First report of a 16SrVI 'Candidatus Phytoplasma trifolii' isolate infecting Norfolk Island pine (Araucaria heterophylla) in India

Norfolk Island pine, Araucaria heterophylla, family Araucariaceae, is a distinctive conifer of global significance used in the timber industry. During 2006-07, about 15% of A. heterophylla plants at the Central Institute of Medicinal and Aromatic Plants, Lucknow, exhibited various symptoms including yellowing, little leaf, bunchy shoots, witches’ broom, branch or apical proliferation and bushy appearance (Fig 1). Leaves and petioles from plants with and without typical symptoms were collected and assayed for possible phytoplasma infection by transmission electron microscopy (TEM).Typical pleomorphic bodies, mostly spherical to oval, ranging from 350 to 800 nm were observed only in sieve elements of plants showing symptoms by TEM, as previously reported (Ajayakumar et al., 2007). In addition, total DNA was extracted and indexed in a nested PCR with universal 16S rDNA phytoplasma primers, P1/P6 followed by R16F2n/R16R2.

PCR products of expected size (~1.5 and ~1^.2 kb, respectively) were obtained from all plants with symptoms (4/4), but not from the symptomless plants (Fig. 2). PCR amplicons were cloned (TOPO TA kit, Invitrogen, Carlsbad, CA, USA), and sequenced. BLAST comparisons of the 16S rDNA consensus sequence, submitted to GenBank (Accession No. FJ002570), showed highest identity (96%) with those of members of group 16SrVI, ‘Candidatus Phytoplasma trifolii’. Phylogenetic analysis (MEGA 4.0) confirmed the Araucaria little leaf phytoplasma as a ‘Ca. Phytoplasma trifolii’-related strain (Fig. 3). The phytoplasma group 16SrVI was previously reported from India with little leaf diseases of aubergine/brinjal (Schneider et al., 1995), Withania somnifera (Zaim and Samad, 1995), Portulaca grandiflora (Samad et al., 2008), and Datura inoxia (Raj et al., 2009). This is the first record of a 16SrVI-related phytoplasma infecting A. heterophylla, suggested as a possible new ‘Ca. Phytoplasma’ species based on sequence analysis.