Untargeted metabolomic analyses support the main phylogenetic groups of the common plant-associated Alternaria fungi isolated from grapevine (Vitis vinifera)

Abstract

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)-high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

Figure 1

Phylogenetic tree of all Alternaria isolates collected from grapevine leaves and clusters and reference Alternaria strains from Woudenberg et al.. The 50% majority rule consensus phylogram inferred from Bayesian analysis of the combined dataset of five loci (rpb2, ITS, Alt a 1, endoPG, OPA10-2). Bayesian posterior probabilities (≥ 0.90) are shown before slashes, ML bootstrap support (≥ 70) is shown after slashes. Isolates representing the lineages of A. alternata (violet) and A. arborescens species complex (AASC, dark green) in this study are shown in bold. Sequences were rooted to A. alternantherae, A. perpunctulata, A. solani, A. porri, A. tagetica, A. macrospora, A. pseudorostrata and A. dauci. Scale bar indicates 1 expected change per branch.

Figure 2

Heatmaps of negative (a) and positive (c) normalized feature areas across samples. On both columns and rows a hierarchical clustering was performed by Ward’s method with Canberra distances. PLS-DA analysis results of negative and positive datasets are shown on panels (b) and (d), respectively.

Figure 3

High-performance liquid chromatography ultraviolet spectrophotometry (HPLC–UV) separations (a, b) of the extracts prepared from Alternaria alternata isolate vvmerl3ml5 (a) and A. arborescens species complex (AASC) isolate vvunid6yl6 (b), and the chemical structures of their main compounds 1 alternarienonic acid, 2 alternerian acid, 3 altenuene, 4 L-tenuazonic acid, 5 altenusin, 6 alternariol, 7 4-hydroxyalternariol methyl ether and 8 alternariol monomethyl ether (λ = 250–600 nm).