2.2.17
CHARACTERIZATION AND ANALYSIS OF A SECOND ISOLATE OF SUGARCANE BACILLIFORM BADNAVIRUS

JR GEIJSKES1, KS BRAITHWAITE1, RM HARDING2, JL DALE2 and GR SMITH1

1David North Plant Research Centre, Bureau of Sugar Experiment Stations, Indooroopilly, Qld, 4068, Australia; 2Centre for Molecular Biotechnology, Queensland University of Technology, Brisbane, Qld, 4001, Australia.

Background and objectives
Sugarcane bacilliform badnavirus (SCBV) is an important pathogen affecting the Australian sugar industry. SCBV is one of the viral pathogens limiting access to imported sugarcane (Saccharum L. interspecific hybrids) germplasm for breeding and cultivation and causes yield loss in some cultivars. Foreign germplasm plays a significant role in both breeding and commercial production of sugarcane in Australia; hence, continued access to exotic germplasm is essential for the long-term productivity, profitability and sustainability of the sugar industry. SCBV is serologically highly variable and initial RFLP studies on PCR-amplified DNA have also shown considerable variability at the nucleotide level [1]. To date, the genome of only one isolate of SCBV has been sequenced [2]. Thus, there is limited information available on the full extent of nucleotide variation between isolates and identification of conserved regions for the development of diagnostic assays is problematic. An understanding of the genome organization and gene functions of SCBV is essential to the successful development of robust detection and control strategies. A second isolate of SCBV has been sequenced and compared with the previous SCBV sequence in order to further the development of assays to diagnose all SCBV variants.

Materials and methods
SCBV was extracted from leaf tissue of the noble cane Ireng Maleng obtained from Meringa in north Queensland, Australia, using the method of Bouhida et al. ([2], with some modifications. The presence of virions was confirmed by immunosorbent electron microscopy. Virion preparations were treated with RNase and DNase to eliminate nonviral nucleic acids. Viral DNA was then extracted from the virion preparation by proteinase K digestion and phenol-chloroform extraction. DNA obtained was confirmed to be viral in origin by Southern blotting. This DNA was then used for enzyme digestion and cloning using the method of Bouhida et al. [2], with some modifications. Deletion mutants and specific primers were used to sequence the full-length SCBV genome. The sequence information was compared with the published sequence of SCBV and other badnaviruses, as well as sequence obtained from PCR products of some other Australian SCBV isolates.

Results and conclusions

The Australian isolate of SCBV from Ireng Maleng is approximately 150 bases longer than the published SCBV sequence. The Ireng Maleng isolate also shows considerable sequence variation from the published SCBV sequence with homologies of 83% for ORF I, 71% for ORF II, 74% for ORF III and 80% for the intergenic region. Significant sequence variability has also been observed in PCR products amplified from other Australian SCBV isolates. Phylogenetic analysis of SCBV isolates shows that the distance between isolates is as great as that between SCBV and other badnaviruses. Combining this recently obtained sequence information with the other available sequence information has allowed some highly conserved regions, up to 100% homology, to be identified. Some of these regions are being used to develop PCR primers or epitopes for antibody production.

References
1. Braithwaite KS, Egeskov NM, Smith GR, 1995. Plant Disease 79, 792-796.
2. Bouhida M, Lockhart BEL, Olszewski, NE, 1993. Journal of General Virology 74, 1-8.