Colletotrichum gloeosporioides species complex (CGSC) is one of the most widely distributed and common plant fungi [48]. They can host among 470 different plants as a pathogen or an endophyte [49]. Most research of CGSC has focused on economic crops [50] or fruits [42]. In this study, we obtained 165 strains belonging to CGSC from three isolation sources (invasive plant A. adenophora healthy leaves and symptomatic leaves, and neighbor plants symptomatic leaves) in 11 geographic sites (belonging to four invasion ages of A. adenophora). We characterized the genetic diversity of these CGSC strains by multiple gene loci sequencing. We found different locus showed distinct haplotype and nucleotide diversity and all 11 geographic populations showed a high level of haplotype and nucleotide diversity based on the concatenated sequences. And haplotype did not cluster according to geographic site, isolation source or invasion age; all geographic populations did not experience population expansion along with A. adenophora invasion.
The results indicated that 5 out of 6 loci showed high nucleotide diversity (Pi > 0.02), only ITS locus had a low nucleotide diversity (Pi = 0.0074). Specifically, gapdh locus had the highest nucleotide diversity (Pi = 0.0705), which is consistent with the result of C. fructicola, a species within CGSC [50], followed by gs (Pi = 0.0341). Our result also supported that gapdh and gs can be used to reliably distinguish most taxa in CGSC [51]. On the contrary, low level nucleotide diversity of ITS explained why single ITS is poor to define CGSC [51, 52]. Thus, multilocus analysis could provide a more precise distinguish in differential genetic diversity from CGSC [23, 53].
Unexpectedly, we found that the genetic diversity of our 165 CGSC isolates was very high, with a haplotype diversity (Hd) ranging from 0.67 to 1 and all nucleotide diversity (Pi) > 0.01 for all 11 geographic populations (Table 1). Since population variability is generally considered to be large when the polymorphism index Pi is more than 0.01 [54], it is concluded that there is a significant genetic variation within these CGSC populations. Previously, Moges, Admassu, Belew, Yesuf, Njuguna, Kyalo and Ghimire [55] found a low level genetic diversity of isolated 163 CGSC strains in two host plants belonging to Citrus from four cultivated orchards in Ethiopia. The high nucleotide and haplotype diversity of CGSC in our study may be the result of diverse host species and wide geographic range (including invasive plant A. adenophora plus 12 neighbor plants from 11 geographic sites, see Table S2), as well as long introduction time of A. adenophora (the longest invasion time more than 80 years, see Table 1), since introduction time and alternative host species diversity may play a role in generating genetic variability within populations [56].
Alternatively, high diversity of our CGSC may suggest a weak selection for infection of CGSC strains by A. adenophora in the invaded range, which is favorable for frequent cross-over transmission among A. adenophora populations in different geographic sites, and between invasive and neighbor plant species. Accordingly, we found that CGSC haplotypes did not clustered by geographic sites, invasion ages or three isolation sources. AMOVA results showed that the major genetic differentiation was within populations (92.7 ~ 99.5%), whenever based on geographical populations or isolation source populations. Moreover, analysis of population genetic distance among geographical populations found that only 9 out of 55 pairs showed significantly genetic differentiation (P < 0.05), and Mantel test also indicated that there was no a significant correlation between geographical distance and genetic distance among geographic populations.
Fungal lifestyle switching between endophyte and pathogen in different host plants even in the same host have been frequently reported [57, 58]. For example, the same haplotype of Fusarium circinatum can be an endophyte colonizing in herbaceous plants but act as a reservoir of a pitch canker disease inoculum in Pinus radiate [59]. Tian et al. (2020) also indicated that Sclerotinia sclerotiorum severed as a widespread pathogen of dicotyledons, while it can protect wheat, rice, barley, maize, and oat against Fusarium head blight, stripe rust, and rice blast by acting as an endophyte [60]. In particular, for C. gloeosporioides, it is common a lifestyle switching between endophyte and necrotrophy [57]; C. gloeosporioides could transform endophytic strategies into saprotrophic lifestyle in the same host Magnolia liliifera [61]. Previously, Colletotrichum strains were isolated from asymptomatic leaves of A. adenophora and symptomatic leaves of neighbor plants, and these strains were nonvirulent to A. adenophora but virulent to some neighbor plants by Koch's postulates [28]. In this case, our multilocus analysis of CGSC further found that, the dominant haplotype H_10 shared by all 11 geographic populations and occurred in both healthy and symptomatic leaves of A. adenophora; another dominant haplotype H_5 were detected in 6 of 11 geographic populations and in all three isolation sources (Fig. 1). The occurrence of these generalist haplotypes of CGSC verified that it was common for fungal lifestyle switches between endophyte and pathogen within host A. adenophora, as well as between A. adenophora and neighbor plants in invaded ecosystem. It thus may be greatly underappreciated of the potential disease risk driven by asymptomatic, endophytic CGSC associated with A. adenophora.
Fungal pathogens shared by the invader and native plants, either through spillover or spillback, may facilitate invasion [3, 4], referred as disease-mediated invasion (DMI) [62]. In reviewing DMIs, Strauss et al. (2012) found that most disease-mediated animal invasions benefit from spillover rather than spillback; in contrast, most disease-mediated plant invasions benefit from spillback because most non-indigenous plants can be introduced by seeds and may not be accompanied by the same avirulent parasites, especially soil pathogens infected in the native ranges[62]. In current, it is difficult to quantify the degree variation that spillover or spillback occurs in plant invasions, because DMIs have been reported only in a few invasive plants so far [13, 63]. A. adenophora mainly disperses into a new scope by its seeds with tiny size and light weight [64]; moreover, there was no molecule evidence that Colletotrichum sp. is seedborne for A. adenophora [65]. In this case, neutrality tests indicated that CGSC gene exchanged among populations but population did not expand with invasion of A. adenophora (Table 4, all P > 0.05). Therefore, if CGSC-mediated invasion can occur in A. adenophora, this process is high possibility through spillback.
In conclusion, our multilocus analysis verifies that Colletotrichum gloeosporioides species complex (CGSC) associated A. adenophora and other neighbor plants in the introduced range are high in genetic diversity, with several dominant haplotypes sharing by most geographic populations, and by healthy and symptomatic leaves of A. adenophora, as well as symptomatic leaves of neighbor plants. The major genetic differentiation is within populations not among populations. It is concluded that there is high possibility that A. adenophora obtain local foliar CGSC from co-occurring neighbor plants as endophyte or pathogens in the introduced range, and can spillback them to neighbor plants. Our results are valuable for making management strategy for crop disease caused by CGSC in the areas invaded with A. adenophora invasion.