Time-Course Transcriptome Analysis Reveals Resistance Genes of Panax ginseng Induced by Cylindrocarpon destructans Infection Using RNA-Seq.
ABSTRACT: Panax ginseng C. A. Meyer is a highly valued medicinal plant. Cylindrocarpon destructans is a destructive pathogen that causes root rot and significantly reduces the quality and yield of P. ginseng. However, an efficient method to control root rot remains unavailable because of insufficient understanding of the molecular mechanism underlying C. destructans-P. ginseng interaction. In this study, C. destructans-induced transcriptomes at different time points were investigated using RNA sequencing (RNA-Seq). De novo assembly produced 73,335 unigenes for the P. ginseng transcriptome after C. destructans infection, in which 3,839 unigenes were up-regulated. Notably, the abundance of the up-regulated unigenes sharply increased at 0.5 d postinoculation to provide effector-triggered immunity. In total, 24 of 26 randomly selected unigenes can be validated using quantitative reverse transcription (qRT)-PCR. Gene ontology enrichment analysis of these unigenes showed that "defense response to fungus", "defense response" and "response to stress" were enriched. In addition, differentially expressed transcription factors involved in the hormone signaling pathways after C. destructans infection were identified. Finally, differentially expressed unigenes involved in reactive oxygen species and ginsenoside biosynthetic pathway during C. destructans infection were indentified. To our knowledge, this study is the first to report on the dynamic transcriptome triggered by C. destructans. These results improve our understanding of disease resistance in P. ginseng and provide a useful resource for quick detection of induced markers in P. ginseng before the comprehensive outbreak of this disease caused by C. destructans.
Project description:<h4>Background</h4> It is estimated that 20–30% of ginseng crops in Canada are lost to root rot each harvest. This disease is commonly caused by fungal infection with Ilyonectria, previously known as Cylindrocarpon. Previous reports have linked the virulence of fungal disease to the production of siderophores, a class of small-molecule iron chelators. However, these siderophores have not been identified in Ilyonectria. <h4>Methods</h4> High-resolution LC–MS/MS was used to screen Ilyonectria and Cylindrocarpon strain extracts for secondary metabolite production. These strains were also tested for their ability to cause root rot in American ginseng and categorized as virulent or avirulent. The differences in detected metabolites between the virulent and avirulent strains were compared with a focus on siderophores. <h4>Results</h4> For the first time, a siderophore N,N?,N?-triacetylfusarinine C (TAFC) has been identified in Ilyonectria, and it appears to be linked to disease virulence. Siderophore production was suppressed as the concentration of iron increased, which is in agreement with previous reports. <h4>Conclusion</h4> The identification of the siderophore produced by Ilyonectria gives us further insight into the root rot disease that heavily affects ginseng crop yields. This research identifies a molecular pathway previously unknown for ginseng root rot and could lead to new disease treatment options.
Project description:Bacillus cereus PgBE311, isolated from the root tissue of a 5-year-old Panax ginseng plant, showed activities against the fungal pathogens Cylindrocarpon destructans and Botrytis cinerea. Here, we report the genome sequence of B. cereus PgBE311. The bacterium contains antibiotic-related gene clusters and has the potential to stimulate plant growth.
Project description:Background:Ginseng (Panax ginseng Meyer) is an invaluable medicinal plant containing various bioactive metabolites (e.g., ginsenosides). Owing to its long cultivation period, ginseng is vulnerable to various biotic constraints. Biological control using endophytes is an important alternative to chemical control. Methods:In this study, endophytic Trichoderma citrinoviride PG87, isolated from mountain-cultivated ginseng, was evaluated for biocontrol activity against six major ginseng pathogens. T. citrinoviride exhibited antagonistic activity with mycoparasitism against all ginseng pathogens, with high endo-1,4-β-D-glucanase activity. Results:T. citrinoviride inoculation significantly reduced the disease symptoms caused by Botrytis cinerea and Cylindrocarpon destructans and induced ginsenoside biosynthesis in ginseng plants. T. citrinoviride was formulated as dustable powder and granules. The formulated agents also exhibited significant biocontrol activity and induced ginsenosides production in the controlled environment and mountain area. Conclusion:Our results revealed that T. citrinoviride has great potential as a biological control agent and elicitor of ginsenoside production.
Project description:Although endophytic bacteria are known to colonize Panax ginseng, little is known about their diversity and roles. We addressed in the present study by comparing endophytic bacterial populations in P. ginseng plants of different ages (2-6 years) and in various tissue types (root, stem, leaf, and flower stalk). A total of 116 strains assigned to 42 species were identified by 16S rDNA sequencing. The predominant phylum was Firmicutes. Two-year-old ginseng plants and root tissues showed the greatest diversity of endophytic bacteria, with Bacillales being the predominant order. The antifungal activity of isolates against two pathogens, Cylindrocarpon destructans and/or Botrytis cinerea, was evaluated in dual-culture assays. In total, 28 strains showed antifungal activity with PgBE14 (Bacillus amyloliquefaciens), PgBE40 (B. megaterium), PgBE39, PgBE45 (Pseudomonas frederiksbergensis), and PgBE42 (Staphylococcus saprophyticus) inhibiting both pathogens. These results improve our understanding of the structure and diversity of endophytic bacterial communities of P. ginseng and identify strains with antifungal activity that have potential applications as biocontrol agents.
Project description:Neonectria is a cosmopolitan genus and it is, in part, defined by its link to the anamorph genus Cylindrocarpon. Neonectria has been divided into informal groups on the basis of combined morphology of anamorph and teleomorph. Previously, Cylindrocarpon was divided into four groups defined by presence or absence of microconidia and chlamydospores. Molecular phylogenetic analyses have indicated that Neonectriasensu stricto and Cylindrocarponsensu stricto are phylogenetically congeneric. In addition, morphological and molecular data accumulated over several years have indicated that Neonectria sensu lato and Cylindrocarponsensu lato do not form a monophyletic group and that the respective informal groups may represent distinct genera. In the present work, a multilocus analysis (act, ITS, LSU, rpb1, tef1, tub) was applied to representatives of the informal groups to determine their level of phylogenetic support as a first step towards taxonomic revision of Neonectriasensu lato. Results show five distinct highly supported clades that correspond to some extent with the informal Neonectria and Cylindrocarpon groups that are here recognised as genera: (1) N. coccinea-group and Cylindrocarpon groups 1 & 4 (Neonectria/Cylindrocarponsensu stricto); (2) N.rugulosa-group (Rugonectria gen. nov.); (3) N. mammoidea/N. veuillotiana-groups and Cylindrocarpon group 2 (Thelonectria gen. nov.); (4) N. radicicola-group and Cylindrocarpon group 3 (Ilyonectria gen. nov.); and (5) anamorph genus Campylocarpon. Characteristics of the anamorphs and teleomorphs correlate with the five genera, three of which are newly described. New combinations are made for species where their classification is confirmed by phylogenetic data.
Project description:Black foot disease is a serious disease of grapevine crops in most areas where vines are grown. Mainly two species of Cylindrocarpon, C. destructans and C. macrodidymum, are associated with this disease. Recent studies have revealed a tremendous molecular variation within the former but only slight molecular variation within the latter, indicating that C. destructans presents a complex of several species The present study elucidates the taxonomic status of C. destructans-like isolates associated with black foot disease of grapevines. Grapevine isolates were studied morphologically, subjected to DNA analyses of their ITS and partial beta-tubulin genes, and were mated in all combinations in vitro. Cylindrocarpon destructans strains isolated from grapevines in Europe and South Africa appeared morphologically and genetically identical, and had identical ITS and partial beta-tubulin gene sequences. Phylogenetic analyses placed these strains in a clade closely related but clearly distinct from other clades with C. destructans-like anamorphs obtained from various herbaceous or woody hosts. Only the ex-type strain of Cylindrocarpon liriodendri had identical sequences to strains isolated from grapevines, and could also not be distinguished by morphological characters. The grapevine isolates are therefore reidentified here as Cylindrocarpon liriodendri. Cylindrocarpn liriodendri formed perithecia in heterothallic conditions and the holomorph of this species is described as Neonectria liriodendri sp. nov. Neonectria liriodendri is genetically distinct from the ex-type strain of Neonectria radicicola, which originated from Cyclamen in Sweden. Both ex-type strains also differ from at least two other clades comprising additional C. destructans-like strains. Many of these strains originated from Panax sp., which is the host of the type of C. destructans. Our phylogenetic analyses indicate that C. destructans is not the anamorph of N. radicicola and that N. liriodendri, N. radicicola and several C. destructans-like taxa may have evolved independently within the same phylogenetic species complex.
Project description:Fungal endophytes isolated from mountain-cultivated ginseng (MCG, Panax ginseng Meyer) were explored for their diversity and biocontrol activity against ginseng pathogens (Alternaria panax, Botrytis cinerea, Cylindrocarpon destructans, Pythium sp. and Rhizoctonia solani). A total of 1,300 isolates were isolated from three tissues (root, stem and leaf) from MCGs grown in 24 different geographic locations in Korea. In total, 129 different fungal isolates were authenticated by molecular identification based on internal transcribed spacer (ITS) sequences. The fungal endophytes belonged to Ascomycota (81.7%), Basidiomycota (7.08%), Zygomycota (10%) and Unknown (1.15%), with 59 genera. Analysis of diversity indices across sampling sites suggested species abundance as a function of geographical and environmental factors of the locations. Shannon diversity index and richness in the different tissues revealed that root tissues are colonized more than stem and leaf tissues, and also certain fungal endophytes are tissue specific. Assessment of the ethyl acetate extracts from 129 fungal isolates for their biocontrol activity against 5 ginseng pathogens revealed that Trichoderma polysporum produces the antimcriobial metabolite against all the pathogens. This result indicates the promise of its potential usage as a biocontrol agent.
Project description:White nose syndrome (WNS) is caused by the psychrophilic fungus Pseudogymnoascus destructans that can grow in the environment saprotrophically or parasitically by infecting hibernating bats. Infections are pathological in many species of North American bats, disrupting hibernation and causing mortality. To determine what fungal pathways are involved in infection of living tissue, we examined fungal gene expression using RNA-Seq. We compared P. destructans gene expression when grown in culture to that during infection of a North American bat species, Myotis lucifugus, that shows high WNS mortality. Cultured P. destructans was grown at 10 to 14 C and P. destructans growing in vivo was presumably exposed to temperatures ranging from 4 to 8 C during torpor and up to 37 C during periodic arousals. We found that when P. destructans is causing WNS, the most significant differentially expressed genes were involved in heat shock responses, cell wall remodeling, and micronutrient acquisition. These results indicate that this fungal pathogen responds to host-pathogen interactions by regulating gene expression in ways that may contribute to evasion of host responses. Alterations in fungal cell wall structures could allow P. destructans to avoid detection by host pattern recognition receptors and antibody responses. This study has also identified several fungal pathways upregulated during WNS infection that may be candidates for mitigating infection pathology. By identifying host-specific pathogen responses, these observations have important implications for host-pathogen evolutionary relationships in WNS and other fungal diseases.
Project description:White-nose syndrome is an emerging disease in North America that has caused substantial declines in hibernating bats. A recently identified fungus (Geomyces destructans) causes skin lesions that are characteristic of this disease. Typical signs of this infection were not observed in bats in North America before white-nose syndrome was detected. However, unconfirmed reports from Europe indicated white fungal growth on hibernating bats without associated deaths. To investigate these differences, hibernating bats were sampled in Germany, Switzerland, and Hungary to determine whether G. destructans is present in Europe. Microscopic observations, fungal culture, and genetic analyses of 43 samples from 23 bats indicated that 21 bats of 5 species in 3 countries were colonized by G. destructans. We hypothesize that G. destructans is present throughout Europe and that bats in Europe may be more immunologically or behaviorally resistant to G. destructans than their congeners in North America because they potentially coevolved with the fungus.
Project description:Ginseng root is an economically valuable crop in Canada at high risk of yield loss caused by the pathogenic fungus Ilyonectria mors-panacis, formerly known as Cylindrocarpon destructans. While this pathogen has been well-characterized from morphological and genetic perspectives, little is known about the secondary metabolites it produces and their role in pathogenicity. We used an untargeted tandem liquid chromatography-mass spectrometry (LC-MS)-based approach paired with global natural products social molecular networking (GNPS) to compare the metabolite profiles of virulent and avirulent Ilyonectria strains. The ethyl acetate extracts of 22 I. mors-panacis strains and closely related species were analyzed by LC-MS/MS. Principal component analysis of LC-MS features resulted in two distinct groups, which corresponded to virulent and avirulent Ilyonectria strains. Virulent strains produced more types of compounds than the avirulent strains. The previously reported I. mors-panacis antifungal compound radicicol was present. Additionally, a number of related resorcyclic acid lactones (RALs) were putatively identified, namely pochonins and several additional derivatives of radicicol. Pochonins have not been previously reported in Ilyonectria spp. and have documented antimicrobial activity. This research contributes to our understanding of I. mors-panacis natural products and its pathogenic relationship with ginseng.