Diversity and pathogenicity of Alternaria species associated with the invasive plant Ageratina adenophora and local plants.

Pathogen accumulation after introduction is unavoidable for exotic plants over a long period of time. Therefore, it is important to understand whether plant invasion promotes novel pathogen emergence and increases the risk of pathogen movement among agricultural, horticultural, and wild native plants. In this study, we used multiple gene analysis to characterize the species composition of 104 isolates of Alternaria obtained from the invasive plant Ageratina adenophora and native plants from Yunnan, Hubei, Guizhou, Sichuan, and Guangxi in China. Phylogenetically, these strains were from A. alternata (88.5%), A. gossypina (10.6%) and A. steviae (0.9%). There was a high amount of sharing between strains associated with A. adenophora and with local plants. Pathogenicity tests indicated that most of these Alternaria strains are generalists; the isolates with a wider host range were more virulent to the plant. Woody plants were more resistant to these strains than herbaceous plants and vines. However, the invasive plant A. adenophora was highly sensitive to these strains. Our data are valuable for understanding how A. adenophora invasion impacts the Alternaria species composition of the native plant and whether A. adenophora invasion causes potential disease risks in invaded ecosystems.

Virulence and community dynamics of fungal species with vertical and horizontal transmission on a plant with multiple infections.

The virulence evolution of multiple infections of parasites from the same species has been modeled widely in evolution theory. However, experimental studies on this topic remain scarce, particularly regarding multiple infections by different parasite species. Here, we characterized the virulence and community dynamics of fungal pathogens on the invasive plant Ageratina adenophora to verify the predictions made by the model. We observed that A. adenophora was highly susceptible to diverse foliar pathogens with mixed vertical and horizontal transmission within leaf spots. The transmission mode mainly determined the pathogen community structure at the leaf spot level. Over time, the pathogen community within a leaf spot showed decreased Shannon diversity; moreover, the vertically transmitted pathogens exhibited decreased virulence to the host A. adenophora, but the horizontally transmitted pathogens exhibited increased virulence to the host. Our results demonstrate that the predictions of classical models for the virulence evolution of multiple infections are still valid in a complex realistic environment and highlight the impact of transmission mode on disease epidemics of foliar fungal pathogens. We also propose that seedborne fungi play an important role in structuring the foliar pathogen community from multiple infections within a leaf spot.

Remotididymella ageratinae sp. nov. and Remotididymella anemophila sp. nov., two novel species isolated from the invasive weed Ageratina adenophora in PR China.

To determine if Ageratina adenophora can accumulate diverse pathogens from surrounding native plants, we intensively sampled fungal communities, including endophytes, leaf spot pathogens and canopy air fungi, associated with Ag. adenophora as well as native plants in its invasive range. In total, we collected 4542 foliar fungal strains from 10 geographic sites, including 1340 from healthy leaves of Ag. adenophora, 2051 from leaf spots of Ag. adenophora and 1151 from leaf spots of 56 species of native plants and crops. Taxonomically, the common fungal genera included Colletotrichum, Diaporthe, Alternaria, Nemania, Xylaria, Neofusicoccum, Nigrospora, Epicoccum, Gibberella, Pestalotiopsis, Irpex, Schizophyllum and Clonostachys. We also isolated the cultivable fungi from 12 air samples collected from six areas in Yunnan Province, PR China. Among the total of 1255 air fungal isolates, the most common genera were Cladosporium, Trichoderma and Epicoccum. Among them, two new Remotididymella species, Remotididymella ageratinae from leaf spot of Ag. adenophora and Remotididymella anemophila from canopy air of Ag. adenophora were found. The two species showed both asexual and sexual reproductive structures. The conidia of R. ageratinae and R. anemophila are larger than those of R. anthropophila and R. destructiva. The size of ascospores of R. ageratinae and R. anemophila also differ from R. bauhiniae. Phylogenetic analysis of the combined ITS, LSU rRNA, rpb2 and tub2 sequences showed that R. ageratinae and R. anemophila each formed a distinct clade, separated from all species previously described in Remotididymella and confirmed them as new species belonging to Remotididymella. Full descriptions of R. ageratinae and R. anemophila are provided in this study.

Quantifying the sharing of foliar fungal pathogens by the invasive plant Ageratina adenophora and its neighbours.

Local pathogens can accumulate as asymptomatic endophytes, making it difficult to detect the impacts of invasive species as propagators of disease in the invaded range. We used the invasive plant Ageratina adenophora to assess such accumulation. We intensively collected foliar fungal endophytes and leaf spot pathogens of A. adenophora and co-occurring neighbours and performed an inoculation experiment to evaluate their pathogenicity and host range. Ageratina adenophora harboured diverse necrotrophic pathogens; its communities of endophytes and leaf spot pathogens were different in composition and shared only a small number of fungal species. In the pathogen communities of local plant hosts, 21% of the operational taxonomic units (OTUs), representing 50% of strains, also occurred as leaf spot pathogens and/or endophytes of A. adenophora. The local pathogen community was more similar to the endophytes than to the pathogens of A. adenophora. The inoculation experiment showed that local pathogens could infect A. adenophora leaves asymptomatically and that local plant hosts were susceptible to both A. adenophora endophytes and pathogens. Ageratina adenophora is a highly competent host for local pathogens, and its asymptomatic latent pathogens are fungi primarily shared with local neighbours. This poses challenges for understanding the long-term ecological consequences of plant invasion.

Characterization of the fungal community in the canopy air of the invasive plant Ageratina adenophora and its potential to cause plant diseases.

Airborne fungi and their ecological functions have been largely ignored in plant invasions. In this study, high-throughput sequencing technology was used to characterize the airborne fungi in the canopy air of the invasive weed Ageratina adenophora. Then, representative phytopathogenic strains were isolated from A. adenophora leaf spots and their virulence to A.adenophora as well as common native plants in the invaded range was tested. The fungal alpha diversities were not different between the sampling sites or between the high/low part of the canopy air, but fungal co-occurrences were less common in the high than in the low part of the canopy air. Interestingly, we found that the phytopathogenic Didymellaceae fungi co-occurred more frequently with themselves than with other fungi. Disease experiments indicated that all 5 Didymellaceae strains could infect A. adenophora as well as the 16 tested native plants and that there was large variation in the virulence and host range. Our data suggested that the diverse pathogens in the canopy air might be a disease infection source that weakens the competition of invasive weeds, a novel phenomenon that remains to be explored in other invasive plants.

Plant-soil-foliage feedbacks on seed germination and seedling growth of the invasive plant Ageratina adenophora.

Some exotic plants become invasive because they partially release from soil-borne enemies and thus benefit from positive plant-soil feedbacks (PSFs) in the introduced range. However, reports that have focused only on PSFs may exaggerate the invader's competitiveness. Here, we conducted three experiments to characterize plant-soil-foliage feedbacks, including mature leaves (ML), leaf litter (LL), rhizosphere soil (RS) and leaves plus soil (LS), on the early growth stages of the invasive plant Ageratina adenophora. In general, the feedbacks from aboveground (ML, LL) adversely affected A. adenophora by delaying germination time, inhibiting germination rate and reducing seedling growth. The increased invasion history exacerbated the adverse effects of LL and LS feedbacks on seedling growth. These adverse effects were partially contributed by more abundant fungi (e.g. Didymella) or/and more virulent fungi (e.g. Fusarium) developed in the aboveground part of A. adenophora during the invasion. Interestingly, the aboveground adverse effects can be weakened by microbes from RSs. Our novel findings emphasize the important role of aboveground feedbacks in the evaluation of plant invasiveness, and their commonness and significance remain to be explored in other invasive systems.

Growth-promoting characteristics of potential nitrogen-fixing bacteria in the root of an invasive plant Ageratina adenophora.

Root endophytic nitrogen-fixing bacteria (reNFB) have been proposed as important contributors to the invasiveness of exotic legumes; however, the reNFB of invasive nonlegumes has received less attention. In particular, the growth-promoting effect of reNFB on invasive plants remains unknown. In this study, 131 strains of potential nitrogen-fixing bacteria were isolated and purified from the roots of the invasive plant, Ageratina adenophora, in Southwest China. Phylogenetically, these reNFB were categorized into three phyla at 97% sequence identity that included Proteobacteria (92.4%), Actinobacteria (4.6%), and Firmicutes (3.1%). The dominant isolates ranked by number were Pseudomonas (80 isolates, 61.1%), Rhizobium (12 isolates, 9.2%), and Duganella (11 isolates, 8.4%). The community composition and diversity of A. adenophora reNFB were markedly different across study regions. The capacity of these reNFB to accumulate indolyl-3-acetic acid (IAA), solubilize phosphate, and produce siderophores was determined. All 131 isolates of reNFB accumulated IAA, 67 isolates solubilized phosphate, and 108 isolates produced siderophores. Among the three dominant genera of reNFB, Pseudomonas had the highest phosphorus solubilization and siderophore production, while the accumulation of IAA in the genus Duganella was the lowest. Interestingly, the calculated reNFB Shannon diversity index of each A. adenophora individual was negatively correlated with the capacity of reNFB to produce growth-promoting products. Six randomly selected isolates from three dominant genera were further used to conduct inoculation experiments, and all isolates showed significant positive growth-promoting effects on A. adenophora seedlings. The contribution of reNFB to the root biomass was higher than that to the shoot biomass. Our results suggest that reNFB, similar to soil or nodular nitrogen-fixing bacteria, can potentially promote plant growth and may play an important role in the invasion of nonleguminous plants. More detailed studies on the correlation between reNFB and invasive plants are necessary.

Enrichment of soil rare bacteria in root by an invasive plant Ageratina adenophora.

The assembly of the root-associated microbiome provides mutual benefits for the host plant and bacteria in soils. It is interesting how invasive plants interact with the local soil microbial community and establish the soil bacterial community in the endosphere of these plants in the short term. In this study, we compared the bacterial community in the rhizosphere with that in the root endosphere of an invasive plant, Ageratina adenophora, using high-throughput sequencing. The results showed that the roots of A. adenophora selectively accumulated the genera Clostridium and Enterobacter, which are rarely distributed in the rhizosphere. This selective accumulation caused a switch in the bacterial composition at the phylum level from Bacteroidetes predominant in the rhizosphere to Proteobacteria dominant in the root endosphere of A. adenophora. Our data indicated the potential existence of a highly conserved signal recognition in which hosts, either invasive or native, enrich the endosphere bacteria, such as Clostridium, Enterobacter, etc., from the rhizosphere. Moreover, the accumulated bacteria were physiologically and genetically different at the strain level and displayed distinct roles in growth between invasive and native plants. The assembly of the bacterial community in the roots may be an advantageous strategy for A. adenophora in competition with native plants.

Tissue-Specific and Geographical Variation in Endophytic Fungi of Ageratina adenophora and Fungal Associations With the Environment.

To understand the distribution of the cultivable fungal community in plant tissues and the associations of these fungi with their surrounding environments during the geographical expansion of an invasive plant, Ageratina adenophora, we isolated the cultivable fungi from 72 plant tissues, 12 soils, and 12 air samples collected from six areas in Yunnan Province, China. A total of 4066 isolates were investigated, including 1641 endophytic fungi, 233 withered leaf fungi, 1255 fungi from air, and 937 fungi from soil. These fungi were divided into 458 and 201 operational taxonomic units (OTUs) with unique and 97% ITS gene sequence identity, respectively. Phylogenetic analysis showed that the fungi belonged to four phyla, including Ascomycota (94.20%), Basidiomycota (2.71%), Mortierellomycota (3.03%), and Mucoromycota (0.07%). The dominant genera of cultivable endophytic fungi were Colletotrichum (34.61%), Diaporthe (17.24%), Allophoma (8.03%), and Fusarium (4.44%). Colletotrichum and Diaporthe were primarily isolated from mature leaves, Allophoma from stems, and Fusarium from roots, indicating that the enrichment of endophytic fungi is tissue-specific and fungi rarely grew systemically within A. adenophora. In the surrounding environment, Alternaria (21.46%), Allophoma (19.31%), Xylaria (18.45%), and Didymella (18.03%) were dominant in the withered leaves, Cladosporium (22.86%), Trichoderma (14.27%), and Epicoccum (9.83%) were dominant in the canopy air, and Trichoderma (27.27%) and Mortierella (20.46%) were dominant in the rhizosphere soils. Further analysis revealed that the cultivable endophytic fungi changed across geographic areas and showed a certain degree of variation in different tissues of A. adenophora. The cultivable fungi in mature and withered leaves fluctuated more than those in roots and stems. We also found that some cultivable endophytic fungi might undergo tissue-to-tissue migration and that the stem could be a transport tissue by which airborne fungi infect roots. Finally, we provided evidence that the fungal community within A. adenophora was partially shared with the contiguous environment. The data suggested a frequent interaction between fungi associated with A. adenophora and those in surrounding environments, reflecting a compromise driven by both functional requirements for plant growth and local environmental conditions.