Molecular characterization of Portuguese populations of the pinewood nematode Bursaphelenchus xylophilus using cytochrome b and cellulase genes

Bursaphelenchus xylophilus is the causal agent of pine wilt disease and a worldwide pest with high economic impact. Since its first diagnosis in Portugal in 1999, it has been subjected to quarantine measures with impact on forest health and ecosystem stability, significantly affecting international trade of wood products. The disease was detected in the north and centre of continental Portugal and, since 2008, the whole country has been considered an affected area. Recently, it was detected in Madeira Island. In order to avoid new outbreaks, it has become of major importance to understand the patterns of spread, introduction points and to characterize the new populations from continental Portugal and Madeira Island. Mitochondrial cytochrome b (cytb) and parasitic cellulase gene sequences were used to evaluate the genetic relationships among isolates that could indicate possible origins of the new outbreaks. Portuguese isolates were compared with isolates from USA, China, Japan and South Korea, in order to investigate possible infection pathways and disease spread patterns in Portugal. Phylogenetic trees based on both genes show that Portuguese isolates group with Asian isolates. Isolates from USA are in a separate position in both gene trees. However, the phylogenetic tree based on the cellulase gene sequences shows higher differentiation among Portuguese isolates than that of cytb. These results agree with those previously obtained using inter-simple sequence repeats (ISSR). This was the first study to use cytb and cellulase genes to characterize pinewood nematode (PWN) populations. This study suggests that cellulase is a better marker than cytb to study genetic diversity in B. xylophilus

Rhizobial Strain Improvement: Genetic Analysis and Modification

Plant genetic engineering has revolutionized our ability to produce genetically improved plant varieties. A large portion of our major crops have undergone genetic improvement through the use of recombinant DNA techniques in which microorganisms play a vital role. The cross-kingdom transfer of genes to incorporate novel phenotypes into plants has utilized microbes at every step-from cloning and characterization of a gene to the production of a genetically engineered plant. This book covers the important aspects of Microbial Biotechnology in Agriculture and Aquaculture with and aim to improve crop yield

Fermentation – Processes, Benefits and Risks

This book covers a variety of topics in the field of fermentation processes. With evolving technology and increasing knowledge regarding the benefits and risks of distinct fermentation processes, new information is available on diverse topics in this broad field. Chapters present information on industrial applications of different fermentation processes and different aspects of fermented foods such as milk, cheese, coffee, and many others. The book compiles current, expert information useful to both students and researchers

Safety conditions and native microbial flora of three processing units in Alentejo , Portugal

Portugal as other Mediterranean countries has a great diversity of dry fermented sausages. This traditional sausage production is highly diverse and products possess very particular organoleptic characteristics, which please consumers. These sensory characteristics are related not only to the manufacturing process, but also to the house microbial flora. On the other hand, the safety of fermented products is always difficult to achieve due to their processing technology and final characteristics, as the process does not have any step able to undoubtedly eliminate pathogens, according to HACCP approach. In this kind of products, coagulase negative staphylococci (CNS) and lactic acid bacteria (LAB) are present in high levels. These bacteria are responsible for the particular taste and flavour and also for extending the shelf life of sausages. LAB are useful particularly for the ability to produce bacteriocins that are generally active towards several foodborne pathogens, such as Escherichia coli, Listeria monocytogenes and Salmonella sp., while CNS are mainly nitrate reducers, thus contributing to flavour development. The aim of this study was to determine the occurrence of Salmonella sp., L. monocytogenes and E. coli in different surfaces of the three visited processing units as well as in meat batters, half ripened and final products (Portuguese traditional sausages). Furthermore, we also evaluated the total number of mesophiles in each surface and product, as well as the number of CNS and LAB. Surfaces were generally well hygienised, only a few presenting with low contamination levels by L. monocytogenes and E. coli. No contamination with Salmonella sp. was detected in the diverse analysed products independently of their processing stage. However, different contaminations levels with L. monocytogenes and E. coli were found in distinct processing units

Molecular Tools in Microbial Ecology

Editors: Laia Diaz and Marta Perez Book Description: The new book presents the latest research on ecology which is the study of the interrelationships between organisms and their environment, including the biotic and abiotic components. There are at least six kinds of ecology: ecosystem, physiological, behavioral, population, and community. Specific topics include: Acid Deposition, Acid Rain Revisited, Biodiversity, Biocomplexity, and Carbon Sequestration in Soils, Coral Reefs, Ecosystem Services, Environmental Justice, Fire Ecology, Floods, Global Climate Change, Hypoxia, and Invasion

Genes commonly involved in acid tolerance are not overexpressed in the plant microsymbiont Mesorhizobium loti MAFF303099 upon acidic shock

Rhizobia are legume bacterial symbionts that fix nitrogen in the root nodules of plants. The aim of the present study was to investigate the global transcriptional response of rhizobia upon an acidic shock. Changes in the transcriptome of cells of Mesorhizobium loti strain MAFF303099 upon an acidic shock at pH 3 for 30 min were analysed. From a total of 7,231 protein-coding genes, 433 were found to be differentially expressed upon acidic shock, of which 322 were overexpressed. Although most of the overexpressed genes encode hypothetical proteins, the two most represented Cluster of Orthologous Group (COG) categories are ‘defence mechanisms’ and ‘transcription’. Differentially expressed genes are dispersed throughout the chromosome, with the exception of the symbiosis island, where most genes remain unchanged. A significant number of transcriptional regulators and ABC transporter genes are overexpressed. No overexpression of genes typically associated to acid tolerance in rhizobia, such as act and exo genes, was detected. Overall, this study suggests a transcriptional response to acidic shock of M. loti distinct from other rhizobia. Additional studies are in course to explore the role of some of the highly overexpressed genes and to further elucidate the molecular bases of acid stress response

Natural populations of chickpea rhizobia evaluated by antibiotic resistance profiles and molecular methods

Abstract The aims of this study were to investigate the hypothesis that intrinsic antibiotic resistance (IAR) profiles of chickpea rhizobia are correlated with the isolates site of origin, and to compare the discriminating power of IAR profiles with molecular approaches in rhizobial strain identification and differentiation. Rhizobial diversity from five Portuguese soils was assessed by IAR profiles and molecular methods [16S rDNA restriction fragment length polymorphism (RFLP) analysis, direct amplified polymorphic DNA (DAPD) fingerprinting, and SDS–PAGE analysis of protein profiles]. For each analysis, a dendrogram was generated using the software BioNumerics. All three molecular methods generated analogous clustering of the isolates, supporting previous results on 16S rDNA sequence-based phylogeny. Clusters obtained with IAR profile are similar to the species groups generated with the molecular methods used. IAR groups do not correlate significantly with the geographic origin of the isolates. These results may indicate a chromosomal location of antibiotic resistance genes, and suggest that IAR is species related. DAPD and IAR profiles proved to be the most discriminating approaches in strain differentiation and can be used as fast methods to screen diversity in new isolates

Addition of vinegar to "cabeça de xara" made from Alentejano pig meat increases shelf life

“Cabeça de xara” is a very typical product from Alentejo region. It’s a very particular type of galantine, made from meat, tongue and connective tissue removed from Alentejano pig breed heads. Usually this product has a parallelepiped shape, as those used in the present study, with 40 cm length and 14 cm height. The aim of this study was to evaluate the use of vinegar to increase the products’ shelf life without negatively affecting the physicochemical and microbiological parameters, as well as its sensorial attributes. Two different treatments were compared, namely control and vinegar, for three months using end product “cabeças de xara”. Three batches with two replicates per treatment were used. One-Way ANOVA was used for statistical analyses. The mean pH of the “cabeças de xara” with vinegar (5.48) was lower than the pH of the control treatment (6.05). However, no significant differences were observed between control and vinegar treatments regarding water activity (aw). The microbiological flora of the “cabeças de xara” showed no significant differences regarding yeasts and moulds. The counts of total mesophiles, psychrophiles, psychrophilic anaerobes and enterobacteria were higher in the control treatment. Only a few samples showed a minor contamination with Listeria monocytogenes. As expected, the counts for all microbial groups increased with time. Regarding sensorial analysis, panellists generally gave higher scores to the control “cabeças de xara”, however no differences were observed between control and vinegar “cabeças de xara” concerning the attributes marbled and colour and aroma intensity. Considering the results of the end product at three months shelf life, the vinegar “cabeças de xara” showed lower microbial counts, probably due to the lower pH values. Our data suggest that the sensorial panel prefers the control “cabeças de xara”; however, the panellists did not taste some of the control products due to contamination with Listeria monocytogenes. To our knowledge, this is the first report on the physicochemical, microbiological and sensorial characteristics of such a traditional product. Acknowledgements This work was supported by national funds through project PRODER (QREN/PRODER/Medida 4.1) and funded by FEDER Funds through the COMPETE and National Funds through FCT under the Strategic Project PEst-C/AGR/UI0115/2011. The authors thank PALADARES ALENTEJANOS, Lda. for their collaboration and A. Oliveira and G. Pias for their technical assistance

Effect of heat and pH stress in the growth of chickpea mesorhizobia

Abstract The development of rhizobial inoculants requires the selection of isolates that are symbiotically efficient as well as adapted to the local environmental conditions. Our aim was to find indigenous chickpea rhizobia tolerant to adverse environmental conditions, such as temperature and pH. Thirteen isolates of chickpea mesorhizobia from southern Portugal were examined. Tolerance to stress temperatures and pH was evaluated by quantification of bacterial growth at 20–37°C and pH 5–9, respectively. Tolerance to heat shocks was studied by submitting isolates to 46°C and 60°C. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis protein analysis revealed qualitative and quantitative differences when isolates were submitted to temperature stress. A 60-kDa protein was overproduced by all isolates under heat stress. Almost all isolates revealed to be more tolerant to 20°C than to 37°C. A positive correlation was found between the maximum growth pH and the isolate origin soil pH. Generally, isolates more tolerant to temperature stress showed a lower symbiotic efficiency

V PhD Students Meeting in Environmental and Agriculture

The V EEDAA is organized by MED – Mediterranean Institute for Agriculture, Environment and Development and IIFA – Institute for Advanced Studies and Research, University of Évora and is supported by UNIMED – Mediterranean Universities Union. The EEDAA focuses on four main areas: Biology and Biochemistry, Veterinary Sciences and Animal Production, Agricultural Sciences and Food Sciences, and Ecology, Environment and Landscape. The meeting will include one invited plenary lecture and several presentations selected from the abstracts submitted by PhD students. In addition, all authors will have the opportunity to present the posters in the two poster sessions included in the program. This meeting intends to stimulate the interaction between PhD students, to streamline scientific discussion and highlight the ones who will become the researchers of the future. Finally, we wish to thank the authors who have contributed to the scientific program