TRIAGEM METABÓLICA POR PKS E NRPS EM ACTINOBACTÉRIAS ENDOFÍTICAS DE Citrus reticulata

Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains.

Initial growth of maize in response to application of rock phosphate, vermicompost and endophytic bacteria

Due to the high energy requirement and demand for non-renewable resources for the production of chemical fertilizers, added also to the environmental impact caused by the use of such products, it is important to intensify research on bio-based agricultural inputs. The use of nitrogen-fixing endophytic and phosphate solubilizing bacteria can provide these nutrients to the plants from the air and poorly soluble phosphorus sources, such as phosphate rock. The objective of this study was to evaluate the nutrition and initial growth of maize (Zea mays L.) in response to the inoculation of nitrogen-fixing and rock phosphate solubilizing endophytic bacteria, in single or mixed formulation, applied with vermicompost. The treatments containing bacteria, both diazotrophic and phosphate solubilizing, when compared to controls, showed higher levels of leaf nitrogen and phosphorus in maize, as well as higher growth characteristics. The application of vermicompost showed synergistic effect when combined with endophytic bacteria. Thus, the innovation of the combination of the studied factors may contribute to the early development of maize.

Larvicidal effects of endophytic and basidiomycete fungus extracts on Aedes and Anopheles larvae (Diptera, Culicidae)

Introduction In vitro bioassays were performed to access the larvicidal activity of crude extracts from the endophytic fungus Pestalotiopsis virgulata (Melanconiales, Amphisphaeriaceae) and the saprophytic fungus Pycnoporus sanguineus (Basidiomycetes, Polyporaceae) against the mosquitoes Aedes aegypti and Anopheles nuneztovari. Methods The extracts were tested at concentrations of 100, 200, 300, 400 and 500ppm. Ethyl acetate mycelia (EAM) extracts and liquid culture media (LCM) from Pe. virgulata and Py. sanguineus were tested against third instar larvae of Ae. aegypti and An. nuneztovari. Results The larvicidal activity of the EAM extracts from Pe. virgulata against Ae. aegypti had an LC50=101.8ppm, and the extract from the basidiomycete fungus Py. sanguineus had an LC50=156.8ppm against the Ae. aegypti larvae. The Pe. virgulata extract had an LC50=16.3ppm against the An. nuneztovari larvae, and the Py. sanguineus extract had an LC50=87.2ppm against these larvae. Conclusions These results highlight the larvicidal effect of EAM extracts from the endophyte Pe. virgulata against the two larval mosquitoes tested. Thus, Pe. virgulata and Py. sanguineus have the potential for the production of bioactive substances against larvae of these two tropical disease vectors, with An. nuneztovari being more susceptible to these extracts.

Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides(Melanconiaceae)

ABSTRACTEndophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by 1H and 13C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Detection of siderophores in endophytic bacteria Methylobacterium spp. associated with Xylella fastidiosa subsp. pauca

The objective of this work was to study the production of siderophores by endophytic bacteria Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp. pauca (Xfp) in citrus plants. The siderophore production of Methylobacterium strains was tested according to chromeazurol agar assay test (CAS), Csáky test (hydroxamate-type) and Arnow test (catechol-type). In addition, the ability of Xfp to use siderophores, in vitro, produced by endophytic bacteria as source of iron, was evaluated. All 37 strains of Methylobacterium spp. tested were CAS-positive for siderophore production. Methylobacterium spp. produced hydroxamate-type, but not catechol-type siderophores. In vitro growth of Xfp was stimulated by the presence of supernatant siderophores of endophytic Methylobacterium mesophilicum.

Enantioselective biotransformation of propranolol to the active metabolite 4-hydroxypropranolol by endophytic fungi

The enantioselective biotransformation of propranolol (Prop) by the endophytic fungi Phomopsis sp., Glomerella cingulata, Penicillium crustosum, Chaetomium globosum and Aspergillus fumigatus was investigated by studying the kinetics of the aromatic hydroxylation reaction with the formation of 4-hydroxypropranolol (4-OH-Prop). Both Prop enantiomers were consumed by the fungi in the biotransformation process, but the 4-hydroxylation reaction yielded preferentially (-)-(S)-4-OH-Prop. The quantity of metabolites biosynthesized varied slightly among the evaluated endophytic fungi. These results show that all investigated endophytic fungi could be used as biosynthetic tools in biotransformation processes to obtain the enantiomers of 4-OH-Prop.

An antimicrobial alkaloid and other metabolites produced by Penicillium sp. An endophytic fungus isolated from Mauritia flexuosa L. f.

The alkaloid glandicoline B (1) and six other compounds: ergosterol (2), brassicasterol (3), ergosterol peroxide (4), cerevisterol (5), mannitol (6) and 1-O-α-D-glucopyranoside (7) were isolated from Penicillium sp. strain PBR.2.2.2, a fungus from Mauritia flexuosa roots. The structures of the isolated metabolites were established by spectral analysis. MeOH extract of the fungal mycelium at 500 µg mL-1 exhibited antimicrobial activity against Staphylococcus aureus and the compound 1 at 100 µg mL-1 was active against S. aureus, Micrococcus luteus and Escherichia coli. The relationship between the bioactive properties of the fungus PBR.2.2.2 and those achieved for glandicoline B, as well the potential of this substance as bactericide is discussed.

Metabolic response induced by endophytic fungi and bacteria in H. marrubioides Epling in vitro microplants

Hyptis marrubioides Epling is a native plant from Brazilian Cerrado. In this paper, the response of in vitro microplants of this species to inoculation with bacterial and fungal endophytic isolates is evaluated. HPLC-DAD analysis showed the presence of 3,4-O-(Z)-dicaffeoylquinic acid and quercetin-7-O-glucoside as the main components. GC/MS analysis demonstrated that the sesquiterpenes τ-cadinol and caryophyllene oxide were only produced in microplants inoculated with endophytic bacteria, while methyl hexadecanoate, methyl heptadecanoate and methyl (Z,Z,Z) 9,12,15-octadecatrienoate and the triterpene methyl 3β-hydroxy-urs-12-en-28-oate were overexpressed only when the microplant was treated with endophytic fungi.

Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. In the present study, we have investigated the attachment to and internal colonization of Phaseolus vulgaris roots by the H. seropedicae wild-type strain SMR1 and by a strain of H. seropedicae expressing a red fluorescent protein (DsRed) to track the bacterium in the plant tissues. Two-day-old P. vulgaris roots were incubated at 30°C for 15 min with 6 x 10(8) CFU/mL H. seropedicae SMR1 or RAM4. Three days after inoculation, 4 x 10(4) cells of endophytic H. seropedicae SMR1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 10(6) CFU/g. The identity of the recovered bacteria was confirmed by amplification and sequencing of the 16SrRNA gene. Furthermore, confocal microscopy of P. vulgaris roots inoculated with H. seropedicae RAM4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that H. seropedicae RAM4 is capable of colonizing the roots of the dicotyledon P. vulgaris. Determination of dry weight of common bean inoculated with H. seropedicae SMR1 suggested that this bacterium has a negative effect on the growth of P. vulgaris.

Diversity of 16S rRNA genes from bacteria of sugarcane rhizosphere soil

Sugarcane is an important agricultural product of Brazil, with a total production of more than 500 million tons. Knowledge of the bacterial community associated with agricultural crops and the soil status is a decisive step towards understanding how microorganisms influence crop productivity. However, most studies aim to isolate endophytic or rhizosphere bacteria associated with the plant by culture-dependent approaches. Culture-independent approaches allow a more comprehensive view of entire bacterial communities in the environment. In the present study, we have used this approach to assess the bacterial community in the rhizosphere soil of sugarcane at different times and under different nitrogen fertilization conditions. At the high taxonomic level, few differences between samples were observed, with the phylum Proteobacteria (29.6%) predominating, followed by Acidobacteria (23.4%), Bacteroidetes (12.1%), Firmicutes (10.2%), and Actinobacteria (5.6%). The exception was the Verrucomicrobia phylum whose prevalence in N-fertilized soils was approximately 0.7% and increased to 5.2% in the non-fertilized soil, suggesting that this group may be an indicator of nitrogen availability in soils. However, at low taxonomic levels a higher diversity was found associated with plants receiving nitrogen fertilizer. Bacillus was the most predominant genus, accounting for 19.7% of all genera observed. Classically reported nitrogen-fixing and/or plant growth-promoting bacterial genera, such as Azospirillum, Rhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia were also found although at a lower prevalence.

A phthalide derivative isolated from endophytic fungiPestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniaeisolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation ofp73, JunB, FKHR, andBim. The results indicate that MP may be a potential treatment for cervical cancer.

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert

Egg laying site selection by a host plant specialist leaf miner moth at two intra-plant levels in the northern Chilean Atacama Desert. The spatial distribution of the immature stages of the leaf miner Angelabella tecomae Vargas & Parra, 2005 was determined at two intra-plant levels (shoot and leaflet) on the shrub Tecoma fulva fulva (Cav.) D. Don (Bignoniaceae) in the Azapa valley, northern Chilean Atacama Desert. An aggregated spatial pattern was detected for all the immature stages along the shoot, with an age dependent relative position: eggs and first instar larvae were clumped at apex; second, third and fourth instar larvae were mostly found at intermediate positions; meanwhile the spinning larva and pupa were clumped at basis. This pattern suggests that the females select new, actively growing leaflets for egg laying. At the leaflet level, the immature stages were found more frequently at underside. Furthermore, survivorship was higher for larvae from underside mines. All these results highlight the importance of an accurate selection of egg laying site in the life history of this highly specialized leaf miner. By contrast, eventual wrong choices in the egg laying site selection may be associated with diminished larval survivorship. The importance of the continuous availability of new plant tissue in this highly human modified arid environment is discussed in relation with the observed patterns.

Avaliação de populações de possíveis rizobactérias em solos sob espécies florestais

Embora estudos recentes relatem a utilização de RCPC (Rizobactérias Promotoras de Crescimento de Plantas) no Brasil, raríssimos trabalhos avaliam a presença natural dessas espécies bacterianas no solo. O objetivo deste trabalho foi avaliar a ocorrência de RPCP em duas amostras de solo sob diferentes tipos de manejo, através da construção e do seqüenciamento de bibliotecas de DNA metagenômico. Utilizaram-se oligonucleotídeos específicos para amplificação da região hipervariável do espaço intergênico dos genes ribossomais 16S-23S de DNA extraído de diferentes solos, sob Eucalyptus sp. e sob mata. Os fragmentos obtidos foram inseridos em vetor e clonados. As bibliotecas geraram 495 clones, que foram seqüenciados e identificados através de comparações realizadas pelo software Blast. O solo sob Eucalyptus sp. apresentou maior número de RPCP do que sob mata. Os filos Actinobacteria e Proteobacteria eram maiores no solo sob Eucalyptus sp., estando o filo Firmicutes ausente no solo sob mata. Somente oito espécies diferentes de RPCP foram detectadas: Bacillus subtilis, Bacillus megaterium, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium sp., Frankia sp., Pseudomonas fluorescens e Pseudomonas gladioli. O trabalho forneceu valiosos dados sobre a presença de RPCP em solos com espécies florestais e sua possível utilização em reflorestamentos, assim como para o melhor conhecimento desses microrganismos nos solos do Brasil.