First report of the association of a ‘Candidatus Phytoplasma asteris‘‐ related strain with Plumbago auriculata leaf yellowing in India

Abstract

Plumbago auriculata (Plumbaginaceae) is a perennial ornamental shrub with many therapeutic values and native to South Africa (Saji & Antony, 5). Several groups of phytoplasma have been reported to infect ornamental and medicinal plants all over the world (Rao et al., 2). However, no phytoplasma has been found so far in P. auriculata. Leaf yellowing symptoms on P. auriculata plants (Fig. 1) were observed in Baramati, Pune, India in March 2016 with a disease incidence of 8-10%. To verify the presence of phytoplasma, three symptom-bearing and three asymptomatic P. auriculata samples were collected and DNA was extracted using the CTAB protocol. PCR was performed in a Mastercycler (Eppendorf, Germany) (Rao et al., 4) and DNA from the sesame phyllody phytoplasma (16SrI group) (GenBank Accession No. KC920747) was used as a positive control. The 16S rRNA gene was amplified from all the symptomatic P. auriculata leaf yellowing (PaLY) samples but not from the asymptomatic samples using P1/P7 (Schneider et al., 6) followed by R16F2n/R2 primer pairs (Gundersen & Lee, 1) in nested PCR. Amplicons of the expected size (c. 1.25 kb) were purified and directly sequenced (MN239503 and MN239504). BLAST analysis showed that these 16Sr RNA gene sequences shared 100% identity with phytoplasmas in the16SrI group (MG252367, MK440284, MK440282, KX15181 & KT957205). A phylogenetic tree was constructed using the neighbour-joining method with MEGA 7.0 (Fig.2). The R16F2n/R2 sequence of the PaLY phytoplasma was subjected to in silico RFLP using the iPhyClassifier online tool (https://plantpathology.ba.ars.usda.gov). The iPhyClassifier analysis indicated that the virtual RFLP patterns derived from the 16S rDNA F2n/R2 fragment of the PaLY phytoplasma strain with restriction enzymes were similar to those of a phytoplasma strain from group 16SrI, ‘Candidatus Phytoplasma asteris’ (M30790), formerly Aster Yellows Group, subgroup B, with a similarity coefficient of 1.0. Based on sequence and RFLP results, the PaLY phytoplasma is classified as a member of the phytoplasma subgroup 16SrI-B. Phytoplasmas have been recorded from plant species in the Plumbaginaceae such as Limonium sinuatum in Canada, Europe and Israel (Rao et al., 2). In India, the ‘Ca. P. asteris’ group is the most widespread group and has been found in 64 plant species (Rao et al., 3). Phytoplasmas in subgroup 16SrI-B have been associated particularly with diseases in pineapple, sesame, sugarcane, squash, rose and fennel. The present study reports P. auriculata as a new host for the 16Sr I-B phytoplasma subgroup worldwide. The authors wish to express sincere thanks to Head, Division of Plant Pathology and Director, Indian Agricultural Research Institute for providing laboratory facilities.