Plant Virus Laboratory, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India

*jawaidkhan@satyam.net.in

Accepted for publication 31/01/06

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.

Figure 1: Upper - Healthy looking sandal (Santalum album) tree (left) and a naturally-infected tree
exhibiting symptoms of sandal spike disease (right). Lower - Leaves from infected (right) and healthy (left) trees.

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). 

Figure 2: Agarose gel showing nested-polymerase chain reaction amplification of 16S rDNA of sandal spike (SAS) phytoplasma of Ca. 1.2 kb. The template consisted of products of direct PCR obtained from crude DNA extracts of symptomatic leaves of five different naturally infected Santalum album (sandal) plants (lanes 1-5); H= healthy control; M= λ DNA digested with EcoRI and HindIII.

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.

Figure 3: Polyacrylamide gel (6%) showing AluI, HinfI and RsaI digested Restriction Fragment Length Polymorphism of nested PCR amplified 16S rDNA (Ca. 1.2 kb) of a phytoplasma derived from symptomatic leaves of five different naturally infected Santalum album (sandal) plants (lanes 1-5); M= λ DNA digested with EcoRI and HindIII; L= 100 bp DNA ladder.

Dijkstra J, Ie TS, 1969. Transmission by dodder of sandal spike disease and the accompanying mycoplasma-like organisms via Vinca rosea. Netherlands Journal of Plant Pathology 75, 374-378.

Hull R, Horne RW, Hayer RM, 1969. Presence of mycoplasma-like bodies associated with sandal spike disease. Nature 224, 1121-1122.

Khan JA, Srivastava P, Singh SK, 2004. Efficacy of nested-PCR for the detection of phytoplasma causing spike disease of sandal. Current Science 86, 1530-1533.

Varma A, Chenulu VV, Raychaudhuri SP, Prakash N, Rao PS, 1969. Mycoplasma-like bodies in tissues infected with sandal spike and brinjal little leaf. Indian Phytopathology 22, 289-291.

The British Society for Plant Pathology