Abstract Cunila galioides Benth. is an aromatic and medicinal plant widely used in the traditional medicine of South Brazil. The essential oil of one of its chemotypes has high citral contents, an oxygenated monoterpene used in perfumery, cosmetics, and food industries. The seeds of C. galioides have low germination percentages and there are few studies regarding its vegetative propagation. Thus, this study aimed to evaluate distinct indole-3-butyric acid concentrations (0 - control, 250, 500, and 1,000 mg∙L-1) on the rooting of herbaceous cuttings of C. galioides and different methods (gibberellic acid, potassium nitrate, and stratification) to overcome a possible dormancy in the seeds. In vegetative propagation, the use of indole-3-butyric acid 250 mg∙L-1 only increased root score relative to the control, and higher IBA concentrations had no effective response on the cuttings, not being necessary to use IBA in C. galioides cuttings. The most effective treatments regarding seed germination were gibberellic acid and potassium nitrate, with germination percentages of 57.8 % and 54.5 %, respectively; stratification was not effective in increasing the germination percentage of the seeds relative to the control.

Resumo O objetivo deste trabalho foi avaliar o efeito do óleo essencial de Eucalyptus staigeriana sobre o crescimento micelial e a germinação de conídios de Colletotrichum gloeosporioides e Greeneria uvicola em uvas, assim como o seu potencial para o controle de podridões no período pós-colheita e o seu efeito nas propriedades organolépticas das uvas. O óleo essencial (OE) apresentou atividade antifúngica in vitro contra ambos os patógenos, com efeito fungicida sobre o crescimento micelial e sobre a germinação de conídios, nas concentrações de 1,0 e 0,5 μL mL−1, respectivamente. Os compostos voláteis do OE exibiram efeito fungistático sobre o crescimento micelial de C. gloeosporioides e efeito fungicida sobre G. uvicola. Na pós-colheita, o OE reduziu a incidência da podridão-madura em até 75 e 86%, nos tratamentos preventivos e curativos, respectivamente, e a incidência da podridão-amarga em até 54% no tratamento curativo. Como o OE não influencia significativamente as propriedades sensoriais das uvas, não afeta a intenção de consumo para uvas tratadas com o OE. O OE de E. staigeriana é eficiente para o controle in vitro de ambos os patógenos; além disso, também é eficiente no controle da incidência de podridões fúngicas no período pós-colheita, principalmente no tratamento curativo.

Abstract The objective of this work was evaluate the effect of Eucalyptus staigeriana essential oil on Colletotrichum gloeosporioides and Greeneria uvicola mycelial growth and conidia germination on grapes, as well as its potential for the control of postharvest rot diseases and its effect on the organoleptic properties of grapes. The essential oil (EO) showed in vitro antifungal activity against both pathogens, with fungicidal effect on mycelial growth and on conidia germination at the concentrations of 1.0 and 0.5 μL mL−1, respectively. The EO volatile compounds had a fungistatic effect on the mycelial growth of C. gloeosporioides and a fungicidal effect on G uvicola. At postharvest, the EO reduced the incidence of ripe rot up to 75% and 86% in the preventive and curative treatments, respectively, and the incidence of bitter rot up to 54% in the curative treatment. Since the EO does not affect significantly grape sensory properties, it does not affect the consumption intention of grapes treated with the EO. The EO of E. staigeriana is efficient in the in vitro control of both pathogens; moreover, it is also efficient in the control of the incidence of postharvest fungal rot diseases, mainly in the curative treatment.

RESUMO: As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.

ABSTRACT: The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.

Abstract The objective of this study was to evaluate the biomass and essential oil production of nine populations of poejo (Cunila galioides) cultivated in five agroecological regions of the state of Rio Grande do Sul, under different edaphoclimatic conditions. The experiments were performed in field conditions in Erechim, Caxias do Sul, Pelotas, São Francisco de Paula, and Santa Vitoria do Palmar. The experimental design was completely randomized, with nine populations, eight plants per plot and four repetitions. The following were evaluated: biomass production and essential oil chemical composition and yield. The data underwent ANOVA, followed by Tukey’s multiple range test. The adaptability and stability of the populations in the different environments were also evaluated by regression analysis. The results showed great differences between the populations and cultivation sites, with genotype vs. environment interaction. Most populations presented the best biomass production results at Erechim. Pelotas and Santa Vitória do Palmar were the worst locations for poejo production, mainly due to a water deficit occurred during the experiment. The Santa Lucia population presented broad stability and the greatest adaptability to the environments for biomass and essential oil production, but its average production was not satisfactory. The André da Rocha population presented the highest average production of essential oil, and was favored in favorable environments. Regarding essential oil chemical composition, the populations kept stable contents of the major compounds at all locations, with a few variations. In some populations, a higher concentration of sesquiterpenes was observed, which can be attributed to environmental stress.

RESUMO Objetivo: Definir escores histológicos de degeneração do disco intervertebral que permitam a identificação de características morfológicas da doença, além de melhorar o conhecimento sobre a discopatia degenerativa lombar por meio de marcadores imuno-histoquímicos. Métodos: As colorações histológicas de hematoxilina e eosina, azul de alcian/PAS, tricrômica de Masson e safranina O/FCF foram utilizadas em cortes de disco intervertebral degenerado de pacientes com discopatia degenerativa lombar. Os marcadores proteicos definidos na imuno-histoquímica permitiram a avaliação da proliferação celular (Ki-67) e da apoptose (p53). Resultados: Os dados do estudo permitiram a determinação da coloração de safranina O/FCF como a mais eficaz para avaliar os parâmetros tais como a área, o diâmetro e o número de agrupamentos de condrócitos. Também se determinou a importância do uso das colorações histológicas de forma associada, como safranina O/FCF, tricrômica de Masson, azul de alcian/PAS e hematoxilina e eosina, uma vez que elas são complementares para a verificação histopatológica da degeneração do disco intervertebral. Pela técnica da expressão de proteínas com técnica imuno-histoquímica, foi possível considerar dois estágios de degeneração do disco: proliferação de células com a formação de agrupamentos de condrócitos, seguida pela indução de apoptose. Conclusão: Este estudo permitiu definir a caracterização histológica e imuno-histoquímica em discopatia degenerativa lombar e seus graus de evolução, determinando novos escores de degeneração discal.

ABSTRACT Objective: To define histological scores for intervertebral disc degeneration that would enable the definition of morphological characteristics of disease, besides improving knowledge of the lumbar degenerative disc disease by means of immunohistochemical markers. Methods: Hematoxylin and Eosin, Alcian/PAS, Masson Trichrome and Safranin O/FCF staining was used on the intervertebral disc degeneration sections of patients with lumbar degenerative disc disease. The protein markers defined in immunohistochemistry were cell proliferation (Ki-67) and apoptosis (p53). Results: The study data enabled the determination of Safranin O/FCF stain as the most effective one for evaluating parameters such as area, diameter, and number of chondrocyte clusters. The importance of using stains in association, such as Safranin O/FCF, Masson Trichrome, Alcian/PAS and Hematoxylin and Eosin, was also determined, as they are complementary for the histopathological verification of intervertebral disc degeneration. By expressing proteins using the immunohistochemistry technique, it was possible to consider two stages of disc degeneration: cell proliferation with chondrocyte cluster formation, and induction of apoptosis. Conclusion: This study enabled the histological and immunohistochemical characterization to be determined for lumbar degenerative disc disease, and its degrees of evolution, by determining new disc degeneration scores.

RESUMEN Objetivo: Definir valores histológicos de degeneración del disco intervertebral que permitan la definición de las características morfológicas de la enfermedad y mejorar el conocimiento de la enfermedad degenerativa del disco lumbar mediante marcadores inmunohistoquímicos. Métodos: Las coloraciones histológicas con hematoxilina y eosina, azul alcián/PAS, tricrómico de Masson y safranina O/FCF se utilizaron en secciones de los discos intervertebrales degenerados de pacientes con enfermedad degenerativa del disco lumbar. Los marcadores de proteínas definidos por inmunohistoquímica permitieron la evaluación de la proliferación celular (Ki-67), y la apoptosis (p53) . Resultados: Los datos de la determinación permitieron establecer la tinción con safranina O/FCF como la más eficaz para evaluar parámetros tales como el área, diámetro y número de agrupaciones de condrocitos. Se determinó también la importancia del uso asociado de diversas tinciones, como safranina O/FCF, tricrómico de Masson, azul de alcián/PAS y hematoxilina y eosina, ya que son complementarias para la verificación histopatológica de la degeneración del disco intervertebral. Por la técnica de la expresión de proteína con análisis inmunohistoquímica, fue posible establecer dos etapas de la degeneración del disco: la proliferación de células con la formación de agrupaciones de condrocitos y la inducción de la apoptosis. Conclusión: Este estudio permitió definir la caracterización histológica e inmunohistoquímica en la enfermedad degenerativa del disco lumbar y sus grados de evolución, mediante la determinación de nuevas puntuaciones de degeneración del disco.

Acanthamoeba are abundant in a wide range of environments, and some species are responsible for cutaneous infections, keratitis, and granulomatous amoebic encephalitis (GAE). The conventional detection and isolation of amoeba from clinical and environmental samples involves sampling and culture on non-nutrient Ágar medium. Although efficient, this system requires several transfers in order to eliminate contaminants, and is not appropriate for the isolation of individual amoeba from samples with a biodiverse community. In this study we propose an alternative method for the isolation of monocystic clones of Acanthamoeba. The propose method involves sampling, enrichment, encystment induction, and direct cysts micromanipulation and culture on Ágar plates.

Promoters are DNA sequences located upstream of the gene region and play a central role in gene expression. Computational techniques show good accuracy in gene prediction but are less successful in predicting promoters, primarily because of the high number of false positives that reflect characteristics of the promoter sequences. Many machine learning methods have been used to address this issue. Neural Networks (NN) have been successfully used in this field because of their ability to recognize imprecise and incomplete patterns characteristic of promoter sequences. In this paper, NN was used to predict and recognize promoter sequences in two data sets: (i) one based on nucleotide sequence information and (ii) another based on stability sequence information. The accuracy was approximately 80% for simulation (i) and 68% for simulation (ii). In the rules extracted, biological consensus motifs were important parts of the NN learning process in both simulations.

Segmentos nodais de plantas adultas de Salvia guaranitica cultivadas em estufa, foram utilizados para avaliar o efeito do meio de cultivo e reguladores de crescimento na micropropagação. A maior taxa de multiplicação foi obtida em meio Murashige and Skoog (MS) suplementado com 2.22 µM de 6-benzilaminopurina (BAP). A melhor condição para enraizamento foi o meio MS acrescido de 2.85 µM de ácido indol-3-acético (IAA). Plântulas enraizadas foram aclimatizadas com sucesso e exibiram desenvolvimento normal até a fase adulta. Utilizando o protocolo descrito, aproximadamente 35 plantas por explante foram obtidas após 3 meses.

Nodal segments from greenhouse grown adult plants of Salvia guaranitica were used to evaluate the effect of culture media and growth regulators on the micropropagation and growth. The highest multiplication rate was obtained on Murashige and Skoog (MS) medium supplemented with 2.22 µM of 6-benzylaminopurine (BAP). The best condition for rooting was MS medium with 2.85 µM indole-3-acetic acid (IAA). Rooted plants were successfully acclimatized and exhibited a normal development until maturity. Using the described protocol, approximately 35 plants per explant were obtained after three months.

The purpose of this work was to stud the biodegradation of citronellol, citronellal and citronellyl acetate by a soil Pseudomonas mendocina strain (IBPse 105) isolated from a Cymbopogon windelandi field. This strain efficiently used citronellol, citronellal, citronellyl acetate and myrcene as sole source of carbon, but was not able to grow on other 15 monoterpenoids evaluated. Gas chromatography-mass spectrometry (GC-MS) analysis of metabolites accumulation during P. medocina IBPse 105 growth on citronellol showed that this strain uses the citronellol catabolic pathway described for other species of the genus. IBPse 105 degradation of citronellyl acetate initiates by its hydrolysis to citronellol. The mini-Tn5 insertion in mutant IBPse 105-303, impaired in citronellol degradation, but able to grow on citronellal, was located in a homologous of the P. aeruginosa atuB gene, that codifies citronellol deshydrogenase.

Proteus mirabilis is one of the most important pathogens associated with complicated urinary tract infections (acute pyelonephritis, bladder infections, kidney stones) and bacteremia, affecting patients with anatomical abnormalities, immunodeficiency, and long-term urinary catheterization. For epidemiological purposes, various molecular typing methods, such as pulse-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen. However, these methods are labor intensive and time-consuming. We evaluated the discriminatory power of several PCR-based fingerprinting methods (RAPD, ISSR, ERIC-PCR, BOX-PCR and rep-PCR) for P. mirabilis clinical isolates. Typing patterns and clustering analysis indicated that RAPD, BOX-PCR and ERIC-PCR differentiated P. mirabilis strains from Escherichia coli, Hafnia alvei, and Morganella morganii. With the exception of rep-PCR, the methods gave medium to high discriminatory efficiency in P. mirabilis. In general, the results obtained with RAPD, BOX-PCR and ERIC-PCR were in good agreement. We concluded that a combination of ERIC-PCR and BOX-PCR results is a rapid and reliable alternative for discrimination among P. mirabilis clinical isolates, contributing to epidemiological studies.

Aeromonas foram isoladas de 27 (6.6%) dos 408 pacientes admitidos com gastroenterite aguda em dois hospitais do Rio Grande do Sul, Brasil. Os isolados foram classificados com A. hydrophila (51.8%), A. caviae (40.8%), e A. veronii biotype sobria (7.4%). A maior prevalência de Aeromonas ocorreu em lactantes e crianças. Genes (aerA -aerolisina/hemolisina, ahpA -serina-protease, satA - glicerofosfolipidio-colesterol aciltransferase, lipA -lipase, e ahyB -elastase) e factores (atividade hemolítica, proteolítica, lipolítica, e formação de biofilme) de virulência foram identificados na maioria dos isolados de A. hydrophila e A. veronii biotype sobria, com freqüências menores em A. caviae. Todos os isolados de Aeromonas apresentaram resistência a ampicilina, ticarcilina/ácido clavulânico, cefalotina e cefazolina, e a maior parte (>70%) exibiram resistência a imipenem, carbenicilina, amoxacilina/sulbactam e piperacilina. Resistência múltipla foi evidenciada em 29,6% dos isolados. Os antibióticos mais eficientes foram as quinolonas (ciprofloxacina e norfloxacina) e os aminoglicosídicos (amicacina e netilmicina).

Aeromonas were isolated from 27 (6.6%) of 408 patients admitted with acute gastroenteritis in two hospitals at Rio Grande do Sul, Brazil. Isolates were classified as A. hydrophila (51.8%), A. caviae (40.8%), and A. veronii biotype sobria (7.4%). The highest prevalence of Aeromonas associated infections occurred in lactants and children. Virulence genes (aerA -aerolysin/hemolysin, ahpA -serine-protease, satA - glycerophospholipid-cholesterol acyltransferase, lipA -lipase, and ahyB -elastase) and virulence factors (hemolytic, proteolitic, lipolitic activities, and biofilm formation) were identified in most A. hydrophila and A. veronii biotype sobria isolates, with lower frequencies on A. caviae. All Aeromonas isolates were resistant to ampicillin, ticarcillin/clavulanic acid, cephalotin, and cephazolin, and most of them (>70%) exhibited resistance to imipenem, carbenicillin, amoxillin/sulbactan, and piperacillin. Multiple-resistance, more than four antibiotics, was evidenced in 29.6% of the isolates. The most efficient antibiotics were the quinolones (ciprofloxacin and norfloxacin), and the aminoglycosides (amikacin and netilmicin).

As relações genéticas entre 11 espécies de Bothrops (sensus stricto) de ocorrência no Brasil foram determinadas através da comparação dos perfis de fragmentos amplificados aleatoriamente (RAPD). Um total de 239 bandas foram obtidas utilizando 20 sequencias iniciadoras (primers). A maior parte das bandas (88,7%) foram polimórficas. A análise filogenética dos dados permitiu a identificação de três grupos dentro do gênero Bothrops: Grupo 1- B. alternatus, B. neuwiedi, B. cotiara, e B. jararacusu; Grupo II- B. insularis, B. jararaca, e B. erythromelas; e Grupo III- B. moojeni, B. leucurus, e B. atrox. Estes grupos coincidem com aqueles obtidos por outros métodos indicando que os marcadores de RAPD podem ser considerados ferramentas úteis para a avaliação de relações a nível interespecífico.

The genetic relationships among 11 species of Bothrops found in Brazil were determined using random amplified fragment (RAPD) information. A total of 239 amplified bands were scored using 20 aleatory primers. Most of the bands (88.7%) were polymorphic. Phylogenetic analysis of the data determined three similarity groups within Bothrops: Group I- B. alternatus, B. neuwiedi, B. cotiara, and B. jararacusu; Group II- B. insularis, B. jararaca, and B. erythromelas; and Group III- B. moojeni, B. leucurus, and B. atrox. These groups coincided with those obtained by other methods, indicating that RAPD's could be a useful tool for the evaluation of genetic relationships at the interspecific level.

O efeito de solutos orgânicos no crescimento de Aeromonas trota e A. hydrophila foi avaliado. A prolina e o ácido glutâmico não apresentaram efeito osmoprotetor, mas a betaina exerceu osmoproteção permitindo o crescimento de ambas bactérias em concentrações inibitórias de NaCl. A cinética do crescimento sugere que a diferença em halotolerância entre as linhagens está associada à síntese de osmolitos, mais do que à capacidade de acúmulo de betaina.

The effect of organic solutes on the growth of Aeromonas trota and A. hydrophila was evaluated. Proline and glutamic acid were not effective as osmoprotectors, but betaine exerts osmoprotection allowing the growth of both strains in inhibitory concentrations of NaCl. Growth kinetics suggests that the halotolerance difference between the strains is associated with the synthesis of osmolytes rather than betaine uptake.

Nos últimos anos, a mosca-africana-do-figo Zaprionus indianus tem se tornado a praga mais importante desta cultura no Brasil. Um processo fermentativo está associado com a infecção dos frutos por esta mosca. Uma única espécie de levedura, Candida tropicalis, foi identificada nos figos infectados. A presença de uma única espécie de levedura e a baixa variabilidade genética (RAPD) dos isolados indica uma relação muito estreita entre C. tropicalis e Z. indianus.

In the last years, the fruit fly Zapronius indianus became the most important plague of Brazilian fig production. A fermentation process is associated with infection of the fruit by this fly. A single yeast species, Candida tropicalis, was identified in all the infected figs. The presence of one species and the low genetic variability (RAPD) of the isolates indicates an uncommonly strict interaction between C. tropicalis and Z. indianus.

Quinze linhagens de Aeromonas pertencentes a treze espécies foram avaliadas através de eletroforese de proteínas totais (SDS-PAGE) e segmentos de DNA amplificados ao acaso (RAPD). A comparação entre os padrões obtidos por ambos métodos permitiu diferenciar todas as linhagens. Agrupamentos formados com base nos dados protéicos mostraram relação com informações bioquímicas e genéticas a respeito das espécies. Os resultados mostraram que análises protéicas têm potencial para diferenciar espécies de Aeromonas, mas a baixa variação qualitativa indica que esta técnica não é eficiente para a caracterização entre linhagens dentro de espécies. Ao contrário, marcadores de RAPD permitem identificar linhagens, mas a alta variabilidade limita seu potencial como método auxiliar na identificação de espécies.

Fifteen well-defined strains of Aeromonas of thirteen species were analyzed by SDS protein electrophoretic analysis (SDS-PAGE) and random amplified polymorphic DNA analysis (RAPD). The comparison between the patterns obtained by both methods allowed differentiating all the strains. Clusters formed by the unweighted pair group method with arithmetic averages applied to protein data correlates with the genetic and biochemical information about the species. The results show that protein fingerprinting has the potential to differentiate Aeromonas species, but the low qualitative variation indicates that this technique is not efficient for the characterization of strains within a species. Conversely, RAPD fingerprinting allows the identification of strains but the high variability limits its potential as an aiding method for species identification.