Identification of a 'Candidatus Phytoplasma asteris'-related strain associated with spike disease of sandal (Santalum album) in India

Sandal (Santalum album) is a hemi-root parasitic tree, famous for its high-valued scented heartwood and oil. It commonly occurs in the dry regions of peninsular India, particularly in Karnataka and Tamil Nadu states. Sandal spike is a major disease of sandal, attributed to phytoplasma aetiology, as shown by three independent groups at the same time (Dijkstra & Ie, 1969; Hull et al., 1969; Varma et al., 1969). The disease is characterised by witches'-broom symptoms, consisting of small, narrow leaves which turn pale-green or yellow on branches. Affected leaves become overcrowded due to internodes shortening and standout stiffly from branches, acquiring a spike-like appearance (Fig. 1). Leaves and branches dry out and affected plants eventually die-off within a couple of years from the first appearance of symptoms. Although a non-specific PCR assay has been developed for the detection of phytoplasmas in sandal (Khan et al., 2004), the associated phytoplasma remains unidentified.

Total DNA extracted separately from leaves of either healthy or diseased sandal was used as template in a nested PCR assay employing phytoplasma rDNA universal primer pairs P1/P7, followed by R16mF2/R16mR1, as previously described (Khan et al., 2004). A nested PCR product of about 1.2 kb was amplified from a diseased plant but not from symptomless plants (Fig. 2).

The PCR products obtained from symptomatic leaves of five separate diseased plants were digested with restriction endonucleases AluI, HinfI and RsaI. The resulting Restriction Fragment Length Polymorphism (RFLP) profile for each enzyme was identical, indicating that all PCR positive plants contained a similar phytoplasma (Fig 3). The PCR product was purified, cloned and sequenced. The sequence was archived in GenBank (accession number DQ0932357). Pair wise comparison of the rDNA sequence by BLAST analysis revealed that it is most similar (99%) to 'Candidatus Phytoplasma asteris'-related strains, previously classified as 16S rDNA RFLP subgroup 16SrI-B members. This is the first definitive identification of a subgroup 16SrI-B phytoplasma associated with sandal spike disease.