Fig 1.
Isolation and virulence verification of leaf spot fungi.
(A) The distribution of A. adenophora pathogens at the family (left) and OTU levels (right). Only families with relative abundance > 2% are shown, and the rest are merged into "Others"; only OTUs with relative abundance > 1% are shown. Each bar represents the abundance of one family (left) or one OTU (right) isolated from A. adenophora at the ~20 y, ~50 y and ~80 y sites, respectively; the part in the red represents those fungi virulent to A. adenophora verified by a disease experiment. (B) The categories of leaf spots with varying pathogenic fungal species. The number in each section represents the number of leaf spots with the corresponding pathogen richness. The green sections contain leaf spots with multiple infections, and the gray section contains those leaf spots with a single infection.
Fig 2.
Shared OTUs between leaf spot fungi and seedborne fungi.
(A) Venn diagram for the number of unique and shared OTUs between leaf spot and seed libraries. The number in parentheses represents the abundance of isolates (for leaf spot fungi, LSF) or sequence reads (for seedborne fungi, SF). The 17 shared OTUs were represented by 311 isolates of leaf spot fungi and 462980 reads of seedborne fungi. (B) Network diagram for the 17 shared OTUs between the leaf spots and the seeds. The lines depict the associations of shared OTUs between leaf spot fungi and seedborne fungi. The abbreviations following each OTU number in the corresponding library represent the family name; Didy, Didymellaceae; Xyle, Xylariales; Nect, Nectriaceae; Pleo, Pleosporaceae; Cera, Ceratobasidiaceae; the numbers in parentheses represent the abundance of isolates (for leaf spot fungi, the number of nonpathogenic fungi is shown in red) or next-generation sequencing reads (for seedborne fungi). Because the ITS sequence obtained by next-generation sequencing technology is short (~250 bp), the alignment is trimmed to this range and clustered to generate novel OTUs. This trimming causes a few OTUs to merge in both libraries, for example, OTU 1_Didy in leaf spot fungi and OTU 125_Xyla in seedborne fungi (see 4.4 Investigation of seedborne fungi of A. adenophora).
Fig 3.
Diversity and structure of the fungal pathogens within leaf spots with multiple infections over the hosts’ invasion history.
Shannon diversity (A) and community structure (B) of fungal pathogens at the leaf spot level at different invasion times. (C) Shannon diversity of seedborne foliar pathogens (SFPs) and nonseedborne pathogens (non-SFPs) at the leaf spot level at different invasion times. Each point represents the pathogen community from one leaf spot with multiple infections. In panels (A) and (C), nonparametric analysis with the Mann-Whitney U test was performed to show that the Shannon diversity index difference was significant among the different invasion times based on the different lowercase letters (p < 0.05). In panel (B), principal coordinate analysis (PCoA) shows the similarity of pathogenic communities among leaf spots with multiple infections. SFP represents a leaf spot containing SFPs that may also cooccur with non-SFPs; non-SFP represents a leaf spot containing only non-SFPs. Percentages of the total explained variation by the PCoA axes in each plot are given in parentheses. (D) The occurrence frequency and relative abundance in leaf spots of SFPs and non-SFPs at different invasion times.
Table 1.
PERMANOVA tests for the differences in microbial community composition.
Fig 4.
Fungal virulence evolution on the host A. adenophora.
(A) Linear analysis of within-spot species diversity and virulence variations. Each spot represents a leaf spot with multiple infections (n = 165). (B) The density plots show the virulence distribution of fungal pathogens to A. adenophora at different invasion times. The boxplots show the differences in the average virulence (top) and the virulence variation (bottom) between different invasion times. (C) Virulence variation within spots of SFPs and non-SFPs at different invasion times. Nonparametric analysis with the Mann-Whitney U test was performed to test the significance of the differences, and different lowercase letters indicate significant differences (p < 0.05). Differences between SFPs and non-SFPs at each invasion time are also given. The leaf spot area (mm2) is shown as square root transformed. The error bar in panel (C) represents the standard deviation.
Fig 5.
The virulence and host range of SFP and non-SFP fungi.
(A) The virulence difference of either SFP or non-SFP fungi to A. adenophora and native plants. (B) The virulence difference and (C) host range of SFP and non-SFP fungi to native plants. Nonparametric analysis with the Mann-Whitney U test was performed to test the significance of the differences, and different lowercase letters indicate significant differences (p < 0.05). Differences between SFPs and non-SFPs at each invasion time are also given. The error bar in panel (A) represents the standard deviation.
Table 2.
Geographical information, population distribution and number of leaf spots at three invasive times.