First report of tomato spotted wilt virus (Orthotospovirus tomatomaculae) and phytoplasma in China aster and development of duplex PCR, LAMP, and qPCR assays for rapid detection.
UNLABELLED: China aster (Callistephus chinensis) is an economically important ornamental crop widely cultivated for cut flowers and landscaping. During field surveys conducted in three districts of Karnataka, India, China aster plants exhibiting chlorotic and necrotic ring spots, leaf deformation, and witches' broom symptoms were collected and analyzed to determine the causal agents. Mechanical inoculation of symptomatic leaf sap onto cowpea (Vigna unguiculata cv. C-152) produced characteristic chlorotic and necrotic ring spots on newly emerging leaves indicating the presence of an infectious viral agent. Serological assay by DAC-ELISA followed by RT-PCR confirmed the presence of tomato spotted wilt virus (TSWV, Orthotospovirus tomatomaculae) in symptomatic plants. Similarly the plants exhibiting witches' broom symptoms tested positive for phytoplasma infection using universal and Nested primers PCR assays targeting the 16S rRNA gene. Sequence analysis of TSWV CP gene revealed more than 97% nucleotide identity with TSWV isolates reported from India and other countries. Based on these results, one representative isolate was selected for complete genome sequencing. The complete sequences of the L, M, S RNA segements were amplified cloned, and sequenced showing more than 97% nucleotide identity with global TSWV isolates available in database. Sequence analysis of 16S rRNA gene of the phytoplasma associated with witches' broom symptoms was identified as 'Candidatus Phytoplasma australasiaticum' belonging to the 16SrII-D subgroup, sharing 99.2% nucleotide identity with previously reported isolates. Phylogenetic analysis further supported the placement of both the TSWV and phytoplasma isolates within their respective taxonomic groups. To facilitate rapid and sensitive diagnosis, quantitative PCR (qPCR) and RT-LAMP assays were developed for TSWV detection. In addition a duplex PCR assay was optimized for simultaneous detection of TSWV and phytoplasma from infected China aster plants in a single reaction. This study represents the first reports of the complete genome characterization of TSWV and phytoplasma infection in China aster in India along with the development of sensitive qPCR, RT-LAMP, and duplex PCR assays for rapid detection of these pathogens providing valuable tools for disease diagnosis, epidemiological studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05038-w.