Unveiling the fungal diversity and associated secondary metabolism on black apples.

Black apples are the result of late-stage microbial decomposition after falling to the ground. This phenomenon is highly comparable from year to year, with the filamentous fungus Monilinia fructigena most commonly being the first invader, followed by Penicillium expansum. Motivated by the fact that only little chemistry has been reported from apple microbiomes, we set out to investigate the chemical diversity and potential ecological roles of secondary metabolites (SMs) in a total of 38 black apples. Metabolomics analyses were conducted on either whole apples or small excisions of fungal biomass derived from black apples. Annotation of fungal SMs in black apple extracts was aided by the cultivation of 15 recently isolated fungal strains on 9 different substrates in a One Strain Many Compounds (OSMAC) approach, leading to the identification of 3,319 unique chemical features. Only 6.4% were attributable to known compounds based on analysis of high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS/MS) data using spectral library matching tools. Of the 1,606 features detected in the black apple extracts, 32% could be assigned as fungal-derived, due to their presence in the OSMAC-based training data set. Notably, the detection of several antifungal compounds indicates the importance of such compounds for the invasion of and control of other microbial competitors on apples. In conclusion, the diversity and abundance of microbial SMs on black apples were found to be much higher than that typically observed for other environmental microbiomes. Detection of SMs known to be produced by the six fungal species tested also highlights a succession of fungal growth following the initial invader M. fructigena.IMPORTANCEMicrobial secondary metabolites constitute a significant reservoir of biologically potent and clinically valuable chemical scaffolds. However, their usefulness is hampered by rapidly developing resistance, resulting in reduced profitability of such research endeavors. Hence, the ecological role of such microbial secondary metabolites must be considered to understand how best to utilize such compounds as chemotherapeutics. Here, we explore an under-investigated environmental microbiome in the case of black apples; a veritable "low-hanging fruit," with relatively high abundances and diversity of microbially produced secondary metabolites. Using both a targeted and untargeted metabolomics approach, the interplay between metabolites, other microbes, and the apple host itself was investigated. This study highlights the surprisingly low incidence of known secondary metabolites in such a system, highlighting the need to study the functionality of secondary metabolites in microbial interactions and complex microbiomes.

Coupling Microplate-Based Antibacterial Assay with Liquid Chromatography for High-Resolution Growth Inhibition Profiling of Crude Extracts: Validation and Proof-of-Concept Study with Staphylococcus aureus.

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.

Antidiabetic xanthones with α-glucosidase inhibitory activities from an endophytic Penicillium canescens.

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens, recovered from fruits of Juniperus polycarpos. The three xanthones 5, 7, and 11 showed inhibitory activities against α-glucosidase with IC50 values of 38.80 ± 1.01 µM, 32.32 ± 1.01 µM, and 75.20 ± 1.02 µM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5, a competitive inhibition for 7, while 11 acted as a non-competitive inhibitor.