Deciphering the mechanisms involved in reduced sensitivity to azoles and fengycin lipopeptide in Venturia inaequalis.

Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis, is currently the most common and damaging disease in apple orchards. Two strains of V. inaequalis (S755 and Rs552) with different sensitivities to azole fungicides and the bacterial metabolite fengycin were compared to determine the mechanisms responsible for these differences. Antifungal activity tests showed that Rs552 had reduced sensitivity to tebuconazole and tetraconazole, as well as to fengycin alone or in a binary mixture with other lipopeptides (iturin A, pumilacidin, lichenysin). S755 was highly sensitive to fengycin, whose activity was close to that of tebuconazole. Unlike fengycin, lipopeptides from the iturin family (mycosubtilin, iturin A) had similar activity on both strains, while those from the surfactin family (lichenysin, pumilacidin) were not active, except in binary mixtures with fengycin. The activity of lipopeptides varies according to their family and structure. Analyses to determine the difference in sensitivity to azoles (which target the CYP51 enzyme involved in the ergosterol biosynthesis pathway) showed that the reduced sensitivity in Rs552 is linked to (i) a constitutive increased expression of the Cyp51A gene caused by insertions in the upstream region and (ii) greater efflux by membrane pumps with the involvement of ABC transporters. Microscopic observations revealed that fengycin, known to interact with plasma membranes, induced morphological and cytological changes in cells from both strains. Sterol and phospholipid analyses showed a higher level of ergosta-7,22-dien-3-ol and a lower level of PI(C16:0/C18:1) in Rs552 compared with S755. These differences could therefore influence the composition of the plasma membrane and explain the differential sensitivity of the strains to fengycin. However, the similar antifungal activities of mycosubtilin and iturin A in the two strains indirectly indicate that sterols are probably not involved in the fengycin resistance mechanism. This leads to the conclusion that different mechanisms are responsible for the difference in susceptibility to azoles or fengycin in the strains studied.

Niche partitioning and plastisphere core microbiomes in the two most plastic polluted zones of the world ocean.

Plastics are offering a new niche for microorganisms colonizing their surface, the so-called "plastisphere," in which diversity and community structure remain to be characterized and compared across ocean pelagic regions. Here, we compared the bacterial diversity of microorganisms living on plastic marine debris (PMD) and the surrounding free-living (FL) and organic particle-attached (PA) lifestyles sampled during the Tara expeditions in two of the most plastic polluted zones in the world ocean, i.e., the North Pacific gyre and the Mediterranean Sea. The 16S rRNA gene sequencing analysis confirmed that PMD are a new anthropogenic ocean habitat for marine microbes at the ocean-basin-scale, with clear niche partitioning compared to FL and PA lifestyles. At an ocean-basin-scale, the composition of the plastisphere communities was mainly driven by environmental selection, rather than polymer types or dispersal effect. A plastisphere "core microbiome" could be identified, mainly dominated by Rhodobacteraceae and Cyanobacteria. Predicted functions indicated the dominance of carbon, nitrogen and sulfur metabolisms on PMD that open new questions on the role of the plastisphere in a large number of important ecological processes in the marine ecosystem.

Bioplastic degradation and assimilation processes by a novel bacterium isolated from the marine plastisphere.

Biosourced and biodegradable plastics offer a promising solution to reduce environmental impacts of plastics for specific applications. Here, we report a novel bacterium named Alteromonas plasticoclasticus MED1 isolated from the marine plastisphere that forms biofilms on foils of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Experiments of degradation halo, plastic matrix weight loss, bacterial oxygen consumption and heterotrophic biosynthetic activity showed that the bacterial isolate MED1 is able to degrade PHBV and to use it as carbon and energy source. The likely entire metabolic pathway specifically expressed by this bacterium grown on PHBV matrices was shown by further genomic and transcriptomic analysis. In addition to a gene coding for a probable secreted depolymerase, a gene cluster was located that encodes characteristic enzymes involved in the complete depolymerization of PHBV, the transport of oligomers, and in the conversion of the monomers into intermediates of central carbon metabolism. The transcriptomic experiments showed the activation of the glyoxylate shunt during PHBV degradation, setting the isocitrate dehydrogenase activity as regulated branching point of the carbon flow entering the tricarboxylic acid cycle. Our study also shows the potential of exploring the natural plastisphere to discover new bacteria with promising metabolic capabilities.

Antifungal Activity of Hop Leaf Extracts and Xanthohumol on Two Strains of Venturia inaequalis with Different Sensitivities to Triazoles.

Hop cones are well-known for their antimicrobial properties, attributed to their specialized metabolites. Thus, this study aimed to determine the in vitro antifungal activity of different hop parts, including by-products such as leaves and stems, and some metabolites against Venturia inaequalis, the causal agent of apple scab. For each plant part, two types of extracts, a crude hydro-ethanolic extract and a dichloromethane sub-extract, were tested on spore germination of two strains with different sensitivities to triazole fungicides. Both extracts of cones, leaves and stems were able to inhibit the two strains, whereas rhizomes did not show activity. The apolar sub-extract of leaves appeared as the most active modality tested with half maximal inhibitory concentrations (IC50) of 5 and 10.5 mg·L-1 on the sensitive strain and the strain with reduced sensitivity, respectively. Differences in activity level between strains were noticed for all active modalities tested. Sub-extracts of leaves were then separated into seven fractions by preparative HPLC and tested on V. inaequalis. One fraction, containing xanthohumol, was especially active on both strains. This prenylated chalcone was then purified by preparative HPLC and showed significant activity against both strains, with IC50 of 1.6 and 5.1 mg·L-1. Therefore, xanthohumol seems to be a promising compound to control V. inaequalis.

Impacts of microplastics and the associated plastisphere on physiological, biochemical, genetic expression and gut microbiota of the filter-feeder amphioxus.

Oceanic plastic pollution is of major concern to marine organisms, especially filter feeders. However, limited is known about the toxic effects of the weathered microplastics instead of the pristine ones. This study evaluates the effects of weathered polystyrene microplastic on a filter-feeder amphioxus under starvation conditions via its exposure to the microplastics previously deployed in the natural seawater allowing for the development of a mature biofilm (so-called plastisphere). The study focused on the integration of physiological, histological, biochemical, molecular, and microbiota impacts on amphioxus. Overall, specific alterations in gene expression of marker genes were observed to be associated with oxidative stresses and immune systems. Negligible impacts were observed on antioxidant biochemical activities and gut microbiota of amphioxus, while we highlighted the potential transfer of 12 bacterial taxa from the plastisphere to the amphioxus gut microbiota. Moreover, the classical perturbation of body shape detected in control animals under starvation conditions (a slim and curved body) but not for amphioxus exposed to microplastic, indicates that the microorganisms colonizing plastics could serve as a nutrient source for this filter-feeder, commitment with the elevated proportions of goblet cell-like structures after the microplastic exposure. The multidisciplinary approach developed in this study underlined the trait of microplastics that acted as vectors for transporting microorganisms from the plastisphere to amphioxus.

Combined LC-MS/MS and Molecular Networking Approach Reveals Antioxidant and Antimicrobial Compounds from Erismadelphus exsul Bark.

Erismadelphus exsul Mildbr bark is widely used in Gabonese folk medicine. However, little is known about the active compounds associated with its biological activities. In the present study, phytochemical profiling of the ethanolic extract of Erismadelphus exsul was performed using a de-replication strategy by coupling HPLC-ESI-Q/TOF with a molecular network approach. Eight families of natural compounds were putatively identified, including cyclopeptide alkaloids, esterified amino acids, isoflavonoid- and flavonoid-type polyphenols, glycerophospholipids, steroids and their derivatives, and quinoline alkaloids. All these compounds were identified for the first time in this plant. The use of molecular networking obtained a detailed phytochemical overview of this species. Furthermore, antioxidant (2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and ferric reducing capacity (FRAP)) and in vitro antimicrobial activities were assessed. The crude extract, as well as fractions obtained from Erismadelphus exsul, showed a better reactivity to FRAP than DPPH. The fractions were two to four times more antioxidant than ascorbic acid while reacting to FRAP, and there was two to nine times less antioxidant than this reference while reacting to DPPH. In addition, several fractions and the crude extract exhibited a significant anti-oomycete activity towards the Solanaceae phytopathogen Phytophthora infestans in vitro, and, at a lower extent, the antifungal activity against the wheat pathogen Zymoseptoria tritici had growth inhibition rates ranging from 0 to 100%, depending on the tested concentration. This study provides new insights into the phytochemical characterization and the bioactivities of ethanolic extract from Erismadelphus exsul bark.

Microbial Diversity and Activity During the Biodegradation in Seawater of Various Substitutes to Conventional Plastic Cotton Swab Sticks.

The European Parliament recently approved a new law banning single-use plastic items for 2021 such as plastic plates, cutlery, straws, cotton swabs, and balloon sticks. Transition to a bioeconomy involves the substitution of these banned products with biodegradable materials. Several materials such as polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), poly(butylene succinate) (PBS), polyhydroxybutyrate-valerate (PHBV), Bioplast, and Mater-Bi could be good candidates to substitute cotton swabs, but their biodegradability needs to be tested under marine conditions. In this study, we described the microbial life growing on these materials, and we evaluated their biodegradability in seawater, compared with controls made of non-biodegradable polypropylene (PP) or biodegradable cellulose. During the first 40 days in seawater, we detected clear changes in bacterial diversity (Illumina sequencing of 16S rRNA gene) and heterotrophic activity (incorporation of 3H-leucine) that coincided with the classic succession of initial colonization, growth, and maturation phases of a biofilm. Biodegradability of the cotton swab sticks was then tested during another 94 days under strict diet conditions with the different plastics as sole carbon source. The drastic decrease of the bacterial activity on PP, PLA, and PBS suggested no bacterial attack of these materials, whereas the bacterial activity in PBAT, Bioplast, Mater-Bi, and PHBV presented similar responses to the cellulose positive control. Interestingly, the different bacterial diversity trends observed for biodegradable vs. non-biodegradable plastics allowed to describe potential new candidates involved in the degradation of these materials under marine conditions. This better understanding of the bacterial diversity and activity dynamics during the colonization and biodegradation processes contributes to an expanding baseline to understand plastic biodegradation in marine conditions and provide a foundation for further decisions on the replacement of the banned single-used plastics.

Relative Influence of Plastic Debris Size and Shape, Chemical Composition and Phytoplankton-Bacteria Interactions in Driving Seawater Plastisphere Abundance, Diversity and Activity.

The thin film of life that inhabits all plastics in the oceans, so-called "plastisphere," has multiple effects on the fate and impacts of plastic in the marine environment. Here, we aimed to evaluate the relative influence of the plastic size, shape, chemical composition, and environmental changes such as a phytoplankton bloom in shaping the plastisphere abundance, diversity and activity. Polyethylene (PE) and polylactide acid (PLA) together with glass controls in the forms of meso-debris (18 mm diameter) and large-microplastics (LMP; 3 mm diameter), as well as small-microplastics (SMP) of 100 µm diameter with spherical or irregular shapes were immerged in seawater during 2 months. Results of bacterial abundance (confocal microscopy) and diversity (16S rRNA Illumina sequencing) indicated that the three classical biofilm colonization phases (primo-colonization after 3 days; growing phase after 10 days; maturation phase after 30 days) were not influenced by the size and the shape of the materials, even when a diatom bloom (Pseudo-nitzschia sp.) occurred after the first month of incubation. However, plastic size and shape had an effect on bacterial activity (3H leucine incorporation). Bacterial communities associated with the material of 100 µm size fraction showed the highest activity compared to all other material sizes. A mature biofilm developed within 30 days on all material types, with higher bacterial abundance on the plastics compared to glass, and distinct bacterial assemblages were detected on each material type. The diatom bloom event had a great impact on the plastisphere of all materials, resulting in a drastic change in diversity and activity. Our results showed that the plastic size and shape had relatively low influence on the plastisphere abundance, diversity, and activity, as compared to the plastic composition or the presence of a phytoplankton bloom.

Antifungal Activities of Bacillus subtilis Lipopeptides to Two Venturia inaequalis Strains Possessing Different Tebuconazole Sensitivity.

Within the framework of biocontrol development, three natural substances produced by Bacillus subtilis, called lipopeptides, have been studied: fengycin (F), surfactin (S), and mycosubtilin (M). Their antifungal properties were tested in vitro, in liquid medium, on two strains of Venturia inaequalis, ascomycete fungi causing apple scab. These two strains were, respectively sensitive and less sensitive to tebuconazole, an active substance of the triazole family. These three molecules were tested on their own, in binary (FS, FM, SM) and ternary mixtures (FSM). The antifungal activities of lipopeptides were estimated by calculating an IC50, compared to tebuconazole chemical substance. In tests involving the sensitive strain, all lipopeptide modalities exhibited antifungal activity. However, modalities involving fengycin and its mixtures exhibited the best antifungal activities; the activity of fengycin alone being very similar to that of tebuconazole. Interestingly, regarding the strain with reduced sensitivity to tebuconazole, surfactin and fengycin alone were not efficient while mycosubtilin and the different mixtures showed interesting antifungal activities. Specifically, the antifungal activity of FS and FSM mixture were equivalent to that of tebuconazole. For both fungal strains, microscopic observations revealed important morphological modifications in the presence of fengycin and in a less important proportion in the presence of surfactin but not in the presence of mycosubtilin. Overall, this study highlights the diversity in mode of action of lipopeptides on apple scab strains.

Microbial Ecotoxicology of Marine Plastic Debris: A Review on Colonization and Biodegradation by the "Plastisphere".

Over the last decades, it has become clear that plastic pollution presents a global societal and environmental challenge given its increasing presence in the oceans. A growing literature has focused on the microbial life growing on the surfaces of these pollutants called the "plastisphere," but the general concepts of microbial ecotoxicology have only rarely been integrated. Microbial ecotoxicology deals with (i) the impact of pollutants on microbial communities and inversely (ii) how much microbes can influence their biodegradation. The goal of this review is to enlighten the growing literature of the last 15 years on microbial ecotoxicology related to plastic pollution in the oceans. First, we focus on the impact of plastic on marine microbial life and on the various functions it ensures in the ecosystems. In this part, we also discuss the driving factors influencing biofilm development on plastic surfaces and the potential role of plastic debris as vector for dispersal of harmful pathogen species. Second, we give a critical view of the extent to which marine microorganisms can participate in the decomposition of plastic in the oceans and of the relevance of current standard tests for plastic biodegradability at sea. We highlight some examples of metabolic pathways of polymer biodegradation. We conclude with several questions regarding gaps in current knowledge of plastic biodegradation by marine microorganisms and the identification of possible directions for future research.

Phenolic Compounds from Humulus lupulus as Natural Antimicrobial Products: New Weapons in the Fight against Methicillin Resistant Staphylococcus aureus, Leishmania mexicana and Trypanosoma brucei Strains.

New anti-infective agents are urgently needed to fight microbial resistance. Methicillin-resistant Staphylococcus aureus (MRSA) strains are particularly responsible for complicated pathologies that are difficult to treat due to their virulence and the formation of persistent biofilms forming a complex protecting shell. Parasitic infections caused by Trypanosoma brucei and Leishmania mexicana are also of global concern, because of the mortality due to the low number of safe and effective treatments. Female inflorescences of hop produce specialized metabolites known for their antimicrobial effects but underexploited to fight against drug-resistant microorganisms. In this study, we assessed the antimicrobial potential of phenolic compounds against MRSA clinical isolates, T. brucei and L. mexicana. By fractionation process, we purified the major prenylated chalcones and acylphloroglucinols, which were quantified by UHPLC-UV in different plant parts, showing their higher content in the active flowers extract. Their potent antibacterial action (MIC < 1 µg/mL for the most active compound) was demonstrated against MRSA strains, through kill curves, post-antibiotic effects, anti-biofilm assays and synergy studies with antibiotics. An antiparasitic activity was also shown for some purified compounds, particularly on T. brucei (IC50 < 1 to 11 µg/mL). Their cytotoxic activity was assessed both on cancer and non-cancer human cell lines.

Colonization of Non-biodegradable and Biodegradable Plastics by Marine Microorganisms.

Plastics are ubiquitous in the oceans and constitute suitable matrices for bacterial attachment and growth. Understanding biofouling mechanisms is a key issue to assessing the ecological impacts and fate of plastics in marine environment. In this study, we investigated the different steps of plastic colonization of polyolefin-based plastics, on the first one hand, including conventional low-density polyethylene (PE), additivated PE with pro-oxidant (OXO), and artificially aged OXO (AA-OXO); and of a polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), on the other hand. We combined measurements of physical surface properties of polymers (hydrophobicity and roughness) with microbiological characterization of the biofilm (cell counts, taxonomic composition, and heterotrophic activity) using a wide range of techniques, with some of them used for the first time on plastics. Our experimental setup using aquariums with natural circulating seawater during 6 weeks allowed us to characterize the successive phases of primo-colonization, growing, and maturation of the biofilms. We highlighted different trends between polymer types with distinct surface properties and composition, the biodegradable AA-OXO and PHBV presenting higher colonization by active and specific bacteria compared to non-biodegradable polymers (PE and OXO). Succession of bacterial population occurred during the three colonization phases, with hydrocarbonoclastic bacteria being highly abundant on all plastic types. This study brings original data that provide new insights on the colonization of non-biodegradable and biodegradable polymers by marine microorganisms.