Phosphate-solubilizing fungi co-inoculated with Bradyrhizobium promote cowpea growth under varying N and P fertilization conditions

We evaluated the compatibility between two nitrogen-fixing Bradyrhizobium inoculant strains and phosphate-solubilizing fungal strains and the effect of co-inoculation of these bacterial and fungal strains on cowpea growth under different N and P conditions. First, the compatibility between Bradyrhizobium strains UFLA03-84 and INPA03-11B and fungi Haematonectria ipomoeae FSA381, Eleutherascus lectardii FSA257a, Pochonia chlamydosporia var. catenulata FSA109, and Acremonium polychromum FSA115 was tested in both solid and liquid media. Cowpea growth and nodulation promotion under two mineral N doses and two P conditions (a low dose of soluble P plus a high dose of Ca3(PO4)2 and another condition with a high dose of soluble P) were tested with two N2 fixing Bradyrhizobium strains co-inoculated with each of the P-solubilizing fungal strains FSA109, FSA115, and FSA381. There was compatibility between each fungal strain and the two Bradyrhizobium strains, except for FSA257a with either of the bacterial strains in liquid medium. When both mineral N and P were limiting, plants were able to grow and accumulate N and P based on biological N2 fixation and solubilization of calcium phosphate in the same amount as the mineral N and soluble phosphate. Even when both nutrients were fully available, the type of co-inoculation promoted plant growth and nutrient accumulation. The responses varied in accordance with the co-inoculated strains, the N source, and the P source, reflecting the enormous complexity of the biological interactions between plants and microorganisms, and the nutrient conditions provided by the environment.

Antifungal compounds produced by Colletotrichum gloeosporioides, an endophytic fungus from Michelia champaca.

In this study, eight endophytic fungi were isolated from the leaves, stems and roots of Michelia champaca. The isolates were screened and evaluated for their antifungal, anticancer and acetylcholinesterase (AChE) inhibitory activities. All of the extracts exhibited potent activity against two evaluated phytopathogenic fungi. Chemical investigation of EtOAc extracts of the endophytic fungus Colletotrichum gloeosporioides resulted in the isolation of one new compound, 2-phenylethyl 1H-indol-3-yl-acetate (1), and seven known compounds: uracil (2), cyclo-(S*-Pro-S*-Tyr) (3), cyclo-(S*-Pro-S*-Val) (4), 2(2-aminophenyl)acetic acid (5), 2(4-hydroxyphenyl)acetic acid (6), 4-hydroxy- benzamide (7) and 2(2-hydroxyphenyl)acetic acid (8). All of the compound structures were elucidated using 1D and 2D NMR and MS analyses. The antifungal and AChE inhibitory activities of compounds 1-8 were evaluated in vitro. Compound 1 exhibited promising activity against Cladosporium cladosporioides and C. sphaerospermum that was comparable to that of the positive control nystatin.

Isolation, identification and antimicrobial activity of propolis-associated fungi.

Propolis is a natural product widely known for its medicinal properties. In this work, fungi present on propolis samples were isolated, identified and tested for the production of antimicrobial metabolites. Twenty-two fungal isolates were obtained, some of which were identified as Alternaria alternata, Aspergillus flavus, Bipolaris hawaiiensis, Fusarium merismoides, Lasiodiplodia theobromae, Penicillium citrinum, Penicillium crustosum, Penicillium janthinellum, Penicillium purpurogenum, Pestalotiopsis palustris, Tetracoccosporium paxianum and Trichoderma koningii. These fungi were grown in liquid media to obtain crude extracts that were evaluated for their antibiotic activity against pathogenic bacteria, yeast and Cladosporium cladosporioides and A. flavus. The most active extract was obtained from L. theobromae (minimum inhibitory concentration = 64 µg/mL against Listeria monocitogenes). Some extracts showed to be more active than the positive control in the inhibition of Staphylococcus aureus and L. monocitogenes. Therefore, propolis is a promising source of fungi, which produces active agents against relevant food poisoning bacteria and crop-associated fungi.

Hydroxylation at carbon-2 of ent-16-oxo-17-norkauran-19-oic acid by Fusarium proliferatum.

A new product of biotransformation of ent-16-oxo-17-norkauran-19-oic acid (1) by Fusarium proliferatum was isolated and identified as a 2beta-hydroxy derivative (2). The structure of 2 was elucidated on the basis of spectroscopic data interpretation and single-crystal X-ray diffraction analysis. The allelopathic activity of compound 2 was evaluated on the growth of radicals and shoots of Lactuca sativa (lettuce). This is the first time that fungal hydroxylation at position C-2 has been reported on an ent-kaurane diterpene skeleton.

Biological activities of the fermentation extract of the endophytic fungus alternaria alternata isolated from coffea arabica L / Atividades biológicas do extrato da fermentação do fungo endofítico alternaria alternata isoladas de coffea arabica L

A total of 22 endophytic fungi isolated from coffee (Coffea arabica L.) were cultivated in vitro and their crude extracts tested. The screening was carried out using the agar diffusion method against Staphylococcus aureus, Escherichia coli and Candida albicans. The most effective isolate was Alternaria alternata, and subsequently, its extract was assayed. The total phenolic content was 3.44 μg GAE/mg of the crude extract. For the antibacterial and antifungal activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal and fungicidal concentrations (MBC and MFC) were determined. The ranges of MIC values were 50-100 μg/mL for S. aureus and 400-800 μg/mL for E. coli. The extract did not show activity in the tested concentrations for C. albicans. The fungal crude extract was assayed for antioxidant activities. Its ability to scavenge DPPH radicals and antioxidant activity by β-carotene/linoleic acid system oxidation was not significant. In addition, antitumor activity was studied using the MTT assay. At a dilution of 400 μg/mL, the extract displayed a cytotoxic activity of approximately 50 percent towards HeLa cells in vitro. The results indicate that endophytic fungi could be a promising source of bioactive compounds and warrant further study.

Total de 22 fungos endofíticos isolados de café (Coffea arabica L.) foi cultivado in vitro e seus extratos testados. A triagem foi conduzida pelo método de difusão em agar contra bactérias Gram-positiva, Gram-negativa e uma levedura. O isolado mais efetivo foi Alternaria alternata e, subsequentemente, seu extrato foi analisado. O conteúdo de fenólicos totais do extrato bruto foi de 3,44 μg EAG/mg de extrato. Para os testes de atividade antimicrobiana, a concentração inibitória mínima (CIM) e concentração bactericida e fungicida mínima (CBM e CFM) contra Staphylococcus aureus, Escherichia coli e Candida albicans foram determinadas. Resultados da CIM variaram entre 50-100 μg/mL para S. aureus e 400-800 μg/mL para E. coli. O extrato bruto não apresentou atividade nas concentrações testadas para C. albicans. Foram analisadas as atividades antioxidantes do extrato bruto. Sua habilidade para seqüestrar radicais DPPH e a atividade antioxidante pela oxidação do sistema β-caroteno/ácido linoléico não foram significativas. Além disso, a atividade antitumoral foi estudada pelo teste do MTT. À diluição de 400 μg/mL, o extrato apresentou atividade de aproximadamente 50 por cento sobre as células HeLa in vitro. Os resultados indicam que fungos endófitos poderiam ser uma fonte promissora de compostos bioativos necessitando de estudos futuros.

Obtenção de protoplastos do fungo filamentoso Aspergillus ochraceus / Production of protoplasts from the filamentous fungus Aspergillus ochraceus

A obtenção de protoplastos é uma ferramenta importante para a transformação genética de fungos. Neste trabalho foi estudada a influência de fatores como idade micelial, tipo e concentração de enzimas e estabilizadores osmóticos na produção de protoplastos de Aspergillus ochraceus. Os melhores resultados de produção de protoplastos foram obtidos utilizando-se NH4Cl 0,8mol L-1 como estabilizador osmótico, micélio com 24h de crescimento e a combinação de Lysing Enzymes e Meicelase ambas, a 20mg mL-1. Entretanto, bons resultados foram também obtidos com a utilização apenas de Lysing Enzymes.

Production of protoplasts is an important tool for genetic transformation of fungi. A protocol for protoplasts production in Aspergillus ochraceus was developed, evaluating culture aging of mycelium, different commercial enzymes and osmotic stabilizers. The best results were obtained with NH4Cl 0.8mol L-1 as osmotic stabilizer, mycelial age of 24 hours and Lysing Enzymes (20mg mL-1) plus Meicelase (20mg mL-1) as lytic enzymes. Good results were also obtained with Lysing Enzymes alone.

Obtenção de protoplastos do fungo filamentoso Aspergillus ochraceus / Production of protoplasts from the filamentous fungus Aspergillus ochraceus

A obtenção de protoplastos é uma ferramenta importante para a transformação genética de fungos. Neste trabalho foi estudada a influência de fatores como idade micelial, tipo e concentração de enzimas e estabilizadores osmóticos na produção de protoplastos de Aspergillus ochraceus. Os melhores resultados de produção de protoplastos foram obtidos utilizando-se NH4Cl 0,8mol L-1 como estabilizador osmótico, micélio com 24h de crescimento e a combinação de Lysing Enzymes e Meicelase ambas, a 20mg mL-1. Entretanto, bons resultados foram também obtidos com a utilização apenas de Lysing Enzymes.(AU)

Production of protoplasts is an important tool for genetic transformation of fungi. A protocol for protoplasts production in Aspergillus ochraceus was developed, evaluating culture aging of mycelium, different commercial enzymes and osmotic stabilizers. The best results were obtained with NH4Cl 0.8mol L-1 as osmotic stabilizer, mycelial age of 24 hours and Lysing Enzymes (20mg mL-1) plus Meicelase (20mg mL-1) as lytic enzymes. Good results were also obtained with Lysing Enzymes alone.(AU)

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.