Cercospora beticola is an economically significant fungal pathogen of sugar beet, and is the causative pathogen of Cercospora leaf spot. Selected host genotypes with contrasting degree of susceptibility to the disease have been exploited to characterize the patterns of metabolite responses to fungal infection, and to devise a pre-symptomatic, non-invasive method of detecting the presence of the pathogen. Sugar beet genotypes were analyzed for metabolite profiles and hyperspectral signatures. Correlation of data matrices from both approaches facilitated identification of candidates for metabolic markers. Hyperspectral imaging was highly predictive with a classification accuracy of 98.5–99.9% in detecting C. beticola. Metabolite analysis revealed metabolites altered by the host as part of a successful defense response: these were L-DOPA, 12-hydroxyjasmonic acid 12-O-β-D-glucoside, pantothenic acid, and 5-O-feruloylquinic acid. The accumulation of glucosylvitexin in the resistant cultivar suggests it acts as a constitutively produced protectant. The study establishes a proof-of-concept for an unbiased, presymptomatic and non-invasive detection system for the presence of C. beticola. The test needs to be validated with a larger set of genotypes, to be scalable to the level of a crop improvement program, aiming to speed up the selection for resistant cultivars of sugar beet. Untargeted metabolic profiling is a valuable tool to identify metabolites which correlate with hyperspectral data.
Mass spectrometry coupled with LC (liquid chromatography) separation has developed into a technique routinely applied for targeted as well as for nontargeted analysis of complex biological samples, not only in plant biochemistry. Earlier on, LC‐MS (liquid chromatography–mass spectrometry) was mostly part of the efforts for identification of one or few unknown metabolites of interest as part of a phytochemical study. As a major strategy, unknown compounds had to be purified in sufficient quantities. The purified fractions were then subjected to LC‐MS/MS as part of the structural elucidation, mostly complemented by NMR (nuclear magnetic resonance) analysis. With the advance of mass spectrometry instrumentation, LC‐MS is now widely applied for analysis of crude plant extracts and large numbers (100s to 1000s) of samples. It has become an essential part of metabolomic studies (see Metabolomics), aiming at the comprehensive coverage of the metabolite profiles of cells, tissues, or organs. Owing to the huge chemical diversity of small molecules, conditions for the extraction will restrict the subfraction of the metabolome, which can be actually analyzed. The conditions for LC have to be adjusted to allow good separation of the particular metabolites from the respective extract. Major consideration will be the selection of an appropriate column and suitable eluents, the establishment of gradient profiles, temperature conditions, and so on.
Technical advances in LC–MS hardware instrumentation and in data analysis software tools have enabled the routine implementation of comprehensive metabolite studies in many biological laboratories. In this chapter, we describe the current types of MS instruments and corresponding applications. LC–MS systems can be coupled to UV or fluorescence detectors and even spatial resolution of metabolic distribution (MALDI‐imaging) is feasible to gain valuable additional information.
LC–MS applications usually divide into targeted or untargeted approaches, whereby the general aims are either to quantify a known substance or to analyze and compare metabolite profiles of two conditions to identify potential biomarkers. For tentative annotation of unknown compounds, LC–MS/MS analysis are conducted.
An incessantly growing number of software tools have been developed to handle the challenge of extracting biologically valuable information from LC–MS analysis, which generates enormous amounts of data. Furthermore, various databases have been set up to facilitate annotation of compounds of interest.
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