First report of ‘Candidatus Phytoplasma trifolii’ associated with little leaf disease of Capsicum chinense from the northeast of India

Abstract

King chilli or bhut jolokia (Capsicum chinense, family Solanaceae), is a variety of capsicum indigenous to northeast India. It is considered one of the hottest chillies in the world and is one of the most valuable spice crops in India, being grown for culinary and medicinal purposes. In April 2022, phytoplasma-associated symptoms such as shortening of leaves, petioles and internodes, and stunting of the whole plant were observed on C. chinense plants in Jorhat, a district in Assam, India (Figure 1). To investigate the possibility of a phytoplasma being associated with the symptoms, total DNA was isolated from leaf samples collected from six diseased and six asymptomatic plants using a CTAB protocol (Kollar et al., 1990). The DNA was analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, and P1/P6 (Deng & Hiruki, 1991) and R16F2n/R16R2 primers (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (1.5 Kb after direct PCR and 1.25 Kb after nested PCR) were obtained from diseased plants only. Nested PCR R16F2n/R16R2 products were Sanger sequenced in each direction, and the consensus sequence deposited in GenBank (Accession No. ON819760). BLASTn analysis revealed that the C. chinense phytoplasma 16Sr DNA sequence shared >99% sequence identity with those of the 16SrVI group including the isolate used to describe ‘Candidatus Phytoplasma trifolii’ (AY390261; Hiruki & Wang, 2004) and many strains found in India (e.g. MN861357 and MN861370). A phylogenetic tree (Figure 2) and an iPhyClassifier-based virtual RFLP analysis of the 16S rRNA gene sequence revealed that the phytoplasma-associated with little leaf of C. chinense belongs to the16SrVI-D subgroup with a similarity coefficient of 1.00. Previously, different phytoplasma subgroups have been associated with Capsicum annuum in India (‘Ca. P. asteris’, 16SrII-D; Sharma et al., 2015). In the present study, we confirmed the association of the ‘Ca. P. trifolii’ subgroup D of phytoplasmas with king chilli. The 16SrVI-D subgroup of phytoplasma has been associated with several other agricultural crops and weeds in northeast India (Dutta et al., 2022). To the best of our knowledge, this is the first report of the association of the 16SrVI-D phytoplasma subgroup with C. chinense and extends the known host range of this phytoplasma in India. In this study, affected crops had about 5–8% disease incidence based on visual observation and bearing in mind the symptoms observed, significant yield losses in commercially grown king chilli may occur. Further epidemiological and insect vector studies are needed to help formulate effective management strategies to prevent further spread.