Detection of pathogenic Verticillium spp. using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Verticillium spp. have been listed by the European and Mediterranean Plant Protection Organization (EPPO) and China as plant quarantine pests. Although attempts have been made to develop a simple routine laboratory assay to detect these organisms, none are routinely used. We describe for the first time a robust assay for reliable identification of Verticillium spp. using protein fingerprinting data obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). Several sample preparation methods and matrices were investigated to improve mass spectra for the routine identification of six species of Verticillium spp.(Verticillium dahiliae, V. alboatrum, V. fungicola, V. nigrescens, and V. lecanii) by MALDI-TOF-MS. Using the optimized experimental method, we constructed a protein fingerprint database for six species of Verticillium and established a analysis criteria of log(Score). This MALDI-TOF-MS protocol should prove useful as a rapid and reliable assay for distinguishing different Verticillium spp.