LAUSR

Acid lime [Citrus aurantifolia (L.) Swingle] is an important citrus cultivar that constitutes nearly 20% of total citrus production in India. During a survey conducted in April 2014 to February 2015, conspicuous symptoms of small chlorotic leaves with highly proliferated shoots and shortened internodes resembling witches’-broom disease of acid lime (WBDL) were observed in four out of 22 acid lime groves in Nagpur district of Maharashtra state, India. Seven representative field samples from symptomatic plants were graft-inoculated to healthy acid lime seedlings under greenhouse conditions and all developed characteristic symptoms 18 to 20 months after grafting. Numerous phytoplasma-like bodies of 100 to 800 nm in diameter and bounded by poorly defined membrane were observed in sieve tubes of diseased samples but not in similar sections from healthy leaves when ultrathin sections of leaf midrib were observed under a JEM 100S transmission electron microscope. PCR was used to confirm the presence of phytoplasma in infected tissues from 20 symptomatic and five asymptomatic plants. Samples were analyzed using P1/P7 primer pairs for the first round of PCR and the products were subsequently amplified with nested primer pairs R16F2n/R16R2 (Lee et al. 2004). An expected amplicon of ∼1.2 kb was obtained from 18 diseased plant tissues while it was not observed in asymptomatic plants. Four amplicons obtained from different field samples using R16F2n/R16R2 were purified, sequenced, and the resultant sequences were deposited in GenBank (KY024345, KY024346, KY024347, KY024348). All obtained sequences were 100% identical. The results of NCBI-BLAST analysis further revealed that the 16S rRNA gene sequences shared 99.9% similarity with that of the ‘Candidatus Phytoplasma cynodontis’ strains (LT558777, KF234570, AB741630). Phylogenetic analysis of obtained sequences showed that the WBDL phytopalsma formed a distinct clade with ‘Ca. Phytoplasma cynodontis’ strain BGWL-C1. In silico RFLP analysis of the aligned 16S rRNA gene sequence of the WBDL phytoplasma was performed with 17 distinct restriction enzymes using iPhyClassifier (Zhao et al. 2009) and the results were compared with each phytoplasma group and subgroup. Based on the analysis, WBDL phytoplasma 16S rRNA gene sequences shared 99.8% similarity with that of the ‘Ca. Phytoplasma cynodontis’ reference strain (AJ550984). Moreover, the virtual RFLP pattern derived from the query 16S rRNA gene F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XIV, subgroup A (AJ550984) (Marcone et al. 2004). Taken together, these results indicate that the phytoplasma associated with WBDL in India is a ‘Ca. Phytoplasma cynodontis’-related strain and belongs to 16SrXIV-A subgroup. To the best of our knowledge, this is the first report of occurrence of 16SrXIV group phytoplasma associated with WBDL not only in India but also in the world. It will be interesting to identify potential plant reservoir and insect vectors of the pathogen in this region. The disease adversely affects fruit yield and has the potential to significantly reduce economic condition of citrus growers of the region.