Biosurfactant production of Piper hispidum endophytic fungi.

AIMS: To evaluate the production of biosurfactants by fungi isolated from the Amazonian species Piper hispidum (Piperaceae), and to determine the physico-chemical properties of the crude biosurfactant obtained from the most promising fungi. METHODS AND RESULTS: A total of 117 endophytic fungi were isolated, and 50 were used to verify the production of biosurfactants. Of these, eight presented positive results in the drop collapse test, and emulsification index ranging from 20 to 78%. The most promising fungi, Ph III 23L and Ph II 22S (identified as Aspergillus niger and Glomerella cingulata, respectively) were recultivated for extraction and analysis of the biosurfactant's physico-chemical characteristics. The cultivation broth that presented the greatest decrease in surface tension (36%) was that of the A. niger, which reduced it from 68·0 to 44·0 mN m-1 . The lowest critical micellar concentration value was found for the same endophyte (14·93 mg ml-1 ). CONCLUSIONS: Endophytes of P. hispidum proved to be interesting producers of biosurfactants and presented promising physico-chemical characteristics for applications in diverse industrial sectors. SIGNIFICANCE AND IMPACT OF THE STUDY: Piper hispidum endophytic fungi can be used as a new source of biosurfactants, as these molecules present a significant market due to their wide industrial applications.

Phytobiotic Activity of Piper Auritum and Ocimum Basilicum on Avian E. Coli

Natural antimicrobials, known as phytobiotics, are used in bacterial infections. The objective of this study was to evaluatethe phytobiotic activity, in vitro and in vivo, of an extract and an essential oil of Piper auritum and Ocimum basilicum on avian Eschericia coli serotype O2 in broiler chickens experimentally infected. For the in vitro test, extracts at 4, 8, 12 and 16% in water-based solvent or alcohol at 70% were prepared from leaves of both plants. In the essential oils, solvents at 10% were used. A concentration of 1×108 CFU mL-1 of bacteria was seeded and on each sense disc, 25 µL of the extract or essential oil were poured, except on the positive or negative control. The diameter of the inhibition zone (DIZ) of bacterial growth was measured. In the in vivo test, 40 chickens were inoculated, by intratracheal route, with a bacterial suspension of 1 × 108 CFU mL-1. E. col was identified and isolated from the organs; mortality, morbidity and relative weight of the organs were measured, and postmortem lesions and histopathologic findings were observed. A completely randomized design and the Kruskal-Wallis test for data analysis was used. By increasing the concentration of the extract, DIZ was greater; at the time of slaughter, differences in body weight (p 0.05) were found and the majority of lesions were observed in lungs. It is concluded that leave extracts of P. auritum and O. basilicum had phytobiotic activity on E. col serotype O2.(AU)

Phytobiotic Activity of Piper Auritum and Ocimum Basilicum on Avian E. Coli

Natural antimicrobials, known as phytobiotics, are used in bacterial infections. The objective of this study was to evaluatethe phytobiotic activity, in vitro and in vivo, of an extract and an essential oil of Piper auritum and Ocimum basilicum on avian Eschericia coli serotype O2 in broiler chickens experimentally infected. For the in vitro test, extracts at 4, 8, 12 and 16% in water-based solvent or alcohol at 70% were prepared from leaves of both plants. In the essential oils, solvents at 10% were used. A concentration of 1×108 CFU mL-1 of bacteria was seeded and on each sense disc, 25 µL of the extract or essential oil were poured, except on the positive or negative control. The diameter of the inhibition zone (DIZ) of bacterial growth was measured. In the in vivo test, 40 chickens were inoculated, by intratracheal route, with a bacterial suspension of 1 × 108 CFU mL-1. E. col was identified and isolated from the organs; mortality, morbidity and relative weight of the organs were measured, and postmortem lesions and histopathologic findings were observed. A completely randomized design and the Kruskal-Wallis test for data analysis was used. By increasing the concentration of the extract, DIZ was greater; at the time of slaughter, differences in body weight (p 0.05) were found and the majority of lesions were observed in lungs. It is concluded that leave extracts of P. auritum and O. basilicum had phytobiotic activity on E. col serotype O2.

Phylogenomics of 2,4-Diacetylphloroglucinol-Producing Pseudomonas and Novel Antiglycation Endophytes from Piper auritum.

2,4-Diacetylphloroglucinol (DAPG) (1) is a phenolic polyketide produced by some plant-associated Pseudomonas species, with many biological activities and ecological functions. Here, we aimed at reconstructing the natural history of DAPG using phylogenomics focused at its biosynthetic gene cluster or phl genes. In addition to around 1500 publically available genomes, we obtained and analyzed the sequences of nine novel Pseudomonas endophytes isolated from the antidiabetic medicinal plant Piper auritum. We found that 29 organisms belonging to six Pseudomonas species contain the phl genes at different frequencies depending on the species. The evolution of the phl genes was then reconstructed, leading to at least two clades postulated to correlate with the known chemical diversity surrounding DAPG biosynthesis. Moreover, two of the newly obtained Pseudomonas endophytes with high antiglycation activity were shown to exert their inhibitory activity against the formation of advanced glycation end-products via DAPG and related congeners. Its isomer, 5-hydroxyferulic acid (2), detected during bioactivity-guided fractionation, together with other DAPG congeners, were found to enhance the detected inhibitory activity. This report provides evidence of a link between the evolution and chemical diversity of DAPG and congeners.

ß-(1→3,1→6)-d-glucans produced by Diaporthe sp. endophytes: Purification, chemical characterization and antiproliferative activity against MCF-7 and HepG2-C3A cells.

This study reports the characterization and antiproliferative activity of exopolysaccharides (EPS) produced by submerged cultures of the endophytes Diaporthe sp. JF766998 and Diaporthe sp. JF767007 isolated from the medicinal plant Piper hispidum Sw. Both strains secreted a crude EPS that, upon size exclusion chromatography, showed to contain a heteropolysaccharide (galactose, glucose and mannose) and a high-molecular weight glucan. Data from methylation analysis, FTIR and NMR spectroscopy (1H, COSY, TOCSY and HSQC-DEPT) indicated that the purified glucan consisted of a main chain of glucopyranosyl ß-(1→3) linkages substituted at O-6 by glucosyl residues. According to MTT assay, some treatments of both ß-glucans have antiproliferative activity against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells after 24 and 48h of treatment, exhibiting a degree of inhibition ratio that reached the highest values at 400µg/mL: 58.0% (24h) and 74.6% (48h) for MCF-7 cells, and 61.0% (24h) and 83.3% (48h) for HepG2-C3A cells. These results represent the first reports on the characterization and antiproliferative effect of ß-glucans from Diaporthe species and also expand the knowledge about bioactive polysaccharides from endophytic sources.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw.

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Endophytic bacteria from Piper tuberculatum Jacq.: isolation, molecular characterization, and in vitro screening for the control of Fusarium solani f. sp piperis, the causal agent of root rot disease in black pepper (Piper nigrum L.).

Endophytic bacteria have been found to colonize internal tissues in many different plants, where they can have several beneficial effects, including defense against pathogens. In this study, we aimed to identify endophytic bacteria associated with roots of the tropical piperaceae Piper tuberculatum, which is known for its resistance to infection by Fusarium solani f. sp piperis, the causal agent of black pepper (Piper nigrum) root rot disease in the Amazon region. Based on 16S rRNA gene sequence analysis, we isolated endophytes belonging to 13 genera: Bacillus, Paenibacillus, Pseudomonas, Enterobacter, Rhizobium, Sinorhizobium, Agrobacterium, Ralstonia, Serratia, Cupriavidus, Mitsuaria, Pantoea, and Staphylococcus. The results showed that 56.52% of isolates were associated with the phylum Proteobacteria, which comprised α, ß, and γ classes. Other bacteria were related to the phylum Firmicutes, including Bacillus, which was the most abundant genus among all isolates. Antagonistic assays revealed that Pt12 and Pt13 isolates, identified as Pseudomonas putida and Pseudomonas sp, respectively, were able to inhibit F. solani f. sp piperis growth in vitro. We describe, for the first time, the molecular identification of 23 endophytic bacteria from P. tuberculatum, among which two Pseudomonas species have the potential to control the pathogen responsible for root rot disease in black pepper in the Amazon region.

Flavonoids from Piper delineatum modulate quorum-sensing-regulated phenotypes in Vibrio harveyi.

Quorum sensing (QS), or bacterial cell-to-cell communication, is a key process for bacterial colonization of substrata through biofilm formation, infections, and production of virulence factors. In an ongoing investigation of bioactive secondary metabolites from Piper species, four new flavonoids (1-4), along with five known ones (5-9) were isolated from the leaves of Piper delineatum. Their stereostructures were established by spectroscopic and spectrometric methods, including 1D and 2D NMR experiments, and comparison with data reported in the literature. The compounds were screened for their ability to interfere with QS signaling in the bacterial model Vibrio harveyi. Four compounds from this series (2, 3, 6, and 7) exhibited remarkable activity in the micromolar range, being compounds 3 and 7 particularly attractive since they did not affect bacterial growth. The results suggest that these flavonoids disrupt QS-mediated bioluminescence by interaction with elements downstream LuxO in the QS circuit of V. harveyi, and also, they exhibited a strong dose-dependent inhibition of biofilm formation. The present findings shed light on the QS inhibition mechanisms of flavonoids, underlining their potential applications.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.(AU)