The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

Eliminação de Clavibacter michiganensis subsp. michiganensisem estacas de bambu infestadas artificialmente

Diferentes tratamentos foram comparados visando a erradicação de Clavibacter michiganensis subsp. michiganensis de estacas de bambu infestadas artificialmente. A imersão das estacas, por 30 minutos, em uma solução de hipoclorito de sódio a 2% foi o tratamento mais eficiente, em comparação à solarização, à exposição direta aos raios solares, à imersão em sulfato de cobre e à fumigação com fosfina ou brometo de metila.

Atividade antimicrobiana de extratos peptídicos de folhas de berinjela na inibição do crescimento de Ralstonia solanacearum e Clavibacter michiganensis subsp. michiganensis

Compostos naturais, incluindo-se peptídios antimicrobianos, vêm se destacando como fontes de agentes de defesa contra fitopatógenos de importância comercial. Esse trabalho visou obter frações peptídicas a partir de extratos de folhas de berinjela e avaliar as atividades antimicrobianas contra duas bactérias fitopatogênicas. Os pools peptídicos catiônicos, PC1 e PC2 e o aniônico PA obtidos de extratos solúvel (ES) e de parede celular (EP) foram analisados em duas concentrações. Para ES e EP, obteve-se inibição do crescimento da Ralstonia solanacearum e da Clavibacter michiganensis subsp. michiganensis superior a 60%, sendo PC2-2X (maior concentração avaliada) a mais efetiva. Os resultados sugerem que extratos de folhas de berinjela apresentam peptídios com potencial aplicação como agentes de defesa de plantas.

Ação de produtos químicos in vitro e in vivo sobre Clavibacter michiganensis subsp. michiganensis, agente causal do cancro bacteriano do tomateiro

O presente trabalho teve como objetivo avaliar a sensibilidade in vitro e in vivo de um isolado de Clavibacter michiganensis subsp. michiganensis (Cmm) aos produtos cloridrato de kasugamicina, fulusulfamide e oxitetraciclina. Para o teste in vitro, foram avaliados os halos de inibição formados ao redor de discos de papel de filtro umedecidos com os produtos, nas concentrações de 0, 1, 10, 100 e 1000 mig mL-1, 24 h após a instalação do ensaio. Quanto à avaliação dos produtos in vivo, dois ensaios foram instalados sob condições de casa-de-vegetação, com plantas de tomateiro cultivar Ângela Hiper, através da inoculação das plantas por dois métodos: pulverização foliar e ferimento no caule. Nos dois ensaios, as plantas foram pulverizadas com os produtos cloridrato de kasugamicina a 0,06 mL L-1, fulusulfamide a 0,025, 0,05 e 0,075 mL L-1 e oxitetraciclina a 0,40 g L-1, duas vezes antes e duas vezes após a inoculação, em intervalos médios de 5-7 dias. Foram avaliados a incidência de folíolos doentes (inoculação foliar) e a severidade dos sintomas nas plantas inoculadas por ferimento no caule. Verificou-se que o isolado de Cmm foi sensível in vitro ao cloridrato de kasugamicina, ao fulusulfamide e à oxitetraciclina, respectivamente a partir das concentrações de 1000, 100 e 10 mig mL-1. Com relação aos ensaios in vivo, apenas oxitetraciclina propiciou menor incidência de folíolos doentes nas plantas inoculadas através de pulverização foliar; nenhum produto teve êxito em controlar a doença nas plantas inoculadas por ferimento no caule. O fulusulfamide, em todas as concentrações, foi fitotóxico aos folíolos das plantas de tomateiro.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Spirura delicata sp. n. (Spiruridae, Spirurinae) from Leontocebus mystax (Callithrichidae) and a check list of other Nematodes of some brazilian primates

Spirura delicata sp. n. from Leontocebus mystax (Six) is proposed and compared to S. guianensis (Ortlepp, 1924) Chitwood, 1938, S. michiganensis Sandground, 1935 and S. nayarani Mirza & Basir, 1938. Their differentiation is based mainly on the size and shape of spicules. Identification of nematode samples recovered from primates along 60 years and presently deposited in the Oswaldo Cruz Helmintological Collection is provided herein through a check list. Some of them are reported in new hsots and/or geographical regions.

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Biogeochemistry and microbiology of groundwater during acetate and bicarbonate amendment to an alluvial aquifer

These data are from a field experiment conducted in a shallow alluvial aquifer along the Colorado River in Rifle, Colorado, USA. In this experiment, bicarbonate-promoted uranium desorption and acetate amendment were combined and compared to an acetate amendment-only experiment in the same experimental plot. Data include names and location data for boreholes, geochemical data for all the boreholes between June 1, 2010 and January 1, 2011, microarray data provided as signal to noise ratio (SNR) for individual microarray probes, microarray data provided as signal to noise ratio (SNR) by Genus.

Recent advances in the molecular biology of Leifsonia xyli subsp xyli, causal organism of ratoon stunting disease

Twelve years ago our understanding of ratoon stunting disease (RSD) was confined almost exclusively to diagnosis of the disease and control via farm hygiene, with little understanding of the biology of the interaction between the causal agent (Leifsonia xyli subsp. xyli) and the host plant sugarcane (Saccharum spp. hybrids). Since then, research has focused on developing the molecular tools to dissect L. xyli subsp. xyli, so that better control strategies can be developed to prevent losses from RSD. Within this review, we give a brief overview of the progression in research on L. xyli subsp. xyli and highlight future challenges. After a brief historical background on RSD, we discuss the development of molecular tools such as transformation and transposon mutagenesis and discuss the apparent lack of genetic diversity within the L. xyli subsp. xyli world population. We go on to discuss the sequencing of the genome of L. xyli subsp. xyli, describe the key findings and suggest some future research based on known deficiencies that will capitalise on this tremendous knowledge base to which we now have access.

DEVELOPMENT OF A 2-DIMENSIONAL FINITE VOLUME MODEL TO ASSESS HYDRODYNAMIC AND MICROBIAL CONTROLS ON DNAPL DISSOLUTION AND DETOXIFICATION

Tetrachloroethene (PCE) and trichloroethene (TCE) form dense non-aqueous phase liquids (DNAPLs), which are persistent groundwater contaminants. DNAPL dissolution can be "bioenhanced" via dissolved contaminant biodegradation at the DNAPL-water interface. This research hypothesized that: (1) competitive interactions between different dehalorespiring strains can significantly impact the bioenhancement effect, and extent of PCE dechlorination; and (2) hydrodynamics will affect the outcome of competition and the potential for bioenhancement and detoxification. A two-dimensional coupled flowtransport model was developed, with a DNAPL pool source and multiple microbial species. In the scenario presented, Dehalococcoides mccartyi 195 competes with Desulfuromonas michiganensis for the electron acceptors PCE and TCE. Simulations under biostimulation and low velocity (vx) conditions suggest that the bioenhancement with Dsm. michiganensis alone was modestly increased by Dhc. mccartyi 195. However, the presence of Dhc. mccartyi 195 enhanced the extent of PCE transformation. Hydrodynamic conditions impacted the results by changing the dominant population under low and high vx conditions.