Novel molecular diagnostic frame work for plant parasitic nematode pest of plant biosecurity concern-better and faster than classical taxonomy?

Phyto-nematodes are one of the four most important agricultural pests worldwide. The annual losses in agricultural production from nematode infection is about USD$120 billion. Although phyto-nematodes cannot move more than a metre per year, human activities have lead to the dispersal of these major pests around the world, and the transportation of infected plant materials from one continent to another can put Australian agriculture at risk. "Plant Biosecurity" requires cutting edge molecular techniques to identify biosecurity pathogens. The aim of this project is to develop new protocols for early detection of exotic phyto-nematodes: this will benefit Australia and help safeguard its borders. A requirement is to reduce the time taken for nematode identification so that pest incursions can be detected and controlled early. Most nematode identification has been done by classical taxonomy. However, this requires trained taxonomists familiar with nematode morphology, and identification is time consuming for an unknown nematode. More recent molecular techniques of nematode diagnostics have shown promising results for nematode identification. Molecular diagnostics has proved to be reliable and rapid compared to classical approaches. In this project, work is being undertaken to compare DNA-based, protein-based and novel methods of nematode identification. So far, DNA and protein diagnostic methods have been developed for a range of root lesion and cyst nematodes. Characteristic sequences of ITS regions have been generated and phylogenetic relations of these nematodes studied. Similarly protein biomarkers have been established that can be used-both to identify species and genera of these nematodes. Although plant nematodes can only move short distances on their own, they have been transported around the world effectively by human activities. The outcome of this project will help reduce entry of exotic nematodes into Australia, and so help reduce potential crop tosses

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

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 nemal:odes, 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 α-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

A novel approach to identify plant-parasitic nematodes using MALDI-TOF mass spectrometry (Abstracts: Society of Nematologists 44th Annual Meeting, Fort Lauderdale, Florida, 9-13 July 2005)

The ability to accurately identify nematodes at the species level requires specific skills in nematode taxonomy. The most common approach used is microscopy, which takes time and requires specific training: molecular approaches to plant nematode identification can also be used. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is being used as a new approach to identify microorganisms. Identification is based on the characteristic protein profile of the organism generated by MALDI-TOF MS. In MALDI-TOF MS, pulses from a UV laser are absorbed by a crystalline matrix to which macromolecules have been adsorbed, and this results in relatively gentle desorption and ionization of the macromolecules, which are then accelerated using a high voltage and travel along the flight tube. The time taken to reach the detector is directly related to the mass/charge ratio of the molecule. MALDI-TOF is a promising approach for rapid identification of organisms because of the simple sample preparation and the rapidity of the technique. 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 MALDI-TOF MS. Two methods have been used: grinding and direct analysis of intact nematodes. Both methods were standardized using the wheat seed-gall nematode Anguina tritici as a model. The standardized methods were applied to analyze the seed-gall nematodes A. tritici and A. funesta and the root-knot nematode, Meloidogyne javanica, which infects many horticultural crops. Characteristic protein profiles and diagnostic peaks were identified for individual nematode species and for mixtures of these species. The results provide proof-of-concept that these nematode species can be identified by protein profiling using MALDI-TOF MS. This approach could be extended to identify other plant and non-plant parasitic nematodes by generating unique species-specific protein profiles