Grapevines (Vitis vinifera L.) were grown on 15,360 ha in Pakistan producing 66,036 tons of grapes in 2014 (FAO 2017). Grapes ranked 10th among the fruit grown in Pakistan (Jaskani et al. 2008). Grapes are grown in all four provinces (Balochistan, Khyber Pakhtunkhwa, Punjab, and Sindh) but the majority of grape production occurs in Balochistan (GoP 2011). The important grape varieties grown in Balochistan are Haita, Kishmish, Sunderkhani, Sahibi and Shekhali (Aujla et al. 2011). In Pakistan, 87% of grape production is used as table grapes and 13% as dried fruit (Aujla et al. 2011). Important cultivars of table grapes are also grown in some districts of Khyber Pakhtunkhwa and annual production is 12,000 tons with an average yield of 19 tons per ha (Bashir et al. 2012). Popular cultivars of table grapes (Italia, Cardinal, Flame Seedless, White Kishmish, Thompson Seedless, Perlette and King’s Ruby) are grown in central area of Punjab (Uddin et al. 2011). In 2008–2009, Pakistan exported grapes valued at approximately US$141,850 to Bahrain, Bangladesh, Germany, United Arab Emirates and United Kingdom (Reisch et al. 2012).

Grapes are a historically important crop that are host to the largest number of intercellular pathogens. More than 70 viruses, viroids, and phytoplasmas infect grapevines and a number of these are known to have negative effects on fruit quality and yield (Martelli 2014; Martelli 2017). During an initial study in 2017, we reported the presence of grapevine Pinot gris virus (GPGV) and grapevine fanleaf virus (GFLV) in Pakistan (Rasool et al. 2017). Due to the lack of additional information about grapevine viruses in Pakistan, the objective of this survey was to provide knowledge about the occurrence and prevalence of the grapevine viruses in different grape-growing regions of Pakistan. Results of this survey will provide baseline information about the sanitary status of vineyards in Pakistan and aid growers and nurseries in their vineyard management decisions.

To analyze the prevalence of grapevine viruses, 257 samples from a range of grapevine cultivars were collected during 2014–2016 from 13 vineyards in three provinces of Pakistan. In the province of Punjab, vineyards collected from included: Sillanwali; National Agriculture Research Center (NARC); Chakwal (Biotrack 1); Chakwal (Biotrack 2); Bahawalpur; Izhar Farm (Kalarkahar); Attock; Okara; Bahawalnager; Lahore SN; and Lahore College for Women University (LCWU), Lahore. Swat and Quetta are in the provinces of Khyber Pakhtunkhwa and Balochistan, respectively. Leaf petioles were collected during the growing season which started in late summer and continued through autumn. No typical virus symptoms were noted at the time of sample collection. The samples were stored at −80 °C until the total RNA was extracted. Total RNA extraction was performed by grinding 300 mg petiole material in guanidine buffer according to Osman et al. (2012) and using GeneJET Plant RNA Purification Mini kit (Thermo Fisher Scientific) according to the manufacturer’s directions. RNA was eluted with 100 μl RNase free water and shipped in ethanol to the United States for analysis. Subsequently, the integrity of RNA was verified using an 18S rRNA assay (Osman and Rowhani 2006) and samples were screened for a panel of 19 viruses, phytoplasmas (universal detection) and Xylella fastidiosa by reverse transcription quantitative PCR (RT-qPCR). Viruses included in the panel were: grapevine leafroll-associated viruses (GLRaV), GLRaV-1, GLRaV-2, GLRaV-2RG, GLRaV-3, GLRaV-4 (including GLRaV-4 strains 5, 6, 9, Pr and Car), and GLRaV-7; tomato ringspot virus (ToRSV); tobacco ringspot virus (TRSV); Arabis mosaic virus (ArMV); grapevine virus A (GVA); grapevine virus B (GVB); grapevine virus D (GVD); grapevine virus E (GVE); grapevine virus F (GVF); grapevine red blotch virus (GRBV); grapevine fleck virus (GFkV); grapevine rupestris stem pitting-associated virus (GRSPaV); GFLV; and GPGV. Primers and probes used for the detection of these viruses were used as described by: GLRaV-1, GLRaV-2, GLRaV-4, GLRaV-4 strain 5, GLRaV-4 strain 9 (Osman et al. 2007), GRSPaV, GVA, GVB, GVD (Osman and Rowhani 2008), and GLRaV-3 (Osman and Rowhani 2006). For all other viruses, primers and probes developed at Foundation Plant Services (FPS, University of California-Davis) were used (Al Rwahnih, unpublished data); these primers and probes are part of the routine testing procedure at FPS. In the case of phytoplasmas, a universal assay that detects all known phytoplasmas was employed (Hodgetts et al. 2009). Lastly, probes and primers designed by Schaad et al. (2002) were used for X. fastidiosa. Positive and negative controls for all grapevine viruses, phytoplasmas and X. fastidiosa were also included. RT-qPCR was carried out in 384-well plates using the TaqMan Fast virus 1-step Master Mix kit and the QuantStudio 6 real-time PCR system (Applied Biosystems). All samples were amplified in 10 μl reaction mix using 2 μl RNA and reagents and cycling conditions as described in the one-step protocol by Osman et al. (2012).

RT-qPCR results showed infection rates, in order from highest to lowest, for GVA (47.8%), GLRaV-2 (37.3%), GRSPaV (36.1%) and GFkV (35%). Lower infection rates were observed for GFLV (11.6%), GLRaV-3 (7%), GLRaV-4 strain 5 (6.6%), GLRaV-4 (6.2%), GLRaV-2RG (5%), GLRaV-7 (4.2%), GVB (4.2%), GLRaV-1 (2.3%), GLRaV-4 strain 6 (1.9%), GLRaV-4 strain Pr (1.9%), GPGV (1.9%), GVE (1.1%), and GVD (0.7%) (Table 1). A total of 187 out of 257 samples (72.7%) were infected with grapevine viruses. Mixed infections were common in these samples; a total of 142 out of 187 infected samples (75.9%) showed multiple infections. The most common mixed infection was a combination of three viruses, GVA + GFkV + GRSPaV, which was found in 16 samples of 142 mixed infected vines. Mixed infections of two, four, five, six and seven viruses in a single plant were also observed. On the other hand, single infections were observed in 45 samples. Positive samples produced cycle threshold (CT) values that ranged from 12.2 to 36 among the different RT-qPCR assays. Pathogens not detected by RT-qPCR in any samples were GLRaV-4 strain 9, GLRaV-4 strain Car, GRBV, ToRSV, TRSV, ArMV, GVF, phytoplasmas and X. fastidiosa. Except for GFLV and GPGV, this is the first report for the detection of grapevine viruses in Pakistan.

Table 1 Results of RT-qPCR assays showing virus incidence in different regions of Pakistan. Pathogens with no positive results (GLRaV-4 strain 9, GLRaV-4 strain Car, GRBV, ToRSV, TRSV, ArMV, GVF, phytoplasmas and X. fastidiosa) are not included in this table
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To confirm the results obtained by the PCR-based assays, 16 selected samples (Table 2) representing the different grape-growing regions in Pakistan were analyzed by high-throughput sequencing (HTS). Briefly, extracted RNA samples were subjected to ribosomal RNA (rRNA) depletion and complementary DNA (cDNA) library construction as described by Al Rwahnih et al. (2016). Later, cDNA libraries were sequenced using the Illumina NextSeq 500 and yielded between 25 and 32 million raw HTS reads (Supplementary Table 1), which were trimmed using the CLC Genomics Workbench (Qiagen). Contiguous consensus sequences (contigs) were generated from the cleaned HTS reads using the de novo assembler of the CLC Genomics Workbench. Finally, contigs were compared against the National Center for Biotechnology Information (NCBI) database of viruses and viroids using the tBLASTx program (Tatusova and Madden 1999) and a custom script. The HTS analysis revealed numerous contigs that ranged in size from 200 to 18,933 nucleotides from the 16 samples, such contigs showed a distant relationship (average identity: 87%) with several virus and viroid species (Table 2). In the case of samples BT47 and BW5 no virus was detected, but contigs resembling different viroids were observed. Near-complete genome sequences were obtained for GVA, GVE, GRSPaV, GFLV, GLRaV-2RG, GLRaV-3 and GLRaV-4. Subsequently, for each sample, all the viruses detected via RT-qPCR were identified during the HTS analysis (Table 3).

Table 2 High-throughput sequencing (HTS) detectable virus and viroid species in 16 selected grapevine samples. Contigs compared against the NCBI database using tBLASTx
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Table 3 Comparison of detected viruses by RT-qPCR and high-throughput sequencing (HTS) in 16 selected grapevine samples
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The occurrence of GVA (47.8%) was the highest. It is not clear whether this high rate of virus spread was initiated by using untested or non-certified propagation material or through a secondary spread by the biological vectors or both. GVA could spread in the vineyards by pseudococcid mealybugs and vine mealybug (Planococcus ficus), which is of economic importance on grapevines in Pakistan (Walton and Pringle 2004). However, the long-distance spread of GVA could be controlled effectively by using healthy propagative material (Martelli et al. 2001). Similar situations have been observed in other countries too. For example, GVA was one of the most commonly detected viruses (97.1%) in a survey conducted in Croatia (Vončina et al. 2017). In different surveys conducted in Jordan (Al-Tamimi et al. 1998) and Egypt (Fattouh et al. 2014), GVA was also found to be widespread, 42.5% and 30%, respectively. The second most significantly prevalent virus in this survey was GLRaV-2 (37.3%). The biological vector for this virus is unknown, therefore, the spread of the virus may be due to the use of virus-infected, non-certified planting material in Pakistan. A total of 93 vines (36.1%) were infected with GRSPaV. Different studies have shown that this virus is widespread throughout the grape growing regions of the world. Meng and Gonsalves (2007) and Rowhani et al. (2000) observed that a high incidence of GRSPaV in vineyards was due to using different GRSPaV-infected rootstock and scion sources for propagation. GRSPaV was also found in pollen and seedlings which could contribute to new infections through breeding lines (Lima et al. 2009). A number of tested samples (35%) were infected with GFkV. While a vector for GFkV is still unknown, reports from Vončina et al. (2017) and Fortusini et al. (1996) suggest that the high incidence of this virus in commercial vineyards might be due to the possibility of natural spread by biological vector(s). However, a detailed study of grapevine virus vectors in Pakistan would be required to make any speculations on vector-mediated transmission. In conclusion, the use of non-certified material may be a contributor to the high incidence of viruses detected in this survey. Other leafroll viruses, vitiviruses, GFLV, and GPGV, were also detected in the tested grapevine samples but the prevalence of these viruses was low. As mentioned before, mixed infections were also detected in the tested vines and mostly observed for the viruses associated with rugose wood complex, GLRaV-2 and GFkV viruses.

HTS analysis has been shown to be efficient and accurate for the detection of plant viruses, including uncharacterized species (Al Rwahnih et al. 2009; Al Rwahnih et al. 2016). Multiple studies have employed HTS to investigate the presence and impact of viruses infecting plants (reviewed in Prabha et al. 2013). In this work, HTS was used to identify viruses and viroids in 16 grapevine samples; thus, validating the results generated by the RT-qPCR testing. Consequently, viruses identified by HTS were compared with the RT-qPCR results and agreement was observed by both detection methods (Table 3). Finally, the obtained HTS results will be used as a basis for a further study to fully characterize virus isolates from Pakistan.

Among viral diseases of grapevines, grapevine leafroll disease (GLD) has been estimated to cause economic losses up to US$226,405 per ha in the grape growing areas of California (Ricketts et al. 2015). Grapevine certification programs have been created in grape-growing regions worldwide to provide the highest quality virus-tested propagation material. Growers and nurseries use healthy, virus-tested planting material originating from these certification programs to reduce spread of disease and thus improve the productivity of grapevine industry (Constable et al. 2010). For example, in the case of GLD a cost-benefit study of California’s north coast vineyards, determined that the economic benefits from a GLRaV-3 testing and clean stock program were US$52.7 million per year for this region (Fuller et al. 2015). Thus, the benefits of clean plant programs are large relative to the costs.

Along with the initial work by Rasool et al. (2017), this is the first time the presence of grapevine viruses has been confirmed in Pakistan. It is evident from the results that nearly all grape growing regions in the country are badly infected by multiple viruses. Results of these studies provide stakeholders with the current knowledge of the status of grapevine viruses in Pakistan. In addition, this research demonstrates the benefit of diagnostic tests for the detection of viruses of grapevines which can continue to be used for routine virus screening. Our results and the results reported by Sharma et al. (2015) indicate that virus presence in a region is not only dependent on localized spread mediated by vectors, but likely a combination of vector spread and the establishment of vineyards with virus-infected plant material. Therefore, implementation of screening programs to produce virus-tested grapevines and maintain healthy vineyards is likely to be of significant benefit to the grape industry in Pakistan. Likewise, in a recent survey of 48 grapevines tested for 31 viruses in Croatia, Vončina et al. (2017) found that among the tested vines not even one vine was virus free. They concluded that the poor sanitary status of the vineyards in Croatia likely resulted from vegetative propagation without consideration of sanitary control. Studies have shown the benefits of establishing a clean plant program for grapevine and providing virus-tested stock material for commercial use, produce higher yield of grapes and reduced virus impact in vines (Fuller et al. 2015). To this end, steps have been taken to begin the implementation of a clean plant program which will greatly improve the sanitary status of grapevine in Pakistan.