Viral Detection in Phalaenopsis Orchids Using High-Throughput Sequencing and One-Step Multiplex RT-PCR

Phalaenopsis orchids are highly valued ornamental plants but are susceptible to viral infections that significantly reduce their commercial value. Traditional detection methods for viruses infecting phalaenopsis are limited in efficiency and scope. In this study, we employed high-throughput sequencing to identify viruses present in 82 samples from three major phalaenopsis cultivars-'Ama' (Phalaenopsis amabilis), 'Haojili' (P. haojili), and 'Hongfuqitian' (P. hongfuqitian)-in Fujian Province, China. We identified six positive-sense single-stranded RNA (+ssRNA) viruses, including Odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV), which are well-known to infect orchids, as well as four viruses not previously reported in phalaenopsis globally: tomato mottle mosaic virus (ToMMV), pepper mild mottle virus (PMMoV), plum pox virus (PPV), and tobacco etch virus (TEV). ORSV exhibited the highest detection rate at 86.59%, followed by TMV at 34.15%. The detection rates of ToMMV, PMMoV, and PPV were 4.88, 3.66, and 2.44%, with TEV having the lowest detection rate at only 1.22%. Coinfection was prevalent, with 37.80% of samples infected by two or more viruses. We designed specific primers and developed a one-step multiplex reverse-transcription polymerase chain reaction detection method optimized for these six viruses, achieving high specificity and efficiency. This method was validated using additional diseased samples, confirming its practicality for rapid virus detection. Our findings enrich the understanding of the phalaenopsis virome and within-host virus diversity and provide a valuable tool for early diagnosis and management of viral diseases, contributing to improved quarantine measures and control strategies in phalaenopsis cultivation.