Genetic Evidence for Colletotrichum gloeosporioides Transmission Between the Invasive Plant Ageratina adenophora and Co-occurring Neighbor Plants.

To understand the disease-mediated invasion of exotic plants and the potential risk of disease transmission in local ecosystems, it is necessary to characterize population genetic structure and spatio-temporal dynamics of fungal community associated with both invasive and co-occurring plants. In this study, multiple genes were used to characterize the genetic diversity of 165 strains of Colletotrichum gloeosporioides species complex (CGSC) isolated from healthy leaves and symptomatic leaves of invasive plant Ageratina adenophora, as well as symptomatic leaves of its neighbor plants from eleven geographic sites in China. The data showed that these CGSC strains had a high genetic diversity in each geographic site (all Hd > 0.67 and Pi > 0.01). Haplotype diversity and nucleotide diversity varied greatly in individual gene locus: gs had the highest haplotype diversity (Hd = 0.8972), gapdh had the highest nucleotide diversity (Pi = 0.0705), and ITS had the lowest nucleotide diversity (Pi = 0.0074). Haplotypes were not clustered by geographic site, invasive age, or isolation source. AMOVA revealed that the genetic variation was mainly from within-populations, regardless of geographic or isolation origin. Both AMOVA and neutrality tests indicated these CGSC strains occurred gene exchange among geographic populations but did not experience population expansion along with A. adenophora invasion progress. Our data indicated that A. adenophora primarily accumulated these CGSC fungi in the introduced range, suggesting a high frequency of CGSC transmission between A. adenophora and co-occurring neighbor plants. This study is valuable for understanding the disease-mediated plant invasion and the potential risk of disease transmission driven by exotic plants in local 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.

Tenuazonic Acid-Triggered Cell Death Is the Essential Prerequisite for Alternaria alternata (Fr.) Keissler to Infect Successfully Host Ageratina adenophora.

The necrotrophic fungus Alternaria alternata contains different pathotypes that produce different mycotoxins. The pathotype Ageratina adenophora secretes the non-host-selective toxin tenuazonic acid (TeA), which can cause necrosis in many plants. Although TeA is thought to be a central virulence factor of the A. adenophora pathotype, the precise role of TeA in different stages of host infection by pathogens remains unclear. Here, an A. alternata wild-type and the toxin-deficient mutant ΔHP001 with a 75% reduction in TeA production were used. It was observed that wild-type pathogens could induce the reactive oxygen species (ROS) bursts in host leaves and killed photosynthetic cells before invading hyphae. The ROS interceptor catalase remarkably inhibited hyphal penetration and invasive hyphal growth and expansion in infected leaves and suppressed necrotic leaf lesion. This suggests that the production of ROS is critical for pathogen invasion and proliferation and disease symptom formation during infection. It was found that the mutant pathogens did not cause the formation of ROS and cell death in host leaves, showing an almost complete loss of disease susceptibility. In addition, the lack of TeA resulted in a significant reduction in the ability of the pathogen to penetrate invasive hyphal growth and spread. The addition of exogenous TeA, AAL-toxin, and bentazone to the mutant ΔHP001 pathogens during inoculation resulted in a significant restoration of pathogenicity by increasing the level of cell death, frequency of hyphal penetration, and extent of invasive hyphal spread. Our results suggest that cell death triggered by TeA is the essential requirement for successful colonization and disease development in host leaves during infection with A. adenophora pathogens.

Two metabolites isolated from endophytic fungus Coniochaeta sp. F-8 in Ageratina adenophora exhibit antioxidative activity and cytotoxicity.

Ageratina adenophora is an invasive plant of global importance and has a broad distribution in the Western Himalayas. Endophtytic fungus Coniochaeta sp.F-8 was found in Ageratina adenophora. In this paper, we aim to investigate the antioxidative activity and cytotoxicity of the metabolites from Coniochaeta sp.F-8. Consequently, two compounds Phomoxanthone A and Penialidin A were isolated from the endophytic fungus of Ageratina adenophora for the first time. The structures of compounds were identified by IR, NMR and ESI-MS methods. Moreover, both of those compounds showed considerable antioxidative activity in vitro and resulted cytotoxicity in mouse embryo fibroblasts cell line Balb/c3T3. The present study provides a theoretical foundation for the development and utilization of endophytic fungi in Ageratina adenophora as a medicinal substance.

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.

Effect of Ageratina adenophora invasion on the composition and diversity of soil microbiome.

In the present study, high throughput 16S rRNA gene sequencing was used to investigate soil invaded by the aggressive weed Ageratina adenophora to determine its effect on the species composition, distribution, and biodiversity of the bacterial communities. Soil samples from 12 micro-sites containing a monoculture of A. adenophora plants, mixtures of A. adenophora and different native plant species, and native species alone were studied. We found that the invasion of this weed resulted in a selection of bacteria belonging to phyla Acidobacteria and Verrucomicrobia and the lack of bacteria belonging to phyla Actinobacteria and Planctomycetes, but did not affect significantly the percentage abundances of members of other phyla. A similar bacterial population selection was also observed at genus or subgroup levels. The NO3--N level was an important factor affecting soil bacterial communities and contributed to the dominance of A. adenophora. However, the numbers of total bacterial species, and the diversity and structure of soil bacterial microbiome did not (P > 0.05) change significantly following invasion by this weed.

Putative Nonribosomal Peptide Synthetase and Cytochrome P450 Genes Responsible for Tentoxin Biosynthesis in Alternaria alternata ZJ33.

Tentoxin, a cyclic tetrapeptide produced by several Alternaria species, inhibits the F1-ATPase activity of chloroplasts, resulting in chlorosis in sensitive plants. In this study, we report two clustered genes, encoding a putative non-ribosome peptide synthetase (NRPS) TES and a cytochrome P450 protein TES1, that are required for tentoxin biosynthesis in Alternaria alternata strain ZJ33, which was isolated from blighted leaves of Eupatorium adenophorum. Using a pair of primers designed according to the consensus sequences of the adenylation domain of NRPSs, two fragments containing putative adenylation domains were amplified from A. alternata ZJ33, and subsequent PCR analyses demonstrated that these fragments belonged to the same NRPS coding sequence. With no introns, TES consists of a single 15,486 base pair open reading frame encoding a predicted 5161 amino acid protein. Meanwhile, the TES1 gene is predicted to contain five introns and encode a 506 amino acid protein. The TES protein is predicted to be comprised of four peptide synthase modules with two additional N-methylation domains, and the number and arrangement of the modules in TES were consistent with the number and arrangement of the amino acid residues of tentoxin, respectively. Notably, both TES and TES1 null mutants generated via homologous recombination failed to produce tentoxin. This study provides the first evidence concerning the biosynthesis of tentoxin in A. alternata.

Changes in arbuscular mycorrhizal fungus community along an exotic plant Eupatorium adenophorum invasion in a Chinese secondary forest.

Knowledge of the changes in arbuscular mycorrhizal (AM) fungi is fundamental for understanding the success of exotic plant invasions in natural ecosystems. In this study, AM fungal colonization and spore community were examined along an invasive gradient of the exotic plant Eupatorium adenophorum in a secondary forest in southwestern China. With increasing E. adenophorum invasion, the density of arbuscules in the roots of E. adenophorum significantly increased, but the AM root colonization rate and the densities of vesicles and hyphal coils in roots of E. adenophorum were not significantly different. A total of 29 AM fungi belonging to nine genera were identified based on spore morphology. Claroideoglomus etunicatum, Funneliformis geosporus, and Glomus aggregatum were the most common AM fungal species. The E. adenophorum invasion significantly decreased the AM fungal spore density in the soil. Furthermore, with increasing of E. adenophorum invasion the spore densities of C. etunicatum, G. aggregatum, and G. arenarium significantly decreased, whereas F. geosporus significantly increased. Nonmetric multidimensional scaling demonstrated that the AM fungus community composition was significantly different (P=0.003) in the different invasive levels of E. adenophorum, and significantly correlated with plant species richness, soil total P, and soil NO3 (-)-N. The results suggest that the alteration in AM fungus community might be caused by E. adenophorum invasion via changing the local plant community and soil properties in a Chinese secondary forest ecosystem.

Microbial community on healthy and diseased leaves of an invasive plant Eupatorium adenophorum in Southwest China.

Invasive plants have caused great economic losses and environmental problems worldwide. Eupatorium adenophorum is one of the most invasive weeds in China. To better understand its invasive mechanisms, in the present paper, the microbial communities of healthy and diseased leaves of E. adenophorum were obtained using both culture-independent and -dependent methods and their diversities were compared. The bacteria obtained from culture-independent method belong to Proteobacteria (95.8%), Actinobacteria (2.1%), and Firmicutes (2.1%) and fungi belong to Ascomycota (65.2%) and Basidiomycota (34.8%). Very few overlapped microbial species were found by culture-dependent and -independent methods. Healthy leaves display higher bacterial diversity than diseased leaves. Phylogenetic structures are very different between healthy and diseased phyllosphere microbial communities. Bacteria close to Acinetobacter and Pseudomonas were dominant on healthy leaves, whereas those close to Shigella were dominant on diseased leaves. 52.9% of fungal clones from healthy leaves were Ustilaginomycetes, close to Rhodotorula phylloplana and uncultured basidomycete; by contrast, 60% of clones from diseased leaves were Lecanoromycetes, close to Umbilicaria muehlenbergii. No bacteria but four fungal strains phylogenetically close to Myrothecium sp. and Alternaria alternate were pathogenic to seedlings and detached leaves of the invasive plant. Therefore, this plant may be resistant to pathogens from bacteria but not fungi in its introduced range.

Characteristics of the microbial community in rhizosphere of Camptotheca acuminata cultured with exotic invasive plant Eupatorium adenophorum.

The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and terminal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possibility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indicated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (delta-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camptothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camptothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bacterial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may benefit the control of overspread of Ea. This study provided theoretical evidence for rhizospheric microbial aspects on substituting Ca for Ea.

[Screening, identification, and antagonism assessment, of dominant bacteria in Ageratina adenophora Sprengel rhizosphere soil].

By using isolation and culture method, 25 strains of dominant bacteria in Ageratina adenophora rhizosphere soil were isolated and identified, of which, 8 strains were assessed for their antagonistic activity. The results showed that Bacillus and Pseudomonas were highly abundant in A. adenophora rhizosphere soil, of which, B. subtilis and B. megaterium were most abundant and occupied 55.6% of the total identified bacteria. These dominant bacteria had different level antagonistic activity to Fusarium oxysporum and Ralstonia solanacearum, and B. subtilis BS-5 and B. thuringiensis BT-1 had the strongest antagonistic effect on F. oxysporum, with the antagonistic activity of their metabolic products being 85.5% and 83.8%, respectively. The metabolic products of the dominant antagonistic bacteria had even more stronger antagonistic effect on pathogens than the dominant antagonistic bacteria themselves. The existence of abundant bacterial groups with strong antagonistic activity in A. adenophora rhizosphere soil could help A. adenophora to resist harmful soil-borne diseases and escape its natural enemies. Through the feedback actions of the beneficial rhizosphere microbes, A. adenophora probably earned its competition superiority directly or indirectly, being favorable to its rapid expansion.