A novel approach to identify plant parasitic nematodes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Plant parasitic nematodes are difficult to identify because different species are morphologically similar, and this makes their control more difficult. The aim of this work was to develop a rapid, simple method to identify plant parasitic nematodes, based on analysis of protein profiles of nematodes generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two methods have been used: grinding and direct analysis of intact nematodes. Both methods were standardised using the nematode Anguina tritici (wheat seed-gall nematode) as a model. Development of the approach involved optimisation of experimental parameters to generate reproducible diagnostic protein profiles for plant parasitic nematodes. With alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix, the most effective solvent extraction was with 90% acetone. With sinapinic acid (SA) as matrix, 90% ethanol was most effective. When intact nematodes were analysed directly by mixing with the matrix solution, 40 min extraction with CHCA matrix solution generated the best protein profiles. The standardised methods were applied to analyse the seed-gall nematodes A. tritici and A. funesta and to the root-knot nematode, Meloidogyne javanica, which infects many horticultural crops. Typical protein profiles and diagnostic peaks were identified for these nematode species and for mixtures of Anguina species. The results provide 'proof-of-concept' that these nematode species can be identified by protein profiling using MALDI-TOFMS. This new approach could be extended to identify other plant and non-plant parasitic nematodes.