In the last decade, next-generation sequencing (NGS) has been extensively employed in the field of plant virology. Here, the transcriptome data of eight samples from leaves and fruits of four lemon varieties, including ‘Alata’, ‘Gulşen’, ‘Kütdiken’, and ‘Uzun’, were utilized for virus and viroid detection. Three viruses (citrus chlorotic dwarf-associated virus (CCDaV), citrus virus A (CiVA), and citrus endogenous pararetrovirus (CitPRV)) and five viroids (hop stunt viroid (HSVd), citrus dwarfing viroid (CDVd), citrus bent leaf viroid (CBLVd), citrus bark cracking viroid (CBCVd), and citrus exocortis viroid (CEVd)) were identified through bioinformatics analysis. However, distinct disparities were observed in the presence and abundance of viral/viroid sequences found in every variety and sample type. Among the detected pathogens, CiVA, CitPRV viruses, and HSVd and CDVd viroids were consistently detected across the various varieties and sample types. In contrast, the remaining viruses and viroids were only presented in specific host-sample type combinations. Several surveys were conducted, and the samples were tested by PCR using specific primer pairs. The data indicated that CitPRV was the most prevalent virus detected, followed by CDVd, CBLVd, CEVd, and CCDaV. Multiple virus-viroid co-infections were detected in 59.2 percent of the samples, with the CDVd + CEVd + CBLVd combination representing the most prevalent association, identified in 33 samples. The findings validated the effectiveness of applying NGS technologies for quick, accurate, and reliable detection of viruses and viroids in sanitation and certification schemes for citrus plants. However, the results can be influenced by tissue type and variety of plants, highlighting the crucial need for establishing standardized procedures for sampling, library preparation, as well as downstream data analysis and validation measures to enable accurate diagnosis of pathogens during routine screening. The findings of this study are essential for the development of effective management strategies aimed at excluding viral diseases, thereby safeguarding the overall health and productivity of crops.
