The Use of Synthetic Microbial Communities to Improve Plant Health.

Despite the numerous benefits plants receive from probiotics, maintaining consistent results across applications is still a challenge. Cultivation-independent methods associated with reduced sequencing costs have considerably improved the overall understanding of microbial ecology in the plant environment. As a result, now, it is possible to engineer a consortium of microbes aiming for improved plant health. Such synthetic microbial communities (SynComs) contain carefully chosen microbial species to produce the desired microbiome function. Microbial biofilm formation, production of secondary metabolites, and ability to induce plant resistance are some of the microbial traits to consider when designing SynComs. Plant-associated microbial communities are not assembled randomly. Ecological theories suggest that these communities have a defined phylogenetic organization structured by general community assembly rules. Using machine learning, we can study these rules and target microbial functions that generate desired plant phenotypes. Well-structured assemblages are more likely to lead to a stable SynCom that thrives under environmental stressors as compared with the classical selection of single microbial activities or taxonomy. However, ensuring microbial colonization and long-term plant phenotype stability is still one of the challenges to overcome with SynComs, as the synthetic community may change over time with microbial horizontal gene transfer and retained mutations. Here, we explored the advances made in SynCom research regarding plant health, focusing on bacteria, as they are the most dominant microbial form compared with other members of the microbiome and the most commonly found in SynCom studies.

Improvement of bioactive metabolite production in microbial cultures-A systems approach by OSMAC and deconvolution-based 1 HNMR quantification.

Traditionally, the screening of metabolites in microbial matrices is performed by monocultures. Nonetheless, the absence of biotic and abiotic interactions generally observed in nature still limit the chemical diversity and leads to "poorer" chemical profiles. Nowadays, several methods have been developed to determine the conditions under which cryptic genes are activated, in an attempt to induce these silenced biosynthetic pathways. Among those, the one strain, many compounds (OSMAC) strategy has been applied to enhance metabolic production by a systematic variation of growth parameters. The complexity of the chemical profiles from OSMAC experiments has required increasingly robust and accurate techniques. In this sense, deconvolution-based 1 HNMR quantification have emerged as a promising methodology to decrease complexity and provide a comprehensive perspective for metabolomics studies. Our present work shows an integrated strategy for the increased production and rapid quantification of compounds from microbial sources. Specifically, an OSMAC design of experiments (DoE) was used to optimize the microbial production of bioactive fusaric acid, cytochalasin D and 3-nitropropionic acid, and Global Spectral Deconvolution (GSD)-based 1 HNMR quantification was carried out for their measurement. The results showed that OSMAC increased the production of the metabolites by up to 33% and that GSD was able to extract accurate NMR integrals even in heavily coalescence spectral regions. Moreover, GSD-1 HNMR quantification was reproducible for all species and exhibited validated results that were more selective and accurate than comparative methods. Overall, this strategy up-regulated important metabolites using a reduced number of experiments and provided fast analyte monitor directly in raw extracts.

Dichlorinated and Brominated Rugulovasines, Ergot Alkaloids Produced by Talaromyces wortmannii.

UHPLC-DAD-HRMS based dereplication guided the detection of new halogenated alkaloids co-produced by Talaromyces wortmannii. From the fungal growth in large scale, the epimers 2,8-dichlororugulovasines A and B were purified and further identified by means of a HPLC-SPE/NMR hyphenated system. Brominated rugulovasines were also detected when the microbial incubation medium was supplemented with bromine sources. Studies from 1D/2D NMR and HRMS spectroscopy data allowed the structural elucidation of the dichlorinated compounds, while tandem MS/HRMS data analysis supported the rationalization of brominated congeners. Preliminary genetic studies revealed evidence that FADH2 dependent halogenase can be involved in the biosynthesis of the produced halocompounds.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Assessing the Biocontrol Potential of Clonostachys Species Isolated as Endophytes from Coffea Species and as Mycoparasites of Hemileia Rusts of Coffee in Africa.

During surveys conducted in South America and Africa to identify natural fungal enemies of coffee leaf rust (CLR), Hemileia vastatrix, over 1500 strains were isolated, either as endophytes from healthy tissues of Coffea species or as mycoparasites growing on rust pustules. Based on morphological data, eight isolates-three isolated from wild or semiwild coffee and five from Hemileia species on coffee, all from Africa-were provisionally assigned to the genus Clonostachys. A polyphasic study of their morphological, cultural and molecular characteristics-including the Tef1 (translation elongation factor 1 alpha), RPB1 (largest subunit of RNA polymerase II), TUB (ß-tubulin) and ACL1 (ATP citrate lyase) regions-confirmed these isolates as belonging to three species of the genus Clonostachys: namely C. byssicola, C. rhizophaga and C. rosea f. rosea. Preliminary assays were also conducted to test the potential of the Clonostachys isolates to reduce CLR severity on coffee under greenhouse conditions. Foliar and soil applications indicated that seven of the isolates had a significant effect (p < 0.05) in reducing CLR severity. In parallel, in vitro tests that involved conidia suspensions of each of the isolates together with urediniospores of H. vastatrix resulted in high levels of inhibition of urediniospore germination. All eight isolates showed their ability to establish as endophytes in C. arabica during this study, and some proved to be mycoparasites of H. vastatrix. In addition to reporting the first records of Clonostachys associated with healthy coffee tissues and with Hemileia rusts of coffee, this work provides the first evidence that Clonostachys isolates have potential as biological control agents against CLR.

Exopolysaccharides produced by Bacillus spp. inhibit biofilm formation by Staphylococcus aureus strains associated with bovine mastitis.

Bovine mastitis is a costly disease in the dairy sector worldwide. Here the objective was to identify and characterize anti-biofilm compounds produced by Bacillus spp. against S. aureus associated with bovine mastitis. Results showed that cell-free supernatants of three Bacillus strains (out of 33 analysed) reduced S. aureus biofilm formation by approximately 40 % without affecting bacterial growth. The anti-biofilm activity was associated with exopolysaccharides (EPS) secreted by Bacillus spp. The EPS decreased S. aureus biofilm formation in a dose-dependent manner, inhibiting biofilm formation by 83 % at 1 mg/mL. The EPS also showed some biofilm disruption activity (up to 36.4 %), which may be partially mediated by increased expression of the aur gene. The characterization of EPS produced by Bacillus velezensis 87 and B. velezensis TR47II revealed macromolecules with molecular weights of 31.2 and 33.7 kDa, respectively. These macromolecules were composed mainly of glucose (mean = 218.5 µg/mg) and mannose (mean = 241.5 µg/mg) and had similar functional groups (pyranose ring, beta-type glycosidic linkage, and alkynes) as revealed by FT-IR. In conclusion, this study shows the potential applications of EPS produced by B. velezensis as an anti-biofilm compound that could contribute to the treatment of bovine mastitis caused by S. aureus.

Chemodiversity and Anti-Leukemia Effect of Metabolites from Penicillium setosum CMLD 18.

Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities.

Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation.

Bacteria, yeasts and filamentous fungi were isolated during natural coffee processing. Bacteria were isolated in greater numbers at the beginning of the fermentation, when the moisture of the coffee beans was around 68%. Gram-positive bacteria represented 85.5% of all bacteria isolated, and Bacillus was the predominant genus (51%). Gram-negative species of the genera Serratia, Enterobacter and Acinetobacter were also found. Approximately 22% of 940 randomly chosen isolates of microorganisms were yeasts. Debaryomyces (27%), Pichia (18.9%) and Candida (8.0%) were the most commonly found genera, and these three genera tended to appear more often as the fruit was fermented and dried. Aspergillus was the most abundant genus besides Penicillium, Fusarium and Cladosporium, with 42.6% of the total fungi isolates. The genera and species identified included members known to have pectinase and cellulase activities. Of the 10 organic acids analyzed and quantified in coffee beans, acetic and lactic acids may have been generated by microbial activity. Butyric acid was not detected in any sample.

Uncovering secondary metabolite evolution and biosynthesis using gene cluster networks and genetic dereplication.

The increased interest in secondary metabolites (SMs) has driven a number of genome sequencing projects to elucidate their biosynthetic pathways. As a result, studies revealed that the number of secondary metabolite gene clusters (SMGCs) greatly outnumbers detected compounds, challenging current methods to dereplicate and categorize this amount of gene clusters on a larger scale. Here, we present an automated workflow for the genetic dereplication and analysis of secondary metabolism genes in fungi. Focusing on the secondary metabolite rich genus Aspergillus, we categorize SMGCs across genomes into SMGC families using network analysis. Our method elucidates the diversity and dynamics of secondary metabolism in section Nigri, showing that SMGC diversity within the section has the same magnitude as within the genus. Using our genome analysis we were able to predict the gene cluster responsible for biosynthesis of malformin, a potentiator of anti-cancer drugs, in 18 strains. To proof the general validity of our predictions, we developed genetic engineering tools in Aspergillus brasiliensis and subsequently verified the genes for biosynthesis of malformin.

Aromatic compounds produced by Periconia atropurpurea, an endophytic fungus associated with Xylopia aromatica.

6,8-Dimethoxy-3-(2'-oxo-propyl)-coumarin (1) and 2,4-dihydroxy-6-[(1'E,3'E)-penta-1',3'-dienyl]-benzaldehyde (2), in addition to the known compound periconicin B (3), were isolated from the ethyl acetate extract of Periconia atropurpurea, an endophytic fungus obtained from the leaves of Xylopia aromatica, a native plant of the Brazilian Cerrado. Their chemical structures were assigned based on analyses of MS, 1D and 2D-NMR spectroscopic experiments. Biological analyses were performed using two mammalian cell lines, human cervix carcinoma (HeLa) and Chinese hamster ovary (CHO). The results showed that compound 1 had no effect when compared to the control group, which was treated with the vehicle (DMSO). Compound 2 was able to induce a slight increase in cell proliferation of HeLa (37% of increase) and CHO (38% of increase) cell lines. Analysis of compound 3 showed that it has potent cytotoxic activity against both cell lines, with an IC50 of 8.0 microM. Biological analyses using the phytopathogenic fungi Cladosporium sphaerospermum and C. cladosporioides revealed that also 2 showed potent antifungal activity compared to nystatin.