First report of Colletotrichum nymphaeae causing anthracnose on Hass avocados in Brazil.

The Hass cultivar is one of the most cultivated and distributed avocado varieties throughout the world, having high productivity, nutritional quality, market acceptance and adaptation to different climatic zones (Dreher and Davenport 2013). Anthracnose affects avocado production in tropical and subtropical regions, causing economic losses, especially post-harvest (Fuentes-Aragón et al. 2020). Correct identification of Colletotrichum species causing anthracnose is essential, as different species vary in important phenotypes such as virulence and sensitivity to fungicides (Chen et al. 2016). Twelve samples from batches of avocados with anthracnose were collected in Minas Gerais State, Brazil, in September 2023. The observed symptoms were brown to black depressed circular spots, ranging from a few millimeters to 3 cm in diameter on the epicarp of the fruits, covered in center by mucilaginous layers of pathogen sporulation. Isolation was performed directly from the spore masses and monoconidial isolates were cultured in PDA at 25°C for 7 days for morphological characterization and preserved in sterile water at 4°C. One of the morphotypes commonly recovered from lesions, represented by isolate UCBV 362 (Culture Collection COAD 3843), formed fast-growing colonies having white aerial mycelium and intense salmon-colored sporulation. The cylindrical conidia were 13 to 17.5 µm long and 4.5 to 7 µm wide (average 14.5 x 5.7 µm, N=100), produced on conidiophores dispersed in the aerial mycelium or aggregated on melanized conidiomata formed on the agar. The partial sequence of the second largest subunit of the RNA polymerase II gene (RPB2) from isolate UCBV 362 (GenBank: PQ034617, 1116 nt) showed 99% of coverage and 99.37% of nucleotide identity with the RPB2 sequence of the ex-epitype strain of Colletotrichum nymphaeae ICMP 17918 (=CBS 515.78) (GenBank: JN985506). In a Maximum Likelihood phylogenetic tree composed with RBP2 sequences from reference strains of the Colletotrichum acutatum species complex, the isolate UCBV 362 formed a highly supported clade with the ex-epitype and other reference strains of Colletotrichum nymphaeae, occupying the Clade 2 of the species complex together with C. scovillae and C. simmondsii (Damm et al. 2012). This result shows the reliability of RPB2 for phylogeny and species delimitation within Colletotrichum. To confirm pathogenicity, 10-mm discs from a 7-day-old colony were inoculated at 3 different points on healthy-looking avocado fruits and incubated at 28°C. Uninoculated fruits served as controls. The first symptoms appeared 5 days after inoculation and were similar to those observed in the original samples, while the fruits in the control group remained asymptomatic. The pathogen was reisolated from the lesions and identified morphologically, fulfilling Koch's postulates. Colletotrichum nymphaeae has been associated with avocado anthracnose in Mexico (Fuentes-Aragón et al. 2020). In Brazil, a study based on molecular phylogeny identified Colletotrichum siamense and C. karsti as etiological agents of this disease (Soares et al. 2021). This report expands the range of species that cause avocado anthracnose in Brazil and provides etiological information for the implementation and monitoring of control methods, especially chemical control.

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.

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.

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.

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.

Endophytic fungi from Brachiaria grasses in Brazil and preliminary screening of Sclerotinia sclerotiorum antagonists

Fungal endophytes of Brachiaria, a nonhost of Sclerotinia sclerotiorum, may harbor species with antagonistic effects against this plant pathogen. The objective of this work was to investigate the diversity of endophytic fungi associated with different Brachiaria species and hybrids and evaluate their potential to inhibit the plant pathogen S. sclerotiorum. Stem samples from 39 Brachiaria spp. plants were collected in pasture fields and experimental areas of three states of Brazil resulting in 74 endophytes isolated. Twenty-eight species were identified by sequences of the Internal Transcribed Spacer (ITS) and 18S rDNA regions. Paraconiothyrium sp. was the most abundant endophyte, accounting for 24 % (14 isolates) of total, and it was isolated from B. ruziziensis, B. decumbens, B. humidicola, and B. brizantha. Phoma sorghina was the second most abundant taxon, followed by Sarocladium strictum, and Plenodomus sp. In vitro analyses showed that Paraconiothyrium sp., Sarocladium kiliense, Acremonium curvulum, Setophoma terrestris, Dissoconium sp., and Cladosporium flabelliforme exhibited antagonistic activity against S. sclerotiorum, with percentages of growth inhibition ranging from 25 to 60 (p < 0.05). Paraconiothyrium sp. BBXE1 (60 %), BBPB4.1 (60 %), BCMT4.1 (54 %), and S. kiliense (54 %) showed the highest values of Antagonism Percentages (AP). Therefore, fungi with inhibitory activity against S. sclerotiorum such as Paraconiothyrium sp. are naturally endophytic in Brachiaria grasses.

Endophytic fungi from Brachiaria grasses in Brazil and preliminary screening of Sclerotinia sclerotiorum antagonists

Fungal endophytes of Brachiaria, a nonhost of Sclerotinia sclerotiorum, may harbor species with antagonistic effects against this plant pathogen. The objective of this work was to investigate the diversity of endophytic fungi associated with different Brachiaria species and hybrids and evaluate their potential to inhibit the plant pathogen S. sclerotiorum. Stem samples from 39 Brachiaria spp. plants were collected in pasture fields and experimental areas of three states of Brazil resulting in 74 endophytes isolated. Twenty-eight species were identified by sequences of the Internal Transcribed Spacer (ITS) and 18S rDNA regions. Paraconiothyrium sp. was the most abundant endophyte, accounting for 24 % (14 isolates) of total, and it was isolated from B. ruziziensis, B. decumbens, B. humidicola, and B. brizantha. Phoma sorghina was the second most abundant taxon, followed by Sarocladium strictum, and Plenodomus sp. In vitro analyses showed that Paraconiothyrium sp., Sarocladium kiliense, Acremonium curvulum, Setophoma terrestris, Dissoconium sp., and Cladosporium flabelliforme exhibited antagonistic activity against S. sclerotiorum, with percentages of growth inhibition ranging from 25 to 60 (p < 0.05). Paraconiothyrium sp. BBXE1 (60 %), BBPB4.1 (60 %), BCMT4.1 (54 %), and S. kiliense (54 %) showed the highest values of Antagonism Percentages (AP). Therefore, fungi with inhibitory activity against S. sclerotiorum such as Paraconiothyrium sp. are naturally endophytic in Brachiaria grasses.(AU)

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.

Fungal endophytes of Panicum maximum and Pennisetum purpureum: isolation, identification, and determination of antifungal potential

This study aimed to isolate and identify endophytic fungi from the forage grass P. maximum and evaluate their ability to inhibit the growth of plant pathogenic fungi. One sample from P. purpureum grass was also included. Surface disinfected stem fragments were used for endophytic fungal isolation. One hundred and twenty-six endophytic fungi were isolated, of which 118 were from P. maximum and eight from P. purpureum. Morphological characteristics and internal transcribed spacer (ITS) and 18S (NS) sequence comparisons identified most isolated endophytic fungi as belonging to the phylum Ascomycota, with Sarocladium being the dominant genus. The isolates were subjected to in vitro antagonism tests against pathogenic fungi, and 31 endophytic fungi inhibited the growth of Bipolaris maydis, Penicillium expansum, and Sclerotinia minor. The results expand our knowledge of the diversity of endophytes associated with tropical grasses and suggest that they may represent new sources of antifungal metabolites for biocontrol and biotechnological purposes.(AU)

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.

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.