Molecular diagnostics insights of little leaf and witches’ broom disease of sunnhemp in Tamil Nadu

Little leaf and witches’ broom disease associated with phytoplasma infection has recently been reported in Tamil Nadu, across all growing seasons. A survey was conducted to assess the incidence of sunnhemp little leaf and witches’ broom disease in various sunnhemp growing regions of Tamil Nadu during 2022-2024, across all seasons. The highest incidence of little leaf and witches broom disease was observed during the Kharif 2023 in Pudukkottai district with an infection rate of 0.55 % to 88.67 %. Infected plants exhibited characteristic symptoms including phyllody (leaf-like structures replacing flowers), virescence (green pigmentation in floral parts), yellowing, reduced leaf size, stunted growth, witches’ broom (proliferation of shoots), leaf curling, bunchy appearance at stem ends, floral gigantism, twig die-back and unseasonal yellowing or reddening of leaves. The disease is caused by Candidatus phytoplasma and is transmitted by leafhoppers. However, the phytoplasma cannot be cultured under in vitro conditions. Therefore, in this study, nested PCR approach was employed as a rapid and reliable molecular diagnostic technique for detecting phytoplasma. Specific second-round primers were used to confirm the presence of phytoplasma in little leaf and witches’ broom infected samples. The infected plant material was analyzed using a 16S rRNA region-specific primer to identify the pathogen. Sequence analysis revealed that the phytoplasma infecting sunnhemp had over 99 % nucleotide similarity with other isolates of Candidatus: Phytoplasma aurantifolia from the NCBI database, confirming the association of Candidatus: Phytoplasma aurantifolia (16SrII group) with sunnhemp little leaf and witches’ broom disease.