Abstract
Metabolomics has emerged as a powerful approach to comprehensively interrogate cellular biochemistry. As such, we applied an untargeted liquid chromatography-mass spectrometry metabolomic strategy to elucidate metabolome changes in the anthracnose-causing hemibiotrophic sorghum pathogen, Colletotrichum sublineolum. An in vitro batch culture study model with different carbon sources, glucose, arabinose and rhamnose, were used to support fungal growth over a period of twelve days. Metabolites representing the intracellular and extracellular (secreted) metabolomes were extracted with methanol and subjected to LC-MS analyses. Chemometric modelling revealed a metabolic variation trajectory, comprising three distinct stages that metabolically describe the adaptation of the fungus to diminishing nutrients. Selected marker gene expression indicated stage one (0–3 d.p.i) as corresponding to the early logarithmic phase. Stage two can be interpreted as an intermediate transitionary stage with stage three corresponding to the stationary phase (9–12 d.p.i). Stage one was characterised by up-regulation of endo-metabolites such as ferricrocin, fatty acids and flavone-conjugates, while stage three was characterised by the secretion of phytotoxins, including colletotrichin and colletotric acid. Ultimately, results from our in vitro model reveal previously unknown insights into the dynamic aspects of metabolome reprogramming in the growth phases of Colletotrichum spp as determined by nutrients obtainable from plant cell walls.
Highlights
Colletotrichum is one of the most widespread and economically detrimental genera of plant pathogenic fungi, and represents a serious threat to global food security and ecosystem health[1,2]
Colletotrichum fungi differentiate specialised infection vesicles, which begin the growth of primary hyphae that develop inside living host cells surrounded by an intact host plasma membrane[6,7]
Understanding nutrient acquisition modes employed by hemibiotrophic phytopathogenic fungi, with carbon metabolism as an essential feature[14], can provide informative insights about the biochemical underpinnings governing fungal adaptation to the host environment[15]
Summary
Colletotrichum is one of the most widespread and economically detrimental genera of plant pathogenic fungi, and represents a serious threat to global food security and ecosystem health[1,2]. Colletotrichum fungi differentiate specialised infection vesicles, which begin the growth of primary hyphae that develop inside living host cells surrounded by an intact host plasma membrane[6,7] This symptomless stage of infection, i.e. biotrophy, is short lived and the fungus switches to a necrotrophic stage, forming differentiated and morphologically distinct secondary hyphae that kill and destroy host tissue for nutrient acquisition. We opted for an in vitro culture system, coupled to a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomic approach to gain information about the underlying biochemistry associated with the different growth stages of C. sublineolum This was achieved by profiling the methanol soluble secondary - and some primary metabolites present in the endo- and exometabolomes that characterise the different stages of growth in batch culture. The uncovered metabolic signatures, accompanied with expression levels of selected marker genes, identified specific biochemical processes involved in the growth stages of the fungus, generating a holistic molecular description of the trophic transition phenomenology
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