Fingerprinting the Australian rhizobial inoculant mother cultures using refined PCR protocols yields beneficial inoculant management applications
A-M. Vachot-Griffin A and J. E. Thies A B C DA Centre for Farming Systems Research, University of Western Sydney — Hawkesbury, Locked Bag 1797, Penrith South DC, NSW 1797, Australia.
B Centre for Biostructural and Biomolecular Research, University of Western Sydney — Hawkesbury, Locked Bag 1797, Penrith South DC, NSW 1797, Australia.
C Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853, USA.
D Corresponding author. Email: jet25@cornell.edu
Australian Journal of Experimental Agriculture 45(3) 141-150 https://doi.org/10.1071/EA04061
Submitted: 28 March 2004 Accepted: 9 February 2005 Published: 14 April 2005
Abstract
Monitoring the success of rhizobial inoculation requires reliable identification of the introduced strains in nodules and when recovered from field soil. The polymerase chain reaction (PCR) coupled with the use of either random or directed primers has increasingly become the molecular method of choice for characterising bacteria at the strain level. We have investigated the use of 5 markers (REP, ERIC, BOXA1R, RPO1 and IGS) commonly used for PCR fingerprinting to characterise rhizobia bacteria used in the manufacture of rhizobial inoculants in Australia. PCR with random primers often yields inconsistent results because most protocols do not specify stringent cycling and non-cycling parameters. We have increased the stringency and improved the specificity of reaction conditions for 4 of the 5 markers tested. Optimised protocols were then used to fingerprint the 39 strains of rhizobia bacteria held in the 1998 mother culture collection of the Australian Legume Inoculant Research Unit (ALIRU). Results for 34 strains using at least one marker are presented. Although the mother cultures of these inoculant strains undergo numerous quality assurance tests annually, it was not until PCR fingerprinting was applied that 2 strains, believed to be unique, were found to be identical. In the subsequent investigation, we determined that the 2 strains were originally unique but that a mix-up in the cultures had occurred at least 3 years before our analysis. Use of serology, plant infection tests and field tests were not sufficient to detect this problem. The use of PCR fingerprinting with optimised protocols has now been incorporated into the annual quality assurance regime used by the ALIRU who monitor strain quality for the Australian rhizobial inoculant industry. Higher quality rhizobial inoculant for use by Australian farmers is a beneficial outcome of this work.
Acknowledgments
The authors are grateful to Elizabeth Hartley and Greg Gemell of ALIRU, Gosford, NSW, for supplying the 1998 and 1999 AIRCS rhizobial mother cultures for our analysis. We thank Alison McInnes, formerly of CSIRO Plant Industry, Brisbane, for revitalising strain CB82 from the CSIRO collection to reinstate it in the ALIRU mother culture collection, and Gary Bullard formerly of Bio-Care Technology Pty Ltd (Sommersby, NSW) for supplying commercial inoculant for our examination. We thank Judy Gray for her technical assistance, and John Brockwell and John Howieson for their assistance with chronicling strain history and application. This work was supported by the Grains Research and Development Corporation grant UWS20.
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