Screening of Secondary Metabolites Produced by Nigrospora sphaerica Associated with the Invasive Weed Cenchrus ciliaris Reveals Two New Structurally Related Compounds.

In the search for new alternative biocontrol strategies, phytopathogenic fungi could represent a new frontier for weed management. In this respect, as part of our ongoing work aiming at using fungal pathogens as an alternative to common herbicides, the foliar pathogen Nigrospora sphaerica has been evaluated to control buffelgrass (Cenchrus ciliaris). In particular, in this work, the isolation and structural elucidation of two new biosynthetically related metabolites, named nigrosphaeritriol (3-(hydroxymethyl)-2-methylpentane-1,4-diol) and nigrosphaerilactol (3-(1-hydroxyethyl)-4-methyltetrahydrofuran-2-ol), from the phytotoxic culture filtrate extract were described, along with the identification of several known metabolites. Moreover, the absolute stereochemistry of (3R,4S,5S)-nigrosphaerilactone, previously reported as (3S,4R,5R)-4-hydroxymethyl-3,5-dimethyldihydro-2-furanone, was determined for the first time by X-ray diffraction analysis. Considering their structural relationship, the determination of the absolute stereochemistry of nigrosphaerilactone allowed us to hypothesize the absolute stereochemistry of nigrosphaeritriol and nigrosphaerilactol.

Comparative Analysis of Secondary Metabolites in Diplodia corticola Strains with Different Virulence Degrees Associated with Canker and Dieback of Quercus spp.

Diplodia corticola is one of the most aggressive fungal pathogens of Quercus species involved in the decline of Mediterranean oak forests. In this study, three strains of D. corticola associated with holm (Quercus ilex) and cork (Quercus suber) oak trees exhibiting dieback symptoms and cankers in Algeria were selected to investigate the production of secondary metabolites. Metabolomic analyses revealed the production of several known compounds, such as sphaeropsidins, diplopyrones and diplofuranones. Moreover, the comparative investigation of secondary metabolites produced by the analyzed strains with different degrees of virulence revealed possible implications of these compounds in the fungal virulence. In particular, sphaeropsidins seem to be the main phytotoxic compounds of D. corticola involved in the infections of Quercus species, with a possible synergistic influence of the less representative compounds in the fungal virulence.

Secondary Metabolites, including a New 5,6-Dihydropyran-2-One, Produced by the Fungus Diplodia corticola. Aphicidal Activity of the Main Metabolite, Sphaeropsidin A.

An undescribed 5,6-dihydropyran-2-one, namely diplopyrone C, was isolated and characterized from the cultures of an isolate of the fungus Diplodia corticola recovered from Quercus suber in Algeria. The structure and relative stereostructure of (5S,6S,7Z,9S,10S)-5-hydroxy-6-(2-(3-methyloxiran-2-yl)vinyl)-5,6-dihydro-2H-pyran-2-one were assigned essentially based on NMR and MS data. Furthermore, ten known compounds were isolated and identified in the same cultures. The most abundant product, the tetracyclic pimarane diterpene sphaeropsidin A, was tested for insecticidal effects against the model sucking aphid, Acyrthosiphon pisum. Results showed a toxic dose-dependent oral activity of sphaeropsidin A, with an LC50 of 9.64 mM.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (Pr3+, Eu3+, Ho3+, Tm3+).

Rare-earth elements (REEs) are in all respect a class of new contaminants that may have toxic effects on organisms and microorganisms and information on their interactions with natural ligands should be of value to predict and control their diffusion in natural environments. In the current study, we investigate interactions of tripositive cations of praseodymium, europium, holmium, and thulium with harzianic acid (H2L), a secondary metabolite produced by selected strains of fungi belonging to the Trichoderma genus. We applied the same techniques and workflow previously employed in an analogous study concerning lanthanum, neodymium, samarium, and gadolinium tripositive cations. Therefore, in the current study, HPLC-ESI-HRMS experiments, circular dichroism (CD), and UV-Vis spectrophotometric absorption data, as well as accurate pH measurements, were applied to characterize bonding interactions between harzianic acid and Pr3+, Eu3+, Ho3+, and Tm3+ cations. Problems connected to the low solubility of harzianic acid in water were overcome by employing a 0.1 M NaClO4/(CH3OH + H2O 50/50 w/w) mixed solvent. For Pr3+, Ho3+, and Tm3+, only the mono complexes PrL+, HoL+, and TmL+ were detected and their formation constant determined. Eu3+ forms almost exclusively the bis complex EuL2- for which the corresponding formation constant is reported; under our experimental conditions, the mono complex EuL+ is irrelevant. Combining the results of the present and previous studies, a picture of interactions of harzianic acid with rare-earth cations extending over 8 of the 17 REEs can be composed. In order to complement chemical information with toxicological information, a battery of bioassays was applied to evaluate the effects of praseodymium, europium, holmium, and thulium tripositive cations on a suite of bioindicators including Aliivibrio fischeri (Gram-negative bacterium), Raphidocelis subcapitata (green alga), and Daphnia magna (microcrustacean), and median effective concentration (EC50) values of Pr3+, Eu3+, Ho3+, and Tm3+ for the tested species were assessed.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (La3+, Nd3+, Sm3+, Gd3+).

Rare-earth elements are emerging contaminants of soil and water bodies which destiny in the environment and effects on organisms is modulated by their interactions with natural ligands produced by bacteria, fungi and plants. Within this framework, coordination by harzianic acid (H2L), a Trichoderma secondary metabolite, of a selection of tripositive rare-earth cations Ln3+ (Ln3+ = La3+, Nd3+, Sm3+, and Gd3+) was investigated at 25 °C, and in a CH3OH/0.1 M NaClO4 (50/50 w/w) solvent, using mass spectrometry, circular dichroism, UV-Vis spectrophotometry, and pH measurements. Experimental data can be satisfactorily explained by assuming, for all investigated cations, the formation of a mono-complex (LnL+) and a bis-complex (LnL2-). Differences were found between the formation constants of complexes of different Ln3+ cations, which can be correlated with ionic radius. Since gadolinium is the element that raises the most concern among lanthanide elements, its effects on organisms at different levels of biological organization were explored, in the presence and absence of harzianic acid. Results of ecotoxicological tests suggest that harzianic acid can decrease gadolinium biotoxicity, presumably because of complex formation with Gd3+.

GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae.

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

The Genus Cladosporium: A Rich Source of Diverse and Bioactive Natural Compounds.

Fungi are renowned as one of the most fruitful sources of chemodiversity and for their ubiquitous occurrence. Among the many taxonomic groupings considered for the implications deriving from their biosynthetic aptitudes, the genus Cladosporium stands out as one of the most common in indoor environments. A better understanding of the impact of these fungi on human health and activities is clearly based on the improvement of our knowledge of the structural aspects and biological properties of their secondary metabolites, which are reviewed in the present paper.

Effect of γ-Aminobutyric Acid (GABA) on the Metabolome of Two Strains of Lasiodiplodia theobromae Isolated from Grapevine.

The effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.

Bivalent Metal-Chelating Properties of Harzianic Acid Produced by Trichoderma pleuroticola Associated to the Gastropod Melarhaphe neritoides.

Harzianic acid is a secondary metabolite of Trichoderma, structurally belonging to the dienyltetramic acid subgroup of the tetramic acids. Biological activities of harzianic acid are of great interest for its antimicrobial and plant growth-promoting activities, which might be related to its chelating properties. In the present work harzianic acid, isolated from cultures of a strain of Trichoderma pleuroticola associated to the gastropod Melarhaphe neritoides, was studied as a complexant agent of a number of biologically relevant transition metals (i.e., Zn2+, Fe2+, Cu2+, and Mn2+), using UV-VIS, potentiometry, MS and NMR techniques. Our findings show the coordination capacity of harzianic acid toward the above cations through the formation of neutral or charged complexes in a variable ratio depending on the metal and pH conditions.

Occurrence and Properties of Thiosilvatins.

The spread of studies on biodiversity in different environmental contexts is particularly fruitful for natural product discovery, with the finding of novel secondary metabolites and structural models, which are sometimes specific to certain organisms. Within the large class of the epipolythiodioxopiperazines, which are typical of fungi, thiosilvatins represent a homogeneous family that, so far, has been reported in low frequency in both marine and terrestrial contexts. However, recent observations indicate that these compounds have been possibly neglected in the metabolomic characterization of fungi, particularly from marine sources. Aspects concerning occurrence, bioactivities, structural, and biosynthetic properties of thiosilvatins are reviewed in this paper.

The Issue of Misidentification of Kojic Acid with Flufuran in Aspergillus flavus.

In the course of investigations on the complex phenomenon of bee decline, Aspergillus flavus was isolated from the haemocoel of worker bees. Observations on the metabolomic profile of this strain showed kojic acid to be the dominant product in cultures on Czapek-Dox broth. However, an accurate review of papers documenting secondary metabolite production in A. flavus also showed that an isomer of kojic acid, identified as 5-(hydroxymethyl)-furan-3-carboxylic acid and named flufuran is reported from this species. The spectroscopic data of kojic acid were almost identical to those reported in the literature for flufuran. This motivated a comparative study of commercial kojic acid and 5-(hydroxymethyl)-furan-3-carboxylic acid, highlighting some differences, for example in the 13C-NMR and UV spectra for the two compounds, indicating that misidentification of the kojic acid as 5-(hydroxymethyl)-furan-3-carboxylic acid has occurred in the past.

Secondary Metabolites of Mangrove-Associated Strains of Talaromyces.

Boosted by the general aim of exploiting the biotechnological potential of the microbial component of biodiversity, research on the secondary metabolite production of endophytic fungi has remarkably increased in the past few decades. Novel compounds and bioactivities have resulted from this work, which has stimulated a more thorough consideration of various natural ecosystems as conducive contexts for the discovery of new drugs. Thriving at the frontier between land and sea, mangrove forests represent one of the most valuable areas in this respect. The present paper offers a review of the research on the characterization and biological activities of secondary metabolites from manglicolous strains of species belonging to the genus Talaromyces. Aspects concerning the opportunity for a more reliable identification of this biological material in the light of recent taxonomic revisions are also discussed.

Structures and Bioactive Properties of Myrtucommulones and Related Acylphloroglucinols from Myrtaceae.

Myrtaceae are a group of plants that include a number of renowned species used in ethnomedicine in many areas worldwide. Their valuable therapeutic properties have stimulated a fruitful research activity addressed to the identification of the bioactive components of their extracts yielding a great diversity of terpenes; polyphenols; and other exclusive products. Among the latter, starting with the discovery of myrtucommulone A from myrtle (Myrtus communis), a series of structurally-related acylphloroglucinol compounds have been characterized from several species that represent the basic active principles to be considered in view of possible drug development. Aspects concerning chemical and biological properties of these products are reviewed in the present paper.

Talarodiolide, a New 12-Membered Macrodiolide, and GC/MS Investigation of Culture Filtrate and Mycelial Extracts of Talaromyces pinophilus.

Talarodiolide, a new 12-membered macrodiolide, was isolated and characterized from the culture filtrate of strain LT6 of Talaromyces pinophilus. The structure of (Z)-4,10-dimethyl-1,7-dioxa-cyclododeca-3,9-diene-2,8-dione was assigned essentially based on NMR and MS data. Furthermore, several known compounds were isolated and identified in the crude extract of the culture filtrate and mycelium of this strain. EI mass spectrum at 70 eV of all isolated metabolites was acquired and compiled in a custom GC/MS library to be employed to detect metabolites in the crude extracts.

A metabolomics footprinting approach using GC-MS to study inhibitory effects of the fungal metabolite diplopyrone C against nosocomial pathogen biofilms.

Seen initially as wonder drugs, the widespread and often inappropriate use of antibiotics led to the development of microbial resistances. As a result, a true emergency has arisen, and a significant need has emerged to discover and develop new safe and valuable antibiotics. The captivating chemical structure of the fungal metabolite diplopyrone C has caught our attention as an excellent candidate for a circumstantial study aimed at revealing its antimicrobial and antibiofilm activities. In this work, we describe the full analytical strategy from the isolation/identification to the evaluation of the metabolomics effect on target microorganisms of this fungal metabolite. Our results show interesting antimicrobial and antibiofilm activities of diplopyrone C against two frequently isolated nosocomial pathogens (i.e., the fungus Candida albicans and the gram-negative bacterium Klebsiella pneumoniae). Moreover, a GC-MS based metabolomics footprinting approach gave an insight into the uptake and excretion of metabolites from and into the culture medium as a response to the presence of this active substance. The workflow employed in this study is suitable to exploit natural resources for the search of lead compounds for drug development.

Evaluation of Potential Probiotic Properties and In Vivo Safety of Lactic Acid Bacteria and Yeast Strains Isolated from Traditional Home-Made Kefir.

Probiotics are known for their health-promoting resources and are considered as beneficial microorganisms. The current study focuses on the isolation, and on a complete in vitro and in vivo characterization, of yeast and lactic acid bacteria acquired from traditional homemade kefir in order to assess their potentiality as probiotic candidates. In particular, the isolates Pichia kudriavzevii Y1, Lactococcus lactis subsp. hordniae LAB1 and Lactococcus lactis subsp. lactis LAB2 were subjected to in vitro characterization to evaluate their suitability as probiotics. Resistance to acid and bile salts, auto-aggregation, co-aggregation, hydrophobicity, and biofilm production capability were examined, as well as their antioxidant activity. A safety assessment was also conducted to confirm the non-pathogenic nature of the isolates, with hemolysis assay and antibiotic resistance assessment. Moreover, mortality in the invertebrate model Galleria mellonella was evaluated. Current findings showed that P. kudriavzevii exhibited estimable probiotic properties, placing them as promising candidates for functional foods. Both lactic acid bacteria isolated in this work could be classified as potential probiotics with advantageous traits, including antimicrobial activity against enteric pathogens and good adhesion ability on intestinal cells. This study revealed that homemade kefir could be a beneficial origin of different probiotic microorganisms that may enhance health and wellness.

Mycotoxin Contamination in Hazelnut: Current Status, Analytical Strategies, and Future Prospects.

Hazelnuts represent a potential source of mycotoxins that pose a public health issue due to their increasing consumption as food ingredients worldwide. Hazelnuts contamination by mycotoxins may derive from fungal infections occurring during fruit development, or in postharvest. The present review considers the available data on mycotoxins detected in hazelnuts, on fungal species reported as infecting hazelnut fruit, and general analytical approaches adopted for mycotoxin investigation. Prompted by the European safety regulation concerning hazelnuts, many analytical methods have focused on the determination of levels of aflatoxin B1 (AFB1) and total aflatoxins. An overview of the available data shows that a multiplicity of fungal species and further mycotoxins have been detected in hazelnuts, including anthraquinones, cyclodepsipeptides, ochratoxins, sterigmatocystins, trichothecenes, and more. Hence, the importance is highlighted in developing suitable methods for the concurrent detection of a broad spectrum of these mycotoxins. Moreover, control strategies to be employed before and after harvest in the aim of controlling the fungal contamination, and in reducing or inactivating mycotoxins in hazelnuts, are discussed.

Anti-Insect Properties of Penicillium Secondary Metabolites.

In connection with their widespread occurrence in diverse environments and ecosystems, fungi in the genus Penicillium are commonly found in association with insects. In addition to some cases possibly implying a mutualistic relationship, this symbiotic interaction has mainly been investigated to verify the entomopathogenic potential in light of its possible exploitation in ecofriendly strategies for pest control. This perspective relies on the assumption that entomopathogenicity is often mediated by fungal products and that Penicillium species are renowned producers of bioactive secondary metabolites. Indeed, a remarkable number of new compounds have been identified and characterized from these fungi in past decades, the properties and possible applications of which in insect pest management are reviewed in this paper.

Metabolites from a Pleurotus tuber-regium collected in Madagascar.

Pleurotus tuber-regium was isolated from a dead trunk of Raphia farinifera (Arecaceae) in a lowland moist forest in Antsohihy, Madagascar, and the species was confirmed by molecular analysis and morphological observations. The main bioactive metabolites of the mycelium extracts were identified by mass spectrometry techniques. Five structural diverse metabolites, tryptophol, pyroglutamic acid, prolyldiketopiperazine B, sporol and RKS-1778, were characterised by LC-MS qTOF analysis of the hydro-alcoholic extract. GC-MS analysis of both chloroform and ethyl acetate extracts revealed the presence of several saturated and -unsaturated fatty acids and their esters derivatives.