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

Abstract

Background: Brown rot of stone and pome fruit is a serious fungal disease that is mainly caused by four species in the genus Monilinia. Of these four species, Monilinia fructicola is the most devastating pathogen and of particular concern because it undergoes sexual recombination and has recently been introduced to Europe. So far, Monilinia diagnosis required a multiplex PCR analysis and gel electrophoresis. In contrast, intact-protein biotyping by mass spectrometry is considerably faster and cheaper. However, it usually requires an in vitro cultivation step prior to the MALDI analysis. It was thus attempted to establish a method for the identification of Monilinia species by MALDI biotyping with fungal material derived directly from infected fruits; without an in vitro cultivation step. Results:To simplify and render MALDI biotyping of fungi more reliable, an improved protocol for the preparation of crude protein extracts and for collecting MALDI-TOF MS data for biotyping was developed. We generated reference spectra for all four Monilinia brown rot fungi and were able to reliably identify Monilinia species based on fungal material that was collected directly from infected fruits. This method allowed the correct, fast and economic identification of M. fructicola and M. laxa, while M. fructigena and M. polystroma could not be distinguished reliably.Conclusions: MALDI biotyping may be used as an economical tool for the routine diagnosis of Monilinia brown rot fungi on infected fruits.

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi

“…Species identification was first attempted by MALDI-TOF as previously described [49]. In cases where MALDI-TOF did not allow species identification, the fungal ITS region was amplified with primers ITS1f [50] and ITS4 [51], PCR products were directly used for sequencing, and all isolates were assigned a species hypothesis according to the UNITE database [52, 53] (see also Table 1).…”

“…In cases where MALDI-TOF did not allow species identification, the fungal ITS region was amplified with primers ITS1f [50] and ITS4 [51], PCR products were directly used for sequencing, and all isolates were assigned a species hypothesis according to the UNITE database [52, 53] (see also Table 1). Crude protein extracts of isolates that were identified based on their ITS sequence were used to generate reference MALDI-TOF spectra for future identifications of the same species [49]. All isolates generated in the course of this study have been deposited and are available at the Culture Collection of Switzerland (CCoS; https://www.ccos.ch; Table 1).…”

“…Vogelgsang, Agroscope-FCr11115 Fusarium crookwellense WheatS. Vogelgsang, Agroscope-11SD14- Monilinia fructicola Infected apricot[49]-106- Rhizoctonia solani [107]ARSEF 1095F52/Met52- Metarhizium brunneum Cydia pomonella Austria …”

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

“…Even Many of these problems can be overcome by generating custom-made, specific MALDI-TOF MS reference spectra for the particular application in question [5]. Since these reference spectra are generated with the same MALD-TOF MS device, settings, and sample preparation as the experimental samples, the scores obtained with these references are usually higher as compared to generic reference databases [5,[17][18][19]. Custom-made reference spectra thus allow ample flexibility with respect to different sample preparation protocols and experimental set-ups in a particular lab or for specific applications.…”

“…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].…”