First evidence of mixed infection of phytoplasmas, “huanglongbing” and two viruses associated with declining disease of pummelo and sweet orange trees in Northeast States of India

Symptoms of little leaf, leaf yellowing with mosaic mottling and chlorotic streaks, stunted growth and decline were recorded in Citrus grandis cv. pummelo plantations at Barapani in Meghalaya and Citrus sinensis cv. sweet orange plantations at Tamenglong in Manipur, during 2021–2022. Five symptomatic leaf samples of both citrus varieties (pummelo and sweet orange) were collected from two locations and processed for verification of phytoplasma, “huanglongbing” and virus(es) presence. PCR and RT-PCR assays were employed using gene specific primers corresponding to 16S rRNA gene for phytoplasmas, omp gene for “huanglongbing” (HLB), coat-protein genes, CTV-cp for Citrus tristeza virus (CTV) and partial cp gene for Citrus yellow vein clearing virus (CYVCV). Expected length fragments of ˜1.25 kb (phytoplasma), ˜809 bp (HLB), ˜672 bp (CTV) and ˜333 bp (CYVCV) were amplified from the symptomatic leaf tissues collected from both citrus species in the two surveyed states. The results of the sequence analysis of 16S rRNA, omp and coat protein genes from pummelo and sweet orange confirmed the presence of mixed infection of ‘Candidatus Phytoplasma asteris’ (16SrI-B), ‘Ca. L. asiaticus’, CTV and CYVCV.