1 1Plant Biotechnology Research Group, School of Biological Sciences and Biotechnology, WA State Agricultural Biotechnology Centre, Murdoch University, Perth, WA 6150, Australia, Department of Agriculture and Food Western Australia,
Biotechnology, 3 Baron-Hay Court, South Perth, WA 6151, Australia;, Email: email@example.com
2 2Plant Biotechnology Research Group, School of Biological Sciences and Biotechnology, WA State Agricultural Biotechnology Centre, Murdoch University, Perth, WA 6150, Australia
3 3South Australian Research and Development Institution, Field Crops Unit, Nematology/Quarantine, Glen Osmond, SA 5064, Australia, Plant Health Australia, FECCA House, Phipps Close, Deakin, ACT 2600, Australia
4 4Department of Agriculture and Food Western Australia, Plant Pathology, 3 Baron-Hay Court, South Perth, WA 6151, Australia
The stem and bulb nematode, Ditylenchus dipsaci, is a serious pest of forage, horticultural and other crops. The two races of D. dipsaci that occur in Australia are the oat and lucerne races. These two races have the 'normal' morphology compared to the 'giant' type that attacks Vicia faba. The oat and lucerne races have been found in eastern Australia but not in Western Australia. There are no morphological or specific molecular tests to differentiate races within closely related 'normal' types of D. dipsaci. The aim of this work was to find protein biomarkers that would differentiate oat and lucerne races of D. dipsaci using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), 2-D PAGE and associated proteomics techniques. Three protein biomarkers at m/z 4313 ± 0.1%, 6300 ± 0.1% and 7460 ± 0.1% were found that discriminate the oat and lucerne races of D. dipsaci using MALDI-TOFMS. The biomarker at m/z 4313 ± 0.1% was the prominent race-specific marker for the lucerne race and is almost absent in the oat race. In addition, proteomic maps were obtained by 2-D PAGE of proteins extracted from oat and lucerne races of D. dipsaci. This analysis allowed a comparison of acetone soluble proteins of oat and lucerne races of D. dipsaci. A prominent protein spot was identified with an isoelectric point (pI) of about 5 and molecular mass ca 4.5 kDa for the lucerne race, which was absent in the oat race. This particular protein was trypsin-digested and analysed by MALDI-TOFMS and MALDI-TOF-TOFMS. The resultant spectra of peptide mass fingerprints (PMF) showed two major peptides at m/z 845 ± 0.1%, 916 ± 0.1% and a less intense peptide at m/z 1258 ± 0.1%. De novo sequencing and MS/MS interpreted amino acid sequences are presented.