Investigation of Viral Agents in Walnut (Juglans spp.) Trees by High Throughput Sequencing from Niğde province, Türkiye

In October 2023, virus-like symptomatic walnut trees in Niğde province were investigated using high-throughput sequencing (HTS) to investigate viral infections. Double-stranded RNA (dsRNA) extraction was conducted, followed by conversion of dsRNA into cDNA. PCR was then performed using barcoding with MID primers for metagenomic analyses. The PCR products from walnut samples B13, B22, B23, and B21 + B5 were sequenced using the Illumina NovaSeq 6000 sequencing platform. The sequencing data underwent bioinformatics analysis using Geneious Prime and CLC Genomic Workbench. In this comparison, more contigs were constructed in CLC Genomic Workbench, resulting in more precise outcomes with BLASTx analysis. According to the map, to reference analyses of B21+B5 HTS data, 29,574 reads matched with cherry leaf roll virus RNA1 (CLRV) RefSeq NC_015414, and 405 reads matched with CLRV RNA2 RefSeq NC_015415. However, 4 reads matched with cucumber mosaic virus (CMV) RefSeq NC_001440 from B23 HTS data. To confirm the presence of these viruses, virus-specific primers were used to test the total RNA extracted from walnuts. CLRV was detected in sample B21 but not in B5. CMV was not detected in sample B23. Improving nucleic acid extraction methods is recommended to enhance the detection of viral agents using high-throughput sequencing.