Phenotypic and genotypic characterization of Paenibacillus larvae isolates

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.     

Resonance of scantlings indicates the stiffness even of small specimens of Eucalyptus from plantations

The aim of this study was to establish the relationship between the stiffness of scantlings, approaching structural size materials, and small specimens of 6-year-old Eucalyptus urophylla?×?grandis wood using a resonance technique. The correlation between the elastic modulus (E) of the scantlings in longitudinal vibration and small specimens in flexural vibration was r?=?75 when comparing the scantling values with the averaged values of the specimens per scantling. However, when the E of each single specimen was compared with its respective scantling, the coefficient of correlation decreased to r?=?0.64 in the longitudinal tests and r?=?0.61 in the flexural tests. A roughly linear correlation (r?=?0.59) between specific modulus and loss tangent was obtained for the small specimens of Eucalyptus. In short, the resonance technique rapidly provided a large, accurate data set of mechanical wood properties as required for high-throughput phenotyping in recent genetic studies.     

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.     

Bioremediation of Mangroves Impacted by Petroleum

The majority of oil from oceanic oil spills (e.g. the recent accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves. Microorganisms are directly involved in biogeochemical cycles as key drivers of the degradation of many carbon sources, including petroleum hydrocarbons. When properly understood and managed, microorganisms provide a wide range of ecosystem services, such as bioremediation, and are a promising alternative for the recovery of impacted environments. Previous studies have been conducted with emphasis on developing and selecting strategies for bioremediation of mangroves, mostly in vitro, with few field applications described in the literature. Many factors can affect the success of bioremediation of oil in mangroves, including the presence and activity of the oil-degrading microorganisms in the sediment, availability and concentration of oil and nutrients, salinity, temperature and oil toxicity. More studies are needed to provide efficient bioremediation strategies to be applicable in large areas of mangroves impacted with oil. A major challenge to mangrove bioremediation is defining pollution levels and measuring recuperation of a mangrove. Typically, chemical parameters of pollution levels, such as polycyclic aromatic hydrocarbons (PAHs), are used but are extremely variable in field measurements. Therefore, meaningful mangrove monitoring strategies must be developed. This review will present the state of the art of bioremediation in oil-contaminated mangroves, new data about the use of different mangrove microcosms with and without tide simulation, the main factors that influence the success of bioremediation in mangroves and new prospects for the use of molecular tools to monitor the bioremediation process. We believe that in some environments, such as mangroves, bioremediation may be the most appropriate approach for cleanup. Because of the peculiarities and heterogeneity of these environments, which hinder the use of other physical and chemical analyses, we suggest that measuring plant recuperation should be considered with reduction in polycyclic aromatic hydrocarbons (PAHs). This is a crucial discussion because these key marine environments are threatened with worldwide disappearance. We highlight the need for and suggest new ways to conserve, protect and restore these environments.     

Molecular phylogeny and morphological characterization of the aetiological agent of sour rot on fruits and vegetables in Brazil

In Brazil, sour rot is an important postharvest disease on fruits and vegetables. Geotrichum candidum (synonym Galactomyces candidus) has been reported as the main species causing this disease. However, the identity of the causal agent is still uncertain. This research aimed to determine the identity of 165 fungal isolates associated with sour rot obtained from fruits and vegetables in Brazil, and to evaluate the effect of different temperatures on the incidence of sour rot on artificially inoculated tomato fruits. Based on the phylogenetic analyses of DNA sequences from the D1/D2 domain of the large subunit (LSU) rRNA gene and morphological analyses, 129 samples belonged to Galactomyces candidus, 15 to G. candidum var. citri-aurantii, 6 to G. phurueaense, 2 to Gal. pseudocandidus, 1 to Hyphopichia burtonii, 1 to H. khmerensis, 3 to Saccharomycopsis crataegensis, 1 to S. vini, 1 to Magnusiomyces tetrasperma, 1 to Trichosporon coremiiforme, and 1 to Zygoascus meyerae. Two new species were found, namely, Geotrichum solani (on potato) and Geotrichum spondiadis (on red mombin). All isolates were pathogenic when inoculated on healthy tomato fruits, including the new species of Geotrichum, which were also inoculated into their respective hosts, that is, potatoes and red mombins. To the best of our knowledge, this study is the first to report the presence of five other genera besides Geotrichum associated with sour rot on fruits and vegetables in Brazil, which demonstrates the diversity of fungi and yeasts associated with this disease.     

Chemical characterization of Cuban propolis by HPLC-PDA, HPLC-MS, and NMR: the brown, red, and yellow Cuban varieties of propolis

Sixty-five samples of propolis were collected from eleven regions of Cuba; methanolic extracts of propolis were prepared from all samples, and a classification method was developed using a combination of NMR, HPLC-PDA, and HPLC-ESI/MS techniques. The analysis of (1)H and (13)C NMR spectra and chromatographic profiles of all propolis extracts allowed the definition of three main types of Cuban propolis directly related to their secondary metabolite classes: brown Cuban propolis (BCP), rich in polyisoprenylated benzophenones, red Cuban propolis (RCP), containing isoflavonoids as the main constituents, and yellow Cuban propolis (YCP), probably with aliphatic compounds. Subsequently, the principal compounds of the brown and red types were characterized by HPLC-ESI/MS analysis. Instrumental techniques used are complementary; NMR was shown to be a quick and informative tool for the rapid analysis of crude propolis polar extracts and allowed the identification of the main class of secondary metabolites, while LC-PDA and LC-MS techniques were useful tools for qualitative and quantitative analysis of marker compounds of Cuban propolis.     

Genetic mapping provides evidence for the role of additive and non-additive QTLs in the response of inter-specific hybrids of <Emphasis Type="Italic">Eucalyptus</Emphasis> to <Emphasis Type="Italic">Puccinia psidii</Emphasis> rust infection

Eucalypts are susceptible to a wide range of diseases. One of the most important diseases that affect Eucalyptus plantations worldwide is caused by the rust fungus Puccinia psidii. Here, we provide evidence on the complex genetic control of rust resistance in Eucalyptus inter-specific hybrids, by analyzing a number of full-sib families that display different patterns of segregation for rust resistance. These families are totally unrelated to those previously used in other inheritance studies of rust resistance. By using a full genome scan with 114 genetic markers (microsatellites and expressed sequence tag derived microsatellites) we also corroborated the existence and segregation of a resistance locus, explaining 11.5% of the phenotypic variation, on linkage group 3, corresponding to Ppr1. This find represents an additional validation of this locus in totally unrelated pedigree. We have also detected significant additive × additive digenic interactions with LOD >10.0 on several linkage groups. The additive and epistatic QTLs identified explain between 29.8 and 44.8% of the phenotypic variability for rust resistance. The recognition that both additive and non-additive genetic variation (epistasis) are important contributors to rust resistance in eucalypts reveals the complexity of this host-pathogen interaction and helps explain the success that breeding has achieved by selecting rust-resistant clones, where all the additive and non-additive effects are readily captured. The positioning of epistatic QTLs also provides starting points to look for the underlying genes or genomic regions controlling this phenotype on the upcoming E. grandis genome sequence.     

A common fold mediates vertebrate defense and bacterial attack

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.