Adaptation and development of culture-independent techniques for the indentification and enumeration of microorganisms in wine fermentations

L'objectiu d'aquesta tesi va ser l'adaptació i validació de diferents tècniques independents de cultiu per a la detecció i quantificació de la microbiota present a la fermentació vínica. Es van assajar la QPCR (PCR quantitativa) per a la quantificació i seguiment d'espècies clau i la diversitat ecològica es va analitzar per DGGE (electroforesi en gel desnaturalitzant) i la clonació d'un fragment ribosomal.Tanmateix es va estudiar l'aplicació de la Hibridació in Situ (FISH) i la QPCR amb colorants específics per tal de diferenciar cèl·lules vives i mortes. Aquestes tècniques es varen aplicar a fermentacions industrials, essent notable la detecció de bacteris acètics i llevats No-Saccharomyces en concentracions superiors a les detectades com a poblacions cultivables. Es van estudiar interaccions entre llevats Saccharomyces i No-Saccharomyces al laboratori, observant-ne la supervivència d'aquestes en estats viables però no cultivables. Aquestes tècniques independents de cultiu indiquen una població i dinàmiques microbianes prèviament desapercebudes.The objective of this thesis was the adaptation and validation of different culture-independent techniques for detection and quantification of the microbiota present in wine fermentation. We tested the QPCR (PCR quantitative) for the quantification and monitoring of key species. The ecological diversity was analyzed by DGGE (denaturing gradient gel electrophoresis) and by cloning of a ribosomal fragment. However, we studied the application of in Situ Hybridization (FISH) and QPCR with specific dyes to differentiate between live and dead cells. These techniques were applied in industrial fermentations, being significant the detection of acetic acid bacteria and Non-Saccharomyces yeast in concentrations higher than those identified as culturable populations. We studied the interactions between Saccharomyces and Non-Saccharomyces yeast in the laboratory, observing their survival in these viable but non-culturable state. These culture-independent techniques indicate a population and microbial dynamics previously unnoticed

Identification of Eurycea Using Cytochrome b

Genomic sequencing is a powerful tool that has many applications for research, one of which is in the field of taxonomy and the identification of species. This thesis discusses the mitochondrial gene cytochrome b and its utility in population genetics and identification of larval amphibians. The development of the Polymerase Chain Reaction and primers are an integral part of the modern DNA sequencing process. The Polymerase Chain Reaction is used to amplify a target DNA sequence, and the protocol for this procedure must be optimized for the specific sequence of target DNA. Primers must also be designed and modified for a selected portion of the DNA to be copied. This thesis also discusses the application of these techniques in a current study of a population of Eurycea. Three species of Eurycea, the Cave Salamander (E. lucifuga), Long-tailed Salamander (E. longicauda longicauda), and Three-lined Salamander (E. guttolineata) were discovered in an abandoned mine shaft near Riverville, Amherst County, Virginia in 1999. The population is unusual, as E. longicauda longicauda and E. lucifuga are outside their normal distribution and this was the first syntopic occurrence in Virginia of E. guttolineata and E. longicauda longicauda, the species usually indigenous to the Piedmont physiographic region. In addition, there is possible hybridization between E. guttolineata and E. longicauda. The larvae of these species are difficult to identify morphologically; so the paper discusses the use of cytochrome b sequencing for species identification among this population of Eurycea

Cyanobacterial diversity in Salar de Huasco, a high altitude saline wetland in Northern Chile, are highly similar to Antarctic cyanobacteria

The diversity of Cyanobacteria in water and sediment samples from four representative sites of the Salar de Huasco was examined using denaturing gradient gel electrophoresis and analysis of clone libraries of 16S rRNA gene PCR products. Salar de Huasco is a high altitude (3800 m altitude) saline wetland located in the Chilean Altiplano. We analyzed samples from a tributary stream (H0) and three shallow lagoons (H1, H4, H6) that contrasted in their physicochemical conditions and associated biota. Seventy-eight phylotypes were identified in a total of 268 clonal sequences deriving from seven clone libraries of water and sediment samples. Oscillatoriales were frequently found in water samples from sites H0, H1 and H4 and in sediment samples from sites H1 and H4. Pleurocapsales were found only at site H0, while Chroococcales were recovered from sediment samples of sites H0 and H1, and from water samples of site H1. Nostocales were found in sediment samples from sites H1 and H4, and water samples from site H1 and were largely represented by sequences highly similar to Nodularia spumigena. We suggest that cyanobacterial communities from Salar de Huasco are unique - they include sequences related to others previously described from the Antarctic, along with others from diverse, but less extreme environments

Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens

Phytopathogenic fungi affecting crop and post-harvested vegetables are a amajor threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. RESULTS: The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. CONCLUSION: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents

Considerable MHC Diversity Suggests That the Functional Extinction of Baiji Is Not Related to Population Genetic Collapse

To further extend our understanding of the mechanism causing the current nearly extinct status of the baiji (Lipotes vexillifer), one of the most critically endangered species in the world, genetic diversity at the major histocompatibility complex (MHC) class II DRB locus was investigated in the baiji. Nine highly divergent DRB alleles were identified in 17 samples, with an average of 28.4 (13.2%) nucleotide difference and 16.7 (23.5%) amino acid difference between alleles. The unexpectedly high levels of DRB allelic diversity in the baiji may partly be attributable to its evolutionary adaptations to the freshwater environment which is regarded to have a higher parasite diversity compared to the marine environment. In addition, balancing selection was found to be the main mechanisms in generating sequence diversity at baiji DRB gene. Considerable sequence variation at the adaptive MHC genes despite of significant loss of neutral genetic variation in baiji genome might suggest that intense selection has overpowered random genetic drift as the main evolutionary forces, which further suggested that the critically endangered or nearly extinct status of the baiji is not an outcome of genetic collapse

Endophytic Bacillus subtilis ZZ120 and its potential application in control of replant diseases

An endophytic bacterial strain ZZ120 that was isolated from healthy stems of Prunus mume (family: Rosaceae) was identified as Bacillus subtilis based on biochemical and physiological assays and 16s rRNA, rpoB and tetB-yyaO / yyaR genes analysis. Both the culture filtrate and the n-butanol extract of strain ZZ120 showed strong growth inhibition activity in vitro against the replant disease phytopathogens Fusarium graminearum, Alternaria alternata, Rhizoctonia solani, Cryphonectria parasitica and Glomerella glycines. The active metabolite in the filtrate was found to be produced 24 h after inoculation and the concentration remained at a high level until 66 h and was quite thermally stable with more than 75% of the antifungal activity even after being held at 121°C for 30 min. In addition, the antifungal activity of the filtrate remained almost unchanged when the culture was exposed to a pH ranging from 1 to 8, but significantly reduced after the filtrate had been exposed to alkali conditions (pH 9 to 14) for 30 min. The antifungal compounds were isolated from n-butanol extract as a mixture of iturins. The strong antifungal activity suggested that the endophytic B. subtilis ZZ120 and its bioactive components might provide an alternative agent for the biocontrol of replant diseases.Key words: Endophytic bacterium, Bacillus subtilis, replant pathogens

Reference genome of Lumpfish Cyclopterus lumpus Linnaeus provides evidence of male heterogametic sex determination through the AMH pathway

acceptedVersio

Controlling Tungiasis in an Impoverished Community: An Intervention Study

Tungiasis is a disease caused by the sand flea Tunga penetrans, a parasite prevalent in many impoverished communities in developing countries. The female sand flea penetrates into the skin of animals and humans where it grows rapidly in size, feeds on the host's blood, produces eggs which are expelled into the environment, and eventually dies in situ. The lesions become frequently superinfected and the infestation is associated with considerable morbidity. Clearly, tungiasis is a neglected disease of neglected populations. We investigated the impact of a package of intervention measures targeted against on-host and off-host stages of T. penetrans in a fishing community in Northeast Brazil. These measures decreased disease occurrence only temporarily, but had a sustained effect on the intensity of the infestation. Since infestation intensity and morbidity are correlated, presumably the intervention also lowered tungiasis-associated morbidity. Control measures similar to the ones used in this study may help to effectively control tungiasis in impoverished communities