Plant-microbe features of Dendrobium fimbriatum (Orchidaceae) fungal community

International audienc

Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in <em>Vitis vinifera</em> tissues

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples. Methods and results: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola. Conclusion: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew. Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations

Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in Vitis vinifera tissues

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples.Methods and results : Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specific for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola.Conclusion : A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew.Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations."Premiers Grands Crus" of BordeauxPeer reviewe

Dynamics of Metabolite Induction in Fungal Co-cultures by Metabolomics at Both Volatile and Non-volatile Levels

Fungal co-cultivation has emerged as a promising way for activating cryptic biosynthetic pathways and discovering novel antimicrobial metabolites. For the success of such studies, a key element remains the development of standardized co-cultivation methods compatible with high-throughput analytical procedures. To efficiently highlight induction processes, it is crucial to acquire a holistic view of intermicrobial communication at the molecular level. To tackle this issue, a strategy was developed based on the miniaturization of fungal cultures that allows for a concomitant survey of induction phenomena in volatile and non-volatile metabolomes. Fungi were directly grown in vials, and each sample was profiled by head space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS), while the corresponding solid culture medium was analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) after solvent extraction. This strategy was implemented for the screening of volatile and non-volatile metabolite inductions in an ecologically relevant fungal co-culture of Eutypa lata (Pers.) Tul. &amp; C. Tul. (Diatrypaceae) and Botryosphaeria obtusa (Schwein.) Shoemaker (Botryosphaeriaceae), two wood-decaying fungi interacting in the context of esca disease of grapevine. For a comprehensive evaluation of the results, a multivariate data analysis combining Analysis of Variance and Partial Least Squares approaches, namely AMOPLS, was used to explore the complex LC-HRMS and GC-MS datasets and highlight dynamically induced compounds. A time-series study was carried out over 9 days, showing characteristic metabolite induction patterns in both volatile and non-volatile dimensions. Relevant links between the dynamics of expression of specific metabolite production were observed. In addition, the antifungal activity of 2-nonanone, a metabolite incrementally produced over time in the volatile fraction, was assessed against Eutypa lata and Botryosphaeria obtusa in an adapted bioassay set for volatile compounds. This compound has shown antifungal activity on both fungi and was found to be co-expressed with a known antifungal compound, O-methylmellein, induced in solid media. This strategy could help elucidate microbial inter- and intra-species cross-talk at various levels. Moreover, it supports the study of concerted defense/communication mechanisms for efficiently identifying original antimicrobials

Comparative metabolomics of fungal foliar endophytes and their long-lived host Astrocaryum sciophilum: a model for exploring the chemodiversity of host-microbe interactions

International audienceIn contrast to the dynamics observed in plant/pathogen interactions, endophytic fungi have the capacity to establish enduring associations within their hosts, leading to the development of a mutually beneficial relationship that relies on specialized chemical interactions. Research indicates that the presence of endophytic fungi has the ability to significantly modify the chemical makeup of the host organism. Our hypothesis proposes the existence of a reciprocal exchange of chemical signals between plants and fungi, facilitated by specialized chemical processes that could potentially manifest within the tissues of the host. This research aimed to precisely quantify the portion of the cumulative fungal endophytic community's metabolome detectable within host leaves, and tentatively evaluate its relevance to the host-endophyte interplay. The understory palm Astrocaryum sciophilum (Miq.) Pulle was used as a interesting host plant because of its notable resilience and prolonged life cycle, in a tropical ecosystem. Using advanced metabolome characterization, including UHPLC-HRMS/MS and molecular networking, the study explored enriched metabolomes of both host leaves and 15 endophytic fungi. The intention was to capture a metabolomic "snapshot" of both host and endophytic community, to achieve a thorough and detailed analysis. This approach yielded an extended MS-based molecular network, integrating diverse metadata for identifying host- and endophyte-derived metabolites. The exploration of such data (>24000 features in positive ionization mode) enabled effective metabolome comparison, yielding insights into cultivable endophyte chemodiversity and occurrence of common metabolites between the holobiont and its fungal communities. Surprisingly, a minor subset of features overlapped between host leaf and fungal samples despite significant plant metabolome enrichment. This indicated that fungal metabolic signatures produced in vitro remain sparingly detectable in the leaf. Several classes of primary metabolites were possibly shared. Specific fungal metabolites and/or compounds of their chemical classes were only occasionally discernible in the leaf, highlighting endophytes partial contribution to the overall holobiont metabolome. To our knowledge, the metabolomic study of a plant host and its microbiome has rarely been performed in such a comprehensive manner. The general analytical strategy proposed in this paper seems well-adapted for any study in the field of microbial- or microbiome-related MS and can be applied to most host-microbe interactions

Characterization of Pseudomonas aeruginosa quorum sensing inhibitors from the endophyte Lasiodiplodia venezuelensis and evaluation of their anti-virulence effects by metabolomics

AbstractNMR data of all isolated compound

Isolation and identification of isocoumarin derivatives with specific inhibitory activity against Wnt pathway Lasiodiplodia venezuelensis and metabolome characterization of

AbstractNMR dat

Generation of novel stilbene antimicrobial agents against multi resistant strains of Staphylococcus aureus through biotransformation by the enzymatic secretome of Botrytis cinerea

AbstractUHPLC-PDA-ELSD-MS analysis of the biotransformation reactions at analytical scale and the NMR and HRMS data for all isolated compounds