Development and application of polymerase chain reaction-based assays for Rathayibacter toxicus and a bacteriophage associated with annual ryegrass (Lolium rigidum) toxicity
M. C. Kowalski A , D. Cahill B , T. J. Doran A and S. M. Colegate A CA Australian Animal Health Laboratories, CSIRO Livestock Industries, Geelong, Vic. 3220, Australia.
B School of Life and Environmental Sciences, Deakin University, Waurn Ponds Campus, Geelong, Vic. 3220, Australia.
C Corresponding author. Email: steve.colegate@csiro.au
Australian Journal of Experimental Agriculture 47(2) 177-183 https://doi.org/10.1071/EA05162
Submitted: 17 June 2005 Accepted: 19 June 2006 Published: 23 January 2007
Abstract
Annual ryegrass toxicity (ARGT) is responsible for significant stock losses in South Australia and Western Australia. The toxicity is caused by corynetoxins produced by the bacterium Rathayibacter toxicus (with the possible involvement of a bacteriophage), which infects annual ryegrass (Lolium rigidum). Polymerase chain reaction (PCR)-based assays, compatible with an existing enzyme-linked immunosorbent assay for the corynetoxins, have been developed and used to screen L. rigidum for both the presence of R. toxicus and for the bacteriophage isolate NCPPB 3778. The results from analysing bacterially infected galls from toxic grain screenings showed a positive correlation between the presence of the bacterium and corynetoxins but not with the bacteriophage. Analysis of pasture-derived samples of annual ryegrass showed about a 50% correlation of corynetoxins with bacterial presence and about a 5% correlation of phage with the presence of the bacterium. These observations support the potential application of the PCR-based assays in providing a useful, complementary tool in the assessment of the likelihood of pasture and feed to cause ARGT and to enable a better understanding of the complex aetiology of ARGT.
Additional keywords: 16S rRNA, BLAST search, DNA sequencing, nested PCR.
Acknowledgements
Dr Jeremy Allen (Department of Agriculture, Western Australia) and Dr Ian Riley (University of Adelaide, South Australia) are gratefully acknowledged for providing material for this study. Neil Anderton and Yu Cao (Plant Toxins Research Group, Geelong) are thanked for their assistance with the gall selections and corynetoxins ELISA respectively. Dr Vicky Stevens and Dr Volker Haring (Australian Animal Health Laboratory, Geelong) are thanked for reviewing this document as part of the CSIRO Livestock Industries internal review process. The project was funded by Deakin University and the CSIRO. The authors sincerely acknowledge the contribution of the anonymous referees in this final version of this report.
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