyme-linked immunosorbent assay (ELISA) indicated that this ZYMV isolate did not infect Dina. However, inoculation of Dina at the cotyledon stage revealed minor symptoms that were confined to a few of the lower leaves, suggesting that Dina is resistant but not immune to this ZYMV isolate. Virus titer in Dina was low and the virus was restricted to the older leaves; whereas in Farol virus titer was considerably higher and the virus spread to all leaves.

Detection of new viruses in alfalfa, weeds and cultivated plants growing adjacent to alfalfa fields in Saudi Arabia

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, Sonchus oleraceus, Chenopodium spp., Hibiscus spp., Cichorium intybus, Convolvulus arvensis, Malva parviflora, Rubus fruticosus, Hippuris vulgaris, and Flaveria trinervia. These viruses were also detected in seven cultivated crop plants growing adjacent to the alfalfa fields including Vigna unguiculata, Solanum tuberosum, Solanum melongena, Phaseolus vulgaris, Cucurbita maxima, Capsicum annuum, and Vicia faba. 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.

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 Kochs 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.

IDENTIFICATION AND CHARACTERIZATION OF COWPEA APHID-BORNE MOSAIC VIRUS ISOLATES IN SAUDI ARABIA

SUMMARY Cowpea (Vigna unguiculata subsp. unguiculata) plants showing mosaic, and other virus‐like symptoms, were noticed in May 2009 in a field located in the Alahsa Governorate (eastern Saudi Arabia). Systemic infections were observed on mechanically inoculated V. unguiculata subsp. unguiculata, V. unguiculata subsp. sesquipedalis, and Nicotiana benthamiana plants, while local infections developed on inoculated leaves of Chenopodium quinoa, C. amaranticolor, and N. occidentalis. Latent infections were detected in inoculated Ocimum basilicum and Cajanus cajan. DAS-ELISA from the above plants gave positive reactions with Cowpea aphid-borne mosaic virus (CABMV) antibodies. The virus was transmitted by Aphis craccivora Koch. in a non-persistent manner and by infected cowpea seeds at a rate of ca. 5%. Electron microscopic examination of leaf-dip preparations from symptomatic cowpea leaves revealed flexuous particles, 750x12 nm. The virus was also identified molecularly by dot-blot hybridization and RT-PCR assays. The coat protein gene of each of the Saudi virus isolates was partially sequenced and compared with those of 18 CABMV strains from different countries. Phylogenetic analysis indicated that the Saudi virus isolates are virtually identical and had the highest nucleotide sequence identity (93.5%) with two CABMV strains from Morocco and one strain from Nigeria, and the lowest identity with two strains from Zimbabwe and a strain from Brazil. The partial nucleotide sequence of only one of these Saudi isolates was deposited in the GenBank under the accession number JQ638520 to represent all other isolates. This is the first report of the presence of CABMV in Saudi Arabia whose strain is denoted CABMV-SA.

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.

Molecular characterization of Criniviruess involved in the etiology of cucumber yellowing disease in Riyadh region, Saudi Arabia

During the growing seasons 2014 and 2015, 134 cucumber and 103 weed samples showing typical yellowing diseases symptoms accompanied by chlorotic spots and interveinal chlorosis were collected from Riyadh region, Saudi Arabia. For this reason, RT-PCR was conducted for detection of two criniviruses, Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) which are implicated in cucurbit yellows disease. Results showed the presence of CCYV and CYSDV in cucumber samples in 61.1% and 19.4%, respectively, whereas 9% of the plants were contained mixed infection. In addition, four weed species were identified, for the first time, as alternative hosts of CCYV; whereas CYSDV was reported only in Malva parviflora plants. Partial nucleotide sequencing analysis was conducted using 13 CCYV and five of CYSDV isolates originated from cucumber and wild hosts. Both viruses exhibited low genetic diversity; however, phylogenetic analysis clustered the obtained CYSDV isolates in a separated group, named the “Eastern subpopulation”, while CCYV isolates were classified into subgroups IIa and IIb, among other published sequences, forming a different evolutionary lineage from group I, which contained an isolate from Iran.