Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity

Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.

Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.

Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.

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Different arbuscular mycorrhizal (AMF) fungal taxa have a differential effect on the growth of co-existing plant species. This means that in order to fully understand the role of these fungi in plant communities, information is needed on whether the symbiosis is specific. In this chapter, I briefly review the ecological consequences of specificity versus non-specificity in the arbuscular mycorrhizal symbiosis on plant ecology. Both from a theoretical approach, and based on observations, there has been an underlying assumption that no specificity exists in the arbuscular mycorrhizal symbiosis. I consider why these assumptions have been made. Direct evidence for or against specificity in the symbiosis is scant and the reason is mainly due to the difficulty in describing AMF community structure in natural communities (see Clapp et al., Chap.8, this Vol.). Here, I take an evolutionary, as well as an ecological, approach to look at the evidence that predicts that evolution of specificity in the arbuscular mycorrhizal symbiosis could occur. I then consider alternative hypotheses and evidence that could explain why the evolution of specificity might not occur. These hypotheses are based on the growth habit, reproductive strategies and foraging behaviour of AMF and on new findings concerning ANF genetics.

Nonself vegetative fusion and genetic exchange in the arbuscular mycorrhizal fungus Glomus intraradices.

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants and form extensive underground hyphal networks simultaneously connecting the roots of different plant species. No empirical evidence exists for either anastomosis between genetically different AMF or genetic exchange.Five isolates of one population of Glomus intraradices were used to study anastomosis between hyphae of germinating spores. We show that genetically distinct AMF, from the same field, anastomose, resulting in viable cytoplasmic connections through which genetic exchange could potentially occur.Pairs of genetically different isolates were then co-cultured in an in vitro system.Freshly produced spores were individually germinated to establish new cultures.Using several molecular tools, we show that genetic exchange occurred between genetically different AMF. Specific genetic markers from each parent were transmitted to the progeny. The progeny were viable, forming symbioses with plant roots. The phenotypes of some of the progeny were significantly different from either parent.Our results indicate that considerable promiscuity could occur in these fungi because nine out of 10 combinations of different isolates anastomosed. The ability to perform genetic crosses between AMF experimentally lays a foundation for understanding the genetics and evolutionary biology of these important plants symbionts.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.

Different arbuscular mycorrhizal fungi alter coexistence and resource distribution between co-occurring plants

It is often thought that the coexistence of plants and plant diversity is determined by resource heterogeneity of the abiotic environment. However, the presence and heterogeneity of biotic plant resources, such as arbuscular mycorrhizal fungi (AMF), could also affect plant species coexistence. In this study, Brachypodium pinnatum and Prunella vulgaris were grown together in pots and biotic resource heterogeneity was simulated by inoculating these pots with one of three different AMF taxa, with a mixture of these three taxa, or pots remained uninoculated. The AMF acted as biotic plant resources since the biomass of plants in pots inoculated with AMF was on average 11.8 times higher than uninoculated pots. The way in which the two plant species coexisted, and the distribution of phosphorus and nitrogen between the plant species, varied strongly depending on which AMF were present. The results showed that the composition of AMF communities determines how plant species coexist and to which plant species nutrients are allocated. Biotic plant resources such as AMF should therefore be considered as one of the factors that determine how plant species coexist and how soil resources are distributed among co-occurring plant species.

Efecte de la recurrència dels incendis sobre la vegetació i el sòl a les zones de matollar del Macizo Central Ourensano

El matollar sec europeu format per Erica australis, Erica arborea, Pterosparum tridentatum, Halimium lasianthum i Calluna vulgaris com a espècies predominants és cremat en les serralades del Massís Central de forma repetida durant el pas dels anys. Això ha sigut, tradicionalment, a causa de la necessitat de mantenir-lo transitable per al bestiar, encara que altres circumstàncies s’han afegit actualment a aquesta necessitat, que incrementen el risc d’incendi d’aquesta comunitat, disparant-se el número d’incendis en els últims 30 anys. Sabent això, vam decidir realitzar un estudi a les zones de matollar que trobem al Massís Central situat a la zona del Concello de Villariño de Conso, a Ourense, per conèixer com es comporten les espècies característiques d’aquest tipus d’ecosistema després dels incendis, mitjançant la realització de mostrejos i perfils del sòl. D’aquesta manera hem volgut veure com es desenvolupen les espècies vegetals de matollar al llarg del temps, després de ser cremats. Gràcies a l’estudi hem pogut veure com el matollar observat te unes característiques que li permet sobreviure als incendis i tindre una capacitat de regeneració elevada després de patir aquesta pertorbació. Això es degut a la capacitat per rebrotar d’aquestes espècies arbustives i a la seva competitivitat. També hem vist com a necessari, donar un valor econòmic a la bona conservació de l’hàbitat, per tal de que la població local abandoni pràctiques de gestió forestal nocives, com el foc.

Convergent evolution of floral signals underlies the success of Neotropical orchids.

The great majority of plant species in the tropics require animals to achieve pollination, but the exact role of floral signals in attraction of animal pollinators is often debated. Many plants provide a floral reward to attract a guild of pollinators, and it has been proposed that floral signals of non-rewarding species may converge on those of rewarding species to exploit the relationship of the latter with their pollinators. In the orchid family (Orchidaceae), pollination is almost universally animal-mediated, but a third of species provide no floral reward, which suggests that deceptive pollination mechanisms are prevalent. Here, we examine floral colour and shape convergence in Neotropical plant communities, focusing on certain food-deceptive Oncidiinae orchids (e.g. Trichocentrum ascendens and Oncidium nebulosum) and rewarding species of Malpighiaceae. We show that the species from these two distantly related families are often more similar in floral colour and shape than expected by chance and propose that a system of multifarious floral mimicry-a form of Batesian mimicry that involves multiple models and is more complex than a simple one model-one mimic system-operates in these orchids. The same mimetic pollination system has evolved at least 14 times within the species-rich Oncidiinae throughout the Neotropics. These results help explain the extraordinary diversification of Neotropical orchids and highlight the complexity of plant-animal interactions.

Jasmonate biochemical pathway.

Plants possess an interrelated family of potent fatty acid-derived regulators-the jasmonates. These compounds, which play roles in both defense and development, are derived from tri-unsaturated fatty acids [alpha-linolenic acid (18:3) or 7Z,10Z,13Z-hexadecatrienoic acid (16:3)]. The lipoxygenase-catalyzed addition of molecular oxygen to alpha-linolenic acid initiates jasmonate synthesis by providing a 13-hydroperoxide substrate for the formation of an unstable allene oxide that is then subject to enzyme-guided cyclization to produce 12-oxo-phytodienoic acid (OPDA). OPDA has several fates, including esterification into plastid lipids or transformation into the 12-carbon co-regulator jasmonic acid (JA). JA, the best-characterized member of the family, regulates both male and female fertility (depending on the plant species) and is an important mediator of defense gene expression. JA is itself a substrate for further diverse modifications. Genetic dissection of the pathway is revealing how the different jasmonates modulate different physiological processes. Each new family member that is discovered provides another key to understanding the fine control of gene expression in immune responses, in the initiation and maintenance of long-distance signal transfer in response to wounding, in the regulation of fertility, and in the turnover, inactivation, and sequestration of jasmonates, among other processes. The Jasmonate Biochemical Pathway provides an overview of the growing jasmonate family, and new members will be included in future versions of the Connections Map.

Guayule and Russian dandelion as alternative sources of natural rubber.

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.

A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants.

BACKGROUND: Genes involved in arbuscular mycorrhizal (AM) symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes (forward genetics). In addition, a number of AM-related genes has been identified by their AM-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches (reverse genetics). However, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. If such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. The goal of this study was to systematically search for AM-related genes with a bioinformatics strategy that is insensitive to these problems. The central element of our approach is based on the fact that many AM-related genes are conserved only among AM-competent species. RESULTS: Our approach involves genome-wide comparisons at the proteome level of AM-competent host species with non-mycorrhizal species. Using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the AM-competent species. Proteins of these clusters were then analyzed in an extended set of 16 plant species and ranked based on their relatedness among AM-competent monocot and dicot species, relative to non-mycorrhizal species. In addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. As a result we present a list of yet uncharacterized proteins that show a strongly AM-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in AM. Among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the S-locus receptor kinases involved in sporophytic self-incompatibility. CONCLUSIONS: We present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. This strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest.

Comunidade de Euglossini (Hymenoptera, Apidae) das dunas litorâneas do Abaeté, Salvador, Bahia, Brasil

Community of Euglossini (Hymenoptera, Apidae) from the coastal sand dunes of Abaeté, Salvador, Bahia, Brazil. The Euglossini community structure was analyzed by attracting males with the scents eucalyptol, eugenol, vanillin, benzyl benzoate and methyl salicylate, and by netting bees on flowers. The samplings took place three times a month along one year from 6:00 a.m. to 6:00 p.m. The scent baits attracted 670 individuals belonging to seven species of three genus. The predominant species were Euglossa cordata (Linnaeus, 1758) (76.6%) and Eulaema nigrita Lepeletier, 1841 (21.8%). Euglossini males visited the scents along the whole year, being more abundant in May and in August. The most efficient fragrance was eucalyptol, attracting 624 individuals of five species. The males abundance fluctuated along the day, being the highest frequency observed between 8:00 a.m. to 10:00 a.m. Forty eigth Euglossini females of four species were netted visiting flowers of 14 plant species belonging to 13 families. Solanaceae and Caesalpiniaceae were the most visited. The species catched on flowers were Euglossa cordata, Eulaema nigrita, Euplusia mussitans (Fabricius, 1787) and Eulaema meriana flavescens Friese 1899. Euglossa cordata was the predominant species on flowers (64.6%), being collected during almost the whole year. Euplusia mussitans was the only species netted on flowers which males were not sampled on the scents.

Efeitos do sexo, do vigor e do tamanho da planta hospedeira sobre a distribuição de insetos indutores de galhas em Baccharis pseudomyriocephala Teodoro (Asteraceae)

Several hypotheses have been proposed to explain the patterns of host plant utilization by herbivorous insects in natural communities. We tested four hypotheses aiming to understand the pattern of attack by gall-inducing insects on the dioecious shrub, Baccharis pseudomyriocephala (Asteraceae). The shrub occurs in the Parque Estadual do Itacolomi, Southeastern Brazil, and supports ten species of galling insects. The following hypotheses were tested: i) male plants are more attacked by galling insects than female plants; ii) larger plant modules are preferentially attacked by galling insects; iii) galling insects perform better on larger modules than on smaller modules; iv) galling insects increase in abundance with meristematic availability. To address these questions, 240 plants (120 of each sex) were sampled in both reproductive and vegetative periods. We recorded the growth rate (4 cm), inflorescence and fruit production, attack rates of the galling insects, and their survivorship and mortality per shoot (module). Modules were separated into size classes (cm) and analyzed by regressions and ANCOVAs. Module size and reproductive effort were positively correlated with host plant size. We did not observe any effect of host plant gender on either variables. In the same way, host plant sex did not show any influence on the abundance and richness of galling insects. Although the abundance of galling insects showed a positive correlation with shoot size, the trend disappeared when the analyses were performed taking into consideration the number of galls per unit of growth (number of galls/cm of shoot) or biomass (number of galls/dry weight). Larval survivorship was not influenced by shoot size. Also, we observed that the abundance of one species of hemipteran galling insect showed a positive relation with leaf biomass. Therefore, we conclude that gender and vigor of this plant species do not influence the community structure of its galling herbivores.

Role of Methyl Salicylate on Oviposition Deterrence in Arabidopsis thaliana.

Plants attacked by herbivores have evolved different strategies that fend off their enemies. Insect eggs deposited on leaves have been shown to inhibit further oviposition through visual or chemical cues. In some plant species, the volatile methyl salicylate (MeSA) repels gravid insects but whether it plays the same role in the model species Arabidopsis thaliana is currently unknown. Here we showed that Pieris brassicae butterflies laid fewer eggs on Arabidopsis plants that were next to a MeSA dispenser or on plants with constitutively high MeSA emission than on control plants. Surprisingly, the MeSA biosynthesis mutant bsmt1-1 treated with egg extract was still repellent to butterflies when compared to untreated bsmt1-1. Moreover, the expression of BSMT1 was not enhanced by egg extract treatment but was induced by herbivory. Altogether, these results provide evidence that the deterring activity of eggs on gravid butterflies is independent of MeSA emission in Arabidopsis, and that MeSA might rather serve as a deterrent in plants challenged by feeding larvae.