Antimicrobial and antioxidant activities of secondary metabolites from endophytic fungus Botryosphaeria fabicerciana (MGN23-3) associated to Morus nigra L.

This study was to evaluate the biological activity of the extract of Botryosphaeria fabicerciana isolated from leaves of Morus nigra. The volatile compounds from the crude extract were analysed by GC-MS which demonstrate that mellein and ß-orcinaldehyde were are the major compounds. The best minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract was observed against Gram-positive bacteria, with a MIC of 15.6 µg/mL towards B. cereus and MIC of 62.5 µg/mL towards S. aureus and B. subtilis. MBC values of 31.25 µg/mL, 62.5 µg/mL, and 250 µg/mL were observed towards B. cereus, B. subtilis, and S. aureus, respectively. The cytotoxicity analyses showed CC50 of 115 µg/mL. The crude extract showed antioxidant activity by the DPPH, ABTS, and FRAP assays. Therefore, the extract of the endophytic fungus presented biotechnological potential as an antibacterial and antioxidant agent.

Assessment of Qigong Effects on Anxiety of High-school Students: A Randomized Controlled Trial.

CONTEXT: Students are vulnerable to developing anxiety, a psychiatric disorder closely related to emotional stress, when systematically stressed by classes, homework, and evaluations. Qigong integrates physical, respiratory, and mental exercises, inducing vegetative biofeedback with significant effects on physiological regulation. OBJECTIVE: The current study aimed to assess the potential effects of specific Qigong exercises on students' anxiety levels and evaluate the feasibility of practical integration in a daily school context. DESIGN: The research team developed a randomized controlled trial. SETTING: This study was performed in Cedros and Horizonte private schools located in Vila Nova de Gaia in Portugal. PARTICIPANTS: Participants were 104 high-school students at the schools. INTERVENTION: Participants were randomly divided into three groups: (1) an intervention group, the Qigong (QG) group (n = 34), which performed Qigong exercises; (2) a control group, the TV documentary (TVD) group (n = 34), which watched a TV documentary; and (3) a second control group, the typical school duties (TSD) group (n = 36), which performed regular school duties. OUTCOME MEASURES: Anxiety levels were assessed through a psychological test, the State-Trait Anxiety Inventory (STAI) and salivary cortisol tests. RESULTS: Psychological and biochemical variables assessed at baseline and postintervention showed a greater decrease in anxiety levels in the QG group than in the other two groups. CONCLUSIONS: Qigong seems to be an efficient tool to reduce anxiety and control the stress of high-school students.

Secondary metabolites of Curvularia sp. G6-32, an endophyte of Sapindus saponaria, with antioxidant and anticholinesterasic properties.

In the present study, the biological activity of an extract of the secondary metabolites (E-G6-32) produced by the Curvularia sp. G6-32 endophyte (isolated from the medicinal plant Sapindus saponaria L.) was investigated. The antioxidant potential was confirmed by the DPPH (22.5%) and ABTS (62.7%) assays, and the total phenolic compound content was 40 µg gallic acid equivalents/mg. The extract E-G6-32 displayed good inhibitory activity toward butyrylcholinesterase (BuChE; IC50 = 110 ± 0.05 µg mL-1). The extract E-G6-32 was subjected to spectroscopic and mass spectrometry analyses. Comparison with the literature data confirmed that (-)-asperpentyn (1) was a major component. Asperpentyn belongs to the epoxyquinone family, which has attractive structural complexity, diverse functional groups, and a broad range of biological activities, including specific enzyme inhibitory activity. Our results suggest that Curvularia sp. G6-32 is a promising source of bioactive secondary metabolites and contains (-)-asperpentyn, which has potential pharmaceutical interest.[Figure: see text].

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

Colletotrichum siamense, a Mycovirus-Carrying Endophyte, as a Biological Control Strategy for Anthracnose in Guarana Plants

Abstract Guarana, the fruit of Paullinia cupana, is known for its stimulating and medicinal properties by the Amazonian indigenous population and communities. However, it presents serious phytopathological problems, such as anthracnose disease caused by Colletotrichum spp. The objective of this study was to verify if C. siamense, a mycovirus-carrying endophytic fungus, could protect guarana seedlings, by reducing or by eliminating characteristic disease symptoms. Other physiological changes in the plant caused by the presence of this endophyte were also evaluated. The cuttings of the Cereçaporanga cultivar were dipped in a biological control suspension and planted in a specific substrate. After four months in the greenhouse, the seedlings were sprayed with a suspension of phytopathogen conidia, and a portion of these seedlings received the fungicide indicated for the crop to be compared with the control seedlings. After 28 days, the number of lesions, morphophysiological and macro characteristics, and leaf micronutrients were evaluated. The seedlings treated with C. siamense showed a lower percentage of lesions and an increased aerial part and root system compared to the other treatments. There were no significant differences between treatments regarding the percentage of macronutrients and micronutrients.

Rickets in white-eared opossums (Didelphis albiventris) / Raquitismo em gambás-de-orelha-branca (Didelphis albiventris)

Two young opossums were necropsied and diagnosed with rickets. This study aims to describe the clinical-pathological aspects of rickets in Didelphis albiventris. Macroscopically, the opossums presented kyphosis and scoliosis, lateral deviation of the limbs in varus, locomotion difficulty, and enlargement with softening of costochondral junctions (rickety rosary). Samples of bones and joints were processed for hematoxylin and eosin staining and Masson's trichrome. Microscopically, we observed thickening of the epiphyseal plate, characterized by irregular and multifocal proliferation of serialized and hypertrophic cartilage zones, which formed circular groups of large, dysplastic chondrocytes towards the spongy zone, often surrounded by non-mineralized osteoid tissue. In the cortical bone, there were pale eosinophilic zones around the Havers channels consistent with non-mineralized osteoid. The staining of Masson's trichrome evidenced the accumulation of osteoid tissue in cortical and trabecular bones. It is possible that a mixed cause of absorption deficiency of vitamin D3 associated with an unbalanced Ca:P diet based on lactose-free milk and fruits may have triggered the disease.(AU)

Dois gambás jovens foram necropsiados e diagnosticados com raquitismo. O objetivo do trabalho é descrever os aspectos clínico-patológicos de raquitismo em Didelphis albiventris. Macroscopicamente os gambás apresentaram cifose e escoliose, desvio lateral dos membros em varus, dificuldade de locomoção e alargamento com amolecimento das junções costocondrais (rosário raquítico). Amostras dos ossos e articulações foram processadas para coloração de hematoxilina e eosina e Tricrômico de Masson. Microscopicamente havia espessamento da placa epifisária, caracterizada pela proliferação irregular e multifocal das zonas de cartilagem seriada e hipertrófica, que formavam grupos circulares de condrócitos grandes, displásicos em direção a zona esponjosa frequentemente cercados por tecido osteoide não mineralizado. No osso cortical haviam zonas eosinofílicas pálidas ao redor dos canais de Havers consistentes com osteoide não mineralizado. A coloração de Tricrômico de Masson evidenciou o acúmulo de tecido osteoide no nosso cortical e trabecular. Acredita-se que uma causa mista de déficit de absorção de vitamina D3 associada a uma dieta desbalanceada em Ca:P a base de leite sem lactose e frutas tenha desencadeado a doença.(AU)

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic effects.

Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Morphophysiological evaluation of Bauhinia divaricata L. (Fabaceae) plants at different water regimes / Avaliação morfofisiológica em plantas de Bauhinia divaricata L. (Fabaceae) em diferentes regimes hídricos

The pata-de-vaca (Bauhinia divaricata L.) tree is a species widely distributed in Brazil, it has a high ornamental and economic value. Its leaves are used in renal inflammations, such as diuretic, hypoglycemic, being considered a medicinal plant of popular use. The research was carried out in a greenhouse belonging to the Laboratório de Ecologia Vegetal, at the Centro de Ciências Agrárias (CCA), from Universidade Federal of Paraíba (UFPB). The objective was to evaluate the morphophysiological characteristics of B. divaricata plants at different water regimes. The experimental design was completely randomized with five water treatments 1 (100% control), 2 (80%), 3 (60%), 4 (40%) and 5 (20%) of the container capacity. The height, number of leaves and stem diameter were evaluated weekly. At the end of the experiment the plants had their organs separated and taken to the greenhouse to obtain the dry matter of the leaves, stem, roots and total dry matter, and also, biomass allocation in the leaves, stem and roots. Plant height did not differ statistically between the treatments. The number of leaves, diameter, leaf, stem and root allocation and root and shoot dry matter were higher under water availability (100% of container capacity). For the production of B. divaricata L. seedlings water regime should be 100% and 80% of the container capacity, but the seedlings grow satisfactorily.

A pata-de-vaca (Bauhinia divaricata L.) é uma espécie arbórea, amplamente distribuída no Brasil, de alto valor ornamental e econômico. Suas folhas são utilizadas nas inflamações renais, como diuréticas, hipoglicemiantes, sendo considerada uma planta medicinal de uso popular. A pesquisa foi desenvolvida em casa de vegetação pertencente ao Laboratório de Ecologia Vegetal, Centro de Ciências Agrárias (CCA), Universidade Federal da Paraíba (UFPB). O Objetivo foi avaliar as características morfofisiológicas em plantas de Bauhinia divaricata em diferentes regimes hídricos. O delineamento experimental foi inteiramente casualizado com cinco tratamentos hídrico 100%, 80%, 60%, 40% e 20% da capacidade de pote. Avaliou-se, semanalmente a altura, número de folhas e diâmetro do caule. Ao final do experimento as plantas tiveram seus órgãos separados e levados à estufa para obtenção da matéria seca das folhas, caule, raízes e massa seca total, e ainda, alocação de biomassa nas folhas, caule e raízes. A altura de plantas não diferiu estatisticamente entre os tratamentos, o número de folhas, diâmetro, alocação de folhas, caules e raízes e massas seca da raiz e parte aérea foram maiores sob maior disponibilidade de água (100% da capacidade do recipiente). Para a produção de mudas de B. divaricata L. o regime hídrico deve ser de 100% e 80% da capacidade do recipiente, porém as mudas crescem satisfatoriamente.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose.

Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46µmolmL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02µmolmL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Curvulin and spirostaphylotrichins R and U from extracts produced by two endophytic Bipolaris sp. associated to aquatic macrophytes with antileishmanial activity.

In the present study, biological activity and chemical composition of two crude extracts of endophytic fungal strains of Bipolaris genera isolated from two species of aquatic macrophytes: Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) were investigated. The nuclear magnetic resonance and mass spectrometry data provided the identification of three main compounds: curvulin (1), spirostaphylotrichin R (2) and U (3). The fragmentation mechanism of the precursor ions towards collision induced dissociation (CID) tandem mass spectrometry experiment (MS/MS) is also proposed. Furthermore, biological screening of the crude extracts displayed antileishmanial activity with IC50 values ranging from 70-84.2 µg.mL-1.

Biosurfactants produced by Microbacterium sp, isolated from aquatic macrophytes in hydrocarbon-contaminated area in the Rio Negro, Manaus, Amazonas / Biossurfactantes produzidos por Microbacterium sp, isolada de macrófita aquática em área impactada por hidrocarbonetos no rio Negro, Manaus, Amazonas

Endophytic bacteria isolated from Eichhornia crassipes (Mart) Solms., collected in oil contaminated wastewater of effluent generated by Petrobras refinery in Manaus were investigated to determine their potential for producing biosurfactants. Assay with 2.6-dichlorophenol indophenol (DCPIP) indicator to verify hydrocarbon biodegradation activity; oil emulsification test; drop-collapse method; surface tension and growth curve of biosurfactant production. The M87 Microbacterium sp. strain chosen for this work was identified by the sequencing of the rDNA region and the chemical characterization was performed by FTIR, UFLC/MS and 1H RMN techniques. The selected bacterial isolate provided 3g L-1 of biosurfactant, using diesel oil as sole carbon source, being efficient in biodegrading oil as demonstrated by the DCPIP test. Fractions obtained by column chromatography were efficient in reducing water surface tension around 40 mN m-1, especially fraction 1, which reduced it to 34.17 mN m-1. The different techniques of chemical analysis used for the identification of the biosurfactant isolate indicated that this is probably a long - chain fatty acid lipid type, which may be used in the future as both biosurfactant in decontamination processes of hydrocarbon-polluted areas or as bioemulsifier in countless processes, since it exhibited no toxicity as determined by Alamar Blue assay.

Foram investigadas bactérias endofíticas isoladas de Eichhornia crassipes (Mart.) Solms., coletadas em águas contaminadas com resíduos de petróleo em um afluente da refinaria da Petrobrás/Manaus, para avaliação da produção de biossurfactantes. Para selecionar o micro-organismos à produção e caracterização de biossurfactantes, foram realizados os seguintes testes: a descoloração do indicador 2,6 indofenol (DCPIP), emulsificação do diesel, colapso da gota, tensão superficial e curva de produção. A caracterização química foi realizada por meio das técnicas de FT-IR, UFLC/MS e RMN1H. A bactéria M87 Microbacterium neste estudo, foi identificada pelo sequenciamento da região rDNA e produziu 3g L-1 de biossurfactantes utilizando o diesel como fonte de carbono, mostrando-se eficiente na ação biodegradadora do petróleo, por meio do teste de Indofenol (DCPIP). As frações obtidas, mostraram-se eficazes na redução da tensão superficial da água abaixo de 40 mN m-1, com destaque para a fração 1 que reduziu a tensão superficial para 34,17 mN m-1. Pelas análises química utilizadas, pode-se inferir que, provavelmente, se trata de um ácido graxo de cadeia longa, que pode ser utilizado futuramente tanto como biossurfactante em processos de descontaminação de ambientes impactados por hidrocarbonetos, assim como bioemulsificante em inúmeros processos uma vez que não apresentou toxicidade por meio do teste realizado.

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.

Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides.

Guarana (Paullinia cupana var. sorbilis) is a plant from the Amazonas region with socio-economic importance. However, guarana production has been increasingly affected by unfavorable conditions resulting from anthracnose, caused by the Colletotrichum fungal genus, which primarily affects mainly the Amazonas region. The aim of the present study was to isolate bacterial endophytes from the seeds of guarana plants obtained from Amazonas region and the Northeast state of Bahia, a region where this disease is not a problem for guarana plantations. The number of bacterial Colony Forming Units (CFU/g seeds) was 2.4 × 10(4) from the Bahia and 2.9 × 10(4) from the Amazonas region. One hundred and two isolated bacteria were evaluated in vitro against the phytopathogenic strain Colletotrichum gloeosporioides L1. These isolates were also analyzed for the enzymatic production of amylase, cellulase, protease, pectinase, lipase and esterase. Approximately 15% of isolates, showing high antagonistic activity, and the production of at least one enzyme were identified through the partial sequencing of 16S rDNA. The genus Bacillus was the most frequently observed, followed by Paenibacillus, Ochrobactrum, Microbacterium and Stenotrophomonas. Proteolytic activity was observed in 24 isolates followed by amylolytic, pectinolytic and cellulolytic activities. No esterase and lipase production was detected. Most of the isolates, showing antagonistic effects against C. gloeosporioides and high enzymatic activities, were isolated from the anthracnose-affected region. A biocontrol method using the endophytes from guarana seeds could be applied in the future, as these bacteria are vertically transferred to guarana seedlings.

Metabólitos Secundários de Interesse Farmacêutico Produzidos por Fungos Endofíticos / Secondary Metabolites Produced by Endophytic Fungi of Pharmaceutical Interest

Fungos endofíticos são grandes produtores de metabólitos secundários. No entanto, essa produção ainda não foi estimada, mas sabe-se que 80% dos fungos endofíticos estudados produzem metabólitos ativos, influenciados por condições ambientais, parâmetros de fermentação, tempo, temperatura, pH e nutrientes. Esses compostos são agrupados em diversas classes, e se apresentam tóxicos e até mesmo letais frente a outros microrganismos. Essas classes de compostos possuem atividades hormonais, antibióticas, antitumorais, antifúngicas, citotóxicas, antivirais, imunossupressoras, antiparasitárias, entre outras. Os produtos naturais obtidos de endófitos incluem principalmente alcalóides, esteróides, terpenóides, isocumarinas, quinonas, fenilpropanóides, ligninas, fenóis e ácidos fenólicos, metabólitos alifáticos, lactonas, citocatalasinas, flavonóides, peptídeos e xantonas. Considerando que os endófitos ainda são pouco estudados, especialmente em espécies tropicais, eles surgem como fonte de novos produtos para a incorporação de medicamentos e como forma de preservação de plantas medicinais. Salienta-se que a produção de metabólitos fúngicos realizada in vitro apresenta um menor custo e melhor otimização, bem como os microrganismos serem fontes facilmente renováveis e reprodutíveis quando comparadas com outras fontes naturais como plantas e animais. Várias técnicas têm sido utilizadas para aprimorar a utilização de microrganismos para a produção de novos princípios ativos, reunindo o conhecimento em química, bioquímica, microbiologia e biologia molecular. Este trabalho tem como objetivo mostrar a diversidade de produtos metabolicamente ativos produzidos por fungos endofíticos, assim como seu potencial farmacológico.

Endophytes are large producing secondary metabolites. Although the production has not yet been estimated, it is known that 80% of endophytic fungi studied produce active metabolites, influenced by environmental conditions, fermentation parameters, time, temperature, nutrients and pH. These compounds are grouped into several classes, which are toxic and even lethal against other microorganisms. These classes of metabolites have hormonal, antibiotic, antitumor, antifungal, cytotoxic, antiviral, immunosuppressive, and antiparasitic activities. The products obtained from natural endophyte mainly include alkaloids, steroids, terpenes, isocumarins, quinones, phenylpropanoids, lignins, phenols and phenolic acids, aliphatic metabolites, lactones cytocatalasins, flavonoids, peptides and xanthones. Whereas the endophytes are still poorly studied, particularly in tropical species, they appear as a source of new products for the incorporation in drugs and as a means of preservation of medicinal plants. It is noted that the production of fungal metabolites performed in vitro has a lower cost and a better optimization. In addition, microorganisms are easily renewable and reproducible sources when compared with other natural sources such as plants and animals. Several techniques have been used to enhance the use of microorganisms for production of new active metabolites, gathering knowledge in chemistry, biochemistry, microbiology and molecular biology. This study aims to show the diversity of products produced by metabolically active endophytes as well as their pharmacological potential.

Acaricidal activity of Palicourea marcgravii, a species from the Amazon forest, on cattle tick Rhipicephalus (Boophilus) microplus.

Leaves of Palicourea marcgravii were extracted successively with hexane, ethyl acetate and ethanol in order to evaluate their acaricidal activity on larvae and adult stages of the cattle tick Rhipicephalus (Boophilus) microplus. The ethyl acetate extract showed the highest bioactivity of the tested extracts, which contained 0.12% monofluoroacetic acid. On engorged female, the ethyl acetate extract showed a lethal concentration 50% - LC(50)=30.08 mg ml(-1), inhibitory concentration 50% - IC(50)=5.79 mg ml(-1) and lethal time 50% - LT(50)=4.72 days; 100% reproduction was controlled at concentrations of 50 mg ml(-1) and on larvae the ethyl acetate extract showed a LC(50)=2.46 mg ml(-1). No alkaloids were detected in any of the extracts. This is the first report on the acaricidal activity of P. marcgravii extracts against R. microplus as well as the acaricidal properties of a plant species containing monofluoroacetic acid.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum).

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Toxicity of Piper aduncum L. (Piperales: Piperaceae) from the Amazon forest for the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The mortality of 14-21-day-old Rhipicephalus (Boophilus) microplus larvae, and the mortality and fertility of groups of engorged adult females exposed to different concentrations of hexane, ethyl acetate and ethanol extracts of spiked pepper (Piper aduncum) were evaluated, using a completely randomized design with five treatment groups, two control groups, and two replicates for the larvae and five replicates for the adult females. Similar methodology was used to investigate the toxicity of the essential oil hydro-distillate (94.84% dillapiole) obtained from the P. aduncum crude hexane extract. The LC(50) of the hexane extract was 9.30 mg ml(-1) for larvae and the reproduction reduction ranged from 12.48% to 54.22%, while 0.1mg/ml(-1) of the essential oil induced 100% mortality in larvae. Literature reports on natural products active against R. microplus were listed and compared with the results presented here. These results indicate that P. aduncum extracts, and particularly its essential oil, are potential alternative control agents for R. microplus.

Endophytic colonization of potato (Solanum tuberosum L.) by a novel competent bacterial endophyte, Pseudomonas putida strain P9, and its effect on associated bacterial communities.

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.