Sample collection and ELISA

Onion plants that  exhibited the symptoms of yellowing, dwarfism, yellow mosaic, and twisted leaves were observed in Riyadh and Al-Baha regions during field surveys in the years 2021-2022 (Figure 1). A total of 81 samples of onion plants were collected from the Al-Baha and Riyadh Regions (Table 1). Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with specific antibodies obtained from LOEWE®, Biochemica, and DSMZ, Germany, were used to test the collected samples against the important allexiviruses including Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C), shallot virus X (ShVX), and potyviruses including Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV) and Shallot yellow stripe virus (SYSV).

The procedure was performed and demonstrated according to the manufacturer’s protocol using BioTek ELx808 ELx808IU Absorbance Microplate Reader96-Well. Samples were considered positive according to Clark and Adams,1977 [18] and Daryono et al., 2003 [19].

Reverse transcription polymerase chain reaction (RT-PCR) 

Total RNA was extracted from all collected leave samples (100mg) using Gene JET Plant RNA Purification Mini Kit and One-step RT-PCR was carried out by following the instruction of the manufacturer’s protocol of Thermo Scientific, USA using degenerate primer for allexiviruses (Allex-CP (+) and Allex-NABP (−) [20]. RT-PCR cycle was performed as follows: 60℃ for 10 min and 98℃ for 2 min followed by 35 cycles, 94℃ for 45 sec, 60℃ for 45 sec, 72℃ for 1 min, 72℃ for 10 min as final extension. For Potyvirus, the degenerate primers (Nib2-F and Nib3-R) were used [21] and the RT-PCR cycles were set to 42°C for 45 minutes, 95°C for 12 seconds, 40 cycles, 95°C for 15 seconds, 48°C for 20 seconds, 72°C for 30 seconds, and 72°C for 1 minute as the final extension.

The RT-PCR products were electrophoresed using 1% agarose gel in TBE buffer (Tris Borate EDTA) [22]. A Gel Documentation system was used to analyze the PCR-amplified products. The expected molecular sizes have been determined using the 50 bp HyperLadder DNA (Bioline Ltd., USA) and the 100 bp DNA Ladder (GeneRuler, Thermo Scientific, USA).

Analysis of phylogenetic trees with nucleotide sequencing

Three and two RT-PCR products were obtained from onion samples by general primers of Allexivirus and Potyvirus groups, respectively. These amplified products were purified and sent to Macrogen Inc. Seoul, South Korea for two-directional Sanger sequencing of the DNA. Sequencing data was aligned using DNA star computer software and the National Centre for Biotechnology Information (NCBI). Using 1000 bootstrap replications and the Maximum-Likelihood method algorithm, the phylogenetic trees were constructed from ClustalW-aligned sequences by MEGA-X to analyze the phylogenetic relationship of the Saudi isolates with the other isolates reported from different countries. Pairwise nucleotide sequence identity percentages were determined using the MegAlign tool in the DNAStar software package by Lasergene and a graphical explanation of pairwise nucleotide percentage identity was presented by Sequence Demarcation Tool (SDT) [23].

Results

Field Observation and ELISA

Eighty-one symptomatic samples exhibited mosaic, dwarfism, yellowing, leaf striping, stunted growth, and deformation of leaves were collected from two main different onion growing regions (Al-Baha and Riyadh) in Saudi Arabia (Figure 1).  The obtained results of ELISA from 81 onion plant samples indicated the occurrence of GarV-A and OYDV with a detection percentage of 26% and 32%, respectively. The obtained ELISA results showed that 47/81 (58%) onion samples were positive for at least one of the tested viruses. As far as a single and mixed infection of GarV-A and OYDV was concerned, 27% of the infected samples were singly infected with either GarV-A or OYDV and 55% of those had mixed infection of both viruses. The GarV-B, GarV-C, and ShVX were not detected in any of the tested samples (Table 1).

RT-PCR Reaction

For the detection of allexiviruses from onion samples using general primers, the amplified RT-PCR products have been of expected size 750 bp (Figure 2 A). In the case of Potyvirus amplification, the expected size of the amplicon was 350 bp (Figure 2 B). RT-PCR amplification was not observed with total RNA isolated from healthy plants as a negative control with any of the primer pairs used in this study. The outcome of these results revealed that ELISA-positive onion samples were infected with at least one of the viruses belonging to Allexivirus and Potyvirus. The presence of the exact virus infection was confirmed after Sanger DNA sequencing of these RT-PCR amplified samples.

Phylogenetic tree analysis with partial nucleotide sequencing

For general allexiviruses, two isolates of GarV-A and one isolate of GarV-D isolated from the onion samples showed a  higher level of sequence identity with GarV-A and GarV-D based on their BLAST (basic local alignment search tool) analysis. After completing the analysis, sequences have been submitted to  GenBank with the following accession numbers: OQ397545 (GarVA-O-Riyadh), OQ397546 (GarVA-O-AlBaha), and OQ397547 (GarVD-O-Riyadh) sequentially. The phylogenetic tree of GarV-A showed that our Saudi isolates grouped with the GarV-A isolates from Russia which were isolated from Allium sativum (JX682827), and China (LC097148) and our GarV-D isolate made a clade with GarV-D isolates from Argentine isolated from A. sativum (KF550407; Figure 3 A). As far as their nucleotide sequence identity percentage is concerned, GarV-A isolates from Riyadh and Al-Baha regions had 100% identity among themselves and 96.7% to 98.5% with the already published isolates in NCBI GenBank databases. The highest percentage identity was observed with GarV-A Chinese isolate (MN059312, MN059313), and the lowest percentage was observed with Poland isolate (KX889752; Figure 4 A). Moreover, GarV-D shared 80.6% to 83.6% of identity with the other available isolates in the NCBI GenBank. The highest percentage was observed with Poland isolate (KX889787), and the lowest percentage was also observed with Poland isolate (HQ681925; Figure 4 B).

For Potyvirus, the nucleotide sequence analysis of the two isolates of Potyvirus isolated from the onion samples in Riyadh and Al-Baha revealed that these sequences were related to OYDV. These sequences were submitted to  GenBank with the accession numbers OQ397548 (OYDV-Riyadh) and OQ397549 (OYDV-AlBaha), respectively. The phylogenetic tree and SDT analysis of these two isolates of OYDV were  constructed (Figures 3 B and Figure 4 C). Both isolates showed 99% to 100% identity between themselves. However, OYDV from Riyadh shared 80% to 95% identity with other isolates from NCBI GenBank, and OYDV from Al-Baha showed 80% to 95.5% similarity with the NCBI GenBank isolates.

Figures & Tables

Discussion

Onion is a major bulbous crop among vegetables and is of global importance [24, 25]. As a result of vegetative propagation, which causes viruses to accumulate and replicate in planting material, viruses are the primary issue in bulb crops like onion and garlic. Although viruses that infect Allium species have been intensively studied. Due to the multiple virus infections, serological and molecular studies were not easy to conduct for the detection of these viruses [26-28]. Viruses are ubiquitous worldwide and cause impacts in the form of crop production loss and reduction in quality [29, 30, 7]. It is tough to identify and estimate yield losses that are precisely attributable to each virus due to the incidence of mixed virus infections.

In eighty-one samples, 47 samples of onion (58%) were positive with GarV-A and OYDV. Hence, a large number of infected samples showed that the exchange of infected planting material from one region to another region could be the main reason for their occurrence in surveyed regions, but the presence of their vectors (aphids and mites) cannot be neglected because they play a significant role in their short distance disseminations within the fields.

Furthermore, in this study, two allexiviruses (GarV-A and GarV-D) and one Potyvirus (OYDV) isolated from onion were amplified by degenerate primers in the Riyadh and Al-Baha regions of Saudi Arabia. Interestingly, GarV-D could not be detected during the ELISA assay (unavailability of antisera) but got amplified in RT-PCR using general allexiviruses primers. This showed along with ELISA tests, a variety of other molecular techniques, RT-PCR, and hybridization must be incorporated into detection methods. Thus, a virus's entire genome can be identified by molecular analysis, and only a small number of viruses can be identified through serological investigation [31-36]. Therefore, molecular analysis is always chosen over serological testing due to their higher sensitivity and robustness.

In RT-PCR, two Saudi Arabian isolates of GarV-A from Riyadh and Al-Baha regions and one Saudi Arabian isolate of GarV-D from Riyadh region showed their nucleotide identities of 96.7% to 98.5% and 80.6% to 83.6%, respectively with already published isolates in GenBank from different countries. Moreover, two Saudi Arabian isolates of OYDV from Riyadh and Al-Baha regions showed 100% nucleotide sequence identity with each other and 80.4% to 95.5% nucleotide identity with the other isolates of different countries. So, natural genetic variation could be the reason for the variation in the nucleotide identity of these isolates. Generally, phylogenetic analyses unveiled that Saudi Arabian onion isolates are normally grouped together, and the phylogenetic similarity relationship between them and their geographical roots is frequently unrelated. The great diversity and swift evolution of these viruses have been associated with the international exchange of vegetative planting materials between states, based on numerous studies conducted in other countries [37-40].

The results of this investigation would be useful in providing information about the evolutionary changes and diversity of these significant viruses. Also, this research may play a significant role in enhancing disease management procedures. Therefore, biological research is necessary to identify the transmission routes and assess the effects of isolated and combined infections of different isolates and different viruses causing mixed infections on onion crops. According to the best of our information, this is the first report of GarV-A, GarV-D, and OYDV infecting onion crops in Saudi Arabia, and this is also the first report of GarV-A from onion in the world.

Acknowledgements

The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through project no. (IFKSUOR3-556-2).

Author Contributions

Mohammed Amir, Mohammed Al-Saleh, and Mahmoud Amer designed the study, prepared the materials, collected the data, conducted the analysis, wrote and edited the manuscript. Ibrahim M. Al-Shahwan and Khadim Hussain conducted the data analysis. 

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

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