MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Abstract

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI-TOF MS-based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer-based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10(9) spores per ml. The best peptide/protein profiles (in terms of signal-to-noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves.

Direct identification of Monilinia brown rot fungi on infected fruits by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

“…For example, Monilinia brown rot fungi have been subjected to different electrophoresis methods as well as to MALDI-TOF MS analysis [22]. Similarly, downy and powdery mildew, caused by Bremia lactucae and Oidium neolycopersici, respectively, were identified by MALDI-TOF MS in vitro as well as on infected plant leaves [36]. Furthermore, MALDI-TOF MS, in combination with a DNA-based phylogenetic analysis, was used to compare and differentiate closely related, plant pathogenic Alternaria species [37] as well as a collection of 45 Brazilian isolates of the genus Clonostachys [38].…”

Intact spore MALDI-TOF mass spectrometry and proteomic analysis of Puccinia pathogenic fungi

“…Suspending Puccinia spores in water according to a previous protocol for IC/IS MALDI-TOF MS of fungal phytopathogenic microorganisms Bremia lactucae and Oidium neolycopersici [19] was rather unsuccessful because of their hydrophobicity. For that reason, water was replaced by the matrix solution: FA/SA, 5:15 mg ml − 1 in ACN/2.5% (v/v) TFA, 7:3, v/v, either alone or diluted with water in a volume ratio of 1:1.…”

“…After washing, the spores were suspended in the matrix solution -alone or after diluting it in a ratio of 1:1 (v/v) with deionized water -to get a required spore concentration. The MALDI matrix used was FA:SA, 5:15 mg ml −1 in ACN/2.5% (v/v) TFA, 7:3, v/v [19]. There were three sample preparation techniques evaluated [14]: dried droplet (1 μl of the suspension was mixed with 1 μl of matrix directly on the target plate), mixed volume (MV; the suspension was premixed with matrix or diluted matrix and then 2 μl were applied on the target plate) or two-layer volume (2LV; after drying out of 2 μl of the sample-matrix solution applied by the MV technique, an additional 1 μl of the matrix solution was pipetted and left to dry).…”

“…Recently, IC/IS MALDI-TOF MS was applied to downy and powdery mildews [19]. The experience gained from the previous work was utilized now in a study with rust fungi.…”

MALDI-TOF mass spectroscopy of yeasts and filamentous fungi for research and diagnostics in the agricultural value chain

“…As in clinical diagnostics, the need to identify fungi often goes along with the goal to identify a particular organism directly in the environment, infected plant tissue, or food, which can harbor complex microbial communities and potentially host tissue. Examples of in situ identifications of microorganisms by MALDI-TOF MS include plant invasive Rhizobia [95], obligate biotrophic fungal pathogens [96,97], or Monilinia brown rot fungi [18]. In food science, MALDI biotyping has been mainly used for the identification of food-borne yeasts, which were pre-cultivated prior to MALDI-TOF MS analysis [87,98,99].…”