Exploring virus presence in field-collected potato leaf samples using RNA sequencing.

BACKGROUND: The quick and accurate identification of viruses is essential for plant disease management. Next-generation sequencing (NGS) technology may allow the discovery, detection, and identification of plant pathogens. This study adopted RNA-sequencing (RNA-Seq) technology to explore the viruses in three potato plants (S3, S4, and S6) growing under field conditions. RESULTS: Potato-known infecting viruses, such as alfalfa mosaic virus (AMV), potato leafroll virus (PLRV), and potato virus Y (PVY), were identified using bioinformatics programs and validated using RT-PCR. The presence of these potato viruses was also confirmed by visual inspection of host symptoms. In addition, the nearly complete genome of PLRV and the complete or partial genome sequence of multipartite virus segments have been identified. Besides the three major potato viruses that BLASTn analysis revealed were present in our samples, BLASTx analysis revealed some reads are derived from other potato viruses, such as potato virus V (PVV), Andean potato latent virus (APLV), and tomato chlorosis virus (ToCV), which are not frequently reported in potato field screenings in Egypt. Other microbial agents, such as bacteria and fungi, were also identified in the examined sample sequences. Some mycovirus sequences derived from ourmia-like viruses and Alternaria alternata chrysovirus were also identified in sample S4, confirming the complexity of the potato microbiome under field conditions. CONCLUSION: NGS quickly and accurately identifies potato plant viruses under field conditions. Implementing this technology on a larger scale is recommended to explore potato fields and imported plants, where symptoms may be absent, unspecific, or only triggered under certain conditions.

Biodiversity and full genome sequence of potato viruses Alfalfa mosaic virus and potato leaf roll virus in Egypt.

Solanum tuberosum (potato) is the second most important vegetable crop in Egypt. It is locally consumed, manufactured or supplied for export to Europe and other Arab countries. Potato is subject to infection by a number of plant viruses, which affect its yield and quality. Potato virus Y (PVY), potato leaf roll virus (PLRV), and Alfalfa mosaic virus (AMV) were detected in major potato-growing areas surveyed. Multiplex-RT-PCR assay was used for the detection of these three viruses in one reaction using three specific primer pairs designed to amplify genomic parts of each virus (1594 bp for PLRV, 795 bp for AMV, 801 bp for PVY). All three viruses were detected in a single reaction mixture in naturally infected field-grown potatoes. Multiplex RT-PCR improved sensitivity necessary for the early detection of infection. Incidence of single, double, or triple infection has been recorded in some locations. Full-length sequencing has been performed for an Egyptian FER isolate of PLRV. Through phylogenetic analysis, it was shown to occupy the same clade with isolate JokerMV10 from Germany. Complete nucleotide sequence of an Egyptian FER isolate of AMV and phylogenetic analysis was also performed; we propose that it is a new distinct strain of AMV belonging to a new subgroup IIC. This is the first complete nucleotide sequence of an Egyptian isolate of AMV. Genetic biodiversity of devastating potato viruses necessitates continuous monitoring of new genetic variants of such viruses.

Occurrence of Diverse Recombinant Strains of Potato virus Y Circulating in Potato Fields in Egypt.

Potato is one of the staple crops in Egypt, grown under irrigation almost continuously year-round. Potato virus Y (PVY) has been reported as one of the main viruses affecting potatoes in Egypt, but limited information is available on PVY strains circulating in potato fields in the country. From 2014 to 2016, virus surveys were conducted in several potato-growing governorates of Egypt, and PVY-positive samples were found to represent at least five distinct recombinant PVY strains, including PVYNTN and PVYN-Wi. Whole genome sequences were determined for four isolates representing strains PVY-SYR-III (Egypt7), PVY-261-4 (Egypt11), PVYNTNa (Egypt35), and a novel recombinant named Egypt24 that combined molecular properties of strains PVY-261-4 and PVY-Wilga156var. At least three recombinants found in Egypt in potato were previously found associated with potato tuber necrotic ringspot disease (PTNRD). The identification of multiple recombinant types of PVY in potato in Egypt, including the novel recombinant Egypt24, suggests a wide presence of PTNRD-inducing virus strains in the country.

Tomato bushy stunt virus (TBSV) infecting Lycopersicon esculentum.

Tomato bushy stunt virus (TBSV) was detected in tomato crop (Lycopersicon esculentum) in Egypt with characteristic mosaic leaf deformation, stunting, and bushy growth symptoms. TBSV infection was confirmed serologically by ELISA and calculated incidence was 25.5%. Basic physicochemical properties of a purified TBSV Egh isolate were identical to known properties of tombusviruses of isometric 30-nm diameter particles, 41-kDa coat protein and the genome of approximately 4800 nt. This is the first TBSV isolate reported in Egypt. Cloning and partial sequencing of the isolate showed that it is more closely related to TBSV-P and TBSV-Ch than TBSV-Nf and TBSV-S strains of the virus. However, it is distinct from the above strains and could be a new strain of the virus which further confirms the genetic diversity of tombusviruses.

Comparison of barley stripe mosaic virus strains.

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.