Use of GC–MS based metabolic fingerprinting for fast exploration of fungicide modes of action

Hu et al. BMC Microbiology (2019) 19:141 https://doi.org/10.1186/s12866-019-1508-5 RESEARCH ARTICLE Open Access Use of GC–MS based metabolic fingerprinting for fast exploration of fungicide modes of action Zhihong Hu, Tan Dai, Lei Li, Pengfei Liu* and Xili Liu Abstract Background: The widespread occurrence of fungicide resistance in fungal plant pathogens requires the development of new compounds with different mode(s) of action (MOA) to avoid cross resistance. This will require a rapid method to identify MOAs. Results: Here, gas chromatography–mass spectrometry (GC–MS) based metabolic fingerprinting was used to elucidate the MOAs of fungicides. Botrytis cinerea, an important pathogen of vegetables and flowers, can be inhibited by a wide range of chemical fungicides with different MOAs. A sensitive strain of B. cinerea was exposed to EC50 concentrations of 13 fungicides with different known MOAs and one with unknown MOA. The mycelial extracts were analyzed for their “metabolic fingerprint” using GC–MS. A comparison among the GC–MS vector’ profiles of cultures treated with fungicides were performeded. A model based on hierarchical clustering was established which allowed these antifungal compounds to be distinguished and classified coinciding with their MOAs. Thus, metabolic fingerprinting represents a rapid, convenient, and information-rich method for classifying the MOAs of antifungal substances. The biomarkers of fungicide MOAs were also established by an analysis of variance and included succinate for succinate dehydrogenase inhibitors and cystathionine for methionine synthesis inhibitors. Using the metabolic model and the common perturbation of metabolites, the new fungicide SYP-14288 was identified as having the same MOA as fluazinam. Conclusion: This study provides a comprehensive database of the metabolic perturbations of B. cinerea induced by diverse MOA inhibitors and highlights the utility of metabolic fingerprinting for defining MOAs, which will assist in the development and optimization of new fungicides. Keywords: Metabolic fingerprinting, GC–MS, Botrytis cinerea, Fungicide, Mode of action Background The fungal pathogen Botrytis cinerea causes serious losses in more than 200 crops worldwide. It can survive for relatively short periods as mycelia and/or conidia and for extended periods as sclerotia in crop debris [1]. The fungus causes grey mold disease, which can be controlled by the application of a wide range of chemical fungicides that act as seven modes of action (MOAs), including β-tubulin assembly inhibitors, respiration inhibitors, uncouplers of oxidative phosphorylation, methionine biosynthesis inhibitors, signal transduction inhibitors, sterol biosynthesis inhibitors, * Correspondence: Department of Plant Pathology, China Agricultural University, Beijing 100193, People’s Republic of China and multi-site inhibitors. Unfortunately, B. cinerea has developed high levels of resistance to most of the fungicides used for its control in the field [2–5]. Although many new fungicides that target B. cinerea have been developed, these may be ineffective if they have MOAs that are similar to those of fungicides to which B. cinerea is already resistant; i.e., there may be cross-resistance between the new fungicides and the previously used fungicides. It is, therefore, important to develop a high-throughput screening method to identify fungicide MOAs. A fast exploration of MOAs is helpful for the scientific application of new fungicides. A series of research methods have been used to reveal fungicide MOAs. The MOA of flumorph was explored by analyzing alterations of hyphal morphology, cell wall © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hu et al. BMC Microbiology (2019) 19:141 deposition patterns, F-actin organization, and other organelles in Phytophthora melonis. Results showed that flumorph may be involved in the impairment of cell polar growth through directly or indirectly disrupting the organization of F-actin [6]. Deuterium-labelling was used to determine that the MOA of metalaxyl involved the inhibition of RNA polymerase I [7]. The researcher found that RNA synthesis of phenylamide-sensitive strains, measured as [3H] uridine incorporation, was inhibited by about 80% (Phytophthora megasperma f. sp. medicaginis) and by about 40% (Phytophthora infestans) by metalaxyl and oxadixyl at a concentration of 1 μg/ml. RNA synthesis of resistant strains was completely insensitive to metalaxyl and oxadixyl at concentrations as high as 200 μg/ml. Additionally, endogenous nuclear RNA polymerase activity of both Phytophthora sensitive isolates appeared to be more sensitive to the phenylamides than of both Phytophthora resistant isolates. These means of cross-resistance could be applied in the bioassay method to determine the MOA by assessing the resistance mechanism. A complex II analysis of mutants of several organisms resistant to succinate dehydrogenase inhibitors (SDHIs), such as carboxin, provided insights into the MOA of SDH-inhibitors [8, 9]. Comparison of the sequence from a carboxinsensitive Ustilago maydis strain of iron-sulphur protein (Ip) subunit of succinate dehydrogenase (Sdh) with that of the Ip allele from a carboxin -resistant strain revealed a two-base difference between the sequences. This mutation led to the substitution of a leucine residue for a histidine residue within the third cysteine-rich cluster of the deduced amino-acid sequence of the Ip allele. This cluster, which is associated with the S3 iron-redox centre, is involved in the transport of electrons from succinate to ubiquinone (Q). Confirmation that this nucleotide change led to enhanced resistance to carboxin was obtained following mutagenesis of the sensitive Ip allele to the resistant form and expression of the mutated allele in U. maydis [8]. A patent proposed the use of affinity chromatography to determine the MOA of oxathiapiprolin [10]. The authors found that the oxathiapiprolin specifically binds to Oomycete oxysterol binding polypeptide in the total protein mixture obtained from Oomycete. The MOA of quinone outside Inhibitors (QoI) was explored using protein crystallization combined with molecular docking. The existence of more than 40 different fungicide MOAs (FRAC, 2019) makes screening by the methods above time-consuming and costly. Thus, fast, robust, and high-throughput screening techniques are required. In (...truncated)