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To document in more details our knowledge of the fungal community associated to the Orchidaceae family, 25 cultivable fungal species were isolated from greenhouse apparently healthy Dendrobium fimbriatum var. oculatum Hook. adult plants. These fungi, were identified based on a molecular approach, belonging predominantly to Fusarium, Trichoderma, Colletotrichum, Curvularia and Didymella genera. To characterize their impact on plant growth and/or plant defense, their ability to produce different metabolites (phytohormones, siderophores) and hydrolytic enzymes, as well as their anti-phytopathogenic activity against Botrytis cinerea, Colletotrichum gloeosporioides and Fusarium oxysporum, were evaluated ex situ. Based on these criteria, three fungi, initially isolated from the roots, Fusarium sp., Trichoderma yunnanense, and Curvularia sp., showed a high potential to act on plant growth. Our results also show that an important number of isolates of the fungal community were able to produce phytohormones, siderophores, and had a similar hydrolytic enzyme production profile. Finally, these fungal abilities, studied ex situ, were analyzed to determine whether they can be used to classify the fungal partners as beneficial or detrimental to their host.

Summary Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Dendrobium fimbriatum Hook is an important medicinal and ornamental orchid that may lead to important drugs and cosmetics. In this study, 2 analytical strategies were combined to describe the detailed composition of a hydroethanolic extract of the whole plant. The CARAMEL approach, based on centrifugal partition chromatography and nuclear magnetic resonance data interpretation, allowed the rapid dereplication of 21 major constituents, mainly including polyols, nucleosides, zwitterionic compounds, phenolic compounds, and organic acids. Further semipreparative high-performance liquid chromatography and spectroscopic analyses led to the isolation of 6 additional compounds; 4 phenanthrenes (plicatol B, hircinol, plicatol A, and plicatol C), 1 bibenzyl (3′,4-dihydroxy-3,5′-dimethoxybibenzyl), and 1 furostanol saponin (protodioscin), as well as a new phenanthrene derivative, plicatol D. A total of 28 metabolites were structurally elucidated, among which 23 are described for this species for the first time.

International audience; Orchids are associated with diverse fungal taxa, including nonmycorrhizal endophytic fungi as well as mycorrhizal fungi. The orchid mycorrhizal (OM) symbiosis is an excellent model for investigating the biological interactions between plants and fungi due to their high dependency on these symbionts for growth and survival. To capture the complexity of OM interactions, significant genomic, numerous transcriptomic, and proteomic studies have been performed, unraveling partly the role of each partner. On the other hand, several papers studied the bioactive metabolites from each partner but rarely interpreted their significance in this symbiotic relationship. In this review, we focus from a biochemical viewpoint on the OM dynamics and its molecular interactions. The ecological functions of OM in plant development and stress resistance are described first, summarizing recent literature. Secondly, because only few studies have specifically looked on OM molecular interactions, the signaling pathways and compounds allowing the establishment/maintenance of mycorrhizal association involved in arbuscular mycorrhiza (AM) are discussed in parallel with OM. Based on mechanistic similarities between OM and AM, and recent findings on orchids’ endophytes, a putative model representing the different molecular strategies that OM fungi might employ to establish this association is proposed. It is hypothesized here that (i) orchids would excrete plant molecule signals such as strigolactones and flavonoids but also other secondary metabolites; (ii) in response, OM fungi would secrete mycorrhizal factors (Myc factors) or similar compounds to activate the common symbiosis genes (CSGs); (iii) overcome the defense mechanism by evasion of the pathogen-associated molecular patterns (PAMPs)-triggered immunity and by secretion of effectors such as small inhibitor proteins; and (iv) finally, secrete phytohormones to help the colonization or disrupt the crosstalk of plant defense phytohormones. To challenge this putative model, targeted and untargeted metabolomics studies with special attention to each partner’s contribution are finally encouraged and some technical approaches are proposed.

The dichloromethane extract of the root bark of the Panamanian plant Cordia anisophylla J.S. Mill. (Boraginaceae) presented antifungal activity against a susceptible strain of Candida albicans in a bioautography primary screening. The susceptible strain was used to detect minor active compounds that would not have been detected using a classical approach. In order to identify the antimicrobial compounds, the active extract was fractionated by semi-preparative high-performance liquid chromatography and the fractions were submitted to the antifungal bioassay. This procedure enabled a precise localization of the antifungal compounds directly in the chromatogram of the crude extract and allowed for an efficient, targeted isolation. Four compounds were isolated, one of which is a new natural product. The structures were elucidated using spectroscopic methods. Their antifungal properties were evaluated by determination of the minimum inhibitory quantity and concentration by bioautography and dilution assay against a wild type strain of C. albicans.

Chemical investigation of a dichloromethane extract of the aerial parts of Waltheria indica led to the isolation and characterization of five polyhydroxymethoxyflavonoids, namely, oxyanin A (1), vitexicarpin (3), chrysosplenol E (4), flindulatin (5), 5-hydroxy-3,7,4'-trimethoxyflavone (6), and six quinolone alkaloids, waltheriones M-Q (2, 7, 8, 10, 11) and 5(R)-vanessine (9). Among these, compounds 2, 7, 8, 10, and 11 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including (1)H and (13)C, HSQC, HMBC, COSY, and NOESY NMR experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by ECD. The isolated constituents and 10 additional quinoline alkaloids previously isolated from the roots of the plant were evaluated for their in vitro antifungal activity against the human fungal pathogen Candida albicans, and 10 compounds (7, 9, 11-16, 18, 21) showed growth inhibitory activity on both planktonic cells and biofilms (MIC ≤ 32 μg/mL). Their spectrum of activity against other pathogenic Candida species and their cytotoxicity against human HeLa cells were also determined. In addition, the cytological effect of the antifungal isolated compounds on the ultrastructure of C. albicans was evaluated by transmission electron microscopy.

As a part of a broad screening of antifungal agents from plant origin, crude extracts from Panamanian plants having related types of constituents displayed significant activities in an agar overlay thin layer chromatography assay against a susceptible strain of Candida albicans. These were the methanolic extract of the leaves of Schefflera systyla and Odontadenia puncticulosa and of the stems of Conostegia speciosa, that are species not previously investigated from a phytochemical viewpoint. For all plants, high-performance liquid chromatography (HPLC) antifungal activity based profiling allowed the rapid localization of antifungal agents that were further obtained by targeted isolation procedure by semi-preparative HPLC or medium pressure liquid chromatography (MPLC) after LC gradient transfer. Different hederagenin saponins and one aglycone were found to be responsible for the antifungal activities of the extracts. Alpha-hederin was the antifungal of S. systyla, pulsatilla saponin D and 3β-O-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosylhederagenin of O. puncticulosa and arjunolic acid of C. speciosa. Their minimal inhibitory concentration (MIC) against planktonic and biofilm cells of C. albicans were determined. Alpha-hederin was the most potent compound with a MIC of 4 µg mL-1. Structurally related compounds (hederagenin, medicagenic acid 3-O-β-D-glucopyranoside and medicagenic acid) were used as standards and tested for comparison purposes. In order to better estimate the potential of these triterpenoids as antifungal agents, their cytological effects on C. albicans were determined by transmission electron microscopy (TEM) and the in vivo activity of alpha-hederin, medicagenic acid 3-O-β-D-glucopyranoside and medicagenic acid was evaluated for the first time in the Galleria mellonella larvae model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS.

This paper reviews the use of TLC-bioautography in the search for antifungal compounds from natural sources. The main methods used for antifungal screening are presented, with special emphasis on bioautography. Different aspects of the technique, including the latest chromatographic developments such as HPTLC and HPLC microfractionation are presented. The present status and recent advances made in antifungal bioautography are discussed, and a comprehensive review of the applications over the last 6 years is presented. Various strategies applied in the search for antifungal compounds from natural sources are discussed, with a highlight on the challenges faced when screening complex crude mixtures. The activities of approximately 100 antifungal compounds of natural origin are presented with their minimum inhibitory quantity. The most active natural source compounds against Candida, Cladosporium, Colletotrichum, and Fusarium species are highlighted, and the compound activities discussed. In addition, perspectives concerning future improvements in bioautography sensitivity and reproducibility are noted.

In natural products research, the efficient purification of molecules from large amount of complex extracts is a key element. In this regard, an integrative strategy for efficient mass spectrometry (MS)-guided isolation of antifungal compounds has been developed. First, at-line HPLC antifungal activity based profiling and HPLC-PDA-MS profiling were used to localise the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of the antifungal principles from the Chinese liverwort Chiloscyphus polyanthos (L.) Cord. This rational methodology has a high potential for the targeted large-scale purification of bioactive compounds avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism (ECD) spectroscopy.